ROLE OF ARABIDOPSIS THALIANA CALMODULIN ISOFORMS IN TOLERANCE TO ABIOTIC STRESS 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. ________________________________________ Nisreen AL-Quraan Certificate of Approval: _______________________ ________________________ Robert D. Locy Narendra K. Singh, Chair Professor Professor Biological Sciences Biological Sciences _______________________ _______________________ Joe H. Cherry Joanna Wysocka-Diller Professor Associate Professor Biological Sciences Biological Sciences ________________________ ________________________ Evert C.Duin George T. Flowers Assistant Professor Interim Dean Chemistry and Biochemistry Graduate school ROLE OF ARABIDOPSIS THALIANA CALMODULIN ISOFORMS IN TOLERANCE TO ABIOTIC STRESS Nisreen AL-Quraan 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 9, 2008 iii ROLE OF ARABIDOPSIS THALIANA CALMODULIN ISOFORMS IN TOLERANCE TO ABIOTIC STRESS Nisreen AL-Quraan 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 of Graduation iv VITA Nisreen AL-Quraan, daughter of Mr. Adnan, AL-Quraan and Mrs. Fayzeh, AL-Quraan was born on June 3, 1976 in Irbid, Jordan. She graduated from AL-Taybeh High School in Irbid, Jordan in 1994. She graduated in 1998 with Bachelor of Science degree from the Department of Biological Sciences, Yarmouk University, Jordan. She joined the graduate program in the Department of Biological sciences, Yarmouk University and received her Master of Science degree in Plant Biochemistry and Molecular biology in 2001. After completion of her MS, she worked as research and teaching assistant for two years in the Department of Biological Sciences, Yarmouk University, Jordan. She was married to Dr. Abdelsalam Talafha, Professor in Large Animal Medicine (Theriogenology) in 2002 and came to the University of Georgia, Athens with her husband. On May, 2004 she joined the Department of Biological Sciences, Auburn University to pursue her PhD degree in Plant Biochemistry and Molecular Biology. She and her husband are gifted with their first child, Morad born in February, 2005. v DISSERTATION ABSTRACT ROLE OF ARABIDOPSIS THALIANA CALMODULIN ISOFORMS IN TOLERANCE TO ABIOTIC STRESS Nisreen AL-Quraan Doctor or Philosophy August 9, 2008 (M.S. Yarmouk University, Irbid, Jordan, 2001) (B.S.Yarmouk University, Irbid, Jordan, 1998) 189 Typed Pages Directed by Narendra K.Singh Calmodulin and Ca 2+ have been implicated in adaptation of plants to changes in its environment. Arabidopsis thaliana (Arabidopsis) genome has nine genetic loci encoding calmodulin protein isoforms. We have used wild type (WT) and T-DNA insertion mutants of seven calmodulin genes in Arabidopsis to determine role of specific isoform of calmodulin in abiotic stress in Arabidopsis. To understand the role of specific CAM genes in heat stress, the steady-state level of mRNA for the nine CAM genes in root and shoot tissues of WT seedlings grown at normal growth temperature (25 o C) and during heat stress at 42 o C for 2 hr was determined using gene specific primers. Normalized steady-state level of all CAM RNA in WT and T-DNA insertional mutant lines (cam) at normal growth temperature (25 o C) and after heat treatment at 42 o C was determined. Compared to growth at 25?C, the mRNA levels vi of all CAM genes were upregulated in both root and shoot after heat treatment with the notable exception of CAM5 in root and shoot, and CAM1 in shoot where the mRNA levels were reduced. At 25 o C all cam mutants showed varying levels of mRNA for corresponding CAM genes with the highest levels of CAM5 mRNA being found in cam5- 1 and cam5-3. CAM5 mRNA was not observed in the cam5-4 allele which harbors a T- DNA insertion in exon II. Compared to wild type, the level of mRNA for all CAM genes varied in each cam mutant, but not in a systematic way. During heat stress at 42 o C the level of CAM gene mRNAs was also variable between insertional mutants, but the level of CAM1 and CAM5 mRNA was consistently greater in response to heat stress in both root and shoot. These results suggest differential tissue specific expression of CAM genes in root and shoot tissues, and specific regulation of CAM mRNA levels by heat. Each of the CAM genes appears to contain noncoding regions that play regulatory roles in the interaction between CAM genes leading to changes in specific CAM gene mRNA levels in Arabidopsis. With respect to expression of specific CAM gene only exonic mutation lead to a loss of function of CAM genes. All cam mutants were screened for phenotypic alterations in seed germination, survival and treatment induced oxidative damage under a variety of environmental stress. Screening of cam mutants demonstrated that cam5-4 and cam6-1 accumulate significantly higher level of melonaldehyde and are sensitive to all abiotic factors tested including heat and low temperature exposures, osmotic and salinity stress. Surprisingly, other cam5 alleles with T-DNA insertions in either 5? UTR or 3?UTR did not show any significant difference from WT in germination, survival and oxidative damage in response to various stresses. This difference in phenotype between cam5-4 and other vii cam5 alleles suggests important role for CaM5 protein in tolerance to abiotic stress in cam5-4. T-DNA insertion in cam6-1 is at the 3?UTR of CAM6 gene with significant residual level of CAM6 mRNA. It seems likely that sensitivity of cam6-1 to all environmental stress tested may be mediated indirectly through a different mechanism. An initial elevation in the level of GABA shunt metabolites followed by a significant and substantial increase in its level after prolonged exposure to same stress or exposure to increasing level of stress was observed in WT and most cam mutants. Root tissues accumulated higher level of metabolites than the shoot tissues in WT and cam mutants after exposure to stress, except for GABA level in shoot tissues at higher level of salinity stress. Level of GABA and glutamate changed more significantly than alanine under stress. Different cam mutants showed significant variations in levels of GABA shunt metabolites. These results suggest that collective amounts of CaM proteins may regulate GAD activity in response to abiotic stress rather thanspecific CaM protein under different stress conditions. Steady-state level of CAM mRNA in WT and cam mutants provides support for some level of compensation in expression CAM genes in Arabidopsis thaliana. viii ACKNOWLEDGEMENTS Nisreen would like to thank her advisor, Dr. Narendra Singh and co-advisor, Dr. Robert Locy for their continued support, understanding and encouragement during her work. They taught her how to ask questions and express her ideas. They showed her ways to approach a research problem and the needs to be persistent to accomplish any goal. She is forever beholden and inspired. She is deeply thankful to her committee members Dr. Joe H. Cherry, Dr. Joanna Wysocka-Diller, and Dr. Evert C. Duin for guidance and helpful discussions. Special thanks to Dr. Joe W. Kloepper for serving as an outside reader of the dissertation and for his valuable comments and discussions. She would like to thank her coworkers in the laboratory, specially Mrs. Chiachen Weng and Mrs.Yuying Sang, and a large number of friends in Auburn, especially Mss. Larrisa Parsley for their support and help in times of need. Lastly, she would like to thank her supportive and loving husband, Dr. Talafha and her sweet son, Morad for their unconditional love, support and understanding without which she would not have completed her dissertation. ix Style manual of journal used: Plant Physiology Computer softwares used: Microsoft Word 2003 Microsoft Excel 2003 Microsoft Office Picture Manager 2003 Minitab 13.0 Sigma Plot 9.0 Adobe Photoshop 6.0 Endnote 10.0 x TABLE OF CONTENTS LIST OF TABLES............................................................................................................ xii LIST OF FIGURES ......................................................................................................... xiii I. LITERATURE REVIEW Introduction..............................................................................................................1 Heat Stress and Thermotolerance ............................................................................2 Low Temperature and Cold Acclimation ................................................................3 Drought and non-ionic Osmotic Stress....................................................................6 Salinity and Ion Toxicity .......................................................................................10 Ultraviolet (UV) Irradiation...................................................................................14 ROS and Oxidative Stress......................................................................................15 Biotic Stress ...........................................................................................................18 Modulation of Calcium (Ca +2 ) effects via Ca +2 -Binding Proteins.........................19 Calmodulin (CaM) ................................................................................................20 CaM Isoforms and Genes in Arabidopsis .................................................21 Mechanisms of Action ..............................................................................22 Other Calcium-binding Proteins ...........................................................................24 Calmodulin Like Proteins (CML) .............................................................24 Calcium Dependent Protein Kinase (CDPK) ............................................24 Calcinurein B (CBL) .................................................................................25 Regulation of GABA Shunt ..................................................................................25 Literature Cited .....................................................................................................34 II. THE REGULATION OF CALMODULIN GENE mRNAs IN WILD TYPE AND CALMODULIN MUTANTS OF ARABIDOPSIS THALIANA UNDER HEAT STRESS Abstract .................................................................................................................56 Introduction ...........................................................................................................58 Material and Methods ...........................................................................................59 Results and Discussion ......................................................................................... 62 Literature Cited .....................................................................................................69 xi III. SENSITIVITY OF CALMODULIN MUTANTS OF ARABIDOPSIS THALIANA TO HIGH TEMRERATURE EXPOSURE Abstract .................................................................................................................79 Introduction............................................................................................................82 Material and Methods ............................................................................................83 Results and Discussion ..........................................................................................87 Literature Cited ......................................................................................................92 IV. SENSITIVITY OF CALMODULIN MUTANTS OF ARABIDOPSIS THALIANA TO LOW TEMPERATURE EXPOSURE Abstract................................................................................................................105 Introduction..........................................................................................................108 Material and Methods ..........................................................................................109 Results and Discussion ........................................................................................111 Literature Cited ....................................................................................................117 V. SENSITIVITY OF CALMODULIN MUTANTS OF ARABIDOPSIS THALIANA TO SALT AND OSMOTIC STRESS Abstract................................................................................................................130 Introduction..........................................................................................................133 Material and Methods ..........................................................................................135 Results and Discussion ........................................................................................137 Literature Cited ....................................................................................................147 APPENDIX I ...................................................................................................................169 APPENDIX II ..................................................................................................................170 APPENDIX III.................................................................................................................173 APPENDIX IV ................................................................................................................174 APPENDIX V .................................................................................................................175 xii LIST OF TABLES Table II.1. Mutant lines with T-DNA insertion in calmodulin genes of A. thaliana used in this study ............................................................................................................................77 Table II.2. Nucleotide sequence of primers for CAM genes and CBP20 gene used as an internal standard for RT-PCR amplification......................................................................78 Table III.1. Mutant lines with T-DNA insertion in calmodulin genes of A. thaliana used in this study......................................................................................................................104 Table IV.1. Mutant lines with T-DNA insertion in calmodulin genes of A. thaliana used in this study......................................................................................................................129 Table V.1. Mutant lines with T-DNA insertion in calmodulin genes of A. thaliana used in this study ..........................................................................................................................168 xiii LIST OF FIGURES Figure II.1. Estimates of normalized levels of steady state mRNA of nine CAM genes in root and shoot tissues of wild type A. thaliana grown at 25 o C (control) (A), and fold increase in the levels of nine CAM genes nRNAs in root and shoot tissues of wild type A. thaliana after 2 hr heat treatment at 42?C compared to growth at 25?C (B). ................... 73 Figure II.2. Extimates of normalized steady state mRNA levels of nine CAM genes in root and shoot tissues of the corresponding cam mutants of A. thaliana at 25?C (A) and the fold increase in the expression of CAM genes in root and shoot tissues of corresponding cam mutants compared to its expression in wild type at 25?C (B) ........... 74 Figure II.3. The fold change of the nine Arabidopsis CAM gene mRNAs in root and shoot tissues of each of the cam alleles of seven of the CAM genes compared to wild type. ...75 Figure II.4. The fold change in the level of mRNA for the nine Arabidopsis CAM gene mRNAs in root and shoot tissues of each of the cam alleles of seven of the CAM genes and wild type after 2 hr heat treatment at 42?C compared to growth at 25?C ...................76 Figure III.1. Germination of A. thaliana seeds of wild type and T-DNA insertion mutant alleles cam5-4 and cam6-1 after exposure to 42 o C temperature........................................96 Figure III.2. Survival of A. thaliana wild type and T-DNA insertion mutant alleles cam5- 4 and cam6-1 seedlings exposed to 42?C heat...................................................................97 Figure III.3. Oxidative damage by high temperature exposure in cam mutants of A. thaliana ..............................................................................................................................98 Figure III.4. The levels of GABA, alanine and glutamate in two-week-old seedlings of cam mutants of A. thaliana after exposure to heat at 42 o C for 30 min, 1 hr and 2 hr ....103 Figure IV.1. Germination of seeds of wild type, cam5-4, and cam6-1 mutants of A. thaliana after exposure to 4 o C for 24 hr ..........................................................................120 Figure IV.2. Oxidative damage in cam mutants of A. thaliana after exposure to low temperature at 4 o C. .........................................................................................................122 Figure IV.3. The level of GABA, alanine and glutamate in root and shoot tissues of two week old seedlings of wild type and cam mutants of A. thaliana was determined after xiv exposure to cold as indicated. Seedlings were treated for 0 hr, 1 hr, 3 hr, 6 hr and 12 hr at 4?C ...................................................................................................................................128 Figure V.1. Germination of WT and cam mutant seeds on various concentrations of NaCl (A) and Mannitol (B) .......................................................................................................151 Figure V.2. Root elongation of cam mutants and WT seedlings after 7 days of growth on agar plates supplemented with 0, 75, 100, and 150 mM NaCl (A) and with 0, 200, 300, 400 mM Mannitol (B)......................................................................................................153 Figure V.3. Oxidative damage in seedlings of cam mutants and wild type A. thaliana after exposure to 150 mM NaCl.......................................................................................155 Figure V.4. Oxidative damage in cam mutants and wild type A. thaliana caused after exposure to 400 mM Mannitol.........................................................................................157 Figure V.5. GABA shunt metabolites in cam mutants of A. thaliana after exposure to 0, 75, 100, and 150mM NaCl were grown under continuous light at 25?C for seven days?? ..........................................................................................................................162 Figure V.6. GABA shunt metabolites in cam mutants and wild type A. thaliana seedlings after exposure to 0, 200, 300 and 400mM mannitol grown under continuous light at 25?C for seven days. .................................................................................................................167 1 I. LITERATURE REVIEW Introduction Environmental fluctuations in temperature and water availability limit crop productivity (Bray et al., 2000). Estimated losses of productivity due to biotic and abiotic stresses range from 65% to 87% worldwide (Bray et al., 2000). Organisms respond to the environment by adaptive mechanisms in growth and development involving a complex network for perception and transmission of stress signals to initiate cellular responses by changes in enzyme activity, gene expression, and transport of molecules across membranes (Boyer, 1982; Mittler, 2006; Shelp et al., 2006). Stress tolerance in plants involves a variety of genetic and molecular adaptive mechanisms (Bohnert et al., 1995) such as osmoregulation and the accumulation of osmoprotectants (McNeil et al., 1999), calcium signaling (Hetherington and Brownlee, 2004), and the accumulation of ?-aminobutyric acid (GABA) (Steward, 1949; Breitkreuz and Shelp, 1995; Mayer et al., 1990; Locy et al., 2000). The pathway that converts glutamate to succinate via GABA is called the GABA shunt. The GABA shunt pathway in plants is composed of a cytosolic enzyme glutamate decarboxylase (GAD), and mitochondrial enzymes GABA transaminase (GABA-TA) and succinate semialdehyde dehydrogenase (SSADH) (Shelp et al., 1999; Bouch? and Fromm, 2004). Rapid accumulation of GABA in response to unfavorable or extreme environments sparked interest in the elucidation of the GABA shunt pathway and its association with mechanisms including pH regulation, TCA cycle fluxes, nitrogen 2 metabolism, protection against oxidative stress, osmoregulation, and stress signaling (Shelp et al., 1999; Kinnersley and Turano, 2000). Heat stress and thermotolerance Plants perceive high temperature in different ways. Adaptation or acclimation to heat occurs differentially over time and across tissue organization. Heat affects a variety of cellular structures and functions (Taiz and Zeiger, 2002). The classical response to heat stress is the production of heat shock proteins (HSPs), which function as molecular chaperons to maintain proper protein folding. Binding of trimeric heat shock factor (HSF) to a heat shock DNA sequence element (HSE) is essential for the transcription of HSP mRNA. As a response to heat stress the synthesis of HSPs enable cells to cope with high levels of cellular protein dentauration during heat stress (Prandl et al., 2001). Membranes and proteins damaged by heat may produce reactive oxygen species (ROS) during photosynthesis possibly causing acceleration of senescence (Larkindale et al., 2005). The oxygen evolving complex, reaction center, and light harvesting complexes can be disrupted by elevated temperature. The low molecular weight HSPs in chloroplast are involved in PS II thermotolerance (Heckathorn et al., 1998). Phytohormones, such as abscisic acid (ABA), salicylic acid (SA), and ethylene have been linked to heat stress signaling (Cherry and Nielsen, 2004). The role of ABA and SA in the induction of thermotolerance and protection against oxidative damage during heat stress either by prevention or rapid repair of oxidative damage has been demonstrated (Larkindale and Knight, 2002; Larkindale and Huang, 2004; Larkindale et al., 2005). Ethylene as a potential signaling molecule in tolerance to heat stress and prevention of heat induced oxidative damage without its involvement in HSP induction 3 during heat stress has been demonstrated (Larkindale and Huang, 2004; Suzuki and Mittler, 2006; Kotak et al., 2007). The role of calcium signaling in heat stress has been debated for many years. In maize calcium (Ca 2+ ) and calmodulin (CaM) induce HSP70 binding to heat shock elements in vitro, suggesting a role for Ca 2+ /CaM in HSP gene expression through the regulation of HSFs (Sun et al., 2000; Li et al., 2004). Liu et al. (2003) suggested that regulation of DNA-binding activity of HSF is mediated by direct binding of CaM to cytoplasmic HSP70 or by regulation of HSF phosphorylation involving a CaM-dependent kinase activity. Yang and Poovaiah (2000) also demonstrated that the chloroplast chaperonin 10 in Arabidopsis is a Ca 2+ /CaM-binding protein and suggested that the calcium signaling system can regulate protein folding in chloroplast. Exposure of Arabidopsis to heat results in increased cytoplasmic Ca 2+ that activates glutamate decarboxylase (GAD) via activation Ca 2+ /CaM and GABA accumulation in roots, and this effect is reversed by CaM and Ca 2+ inhibitors (Locy at al., 2000). Calcium channel blocker and CaM inhibitors induce oxidative damage to membranes and enhance reactive oxygen substance (ROS) production while pre-treatment with H 2 O 2 can enhance thermotolerance (Larkindale et al., 2005; Larkindale and Knight, 2002; Suzuki and Mittler, 2006). Low temperature and cold acclimation Plants exhibit extensive changes in lipid composition, soluble protein content, and simple sugars type and content in response to low temperature. Genetic analysis in plants suggests the involvement of many genes in cold tolerance (Thomashow et al., 1999). Plants can sense cold stress through low temperature induced changes in membrane 4 fluidity, conformational changes in macromolecules, and metabolite concentration. Pharmacological analyses have shown that plasma membrane rigidification induces COR (cold responsive) genes and results in cold acclimation in alfalfa and Brassica napus (Orvar et al., 2000; Sangwan et al., 2001). CBF (C-repeat binding factors) regulates the expression of genes involved in phosphoinositide metabolism, transcription of many Cor genes, osmolyte biosynthesis, ROS detoxification, membrane transport, hormone metabolism, signaling, and a number of additional protective functions. CBFs, also