Studies On The Different Mechanism Of Sucrose Accumulation In Fruits Of Nanguo Pear And Its High Sugar Accumulated Bud Sport

Pear is a very important horticultural crop in the world,and improving the quality of pear fruit is a major goal in horticultural crop breeding.Sweetness is one of the main factors of fruit quality and it has been recognized as an important driver of consumer preference.However,the mechanisms underlying the sugar accumulation in pear fruit has not been studied intensively yet.Nanguo(NG)is much sought after by growers and consumers because of its cold resistance,good taste and nice aroma.In this study,NG and its bud sport(BNG)varieties were used to study the mechanism underlying the sugar accumulation.We compared the transcriptomes of NG and BNG fruits and identified a sucrose transporter Pu SWEET15 and a WRKY transcription factor Pu WRKY31 which are expressed at higher levels in BNG fruit.We found that Pu WRKY31 bound to the Pu SWEET15 promoter and induce its expression.Moreover,the high acetylation level of the Pu WRKY31 promoter was associated with its high expression level in BNG fruit.The main results are as follows:1.The sucrose content in BNG fruit is higher than that in NG fruit.We measured the total soluble solid content and the sugar content in NG and BNG pear fruits and we found that the total soluble solid content was higher in BNG than in NG.And we observed a significantly higher sucrose content in BNG fruit than in NG fruit.These findings suggested that the higher BNG sugar content and sweeter taste was due to a higher sucrose accumulation.2.The sucrose transporter,Pu SWEET15,is highly expressed in BNG fruit.To identify genes that might contribute to the higher sucrose accumulation in BNG fruit,we compared the transcriptomes of NG and BNG fruits using RNA-seq,and identified a SWEET gene,Pu SWEET15,as being expressed at higher levels in BNG fruit.The expression profile of Pu SWEET15 was investigated in NG and BNG fruits during development,and we found that it was expressed at significantly higher levels in BNG fruit,consistent with the change in sucrose content.Heterologous expression of Pu SWEET15 in a SUSY7/ura(Saccharomyces cerevisiae)yeast strain showed that Pu SWEET15 was an active sucrose transporter.Overexpression of Pu SWEET15 in NG pear fruit resulted in increases in the sucrose content,while silencing of Pu SWEET15 in BNG fruit resulted in decreases of the sucrose content.These results were all consistent with Pu SWEET15 contributing to sucrose transport.3.Pu WRKY31 promotes the expression of Pu SWEET15.Pu SWEET15 is essential for sucrose accumulation in pear fruit,to elucidate the Pu SWEET15 expression profiles in NG and BNG fruits,we compared the CDS of Pu SWEET15 from each;however,no difference was found.Moreover,no differences were observed in the Pu SWEET15 promoter from NG and BNG fruits,and the methylation levels and the histone acetylation levels of the Pu SWEET15 promoters were also almost identical.We then analyzed the cis-elements of the Pu SWEET15 promoter and identified binding sites of transcription factors such as WRKY,DOF(DNA-binding one finger)and MYB.In combination with the RNA-seq results,a WRKY transcription factor,Pu WRKY31,was observed to be more highly expressed in BNG fruit than in NG fruit.The expression profile of Pu WRKY31 was confirmed by quantitative reverse transcription PCR(q RT-PCR).To investigate the function of Pu WRKY31,we overexpressed it in NG fruit(Pu WRKY31-OE),and the higher expression of Pu WRKY31 in Pu WRKY31-OE fruit was verified by q RT-PCR.We detected that the sucrose content in Pu WRKY31-OE fruit was significantly higher than that in the control fruit.Notably,the expression level of Pu SWEET15 was also observed to be higher in Pu WRKY31-OE fruit,suggesting that Pu WRKY31 might play a role in sucrose transport by regulating the expression of Pu SWEET15.To investigate whether Pu SWEET15 is a direct target of Pu WRKY31,we performed an EMSA.Next,we used Ch IP PCR to investigate the in vivo binding of Pu WRKY31 to the Pu SWEET15 promoter.We then investigated the regulation by Pu WRKY31 of the Pu SWEET15 promoter using a ?-glucuronidase(GUS)activation assay in wild tobacco leaves.Collectively,these results suggested that Pu WRKY31 bound to the Pu SWEET15 promoter and promoted its transcription.4.Pu HLS1 directly interacts with the CDS of Pu WRKY31.To investigate the Pu WRKY31 expression profiles in NG and BNG fruits,we compared its CDS,promoter sequences and methylation levels of the promoter regions.However,no significant differences were observed.We hypothesized that the Pu WRKY31 expression pattern might correlate with a change in histone modification,and so examined the histone acetylation levels of the Pu WRKY31 promoter in NG and BNG fruits and we found that the acetylation levels of the Pu WRKY31 promoter were significantly higher in BNG than in NG fruit.To investigate what causes the higher acetylation level of Pu WRKY31 promoter in BNG fruit,we identified a histone acetyltransferase Pu HLS1(HOOKLESS 1),from the RNA-seq results.Pu HLS1 was expressed higher in BNG fruit than in NG fruit.EMSA analysis was performed to confirm that Pu HLS1 bound to the CDS of Pu WRKY31 in vitro.In summary,Pu SWEET15 was expressed higher in BNG fruit than in NG fruit and Pu WRKY31 bound to the Pu SWEET15 promoter to induce its expression.Moreover,the high acetylation level of the Pu WRKY31 promoter was found to be associated with its high expression level in BNG fruit.The results above indicate that the acetylation level of the Pu WRKY31 promoter in these two pear fruits led to the higher sucrose accumulation in BNG.