Regulation Mechanism Of 1-MCP On Fresh Common Bean Lignocellulosis

Fresh common bean（Phaseolus vulgaris L.）is popular due to fresh,tender,thickening,delicious,and nutrition in pod.However,postharvest fresh common beans are prone to quality deterioration,such as water loss,yellowing,rust,and lignocellulosis,especially lignocellulosis leading to the increase in length of pod tendon and the tissue rough,which seriously affect its edible quality.Previous studies have found that 1-MCP treatment can significantly inhibit postharvest lignocellulosis of fresh common bean,ethylene may be involved in its regulation,but its regulatory mechanism is still unclear.Therefore,we systematically explored the changes of endogenous hormones and cell wall metabolism and their relationship of fresh common bean during pod development,as well as the effects of ethylene and 1-MCP on the metabolism related to postharvest lignocellulosis and the regulatory genes responding to 1-MCP in fresh common bean,and identified the key transcription factors and mi RNAs and their possible regulatory effects.The main conclusions are as follows:1.The changes of microstructure,endogenous hormones and cell wall metabolism during the development of fresh vegetable bean pods were studied by morphological observation and biochemical analysis.The results showed that the content of Gibberellins（GA₃）and 6-benzylaminopurine（6-BA）decreased,the of ethylene production rate and jasmonate（JA）content decreased firstly and then increased during the development of pods.The pods were mainly elongated before the 10^th day after anthesis,and the thickening of pods after the 10^th day after anthesis.Sucrose synthetase（Su Sy）promoted sucrose synthesis before 10 days after anthesis.Sucrose content decreased and cellulose content increased with the activity decrease of Su Sy and cellulase（Cx）.G-lignin deposition in xylem at 5 days after anthesis,G-lignin and S-lignin were deposited in phloem,gradually deepened and diffused to both sides,S-lignin only deposited in phloem.The high phenylalanine ammonia lyase（PAL）activity provided the substrate for lignin biosynthesis at the 5th day after anthesis,and then the activity increase of cinnamyl alcohol dehydrogenase（CAD）promoted lignin synthesis,the activity increase of superoxide dismutase（SOD）promoted the conversion of superoxide anion(O₂^·-)to hydrogen peroxide（H₂O₂）,and the polymerization of lignin with peroxidase（POD）activity.Polygalacturonase（PG）activity,sodium-carbonate-soluble-pectin（SSP）and water soluble pectin（WSP）content increased firstly and then decreased,pectin methylesterase（PME）activity decreased,chelator soluble pectin（CSP）content decreased firstly and then increased.Correlation analysis showed that ET,GA₃,6-BA and abscisic acid（ABA）may promote lignin synthesis by enhancing PAL and POD activity,JA may inhibit lignin polymerization by inhibiting the transformation of O₂^·-to H₂O₂ and POD activity.IAA and SA may be involved in pectin regulation metabolism.These results indicated that the lignocellulosis of fresh common bean is the result of the coordinated regulation of various endogenous hormones from substrate supply,key enzymes of biosynthesis to oxidative polymerization,among which ET plays an important role in the lignocellulosis of fresh common bean.2.To clarify the regulatory effect of ET on postharvest lignocellulosis of fresh common bean,the structure and physiological basis of 1-MCP regulating its lignocellulosis was studied by analyzing the effects of exogenous ethylene,ethylene inhibitor 1-MCP and their concentrations on the microstructure,components and key enzyme activities related to lignocellulosis metabolism of main fresh common beans during storage.The results showed that ethylene treatment promoted the respiration,the activities of key enzymes of cellulose metabolism and degradation（SPS,Su Sy and Cx）,and the activities of key enzymes of Lignin Metabolism（PAL,CAD and POD）,resulting in the increase of lignified cell group,thinning of sclerenchyma,thickening of vascular bundle and increase of lignified cells,thus promoting the lignocellulosis,while 1-MCP did the opposite.Compared with the control,high concentration(1.5μL^-1)of1-MCP accelerated the lignocellulosis by promoting senescence,while low concentration of 1-MCP(0.5μL^-1)inhibited the respiration rate,the activities of enzymes（PAL,CAD,POD and Cx）related to lignocellulosis metabolism,inhibited the conversion of reducing sugar to cellulose,delayed the increase of tendon length,the decrease of pod relative thickness and hardness,and delayed lignocellulosis of fresh