Abstract
Exposure of plants to different biotic and abiotic stress condition instigates significant change in the cellular redox status; resulting in the elevation of reactive nitrogen species that play signaling role in mediating defense responses. Heavy metal associated (HMA) domain containing genes are required for spatio-temporal transportation of metal ions that bind with various enzymes and co-factors within the cell. To uncover the underlying mechanisms mediated by AtHMA genes, we identified 14 Arabidopsis HMA genes that were differentially expressed in response to nitrosative stress through RNA-seq analysis. Of those 14 genes, the expression of eight HMA genes was significantly increased, whereas that of six genes was significantly reduced. We further validated the RNA-seq results through quantitative real-time PCR analysis. Gene ontology analysis revealed the involvement of these genes in biological processes such as hemostasis and transport. The majority of these nitric oxide (NO)-responsive AtHMA gene products are carrier/transport proteins. AtHMAD1 (At1g51090) showed the highest fold change to S-nitrosocystein. We therefore, further investigated its role in oxidative and nitrosative mediated stress conditions and found that AtHMAD1 has antagonistic role in shoot and root growth. Characterization of AtHMAD1 through functional genomics showed that the knock out mutant athmad1 plants were resistant to virulent Pseudomonas syringae (DC3000) and showed early induction and high transcript accumulation of pathogenesis related gene. Furthermore, inoculation of athamd1 with avirulent strain of the same bacteria showed negative regulation of R-gene mediated resistance. These results were supported by hypersensitive cell death response and cell death induced electrolyte leakage. AtHMAD1 was also observed to negatively regulate systemic acquired resistance SAR as the KO mutant showed induction of SAR marker genes. Overall, these results imply that NO-responsive AtHMA domain containing genes may play an important role in plant development and immunity.
Highlights
Nitric oxide (NO), one of the smallest diatomic molecules is a highly reactive gaseous free radical and is the center of attention for most of the plant scientists due to its marvelous regulatory role in both plants and animals (Tuteja et al, 2004)
The results indicated that 93.30% of genes were involved in cellular processes, 2% in metabolic processes, 33% in biological regulation (Hemostasis), and 93.30 % are involved in localizaition [transport (Figure 1C)]
Our results showed that the first domain (1–80 amino acids) of AtHMAD1 can bind to different metal ligands at different rates (Supplementary Figure S1D), as represented by pocket multiplicity (PM; the frequency at which a particular pocket was found in a set of ligand- protein structures)
Summary
Nitric oxide (NO), one of the smallest diatomic molecules is a highly reactive gaseous free radical and is the center of attention for most of the plant scientists due to its marvelous regulatory role in both plants and animals (Tuteja et al, 2004). Two major players of the negative interaction, biotic and abiotic stresses are the prime challenges for plants These stresses lead to the production of increased reactive oxygen species (ROS) such as hydrogen peroxide, superoxide and hydroxyl radicals in the cell (Sudhakar et al, 2001; Xiong et al, 2002). Another type of important molecules produced during stress responses are reactive nitrogen species (RNS) (Delledonne et al, 1998) that has been shown to regulate a plethora of physiological activities and plant functions including plant defense and development (Delledonne et al, 1998; Durner et al, 1998). The enzyme GSNO reductase (GSNOR) is the primary enzyme that controls intracellular levels of GSNO and S-nitrosylated proteins in eukaryotes through de-nitrosylation (Feechan et al, 2005; Espunya et al, 2012)
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