Study On The Roles Of Arabidopsis MDN1 In Regulating Ribosomal Biogenesis,plant Growth And Dvelopment

Ribosomal biosynthesis is a complex assembly process,which relies on many ribosome biogenesis factors(RBFs).In Arabidopsis(Arabidopsis thaliana),homologous to about 75%of yeast RBFs were identified by bioinformatics methods,but only a small fraction of these RBFs has been functionally characterized.In yeast,AAA ATPase Midasin 1(MDN1)is involved in the processing of the 60S ribosome subunit precursor(pre-60S),but the molecular function of Arabidopsis MDN1(AtMDN1)in ribosomal biogenesis is not clear.In this study,cell biology and molecular biology,etc.methods were used to investigate this question.A large amount of gene expression and energy in cells are devoted to ribosomal biogenesis,therefore ribosomal biogenesis can not only drive the growth of plant cells but also might be involved in the regulation of plant growth and development.Normally,ribosome-deficient mutants always show characteristic growth defects phenotypes,such as short root and abnormal embryogenesis,etc.,suggesting that plant growth and development might be sensitive to and be regulated by the process of ribosomal biogenesis.Arabidopsis mdnl-1 mutant displays an auxin deficiency phenotype,such as short root,abnormal leaf vein development,pointed and serrated leaves,and insensitive to auxin treatment,and the short siliques and aborted seeds and delayed flowering time phenotypes.To explore the roles of ribosome biogenesis on plant growth and development,I used the mdnl-1 mutant to systematically analyze the relationship between ribosome deficiency and auxin response,embryo development,and flowering time regulationIn Arabidopsis,multiple divergent ribosomal proteins(RPs)are synchronously expressed and incorporated into different cytosolic ribosomes,which leads to a high level of ribosome heterogeneity.However,the function of specific RP and specific RP-containing ribosomes remains unclear.In this study,I carried out a strategy by overexpressing specific RPs to study its function and the function of specific RP-containing ribosomes.The establishment of efficient embryogenic callus induction and shoot regeneration system is essential for crop genetic improvement using genetic modification.In this study,using a glucocorticoid receptor-dexamethasone(GR-DEX)inducible system,we investigate the function of co-expression of embryonic transcription factor and cytokinin synthesis enzyme genes in the establishment of embryogenic callus induction and shoot regeneration system1.AtMDNl regulates 60S ribosomal biogenesis.AtMDN1 is highly expressed in proliferating tissues,mdn1-1 mutant is a weak allele with a single base substitution,the mdnl-2 mutant is a T-DNA insertion mutant,and homozygous mdnl-2 is lethal.Dot-immunoblotting showed that AtMDN1 was localized in the nucleus,but some of the AtMDNl proteins appeared in the cytoplasm in the mdnl-1 mutant.The GFP fluorescence distribution of ribosomal protein of large subunit(RPL16D-GFP)and ribosomal protein of small subunit(RPS13A-GFP)reporters indicated that the nuclear export of pre-60S was impaired in the mdnl-1 mutant.The accumulation of rRNA precursors(pre-rRNAs)showed that the accumulation of 35S,27SB,and 20S pre-rRNA was accumulated in the mdnl-1 mutant.Protein interaction experiments showed that AtMDNl interacted with two other homologs of yeast pre-60S biosynthesis RBFs(PESCADILL02/PES2 and NOCHELESS/NLE).The mdnl-1 mutant was hypersensitive to eukaryotic translation inhibitor(cycloheximide),indicating that the translation efficiency of the mdnl-1 mutant was decreased.Taken together,our results indicated that Arabidopsis MDN1 is involved in 60S ribosomal biosynthesis.2.Ribosomal biogenesis and auxin form a feedback loop to regulate plant growth Ribosome-deficiency mutants usually display auxin-deficiency phenotypes,but the relationship between ribosome deficiency and auxin is not clear.In this study,I systematically analyzed auxin-deficiency phenotypes of the mdnl-1 mutant,including short primary root,abnormal leaf veins,pointed leaves,and insensitivity to auxin treatment.Different from other ribosome-deficiency mutants,the mdnl-1 mutant also showed increased lateral root and root hair density.Auxin response and response distribution in the cotyledon,primary root,and leaf margin of the mdn1-1 mutant were abnormal.Flow cytometry analysis showed that more cells of the mdn1-1 mutant root were at the G0/G1 phase than that of wild type.The expression of auxin synthesis genes(YUC3,YUC5,YUC7,YUC8,YUC9,and TAA1)was decreased in the mdnl-1 mutant.In the mdn1-1 mutant,the protein accumulation of two auxin polar transporters(AUX1 and PIN1)was decreased,whereas that of PIN2 was increased.In addition,the expression of PIN3 and PIN7 were decreased in the mdnl-1 mutant.Transcriptome analysis showed that the expression of most of the auxin synthesis,signaling,and auxin response-related genes was decreased.The expression of root growth regulators(PLT1,PLT2,SCR,and SHR)was decreased in the mdn1-1 mutant.Auxin can activate the expression of AtMDN1,and yeast one-hybrid results suggested that seven ARFs might directly bind to the AtMDN1 promoter.The expression of AtMDN1 was decreased in the mdnl-1 mutant.Taken together,our results show that ribosome deficiency regulates auxin signal,while auxin regulates ribosomal biogenesis through modulating the expression of RBFs to modulate plant growth and development3.AtMDN1 regulates embryo development.The siliques were short,the number of mature seeds was decreased,and some aborted seeds were observed in the mdn1-1 mutant,suggesting that the MDN1 mutation led to abnormal embryonic development,but how ribosomal biogenesis deficiency affected embryonic development was unclear The homozygous mdnl-2 mutant was lethal.Some aborted ovules were found in the siliques of the heterozygous mdnl-2/+mutant and the embryogenesis was arrested at the early globular stage.The process of global embryo development of the mdn1-1 mutant was observed and displayed delayed embryo development and four kinds of abnormal embryos at the early globular stage(?