Research On The Mechanism Of Fruit Cracking And Its Regulation By Exogenous Calcium In Vitis Vinifera

Grape is widely planted in China because of its wonderful flavors,rich nutrition and high economic value.However,because of the high temperature and humidity,and heavy rainfalls in South China,the cracking rate of some grape cultivars may reach up to 90% during the ripening stage.Fruit cracking is a physiological disorder in which cracks appear in the skin and flesh.This disorder affects the appearance of the fruit,increases its susceptibility to pathogen infection,and decrease storage and shelf life,resulting in enormous commercial losses to growers and restricting the development of grape industry in South China.Calcium is an essential macro element for grape growth.Our previous study found that dipping fruit with calcium solution could significantly reduce grape cracking rate,but the mechanism of reducing grape berry cracking is still unclear.To reveal the mechanism of fruit cracking and its prevention by calcium,we firstly investigated the fruit cracking rate and berry physiological indexes of 22 grape varieties in the field,and analyzed the relationship between cracking-resistance and fruit physiological indexes;the cracking-susceptible and cracking-resistant varieties were screened out,and the mechanism of grape cracking was deeply explored based on the characteristics of grape skins;then,based on the material susceptible �Xiangfei',the effect of calcium dips on fruit cracking and underlying mechanism was studied at subcellular,physiological,biochemical and transcriptional level.By analyzing the RNA-seq data based on the physiological indexes,VvPL1 was screened as the important candidate gene influencing fruit cracking;then the function of VvPL1 in fruit cracking was verified by transgenic method.This study can further rich grape berry cracking mechanism and provide scientific basis for prevention and improvement of grape berry cracking in the future.The main results of this experiment are presented as follows:(1)In an invistigation of 22 grape cultivars in Sounth China,our results showed that the cracking-resistance of different grape varieties was obviously different,including: 8 grape varieties with no crack,and the other varieties cracked at different degrees;for example,the average fruit cracking incidence of susceptible �Xiangfei' and �Aishenmeigui' was about 76.22% and 62.74% in successive two years;the moderately susceptible �Ruiduzaohong' and�Ruiduxiangyu'was 32.96% and 15.12%;the others were below 2%.The grape berry cracking types include: ring cracking,longitudinal cracking,ring-longitudinal cracking,apical end cracking.Moreover,the cracking incidence in V.vinifera was significantly higher than that in hybrids of V.vinifera and V.labrusca;the grape berry cracking was largely influenced by water;the berry cracking in 2017 was more serious than that in 2018,and the cracking incidence of different ripening time was early>middle>late.Further,the correlation analysis found that the skin break force and elasticity was most closely related to fruit cracking,and the skin thickness,total soluble solids and fruit shape showed no significant correlation with fruit cracking.(2)In the comparative study between cracking-susceptible �Xiangfei'(XF)and cracking-resistant �Shennongjinhuanghou'(SN),we found the cracking incidence of cracking-resistant �SN' was significantly lower than that of cracking-susceptible �XF' during the whole fruit development.In terms of tissue structure,the cuticle thickness,the size of subepidermal cells and the thickness of cell wall were significantly higher in cracking-resistant �SN',and the lateral and wall-mounted cell arrangement had a stronger ability to buffer the internal pressure on skin.From the cell wall metabolism,the cracking-resistant �SN' had higher pectinmethylesterase(PME)activity in early stage,and lower polygalacturonase(PG),?-galactosidase(?-Gal),pectate lyase(PL)and cellulase(Cx)during the whole fruit developmental stages,which resulted in formation of higher calcium-binding pectin,covalently bound pectin and hemicellulose,and lower water soluble pectin at the presence of higher calcium and boron content in cracking-resistant �SN'.Therefore,the stronger cell wall,skin break force and elasticity made �SN' more resistant to fruit cracking.Besides,compared to the equator and apical-end region,the obviously lower skin break force and elasticity of �XF' and largely accumulated water at stem-end may contributed to the ring-cracking.(3)Based on the material of cracking-susceptible cultivar �Xiangfei',we further try to elucidate the underlying physiological and molecular mechanism and explore the key genes related to fruit cracking.The results showed: dipping fruit with 5 g/L Ca Cl2 effectively enhanced calcium pools in the vacuole and cell wall,inhibited production of WSP,and delayed degradation of protopectin(ionically-bound pectin(CSP)and covalently bound pectin(SSP))by reducing the enzyme activities of PME,PG,PL and?-Gal,then enhanced the skin break force and,ultimately,significantly reduced the frequency and degree of fruit cracking.Further,through transcriptional analysis of genes involved in cell wall modification in berry skin under calcium treatment,30 genes associated with cell wall modification were identified,including: ten PME,two PMEI(PME inhibitor),five PG,four PL,four ?-Gal,one AFase(alpha-L-arabinofuranosidase),three EG(endoglucanase),and one ?-Glu(Beta-glucosidase).In general,most of the genes were significantly down-regulated by calcium treatment,among which VIT₀5s0051g00590(PL)with a higher expression level and fold changes(significantly down-regulated by 3.36 and 3.78 times at 47 and 54 DAFB under calcium)were detected,which may be the key functional gene related to berry cracking.(4)To study the relationship between VvPL1 and fruit cracking,we constructed the overexpression vector PC1300s-VvPL1,and heterogenously expressed VvPL1 on Alisa Craig.Over-expression of VvPL1 enhanced the PL activity from breaker stage and demonstrated higher resistant to fruit cracking.Consistent with this,compared with WT,over-expressed lines had lower level of CSP,SSP,cellulose and hemicellulose,and higher level of WSP at red-ripe stage,which resulted in significant reduction in skin break force and flesh firmness,and increased fruit cracking incidence.