know as dehydration-responsive element-binding proteins (DREBs), signaling is conserved in plants, but varies during cold tolerance versus freezing tolerance (Fowler and Thomashow, 2002; Lee et al., 2005; Maruyama et al., 2004). Central young leaves of Arabidopsis rosettes exhibit a faster and greater freezing tolerance than peripheral mature leaves during cold acclimation (Takagi et al., 2003). Sugar accumulation contributes to this differential freezing tolerance during the early days of cold treatment, but the degree of freezing tolerance in different leaves was not associated with the CBF/DREB1 family proteins or with the COR78/RD29A protein (Kawamura and Uemura, 2003). Proteins induced in the central leaves are associated with membrane repair, protection of the membrane against osmotic stress, enhancement of carbon dioxide fixation, and proteolysis (Kawamura and Uemura, 2003) Cold-regulated genes in Arabidopsis constitute between 4% and 20 % of the genome (Lee et al., 2005). Expression of CBFs and APETALA2/ETHYLENE RESPONSE FACTOR family transcription factors is induced by cold exposure. The above transcription factors can bind to cis-elements in the promoters of COR genes to increase expression. Transgenic plants with ectopic expression of CBF induce expression 5 of COR genes and cold acclimation even at normal growth temperatures (Stockinger et al., 1997; Liu et al., 1998; Chinnusamy et al., 2007). An increase in the level of ABA during cold exposure has been demonstrated (reference), and pretreatment of plants with ABA can cause the freezing tolerance of plants to increase suggesting a role for ABA in low temperature stress (Gilmour et al., 1991). CIPK3 is a Serine/Therionine kinase associated with calcineurin B-like calcium sensor and regulates ABA responses during seed germination and gene expression in Arabidopsis (Kim et al., 2003). The expression of the CIPK3 gene is responsive to ABA and cold (Wasilewsk et al., 2008). These findings suggest a link between low temperature exposure, ABA, and gene expression during stress cold stresses. The activity and transcription of genes encoding ROS-scavenging enzymes are increased by cold (O`Kane et al., 1996). A number of cold responsive genes such as RD29A, KIN2, COR15A, COR47, DREB1A, DREB2A, and ERD10 have been characterized in Arabidopsis (Thomashow, 1999). Some of the COR gene products may be involved in ROS scavenging (Lee et al., 2002). The Arabidopsis frostbite1 (fro1) mutant displayed reduced expression of cold-responsive genes such as RD29A, KIN1, COR15A, and COR47, and constitutively accumulated ROS. The FOR1 gene encodes a mitochondrial complex I protein, suggesting that expression of the cold-responsive genes and ROS accumulation might be modulated by disruption of a mitochondrial function (Suzuki and Mittler, 2006). Such studies demonstrate a close link between ROS, ROS signaling, and the cold stress response. Cytosolic calcium in Arabidopsis seedlings increases significantly at the start of cooling (Larkindale and Knight, 2002). This increase is likely mediated through 6 activation of calcium channels and phospholipid signaling (Vergnolle et al., 2005; Williams et al., 2005; Chinnusamy et al., 2006; Komatsu et al., 2007). Involvement of CaM in cold induction of COR genes and the induction of CaM by low temperature has been shown (Braam and Davis, 1990). Over-expression of CAM3 in Arabidopsis resulted in decreased expression of cold-induced COR genes suggesting a negative regulatory role for CaM in regulation of COR expression in plants (Townely and Knight, 2002). Since GAD is a Ca 2+ /CaM-regulated enzyme (Snedden et al., 1996), Ca 2+ influx following a temperature drop (Monroy and Dhindsa, 1995) could be responsible for the activation of the GAD enzyme during cold stress. Drought and non-ionic osmotic stress Drought induces a number of metabolic pathways in various plants. Changes in the metabolism and accumulation of sugars (such as raffinose, sucrose, and trehalose), sugar alcohols (such as mannitol, and sorbitol), amino acids (such as proline, glutamate, alanine, and GABA), and amines (such as glycine betaine and polyamines) are well documented durning osmotic stresses (Taji et al, 2002; Bartels and Sunkar, 2005). Many of these metabolites are believed to function as compatible osmolytes and antioxidants by protecting cellular functions or maintaining structural components of cells (Umezawa et al., 2006, Seki et al., 2007). Drought-related water deficit triggers the production of Abscisic acid (ABA), which causes stomatal closure and enhances the expression of a number of specific osmotic ssstress-related pdofsins such as ABA-responsive element binding protein1 (AREB1), dehydration-responsive element binding protein 2A (DREB2A), Receptor-like kinase1(RPK1), SNF1-related protein kinase2C (SRK2C), and guard cell-expressed 7 calcium-dependent protein kinases 3 and 16 CPK3 and CPK6 (Umezawa et al., 2004; Fujita et al., 2005; Osakabe et al., 2005; Furihata et al., 2006; Mori et al., 2006). Calcineurin B-like (CBL) proteins are calcium sensors that are induced by a number of stresses (Luan et al., 2002). Gene disruption of CBL1 in Arabidopsis suggests that CBL1 may be a positive regulator of osmotic stress and a negative regulator of cold responses in plants (Cheong et al., 2003). The cellular responses to osmotic stress involve water channels (aquaporin), electrolyte transport, the accumulation of osmolytes (Booth and Lewis, 1999), and the protection of proteins and sub-cellular structures (Nuccio et al., 1999; Mager et al., 2000). Upon exposure to osmotic stress, plants exhibit a wide range of response at the molecular, cellular, and whole plant levels (Hasegawa et al., 2000). These include, morphological and developmental changes (such as life cycle, inhibition of shoot growth, and enhancement of root growth), adjustment in ion transport (such as uptake, extrusion, and sequestration of ions), and metabolic changes (such as carbon metabolism, the synthesis of compatible solutes). Some of these responses are triggered by the primary osmotic signals, whereas others may result from secondary stresses/signals caused by the primary signals. These secondary signals can be phytohormones (ABA and ethylene), ROS, and intracellular second messengers (phospholipids). Some of these secondary signals may not be confined to the primary stress sites such as the root, and their ability to move to other parts of the plant contributes to the co-ordination of whole plant responses to stress conditions (Xiong and Zhu, 2002). To cope with osmotic stress plants accumulate compatible osmolytes to function in osmotic adjustment by lowering the cellular osmotic potential to facilitate water 8 absorption. Compatible osmolytes are synthesized by virtually all organisms under hyperosmotic stress (Yancey et al., 1982). Common osmolytes include sugars, polyols, quaternary ammonium compounds, and amino acids or their derivatives. These compounds appear to have little interference in cellular function, even at high concentrations (Nuccio et al., 1999; Zhu, 2002). Many plant species accumulate glycine betaine (GB), which is a quaternary ammonium compound that is abundant mainly in the chloroplast. GB plays a vital role in adjustment and protection of thylakoid membranes and maintains photosynthetic efficiency (Bray et al., 2000). GB is known to accumulate in response to stress in various plant species (Yang et al., 2003), while some plants such as rice (Oryza sativa), tobacco (Nicotiana tabaccum), and Arabidopsis (Arabidoposis thaliana) naturally do not produce GB under stress or non-stress conditions. In these species, transgenic plants overexpressing GB-synthesizing genes exhibited increases in the production of GB and an enhancement in tolerance to salt, osmotic, cold, or high temperature stress (Rhodes and Hansen, 1993; Ashraf and Foolad, 2007). In cultured tobacco cells accumulation of sucrose, GABA, alanine, glutamate, proline, malate, citrate, GB, K + , NO 3 - , Na + , and Cl - increased with decreased external water potential (Handa et al., 1983). Water deficit in barley roots led to electrolyte leakage from the leaf due to dehydration-induced membrane injury (Kocheva et al., 2004). In yeast and mammalian systems, protein phosphorylation is central to osmotic stress signaling. In Saccharomyces cerevisiae, the osmoregulatory pathways begin with either a Src- homology3 (SH3)-domain-containing membrane protein or a two-component histidine 9 kinase, which activates a mitogen-activated protein kinase (MAPK) cascade and leads to increased osmolyte synthesis and accumulation (Gustin et al., 1998; Zhu, 2001). Plants also have several MAPKs that are activated by hyperosmotic stress. In alfalfa cells, a 46-kDa MAPK, named Stress-Inducible MAP Kinase (SIMK), is activated by another kinase called SIMKK upon hyperosmotic stress (Munnik et al., 1999, Kiegerl et al., 2000; Cardinale et al., 2002). In tobacco cells, a similar MAPK named SIPK (Salicylic-acid-induced protein kinase) is activated by hyperosmotic stress (Mikolajczyk et al., 2000). This tobacco MAPK is also activated by hypoosmotic stress, salicylic acid, or fungal elicitors (Droillard et al., 2000). Two Arabidopsis kinases, AtMPK4 and AtMPK6, are activated by cold, drought, salt, and osmotic stress suggesting a role for these two kinases in osmotic stress (Ichimura et al., 2000). Involvement of a MAPK cascade has been demonstrated in a protoplast transient assay system in response to cold and salt stress (Teige et al. 2004). A MAPKs signaling cascade is also activated in response to biotic stresses such as fungal elicitors. This provides evidence for the key position of the MAPKs at the crossroads of signaling pathways (Boudsocq and Lauriere, 2005). Transgenic tobacco plants overexpressing the NtC7 receptor-like kinase showed greater tolerance to osmotic stress (Yoshiba et al., 1997). The function of NtC7 was attributed to the regulation of compatible solutes (Yoshiba et al., 1997; Tamura et al., 2003). An Arabidopsis srf6 mutant is impaired in the accumulation of cold proteins and fails to acclimate during freezing and drought, suggesting that SRF6 is involved in both osmotic and cold stress signaling (Boyce et al., 2003). 10 CaM and CaM-binding proteins (CaMBPs) interact with target proteins implicated in osmotic stress responses. Such interactions between CaM/CaMBPs proteins have positive or negative effects on stress tolerance (Snedden and Fromm, 2001; Yang and Poovaiah, 2003). Perruc et al. (2004) identified a stress-responsive CaM- binding protein, AtCaMBP25, in Arabidopsis that is a nuclear localized, regulatory protein involved in osmotic stress signaling. Reduction in the expression of AtCaMBP25 protein resulted in enhanced tolerance to salt and osmotic stress, suggesting its role as a negative regulator of salt and osmotic stress tolerance with a less important role in cold tolerance (Perruc et al., 2004). Salinity and ion toxicity Nearly three quarters of earth is covered by salt water. Hence, it is not surprising that salt in soil affects a significant proportion of the world?s land surface. These salt- affected areas fall broadly into sodic and saline categories. Sodic soils are dominated by excess sodium on exchange sites, a high concentration of carbonate/bicarbonate anions with pH greater than 8.5 and up to 10.8, high sodium absorption ratio, and poor soil structure. Saline soils are generally dominated by sodium ions with chloride and sulphate anions, and a lower pH and sodium absorption ratio, and higher electrical conductivity. Salt-affected soils contain sufficient concentrations of soluble salts to reduce the growth of most plant species. Salt-tolerant plants have evolved to grow on these soils with a wide range of adaptations. However, most agronomically important crops are salt- sensitive (Flowers and Flowers, 2005). Salinity effects in plants are caused by ionic and osmotic stress. Ionic stress is caused when the amount of free ions disrupt cellular function. The plant deals with 11 salinity and associated ion toxicity by various detoxification processes reestablishing normal cellular functions (Denby and Gehring, 2005). Salt-tolerant plants differ from salt-sensitive ones by having a low rate of Na + and Cl - transport to leaves. The ability to compartmentalize these ions into vacuoles is critical to preventing their build-up in the cytoplasm, and thus avoiding salt toxicity (Munns, 2002). A secondary aspect of salt stress is the production of ROS, including superoxide radicals (O 2 ??), hydrogen peroxide (H 2 O 2 ), and hydroxyl radicals (OH?). ROS may be products of chloroplast and mitochondrial metabolism during stress, and can also be produced by other processes. ROS cause oxidative damage to cellular components including membrane lipids, proteins, and nucleic acids (Haliwell and Gutteridge, 1986). Plants use low molecular mass antioxidants such as ascorbic acid and reduced glutathione and employ a diverse array of enzymes such as superoxide dimutases (SOD), catalases (CAT), ascorbate peroxidases (APX), glutathione S-transferases (GST), and glutathione peroxidases (GPX) to scavenge ROS. Transgenic tobacco plants overexpressing both GST and GPX displayed improved seed germination and seedling growth under stress (Roxas et al., 1997; Apse and Blumwald, 2002). Salinity leads to ABA accumulation (Xiong and Zhu, 2003). Turgor reduction induced by the hyperosmotic component of salt stress is responsible for the increase in endogenous ABA production, particularly in root (Jia et al., 2002). Accumulation of ABA is mediated by induction of ABA biosynthesis genes and suppression of ABA degradation (Xiong and Zhu, 2003). Changes in the level of ABA regulate stomatal closure and water loss and regulate water balance by inducing genes involved in osmolytes and compatible solute biosynthesis (Hasegawa et al., 2000; Zhu, 2002). 12 Plant cells accumulate compatible solutes in response to salinity such as essential elemental ions like K + , simple sugars (mainly fructose and glucose), complex sugars (trehalose, raffinose, and fructans), quaternary aminonium derivatives (glycine betaine, ?- alanine betaine, proline betaine), and amino acids (proline, GABA, alanine) (Bohnert and Jensens, 1996; Zhu, 2001). The synthesis of these compatible osmolytes is often achieved by diversion of basic intermediary metabolites into unique biochemical reactions without disturbing intracellular metabolic pathways. These compatible solutes have the capacity to preserve the activity of enzymes with minimal effect on pH or charge balance of the cytosol or lumenal compartments of cellular organelles (Binzel et al., 1987; Bohnert and Jensens, 1996). Three genetically linked Arabidopsis loci (SOS1, SOS2 and SOS3) are components of a stress-signaling pathway, also called Salt-Overly-Sensitive signal transduction pathway that controls ion homeostasis and salt tolerance (Hasegawa et al., 2000a; Sanders, 2000; Zhu, 2000; Zhu, 2001). Genetic analysis of Na + /Li + sensitivity established that SOS1 is epistatic to SOS2 and SOS3 (Zhu, 2000; Zhu, 2002). These sos mutants also exhibit a K + deficient phenotype in media supplemented with 50 ?M K + and Ca 2+ . K + , and the K + deficiency of sos2 was suppressed with 100 mM Ca 2+ (Zhu et al., 1998). The sos1 mutant exhibits hyperosmotic sensitivity unlike sos2 and sos3. Together these results indicate that the SOS signaling pathway regulates Na + and K + homeostasis, and it is Ca 2+ activated. SOS3 encodes a Ca 2+ binding protein with sequence similarity to the regulatory B subunit of calcineurin (protein phosphatase2B) and neuronal Ca 2+ sensors (Liu and Zhu, 1998). Interaction of SOS3 with the SOS2 kinase (Liu et al., 2000) and SOS2 activation is Ca 2+ dependent (Halfter et al., 2000). 13 ACA4 is a novel member of the subfamily of N-terminal CaM-regulated Ca 2+ - ATPases, and it is localized in the membrane of Arabidopsis vacuoles. ACA4 with vacuolar H + /Ca 2+ antiporter CAX1 might adjust cytosolic calcium concentrations by filling vacuolar compartments where calcium is stored (Hirschi et al., 1996; Muir and Sanders, 1997). Overexpression of ACA4 in yeast increased salt tolerance indirectly via the reestablishment of intracellular calcium level (Geisler et al., 2000). The in planta function of SOS3 as a salt tolerance determinant is dependent on Ca 2+ binding and N- myristoylation (Ishitani et al., 2000). The SOS2 protein is a Serine/Therionine kinase (446 amino acids) and has a 267 amino acid N-terminal catalytic domain that is similar in sequence to yeast SNF1 (sucrose non-fermenting) kinase and the mammalian AMPK (AMP-activated protein kinase) (Liu et al., 2000; Zhu, 2000; Zhu, 2002). The kinase activity of SOS2 is essential for its tolerance determinant function. The C-terminal regulatory domain of SOS2 interacts with the kinase domain to cause auto-inhibition. A 21 amino acids motif in the regulatory domain of SOS2 is the site where SOS3 interacts with the kinase and is the auto-inhibitory domain of the kinase (Guo et al., 2001). Binding of SOS3 to the 21 amino acid motif blocks the SOS2 auto- inhibitory kinase activity. Deletion of the auto-inhibitory domain results in constitutive SOS2 activation, independent of SOS3, and also a threionine (168) to aspartate mutation in the activation loop of the kinase domain constitutively activates SOS2. The plasma membrane localized Na + /H + antiporter SOS1 is controlled by the SOS pathway at the transcriptional and post-transcriptional level (Guo et al., 2001; Zhu, 2002). The compartementation of Na + into vacuoles provides an efficient mechanism to minimize the toxic effect of Na + in the cytosol. The transport of Na + into vacuoles is 14 mediated by an Na + /H + antiporter that is driven by the electrochemical gradient of protons generated by the vacuolar H + -translocating enzymes, H + -ATPase and H + - pyrophosphatase (H + -PPase) (Blumwald, 1987). The overexpression of AtHK1, a vacuolar Na + /H + antiporter from Arabidopsis, in Arabidopsis resulted in transgenic plants that were able to grow in high salt concentration (Aspe et al., 1999). Overexpression of Arabidopsis plasma membrane Na + /H + antiporter SOS1 improves plant tolerance to salt by limiting Na + accumulation (Shi et al., 2003). Disruption of AtHK1 suppresses the salt sensitive phenotype of sos3-1, indicating that SOS pathway negatively controls this Na + influx system in plant cells (Rus et al., 2001). Such studies indicated that the Ca +2 and SOS signaling pathways are essential in regulation of ion homeostasis in salt tolerance (Yokoi et al., 2002; Zhu, 2002). Ultraviolet (UV) irradiation Heat-induced cellular damage was enhanced by growth in thelight compared to that in the dark (Jeong et al., 2002; Larkindale and Knight, 2002). High light results in increased ROS production potentially damaging to the photosynthetic apparatus (Niyogi, 1999). UV-A, UV-B, and blue light are important factors in perception of the light signal and in the regulation of genes in flavonoid biosynthesis, such as chalcone synthase (CHS), This may involve Ca 2+ signaling (Preisig and Moreau, 1994). The UV-A and UV-B/ blue light signaling pathways are distinct and involve reversible protein phosphorylation and de novo protein synthesis (Christie and Jenkins, 1996). An Arabidopsis succinate semialdehyde dehydrogenase mutant (ssadh) showed a rapid increase in hydrogen peroxide production when subjected to UV irradiation leading to necrosis (Bouch?` et al., 2003). Mutants deficient in ascorbate, zeaxanthinin or 15 glutathione had dramatically reduced survival under high light and heat (Larkindale et al., 2005). Such results suggest that ROS-scavenging enzymes play an important role in preventing photo-oxidative damage under a combination of temperature and high-light stress (Suzuki and Mittler, 2006). ROS and oxidative stress ROS (Reactive Oxygen Species) such as singlet oxygen ( 1 O 2 ), superoxide (O 2 ??), hydrogen peroxide (H 2 O 2 ), hydroxyl radical (OH?), and nitric oxide (NO?) are critical parts of many physiological processes, and usually exist in the cell in a balance with biochemical antioxidants. Increases in ROS resulting from exposure to environmental oxidants, toxicants, radiation, or stresses disturb cellular oxidation-reduction (redox) balance to a more oxidized state affecting biological functions. This condition is referred to as ?oxidative stress? and may be detrimental to the organism and lead to cell death (Scandalios, 2001). There are many potential sources of ROS in plants. ROS are unavoidable by-products of aerobic metabolism (Dat et al., 2000; Mittler, 2002). Other sources of ROS are glycolate oxidase in peroxisomes during photorespiration, NADPH oxidases, amine oxidases, and cell-wall-bound peroxidases. These are highly regulated processes and generate ROS leading to programmed cell death (PCD) (Hammond- Kosack and Jones, 1996; Dat et al., 2000; Grant and Loake, 2000). Primary sources of ROS production are the Mehler reaction in antenna pigments of chloroplast (Asada and Takahashi, 1987). Production of ROS is enhanced by conditions limiting CO 2 fixation, such as drought, salt, temperature stress, and a combination of these conditions with high light. Limiting CO 2 in C 3 plants activates the photorespiratory pathway resulting in generation of H 2 O 2 in peroxisomes by glycolate 16 oxidase (Mittler, 2002). Over-reduction of the electron transport chain is the main source of O 2 ?? production under specific stress conditions in mitochondria (Moller, 2001). Additional sources of ROS in plant cells include the detoxifying reactions catalyzed by cytochromes in both the cytoplasm and the endoplasmic reticulum (Urban et al., 1997). Major ROS-scavenging enzymes of plants include superoxide dimutase (SOD), ascorbate peroxidase (APX), catalase (CAT), glutathione peroxidase (GPX), and peroxiredoxin (PrxR). Together with the antioxidants ascorbic acid and glutathione (Noctor and Foyer, 1998), these enzymes provide cells with highly efficient machinery for detoxifying O 2 ?? and H 2 O 2 . The balance between SODs and the different H 2 O 2 - sacvenging enzymes in cells is considered to be crucial in determining the steady-state level of O 2 ?? and H 2 O 2 (Apel and Hirt, 2004; Mittler et al., 2004). Compatible solutes such as sorbitol, myo-inositol, and proline are effective scavengers of hydroxyl radicals and could protect cells against internally generated hydroxyl radicals (Smirnoff and Cumbes, 1989). Recent studies in Arabidopsis have uncovered some of the key components involved in the ROS signal transduction pathway in plants. It is suggested that plant cells sense ROS via at least three different mechanisms: (i) unidentified receptors proteins; (ii) oxidation-reduction-sensitive transcription factors; and (iii) direct inhibition of phosphatases by ROS (Mittler, 2002; Neill et al., 2002; Vranova et al., 2002; Apel and Hirt, 2004). H 2 O 2 activates several MAPks in Arabidopsis including MPK3, and MPK6 via a MAPK cascade (Kovtun et al., 2000). H 2 O 2 also increases expression of the Arabidopsis nucleotide diphosphate (NDP) Kinase2 (Moon et al., 2003). Overexpression of AtNDPK2 reduced the levels of H 2 O 2 and enhanced tolerance to multiple stresses 17 including cold, salt, and oxidative stress. These studies suggest ROS-induced activation of MAPK appears to be central in mediating cellular responses to multiple stresses (Mitller, 2002; Mittler et al., 2004, Suzuki and Mittler, 2006). Downstream signaling events associated with ROS sensing involve Ca 2+ and Ca 2+ - binding proteins (Kinght and Knight, 2001), the activation of G-protein (Baxter-Burrell et al., 2002), and the activation of phospholipids signaling (Anthony et al., 2004; Rentel et al., 2004). It is possible that the localization of ROS signals in specific cellular sites is similar to that of Ca 2+ signals in response to external stimuli (Coelho et al., 2002; Mittler et al., 2004). H 2 O 2 treatments induce elevation in Ca +2 (Rentel and Knight, 2004; Hu et al., 2007) and the expression of genes encoding calmodulin in plants (Desikan et al., 2001). Calmodulin stimulates stomatal closure through activation of heterotrimeric G protein and subsequent production of H 2 O 2 and intracellular Ca +2 elevations in Arabidopsis (Chen et al., 2004). Yang and Poovaiah (2002) showed that catalase regulates H 2 O 2 level in Arabidopsis stimulated by Ca +2 /CaM to maintain H 