ISOLATION OF GENES FROM COLD ACCLIMATED Poncirus trifoliata AND Citrus unshiu Except where reference is made to the work of others, the work described in this dissertation is my own or was done in collaboration with my advisory committee. This dissertation does not include proprietary or classified information. Cankui Zhang Certificate of Approval: Fenny Dane, Co-Chair Associate Professor Horticulture Robert D. Locy Professor Biological Sciences Narendra K. Singh Professor Biological Sciences Robert C. Ebel, Co-Chair Associate Professor Horticulture Zhanjiang (John) Liu Professor Fisheries and Allied Aquacultures Stephen L. McFarland Dean Graduate School ISOLATION OF GENES FROM COLD ACCLIMATED Poncirus trifoliata AND Citrus unshiu Cankui Zhang A dissertation Submitted to the Graduate Faculty of Auburn University in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy Auburn, Alabama August 8, 2005 ISOLATION OF GENES FROM COLD ACCLIMATED Poncirus trifoliata AND Citrus unshiu Cankui Zhang Permission is granted to Auburn University to make copies of this dissertation at its discretion, upon request of individuals or institutions and at their expense. The author reserves all publication rights. Signature of Author Date iii VITA Cankui Zhang, son of Tianqi Zhang and Yueping Song, was born on November 18, 1971 in Luoyang city, Henan Province, the Peoples? Republic of China. He graduated from Mengjin No. 1 High School in 1990. He entered Henan University in September 1990 and earned a Bachelor of Science degree in Biology in July 1994. He entered Wuhan Institute of Botany, the Chinese Academy of Sciences in September 1994, and earned a Master of Science degree in Botany in July 1997. From 1997 to 2000, he worked as a research assistant professor in Wuhan Institute of Botany, the Chinese Academy of Sciences. In August 2000, he entered the University of Arkansas at Fayetteville to pursue a Doctor of Philosophy degree in Entomology. In August 2001, he transferred to Auburn University to pursue a Doctor of Philosophy degree in Horticulture. He married Ping Lang in November 1998. They have a son, Jingzhe Zhang, and a daughter, Jingran (zoe) Zhang. iv DISSERTATION ABSTRACT ISOLATION OF GENES FROM COLD ACCLIMATED Poncirus trifoliata AND Citrus unshiu Cankui Zhang Doctor of Philosophy, August 8, 2005 (M.S., Wuhan Institute of Botany, the Chinese Academy of Sciences, 1997) (B.S., Henan University, 1994) 131 Typed pages Directed by Fenny Dane and Robert C. Ebel Many adverse environmental conditions can affect the productivity and distribution of plant species. Low temperature is one of the most common stresses faced by many plants. Many plants are able to increase their freezing tolerance ability in response to a period of low, non-freezing temperature, a process called cold acclimation. During this process, various physiological and biochemical changes occurred in order to impart cold tolerance capacity to plants. Cell metabolom and proteome changes due to the adjustment at transcription level have been postulated and investigated in the process of cold acclimation in many plants, especially in Arabidopsis. Many genes have been isolated in response to low temperature stress in many herbaceous plants, while very limited information is available for woody plants, including economically important fruit crops, such as citrus. Citrus species are cold-tender evergreen plants with a tropical and subtropical origin. Citrus unshiu (one kind of Satsuma) is one of the most cold hardy commercial v Citrus species which can tolerate temperature as low as ?9 to ?10 degree C. Poncirus trifoliata, with a maximum freeze tolerance ability of ?30 degree C, is a deciduous relative of Citrus that is often used as rootstock to enhance freeze tolerance of the other Citrus species. In order to gain an understanding of the molecular mechanisms of these two species under cold temperature and compare their responses to low temperature, mRNA differential display and cDNA amplified fragment length polymophism (cDNA- AFLP) were used to identify the cold responsive genes under a gradually declined temperature regime. With relative quantitative RT-PCR, genes as follows were identified as differentially expressed in P. trifoliata and C. unshiu: betaine/proline transporter, water channel protein, aldo-keto reductase, early light induced protein, nitrate transporter, tetratricopeptide-repeat protein, F-box protein, ribosomal protein L15, chlorophyll a/b binding protein, photosystem II OEC 23, carbonic anhydrase, tumor related protein, pyrrolidone-carboxylate peptidase, ?-galactosidase, translation initiaton factor eIF1, cytochrome C, trigger factor type chaperone family protein, polyprotein, leucine-rich repeat transmembrane protein kinase/receptor-like protein kinase, PAZ/PIWI domain containing protein, 40S ribosomal protein S23, amino acid permease 6, miraculin-like protein 2, 14-3-3 d-2 protein, nucleoside diphosphate kinase III, regulator of chromosome condensation like protein and glutathione S-transferase C-terminal domain containing protein. Osmotic adjustment, photo-oxidative protection and photosynthesis adaptation were suggested to be the main mechanisms for these plants to acclimate to low temperatures. The full length sequences of carbonic anhydrase, proline transporter and nitrate transporter have been obtained. The detailed characterization of these genes are ongoing. vi ACKNOWLEDGEMENTS The author would like to thank the members of his research committee Dr. Fenny Dane, Dr. Robert C. Ebel, Dr. Narendra K. Singh, Dr. Robert Locy and Dr. Zhanjiang Liu for their constructive advices and complete support during his research. He would also like to thank Dr. William Dozier, Mr. Brandon Hockema, Mr. Bryan Wilkins and Mr. Monte Nesbitt for their help in his work. Thanks are also given to his group member, Ms. Rasima Bakhtiyarova, and other faculty and graduate students who were able to given their helps during this research. His deepest gratefulness goes to his parents, Tianqi Zhang and Yueping Song, and wife, Ping Lang, who always support and encourage him during his research. vii Style manual of journal used Plant Cell Reports Computer software used Microsoft word 98, Vector NTI viii TABLE OF CONTENTS LIST OF TABLES ix LIST OF FIGURES xi I LITERATURE REVIEW ?????????????????????...1 II COLD ACCLIMATION INDUCED GENES OF TRIFOLIATE ORANGE (Poncirus trifoliata) ???????????????????????..29 III COLD ACCLIMATION DOWN REGULATED GENES IN Poncirus trifoliate????????????????????????????..50 IV IDENTIFICATION OF COLD ACCLIMATED GENES IN LEAVES OF Citrus unshiu BY mRNA DIFFERENTIAL DISPLAY????????????...75 V COLD ACCLIMATION GENES IN Citrus unshiu ?OWARI???????..100 VI CONCLUSIONS, ONGOING RESEARCH AND FUTURE PERSPECTIVES????????????????????????.116 ix LIST OF TABLES II. 1. Oligonucleotide primer sequences, PCR conditions and cycle numbers for the confirmation of differential displayed products with quantitative RT-PCR??..47 2. Isolated DD products, accession number and percentage similarity to known proteins by BLASTx search in NCBI ???..?????????????.48 III. 1. Oligonucleotide primer sequences, PCR conditions and cycle numbers for the confirmation of different displayed products with quantitative RT-PCR ???.71 2. Isolated DD products, accession number and percentage similarity to known genes by BLASTx search in NCBI ???..?????????????.....72 IV. 1. Oligonucleotide primer sequences, PCR conditions and cycle numbers for the confirmation of different displayed products with quantitative RT-PCR???..96 2. Isolated DD products, accession number and percentage similarity to known genes by BLASTx search in NCBI ???..??????????????.97 V. 1. Oligonucleotide primer sequences, PCR conditions and cycle numbers for the confirmation of differentially expressed products with quantitative RT-PCR....113 2. Isolated cDNA AFLP products, accession number in Genbank and percentage similarity to known genes by BLASTx search in NCBI???..??????114 x LIST OF FIGURES II. 1. (Top): Confirmation of differential expression of 8 DD products using relative quantitative RT-PCR with RNA from control and cold acclimated leaves. The cDNAs were synthesized from total RNAs isolated from control leaves (end of second week) and cold acclimation leaves (end of fifth week). Actin mRNA was used as an internal control. Specific RT-PCR primer pair information for all DD products is listed in table 1. (Bottom): A histogram showing the relative abundance of 8 DD products between control and cold acclimated plants. The intensities in each control and cold acclimation pair were normalized by setting the intensity of actin gene to 100. The values are the means of three independent experiments +SE.???? ?????.49 III. 1. Example of DDRT-PCR results. cDNAs were amplified from two separately isolated total RNAs by primer combination of HT 11 A and HAP 1. C 1 and C 2 are cDNAs amplified from the leaves collected at the end of second week (control); T 1 and T 2 are cDNAs amplified from the leaves collected at the end of fifth week (cold acclimated). Arrows 1 and 2 indicate two up regulated cDNA fragments; Arrows 3 indicates one down regulated cDNA fragment. ????.???????????????...73 2. (top): Confirmation of differential expression of 6 DD products using relative quantitative RT-PCR with RNA from control and cold acclimated leaves. The xi cDNAs were synthesized from total RNAs isolated from control leaves (end of first week) and cold acclimation leaves (end of fifth week). Actin gene was used as an internal control. Specific RT-PCR primer pair information for all DD products was listed in table 1. (Bottom): A histogram showing the relative abundance of 6 DD products between control and cold acclimated plants. The intensities in each control and cold acclimation pair were normalized by setting the intensity of actin gene to 100. The values are the means of three independent experiments + SE?????.74 IV. 1. Example of DDRT-PCR results. cDNAs were amplified from two separately isolated total RNAs by primer combination of HT 11 C and HAP 1. C 1 and C 2 are cDNAs amplified from the leaves collected at the end of second week (control); T 1 and T 2 are cDNAs amplified from the leaves collected at the end of fifth week (cold acclimated). Arrow 1 indicates one up regulated cDNA fragment; Arrow 2 indicates one down regulated cDNA fragment??????????????????????....98 2. 2a: Confirmation of differential expression of 8 differential displayed (DD) products using relative quantitative RT-PCR with RNA from control and cold acclimated leaves. The cDNAs were synthesized from total RNAs isolated from control leaves (end of second week) and cold acclimation leaves (end of fifth week). Actin mRNA was used as an internal control. Specific RT-PCR primer pair information for all DD products is listed in table 1 2b: A histogram showing the relative abundance of 8 DD products between control and cold acclimated plants. The intensities in each control and cold acclimation pair xii were normalized by setting the intensity of actin mRNA to 100. The values are the means of three independent experiments + SE.??????????????...99 V. 1. (Top): Confirmation of differential expression of 6 cDNA AFLP products using relative quantitative RT-PCR with RNA from control and cold acclimated leaves. The cDNAs were synthesized from total RNAs isolated from control leaves (end of second week) and cold acclimation leaves (end of fifth week). Actin gene was used as an internal control. Specific RT-PCR primer pair information for all cDNA AFLP products was listed in table 1. (Bottom): A histogram showing the relative abundance of 6 cDNA AFLP products between control and cold acclimated plants. The intensities in each control and cold acclimation pair were normalized by setting the intensity of actin gene to 100. The values are the means of three independent experiments + SE???????????????...........................................115 xiii I. LITERATURE REVIEW 1.1 Cold acclimation Plants are sessile organisms. Adverse environmental conditions such as salinity, drought, nutritional deficiency and extreme temperature (high and low), can potentially cause significant losses of plant productivity and constrain the distribution of some major crops. Low temperature is one of the most common stresses faced by many plants. The ability of plants to respond to cold temperatures is an important factor in their ecological and evolutionary dynamics. Plants vary greatly in their ability to survive freezing temperatures. Many tropical and subtropical plant species cannot tolerate low temperatures and their biogeographical range is limited. In contrast, temperate herbaceous species or perennials can survive freezing temperatures ranging from ?5?C to ?30?C or even lower (Thomashow 1998). Temperate plants have evolved mechanisms by which they can increase their ability to withstand such low freezing temperatures after a period of pre-exposure under low but non-freezing temperatures, a process called cold acclimation (Levitt 1980). During the cold acclimation process, cold hardening develops, providing the plant with a tolerance to low temperatures that would be lethal to an unhardened plant (Howarth and Ougham 1993). For some perennial plants such as grape, the combination of declining day length and decreasing temperatures in autumn are important factors influencing acclimation and cold hardiness (Wample et al. 2000), which is thought to be a gradual process. 1 1.2 Freezing injuries and tolerance mechanisms 1.2.1. Membranes When exposed to low temperatures, plant cells encounter three main metabolic constraints: changes in the spatial organization of biological membranes, a retardation of biochemical and chemical reactions, and alterations in the availability and status of water (V?zina et al. 1997). Many studies have indicated that membrane disruption is the most severe injury caused by freezing (Levitt 1980; Steponkus 1984). Ice generally forms in intercellular spaces, mainly due to lower solute concentration of extracellular fluid and a higher freezing point (Thomashow 1999). The lower chemical potential of ice formed extracellularly is a driving force for the movement of water molecules from inside the cell, thus causing symplastic dehydration. Freezing injury is regarded to be a consequence of membrane lesions caused by freeze-induced dehydration (Steponkus 1984), although other factors may also contribute to cellular damage (Thomashow 1999). During thawing, the water potential gradient is reversed, and the cell?s cytosol can be rehydrated. This dehydration/rehydration cycle has significant effects on cellular ultrastructure (Hincha and Schmitt 1992). There are three types of damages based on the ways that low temperature caused to cell membrane. ?Expansion-induced lysis?, a process caused by the osmotic contraction and expansion cycle during freezing and thawing, occurs in non-acclimated plants. ?Lamellar-to-hexagonal II? phase transitions is another main injury in non-acclimated plants, a process involving the fusion of many cellular membranes. ?Fracture-jump lesions? occur due to the low water potential and severe dehydration due to extreme freezing (Steponkus et al. 1993). In addition membrane lesions, many loosely bound, peripheral membrane proteins are released 2 during freezing under injurious conditions (Volger et al. 1978; Hincha and Schmitt 1985), thus causing negative effects to cellular metabolism. 1.2.2. Oxidative stress At low temperatures, the balance between photosynthetic energy conversion and consumption is disrupted. When temperature declined, the ability of the plant to utilize captured light energy is substantially reduced, while the energy absorption and electron flow are less retarded, thus resulting in extra energy which can react with O 2 to produce 1 O 2 , hydrogen peroxide, and superoxide radicals O 2 ? (Asada 1994; Prasad et al. 1994; Fadzillah et al. 1996; O'Kane et al. 1996; Foyer 1997), which damage cellular components (Elstner 1991; McKersie 1991). Acclimation to low temperature may be partly related to an enhanced antioxidant system that would prevent the accumulation of these reactive oxygen species (ROS) (Prasad 1996). Different plant species have evolved different mechanisms to cope with low temperature related oxidative stress. Under natural conditions, low temperature induced accumulation of glutathione (GSH) has been observed in spruce (Picea abies L.) and white pine (Pinus strobes L.) during winter time (Esterbauer and Grill 1978; Anderson et al. 1992). Under experimental conditions, GSH was also found to be induced in response to low temperature in soybean, squash and wheat (Vierheller and Smith 1990; Wang 1995; Kocsy et al. 2000). It also has been found that chilling-tolerant plants increase endogenous polyamine (PA) levels in response to chilling to a much greater extent than chilling-sensitive ones (Guye et al. 1986; Lee 1997; Shen et al. 2000). In a chilling sensitive cucumber cultivar, pretreatment of leaves with PA alleviated chilling injuries, while in a chilling tolerant cucumber cultivar, pretreatment of leaves with PA synthesis inhibitor enhanced chilling injuries. 3 The primary function of PA is probably inhibition of chill-induced activation of microsomal NADPH oxidase and consequential ROS generation (Shen et al. 2000). Because of their polycationic nature at physiological pH, PA can bind strongly to negatively charged cellular components such as nucleic acids, proteins, and phospholipids. Interactions of PAs with membrane phospholipids may stabilize membranes under conditions of stress (Smith 1985; Roberts et al. 1986). In addition to the above responses to deal with the oxidative stress under low temperature, plant cells can synthesize lipid-soluble antioxidants ( -tocopherol and ?-carotene), water-soluble reductants (ascorbate and glutathione) and enzymes such as superoxide dismutase (SOD; EC 1.15.1.1), catalase (CAT; EC 1.11.1.6), ascorbate peroxidase (APX; EC 1.11.1.11), and glutathione reductase (GR; EC 1.6.4.2) (Zhang et al. 1995). These enzymes have important roles in detoxification of ROS. For instance, SOD can catalyze dismutation of superoxide to hydrogen peroxide and molecular oxygen (Bowler et al. 1992). Transgenic plants overexpressing SOD have exhibited enhanced tolerance to oxidative stress (Bowler et al. 1991; Perl et al. 1993). Sen Gupta et al. (1993) transformed a Cu-Zn-SOD from pea to tobacco and found that the photosynthetic rates of transgenic tobacco plants were approximately 20% higher than nontransformed plants when subjected to chilling temperature with moderate light intensity. Mn-SOD cDNA was transformed into alfalfa, resulting in an increase in freezing tolerance. Mn-SOD functions to minimize the deleterious accumulation of active oxygen and the enhanced SOD enzymatic activity may enable the injury to be contained within a few cells (McKersie et al. 1993). 4 1.3. Expression changes of genes and proteins in response to low temperature 1.3.1. Lipid composition The lipid composition of the plasma membrane has to be changed under low temperatures in order to maintain membrane integrity and membrane associated protein with proper function. In rye seedlings, the ratio of unsaturated phosphatidylcholine and phophatidylethanolamine increased upon cold acclimation (Lynch and Steponkus 1987). Increased unsaturation of the membrane lipids in response to low temperature was also observed in cyanobacteria (Sato and Murata 1980). The increased unsaturation of membrane lipids might be due to the up-regulation of desaturase genes under cold stress (Los et al. 1993; Sakamoto and Bryant 1997). In addition to the abundant information devoted to lipid membrane composition in response to low temperature, research also indicated that the membranes of individual cellular compartments could experience a similar modification upon temperature stress. In Brassica napus, the content of the unsaturated fatty acid, linolenic acid (18:3) in the endoplasmic reticulum (ER) membrane increased about two fold upon exposure to low temperature, but the oleate desaturase and linoleate desaturase genes were only transiently regulated in response to low temperature. This suggests that enhanced translation or enhanced enzymatic activities are involved in ER lipid composition changes in response to cold (Tasseva et al. 2004). In green alga, the percentage of membrane polyunsaturated fatty acids was higher in winter time than in summer time. This increase in the degree of unsaturation might be important for green alga in order to decrease their threshold of low temperature survival and acclimate net photosynthesis and dark respiration rates to winter temperatures (Terrados and Lopez- 5 Jimenez 1996). The most direct evidence on lipid composition changes in the cold acclimation process was reported by Steponkus et al. (1988) in rye. 1.3.2. Heat shock proteins (HSPs) HSPs are induced not only by high temperature but also by other stresses such as cold, drought, or salinity (Anderson et al. 1994; Coca et al. 1994). HSPs are part of a group of proteins induced by environmental stress either to protect the plant from damage or to help repair damage caused by the stress. There is very likely some overlap in function among the different stress proteins since one stress can induce protection against another (Lurie et al. 1994; Leshem and Kuiper 1996). In tomato, the expression of two heat shock-induced proteins, tom66 and tom111, was found to be first decreased then increased in response to low temperature. A clear correlation between the induction of these two genes and low temperature tolerance of the tissue was found (Sabehat et al. 1998). A similar mechanism of heat-shock-induced tolerance to chilling injury may exist in all plant organs. Protection against chilling injury by high-temperature treatment has been found in mung bean hypocotyls (Collins et al. 1995) and cucumber cotyledons and seeds (Lafuente et al. 1991; Jennings and Saltveit 1994). In these studies loss of protection was correlated with the disappearance of HSPs from the tissue (Lafuente et al. 1991; Collins et al. 1995). Presently, 11 heat shock protein families are known in plants (Nover and Scharf 1997). The function of heat shock protein is related to their molecular chaperon characteristics. Heat shock proteins can stabilize native proteins (Anderson and Guy 1995), refold stress-denatured proteins (Gaitanaris et al. 1990) and prevent aggregation of denatured proteins (Ellis and van der Vies 1991). Many members of the hsp70 family were found to be up-regulated in response to both heat shock and low 6 temperature in tomato and spinach. The timing of the increased up-regulation of heat shock proteins is consistent with the hypothesis that protein biogenesis or protein conformation are adversely affected by low temperatures (Li et al. 1999), indicating a possible role associated with heat shock proteins. 1.3.3. Antifreeze proteins (AFPs) Antifreeze proteins are found in a wide range of overwintering plants and located in the apoplast where they inhibit the growth and recrystallization of ice that forms in intercellular spaces. During cold acclimation, the accumulations of AFPs is correlated with increased freeze tolerance in rye, wheat, and barley (Marentes et al. 1993; Antikainen and Griffith 1997). When AFPs bind to the surface of ice, they adsorb irreversibly onto a specific plane of the crystal lattice (Knight et al. 1991; Knight et al. 1995). Specific activities of AFPs include changes in ice crystal shape, thermal hysteresis, or inhibition of ice recrystallization, which varies among overwintering organisms and could be related to their freezing strategies (Griffith and Yaish 2004). Ice crystals grown in water or in a solution of substances that do not interact with ice are disc-shaped. By contrast, most AFPs bind to the prism face of ice, creating hexagonally shaped crystals (Griffith and Yaish 2004). At high concentrations, AFPs depress the freezing temperature of a solution noncolligatively without affect the melting temperature, a process known as thermal hysteresis (DeVries 1986). In bittersweet nightshade, a thermal hysteresis protein gene (STHP-64) was isolated and found to contain 10 consecutive 13-mer repeats at its C-terminus, a common feature of animal antifreeze proteins. Northern blots demonstrated that the STHP-64 transcript was not present in leaves until November and December, suggesting that cold acclimation induces 7 STHP-64 production (Huang and Duman 2002). At low concentrations, AFPs can inhibit the recrystallization of ice (Knight et al. 1984), which is the growth of larger ice crystals at the expense of smaller ice crystals. Larger ice crystals increase the possibility of physical damage within frozen plant tissues (Griffith et al. 1997). Two AFPs (TaIRI-1 and TaIRI-2) were identified to be up-regulated in wheat in response to low temperature. TaIRI-1 protein was found to be able to inhibit the growth of ice crystals (Tremblay et al. 2005). Amino-terminal sequence comparisons, immuno-cross-reactions, and enzymatic activity analysis of AFPs in winter rye showed similarity between AFPs and pathogenesis-related (PR) proteins (Hon et al. 1995). Because the AFPs retain their enzymatic activities, they may also have antifungal properties that are important in disease resistance, particularly against low-temperature pathogens such as snow molds (Ergon et al. 1998; Hiilovaara-Teijo et al. 1999). 