| Ca2+is a large amount of element and second messenger necessary for plant growth and development.Plants in karst areas with high calcium need specific high calcium adaptation,but the metabolic and molecular mechanisms of these plants’high calcium adaptation are currently poorly understood.The preliminary research team found that woodland strawberry（Fragaria vesac）is a high calcium plant and an ideal material for studying the high calcium adaptation of plants in karst areas.Therefore,the key metabolic pathways and candidate genes of high calcium response in F.vesac were explored through the integrated analysis of transcriptome and metabonome.Meanwhile,in order to fill the gap in the transient transformation of F.vesac roots,we established an efficient system for Agrobacterium rhizogenes-mediated transient transformation.The research results can provide a theoretical basis for the high calcium adaptability of plants in karst areas and provide technical support for rapid research on gene function.The main results are as follows:1 Transcriptome and metabolome sequencing were performed on the7 d seedlings of Yellow Wonder（YW）,treated with five concentrations of Ca Cl2,such as 1.5,10,30,50,and 70 m M.A total of 3707 Differentially Expressed Genes（DEGs）were identified.GO and KEGG analysis showed that these DEGs were mainly enriched in transport activity,transcription factor activity,sensor activity,and other processes,mainly involved in phenylpropanoid biosynthesis,plant hormone signal transduction,and MAPK signaling pathway-plant metabolic pathways.The metabolome detected 951 Differential Metabolites（DEMs）,mainly involved in metabolic processes such as arginine and proline metabolism,lysine degradation,and zeatin biosynthesis etc.Integrated analysis of transcriptome and metabolome,based on the correlation coefficient（PCC）,WGCNA,K-means cluster analysis,four possible high calcium response pathways were discovered:1)Calcium signal transduction pathway:According to ICP-MS detection,the content of Ca2+and Mn2+etc in plants under different treatments was compared.The results showed that as the concentration of Ca2+treatment increased,the Ca2+content in YW seedlings increased,while the Mn2+content was the opposite.Calmodulin protein（CML）can respond to high calcium stimulation by increasing cytosolic Ca2+concentration in the calcium signal transduction pathway.The expression of four CMLs（FveCML27/36/44/45）and their co-expressed four TFs（FveWRKY46,Fveb HLH100,FveMYB108,FveMYB4）were induced by high calcium through gene expression analysis such as transcriptome and q RT-PCR.It is speculated that F.vesca improve their high calcium adaptability through CMLs gene transduction of excess Ca2+.2)ABA signal transduction pathway:ABA can adapt to abiotic stress and other stresses by stimulating stomatal closure.Metabolome comparison showed that the content of ABA in YW seedlings increased with the increase of Ca2+concentration.Compared with the control(1.5m M Ca2+),the leaf stomata of YW seedlings under high calcium stress(treated with 50 and 70 m M Ca2+for 30 d)closed.The expression of the ABA signaling pathway related genes FvePP2C6/24/51 is upregulated under high calcium stress,suggesting that F.vesca may promote ABA accumulation by regulating the expression of PP2Cs genes to improve their high calcium adaptability.3)Anthocyanin biosynthesis pathway:After high calcium treatment(70 m M Ca2+),the anthocyanin content in YW seedlings was 11.58 times higher than that in the control(1.5 m M Ca2+).Integrated analysis showed that the anthocyanin synthesis genes FveANR and FveF3H were highly correlated with five DEMs（procyanidin B1,procyanidin B3,procyanidin C1,procyanidin C1,and peonidin-3-O-glucoside）,and co-expressed with transcription factors such as FveHSFA6b,FveNAC021,Fveb HLH12,and FveMYB3.Expression analysis showed that FveANR,FveFveF3H,FveNAC021,Fveb HLH12,and FveMYB3 were upregulated under high calcium stress,while FveHSFA6b was downregulated.It suggests that it may enhance the high calcium adaptability of F.vesca by promoting the accumulation of anthocyanins.4)Phenylpropane biosynthesis pathway:Safranine staining revealed a significant increase in lignin content in the root elongation zone of YW seedlings after high calcium treatment.The key genes for lignin synthesis,FveCAD1 and FveHCT13,are co-correlated with DEMs such as L-phenylalanine,p-Coumaric acid,Caffeic aldehyde,Sinapyl alcohol,Coniferyl alcohol and Caffeic acid.FveCAD1 is co-expressed with transcription factors such as Fveb ZIP44 and FveNAC2,while FveHCT13is co-expressed with transcription factors such as FveWRKY40 and FveWRKY46.Expression analysis showed that the expression levels of FveCAD1 and FveNAC2 decreased first and then increased after high calcium stress.FveHCT13,FveWRKY40,and FveWRKY46 were upregulated with increasing Ca2+concentration.It is speculated that these transcription factors may promote lignin accumulation by regulating the expression of FveCAD1 and FveHCT13,thereby improving the high calcium adaptability of F.vesca.2 In order to establish a system for transient transformation of F.vesca roots,F.vesca seedling YW5AF7 was used as the material to compare the effects of Agrobacterium rhizogenes,explant types,and transformation conditions on the transient transformation of F.vesca roots.Finally,C58C1 and MSU440 were determined as the suitable strains for transient transformation of YW roots,while hypocotyls and seedlings without root were the most suitable explant types.The concentration of bacterial solution was 0.7,the infection time was 20 min,and the co-cultivation time was 3 d as the optimal transformation conditions.The activity of the root specific promoter DR5::GFP and the function of the exogenous calcium indicator gene 35S::j GCa MP7c were successfully detected using the established root transient transformation system.The established system can provide technical support for rapid research on the function of F.vesca genes in roots in the future. |
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