Role of Arabidopsis Thaliana Calmodulin Isoforms in Tolerance to Abiotic Stress

Abstract

Calmodulin and Ca2+ 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 (cam) 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 seedlings grown at normal growth temperature (25oC) and during heat stress at 42oC 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 25ºC, 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 25oC 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 42oC 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. 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 are sensitive to all abiotic factors tested including heat and low temperature exposures, osmotic and salinity stress. Surprisingly, other cam5 alleles, which have 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 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 appreciable expression of CAM6. It seems likely that sensitivity of cam6-1 to all environmental stress tested may be mediated indirectly through other alternative mechanism. Levels of GABA metabolites are nearly similar in WT and cam mutants, respectively. An initial elevation in the level of GABA shunt metabolites followed by a significant 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. Differences in metabolite levels between WT and cam mutants were observed. This result suggests that individual CaM protein may not be involved in regulation of GABA shunt pathway; rather collective amounts of CaM proteins may regulate GAD activity in response to abiotic stress. Our result with mRNA level analysis of the 9 CAM genes in the calmodulin mutants (cam) used in this study suggests some level of compensation in expression CAM genes in Arabidopsis thaliana calmodulin (cam) mutants.