2 O 2 homeostasis. Kwon et al. (2007) have suggested that radish catalase is involved in defense against stress induced ROS. These studies indicate that CaM is involved in ROS signaling in plants. Involvement of Ca 2+ in H 2 O 2 signaling is shown by the expression of Glutathione- S-Transferase (GST1) in Arabidopsis. The magnitude of Ca 2+ response is correlated with the level of GST1 expression (Rentel and Knight, 2004). ABA induced an increase in the concentration of cytosolic Ca 2+ and expression of CAM1 and antioxidant genes such as superoxide dismutase4 (SOD4), cytosolic ascorbate peroxidase (cAPX), and glutathione 18 reductase (GR1). Hu et al. (2007) suggest that Ca 2+ -CaM is involved in ABA-induced antioxidant defense and ABA signaling in response to H 2 O 2 and ROS productions. Arabidopsis mutants deficient in SSADH enzyme showed inhibition of gamma- hydroxybutyric acid (GHB), ROS accumulations, and improvement in growth (Fait et al. 2005). This result is in agreement with Bouch? et al. (2003) showed that high level of ROS is a major cause of ssadh aberrant phenotype. Oxidation of polyunsaturated fatty acids, lipid peroxidation, and production of ROS has been used as an index for oxidative damage in plant tissues (Hodges et al., 1999, Rael et al., 2004). Biotic stress The resistance of plants to invading pathogens is accomplished by the deployment of a complex array of defense responses. These responses include rapid death of challenged cells leading to the formation of local lesions (hypersensitive response, HR), and non-specific immunity to subsequent infection by a variety of pathogens known as systemic acquired resistance (SAR). Typical rapid response to pathogen attack include ion fluxes, production of ROS, cell wall fortification, synthesis of defense compounds, and changes in gene expression (Dangel and Jones, 2001). Ca 2+ influx into the cytosol is among the earliest of these responses and likely serves as a messenger to regulate specific downstream defense pathways. CaM and CaM-like proteins (CaML) are components of plant defense signal transduction. SCaM4 and SCaM5 in soybean are rapidly induced by elicitor, and this induction is enhanced by using Ca 2+ ionophores and prevented by Ca 2+ chelators (Heo et al., 1999; Park et al., 2004). In tomato CaM transcripts increased in response to wounding or the wound signal molecule system and were constitutively high in transgenic plant overexpressing system 19 (Bergey and Ryan, 1999). Expression profile of Arabidopsis identified CaM-like protein (CaML9) as one of the earliest genes induced after bacterial infection (de-Torres et al., 2003). Modulation of Calcium (Ca +2 ) effect via Ca +2 -binding proteins Calcium (Ca 2+ ) is a universal second messenger and acts as a mediator of stimulus responses coupling regulation of diverse cellular functions (Trewavas and Malho, 1998; Knight, 2000; Allen and Schroeder, 2001). Cytosolic Ca 2+ in plants is rapidly elevated via an increased Ca 2+ influx in response to stimuli (Reddy, 2001). Transient Ca 2+ elevations are sensed by several Ca 2+ sensors or Ca 2+ -binding proteins, which contain so called EF-hand motif(s), a helix-loop-helix structure (Snedden and Fromm, 2001; Yang and Poovaiah, 2003). In Arabidopsis, it is estimated that there are around 250 putative EF-hand proteins (Day et al., 2002). CaM is one of the most conserved Ca 2+ -binding proteins in eukaryotes. Like animals, plant CaM has four EF-hands embedded within two separate globular regions in the N-and C-terminal regions separated by flexible central helix (Chin and Means, 2000). Once calcium binds to a CaM EF-hand through electrostatic interactions (Hoeflich and Ikura, 2002), t\he Ca 2+ -CaM complex interacts with GAD to change its conformation to the Ca 2+ -CaM/GAD active complex, which activates the first step of GABA shunt in plants (Snedden et al., 1995). The first evidence for Ca 2+ -stimulated GAD activity was obtained by screening a Petunia cDNA expression library with 35 S-labeled calmodulin (Baum et al., 1993) where Ca 2+ -CaM/GAD complex was activated at neutral pH (Snedden et al., 1996). Either Ca 2+ or H + elevation appears to be sufficient to activate GAD and GABA synthesis (Shelp et al., 1999). 20 Calmodulin (CaM) Calmodulins (CaM) are highly conserved, small acidic proteins present in all eukaryotes (Snedden and Fromm, 2001). CaM protein is 149-150 amino acids long with pI 3.92. The CaM prototype is comprised of two globular domains connected with a long flexible helix. Each globular domain contains a pair of intimately linked EF-hands. One EF-hand motif is composed of a specialized helix-loop-helix structure that binds one molecule of Ca 2+ (Hoeflich and Ikura, 2002). CaM binds to a short and specific amino acid sequence within target proteins. Conformation of the target protein changes after binding to CaM, resulting in activation of the target protein in response to intracellular Ca 2+ concentration (McCormack et al., 2005). To facilitate this interaction, the central region of CaM forms a random coil. The globular domains at the N- and C- terminus of the molecule can then wrap around and engulf the target peptide (Bouch?` et al., 2005). In plants there is an extended family of CaM isoforms and CaM-like proteins (CaML). In addition, CaM-like proteins are harboring an EF-hand structure like CaM, but differences in functions such as target specificity, subcellular localization, and Ca +2 affinities are known to play an important role in the regulation of physiological processes (Luan et al., 2002; Reddy et al., 2002; McCormack and Braam, 2003; McCormack et al., 2005). CaM and CaML proteins have varied EF-hands, from the three EF-hand in wheat CaM-III (Yang et al., 1996) to the six Arabidopsis TCH3 (Sistrunk et al., 1994). Calmodulin genes (CAM) show differential temporal and spatial expression in response to external stimuli such as touch, heat shock, cold, light, and pathogens and to internal stimuli such as hormones. CAM expression was elevated in embryo and reduced 21 in aleurone cells, starchy endosperm, and developed root and shoot tissues of wheat (Yang et al., 1998). In Pisum staivum L, PsCAM1 and PsCAM2 were detected in dry axes and cotyledons with highest accumulation during imbibition and prior to radicle protrusion, while PsCAM3 was detected only upon radicle protrusion (Duval et al., 2002). In Nicotiana plumbaginifolia, the level of NpCAM1 was high in response to cold and high wind, while NpCAM2 was not induced by either of the treatments (Van der Luit et al., 1999). Liu et al. (2005) demonstrated increased expression of AtCAM3 and AtCAM7 genes in Arabidopsis after heat stress at 37?C. Increased expression of CAM3 in Arabidopsis seedlings inhibits the expression of COR gene in response to cold (Townley and Knight, 2002). In soybean, SCAM4 expression was induced within 30 min of pathogen or NaCl exposure (Park et al., 2004). CaM isoforms and CAM genes in Arabidopsis: The genomic database of Arabidopsis thaliana shows 9 CAM gene loci. The first 7 calmodulins, (CaM1-CaM7), are 149 amino acids long, while CaM8 and CaM9 are predicted to be 151 amino acids long (TAIR web site). CAM genes share a high level of sequence identity to vertebrate CaM isoforms (McCorrmack and Braam, 2003). The first seven CAM genes in Arabidopsis encode for four CaM protein isoforms. The first isoform is encoded by CAM1 and CAM4, the second isoform is encoded by CAM2, CAM3 and CAM5, the third isoform is encoded by CAM6, and CAM7 encodes the fourth CaM isoform. CaM1 and CaM4 differ from CaM7 by four amino acids; CaM2, CaM3, CaM5 and CaM6 differ from CaM7 by a single amino acid (Kretsinger, 1998). The Arabidopsis CaM isoforms are highly similar but minor changes in CaM might contribute to target specificity (Bhattacharya et al., 2004). 22 CaM genes are differentially expressed in response to numerous external stimuli such as touch, heat shock, cold, light, and pathogens and to internal stimuli like hormones (Townley and Knight, 2002). CaMs are also differentially expressed in different stages as well as in different tissue and cell type (Snedden and Fromm, 2001; Yang and Poovaiah, 2003). Mechanisms of CaM action: Three CaM-activating mechanisms have been studied in animal systems (Hoeflich and Ikura, 2002). The first activation mechanism involves relieving auto-inhibition. In this mechanism, a CaM binding site is adjacent to or within an auto-inhibitory domain of an enzyme and binding of CaM induces a conformational change that displaces the pseudosubstrate inhibitory domain and allows full enzyme activity (Chin and Means, 2000). The second activation mechanism is active site remodeling. This mechanism was found in anthrax adenylyl cyclase (oedema factor) where four regions of the oedema factor form a surface that recognizes an extended conformation of CaM. Upon CaM binding, a helical domain of the oedema factor undergoes a 30? rotation away from the catalytic core, stabilizing a disordered loop and enzyme activation occurs (Drum et al., 2002). The third activation mechanism is CaM- induced dimerization. Two CaM molecules interact with two K + channel domains of the Ca 2+ -activated potassium channels upon Ca 2+ -binding. The C-terminal EF-hands mediate tethering to the channel and the N-terminal EF-hands are responsible for Ca 2+ -induced dimerization leading to channel gating (Schumacher et al., 2001). In plant systems the best studied CaM system involves the activation of glutamate decarboxylase. The C-terminal CaM-binding domain of glutamate decaroxylase (GAD) binds to a CaM and changes its conformation to GAD/CaM active complex in plant (Yap 23 et al., 2003). CaM proteins are composed of two pairs of Ca 2+ -binding sites joined by a linker domain (Chin and Means, 2002). Each binding site is composed of two helices, called the E and F helices, flanking a Ca 2+ -binding loop called the EF- hand.Conformational changes in CaM affects interactions of CaM with target proteins for modulation of the activity of the target (Snedden and Fromm, 2001; Day et al., 2002; McCormack et al., 2005). The ability of CaM to regulate the target enzyme activity resides in hydrophobic regions that are exposed after conformational change caused by Ca 2+ binding to CaM (Hoeflich and Ikura, 2002). In addition, electrostatic interactions play a role in CaM-binding, such as interaction between basic helix-loop-helix DNA- binding proteins with CaM (Onions et al., 2000). CaM is a cytosolic protein. However, it has been found in the nucleus (Van Der Luit et al., 1999), peroxisome (Yang and Poovaiah, 2002), and extracellular matrix (Ma et al., 1999). The necessity of multiple sub-cellular locales for CaM is expected because the CaM-target proteins are present in different subcellular locations. The regulatory activities of CaMs are result from its ability to modulate certain sets of enzymes. CaM has a diverse set of target proteins that are involved in a variety of processes, including ion transport (Arazi et al., 1999; Sze et al., 2000), gene regulation (Yang and Poovaiah, 2002; Bouch?` et al., 2002), cytoskeleton organization (Reddy, 2001), disease resistance (Kim et al., 2002), and metabolism and stress tolerance (Reddy, 2001; Snedden and Fromm, 2001). The CaM-binding proteins have been classified into three groups. The first group is plant-specific CaM-binding proteins (e.g., auxin responsive SAURs and pollen-specific MPCBP) (Yang and Poovaiah, 2000; Safadi et al., 2000). The second group is composed of proteins that are similar to animal homologs but have an additional 24 CaM-binding domain (e.g ,GAD, kinesin, and catalase) (Snedden and Fromm, 2001; Reddy, 2001; Yang and Poovaiah, 2002). The third group is composed of proteins that are similar to the CaM homologs in animals (e.g , CGCG protein, transcription factor, and nuclear proteins) (Bouch?` et al., 2002). Many CaM-binding proteins are plant- specific. Other Calcium-binding Proteins Calmodulin Like proteins (CML): CaM-like proteins (CML) are characterized by the presence of EF-hand Ca 2+ -binding motifs. Plants have a divergent and large number of CML proteins. The Arabidopsis genome has circa 50 CML genes. The divergent CML family is likely to have evolved from progenitor CaMs (McCormack and Braam, 2003). Many of the CMLs appear to have undergone significant subfunctionalization. Several CMLs have expression limited to a small subset of organs and have dramatic expression changes in response to a variety of biotic and abiotic stimuli (White and Broadley, 2003). Even the CMLs with closely related paralogs are generally found to display distinct expression characteristics. Thus, the different CML family members might have evolved in ways that enable their continued presence in the Arabidopsis genome to be under selective pressure (McCormack et al., 2005). Determination of the biochemical and physiological functions of this large family of potential Ca 2+ signal sensors remains an important challenge. Calcium Dependent Protein Kinase (CDPK): CDPKs form a large subfamily of protein kinases in plants and have been implicated in the control of aspects of plant growth and development (Cheng et al., 2002). The CDPKs have four well characterized conserved motifs: 1) an ATP-binding domain; 2) a catalytic domain; 3) an auto-inhibitor 25 domain is predicted in the region immediately following the kinase domain; and 4) a number of functional calcium-binding EF-hands in a C-terminal regulatory domain. CDPKs modulate other proteins during calcium signaling under various abiotic stress conditions in plants (Roberts, 1993; Xiong et al., 2002). Calcinurein B (CBL): CalcineurinB is a Ca 2+ /calmodulin-dependent serine- therionine phosphatase that plays an important role in transducing Ca 2+ -dependent signals in a variety of cell types. It is a heterodimer of two subunits: calcineurin B/CBL, the 19- kDa Ca 2+ -binding and regulatory subunit, and calcineurin A/CAL, ~61-kDa catalytic subunits. Multiple catalytic subunits of calcineurin are derived from at least two structural genes, type one (calcineurin A-alpha) and type two (calcineurin A-beta), each of which can produce additional alternatively spliced transcripts (Sharma and Deswal, 2004). CBL belongs to the family of EF-hand Ca 2+ -binding proteins. Both CBL and CaM are important for the activation of the phosphatase activity of calcineurin (Kim et al. 2007). Arabidopsis SOS3 is a CBL-like protein involved in the regulation of salt tolerance. SOS3 activates the SOS2 protein kinase, which is a regulator of SOS1 and AtNHX1, the plasma membrane, and tonoplast Na + /H + antiporter (Wang et al., 2007; Xu et al., 2006; Ma et al., 2005). Regulation of the GABA shunt The non-protein amino acid, ?-Aminobutyric acid (GABA), is a four carbon amino acid found in all organisms. GABA is a zwitterion with the amino group on the ?- carbon and exists in an unbound water soluble from in the cell (Shelp et al., 1999). GABA is synthesized from glutamate by glutamate decarboxylase (GAD) and serves as a neurotransmitter inhibitor in the nervous system of vertebrates and some invertebrates 26 (Sattelle, 1990; Bormann, 2000). GABA is synthesized from glutamate and polyamines in fungi (Kumar and Punekar, 1997). Some of the proposed functions of GABA in fungi include nutritional roles, involvement in conidiation and germination, acidogenesis (Kumar and Punekar, 1997), and as a source of nitrogen and carbon during stress (Solomon and Oliver, 2002). Glutamate decarboxylase (GAD) (EC 4.1.1.15) catalyzes irreversible decarboxylation of glutamate to GABA in the presence of cofactor pyridoxal phosphate (PLP). GAD is a cytosolic enzyme (Breitkreuz and Shelp, 1995) involved in stress- induced GABA synthesis caused by cytosolic acidosis and the consequent stimulation of GAD as a mechanism to regulate cell pH (Snedden et al., 1992). Five putative genes encoding GAD in Arabidopsis thaliana have been identified (Shelp et al., 1999). Two A. thaliana genes encoding GAD isoforms, GAD1 and GAD2 have differential tissue specific expression; GAD1 is expressed in root, whereas GAD2 is expressed in all tissues (Turano anf Fang, 1998; Zik et al., 1998; Bouch? et al., 2004). Arabidopsis GAD1 is a 57.1 KDa protein while GAD2 is a 56.1 kDa protein with 82% identity at the level of amino acids sequence (Zik et al., 1998). Both GAD1 and GAD2 are calcium/ calmodulin (Ca 2+ /CaM) regulated enzymes and their calmodulin (CaM)-binding domain differs in their potential phosphorylation sites (Turano and Fang, 1998). GAD2 lacks all four serine and therionine residues present in the CaM-binding domain of GAD1, suggesting differences in regulatory mechanisms (Yevtushenko et al., 2003; Bouch?` et al., 2004). In rice (Oryza sativa) two GAD isoforms were identified, OsGAD1 and OsGAD2, which encode polypeptides of 501 and 500 amino acids, respectively. These proteins share 69% amino acid identity. 27 OsGAD2 is not activated by Ca 2+ /CaM in vitro (Akama et al., 2001; Yevtushenko et al., 2003). At least four tobacco (Nicotiana tabacum) GAD genes are highly conserved and do not have CaM binding domains (McLean et al., 2003). GAD is a homohexameric enzyme of about 330 kDa with three types of domains, conserved catalytic domain (~250 amino acids), Pyridoxal-5`-phosphate (PLP) binding domain (~10-20 amino acids), and the C-terminal CaM-binding domain (~33-50 amino acids). Plant GAD enzymes with Ca 2+ -dependent CaM binding domain (Baum et al., 1993) differ from bacterial enzyme in having a C-terminal extension of 33 amino acids within which resides CaM binding domain with in vitro maximal activity at pH 5.8 (Ling et al., 1994; Arazi et al., 1995; Yuan and Vogel, 1998; Zik et al., 2006). GAD activity was found in extracts of various plant species to be modulated by Ca 2+ /CaM by binding to GAD CaM-binding domain and to change its conformation to GAD/CaM active complex by inducing the enzyme complex dimerization (Yuan and Vogel, 1998; Yap et al., 2003). Plant GAD is specific for L-glutamate, requires PLP, is inhibited by reagents known to react with Sylfhydryl groups, possesses a CaM-binding domain, and exhibits a sharp acidic pH optimum of 5.8 (Baum et al., 1993; Shelp et al., 1999). GAD enzyme is widely distributed among eukaryotes and prokaryotes, but its function varies in different organisms. In animals, especially in vertebrates, GAD has an important role in the central nervous system where it is responsible for the synthesis of GABA, the major inhibitory neurotransmitter. The majority of vertebrates GAD occur in two isoforms, GAD65 and GAD67, and both are active at neutral pH (Soghomonian and Martin, 1998). In Saccharomyces cerevisiae GAD is Ca 2+ /CaM dependent enzyme. It 28 has a C-terminal CaM-binding domain (~40 amino acids) and its expression is required in abatement of oxidative stress (Coleman et al., 2001). The bacterial GAD is a homohexamer of about 330 kDa, and its maximal activity is at acidic pH of 3.8-4.6. The activation of GAD in bacteria at acidic pH involves association of the hexameric complex with membranes, a process that involves the N- terminus of the enzyme (Capitani et al., 2003). Bacterial GAD has some features similar to those of the plant enzyme: it exhibits an acidic pH optimum (3.8-4.6) (Sukhareva et al., 1994), forms hexamer, and is expressed in response to environmental stresses (Blankenhorn et al., 1999; De Biase et al., 1999; Shelp et al., 2006). GAD is a cytosolic enzyme and stress-induced GABA synthesis is thought to be the result of cytosol acidosis (Shelp et al., 1992; Shelp et al., 1999). Stress factors such as heat or cold shock, which stimulates GABA level, are also known to increase cytosolic Ca 2+ levels (Kinght et al., 1991; Breitkreuz and Shelp, 1995), which stimulate the activity of Ca 2+ -CaM/GAD complex and GABA synthesis. Rapid accumulation of GABA within 1 min without change in pH suggests that either Ca 2+ or H + is sufficient to stimulate GABA synthesis (Bown and Shelp, 1997; Kinnersley and Turano, 2000; Bouch? and Fromm, 2004). GABA is converted reversibly to succinate semialdehyde (SSA) by two types of GABA-transaminases (GABA-TA) (EC 2.6.1.19) inside the mitochondrial matrix. GABA-TA uses either ?-ketoglutarate (GABA-TK) or pyruvate (GABA-TP) as amino group acceptors, producing glutamate or alanine (Shelp et al., 1999; Bouch? and Fromm, 2004). In mammals, only GABA-TK is functionally active, while both GABA-TK and GABA-TP are present in tobacco leaf and can be separated from each other using ion 29 exchange chromatography. GABA-TK and GABA-TP activities exhibit a broad pH optimum from 8 to 10 (Van Cauwenberghe et al., 2002). Knockout mutants of A. thaliana in GABA-TA gene have elevated GABA level by 100-fold in flowers suggesting that GABA-TP is the functional enzyme in vivo with specific function in pollen tube guidance (Palanivelu et al., 2003). Succinate semialdehyde dehydrogenase (SSADH) (EC 1.2.1.16) is the third enzyme in the GABA shunt pathway. SSADH irreversibly oxidizes SSA to succinate inside the mitochondria where succinate enters the TCA cycle. Plant SSADH has been localized to the mitochondria (Breitkreuz and Shelp, 1995), while SSADH in yeast is localized in the cytosol (Coleman et al., 2001). SSADH is specific for SSA oxidation and reduction of NAD + to produce NADH with optimal pH~9. Both ATP and NADH negatively regulates SSADH activity (Bouch? and Fromm, 2004). There is a locus in Arabidopsis genome encoding for SSADH (ssadh1). SSADH1 cDNA encodes a protein of 528 amino acids (56 kDa) with high homology to E. coli and human SSADH (Busch and Fromm, 1999). The recombinant Arabidopsis SSADH protein is similar to the multimeric SSADHs of other eukaryotes with the 53 kDa subunit assembled into a native enzymes of 197 kDa implying that the native enz;yme is a homotetramer similar to SSADH from barley and animals (Chambliss and Gibson, 1992; Busch and Fromm, 1999). Disruption of SSADH in Arabidopsis results in necrotic cell death in response to UV-B irradiation or heat stress apparently due to the accumulation of reactive oxygen species (Bouch? et al., 2003). ?-Hydroxybutyrate dehydrogense (GHBDH) is another enzyme responsible for the conversion of SSA to ?-hydroxybutyrate (GHB). In a mammalian brain, GHB is 30 present in small quantities relative to the GABA level (Maitre, 1997). GHBDH in Arabidopsis is a 289 amino acids polypeptide containing a NADP-binding domain, showing increased activity in response to flooding and oxygen deficiency (Breitkreuz et al, 2003). Oxygen deficiency increases the production of NADPH and reactive oxygen species (ROS) such as superoxides and hydrogen peroxide in plants (Bouch? et al., 2003). However, the oxidation of NADPH as well as NADH via the mitochondrial respiratory chain is limited. These findings suggest that GHBDH and SSADH activities in plants are regulated in a complementary fashion by Redox balance and that GHB functions in oxidative stress tolerance (Breitkreuz et al, 2003; Bouch? and Fromm, 2004). GABA serves as a source of carbon and nitrogen in bacteria (Jin et al., 1990). The integrity of GABA synthesis is essential for coping with acidic stress (Cotter et al., 2001). The GAD controls acidification of cytosol by decarboxylation of an acidic glutamate into a neutral GABA via incorporation of H + ion (Meng and Bennett, 1992; Cotter et al., 2001). Rapid accumulation of GABA in plants in response to heat, cold, drought, salinity, anoxia, and wounding has been demonstrated (Steward. 