Omics-based Studies On The Effect Of Posthavested Quality Of Pear Fruit Treated By Low Temperature And 1-MCP

Pear is respiratory climacteric fruit and prone to softening and rot at room temperature.1-MCP treatment and low-temperature storage are the main methods for prolonging the storage time of pear fruit.Fruit ripening and senescence is an indispensable and important process in the life activities of fruit tree.However,the molecular mechanism of different treatments to delay fruit senescence has not been thoroughly studied at present.Therefore,it is of great scientific value to carry out in-depth exploration and research on the aging of pear fruit.In this paper,the effects of two commonly used fresh-keeping methods,1-MCP and cold storage on gene expression,metabolite and miRNA expression of pear fruit during postharvest storage were studied.The molecular mechanism of two kinds of preservation treatment to delay the senescence of pear fruit was summarised.The effects of external ATP treatment on maintaining the quality of cv.HS,in which the optimum of exogenous ATP concentration to prolong the normal temperature preservation of pear cultivar Housui was found.The main results obtained were as follows:1.Ripening fruits(RF)of pear cultivar Housui(HS;Pyrus pyrifolia)were harvested and soaked into ethephon solutions,and was treated by 1-MCP.When 30%of whole fruits were decayed under ethephon treatment,the pre-decayed fruits(ED)were collected,as well as the control fruits(EC).Similarly,when 30%of whole fruits were decayed in control,the pre-decayed fruits(ND)were collected,as well as the 1-MCP treated fruits(MC).Moreover,when 30%of whole fruits were decayed under 1-MCP treatment,the pre-decayed fruits(MD)were collected.To reveal the related genes involved in fruit senescence,the RNA-seq of the fruit from these 6 time points of different treatments mentioned above were conducted.Among natural,ethephon,and 1-MCP treated fruits,500?L/L ethephon treated fruits were respectively prior decayed to non-treated fruits about seven days,while 1.5 ?L/L 1-MCP treated fruits were individually delay decayed about nine days.To reveal the genes related to fruit senescence,expression levels of all predicted genes in RF were respectively compared to that in pre-decayed fruits(ED,ND,and MD).As a result,1546 and 1810 genes were respectively up-and down-regulated in ED,1522 and 1424 genes were individually up-and down-regulated in ND,and 793 and 637 genes were separately up-and down-regulated in MD.In summary,455 and 496 genes were commonly up-and down-regulated in pre-decayed fruits,respectively.To unveil the mediation of ethylene during fruit senescence,differentially expressed genes(DEGs)were analyzed between ED and EC and between ND and MC.As a result,a total of 573 and 381 genes were individually up-and down-regulated in ED.Meanwhile,a total of 313 and 265 genes were up-and down-regulated in ND,respectively.Due to ethephon and 1-MCP were associated with ethylene production,the DEGs induced by ethephon and 1-MCP were further compared.The result showed that 136 and 70 genes were commonly up-and down-regulated in ED and ND,respectively.Of these DEGs,61 up-regulated and 16 down-regulated genes were induced by ethylene.However,only one novel ERF genes(Pbr022708.1)was induced by ethylene in post-harvested fruits during storage.Moreover,based on the changes of fruit firmness,two ethylene-induced genes that individually encode polygalacturonase(Pbr010853.1)and xyloglucan endotransglucosylase/hydrolase(Pbr040203.1)were isolated to be associated with fruit softening during post-harvest storage.Besides auxin signal and stress tolerance were likely involved in fruit senescence.These results will be available for understanding gene regulation of post-harvested fruits during storage.2.High-temperature and low-temperature treatments of the fruits of pear cv.HS enhanced and delayed fruit senescence respectively.Ripening fruits(RF)of cv.HS were harvested and divided into three groups.The three groups contain 120,180,and 300 fruits that were stored at 40�2?(high temperature),25�1?(room temperature),and 4�1?(low temperature),respectively.Since more than 30%of the fruits treated by high,room,and low temperatures were individually decayed at 15,19,and 105 days after treatment(DAT),the fruits treated by high temperature were collected at 10(HT-10)and 15 DAT(HT-15).The fruits treated by room temperature were collected at 10(RT-10),15(RT-15),and 19 DAT(RT-19).And the fruits treated by low temperature were collected at 10(LT-10),15(LT-15),19(LT-19),79(LT-79),and 105 DAT(LT-105).It was found that the storage period of high-temperature treatment was reduced by four days compared with the control treatment,while the storage period of low-temperature treatment was extended by 86 days compared with the control treatment.The related genes and metabolites related to temperature-induced