1.3.4. Dehydrins Dehydrin, also known as a group-2 late embryogenesis abundant (LEA) protein, is one of several ubiquitous water-stress-responsive proteins in plants (Ingram and Bartels 1996). Dehydrins are highly hydrophilic, glycine-rich and boiling-stable proteins. They possess repeated sequence structures containing lysine-rich motifs which are speculated to form putative amphiphilic ? helices. Dyhydrins are thought to bind to membranes and cellular proteins by hydrophobic interactions, and thus protect the functions of intracellular molecules by preventing their coagulation during symplastic dehydration (Close 1997). These proteins may accumulate during drought, salinization, late stages of seed development, freezing and in response to exogenous ABA (Close et al. 1993; Welin et al. 1994; Campbell and Close 1997; Close 1997). Many dehydrins have been isolated and 8 characterized not only in herbaceous plants but also in several woody species including Prunus persica (Arora and Wisniewski 1994), birch (Rinne et al. 1999), blueberry (Muthalif and Rowland 1994) and citrus (Hara et al. 2001). CuCOR19, a dehydrin isolated from Citrus unshiu, showed cryoprotective activities for lactate dehydrogenase (LDH) and catalase. The protective role of the CuCOR19 protein is thought to be related to its random coil structure, which can form a layer cohesive to other structures. Low temperatures can denature the association of some enzymes irreversibly. The binding of the coil structure in the CuCOR19 could prevent the disassociation of the enzymes. In addition to its cryoprotective role, CuCOR19 can bind water molecules which might be necessary to maintain water in the cell during symplastic dehydration (Hara et al. 2001). PCA60 is a dehydrin isolated from winter bark tissue of peach (Wisniewski et al. 1999). The extracted protein has been shown to preserve the in vitro enzymatic activity of lactate dehydrogenase during repeated freeze-thaw cycles. Another surprising characteristic of this dehydrin is its antifreeze activity shown by its involvement with ice crystal morphology and thermal hysteresis, the first time that antifreeze activity was demonstrated for a dehydrin (Wisniewski et al. 1999). In addition to its water-binding, macromolecule-stabilizing, and cryoprotective activities of dehydrins, at least one report showed that dehydrins may be involved in free radical scavenging (Hara et al. 2003). The overexpressed citrus dehydrin CuCOR19 in tobacco causes an increase in the cold tolerance ability of transformed plants. Further analysis correlated this cold tolerance enhancement to the antioxidative activity imposed by the expressed protein (Hara et al. 2003). Another notable work about dehydrin was done by Puhakainen et al. (2004) in Arabidopsis. Chimeric double constructs with dehydrins RAB18 and COR47 or LTI29 9 and LTI30 were introduced into Arabidopsis and resulted in increased accumulation of related dehydrin proteins. Compared to control plants, transgenic plants have more tolerance to cold temperature, and this increased resistance to cold temperature was speculated to be partly due to their protective role of cell membranes (Puhakainen et al. 2004). 1.3.5. Compatible solutes Under environmental stresses including salinity, drought and low temperature conditions, plants can accumulate certain organic metabolites of low molecular weight known collectively as compatible solutes (Bohnert et al. 1995). Compatible solutes mainly include polyhydroxylated sugar alcohols, amino acids and their derivatives, tertiary sulphonium compounds and quaternary ammonium compounds (Bohnert and Jensen 1996). The main functions of compatible solutes involve membrane protection (Rudolph and Crowe 1985), cryoprotection of proteins (Carpenter and Crowe 1988), maintenance of osmotic potential (Yancey et al. 1982), and scavenging of free radicals (Smirnoff and Cumbes 1989). The levels of proline and sucrose increase in Arabidopsis (McKown et al. 1996), and spinach (Guy et al. 1992) during cold acclimation. The increase in synthesis of proline and sucrose might be associated with freezing tolerance enhancement (Stitt and Hurry 2002). The stress tolerance ability of Arabidopsis was increased by the suppression of proline dehydrogenase, an enzyme to catalyze the proline degradation, with its antisense cDNA (Nanjo et al. 1999), further demonstrating the protective role of proline during cold acclimation. In comparison of three wheat cultivars with different cold tolerance ability, the accumulation of sugars, amino acids and glycine betaine were the highest in the most cold tolerant cultivar, the lowest in the most cold sensitive cultivar 10 (Kamata and Uemura 2004). In Arabidopsis, levels of endogenous glycine betaine in the leaves were found to be greatly induced in response to cold acclimation, water stress and exogenous ABA application, indicating the involvement of glycine betaine in cold acclimation and water stress (Xing and Rajashekar 2001). The relationship between sugar content and freezing tolerance was investigated in cabbage. Concentration of sucrose, glucose, and fructose gradually increased during cold acclimation. However, the induced freezing tolerance was lost after only 1 day of deacclimation at control temperatures, and this change was associated with a large reduction in sugar content (Sasaki et al. 1996). Genetic engineering to increase levels of some compatible solutes, such as mannitol and proline, has been proven to be a promising approach to increase the ability of plants to tolerate environmental stress (Hayashi and Murata 1998). 1.4. Regulation of the cold acclimation response 1.4.1. The ICE/CBF/DREB1 regulatory pathway Several cold responsive genes have been identified and characterized in Arabidopsis. These genes were designated as COR (cold-responsive or regulated), LTI (low temperature induced), KIN (cold inducible), RD (responsive to desiccation), and ERD (early dehydration inducible) (Thomashow 1994). Studies have shown that the promoter of some of these genes has a core sequence ?CCGAC? designated as CRT (C-repeat) or DRE (dehydration responsive element) (Baker et al. 1994; Yamaguchi-Shinozaki and Shinozaki 1994). A family of transcription factors in Arabidopsis known either as C- repeat-binding factor (CBF1, CBF2, and CBF3) (Stockinger et al. 1997; Gilmour et al. 1998) or dehydration-responsive element-binding factor (DREB1B, DREB1C, and DREB1A)(Liu et al. 1998) have been identified. These transcription factors can bind to 11 the cis element and activate the transcription of the down stream cold responsive or dehydration responsive genes. Transgenic overexpression of the CBF/DREB gene in other plants turns on the expression of down stream cold responsive genes without cold acclimation and can increase the low temperature resistance ability (Jaglo-Ottosen et al. 1998; Liu et al. 1998). Since CBF transcripts begin accumulating within 15 min of plants' exposure to cold, Gilmour et al. (1998) proposed that another transcription factor constitutively exists, which can bind to the cis element of CBF gene under cold stimulus. Gilmour et al. (1998) named the unknown activator(s) "ICE" (inducer of CBF expression) protein(s). This ICE gene has been identified by another group and was found to encode a MYC-like bHLH (helix loop helix) transcription factor. ICE is a positive regulator of CBF3 and has a critical role in cold acclimation (Chinnusamy et al. 2003). CBF3 promoter includes five putative MYC recognition sequences, while CBF1 and CBF2 only includes one (Shinwari et al. 1998). The ice1 mutation abolishes CBF3 expression, and reduces the expression of CBF-target genes in the cold (Chinnusamy et al. 2003). Although ICE has a strong effect on the regulation of CBF3, it only slightly affects the induction of CBF2 and CBF1. Actually, the expression of CBF2 is enhanced in the ice1 mutant after 6 and 12 h of cold treatment (Chinnusamy et al. 2003). The potential negative regulation of the CBF transcription factor genes may be important for ensuring that their expression is transient and tightly controlled (Novillo et al. 2004). Novillo et al. (2004) found a CBF2/DREB1C Arabidopsis mutant with a higher capacity to tolerate freezing than wild type plants before and after cold acclimation and higher tolerance to dehydration and salt stress. The increased cold tolerance was due to the enhanced expression of the CBF1/DREB1B and CBF3/DREB1A and the downstream 12 regulated COR genes. These results indicate that CBF2/DREB1C negatively regulates CBF1/DREB1B and CBF3/DREB1A, ensuring that their expression is transient and tightly controlled (Novillo et al. 2004). In addition to the CBF/DREB signaling pathway in plant cold acclimation, there is evidence that multiple pathways may be involved in the acclimation process. Analysis of the eskimo1 (esk1) mutant of Arabidopsis revealed that considerable freezing tolerance can be achieved in the absence of COR gene expression (without CBF/DREB signaling activation) (Xin and Browse 1998). Studies from the analysis of sensitive-to-freezing (sfr) mutants that are not able to fully acclimate also support the theory that CBF/DREB is not the only signaling pathway for plant cold acclimation. sfr mutants do not fully cold acclimated, and only retain about 50% of the wild-type capacity for cold acclimation, which means that the sfr mutation blocks some signaling pathway. Therefore, each mutant is still able to partially cold acclimate through signaling pathways that are not disrupted (Warren et al. 1996). 1.4.2. ABA-dependent and ABA-independent pathway Expression of genes in response to low temperature is thought to be regulated by both ABA-dependent and ABA-independent signalling pathways. In Arabidopsis, ABA synthesis mutants, aba1 or abi, are less freezing tolerant than wild-type plants (Heino et al. 1990; Gilmour and Thomashow 1991). The expression of many genes has been reported to be associated with ABA (Shinozaki and Yamaguchi-Shinozaki 2000; Ramanulu and Bartels 2002). The analysis of the promoter region of these ABA responsive genes reveals that many of the genes contain a fragment of (C/T)ACGTGGC consensus sequence, generally known as ?ABA-responsive element (ABRE)? (Busk and 13 Pages 1998; Rock 2000). bZIP transcription factors are likely to bind to these elements in cold regulated genes and activate gene expression (Thomashow 1999). Contrary to the involvement of ABA in the changes of expression of specific genes, some genes are not dependent on ABA regulation. In the aba1 mutant, cold induced expression of COR78, COR47, and COR6.6 is normal (Gilmour and Thomashow 1991). Thus, Gilmour and Thomashow proposed that cold regulated expression of these genes occurs through an ABA-independent pathway. The important CBF/DREB transcription factor and its regulon are also not changed in response to exogenous ABA, further pointing to the existence of the ABA-independent regulation pathway under cold acclimation (Yamaguchi-Shinozaki and Shinozaki 1994). In moss Physcomitrella patens, ABA treated cells had slender chloroplasts and reduced amount of starch grains. When frozen to -4 ?C, freezing injury-associated ultrastructural changes such as formation of a particulate domains and fracture-jump lesions were frequently observed in the plasma membrane of non-treated protonema cells but not ABA-treated cells. The ABA treated cells also had higher accumulation of free soluble sugars (Nagao et al. 2005). In another work from the same group, surprisingly, determination of ABA content by GC?MS revealed that low-temperature treatment did not increase accumulation of ABA, but that levels of freezing tolerance increased. These results suggest that P. patens does not require increases in levels of ABA for cold acclimation and possesses an ABA- independent cold-signaling pathway leading to the development of freezing tolerance (Minami et al. 2005). Whether and how ABA relates to the activation of expression of cold responsive genes is still unsolved. A clear conclusion about ABA?s function in environmental stresses beyond its traditional function in plant development and growth 14 requires a total understanding of the cold acclimation mechanisms and whether ABA plays a critical role in regulating the activity of the mechanisms (Thomashow 1999). 1.4.3. Participation of Calcium in cold acclimation The cytosolic concentration of the intracellular second messenger calcium (Ca 2+ ) can be transiently increased in response to low temperature in many plant species, including Arabidopsis (Lewis et al. 1997), tobacco (Knight et al. 1991), and tomato (Sebastiani et al. 1999). Elevation of calcium levels is mainly due to an influx of Ca 2+ from external sources (Monroy and Dhindsa 1995), but evidence has shown that some calcium can be released from vacuole (Knight et al. 1996). In Arabidopsis, the calcium chelater (EGTA) blocks plasma-membrane calcium channels (La Gd3+), thus inhibiting cold acclimation as well as kin gene expression. Ruthenium red, an inhibitor of calcium release from intracellular storages, partially inhibited kin gene expression and development of freezing tolerance (T?htiharju et al. 1997). In Populus tomentosa cuttings, treatment with CaCl 2 at the time of freezing acclimation enhanced the effect of freezing acclimation, but this enhancement was abolished by Ca 2+ chelator EGTA, Ca 2+ channel inhibitor LaCl 3 or CaM (calmodulin) antagonist chlorpromazine, indicating that the calcium-calmodulin messenger system was involved in the course of freezing resistance development (Lin et al. 2004). 1.5. Biology of citrus Commercial citrus species and related genera are primarily evergreen species of subtropical and tropical origins belonging to the family Rutaceae (Swingle and Reece 1967). Citrus and related genera within the true citrus group are diploid (2n=18). Some triploids, tetraploids and hexaploids exist but generally occur at low percentages in the 15 population (Raghuvanshi 1968). The progenitor of the Satsuma group of mandarins (C. unshiu Marc.) probably originated in China but was transported to Japan (Saunt 1990). There are over 100 cultivars of Satsuma, which differ from each other primarily in time of maturity, fruit shape and fruit quality (Saunt 1990). Satsuma foliage and wood are the most freeze hardy of all commercially grown citrus cultivars, withstanding minimum wood temperatures of ?9?C when fully acclimated (Yelenosky 1985). Poncirus trifoliata is a small tree with trifoliate, deciduous leaves, and can tolerate temperature as low as ? 30?C when fully acclimated (Yelenosky 1985). It is native to northern China, where low temperatures are common during winter months. P. trifoliata trees are used as rootstocks in commercial citrus production (Davis and Albrigo 1994). Selection of new citrus cultivars has occurred for thousands of years in ancient China. Citrus breeding is difficult, slow and time consuming. Most citrus and related species are very heterozygous and few important traits show single gene inheritance patterns (Davis and Albrigo 1994). Most citrus breeding programs have relied on traditional methods for developing new cultivars or rootstocks based on controlled hand crosses and selection of superior types from literally thousands of seedlings in the field (Hearn 1985). The entire process may take over 20 years. Therefore, many citrus breeders are searching for new methods to shorten this costly and time-consuming process. Molecular biotechnology is one of the most promising potential methods to do this. Much effort has been made to transfer disease resistance genes into citrus plants, thus far without much success due to the complexity of the techniques, and the specificity of citrus species (Ghorbel et al. 2000; Febres et al. 2003), and lack of knowledge about the resistance or acclimation mechanisms of woody plants at the molecular level. Thus, a better elucidation of the 16 responses at the molecular level of citrus plants under environmental stress will be a prerequisite for further transgenic effort. So far, a few genes have been identified and characterized in citrus or related species. Most of the identified genes belong to the dehydrin family, and were found to serve as free radical scavengers to protect cell membranes (Hara et al. 2003; Hara et al. 2004) and as cryoprotectants (Sanchez-Ballesta et al. 2004). Yet, most of these experiments were conducted following cold shock treatment in which plants were moved from warm temperature (25?C) to low temperature (4?C) directly, a process that does not occur in nature. A more objective transcriptome profiling analysis under a gradually declined temperature regime that mimics the natural decline in air temperature is necessary to fully understand the mechanisms of citrus plant in response to low temperature. 1.6. Techniques used to isolate genes Many gene profiling techniques can be used to study the changes of transcripts in response to environmental stresses, such as Differential Display Reverse Transcription PCR (DDRT-PCR), cDNA Amplified Fragment Length Polymorphism (cDNA-AFLP), Serial Analysis of Gene Expression (SAGE), subtractive hybridization, DNA-chip, and cDNA microarray. Differential display and cDNA-AFLP have been widely used to identify genes whose expression levels have been altered under different environmental conditions because both techniques are straight forward and especially, don?t need previous genomic information of the species of interest (Liang and Pardee 1992; Vos et al. 1995). Due to the lack of genomic information for P. trifoliata and C. unshiu, both techniques were utilized for the current research. 17 References Anderson JV, Chevone BI, Hess JL (1992) Seasonal variation in the antioxidant system of eastern white pine needles. Plant Physiol 98: 501-508 Anderson JV, Guy CL (1995) Spinach leaf 70-kilodalton heat-shock cognate stabilizes bovine adrenal glucose-6-phosphate dehydrogenase in vitro without apparent stable binding. Planta 196: 303?310 Anderson JV, Li QB, Haskell DW, Guy CL (1994) Structural organization of the spinach endoplasmic reticulum-luminal 70-kilodalton heat-shock cognate gene and expression of 70-kilodalton heat-shock genes during cold acclimation. Plant Physiol 104: 1359-1370 Antikainen M, Griffith M (1997) Antifreeze accumulation in freezing tolerant cereals. Physiol Plant 99: 423?432 Arora R, Wisniewski ME (1994) Cold acclimation in genetically related (sibling) deciduous and evergreen peach (Prunus persica [L.] Batsch). Plant Physiol 105: 95-101 Asada K (1994) Production and action of active oxygen species in photosynthetic tissues. In: Mullineaux P.M. (Ed.), Causes of photo-oxidative stress and amelioration of defence systems in plants. CRC Press, Boca Raton, FL, 77?104 Baker SS, Wilhelm KS, Thomashow MF (1994) The 5'-region of Arabidopsis thaliana cor15a has cis-acting elements that confer cold-, drought- and ABA-regulated gene expression. Plant Mol Biol 24: 701-713 Bohnert HJ, Jensen RG (1996) Strategies for engineering water-stress tolerance in plants. Trends in Biotech 14: 89?97 Bohnert HJ, Nelson DE, Jensen RG (1995) Adaptations to environmental stresses. Plant Cell 7: 1099-1111 Bowler C, Slooten L, Vandenbranden S, De Rycke R, Botterman J, Sybesma C, Van Montagu M, Inze D (1991) Manganese super-oxide dismutase can reduce cellular damage mediated by oxygen radicals in transgenic plants. EMBO J 10: 1723- 1732 Bowler C, Van Montague M, Inze D (1992) Superoxide dismutase and stress tolerance. Annu Rev Plant Physiol Plant Mol Biol 43: 83-116 Busk PK, Pages M (1998) Regulation of abscisic acid-induced transcription. Plant Mol Biol 37: 425-435 18 Campbell SA, Close TJ (1997) Dehydrins: genes, proteins, and associations with phenotypic traits. New Phytol 137: 61?74 Carpenter JF, Crowe JH (1988) The mechanism of cryoprotection of proteins by solutes. Cryobiology 25: 244-255 Chinnusamy V, Ohta M, Kanrar S, Lee B-h, Hong X, Agarwal M, Zhu J-K (2003) ICE1: a regulator of cold-induced transcriptome and freezing tolerance in Arabidopsis. Genes Dev 17: 1043-1054 Close TJ (1997) Dehydrins: a commonalty in the response of plants to dehydration and low temperature. Physiol Plant 100: 291?296 Close TJ, Fenton RD, Yang A, Asghar R, DeMason DA, Crone DE, Meyer NC, Moonan F (1993) Dehydrin: The protein. In: Bray E.A. (Ed.), Plant reponses to cellular dehydration during environmental stress. American Society of Plant Physiologists, Rockville., 104?118 Coca MA, Almoguera C, Jordano J (1994) Expression of sunflower low-molecular- weight heat-shock proteins during embryogenesis and persistence after germination: localization and possible functional implications. Plant Mol Biol 25: 479-492 Collins G, Nie XL, Saltveit M (1995) Heat shock proteins and chilling sensitivity of mung bean hypocotyls. J Exp Biol 46: 479-492 Davis FS, Albrigo LG Citrus. CAB INTERNATIONAL, Wallingford, 1994 DeVries AL (1986) Antifreeze glycopeptides and peptides: interactions with ice and water. Methods Enzymol 127: 293-303 Ellis RJ, van der Vies SM (1991) Molecular chaperones. Annu Rev Biochem 60: 321? 347 Elstner EF (1991) Mechanism of oxygen activation in different compartments of plant cells. In: Steffen K.L. (Ed.), Active oxygen/oxidative stress and plant metabolism. American Society of Plant Physiologists, Rockville, MD, 13-25 Ergon A, Klemsdal SS, Tronsmo AM (1998) Interactions between cold hardening and microdochium nivale infection on expression of pathogenesis-related genes in winter wheat. Physiol Mol Plant Pathol 53: 301-310 Esterbauer H, Grill D (1978) Seasonal variation of glutathione and glutathione reductase in needles of Picea abies. Plant Physiol 61: 119-121 Fadzillah NM, Gill V, Finch RP, Burdon RH (1996) Chilling, oxidative stress and antioxidant responses in shoot cultures of rice. Planta 199: 552-556 19 Febres VJ, Niblett CL, Lee RF, Moore GA (2003) Characterization of grapefruit plants (Citrus paradisi Macf.) transformed with citrus tristeza closterovirus genes. Plant Cell Rep 421-428 Foyer CH (1997) Oxygen metabolism and electron transport in photosynthesis. In: Scandalios J.G. (Ed.), Oxidative stress and the molecular biology of antioxidant defenses,. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 587?621 Gaitanaris GA, Papavassiliou AG, Rubock P, Silverstein SJ, Gottesman M E (1990) Renaturation of denatured repressor requires heat shock proteins. Cell 61: 1013- 1020 Ghorbel R, Dominguez A, Navarro L, Penna L (2000) High efficiency genetic transformation of sour orange (Citrus aurantium) and production of transgenic trees containing the coat protein gene of citrus tristeza virus. Tree Physiol 1183- 1189 Gilmour SJ, Thomashow MF (1991) Cold acclimation and cold-regulated gene expression in ABA mutants of Arabidopsis thaliana. Plant Mol Biol 17: 1233- 1240 Gilmour SJ, Zarka DG, Stockinger EJ, Salazar MP, Houghton JM, Thomashow MF (1998) Low temperature regulation of the Arabidopsis CBF family of AP2 transcriptional activators as an early step in cold-induced COR gene expression. Plant J 16: 433-442 Griffith M, Antikainen M, Hon W-C, Pihakaski-Maunsbach K, Yu X-M, Chun JU, Yang DSC (1997) Antifreeze proteins in winter rye. Physiol Plant 100: 327?332 Griffith M, Yaish MWF (2004) Antifreeze proteins in overwintering plants: a tale of two activities. Trends in Plant Sci 9: 399-405 Guy CL, Huber JLA, Huber SC (1992) Sucrose phosphate synthase and sucrose accumulation at low temperature. Plant Physiol 100: 502-508 Guye MG, Vigh L, Wilson JM (1986) Polyamine titre in relation to chilling-sensitivity in Phaseolus sp. J Exp Bot 37: 1036-1043 Hara M, Fujinaga M, Kuboi T (2004) Radical scavenging activity and oxidative modification of citrus dehydrin. Plant Physiol Biochem 42: 657-662 Hara M, Terashima S, Fukaya T, Kuboi T (2003) Enhancement of cold tolerance and inhibition of lipid peroxidation by citrus dehydrin in transgenic tobacco. Planta 217: 290-298. Hara M, Terashima S, Kuboi T (2001) Characterization and cryoprotective activity of cold-responsive dehydrin from Citrus unshiu. J Plant Physiol 158: 1333?1339. 20 Hayashi H, Murata N (1998) Genetically engineered enhancement of salt tolerance in higher plants. In: Satoh K M.N. (Ed.), Stress responses of photosynthetic organisms. Elsevier, Amsterdam, 133?148. Hearn CL (1985) Citrus scion improvement program. Fruit Variety Journal 39: 34-37. Heino P, Sandman G, L?ng V, Nordin K, Palva ET (1990) Abscisic acid deficiency prevents development of freezing tolerance in Arabidopsis thaliana (L.) Heynh. Theor Appl Genet 79: 801-806. Hiilovaara-Teijo M, Hannukkala A, Griffith M, Yu X-M, Pihakaski-Maunsbach K (1999) Snow-mold-induced apoplastic proteins in winter rye leaves lack antifreeze activity. Plant Physiol 121: 665-674. Hincha DK, Schmitt JM (1985) Mechanical and chemical injury to thylakoid membranes during freezing in vitro. Biochim Biophy Acta 812: 173-180. Hincha DK, Schmitt JM (1992) Freeze-thaw injury and cryoprotection of thylakoid membranes. In: Bolis C.L. (Ed.), Water and life: Comparative analysis of water relationships at the organismic, cellular and molecular levels. Springer-Verlag, Heidelberg, 316-337. Hon WC, Griffith M, Mlynarz A, Kwok YC, Yang D (1995) Antifreeze Proteins in Winter Rye Are Similar to Pathogenesis-Related Proteins. Plant Physiol 109: 879- 889. Howarth C, Ougham H (1993) Gene expression under temperature stress. New Phytol 125: 1-26. Huang T, Duman JG (2002) Cloning and characterization of a thermal hysteresis (antifreeze) protein with DNA-binding activity from winter bittersweet nightshade, Solanum dulcamara. Plant Mol Biol 48: 339-350. Ingram J, Bartels D (1996) The molecular basis of cellular dehydration tolerance in plants. Annu Rev Plant Physiol Plant Mol Biol 47: 377-403. Jaglo-Ottosen K, Gilmour SJ, Zarka D, Schabenberger O, Thomashow MF (1998) Arabidopsis CBF1 overexpression induce COR genes and enhances freezing tolerance. Science 280: 104-106. Jennings P, Saltveit ME (1994) Temperature and chemical shocks induce chilling tolerance in germinating Cucumis sativus (cv. Poinsett 76) seeds. Physiol Plant 91: 703-707. Kamata T, Uemura M (2004) Solute accumulation in wheat seedlings during cold acclimation: Contribution to increased freezing tolerance. Cryoletters 25: 311- 322. 