Nitrosative stress-mediated inhibition of OsDHODH1 gene expression suggests roots growth reduction in rice (Oryza sativa L.)

Abstract

This study monitored the transcriptional response of OsDHODH1 under nitrosative stress conditions relative to the transcripts accumulations for the core mitochondrial cytochrome c oxidase1 (CcOX1) subunit, nuclear CcOX subunits 5b and 5c, two rice nitrate reductases (OsNIA1 and OsNIA2), and nitric oxide excess 1 (OsNOE1) genes. Our findings reveal that short-term exposure of rice seedlings to 1 mM SNP (Nitric oxide donor) applied exogenously for 1 h resulted in significant down-regulation of OsDHODH1 expression in all rice cultivars. In addition, the transcriptional patterns for the CcOX subunits, which are known to have a high affinity for nitric oxide, showed that the core catalytic subunit (OsCcOX1) and the nuclear subunit (OsCcOX5b) were up-regulated, while the nuclear subunit (OsCcOX5c) gene expression was suppressed. OsGSNOR1 expression was enhanced or decreased concomitant with a decrease or increase in SNO accumulation, particularly at the basal level. Moreover, high OsNIA1 expression was consistent with impaired root development, whereas low transcript accumulation matched a balanced root-growth pattern. This suggests that OsNIA1 expression would prevail over OsNIA2 expression under nitrosative stress response in rice. The level of malondialdehyde (MDA) content increased with the increase in SNP concentration, translating enhanced oxidative damage to the cell. We also observed increased catalase activity in response to 5 mM SNP suggesting that potential cross-talk exist between nitrosative and oxidative stress. These results collectively suggest a possible role of OsDHODH1 and OsCcOX5b role in plant root growth during nitrosative stress responses.

Keywords: CAT; CcOX; Lipid peroxidation; Nitric Oxide; OsDHODH1; OsNIA1; OsNIA2; Rice.

Fig. 1

SNP-induced nitrosative stress modulates root development in rice. a The changes observed in root development of rice seedlings exposed to 1 mM and 5 mM SNP for 10 days. b The phenotypic response to nitrosative stress is shown to be genotype-dependent. White bars represent the seedlings grown solely in Hoagland nutrient solution; the grey bars indicate rice seedlings exposed to 1 mM SNP and black bars show seedlings under 5 mM SNP. LSD test revealed a highly significant inhibitory effect of 1 mM SNP compared to 5 mM. Error bars represent ± SD of the means. *P < 0.05, **P < 0.01, ***P < 0.001 and ns non-significant

Fig. 2

Root: Shoot ratio suggests enhanced biomass under SNP. a The pattern of shoot weight and root weight b measured on 14-day old rice seedlings from six cultivars 10 days after 1 mM SNP application. c The ratio between the root and shoot weight refers to the root system development in relation to the shoot growth pattern in response to SNP-induced nitrosative stress. White bars represent unstressed seedlings, while black bars show SNP-treated seedlings. All bars are mean values of three replications ± SD and means are compared between untreated and treated samples. Asterisks and ns marks on top of bars show the significance level. *P < 0.05, **P < 0.01, ***P < 0.001 and ns non-significant

Fig. 3

The transcriptional nitrosative response of OsDHODH1 in relation to CcOX and NR genes. a The transcriptional nitrosative response of OsDHODH1, b the core mitochondrial CcOX encoding subunit 1, c the nuclear subunit (OsCcOX5b), d the nuclear subunit (OsCcOX5c). e Nitrate reductase genes, OsNIA1, f OsNIA2 and g the rice catalase encoded by OsNOE1 in the roots of 14-day old rice seedlings exposed to 1 mM SNP for 1 h. The expression values were normalized to rice Actin1. Student’s t tests revealed highly significant effects of SNP on the relative gene expression. All means are compared with their corresponding controls. ns non-significant and *** P < 0.001

Fig. 4

Intracellular SNO levels and OsGSNOR1 expression in roots 1 h after SNP application. a The increased basal SNO accumulation of level in three different rice genotypes (white bars) concomitant with the reduced pattern of OsGSNOR1 expression at basal level (white bar) b. The bars in both panels (a) and (b), respectively represent the SNO levels and OsGSNOR1 gene expression in the roots of seedlings exposed to 1 mM SNP for 1 h. White bars are means of non-treated samples and black bars represent SNO levels in treated samples ± SD. Asterisks indicate the significant level based on Student’s t test

Fig. 5

Proposed model of the functional interaction of NO with the CcOX subunits. The above figure proposes a model of nitric oxide (NO) interaction with the key core cytochrome c oxidase subunits (CcOX1) harboring the binuclear center, the active site where NO and cyanide (CN) are reported to competitively bind. Continuous lines show the recorded expression of target genes in the current study, while the discontinuous dotted lines indicate reported evidences by other research groups showing inhibition of the CcOX either by NO or CN by binding of NO or CN, not observed in the present study. The lower horizontal bar with different compartments identified by their colors represent the four complexes of the mitochondrial electron transport chain

Fig. 6

Induction of catalase enzyme activity induced under nitrosative stress. The above figure shows the catalase enzyme activity induced 1 h after SNP-induced nitrosative stress was imposed to rice seedlings. White and black bars are. Respectively, means from control and SNP-treated seedlings ± SD. Means from treated seedlings are compared to their corresponding controls and the statistical significance at P < 0.05 is indicated on top of each bar. ns non-significant and ***highly significance

Fig. 7

Change in malondialdehyde (MDA) content 1 h after 1 mM SNP treatment. The extinct of lipid peroxidation was investigated by measuring the malondialdehyde (MDA) content at early time point of 1 mM SNP application. White and black bars are respectively means from control and SNP-treated seedlings ± SD. Means from trea66ted seedlings are compared to their corresponding controls and the statistical significance at P < 0.05 is indicated on top of each bar. ns non-significant