Studying The Role Of Citrus Atp-citrate Lyase(ACL) In The Accumulation Of Citrate And Its Relative Metabolites,as Well As ACL Action Mechanism And ACL-affecting Factors

ATP-citrate lyase(ACL),catalyzes the citrate into oxaloacetate and acetyl-CoA,is considered to be the key enzyme that decides the carbon flux towards different metabolite biosynthesis pathways,such as carbohydrates,fatty acids,flavonoids,and isoprenoids.Although the previous researches in citrus confirmed that ACL participates in citrate utilization during fruit ripening,the role of ACL on the accumulation of soluble sugars,organic acids,and other metabolites as well as the response of abiotic factors on ACL are still unclear.In this study,ACL activity and the content of soluble sugars,organic acids,flavonoids,and other metabolites were assessed during the development and ripening of different citrus cultivars,and the function of ACL was verified by transgenic experiments.Through environmental factor treatments,the effect of different environmental conditions on ACL activity was examined.The main results are as following1.The increase in ACL activity may be an important reason for the low accumulation of citrate and high accumulation of some secondary metabolites in'Honganliu'(HAL,Citrus sinensis cv.Honganliu)fruit juice sacs(JS).By comparing the changes of metabolites,ACL genes expression,and activity between 'Anliu' orange(AL,C.sinensis cv.Anliu)and its low-acid mutant 'HAL',it was observed that the citrate content in 'HAL' fruit juice sacs was significantly lower than in 'AL',soluble sugar and many secondary metabolites were significantly higher than in 'AL',while the ACL genes expression and activity were significantly higher than in 'AL'.It indicated that the enhanced ACL activity in 'HAL' promoted the utilization of citrate,increased the gluconeogenesis pathway and the secondary metabolite biosynthesis2.The relationship between changes in ACL activity and the accumulation of some metabolites in different citrus cultivars is different.Changes of ACL and cytoplasmic aconitase(cyt-ACO)activities,and the contents of citrate,flavonoids,carotenoids,and other metabolites were systematically compared among the fruits of 'AL','Newhall'orange(C.sinensis cv.Newhall),'Guoqing No.1' Satsuma mandarin(C.unshiu cv Guoqing No.1),'Huagan No.2' ponkan(C.reticulata cv.Huagan No.2),'Zaoxiang'pomelo(C.grandis cv.Zaoxiang)and 'HB' pomelo(C.grandis cv.'Hirado Buntan')during development and ripening.The results showed that the correlation between the citrate content and ACL or cyt-ACO activity varied greatly among cultivars,while the activities of ACL and cyt-ACO had a significantly negative correlation(r=-0.443).Besides,a significant correlation was only observed between the ACL activity and the content of cytosolic acetyl-CoA(r=0.433).ACL overexpression or RNA interference in citrus callus showed that increasing or decreasing ACL activity can reduce or induce cyt-ACO activity,respectively.It shows that ACL and cyt-ACO synergistically control the citrate fate for the biosynthesis of other metabolites,but they are not the key determinants for the accumulation of citrate,as well as other metabolites in citrus fruits.3.The significant difference in ACL activity may be an important reason for the difference in the accumulation of metabolites in citrus mature leaves(ML)and fruit JS.The differences of ACL genes expression and enzyme activity,citrate and cytoplasmic acetyl-CoA content in ML and JS among seven citrus cultivars were compared.The results showed that ACL genes expression,enzyme activity and cytoplasmic acetyl-CoA content in ML of the seven citrus cultivars were significantly higher than in JS,while the citrate content was significantly lower than in JS.Furthermore,a comprehensive comparison was carried out between the differences in metabolites and gene transcription levels of ML and JS in 'HB' pomelo,and finally,227 differentially expressed metabolites(DEMs)and 10040 differentially expressed genes(DEGs)were identified.Compared with JS,starch and some DEMs belonging to flavonoids,fatty acids,and glycerolipids,and certain specific DEGs in the photosynthesis pathway were up-regulated in ML;while soluble sugar,citrate and vitamin C were down-regulated.The joint analysis showed that the differences in starch,sucrose,citrate,flavonoids,carotenoids,vitamin C and jasmonic acid between ML and JS were regulated by the expression levels of certain genes in their respective metabolic pathways.It showed that the high ACL activity of ML not only increases the intermediate precursor substances of many secondary metabolites synthesized but also might regulate the synthesis or accumulation of these substances by affecting the expression levels of some key genes4.Transgenic studies have confirmed that changes in ACL activity can indeed affect the content of related metabolites,as well as the expression levels or enzyme activities of some genes.Subcellular localization analysis revealed that CitACLs were localized in the cytoplasm and nucleus.Overexpression or RNA interference of ACL significantly increased or decreased the citrate content in citrus callus.Further expression levels analysis of citrate metabolism-related genes revealed that the increase of citrate accumulation under overexpression of ACL was mainly related to the suppression of ACO expression level and the increased of citrate synthase(CS)expression level;the decrease of citrate content under RNA interference of ACL was mainly due to the induction of ACO expression level and the suppression of CS expression level Cytoplasmic acetyl-CoA,as the central metabolite of citrate utilization catalyzed by ACL,was accumulated significantly in ACL overexpression citrus callus;its content was decreased significantly in ACL interference citrus callus.In addition,the flavonoids content in ACL overexpression citrus callus was increased significantly,and the carotenoids content was decreased significantly.The flavonoids and carotenoids contents in ACL interference citrus callus were decreased significantly.This showed that the effect of ACL on flavonoid content might be greater than carotenoids.Besides,the ACL overexpression vector was transformed to produce transgenic plants of tomato.The results showed that the citrate content of transgenic tomato fruits and leaves were significantly reduced,and the cytoplasmic acetyl-CoA content was significantly increased.Analysis of the expression levels of endogenous genes related to citrate metabolism showed that overexpression of ACL could promote the expression level of SlACOs and SlACLs genes and inhibit the expression level of phosphoenolpyruvate carboxylase(SIPEPC2)gene,thus reducing the citrate accumulation5.ACL activity can be influenced by nutrients and environmental factors.(1)Foliar application of nitrogen fertilizer and shade treatment significantly reduced the ACL activity and increased the citrate content in fruits,while the contents of cytoplasmic acetyl-CoA and flavonoids were decreased;(2)Foliar application of phosphorus,potassium fertilizers enhanced the ACL activity,reduced the citrate content and increased the cytoplasmic acetyl-CoA content in fruits;(3)Foliar application of magnesium fertilizers enhanced the ACL activity,reduced the citrate content and increased the contents of cytoplasmic acetyl-CoA and flavonoids in fruits;(4)At low pH(pH 4.8)treatment of 'Guoqing No.1' Satsuma mandarin callus,ACL activity was enhanced,citrate content was reduced,and the content of cytoplasmic acetyl-CoA and flavonoids were increased;(4)At low-temperature(4?)treatment of callus,ACL activity was enhanced,and the content of citrate,cytoplasmic acetyl-CoA and flavonoids were increased.