21 Knight CA, DeVries AL, Oolman LD (1984) Fish antifreeze protein and the freezing and recrystallization of ice. Nature 208: 295-296. Knight CA, Wen DY, Laursen RA (1995) Nonequilibrium antifreeze peptides and the recrystallization of ice. Cryobiology 32: 23?34. Knight H, Trewavas AJ, Knight MR (1996) Cold calcium signaling in Arabidopsis involves two cellular pools and a change in calcium signature after acclimation. Plant Cell 8: 489?503. Knight MR, Campbell AK, Smith SM, Trewavas AJ (1991) Transgenic plant aequorin reports the effects of touch and cold-shock and elicitors on cytoplasmic calcium. Nature 352: 524?526. Kocsy G, Szalai G, V?g?jfalvi A, St?hli L, Orosz G, Galiba G (2000) Inhibition of glutathione synthesis reduces chilling tolerance in maize. Planta 211: 528-536. Lafuente MT, Belver A, Guye MG, Saltveit M (1991) Effect of temperature conditioning on chilling injury of cucumber cotyledons. Plant Physiol 95: 443-449. Lee TM (1997) Polyamine regulation of growth and chilling tolerance of rice (Oryza sativa L.) roots cultured in vitro. Plant Sci 122: 111-117. Leshem Y and Kuiper P (1996) Is there a GAS (general adaption syndrome) response to various types of environmental stress? Biol Plant 38: 1-18. Levitt J (1980) Responses of plants to environmental stresses, 2nd ed. Academic Press, New York, NY. Lewis BD, Karlin-Neumann G, Davis RW, Spalding EP (1997) Ca2+-activated anion channels and membrane depolarizations induced by blue light and cold in Arabidopsis seedlings. Plant Physiol Biochem 114: 1327?1334. Li QB, Haskell DW, Guy CL (1999) Coordinate and non-coordinate expression of the stress 70 family and other molecular chaperones at high and low temperature in spinach and tomato. Plant Mol Biol 39: 21-34. Liang P, Pardee AB (1992) Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction. Science 257: 967-971. Lin SZ, Zhang ZY, Lin YZ, Zhang Q, Guo H ( 2004) The role of calcium and calmodulin in freezing-induced freezing resistance of Populus tomentosa cuttings. Zhi Wu Sheng Li Yu Fen Zi Sheng Wu Xue Xue Bao 30: 59-68. Liu Q, Kasuga M, Sakuma Y, Abe H, Miura S, Yamaguchi-Shinozaki K, Shinozaki K (1998) Two transcription factors, DREB1 and DREB2, with an EREBP/AP2 DNA binding domain separate two cellular signal transdcution pathways in 22 drought- and low-temperature-responsive gene expression, respectively, in Arabidopsis. Plant Cell 10: 1391-1406. Los DA, Horvath I, Vigh L, Murata N (1993) The temperature-dependent expression of the desaturase gene desA in Synechocystis PCC6803. FEBS Lett 318: 57. Lurie S, Klein JD, Fallik E (1994) Cross protection of one stress by another. In: Cherry J. (Ed.), Biochemical and cellular mechanisms of stress Tolerance of Plants. Springer Verlag, Berlin, 201-212. Lynch DV, Steponkus P (1987) Plasma membrane lipid alterations associated with cold acclimation of winter rye seedlings (Secale cereale L. cv Puma). Plant Physiol 83: 761. Marentes E, Griffith M, Mlynarz A, Brush RE (1993) Proteins accumulate in the apoplast of winter rye leaves during cold acclimation. Physiol Plant 87: 499?507. McKersie BD (1991) The role of oxygen free radicals and desiccation stress in plants. In: Pell E.J., Steffen, K. L. (Ed.), Active oxygen/oxidative stress and plant metabolism. American Society of Plant Physiologists, Rockville, MD, 107-118. McKersie BD, Chen Y, de Beus M, Bowley SR, Bowler C, Inze D, D'Halluin K, Botterman J (1993) Superoxide dismutase enhances tolerance of freezing stress in transgenic alfalfa (Medicago sativa L.). Plant Physiol 103: 1155-1163. McKown R, Kuroki G, Warren G (1996) Cold responses of Arabidopsis mutants impaired in freezing tolerance. J Exp Bot 47: 1919-1925. Minami A, Nagao M, Ikegami K, Koshiba T, Arakawa K, Fujikawa S, Takezawa D (2005) Cold acclimation in bryophytes: low-temperature-induced freezing tolerance in Physcomitrella patens is associated with increases in expression levels of stress-related genes but not with increase in level of endogenous abscisic acid. Planta 220: 414-423. Monroy AF, Dhindsa RS (1995) Low-temperature signal transduction: induction of cold acclimation-specific genes of alfalfa by calcium at 25 degrees C. Plant Cell 7: 321?331. Muthalif MM, Rowland LJ (1994) Identification of dehydrin-like proteins responsive to chilling in floral buds of blueberry (Vaccinium, section Cyanococcus). Plant Physiol 104: 1439-1447. Nagao M, Minami A, Arakawa K, Fujikawa S, Takezawa D (2005) Rapid degradation of starch in chloroplasts and concomitant accumulation of soluble sugars associated with ABA-induced freezing tolerance in the moss Physcomitrella patens. J Plant Physiol 162: 169-180. 23 Nanjo T, Kobayashi M, Yoshiba Y, Kakubari Y, Yamaguchi-Shinozaki K, Shinozaki K (1999) Antisense suppression of proline degradation improves tolerance to freezing and salinity in Arabidopsis thaliana. FEBS Lett 461: 205-210. Nover L, Scharf KD (1997) Heat stress proteins and transcription factors. Cell Mol Life Sci 53: 80-103. Novillo F, Alonso JM, Ecker JR, Salinas J (2004) CBF2/DREB1C is a negative regulator of CBF1/DREB1B and CBF3/DREB1A expression and plays a central role in stress tolerance in Arabidopsis. Proc Natl Acad Sci USA 101: 3985-3990. O'Kane D, Gill V, Boyd P, Burdon R (1996) Chilling, oxidative stress and antioxidant responses in Arabidopsis thaliana callus. Planta 198: 371-377. Perl A, Perl-Treves R, Galili G, Aviv D, Shalgi E, Malkin S, Galun E (1993) Enhanced oxidative stress defense in transgenic potato expressing tomato Cu, Zn superoxide dismutases. Theor Appl Genet 85: 568-576. Prasad TK (1996) Mechanisms of chilling-induced oxidative stress injury and tolerance in developing maize seedlings: changes in antioxidant system, oxidation of proteins and lipids, and protease activities. Plant J 10: 1017-1026. Prasad TK, Anderson MD, Martin BA, Stewart CR (1994) Evidence for chilling-induced oxidative stress in maize seedlings and a regulatory role for hydrogen peroxide. Plant Cell 6: 65-74. Puhakainen T, Hess MW, Makela P, Svensson J, Heino P, Palva ET (2004) Overexpression of multiple dehydrin genes enhances tolerance to freezing stress in Arabidopsis. Plant Mol Biol 54: 743-753. Raghuvanshi SS (1968) Cytological evidence bearing on evolution in citrus., Proceedings of the First International Citrus Symposium, pp. 207-214. Ramanulu S, Bartels D (2002) Drought- and desiccation-induced modulation of gene expression in plants. Plant Cell Environ 25: 141-151. Rinne PL, Kaikuranta PL, van der Plas LH, van der Schoot C (1999) Dehydrins in cold- acclimated apices of birch (Betula pubescens ehrh. ): production, localization and potential role in rescuing enzyme function during dehydration. Planta 209: 377- 388. Roberts DR, Dumdroff EB, Thompson JE (1986) Exogenous polyamines alter membrane fluidity in bean leaves: a basis for potential misinterpretation of their true physiological role. Planta 167: 395-401. Rock C (2000) Pathways to abscisic acid-regulated gene expression. New Phytol 148: 357-396. 24 Rudolph AS, Crowe JH (1985) Membrane stabilization during freezing: the role of two natural cryoprotectants, trehalose and proline. Cryobiology 22: 367-377. Sabehat A, Lurie S, Weiss D (1998) Expression of small heat-shock proteins at low temperatures . A possible role in protecting against chilling injuries. Plant Physiol 117: 651-658. Sakamoto T, Bryant DA (1997) Temperature-regulated mRNA accumulation and stabilization for fatty acid desaturase genes in the cyanobacterium Synechococcus sp. strain PCC 7002. Mol Microbiol 23: 1281. Sanchez-Ballesta MT, Rodrigo MJ, Lafuente MT, Granell A, Zacarias L ( 2004) Dehydrin from citrus, which confers in vitro dehydration and freezing protection activity, is constitutive and highly expressed in the flavedo of fruit but responsive to cold and water stress in leaves. J Agric Food Chem 52: 1950-1957. Sasaki H, Ichimura K, Oda M (1996) Changes in sugar content during cold acclimation and deacclimation of cabbage seedlings. Annal Bot 78: 365-369. Sato N, Murata N (1980) Temperature shift induced responses in lipids in the blue-green alga, Anabaena variabilis: The central role of diacylmonogalactosylglycerol in thermo-adaptation. Biochi Biophy Acta 619: 353. Saunt J Citrus cultivars of the world. Sinclair International, UK, 1990. Sebastiani L, Lindberg S, Vitagliano C (1999) Cytoplasmic free Ca2+ dynamics in single tomato (Lycopersicon esculentum) protoplasts subjected to chilling temperatures. Physiol Plant 105: 239?245. Sen Gupta A, Heinen JL, Holoday AS, Burke JJ, Allen RD (1993) Increased resistance to oxidative stress in transgenic plants that overexpress Chloroplastic Cu/Zn superoxide dismutase. Proc Natl Acad Sci USA 90: 1629-1633. Shen W, Nada K, Tachibana S (2000) Involvement of polyamines in the chilling tolerance of cucumber cultivars. Plant Physiol 124: 431-440. Shinozaki K, Yamaguchi-Shinozaki K (2000) Molecular responses to dehydration and low temperature: differences and cross-talk between two stress signalling pathways. Curr Opin Plant Biol 3: 217-223. Shinwari ZK, Nakashima K, Miura S, Kasuga M, Seki M, Yamaguchi-Shinozaki K, Shinozaki K (1998) An Arabidopsis gene family encoding DRE/CRT binding proteins involved in low-temperature-responsive gene expression. Biochem Biophys Res Commun 250: 161-170. Smirnoff N, Cumbes QJ (1989) Hydroxyl radical scavenging activity of compatible solutes. Phytochemistry 28: 1057-1060. 25 Smith TA (1985) Polyamines. Annu Rev Plant Physiol 36: 117-143. Steponkus P (1984) Role of the plasma membrane in freezing injury and cold acclimation. Annu Rev Plant Physiol 35: 543-584. Steponkus PL, Uemura M, Balsamo RA, Arvinte T, V. LD (1988) Transformation of the cryobehavior of rye protoplasts by modification of the plasma membrane lipid composition. Proc Natl Acad Sci USA 85: 9026-9030. Steponkus PL, Uemura M, Webb MS (1993) A contrast of the cryostability of the plasma membrane of winter rye and spring oat. Two species that widely differ in their freezing tolerance and plasma membrane lipid composition. In: Steponkus P.L. (Ed.), Advances in low temperature biology. JAI Press, London, 211-312. Stitt M, Hurry V (2002) A plant for all seasons: alterations in photosynthetic carbon metabolism during cold acclimation in Arabidopsis. Curr Opin Plant Biol 5:199- 206. Stockinger EJ, Gilmour SJ, Thomashow MF (1997) Arabidopsis thaliana CBF1 encodes an AP2 domain-containing transcriptional activator that binds to the C- repeat/DRE, a cis-acting DNA regulatory element that stimulates transcription in response to low temperature and water deficit. Proc Natl Acad Sci USA 94: 1035- 1040. Swingle WT, Reece PC (1967) The botany of citrus and its wild relatives. In: Webb H.J. (Ed.), The Citrus Industry. University of California Press, California, 190-340. T?htiharju S, Sangwan V, Monroy AF, Dhindsa RS, Borg M (1997) The Induction of kin genes in cold-acclimating Arabidopsis thaliana. Evidence of a role for calcium. Planta 203: 442?447. Tasseva G, Davy de Virville J, Cantrel C, Moreau F, Zachowski A (2004) Changes in the endoplasmic reticulum lipid properties in response to low temperature in Brassica napus. Plant Physiol Biochem 42: 811-822. Terrados J, Lopez-Jimenez JA (1996) Fatty acid composition and chilling resistance in the green alga Caulerpa prolifera (Forrskal) Lamouroux (Chlorophyta, Caulerpales). Biochem Mol Biol Int 39: 863-869. Thomashow M (1994) Arabidopsis thaliana as a model for studying mechanisms of plant cold tolerance. In: Somerville C. (Ed.), Arabidopsis. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 807-834. Thomashow M (1999) Plant cold acclimation: Freezing tolerance genes and regulatory mechanisms. Annu Rev Plant Physiol Plant Mol Biol 50: 571-599. 26 Thomashow MF (1998) Role of cold-responsive genes in plant freezing tolerance. Plant Physiol 118: 1-8. Tremblay K, Ouellet F, Fournier J, Danyluk J, Sarhan F (2005) Molecular characterization and origin of novel bipartite cold-regulated ice recrystallisation inhibition proteins from cereals. Plant Cell Physiol V?zina L, Ferullo J, Lalibert? G, Laberge S, Willemot C (1997) Chilling and freezing. In: MNV P. (Ed.), Plant Ecophysiology. John Wiley & Sons, Inc., New York, p 61- 100. Vierheller TL, Smith IK (1990) Effect of chilling on glutathione reductase and total glutathione in soybean leaves (Glycine max (L) Merr). In: Stulen I. (Ed.), Sulfur nutrition and sulfur assimilation in higher plants. SPB Academic Publishing., The Hague, 261-265. Volger H, Heber U, Berzborn RJ (1978) Loss of function of biomembranes and solubilization of membrane proteins during freezing. Biochim Biophy Acta 511: 455-469. Vos P, Hogers R, Bleeker M, Reijans M, van de Lee T, Hornes M, Frijters A, Pot J, Peleman J, Kuiper M, Zabeau M (1995) AFLP: a new technique for DNA fingerprinting. Nucl Acids Res 23: 4407-4414. Wample RL, Hartley S, Mills L (2000) Dynamics of grapevine cold hardiness. Proceedings of the ASEV 50th Anniversary Meeting, Seattle, Washington. 81-93. Wang CY (1995) Temperature preconditioning affects glutathione content and glutathione reductase activity in chilled zucchini squash. J Plant Physiol 145: 148- 152. Warren G, McKown R, Marin A, Teutonico R (1996) Isolation of mutations affecting the development of freezing tolerance in Arabidopsis thaliana (L.) Heynh. Plant Physiol 111: 1011?1019. Welin BA, Olson A, Nylander M, Palva ET (1994) Characterization and differential expression of dhn/lea/rab-like genes during cold acclimation and drought stress in Arabidopsis thaliana. Plant Mol Biol 26: 131?144. Wisniewski M, Webb R, Balsamo R, Close TJ, Yu X-M, Griffith M (1999) Purification, immunolocalization, cryoprotective, and antifreeze activity of PCA60: A dehydrin from peach (Prunus persica). Physiol Plant 105: 600-608. Xin Z, Browse J (1998) eskimo1 mutants of Arabidopsis are constitutively freezing- tolerant. Proc Natl Acad Sci USA 95: 7799?7804. 27 28 Xing WB, Rajashekar CB (2001) Glycine betaine involvement in freezing tolerance and water stress in Arabidopsis thaliana. Environ Exp Bot 46: 21-28. Yamaguchi-Shinozaki K, Shinozaki K (1994) A novel cis-acting element in an Arabidopsis gene is involved in responsiveness to drought, low-temperature, or high-salt stress. Plant Cell 6: 251-264. Yancey PH, Clark ME, Hand SC, Bowlus RD, Somero GN (1982) Living with water stress: evolution of osmolyte systems. Science 217: 1214?1222. Yelenosky G (1985) Cold hardiness in Citrus. Hort Rev VII: 201-238. Zhang J, Cui S, Li J, Wei J, Kirkham MB (1995) Protoplasmic factors, antioxidants responses, and chilling resistance in maize. Plant Physiol Biochem 33: 567-575. II. Cold acclimation induced genes of trifoliate orange (Poncirus trifoliata) 1 ABSTRACT Commercial citrus varieties are sensitive to low temperature. Poncirus trifoliata is a close relative of Citrus species, and has been widely used as a cold hardy rootstock for citrus production in low temperature environments. mRNA differential display- polymerase chain reaction (DDRT-PCR) and quantitative relative RT-PCR were used to study gene expression of P. trifoliata under a gradual cold-acclimation temperature regime. Eight up-regulated cDNA fragments were isolated and sequenced. These fragments showed high similarities at the amino acid level to the following genes with known functions: betaine/proline transporter, water channel protein, aldo-keto reductase, early light induced protein, nitrate transporter, tetratricopeptide-repeat protein, F-box protein and ribosomal protein L15. These cold acclimation up-regulated genes in P. trifoliata are also regulated by osmotic and photo-oxidative signals in other plants. Keywords Cold acclimation ? differential display ? gene expression ? Poncirus trifoliata ? quantitative relative RT-PCR 1 The nucleotide sequences reported in this paper have been submitted to Genbank under accession numbers of CN779663 (P1), CN779664 (P2), CN779665 (P3), CN779666 (P4), CN779667 (P5), CN779668 (P6), CN779669 (P7) and CN779670 (P8). Introduction Environmental factors such as freezing, drought, and high salt affect the growth, productivity and distribution of plants (Kasuga et al. 1999). Plants have evolved a wide variety of mechanisms that allow them to thrive in hostile environments, albeit their ability to survive in adverse environments varies greatly (Garwe et al. 2003). Distribution of many temperate fruit crops is restricted by low temperature conditions (Owens et al. 2002). Low temperatures can cause extracellular ice formation and damage to cell membrane (Gilmour et al. 1988), transporters and receptor proteins (Hazel 1995). Many plants can acclimate to freezing by pre-exposure to low but nonfreezing temperatures (Fowler and Thomashow 2002). The cold acclimation process requires synthesis of new proteins (Tseng and Li 1991), alterations in lipid and carbohydrate composition, the accumulation of compatible osmolytes such as proline, betaine, and soluble sugars (Bohnert et al. 1995; Lynch and Steponkus 1987; Thomashow 1994), and activation of ion channels (Knight et al. 1996). Several plant genes induced by low temperature have been identified in alfalfa (Wolfraim et al. 1993), Arabidopsis thaliana (Gilmour et al. 1992) and strawberry (Yubero-Serrano et al. 2003). A low-temperature responsive dehydrin-like protein, wcor410, is involved in cryoprotection of the plasma membrane against freezing or dehydration stress (Danyluk et al. 1994). Several cold regulated (COR) proteins from different plants are involved in membrane stability (Thomashow 1998). COR proteins are hydrophilic and have been suggested to protect cells from low temperature, water deficit, and high salt stress conditions (Artus et al. 1996; Steponkus et al. 1998). C-repeat binding factor 1 (CBF1) is the DNA binding protein isolated from A. thaliana. The binding of this transcription factor to the C- 30 repeat/dehydration response element (CRT/DRE-motif) can activate the co-ordinate expression of several COR genes (Jaglo-Ottosen et al. 1998). Expression of CBF1 in transgenic plants resulted in enhanced freezing tolerance (Jaglo-Ottosen et al. 1998; Liu et al. 1998). Citrus is an economically important crop throughout the world, and its productivity is seriously affected by low temperature (Porat et al. 2002). Traditional breeding methods of major crops for improving freezing tolerance have had limited success in commercial Citrus species (Yelenosky, 1985). An alternative approach is to identify and characterize important cold induced genes and introduce these genes into susceptible crops to enhance freezing tolerance (Owens et al. 2002). P. trifoliata is a popular rootstock used in the citrus industry to impart greater cold tolerance to the scion (Yelenosky 1985). Dehydrins have been identified from Citrus and related species under low temperature stress (Cai et al. 1995; Hara et al. 1999; Porat et al. 2002). Treatment approaches for low temperature studies in plants are usually ?cold shock? in which plants are moved from control temperature to 4 ?C directly, versus ?cold acclimation? which more closely resembles the natural conditions in the Southeastern United States. In our experiment, one year old plants were cold acclimated using a gradually declining day/night temperature regime as outlined by Yelenosky (1979) and Nesbitt et al. (2002) for citrus. This study was undertaken to elucidate the genetic basis of cold acclimation in P. trifoliata in order to develop a transgenic approach toward enhancing cold hardiness in Citrus cultivars. 31 Materials and methods Plant growth conditions One year old P. trifoliata plants were grown for 6 weeks in a growth chamber with a 12 h light period at 400 ?mol m -2 s -1 intensity. The regimen for temperature decline was as follows: 32?C day/21?C night for 14 days; 27?C day/16?C night for 7 days; 24?C day/13?C night for 7 days and 18?C day/7?C night for 7 days. Plants were uniformly watered every day. RNA extraction and mRNA differential display Expanded leaves at the end of the second and the fifth week were collected, immediately immersed in liquid nitrogen and stored at ?80?C for later use. RNA was extracted from leaves according to the RiboPure kit protocol (Ambion, Austin, TX). Extracted RNA was mixed with 1/9 volume of 10X DNase buffer and 4?l DNase I (2U/?l) and incubated for 30 min at 37?C to digest the remaining genomic DNA. Digested RNA was treated with DNase inactivation reagent (20% volume) for 2 min, followed by centrifugation for 1 min at 14000g and transferred to a new tube. The concentration of isolated RNA was measured using an Eppendorf Biophotometer (Brinkmann Instruments, NY). The quality of RNA was checked using formaldehyde-agarose gel electrophoresis. RNA from the end of second week was used as unacclimated control and RNA from the end of fifth week as treatment. mRNA differential display was performed using RNAimage kits and 144 primer pairs according to the protocol supplied with this kit (GenHunter, TN). 0.2?g of RNA was reverse transcribed in a 20?l reaction mixture at 42?C for 60 min with M- MuLV reverse transcriptase (GenHunter). Amplification of cDNA fragments was 32 performed in a 20?l reaction mixture containing 2?l of the reverse transcribed cDNA, 0.2?M arbitrary primer (GenHunter), 0.2?M anchored oligo (dT)-primers (H-T11M, where M=A, G, C), 2?M of each dNTP, 10mM Tris-Cl, pH8.4, 50mM KCl, 1.5mM MgCl 2 , 0.001% gelatin, 1?l ?-[S 35 ] dATP (1000Ci/mmole) and 1 U Taq DNA polymerase (Qiagen, CA). The PCR program consisted of 40 cycles: 30 s at 94?C, 2 min at 40?C, 1 min at 72?C; and a final 5 min elongation step at 72?C. Amplified PCR products were separated on a 6% denaturing PAGE gel. Gel was transferred to a filter paper (Whatman, England) and dried at 80?C for 1 hour in a gel dryer (BioRad, CA). PCR products on filter paper were exposed to BioMax Kodak film covered with two intensifying screens for 24 to 72 hours in a ?80?C freezer. The film was developed and the differentially expressed bands between control and treatment were excised from the filter paper according to the pattern on the film. The PCR products were extracted according to the GenHunter protocol, and reamplified using the original primer pair. Cloning and sequence analysis of DNA fragments Selected amplified DNA fragments were ligated directly into a PCR-Trap Vector (GenHunter) and transformed into competent Escherichia coli (GenHunter). Ten colonies were selected for each transformation event. 20?l lysis PCR was carried out according to GenHunter protocol. 10?l of lysis PCR products were separated on 1.5% agarose gel. Remaining 10?l of PCR product containing correct size inserts were digested with 0.2?l of TaqI (10U/?l), 10mM Tris-HCl, pH8.4, 50mM KCl, 1.5mM MgCl 2 , 0.001% gelatin, and incubated at 65?C overnight and products analyzed in agarose gel. Differentially restricted DNA fragments were used for plasmid isolation. Only fragments larger than 33 250bp were selected and sequenced in both directions using Rseq and Lseq primers (GenHunter) with ABI 3100 DNA sequencer (AU Genomics Lab). Analysis of nucleotide sequence of selected fragments was carried out using National Center for Biotechnology Information BLASTx search tool. Quantitative Relative Reverse Transcription Polymerase Chain Reaction (RT-PCR) Quantitative relative RT-PCR was used to confirm the differential expression of DNA fragments isolated from control and cold acclimated plants. 2.5?g total RNA was reverse transcribed with 0.5mM dNTP, 5mM oligo(dT)-primers, 10mM Tris-HCl, pH 8.3, 50 mM KCl, 15mM MgCl 2 , 1?l RNase inhibitor, and 100U M-MuLV-RT (Ambion). The mixture (20?l total reaction volume) was incubated at 42?C for 1 hour. 1?l RT reaction was amplified in a 25?l solution with 10mM Tris-HCl, pH8.4, 50mM KCl, 1.5mM MgCl 2 , 2.5mM dNTPs, 0.3?M actin primer, 0.6?M actin competitor, 0.5mM of each gene specific primers (Ambion) and Taq polymerase (Qiagen). PCR cycle program was as follows: 30 s at 94?C, 30 s at 55?C to 60?C and 30 s at 72?C for 30 to 35 cycles. Specific oligonucleotide primers were designed for each DD product for RT-PCR. The optimal PCR annealing temperatures and the cycle numbers are shown in Table 1. 10?l of the amplification products were separated using 1.8 % agarose gel electrophoresis and stained with ethidium bromide. The stained gel was used for quantitation of each band using a BioRad photo documentation system. A constitutively expressed actin gene was used as an internal standard in each reaction. 