Physiological And Molecular Mechanism Of Salicylic Acid In Regulating Growth Of Oryza Sativa And Toxicity Identification Under Quinclorac Stress

Quinclorac is a new class of highly selective auxin herbicides that have been used as pre-and post-application in transplanted and directly seeded rice.But excessive use of herbicides can cause higher risks to the growth of rice at early stage.Salicylic acid(SA)is a kind of endogenous signal molecule in plant and plays a vital role in plant defense system.This thesis mainly studied the ameliorating role of SA in rice(Xiushui134 and Zhejing 88)on morphological,physio-biochemical processes under quinclorac stress.We also tried to build quinclorac toxicity identification in rice with hyperspectral image technology.Then RNA-Seq and iTRAQ methods were used to analyse the expression level in genes and proteins.The major results are as follows:1.The sensibility test of four common used herbicides(bensulfuron methyl,quinclorac,penoxsulam,butachlor)in rice cultivar Xiushui 134 was investigated.The influence level on rice biomass was quinclorac>butachlor>bensulfuron methyl>penoxsulam.The recommended dose of other three herbicides had no significant effects on fresh weight except quinclorac(1 g/L).The changes in antioxidant enzymes after 6 h,12 h and 72 h herbicide treament were studied.Superoxide dismutase(SOD)was qiuckly first expressed after 6 h.Ascorbic acid peroxidase(APX)was accumulated more in leaf while catalase(CAT)was more in root.2.The effects of SA(10 mg/L)on morphological,physio-biochemical changes and abscisic acid(ABA)related gene expressions in four-leaf stage rice seedlings under quinclorac stress were studied.Quinclorac with 0.1 g/L concentration had slight effects on rice while 0.5 g/L significantly inhibited the growth.Quinclorac stress induced the accumulation of malonaldehyde(MDA)and reactive oxygen species(ROS)and destroyed the structure of chloroplasts and mitochondria.Meanwhile,ABA related genes(OsNECD and OsABAox)were up-regulated to restrain the growth.SA pre-treatment remarkably increased the activities of antioxidant enzymes,glutathione s-transferase(GSTs)and reduced glutathione(GSH),decreased the contents of MDA and ROS,downregulated ABA related gene expressions to mediate quinclorac toxicity.The cultivar Xiushui 134(XS134)is more tolerant than Zhejing 88(ZJ88)to quiclorac.3.The feasibility of quinclorac toxicity detection in rice cultivars(Xiushui 134 and Zhejing 88)was discussed.We selectd spectrum from 390-1030 nm to identify quinclorac toxicity.Besides the general physiological parameters analysis,we extracted the total spectrum and average spectrum,chose the optimal wavelength based on PCA analysis and bulit SVM model to get prediction map.The discriminant results of SVM models showed the classification accuracy of XS134 and ZJ88 were 84.6%and 92.5%,respectively.The roots were easier to distinguish thant leaves.Outcomes demonstrated the good performance of hyspectral imaging to discern quinclorac toxicity and alleviation of SA.4.RNA-Seq for gene identification and transcriptional profiling of the mechanism of salicylic acid alleviation under quinclorac stress in rice cultivar Xiushui 134 was analysed.Quinclorac induced 2207 differentially expressed genes(DEGs)(1427 up-regulated,780 down-regulated)and they were enriched in GSH metabolism,porphyrin and chlorophyll metabolism,biosynthesis of secondary metabolites,amino acids metabolism,glyoxylate and dicarboxylate metabolism,carbon fixation and so on.It also influenced AP2-EREBP family,MYB family and WRKY family transcription factors.After SA pre-treamt,697 genes were dfferentially expressed including 382 up-regulated genes and 315 down-regulated genes.These genes were enriched in GSH metabolism,biosynthesis of secondary metabolites,phenylpropanoid biosynthesis,fatty acid metabolism,flavonoid biosynthesis,glyoxylate and dicarboxylate metabolism.Transcription factors were distributed in bHLH,MYB,Tify and WRKY families.Quantitative real-time PCR resluts revealed quinclorac stress induced the expression of glutathion reductase(GR)genes(OsGR2,OsGR3),SA further up-regulated the expression.Quinclorac also mediated the accumulation of acetaldehyde in rice while SA enhanced the expression of OsALDH2B5 and OsALDH7 to accelerate the metabolism to protect rice.Quinclorac induced AP2/EREBP family transcription factors to express.SA was found to up-regulate OsWRKY45 and OsWRKY76 expression.5.iTRAQ method was used to analyse proteome data of the mechanism of salicylic acid alleviation under quinclorac stress in rice cultivar Xiushui 134.Results showed that quinclorac induced 49 differentially expressed proteins to up-regulate,98 proteins to down-regulate.Pathway enrichment showed these proteins were related with cysteine and methionine metabolism,glyoxylate and dicarboxylate metabolism and phenylpropanoid biosynthesis.SA stimulated 56 proteins to up-regulate and 68 proteins to down-regulate.The most abundant pathways were alanine,aspartate and glutamate metabolism,riboflavin metabolism,arginine biosynthesis,terpenoid backbone biosynthesis and 2-oxocarboxylic acid metabolism.Correlation analysis between transcriptome and proteomics exhibited that under quinclorac stress,proteins related with synthesis of chlorophyll such as Mg-chelatase H subunit,CAT,Geranylgeranyl Reductase and protochlorophyllide oxidoreductase B were inhibited.SA regulated phenylalanine ammonia-lyase,rice ferritin and Mn-SOD family proteins.