The Functional Analysis Of Arabidopsis Thaliana Ribosomal Gene RPS9M

Ribosomes are cellular machines that translate the genetic codes in mRNAs and catalyze protein synthesis in all organisms.As essential component of the ribosome,ribosomal proteins play important role in plant development and growth by sustaining the stability and biogenesis function of the ribosome and regulating protein synthesis.According to their locations,ribosomal proteins in Arabidopsis can be divided into cytoribosomal proteins,chloroplast ribosomal proteins and mitoribosomal proteins.During the past years,the functions of some members of ribosomal proteins in Arabidopsis have been revealed,however,the functions of RPS9M in Arabidopsis were still unknown.Thus,studies on RPS9M would deepen our understanding of functions of ribosomal proteins and regulation mechanism of the ribosomal protein in controlling plant development.In this project,we performed a Y2H screening by using ANK6,an key regulatory factor during fertilization we reported previously,as a bait to screen a prey cDNA library and RPS9M were identified.By using RPS9M deletion mutant and T-DNA insertion mutant,functions of RPS9M were analyzed with the methods of biochemical,molecular,genetic and cellular biological technology.The main results are as follows:Firstly,we confirmed the protein-protein interaction between RPS9M and ANK6 using the yeast two-hybrid(Y21H)system and bimolecular fluorescence complementation(BiFC)analysis.RPS9M protein contained a Ribosomal_S9 domain at the C-terminus and a nonconservedregion at the N-terminus.Y2H analysis revealed that ANK6 interacts with the C-terminus Ribosomal_S9 domain of RPS9M,while the N-terminus of RPS9M participates in the regulation of interaction between RPS9M and ANK6.BiFC analysis confirmed that RPS9M and ANK6 indeed interacted with each other in vivo and their interactions took place in mitochondria.Secondly,the subcellular localization of RPS9M protein was analyzed,and the results indicating that RPS9M was targeted to mitochondria,which was in conformity with the BiFC results.Thirdly,the tissues expression pattern of RPS9M were analyzed.RPS9M is highly expressed in anthers,pistils and seeds during the whole developmental stage,especially in mature pollen grains and mature embryo sacs.RPS9Mwas also expressed during vegetative growth stages,mainly in young leaves and root tips.Fourthly,two RPS9M deletion mutants were obtained and named rps9m-1 and rps9m-2.No homozygous mutants were obtained from the progenies of both +/rps9m-1 and +/rps9m-2.The heterozygous plants showed 50%ovules aborted in both +/rps9m-1 and +/rps9m-2 siliques at early stage after fertilization.Reciprocal cross tests between +/rps9m-1,+/rps9m-2 and wild type plants revealed that the transmission of rps9m allele through female gametophyte were reduced to 3%and the transmission efficiency of the rps9m through male gametophyte were reduced to 21%.These results demonstrate that RPS9M play an important role during reproductive development,loss of RPS9M function cause defects in male and female development.Then,we analyzed the defects caused by RPS9M mutation during male,female gametophyte development,double fertilization processes and seed formation in detail.Nearly 30%pollens from +/rps9m-1 anthers were aborted with no nucleus,wizened shapes,unclear exine patterning and undistinguishable germinal furrow.During female gametophyte development,the two polar nuclei in mutant ovules couldn't fuse into one central nuclear,moreover,the central cell vacuole in mutant ovules were growing slowly and the final vacuole size were smaller than that of the wild type;indicating that mutation in RPS9M affect polar nuclei fusion and central cell vacuole development.Further analysis revealed that rps9m ovules do not affect pollen tube guidance and fertilization processes,but the embryo development and endosperm proliferation processes were severely affected.The embryos were arrested at the zygote stage or 2-cell embryo stage;and the fertilized polar nuclei can divide once or twice to form two or four endosperms before abortion,and finally leading to seed abortions.The seed aborted phenotype even couldn't be rescue by fertilized with wild type pollens,suggesting that the seed abortion may a result from female gametophyte defects.In addition,a homozygous knock-down mutant of RPS9M was obtainedand named rps9m-3.The expression level of RPS9M protein was reduced to 30%in compared with the wild type.Phenotype analysis revealed that suppressed expression of RPSPM also impacts male and female gametophyte development in rps9m-3 mutant.Most pollens in rps9m-3 mutant contained three nuclei,but the shapes were abnormal and the pollen surface were shrunken;and the pollen germination and pollen tube growth were also delayed in compared with wild type.Some female gametophytes in rps9m-3 mutant were also defective and the abortion phenotypes were more serious than that in rps9m-l and rps9m-2.Mutation in RPS9M has affects on seed development and embryo patterning.During seed formation,rps9m-3 showed delayed embryo development and endosperm proliferation,and the synchrony of embryo in per silique was also affected.Moreover,the embryo patterning was also affected during rps9m-3 embryo development.During the early embryo development stage,the apical domain and the suspensor showed aberrant cell divisions which leading to altered proportion of protoderm and the inner cell tissues,enlarged suspensor and aberrations in the basal embryo region and abnormal cotyledon primordium formation and proliferation to form unwell developed cotyledons,fused cotyledons,single cotyledons,three or more cotyledons.RPS9M also has effects on vagetative growth.During germination stage,rps9m-3 seeds showed delayed germination and lower germination rates in compared with WT.The root length of rps9m-3 seedlings were also shorter.When these seedlings were transplanted to soil and continue to growth for 4 weeks,rps9m-3 showed delayed vegetative growth,smaller whole rosette diameters and smaller leaves;after bolting,the stem of rps9m-3 was also shorter and thinner.These results revealed that RPS9M play a role in seed germination,root,leaf and stem development.In the end,the effect of RPS9M on mitochondrial function was analyzed using rps9m-3 mutant.The abundance of mitochondrial large and small subunit rRNAs were investigated,results showed that the abundance of mt 26S rRNA was significantly increased in rps9m-3 plants and the ratio of mt 26S rRNA to mt 18S rRNA for mitochondrial ribosome was significantly increased in rps9m-3 mutant,suggesting a defect in mitochondrial ribosome biogenesis.Further analysis showed that the rps9m-3 plants accumulated higher level of ROS species in compared with wild type,suggesting rps9m-3 may suffer from oxidative damages.RT-PCR analysis revealed that the expression levels of two mitochondrial disfunction regulon and mitochondrial oxidative stress induced genes AOX1a and NDB4 were significantly increased in rps9m-3 plants.These results indicated that RPS9M indeed affect mitochondrial function.In conclusion,RPS9M not only play very important roles in reproductive development,but also function on root,leaf and stem growth.RPS9M may work together with ANK6 in controlling mitochondrial ribosomal functions,then affect some mitochondrial genes expression and mitochondrial functions,and finally promote plant growth.