Research On The Transcriptional Regulation Mechanism Of CaPLD?4 Gene In Bell Pepper During Chilling Injury Based On Transcriptome Sequencing

Green pepper(Capsicum annuum L.),also known as bell pepper,evolves from the tropical South American pepper.Because of its sweet taste,thick flesh but not spicy,it is loved by people.In addition,pepper fruit is rich in vitamin C,vitamin A and phenolic substances,which has a high edible value and economic value,and its annual consumption remains high in the world.For a long time,low temperature storage and transportation is the main means to keep the quality and prolong the shelf life of the bell pepper fruit.However,due to the sensitive to low temperature,pepper fruit is likely to suffer from chilling injury(CI),showing seeds and calyx browning,pitting,especially in the range of 0? to 7?,which seriously affects the quality and value.Therefore,it is of great theoretical and practical significance to study the mechanism of CI and explore the corresponding control technology in bell pepper under low temperature.In this paper,the CI mechanism and the changes of related indicators in bell pepper under low temperature stress were studied through the observation and analysis the morphological,physiological and biochemical indexes.Then a comprehensive study of differentially expressed genes(DEGs)and transcription factors(TFs)between CI and non-CI bell pepper fruit were analyzed via transcriptome sequencing.Meanwhile,we conducted bioinformatics analysis and found the metabolic pathways that DEGs involved in.On this basis,we focused on the key structure genes in membrane lipid metabolism and screened its targeted TFs with the help of yeast one-hybrid(Y1H),electrophoretic mobility shift assays(EMSA)and ?-glucuronidase reporter assays(GUS),which explored the transcriptional regulation mechanism.Based on the above mechanism,the effects of control technology on pepper CI at low temperature were discussed.The results of this study provide a reference for other studies on the CI mechanism of fruits and vegetables and also offer a theoretical basis for the research and development of the CI control technology in bell pepper.The main results of this research are as follows:The changes of pepper apparent,the anatomical structure,cell structure,water distribution,membrane lipid components and enzyme activities related to membrane lipid metabolism of bell pepper stored at 4? and 10? were different.It was found that the bell pepper fruit displayed CI symptoms at 4?,and the cell membranes damaged seriously.The organelles collapse,water deficit and the drop of free water content were obvious.The relative conductivity,malonaldehyde(MDA)and proline content increased with the extension of storage time.Besides,membrane lipid content significantly degraded,among of which,monogalactosyldiacylglycerol(MGDG),digalactosyldiacylglycerol(DGDG),phosphatidylcholine(PC)and phosphatidyl ethanolamine(PE)and phosphatidic acid(PA)have been found to response to low temperature.The proportion of unsaturated fatty acids such as linoleic acid and linolenic acid decreased along with the extension of time,whereas the saturated fatty acid including palmitic acid and stearic acid increased.At the same time,the activities of phospholipase D(PLD)and lipoxygenase(LOX)are significantly higher at 4?than that at 10?.The results confirmed Ca PLD and Ca LOX enzymes were activated under low temperature stress to participate in the membrane lipid metabolism path,as a result,the membrane lipid composition and cell membrane properties were changed.Then process of membrane lipid peroxide degradation was triggered,which leads to the imbalance of intracellular reactive oxygen metabolism and causes a series of secondary metabolic reactions,resulting in the CI of bell pepper.Transcriptome sequencing(RNA-seq)was performed on bell pepper samples stored at4?,10? and the day after harvest.A total of 4,437 new genes were discovered,of which3,231 were functionally annotated.A total of 3863 DEGs were produced by comparing the data of 4? and 10? groups.The gene ontology(GO)classification showed that “cellular process”,“metabolic process”,“response to stimulus”,“organelle”,“membrane”,“binding”and “nucleic acid binding translation factor activity” were the main process that DEGs involved in during the CI of bell pepper fruit.The GO enrichment analysis results showed that the process of “plant-type hypersensitive response”,“external side of plasma membrane”,“chloroplast thylakoid membrane” and “protein binding” DEGs participated in were highly significant.In addition,the kyoto encyclopedia of genes and genomes(KEGG)metabolic pathway analysis results showed that “glycero lipid metabolism”,“glycerophospholipid metabolism”,“fatty acid metabolism”,“arginine and proline metabolism”,“glycosphingolipid biosynthesis-globo series”,“plant hormone signal transduction” and “glutathione metabolism”were the main metabolic pathway DEGs involved in.Additionally,250 differentially expressed TFs including 41 families such as AP2/ERF,MYB,NAC,C2H2 and WRKY were identified by comparing DEGs produced at 4? and 10?.Based on the physiological analysis and transcriptome analysis results of CI in green pepper,the key gene Ca PLD?4 involved in membrane lipid metabolism pathway were identified,and the upstream transcription factors and their regulatory patterns were studied.The length of 1.3kb upstream promoter sequence of Ca PLD?4 gene was cloned,containing multiple transcription factor binding sites related to abiotic stress.Then the regulation pattern between TF and Ca PLD?4 was explored via Y1 H,EMSA and GUS.It was proved that the TF Ca NAC1 can specifically bind to the cis-acting element CTGCAG to regulate thetranscription of Ca PLD?4 gene,participating in the regulation of phospholipid degradation.Moreover,Ca NAC1 was an ubiquitinated protein containing 7 ubiquitinated peptides.Furthermore,the result of subcellular localization indicated that Ca NAC1 was located in the nucleus.Protein domain comparison analysis showed that Ca NAC1 had a conserved NAM domain,and the phylogenetic tree results showed that the protein sequence of Ca NAC1 was close to the Ca NAC1 sequence of hot pepper and the At NTL6 sequence of Arabidopsis.This result confirmed that Ca NAC1 positively regulated the transcription of Ca PLD?4 gene by targeting cis-acting elements in the promoter of Ca PLD?4 under low temperature stress,in return participating in the process of membrane lipids degradation.Based on the results of transcriptional regulation in bell pepper during CI,the effects of melatonin on the CI of bell pepper during low temperature storage were further discussed.The results showed that at 4?,the occurrence time of CI in bell pepper treated with melatonin was delayed,the CI incidence and CI index were both reduced,and the pulp cells and the cell structure were more complete and full.In addition,the relative conductivity and MDA content of melatonin-treated bell pepper decreased significantly,whereas the content of proline increased.Moreover,the degradation of PC and PE in the cell membrane was reduced after melatonin treatment,and the unsaturation of fatty acids was increased.In the treatment group,the transcription levels of Ca NAC1,Ca PLD and Ca LOX genes as well as the activities of Ca PLD and Ca LOX enzyme were significantly decreased.Moreover,the expression level of gene coding the superoxide dismutase(SOD),peroxidase(POD),catalase(CAT)and ascorbate peroxidase(APX)increased obviously in melatonin-treated pepper,which suggested that melatonin treatment maybe triggered the antioxidant system in pepper to reduce the membrane lipid peroxidation induced by low temperature,thus increased the tolerance ability to the low temperature stress.