-type,?-type,?-type,and ?-type).AtPES2 and AtNLE interact with AtMDN1 in nucleolus and nucleoplasm,respectively.In addition,homozygous pes2-1 and nle-1 mutants were lethal.Some aborted ovules were found in the siliques of the heterozygous pes2-1/+and nle-1/+mutants and the embryogenesis were arrested at the early globular stage.These results indicate that MDN1,PES2,and NLE play an important role in early embryonic development.The abnormal development of the globular embryo might be related to the rapid cell division at this stage which needs a large number of ribosomes to produce protein,and the role of MDN1,PES2,and NLE function in ribosomal biogenesis.Observation of the DR5rev:GFP fluorescence in embryos showed that the auxin response and auxin response distribution during the globular embryo stage were obviously affected in the mdn1-1 mutant.These results indicating that MDN1 plays an important role in maintaining the homeostasis of auxin in the embryo at the globular stage4.AtMDN1 regulates flowering time.In general,the mutations in ribosomal proteins(RPs)and RBFs lead to retarded growth and delayed flowering.However,early flowering resulted from ribosomal biogenesis deficiency has not been reported yet.In this study,we found that the mdn1-1 mutant was early flowering.Transcriptomic analysis indicated that the up-regulated expression of autonomous pathway genes and AB15,and down-regulated expression of FLC might be responsible for the early flowering in mdn1-1 mutant.mdn1-1 flk and mdn1-1 fve-4 double mutants inhibit the early flowering phenotype of mdn1-1,indicating that the downregulation of FLC plays an important role in the early flowering of mdn1-1.mdn1-1 flk and mdn1-1 fve-4 significantly restore the late flowering of flk and fve-4,indicating that MDN1 mutation simultaneously produce a FLC-independent regulatory network.The flowering time of mdn1-1 flc-3 double mutants was later than that of flc-3,indicating that FLC plays an important role in the flowering regulation of mdn1-1.After FLC mutation,FLC-independent pathway may not be sufficient to regulate the early-flowering in mdn1-1.The flowering time of n2dn1-1 abi5 double mutants was later than that of wild-type and mdn1-1,indicating that ABI5 is crucial for the early flowering in mdn1-1.The flowering time of mdn1-1 clf-28 was earlier than mdn1-1 but later than clf-28,which may be due to the downregulation of FT in mdn1-1,and increasing FT expression in mdn1-1 could further promote the flowering time in mdn1-1 mdn1-1 co and mdn1-1 ft-10 double mutants delay the flowering time of mdn1-1 mutants,indicating that the photoperiodic pathway affected the flowering time of mdn1-1.5.RPL16D balances plant growth and immune.Here,we found that overexpressed RPL16D-GFP(L16D-OEs)led to curled leaves and shortened petioles.Transcriptomic and proteomic analysis showed that the accumulation of proteins that regulated plant growth was decreased.In addition,the expression of immune defense-related genes and proteins was significantly up-regulated,suggested that L16D-OEs have enhanced pathogen resistance.Bacterial infection indicated that L16D-OEs have enhanced resistance to Pst DC3000.Our results indicated that RPL16D overexpression reduces plant growth,while enhances pathogen resistance,suggesting that RPL16D may play an important role in balancing plant growth and immune.Therefore,appropriately increasing the RPL16D protein dosage might have a promising application in the genetic improvement of crops6.Innovation and application--establish a simple and efficient embryogenic callus induction and shoot regeneration system.LEAF COTYLEDON2(LEC2)is responsible for maintaining the embryogenic identity throughout embryogenesis and maturation stage.Our previous study showed that induced expression of Arabidopsis LEC2(AtLEC2)by a GR-DEX inducible system(AtLEC2-GR)triggers the formation of embryogenic callus in transgenic tobacco(Nicotiana tabacum),but with a low shoot regeneration efficiency.Generally,cytokinin could enhance shoot regeneration efficiency,therefore,adenosine phosphate isopentenyltransferase genes AtIPT3 and A tIPT7 and tRNA isopentenyltransferase gene AtIPT9 were overexpressed in AtLEC2-GR transgenic tobacco.Under DEX induction,high-quality embryogenic callus was obtained in AtIPT7-OE AtLEC2-GR and AtIPT9-OE AtLEC2-GR seedlings,and the shoot regeneration efficiency was 2-fold and 3.5-fold higher than that of A tLEC2-GR In this study,the GR-DEX system was used to construct a simple and efficient embryogenic callus induction and shoot regeneration system.Induced expression of A tLEC2 promoted embryogenic callus formation,while overexpression of AtIPT7 and AtIPT9 increased shoot regeneration efficiency.