Functional Analysis Of Key Genes AtSLO8,GRP23,ZmPPR21,and ZmPPR26 In Growth And Development Of Arabidopsis And Maize

The nature of plant growth and development is the physiological and biochemical activities and morphological changes caused by the selective expression of genes in different periods and different spaces.This process is jointly regulated by internal genetic basis and external environmental factors.The growth and development of plants starts from a fertilized egg,and then the zygote develops into an embryo containing tissue and organ primordium through a series of developmental processes such as zygotic activation,polarity establishment,and organ differentiation.Postembryonic development is subsequently carried out through the co-regulation of internal developmental signals and external environmental signals.This thesis reveals the functions of four key genes in plant growth and development by means of physiology,cell biology,genetics,and molecular biology,which encode Arabidopsis small peptide AtSLO8,special PPR(Pentatricopeptide repeat)protein GRP23(GLUTAMINE-RICH PROTEIN 23),maize PPR proteins ZmPPR21 and ZmPPR26,respectively.1.The information exchange between endomembrane organelles plays an important role in plant growth and development and plant responses to environmental changes.In this chapter,a preliminary study of AtSLO8,a small peptide protein localized to the endomembrane system in Arabidopsis,was conducted.The results show:(1)AtSLO8 encodes a plant-specific small peptide.AtSLO8 consists of 57 amino acids and contains a single transmembrane domain at its N-terminus.Subcellular localization results showed that AtSLO8 was localized in cis-and trans-Golgi bodies and endosomes.(2)Mutation of AtSLO8 affects multiple processes of growth and development in Arabidopsis,including embryonic development,hypocotyl elongation,root growth,and leaf development.Overexpression of AtSLO8 in Arabidopsis resulted in larger seeds,increased root length,larger leaves and increased plant height.(3)The results of transmission electron microscopy showed that compared with the wild type,the structure of the Golgi apparatus and endosome in the root cells of the atslo8 mutant was abnormal.A large number of small vesicles.The small vacuoles contained a large number of undegraded vesicles surrounded by a monolayer membrane,and a large number of vesicles accumulated near the plasma membrane.(4)Co-immunoprecipitation and TurboID proximity markers identified multiple AtSLO8 interacting proteins localized in the inner membrane system,including AtSAR1B(Secretion-associated and Ras-related protein 1B),a component of COP Ⅱvesicle.Further experiments demonstrated that AtSAR1B colocalized with AtSLO8.The interaction between AtSLO8 and AtSAR1B was demonstrated by split-ubiquitin mediated DUAL membrane yeast two-hybrid experiments,bimolecular fluorescence complementation(BiFC)and pull-down experiments.Whether the interaction between AtSLO8 and AtSAR1B affects Arabidopsis growth and development or stress response needs further confirmation.Based on the above results,AtSLO8 may affect the growth and development of plants by participating in the transport of substances in the endomembrane system of plants,but its specific molecular mechanism needs to be further analyzed.2.PPR proteins participate in the post-transcriptional processing of organelle RNA and play an important role in the regulation of plant growth and development.Previous studies have shown that the Arabidopsis PPR protein GRP23(GLUTAMINE-RICH PROTEIN 23)is localized in the nucleus and functions as a transcriptional regulator to regulate seed development.Our previous studies showed that GRP23 is simultaneously localized in mitochondria,chloroplast and nucleus,and is involved in the C-to-U editing at 352 sites in mitochondrial and 6 sites in chloroplast.However,the mechanism by which GRP23 participate in editing is unclear.In this chapter,we found that GRP23 is involved in mitochondrial RNA editing by interacting with atypical PPR-DYWs and MORF(Multiple Organellar RNA editing Factors)proteins.The main results are as follows:(1)TurboID proximity markers identified three classes of GRP23-interacting proteins in Arabidopsis:atypical PPR-DYW proteins(DYW2,MEF8,and MEF8S),MORF proteins(MORF1 and MORF8),and specific P-class PPR proteins(NUWA and MEF8S)GRP23 itself).