| Alternation of generations is a representative cycle in the life history of land plants,which is characterized by alternation between a haploid gametophyte and a diploid sporophyte.Taking the model plant Arabidopsis as an example,through double fertilization,the embryo originates from the fusion of one sperm cell with an egg cell,while the endosperm comes from the fusion of the second sperm cell with a central cell,thus initiating the development of the sporophyte.For this reason,the generation of gametophytes and the development of embryo and endosperm are crucial for plant development.As a member of SMC(Structural Maintenance of Chromosome)protein complexes,SMC5/6 complex has been extensively studied in yeast and mammals.A widespread view is that the SMC5/6 complex participates in maintaining genome stability,and it is indispensable to many biological processes,including the DNA damages repair and DNA replication.According to the research of mammals,deficiencies in some subunits of the SMC5/6 complex will seriously impact gametophyte generation and embryo development.However,there are few studies on the biological function of this complex in plant development,especially during reproduction.In this study,we used 45 mutants of 8 subunits in the SMC5/6 complex as test materials,and utilized various biological methods to reveal the influence of the SMC5/6 complex in reproduction development of Arabidopsis,especially the important function of AtSMC6A and AtSMC6B in seed and gametophytic development.In addition,we also investigated the subunit interactions of the SMC5/6 complex in Arabidopsis.We summarized the results as followed:1.The 45 T-DNA insertion mutants in eight subunits of the SMC5/6 complex were obtained from ABRC and NASC.We identified ten T-DNA insertion mutants of AtNSE4A and AtNSE4B genes,but one of these mutants was unable to germinate,and the T-DNA insertion sites of other four mutants couldn’t be identified.Although the remaining five mutants were able to obtain the homozygous offsprings,transcriptional analysis revealed that AtNSE4A and AtNSE4B gene expression in the five mutants were not obviously different from wild type,and there were no phenotypic alterations in these mutants compared with wild type plant.In two mutants of AtSMC5,the homozygous offsprings couldn’t be obtained,and in the silique of heterozygous plants,the white and undeveloped abnormal ovules were found at rates around of 40%.Complemental assay showed that AtSMC5 could only restore about 25%aborted seeds,and 15%undeveloped ovules remained in the mutant silique.These results implied that AtSMC5 was essential for seed development,and the undeveloped ovules that still existed might be caused by other genes deficiency.We identified nine T-DNA insertion mutants in the AtSMC6A and AtSMC6B genes.RT-PCR analysis showed that the transcript levels of AtSMC6A(or AtSMC6B)was absent in smc6a-4,smc6b-1 and smc6b-2 homozygous plants,but the knock-out mutations of AtSMC6A or AtSMC6B did not cause any obvious reproductive defects compared with wild type plants.Thus,we made a cross between smc6a-4 and smc6b-2 homozygous plants,and found that the smc6a-4 smc6b-2 double mutants were embryo lethal.Complemental assay showed that either AtSMC6A or AtSMC6B could completely restore the aborted seeds,indicating that AtSMC6A and AtSMC6B genes were functionally redundant in seed development.2.By observing the embryo developmental process,we found that the early embryo development was delayed in the AtSMC6A and AtSMC6B double mutant siliques at 1-2 days after pollination,and the phenotype was more serious in the smc6a-4/+ smc6b-2 than in the smc6a-4 smc6b-2/+ genotypic siliques.By observing the embryo development during 3 to 7 DAP,we found that the cell division planes were disturbed and embryo patterning was incomplete in the smc6a-4 smc6b-2 mutant embryos,and finally the mutant seeds aborted.The ultrastructure of embryonic cells showed that a large number of embryo cells in smc6a-4 smc6b-2 mutant ovules at the globular stage started to produce either vacuolar programmed cell death(vPCD)or necrosis programmed cell death(nPCD).Moreover,the autofluorescence of endosperm displayed that mutations of AtSMC6A and AtSMC6B disturbed the endosperm nucleus division and delayed the endosperm cellularization.Through the qRT-PCR analysis,it was suggested that the expression of genes which participating in embryo patterning,endosperm cellularization,programmed cell death,DNA damages repair,cell cycle regulation and DNA replication were obviously changed in the smc6a-4 smc6b-2 ovules.All of these results implied that knocking out both AtSMC6A and AtSMC6B might cause the accumulation of DNA damage and trigger the programmed cell death,and finally result in abortion of the mutant seeds.From the phenotype of smc6b-2 SMC6A RNAi double mutants,we found that the knock-out of AtSMC6B and partially silenced expression of AtSMC6A could decrease the fertility of plants,and result in the female and male gametophyte sterility.In addition,the qRT-PCR analysis showed that only the expression of AtSMC6B was up-regulated in the AtSOG1 over-expression lines,while the expression of AtSMC6A wasn’t influenced by AtSOGl over-expression,which suggested that AtSMC6A and AtSMC6B genes have differentiation of their functions,and that SMC5/6 complex might participate in the AtSOG1-dependent DNA damages repair pathway,and AtSMC6B might have more important function than AtSMC6A in Arabidopsis development..3.We used the BiFC(Bimolecular Fluorescence Complementation)technique to detect the interaction relationship of subunits in SMC5/6 complex.These results showed that AtNSE1 AtNSE3 and AtNSE4A(or AtNSE4B)could interact with each other to form sub-complexes,and then,the sub-complexes through AtNSE4A or AtNSE4B to connect to the ATPase domain of AtSMC5.Meanwhile,AtNSE2(AtMMS21)depended on its N-terminal a-helix bundle to interact with the RING-like domain in the C-terminal of AtNSE1,the N-terminal domain of AtNSE4A,and the Walker A domain of AtSMC5.According to our study,the construction of the SMC5/6 complex in Arabidopsis was different from yeast and mammals,suggesting that the construction and function of SMC5/6 have changed along with the organismic evolution. |
