| Cold stress can affect the changes of fatty acids and lipid components,and then destroy the cell membrane integrity and fluidity,which is also an important reason for the chilling injury of cold-sensitive fruits and vegetables.The unsaturated fatty acids as well as phosphatidic acid(PA)produced by phospholipids degradation largely determines the cell membrane integrity and reflects the damage degree of cold-sensitive fruits and vegetables in response to cold stress.Therefore,it is of great significance to study the transcription and post-transcriptional regulation mechanism of fatty acid desaturation and phospholipids degradation induced by cold stress.Banana,as a typical tropical and subtropical fruit,is prone to chilling injury when stored below 13 ℃,mainly manifested as peel browning,flesh powdering,abnormal maturity and easy to rot,which is also the main limiting factor for banana cold storage.Thus,the better understanding of physiological response to cold stress of harvested banana fruits and improving techniques to induce chilling tolerance is in urgent demand for its storage and cold chain logistics.Previous studies have found that pre-cooling treatment can enhance the cold tolerance of banana fruits by inhibiting the reduction of membrane lipid unsaturated fatty acids,fatty acid unsaturated index and fatty acid unsaturation,showing that desaturation of fatty acids is related to cold tolerance of bananas.However,there are few reports on the transcriptional and post-transcriptional regulatory mechanisms of fatty acid desaturation in bananas in response to cold stress.In addition,exogenous propylene,an ethylene functional analog,was involved in Ma NAC1 regulating the ICE1-CBF cold signal pathway to improve cold tolerance in banana fruit.NAC TFs,as one of the largest family of plant transcription factors,play a key regulatory role in the complex signal network involved in plant abiotic stress response and respond to various plant hormones.However,ethylene may be involved in the regulation of multiple metabolic pathways(such as PA production)by NAC TFs to induce cold tolerance in banana fruit,and the induction mechanism is not deep enough.Therefore,this study reveals for the first time the epigenetic regulation mechanism of fatty acid desaturation during banana chilling and the transcriptional regulation mechanism of phospholipids degradation to produce PA in ethylene-mediated banana cold tolerance.The main results are as follows:1.During the banana fruit response to cold stress,the relative conductivity and malondialdehyde(MDA)content were significantly increased,the reduction rate of unsaturated fatty acids linoleic acid(LA,C18:2)and α-linolenic acid(ALA,C18:3)was significantly weakened,and ALA/LA ratio as well as transcriptional levels and histone H3 and H4 levels of ω-3 Ma FADs were significantly increased.Ma MYB4 inhibited their expression by binding to the ω-3 Ma FADs(Ma FAD3-1,Ma FAD3-3,Ma FAD3-4 and Ma FAD3-7)promoters.The interaction between the histone deacetylase Ma HDA2 and Ma MYB4 enhanced Ma MYB4-mediated suppression of ω-3 Ma FADs transcription.In short,Ma MYB4 recruited the histone deacetylase Ma HDA2 to regulate the expression ofω-3 fatty acid desaturase genes,which in turn affected changes in fatty acid components during banana chilling.2.During the banana fruit response to cold stress,the relative conductivity and MDA content were significantly increased,catalase(CAT),super oxide dimutese(SOD),and peroxidase(POD)activities were significantly decreased,PA in the phospholipid component was gradually accumulated,accompanied by the decrease in phosphatidylethanolamine(PE),phosphatidylcholine(PC)and phosphatidylinositol(PI),which damaged cell membrane stability and fluidity,eventually leading to chilling injury in banana fruit.However,exogenous ethylene treatment inhibited the increase of relative conductivity and MDA content and the decrease of CAT,SOD and POD activities,especially reduced PA production,maintaining the stability of cell membrane structure and keeping the good appearance of banana fruit.Therefore,PA production plays an important role in banana fruit response to cold stress,and ethylene may participate in the transcriptional regulation process of PA produced by degradation of phospholipids to induce cold tolerance in banana fruit.3.Phospholipids were mainly degradated to produce PA through phospholipase D(PLD)pathway and diacylglycerol kinase(DGK)/phospholipase C(PLC)pathway.Based on banana genome and transcriptome data,we found 13 PA production key enzyme genes,whose expressions were up-regulated by cold stress and inhibited by ethylene,including 8Ma PLDs(Ma PLDα1/4,Ma PLDβ1/2/3,Ma PLDδ1/2/5),3 Ma DGKs(Ma DGK1/2/3),and 2Ma PLCs(Ma PLC1/2)named after evolutionary tree analysis.Furthermore,we identified two NAC TFs with similar expression patterns to PA synthesis genes,named Ma NAC25 and Ma NAC28,which were nuclear localized proteins from different subgroups.Cold-responsive Ma NAC25 and Ma NAC28 could positively regulate PA producing genes in banana fruits,and promote PA accumulation,affecting the stability of cell membrane structure and promoting the occurrence of chilling injury in banana fruit.4.The mechanism of NAC TFs regulating ethylene-mediated cold tolerance of banana fruit by modulating PA production was analyzed in depth,and its function was verified by genetically modified tomato technology.The results showed that exogenous ethylene inhibited the cold-induced upregulation of Ma NAC25,Ma NAC28,and PA producing genes expression,reduced the transcriptional activation effect of Ma NAC25 and Ma NAC28 on PA producing genes,and thereby enhanced cold tolerance of banana fruit.Meanwhile,exogenous ethylene also repressed ebhanced cold sensitivity of tomato fruit confirred by overexpression of Ma NAC25 and Ma NAC28 by inhibiting PA production in transgenic tomato fruit.5.DAP-Seq analysis showed that the two biologically duplicated Ma NAC25 and Ma NAC28 binding regions(17407 and 37606 overlapping peaks,respectively)had high confidence,most of Ma NAC25(49%)and Ma NAC28(68%)binding sites were located in the promoter region,and the binding signals were the strongest in the region adjacent to TSS,indicating that DAP-Seq could analyze the binding sites of Ma NAC25 and Ma NAC28 on the whole genome to a certain extent.Moreover,through strict MEME-Ch IP analysis and screening,we found the three rich binding motifs of Ma NAC28 with high significance(E-value = 4.1e-450,9.5e-336 and 2.5e-231,respectively),but not containing the core binding sequence CACG/CGTG of NACs.And EMSA verified that Ma NAC25 and Ma NAC28 could all recognize three rich binding motifs of Ma NAC28,which may be the new binding motifs for NAC TFs response to cold stress,providing further selectivity for target genes regulated by different NAC TFs.6.Ma NAC25 and Ma NAC28 could interact to form a positive feedback loop,which positively regulated PA production.Interestingly,cold stress promoted the positive regulation of PA production by the feedback loop formed by Ma NAC25 and Ma NAC28,and then negatively regulated the cold tolerance of banana fruit.However,exogenous ethylene inhibited the promotion effect of cold stress to enhance cold tolerance of banana fruit.This helps us understand the co-regulation mechanism of PA production by NACs TFs and the mechanism of ethylene on improving cold tolerance of cold-sensitive fruits.In summary,the results of this study provide new evidence for understanding the mechanism of NAC TFs regulating ethylene-mediated cold tolerance of banana fruits. |
