Genome-wide Identification Of The 1-aminocyclopropane-1-carboxylic Acid Synthase(ACS) Genes And Their Possible Roles In Fruit Ripening Of Sand Pear(Pyrus Pyrifolia)

Sand pear（Pyrus pyrifolia Nakai）is the main cultivated species of Pyrus L.in China.Short shelf life and poor storage are the main bottlenecks of sand pear industry.Ethylene production is negatively associated with storage life of sand pear,particularly at the time of fruit harvest.1-Aminocyclopropane-1-carboxylic acid synthase（ACS）is the rate-limiting enzyme in ethylene biosynthesis and is considered to be very important for fruit storage life.However,the candidate ACS genes and their roles in sand pear remain unclear.Previous studies showed that there were extremely significant differences in ethylene release during fruit ripening of different sand pear cultivars,that is,there were both climacteric and non-climacteric characteristics.Therefore,in addition to postharvest storage and preservation technology,the influence of climacteric characteristics of different pear varieties on their storage is more critical.Clarifying the ripening mechanism of pear fruit and breeding storage-resistant pear varieties are the main technical means to fundamentally and effectively solve this problem.The ethylene release during fruit ripening is a key factor affecting storage.ACS is a rate-limiting enzyme in ethylene biosynthesis.In this study,early maturity and low ethylene specific cultivar’Eli2’and early maturity and high ethylene cultivar’Ninomiyahakuri’were selected and used to explore the mechanism of different members of ACS family during fruit ripening by RNA-seq and qRT-PCR,which may provide a scientific basis for fruit ripening regulation of sand pear.The results are as follows:1.Analysis of fruit maturation characteristics of sand pear germplasm resources.Through the determination and analysis of the fruit development period,shelf life,soluble solid content,flesh firmness and ethylene release of 26 sand pear cultivars,it was found that the peak value of ethylene release during shelf life was 0.1-214.4μL·kg^-1·h^-1,and the ethylene release of early-maturing varieties was generally high,showing the characteristics of respiratory climacteric,while the ethylene release of mid-late maturing varieties of sand pear during shelf life was generally lower and no obvious climacteric peak,showing the characteristics of non-respiratory climacteric.The fruit development period of different varieties was significantly positively correlated with fruit shelf life and ethylene production,and was significantly negatively correlated with ethylene production.There was a significant negative correlation between fruit shelf life and ethylene release.Two specific cultivars were screened,early maturing non-climacteric cultivar’Eli 2’and late maturing mutant cultivar’Jinshui 1’.2.Genome-wide identification of ACS gene family in Pyrus pyrifolia and its related Rosaceae species.Through genome-wide screening,56 ACS genes were identified from 5latest versions of Rosaceae genomes:13 genes from apple and 8 from peach,10 genes from Chinese white pear,12 genes from European pear as well as 13 genes from sand pear.Gene evolution pattern analysis showed that WGD was the main driving force of ACS gene family expansion.The number of ACS genes in P.pyrifolia,P.bretschneideri,P.communis and Malus domestica belonging to Maloideae is roughly equal,and is close to twice that of peach,which may be related to the fact that peach has not experienced modern genome-wide replication.ACSs were phylogenetically categorized into four subgroups that perfectly fit their types.The number of putative AAT ACS is the most primitive and conservative.Nine of the 13 PpyACSs identified in P.pyrifolia were first reported in this study.The results of chromosome localization and collinearity analysis showed that the ACS gene was the most distributed in Chr15 which included the most critical PpyACS1 and PpyACS2.Therefore,this region may be the key region for the regulation of ethylene production in sand pear fruit ripening.The results of subcellular localization showed that PpyACSs were all located in chloroplast except Type II ACS located in the cytoplasm.Promoter cis-element prediction found that PpyACSs promoter region has a large number of hormone and stress response elements.Tissue-specific expression analysis showed that PpyACSs were mainly expressed in mature fruits and flowers.3.The expression analysis of ACS during fruit development and ripening of different mutant fruits.The fruits before and after ripening of the climacteric cultivar’Ninomiyahakuri’and the non-climacteric cultivar’Eli No.2’were used as test materials.RNA-seq and q RT-PCR analysis showed that the differentially expressed genes were mainly enriched in the plant hormone response pathway,and were higher in the climacteric cultivar than in the non-climacteric cultivar,and higher after maturation than before maturation.Seven members of PpyACSs were involved in fruit ripening,five of which were first reported.PpyACS8,PpyACS12,and PpyACS13 were responsible for ethylene production before fruit maturation,while PpyACS1,PpyACS2,PpyACS3 and PpyACS9 were mainly responsible for ethylene production after fruit maturation,and PpyACS1 was especially responsible for system 2 ripening-associated ethylene production.4.Cloning and cis-element analysis of PpyACS1 promoter.Cloning and sequencing analysis of PpyACS1 promoter of 18 pear cultivars with different ethylene release capacities showed that there was a 285 bp indel at-855 bp upstream of the initiation codon of PpyACS1 in P.communis,and the same promoter sequence was found in the high ethylene cultivars and the low ethylene cultivars of oriental pears,respectively.Six hormones and stress response type cis-acting element included ABRE,G-box,ARE,TCA-element,WUN-motif and TCT-motif,were identified to be correlated with ethylene release capacity.In summary,ACS gene family information of Pyrus pyrifolia and other four related species in Rosaceae was first identified and comprehensive analysied,among which nine of the 13 members of the ACS gene family of Pyrus pyrifolia were first reported.An early ripening and low ethylene specific germplasm’Eli No.2’was screened and used as materials to clarify the ACS gene family and its expression patterns in different stages of fruit ripening of different climacteric cultivars.In addition,it was also found that the Putative AAT type ACS genes that were previously excluded from the ACS family because of no ACS enzyme activity still play a role in ethylene synthesis.