1949; Mayer et al., 1990; Breitkreuz and Shelp, 1995; Bown and Shelp, 1997; Shelp et al., 1999; Locy et al., 2000). Also, a possible role of GABA as a signaling molecule in plants has been proposed (Kinnersley and Turano, 2000; Bouch? et al., 2003; Shelp et al., 2006). GABA has been considered as a metabolite that accumulated in response to stress in plants (Mayer et al. 1990; Chung et al., 1992; Shelp et al. 1999). An increase in GABA level in wheat and barley during cold stress is caused by GAD activation and accompanied with induction of the transcription of genes encoding GABA-shunt enzymes (Mazzucotelli et al., 2006). Accumulation of GABA in response to cytosolic 31 acidification supports the involvement of GABA in pH regulation (Crawford et al., 1994). In soybean leaves the accumulation of GABA in response to low temperature likely results from activation of glutamate decarboxylase activation or lowering of protoplasmic pH (Wallace et al., 1984). Increases in GABA under hypoxia and no change in its level under drought suggests different mechanisms of GABA accumulations under these two stresses in soybean (Serraj et al., 1998). GABA shunt metabolites accumulated in rice root in response to anoxia, but its level was depleted after treatments with Ca 2+ -channel blockers and calmodulin antagonists (Reggiani et al., 1988). The accumulation of GABA under hypoxia provides an immediate substrate upon recovery from stress (Wallace et al., 1984; Shelp et al., 1995). GABA may be involved in recycling nitrogen and carbon in plant growth, development, and defense (Micallef and Shelp, 1989), and as a compatible osmolyte (Shelp et al., 1999). Polyamines may be involved in higher plant responses to salt stress, playing a key role in ion balance, anti-senescence, free radical scavenging, and membrane stabilization (Bouchereau et al., 1999). Xing et al. (2007) proposed that polyamines could perform their function through their oxidative products such as GABA under salinity in soybean. High levels of GABA in soybean root along with increases in diamine oxidase (DAO) activity in response to salt treatment, and reductions in GABA levels after treatment with aminoguanidine (AG, a specific inhibitor of diamine oxidase) during salt stress resulted in the reduction of GABA by 40%. This result suggests a contribution of polyamine oxidation to GABA leavels via DAO (Xing et al., 2007). 32 GAD1 protein is required for normal oxidative stress tolerance in yeast (Coleman et al. 2001). GAD1 protein increases resistance to H 2 O 2 and diamide oxidants, suggesting that GAD and glutamate catabolism via the GABA shunt pathway is required to buffer redox changes in the cell in response to oxidative stress in yeast. GABA accumulation has been demonstrated in response to mechanical stress and wound healing (Petrivalsky et al., 2007). GABA ingestion in oblique-banded leaf-roller larvae showed reduced growth and survival of larvae suggesting GABA as a first line of plant defense (Ramputh and Bown, 1996; Bown et al., 2006). Beside its role as a stress metabolite, GABA plays a critical role in the regulation of gene expression, including the expression of the 14-3-3 gene family in plants\(Lancien and Roberts, 2006). Recent studies with Arabidopsis suggest a possible role of GABA as a signal molecule (Bouch? and Fromm, 2003; Palanivelu et al. 2003). Pollen tubes carrying sperm cells navigate through several female tissues during reproductive development in plant (Mascarenhas, 1993). POP2 (pollen-pistil interaction2) protein in Arabidopsis appears to mediate interaction on both male and female sides (Wilhelmi and Preuss, 1996). The homozygous pop2 mutant is self-sterile even though the pollen tube can germinate and penetrate the stigma, but its guidance is abnormal (Ma, 2003). Pollen tube guidance was investigated in pop2 mutant (Palanivelu et al. 2003). POP2 encodes a GABA transaminase (GABA-TA) that degrades GABA (Van Cauwenberghe et al. 2002). Increases in the level of GABA occured along the pollen tube path in wild type, while the pop2 mutant showed a disturbed gradient of GABA. This disturbance lead to the inhibition and misguidance of pollen tubes in pop2 pistils (Bouch? et al. 2003). The 33 pop2 phenotype demonstrates the role of GABA in plant reproduction and development (Bouch? et al., 2003; Ma, 2003; Palanivelu et al., 2003; Bouch? et al., 2005). In duckweed (Lema minor), promotion of growth by GABA was associated with an increase in mineral content of treated plants in a dose dependent manner (Kinnersley and Lin, 2000), while in transgenic tobacco plants expressing a mutant GAD that lacked an auto-inhibitory CaM-binding domain, higher GABA levels, lower glutamate levels, and less stem elongation resulted (Baum et al., 1996). Since the intracellular Ca 2+ pools in these plants were unaltered, growth inhibition may result from altered GABA metabolism. Brassinosteroid (BR) promotes stem elongation and cell divisions (Clouse, 2002). BR plays an important role in coupling environmental factors, especially light, with plant growth and development (Nemhauser and Chory, 2004). Arabidopsis DWARF1 (DWF1) is responsible for an early step in brassinosteroid biosynthesis that converts 24- methylenecholesterol to Campesterol (Choe et al., 1999). DWF1 is a Ca 2+ /CaM-binding protein and this binding is critical for function. Loss of CaM binding completely abolished the function of DWF1, whereas partial loss of calmodulin binding resulted in partial dwarf phenotype in Arabidopsis (Du and Poovaiah, 2005). Several abiotic stress pathways share common elements that have potential for cross-talk. Cross talk can also occur between pathways in different organs of the plant when a systemic signal such as hydrogen peroxide moves from a stimulated cell into another tissue to elicit a response (Knight and Knight, 2001). Genetic analysis and biochemical characterization will be required to determine specificity and cross-talk in abiotic stress signaling pathways in plants (Chinnusamy et al., 2004). 34 The Ca 2+ /CaM-mediated signal network affects many aspects of plant growth, development, and response to environmental changes (Chinnusamy et al., 2004; Yang and Poovaiah, 2003). GABA accumulation in response to diverse stimuli is associated with various physiological responses and signaling cascades in plants (Bown and Shelp, 1997; Kinnersley and Turano, 2000; Bouch? and Fromm, 2004). The GABA shunt and its components (Ca 2+ /CaM, shunt enzymes, and metabolites) may have both metabolic and signaling function in plants to create a link in the chain of events leading from perception of environmental stimuli to precise physiological responses. 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Annu Rev Plant Biol 53: 247-273 Zhu J-K, Liu J, Xiong L (1998) Genetics Analysis of Salt Toerance in Arabidopsis: Evidence for a Critical Role to Potassium Nutrition. Plant Cell 10: 1181-1191 55 Zik M, Arazi T, Snedden WA, Fromm H (1998) Two isoforms of Glutamate Decaroxylase in Arabidopsis are regulated by Clacium/Calmodulin and Differ in organ Distribution. Plant Mol Biol 37: 967-975 Zik M, Fridmann-Sirkis Y, Fromm H (2006) C-terminal Residues of Plant Glutamate Decarboxylase are Required for Oligomerization of a high-molecular weight Complex and for Activation by Calcium/Calmodulin. Biochem Biophys Acta 1764:872-876 56 II. THE REGULATION OF CALMODULIN GENE mRNAs IN WILD TYPE AND CALMODULIN MUTANTS OF ARABIDOPSIS THALIANA UNDER HEAT STRESS ABSTRACT Calmodulin protein (CaM) regulates the activity of a number of key enzymes and plays important roles in cellular response to environmental changes. The A. thaliana genome contains nine calmodulin (CAM) genes. To understand the role of specific CAM genes in heat stress, the steady-state level of mRNA for the nine CAM genes in root and shoot tissues of seedlings grown at normal growth temperature (25 o C) and during heat stress at 42 o C for 2 hr was compared in T-DNA insertional mutant lines of 7 CAM genes and the wild type using gene specific primers and RT-PCR. Compared to growth at 25?C, the mRNA levels of all CAM genes were upregulated in both root and shoot after heat treatment with the notable exception of CAM5 in root and shoot, and CAM1 in shoot where the mRNA levels were reduced. At 25 o C all cam mutants showed varying levels of mRNA for corresponding CAM genes with the highest levels of CAM5 mRNA being found in cam5-1 and cam5-3. CAM5 mRNA was not observed in the cam5-4 allele which harbors a T-DNA insertion in exon II. The level of respective CAM gene mRNAs were reduced in all cam alleles compared to levels in wild type except for increased expression of CAM5 in roots and shoots of cam5-1 and cam5-3. Compared to wild type, the level of mRNA for all CAM genes varied in each cam mutant, but not in a systematic 57 way. In general any nonexonic T-DNA insertion produced a decrease in the mRNA level of the CAM2 and CAM3 genes, and the levels of CAM gene mRNAs was the same as wild type or lower in the cam1, cam4, cam5-2, and cam6-1 nonexonic mutant alleles. However the level of mRNA for all genes except CAM2 and CAM3 genes was upregulated in all cam2 and cam3 alleles and in the cam5-1 and cam5-3 alleles. During heat stress at 42 o C the level of CAM gene mRNAs was also variable between insertional mutants, but the level of CAM1 and CAM5 mRNA was consistently greater in response to heat stress in both root and shoot. These results suggest differential tissue specific expression of CAM genes in root and shoot tissues, and specific regulation of CAM mRNA levels by heat. Each of the CAM genes appears to contain noncoding regions that play regulatory roles in the interaction between CAM genes leading to changes in specific CAM gene mRNA levels in Arabidopsis. With respect to CAM gene expression only exonic mutations lead to a loss of function of CAM genes. Key words: Arabidopsis, Calmodulin, CAM, CaM, Ca +2 , Expression, Heat stress, RT-PCR. Abbreviations: CAM: Calmodulin gene CaM: Calmodulin protein Ca +2 /CaM: Calcium/Calmodulin complex RT-PCR: Reverse transcriptase- Polymerase Chain Reaction UTR: 3` or 5` gene Un-Translated Region 58 INTRODUCTION Calmodulins (CaM) are highly conserved and ubiquitous transducers of Ca +2 signals in all eukaryotes. The regulatory abilities of CaMs are revealed by their ability to modulate the activities of enzymes involved in various physiological responses including growth, gravitropism, phototropism, environmental stress, and biotic defense responses (McCormack and Braam, 2003; Yang and Poovaiah, 2003; McCormack et al. , 2005). CaM harbors no intrinsic enzyme activity of its own, but calmodulin proteins are highly conserved with 4 repeating units called EF-hands. Each EF-hand binds a single Ca +2 ion (Strynadka and James, 1989; Crivici and Ikura, 1995). Upon Ca +2 binding to the EF- hand domains, CaM alters its structure (Wriggers et al., 1998) by revealing hydrophobic surfaces that serve to interact with target proteins. In this way the Ca +2 -dependent regulation of target proteins is mediated. The genomic database of A. thaliana reveals 9 CAM gene loci. The first 7 calmodulins proteins, (CAM1-CAM7) produce proteins consisting of 149 amino acids each, While CaM8 and CaM9 are predicted to code for proteins that are 151 amino acids each (http://www.arabidopsis.org). Calmodulins are generally considered cytosolic proteins, but they have also been found in the nucleus, mitochondria, chloroplasts, the extracellular matrix, and perixosomes (Jarrett et al. , 1982; Roberts et al. , 1983; Ma et al. , 1999; Van der Luit et al. , 1999; Yang and Poovaiah, 2002). Some CAM genes show differential temporal and spatial expression in response to external stimuli such as touch, heat shock, cold, light, pathogens, and hormones (Yang et al., 1998; Duval et al. , 2002; Van der Luit et al. , 1999; Townley and Knight, 2002; Park et al. , 2004). 59 Exposure to high temperature increases the level of CaM protein in maize seedlings (Gong et al., 1997). Expression of the CaM-related touch (TCH) genes in heat shocked Arabidopsis cells has also been shown (Braam, 1992). In maize, HSP70 binds CaM inside the cytoplasm in a Ca +2 -dependent manner (Sun et al, 2000). Ca +2 /CaM complexes activate the binding of heat shock factor (HSF) to DNA resulting in the induction of HSP (Li et al. , 2004; Liu et al. , 2007), and increases in the intracellular Ca +2 level during heat stress have been shown to result in the induction of CAM gene expression and the synthesis of HSP under heat stress in wheat (Fan et al., 2000; Liu et al., 2003). Liu et al. (2005) demonstrated increased expression of CAM3 and CAM7 genes in Arabidopsis after heat stress at 37?C. To elucidate the role of calmodulin isoforms in tolerance to heat stress in plants, we have examined the steady state levels of the nine Arabidopsis CAM genes in wild type and in a series of insertion mutations in seven of the 9 CAM genes in Arabidopsis seedlings in response to heat stress. MATERIALS AND METHODS Plant material and growth conditions The wild type and mutant lines of Arabidopsis (Arabidopsis thaliana Ecotype Columbia) bearing T-DNA insertions in various calmodulin genes (cam) used in this study are listed in Table 1. Wild type, cam1 and cam4 seeds were obtained from Arabidopsis Biological Research Stock Center, Ohio State University, Columbus, OH, while seeds of all other mutants were obtained from Dr. Janet Braam, Rice University, Texas. Homozygous seed stocks were propagated for this study. Seeds were surface sterilized with bleach (v/v, 6% sodium hypochlorite) for 10 min followed by five washes 60 with sterile distilled water. Seeds were plated in Petri dishes on sterile 1X Murashige and Skoog (1962) medium (pH 5. 7) supplemented with 2% (w/v) sucrose, solidified with 1. 2% (w/v) agar, Seedlings were grown under continuous illumination (40?mol m -2 s -1 ) provided by cool white fluorescent lamps at 25 ?C. Heat treatment Two-week-old Arabidopsis seedlings of wild type and cam mutant lines were grown at 25?C (control) and at 42?C for 2 hr (heat treated) (Locy et al. , 2000). Root and shoot tissues of all control and heat treated samples were harvested separately, frozen in liquid nitrogen (LN 2 ) and used for total RNA extraction. Three replicates of each control or treated sample consisting of 50 seedlings each were collected. RNA extraction Total RNA from frozen samples was extracted using the RNeasy Plant Mini kit (Qiagen,) according to manufacturer instructions. Total RNA was suspended in RNase- free water. RNA concentrations were determined be their absorbance A 260 using GeneQuantproRNA/DNA calculator (New England Biolab), and the integrity of RNA was determined after separation of RNA on a 1% (w/v) agarose gel after electrophoresis and staining with ethidium bromide. Estimation of steady state levels of mRNA by reverse transcriptase-PCR Gene specific primers for each of the nine A. thaliana calmodulin genes (CAM) were designed for RT-PCR analysis of steady state mRNA levels as shown in Table 2. One-step Reverse Transcriptase-PCR (RT-PCR) reaction was performed using primer pairs, SuperScript TM III One-step RT-PCR system with platinum? Taq DNA polymerase 61 (Invitrogen, Inc.) according to manufacturer?s instruction, and 1?g total RNA as determined above. Primers were used at a concentration of 0.2?M in a reaction volume of 15?L. The annealing temperatures were calculated on the basis of the Tm values of each primer pair (Table 2). The following temperature program was used for RT-PCR in a PTC-100 thermocycler (MJ Research): 30 min cDNA synthesis at 50 o C, 2 min dentauration at 94C, followed by 40 cycles each of dentauration at 94?C for 1 min, primer annealing for 1 min at 45 o C to 55?C (based on the Tm of the primers), and extension at 68?C for 1 min. The program ended with a 5 min final extension at 68?C. A primer pair for the CBP20 protein (Mar?n-Vinader et al. 2006) was used as an internal control to normalize RNA concentrations in each reaction tube. RT-PCR amplification products were separated on 1% agarose gels and stained with ethidium bromide. The expression level of each calmodulin (CAM) gene in all cam mutant lines and the wild type were determined by measuring the fluorescence of RT-PCR amplicon bands using the Gel Documentation and Image Analysis System (Alpha Innotech, CA). The amount of fluorescence in a cDNA amplicon representing specific RNA in each sample was used as a measure of the level of expression. The level of RNA in each tube was normalized with respect to the fluorescence of the CBP20 internal control. The background fluorescence on agarose gels was subtracted from the fluorescence value of each DNA band. Each determination represents an average of three different biological replicates with standard deviation. Data was represented as Log 2 Fold change in the level of CAM gene expression after heat treatment compared to the normal growth (Control). 62 RESULTS AND DISCUSSION Comparative steady state levels of CAM mRNAs in heat treated Arabidopsis seedlings The steady state level of mRNA for the 9 CAM genes was estimated in root and shoot tissues of wild type Arabidopsis at 25 o C (Figure 1A) using RT-PCR. RNA levels of the CAM genes in root and shoot tissue was approximately equal for all genes except CAM2 where the level in shoot was only 60% of the level in root. The levels of individual CAM mRNAs in wild type at 25 o C were variable. The lowest mRNA level was observed for CAM7 and CAM1 in both root and shoot tissues. While the highest mRNA levels were observed for CAM2 in root, and CAM3 in root or shoot tissues. All other CAM genes showed roughly equivalent mRNA levels. The change in CAM mRNA levels after a 2 hr heat treatment at 42 o C was also determined (Figure 1B). All CAM genes showed a two to eight-fold increase in expression after heat treatment in root and shoot tissues with the notable exception of CAM1 in shoot and CAM5 in both root and shoot (Figure 1B) which decreased in expression by as much as two to over 50-fold. The level of CAM2, CAM3, CAM7, and CAM8 mRNAs in root was higher than the level of mRNAs for CAM1, CAM4, CAM6, and CAM9 after heat treatment (Figure 1B). CAM5 mRNA was not detected in root tissue after heat treatment. The highest levels of CAM2, CAM3, CAM8, and CAM9 mRNAs were observed in shoot followed by the levels of mRNAs for the CAM7, CAM4 and CAM6 genes, respectively. The level of CAM5 mRNA in shoot was greatly reduced under heat as was the level of CAM1 mRNA (Figure 1B). It can be concluded that heat stress modulates tissue-specific expression of CAM genes in Arabidopsis. 63 Members of the gene family are usually differentially regulated to accomplish specific functions in spatial or temporal manner. Differential expression of CAM genes in response to environmental stimuli such as touch, heat shock, cold, light, pathogens and hormones have been shown in plants (Perera and Zielinski, 1992; Botella and Arteca, 1994; Kudla et al. , 1999; Heo et al. , 1999; Yang and Poovaiah; 2000; Yamakawa et al. , 2001; Pandey et al. , 2002; Ali et al. , 2003; Park et al. , 2004; Takabatake et al. , 2007). Accumulation of CaM protein during heat stress has been shown in maize seedlings (Gong et al. , 1997; Sun et al. , 2000). Differential induction of specific CAM genes by high temperature was demonstrated in wheat (Fan et al. , 2000; Liu et al. , 2003) and rice (Pheanopas et al. , 2005). In Arabidopsis all CAM genes including the divergent forms, CAM8 and CAM9, are ubiquitously expressed (Zielinski, 2001). Expression level of CAM3 and CAM7 mRNA increased in Arabidopsis in response to a 37 o C heat treatment (Liu et al. 2005). This observation was supported by the results presented here that showed that at 42 ? C (still a nonlethal temperature) CAM3 and CAM7 were among those CAM genes that increased in expression (Figure 1A). The level of CAM2 and CAM5 mRNA was reduced in response to heat stress at 37 o C (Liu et al. 2005). For CAM5 this observation was also supported by the observations presented here. However, the CAM5 expression was measured as total plant tissue expression under heat stress at 37C. Expression of CAM genes in the corresponding cam mutants To potentially elucidate a role of specific CAM genes and/or specific isoform(s) of CaM in heat stress, we have used a set of available T-DNA insertion mutants in the first 7 CAM genes in Arabidopsis (see Table 1). It should be noted that all cam mutants have 64 T-DNA insertions in either the 5?UTR, 3? UTR or in the single intron of a CAM gene, except cam5-4, which contains an insertion in the cam5 exon II. The level of mRNA observed for each of the CAM genes was determined in root and shoot tissues in corresponding cam mutants grown at 25 o C (Figure 2A). The steady state levels of CAM gene mRNAs in corresponding cam mutants were variously affected with the highest levels of CAM5 mRNA being found in root and shoot tissues of cam5-1 and cam5-3. CAM5 expression was not detected in the cam5-4 mutant. The level of mRNA of each of the CAM genes in the corresponding cam mutants was compared to its expression in wild type, and the increase in mRNA levels is shown in Figure 2B. The level of the CAM5 gene mRNA in various cam5 alleles was variable depending upon location of insertion in the CAM5 gene. In the cam5-4 allele (bearing the exonII insertion) mRNA was not detected in root or shoot tissues consistent with the cam5-4 allele being a true knock-out mutation. A higher than wild type level of CAM5 mRNA in the cam5-1 allele (bearing an insertion in the 3`UTR) and in the cam5-3 allele (bearing an insertion in 5`UTR) was seen in both root and shoot tissues, while the cam5-2 allele (bearing a different 3`UTR insertion) showed a 15-fold reduction in the expression of CAM5 in root and an 8-fold reduction in shoot tissues. The level of CAM7 gene mRNA was increased in the shoot of cam7-1 compared to its level in wild type which also bears a 3`UTR insertion. The level of mRNA for all other CAM genes was reduced in the corresponding cam mutants compared to their levels in wild type (Figure 2B). Thus this series of non- exonic T-DNA insertions did not completely eliminate CAM expression as anticipated, although insertions in several of the UTRs and introns dramatically reduced 65 corresponding mRNA levels in some tissues. Only the cam5-4 exonic insertion allele completely eliminated CAM5 expression in both root and shoot tissues. Depending upon the location of insertion, the level of the respective CAM gene was variable, but the expression of most of the CAM genes was reduced in cam mutants compared to the respective level in wild type, with the exception of some cam5 alleles and cam7-1 allele where mutations produced tissue specific increases in mRNA level. Alleles of CAM genes have pleiotrophic effects on the expression of the other CAM genes It is striking that the steady state level of most CAM gene mRNAs in all of the T- DNA insertional alleles of the Arabidopsis CAM genes is dramatically lower than the mRNA levels in wild type (Figure 3) except for the cam2, cam3 alleles, and cam5-1and cam5-3 alleles. This particularly applies to the level of CAM1 and CAM7 mRNA levels which demonstrate the highest fold increases observed as a result of T-DNA insertion in a CAM gene. Additionally, modest increases in the mRNA levels for CAM4, CAM5, and CAM6 are shown in most of the mutant alleles listed above. The levels of CAM8 and CAM9 mRNA in various alleles are more variable, particularly in root, but in shoots these CAM genes follow the same trend as the other CAM genes except for the cam2-1 and cam3-3 alleles. In contrast to the above T-DNA insertion alleles, the cam1, cam4, cam5-2, cam5- 4, cam6-1, and cam7-1 insertion alleles all demonstrate lower or no change in the overall expression of all of the CAM genes except for slight increases in the level of mRNA for CAM7 in the cam4, cam5-2 , and cam7-1 alleles in shoots and for the CAM1 gene in the 66 cam5-3 allele and cam7-1 allele in roots. The cam5-3 allele also demonstrates higher levels of mRNA for the CAM6 and CAM7 gene in roots. These observations suggest that there are complex interactions involving either the expression of CAM genes or the processing and stability of CAM gene mRNAs. It is clear that insertion in any CAM gene that was examined in this study lowers the steady state mRNA levels of the CAM2 and CAM3 genes. In most of the alleles the levels of CAM8 and CAM9 mRNA were similarly affected. While there are a few allele septic effects in root versus shoot, the general pattern of increased mRNA levels of the CAM1, CAM4, CAM5, CAM6 and CAM7 genes and decreased mRNA levels in CAM2, CAM3, CAM8 and CAM9 in both root and shoot in those alleles which did not lead to lower expression of all CAM genes suggests that there is a critical regulatory function for CAM genes located in the 3?-UTR of these alleles, since all of the alleles which show some upregulation are insertions in the 3?-UTR while almost all of the other strictly down regulated CAM mRNA level alleles involve insertions in regions other than the 3?