Physiological And Molecular Mechanism Of Affecting Sugar Biosynthesis And Distribution In Pear Leaves And Fruit By Potassium Supply

Pear(Pyrus L.)is one of the leading cultivated fruit trees of temperate regions in China following apple and citrus in both planting areas and fruit yield.Potassium(K)is one of the most abundant elements in pear fruit,and generally considered to be the quality element.It is closely related to leaf photosynthesis,flower bud quality,fruit size,color and flavor.Farmers tend to apply excessive or insufficient K fertilizers in pear orchards in China,which usually resulted in the deterioration of fruit quality and the aggravation of physiological disease.Balanced fertilization can improve the total soluble sugar content of the fruit,increase the fruit weight,and promote the fruit color,which is a commonly measure the increase of the 'quality and efficiency'.However,little research has been reported on the mechanism of how to improve the quality by K.In this experiment,through reasonable management of K fertilizer management,field experiments(0(K0),150(K150),300(K300),450(K450)kg·ha-1)and pot culture experiments(0(K0),0.4(K0.4)and 0.8(K0.8)g·kg-1 soil)were combined to evaluate the effect of different K levels on pear vegetative growth,leaf photosynthetic characteristics in pear,and the accumulation process of primary metabolites in leaves and fruit and the expression patterns of multiple members of gene families encoding key enzymes or transporters involved in these metabolites in different organs.At same time,based on multivariate and bioinformatics analyses of the leaves and fruit in the middle and later stage of development,we discussed the genes associated with K transport and sugar metabolism and the associated signalling pathways mediated by K,to clear the regulation mechanism of K application on the distribution of nutrients and sugar in leaves and fruit,and to reveal the molecular physiological mechanism of K application to improve fruit quality.The main results are listed as follows:(1)The results of field experiments demonstrated that the K content and accumulation in the pear leaves and fruit,the net photosynthetic rate and SPAD value of leaves were found to be increased with the increase of K application rates.Increasing K application rates also led to promotion effectiveness on the accumulation of glucose,fructose sorbitol and sucrose in the fruit.The fruit single weight,yield and fruit sugar-to-acid ratio in the K450 treatment were significantly higher than that in the control(K0).K application significantly improved the fruit quality,especially the sugar to acid ratio,which under K450 treatment was significantly increased by 27.2%and 30.1%,respectively,comparing with the Control.During the early fruit development stage,all sugar components in the leaves were increased with K application rates,correlating with the up-regulation expression of gene acid invertase(AIV)and sorbitol 6-phosphatedehydrogenase(S6PDH)Furthermore,with the fruit development,the expression of AIVI,sucrose phosphate synthase(SPS1)and sucrose synthase(SUS),S6PDH and sorbitol dehydrogenase(SDH3)involved in sugar metabolism in the leaves were up-regulated by increasing the K application rates,resulting in higher accumulation of sugar components in the leaves.Interestingly,at the fruit maturity stage,the expression levels of SUT in the leaves,SPS1,SUS and sucrose transporter(SUT)in the fruit,which were involved in sucrose metabolism,were found to be significantly up-regulated,leading to higher sucrose accumulation in the fruit.Our data indicated that the accumulation of fruit sugar components were not only affected by the source-sink strength but also regulated by the inner regulating mechanism between sorbitol and sucrose metabolism in both leaves and fruit.K plays a key role on promoting the sugar assimilation and transportation from leaves to fruit,and up-regulating the expression levels of key genes involved in sugar metabolism in leaves and fruit.(2)K enhances sorbitol metabolism,and promoted sorbitol assimilation in pear leaf and fruit and the sorbitol distribution from source to sink.The expression patterns of different K levels on the three sorbitol-6-phosphate dehydrogenase genes(PbS6PDHs),eight sorbitol dehydrogenase genes(PbSDHs),and two sorbitol transporter genes(PbSOTs)were analyzed.The results showed that the K accumulation in leaves and fruit was significantly increased by the K treatments,as well as those of sorbitol,fructose and glucose in fruit.At all stages of development,the expression of PbS6PDHl and PbSDH14 in leaves was up-regulated by K,while PbS6PDH3,PbSDH2,PbSDH13 and PbSOT22 were down-regulated.During the young fruit stage,the expression of PbSDH2,PbSDH4 and PbSOT22 was up-regulated by K,while it was on the contrary at maturity,suggesting an important role they played in the sorbitol metabolism in fruit.Meanwhile,the expression of PbS6PDHl,PbS6PDH3 and PbSDH13 in fruit was all promoted by K during the expansion stage ?,indicating that the transport of sorbitol between source and sink was regulated by K.In conclusion,K could regulate expression of key genes involved in sorbitol metabolism in both source and sink for the improvement of fruit quality.