34 Results and discussion Differential display polymerase chain reaction (DDRT-PCR) was used to study the responses of P. trifoliata to low temperature acclimation. A total of 144 different primer combinations were used and about 15,000 cDNA fragments were analyzed after autoradiography. Two hundred fifty five putative differentially expressed DNA fragments were cloned, sequenced and analyzed. DDRT-PCR can be a powerful technique to study differentially expressed genes. However, this technique can produce some spurious results (Marty et al. 1997). For example, we found two or three unique DNA fragments in some single bands. Because of this comigration of several separate fragments, ten clones obtained from each band on the gel were subsequently examined. These colonies were randomly selected from each transformation, and the sequence of the cloned insert in each of the colonies was determined. Of the 255 putative differentially expressed DNA fragments, 14 (8 up regulated and 6 down regulated) fragments were confirmed to be differentially expressed in treated plants as determined by quantitative relative RT-PCR and show significant homology to known genes in the GenBank database using the BLASTx search utility. The nucleotide sequence of selected clones has been deposited in GenBank and their accession numbers along with the results of the BLASTx search are shown in Table 2. The subsequent results presented in this paper summarize our characterization of the 8 up-regulated transcripts. Quantitative RT-PCR has been used extensively to study gene expression because of its high sensitivity and reproducibility (Taylor and Harrier 2003). Three different sets of independently isolated RNA were used to confirm the differential display results with 35 RT-PCR. Only reproducible differences between control and treatment in all three sets were considered positive. Agarose gel electrophoresis pattern of eight up-regulated differential display (DD) genes by RT-PCR with actin as an internal standard and a histogram representing relative quantity of each of the amplified bands on the gel are presented in Fig 1. The inferred amino acid sequence of P1 shows 71% similarity to a betaine/proline transporter from Avicennia marina. Plants synthesize compatible osmolytes such as proline, glycine betaine and sugar under stress (Ishitani et al. 1993). In bacteria and animals, transporters have been identified that mediate the transport of either proline and betaine or GABA and betaine (Csonka 1989; Yamauchi et al. 1992). Increased level of proline in phloem sap during water stress in alfalfa suggests a role of transporter (Guerrier 1996). HvProT in barley encodes for proline transporter under salinity (Ueda et al. 2001). It is not only important to synthesize solutes, but its transport within cell may be equally important in stress abatement. These reports indicate a role for transport of proline and/or betaine in adaptation of plant cells to low water potential. The inferred amino acid sequence of P2 shows 94% similarity to an A. thaliana water channel protein (aquaporin). Aquaporins are 25-30 kDa polypeptides with six putative membrane-spanning segments (Verkman et al. 1996), which facilitate movement of water across membrane by forming water-specific pores (Sch?ffner 1998). Ice plant aquaporin is down-regulated under low water potential (Yamada et al. 1995) and the down regulation of aquaporin might allow for cellular water conservation (Johansson et al. 1998). In contrast, the rd28 gene in A. thaliana is up-regulated under water stress 36 (Yamaguchi-Shinozaki K et al. 1992). Because of their role in water transport, it is logical to assume an important role for aquaporins in abatement of stress in plants. The inferred amino acid sequences of P3 and P4 share 85% similarity with an aldo-keto reductase protein and 85% similarity with an early light inducible protein (ELIP) in A. thaliana, respectively. Water deficiency and low temperature can impose oxidative stress and increase production of reactive oxygen species (ROS) including the free radical superoxide (Moran et al. 1994; Powles et al. 1983). Free radicals can further react with cellular constituents such as lipids. Both aldo-keto reductase and early light inducible protein can detoxify these toxic chemicals (Burczynski et al. 2001; Hutin et al. 2003). The inferred amino acid sequence of P5 demonstrates 55% similarity with an Oryza sativa nitrate transporter, NRT1-5. Nitrate transporters have been studied in species such as A. thaliana (Tsay et al. 1993) and ice plant (Popova et al. 2003). AtNRT1.1 is a low-affinity nitrate transporter in A. thaliana (Tsay et al. 1993) and is associated with stomatal opening and drought susceptibility (Guo et al. 2003), while McNRT1 nitrate transporter from ice plant is dependent on salt (Popova et al. 2003). Recent research on nitrate assimilation in Synechococcus sp. PCC7002 indicates that low temperature can inhibit the uptake of nitrate (Sakamoto and Bryant, 1998). The strongly up regulated expression of this gene in Poncirus during low temperature might indicate an active transport of nitrate which could be helpful to acclimate plants under adverse environment. The inferred amino acid sequence of P6 shows 54% similarity with a putative tetratricopeptide-repeat containing protein (TPR) from O. sativa. The repeat motif serves 37 as protein-protein interaction module found in a number of functionally different proteins which facilitates specific interactions with a partner protein (Das et al. 1998). Soybean gmsti with a TPR containing motif is highly expressed on exposure to elevated temperature and plays a role in mediating the heat shock response with HSP70 proteins (Torres et al. 1995). Low temperature exposures are equally relevant for protein denaturation and folding. Hence, the role of TPR containing proteins in cold tolerance may be important. The inferred amino acid sequences of P7 and P8 show 78% similarity to the F- Box protein family in A. thaliana and 94% similarity to ribosomal protein L15 in O. sativa, respectively. There is very limited information about these two genes related to low temperature, salinity and drought stresses. F-box proteins include protein-protein interaction domains that confer substrate specificity for ubiquitination. Ubiquitin- dependent proteolysis has been reported to selectively degrade certain proteins (Callis and Vierstra, 2000). Ribosomal protein plays a major role in controlling cell growth, division and development (Barakat et al. 2001). A low temperature induction of F-box protein and ribosomal L15 suggests the adjustment of cell metabolism at translation level in which certain proteins are selectively degraded and synthesized according to the need of cell. P. trifoliata is an extensively used rootstock in the citrus industry. Citrus species are cold-tender evergreen plants with a tropical and subtropical origin and their capacity to survive freezing temperature does not approach that of other woody plants (Webber et al. 1967). P. trifoliata is a deciduous relative of Citrus and is a cold hardy plant (Yelenosky 1985). In our experiment, we have used a treatment condition that mimics 38 natural declines of temperature in the Southeast U.S. Effects of gradually declining temperature (acclimation) and sudden decrease in temperature (shock) were compared in a preliminary experiment using differential display. RNA was isolated from acclimated leaves and cold shocked leaves. RT-PCR using similar set of primers and RNA from control, cold shocked and cold acclimated plants was performed, and products were analyzed using denaturing PAGE in adjacent lanes (data not shown). As anticipated, DNA fragment representing expression patterns of cold shock and cold acclimation were different. We observed differential pattern of expression under cold acclimation and cold shock. Salinity, drought, and cold are known to cause osmotic/dehydration stress in plants. In this study, three of the induced genes encountered during cold acclimation, have also been reported as important for the maintenance of osmotic balance in plant cells under salt or drought stress. This reinforces the notion that low temperature acclimation of plants involves some mechanisms required for growth and survival under low water potential. Plants can synthesize osmotic chemicals during salinity and drought stresses. Many plant transgenic researches have been focusing on the synthesis of these osmoprotectants but the stress tolerance abilities of transgenic plants have not been increased very efficiently even with overexpressed synthesis enzyme. It?s reasonable to infer that plant might not have enough transporters to transport these newly synthesized chemicals to the function place in plant. The comparison of the cold tolerance ability between a transgenic plant with a synthesis enzyme such as P5CS and a transgenic plant with both the synthesis enzyme and transporter genes may shed more light on this aspect. 39 Besides the dehydration damage due to stress, another very serious problem for the plant cell is stress induced oxidative damage. As discussed, low temperature and high light induce oxidative stress in plants. Stresses like drought, salt, ozone and UV irradiation similarly can induce oxidative stress, indicating a common damage factor in plants exposed to environmental stress (Desikan et al. 2001). Plants possess capabilities to cope with oxidative stress through the use of ROS-scavenging enzymes such as superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT), and low molecular weight antioxidants including ascorbic acid, glutathione and phenolic compounds (Asada 1999). Three of the eight up regulated genes isolated have functions related to ROS scavenging. The damages related to certain stresses were reduced in transgenic tobacco with alfalfa aldose?aldehyde reductase (MsALR) and transgenic Arabidopsis with early light inducible protein indicate the participation of these proteins to adapt plants under stress conditions (Bartels 2001; Hutin et al. 2003). The correlation of the accumulation of ELIP and the tolerance to chilling-induced photooxidation in barley shed more light on this aspect (Bei-Paraskevopoulou and Kloppstech 1999). Transformation of model plants with the Poncirus early light inducible protein gene and aldo-keto reductase and the correlated concentration of the reactive chemicals and their derivatives should provide more understanding about the role of these genes in plants. Low temperature is a serious problem for many plants, especially horticulturally important woody plants such as Citrus, but our understanding of the mechanisms of low temperature abatement at the molecular level is limited. Cold acclimation resulted in the up-regulation of several genes involved in maintenance of osmotic balance, scavenging of reactive oxygen species and photo-oxidative protection of plants. Recognition of 40 specific genes and their relevance in the process of stress abatement will allow their use in improvement of stress tolerance ability in Citrus. Acknowledgements We are grateful to Brandon Hockema, Bryan Wilkins and Monte Nesbitt for their helps in sample preparations. This research was funded in part by USDA CSREES Special Research Grants OEP 2001-03124 and 2002-06162 and the Alabama Agricultural Experiment Station. 41 References Artus N, Uemura M, Steponkus PL, Gilmour SJ, Lin C, Thomashow MF (1996) Constitutive expression of the cold-regulated Arabidopsis thaliana COR15 gene affects both chloroplast and protoplast freezing tolerance. Proc. Natl Acad. Sci. USA. 93: 13404-13409. Asada K (1999) The water-water cycle in chloroplasts: scavenging of active oxygens and dissipation of excess photons. Annu. Rev. Plant Physiol. Plant Mol. Biol. 50: 601- 639. Barakat A, Szick-Miranda K, Chang I-F, Guyot R, Blanc G, Cooke R, Delseny M, Bailey-Serres J (2001) The organization of cytoplasmic ribosomal protein genes in the Arabidopsis genome. Plant Physiol. 127: 398-415. Bartels D (2001) Targeting detoxification pathways: an efficient approach to obtain plants with multiple stress tolerance? Trends Plant Sci 6: 284-286. Bei-Paraskevopoulou T, Kloppstech K (1999) The expression of early light-inducible proteins (ELIPs) under high light stress as defense marker in Northern- and Southern European cultivars of barley (Hordeum vulgare). Physiol. Plant. 106: 105-111. Bohnert HJ, Nelson DE, Jensen RG (1995) Adaption to environmental stresses. Plant Cell 7: 1099-1111. Burczynski ME, Sridhar GR, Palackal NT, Penning TM (2001) The reactive oxygen species- and michael acceptor-inducible human aldo-keto reductase AKR1C1 reduces the alpha ,beta -unsaturated aldehyde 4-hydroxy-2-nonenal to 1,4- dihydroxy-2-nonene. J. Biol. Chem. 276: 2890-2897. Cai Q, Moore GA, Guy CL (1995) An unusual group 2 LEA gene family in citrus responsive to low temperature. Plant Mol. Biol. 29: 11-23. Callis J, Vierstra RD (2000) Protein degradation in signaling. Curr Opin Plant Biol 3(5): 381-386. Csonka LN (1989) Physiological and genetic responses of bacteria to osmotic stress. Micobiol. Rev. 53: 121-147. Danyluk J, Houde M, Sarhan F (1994) Differential expression of a gene encoding an acidic dehydrin in chilling-sensitive and freezing -tolerant Gramineae species. FEBS Lett. 344: 20-24. 42 Das AK, Cohen PW, Barford D (1998) The structure of the tetratricopeptide repeats of protein phosphatase 5: Implications for TPR-mediated protein-protein interactions. EMBO J. 17: 1192?1199. Desikan R A-H, Mackerness S, Hancock JT, Neill SJ (2001) Regulation of the Arabidopsis transcriptome by oxidative stress. Plant Physiol. 127: 159-172 Fowler S, Thomashow MF (2002) Arabidopsis transcriptome profiling indicates that multiple regulatory pathways are activated during cold acclimation in addition to the CBF cold response pathway. Plant Cell 14: 1675-1690. Garwe D, Thomson JA, Mundree SG (2003) Molecular characterization of XVSAP1, a stress-responsive gene from the resurrection plant Xerophyta viscosa Baker1. J. Exp. Bot. 54: 191-201. Gilmour SJ, Artus N, Thomashow MF (1992) cNDA sequence analysis and expression of two cold-regulated genes of Arabidopsis thaliana. Plant Mol. Biol. 18: 13-21. Gilmour SJ, Hajela R, Thomashow MF (1988) Cold acclimation in Arabidopsis. Plant Physiol. 87: 745-750. Guerrier G (1996) Fluxes of Na + , K + and Cl ? and osmotic adjustment in Lycopersicon pimpinellifolium and L. esculentum during short- and long-term exposures to NaCl. Physiol. Plant. 97: 583?591. Guo F-Q, Young J, Crawford NM (2003) The nitrate transporter AtNRT1.1 (CHL1) functions in stomatal opening and contributes to drought susceptibility in Arabidopsis. Plant Cell 15: 107-117. Hara M, Wakasugi Y, Ikoma Y, Yano M, Ogawa K, Kuboi T (1999) cDNA sequence and expression of a cold-responsive gene in Citrus unshiu. Biosci. Biotech. Biochem. 63: 433-437. Hazel JR (1995) Thermal adaption in biological membranes: is homeoviscous adaption the explanation? Annu. Rev. Physiol. 75: 19-42. Hutin C, Nussaume L, Moise N, Moya I, Kloppstech K, Havaux M (2003) Early light- induced proteins protect Arabidopsis from photooxidative stress. Proc. Natl Acad. Sci. USA. 100: 4921-4926. Ishitani M, Arakawa K, Mizuno K, Kishitani S, Takabe T (1993) Betaine aldehyde dehydrogenase in the Gramineae: levels in leaves of both betaine-accumulating and nonaccumulating cereal plants. Plant Cell Physiol. 34: 493?495. 43 Jaglo-Ottosen K, Gilmour SJ, Zarka D, Schabenberger O, Thomashow MF (1998) Arabidopsis CBF1 overexpression induce COR genes and enhances freezing tolerance. Science 280: 104-106. Johansson I, Karlsson M, Shukla VK, Chrispeels MJ, Larsson C, Kjellbom, P. (1998) Water transport activity of the plasma membrane aquaporin PM28A is regulated by phosphorylation. Plant Cell 10: 451?459. Kasuga M, Liu Q, Miura S, Yamaguchi-Shinozaki K, Shinozaki K (1999) Improving plant drought, salt and freezing tolerance by gene transfer of a single stress- inducible transcription factor. Nat Biotechnol. 17: 287-291. Knight H, Trewavas AJ, Knight MR (1996) Cold calcium signaling in Arabidopsis involves two cellular pools and a change in calcium signature after acclimation. Plant Cell 8: 489-503. Liu Q, Kasuga M, Sakuma Y, Abe H, Miura S, Yamaguchi-Shinozaki K, Shinozaki K (1998) Two transcription factors, DREB1 and DREB2, with an EREBP/AP2 DNA binding domain separate two cellular signal transduction pathways in drought- and low-temperature-responsive gene expression, respectively, in Arabidopsis. Plant Cell 10: 1391-1406. Lynch DV, Steponkus PL (1987) Plasma membrane lipid alterations associated with cold acclimation of winter rye seedlings (Secale cereale L.cv Puma). Plant Physiol. 83: 761-767. Marty C, Jones B, Bouquin T, Latche A, Pech JC, Bouzayen M (1997) Improved screening of cDNAs generated by mRNA differential display enables the selection of true positives and the isolation of weakly expressed messages. Plant Mol. Biol. Rep. 15: 236-245. Moran JF, Becana M, Iturbe-Ormaetxe I, Frechilla S, Klucas RV, Aparicio-Tejo P (1994) Drought induces oxidative stress in pea plants. Planta 194: 346-352. Nesbitt ML, Ebel RC, Findley D, Wilkins B, Woods F, Himelrick D (2002) Assays to assess freeze injury of Satsuma mandarin (Citrus unshiu). HortScience. 37: 871- 877. Owens CL, Thomashow MF, Hancock JF, Iezzoni AF (2002) CBF1 orthologs in sour cherry and strawberry and the heterologous expression of CBF1 in strawberry. J. Am. Soc. Hort. Sci. 127: 489-494. Popova OV, Dietz KJ, Golldack D (2003) Salt-dependent expression of a nitrate transporter and two amino acid transporter genes in Mesembryanthemum crystallinum. Plant Mol. Biol. 52: 569-578. 44 Porat R, Pavoncello D, Lurie S, Mcollum TG (2002) Identification of a grapefruit cDNA belonging to a unique class of citrus dehydrins and characterization of its expression patterns under temperature stress conditions. Physiol Plant. 115: 598- 603. Powles S, Berry J, Bjorkman O (1983) Interaction between light and chilling temperature on the inhibition of photosynthesis in chilling-sensitive plants. Plant Cell Environ. 6: 117?123. Sakamoto T, Bryant DA (1998) Growth at low temperature causes nitrogen limitation in the cyanobacterium Synechococcus sp. PCC7002. Arch Microbiol. 169(1): 10-19 Sch?ffner AR (1998) Aquaporin function, structure, and expression: Are there more surprises to surface in water relations? Planta 204: 131?139. Steponkus PL, Uemura M, Joseph R, Gilmour SJ, Thomashow MF (1998) Mode of action of the COR15a gene on the freezing tolerance of Arabidopsis thaliana. Proc. Natl Acad. Sci. USA. 95: 14570-14575. Taylor J, Harrier LA (2003) Expression studies of plant genes differentially expressed in leaf and root tissues of tomato colonised by arbuscular mycorrhizal fungus Glomus mosseae. Plant Mol. Biol. 51: 619-629. Thomashow MF (1994) Arabidopsis thaliana as a model for studying mechanisms of plant cold tolerance. In Meyeowitz, E.a.S., C. (ed.), In Arabidopsis. Cold Spring Harbor Press, Cold Spring Harbor, USA pp. 807-834. Thomashow MF (1998) Role of cold-responsive genes in plant freezing tolerance. Plant Physiol. 118: 1-8. Torres JH, Chatellard P, Stutz E (1995) Isolation and characterization of gmsti, a stress- inducible gene from soybean (Glycine max) coding for a protein belonging to the TPR (tetratricopeptide repeats) family. Plant Mol. Biol. 27: 1221-1226. Tsay Y, Schroeder J, Feldmann K, Crawford N (1993) The herbicide sensitivity gene CHL1 of Arabidopsis encodes a nitrate-inducible nitrate transporter. Cell 72: 705? 713. Tseng MJ, Li PH (1991) Changes in protein synthesis and translatable messenger RNA populations associated with ABA-induced cold hardiness in potato (Solanum commersonii). Physiol. Plant. 81: 349-358. Ueda A, Shi W, Sanmiya K, Shono M, Takabe T (2001) Functional analysis of salt- inducible proline transporter of barley roots. Plant Cell Physiol. 42: 1282-1289. 45 Verkman AS, van Hoek AN, Ma T, Frigeri A, Skach WR (1996) Water transport across mammalian cell membranes. Am. J. Physiol. 270: C12-30. Webber HJ, Reuther W, Lawton HW (1967) History and development of the citrus industry. In Reuther, W., Webber, H.J., Batchelor, L.D. (ed.), The citrus industry. University of California, Berkeley, USA Vol. 1, pp. 1-39. Wolfraim L, Langis R, Tyson H, Dhindsa R (1993) cDNA sequence, expression and transcript stability of a cold acclimation-specific gene, cas18, of alfalfa (Medicago falcata) cells. Plant Physiol. 101: 1275-1282. Yamada S, Katsuhara M, Kelly WB, Michalowski CB, Bohnert, HJ (1995) A family of transcripts encoding water channel proteins: tissue-specific expression in the common ice plant. Plant Cell 7: 1129-1142. Yamaguchi-Shinozaki K, Koizumi M, Urao S, Shinozaki K (1992) Molecular cloning and characterization of 9 cDNAs for genes that are responsive to desiccation in Arabidopsis thaliana: sequence analysis of one cDNA clone that encodes a putative transmembrane channel protein. Plant Cell Physiol. 33: 217-224. Yamauchi A, Uchida S, Kwon H, Preston A, Robey R, Garcia-Perez A, Burg M, Handler J (1992) Cloning of a Na + - and Cl - -dependent betaine transporter that is regulated by hypertonicity. J. Biol. Chem. 267: 649-652. Yelenosky G (1979) Accumulation of free proline in citrus leaves during cold hardening of young trees in controlled temperature regimes. Plant Physiol. 64: 425-427. Yelenosky G (1985) Cold hardiness in Citrus. Hort Rev 7: 201-238. Yubero-Serrano E, Moyano E, Medina-Escobar N, Munoz-Blanco J, Caballero J (2003) Identification of a strawberry gene encoding a non-specific lipid transfer protein that responds to ABA, wounding and cold stress. J. Exp. Bot. 54: 1865-1877 46 Table 1. Oligonucleotide primer sequences, PCR conditions and cycle numbers for the confirmation of differential displayed products with quantitative RT-PCR DD product Forward Primer (5?-3?) Reverse Primer (5?-3?) PCR annealing temperature Cycle numbers P1 AAGCTTCCTGCAATCAGTCAT GGCAATTGGAGAATACACTTGA 55?C 33 P2 GCCATCCCATTCAAGTCCAA GAATCGGCCATGAACATGTA 57?C 32 P3 CGTATGGCACAGTAATGGGTG ACTCCAAGCAAATCCTTCCC 58?C 30 P4 GGTTGCGTCCCTGGTTCCTT AGATTTATTTACAGTAGGAT 55?C 30 P5 ATAAAGGGAGTGGTGACGGC AACACAGTATCCGGATTCCG 57?C 35 P6 TGATGGAAGTCAAGCAACTGG CCACAAACATTTGGGAAGCA 58?C 33 P7 ATGAATGTGGCAGACTTGAGTC AGCTGAGCATGTGTCATTGC 55?C 35 P8 ACGACCTTCAAGAAGGGCA CCGCACATTATTATCACCTTC 54?C 35 47 Table 2. Isolated DD products, accession number and percentage similarity to known proteins by BLASTx search in NCBI DD product Accession Number Length (bp) Plant protein similarity Function P1 CN779663 529 betaine/proline transporter 71% Osmotic, Oxidative P2 CN779664 259 water channel protein 94% Osmotic P3 CN779665 508 aldo-keto reductase 85% Oxidative P4 CN779666 313 early light inducible protein 85% Oxidative P5 CN779667 389 nitrate transporter NRT1 55% Osmotic P6 CN779668 645 tetratricopeptide repeat containing protein (TPR) 54% Multifunction P7 CN779669 444 F-Box protein family 78% Multifunction P8 CN779670 464 ribosomal protein L15 94% Protein synthesis 48 P1 P2 P3 P4 P5 P6 P7 P8 C T C T C T C T C T C T C T C T Actin Gene of interest 0 100 200 300 400 500 600 700 800 p1 p2 p3 p4 p5 p6 p7 p8 DD Products Differential expression detected by RT-PCR Control Treatment Fig. 1 (Top): Confirmation of differential expression of 8 DD products using relative quantitative RT-PCR with RNA from control and cold acclimated leaves. The cDNAs were synthesized from total RNAs isolated from control leaves (end of second week) and cold acclimation leaves (end of fifth week). Actin mRNA was used as an internal control. Specific RT-PCR primer pair information for all DD products is listed in table 1. (Bottom): A histogram showing the relative abundance of 8 DD products between control and cold acclimated plants. The intensities in each control and cold acclimation pair were normalized by setting the intensity of actin gene to 100. The values are the means of three independent experim ents + SE. 49 III. Down-regulated gene expression of cold acclimated Poncirus trifoliata 1 ABSTRACT Citrus sp. are important commercial fruit crops throughout the world that are occasionally devastated by subfreezing temperatures. Poncirus trifoliata (maximum freeze tolerance of -26?C) is a close relative of commercial Citrus sp. (maximum freeze tolerance of -10?C) that has been used in breeding programs to develop more cold-hardy genotypes and as a rootstock to enhance freeze tolerance of the scion. Species with greater freeze tolerance vary in gene expression during cold acclimation temperatures. mRNA differential display (DDRT-PCR) and quantitative relative RT-PCR were used to study the down regulation of gene expression in intact P. trifoliata exposed to a gradual cold acclimation regime to enhance our understanding of the mechanism that makes this species so freeze tolerant. Six down regulated genes were isolated and sequenced. These down regulated genes showed high homology to the following known function genes: chlorophyll a/b binding protein, photosystem II OEC 23, carbonic anhydrase, tumor related protein, pyrrolidone-carboxylate peptidase and ?