-UTR. It is also clear that the complete loss of function of the CAM5 gene (cam5-4 exonic insertion allele), leads to the down regulation of mRNAs of nearly all of the CAM genes in both root and shoot tissues. Other alleles also lead to such strong downregulation of CAM gene mRNA levels (cam1, cam4, cam5-2, and cam6-1). However, none of these alleles demonstrate a strong phenotype at 25 C. Expression of nine CAM genes in Arabidopsis cam mutants after heat treatment To better understand how each of the nine CAM genes are regulated by heat and whether CAM genes interact during heat stress, we determined the steady state mRNA levels for the nine CAM genes in root and shoot tissues of all of the cam mutant alleles 67 after heat treatment and compared this level to the level in plants grown at 25 C. The results are shown in Figure 4 (color plot diagram) as the fold increase in mRNA level at 42 o C compared to the level in plants grown at 25 o C. In wild type the mRNA levels of all nine CAM genes are upregulated between 1.8 and 8 fold by heat treatment in both root and shoot except for CAM5 mRNA in roots which is 1.6-fold downregulated, and CAM1 and CAM5 mRNAs in shoots which are downregulated 3.1-fold and 52-fold respectively. Although variable from mutant to mutant, it is apparent that none of the mutant alleles examined in this study show an expression pattern during heat-stress that is similar to the wild type. Most notable among the observe differences is the level of CAM5 mRNA induced by heat stress in all of the cam2, cam3, cam4, and cam5 mutant alleles (except the exon insertion cam5-4) where instead of decreasing as in wild type the mRNA level for the CAM5 gene increases by as much as much as 420-fold. There is no detectable CAM5 mRNA found in the cam5-4 allele, but the expression of all other CAM genes is downregulated during heat-stress by as much as 2- to 129-fold in roots, and 2- to 325-fold in shoots. The mutant alleles which are most distinct from this wild type pattern of heat- stress CAM gene mRNA levels and which share the down regulation of CAM genes with the cam5-4 allele are: cam1, where the mRNAs for all CAM genes are significantly downregulated in roots and shoots except that CAM1 and CAM5 mRNAs are upregulated in shoots; cam6-1, where all CAM gene mRNAs are downregulated except CAM3 mRNA in root and CAM5 mRNA in shoot; and CAM7-1, where all CAM gene mRNAs are downregulated except CAM5 mRNA in shoot which is upregulated by 2-fold. 68 The expression of nine Arabidopsis CAM genes in various cam mutants presented here provides evidence for complex interactions between CAM gene leading to the regulation of the mRNA levels of other CAM loci and the modulation of such interactions by heat. This finding is unexpected as T-DNA insertions in 3-UTRs, 5-UTRs, or introns often lead to loss of gene function. The data presented here elucidate a role of the 3?- UTR in many of the mutant alleles in the regulation of calmodulin mRNAs by heat stress. Additionally, both the expression of CAM5 during heat stress and the regulation of CAM5 gene mRNA by other CAM genes suggests the importance of this calmodulin gene during heat stress. Calmodulins are involved in the regulation of many proteins under stress (Ma et al., 2004; Bouche et al. , 2005). Arabidopsis PP7 (Ser/Ther Phosphatase) interaction with CaM regulates expression of HSP genes during heat stress (Liu et al. , 2007). Functional and regulatory interaction between phyA and phyB in light signaling was demonstrated in Arabidopsis (Casal and Mazella, 1998; Hennig et al., 1999; Neff and Chory, 1999). Because of a central role for CaM in plants under stress, it may be necessary to compensate the amount of CaM in cam mutants as an evolutionary necessity for plants to cope with continuously changing environmental cues. 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Planta 214: 446-455 A. 0 0.5 1 1.5 2 2.5 C AM 1 CAM 2 CAM 3 CAM 4 CA M 5 C AM 6 CAM 7 CAM 8 CAM 9 Relat i v e amo unt of mRN A Root Shoot B. -7 -6 -5 -4 -3 -2 -1 0 1 2 3 4 CA M1 CAM 2 C AM 3 CAM 4 C AM 5 CAM 6 CAM 7 CAM 8 CA M9 Lo g 2 f o l d in crease in expressi on Root Shoot Figure 1. Estimates of normalized levels of steady state mRNA of nine CAM genes in root and shoot tissues of wild type A. thaliana grown at 25 o C (control) (A), and fold increase in the levels of nine CAM genes nRNAs in root and shoot tissues of wild type A. thaliana after 2 hr heat treatment at 42?C compared to growth at 25?C (B). Total RNA from root and shoot tissues of 2 week-old seedlings was used to perform RT-PCR according to procedure outlined in Materials and Methods. CPB20 was used as an internal standard, and all mRNA levels were normalized to the save level of CPB20 mRNA. The expression level of each CAM gene was calculated as outlined in materials and methods. Error bars represent SD of three replicate samples from which RNA was isolated. 73 A. 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 ca m 1 ca m 2- 1 ca m 2- 2 ca m 3- 2 ca m 3- 3 ca m 4 ca m 5- 1 ca m 5- 2 ca m 5- 3 ca m5 -4 ca m 6- 1 ca m 7- 1 R e lat i ve amou nt of mR N A Root Shoot B. -5 -4 -3 -2 -1 0 1 2 3 ca m 1 ca m 2- 1 ca m 2- 2 ca m 3- 2 ca m 3- 3 ca m 4 ca m 5- 1 ca m 5- 2 ca m 5- 3 ca m 5- 4 ca m 6- 1 ca m 7- 1 Lo g 2 fo ld i n creas e i n ex p r ess i o n comp ared to WT a t 25 o C Root Shoot Figure 2. Extimates of normalized steady state mRNA levels of nine CAM genes in root and shoot tissues of the corresponding cam mutants of A. thaliana at 25?C (A) and the fold increase in the expression of CAM genes in root and shoot tissues of corresponding cam mutants compared to its expression in wild type at 25?C (B). Total RNA from root and shoot tissues of 2 week-old control and heat-treated seedlings were used to perform RT-PCR using gene specific primers as described in Materials and Mehtods. CPB20 was used as an internal control. 74 Figure 3. The fold change of the nine Arabidopsis CAM gene mRNAs in root and shoot tissues of each of the cam alleles of seven of the CAM genes compared to wild type. The color scale show indicates the colors corresponding to a decrease in mRNA level in the mutant relative to wild type (green) or an increase in mRNA level in mutant relative to wild type (red). mRNA levels were measured as described in Materials and methods using CPB20 as an internal standard. 75 76 vel in mutant relative to wild type (red). mRNA levels were measured as described in aterials and methods using CPB20 as an internal standard. Figure 4 . The fold change in the level of mRNA for the nine Arabidopsis CAM gene mRNAs in root and shoot tissues of each of the cam alleles of seven of the CAM genes and wild type after 2 hr heat treatment at 42?C compared to growth at 25?C. The color scale show indicates the colors corresponding to a decrease in mRNA level in the mutant relative to wild type (green) or an increase in mRNA le M 77 es were obtained from Dr. Janet Braam, Rice University, except cam1 and cam4 seeds ere obtained from the Arabidopsis Biological Research Stock Center, Ohio State University, olumbus. Table 1. Mutant lines with T-DNA insertion in calmodulin genes of A. thaliana used in this study. All seeds lin w C Mutant Insertion Location SALK Line # cam1 3`UTR SALK_107507 cam2-1 Intron SALK_066990 cam2-2 Intron SALK_089283 cam3-2 5`UTR SALK_075669 cam3-3 3`UTR SALK_042391 cam4 5`UTR SALK_149142 cam5-1 3`UTR SALK_007371 cam5-2 3`UTR SALK_073480 cam5-3 5`UTR SALK_138758 cam5-4 Exon II SALK_027181 cam6-1 3`UTR SALK_071609 cam7-1 3`UTR SALK_074336 78 able 2. Nucleotide sequence of primers for CAM genes and CBP20 gene used as an internal i ene l us T standard for RT-PCR amplificat on. CAM gene G oc Primer sequences CAM1 A F: 5`AAAGAGAGCGACTCTGAAT 3` t5g37780 t2g41110 t3g56800 t1g66410 t2g27030 t3g43810 t4g14640 CC 3` t3g51920 R: 5`CGACCGAAAAAAATCATTG 3` BP20 At5g44200 F: 5`ATGGCTTCTTTGTTCAAGGAGC 3? R: 5`TTAAGATCTTCTCTTCCGATCATC3? R: 5`AAAGCAACAAAATGTGGGTA3` CAM2 A F: 5`AGCTGATCTTCAGGACTTGA 3` R: 5`TCAGACCCTATTGGCATAAA 3` CAM3 A F: 5`TTTCCAGCAGAGACACTTTT 3` R: 5`CGCAAAAACATTCGCGTCTT 3` CAM4 A F: 5`CTTCTCTGAAACGAAAAACC 3` R: 5`CCGAAATCTAAACAGCCATT 3` CAM5 A F: 5`ACACACACCAACGTTGATT 3` R: 5`CCTGGTACTTCACATAATAT 3` CAM6 At5g21274 F: 5`GAGAGGCAAATAATATATTC 3` R: 5`AGGGCATTGCTTTAAGATAA 3` CAM7 A F: 5`AAAGGAGAAGGAACATTCAC 3` R: 5`AAAGCAAATGATGAGTGCAA 3` CAM8 A F: 5`CAGAGATTTAAGCACTTCAAA R: 5CACGAAGTAGTAACAACTAACAAGG3` CAM9 A F: 5`CATGAGTATTGACCTTGACCG 3` C 79 III. SENSITIVITY OF CALMODULIN MUTANTS OF ARABIDOPSIS THALIANA TO HIGH TEMRERATURE EXPOSURE ABSTRACT Arabidopsis mutants with T-DNA insertion in seven calmodulin genes (CAM) were used to determine the role of specific CAM genes in tolerance of plants to high temperature. Arabidopsis calmodulin mutant alleles (cam) were screened for survival, seed germination, heat-induced oxidative damage, and GABA shunt metabolite levels. Exposure of cam seedlings to 42 o C for 2 hr showed reduction in seed germination and survival of seedlings in cam5-4 and cam6-1 mutants compared to wild type (WT). Oxidative damage by heat measured as the level of malonaldehyde (MDA) was detected in root and shoot of most cam mutants with highest levels in cam5-4 and cam6-1. The MDA level was reduced slightly in the root and shoot of cam1, the root of cam2-2, and the shoot of cam4. GABA shunt metabolites in both root and shoot were generally elevated after 30 min and 1 hr treatment at 42 o C, and increased substantially after 2 hr at 42 o C comparing to the control (no treatment). GABA and glutamate levels were increased significantly more than alanine in root and shoot tissues of all cam mutants and wild type compared to the control. After 30 min, the level of GABA and glutamate increased 0.2-1.5-fold and 0.2-2.5-fold respectively in all cam mutants and in wild type root and shoot tissues compared to the control. The shoot of cam1 and cam7-1 mutants 80 st that regulation by factors other than CaM is likely, and that this factor may relate to the regulation of GAD by intracellular pH and/or metabolite partitioning under heat stress. were exceptions to this trend. After 1 hr, the level of GABA and glutamate increased 0.7-3-fold and 1-4.3-fold respectively in all cam mutants and wild type root and shoot tissues. The root of cam5-4 was an exception to this trend. After 2 hr at 42 o C, GABA levels increased in all cam mutants and WT up to 5-fold in the root cam7-1 and in the shoot of cam2-2, cam5-1, cam5-2, and cam5-4. The glutamate level increased 3-11-fold in all cam mutants and WT with the highest levels observed in root of cam5-4 and cam6- 1 (11 fold) and the shoot of cam1 (3 fold). Alanine levels showed significant decreases in all cam mutants and in WT for 30 and 60 min of heat stress. Initial general elevation in GABA shunt metabolites after 30 min heat treatment in cam mutants suggests regulation of GABA level by CaM. A significant increase in GABA levels in cam mutants after 2 hr of heat treatment was observed. These data sugge 81 rabidopsis, Calmodulin, CAM, CaM, Ca +2 , GABA, GAD, Heat stress, Metabolism, n complex oxylse ADP + : Nicotinamide adenine dinucleotide phosphate BARS: Thiobarbiturate reactive substances Key words: A Thermotolerance Abbreviations: CAM: Calmodulin gene CaM: Calmodulin protein Ca +2 /CaM: Calcium/Calmoduli GAD: Glutamate Decarb GABA: ?-Aminobutyric Acid MDA: Malonaledehyde NAD + : Nicotinamide adenine dinucleotide N T 82 INTRO and Vierling, 2000, 2001; Hong et al., 2003) and Zea mays (Nieto- Sotelo pea cells (Mayer et al., 1990) a succinate semialdehyde dehydr DUCTION Plants have evolved strategies to prevent and repair damage caused by rapid changes in the surrounding temperature. Acquired thermotolerance is one such strategy and involves production of heat shock proteins (Vierling, 1991; Larkindale et al., 2005). Heat shock proteins act as molecular chaperons to protect cellular proteins against heat- induced dentauration and to facilitate refolding of heat-damaged proteins (Boston et al., 1996). The Hsp100 family proteins are essentials for the acquisition of thermotolerance in Arabidopsis (Hong et al., 1999). Activation of GABA shunt pathway in response to various stresses has been shown (Mayer et al., 1990; Bown and Shelp, 1997; Shelp et al., 1999; Locy et al., 2000). A rapid increase in GABA level in response heat in cultured cow nd Arabidopsis (Locy et al., 2000), have been reported. GABA is synthesized by a cytosol-localized Ca 2+ -calmnodulin-dependent glutamate decarboxylase (GAD) protein (Baum et al., 1993; Snedden et al., 1996). Following irreversible decarboxylation from glutamate, GABA is metabolized to succinate semialdehyde bypassing two steps in the TCA cycle. This pathway is referred to as the GABA shunt. In plants, the last 2 steps of the GABA shunt take place in mitochondria. These steps are catalyzed by a GABA-transaminase using either ?- ketoglutarate or pyruvate as amino group acceptor and ogenase (Shelp et al., 1999, Bouche and Fromm, 2004). Ca 2+ /CaM provides a possible level of control for the activation of the GAD enzyme activity (Shelp et al., 1997; Bouche and Fromm, 2004) since this protein is 83 Meyer et al., 2006) may play a role in intercellular or intracel mination, oxidative damage, and changes in the levels of GABA shunt metabolites. Plant m demonstrates a calmodulin binding domain (Zik et al., 1998). Such activation could account for the control of the GABA shunt pathway and the concomitant accumulation of GABA during stress. Possible roles of the GABA shunt in processes such as carbon- nitrogen balance, cytosolic pH regulation, protection from damage by reactive oxygen, defense against insects, and plant growth and development have been suggested (Shelp et al., 1997; Kinnersley and Turano, 2000; Bouche and Fromm, 2004). A proline transporter (AtProT2 and LeProT1) is able to recognize GABA as a substrate (Schwacke et al., 1999; Grallath et al., 2005), and the presence of an Arabidopsis high affinity GABA transporter (AtGAT1) ( lular GABA transport. We have used calmodulin T-DNA insertion mutants (cam) of Arabidopsis to examine the role of specific CAM genes in high temperature tolerance with respect to survival, ger MATERIALS AND METHODS aterial and growth conditions The wild type and mutant lines of Arabidopsis (Arabidopsis thaliana Ecotype Columbia) bearing T-DNA insertions in various calmodulin genes (cam) used in this study are listed in Table 1. Wild type, cam1 and cam4 seeds were obtained from Arabidopsis Biological Research Stock Center, Ohio State University, Columbus, OH, while seeds of all other mutants were obtained from Dr. Janet Braam, Rice University, Texas. Homozygous seed stocks were propagated for this study. Seeds were surface 84 uous illumination (40?mol m -2 s -1 ) ite fluorescent lamps at 25 ?C. Seed g compared to WT. The average of three replicate each treatment. Seedlin An average of 3 replicates with fifty seeds on each plate were used. Oxidat sterilized with bleach (v/v, 6% sodium hypochlorite) for 10 min followed by five washes with sterile distilled water. Seeds were plated in Petri dishes on sterile 1X Murashige and Skoog (1962) medium (pH 5. 7) supplemented with 2% (w/v) sucrose, solidified with 1. 2% (w/v) agar, Seedlings were grown under contin provided by cool wh ermination Surface sterilized seeds of WT and each of the cam mutants were suspended in 500 ?L sterile distilled water and incubated at 42?C in a water bath block for 2 hr. Each tube sample contained 50 seeds. Immediately after heat treatment seeds were plated on square Petri dishes containing media as described above and allowed to grow vertically under continuous light (40?mol m -2 s -1 ) at 25 ?C for 7 days. Emergence of radicle from germinating seeds was recorded and compared to control without heat treatment. Percent germination of each cam mutant was plates was used for g survival Two week old seedlings of WT and cam mutants grown under continuous light (40?mol m -2 s -1 ) at 25 ?C were incubated at 42?C 30 min, 1 hr, and 2 hr in a growth chamber. Heat treated seedlings were transferred to growth chambers for 5 days at 25 o C under continuous light before scoring for the survival. Green and growing seedlings were scored as surviving. ive damage Two sets of two week old seedlings of WT and cam mutants were heat treated at 42?C for 2 hr. One set was used immediately, while the second set of seedlings was 85 1968). Three plates w etermined as nmol/mgFW from a standard curve of MDA. GABA of metabolites. Three plates w placed under continuous light at 25 o C for a 2 day recovery period. The level of malonaldehyce (MDA) in root and shoot tissues of seedlings exposed to heat and after recovery was determined using the TBARS assay (Heath and Packer, ith 50 seeds each were used in each replicate of each treatment. Root and shoot tissues after heat treatment were separated and frozen in liquid nitrogen. Tissue (0.50g) was ground in a 1.5mL microfuge tube, and 0.5mL of 0.5% (w/v) thiobarbituric acid in 20% (w/v) trichloroacetic acid and 0.5mL 175 mM NaCl in 50 mM Tris-HCl, pH 8 was added to the ground tissues. Tubes were heated to 90?C for 25 min. The supernatant was collected after the samples were centrifuged for 20 min at full speed. The absorbance of the supernatant was measured at 532 nm. The level of malonaldehyde (MDA) was d -shunt metabolites WT and cam seedlings of Arabidopsis grown at 25 o C for two week in continuous light were exposed to 42?C in a growth chamber for 30 min, 1 hr, and 2 hr. Root and shoot tissues were separated and used for extraction for analysis ith 50 seedlings on each plate were used for each sample. Metabolites were extracted according to Zhang and Bown (1997) with the following modifications. Separately harvested root and shoot tissues were ground in 1.5 mL microfuge tubes under LN 2 until a fine powder was obtained. To each tube 400?L methanol was added, and the samples were mixed for 10 min. Liquid from samples were removed by vacuum drying. Five hundred ?L of 70 mM lanthanum chloride was added to each tube: the tubes were mixed for 15min, and subsequently centrifuged at full speed for 5min. Supernatants was removed to new tubes and mixed with 160?L of 1 M KOH. 86 as transferred to a new tube and used to determine the quantity of spec SA). The level GABA nmol/mgFW was determined using a . The level of alanine as nmol/mgFW was determined using an NADH dehydrogenase enzyme suspension (Sigma-Aldrich). The level of glutamate was After 10min mixing, tubes were centrifuged at full speed for 5min. The supernatant containing metabolites w ific metabolites. GABA (?-aminobutyric acid) was measured according to Zhang and Bown (1997) with the following modifications. The reaction mixture contained 50?L of sample extract, 14 ?L of 4 mM NADP + , 19 ?L of 0.5 M potassium pyrophosphate, pH (8.6), 10 ?L of (2 u/?L) GABASE enzyme (GABASE enzyme was suspended in 0.1 M potassium pyrophosphate, pH 7.2 containing 12.5% Glycerol and 5 mM ?-marcaptoethanol), and 10 ?L of ?-ketoglutarate. The change in absorbance at 340 nm after addition of ?- ketoglutarate was recorded after 90 min incubation at 25 o C using the microplate reader (BioTek power wave, Max200R, U n NADPH standard curve. Alanine was measured according to Bergmeyer (1983) with the following modifications: the reaction contained 180 ?L of 0.05 M Na-carbonate buffer pH (10), 7 ?L of 30 mM ?-NAD + , 10 ?L of sample extract, and 0.3 units of alanine dehydrogenase (Sigma-Aldrich) enzyme suspension. Changes in absorbance at 340 nm after addition of alanine dehydrogenase was recorded after 60 min incubation at 25 o C using a microplate reader (make and model) standard curve. Glutamate was measured according to Bergmeyer (1983) with the following modifications: the deamination reaction contained 180 ?L of 0.1 M Tris-HCl, pH 8.3, 8 ?L of 7.5 mM ?-NAD + , 10 ?L of sample extract, and 0.8u nits of glutamate 87 determined at 340nm after 60 min incubation using a microplate reader. The level of glutamate was determined as nmol/mgFW using an NADH standard curve. Data Analysis Each data point is expressed as a mean ? standard deviation of the three independent experiments. The values were compared and analyzed by two-way ANOVA (analysis of variance) using LSD multiple comparison tests on the means. Where differences are reported, they are at the 95% confidence level (P<0.05). RESULTS AND DISCUSSION Sensitivity of cam seeds and seedlings to heat exposure Except for cam5-4 and cam6-1, seeds of the other Arabidopsis cam mutants exposed to 42?C for 2 hr germinated normally (data not shown). Germination of cam5-4 and cam6-1 seeds was inhibited by 45% and 50% after exposure to 42?C for 2 hr, respectively (Figure 1). Survival of 2 hr heat treated seedlings after 5 days of recovery at 25 o C showed a significant reduction in seedlings survival of cam5-4 and cam6-1 (Figure 2). Survival of cam5-4 seedlings after 2 hr of growth at 42?C was reduced by 40% while cam6-1 seedlings showed a reduction in survival by 30% after 2 hr at 42?C. Seedling survival of all other cam mutants was comparable to wild type seedlings (data not shown). The significant (P<0.05) sensitivity of cam6-1 and cam5-4 alleles may be explained by the fact that the protein products of the CAM5 and CAM6 genes may be involved in the protection of plants from heat stress involved in protective pathways that contribute to germination and survival of plants at high temperatures. 88 The nature of the cam5-4 mutant allele was investigated by examining the mRNA level of all of the CAM genes produced in this mutant, and it was observed that the cam5- 4 exonic insertion mutant fails to produce detectable levels of CAM5 mRNA and produces either the same or reduced levels of the other CAM gene mRNAs (see Chapter II, Figure 4). Although the level of CAM6 mRNAs in the cam6-1 mutant is reduced it is not eliminated, but the levels of other CAM gene mRNAs are either equal to wild type levels or reduced comparable to those in cam5-4 (see chapter II, Figure 4). However, the CAM gene mRNA levels observed in cam1 and cam4 alleles are equally low overall, and the results do not allow an explanation of the seed germination phenotype of cam6-1 on the basis of CAM gene expression alone (see Chapter II, Figure 4). The sensitivity of the cam5-4 mutant to heat treatment may due to the absence of CaM5 protein in this mutant, but since the mutation in the cam6-1 allele did not eliminate CAM6 gene expression the heat stress sensitivity of the cam6-1 mutant (and possibly the cam5-4 allele) may be due to pleiotrophic effects of one or both of these mutations rather than a direct effect on the expression of these genes. Oxidative damage by high temperature in cam mutants Malonaldehyde (MDA) is a secondary end product of the oxidation of polyunsaturated fatty acids and is a useful index of general lipid peroxidation and ROS production in situ (Hodges et al., 1999, Rael et al., 2004). WT and cam mutants were analyzed for the accumulation of malonaldehyde (MDA) as nmol/mgFW. The level of MDA was determined in root and shoot tissues immediately after heat treatment and after recovery for 2 days at 25 o C under continuous light. Seedlings of cam5-4 and cam6-1 demonstraed dramatically higher levels of MDA in both root and shoot, and the level of 89 MDA remained significantly higher after 2 days of recovery (Figure 3). Root tissues of cam2-1, cam3-2, and cam3-3 demonstrated elevated levels of MDA after heat treatment and recovery compared to wild type, while roots of cam4 and cam7-1showed reduced MDA production after the recovery period. The accumulation of MDA in roots of cam1 and cam2-2 was appreciably lower than wild type after the stress period, but was equivalent to wild type after recovery. Shoot tissues of cam5-2, cam5-3 and cam7-1 had higher MDA after heat treatment and recovery. While cam2-1, cam2-2, cam3-2, cam3-3, and cam5-1 shoots showed lower MDA after recovery. Shoots of cam1 and cam4 had reduced MDA compared to wild type after heat treatment and recovery. The production of ROS and the oxidative damage phenotype of cam5-4 and cam6-1 as a result of heat treatment are consistent with the germination and survival sensitivity phenotype of the alleles examined. These results possibly delineate ROS production as a part of the pleiotrophic mechanism involved in reduced thermotolerance of cam5-4 and cam6-1. Photo-peroxidation damage and ROS production have been observed after a wide range of stresses in other system, and thus this finding is consistent with these previously presented results (Foyer et al., 1994; Harndahl et al., 1998; Lu and Zhang, 1999). Under heat stress oxidative damage has been observed in creeping bentgrass (Agrostis stolonifera) (Larkindale and Huang, 2004), and Arabidopsis seedlings (Larkindale and Knight, 2002; Larkindale et al., 2005; Larkindale and Vierlang, 2008). Heat induced oxidative damage increased in Arabidopsis seedlings pretreated with Ca +2 /CaM inhibitors (Larkindale et al., 2002), and higher CaM proteins levels have been linked to lower levels of heat-induced membrane damage in maize (Gong et al., 1997). The oxidative damage (high MDA level) in the cam5-4 is directly consistent with these 90 observations provided that the CAM5 gene product is the only CAM required for thermal protection. However, the results obtained here with the cam6-1 mutant suggest a more pleotrophic explanation of the results presented here. Levels of GABA shunt metabolites in response to high temperature in cam mutants The levels of GABA shunt metabolites (L-glutamate, GABA, and L-alanine) in wild type and 11 alleles of seven of the nine Arabidopsis CAM genes were determined after 0 min, 30 min, 1 hr, and 2 hr at 42?C. GABA shunt metabolites in both root and shoot were generally elevated after 30 min and 1 hr of heat treatment at 42 o C, and increased substantially after 2 hr at 42 o C. Furthermore, GABA and glutamate levels were increased significantly more than alanine in root and shoot tissues of wild type and all cam mutants. After 30 min, the level of GABA (0.2-1.5 fold increase) and glutamate (0.2-2.5 fold increase) were significantly increased in all cam mutants and in wild type root and shoot tissues compared to the control except in the shoot of cam1 and cam7-1 mutants (Figure 4B). After 1 hr, level of GABA (0.7-3 fold increase) and glutamate (1-4.3 fold increase) were significantly increased in all cam mutants and wild type root and shoot tissues compared to the control except in root of cam5-4 (Figure 4C). After 2 hr at 42 o C, the GABA level increased in all cam mutants, and WT increased up to 5-fold in root cam7-1 and shoot of cam2-2, cam5-1, cam5-2, and cam5-4 compared to the control. Glutamate levels increased in all cam mutants and WT (3-11 fold increase) with highest level in root of cam5-4 and cam6-1 (11-fold) and shoot of cam1 (3-fold) by comparison to the glutamate level in the untreated sample (figure 4D). 