Mechanism Of Gamma-Aminobutyric Acid Shunt Regulating Citrate Metabolism In Citrus Fruit

Organic acid is one of the most important components for fruit flavor of citrus.The changes of organic acid level can not only influence the fruit senescence process,but also affect the shelf life of fresh fruits.During postharvest storage,the consumption of organic acid caused by respiration and energy metabolism results in the decline of flavor and quality,which leads to great economic loss in citrus production.Fruit inner quality,including the content of organic acids,is affected by genetics,environmental factors and cultural practices.As the main organic acid in citrus fruit,citrate content is determined by the balance between its synthesis,catabolism,transport and vacuole storage.Previous studies on organic acid metabolism were focused on the regulatory effects of genes,which were functioned in TCA cycle.In recent years,there have been increasing evidences suggesting that cytoplasm degradation and vacuole storage process play important roles in determination of organic acid level.GABA shunt is one of the major pathways participating in citrate utilization;meanwhile,succinate which is the end-product of GABA shunt could flow back to TCA cycle and provide the carbon skeleton.Hence,the compartmentation of GABA metabolism in cytosol or mitochondrion can not only influence the citrate degradation,but also affect the substrate supply of TCA cycle.Mitochondrial GABP,a key regulator linking the GABA metabolism and TCA,controls the translocation of GABA from cytosol into mitochondrion.However,there is still limited study concerning regulation mechanism of GABP on citrate metabolism,and the physiological and biochemical reactions caused by the transportation of GABA into mitochondrion under GABP function are still remaining unknown.In our study,we used different both high-and low-acid citrus fruit to analyze the effects of GABA shunt on citrate level.The candidate gene,CgGABP,its potential roles in regulating citrate metabolism was further researched.The main results were as follows:1.Exogenous GABA treatment significantly increased citrate content in Valencia(Citrus sinensis Osbeck ‘Valencia')and Newhall(Citrus sinensis Osbeck‘Newhall')fruit during postharvest storage.The results showed that the endogenous GABA obviously increased under exogenous GABA treatment.Gene expression analysis indicated that the increase of citrate was primarily attributed to the lower expression of GAD.Meanwhile,the expression of GABP was also decreased in treated fruit at earlier storage period.Contents of amino acids that related to citrate metabolism including glutamate,aspartic acid,serine,valine and proline were also increased under treatment.Moreover,GABA treatment enhanced the POD activity and increased the ATP content.Importantly,the fruit rot rate of treated fruits was significantly lower than that of control;and the fruit peel color change of Newhall fruit from orange to red was accelerated by treatment,which are two parameters closely connected to the fruit quality and storageperformance.As one of the GRAS(Generally Recognized as Safe)compounds,our research provided a effective and safety approach for maintenance of postharvest quality and improvement of storage performance in citrus production.2.GABA pathway rate-limit citrate degradation in postharvest citrus fruit evidence from HB Pumelo(Citrus grandis Osbeck ‘Hirado Buntan')× Fairchild(Citrus reticulata × Citrus grandis ‘Fairchild')hybrid population.The quality characteristics were traced for three years,we found a stable transgressive level of TA content in hybrid populations.And the fruit weight loss was significantly negatively correlated with the TA content during postharvest storage.Two prolific hybrid plants exhibiting a stable phenotype of high acid and low acid were respectively chosen for further analysis.The TA content generally showed a decreasing trend during postharvest storage,but obviously that the significantly higher acid trait maintained throughout the storage period.Unlike that of TA,the content of TSS maintained at a relatively stable level.Analysis of primary metabolites indicated that the transgressive content of TA was attributed to citrate accumulation.The contents of amino acids did not follow a certain pattern as the content of citrate,except for that of aspartate and asparagine.It is worth noting that the characteristics of these metabolites contents at mature stage consistently maintained throughout the storage period.Expression of genes related to citrate metabolism indicated that most of GABA shunt genes exhibited lower