Mining Of The Proton Pump Genes Related To The Citrus Citric Acid Accumulation And Function Analysis Of The Key Gene

There are three kinds of proton pumps on the plant cell tonoplast associated with the accumulation of organic acid,namely,P-type H⁺-ATPase?PHA?,V-type H⁺-ATPase and V-type H⁺-PPase.These proton pumps can transport H⁺from the cytoplasm into the vacuole and are important factors controlling citrate accumulation.Based on the citrus genome databases,we cloned all the proton pump genes and analyzed their sequences and structures.Taking the‘Anliu'orange?Citrus sinensis cv.Anliu?and its low acid mutant‘Hong Anliu'orange?C.sinensis cv.Hong Anliu?as research materials,the relationship between the expression levels of proton pump genes and the citric acid content of fruits were systematically studied,as well as the response of these genes to ABA teatment.On the other hand,the relationship between the changes of organic acid content and all genes,especially CsPH8,related to citrate metabolism were systematically analyzed between the fruits of AL and HAL,among fruits of different citrus cultivars and between fruits and leaves.Combined with transgenic identification,it was clear that CsPH8 may be the critical gene responsive for the differential accumulation of citrate;moreover,CsPH8low-expression is an important reason for the low accumulation of citrate in HAL fruits or citrus leaves.These results are listed in detail as follows:1.All proton pump genes associated with citric acid accumulation were successfully identified from the citrus genome.Of them,there were 20 genes?V₁-A,V₁-B,V₁-C,V₁-D,V₁-E1,V₁-E2,V₁-F1,V₁-F2,V₁-H1,V₁-H2,V₁-G,V₀-a1,V₀-a2,V₀-c1,V₀-c2,V₀-c3,V₀-c4,V₀-c",V₀-d and V₀-e?encoding VHA subunits,4 genes?VHP1,VHP2,VHP3and VHP4?encoding VHP,and 8 genes?CsPH1,CsPH2,CsPH3,CsPH4,CsPH5,CsPH6,CsPH7 and CsPH8?encoding PHA.2.By analyzing the relationship between the content of citric acid and the expression profiles of VHA and VHP genes,it was confirmed that VHA and VHP are involved in the regulation of citric acid accumulation in cell vacuole.In detail,it was found that the citric acid content of AL was significantly higher than that of HAL duringthe fruit development,and the ABA treatment can promote the accumulation of citric acid.On the other hand,gene expression analysis indicated that expression levels of V₁-A,V₁-C,V₁-E1,V₁-H2,V₀-a2,V₀-c4 and V₀-d,and the four genes of VHP were significantly higher in AL than in HAL.In addition,expression levels of these genes were also significantly induced by ABA.These results suggested that citric acid accumulation is associated with the high expression of these genes,namely,the accumulation of citric acid is indeed related to the activities of VHA and VHP.3.By analyzing the expression of all PHA genes in different tissues of AL,between the fruits of AL and HAL,the fruits of acid-less pomelo and HB pomelo?or Zaoxiang pomelo?,and the ABA treatment fruits,only CsPH8 was found highly expressed in juice sacs,and its expression level was significantly higher in high acid cultivars?AL,HB pomelo or Zaoxiang pomelo?than that of the low acid cultivars?HAL and Acid-less pomelo?;In addition,ABA treatment can significantly induce the expression level of CsPH8.Taken together,the present results provided the preliminary evidence that the low expression level of CsPH8 may be an important reason for the low accumulation of citric acid in HAL and acid-less pomelo;moreover,ABA treatment promoting the accumulation of citric acid is also associated to the significant increase of CsPH8 expression level.4.The metabolites and the expression levels of genes related to citrate metabolism were systematically compared between the fruits of AL and HAL.Results indicated that the enzyme activities and the expression levels of genes related to citrate synthesis and utilization were significant higher in HAL than AL although HAL accumulated less citric acid in juice sacs.Moreover,the expression levels of proton pump genes were significantly lower in HAL than AL.These results further confirmed that the low expression of CsPH8 is an important reason for the low accumulation of citric acid in HAL.5.The citric acid content of citrus fruits was significantly higher than that in the leaves.It was found that the expression levels of CsPH8 in most fruits were significantly higher than that in leaves by comparing citrate acid content and the gene expression level, which indicated that the low expression of CsPH8 may be an important reason for the low accumulation of citric acid in leaves.6.The significant correlation was found between the expression level of CsPH8and citric acid content by analyzing the relationship between the citric acid content and CsPH8 expression level during fruit development of six citrus cultivars,and the titratable acid content and CsPH8 expression in 21 citrus fruit samples.Also,transient overexpression in HAL leaves,strawberry fruits,and juice sacs of acid-less pomelo indicated that increase of CsPH8 expression level therein could significantly increase citric acid content,respectively,while transient RNA interference in HAL leaves and juice sacs of Zaoxiang pomelo significantly decreased CsPH8 expression level,as well as citric acid content.CsPH8 heterologous overexpression was also conducted in tomato.Results showed that CsPH8 expression levels and citric acid contents were significantly increased in the transformed tomato fruits and leaves.These results confirmed CsPH8 plays a pivotal role in citric acid differential accumulation in citrus fruits or leaves.On the whole,citric acid accumulation was regulated by proton pump,and the increase of citric acid content induced by ABA treatment was also closely related to the increase of the expression level of proton pump genes.Through extensive analysis of the relationship between changes in citric acid content and the expression levels of proton pump genes,combined with transgenic experiments,it was confirmed that the differential expression of CsPH8 is the main reason for the differential accumulation of citric acid in different citrus cultivars,while the low expression of CsPH8 is the main reason for the low accumulation of citric acid in HAL and citrus leaves of most cultivars.