91 Alanine level did not increase in WT or any of the cam mutants during heat treatment (Figure 4). Our results showed that CaM may involve the regulation and partitioning of GABA shunt metabolites in root and shoot tissue under heat treatments. GABA accumulation under various abiotic stresses is well documented and studied in various plants system (Hanower and Brazozowska, 1975; Wallace et al., 1984; Mayer et al., 1990; Locy et al., 2000; Mazzucotelli et al., 2006; Kaplan, 2007). The data presented here are consistent with the regulation of GABA shunt pathway by calcium and calmodulin by several possible regulatory mechanisms such as: regulation of GAD activity to produce GABA in response to heat stress in the cytosol, regulation of the translocation of GABA across the mitochondrial membrane for conversion into alanine or glutamate, regulation of the level and the pattern of GABA, alanine and glutamate accumulation in root and shoot tissues, and involvement of the translocation of metabolites between the root and shoot under heat stress. The GABA shunt and Ca +2 /CaM signaling through CaM isoforms may function in multiple pathways acting together to allow plant survival at high temperature. 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Plant Mol Biol 37: 967-975 0 20 40 60 80 100 120 WT cam5-4 cam6-1 Germinat ion % No treatment 42?C, 2 hr ** WT cam5-4 cam6-1 Figure 1: Germination of A. thaliana seeds of wild type and T-DNA insertion mutant alleles cam5-4 and cam6-1 after exposure to 42 o C temperature. Fifty seeds each of genotype were surface sterilized and exposed to 42?C for 2 hr. Heat treated and control seeds were allowed to germinate described in Materials and Methods. The number of germinating seeds was scored. Error bars represent the SD over three replicate plates. 96 0 10 20 30 40 50 60 70 80 90 100 110 WT cam5-4 cam6-1 Su r vival % No treatment 42?C, 30min 42?C, 1 hr 42?C, 2hr * * * * * * Figure 2: Survival of A. thaliana wild type and T-DNA insertion mutant alleles cam5-4 and cam6-1 seedlings exposed to 42?C heat. Two week old seedlings of each genotype were grown as described in Materials and Methods and exposed to 42 o C for 30 min, 1 hr, and 2 hr. After heat treatment, plates containing seedlings were transferred to a growth chamber at 25 o C with continuous light for 5 days. Percent survival of was determined. Error bars represent the SD over three replicate plates. Each plate contained 50 seedlings. 97 Root 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 cam1 cam2 -1 cam 2 -2 cam3 -2 cam 3 -3 ca m4 ca m5 -1 cam 5 -2 cam5 -3 cam 5 -4 cam6 -1 cam 7 -1 WT MDA l evel n m ol /mg F W No treatment 42?C, 2hr 42?C, 2hr- recovery * * * * * ***** *** Shoot 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 ca m1 ca m2- 1 ca m2- 2 ca m3- 2 ca m3- 3 ca m4 ca m5- 1 c am 5 - 2 ca m5- 3 ca m5- 4 c am 6 - 1 ca m7- 1 WT MDA l evel nmol /mgFW No treatment 42?C, 2hr 42?C, 2hr- recovery **** ** * * ******** Figure 3: Oxidative damage by high temperature exposure in cam mutants of A. thaliana. The MDA level was determined in root and shoot tissues by the TBARS assay describe in materials and methods. Two set of seedlings were treated at 42?C for 2 hr. One set was used immediately for measurement of MDA level, while the other set was allowed to recover for 2 days under continuous light at 25 o C before determination of MDA level. Percent of MDA level in cam mutants and wild type in root and shoot tissues were determined. Error bars represent the SD over three replicate plates with 50 seedlings on each plate. Mutants with (*) represent the significant oxidative damage (P <0.05). 98 A. Root: No treatment 0 1 2 3 4 5 cam 1 c am 2 -1 ca m 2-2 cam 3 -2 cam 3 -3 c am4 cam 5-1 c am 5-2 cam 5-3 ca m 5-4 cam 6 -1 ca m 7-1 WT Meta b o l i t es l e vel n m ol/mgFW GABA Alanine Glutamate Shoot: No treatment 0 1 2 3 4 5 6 c am 1 cam 2-1 c am 2 -2 ca m 3-2 cam 3 -3 c am 4 cam 5-1 cam 5-2 c am 5 -3 ca m 5-4 cam 6 -1 ca m 7-1 WT Met ab o l i tes l eve l nmol/mgF W GABA Alanine Glutamate 99 B. Root: 30min 0.0 0.5 1.0 1.5 2.0 2.5 ca m1 GABA Alanine 100 ca c ca ca ca ca c ca ca c m2 -1 a m2- 2 m3- 2 m3 -3 cam4 m5- 1 m5 -2 a m5- 3 m5- 4 m6 -1 a m7- 1 W T Metabolites l e vel nmol/ m gFW fold inc r ea se Glutamate * * * * * * * * * * * * * * * * * * * * * * * * * Shoot: 30min 0.0 0.2 0.4 0.6 0.8 1.0 1.2 c am1 c am2 - 1 c am2 - 2 c am3 - 2 c am3 - 3 c am4 c am5 - 1 c am5 - 2 c a m5 - 3 c am5 - 4 c am6 - 1 c a m7 - 1 W T M et abol i t es l evel nm ol / m gFW f o l d i n cr ease GABA Alanine Glutamate * * * * * * * * * * * * * * * * * * * * * * * C. Root: 1 hr 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 c a m1 c a m 2 -1 ca m 2 - 2 c a m 3-2 ca m 3 -3 ca m 4 c a m 5-1 c am 5-2 c a m 5 - 3 ca m 5 - 4 c a m 6-1 c a m 7 -1 WT Metabolites level nmol /mgFW fold i n cr e ase GABA Alanine Glutamate ** ** * * * * ** * * * * ** ** * * * * * * 101 Shoot: 1 hr 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 c am1 c a m2 - 1 cam2 - 2 c am3 - 2 c a m3 - 3 cam4 c am 5- 1 c am5 - 2 c a m5 - 3 c am5 - 4 c a m6 - 1 cam7 - 1 W T Me t a bolite s le ve l nm ol/mgFW fol d inc r ea se GABA Alanine Glutamate ** ** **** ** **** ******** ** ** D. Root: 2 hr 0 2 4 6 8 10 12 14 ca m1 ca m2 -1 c a m2- 2 ca m3- 2 ca m3 -3 cam4 c a m5- 1 ca m5- 2 ca m5 -3 c a m5- 4 ca m6- 1 ca m7 -1 WT Met abol i te s l evel nm ol / m gFW f o l d i n c r ease GABA Alanine Glutamate * * * * * * * * * * * * * * * * * * *** * * * * * * Shoot: 2 hr 0 1 2 3 4 5 6 cam1 cam2 - 1 ca m2-2 cam3-2c am3-3 cam4 cam5 - 1 ca m5-2 c am5-3 c am5-4 cam6-1cam7 - 1 W T Me tabolite s l e v e l nm ol/mgFW fold increas e GABA Alanine Glutamate * * * * * * * * * * * * * * * * * * * * * * * * * * * 102 103 Figure 4: The levels of GABA, alanine and glutamate in two-week-old seedlings of cam mutants of A. thaliana after exposure to heat at 42 o C for 30 min, 1 hr and 2 hr. Root and shoot tissues were harvested separately and frozen in liquid nitrogen for determination of the level of metabolites as described in materials and methods. Metabolite levels in wild type and cam mutants were calculated and expressed as nmol/mgFW fold increase in root and shoot tissues. A, No treatment B, 30 min. C, 1 hr. D, 2 hr. Error bars represent SD over three replicate plates. Each sample contained 50 seedlings. Mutants with (*) represent the significant level of changes in metabolite levels (P <0.05). Table 1. Mutant lines with T-DNA insertion in calmodulin genes of A. thaliana used in this study. All seeds lines were obtained from Dr.Janet Braam, Rice University, except cam1 and cam4 seeds were obtained from the Arabidopsis Biological Research Stock Center, Ohio State University, Columbus. Mutant Insertion Location SALK Line # cam1 3`UTR SALK_107507 cam2-1 Intron SALK_066990 cam2-2 Intron SALK_089283 cam3-2 5`UTR SALK_075669 cam3-3 3`UTR SALK_042391 cam4 5`UTR SALK_149142 cam5-1 3`UTR SALK_007371 cam5-2 3`UTR SALK_073480 cam5-3 5`UTR SALK_138758 cam5-4 Exon II SALK_027181 cam6-1 3`UTR SALK_071609 cam7-1 3`UTR SALK_074336 104 105 IV. SENSITIVITY OF CALMODULIN MUTANTS OF ARABIDOPSIS THALIANA TO LOW TEMPERATURE EXPOSURE ABSTRACT Plants have evolved mechanisms to cope with changes in surrounding temperatures. T-DNA insertions in seven calmodulin genes of Arabidopsis thaliana were used to investigate the role of specific calmodulin isoforms in tolerance of plants to low temperature for survival, seed germination, susceptibility to low temperature induced oxidative damage, and changes in the levels of GABA shunt metabolites. Exposure of wild type (WT) and cam mutant seeds at 4 o C showed reduction in germination of cam5-4 and cam6-1 seeds. Survival of WT and cam seedlings was not affected by exposure to low temperature. After treatment at 4 o C, MDA levels increased in root tissues of cam2-2, cam2-3, cam4, cam5-4 and cam6-1, and in shoot tissues of cam5-4 and cam6-1. Level of GABA shunt metabolites in seedlings were gradually increased after 1 hr and 3 hr treatments with maximum level after 6 hr and 12 hr. After 1 hr, the GABA shunt metabolite levels increased up to 1-fold compared to the control (no treatment) in root and shoot tissues of all cam mutants and the wild type. After 3 hr treatment at 4?C, GABA level increased 0.65-2.5-fold while alanine and glutamate shoed more modest increases. In response to 6 hr incubation at 4?C, GABA and glutamate increased 1.5-5- fold and alanine increased only 1-fold in root tissues of all cam mutants and the wild type compared to the control. In shoot tissues, alanine and glutamate increased at the same 106 level (0.5-fold) while GABA increased up to 2-fold in all mutants with maximum levels in shoot of cam2-2, cam3-2, cam3-3, cam4, cam5-1, cam5-2 and cam5-4 mutants. After 12 hr treatment at 4?C, GABA and glutamate increased 2-7-fold, and alanine levels increased 0.2-0.5-fold in root tissues of all cam mutants and the wild type. Similarly in shoot tissues, GABA and glutamate increased 0.5-3.5-fold, and alanine increased 0.2-0.5- fold in shoots of all the mutants and the wild type compared to the control. GABA levels increased 0.5-1.5-fold in shoots of cam4, cam5-1, cam5-2, and cam5-4 mutants. Glutamate level increased (1-3.5-fold) in shoots of cam1, cam2-2, cam3-2, cam3-3, cam4, cam5-1, cam5-2, cam5-3, cam5-4, cam6-1 and cam7-1 mutants and the wild type. Sensitivity of cam5-4 and cam6-1 to low temperatures suggests a role of the CAM5 and CAM6 genes in seed germination and protection against cold induced oxidative damage. Increases in the level of GABA shunt metabolites in response to cold treatment after initial reduction in some cam mutants suggests a role for CaM in the activation of GAD after exposure to cold, while increased metabolite levels may indicate involvement of other factors like reduction in cytoplasmic pH in cold regulation. 107 Key words: Arabidopsis, Calmodulin, CAM, CaM, Ca +2 , GABA, Cold stress, Metabolism, low Temperature Abbreviations: CAM: Calmodulin gene CaM: Calmodulin protein Ca +2 /CaM: Calcium/Calmodulin complex GAD: Glutamate Decarboxylase GABA: ?-Aminobutyric Acid MDA: Malonaledehyde NAD + : Nicotinamide adenine dinucleotide NADP + : Nicotinamide adenine dinucleotide phosphate TBARS: Thiobarbiturate reactive substances 108 INTRODUCTION Plants are exposed to either rapid or gradual changes in surrounding temperature. Adaptation to low temperature is associated with many physiological and metabolic processes, which require changes at molecular and biochemical levels, and rearrangement of cell metabolism (Kaye and Guy, 1995). During cold acclimation changes in gene expression occur, especially increased expression of cold-responsive genes (COR). A great number of COR genes have been characterized from a variety of plant species, but only some of them code for proteins whose biochemical function is known, or can be predicted based on sequence similarity (Guy et al., 1985; Jung et al., 2003). Other factors that contribute to low temperature acclimation or tolerance such as changes in membrane lipid composition and accumulation of sugars do not necessarily rely on changes in gene expression and may involve post-translational modulation of enzymes (Cossins et al., 2002, Stitt and Hurry, 2002). Activation of GABA shunt pathway in response to various stresses has been shown (Mayer et al., 1989; Bown and Shelp, 1997; Shelp et al., 1999; Locy et al., 2000). A rapid increase in GABA level in response to draught in cotton (Hanower and Brzozowska, 1975), heat in cultured cowpea cells (Mayer et al., 1990) and Arabidopsis (Locy et al., 2000), cold stress and mechanical damage in soybean (Wallace et al., 1984) and in wheat and barely (Mazzucotelli et al., 2006) have been reported. GABA is synthesized by a cytosol-localized Ca 2+ -calmnodulin-dependent glutamate decarboxylase (GAD) protein (Baum et al., 1993; Snedden et al., 1996). Following irreversible decarboxylation from glutamate, GABA is metabolized to succinate semialdehyde by the GABA shunt pathway which bypasses two steps in the 109 TCA cycle. The last two enzymes of the GABA shunt, a GABA-transaminase using either ?-ketoglutarate or pyruvate as amino group acceptor and succinate semialdehyde dehydrogenase (Shelp et al., 1999, Bouche and Fromm, 2004), are located in the mitochondria. Ca 2+ /CaM-dependent modulation of GAD provides activation of GAD enzyme activity, and thus GABA shunt pathway, leading to GABA accumulation during stress. We have used calmodulin T-DNA insertion mutants of A. thaliana to examine the role of specific calmodulin protein in tolerance of Arabidopsis to low temperature with respect to survival of seedlings, seed germination, and oxidative damage and GABA shunt metabolite. MATERIALS AND METHODS Plant material Wild type (WT) (Arabidopsis thaliana Ecotype Columbia) and T-DNA insertions in seven calmodulin genes (cam) (also in Arabidopsis thaliana Ecotype Columbia) used in this study are listed in Table 1. Seeds were obtained from Arabidopsis Biological Research Stock Center, Ohio State University, Columbus, OH, and from Dr. Janet Braam, Rice University, TX. Homozygous stocks were propagated for use in these stduies. Seeds were surface sterilized with 100% bleach (v/v, 6% sodium hypochlorite) for 10 min followed by five washes with sterile distilled water. Seeds were plated on sterile 1XMS (Murashige and Skoog, 1962) medium (pH 5.7) on Petri dishes supplemented with 2% (w/v) sucrose and solidified with 1.2% (w/v) agar. Petri dishes were placed under continuous light (40?mol m -2 s -1 ) at 25 ?C for seed germination and growth of seedlings. 110 Seed germination Surface sterilized seeds of WT and cam mutants were suspended in 500 ?L of sterile distilled water and incubated at 4?C for 24 hr. Treated seeds were plated on 1X MS Petri dish plates. Plates were vertically placed under continuous light for 7 days at 25 o C. Radicle emergence was recorded after 7 days and compared to control seeds without treatment and WT. For each line average of three plates each with 50 seeds were used. Seedling survival Two week old seedlings of WT and cam mutants were incubated at 4?C for 1 hr, 3 hr, 6 hr, 12 hr, and 24 hr. Seedlings were allowed to recover under continuous light for 5 days at 25 o C. Healthy seedlings with green leaves were scored as surviving. For each mutant and WT the average of three plates each with 50 seeds was determined. Oxidative damage Two set of two week old seedlings of WT and cam mutants were exposed to 4?C for 12 hr in growth chamber. Seedlings from one set were used immediately after treatment for determination of malonaldehyde (MDA) level using the TBARS assay in root and shoot tissues (Heath and Packer, 1968). The second set of seedlings was allowed to recover under continuous light for 2 days at 25 o C before determination of MDA levels. Three plates with 50 seedlings each were used for each sample. Root and shoot tissues were separated and frozen in liquid nitrogen before extraction and determination of MDA level. 111 GABA-shunt metabolites Two weeks old seedlings of WT and cam mutants grown under continuous light on 1X MS agar plates at 25 o C were transferred to growth chamber at 4?C for 1 hr, 3 hr, 6 hr and 12 hr. Root and shoot tissues were separated and used for metabolite analysis. Three plates with 50 seeds each were used for each sample. Metabolites from frozen tissues were extracted according to Zhang and Bown (1997). Alanine and glutamate levels were determined according to Bergmeyer (1983) and the level of GABA was determined according to Zhang and Bown (1997) at 25 o C using a microplate reader. Data Analysis Each data point is expressed as means ?SD of the three independent experiments. The values were compared and analyzed by two-way ANOVA (analysis of variance) using LSD multiple comparison tests on the means. Where differences are reported, they are at the 95% confidence level (P<0.05). RESULTS AND DISCUSSION Sensitivity of cam Mutants to cold treatment Seeds of the cam6-1 and cam5-4 alleles exposed to low temperature at 4 o C for 24 hr showed 65% and 50% reduction in germination respectively, while seeds of all other cam mutants and WT showed germination rates between 85-93% (Figure 1). Exposure of two-week-old seedlings of WT and cam mutants to 4 o C for 24 hr showed no significant between WT and any of the cam mutants. 112 The significant (P<0.05) sensitivity of cam6-1 and cam5-4 alleles to low temperature germination suggests that the CAM5 and CAM6 genes may be directly or indirectly involve in cold stress tolerance in Arabidopsis. The nature of the cam5-4 mutant allele was investigated by examining the mRNA level of all of the CAM genes produced in this mutant, and it was observed that the cam5- 4 exonic insertion mutant fails to produce detectable levels of CAM5 mRNA and produces either the same or reduced levels of the other CAM gene mRNAs (see Chapter II, Figure 4). Although the level of CAM6 mRNAs in the cam6-1 mutant is reduced it is not eliminated, but the levels of other CAM gene mRNAs are either equal to wild type levels or reduced comparable to those in cam5-4 (see chapter II, Figure 4). However, the CAM gene mRNA levels observed in cam1 and cam4 alleles are equally low overall, and the results do not allow an explanation of the seed germination phenotype of cam6-1 on the basis of CAM gene expression alone (see Chapter II, Figure 4). Since CAM5 is not expressed in the cam5-4 the results presented here are consistent with CAM5 having a direct role in cold tolerance, while CAM6 acts pleiotrophically rather than directly in thermotolerance. Oxidative damage by low temperature in cam mutants All cam mutants and the wild type were assayed for the accumulation of thiobarbituric acid reactive substances (TBARS) by measuring the accumulation of malonaldehyde (MDA) in root and shoot under cold treatment at 4?C for 12 hrs and after recovery for 2 days under continuous light. In response to cold treatment MDA level accumulated significantly in many cam mutants both immediately and after recovery in root and shoot tissues (Figure 2). 113 A significant MDA accumulation was not observed in the cam1 mutant while in cam2 alleles only cam2-1 accumulated MDA in root tissue up to 0.4 fold after recovery and the cam2-2 allele accumulated MDA 1.5-fold, (p<0.05) only in root compared to wild type. The cam3-3 allele accumulated MDA 3.5-4-fold (P<0.05) in root after immediate assay compared to wild type, and the cam4 mutant showed 4?fold (P<0.05) in roots immediately and showed a 0.4-fold increase in the recovery assay. The cam5-1, cam5-2, and cam5-3 mutants accumulated MDA up to 0.5-fold in root after the recovery assay while the cam5-4 allele showed a significant 4 to 5 fold after recovery in both root and shoot when compared to wild type. For cam6-1 MDA accumulated 1.5-fold in both root and shoot tissues but not when assayed after recovery. The cam7-1 allele had no phenotype in either the root or shoot (Figure 2). The production of ROS by the cam5-4 allele during cold treatment was consistent with the germination phenotype of this mutant. However, the production of TBARS reactive substances does not produce a reasonable explanation of the increased cold sensitivity of cam5-4 allele since the accumulation of MDA was not greater in tissues of cam5-4 than in other cam mutants which were not more thermo-sensitive. The MDA accumulation that we observed in cam mutants and wild type especially in root tissues might be modulated by disruption of a mitochondrial function and changes in membrane fluidity as a result of cold treatment (Suzuki and Mittler, 2006). Level of GABA shunt metabolites in cam mutants after cold treatment Since GABA shunt pathway has been reported to be activated following cold stress in plant systems (Wallace et al., 1984; Mazzucotelli et al, 2006), the levels of 114 glutamate, alanine, and GABA in roots and shoots during cold treatment at 4?C was determined (figure 3). The levels of the three amino acids gradually increased after 1 hr of cold treatment with a maximum level reached after 6 hr and 12 hr of treatment. After 1 hr, GABA shunt metabolite levels increased up to 1-fold compared to the control (no treatment) in root and shoot tissues of all cam mutants and the wild type. In root tissue, glutamate levels increased (0.8-1-fold) while GABA and alanine levels showed a smaller increase (0.2-0.6-fold). The maximum increase occurred in root of cam2-1, cam3-3, cam4, cam5-1, cam5-2, cam6-1 and the WT (Figure 3B). In shoot tissue, GABA levels increased (0.7-fold increase) to a greater extent than alanine and glutamate (0.2-0.5-fold increase) with higher levels in shoot of cam2-2, cam4, cam5-1, cam5-2 and cam5-4 mutants (Figure 3B). After a 3 hr cold treatment at 4?C, GABA levels increased 0.65-2.5-fold while alanine and glutamate increased less (0.5-1.5-fold) in all mutants and the wild type and in both root and shoot tissue compared with the control. GABA increased in root tissue of cam1, cam2-1, cam5-1 and cam7-1 and in shoot tissue of cam2-2, cam3-2, cam4, cam5- 1, cam5-2, and cam5-4 mutants. Alanine and glutamate increased in all cam mutants and the wild type greatest increases in roots of cam3-3, cam4, cam5-1, cam5-2 and cam6-1 and shoots of cam2-2, cam3-2, cam4, cam5-1, cam5-2 and cam5-4 mutants (Figure 3C). After 6 hr incubation at 4?C, GABA and glutamate were increased 1.5-5-fold while the alanine level only increased 1-fold in root tissues of all cam mutants and the wild type compared to the cold untreated control. GABA levels increased 2-3-fold in roots of cam1, cam2-1, cam4, cam5-1, and cam7-1 mutants. Glutamate levels increased 115 3-5-fold in roots of the cam1, cam3-3, cam4, cam5-1, cam6-1 and cam7-1 mutants (Figure 3D). In shoot tissues, alanine and glutamate increased 0.5-fold while GABA increased up to 2-fold in all mutants with maximum increases in shoots of the cam2-2, cam3-2, cam3-3, cam4, cam5-1, cam5-2 and cam5-4 mutants as shown in Figure 3D. After 12 hr of cold treatment at 4?C, GABA and glutamate accumulated 2-7-fold while alanine accumulated to a lower level of 0.2-0.5-fold increase in root tissues of all cam mutants and the wild type compared to the cold untreated control. GABA levels increased 2-7-fold in roots of cam1, cam2-1, cam4, cam5-1, cam5-4 and cam7-1 mutants. Glutamate levels increased 3-7-fold in roots of cam3-3, cam5-1, cam5-2, cam6-1 and cam7-1 mutants (Figure 3E). Similarly in shoot tissues, GABA and glutamate increased 0.5-3.5-fold while alanine increased 0.2-0.5-fold in shoots of all the mutants and the wild type compared to the control. GABA levels increased 0.5-1.5-fold in shoots of cam4, cam5-1, cam5-2, and cam5-4 mutants. Glutamate levels increased 1-3.5-fold in shoots of cam1, cam2-2, cam3-2, cam3-3, cam4, cam5-1, cam5-2, cam5-3, cam5-4, cam6-1 and cam7-1 mutants and the wild type as shown in Figure 3E. During cold treatment almost all of the mutants showed a major changes in the levels of glutamate, alanine, and GABA in both root and shoot tissues (Figure 3). The accumulation of GABA in response to low temperature may result from an altered intracellular compartementation of glutamate, alteration glutamate decarboxylase activity, or from the intracellular damage and lowering of protoplasmic pH that might lead to the activation of glutamate decarboxylase. CaM functions as a ubiquitous Ca +2 -binding protein that may be involved in GABA shunt pathway regulation by various possible mechanisms such as: regulation of 116 GAD activity in response to cytosol calcium accumulation, regulation of the translocation of GABA across the mitochondrial membrane, regulation of the level and the pattern of GABA, alanine and glutamate pools, and regulating translocation of GABA, alanine and/or glutamate between roots and shoots. Mazzucotelli et al (2006) in their study for characterization of GABA shunt metabolites and GABA shunt genes during cold acclimation and freezing in wheat and barely found that GABA shunt metabolite accumulation and shunt gene expression were activated in response to low temperature, while GABA accumulation, glutamate availability, and GAD activity were correlated to the severity of the stress. Since GABA shunt may play a protective role in a pH-stat mechanism counteracting cytosol acidification due to membrane leakage as a result of cold stress, CaM signaling through activation of functional GAD (H + -consuming properties) may be involved in regulating the accumulation rate of GABA and subsequently alanine and glutamate during cold stress. Increased levels of GABA shunt metabolites in response to cold treatment after an initial reduction in some cam mutants suggests that activation of GAD does occur after exposure to cold stress in response to the intracellular damage and low cytoplasmic pH. Since GAD is a Ca 2+ /CaM-regulated enzyme; the Ca 2+ influx that occurs following a temperature drop could be responsible for the activation of the CaM/GAD complex and induction of GABA shunt pathway in response to cold stress. 