Effect And Mechanism Of High Calcium On Seedlings And Fruits In Fragaria Vesca

One of the main feature of soil in karst area is its high content of calcium.Ca²⁺is a macroelement necessary for plant growth and development.It does not only act as a nutrient to stabilize cell structure but also as a signal molecule to help plants adapt to the changes in the external environment.Excessive Ca²⁺in the environment will cause Ca²⁺poisoning in plants,including seedlings and fruits.The woodland strawberry（Fragaria vesca）is a model for studying Rosaceae fruit trees.At present,little is known about the effect and mechanism of high calcium on F.vesca seedlings and fruits.Therefore,we treated F.vesca seedlings and fruits with high calcium content,respectively,and studied the effect and mechanism of high calcium content on F.vesca seedlings and fruits through phenotypical analysis,bioinformatics study,and gene function.This study can provide a theoretical basis and genetic resources for applying Rosaceae fruit trees in karst areas.The main results are as follows:1 Effect and mechanism of high calcium content on F.vesca seedlings1.1 F.vesca seedlings were treated with Ca²⁺concentration gradients（1.5,10,20,30,40,50,70,100 mM）.Compared with 1.5 mM Ca²⁺（1/2MS）,the root length of seedlings was significantly increased at 10～30mM Ca²⁺concentrations.With the increase of Ca²⁺concentration to 50mM,seedling growth was inhibited and elongation of root length was reduced by 45%.When the concentration of Ca²⁺reached 70、100 mM,the growth of seedlings was repressed seriously and root length almost stopped extending.These results showed that seedlings could adapt to high calcium concentration of 30 mM,indicating that F.vesca is a high calcium plant.This results showed that 10 mM Ca²⁺can be used as the suitable growth concentration for F.vesca,and the seedlings can adapt to the high calcium concentration of 30 mM,which indicates that F.vesca is a high calcium plant.1.2 Comparative analysis of the transcriptomes of F.vesca seedlings treated with the suitable growth concentration of 10 mM versus the high calcium stress concentration of 70 mM revealed 1850 DEGs,of which1045 were up-regulated genes and 805 were down regulated genes.GO analysis identified that the up-regulated genes were mainly enriched in processes involved in antioxidant activity,molecular sensors,signal transducer activity,indicating that F.vesca seedlings can adapt to high calcium stress by participating in biological processes such as antioxidant and signal transduction.Similarly,their KEGG analysis revealed that the up-regulated DEGs were mainly enriched in the pathways of plant pathogen interaction,biosynthesis of phenylpropanoids,plant MAPK signaling pathway,starch and sucrose metabolism,glutathione metabolism,biosynthesis of cutin,softwood fat and wax and selenium compound metabolism,implying that F.vesca seedlings may respond to high calcium stress through the above-mentioned secondary metabolic pathways.1.3 As a calcium responsive protein,calcium-dependent protein kinase（CDPK）participates in Ca²⁺decoding and often plays an important role in plant adaptation to calcium stress,but the CDPK superfamily in F.vesca is still unknown.Firstly,28 CDPK superfamily members were identified from the whole genome data of F.vesca.Then,the conventional biological information such as gene structure,conserved domain,evolutionary relationship,and spatiotemporal expression profile in the whole development stage was analyzed.Subcellular localization showed that most CDPK superfamily genes were located on the cell membrane,corresponding to palmitoylation and myristoylation sites.Finally,the transcriptome and qRT-PCR results showed that FvCDPK1/5/6/8/16/17 and FvPEPRK2 were upregulated under high calcium stress,suggesting that it may be involved in the response of F.vesca seedlings to calcium stress.Finally,a comparison of the expression of the FvCDPK superfamily at 10 mM and 70 mM Ca²⁺concentrations by transcriptome and qRT-PCR showed that FvCDPK1/5/6/8/16/17 and FvPEPRK2 responded to high calcium stress,and may play a role in the adaptation of F.vesca to calcium stress.1.4 M2 generation mutant seedlings were screened using an existing M1 generation fast neutron mutant library with 50 mM Ca²⁺that caused a45%reduction in root length elongation as a screening concentration.According to their phenotype of these M₂seedlings on 50 mM Ca²⁺,a calciphobe mutant FN-1-046 and four calciphile mutants（FN-1-026/-028/-052/-082）were preliminarily identified.The acquisition of these candidate mutants would provide experimental material for further study on the high calcium stress adaptation genes in F.vesca.2 Effect and mechanism of high calcium on F.vesca fruits2.1 90 mM Ca²⁺was used as a high calcium treatment concentration for cultivated strawberry fruits,so we soaked the turning stage（18～20DAP）fruits of F.vesca with 90 mM Ca²⁺concentration.Compared with1/2 MS control,there was no obvious difference observed in fruit appearance and morphology,but the total pectin content and fruit firmness increased significantly,suggesting that high calcium can inhibit fruit softening.2.2 Pectin lyase（PL）is a critical enzyme during fruit softening,but it is not clear which members of the PL gene family are involved in fruit softening in F.vesca.So we first isolated and identified 16 FvPL genes based on the genome data of F.vesca;Then,protein properties,conserved motifs,gene structure,evolutionary relationship,and expression profile of these FvPLs were analyzed.The results showed that the expression levels of FvPL1,FvPL4,and FvPL7 were significantly up-regulated in turning stage fruit compared with that in green fruit;Further in situ hybridization showed that all the three FvPL genes were enriched in the parenchyma of fruits after the turning stage,suggesting that these three FvPL genes may be involved in fruit softening.Further subcellar location analysis showed that FvPL1 and FvPL4 were both located on the cell membrane,while FvPL7 was located either on the cell membrane or in the chloroplast.Finally,overexpression of FvPL/4/7 could accelerate the degradation of cell wall and make fruit soft,respectively.On the contrary,when respectively knock down the expression of FvPL1/4/7 by RNA interference,the fruit was harder,cell wall degradation was inhibited,and cell structure was complete and compact.These results suggested that FvPL1/4/7 involved in promoting fruit softening in woodland strawberry.2.3 Ca²⁺can inhibit fruit softening of cultivated strawberry.In order to test whether Ca²⁺inhibit fruit softening by mediating FvPL,exogenous Ca²⁺was used to treat F.vesca fruits at truning stage.The expression of FvPL4 and FvPG were significantly down-regulated in fruits treated with Ca²⁺for 4 d,although the expression of FvPL1,FvPL7 and FvPME39were up-regulated,the variation range of FvPL1 and FvPL7 gene expression also showed a downward trend between untreated and Ca²⁺treated fruits.These results indicated that exogenous Ca²⁺may directly inhibited the expression of FvPL4 and FvPG,and decreased the expression increase of FvPL1 and FvPL7 at fruit mature stage,thus improving the fruit firmness of F.vesca.