(2)Yeast two-hybrid and bimolecular fluorescence complementation experiments showed that GRP23 interacts with atypical PPR-DYWs and MORF proteins,and there is also interaction between atypical PPR-DYWs and MORF proteins.(3)The results of yeast two-hybrid analysis with truncated GRP23 proteins indicated that the N-terminal domain of GRP23 mediates its interaction with three atypical PPR-DYW proteins,and the carboxy-terminal domain mediates its interaction with MORF proteins.(4)Bimolecular fluorescence complementation experiments showed that PPR-E proteins had weak interaction with MEF8/MEF8S protein,while the simultaneous expression of GRP23 enhanced the interaction between PPR-Es and MEF8/MEF8S.It indicates that PPR-E proteins perform deamination reaction by recruiting MEF8/MEF8S as trans deaminase,and GRP23 enhances the stability of PPR-EMEF8/MEF8S editing complex.(5)The results of bimolecular fluorescence complementation experiments or luciferase complementation experiments showed that GRP23 formed homodimers or heterodimers with NUWA.Yeast two-hybrid results indicated that NUWA also interacted with atypical PPR-DYW proteins.(6)The sequence alignment showed that the amino acid sequence of GRP23 was similar to that of NUWA,especially in the N-terminus and PPR domain.Genetic complementation experiments showed that the CC(coiled-coil)domain and the WQQ domain at the C-terminus of GRP23 have similar functions to the carboxy-terminal domain of NUWA.Based on these results,we propose a possible model of the mitochondrial editosome,that is,PPR-E proteins specifically recruit MEF8/MEF8S whereas PPR-E+proteins specifically recruit DYW2 as the trans deaminase,and then GRP23 and/or NUWA,and MORFs facilitate and stabilize the E-or E+-type editosome formation.3.Maize genome encodes hundreds of PPR proteins that are mainly localized in mitochondria or chloroplasts.Since mitochondrial function is critical for maize embryogenesis and endosperm development,mutants of key mitochondrial PPR genes are often seed lethal.In this chapter,we characterized the functional a mitochondrialocalized P-type PPR protein ZmPPR21,in maize embryogenesis and endosperm development.Mutation of ZmPPR21 leads to empty pericarp phenotype.Analysis of mitochondrial transcripts revealed that null mutation of ZmPPR21 reduces the splicing of nad2-introns 1,2,and 4,resulting in a severe reduction in the content of mature nad2 transcripts.The mitochondrial respiratory chain complex I fails to assemble and its activity is severely reduced in the mutant,leading to seed abortion.Our study also found that ZmPPR21 interacts with two specific P-class PPR proteins,PPR-SMR1 and SPR2.Another P-class PPR protein,EMP12,which is also responsible for the splicing of nad2-introns 1,2,and 4,also interacts with PPR-SMR1 and SPR2.PPR-SMR1 and SPR2 are responsible for the splicing of more than half of mitochondrial introns and also interact with each other.These conclusions suggest that there may be a major splicing pattern consisting of PPR-SMR1/SPR2/P-type PPR proteins in mitochondria.4.The function of chloroplast-targeted PPR proteins involved RNA editing is rarely reported in maize.In this chapter,the function of a chloroplast-localized PPRDYW protein was studied.The null mutation of ZmPPR26 leads to albino seedlinglethal phenotype.The editing of atpA-1148 was completely absent and the editing efficiency of ndhF-62,rpl20-308,rpl2-2,rpoC2-2774,petB-668,rps8-182 and ndhA50 sites was reduced in the zmppr26.Further functional studies showed that abolished editing at atpA-1148 results in abnormal assembly of chloroplast ATPase and a significantly decreased accumulation of photosynthetic proteins.Our results suggest that ZmPPR26 is required for the editing at atpA-1148,which is critical for function of AtpA,assembly of ATPase and chloroplast biogenesis in maize.