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Except for cam5-4 and cam6-1, all cam mutants showed germination of seeds comparable to wild type. 120 121 Root 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 c am 1 ca m 2- 1 c a m 2- 2 ca m3- 2 c a m 3- 3 c a m4 c a m 5- 1 ca m5- 2 ca m5- 3 ca m5- 4 ca m6- 1 ca m7- 1 WT M D A le vel n mo l /m gF W No treatment 4?C, 12hr 4?C, 12hr- recover ** * *** * **** ** y Shoot 0.0 0.1 0.1 0.2 0.2 0.3 0.3 0.4 0.4 ca m1 ca m 2 -1 ca m2- 2 ca m3- 2 ca m3- 3 ca m4 ca m5- 1 ca m 5- 2 ca m5- 3 ca m 5- 4 ca m6- 1 ca m7- 1 W T MDA level nm ol /m gFW No treatment 4?C, 12hr 4?C, 12hr- recovery ** ** * ** ** ** ** ** ** * * * * ** ** 122 Figure 2. Oxidative damage in cam mutants of A. thaliana after exposure to low temperature at 4 o C. MDA level was determined in root and shoot tissues by the TBARS assay describe in materials and methods. Two sets of seedlings were treated at 4?C for 12 hr. One set was used immediately for measurement of MDA level while the other set was allowed to recover for 2 days under continuous light at 25 o C before determination of MDA level. Percent of MDA level in cam mutants and the wild type were determined. Error bars represent the SD over three replicate plates with 50 seedlings on each plate. Mutants with (*) represent the significant oxidative damage (P <0.05). A. Root: No treatment 0 1 2 3 4 5 cam 1 c am 2 -1 ca m 2-2 cam 3 -2 cam 3 -3 c am4 cam 5-1 c am 5-2 cam 5-3 ca m 5-4 cam 6 -1 ca m 7-1 WT Meta b o l i t es l e vel n m ol/mgFW GABA Alanine Glutamate Shoot: No treatment 0 1 2 3 4 5 6 c am 1 cam 2-1 c am 2 -2 ca m 3-2 cam 3 -3 c am 4 cam 5-1 cam 5-2 c am 5 -3 ca m 5-4 cam 6 -1 ca m 7-1 WT Met ab o l i tes l eve l nmol/mgF W GABA Alanine Glutamate 123 B. Root: 1 hr 0.0 0.2 0.4 0.6 0.8 1.0 1.2 c am1 c a m2- 1 c am2 - 2 c am3 - 2 c a m3- 3 cam4 c am 5- 1 cam5 - 2 c am5 - 3 c am 5- 4 cam6 - 1 c am7 - 1 W T Metabolites level nmol /mgFW f o ld i n crea se GABA Alanine Glutamate *** *** ** ** ** *** *** *** * * * * * * Shoot: 1 hr 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 c a m1 c am2 - 1 cam2 - 2 c am3 - 2 c am3 - 3 c am4 c am5 - 1 c am5 - 2 c a m5- 3 c am5 - 4 c a m6- 1 c a m7- 1 WT M e tabol ites le ve l n m ol/m gFW fol d inc r ea se GABA Alanine Glutamate ** * * ** * * *** *** *** * ** * * * 124 C. Root: 3 hr 0.0 0.5 1.0 1.5 2.0 2.5 3.0 ca m1 ca m 2- 1 ca m 2- 2 c am 3- 2 ca m 3 - 3 ca m4 c am 5- 1 ca m 5 - 2 ca m 5- 3 c am 5- 4 ca m 6 - 1 ca m 7- 1 W T Metabolites l e vel nmol /mgFW fold i n cre ase GABA Alanine Glutamate *** *** *** * *** *** *** *** * * * * * * Shoot: 3 hr 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 cam1 c am2 - 1 c a m2- 2 c am3 - 2 c a m3- 3 c am 4 cam5 - 1 c am5 - 2 c am5 - 3 c am5 - 4 c am6 - 1 c a m7- 1 WT Met abol i t es l evel nm ol / m gFW f o l d i n cr ease GABA Alanine Glutamate ** * * ** * ** ** * *** *** * * ** * * * 125 D. Root: 6 hr 0 1 2 3 4 5 6 ca m 1 c am 2 - 1 ca m 2 -2 c am 3- 2 ca m 3 - 3 c am 4 ca m 5 -1 ca m 5 -2 c am 5- 3 ca m 5 - 4 ca m 6 -1 c am 7 - 1 W T Metabolites level nmol /mgFW fold i n cre ase GABA Alanine Glutamate * * * * * * * * * * * * * * * * * * * * * * * * * * Shoot: 6 hr 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 cam1 c am2 - 1 c am2 - 2 c a m3- 2 c am 3- 3 c a m4 c a m5- 1 c a m5- 2 cam5 - 3 cam5 - 4 c am6 - 1 c am7 - 1 W T M e tabolites level nmol/ m gFW fol d i n cr ea se GABA Alanine Glutamate * * *** *** ** * * ** * ** * ** * * * * * 126 E. Root: 12 hr 0 1 2 3 4 5 6 7 8 cam1 c a m2- 1 ca m2- 2 ca m3 -2 ca m3 -3 cam4 c a m5- 1 ca m5- 2 ca m5 -3 ca m5- 4 ca m6- 1 c a m7- 1 W T Me ta boli t e s le ve l nm ol/mg F W fol d i n crea se GABA Alanine Glutamate *** *** *** *** *** *** *** *** * * * ** * * ** ** * * Shoot: 12 hr 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 c a m1 cam2 - 1 c am2-2 ca m3-2 cam3 - 3 ca m4 ca m5-1 cam5 - 2 cam5-3cam5-4c am6-1 cam7 - 1 W T Metaboli t es level nm ol/m gFW fol d i n crea se GABA Alanine Glutamate * *** ** * ****** *** ** * * 127 128 Figure 3. The level of GABA, alanine and glutamate in root and shoot tissues of two week old seedlings of wild type and cam mutants of A. thaliana was determined after exposure to cold as indicated. Seedlings were treated for 0 hr, 1 hr, 3 hr, 6 hr and 12 hr at 4?C. Root and shoot tissues were harvested separately and frozen in liquid nitrogen for determination of metabolite levels as described in materials and methods. Metabolite levels were determined and expressed as nmol/mgFW fold increase. A, No treatment (0 hr). B, 1 hr. C, 3 hr. D, 6 hr. E, 12 hr. Error bars represent SD over three replicate plates, each containing 50 seedlings each of WT and cam mutants. Table 1. Mutant lines with T-DNA insertion in calmodulin genes of A. thaliana used in this study. All seeds lines were obtained from Dr.Janet Braam, Rice University, except cam1 and cam4 seeds were obtained from the Arabidopsis Biological Research Stock Center, Ohio State University, Columbus. Mutant Insertion Location SALK Line # cam1 3`UTR SALK_107507 cam2-1 Intron SALK_066990 cam2-2 Intron SALK_089283 cam3-2 5`UTR SALK_075669 cam3-3 3`UTR SALK_042391 cam4 5`UTR SALK_149142 cam5-1 3`UTR SALK_007371 cam5-2 3`UTR SALK_073480 cam5-3 5`UTR SALK_138758 cam5-4 Exon II SALK_027181 cam6-1 3`UTR SALK_071609 cam7-1 3`UTR SALK_074336 129 130 V. SENSITIVITY OF CALMODULIN MUTANTS OF ARABIDOPSIS THALIANA TO SALT AND OSMOTIC STRESS ABSTRACT Plants accumulate compatible solutes in response to low water potential caused by ionic and nonionic osmotic stress. T-DNA insertion mutants in seven calmodulin genes of Arabidopsis thaliana (Arabidopsis) were used to determine the roles of specific CAM genes in tolerance of plants to salinity and osmotic stress induced by NaCl and mannitol. Canges were measured in seed germination, growth, stress induced oxidative damage, and changes in the levels of GABA shunt metabolites. Seed germination and seedling growth was affected by ionic and nonionic stresses in cam5-4 and cam6-1 mutants. Oxidative damage measured as the level of malonaldehyde (MDA) was significantly higher in root and shoot of cam4 and cam6-1 in response to salinity stress. Exposure to nonionic osmotic stress by mannitol increased the MDA level in root of cam mutants except cam1, cam2-1, cam2-2, and cam7-1 and shoot of cam5-4 and cam6-1. All metabolites levels in both root and shoot were generally increased in response to all salt (NaCl) treatments compared to the control. In response to 75 and 100mM NaCl treatments; GABA, alanine and glutamate level were increased significantly in root tissue of cam1, cam5-4, cam7-1, and shoot tissue of cam1 and cam5-4 mutants. All cam mutants except the wild type accumulated alanine and glutamate to a greater extent than 131 GABA in root and shoot tissues at 150mM NaCl treatment. In General, all the shunt metabolites level were accumulated in root more than shoot in response to non-ionic osmotic stress. GABA, alanine, and glutamate levels was significantly increased in root and shoot tissues of cam1, cam4, cam5-4, cam6-1 and cam7-1 mutants in response to treatments of seedlings to all mannitol concentrations. Our results show that cam5-4 and cam6-1 mutants are sensitive to salt and osmotic stress for seed germination, seedling growth and oxidative damage. High accumulation of GABA, alanine and glutamate under salt and osmotic stress suggest that GABA shunt metabolites may function in osmoregulation and signaling in response to salinity and non-ionic osmotic stress, and may serve as an osmoprotectants in response to low water availability. These results suggest GABA shunt metabolites are differentially used for ionic and non-ionic stress in Arabidopsis. 132 Key words: Arabidopsis, Calmodulin, CAM, CaM, Ca +2 , GABA, NaCl, Mannitol, Salt stress, Osmotic stress, Metabolism Abbreviations: CAM: Calmodulin gene CaM: Calmodulin protein Ca +2 /CaM: Calcium/Calmodulin complex GAD: Glutamate Decarboxylse GABA: ?-Aminobutyric Acid MDA: Malonaledehyde NAD + : Nicotinamide adenine dinucleotide NADP + : Nicotinamide adenine dinucleotide phosphate TBARS: Thiobarbiturate reactive substances 133 INTRODUCTION Membrane disorganization, reactive oxygen species (ROS) production, metabolic toxicity, and inhibition of photosynthesis are some of the consequences of salt stress on the plants (Yeo, 1998). Plants respond to salt stress by changing in gene expression, metabolic activity, and ion and water transport to minimize stress damage and to reestablish ion and water homeostasis (Zhu, 2001; Shi et al., 2002; Zhu, 2002). Upon exposure to osmotic stress, plants exhibit a wide range of responses at the molecular, cellular, and whole plant levels (Xiong and Zhu, 2002). Altered water status most likely brings about initial growth reduction and induces the inhibition of cell division and accelerates cell death (Hasegawa et al., 2000). To withstand osmotic stress, certain plants have evolved a high capacity to synthesize and accumulate non-toxic solutes (osmoprotectants or compatible solutes), predominantly in the cytoplasm, as part of an overall mechanism to reduce intracellular osmotic potenital and thereby maintain both Turgor and drive an osmotic gradient for water uptake (LeRudulier et al., 1984; Smirnoff, 1998). Being non-toxic, osmoprotectants can accumulate to osmotically significant levels without disturbing metabolic pathways inside the cell. Osmoprotectants can protect enzymes and membranes inside the cell against damage from high salt concentration and oxidative damage (Nuccio et al., 1999; Ashraf and Foolad, 2007). One of the earliest responses to osmotic and salinity stress in plant cells is a transient increases in cytosolic free Ca +2 (Knight, 2000). Experimental evidence implicates Ca +2 function in salt and osmotic adaptation (Knight and Knight, 2001). Ca +2 as a second messenger in abiotic stress (Rudd and Franklin-Tong, 2001; Sanders et al., 134 2002) is required for the expression of some stress-induced genes (Knight et al., 1996) and activation of a stress-associated gene promoters in plants (Sheen, 1996). Since Ca +2 transduces the stress signal by interacting with protein sensors, Ca +2 sensors such as CaM, CaM-dependent protein kinases (CDPK), and Calcineurin B-like proteins (CBLs) are prime candidates that link Ca +2 signal to downstream responses during salt and osmotic stress in plants (Zhu, 2002; Cheong et al., 2003; Perruc et al., 2004). CaM is known to couple Ca +2 signals to changes in the activity of downstream target proteins via direct interaction between the Ca +2 /CaM complex and target molecules (Bouche et al., 2005). Glutamate decarboxylase (GAD) is one of the target proteins that can be activated by Ca +2 /CaM complex (Arazi et al., 1995; Snedden et al., 1996; Yuan and Vogel., 1998; Zik et al., 2006). In response to various abiotic stresses GAD decarboxylates glutamate to produce GABA which subsequently is transported into the mitochondrial matrix and degraded via succinic semialdehyde to succinate, a pathway that is called the GABA shunt (Shelp et al., 1999; Kinneresly and Turano, 2000). During salt and osmotic stress, GABA has been suggested to be involved in osmoregulation, regulation of cytosolic pH, and the regulation of detoxification of reactive oxygen radicals (Shelp et al., 1999; Bouche and Fromm, 2004; Xing et al., 2007). GABA might also act as a compatible solute and be involved in intracellular signaling during salt and osmotic stress. However, the specific role of GABA in signaling and osmoregulation is not well understood (Bouche et al., 2003). Therefore, the characterization of the role of CaM in GABA shunt metabolite accumulation is crucial to understanding the possible roles of the GABA shunt activation in Arabidopsis seedlings under the osmotic and salt treatments. 135 T-DNA insertion mutants in the calmodulin genes of A. thaliana have been used to examine the roles of specific calmodulin genes in tolerance to high salt and osmotic treatments with respect to seed germination, seedlings growth, oxidative damage and GABA shunt metabolite levels. MATERIALS AND METHODS Plant material Wild type (WT) (Arabidopsis thaliana Ecotype Columbia) and T-DNA insertions in seven calmodulin genes (cam) (also in Arabidopsis thaliana Ecotype Columbia) used in this study are listed in Table 1. Seeds were obtained from the Arabidopsis Biological Research Stock Center, Ohio State University, Columbus, OH and from Dr. Janet Braam, Rice University, Texas. Homozygous stocks were propagated for use in these studies. Seeds were surface sterilized with 100% bleach (v/v, 6% sodium hypochlorite) for 10 min followed by five washes with sterile distilled water. Seeds were plated on sterile 1XMS (Murashige and Skoog, 1962) medium (pH 5.7) on Petri dishes supplemented with 2% (w/v) sucrose and solidified with 1.2% (w/v) agar. Petri dishes were placed under continuous light (40?mol m -2 s -1 ) at 25 ?C for seed germination and growth of seedlings. Seed germination 150 surface sterilized seeds of WT and each of cam mutants were plated on 1X MS medium supplemented with 0, 75, 100, and 150 mM NaCl and allowed to grow under continuous light at 25 o C. A similar set of seeds were plated on 1X MS Petri dish plates supplemented with 0, 200, 300, and 400 mM mannitol and allowed to grow under continuous light at 25 o C. Emergences of radicle from germinating seeds were scored 136 after 10 days. The effect of salt and mannitol on seed germination was calculated for WT and cam mutants. Root elongation Seven-day-old seedlings of WT and cam mutants grown on 1X MS medium under continuous light were transferred onto 1X MS medium supplemented with 0, 75, 100, or 150 mM NaCl, and grown under continuous light for seven days before measurement of increase in root lengths in response to varying salt concentrations. For determination of effect of osmotic stress, seven-day-old seedlings were transferred onto new 1X MS medium supplemented with 0, 200, 300, or 400 mM mannitol and seedlings were allowed to grow for next seven days in continuous light before measurement of the increase in the length of primary root in response to varying concentrations of mannitol. Each plate contained 15 seedlings. Three replicate plates were used for each treatment. The increase in primary root length (mm) was measured after 7 days of growth. Oxidative damage Seven-day-old seedlings grown on 1X MS medium under continuous light were transferred onto 1X MS plates supplemented with 0, 150 mM NaCl and 400 mM mannitol. Plates were incubated under continuous light at 25 o C for the next seven days. Root and shoot tissues were separated for determination of malonaldehyde (MDA) level by the TBARS assay (Heath and Packer, 1968). Three replicate plates with 15 seedlings on each plate were used. 137 GABA shunt metabolites Seven-day-old WT and cam seedlings grown under continuous light at 25 o C were transferred to 1X MS medium supplemented with 0, 75, 100 and 150mM NaCl, and 0, 200, 300 and 400mM mannitol, respectively and grown under continuous light for seven days. The levels of L-glutamate, GABA, and L-alanine in seedlings grown on control and supplemented medium were determined in root and shoot tissues. Three plates with 15 seedlings on each plate were used for each sample. Metabolites from each sample were extracted according to Zhang and Bown (1997). Glutamate and alanine levels were measured according to Bergmeyer (1983) and GABA level was measured according to Zhang and Bown (1997) using microplate reader. Data Analysis Each data point is given as a mean ?SD of the three independent experiments. The values were compared and analyzed by two-way ANOVA (analysis of variance) using LSD multiple comparison tests on the means. Where differences are reported, they are at the 95% confidence level (P<0.05). RESULTS AND DISCUSSION Sensitivity of cam mutants to salt and osmotic stress To test the sensitivity/tolerance of cam mutants to salt and osmotic stress, all the mutants and wild type plants were grown on media containing various concentrations of NaCl (0, 75, 100, 150mM) or Mannitol (0, 200, 300, 400mM). The germination rate and the growth as increase in root length were monitored. As shown in Figure (1A) the germination of cam6-1 and cam5-4 was inhibited and significantly (P<0.05) as a results 138 of salt toxicity while there was no significant difference between wild type and the other cam mutants. On 75mM, 100mM and 150mM NaCl the germination of cam5-4 and cam6-1 seeds reached 75%, 65% and 60% germination respectively after 8-10 days (Figure 1A), while wild type and the other cam mutant seeds germinated normally (85- 95%) after 4 days on the same treatments (data not shown). The same pattern of germination tolerance/sensitivity was observed under mannitol treatments (Figure 1B). Seed germination was inhibited and significantly reduced (P<0.05) in cam5-4 and cam6-1 seeds in response to osmotic stress. Consequently; cam5-4 and cam6-1 seeds germinated 75% at 200mM Mannitol, 60-65% at 300mM Mannitol, and 56-60% at 400mM Mannitol after 10 days. However, wild type and the other cam mutants were able to fully germinate at 200 mM Mannitol after 4 days, 300 mM Mannitol after 6 days, and 300 mM Mannitol after 8 days over the same period. The tolerance/sensitivity of cam mutants to salt and osmotic stresses was also examined at the seedling stage (Figure 2). To this end, 7 days-old seedlings grown on 1X MS medium under continuous light were transferred to media containing 0, 75, 100, or 150 mM NaCl or 0, 200, 300, or 400mM mannitol. Root elongation (mm) was measured after 7additional days. As shown in Figure 2; root growth of all of the mutants and the wild type was retarded on NaCl or mannitol-containing media compared to control media without salt or mannitol treatments. However, most of the mutants showed no significant differences in seed germination assay compared to wild type on 75mM and 100 mM NaCl or 200 mM and 300 mM mannitol after 7 days, while at 75, 100, and 150 mM NaCl or 200, 300, and 400 mM mannitol treatments the root length was significantly (P<0.05) retarded and reduced for all cam mutants compared to wild type (Figure 2). Only, cam6- 139 1 and cam5-4 seedlings showed greater sensitivity to NaCl treatments and a significant (P<0.05) reduction in root length at 75, 100 and 150 mM NaCl (Figure 2A) or 200, 300 and 400 mM mannitol (Figure 2B) in comparison with all the other mutants and the wild type. Furthermore cam6-1 and cam5-4 seedlings showed a highly significant (*,P<0.05) sensitivity to 150mM NaCl or 400mM Mannitol demonstrating a reduction of more than 50% in root growth after 7 days. Taken together, these data are consistent with 2 mutants, cam5-4 and cam6-1, demonstrating greater sensitivity to salt and osmotic stress at both the germination and seedling stages than wild type or any of the other CAM mutations studied here. This suggests that CaM5 and CaM6 may be directly or indirectly involved in tolerance and signaling pathways during early development and seedlings growth leading to tolerance to salt and osmotic stress treatments. We investigated the nature of the cam5-4 mutant allele by looking at the mRNA levels in the wild type and the mutants, and we find that the cam5-4 mutant does not show any steady state level of CAM5 mRNA and the levels of all other CAM genes are reduced (refer to Chapter II) while the cam6-1 mutation demonstrates a reduced level of mRNA of CAM6 in shoot up to 50% and overall reduced levels of all other CAM genes as well (refer to Chapter II). The absence of the CAM5 mRNA in the cam5-4 mutant may indicate the loss of a critical CaM protein that directly functions in salt and osmotic stress tolerance possibly in signaling pathways or GABA synthesis during seeds germination and seedling growth. However, the incomplete loss of mRNA for CAM6 in the cam6-1 mutant (refer to Chapter II) despite the fact that this mutant demonstrates a germination and seedling growth phenotype on media containing salt and mannitol could means that this phenotype results not from the loss of CaM6 140 protein but is related to pleiotrophic effects of the lowered level of CaM6 protein or other proteins including the products of other CaM genes. The exact nature of these effects has not been elucidated at this time. In Arabidopsis, Ca 2+ -binding protein CML24 -also known as TCH2 and shares 40% identity with CaM proteins was strongly suggested to positively regulate ABA inhibition of germination and seedling development and negatively regulates ion sequestering under high salt treatment, which implicates its possible role in ion homeostasis (Braam, 1992; Delk et al., 2005). In other studies, the transcription of CAM genes was induced when cultured cells from tomato or bean (Vigna radiate) are exposed to salinity (Botella and Arteca, 1994; Delumeau et al., 2000). On the other hand, transgenic tobacco plants expressing bovine CAM germinate faster on media containing high levels of NaCl comparing to the control (Olsson et al., 2004). Conversely, Arabidopsis CaM binding protein of 25kDa (AtCaMBP25) overexpression exhibits an increased sensitivity to both ionic (NaCl) and non-ionic (Mannitol) osmotic stress during seed germination and seedling growth (Perruc et al., 2004). In agreement with the data presented here CaM might be a regulatory component during seed germination and plant growth during osmotic and salinity stress signaling in Arabidopsis. Oxidative damage in cam mutants by salt and osmotic stress In order to test the effects of high salt and osmotic stress treatments on oxidative damage and ROS levels in Arabidopsis seedlings. One-week-old seedlings of all cam mutants and the wild type were subject to 150 mM NaCl or 400 mM mannitol for 7 days. 141 After 7 days, the MDA levels (nm/mgFW) were determined in both root and shoot separately using the TBARS assay as shown in Figure 3 and 4. The MDA levels were significantly reduced in root and shoot tissues of most cam mutants under high salt and osmotic treatments. The cam1 mutant accumulated 0.4-fold more MDA (P<0.05) only in shoot tissues at 150 mM NaCl, and the cam5-4 mutant accumulated 1.2-fold higher levels of MDA (P<0.05) only in shoot under the same treatment. The cam6-1 allele accumulated 1.6-fold and 0.4-fold higher MDA levels in both root and shoot respectively under NaCl treatment (Figure 3). As a Result of high Mannitol treatment (Figure 4): cam3-2 and cam3-3 accumulated 0.35-fold more MDA in root at 400 mM mannitol. The cam4 mutant accumulated 0.5-fold more MDA in root (P<0.05), while the cam6-1 mutant showed a similar phenotype in root (0.85-fold increase) and in shoot (0.3-fold increase) at 400 mM mannitol treatment. All cam5 alleles showed significant accumulation of MDA in root (1-fold, P<0.05) at 400 mM mannitol, while cam5-4 is the only cam allele that accumulated higher level MDA in the shoot (0.6-fold) under the same mannitol treatment after 7 days incubation (Figure 4). Taken together, these data allow the inference that CaM may be involved in oxidative damage protection during salt and osmotic stress. CaM might indirectly be involved in scavenging hydroxyl radicals and superoxides ions as a second response under salt and osmotic stress. The distinctive phenotype of cam5-4 and cam6-1 implicates the possible interactions between different CaM isoforms in signaling and acquired tolerance to oxidative damage in response to high salinity and hyperosmolarity. 