Heat and cold stresses phenotypes of Arabidopsis thaliana calmodulin mutants: regulation of gamma-aminobutyric acid shunt pathway under temperature stress

Abstract

Plants have evolved mechanisms to cope with changes in surrounding temperatures. T-DNA insertions in seven calmodulin genes of <em>Arabidopsis thaliana</em> were used to investigate the role of specific calmodulin isoforms in tolerance of plants to low and high temperature for seed germination, susceptibility to low and high temperature induced oxidative damage, and changes in the levels of gammaaminobutyric acid (GABA) shunt metabolites in response to temperature stress. Exposure of wild type (WT) and <em>cam</em> mutant seeds at 4°C showed reduction in germination of <em>cam5-4</em> and <em>cam6-1</em> seeds. Exposure of cam seedlings to 42°C for 2 hr showed reduction in seed germination and survival of seedlings in <em>cam5-4</em> and <em>cam6-1</em> mutants compared to WT and other <em>cam</em> mutants. Oxidative damage by heat and cold stress measured as the level of malonaldehyde (MDA) was detected increased in root and shoot tissues of cam5- 4 and cam6-1. Oxidative damage by heat measured as the level of MDA was detected in root and shoot of most cam mutants with highest levels in <em>cam5-4</em> and <em>cam6-1</em>. Level of GABA shunt metabolites in seedlings were gradually increased after 1 hr and 3 hr with maximum level after 6 hr and 12 hr treatments at 4ºC. GABA shunt metabolites in both root and shoot were generally elevated after 30 min and 1 hr treatment at 42°C, and increased substantially after 2 hr at 42°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. Alanine levels showed significant decreases in all cam mutants and in WT for 30 and 60 min of heat stress. Sensitivity of <em>cam5-4 </em>and <em>cam6-1</em> to low temperatures suggests a role of the <em>CAM5</em> and <em>CAM6</em> 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 calmodulin protein (<em>cam</em>) in the activation of glutamate decarboxylase (GAD) after exposure to cold, while increased metabolite levels may indicate involvement of other factors like reduction in cytoplasmic pH in cold regulation. Initial general elevation in GABA shunt metabolites after 30 min heat treatment in cam mutants suggests regulation of GABA level by <em>cam</em>. These data suggest 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.