Physiological And Molecular Mechanism Of Watermelon In Response To Low Boron And Boron Absorption Regulation By Rootstock

Watermelon[Citrullus lanatus(Thunb).Matsum.&Nakai]is an important fruitvegetable commercially cultivated worldwide.China being the largest producer of watermelon shares about 67.60%in world's production.Boron(B)is an essential element required for proper plant growth and deficiency of B is a nutritional disorder that disturbs plant growth.The transport of B from soil solution to aboveground plant parts is important for optimum plant growth under scarce B condition.The proper plant growth under less available B condition can be obtained by foliar or soil amendment of B fertilizers or by modifying root architecture.Grafting is an efficient technique used to increase soil borne diseases and abiotic stress tolerance,and has been widely used in watermelon production.In this study,we observed the effect of low B on watermelon plant growth and used grafting to improve B absorption under less available B conditions.The results of our study are presented below:1.The first experiment was conducted to optimize the dose of B for watermelon,and screening of low B tolerant and sensitive genotype under limited B conditions.In B dose optimization experiment,a commercial watermelon genotype‘Zaojia 8424'as planting material was exposed to six B levels(0?M,25?M,46?M,75?M,100?M and150?M)under hydroponic condition.Plants subjected to 75?M showed a significant improvement in plant fresh weight,dry weight,relative chlorophyll content,root length,root surface area,root volume and plant tissue B concentration compared with any other level of B supply.For genotype screening experiments,initially eight watermelon genotypes‘1553',‘Feizhou Huangzi(FH)',‘Hongziao Yu(HY)',‘Feihong Chuan(FC)',‘Wanfu Lai(WL)',‘Ju Feng(JF)',‘Zaojia 8424(ZJ)'‘Zaochun Honggyu(ZH)'and three B levels(0.25?M,25?M and 75?M)were used.In the second experiment,four genotypes namely‘FH','WL',‘JF',and‘ZJ'were exposed to two B levels(0.25?M and75?M).The results of both experiments showed restricted plant growth of all genotypes at 0.25?M B level compared with control(75?M B).However,the level of severity was significant for‘WL'exhibited by reduced root and shoot growth.Moreover,less tissue B accumulation showed the limited ability of‘WL'for B absorption and transport under limited B conditions.‘FH'showed more tolerance to low B treatment(0.25?M B)in term of improved fresh and dry weight,F_v/F_m,root morphology,and higher B accumulation in root and leaf tissues compared with other genotypes under low B treatment.Based on these experimental results,we considered‘FH'as low B tolerant and‘WL'as low B sensitive watermelon genotype.2.The combination of vegetative and reproductive growth parameters,and transcriptome analysis was employed to study the response of low B sensitive(Wanfu Lai)and low B tolerant(Feizhou Huangzi)watermelon genotype under 0.25?M B(low B)concentration.Low B treatment severely restricted the plant growth of‘Wanfu Lai'evidenced by less dry mass production,reduction of photosynthetic capacity and root growth inhibition,whereas,plants of‘Feizhou Huangzi'showed less growth defects under low B conditions.The results of scanning electron microscopic(SEM)analysis showed less structural defects in shape of stamen,pollen topography and stigma's papilla cell integration of‘Feizhou Huangzi'compared with‘Wanfu Lai'under low B conditions.Based on transcriptomic results,we identified that low B tolerance mechanism of Feizhou Huangzi'was closely associated with the more up-regulation of DEGs in roots(70%)and leaves(44%)with particular identification of more B transporter genes(NIP5;1,BOR2)compared with‘Wanfu Lai'.Moreover,more DEG enrichment in peroxidase(POD),cellular oxidant detoxification process and plant hormonal signalling pathway,high transcript of invertase/pectin methylesterase inhibitor family protein(PMEI),and high gene expression of BOR2 gene helped in inhibition of ROS signalling cascade that reduced the root growth inhibition of‘Feizhou Huangzi'and promoted the continuous uptake of B from root and its transport towards shoot.Taken together,we conclude that low B stress tolerance was mainly mediated by NIP5;1 and BOR2 signalling network that promoted the root growth,restricted the H₂O₂ accumulation in root,enhanced B uptake,and transported it to shoot where it was utilized in plant reproductive organ development,and photosynthesis that ultimately improved watermelon's plant growth under low B conditions.3.This experiment was conducted to investigate the potential of low B tolerant watermelon rootstock‘Feizhou Huangzi'to improve the growth of low B sensitive watermelon genotype‘Wanfu Lai'under less available B conditions.In this study,two grafting combinations were made as 1)self-grafted low B sensitive watermelon genotype‘Wanfu Lai'(S/S),and 2)low B sensitive watermelon genotype grafted onto low B tolerant watermelon rootstock‘Feizhou Huangzi'(S/T).Grafted watermelon plants were exposed to two level of B supply[0.25?M(treatment)and 75?M(control)].According to results,S/T plants showed improved plant dry mass,root mass,number of root tips and root activity,and less H₂O₂ accumulation and lipid peroxidation,and higher antioxidant enzymatic activities in the roots and leaves compared with S/S plants under 0.25?M B conditions.Moreover,up-regulation of watermelon aquaporin and B transporter genes(Cla.NIP5;1,Cla.NIP6;1;Cla.BOR1;Cla.BOR2)helped enhance the B uptake and supply to the plant tissues and cell wall of S/T plants under limited B conditions compared with S/S plants.The FTIR analysis results showed less protein structure destruction and wax accumulation in cell wall of S/T plants evidenced by less uronic acid content,cellulose content,and high expression of cell wall related gene(Cla.XTH27)under limited B condition.In conclusion,the up-regulation of Cla.NIP5;1,Cla.BOR1 and Cla.BOR2genes in S/T plants promoted B absorption that increased the B-RG-II complex and decreased the cell wall structural defects that ultimately increased low B tolerance.4.In this study,a commercial watermelon cultivar“Zaojia 8424”[Citrullus lanatus(Thunb.)Matsum.and Nakai.]was grafted onto pumpkin(Cucurbita maxima×Cucurbita moschata)rootstock cv.“Qingyan Zhenmu No.1”with an aim to investigate the response of grafted plants to different levels of B supply(0.25?M,25?M and 75?M B)in the nutrient solution.Self-grafted watermelon plants were used as control.Pumpkin rootstock improved the plant growth,B accumulation,chlorophyll and carotenoid contents,photosynthetic assimilation,stomatal conductance,and transpiration rate,accompanied by up-regulating the expression of aquaporins(NIP5;1,NIP6;1)and B transporter(BOR2)genes in the roots and leaves,and down-regulating expression of chlorophyll degradation gene(NOL)expression at 25?M B compared with self-grafted watermelon plants.Pumpkin grafting reduced the oxidative stress and cell damage by reducing H₂O₂ and MDA contents,and down-regulating the expression of PDCD2-1,PDCD2-2 genes.Moreover,grafting watermelon onto pumpkin enhanced the antioxidant activity of watermelon by up-regulating the expression of SOD1,SOD2,CAT2-1,and CAT2-2 genes.Based on these observations,we concluded that pumpkin rootstock grafted watermelon plants increase their tolerance to low B via mechanism of up-regulating expression of aquaporins(NIP5;1,NIP6;1)and B transporter(BOR2)genes,inhibition of ROS signalling cascade and promoting of SOD and CAT activities.