common bean.The effects of 1-MCP on lignocellulosis of different varieties of fresh common beans were different,1-MCP treatment inhibited respiration,1-MCP inhibited the conversion of reducing sugar to cellulose by reducing the activities of sucrose phosphate synthase（SPS）and Su Sy,1-MCP treatment reduced the synthesis of lignin monomers by inhibiting the activities of PAL,4-coumarate:coenzyme A ligase（4CL）,CAD,and inhibited the polymerization of lignin monomers by suppressing the POD activity and the conversion of O₂^·-to H₂O₂,and ultimately inhibiting the lignocellulosis of fresh common bean.However,the transcriptional regulation mechanism of 1-MCP on its lignocellulosis is still unclear.3.In order to further clarify the transcriptional regulation mechanism of 1-MCP on postharvest lignocellulosis of fresh common beans,the physiological effects of 1-MCP on postharvest lignocellulosis were analyzed.The results showed that the 1-MCP treatment group had significant difference from the control group at 15 days of storage.Therefore,Transcriptomics was used to analyze the samples stored at harvest（CK0）,control（CK15）and 1-MCP treatment（T15）15 days of storage.The results showed that there were 5882 co-expressed genes in CK15/CK0 and T15/CK0.Genes in lignin synthesis pathway were inhibited by 1-MCP,including PAL,CCR,4CL,COMT,HCT,CAD,POD and ROBH,but there was no significant difference in LAC genes.It was also found that 1-MCP regulated the expression of key enzyme coding genes of lignin synthesis intermediate products and monomer decomposition,including coniferyl-alcohol glucosyltransferase（CAGT）and coniferyl-aldehyde dehydrogenase（ALDH）.1-MCP also significantly inhibited the expression of cellulose synthesis genes,hemicellulose synthesis genes and cellulose degradation related genes,including Su Sy,SPS,INV,CESA,USP,IRX,BGLU,EG and GN.In addition,1-MCP significantly inhibited the expression of key genes of pectin degradation,including PE,PG and PL.In other words,1-MCP not only inhibits the expression of key genes,but also promotes the expression of key genes in the degradation of intermediates and products in the biosynthesis pathway to inhibit the lignocellulosis of fresh common bean.4.KEGG enrichment analysis of the significantly differentially expressed genes found that 1-MCP treatment regulated genes related to phytohormone synthesis and signal transduction pathway.1-MCP significantly down-regulated the expression of ethylene synthesis genes ACS and ethylene response factor ERF1/2,and up-regulated the expression of ethylene downstream gene EIN3.1-MCP treatment down-regulated the JA synthesis OPCL1 and JA receptor genes MYC2 and VSP2,also inhibited the expression of gibberellin synthesis gene GA2OX3 and G44OX and signal transduction gene PIF4.In addition,1-MCP significantly down regulated ABA synthesis ZEP and CYP707A and signal transduction gene PYR/PYL,indicating that ABA may play an important role in the regulation lignocellulosis of fresh common bean by 1-MCP.These results suggest that 1-MCP may inhibit the lignocellulosis of fresh kidney bean by regulating multiple hormones.Moreover,transcription factors such as MYB and WRKY may be important regulators of 1-MCP in the regulation lignocellulosis of fresh common bean,while AP2/ERF and LIM play a regulatory role in the inhibition of 1-MCP on postharvest lignocellulosis in fresh common bean.At the same time,a number of transcriptional regulatory factors such as TCP4 and ARF are regulated by1-MCP.Whether 1-MCP controls the lignocellulosis of fresh common bean by regulating mi RNA remains to be further studied.5.To further understand whether lignocellulosis in fresh common bean is regulated of by 1-MCP at posttranscription,the expression of mi RNA in CK0,CK15 and T15 and posttranscriptional regulation effect of was analyzed.220 conserved mi RNAs and 55 novel mi RNAs were identified during fresh common bean storage.Among them,50 and 9 were significantly regulated by 1-MCP.Target genes of differentially expressed mi RNAs may be involved in hormone signal transduction,secondary wall metabolism and transport.mi R170-5p,mi R319a-3p,mi R397a-3,mi R399e and mi R408d may be the key mi RNAs for 1-MCP to directly regulate the lignocellulosis of fresh common bean by the association analysis of transcriptome and micro RNAs.q RT-PCR quantitative expression and dual luciferase reporter gene system study found that 1-MCP may regulate the secondary wall metabolic switch transcription factor TCP4 by regulating mi R319a-3p,so as to achieve the regulation of inhibiting the lignocellulosis of fresh common bean.