Nitric Oxide Regulates The Adaptation Of Arabidopsis Plant To Sulfur Dioxide Stress

In plant,nitric oxide?NO?functions as an important messenger in plant growth and development,and was induced by multiple environmental stresses to mediate plant responses.High concentrations of sulfur dioxide?SO₂?in the environment can affect plant growth and development.In order to further understanding the function of NO in plant response to SO₂,an Arabidopsis mutant nialnia2?for nitrate reductase[NR]-defective double mutant?and wildtype plants were selected as the materials,we investigated the levels of antioxidants,the activities of enzymes in tricarboxylic acid cycle?TCA cycle?,stomatal aperture,defense-related gene and microRNA?miRNA?gene expression pattern in Arabidopsis exposed to 30 mg/m-3 SO₂.In this study,we use nia1nia2 as a loss-of-NO mutant to analysis the role of NO in the tolerance of Arabidopsis plant to SO₂ stress.The results showed that the activities of superoxide dismutase?SOD?,peroxidase?POD?,glutathione peroxidase?GPX?and glutathione S-transferase?GST?in wildtype plants were higher than those in nialnia2 mutant.Exposure to higher concentrations of SO₂ increased the contents of hydrogen peroxide?H2O₂?and malondialdehyde?MDA?,but the increments of MDA in Arabidopsis nialnia2 mutant was higher than in wildtype.Furthermore,exposure of SO₂ significantly increased both the contents of sulfur-containing compounds such as cysteine?Cys?and glutathione?GSH?,and the activities of enzymes such as SOD,POD,GST and GPX.The increments of these six indicators in Arabidopsis wildtype plants were higher than those in nialnia2 mutant.NO donor sodium nitroprusside?SNP?was able to increase the GSH content and the activites of SOD and GST in nia1nia2 mutant under SO₂ stress.Besides,some defense-related genes such as CAT3,CYP81D1,GSTU24,GPX7,PR4 and PAL3 were differentially expressed at the mRNA level between wildtype and nia1nia2 mutant after plants were exposed to SO₂.These results indicated that NO was involved in the regulation of antioxidant levels and defense-related gene transcription.NR deficiency could affect antioxidants synthesis and the transcriptional levels of defense genes,especially during plant response to SO₂ stress.Collectively,NO can mediate the response of plants to environmental stress through regulating the activities of antioxidant enzymes and the levels of sulfur-containing antioxidants and then related reduction-oxidation reaction and cellular detoxification.The activity of pyruvate dehydrogenase?PDH?in nia1nia2 mutant had no significantly difference from wildtype plants,but activities of isocitrate dehydrogenase?IDH?and a-oxoxglutarate dehydrogenase?OGDH?in wildtype plants were higher than those in nialnia2 mutant.SO₂-fumigated Arabidopsis showed increased enzymes activities,consistent with antioxidant enzymes,wildtype plants had higher increments of TCA cycle critical enzymes than nia1nia2 mutant.These date suggest that NO was involved in the regulation of TCA cycle.Since the TCA cycle provides plant energy and some substances,NO could affect the energy generation and plant metabolism during Arabidopsis adaptation to SO₂ stress.RT-PCR analysis showed that the expression level of MIR398 was decreased along with the increase in transcriptional level of target gene CSD1 and CSD2.The decrease of MIR398c and the increase of CSD2 in widetype plants were higher than in nialnia2 mutant.In addition,the expression level of MIR393 and MIR167 were increased while their target gene AFB2,AFB3,TIR1 and ARF6,ARF8 decreased.We also confirmed that SO₂-fumigated wildtype Arabidopsis showed different gene expression pattern from nialnia2 mutant,like MIR393b,MIR167a,MIR167d and TIR1,AFB3,ARF6.The experimental results indicated that NO can regulate the expression of miRNA in Arabidopsis plants mediating adaption under SO₂ stress.It is confirmed that wildtype Arabidopsis epidermis stomatal aperture was prominently decreased after exposed to SO₂,while no significantly change in nialnia2 mutant.SO₂ stress can induce heat shock,as a countermeasure,heat shock transcription factors?Hsfs?and heat shock proteins?Hsp?were up-regulated,and the expression of HsfA2 in nia1nia2 mutant is not higher than in wildtype.It is demonstrated that NO was involved in stomatal movement,mediating heat shock genes transcription.Collectively,NO can mediate the responses of plants to environmental stress through regulating stomatal movement,inducing both reduction-oxidation reaction and TCA cycle,regulating defense-related genes and miRNA transcription,which resulted in reduced SO₂ uptake and enhanced cellular detoxification to strengthen plants adaption to environment stress.