Effect Of CPPU At Different Concentrations On Cucurbitacin B Synthesis In Oriental Melon(Cucumis Melo Var.Makuwa Makino)Fruit

Melon(Cucumis melo L.),widely cultivated in China,the high levels of sugars and unique aroma recognized by consumers.However,several factors,such as varieties,environment and cultivation technique,may enhance the synthesis of cucurbitacins and lead to bitterness in fruit,which has a considerable negative impact on the quality of melon.Bitterness is one of important traits that caused by cucurbitacins.CPPU is a cytokinin-like plant growth regulator,which can promote cell division and expansion.During the cultivation of melon,high concentrations of CPPU will increase the bitterness of fruit.However,more information is needed about the physiological and molecular mechanisms of bitter fruit regulated by CPPU.Therefore,further clarifying the regulation mechanism of CPPU on the synthesis of cucurbitin B in melon is of great significance for improving the quality of melon fruit.In this study,two oriental melon cultivars 'YMR'(easy to present bitter fruit)and'HDB'(hardly produce bitter fruit)were used.The effect of cucurbitacin B(Cu B)accumulation in melon fruit treated by CPPU at different developmental stages was investigated,and CmBt by transient overexpression in fruit treated by 20 mg·L-1 CPPU,transcription analysis was evaluated.The results indicate that high CPPU concentrations promoted Cu B accumulation in melon fruit by inducing transcription factor CmBt.The main results are as follows:1.To investigate the Cu B accumulation pattern in melon,we measure Cu B content in different plant tissues including cotyledons,roots,stems,leaves,female and male flowers,tendrils and fruit at 7 DAF in two cultivars using HPLC analysis.The relative expression of the Cu B related genes(CmBi,Cm160,Cm170,Cm180,Cm490,Cm710,Cm890,CmACT,CmBr and CmBt)was quantified by qRT-PCR Cu B in two varieties accumulates mainly in the roots,followed by tendrils and fruit.None was detected in the cotyledons.Interestingly,Cu B content in the 'YMR' tissues was about 2-3 times higher than that of‘HDB',which is presumed to be the reason for occurrence of bitter melon in 'YMR'.The Cu B biosynthetic genes expression patterns were found to be consistent with Cu B content in plant tissues of two varieties.2.In order to reveal effect of CPPU at different concentrations on Cu B content and related genes,four concentrations of CPPU(2.5,5,10 and 20 mg·L-1)were set and hand pollination was used as control.Fruit of two cultivars('YMR' and 'HDB')were harvested at developmental(7,14,21 and 28 DAF)and mature stage(35 DAF)for analysis.Cu B accumulation was highest at 7 DAF,but moderate and disappeared from 14 to 35 DAF in two varieties.Cu B content of two varieties significantly increased with CPPU at different concentration on young fruit(7 DAF),which 20 mg·L-1 CPPU was obviously accelerated Cu B accumulation of 'YMR'.During fruit maturation,Cu B content was still detected in 'YMR'fruit treated by 20 mg·L-1 CPPU,while disappeared in 'HDB' fruit.The expression levels of Cu B biosynthetic genes(CmBi,Cm710 and CmACT)and CmBt,the fruit-specific transcription factors,were significantly induced by 20 mg·L-1 CPPU.3.Cu B content in different parts(pedicle pulp;middle pulp;navel pulp;stalk;peel)of'YMR' melon fruit treated by 20 mg·L-1 CPPU and hand pollination at different development stages were detected.In the early developmental stages(7?14 DAF),Cu B content was the highest in the stalk,followed by pedicle pulp,middle pulp,navel pulp,peel.Interestingly,Cu B content was present the lowest in the stalk at the middle developmental stages and maturation(21?35 DAF),which mainly accumulated the pedicle pulp and peel.Cu B content in different parts of 'YMR' melon fruit treated by 20 mg·L-1 CPPU were higher than that of controls in each developmental stages.The results suggest that 20 mg·L-1 CPPU increased the content of Cu B in melon fruit.Meanwhile,to study whether high CPPU concentrations produced bitter fruit during maturation,total eighty fruit treated by 20 mg·L-1 CPPU and hand pollination was investigated.The highest proportion of bitter fruit treated by 20 mg·L-1 CPPU was 76.25%during maturation.However,non-bitterness fruit were identified in all fruit after hand pollination.Moreover,20 mg·L-1 CPPU increased the probability of bitterness in mature fruit.4.An oriental melon 'YMR' at 10 DAF and 20 DAF after hand pollination was used as a test material.2.5,5,10 and 20 mg·L-1 CPPU treatment was set and mannitol buffer without CPPU was used as a control.Cu B increased,reduced and then increased.Cu B content of fruit treated by 20 mg·L-1 CPPU was significantly higher than that of control at 2 h and 12 h after treatments.5.The coding DNA sequence(CDS)of CmBt was amplified and generated the LUC-CmBt for over-expression.A transient expression assay was performed by injecting the construct into the young fruit treated by 20 mg·L-1 CPPU after 7 DAF.After injection of the agrobacterium-mediated LUC-CmBt construct into the young fruit,the expression of CmBt significantly increased in over-expression fruit and accelerated Cu B accumulation,which was higher than that of control in 3 and 4 days post injection.The transcripts of CmBi,CmACT,Cm710,Cm890 initially showed significant increase in over-expression fruit compared with controls at 1 days post injection.These results suggest that CmBt respond to 20 mg·L-1 CPPU and activated Cu B biosynthetic genes to some extent.In a conclusion,Cu B accumulated mainly in roots and fruit of oriental melon.Furthermore,20 mg·L-1 CPPU was obviously promoted Cu B accumulation.Transient overexpression of CmBt gene in cultivated melon fruit promoted Cu B accumulation by activating Cu B biosynthetic genes(CmBi,CmACT,Cm710 and Cm890).Taken together,this study demonstrated that high CPPU concentrations promoted Cu B accumulation that CmBt activated Cu B biosynthetic genes.