Effects Of SO₂ On MicroRNA Transcription In Plants And Postharvest Preservation Of Table Grape

Sulfur dioxide?SO₂?is a common air pollutant that has complex impacts on plants.Low concentrations of SO₂ can be taken in as sulfur nutrition to promote plant growth.However,higher concentrations of SO₂ can cause leaf chlorosis and necrosis,photosynthesis decline,growth inhibition,and even death.Plants have developed efficient defence mechanisms,including activating stomatal closure,increasing antioxidant enzyme activity and activating gene transcriptional regulation,in response to SO₂ stress.Transcriptional regulation is a key step in the activation of plant defence responses.Studies have shown that the transcriptional response in response to SO₂ stress is related to the alteration of DNA cytosine methylation in genome,and is also regulated by microRNAs?miRNAs?.MicroRNAs?miRNAs?are a class of endogenous small non-coding RNAs of 21²3nt in length,which regulate gene expression at the post-transcriptional level by mRNA leavage and/or translation inhibition.The functions of plant miRNAs range from controlling a wide range of biological and metabolic processes,nutrient homeostasis,and response to biotic and abiotic stresses.Investigations have shown that many abiotic stresses,such as drought,high salinity,low temperature and nutritional deficiency induce differential expression of miRNAs in plants.However,plant miRNA in response to SO₂stress and the regulatory mechanisms are unclear.Another important physiological function of SO₂ is used as the single-most effective preservative in the table grape industry.In order to find a more appropriate preservative for grape storage,transcriptome analysis was carried out on grape berries treated with SO₂.The research found that SO₂ evoked a large scale reprogramming of the grape berry transcriptome associated with oxidative signalling and biotic defence responses.Bioinformatics analysis showed that many of the differentially expressed genes were miRNAs'target genes in Arabidopsis thaliana.Therefore,the preservation effect of SO₂may be related to the regulation of miRNAs.In this study,we used model plant Arabidopsis thaliana and local grapes?Vitis vinifera L.?as experimental materials,to investigate the mechanism of miRNAs in response to SO₂ and its role in plants resistance to infection.Results demonstrated that:1.Deep sequencing?Illumina Hiseq2000?was used for genome-wide identification of miRNAs and their expression profiles in response to SO₂ stress in Arabidopsis thaliana.A total of 27 known miRNAs and 5 novel miRNAs were found to be differentially expressed under SO₂ stress.KEGG pathway analysis on 58 target genes of differentially expressed miRNAs showed that miRNAs are involved in plant hormone signal transduction,secondary metabolitsm,etc.RT-PCR analysis showed negative correlation between miR160,miR393 accumulation and their target genes ARF10/16/17?Auxin Response Factor 10/16/17?and TIR1?Transport Inhibitor Response 1?,AFB2/3?Auxin Signaling F-Box 2/3?,suggesting regulatory roles of miRNAs-regulated auxin signaling pathway during SO₂ exposure.2.The effect of prior exposure to 30 mg·m^-3 SO₂ on defence against Botrytis cinerea?B.cinerea?in Arabidopsis thaliana and the possible action mechanism were investigated.The results indicated that pre-exposure to 30 mg·m^-3 SO₂ resulted in significantly enhanced resistance to B.cinerea infection.SO₂ pre-treatment significantly enhanced the activities of defence-related enzymes including phenylalanine ammonia-lyase?PAL?,polyphenol oxidase?PPO?,?