-galactosidase. Photoprotection and the global control of expression of genes related to photosynthesis appear to be important mechanisms for cold acclimation of P. trifoliata. Key words: Differential display, down regulated genes, Poncirus trifoliata, cold acclimation and quantitative relative RT-PCR 1 The nucleotide sequences reported in this paper have been submitted in Genbank under accession numbers of CN807022 (PD1), CN807023 (PD2), CN807024 (PD3), CN807025 (PD4), CN807026 (PD5), CN807027 (PD6). INTRODUCTION The geographical distribution of plants is a function of their maximum freeze tolerance. Freezing temperatures can cause extracellular ice formation, which lowers apoplastic water potential, dehydrates the symplast, and destabilizes cellular membranes (Steponkus 1984). Plants have evolved a wide variety of mechanisms that allow them to thrive in hostile environments even though their ability to survive in adverse environments varies greatly among species (Garwe et al. 2003). Many plants acquire freeze tolerance when subjected to low, nonfreezing temperatures, a phenomenon known as cold acclimation (Levitt 1980; Guy 1990). Acquisition of freezing tolerance requires physiological and biochemical changes including changes induced by altered gene expression (Guy et al. 1985; Thomashow 1998). A number of up-regulated genes have been identified in several plant species exposed to cold acclimating temperatures including alfalfa (Wolfraim et al. 1993), Arabidopsis thaliana (Gilmour et al. 1992) and barley (Sutton et al. 1992). Considerably less research has been devoted to down-regulation of genes during cold acclimation, even though studies have shown suppression of metabolic activity may be an important component of plant adaptation to low temperature. Arabidopsis transcriptome-profiling resulted in 306 genes identified as cold responsive, 88 of which were found to be down- regulated in response to low temperature (Fowler and Thomashow 2002). Several Citrus sp. are important fruit crops throughout the world, but productivity is occasionally devastated by freezing temperatures in freeze-prone regions (Yelenosky 1985). Poncirus trifoliata (L.) Raf is a close relative of Citrus sp. in the Rutaceae family. P. trifoliata is used as a rootstock for commercial Citrus sp. to impart greater 51 cold tolerance to the scion, and has been crossed with commercial Citrus sp. in breeding programs to produce more cold-hardy germplasm, although these crosses have not led to genotypes with commercially acceptable fruit (Barrett 1978, 1982; Yelenosky 1985; Yelenosky et al. 1993). P. trifoliata is freeze tolerant to -26?C (Spiegel-Roy and Goldschmidt 1996), whereas commercially grown Citrus sp. are freeze tolerant to at most -10?C (Yelenosky 1985), a level that is very similar to that of many herbaceous species such as arabidopsis, which also has a maximum freeze tolerance of -10?C (Gilmour et al. 1988). Gene expression during cold acclimation of woody plants that exhibit such low freeze tolerance is not well understood. Because P. trifoliata is used in breeding programs and as a rootstock for Citrus sp., there must be significant genetic overlap among these species, yet differences in freeze tolerance likely result from significant differences in gene expression during cold acclimation. We are studying gene expression of P. trifoliata during cold acclimation to identify genes that are involved in its greater freeze tolerance. Transcriptome profiling of plants to environmental stresses can be studied via several different techniques, including differential display reverse transcription PCR (DDRT-PCR), serial analysis of gene expression (SAGE), subtractive hybridization, DNA-chip and cDNA microarray (Donson et al. 2002). DDRT-PCR, the technique we used in the present study, is a useful technique for gene expression studies in non-model organisms because detailed genomic information of the organism is not necessary and it is technically simple (Liang and Pardee 1992). In our study, we used a gradual cold acclimation regime that simulated natural declines in temperature during autumn in subtropical regions. Other research on Citrus sp. and other related species utilized a 52 much faster cold treatment that was equivalent to cold shock (Cai et al. 1995; Hara et al. 1999; Porat et al. 2002). Our research approach compliments theirs in helping identify differences in gene expression under different temperature acclimation regimes. We examined gene regulation by DDRT-PCR during a gradual process of cold acclimation in P. trifoliata. The genes identified by comparing DNA sequences provide insight into the process of cold acclimation and freeze tolerance of citrus. MATERIALS AND METHODS Plant culture One-year-old P. trifoliata (L.) Raf. plants were grown for 5 weeks in a growth chamber with a 12 h light period at 400 ?mol m -2 s -1 intensity. The regimen for temperature decline was as follows: 32?C day/21?C night for 14 d; 27?C day/16?C night for 7 d; 24?C day/13?C night for 7 d and 18?C day/7?C night for 7 d. These temperature treatments have been shown to increase freezing tolerance of P. trifoliate and improve plant response to freezing temperatures (Young and Peynado 1962; Stathakopoulos and Erickson 1966). Plants were uniformly watered every day. RNA extraction and mRNA differential display Full expanded leaves were collected at the end of the 2 nd and the 5 th weeks at the same time of day (10 th hour in day time cycle), immediately immersed in liquid nitrogen and stored at ?80?C for later use. RNA was extracted from leaves according to the RiboPure kit protocol (Ambion, Austin, TX). Extracted RNA was mixed with 1/9 volume of 10X DNase buffer and 4?l DNase I (2U/?l) and incubated for 30 min at 37?C to digest the 53 remaining genomic DNA. The resulting preparation treated with DNase inactivation reagent (20% volume) for 2 min, followed by centrifugation for 1 min at 14,000g. The resulting supernatant was transferred to a new tube, and the concentration of isolated RNA was measured using an Eppendorf Biophotometer (Brinkmann Instruments, NY). The quality of RNA was checked using formaldehyde-agarose gel electrophoresis. Total RNA prepared from leaves at the end of the second week of treatment was used as the unacclimated control and RNA from leaves harvested at the end of fifth week of cold acclimation was used as the treatment. mRNA differential display was performed using RNAimage kits and 144 primer pairs according to the protocol supplied with this kit (GenHunter, TN). RNA (0.2?g) was reverse transcribed in a 20?l reaction mixture at 42?C for 60 min with M-MuLV reverse transcriptase (GenHunter). Amplification of cDNA fragments was performed in a 20?l reaction mixture containing 2?l of the reverse transcribed cDNA, 0.2?M arbitrary primer (GenHunter), 0.2?M anchored oligo (dT)- primers (H-T11M, where M=A, G, C), 2?M of each dNTP, 10mM Tris-Cl, pH8.4, 50mM KCl, 1.5mM MgCl 2 , 0.001% gelatin, 1?l ?-[S 35 ] dATP (1000Ci/mmole) and 1 U Taq DNA polymerase (Qiagen, CA). The PCR program consisted of 40 cycles: 30 s at 94?C, 2 min at 40?C, 1 min at 72?C; and a final 5 min elongation step at 72?C. Amplified PCR products were separated on a 6% denaturing PAGE gel. The gel was transferred to a filter paper (Whatman, England) and dried at 80?C for 1 hour in a gel dryer (BioRad, CA). PCR products were detected by exposing the filter paper to BioMax Kodak film for 24 to 72 hours in a ?80?C freezer. The film was developed and the differentially expressed bands between control and treatment were excised from the filter paper 54 according to the pattern on the film. The PCR products were extracted according to the GenHunter protocol, and reamplified using the original primer pair. Cloning and sequence analysis of DNA fragments Selected, amplified DNA fragments were ligated directly into a PCR-Trap Vector (GenHunter) and transformed into competent Escherichia coli (GenHunter). Ten colonies were selected for each transformation event, and a 20?l PCR reaction was carried out on the lysed colonies according to the GenHunter protocol. Half of this PCR product was separated on a 1.5% agarose gel, and the remaining 10?l of PCR product (from colonies containing correct size inserts) were digested with 0.2?l of TaqI (10U/?l), 10mM Tris- HCl, pH8.4, 50mM KCl, 1.5mM MgCl 2 , 0.001% gelatin, and incubated at 65?C overnight and products analyzed on agarose gel. Differentially restricted DNA fragments were used for plasmid isolation. Only fragments larger than 250bp were selected and sequenced in both directions using Rseq and Lseq primers (GenHunter) with ABI 3100 DNA sequencer (AU Genomics Lab). Analysis of nucleotide sequence of selected fragments was carried out using National Center for Biotechnology Information BLASTx search tool. Quantitative Relative Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) Quantitative relative RT-PCR was used to confirm the differential expression of DNA fragments isolated from control and cold acclimated plants. Total RNA (2.5?g) was reverse transcribed with 0.5mM dNTP, 5mM oligo(dT)-primers, 10mM Tris-HCl, pH 8.3, 50 mM KCl, 15mM MgCl 2 , 1?l RNase inhibitor, and 100U M-MuLV-RT reverse 55 transcriptase (Ambion). The mixture (20?l total reaction volume) was incubated at 42?C for 1 hour. 1 ?l of the reverse transcriptase reaction was amplified in a 25?l reaction with 10mM Tris-HCl, pH8.4, 50mM KCl, 1.5mM MgCl 2 , 2.5mM dNTPs, 0.3?M actin primer, 0.6?M actin competitor, 0.5mM of each gene specific primer and Taq polymerase (Qiagen). The PCR cycle program was as follows: 30 s at 94?C, 30 s at 55?C to 60?C and 30 s at 72?C for 30 to 35 cycles. Specific oligonucleotide primers were designed for each DD product for RT-PCR. The optimal PCR annealing temperatures and the cycle numbers are shown in Table 1. 10?l of the amplification products were separated using 1.8 % agarose gel electrophoresis and stained with ethidium bromide. The stained gel was used for quantitation of each band using a BioRad photo documentation system. A constitutively expressed actin gene was used as an internal standard in each reaction. RESULTS AND DISCUSSION Citrus sp. are some of the major fruit crops of the world, and are classified as cold-tender evergreens with tropical and subtropical origins (Webber et al. 1967). The distribution of citrus is mainly affected by temperature, especially subfreezing temperatures (Young 1961). Its capacity to survive freezing temperature does not approach that of northern woody plants, yet commercial citrus crops are grown in freeze-prone areas throughout the world. During the late 1970s to mid 1980s, freeze events were especially devastating and caused extensive damage to the citrus industry in the Southeastern US (Yelenosky 1985). P. trifoliata, a very cold-hardy relative of Citrus sp., has been extensively used in breeding programs to develop more cold-hardy genotypes and as a rootstock to impart a degree of cold tolerance to the citrus scion (Yelenosky 1985). Little research has been 56 conducted on this cold-hardy plant to gain an understanding of the molecular processes associated with cold acclimation. mRNA Differential Display In this study, DDRT-PCR was used to study low temperature acclimation in P. trifoliata. A total of 144 different primer combinations were used. Six down regulated genes were identified and pattern of expression confirmed by quantitative relative RT-PCR using comparisons between control (non-acclimated) and acclimated plants. As an example, the banding pattern of cDNA fragments amplified by the combination of one primer pair is shown in Fig. 1. To reduce the possibility of false positives associated with DDRT- PCR (Marty et al. 1997), each primer pair was used to amplify two different sets of RNA isolated from different Poncirus plants. RT-PCR products were applied in adjacent lanes on a 6% denatured PAGE gel. Differentially regulated cDNA fragments from both sets of RNA were extracted, cloned and sequenced. All sequenced fragments showed an 11A or 11 T tail (corresponding to the reverse transcription primer HT11M in the RNAimage kit from Genhunter, TN, USA) and the right arbitrary primer sequence, indicating successful cloning of the re-amplified cDNA isolated from filter paper. The results of sequence homology are shown in Table 2. Quantitative RT-PCR has been used extensively to study gene expression because of its high sensitivity and reproducibility (Okamoto et al. 2003; Pires-Alves et al. 2003; Taylor and Harrier 2003). RNA isolated from three different groups of plants was used to confirm results of the differential display using RT-PCR. Only consistent differences between control and treatment in the three sets of RNA were considered significantly 57 different. Fig. 2 (top) shows the agarose gel electrophoresis pattern of six down- regulated genes confirmed by RT-PCR using actin sequence as an internal standard. Fig. 2 (bottom) shows histogram of RT-PCR results. Chlorophyll a/b binding protein (LHC) The inferred amino acid sequence of PD1 shows 96% similarity to Chlorophyll a/b binding protein (LHC) in cucumber seedlings. Light-harvesting chlorophyll a/b (LHC) proteins are major components of light-harvesting antennae of photo system II (PSII) in higher plants. LHC levels are known to change by the intensity of irradiance (Anderson et al. 1995). LHC RNA level in Arabidopsis was suppressed when plants were exposed to low temperature (Strand et al. 1997). In Ammopiptanthus mongolicus, a drought tolerant plant, expression of LHC and other photosynthetic genes were reduced under water stress suggesting reduction in energy requirement under water stress condition (Zhang et al. 2002). In maize (Hao et al. 1999) and salt meadow reed, Phragmites communis [P. australis] (Wang et al. 1998), LHC II gene expression was markedly reduced during osmotic stress suggesting that the down-regulation of photosynthesis related components might help with the dissipation of excess excitation energy. Plants respond to stress by slowing down the metabolic process, thus lowering the need for energy generated by photosynthesis. If photosynthetic energy levels are not changed accordingly, damaging active oxygen species could be produced. 58 Photo system II OEC 23 The inferred amino acid sequence of PD2 shows 91% similarity to Photo system II OEC 23 in Arabidopsis. Photo system II (PSII) is a multi-subunit pigment-protein complex that catalyzes the splitting of water and releases molecular oxygen in the biochemical pathway of photosynthesis. The 23kDa protein is an extrinsic protein of PSII and part of the structure of oxygen-evolving complex in higher plants (Michael and Peter 1997). Murota et al. (1994) found almost complete dissociation of the 23kDa protein from isolated PSII, inhibition of photochemical reactions, and damage of the oxygen-evolving complex in tobacco grown under high concentration of NaCl. To date most research on OEC 23 has focused mainly on the dissociation and reassociation of the OEC. Our study provides the first evidence that changes in the transcriptional level of this component of the photosynthetic apparatus during cold acclimation. Because PSII drives a strong oxidizing reaction that is responsible for splitting water that generates oxygen, down regulation of OEC23 may result in partial loss of function of PSII. This in turn might correlate with lower demand for energy minimizing the production of potentially damaging reactants under the stress situation. Carbonic anhydrase The inferred amino acid sequence of PD3 shows 82% similarity to carbonic anhydrase (CA) in mechanically wounded tobacco plants. Carbonic anhydrase is a zinc-containing enzyme which catalyses the reversible hydration of CO 2 to produce bicarbonate (Larsson et al. 1997). Known CAs can be grouped into -CA, -CA, and -CA types and plants appear to contain all three types (Hewett-Emmett and Tashian 1996). CA was proposed 59 to expedite diffusion of CO 2 into the chloroplast acting as a partner with Rubisco in CO 2 fixation (Graham et al. 1984), and to play a role in the buffering capacity of the chloroplast stroma by enhancing the rates of the dehydration/hydration reactions (Mayeau and Coleman 1991). In addition to reversibly converting CO 2 to bicarbonate, CA in tobacco also appears to have antioxidant activity as a plant defense response to biotic stress (Slaymaker et al. 2002). The authors further suggested that oxidative stress protection and the CA enzymatic activity are independent functions of plant CA. It has also been reported that wounding can cause changes in the expression of CA transcripts, indicating a new function related to maintenance of plant cellular homeostasis (Hara et al. 2000). The CA found in this experiment was strongly expressed in control plants, but was undetectable in cold acclimated Poncirus plants. Pea CA transcripts were reported to be light-regulated (Mayeau and Coleman 1991). Our plant samples were collected at the same time during the day/night cycle, excluding the possibility of a confounding effect of light on CA expression. This is the first report about the potential role of CA in the adaptation of plants to low temperature. Whether this role is related to CA?s participation in photosynthesis or the antioxidant process, or both, is not clear at this time. Tumor related protein The inferred amino acid sequence of PD4 shows 47% similarity with a tumor related protein expressed in tobacco callus and tumors. The function of this protein in callus and tumors is still not clear (Fujita et al. 1994). PD4 also shows 45% similarity to miraculin from grapefruit and trypsin inhibitor from Theobroma obovatum, respectively. Miraculin 60 is a taste modifier protein which can alters human taste perception (Theerasilp et al. 1989). Both miraculin and trypsin inhibitor are likely involved in biotic stresses. LeMir, a putative miraculin gene in tomato, was induced by a root-knot nematode. The encoded protein product might involve in the inhibition of the penetration of microorganisms around tomato root (Brenner et al. 1998). Transgenic tobacco plants expressing a cowpea trypsin inhibitor gene showed enhanced levels of insect resistance to a variety of insect pests (Boulter et al. 1989). Trypsin inhibitor activity was critical for the inhibition of growth and development of cyst nematodes in sugar beet hairy roots expressing the sporamin (trypsin inhibitor) gene (Cai et al. 2003). Pyrrolidone-carboxylate peptidase The inferred amino acid sequence of PD5 shows 72% similarity to pyrrolidone-carboxyl peptidase-like protein. The pyrrolidone carboxyl peptidases (Pcps) are a group of exopeptidases responsible for the removal of N-terminal pyroglutamate residues from a variety of peptides and proteins (Doolittle and Armentrout 1968). This enzyme was found to be widely distributed (Szewczuk and Kwiatkowska 1970), but its physiological role is still not clear. Pcp activity in archaea and eubacteria is thought to be involved in detoxification processes and nutrient metabolism (Awad? et al. 1994). In eukaryotic organisms, the enzyme is involved in the processing of biologically active peptides (Griffiths et al. 1980). Down-regulation of this gene in Poncirus under cold acclimation might indicate a new role for this gene in the plants. 61 ?-galactosidase The inferred amino acid sequence of PD6 shows 97% and 86% similarity with Beta- galactosidases from Citrus sinensis and Lycopersicon esculentum, respectively. ?- galactosidase modifies cell walls during development of many fruit crops such as apple (Yoshioka et al. 1994), kiwifruit (Ross et al. 1993), mango (Mangifera indica) (Ali et al. 1995) and Japanese pear (Tateishi et al. 2001). ?-galactosidase can cleave the ?-(1 4)- galactan bond directly and produce galactosyl residues from the cell wall during ripening (Smith et al. 1998), with free galactose (GAL) increasing simultaneously during this process (Gross and Sams 1984). Contrary to our observation of down regulation of ?- galactosidase in response to environmental stress, Kreps et al. (2002) also showed that in Arabidopsis, a putative ?-galactosidase was induced 3.2 to 7.7 folds in response to cold, osmotic, and salt stress. It?s unclear whether cell wall modification can help the plant increase its tolerance to stress or if other indirect functions exist for ?-galactosidase. Additionally, the down regulation of this gene may not correlate with changes in the level of translation of the gene, and thus the amount of functional protein may not directly relate to the transcriptional regulation observed in this study. The down-regulation of this gene in transgenic plants using a ?reverse genetics? technique might help answer this question. Research on cold stress in Arabidopsis showed induction of genes involved with protein synthesis, metabolism, transport facilitation, and protein targeting, while most of the down-regulated genes were related to photosyntheis (Jung et al. 2003). Arabidopsis transcriptome profiling identified 88 down-regulated genes in response to cold suggesting an important role for down regulated genes in cold tolerance. These down regulated 62 genes represent a wide range of functions, including transcription, signaling, cell wall biogenesis, defense, and photosynthesis (Fowler and Thomashow 2002). In our study, three of the six down-regulated genes are associated with photosynthesis, one gene with cell wall biogenesis and two genes with biotic defense. Low temperatures have been found to slow the energy-consuming Calvin Cycle enzymes more than the energy-transducing light reactions (Wise 1995). Thus, low temperature stress may result in light stress. The excess of reducing power generated by light under low temperature stress conditions imposes oxidative stress and increases the production of reactive oxygen in photosynthetic organisms (Powles et al. 1983). Down regulation of the LHC and OEC23 genes might reduce photosynthesis efficiency, thus avoiding oxidative damage to plant cells. However, production of reactive oxygen species cannot be totally avoided. Production of photo-oxidative protection chemicals can serve as an alternative mechanism for adaptation of plants to light stress resulting from low temperature stress. High light intensities are known to inhibit the transcription of LHC genes while activating the synthesis of the ELIPs (a sequence homolog of LHC) (Potter and Kloppstech 1993). It has been postulated that ELIPs function as substitutes for the inner LHC proteins, possibly in both PSI and PSII, when plants are grown under potentially harmful light conditions (Krol et al. 1995; Potter and Kloppstech 1993). A recent discovery of the photo-oxidative protection role of ELIPs in Arabidopsis sheds more light on this process (Hutin et al. 2003). The inverse relationship between LHC genes and ELIPs was also found in our study (Zhang et al., manuscript in preparation) and further demonstrates that the down regulation of some genes is an active adaptation mechanism of plants under adverse environments. The induction and repression of some 63 important genes related to photosynthesis are ?balanced? in the plant cell and their global expression control can adapt the plant to stressful environments. Biotic stresses also induce physiological changes during plant growth and development. Carbonic anhydrase and tumor-related protein, detected in Poncirus after cold acclimation, has been associated with abiotic and biotic stresses in other plants, demonstrating similarity at the molecular level. Subfreezing temperatures are a serious abiotic stress for perennial woody plants grown in most parts of the world. The mechanisms associated with low temperature adaptation at the molecular level, with respect to the down regulation of globally controlled genes are not very well understood. Several interacting pathways are activated during cold acclimation and ensure the winter survival of plants. A better understanding of these pathways should provide insight into strategies that impart higher levels of freezing tolerance to economically important fruit crops. ACKNOWLEDGEMENTS We are grateful to Brandon Hockema, Bryan Wilkins and Monte Nesbitt for their helps in sample preparations. This research was funded in part by USDA CSREES Special Research Grants OEP 2001-03124 and 2002-06162 and the Alabama Agricultural Experiment Station. 64 References Ali ZM, Armugam S, Lazan H (1995) Beta-galactosidase and its significance in ripening mango fruit. Phytochemistry 38: 1109-1114. Anderson JM, Chow WS, Goodchild DJ (1995) The grand design of photosynthesis: acclimation of the photosynthetic apparatus to environmental cues. Photosynth. Res. 46: 129-139. Artus N, Uemura M, Steponkus PL, Gilmour SJ, Lin C, Thomashow MF (1996) Constitutive expression of the cold-regulated Arabidopsis thaliana COR15 gene affects both chloroplast and protoplast freezing tolerance. Proc. Natl Acad. Sci. USA 93: 13404-13409. Awad? AC, Cleuziat P, Gonzales T, Robert-Baudouy J (1994) Pyrrolidone carboxyl peptidase (Pcp): an enzyme that removes pyroglutamic acid (pGlu) from pGlu- peptides and pGlu-proteins. Proteins Struct Funct Genet 20: 34-51. Barrett HC (1978) Intergeneric hybridization of Citrus and other genera in citrus improvement. Pages 586-589 in W. Grierson, ed. Proc Int Soc Citriculture, 1977, Lake Alfred, FL. Barrett HC (1982) Breeding cold-hardy citrus scion cultivars. Pages 61-66 in K. Matsumoto, ed. Proc Int Soc Citriculture, 1977. Okitsu Fruit Tree Research Station, Okitsu, Shizuoka Japan. Boulter D, Gatehouse AM, Hilder V (1989) Use of cowpea trypsin inhibitor (CpTI) to protect plants against insect predation. Biotechnol Adv 7: 489-497. Brenner ED, Lambert KN, Kaloshian I, Williamson VM (1998) Characterization of LeMir, a root-knot nematode-induced gene in tomato with an encoded product secreted from the root. Plant Physiol 118: 237-247. Cai D, Thurau T, Tian Y, Lange T, Yeh KW, Jung C (2003) Sporamin-mediated resistance to beet cyst nematodes (Heterodera schachtii Schm.) is dependent on trypsin inhibitory activity in sugar beet (Beta vulgaris L.) hairy roots. Plant Mol Biol 51: 839-849. Cai Q, Moore GA, Guy CL (1995) An unusual group 2 LEA gene family in citrus responsive to low temperature. Plant Mol Biol 29: 11-23. Donson J, Fang Y, Espiritu-Santo G, Xing W, Salazar A, Miyamoto S, Armendarez V, Volkmuth W (2002) Comprehensive gene expression analysis by transcript profiling. Plant Mol Biol 48: 75-97. 