expression in high-acid fruits during postharvest storage,which mainly contributed to the high citrate accumulation.Moreover,we found that the respiratory rate showed a decrease tendency during storage,and was lower in high-acid fruits than low-acid fruits,indicating that the consumption of citrate by respiratory rate was lower in high-acid fruits.Further analysis of the fruit weight loss suggested that the TA level was significantly negatively correlated with weight loss during postharvest storage,implying a close relationship between organic acid and water metabolism.This study reveals that the importance of genetic effect on organic acid level in postharvest citrus fruit,highlighting the significance of GABA metabolism pathway.3.The CgGABP gene from HB Pumelo(Citrus grandis Osbeck ‘Hirado Buntan')participates in citrate metabolism through regulating the expression of related genes,and the transcriptional factors CgbHLH13 and CgBBX32 can modulate the citrate level through negatively regulate the GABP expression.The common high-acid HB Pumelo and low-acid acid-free Pumelo(Citrus grandis Osbeck)with significantly difference in citrate content were used in this study.We also found that the GABA shunt genes were all significantly lower expressed in HB than in acid-free Pumelo,indicating GABA metabolism pathway also participated in determination of citrate level in HB and acid-free Pumelo fruit.Hence,we focused our research on function of CgGABP and its regulation role on citrate metabolism.Cluster analysis showed that CgGABP was classified as member of BAT family under APC superfamily.Similar to the reportedAtGABP,CgGABP is predicted to have 12 transmembrane domains with internal N and C termini.Topology superimposition of CgGABP with AtGABP implies high topological similarity.Subcellular localization showed that CgGABP was located in mitochondrion,indicating that CgGABP is a mitochondrial GABA transporter.The CgGABP overexpression tomato lines were successfully obtained.Primary metabolites analysis indicated that the content of citrate was significantly increased in transgenic tomato fruits.Additionally,it also exhibited higher contents of glutamate,glutamine,palmitic acid,stearic acid and lower contents of proline,alanine,valine,aspartate,asparagine,succinate and sucrose in transgenic tomato fruits.Further gene expression detection showed that the accumulation of citrate was mainly attributed to the lower expression of SlAco3 a and Sl Aco3 b,and partially attributed to the higher expression of SlCS and SlPEPC.Two transcriptional factors were identified through yeast one-hybrid screening.One is a member of bHLH family,named CgbHLH13;the other is the member of BBX family,named CgBBX32.Subcellular localization analysis showed that CgbHLH13 and CgBBX32 were located in nucleus.Further yeast one-hybrid experiment proved that CgbHLH13 and CgBBX32 could interact with CgGABP promoter.Dual-luciferase reporter assay indicated that CgbHLH13 and CgBBX32 negatively regulated the expression of CgGABP.Overexpression of CgbHLH13 and CgBBX32 in citrus callus significantly reduced the expression of GABP gene,and simultaneously decreased the content of citrate.Moreover,the contents of proline,ornithine,GABA,alanine,valine,glycine and sucrose were all significantly higher in transgenic callus than that in WT;the contents of glutamate,palmitic acid and stearic acid were significantly lower.That was just the opposite of that in transgenic tomato,which was consistent with the negatively regulation of CgbHLH13 and CgBBX32 on GABP.In addition,the expression of Aco was increased,and PEPC and CS were decreased in transgenic citrus callus,which could also explain the decline of citrate content.Moreover,overexpressed CgGABP activated the GABA shunt,and glutamate flux was primarily turn to GABA metabolism;on the contrary,overexpressed transcriptional repressors blocked the GABA metabolism pathway,and resulted in metabolism shift of glutamate flux into proline,ornithine and others.Taken together,these results demonstrate that GABP could regulate the citrate accumulation through reconfiguring the central C and N metabolism,and glutamate flux played important role in this process.In this study,we demonstrated that the GABP could regulate citrate metabolism through overexpression of CgGABP and its transcriptional repressors.We firstly proved that the transcriptional factors,CgbHLH13 and CgBBX32 can negatively regulate citrate accumulation through directly down-regulating the GABP expression.Our results providepossible targets for manipulation of the citrate accumulation in horticultural crops,especially in fruits.