142 Catalase enzyme, which is mainly localized in peroxisomes and glyoxysomes, was found to be activated by Ca +2 /CaM to down regulate hydrogen peroxide (H 2 O 2 ) and maintain H 2 O 2 homeostasis levels in plants (Yang and Pooviah, 2002). Transgenic plants with reduced catalase activity showed increased sensitivity to salt stress (Willekens et al., 1997). Furthermore, it is strongly indicating that CaM and CaM-signaling are involved in regulating the ROS levels and ROS signaling under salt and osmotic stress in Arabidopsis. GABA shunt metabolites in cam mutants in response to salt and osmotic stress To investigate changes in the level of GABA, it?s substrate glutamate, and alanine, the product of GABA degradation under salt stress levels of these compounds were determined in root and shoot separately of all cam mutants and the wild type after seedlings were grown on 0, 75, 100, or 150 mM NaCl for 7 days. Generally, glutamate, GABA, and alanine all accumulated in root and shoot in all cam mutants and wild type compared to the control in response to all salt (NaCl) treatments (Figure 5). In response to 75mM NaCl treatment, GABA increased 0.5-3.5- fold, alanine increased 1-2.4-fold, and glutamate increased 1.5-4.5-fold increased in root tissues of all cam mutants and the wild type with a 6.5-fold increase of alanine and an 8.7-fold increase glutamate in roots of the cam5-4 mutant (Figure 5B). In shoot tissues under the same treatment, alanine and glutamate increased 1-2.5-fold in all cam mutants and wild type while the GABA level increased 0.1-1-fold when compared to the control (Figure 5B). At 100 mM NaCl exposure; glutamate, GABA, and alanine increased in root tissues. GABA, alanine and glutamate level increased up to 8-fold in root tissues of all 143 mutants and the wild type compared to the control with maximum accumulation in roots of cam1, cam5-4, and cam7-1 mutants to 10-, 11-, and 12-fold increase respectively as shown in Figure 5C. On the other hand, in shoot tissues GABA showed higher accumulation levels (5-33-fold increase) than alanine and glutamate (1-4-fold increase) in all cam mutants but not in wild type. The maximum GABA, alanine, and glutamate levels were observed in shoot of cam1 and cam5-4 mutants (Figure 5C). After 7 days of seedlings exposure to 150 mM NaCl, GABA accumulated (P<0.05) to greater levels than alanine and glutamate in roots and shoots of all cam mutants compared to the control except in shoot tissues of wild type (Figure 5D). In root tissues, GABA accumulated up to 25-fold while alanine and glutamate levels reached 14- fold in roots of the cam1, cam2-1, cam5-4, and cam7-1 mutants. Furthermore, all cam mutants showed increases up to 55-fold in GABA levels. This was higher than the levels of alanine and glutamate that were observed (1-3-fold) especially in shoots of cam1 and cam5-4 mutants as shown in Figure 5D. Wild type shoot tissues did not accumulate glutamate, GABA, or alanine under 150 mM NaCl treatment (Figure 5D). To investigate the GABA shunt and its metabolites levels under osmotic stress; GABA, alanine, and glutamate levels were determined in roots and shoots separately for all cam mutants and the wild type after 7 days on media containing 0, 200, 300, or 400 mM mannitol. Over all glutamate, GABA, and alanine increased to a greater extent in roots of all cam mutants and the wild type than in shoot tissues compared to the unstressed control under all mannitol treatments (Figure 6). In response to 200 mM mannitol; GABA increased 3.5-18-fold while alanine and glutamate increased 2-4-fold in all cam mutants 144 and the wild type root tissues. Significant accumulation of GABA (17 fold), alanine (17 fold) and glutamate (28 fold) were observed in roots of cam1, cam4 and cam5-4 mutants (Figure 6B). In shoot tissues, glutamate, GABA, and alanine accumulated in all cam mutants and the wild type compared to the control where GABA, alanine, and glutamate levels reached up to 1.7-fold in shoots of the cam1, cam3-2, and cam5-4 mutants (Figure 6B). At 300 mM mannitol treatment; GABA, alanine and glutamate increased up to 25-30-fold in roots of all cam mutants and the wild type compared to the control as shown in Figure 6C. GABA increased 8-30-fold; alanine increased 4-22-fold; and glutamate increased 4.5-32-fold. The maximum levels of all metabolites were reached in roots of the cam1, cam4, cam6-1, and cam7-1 mutants. In shoot tissues, glutamate, GABA, and alanine increased 1-5-fold of all cam mutants and the wild type especially in shoots of cam1, cam3-2, cam5-1, cam5-2, cam5-4, cam6-1 and cam7-1 mutants as shown in Figure 6C. In response to 400 mM mannitol treatment for 7 days glutamate (7-37 fold increase), GABA (12-52 fold increase), and alanine (5-23 fold increase) accumulated significantly in all mutants and the wild type root tissues compared to the control and to the other mannitol treatments (Figure 6D). The maximum accumulation occurred in root tissues of cam1, cam4, cam5-4, cam6-1 and cam7-1 mutants. On the other hand, GABA increased 11-fold, alanine increased 1.5-4.5-fold, and glutamate increased 1.5-6-fold in shoot tissues under the same treatment. GABA level reached maximum levels in shoots of the cam5-1 and cam5-2 mutants while alanine and glutamate reached maximum levels 145 only in the shoots of cam6-1 mutants compared to the control under 400 mM mannitol treatment as shown in Figure 6D. GABA shunt pathway has been suggested to be involved in osmoregulation and ion homeostasis under salt and osmotic stress (Shelp et al., 1999; Bouche and Fromm, 2004; Xing et al., 2007). GABA, and the other shunt metabolite levels vary between genotypes, and with salt and osmotic stresses in root versus shoot tissues. High accumulation of GABA under salt stress suggested that GABA might be involved in osmoregulation and signaling in response to salinity. High accumulation of GABA, alanine and glutamate under osmotic stress especially in root tissues suggested that GABA, alanine and glutamate may serve as an osmoprotectants in response to high osmolarity and low water availability under mannitol treatments. High accumulation of GABA shunt metabolites in cam5-4 mutant?s tissues under salt stress suggests that the CaM5 isoform might play a role in metabolite accumulation and partitioning under salt but not necessarily under osmotic stress. Beside its involvement in osmoregulation; GABA may be involved in ion balance and homeostasis. Kinnersley and Lin (2000) found that promotion of lemna minor growth by GABA application was associated with an increase in mineral contents of treated plants. On the other hand, higher GABA accumulation in the roots of soybean (Glycine max L.) plants under salt stress was found to be the result of polyamine degradation (PA) to maintain ion balance and enhance salt tolerance (Xing et al., 2007). Arabidopsis CaM-regulated Ca +2 -ATPase (ACA4), is a calmodulin regulated calcium pump that is located on the vacuole membrane. This protein conferred protection against high NaCl and mannitol treatments in yeast (Geisler et al., 2000). 146 Since cam5-4 mutants showed a significant elevation in GABA shunt metabolites under salt stress, it is possible that CaM5 might be involved in Ca +2 influx/outflow between the cytosol and other cellular organelles via regulating the membrane localized Ca +2 pump, which in turn regulates the GABA shunt in response to salt stress. 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Biochem Biophys Acta 1764: 872-876 A. 0 10 20 30 40 50 60 70 80 90 100 110 No treatment 75mM NaCl 100mM NaCl 150mM NaCl Ge rmin ation % WT cam5-4 cam6-1 * * ** * * B. 0 10 20 30 40 50 60 70 80 90 100 110 No treatment 200mM Mannitol 300mM Mannitol 400mM Mannitol Ge rm in ation % WT cam5-4 cam6-1 * * * * * * 150 151 Figure 1: Germination of WT and cam mutant seeds on various concentrations of NaCl (A) and Mannitol (B). 150 WT and each of the cam mutants seeds were surface sterilized and grown on medium containing 0, 75, 100, or 150 mM NaCl, or 0, 200, 300, or 400 mM Mannitol. Germinating seeds were scored after 10 days. Results are presented as percent of germination. Error bars represent the SD over three replicate plates for each of the mutants. All cam mutants were similar to WT under all concentrations of NaCl and Mannitol except cam5-4 and cam6-1. A. 0 10 20 30 40 50 60 No treatment 75mM NaCl 100 mM NaCl 150 mM NaCl Roo t L e ngt h (mm) WT cam5-4 cam6-1 * * * * ** * ** B. 0 10 20 30 40 50 60 No treatment 200 mM Mannitol 300 mM Mannitol 400 mM Mannitol Ro o t Le ng t h ( mm) WT cam5-4 cam6-1 * * * ** ** ** 152 153 Figure 2. Root elongation of cam mutants and WT seedlings after 7 days of growth on agar plates supplemented with 0, 75, 100, and 150 mM NaCl (A) and with 0, 200, 300, 400 mM Mannitol (B). Results are presented as root length (mm) for WT and cam mutants. Error bars represent the SD over three replicate plates. WT and cam seedlings had similar sensitivity to different concentrations of NaCl and Mannitol treatments except for cam5-4 and cam6-1. Root 0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14 0.16 0.18 0.20 ca m1 c a m2 - 1 c am 2 - 2 c a m3 - 2 c am 3 - 3 ca m4 c a m5 - 1 c am 5 - 2 ca m 5- 3 c a m5 - 4 ca m 6 - 1 c a m7 - 1 WT M D A level n m ol /m g F W No treatment 150 mM NaCl * * * * * * * * * * ** * * Shoot 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 c am 1 ca m 2- 1 c a m2 -2 c a m3 -2 ca m3 - 3 cam4 ca m5- 1 ca m 5- 2 ca m 5- 3 c a m5 -4 c a m6 -1 ca m7 - 1 WT MDA l evel nmol /m gFW No treatment 150 mM NaCl *** ** * ****** * 154 155 Figure 3. Oxidative damage in seedlings of cam mutants and wild type A. thaliana after exposure to 150 mM NaCl. MDA level was determined in root and shoot tissues by the TBARS assay described in materials and methods. Two sets of one week old seedlings were subjected to two 150 mM NaCl for 7 days under continuous light at 25?C. After 7 days root and shoot tissues were used for MDA level measurement. Percent of MDA level in cam mutants and the wild type in root and shoot tissues were determined. Error bars represent the SD over three replicate plates. Mutants with (*) represent the significant oxidative damage (P <0.05). Root 0.00 0.02 0.04 0.06 0.08 0.10 c a m1 c a m2 -1 ca m2 - 2 c a m3 -2 ca m3- 3 c a m4 ca m5- 1 c a m5 -2 ca m 5- 3 c a m5 -4 c a m6 -1 ca m7 - 1 WT MD A le v e l nm ol /m g F W No treatment 400 mM Mannitol * **** **** *** * Shoot 0.00 0.05 0.10 0.15 0.20 0.25 0.30 cam1 c a m2 - 1 ca m2- 2 ca m3- 2 ca m 3- 3 c a m4 c a m5 -1 ca m5- 2 c a m5 - 3 ca m5- 4 ca m6- 1 ca m 7- 1 WT MDA level nm ol/ m gF W No treatment 400 mM Mannitol ** * * ** ** * ** ** ** ** ** ** * 156 157 Figure 4. Oxidative damage in cam mutants and wild type A. thaliana caused after exposure to 400 mM Mannitol. MDA level was determined in root and shoot tissues by the TBARS assay described in materials and methods. Two sets of one week old seedlings were subjected to 400 mM Mannitol for 7 days under continuous light at 25?C. After 7 days root and shoot tissues were used for MDA level measurement. Percent of MDA level in cam mutants and the wild type in root and shoot tissues were determined. Error bars represent the SD over three replicate plates. Mutants with (*) represent the significant oxidative damage (P <0.05). A. Root: No treatment 0 1 2 3 4 5 cam 1 c am 2 -1 ca m 2-2 cam 3 -2 cam 3 -3 c am4 cam 5-1 c am 5-2 cam 5-3 ca m 5-4 cam 6 -1 ca m 7-1 WT Meta b o l i t es l e vel n m ol/mgFW GABA Alanine Glutamate Shoot: No treatment 0 1 2 3 4 5 6 c am 1 cam 2-1 c am 2 -2 ca m 3-2 cam 3 -3 c am 4 cam 5-1 cam 5-2 c am 5 -3 ca m 5-4 cam 6 -1 ca m 7-1 WT Met ab o l i tes l eve l nmol/mgF W GABA Alanine Glutamate 158 B. Root: 75mM NaCl 0 1 2 3 4 5 6 7 8 9 10 ca m 1 ca m 2 -1 ca m 2 -2 ca m 3 -2 ca m 3 -3 ca m 4 ca m 5 -1 ca m 5 -2 ca m 5 -3 ca m 5 -4 ca m 6 -1 ca m 7 -1 W T Metabolites l e vel nmol/ m gFW f o ld incr ea se GABA Alanine Glutamate ** * * * * * * * * *** ** * * * Shoot: 75mM NaCl 0.0 0.5 1.0 1.5 2.0 2.5 c a m1 c am2 - 1 c a m2- 2 cam3 - 2 cam3 - 3 c am4 c a m5- 1 cam5 - 2 c am5 - 3 c am5 - 4 c a m6- 1 cam7 - 1 W T Me ta boli t e s le ve l nm ol/mg F W fol d i n crea se GABA Alanine Glutamate ** ** ** ** ** ** ** * * ** *** ** ** ** 159 C. Root: 100mM NaCl 0 2 4 6 8 10 12 14 16 c am1 c a m2- 1 c am2 - 2 c a m3- 2 c am 3- 3 c a m4 c a m5- 1 c am5 - 2 c a m5- 3 c am 5- 4 cam6 - 1 c a m7- 1 WT M e tabol ites level nmol/ m gFW fold i n cre a s e GABA Alanine Glutamate *** *** *** *** *** ** *** *** ** *** ** *** ** Shoot: 100mM NaCl 0 5 10 15 20 25 30 35 40 cam1 ca m2- 1 c a m2- 2 ca m3- 2 ca m3- 3 cam4 ca m5- 1 ca m5- 2 c a m5- 3 ca m5- 4 c a m6- 1 c a m7- 1 W T M e t a b ol i t es level nmo l / m gF W fol d i n crease GABA Alanine Glutamate *** * * * * * * * * *** * * 160 D. Root: 150mM NaCl 0 5 10 15 20 25 30 c am1 c am 2- 1 cam2 - 2 c am3 - 2 c am3 - 3 c a m4 c a m5- 1 cam5 - 2 c am5 - 3 c am5 - 4 c a m6- 1 c am7 - 1 W T Metaboli t es level nm ol/m gFW fol d i n crea se GABA Alanine Glutamate *** *** *** *** *** *** *** *** *** *** *** *** * Shoot: 150mM NaCl 0 10 20 30 40 50 60 70 cam1 c am2 - 1 c am2 - 2 c a m3- 2 c am3 - 3 c a m4 c am 5- 1 c am5 - 2 c a m5- 3 c am5 - 4 c a m6- 1 c a m7- 1 WT Me ta boli t e s le ve l nm ol/mg F W fol d i n crea se GABA Alanine Glutamate ************** 161 162 Figure 5. GABA shunt metabolites in cam mutants of A. thaliana after exposure to 0, 75, 100, and 150mM NaCl were grown under continuous light at 25?C for seven days. Root and shoot tissues were harvested and frozen in liquid nitrogen for determination of the level of metabolites as described in materials and methods. Metabolite levels in mutants were calculated as nmol/mgFW and are presented as fold increase in root and shoot tissues of all cam mutants and wild type compared to the 0 NaCl control treatment. A, No treatment. B, 75mM NaCl. C, 100mM NaCl. D, 150mM NaCl. Error bars represent the SD over three replicate plates each with 50 seedlings each. Mutants with (*) represent the significant level of changes in metabolite levels (P <0.05). A. Root: No treatment 0 1 2 3 4 5 cam 1 c am 2 -1 ca m 2-2 cam 3 -2 cam 3 -3 c am4 cam 5-1 c am 5-2 cam 5-3 ca m 5-4 cam 6 -1 ca m 7-1 WT Meta b o l i t es l e vel n m ol/mgFW GABA Alanine Glutamate Shoot: No treatment 0 1 2 3 4 5 6 c am 1 cam 2-1 c am 2 -2 ca m 3-2 cam 3 -3 c am 4 cam 5-1 cam 5-2 c am 5 -3 ca m 5-4 cam 6 -1 ca m 7-1 WT Met ab o l i tes l eve l nmol/mgF W GABA Alanine Glutamate 163 B. 164 Root: 200mM Mannitol 0 5 10 15 20 25 30 35 c am1 c a m2- 1 cam2 - 2 c a m3- 2 c am3 - 3 c am4 c a m5- 1 c am5 - 2 c a m5- 3 cam5 - 4 cam6 - 1 c am7 - 1 W T Meta bolite s l v e l nm ol/m gFW f o ld i n cr ea se GABA Alanine Glutamate *** *** *** *** * *** *** *** *** *** *** *** *** Shoot: 200mM Mannitol 0.0 0.5 1.0 1.5 2.0 cam1 ca m2- 1 c a m2- 2 ca m3- 2 ca m3- 3 c am4 ca m5- 1 ca m5 -2 c a m5- 3 ca m5- 4 c a m6- 1 c a m7- 1 W T M e t a bolite s le ve l nmol /mgFW fol d inc r ea se GABA Alanine Glutamate *** *** *** *** *** *** *** *** *** *** *** *** *** C. 165 Root: 300mM Mannitol 0 5 10 15 20 25 30 35 40 cam1 cam2 - 1 cam2 - 2 c a m3- 2 c am3 - 3 c am4 c a m5- 1 c am 5- 2 c am5 - 3 c am5 - 4 cam6 - 1 cam7 - 1 W T Me tabolite s l e v e l nm ol/mgFW fold increas e GABA Alanine Glutamate *** *** *** *** *** *** *** *** *** *** *** *** *** Shoot: 300mM Mannitol 0.0 1.0 2.0 3.0 4.0 5.0 6.0 ca m1 c am2-1 c am2-2 ca m3-2 ca m3- 3 c a m4 cam5-1ca m5-2 cam5 - 3 cam5 - 4 cam6-1cam7-1 W T Metabolites l e vel nmol/ m gFW f o ld incr ea se GABA Alanine Glutamate *** *** *** *** *** *** *** *** *** *** *** *** *** D. 166 Root: 400mM Mannitol 0 10 20 30 40 50 60 c am1 c a m2- 1 cam2 - 2 c am3 - 2 c am 3- 3 c a m4 c am5 - 1 cam5 - 2 c am5 - 3 c a m5- 4 cam6 - 1 c am7 - 1 W T Met a bol i t es l evel nm ol / m gFW f o l d i n cr ea se GABA Alanine Glutamate *** *** *** *** *** *** *** *** *** *** *** *** *** Shoot: 400mM Mannitol 0 2 4 6 8 10 12 14 ca m1 ca m 2- 1 ca m 2 - 2 ca m 3- 2 ca m 3- 3 ca m4 ca m 5- 1 ca m 5- 2 ca m 5- 3 ca m 5- 4 c am 6- 1 ca m 7- 1 WT Metabolites level nmol /mgFW fold i n cre ase GABA Alanine Glutamate *** *** *** *** *** *** *** *** *** *** *** *** *** 167 Figure 6. GABA shunt metabolites in cam mutants and wild type A. thaliana seedlings after exposure to 0, 200, 300 and 400mM mannitol grown under continuous light at 25?C for seven days. Root and shoot tissues were harvested and frozen in liquid nitrogen for determination of the level of metabolites as described in materials and methods. Metabolite levels in mutants were calculated and expressed as nmol/mgFW or fold increase relative to the 0 mM mannitol control in root and shoot tissues of all cam mutants and wild type. A, No treatment. B, 200mM Mannitol. C, 300mM Mannitol. D, 400mM Mannitol. Error bars represent the SD over three replicate plates each with 50 one week old seedlings transferred after growth on normal medium. Mutants with (*) represent the significant level of changes in metabolite levels (P <0.05). Table 1. Mutant lines with T-DNA insertion in calmodulin genes of A. thaliana used in this study. All seeds lines were obtained from Dr.Janet Braam, Rice University, except cam1 and cam4 seeds were obtained from the Arabidopsis Biological Research Stock Center, Ohio State University, Columbus. Mutant Insertion Location SALK Line # cam1 3`UTR SALK_107507 cam2-1 Intron SALK_066990 cam2-2 Intron SALK_089283 cam3-2 5`UTR SALK_075669 cam3-3 3`UTR SALK_042391 cam4 5`UTR SALK_149142 cam5-1 3`UTR SALK_007371 cam5-2 3`UTR SALK_073480 cam5-3 5`UTR SALK_138758 cam5-4 Exon II SALK_027181 cam6-1 3`UTR SALK_071609 cam7-1 3`UTR SALK_074336 168 169 APPENDIX I Summary of the normalized expression in root and shoot tissues of A. thaliana wild type at 25?C normal growth and 42?C heat treatment for 2 hr. Root Shoot Gene Control (25?C) Heat Treated (42?C) Control (25?C) Heat Treated (42?C) CAM1 0.3946 1.3834 0.473 0.152 CAM2 2.205 10.6899 1.301 10.3431 CAM3 1.7842 10.8173 1.9786 9.1667 CAM4 1.19 3.0732 1.11 2.6178 CAM5 1.2107 0.7667 1.0873 0.021 CAM6 1.0456 3.3122 1.11 2.0277 CAM7 0.1077 0.6376 0.1083 0.3944 CAM8 1.0233 8.3813 1.0072 7.3041 CAM9 1.1772 4.1119 1.0676 3.9521 170 APPENDIX II Summary of Log 2 fold increase in the expression level of nine CAM genes in root and tissues of cam mutants after 2 hr treatment at 42?C compared to normal growth at 25?C. Numbers in parenthesis represent Log 2 fold change. Change that is less than 1 fold was not considered as a significant change. Root Shoot Genes Increase Decrease Increase Decrease CAM1 cam1 (5) cam3-2 (3) cam5-2 (2) cam1 (3) cam4 (3) cam3-3 (8) cam5-4 (3) cam2-2 (2) cam5-2 (8) cam5-1 (11) cam6-1 (3) cam3-2 (1) cam5-4 (3) cam7-1 (2.4) cam3-3(1) cam6-1(5) cam5-1 (1.2) cam7-1(5) cam5-3 (1) WT (2) WT (2) CAM2 cam1(13) cam3-3 (1) cam1 (1) None cam2-1(2) cam5-1 (1) cam2-1 (1) cam2-2 (2) cam2-2 (1) cam3-2 (4) cam3-2 (3) cam4 (6) cam3-3 (3) cam5-2 (3) cam4 (3) cam5-3 (4) cam5-1 (3) cam5-4 (5) cam5-2 (1) cam6-1 (4) cam5-3 (4) cam7-1 (6) cam5-4 (3) WT (3) cam6-1 (3) cam7-1 (3) WT (2.3) CAM3 cam1 (6) cam3-3 (4) cam1 (3) None cam2-1 (2) cam4 (1) cam2-1 (1) cam2-2 (4) cam5-1 (3) cam2-2 (1) cam3-2 (1) cam6-1 (1) cam3-2 (1) 171 cam5-2 (7) cam3-3 (2) cam5-3 (3) cam4 (4) cam5-4 (4) None cam5-1 (2) cam7-1 (4) cam5-2 (4) WT (3) cam5-3 (2) cam5-4 (5) cam6-1 (4) cam7-1 (6) WT (2) CAM4 cam1 (1) cam2-2 (2) cam1 (1) cam2-1(1) cam2-1 (2) cam3-2 (1) cam4 (1) cam2-2 (1) cam4 (3) cam3-3 (4) cam5-2 (2) cam3-2(1) cam5-2 (6) cam5-1 (3) cam5-4 (1) cam3-3 (1) cam5-4 (2) cam6-1 (1) cam5-1 (1) cam6-1 (3) cam7-1 (2) cam7-1 (2) WT (1) WT (2) CAM5 cam1 (1) cam2-1 (3) None cam1 (1) cam7-1 (1) cam2-2 (3) cam2-1 (7) cam3-2 (4) cam2-2 (5) cam3-3 (9) cam3-2 (7) cam4 (2) cam3-3 (6) cam5-1 (8) cam4 (2) cam5-2 (6) cam5-1 (4) cam5-3 (1) cam5-2 (7) WT (1) cam5-3 (3) cam6-1 (1) cam7-1 (2) WT (2) CAM6 cam1 (5) cam2-1 (3) cam1 (3) cam3-3 (2) cam4 (1) cam2-2 (2) cam3-2 (1) cam5-1(1) cam5-2 (8) cam3-2 (3) cam4 (2) cam5-3 (1) cam5-4 (3) cam3-3 (2) cam5-2 (3) 172 cam6-1 (3) cam5-1 (1) cam5-4 (2) cam7-1 (4) cam5-3 (1) cam6-1 (3) WT (2) cam7-1 (2) WT (1) CAM7 cam1 (13) cam2-2 (1) cam1 (4) None cam4 (10) cam3-2 (1.4) cam2-1 (3) cam5-2 (8) cam3-3 (1) cam2-2 (1.4) cam5-4 (10) cam5-1(1) cam3-3 (2) cam6-1 (10) cam4 (3) cam7-1 (7) cam5-1 (1) WT (3) cam5-2 (2) cam5-3 (2) cam5-4 (4) cam6-1 (3) cam7-1 (2) WT (2) CAM8 cam1(9) cam3-2 (1) cam1 (9.4) cam3-2 (3) cam2-2 (2) cam3-3 (1) cam2-1 (11) cam3-3 (1) cam4 (3) cam5-1 (2) cam4 (4.4) cam5-1 (2) cam5-2 (8) cam5-2 (5.4) cam5-3 (1) cam5-4 (7) cam5-4 (8) cam6-1 (8) cam6-1 (9) cam7-1 (6) cam7-1 (9) WT (3) WT (3) CAM9 cam1 (7) cam3-2 (2) cam1 (8) cam2-2 (1) cam2-2 (1) cam3-3 (3) cam2-1 (1.1) cam3-2 (1) cam4 (2) cam5-1 (5) cam3-3 (1) cam5-3 (8) cam5-2 (3) cam4 (5) cam5-3 (1.2) cam5-2 (5) cam5-4 (6) cam5-4 (4) cam6-1 (5) cam6-1 (5) cam7-1 (6) cam7-1 (5) WT (2) WT (2) 173 APPENDIX III Summary of the normalized expression in root and shoot tissues of A. thaliana cam mutants with the corresponding primer at 25?C normal growth and 42?C heat treatment for 2 hr. Root Shoot Control Heat Treated Control Heat Treated Mutant (25?C) (42?C) (25?C) (42?C) cam1 0.0631 1.8384 0.3043 0.033 cam2-1 1.1035 4.0001 1.0756 2.4115 cam2-2 0.8534 3.7809 0.9692 2.1136 cam3-2 0.4221 0.7786 0.4327 2.1283 cam3-3 0.143 0.0333 0.5586 2.3187 cam4 1.22 11.3113 1 2.3462 cam5-1 2.4133 0.0113 4.5469 0.2498 cam5-2 0.0807 0.001 0.1076 0.001 cam5-3 1.6362 0.7901 3.4744 0.3276 cam5-4 0 0 0 0 cam6-1 0.9875 7.4035 0.43 4.259 cam7-1 0.0193 1.8125 0.19 0.5849 APPENDIX IV. GABA SHUNT METABOLIC PATHWAY IN PLANT AND ITS RELATION WITH OTHER METABOLIC PATHWAYS ? The major metabolites and enzymes that are specifically associated with the shunt pathway are indicated by green color. (Copyright?: Dr.Robert D.Locy) MITOCHONDRIAITOCHONDRI CYTOSOLCYTOSOL PyruvatePyru CoA Acetyletyl-CoA Citrateate Oxaloacetatealoacetate IsocitrateIsocitra ADP+P i CoACoA 2-ketoglutarateogl 2-ketoglutarateogluta CO 2 NADNAD + NADH+H + SuccinylSu in -CoACoA NADNAD + NADH+HNADH+H +CO 2 NADNAD + NADH+HNADH+H + SuccinateSu inat FADH 2 FAD FumarateFuma te Malate H 2 O Pyruvate 174 Succinyl- semialdehyde Su ininyl- lsemial hyde GABAGABA GAB H + CO 2 Glutamatema t t ATP CoA CO 2 NADH+HNADH+H + NADNAD + Isocitratetra CO 2 NADPNAD + NADPH + HNADPH + H + GABAGABA GAB NADH+HH+H + NAD + Alanineanine anin AlanineAlanineanin GAD GABA-TP (1) (2) (3)(3) S S A D H AAT cyt TCA CycleCycl Mi t o cho n d r i a l M i t o c h on dr i al i n n e r me mb ra ne i nne r m e m b r a n e GHBGHB Succinyl- semialdehyde Su in - NADPNAD + NADPHPH + H + Mi t o c h o n d r i a l M i t o c h on dr i al inne r m e m b r a n e i nne r m e m b r a n e semial hydel G H B D H APPENDIX V. T-DNA INSERTION DIAGHRAMS ON ARABIDOPSIS THALIANA CAM GENE OF CAM MUTANTS USED IN THIS STUDY cam3-3 cam5-2 cam7-1 5`UTR Exon I Intron Exon II 3`UTR (65-140 bp) (76 bp) (350-1200 bp) (374 bp) (242-320 bp) cam4 cam5-1 cam2-1 cam2-2 cam1 cam5-3 cam5-4 cam6-1 cam3-2 ? 5`UTR: ~ 65-140bp ? Exon I: ~ 76bp ? Intron: ~ 350-1200bp ? Exon II: ~ 374bp ? 3`UTR: ~ 242-320bp 175