65 Doolittle RF, Armentrout RW (1968) Pyrrolidone peptidase. An enzyme for the selective removal of pyrrolidone carboxylic acid residues from polypeptides. Biochemistry 7: 516-521. Fowler S, Thomashow MF (2002) Arabidopsis transcriptome profiling indicates that multiple regulatory pathways are activated during cold acclimation in addition to the CBF cold response pathway. Plant Cell 14: 1675-1690. Fujita T, Kouchi H, Ichikawa T, Syono K (1994) Cloning of cDNAs for genes that are specifically or preferentially expressed during the development of tobacco genetic tumors. Plant J 5: 645-654. Garwe D, Thomson JA, Mundree SG (2003) Molecular characterization of XVSAP1, a stress-responsive gene from the resurrection plant Xerophyta viscosa Baker. J Exp Bot 54: 191-201. Gilmour SJ, Artus N, Thomashow MF (1992) cNDA sequence analysis and expression of two cold-regulated genes of Arabidopsis thaliana. Plant Mol Biol 18: 13-21. Gilmour SJ, Hajela R, Thomashow MF (1988) cold acclimation in Arabidopsis. Plant Physiol 87: 745-750. Graham D, Reed ML, Patterson BD, Hockley DG, Dwyer MR (1984) Chemical properties, distribution, and physiology of plant and algal carbonic anhydrases. Ann NY Aca Sci 429: 222-237. Griffiths EC, Kelly JA, With N, Jeffcoate SL (1980) Further studies on the inactivation of thyrotropin-releasing hormone (TRH) by enzymes in the rat hypothalamus. Acta Endocrinol 93: 385-391. Gross KC, Sams CE (1984) Changes in cell wall neutral sugar composition during fruit ripening: a species survey. Phytochemistry 23: 2457-2461. Guy, CL (1990) Cold acclimation and freezing stress tolerance: role of protein metabolism. Annu Rev Plant Pphysiol Plant Mol Biol 41: 187-223. Guy CL, Niemi KJ, Brambl R (1985) Altered gene expression during cold acclimation of spinach. Proc Natl Acad Sci USA 82: 3673-3677. Hao L, Wang H, Liang H (1999) Effects of rewatering on light-harvesting chlorophyll a/b-protein complex of photosystem II in Zea mays. Acta Bot Sin 41: 613-616. Hara K, Yagi M, Koizumi N, Kusano T, Sano H (2000) Screening of wound-responsive genes identifies an immediate-early expressed gene encoding a highly charged protein in mechanically wounded tobacco plants. Plant Cell Physiol 41: 684-691. 66 Hara M, Wakasugi Y, Ikoma Y, Yano M, Ogawa K, Kuboi T (1999) cDNA sequence and expression of a cold-responsive gene in Citrus unshiu. Biosci Biotech Biochem 63: 433-437. Hewett-Emmett D, Tashian RE (1996) Functional diversity, conservation, and convergence in the evolution of the alpha-, beta-, and gamma-carbonic anhydrase gene families. Mol Phylo Evol 5: 50-77. Hutin C, Nussaume L, Moise N, Moya I, Kloppstech K, Havaux M (2003) Early light- induced proteins protect Arabidopsis from photooxidative stress. Proc Natl Acad Sci USA 100: 4921-4926. Jaglo-Ottosen K, Gilmour SJ, Zarka D, Schabenberger O, Thomashow MF (1998) Arabidopsis CBF1 overexpression induce COR genes and enhances freezing tolerance. Science 280: 104-106. Jung S-H, Lee J-Y, Lee D-H (2003) Use of SAGE technology to reveal changes in gene expression in Arabidopsis leaves undergoing cold stress. Plant Mol Biol 52: 553- 567. Krol M, Spangfort M D, Huner N, Oquist G, Gustafsson P, Jansson S (1995) Chlorophyll a/b-binding proteins, pigment conversions, and early light-induced proteins in a chlorophyll b-less barley mutant. Plant Physiol 107: 873-883. Kasuga M, Liu Q, Miura S, Yamaguchi-Shinozaki K, Shinozaki K (1999) Improving plant drought, salt and freezing tolerance by gene transfer of a single stress- inducible transcription factor. Nature Biotech 17: 287-291. Kreps JA, Wu Y, Chang HS, Zhu T, Wang X, Harper JF (2002) Transcriptome changes for Arabidopsis in response to salt, osmotic, and cold stress. Plant Physiol 130: 2129-2141. Larsson S, Bj?rkbacka H, Forsman C, Samuelsson G (1997) Molecular cloning and biochemical characterization of carbonic anhydrase from Populus tremula ? tremuloides. Plant Mol Biol 34: 583-592. Levitt, J (1980) Responses of plants to environmental stresses. 2 nd , Vol 1. Chilling, freezing, and high temperature stresses. Academic press, New York, NY. Liang P, Pardee AB (1992) Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction. Science 257: 967-971. 67 Marty C, Jones B, Bouquin T, Latche A, Pech JC, Bouzayen M (1997) Improved screening of cDNAs generated by mRNA differential display enables the selection of true positives and the isolation of weakly expressed messages. Plant Mol Biol Rep 15: 236-245. Mayeau N, Coleman JR (1991) Isolation and characterization of a cDNA coding for pea chloroplastic carbonic anhydrase. Plant Physiol 95: 264-268. Michael UH, Peter W (1997) Potato psbP transcript encoding the 23 kDa protein of the oxygen-evolving complex of photosystem II (accession No. X99320) (PGR97- 033). Plant Physiol 113: 665. Murota K, Ohshita Y, Watanabe A, Aso S, Sato F, Yamada Y (1994) Changes related to salt tolerance in thylakoid membranes of photoautotrophically cultured green tobacco cells. Plant Cell Physiol 35: 107-113. Okamoto M, Vidmar JJ, Glass ADM (2003) Regulation of NRT1 and NRT2 gene families of Arabidopsis thaliana: responses to nitrate provision. Plant Cell Physiol 44: 304-317. Pires-Alves M, Grossi-de-Sa MF, Barcellos GBS, Carlini CR, Moraes MG (2003) Characterization and expression of a novel member (JBURE-II) of the urease gene family from jackbean [Canavalia ensiformis (L.) DC]. Plant Cell Physiol 44: 139-145. Porat R, Pavoncello D, Lurie S, Mccollum TG (2002) Identification of a grapefruit cDNA beloning to a unique class of citrus dehydrins and characterization of its expression patterns under temperature stress conditions. Physiol Plant 115: 598- 603. Potter E, Kloppstech K (1993) Effects of light stress on the expression of early light- inducible proteins in barley. Eur J Biochem 214: 779-786. Powles S, Berry J, Bjo?rkman O (1983) Interaction between lightand chilling temperature on the inhibition of photosynthesis inchilling-sensitive plants. Plant Cell Environ 6: 117?123. Ross GS, Redgwell RJ, MacRae EA (1993) Kiwifruit beta-galactosidase: isolation and activity against specific fruit cell-wall polysaccharides. Planta 189: 499-506. Slaymaker DH, Navarre DA, Clark D, del Pozo O, Martin GB, Klessig DF (2002) The tobacco salicylic acid-binding protein 3 (SABP3) is the chloroplast carbonic anhydrase, which exhibits antioxidant activity and plays a role in the hypersensitive defense response. Proc Natl Acad Sci USA 99: 11640-11645. Smith DL, Starrett DA, Gross KC (1998) A gene coding tomato fruit beta-galactosidase II is expressed during fruit ripening. Plant Physiol 117: 417-423. Spiegel-Roy P, Goldschmidt EE (1996) Biology of citrus. Cambridge University Press, Cambridge, UK. P.30. 68 Stathakopoulos NP, Erickson LC (1966) Induction of dormancy in Poncirus trifoliata (L.) Raf. By controlled environmental conditions. Pro Am Soc Hort Sci 89: 216- 221. Steponkus PL (1984) Role of the plasma membrane in freezing injury and cold acclimation. Annu Rev Plant Physiol 35: 543-584. Steponkus PL, Uemura M, Joseph R, Gilmour SJ, Thomashow MF (1998) Mode of action of the COR15a gene on the freezing tolerance of Arabidopsis thaliana. Proc Natl Acad Sci USA 95: 14570-14575. Strand A, Hurry V, Gustafsson P, Gardestrom P (1997) Development of Arabidopsis thaliana leaves at low temperatures releases the suppression of photosynthesis and photosynthetic gene expression despite the accumulation of soluble carbohydrates. Plant J 12: 605-614. Sutton F, Ding X, Kenefick D (1992) Group 3 LEA gene HVA1 regulation by cold acclimation and deacclimation in two barley cultivars with varying freezing resistance. Plant Physiol 99: 338-340. Szewczuk A, Kwiatkowska J (1970) Pyrrolidonyl peptidase in animal, plant and human tissues: occurrence and some properties of the enzyme. Eur J Biochem 15: 92-96. Tateishi A, Inoue H, Shiba H, Yamaki S (2001) Molecular cloning of beta-galactosidase from japanese pear (Pyrus pyrifolia) and its gene expression with fruit ripening. Plant Cell Physiol 42: 492-498. Taylor J, Harrier LA (2003) Expression studies of plant genes differentially expressed in leaf and root tissues of tomato colonised by arbuscular mycorrhizal fungus Glomus mosseae. Plant Mol Biol 51: 619-629. Theerasilp S, Hitotsuya H, Nakajo S, Nakaya K, Nakamura Y, Kurihara Y (1989) Complete amino acid sequence and structure characterization of a taste-modifying protein, miraculin. J Biol Chem 264: 6655-6659. Thomashow MF (1998) Role of cold-responsive genes in plant freezing tolerance. Plant Physiol 118: 1-7. Wang HL, Hao LM, Wen JQ, Zhang CL, Liang HG (1998) Differential expression of photosynthesis-related genes of reed ecotypes in response to drought and saline habitats. Photosynthetica 35: 61-69. Webber HJ, Reuther W, Lawton HW (1967) History and development of the citrus industry. In: Reuther W., Webber, H.J., Batchelor, L.D. (Ed.), The citrus industry. University of Califania, Berkeley, 1-39. 69 Wise R (1995) Chilling-enhanced photooxidation - the production, action and study of reactive oxygen species produced during chilling in the light. Photosynth Res 45: 79-97. Wolfraim LA, Langis R, Tyson H, Dhindsa RS (1993) cDNA sequence, expression, and transcript stability of a cold acclimation-specific gene, cas18, of alfalfa (Medicago falcata) cells. Plant Physiol 101: 1275-1282. Yelenosky G (1985) Cold hardiness in Citrus. Hort Rev 7: 201-238. Yelenosky G (1993) Freezing resistance of progeny from open-pollinated Pummelo x Trifolate orange hybrids. HortSci 28:1120-1121. Young RH (1961) Influnce of day length, light intensity, and temperature on growth, dormancy, and cold-hardiness of Red Blush Grapefruit trees. Proc Amer Soc Hort Sci 78: 174-180. Young RH, Peynado A (1962) Growth and cold hardiness of citrus and related species when exposed to different night temperatures. Proc Amer Soc Hort Sci 81: 238- 243. 70 Table 1. Oligonucleotide primer sequences, PCR conditions and cycle numbers for the confirmation of different displayed products with quantitative RT-PCR DD product Forward Primer (5?-3?) Reverse Primer (5?-3?) PCR annealing temperature Cycle numbers PD1 GCTTACCAGGTGGTCTTGATG CCCTCGAACTGGTAGTAGCAGTA 55 ?C 35 PD2 TGGAGATGAAGGTGGCAAGC GGGAAAGATGGGAGTAGACAAT 52 ?C 33 PD3 AAACTCTGGCACTCAAGGGT GATTCCCCAACAGGACTCTG 52 ?C 35 PD4 GCAACGAACCAACAGTATGG GCTAGCCCGATCCATTATTT 50 ?C 30 PD5 ACAGACATCTCTATCCACTGAAGC CTCCAAGAGTGTGGCTACAAAT 55 ?C 33 PD6 GCTTCTCAACGATGGTATCACG GAAACCCAGCAAGCCTACAT 52 ?C 31 71 Table 2. Isolated DD products, accession number and percentage similarity to known genes by BLASTx search in NCBI DD product Accession Number Length (bp) Plant gene Similarity Function PD1 CN807022 468 Chlorophyll a/b binding protein (LHC) 96% photosynthesis PD2 CN807023 297 Photosystem II OEC 23 91% photosynthesis PD3 CN807024 331 Carbonic anhydrase 82% Photosynthesis, antioxidant PD4 CN807025 508 Tumor related protein 47% biotic stress PD5 CN807026 469 Pyrrolidone-carboxylate peptidase 72% multiple PD6 CN807027 482 Beta-galactosidase 97% cell wall metabolism 72 C C T T 1 2 3 Fig. 1. Example of DDRT-PCR results. cDNAs were amplified from two separately isolated total RNAs by primer combination of HT 11 A and HAP 1. C 1 and C 2 are cDNAs amplified from the leaves collected at the end of second week (control); T 1 and T 2 are cDNAs amplified from the leaves collected at the end of fifth week (cold acclimated). Arrows 1 and 2 indicate two up regulated cDNA fragments; Arrows 3 indicates one down regulated cDNA fragment. 73 PD1 PD2 PD3 PD4 PD5 PD6 C T C T C T C T C T C T Gene of interest Actin 0 100 200 300 400 500 600 PD1 PD2 PD3 PD4 PD5 PD6 DD Products Differential expression detected by RT-PCR Control Treatment Fig. 2 (top): Confirmation of differential expression of 6 DD products using relative quantitative RT-PCR with RNA from control and cold acclimated leaves. The cDNAs were synthesized from total RNAs isolated from control leaves (end of first week) and cold acclimation leaves (end of fifth week). Actin gene was used as an internal control. Specific RT-PCR primer pair information for all DD products was listed in table 1. (Bottom): A histogram showing the relative abundance of 6 DD products between control and cold acclimated plants. The intensities in each control and cold acclimation pair were normalized by setting the intensity of actin gene to 100. The values are the means of three independent experiments + SE. 74 IV. Identification of cold acclimated genes in leaves of Citrus unshiu by mRNA differential display ABSTRACT Citrus unshiu is freeze tolerant to ?10?C when fully acclimated after exposure to cold, nonfreezing temperatures. To gain an understanding of its cold tolerance mechanism, mRNA differential display reverse transcriptase polymerase chain reaction (DDRT-PCR) and quantitative relative RT-PCR were used to study gene expression of C. unshiu under a gradual cold-acclimation temperature regime. Six up-regulated and two down regulated genes were identified based on their amino acid sequences. The identified proteins encoded by the up-regulated genes were: 14-3-3 protein, 40S ribosomal protein S23, putative 60S ribosomal protein L15, nucleoside diphosphate kinase III protein, regulator of chromosome condensation-like protein, and amino acid permease 6. The proteins encoded by the two down-regulated genes were: miraculin-like protein and beta-galactosidase. Their individual function has been briefly reviewed based on published information. In addition to the findings in this study, we compared the function of cold responsive genes of Poncirus trifoliata, a very cold hardy relative of Citrus species that is freeze tolerant to ?30?C when fully acclimated, to the function of those genes in this study. 75 Keywords: Gene expression; Cold acclimation; 14-3-3 protein; nucleoside diphosphate kinase; amino acid permease Abbreviations: CBF, C repeat-binding proteins; cDNA, DNA complementary to RNA; AFLP, amplified fragment length polymorphism; DDRT-PCR, differential display reverse transcriptase polymerase chain reaction; DNase, deoxyribonuclease; dNTP, deoxyribonucleoside triphosphate; M- MuLV, moloney murine leukemia virus; Oligo(dT), oligodeoxyribonucleotide thymidine; RNase, ribonuclease; RT, reverse transcriptase; SAGE, serial analysis of gene expression 76 1. Introduction Crop quality and productivity are negatively affected by biotic and abiotic stresses. Low temperature is one of the most common environmental stresses and can potentially cause severe losses to major economically important plants. Disruption of cell membranes is the primary injury associated with freeze-induced dehydration (Thomashow 1998). Many plants increase their freezing tolerance in response to low, nonfreezing temperatures, a phenomenon known as cold acclimation. Changes at gene expression level were suggested to be associated with this process, and many genes related to cold acclimation have been cloned from several plants, including Arabidopsis (Gilmour et al. 1992), Brassica napus (Orr et al. 1992), and Poncirus trifoliata (Zhang et al. 2004). A number of the C repeat binding proteins (CBF) from Arabidopsis have been characterized. Overexpression of the gene encoding the protein was shown to increase tolerance of transformed plants to environmental stresses (Jaglo-Ottosen et al. 1998). Although efforts have been taken to elucidate the cold adaptation mechanism of herbaceous plants, very limited information is available for woody plants under low temperatures. Citrus sp. are some of the most important fruit crops throughout the world. Yet, some of the most valued citrus crops are grown in relatively high-risk freeze areas (Yelenosky 1985). Research has been conducted in some Citrus varieties and relatives, and a few genes have been cloned (Cai et al. 1995; Hara et al. 1999; Sanchez-Ballesta et al. 2003; Zhang et al. 2004; Zhang et al. in press). Citrus unshiu is considered as one of the most cold hardy commercial Citrus species (Yelenosky 1985), but the changes in gene expression exposed to a gradually declined temperature regime, which mimics the natural temperature changes in the southeastern-U.S., have not been studied. 77 Transcriptome profiling of plants to environmental stresses can be studied using different techniques, which include differential display reverse transcription PCR (DDRT-PCR), serial analysis of gene expression (SAGE), subtractive hybridization, DNA-chip, and cDNA microarray. mRNA differential display has been widely used to identify genes whose expression levels have been altered under different environmental conditions because of its technical simplicity and lack of requirement for previous genomic information of the species of interest (Liang and Pardee 1992; Carginale et al. 2004). DDRT-PCR was used to clone genes in C. unshiu following a gradual cold acclimation regime. The identified genes will provide insights into the adaptation of important fruit crops to low temperature and may lead to strategies to develop transgenic citrus with enhanced levels of cold tolerance. 2. Materials and methods 2.1. Plant growth conditions One year old Citrus unshiu plants were grown for 5 weeks in a growth chamber with a 12 h light period at 400 ?mol m -2 s -1 intensity. The regimen for temperature decline was as follows: 32?C day/21?C night for 14 days; 27?C day/16?C night for 7 days; 24?C day/13?C night for 7 days and 18?C day/7?C night for 7 days. Plants were uniformly watered every day. 78 2.2. RNA extraction and mRNA differential display Fully expanded leaves at the end of the second and fifth weeks were collected, immediately immersed in liquid nitrogen and stored at ?80?C for later use. RNA was extracted from leaves according to the RiboPure kit protocol (Ambion, Austin, TX). Extracted RNA was mixed with 1/9 volume of 10X DNase buffer and 4?l DNase I (2U/?l) and incubated for 30 min at 37?C to digest the remaining genomic DNA. Digested RNA was treated with DNase inactivation reagent (20% volume) for 2 min, followed by centrifugation for 1 min at 14000g and transferred to a new tube. The concentration of isolated RNA was measured using an Eppendorf Biophotometer (Brinkmann Instruments, NY). The quality of RNA was checked using formaldehyde- agarose gel electrophoresis. RNA from the end of second week was used as an unacclimated control and RNA from the end of fifth week as treatment. mRNA differential display was performed using RNAimage kits and 64 primer pairs according to the protocol supplied with this kit (GenHunter, TN). 0.2?g of RNA was reverse transcribed in a 20?l reaction mixture at 42?C for 60 min with M-MuLV reverse transcriptase (GenHunter). Amplification of cDNA fragments was performed in a 20?l reaction mixture containing 2?l of the reverse transcribed cDNA, 0.2?M arbitrary primer (GenHunter), 0.2?M anchored oligo (dT)-primers (H-T11M, where M=A, G, C), 2?M of each dNTP, 10mM Tris-Cl, pH8.4, 50mM KCl, 1.5mM MgCl 2 , 0.001% gelatin, 1?l ?- [S 35 ] dATP and 1 U Taq DNA polymerase (Qiagen, CA). The PCR program consisted of 40 cycles: 30 s at 94?C, 2 min at 40?C, 1 min at 72?C; and a final 5 min elongation step at 72?C. Amplified PCR products were separated in a 6% denaturing PAGE gel. The gel 79 was transferred to filter paper (Whatman, England) and dried at 80?C for 1 hour in a gel dryer (BioRad, CA). PCR products on filter paper were exposed to BioMax Kodak film covered with two intensifying screens for 24 to 72 hours in a ?80?C freezer. The film was developed and the differentially expressed bands between control and treatment were excised from the filter paper according to the pattern on the film. The PCR products were extracted according to the GenHunter protocol, and reamplified using the original primer pair. 2.3. cDNA-AFLP analysis cDNA was synthesized using RETROscript TM (Ambion, TX) according to the manufacturer?s instructions, and digested using the MseI/EcoRI enzyme combination. AFLP analysis was conducted according to the protocol of AFLP kit from Li-COR (Li- COR Biosciences, NE). Sequences of the adapters and primers used for cDNA AFLP analysis were: 5?-GACGATGAGTCCTGAG-3? (MseI adapter 1); 5?TACTCAGGACT CAT-3? (MseI adapter 2); 5?-CTCGTAGACTGCGTACC-3? (EcoRI adapter 1); 5?- AATTGGTACGCAGTCTAC-3? (EcoRI adapter 2); 5?-GATGAGTCCTGAGTAAC-3? (non-selective primer for MseI); 5?-GACTGCGTACCAATTCA-3? (non-selective primer for EcoRI); 5?-GATGAGTCCTGAGTAACNN-3? (selective primer for MseI, and NN represents 2bp extension); 5?-GACTGCGTACCAATTCANN-3? (selective primer for EcoRI, and NN represents 2bp extension). Pre-amplification was performed in a 25 ?l reaction solution, containing 0.2 mM dNTPs, 0.2 ?M non-selective primers, 1.2mM MgCl 2 , 0.5U Taq polymerase (Invotrogen, CA) and 10 ?l ligated cDNA fragments. The 80 PCR program consists 20cycles: 30 s at 94 ?C, 60 s at 56 ?C and 60 s at 72 ?C. Selective amplification was performed in a 10?l reaction solution, containing 0.1mM dNTPs, 0.15 ?M EcoRI primer and 0.3 ?M MseI primer, 1.2mM MgCl 2 , 0.5 U Taq polymerase (Invitrogen) and 2.5 ?l pre-amplification solution (diluted at 1:40). The PCR program was carried out with the following procedure: one cycle at 94 ?C for 30 s, 65 ?C for 30 s, and 72 ?C for 60 s; 13 cycles with annealing temperature decreasing 0.7 ?C per cycle followed by 23 cycles at 94 ?C for 30 s, 56 ?C for 30 s and 72 ?C for 60 s. Selective reaction products were run on 6% polyacrylamide sequencing gel at 85 W for 3 hrs. Fragments were visualized by silver staining according to the Silver Sequence TM DNA Sequencing System Technical Manual (Promega, WI). 2.4. Cloning and sequence analysis of DNA fragments Selected amplified DNA fragments were ligated directly into a PCR-Trap Vector (GenHunter) and transformed into competent Escherichia coli (GenHunter). Ten colonies were selected for each transformation event. 20?l lysis PCR was carried out according to the GenHunter protocol. 10?l of lysis PCR products were separated in 1.5% agarose gel. The remaining 10?l of PCR product containing correct size inserts were digested with 0.2?l of TaqI (10U/?l), 10mM Tris-HCl, pH8.4, 50mM KCl, 1.5mM MgCl 2 , 0.001% gelatin, and incubated at 65?C overnight. Products were analyzed using agarose gel electrophoresis. Differentially restricted DNA fragments were used for plasmid isolation. Only fragments larger than 250 bp were selected and sequenced in both directions using 81 Rseq and Lseq primers (GenHunter) with ABI 3100 DNA sequencer (AU Genomics Lab). Analysis of nucleotide sequence of selected fragments was carried out using the National Center for Biotechnology Information BLASTx search tool. 2.5. Quantitative Relative Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) Quantitative relative RT-PCR was used to confirm the differential expression of DNA fragments isolated from control and cold acclimated plants. 2.5?g total RNA was reverse transcribed with 0.5mM dNTP, 5mM oligo(dT)-primers, 10mM Tris-HCl, pH 8.3, 50 mM KCl, 15mM MgCl 2 , 1?l RNase inhibitor, and 100U M-MuLV-RT (Ambion). The mixture (20?l total reaction volume) was incubated at 42?C for 1 hour. 1?l RT reaction was amplified in a 25?l solution with 10mM Tris-HCl, pH8.4, 50mM KCl, 1.5mM MgCl 2 , 2.5mM dNTPs, 0.3?M actin primer, 0.6?M actin competitor, 0.5mM of each gene specific primers (Ambion) and Taq polymerase (Qiagen). The PCR cycle program was as followed: 30 s at 94?C, 30 s at 55?C to 60?C and 30 s at 72?C for 30 to 35 cycles. Specific oligonucleotide primers were designed for each DD product for RT-PCR. The optimal PCR annealing temperatures and the cycle numbers are shown in Table 1. 10?l of the amplification products were separated using 1.8 % agarose gel electrophoresis and stained with ethidium bromide. The stained gel was used for quantitation of each band using a BioRad photo documentation system. A constitutively expressed actin gene was used as an internal standard in each reaction. 82 3. Results and discussion 3.1. Identification and confirmation of differentially expressed transcripts in C. unshiu in response to low temperature Understanding a plant?s responses to a stress will require a comprehensive evaluation of stress-induced changes in gene expression. mRNA differential display, cDNA-AFLP and quantitative RT-PCR were used to study the responses of C. unshiu to low temperature stress. Sixty-four primer pair combinations were used and about 8,000 cDNA fragments were analyzed after autoradiography. The banding pattern of cDNA fragments amplified by the combination of one primer group is shown in Fig. 1. To decrease the ?false positive? rate of mRNA differential display, each primer pair was used to amplify two different sets of RNA isolated from different C. unshiu plants. RT- PCR products were loaded in adjacent lanes in a 6% denatured PAGE gel. One hundred and sixty-five putative differentially expressed DNA fragments were cloned, sequenced, and analyzed. Because of the possibility of comigration associated with DDRT-PCR (Zhang et al. 2004), ten clones, obtained from each band on the gel, were examined. These colonies were randomly selected from each transformation, and the sequence of the cloned insert in each of the colonies was determined. Six genes were confirmed as up-regulated and two genes were confirmed as down-regulated in cold acclimated plants with quantitative RT-PCR. The genes show significant homology to known genes in the GenBank database using the BLAST X search utility. The nucleotide sequences of selected 83 clones have been deposited in GenBank, and their accession numbers along with the results of the BLAST X search are shown in Table 2. Three different sets of independently isolated RNA were used to confirm the DD results with RT-PCR. Only reproducible differences in banding patterns between the control and treatment in all three sets were considered to be positive. The agarose gel electrophoresis pattern of six up-regulated and two down-regulated DD genes using actin mRNA as an internal standard and a histogram representing the relative quantity of each of the amplified bands on the gel are presented in Fig 2. 