| In plant kingdom,most plants contain calcium oxalate biomineral,which often possess inter-species diversity and species-specific characteristics.Calcium oxalate,as a metabolite in plants,has many important biological functions,such as calcium regulation,light reflection and defense against pests.The formation of calcium oxalate crystal in plants is precisely controlled not only by intracellular biomacromolecules(polysaccharides and proteins),but also by calcium concentration in their environment.However,the mechanism of calcium oxalate formation induced by high calcium in plants remains unclear.Additionally,although the accumulation of calcium oxalate crystals in plants under drought conditions has been reported,the roles of calcium oxalate in drought tolerance are not clear yet.In this paper,the accumulation of calcium oxalate in Medicago lupulina leaf under high calcium treatment and its role in drought tolerance were studied.Moreover,the changes of metabolites and related gene expression in oxalate biosynthesis pathway under high calcium were analyzed,and the expression profiles of gene related to calcium transport systems were also obtained.Finally,the expression of CDPK gene family in high calcium and drought conditions were fulfilled.This study focus on the molecular mechanism of calcium oxalate formation induced by high calcium in plants and its role in drought tolerance.The main results are sumnarized as follows:(1)High calcium promotes the accumulation of calcium oxalate in M.lupulina leaves and enhances plant drought tolerance.In high calcium(25 mM Ca2+)condition,calcium oxalate crystals were significantly accumulated in main vein of M.lupulina leaf.The component of the crystal was confirmed by X-ray diffraction(XRD)and Fourier transformation infrared(FTIR)analysis.The shape of crystals was observed by scanning electron microscopy(SEM),and it was classified as a prismatic crystal.Calcium oxalate crystals were not found in the model plant Arabidopsis thaliana leaves.Under drought stress,M.lupulina,which have more calcium oxalate induced by high calcium,is more drought tolerance with much lower leaf temperature than that in A.thaliana.Several Medicago species with different calcium oxalate levels were used to analyze the relationship between calcium oxalate content and drought tolerance.The result indicated that the species with high calcium oxalate content could tolerate more serve drought stress.Furthermore,relative water content,stomatal conductance,transpiration rate and osmotic potential for soluble ion in M.lupulina leaves were not significantly changed,however the water potential were decreased significantly,suggesting that the most volumes of vacuole were occupied by the calcium oxalate crystals which was increased by numbers and volumes under high calcium condition,leading to the increase of solution concentration and reduction of osmotic potential.As a result,the water potential in leaves was decreased,which enable plant to keep or to uptake more water under drought condition.Finally,M.lupulina could tolerate more drought stress.(2)High calcium alters the metabolites in ascorbate metabolic pathway and promotes oxalate production.The leaves of M lupulina cultured in high calcium were used to analyze the metabolites changes by using metabolomic technique.In addition,the principal component analysis(PCA)and orthogonal projections to latent structures-discriminate analysis(OPLS-DA)were applied to screen the differential metabolites.By metabolic pathway annotation and cluster analysis,the results showed that high calcium mainly affected the accumulation of carbohydrate(such as fructose and mannose)and organic acids(such as citric acid and threonic acid).Besides,the accumulation of hydrogen peroxide in M.lupulina leaves was also induced by high calcium.Dehydroascorbate were formed from ascorbate by reacting with hydrogen peroxide,then rapid to produce oxalate.(3)High calcium induces the expression of gene involved in ascorbic acid metabolic pathway and calcium transport system and promotes calcium oxalate crystals formation.The leaves of M.lupulina cultured in high calcium were used to RNA sequencing.Sequences assembly,functional annotation and metabolic pathway annotation were carried out.Then,the differentially expressed genes were screened from the Unigene bank.The results showed that the gene encoding ascorbate oxidase in the ascorbate cycle was significantly up-regulated under high calcium,implying the ascorbate cycle is susceptible to calcium.Similarly,the gene encoding respiratory burst oxidase was also significantly up-regulated under high calcium,suggesting hydrogen peroxide was profoundly induced by high calcium.Additionally,the gene encoding calcium transport system(such as calcium channel and calcium pump and calcium exchanger)were significantly up-regulated under high calcium,resulting in more calcium were transported into cell and were eventually transported into vacuole,then finally to form more calcium oxalate crystals.Furthermore,high calcium also induced the gene expression encoding calreticulin,which could buffer the high concentrations of calcium in the cell,to avoid the serious cell damage.(4)High calcium up-regulates the gene expression of CDPK gene family and increase the tolerance of M lupulina to drought.Bioinformatic analysis was used to identify CDPK gene family based on transcriptome database.The result showed that 16 CDPK genes and related gene families were identified from the transcriptome database,including 10 CDPK genes,4 CRK genes and 2 PEPRK genes.Their biochemical information,phylogenetic tree,conserved region and protein domain were analyzed.Finally,the expression patterns of these genes in different tissues,high calcium and drought conditions were determined by qRT-PCR.The results showed that the expression levels of MlCDPK1,MICDPK7,MlCRK1 and MlPEPRK1 were significantly up-regulated under high calcium.Under drought conditions,many MlCDPKs and related gene family were up-regulated,indicating that these genes play an important role in drought tolerance of M lupulina under high calcium. |
PDF Full Text Request