Research On Molecular Mechanism Of Pumpkin Rootstock Effects On Fruit Quality Of Grafted Watermelon

Watermelon(Citrullus lanatus L.)belongs to the gourd family,is an important horticultural crop,originated in equatorial Africa and the Mediterranean coast,because of its sweet and juicy,loved by consumers.The production of watermelon is huge in China,accounting for more than 60%of global output.Watermelon planting benefit is high,but limited by the watermelon Fusarium wilt disease.The application of grafting technology has successfully solved the problem.However,watermelon can not be continuous cropping on a plot.Currently,many reports mainly concentrated in screening of watermelon rootstock varieties,exploration of grafting methods,physiological and biochemical study related to disease resistant and stress resistant,etc..Only a few scholars studied on pumpkin rootstock influence on grafted watermelon quality,especially lacked of in-depth analysis on physiological biochemical and molecular mechanism.These will severely restrict the application of grafting technology in the production of high quality watermelon.Therefore,this study used pumpkin 'Jingxinzhen No.4'as rootstock,and watermelon'WM022' as scion.Three treatments were used,including' no-grafted watermelon(NGW),self-grafted watermelon(SGW)with scion as grafting rootstock,and pumpkin-grafted watermelon(PGW)with 'Jingxinzhen No.4' pumpkin as grafting rootstock.The effects of the node order of female flower,the fruit setting rate,the production and the morphology characteristics of pumpkin grafting on watermelon fruit were studied,such as,fruit size,fruit shape,pericarp thickness,fresh firmness,and so on.Then the effects of pumpkin grafting on the dynamic changes of related substances in watermelon fruit were analyzed by physiological and biochemical methods,such as dry matter,soluble sugar,organic acid,lycopene.Further the dynamic changes of key metabolic enzymes regulated sugar and acid content of grafted watermelon fruit were also investigated.In addition,the differentially expressed genes between self grafted watermelon and pumpkin grafted watermelon fruit were analyzed by high-throughput technology(RNA-Seq).The genes involved in regulation the lycopene biosynthesis in pumpkin grafted watermelon were focused.The main results are as follows:1.Influence on morphology characteristics of the grafted watermelon fruit on pumpkin rootstockThe fruit changes of NGW,SGW and PGW were studied in the fruit growth periods.The results showed that there was no significant difference in fruit shape index between NGW,SGW and PGW,and the fruit shape index of these three was 1 at maturity,which illustrated pumpkin rootstock 'Jingxinzhen No.4' did not significantly change the shape of gafted watermelon 'WM022'.At maturity,the central soluble solids of the three were about 13%,while the marginal soluble solids were about 9.8%,which illustrated that the soluble solids content of the fruit was not significantly improved by pumpkin grafting.In terms of single fruit weight,NGW,SGW and PGW had little difference,but did not reach a significant level,and at maturity these were about 5kg,which indicating that pumpkin grafting did not significantly reduce the fruit weight of watermelon.In the aspect of flesh hardness,except 26DAP,PGW,NGW and SGW was not significant difference in the other three periods,which indicated that pumpkin grafting had no significant effect on the flesh hardness of watermelon.There was no much differentiation in the three sample groups on the fruit setting rate and the total yield,which demonstrated that grafting did not significantly effect on fruit setting rate and total yield of watermelon.2.The effect of main quality indexes of grafted watermelon on pumpkin rootstockIn this study,the dynamic changes of some matters related to fruit quality were studied in fruit pulp of NGW,SGW and PGW during the fruit development periods,such as dry matter,soluble sugar,organic acid and lycopene.The results showed that grafting increased total dry matter content of watermelon fruit.At maturity period,the total soluble sugar content of PGW,SGW and NGW were 86 mg·g-1FW-1,77 mg·g-1FW-1,70 mg·g-1FW-1 respectively,which indicated that grafting increased the soluble sugar content of watermelon.Grafting had some influence on the component content of soluble sugar.SGW and PGW showed similar rules in the changes of organic acid in the