3.2. Up-regulated genes in C. unshiu in response to low temperature The inferred amino acid sequence of O10 showed 96% similarity to a 14-3-3 protein from Nicotiana tabacum. The 14-3-3 proteins are small, highly conserved eukaryotic proteins involved in regulating multiple cellular enzymes, including plant calcium dependent protein kinases (Camoni et al. 1998) and plant plasma membrane H + - ATPase (Camoni et al. 2000). In Arabidopsis, the binding of 14-3-3 protein to the calcium dependent protein kinase (CDPK) can activate the activity of the enzyme, and thus probably further activates CDPK signal transduction pathways in this plant (Camoni et al. 1998). In maize, 14-3-3 proteins are able to interact with the H + -ATPase and the interaction depends on the phosphorylation status of the proton pump (Camoni et al. 2000). In Arabidopsis, two genes encoding 14-3-3 proteins were induced in response to low temperature. The 14-3-3 protein accumulation is correlated with an increase of cold tolerance ability and the genes were thought to have an adaptive role in this process 84 (Jarillo et al. 1994). The 14-3-3 in cold acclimated C. unshiu was induced about 2.3 fold in comparison to non-acclimated control. Considering its central role in signal transduction, the induced transcript might produce more 14-3-3 protein, and result in an increased down stream effect in the signal transduction cascade. The inferred amino acid sequences of O26 and O151 showed 98% and 92% similarity to a 40S ribosomal protein S23 and a putative 60S ribosomal protein L15 from Hyacinthus orientalis and Oryza sativa, respectively. The RPL15 transcript in Syntrichia ruralis, a desiccation tolerant moss, was stably maintained in desiccated and rehydrated gametophytes and may be involved in the maintenance of ribosomal integrity within the desiccated state and re-establishment of translational efficiency upon rehydration (Zeng and Wood 2000). Ribosomal L15 gene was reported to be up-regulated in a citrus relative, P. trifoliata, under a similar low temperature acclimation process (Zhang et al. 2004). The participation of ribosomal proteins in cold acclimation process indicates that de novo protein synthesis is necessary for plants under adverse environments. The inferred amino acid sequence of O53 showed 90% similarity to a nucleoside diphosphate kinase III protein (NDPK) from Spinacia oleracea. The NDPKs are enzymes whose major role is to maintain the balance between adenine and non-adenine triphosphates. In cultured sugarcane cells, the activity of NDPK was enhanced by heat shock (Moisyadi et al. 1994). It?s postulated that under heat shock, NDPK can help plant cells maintain the nucleoside triphosphate pools which is an essential activity for plant survival. In peas, the expression of mtNDPK was not directly affected by different stresses including cold temperature, but the interaction of the protein encoded by this gene with another heat stress induced protein was detected, indicating a role of this 85 protein under stress condition (Escobar Galvis et al. 2001). The NDPK gene in the current study was induced almost 5 fold in cold acclimated C. unshiu. Under temperature stress conditions (low and high), plant cells might need to increase the synthesis of the NDPK protein so that cells can maintain the nucleoside triphosphate supply. The participation of NDPKs in the signal transduction pathway (Tanaka et al. 1998) in other organisms might indicate another reason for the up-regulated expression of this gene under stress conditions. The inferred amino acid sequence of O58 showed 75% similarity to a regulator of chromosome condensation-like protein from Arabidopsis. The regulator of chromosome condensation protein is involved in the maintenance of chromatin conformation and can regulate chromosome condensation, and might be involved in monitoring and signaling of DNA replication (Enoch and Nurse 1990). The Arabidopsis gene expression profile under environmental stresses shows that regulator of chromosome condensation-like protein was highly induced by low temperature, indicating a role of this gene in plant low temperature adaptation (Seki et al. 2002). The role of this gene at low temperature stress is currently not clear, although the strongly up-regulated expression level in C. unshiu and Arabidopsis might indicate that adjustment of chromosome structure may be needed under stress conditions. The inferred amino acid sequence of O37 showed 78% similarity to an amino acid permease 6 (AAP6) from Arabidopsis, 61% similarity to an AAP1 from Brassica napus and 52% similarity to AAP2 from Arabidopsis. In higher plants, inorganic nitrogen assimilated from roots and leaves was reduced to its organic form such as amino acids and these amino acids were transported to metabolically active organs for the plant to 86 utilize. Under desiccation and salinity conditions, mRNA levels of AAP6 were reduced (Rentsch et al. 1996). In Mesembryanthemum crystallinum, expression of AAP2 was induced in roots in response to salinity, while expression of AAP1 was induced in leaves in response to salinity (Popova et al. 2003). Although the exact roles of the proteins encoded by these genes under environmental stresses are still not clear, the induced level of the amino acid permease in C. unshiu indicates that active transport and metabolism of amino acids are necessary under low temperature conditions. 3.3. Down-regulated genes in C. unshiu in response to low temperature The inferred amino acid sequence of O71 showed 95% similarity to a miraculin- like protein 2 from Citrus x Paradisi, 48% to tumor-related protein from tobacco, and 49% to aspartic protease inhibitor from potato. Miraculin is a taste modifier protein isolated from ?miracle fruit?. A putative miraculin gene in tomato, LeMir, was induced by a root-knot nematode, and this LeMir may have a role in defense against nematodes or other pathogens/pests (Brenner et al. 1998). In P. trifoliata, a similar gene was identified under cold acclimation conditions (Zhang et al. in press). Unlike the increased expression level of this protein under biotic stress or plant hormone treatment, the decreased expression level of this gene in Citrus and P. trifoliata might indicate a different function of the protein encoded by this gene under abiotic stress. The inferred amino acid sequence of O8 showed 97% similarity to a beta- galactosidase from Citrus sinensis. Beta-galactosidase has been most studied for its role in fruit development. Down-regulation of this gene in tomato delays fruit softening 87 (Smith et al. 2002), but evidence indicates that this gene may be involved in plant stress adaptation. In Arabidopsis, a putative ?-galactosidase was induced 3.2 to 7.7 fold in response to cold, osmotic, and salt stress (Kreps et al. 2002). Contrarily, the mRNA transcript was repressed under a gradually declined temperature regime in P. trifoliata (Zhang et al. in press). The decreased expression level of this gene in Citrus and Poncirus might indicate a unique role of this gene in woody plants under stress conditions. 3.4. Comparison of the function of differentially expressed genes between P. trifoliata and C. unshiu Commercial Citrus species are cold-tender evergreen crops with tropical and subtropical origins. C. unshiu is one of the most cold hardy commercial Citrus species. P. trifoliata is a facultative deciduous relative of Citrus in the Rutaceae family that is often used in citrus breeding programs to develop cold hardy genotypes, and is used as a rootstock to enhance freeze tolerance of the scion in freeze-prone areas. P. trifoliata has a maximum freeze tolerance of about ?30 ?C (Williams 1911). In order to gain an understanding of the molecular mechanisms of these two species under cold temperature and compare their different responses to low temperature, mRNA differential display was used to identify the cold responsive genes under a gradually declined temperature regime. Thus far in P. trifoliata, three groups of genes have been detected as related to cold temperature responses (Zhang et al. 2004; Zhang et al. in press). The first group include up-regulated proline/betaine transporter, nitrate transporter and water channel protein. 88 The proteins encoded by these genes are known to be associated with osmotic adjustment of plant cells under adverse environment such as drought, salinity and cold temperature. The second group include up-regulation of early light inducible protein and aldo-keto reductase. Proteins encoded by these genes were reported to alleviate the damages caused by photooxidative stress, which simultaneously exits in combination with other environmental stresses. The third group include down-regulation of chlorophyll a/b binding protein, photosystem II OEC23 and carbonic anhydrase. The proteins encoded by these genes are related to adjustment of plant photosynthesis under stress condition (Zhang et al. 2004; Zhang et al. in press). Down regulation of these genes can lower photosynthesis efficiency, and probably decreases generation of excess energy, which can produce reactive oxygen species that are toxic to plants. In C. unshiu, an amino acid transporter was isolated, which may indicate that active N-containing molecule translocation is necessary for Citrus and Poncirus to cope with stress. Genes encoding the regulator of chromosome condensation protein (only in C. unshiu) and ribosome proteins (in C. unshiu and P. trifoliata) were found to be up-regulated, indicating adjustments at the transcription and translation level are needed for plants to survive adverse conditions. Two genes (miraculin-like protein and beta-galactosidase) were down-regulated in both species. These genes were reported to be associated with biotic stresses and plant cell wall modification, but our work shows that these genes may have a potential role in plants under abiotic stress conditions and further demonstrates the similarity between biotic and abiotic stresses at the molecular level. Unlike P. trifoliata, genes involved in osmotic adjustment, photooxidative protection and photosynthesis repression were not detected in C. unshiu. This might be due to the limited number of genes that have been 89 isolated using DDRT-PCR or to differences in cold acclimation between these two species. Further work with northern blotting using low stringency conditions may provide more information. 3.5. Comparison of C. unshiu cold acclimated genes isolated with mRNA differential display and cDNA-AFLP Due to the extensively reported high false positive rate characteristic of the differential display procedure, cDNA amplified fragment length polymorphism (cDNA- AFLP) was performed to further explore the alteration of gene expression in C. unshiu exposed to cold acclimation temperatures (Zhang, data unpublished). Two genes (translation initiation factor eIF1 and cytochrome C) were identified as up-regulated and four genes (trigger factor type chaperone family protein, polyprotein, leucine-rich repeat transmembrane protein kinase/receptor-like protein kinase and PAZ/PIWI domain containing protein) were identified to be down-regulated. Translation initiation factor eIF1 was reported to be related to low temperature (Berberich et al. 1995). Similar to ribosomal proteins detected with mRNA differential display, eIF1 is also an important component of the protein synthesis machinery in plant cells. It is likely that de novo synthesis of proteins and the maintenance of the protein synthesis machinery integrity is necessary for plant cells to tolerate environmental stress. The PAZ/PIWI domain containing protein was also reported to be associated with translational regulation in certain species (Morel et al. 2002), further demonstrating that the modulation at the translation level is important for plant adaptation to stress. A receptor-like protein kinase 90 with leucine-rich repeat transmembrane domain was induced by ABA, dehydration, high salt and cold treatment in Arabidopsis, suggesting that the protein encoded by this gene may function in the transmission of ABA and various environmental stress signals into intracellular reactions (Hong et al. 1997). Up-regulation of 14-3-3 and nucleoside diphosphate kinase in C. unshiu under low temperature further indicate that sensing and transduction of environmental signals are needed for plants to adapt to their environment. The low overlap of the identified genes by these two techniques demonstrates that more cold acclimated genes need to be identified to completely understand the adaptation mechanisms at the molecular level of C. unshiu exposed to cold acclimation temperatures. These results might also indicate that these two techniques can complement each other in order to get a complete gene expression profile of cold acclimated C. unshiu. 4. Conclusions - Sixty-four primer pairs were used to generate about 8,000 cDNA fragments. One hundred and six-five putative differentially expressed fragments were cloned and sequenced. Eight genes (six up-regulated and two down-regulated) were confirmed as differentially expressed. - The identified genes were mainly related to signal transduction (14-3-3), protein synthesis (S23 and L15), amino acid transport (AAP6), adjustment of chromosome structure (regulator of chromosome condensation-like protein), plant defense (miraculin) and cell wall metabolism (beta-galactosidase). 91 - The predicted functions of the genes identified from C. unshiu are different from the functions of the genes identified from its close relative, P. trifoliata. Osmotic modulation, photo-oxidative protection and photosynthesis adjustment were predicted as three main mechanisms for P. trifoliata in response to low temperature. - The genes identified with differential display are different from the genes identified with cDNA-AFLP, indicating the two techniques can complement each other. - More genes should be isolated with cDNA-AFLP, a more reliable transcriptome profiling technique, in order to get a more complete understanding of the molecular mechanisms of citrus and related species. - More detailed characterization needs to be performed for some genes identified in both species. For example, the proline transporter gene identified in P. trifoliata might indicate that transport of proline is also important for plant adaptation to stress in addition to proline synthesis. The detailed characterization of the signal transduction important 14-3-3 gene identified in C. unshiu will also shed light on the understanding of the responsive mechanisms under cold acclimation. 92 References Berberich T, Sugawara K, Harada M, Kusano T (1995) Molecular cloning, characterization and expression of an elongation factor 1 gene in maize. Plant Mol Biol 29, 611-615. Brenner ED, Lambert KN, Kaloshian I, Williamson VM (1998) Characterization of LeMir, a root-knot nematode-induced gene in tomato with an encoded product secreted from the root. Plant Physiol 118: 237-247. Cai Q, Moore GA, Guy CL (1995) An unusual group 2 LEA gene family in citrus responsive to low temperature. Plant Mol Biol 29: 11-23. Camoni L, Harper JF, Palmgren MG (1998) 14-3-3 proteins activate a plant calcium- dependent protein kinase (CDPK). FEBS Letters 430: 381-384. Camoni L, Iori V, Marra M, Aducci P (2000) Phosphorylation-dependent interaction between plant plasma membrane H (+) -ATPase and 14-3-3 proteins. J Biol Chem 275: 9919-9923. Carginale V, Maria G, Capasso C, Ionata E, La Cara F, Pastore M, Bertaccini A, Capasso A (2004) Identification of genes expressed in response to phytoplasma infection in leaves of Prunus armeniaca by messenger RNA differential display. Gene 332: 29-34. Enoch T, Nurse P (1990) Mutation of fission yeast cell cycle control genes abolishes dependence of mitosis on DNA replication. Cell 60: 665-673. Enoch T, Nurse P (1991) Coupling M phase and S phase: controls maintaing the dependence of motosis on chromosome replication. Cell 65: 921-923. Escobar Galvis ML, Marttila S, Hakansson G, Forsberg J, Knorpp C (2001) Heat stress response in pea involves interaction of mitochondrial nucleoside diphosphate kinase with a novel 86-kilodalton protein. Plant Physiol 126: 69-77. Gilmour SJ, Artus NN, Thomashow MF (1992) cDNA sequence analysis and expression of two cold-regulated genes of Arabidopsis thaliana. Plant Mol Biol 18: 13-21. Hong SW, Jon JH, Kwak JM, Nam HG (1997) Identification of a receptor-like protein kinase gene rapidly induced by abscisic acid, dehydration, high salt, and cold treatments in Arabidopsis thaliana. Plant Physiol 113: 1203-1212. Jaglo-Ottosen K, Gilmour SJ, Zarka D, Schabenberger O, Thomashow MF (1998) Arabidopsis CBF1 overexpression induce COR genes and enhances freezing tolerance. Science 280: 104-106. 93 Jarillo J A, Capel J, Levya A, Martinez-Zapater JM, Salinas J (1994) Two related low- temperature-inducible genes of Arabidopsis encode proteins showing high homolgy to 14-3-3 protein, a family of putative kinase regulators. Plant Mol Biol 25: 693-704. Kreps JA, Wu Y, Chang H-S, Zhu T, Wang X, Harper JF (2002) Transcriptome changes for Arabidopsis in response to salt, osmotic, and cold stress. Plant Physiol 130: 2129-2141. Liang P, Pardee AB (1992) Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction. Science 257: 967-971. Moisyadi S, Dharmasiri S, Harrington HM, Lukas TJ (1994) Characterization of a low molecular mass autohosphorylated protein in cultured sugarcane cells and its identifications as a nucleoside diphosphate kinase. Plant Physiol 104: 1401-1409. Morel JB, Godon C, Mourrain P, Beclin C, Boutet S, Feuerbach F, Proux F, Vaucheret H (2002) Fertile hypomorphic ARGONAUTE (ago1) mutants impaired in post- transcriptional gene silencing and virus resistance. Plant Cell 14: 629-639. Orr W, Iu B, White TC, Robert LS, Singh J (1992) Complementary DNA sequence of a low temperature-induced Brassica napus gene with homology to the Arabidopsis thaliana kin1 gene. Plant Physiol 98: 1532-1534. Popova OV, Dietz KJ, Golldack D (2003) Salt-dependent expression of a nitrate transporter and two amino acid transporter genes in Mesembryanthemum crystallinum. Plant Mol Biol 52: 569-578. Rentsch D, Hirner B, Schmelzer E, Frommer WB (1996) Salt stress-induced proline transporters and salt stress-repressed broad specificity amino acid permeases identified by suppression of a yeast amino acid permease-targeting mutant. Plant Cell 8: 1437-1446. Sanchez-Ballesta MT, Lluch Y, Gosalbes MJ, Zacarias L, Granell A, Lafuente MT (2003) A survey of genes differentially expressed during long-term heat-induced chilling tolerance in citrus fruit. Planta 218: 65-70. Seki M, Narusaka M, Ishida J, Nanjo T, Fujita M, Oono Y, Kamiya A, Nakajima M, Enju A, Sakurai T, Satou M, Akiyama K, Taji T, Yamaguchi-Shinozaki K, Carninci P, Kawai J, Hayashizaki Y, Shinozaki K (2002) Monitoring the expression profiles of 7000 Arabidopsis genes under drought, cold and high-salinity stresses using a full-length cDNA microarray. Plant J 31: 279-292. Smith DL, Abbott JA, Gross KC (2002) Down-regulation of tomato beta- galactosidase results in decreased fruit softening. Plant Physiol 129: 1755-1762. 94 Tanaka N, Ogura T, Noguchi T, Hirano H, Yabe N, Hasunuma K (1998) Phytochrome- mediated light signals are transduced to nucleoside diphosphate kinase in Pisum sativum L. cv. Alaska. J Photochem Photobiol B: Biology 45: 113-121. Thomashow MF (1998) Role of cold-responsive genes in plant freezing tolerance. Plant Physiol 118: 1-8. Williams PF (1911) The Satsuma orange. Alabama Agricultural Experiment Station, Alabama Polytechnic Institute, Auburn, AL. Bulletin No. 157. Yelenosky G (1985) Cold hardiness in Citrus. Hort Reviews 7: 201-238. Zeng Q, Wood A J (2000) A cDNA encoding ribosomal protein RPL15 from the desiccation-tolerant bryophyte Tortula ruralis: mRNA transcripts are stably maintained in desiccated and rehydrated gametophytes. Biosci Biotech Biochem 64: 2221-2224. Zhang C, Lang P, Dane F, Ebel RC, Singh NK, Locy RD, Dozier WA (2005) Cold acclimation induced genes of trifoliate orange (Poncirus trifoliata). Plant Cell Rep 23: 764-769 Zhang C, Lang P, Dane F, Ebel RC, Singh NK, Locy RD, Dozier WA (2005) Cold acclimation down regulated genes in Poncirus trifoliata. Can J Plant Sci (in press) 95 Table 1. Oligonucleotide primer sequences, PCR conditions and cycle numbers for the confirmation of different displayed products with quantitative RT-PCR DD product Forward Primer (5?-3?) Reverse Primer (5?-3?) PCR annealing temperature Cycle numbers O10 TTGCACCATGGCCAAACAG GGTTGCCAGAATTGGTATGTAG 55 ?C 32 O26 AGCTTTTGATCCAAATGATG TTGAGACAAGTGAGCAATT 55 ?C 32 O151 AAGCTTCTGCTGGGAAGA GCAGAAATATCACTCCCC 55 ?C 30 O53 CTTGCCATGGTATGGAAAG TGGAGTCCATAAAGAGGTG 55 ?C 30 O58 TTCTGCTGGGGATGGAAC TTTTGGACAAAATCATGTTG 57 ?C 32 O37 ATTCCGGCCATTAACAGTT GGAAAAGTTGTAGCTGACAA 55 ?C 32 O71 AAGCTTTCATATGGTGGT CACAGGCGGTGTTATTTT 55 ?C 32 O8 GCTTCTCAACGATGGTATCACG GCCTACATGAATGTTTGAGG 55 ?C 32 96 Table 2. Isolated DD products, accession number and percentage similarity to known genes by BLASTx search in NCBI DD product Accession Number Length (bp) Plant gene Similarity Function O10 CV736155 452 bp 14-3-3 d-2 protein 96 % Signal transduction O26 CV736156 420 bp 40S ribosomal protein S23 98 % Protein synthesis O151 CV736157 429 bp putative 60s ribosomal protein L15 92 % Protein synthesis O53 CV736158 442 bp nucleoside diphosphate kinase III 90 % Abiotic stress, signal transduction O58 CV736159 375 bp regulator of chromosome condensation-like protein 75 % DNA replication, abiotic stress O37 CV736160 360 bp amino acid permease 6 78 % Amino acid translocation O71 CV736161 403 bp miraculin-like protein 2 95 % Biotic stress O8 CV736162 503 bp beta-galactosidase 97 % Cell wall metabolism 97 1 Fig. 1. C 1 C 2 T 1 T 2 2 Fig. 1. Example of DDRT-PCR results. cDNAs were amplified from two separately isolated total RNAs by primer combination of HT 11 C and HAP 1. C 1 and C 2 are cDNAs amplified from the leaves collected at the end of second week (control); T 1 and T 2 are cDNAs amplified from the leaves collected at the end of fifth week (cold acclimated). Arrow 1 indicates one up regulated cDNA fragment; Arrow 2 indicates one down regulated cDNA fragment. 98 O10 O26 O151 O53 O58 O37 O71 O8 C T C T C T C T C T C T C T C T Fig. 2a. Actin Gene of interest 0 100 200 300 400 500 600 O10 O26 O151 O53 O58 O37 O71 O8 DD Products Differential expression detected by RT-PCR control treatment Fig. 2b. Fig. 2a: Confirmation of differential expression of 8 differential displayed (DD) products using relative quantitative RT-PCR with RNA from control and cold acclimated leaves. The cDNAs were synthesized from total RNAs isolated from control leaves (end of second week) and cold acclimation leaves (end of fifth week). Actin mRNA was used as an internal control. Specific RT-PCR primer pair information for all DD products is listed in table 1. Fig. 2b: A histogram showing the relative abundance of 8 DD products between control and cold acclimated plants. The intensities in each control and cold acclimation pair were norm means of three independent experim alized by setting the intensity of actin mRNA to 100. The values are the ents + SE. 99 V. Cold acclimation genes in Citrus unshiu ?Owari? 1 ABSTRACT Citrus unshiu is a moderately cold hardy species, but low temperature affects its productivity. Very little is known about the mechanism of adjustment to low temperature stress in this species. cDNA-AFLP and RT-PCR were used to study changes in gene expression of this species during gradual cold-acclimation. Two up regulated and four down regulated genes were identified. The up-regulated genes show high similarities to translation initiation factor eIF1 and cytochrome C. The down-regulated genes show high similarities to trigger factor type chaperone family protein, polyprotein, leucine-rich repeat transmembrane protein kinase/receptor-like protein kinase and PAZ/PIWI domain containing protein. Expression of some of these cold responsive genes is also regulated by other stresses. The differences of gene expression between Owari and Poncirus during low temperature were also briefly discussed. Key words: Citrus unshiu, cDNA-AFLP, cold acclimation and gene expression 1 The nucleotide sequences reported in this paper have been submitted in Genbank under accession numbers of CO578908 (OA123), CO578909 (OA36), CO578910 (OA120), CO578911 (OA68), CO578912 (OA69), CO578913 (OA87). 