The Regulation In Calcium Addition On The Drought Resistance Physiology Of Mulberry Seedlings And Its Molecular Mechanism

In arid and semi-arid regions,calcium（Ca）as a more heterogeneous element affects plant growth together with water.Mulberry（Morus alba L.）is an important ecological and economic forest tree species in semi-arid regions.To investigate the dynamic effects of calcium addition on growth,morphology,non-structural carbohydrates and photosynthetic of mulberry seedlings under drought stress,mulberry seedlings are treated with water treatments（normal water supply:70±5%of maximum water holding capacity,drought stress:40±5%of maximum water holding capacity）and Ca addition(Ca0:0 mg·kg^-1 Ca²⁺;Ca200:200 mg·kg^-1 Ca²⁺;Ca400:400 mg·kg^-1 Ca²⁺).Three samplings（S1,S2,S3）are conducted in the first growing season and two samplings（S4,S5）are conducted in the second growing season of mulberry seedlings.To explain the metabolic regulatory mechanisms of mulberry seedlings in response to drought stress and Ca addition,transcriptome and metabolome analyses are performed on leaves of mulberry seedlings treated with Ca0,Ca400 under normal water supply conditions and Ca0,Ca400 under drought stress at the S2 growth stage.The main results are as follows:（1）Under well-watered conditions,Ca addition reduce the height and biomass of mulberry seedlings in the first growing season,while the growth rate of mulberry seedlings of Ca addition start to be significantly higher than Ca0 in S3 stage;compare with S4 stage,the height of Ca0,Ca200,and Ca400 of S5 stage increase by 20.75%,34.47%,and 58.64%,respectively;Ca400promoted the growth of mulberry seedlings more significant than Ca200 in the second growing season.Ca200 increase lateral root mass fraction,root length,root surface area,and root volume of mulberry seedlings throughout the growth phase,while Ca400 treatment showed promotion of root growth only in the second growing season under normal water conditions.The growth and biomass accumulation of mulberry seedlings are significantly reduced under drought stress,but mulberry seedlings could mitigate drought stress through strategies such as reducing SLA,increasing SRL,and increasing investment in the root system.Under drought stress,the growth dynamics of mulberry seedlings at Ca200 are similar to Ca200 under normal water,while Ca400always inhibit the growth of mulberry seedlings.（2）At the early stage of drought stress（S1 stage）,the NSC concentration of each organ of mulberry seedlings increase significantly.With the rapid growth of mulberry seedlings（S2stage）,the soluble sugar,starch,and NSC concentration of main roots and stems increase significantly under drought stress,and the soluble sugar,starch,and NSC concentration of leaves and lateral roots decrease significantly.the NSC concentration of stems and leaves significantly decrease and the NSC concentration of lateral roots increase,mulberry seedlings allocated more carbon to the ground to be stored as starch before overwintering（S3 stage）.Under drought stress,Ca400 significantly decrease the whole starch concentration of mulberry seedlings in the first growing season with little change in soluble sugar concentration,and soluble sugar concentration increase significantly in the second growing season.Mulberry seedlings increased soluble sugar concentration at the expense of starch accumulation and growth in order to improve their survival at Ca400 under drought conditions.（3）Under well-watered conditions,Ca200 significantly reduce Pn and Tr,Ca400significantly reduce Pn,Gs,Tr,RC/CS,and increase ABS/RC and ETo/RC at S1 stage.Ca200increase Sm,ψPo andδRo,enhancing electron transfer capacity and PSI electron accepting,Ca400 increased Sm and increased photosynthetic electron transport capacity at S2 stage.Ca addition significantly increase Pn,Gs,Tr,ψPo andδRo in mulberry seedlings at S3,S4 and S5stages,Ca400 promote Pn significantly than Ca200 in mulberry seedlings.Drought stress significantly decrease Pn,Gs,Tr of mulberry seedlings;drought stress significantly decrease Sm,Fm,ETo/CS,ABS/CS,RC/CS,ψEo,φEo,ψPo,δRo,φRo,and increased ABS/RC with the extension of drought stress,indicating that drought stress lead to the inactivation of PSII reaction center,the reduction of PSII electron transfer capacity,and the reduction in PSI electron accepting capacity.DIo/RC,DIo/CS increase throughout the process of drought suggest that mulberry seedlings alleviate the damage to photosystem by increasing heat dissipation,mulberry seedlings enhance drought tolerance by increasing WUE.Under drought conditions,Ca200 increasedψPo,δRo,andφRo at stage S2,Fm,Sm,RC/CS,and ETo/CS at stage S3,and ABS/CS at stage S4,indicating that Ca200 mitigates drought stress by gradually enhancing the electron accepting capacity of PSI,electron transfer capacity of PSII,and light trapping capacity of mulberry seedlings.Ca400 treatment deepen the extent to which drought stress led to inactivation of PSII reaction centers,reduced PSII electron transfer capacity,and reduced PSI electron acceptance capacity,and severely inhibit the photosynthetic capacity of mulberry seedlings.（4）Under Ca400 treatment,threonine,citrulline,7 flavonoids such as apigenin,luteolin,quercetin,rutin and chlorogenic acid are down-regulated,and subunit XI related genes on PSⅠis up-regulated,these results indicating that Ca400 is beneficial to the growth of mulberry seedlings.Under drought condition,1 photosystem electron transfer protein regulatory related gene is down-regulated and 2 subunit XI-related genes on PSⅠare down-regulated,indicating that drought inhibit the photosynthetic ability of mulberry seedlings;although the flavonoids quercetin and rutin are down-regulated,flavonoid biosynthesis related genes are up-regulated indicating that mulberry seedlings could improve the drought resistance of mulberry seedlings by promoting the biosynthesis of flavonoids.Under drought stress,Ca400 treatment resulted in down-regulation of 20 PSⅠand PSⅡlight-harvesting protein related genes,16 down-regulation of genes related to subunits on PSⅠand PSⅡ,and down-regulation of ATP,which severely inhibit photosynthetic ability of mulberry seedlings.Moreover,down-regulation of 4metabolites and 12 related genes of photosynthetic carbon fixation pathway,up-regulation of 6related genes of glycolysis pathway,up-regulation of 4 related genes of TCA cycle,up-regulation of phenylpropanoid,the above results indicate that Ca400 treatment under drought stress accelerates TCA cycle under poor carbon fixation to promote amino acid and phenylpropanoid synthesis,which temporarily improves the resistance of mulberry seedlings but leads to disruption of the whole energy metabolism pathway.In summary,mulberry seedlings improve their survival through strategies such as reducing SLA and increasing SRL,WUE,and NSC content under drought stress.The promotion of mulberry seedling growth by Ca200 is associated with improved photosynthetic capacity and increased investment in the root system under both water regimes,and Ca400 promote mulberry seedlings better than Ca200 under well-water conditions.Although Ca400 briefly improve the survival of mulberry seedlings under drought stress by regulating resistance mechanisms,it still severely inhibit the growth of mulberry seedlings.The above results deepen the understanding of the adaptation response of mulberry seedlings to drought-calcium heterogeneity,and provide theoretical guidance for mulberry afforestation and forest management in semi-arid areas.