process of fruit development,while NGW was different from them.When the fruit was ripe,the total acid content of NGW was 12.5 mg-g-1FW-1,and SGW and PGW were 13.4 mg·g-1FW-1,13.8 mg·g-1FW-1 respectively.The total acid content of the three groups was the same,which showed grafting had no obvious significance on the organic acid.In the mature stage,the content of lycopene of NGW,SGW and PGW were 35.2 mg·g-1FW-1,34.8 mg·g-1FW-1,24.4 mg·g-1FW-1 respectively,which showed that self grafting had no effect on the content of lycopene in watermelon fruit,but pumpkin grafting significantly reduced the content of lycopene.3.Metabolic enzymes regulation mechanism involved in pumpkin rootstock on sugar and acid quality of grafted watermelonIn this study,the dynamic changes of the key enzymes of sugar and acid metabolism were studied in NGW,SGW and PGW during fruit development.The results showed that the invertase(Inv)activity of PGW was lower than that of NGW at maturity.At 34 DAP,the activity of PGW sucrose synthase(SS)was the highest(17.9 mg·h-1·g-1FW-1),followed by SGW(1.5.5 mg·h-1·g-1FW-1),and NGW was the lowest(8.3 mg·h-1g-1FW-1).On the change of sucrose phosphate synthetase(SPS)activity,the peak value of PGW and NGW was at 26 days after pollination(DAP),which were 169.8 mg·h-1·g-1FW-1 and 181.3 mg·h-1·g-1FW-1 respectively,while the peak value of SGW was 18DAP,at 127.0 mg·h-1·g-1FW-1.The sucrose phosphate synthase(SPS)activity of PGW was higher than that of two controls at maturity,and the SS activity was not significantly different from that of two controls.The activity of invertase decreased,and the activity of sucrose synthase and sucrose phosphate synthase increased,which may result to sugar content increase during fruit development,and the grafted watermelon sugar accumulation may be affected by Inv,SPS and SS.The trend of citrate synthase(CS)was basically same in NGW,SGW and PGW,which rising in the process of fruit development,and it was in accord with the change trend of the citric acid content,indicating that CS played a key role in the synthesis of citric acid.In this study,the NAD-dependent malate dehydrogenase(NAD-MDH)activity decreased,the content of malic acid of watermelon increased,which demonstrated that NAD-MDH major played decomposition.Phosphoenolpyruvate carboxylase(PEPC)showed a trend of first increasing and then decreasing in NGW,SGW and PGW.The total acid content of NGW,SGW and PGW was not significant difference in the mature stage,which may be the result of the interaction of CS,PEPC,NAD-MDH and other enzymes.4.Gene regulation mechanism of pumpkin rootstock on quality of grafted watermelonA total of 929 differentially expressed genes(DEGs)between the self-grafted control and pumpkin-grafted watermelon libraries were significant during four fruit developmental stages,64%up-regulated and 36%down-regulated.Gene ontology annotation and KEGG pathway enrichment analysis of these genes indicated that most of the DEGs were involved in biological process related to fruit quality,such as carotenoid biosynthesis,flavonoid biosynthesis,amino sugar and nucleotide sugar metabolism,pentose and glucuronate interconversions,and starch and sucrose metabolism,etc.in early stage of fruit development.In the maturity stage,all the DEGs were not significantly enriched in KEGG pathway.The results of this study provided new insights into the critical factors associated with grafting influence on fruit development and fruit quality and will facilitate further research aimed at understanding the molecular mechanisms on grafting watermelon.5.Gene expression regulation of lycopene biosynthesis related to quality of pumpkingrafted watermelonThe results showed the transcriptome profiling data provided new information on the grafting-induced gene regulation of lycopene biosynthesis during fruit growth and development.The expression levels of 33 genes from 8 gene families(GGPS,PSY,PDS,ZDS,CRTISO,LCYb,LCYe and CHY)related to lycopene biosynthesis,were monitored during the four periods of fruit development in watermelon.14 genes were differentially expressed in pumpkin-grafted watermelon during fruit development,suggesting that these genes possibly help to mediate lycopene biosynthesis in grafted watermelon fruit.This work provided some novel insights into grafting-responsive carotenoid metabolism and its potential roles during pumpkin-grafted watermelon fruit development and ripening.