(3)Pear leaves and fruit under different K levels were harvested at the enlargement and maturity stage after treatment,and their transcriptomes were analyzed by high throughput sequencing.We identified 36,444 transcripts from leaves and fruit using transcriptome sequencing technology.From 105 days after full blooming(DAB)to 129 DAB,the number of differentially expressed genes(DEGs)in leaves and fruit in response to low K(K0)increased,while in response to high K,the number of DEGs in leaves and fruit decreased.At 129 DAB,1801 genes were differentially expressed following low-K treatment compared to middle-K treatment in leaves,including 1136 down-regulated DEGs.In fruit,3290 of 5185 DEGs were down-regulated.We selected 17 of these DEGs for qRT-PCR analysis to confirm the RNA sequencing results.Based on GO enrichment and KEGG pathway analysis,we found that under low and high K,the DEGs in leaves and fruit were significantly enriched in "response to stimulus","single-organism metabolic process","response to oxygen-containing compound","metabolic pathways","biosynthesis of secondary metabolites" and "plant hormone signal transduction" pathways.Low-K treatment significantly reduced K nutrient and carbohydrate metabolism of the leaves and fruit compared with the Control treatment.During the fruit development stages,AKT1(gene39320)played an important role on K+transport of the leaves and fruit response to K stress.At maturity,sucrose and acid metabolic pathways were inhibited by low K.The up-regulation of the expression of three SDH(gene33410,gene33408 and gene20259)and two S6PDH(gene27055 and gene27053)genes involved in sorbitol metabolism was induced by low K,promoting the fructose accumulation.Simultaneously,higher expression was found for genes encoding amylase under low K,promoting the decomposition of the starch and leading the glucose accumulation.High K could enhance leaf photosynthesis,and improve the distribution of the nutrient and carbohydrate from leaf to fruit.Sugar components of the leaves and fruit under low K were regulated by the expression of genes encoding 8 types of hormone signals and reactive oxygen species(ROS).High K(K0.8)can enhance the leaf photosynthesis,and improved the distribution of nutrients and carbohydrates from leaves to fruit.Sugar components of the leaves and fruit under low K were regulated by the expression of genes encoding 8 types of hormone signals and reactive oxygen species(ROS),and the expression of these DEGs was the same under middle-and high-K treatments.Our data revealed the gene expression patterns of leaves and fruit in response to different K levels during the middle and late stages of fruit development as well as the molecular mechanism of improvement of fruit sugar levels by K and provided a scientific basis for improving fruit quality with supplemental K fertilizers.(4)Based on the analysis of transcriptome sequencing,19 candidates genes related to mineral nutrients(K,calcium(Ca)and magnesium(Mg))and sugar(sucrose and sorbitol)transport was selected s for qRT-PCR analysis.The results showed that K supply promoted the development of petiole and fruit stalk,enhanced the distribution of nutrients and sugar from leaf to fruit.Compared with middle K treatment,the content of leaf K and Ca under the low K treatment was decreased,which the content of leaf Mg was significantly increased.While,the content of fruit K,Ca,and Mg gradually increased with the increase of K application rate.K increased the expression of genes related to K and Ca transport system in leaves and fruit,and promoted the absorption of K and Ca in leaves and fruit.However,leaf Mg and Ca were antagonistic to K,which may be related to the up-regulation of MRS2-1(magnesium transporters)and MRS2-3 expression under low K treatment.The improvement of sugar content was closely related to the up-regulation of the expression of SUTs and SOTs genes.This indicated that K fertilization improved the development of the vasculars in pear petiole and fruit stalk,and enhanced the synergistic effect of the genes involed in nutrient and sugar transport,and promoted the transport of the nutrient and sugar from the source(leaves)to sink(fruit)to improve the fruit quality.In summary,the K nutrient levels in leaves and fruit significantly was increased by K supply.K increased the expression of genes involved in sugar metabolism,leading to an increase of the synthesis of sugar components(sorbitol and sucrose).K promoted the structure of vascular bundles in petiole and fruit stalk by regulating the express levels of the genes involed in nutrient and sugar transport in in leaves and fruis,and increased the distribution and accumulation of the nutrient and sugar from from source to sink,resulting in the increase of the total sugar concentration in fruit.