100 Introduction Since plants cannot escape from exposure to a number of environmental extremes, plants have evolved mechanisms for adaptation to their surroundings. Subfreezing temperature is a stress to plants which results in serious inhibition of growth and development. One of the most serious injuries induced by cold temperature is cell dehydration associated with ice formation under freezing temperature (Levitt 1980). Ice formation usually initiates in extracellular space. The lower water potential of ice will be the driving force for dragging water from cytoplasm to the extracellular space, thus causing cell dehydration. Most plants from temperate regions can increase their cold tolerance ability upon exposure to a period of low, nonfreezing temperature, a process called ?cold acclimation? (Guy 1990). During this process, many physiological and biochemical changes occur such as synthesis of new proteins (Tseng and Li 1991) and accumulation of compatible osmolytes (Bohnert et al. 1995). Several cold responsive genes have been identified in different plant species such as the CBF family in Arabidopsis thaliana (Gilmour et al. 1998), hsp90 in Brassica napus (Krishna et al. 1995) and cas15 in alfalfa (Monroy et al. 1993). Considerable efforts have been taken to clarify the molecular mechanisms of herbaceous plants under cold acclimation, but our understanding of the molecular mechanisms of cold acclimation in woody plants is limited. Citrus is an economically important crop throughout the world, and its productivity is seriously affected by low temperature (Porat et al. 2002). Some preliminary cold acclimation molecular studies have been conducted on Citrus and closely related species, and genes such as dehydrin have been isolated (Cai et al. 1995; Hara et al. 1999; Porat et al. 2002). The treatment 101 approach used by these researchers was a ?cold shock? (plants were moved from warmer temperature to low temperature directly) versus a ?cold acclimation? temperature regime, which more closely approximates natural conditions. Citrus unshiu is one of the most cold hardy commercial Citrus species (Yelenosky 1985). We show both up and down- regulated genes during gradual process of cold acclimation in C. unshiu identified by cDNA-AFLP. The identified cold acclimated genes in C. unshiu shed more light to the understanding of the regulatory mechanisms of woody plant cold acclimation, and might also have the potential to enhance the cold tolerance ability of cold sensitive species via transgenic approaches. Materials and methods Plants: One year old C. unshiu plants were grown for 5 weeks in a growth chamber with a 12 h light period at 400 ?mol m -2 s -1 intensity. The regimen for temperature decline was as follows: 32?C day/21?C night for 14 days; 27?C day/16?C night for 7 days; 24?C day/13?C night for 7 days and 18?C day/7?C night for 7 days. Plants were uniformly watered every day. RNA extraction: Expanded leaves at the end of the second and the fifth week were collected, immediately immersed in liquid nitrogen and stored at ?80?C for later use. RNA was extracted from leaves according to the RiboPure kit protocol (Ambion, Austin, TX). Extracted RNA was mixed with 1/9 volume of 10X DNase buffer and 4?l DNase I (2U/?l) and incubated for 30 min at 37?C to digest the remaining genomic DNA. 102 Digested RNA was treated with DNase inactivation reagent (20% volume) for 2 min, followed by centrifugation for 1 min at 14000g and transferred to a new tube. The concentration of isolated RNA was measured using an Eppendorf Biophotometer (Brinkmann Instruments, NY). The quality of RNA was checked using formaldehyde- agarose gel electrophoresis. RNA from the end of second week was used as unacclimated control and RNA from the end of fifth week as treatment. cDNA AFLP analysis: cDNA was synthesized using RETROscript TM (Ambion, TX) according to the manufacturer?s instructions, and digested using the MseI/EcoRI enzyme combination. AFLP analysis was conducted according to the methods of Bachem et al. (1996). Sequences of the adapters and primers used for cDNA AFLP analysis were: 5?- GACGATGAGTCCTGAG-3? (MseI adapter 1); 5?-TACTCAGGACTCAT-3? (MseI adapter 2); 5?-CTCGTAGACTGCGTACC-3? (EcoRI adapter 1); 5?- AATTGGTACGCAGTCTAC-3? (EcoRI adapter 2); 5?-GATGAGTCCTGAGTAAC-3? (non-selective primer for MseI); 5?-GACTGCGTACCAATTCA-3? (non-selective primer for EcoRI); 5?-GATGAGTCCTGAGTAACNN-3? (selective primer for MseI, and NN represents 2bp extension); 5?-GACTGCGTACCAATTCANN-3? (selective primer for EcoRI, and NN represents 2bp extension). Pre-amplification was performed in a 25 ?l reaction solution, containing 0.2 mM dNTPs, 0.2 ?M non-selective primers, 1.2mM MgCl 2 , 0.5U Tag polymerase and 10 ?l ligated cDNA fragments. The PCR program consists 20cycles: 30 s at 94 ?C, 60 s at 56 ?C and 60 s at 72 ?C. Selective amplification was performed in a 10?l reaction solution, containing 0.1mM dNTPs, 0.15 ?M EcoRI primer and 0.3 ?M MseI primer, 1.2mM MgCl 2 , 0.5 U Tag polymerase and 2.5 ?l pre- 103 amplification solution (diluted at 1:40). The PCR program was carried out with the following procedure: one cycle at 94 ?C for 30 s, 65 ?C for 30 s, and 72 ?C for 60 s; 13 cycles with annealing temperature decreasing 0.7 ?C per cycle followed by 23 cycles at 94 ?C for 30 s, 56 ?C for 30 s and 72 ?C for 60 s. Selective reaction products were run on 6% polyacrylamide sequencing gel at 85 W for 3 hrs. Fragments were visualized by silver staining according to the Silver Sequence TM DNA Sequencing System Technical Manual (Promega, WI). Cloning and sequence analysis of DNA fragments: DNA fragments extracted from the gel were ligated with pGEM-T Easy vector system (Promega, WI) and introduced into Escherichia coli DH5 cells. cDNA fragments were sequenced using the ABI3100 sequencer at AU genomics lab. Analysis of nucleotide sequence of selected fragments was carried out using National Center for Biotechnology Information BLASTx search tool. Quantitative RT-PCR: Quantitative relative RT-PCR was used to confirm the differential expression of DNA fragments isolated from control and cold acclimated plants. 2.5?g total RNA was reverse transcribed with 0.5mM dNTP, 5mM oligo(dT)- primers, 10mM Tris-HCl, pH 8.3, 50 mM KCl, 15mM MgCl 2 , 1?l RNase inhibitor, and 100U M-MuLV-RT (Ambion). The mixture (20?l total reaction volume) was incubated at 42?C for 1 hour. 1?l RT reaction was amplified in a 25?l solution with 10mM Tris- HCl, pH8.4, 50mM KCl, 1.5mM MgCl 2 , 2.5mM dNTPs, 0.3?M actin primer, 0.6?M actin competitor, 0.5mM of each gene specific primers and Taq polymerase (Qiagen). 104 PCR cycle program was as follows: 30 s at 94?C, 30 s at 55?C to 60?C and 30 s at 72?C for 30 to 35 cycles. Specific oligonucleotide primers were designed for each DD product for RT-PCR. The optimal PCR annealing temperatures and the cycle numbers are shown in Table 1. 10?l of the amplification products were separated using 1.8 % agarose gel electrophoresis and stained with ethidium bromide. The stained gel was used for quantitation of each band using a BioRad photo documentation system. A constitutively expressed actin gene was used as an internal standard in each reaction. The differential expression of genes in response to cold acclimation is shown in Table 2. Figure 1 (top) shows the agarose gel electrophoresis pattern of 6 low temperature responsive genes. Figure 1 (bottom) shows histogram of RT-PCR results. Results and discussion Transcriptome profiling of plants to environmental stresses can be studied via DDRT- PCR, cDNA-AFLP, SAGE, subtractive hybridization, DNA-chip and cDNA microarray (Donson et al. 2002). cDNA AFLP analysis was used in this study due to its high reliability and non-requirement of prior genetic information of studied organism. A total of 32 primer pairs were used in this study. An average of 20 to 50 bands were obtained. Up-regulated genes in cold acclimated ?Owari?: Cold acclimation is a complex and global process involving many physiological and biochemical changes. To acquire freezing tolerance, de novo protein synthesis is necessary for cold response (Guy 1990). The inferred amino acid sequence of OA 123 shows 96% similarity with the homolog of 105 translation initiation factor in Coffea arabica and rice, and 95% similarity with the translational initiation factor eIF1 from Porteresia coarctata. eIF1 in resurrection grass Sporobolus stapfianus was found to be induced during drought stress (Neale et al. 2000). A correlation between eIF1A and low temperature stress was shown in barley and maize (Berberich et al. 1995; Dunn et al. 1993). It can be postulated that the extensively de novo synthesis of proteins will help plant cells tolerate adverse environments. The inferred amino acid sequence of OA36 shows 95% similarity with cytochrome c in Arabisopsis thaliana. Cytochrome c is a main component of the respiratory chain. In sunflower, it has been demonstrated that the gene encoding cytochrome c is regulated by both tissue type and environmental factors (Felitti and Gonzalez, 1998). In the green alga Chlamydomonas reinhardtii, cytochrome c responds to metabolic regulation (Felitti et al. 2000). The up-regulated expression of cytochrome c might indicate cell need to maintain energy production in order to meet the requirements of cellular metabolism during stress. Down-regulated genes in ?Owari? in response to cold acclimation: Although scientists have focused on up-regulated genes, down-regulation of gene expression also contributes to the adaptation of plants to stress. The inferred amino acid sequence of OA 120 shows 55% similarity with polyprotein in A. thaliana. In sunflower, a putative gene encoding polyprotein was down regulated in shoots and up regulated in roots by salinity stress, but under drought, the expression was down regulated in roots and up regulated in shoots and leaves (Liu and Baird 2003). Differences in expression of retrotransposons in response to 106 different biotic and abiotic stresses can result in genetic changes and alteration of gene expression and thus help the host adapt to stresses (Grandbastien 1998; Wessler 1996). The inferred amino acid sequence of OA 68 shows 51% similarity with trigger factor type chaperone family protein in A. thaliana. Protein denaturation is a common consequence under stress condition. Chaperone family protein could help protein refolding. In Bacillus subtilis, trigger factor functions as a peptidyl-prolyl cis-trans isomerase, which catalyzes the in vitro refolding of ribonuclease T1 (G?thel et al. 1998). In wheat, the FKBP-type peptidyl-prolyl cis-trans isomerase (trigger factor) is heat induced and developmentally regulated (Kurek et al. 1999). Compared to the protective role of chaperone proteins in other species, the down regulation of this protein in Owari during low temperature might indicate that the denaturation of some proteins which are not suited to the acclimation of plant cells is needed. The inferred amino acid sequence of OA 69 shows 52% similarity with leucine- rich repeat transmembrane protein kinase and a receptor-like protein kinase in A. thaliana. The mechanism of perception of extracellular signals in plants is largely unknown. A receptor-like protein kinase with a leucine-rich repeat transmembrane domain was induced by ABA, dehydration, high salt and cold treatment in Arabidopsis, suggesting that this gene may function in the transmission of ABA and various environmental stress signals into intracellular reactions (Hong et al. 1997). However, our finding, that expression of the receptor protein kinase was specifically down regulated, signifies that its role in low temperature stress in ?Owari? is likely to be complicated. 107 The inferred amino acid sequence of OA 87 shows 76% similarity with the PIWI domain containing protein in Oryza sativa and 70% similarity with the PAZ/PIWI domain containing protein in Arabidopsis. The PIWI/PAZ family of genes, which contain conserved PIWI/PAZ domains, play important roles in stem cell self-renewal, RNA silencing, and translational regulation in various organisms (Cerutti et al. 2000; Kuramochi-Miyagawa et al. 2004; Morel et al. 2002). The function of PIWI/PAZ family proteins in plant cell exposed to environmental stresses is unknown. These recent results, together with our finding, show that it may be of importance to down regulate this gene in order to adapt plant cell to stress condition at the post transcription and translation level. Poncirus trifoliata is a close relative of Citrus and is mainly used as the rootstock in citrus industry due to the cold tolerance ability it imparts to the scion. Our previous study of cold acclimation regulated genes in P. trifoliata by differential display RT-PCR have shown that genes with substantial homologies to betaine/proline transporter, water channel protein, aldo-keto reductase, early light inducible protein, nitrate transporter, tetratricopeptide-repeat protein, F-box protein and ribosomal protein L15 are induced during cold acclimation (Zhang et al. 2005a). In addition, we have shown that genes with homology to chlorophyll a/b binding protein, photosystem II OEC 23, carbonic anhydrase, tumor related protein, pyrrolidone-carboxylate peptidase and ?- galactosidase are down-regulated during cold acclimation (Zhang et al. 2005b). Salinity, drought, and cold are known to cause osmotic/dehydration stress in plants. Three of the induced genes encountered during cold acclimation in P. trifoliata were reported as important to deal with cell dehydration caused by extracellular ice formation (Zhang et al. 108 2005a). In addition to dehydration, the accumulation of reactive oxygen species (ROS) is another common consequence of environmental stresses (Smirnoff 1993). Three of the eight up regulated genes isolated from cold acclimated P. trifoliata have functions related to ROS scavenging (Zhang et al. 2005a). Studies have shown that photosynthesis-related proteins are down regulated and it?s postulated that these proteins might not be suitable to the new physiological condition caused by dehydration stress (Chandler and Robertson 1994). One up and two down regulated photosynthesis related genes were detected in cold acclimated P. trifoliata. Compared to those genes detected in cold acclimated P. trifoliata, genes isolated from cold acclimated C. unshiu were not involved in dehydration, photooxidative protection and photosynthesis, but involved in protein translation, respiration, cell metabolism, signal transduction and protein refolding. In summary, the different types of genes detected in response to low temperature between P. trifoliata and C. unshiu might be an indication of their different stress tolerance ability, although the numbers of low temperature responsive genes detected in both species are a bit limited. More low temperature responsive genes should be isolated and the full-length cloning of the cDNA fragments and the detailed characterization of the obtained genes will be necessary to understand the underlined response mechanisms of plant cells to environmental stresses for both species. 109 Acknowledgement: We are grateful to Brandon Hockema, Bryan Wilkins and Monte Nesbitt for their helps in sample preparations. Special thanks give to Drs. Singh and Locy in Department of Biological Sciences at Auburn University for their critical reading of this manuscript. This research was funded in part by USDA CSREES Special Research Grants OEP 2001-03124 and 2002-06162 and the Alabama Agricultural Experiment Station. References Bachem CM, Van der Hoeven RS, de Bruijn S, Vreugdenhil D, Zabeau M, Visser RG (1996) Visualization of differential gene expression using a novel method of RNA fingerprinting based on AFLP: analysis of gene expression during potato tuber development. Plant J 9: 745-753. Berberich T, Sugawara K, Harada M, Kusano T (1995) Molecular cloning, characterization and expression of an elongation factor 1 gene in maize. Plant Mol Biol 29: 611-615. Bohnert HJ, Nelson DE, Jensen RG (1995) Adaptation to environmental stresses. Plant Cell 7: 1099-1111. Cai Q, Moore GA, Guy CL (1995) An unusual group 2 LEA gene family in citrus responsive to low temperature. Plant Mol Biol 29: 11-23. Cerutti L, Mian N, Bateman A (2000) Domains in gene silencing and cell differentiation proteins: the novel PAZ domain and redefinition of the Piwi domain. Trends Biochem Sci 25: 481-482. Chandler P, Robertson M (1994) Gene expression regulated by abscisic acid and its relation to stress tolerance. Annu Rev Plant Physiol Plant Mol Biol 25: 113-141. Donson J, Fang Y, Espiritu-Santo G, Xing W, Salazar A, Miyamoto S, Armendarez V, Volkmuth W (2002) Comprehensive gene expression analysis by transcript profiling. Plant Mol Biol 48: 75-97. Dunn MA, Morris A, Jack PL, Hughes MA (1993) A low temperature-responsive translation elongation factor 1 from barley (Hordeum vulgare L.). Plant Mol Biol 23: 221-225. 110 Felitti SA, Chan RL, Sierra MG, Gonzalez DH (2000) The cytochrome c gene from the green alga Chlamydomonas reinhardtii. structure and expression in wild-type cells and in obligate photoautotrophic (dk) mutants. Plant Cell Physiol 41: 1149- 1156. Felitti SA, Gonzalez DH (1998) Carbohydrates modulate the expression of the sunflower cytochrome c gene at the mRNA level. Planta 206: 410?415. Gilmour SJ, Zarka DG, Stockinger EJ, Salazar MP, Houghton JM, Thomashow MF (1998) Low temperature regulation of the Arabidopsis CBF family of AP2 transcriptional activators as an early step in cold-induced COR gene expression. Plant J 16: 433-442. G?thel SF, Scholz C, Schmid FX, Marahiel MA (1998) Cyclophilin and trigger factor from bacillus subtilis catalyze in vitro protein folding and are necessary for viability under starvation conditions. Biochemistry 37: 13392 -13399. Grandbastien MA (1998) Activation of plant retrotransposons under stress conditions. Trends Plant Sci 3: 181-187. Guy CL (1990) Cold acclimation and freezing stress tolerance: role of protein metabolism. Annu. Rev. Plant Physiol. Plant Mol Biol 41: 187-223. Hong SW, Jon JH, Kwak JM, Nam HG (1997) Identification of a receptor-like protein kinase gene rapidly induced by abscisic acid, dehydration, high salt, and cold treatments in Arabidopsis thaliana. Plant Physiol 113: 1203-1212. Krishna P, Sacco M, Cherutti JF, Hill S (1995) Cold-Induced accumulation of hsp90 transcripts in brassica napus. Plant Physiol 107: 915-923. Kuramochi-Miyagawa S, Kimura T, Ijiri TW, Isobe T, Asada N, Fujita Y, Ikawa M, Iwai N, Okabe M, Deng W, Lin H, Matsuda Y, Nakano T (2004) Mili, a mammalian member of piwi family gene, is essential for spermatogenesis. Development 131: 839-849. Kurek I, Aviezer K, Erel N, Herman E, Breiman A (1999) The wheat peptidyl prolyl cis- trans-isomerase FKBP77 is heat induced and developmentally regulated. Plant Physiol 119: 693-704. Liu X, Baird VW (2003) Differential expression of genes regulated in response to drought or salinity stress in sunflower. Crop Sci 43: 678-687. Monroy AF, Castonguay Y, Laberge S, Sarhan F, Vezina LP, Dhindsa RS (1993) A new cold-induced alfalfa gene is associated with enhanced hardening at subzero temperature. Plant Physiol 102: 873-79. 111 Morel J-B, Godon C, Mourrain P, Beclin C, Boutet S, Feuerbach F, Proux F, Vaucheret H (2002) Fertile hypomorphic ARGONAUTE (ago1) mutants impaired in post- transcriptional gene silencing and virus resistance. Plant Cell 14: 629-639. Neale AD, Blomstedt CK, Bronson P, Le T-N, Guthridge K, Evans J, Gaff DF, Hamill JD (2000) The isolation of genes from the resurrection grass Sporobolus stapfianus which are induced during severe drought stress. Plant Cell Environ 23: 265-277. Porat R, Pavoncello D, Lurie S, Mccollum TG (2002) Identification of a grapefruit cDNA belonging to a unique class of Citrus dehydrins and characterization of its expression patterns under temperature stress conditions. Physiol Plant 115: 598- 603. Smirnoff N (1993) The role of active oxygen in the response of plants to water deficit and desiccation. New Phyto 25: 27-58. Tseng MJ, Li PH (1991) Changes in protein synthesis and translatable messenger RNA populations associated with ABA-induced cold hardienss in potato (Solanum commersonii). Physiol Plant 81: 349-358. Wessler SR (1996) Turned on by stress. Plant retrotransposons. Current Bio: CB 6: 959- 961. Yelenosky G (1985) Cold hardiness in Citrus. Hort Rev 7: 201-238. Zhang C, Lang P, Dane F, Ebel RC, Singh NK, Locy RD, Dozier WA (2005a) Cold acclimation induced genes of trifoliate orange (Poncirus trifoliata). Plant Cell Reports 23: 764-769 Zhang C, Lang P, Ebel RC, Dane F., Singh NK, Locy RD, Dozier WA (2005b) Down- regulated gene expression of cold acclimated Poncirus trifoliata. Can J. Plant Sci 112 Table 1. Oligonucleotide primer sequences, PCR conditions and cycle numbers for the confirmation of differentially expressed products with quantitative RT-PCR Differentially expressed product Forward Primer (5?-3?) Reverse Primer (5?-3?) PCR annealing temperature Cycle numbers OA123 GCTACAACAAGATTCTCAAGGA CCAATAGTTGGAGATGGATAAA 57?C 27 OA36 CCGCCATGTTCTTGTTAGC GCGTCTTCTTTCTCTCTTTGG 56?C 28 OA120 TCATGGTTGGTACACAAAGG GCTAACAATGCCTTGAACAA 58?C 33 OA68 ATCTCCATCAACCTCAATTTCA GAATCTTCCCAGGACGAAAT 55?C 29 OA69 ACGGCGAATTGCAGAGAG CTCTCATGGGTTCTGCAAA 55?C 33 OA87 GGAAATGATGGAAGAAGGATC GCACCAGAAAGCATAGAAGAA 58?C 33 113 Table 2. Isolated cDNA AFLP products, accession number in Genbank and percentage similarity to known genes by BLASTx search in NCBI 114 Differentially expressed product Accession Number Length (bp) Plant gene Similarity Function OA123 CO578908 263 translation initiation factor 96% protein synthesis OA36 CO578909 292 cytochrome c 95% cell respiration OA120 CO578910 313 polyprotein 55% stress-related OA68 CO578911 302 trigger factor type chaperone family protein 51% protein refolding OA69 CO578912 306 leucine-rich repeat transmembrane protein kinase 52% signal transduction OA87 CO578913 287 PIWI domain containing protein 76% translation regulation OA123 OA36 OA120 OA68 OA69 OA87 Fig. 1 (Top): Confirm relative quantitative RT-PCR with RNA from cDNAs were synthesized from week) and cold acclim control. Specific RT-PCR prim listed in table 1. (Bottom products between control and cold acclim cold acclim values are the m 115 C T C T C T C T C T C T Gene of interest Actin OA123 OA36 OA120 OA68 OA69 OA8 cDNA-AFLP product ation of differential expression of 6 cDNA AFLP products using control and cold acclimated leaves. The total RNAs isolated from control leaves (end of second ation leaves (end of fifth week). Actin gene was used as an internal er pair information for all cDNA AFLP products was ): A histogram showing the relative abundance of 6 cDNA AFLP ated plants. The intensities in each control and ation pair were normalized by setting the intensity of actin gene to 100. The eans of three independent experiments + SE. 0 50 100 150 200 250 7 Differential expression detected by RT-PCR Control Treatment VI. Conclusions, ongoing research and future perspectives 1. Conclusions mRNA differential display was used to study the molecular mechanisms of Citrus unshiu and its relative, Poncirus trifoliata, under a gradually declined temperature regime. 144 primer pairs were used and about 15,000 cDNA fragments were analyzed after autoradiography in P. trifoliata, while 64 primer pairs were used and about 8,000 cDNA fragments were analyzed after autoradiograph in C. unshiu. In P. trifoliata, 14 cDNA fragments were confirmed to be positive with relative quantitative RT-PCR. Osmotic modulation, photooxidative protection and photosynthesis adjustment were postulated to be three of the main mechanisms in P. trifoliata under cold acclimation. In C. unshiu, 8 cDNA fragments were confirmed to be positive using relative RT-PCR, and these genes were reported to be associated with signal transduction, protein synthesis, amino acid transport, and cell wall metabolism. In addition to differential display, another technique, cDNA-AFLP was also used to detect differentially expressed transcripts in C. unshiu. A total of 32 primer pairs were used in the study and about 10,000 bands were visualized upon silver staining. Six genes were confirmed to be positive using quantitative RT-PCR. The identified genes were related to protein translation, energy production, protein refolding and signal transduction. 116 2. Ongoing research The isolation of genes with a new cDNA-AFLP protocol is still ongoing. A higher number of isolated genes will shed more light on the mechanisms of cold acclimation of C. unshiu in response to low temperature. Using RACE, full length sequences of carbonic anhydrase, proline transporter and nitrate transporter have been obtained. Carbonic anhydrase (PtCA) cDNA contains an open reading frame of 269 amino acids. The yeast complementation test for this gene is ongoing. Nitrate transporter (PtNRT) cDNA contains an open reading frame of 588 amino acids. 12 transmembrane domains have been predicted for this protein. Chloroplast membrane was predicted to be the functional site. A gene family including four members has been found for Poncirus proline transporter (PtProT). Their cDNAs contain an open reading frame around 391-400 amino acids. 10 transmembrane domains have been predicted for this protein. 3. Future research plan - Due to the limited number of genes isolated by differential display, cDNA-AFLP should be utilized to isolate more genes in order to obtain a more comprehensive picture of the cold acclimation mechanisms in both P. trifoliata and C. unshiu. The comparison of the functions of genes from these two related species might help explain the differences in cold tolerance ability between these two species. - Since no reports have been published to correlate the function of nitrate transporter to low temperature stress, functional analysis of this gene in oocytes, a plant transporter study model, will be indispensable to confirm the involvement of this gene under low temperature stress. Proline and proline synthesis have been 117 118 recognized for many years as important for plants to adapt to adverse environment, but very limited information is available to assign a role of the transport of this chemical in the process of plant cold acclimation. Comparison of the electrolyte leakage rate of transgenic Arabidopsis with P5CS (key enzyme related to proline synthesis), proline transporter gene and P5CS+ proline tranporter gene will be necessary to assign an exact role of the product encoded by proline transporter at the physiological level. Functional analysis of other interesting genes such as 14-3-3 will be helpful to understand the underlined mechanisms of these plant species in response to low temperature. - Gene profiling works have been conducted in many species in response to low temperature, and many genes have been isolated and some of the functions of the isolated genes were also characterized. By contrast, less is known about stress responses of plants at the metabolite and metabolome level, thus a further profiling analysis of metabolites in these two species will enrich our understanding of cold acclimation mechanisms at a different level and may provide new insight into mechanisms utilized by plant species to cope with low temperatures stress.