Abstract
Nitric oxide (NO) is an important signaling molecule involved in many physiological processes in plants. We evaluated the role of NO in cold acclimation and freezing tolerance using Arabidopsis (Arabidopsis thaliana) wild type and mutants nia1nia2 (for nitrate reductase [NR]-defective double mutant) and Atnoa1/rif1 (for nitric oxide associated1/resistant to inhibition by fosmidomycin1) that exhibit defects in NR and reduced NO production, respectively. Cold acclimation induced an increase in endogenous NO production in wild-type and Atnoa1/rif1 leaves, while endogenous NO level in nia1nia2 leaves was lower than in wild-type ones and was little changed during cold acclimation. Cold acclimation stimulated NR activity and induced up-regulation of NIA1 gene expression. In contrast, cold acclimation reduced the quantity of NOA1/RIF1 protein and inhibited NO synthase (NOS) activity. These results indicate that up-regulation of NR-dependent NO synthesis underpins cold acclimation-induced NO production. Seedlings of nia1nia2 were less tolerant to freezing than wild-type plants. Pharmacological studies using NR inhibitor, NO scavenger, and NO donor showed that NR-dependent NO level was positively correlated with freezing tolerance. Furthermore, cold acclimation up- and down-regulated expression of P5CS1 and ProDH genes, respectively, resulting in enhanced accumulation of proline (Pro) in wild-type plants. The stimulation of Pro accumulation by cold acclimation was reduced by NR inhibitor and NO scavenger, while Pro accumulation by cold acclimation was not affected by the NOS inhibitor. In contrast to wild-type plants, cold acclimation up-regulated ProDH gene expression in nia1nia2 plants, leading to less accumulation in nia1nia2 plants than in wild-type plants. These findings demonstrate that NR-dependent NO production plays an important role in cold acclimation-induced increase in freezing tolerance by modulating Pro accumulation in Arabidopsis.
Highlights
Nitric oxide (NO) is an important signaling molecule involved in many physiological processes in plants
The DAF-FM-dependent fluorescence intensity in detached Arabidopsis leaves was abolished by the NO scavenger 2-(4carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1-1oxyl-3-oxide, and the fluorescence was markedly increased by treatment with the NO donor sodium nitroprusside (SNP; Supplemental Fig. S1)
Because nitrate reductase (NR) and NO synthase (NOS) are two key enzymes responsible for plant NO biosynthesis in plants, we examined the effects of the NR inhibitor okadaic acid (OA; Kaiser et al, 2002; Rockel et al, 2002) and the NOS inhibitors Nv-nitroL- arginine (L-NNA) and L-NG-nitroarginine methyl ester (L-NAME; Furchgott, 1995) on NO production during cold acclimation
Summary
Nitric oxide (NO) is an important signaling molecule involved in many physiological processes in plants. Cold acclimation reduced the quantity of NOA1/RIF1 protein and inhibited NO synthase (NOS) activity These results indicate that up-regulation of NR-dependent NO synthesis underpins cold acclimation-induced NO production. In contrast to wild-type plants, cold acclimation up-regulated ProDH gene expression in nia1nia plants, leading to less accumulation in nia1nia plants than in wild-type plants These findings demonstrate that NR-dependent NO production plays an important role in cold acclimation-induced increase in freezing tolerance by modulating Pro accumulation in Arabidopsis. There are numerous changes in biochemical and physiological processes during cold acclimation, ranging from accumulation of osmolytes and cryoprotectants (Xin and Browse, 1998) to disruption of reactive oxygen species homeostasis (Suzuki and Mittler, 2006) Several messenger molecules such as abscisic acid (ABA; Xiong et al, 2001), hydrogen et al, 1994), and cytosolic Ca2+. There has been no detailed study to evaluate the role of NO in cold acclimation and freezing tolerance in plants
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