Mechanism Of Fragaria Viridis Self-incompatibility Based On 'Omics' And Functional Analysis Of Gene FvGLO?

Recent studies showed genus Fragaria owned the gametophyticl self-incompatibility(GSI)mechanism which was controlled by two independent loci(S and T)and a non-S loci.However,the detailed information of these loci and how they worked in the SI system remains unclear.Thus,in the present study,we used a SI Fragaria species(Fragaria viridis(Duchesne)Weston,Greenish strawberry)as material and studied the biological features of F.viridis under self-and cross-pollinations.Then we performed an integrative analysis of transcriptome and proteome for exploring the pollen-pistil interaction,we cloned and identified two F.viridis candidate S-RNase genes(Sa-RNase and Sb-RNase).Besides,the F.viridis FvGLO I gene was cloned and transferred to Arabidopsis thaliana for functional analysis.The detailed results of this research were described as bellow:1.We analysised the reasons for self-incompatibilty in F.viridis:firstly,pollen grains were viable which might not confer the self-incompatibilty.Then we found most of the pollen tubes were arrest at the 2/3 of pistil after self-pollination for 24 h,and the furit setting rate(20.1%)and achene germination rate(22.3%)were both quite low.According to these results,we believed the pre-and post-zygotic SI barrier was existed which mainly resulted the self-incompatibility in F.viridis.Five inbred lines were obtained after self-pollination and progeny Ls-Si-02 showed a relative higher fruit setting rate.Thus,we performed self-pollination on this progeny and obtained the second inbred generation of F.viridis with 86 family members.These progenies showed a significant segregation of SI and SC by evaluating the fruit setting rate,especially,the Ls-S2-76 showed a high fruit setting rate(92.5%)and chene germination rate(82.5%),while that of Ls-S2-53 was poor(5.8%)and no achene was germinated.By performing the cross and reciprocal cross among F.virisis,F.vesca L and F.mandshurica Staudt,we found a few pistls in a gynoecium of F.viridis might be self-compatible,which showed the SnSnTnTn type.Besdies,a heterozygous non-S loci(Tc,Mm)might be existed in controlling the F.viridis self-incompatibility.2.We performed an integrative analysis of transcriptome and proteome exploring the strawberry pollen-pistils interaction.A vast number of differentially expressed genes and proteins were identified between pistil harvested at 0 and 24 hours after pollination,including 2,181 differentially expressed genes(Fold change>2,p-value ? 0.05),including 1,355 down-regulated geneds and 826 up-regulated genes,and 200 differentially expressed proteins(Fold change>1.5,p-value<0.05)including 91 down-regulated proteins and 109 up-regulated proteins,these genes and proteins were widely involved in ubiquitin mediated proteolysis,plant pathogen interaction,Ca2+signaling,hormone signal transduction,ABC transport,programmed cell death(PCD)and stress response.Twenty-three genes were identified with significant changes at both transcript and proteomic levels,20 of these genes(87%)showed similar expressional trends at mRNA and proteomic levels which indicated genes were less influenced by the transcriptional regulation during the SP process.Further,two putative S-RNases(Sa-RNase and Sb-RNase)were identified in the transcript data.3.The full length nucleotide sequences of two putative F.viridis S-RNase(Sa-RNase and Sb-RNase)were cloned.Two genes were highly homologous with a similar nucleotide sequence length,which is an important feature of S-RNase.The deduced amino acid sequence analysis showed Sa-and Sb-RNase shared a similar molecular weight and higher isoelectric point(pI),which matched the characteristics of F.viridis S-RNase.Phylogenetic tree analysis showed Sa-and Sb-RNase had a close relationship with those S-RNase of Prunus,which indicated the S-RNases of Fagaria and Prunus might be evolved from a same ancestor.During further evolution,S-RNase of F.viridis evolved in a new phylogenetic branch compared to Prunus,which might result in the SI mechanism of F.viridis being controlled by two independent loci.According to amino acid sequence structure of Prunus S-RNase,both Sa-and Sb-RNase had five conservative regions(C1,C2,C3,RC4 and C5)and one hypervariable region(RHV).In addition,the region RC4 was less conserved which could be regard as the specific S-RNase region of Fragaria distinguished from Prunus.Sa-and Sb-RNase also specifically expressed in pistils of self-incompatibility Fragaria species(F.viridis and Ls-S2-53),but not in those of the self-compatibility species(F.vescas,F.×ananassa 'Benihoppe' and Ls-S2-76),as we failed to obtain any sequence by genome database blast or sequence cloning of two S-RNases in these species,this might be caused by the loss of S-RNase in these self-compatible species.Further,Sa-and Sb-RNase also showed a similar expressional pattern that both genes were highly expressed after self-polliantion for 12 h when the growth of most pollen tubes were inhibited in previous observation.In clusion,Sa-and Sb-RNase were two significant candidate S-RNases for F.viridis pistil determinant during self-polliation.4.We cloned the F.viridis glyoxalase ?(FvGLO ?)and constructed the overexpressing vector(pYH4215-FvGLO ?)successfully.Then the combined vector was transferred to wild type Arabidopsis thaliana.We observed and analyzed the phenotype of the FvGLO ?transgenic lines druing different development stages and the response of plant under abiotic stress treatment.The results indicated that the FvGLO ? was significantly expressed during the seeding stage,flowering stage and the early silique development stage.The phenotype of flowering stage showed the abnormal growth of pistil and anther(both pistil and stamen were fertile)which resulted in the incomplete pollination or totally pollination failure and finally leading to the seed absence in silique,even silique abortion.We treated the seeds and leaves of FvGLO ? transgenic lines with NaCl,methylglyoxal,glutathiol(GSSG),respectively.The result showed FvGLO ? transgenic lines had stronger resistance to abiotic stress compared to wild type Arabidopsis thaliana,and we suggested FvGLO ? could promote the accumulation of glutathione(GSH)and maintained the steady GSH:GSSG ratio,the accumulated GSH also could assist with glyoxalase ? in degradation of thecytotoxic methylglyoxal and reduced the damage from abotic stress to cells.These results mentioned above suggested that FvGLO ? could not only participated in the development of pistil and anther and remarkably influenced the pollination,but also enhanced the resistance of transgenic Arabidopsis thaliana lines to abiotic stress.