| Polyploidy is a widespread phenomenon in nature.Many field crops are polyploids,including wheat,rape,cotton and potatoes.Polyploids are also prevalent in fruit crops,such as banana,strawberry,kiwifruit,and persimmon.Phenotypic variations following polyploidization possess the potential to improve agricultural productivity and efficiency,especially in increased biomass and stress tolerance.Moreover,polyploidy is one of major force during plant evolution.Most flowering speices have experienced at least one round of polyploidy event during their evolutionary progress.The existing diploids including Arabidopsis thaliana,maize,rice and soybean,are actually paleopolyploids.Previous studies mainly focused on morphological and physiological alterations following polyploidization and the related genomic,regulatory and epigenetic mechanisms.Metabolites are closely related to plant growth,development and environmental adaptability.Moreover,many metabolites in plants such as sugars,organic acids,Vc,amino acids,carotenoids,flavonoids,etc.,are essential nutrients for human.However,the global effects of polyploidy on plant metabolism and its molecular basis are still largely unclear.In order to explore the occurrence and extent of metabolic alterations caused by polyploidy,we performed comparative morphological,metabolic and transcriptional analysis in mature leaves between doubled diploid(4x)and their corresponding diploids(2x)of four citrus rootstocks,namely Ziyang xiangcheng(Citrus junos Sieb.ex Tanaka),red tangerine(C.reticulata Blanco),trifoliate orange(Poncirus trifoliata L.Raf.)and precocious trifoliate orange(P.trifoliata)and in fruit pulp between Ponkan(C.reticulata Blanco)4x and its 2x parent.1.Genome doubling enhanced the accumulation of stress-related metabolites and the expression of stress-related genes in Ziyang xiangcheng,which could be beneficial for its stress tolerance.Ziyang xiangcheng 4x has typical morphological and anatomical features,such as shorter plant height,larger and thicker leaves,bigger stomata and lower stomatal density,compared with its 2x parent.GC-MS analysis revealed that polyploidy has an activation effect on the accumulation of primary metabolites;many stress-related metabolites such as sucrose,proline and?-aminobutyric acid was remarkably up-regulated in 4x.However,LC-MS analysis demonstrated that polyploidy has an inhibition effect on the accumulation of secondary metabolites in leaves;all the 33 flavones were down-regulated in 4x.By RNA-seq analysis,only 212 genes(0.8%of detected genes)were found significantly differentially expressed between 2x and 4x leaves.Notably,those genes were highly related to stress-response functions,including responses to salt stress,water and abscisic acid.Interestingly,the transcriptional divergence could not explain the metabolic changes,probably due to post-transcriptional regulation.2.The metabolic variations induced by genome doubling in red tangerine,trifoliate orange and precocious trifoliate orange are not entirely stochastic.Genome doubling promotes the accumulation of TCA cycle intermediates and inhibits the accumulation of secondary metabolites.The three 4x have typical polyploid morphological features,including dwarfing plants,enlarged and thickened leaves,increased stomata size and reduced stomatal density,suggesting a common morphological effect induced by genome doubling in genera Citrus and Poncirus.Non-targeted metabolomics profiling was performed in the three 4x and their 2x parents.The principal component analysis effectively clustered biological replicates of the metabolomes of the4 x and its corresponding 2x parents into two categories,demonstrating extensive metabolic changes following genome doubling.About 11%-34%of the total detected metabolites were differentially expressed in three groups of 4x and 2x,and mostly by less than fivefold,indicating that polyploidy has a limited effect on metabolism.Primary metabolism was enhanced following genome doubling,especially the TCA cycle intermediates.The levels of citric acid,malic acid,fumaric acid,and succinic acid were higher in the three 4x than their corresponding 2x parents.Therefore,the metabolic changes induced by genome doubling are not entirely stochastic,and the TCA cycle might play an important role in response to genome doubling.However,secondary metabolism was inhibited following genome doubling and the contents of the most phenylpropanoids and terpenoids in the three 4x were lower than those of the 2x parents.In addition,the C/N ratios of the three groups were all significantly reduced following genome doubling.We conclude that in order to cope with genomic stress caused by genome doubling and maintain vitality and growth,plant tends to enhance primary metabolism for providing more carbon source and energy,and to inhibit secondary metabolism for saving carbon source and energy consumption.3.Genome doubling enhances citric acid accumulation in Ponkan fruit by inhibiting the transportation and degradation of citric acid in the late mature stage.Compared with the 2x parent,Ponkan 4x has significantly different morphology including larger leaves,floral organs,fruits and seeds.It is worth noting that the seed number in the4 x fruit is greatly reduced.Fruit quality analysis showed that the contents of titratable acid(TA),ascorbic acid and total phenolics in the 4x fruit were significantly higher than those of the 2x parent in three consecutive years.Citric acid is the main organic acid in Ponkan fruit.Higher TA content is due to more accumulation of citric acid in 4x fruits.There was no significant difference in the levels of soluble sugar and amino acid between the 4x and 2x fruits,indicating that the increased level of citric acid in 4x did not affect the accumulations of sugars and amino acids.The carotenoid and flavonoid profiling showed that the contents of most carotenoids and flavonoids in 4x were significantly lower than those of its 2x parent,suggesting that secondary metabolites accumulation tended to be inhibited following genome doubling.Gene expression analysis showed no difference in the expression of citric acid synthesis-related genes between 4x and 2x during fruit ripening.It indicated that higher citric acid accumulation in 4x was not due to more citric acid production.Citric acid transport-related genes,including vacuolar H~+-ATPase(VHA)vacuolar H~+-PPase(VHP)and H~+/citrate symporter(CsCit1),were significantly down-regulated in the 4x during the later stages of fruit ripening,indicating a lower citric acid transport capacity in 4x.Moreover,the genes of the GABA pathway,including glutamate decarboxylase(GAD),?-aminobutyric acid permease(GABP)and?-aminobutyrate transaminase(GABA-T)were significantly down-regulated in 4x during the later stages of fruit ripening.A similar trend was observed in the ACL pathway genes,including citrate lyase(ACL),phosphoenolpyruvate carboxykinase(PEPCK)and fructose-1,6-bisphosphatase(FBPase).These results indicated 4x had lower citric acid degradation and utilization efficiency,compared with the 2x parent during the later stages of fruit ripening.Therefore,compared with the 2x parent,4x fruit accumulates more citric acid was due to its lower transport capacity and degradation and utilization efficiency of citric acid.Generally,this study has clearly clarified the effect of genome doubling on citrus metabolism.It enriches the theoretical basis for polyploidy of plants and provides important theoretical support for citrus polyploid breeding. |
PDF Full Text Request