fruit senescence were investigated by RNA-seq analysis.Thirty-six up-regulated and 1821 down-regulated genes associated with high-temperature induction were found.Besides,19 up-regulated genes related to hypothermia induction were discovered,while 1,279 low-temperature and fruit senescence-related down-regulated key genes were identified.To clarify physiological mechanism during fruit senescence respectively induced by high and low temperatures,non-targeted metabolic profiling was performed on each sample collected by ultra performance liquid chromatography(UPLC).A total of 2,138 distinct metabolic traits were detected in the ripening and treated fruits.Of these traits,53 and 97 were individually increased and decreased in the pre-decayed fruits(both RT-19,HT-15,and LT-105),compared to that in RF.In these 150 traits related to fruit senescence,30 traits were in response to high temperature,while 35 traits response at low temperature and of the nine traits were also induced by both high and low temperature.These traits associated with fruit senescence induced by high/low temperature were named TSHs/TSLs,which were used as correlation indicator for coding and non-coding RNAs during fruit senescence in pear.3.By combining bioinformatics analysis with next-generation high-throughput sequencing,miRNA related to temperature regulation of fruit senescence was investigated.To get the miRNAs involved in temperature-induced fruit senescence,small RNA sequencing was conducted to predict miRNA and its expression in each sample.(A combination of bioinformatics analysis and next-generation high-throughput sequencing was used to investigate the temperature-regulated miRNAs associated with fruit senescence.To obtain miRNAs involved in temperature-induced fruit senescence,small RNA sequencing was performed to predict miRNA and its expression in each sample.)Approximately 64.15%of the total 122.39 million reads were assembled to 167 predicted miRNAs(including 48 known and 119 novel precursors).Of the 113 miRNAs that target to 728 genes,14 miRNAs could target to 14 genes induced by high and/or low temperatures,respectively.Moreover,due to the diverse regulatory pathways of miRNAs,we analyzed the relationship between miRNAs and the traits/genes associated with temperature-induced fruit senescence.A total of 4 and 12 miRNAs were individually up-and down-regulated in the pre-decayed fruits(both RT-19,HT-15,and LT-105),and thus probably related to fruit senescence.In the differentially expressed miRNAs,miRNAs that were also differentially expressed between HT-15 and RT-15 were induced by high temperature.However,only the Novel₁15 and 9 of its correlated GSHs were positively correlated to POS0539 that is a TSH(FDR=0.03<0.05),indicating that the Novel₁15 is likely involved in fruit senescence by affecting production of the POS0539.Likewise,nine miRNAs that were also differentially expressed between LT-19 and RT-19 were induced by low temperature.Only four miRNAs could be anchored by 288 GSLs,and of the Novel₁13 and its correlated GSLs were anchored on 10 TSLs.while the Novel₇6 and 174 of its correlated GSLs were anchored on seven TSLs.The results indicated that these two miRNAs were likely involved in fruit senescence induced by low temperature and thus designated as MSLs.Taken together,three,twelve,and one miRNAs were individually associated with fruit senescence induced by high,low,and both high and low temperatures.It was found that under high temperature treatment,miR156x and miR395 in pear fruits acted on protein kinase and pectin lyase respectively,which damaged the membrane structure of pear fruits by stress,participating in the aging process of cv.HS.Under low temperature treatment,11 miRNAs in pear fruits,such as miR156p,miR172i and Novel₁07,respectively affect SBP transcription factor,ERF transcription factor,MYB transcription factor and other target genes,which participate in the fruit aging process by inhibiting the expression of ethylene synthetic genes.Meanwhile,the temperature-regulated mdm-miR7124a target gene was identified to be an ATP enzyme(P type)gene.The results indicated that miRNA related to temperature regulation of energy metabolism,membrane metabolism and aging response factors should be involved in the aging process of pear fruits.4.Three different concentrations(1.0,1.5,2.0mM)of exogenous ATP were used to treat pear fruits,which delayed the senescence of pear fruit.The results showed that the storage time of pear cv.HS treated with the 2.0mm concentration of exogenous ATP was eight days longer than that treated with control.At the same time,the treatment with exogenous ATP of 2.0mM concentration delayed the reduction of fruit hardness and skin browning of pear cv.HS,but the content of soluble solids was lower than other treatments during the same period.It showed that exogenous ATP treatment delayed the senescence of pear fruits.