-1,3-glucanase?BGL?and chitinase?CHI?.Transcripts of the defence-related genes PAL,PPO,PR2,and PR3,encoding PAL,PPO,BGL and CHI,respectively,were markedly elevated in Arabidopsis plants pre-exposed to SO₂ and subsequently inoculated with B.cinerea?SO₂+treatment group?compared with those that were only treated with SO₂?SO₂?or inoculated with B.cinerea?CK+?.The contents of lignin,flavonoid and total phenol in the SO₂+group increased significantly.Moreover,SO₂ pre-exposure also led to significant increases in the expression levels of MIR393,MIR160 and MIR167 in Arabidopsis.Meanwhile,the expression of known targets involved in the auxin signalling pathway,was negatively correlated with their corresponding miRNAs.Additionally,the transcript levels of the primary auxin-response genes GH3-like,BDL/IAA12,and AXR3/IAA17 were markedly repressed.Our results demonstrated that 30 mg·m^-3 SO₂ can effectively induce disease resistance against B.cinerea in Arabidopsis.The SO₂-induced disease resistance is associated with inducing defense responses and miRNA-regulated suppression of the auxin signal pathway.3.Seven sRNA libraries?C0,C1,C2,C3,T1,T2,T3?were generated from control?C?and SO₂-treated?T?grape berries of‘Muscat Hamburg'?Vitis vinifera L.?at four stages?0 d,20 d,40 d,60 d?during storage?under commercial conditions?.A total of 148known miRNAs and 49 putative novel miRNAs were identified from seven sRNA libraries via high-throughput sequencing.Sixty-one miRNAs?54 known miRNAs and 7novel miRNAs?were differentially expressed in response to SO₂ fumigation.Among these61 differentially expressed miRNAs?DEMs?,only miR3635-3p had no predicted targets.A total of 543 target genes with functional annotations were predicted from the rest of 60miRNAs.Gene Ontology?GO?analysis showed that 17 categories in biological processes were classified by the target genes,followed by 15 categories in cellular component and11 categories in molecular function.Based on the DEM analysis in the seven libraries,six DEMs were selected to verify their transcription levels.qRT-PCR and Pearson's correlation coefficient analysis validated the negative correlation expression patterns between six DEMs and their target genes.The 6 pairs of miRNA-target genes were found to be involved in fruit softening,senescence,ethylene synthesis,secondary metabolism and so on during storage.These DEMs regulated genes involving in inhibiting fruit physiological aging and resisting pathogen infection during storage.A network of miRNA-targets that regulate SO₂ preservation has been proposed.Our results indicated that a diverse set of miRNAs in grape berries are SO₂-responsive,and the secondary metabolic pathway regulated by miRNAs plays an important role during SO₂ preservation.4.The effect of a postharvest treatment with SO₂ under commercial conditions on table grapes?‘Red Globe'and‘Muscat Hamburg'?quality and the possible action mechanisms were investigated.The results indicated that SO₂ could reduce the pH value of the fruit surface,thus effectively inhibiting the growth of B.cinerea,reducing the fruit threshing and decay and maintaining the fruit quality.The good fruit rate of SO₂ treatment group was significantly higher than that of the control group.Furthermore,SO₂ markedly enhanced activity of PAL,promoted the accumulation of phenolic compounds including total phenol,flavonoid and lignin,and improved the PPO activity.Moreover,SO₂significantly elevated transcription levels of pathogenesis-related?PR?proteins genes chitinase3?CHI3?,CHI1b and?-1,3-glucanase?PR2?.Consistently,the activities of CHI and BGL remarkably increased in SO₂ treatment group.Our founding indicated that SO₂could activate defense responses associated with secondary metabolism and PR proteins for enhancing disease resistance and thereby extending the storage life of table grapes.In summary,SO₂ induced changes in the expression levels of miRNAs in Arabidopsis and grape berries.The target genes of SO₂-responsive miRNAs encode transcription factors and proteins that regulate plant hormone signal transduction,secondary metabolitsm,etc.SO₂ enhanced disease resistance against B.cinerea associated with inducing defense responses related to secondary metabolitsm,and miRNA-regulated suppression of the auxin signal pathway.During SO₂ preservation,miRNA could regulate grape fruit disease resistance response,inhibit fruit physiological senescence,maintain fruit taste,promote fruit preservation and extend postharvest life of table grapes.