Decreased Vascular Bundle 1 Affects Mitochondrial Development And Auxin Synthesis In Rice

Rice is an important model crop for plant genome function research,and it is also one of the important food crops.Therefore,the research on rice is particularly important.Auxin is a plant hormone with very little content but can produce great physiological functions.It has important regulating effects on plant growth and development.When it plays roles,it has a dual effect:it promotes growth at low concentrations and inhibits growth at high concentrations.Therefore,maintaining auxin at a suitable cellular level is extremely important for plants.Previous studies have shown that the mitochondria may associated with auxin to regulate plant growth and development.However,there are not many studies on how this.In this study,a mutant dvb1（decreased vascular bundle 1）was screened and isolated from a library of mutants of‘Jinhui 10’mutagenized by ethyl methane sulfonate（EMS）.Phenotypic identification,histological analysis,map-based cloning,functional complementation,expression analysis,subcellular localization,and functional analysis revealed that dvb1 eventually caused the corresponding phenotype due to the abnormal mitochondrial development affecting auxin synthesis.The main findings contain belows:1.Phenotypic identification and histological analysis of dvb1 mutantCompared with the wild type‘Jinhui 10’,the differences of dvb1 mutant are concentrated on the later stages.At the tillering stage,the dvb1 mutant showed overall dwarfing and increased tillering number,and the internodes were significantly shorter and thinner.Scanning electron microscope observation of the sheath inner surface revealed that dvb1 mutant cells were small and irregular.The leaves in dvb1 mutant was narrower and the degree of this narrowing becomes more pronounced over time.At the booting stage,the anther of the dvb1 mutant became whiter and smaller.No significant starch accumulation was found in dvb1 mutant pollen with iodinepotassium iodide（I₂/KI）staing.At the same time,the number of flower primordia,spikelets,and branches of dvb1 mutants decreased.Furthermore,the number of large and small vascular bundles in dvb1 mutant were reduced through paraffin sections of different tissue,but the distance between vascular bundles did not decrease.Map-based cloning and complementation verification of DVB1 geneThe dvb1 mutant was crossed with‘Xida 1A’,and all F₁ generation showed normal plants.In the F₂ generation obtained from the F₁ generation inbred,the ratio of normal plants to dwarfed plants is 3:1,which fully indicates that the mutant phenotype of the dvb1 mutant is controlled by a ressive nuclear gene.Further,the mutant plants in the F₂generation were used as a localization population,and SRR and In Del markers were used to locate the DVB1 gene between the long arm markers W3-100 and In D3-2 of chromosome 3 with a physical distance of 52 Kb.Information analysis and sequencing comparison finally mapped DVB1 gene to LOC＿Os03g62420.Phylogenetic tree analysis indicates that DVB1 belongs to the Mitochondrial Contact Site and Cristae Organizing System（MICOS）complex and is involved in helping the formation of mitochondrial cristae.The‘G’base at the second exon of the LOC＿Os03g62420 was mutated to an‘A’base,which caused the third amino acid of the core sequence G*G*G*G to convert from glycine to glutamic acid.In order to prove whether the DVB1was the LOC＿Os03g62420,we transferred the vector containing the promoter,the complete gene and the termination sequence of the LOC＿Os03g62420 into the dvb1mutant and all the positive transgenes obtained were restored certain phenotype.It was finally confirmed that the DVB1 gene was LOC＿Os03g62420.3.Analysis of DVB1 gene expression patternThe relative expression of DVB1 gene in different periods and different tissues of wild-type plants was analyzed by q RT-PCR,and it was found that DVB1 gene exhibited a non-specific spatiotemporal and tissue expression pattern.In order to analyze the specific expression pattern of DVB1 gene,we analyzed m RNA in situ expression of DVB1 gene in roots,culms,leaves,sheaths,spikelet and so on.In these tissues,the DVB1 gene is mainly expressed in vascular bundle,demonstrating that the DVB1 plays an important role in vascular bundle development.4.Subcellular localization of DVB1 proteinIn order to investigate the localization of DVB1 in cells,we transferred the constructed DVB1-GFP vector into rice protoplasts,and observed the localization of DVB1 in mitochondria and a non-mitochondrial and ambiguous organelle in the cytoplasm.5.Functional analysis of DVB1 proteinDVB1 is a mitochondrial protein.Mutations in this gene may cause the mitochondrial abnormalities.By transmission electron microscope,most of the mitochondria of wild-type cells were rod-shaped structures,and the inner membrane was fully folded to form cristae.Compared with the wild type,the dvb1 mitochondria showed a round structure,abnormal endometrial folding with a concentric circle structure,and no obvious folded cristae structure was found.Statistics of normal and abnormal mitochondria in wild-type and dvb1 mutant were taken,and abnormal mitochondria were up to 82%in dvb1 mutant.Mitochondria is the main site of ATP production in cells.In order to explore whether mitochondrial function changes in the dvb1 mutant,we then measured the ATP content and ATPase activity and it was found that the ATP content and ATPase activity in the dvb1 mutant were significantly reduced which suggests that mitochondria lost normal functions in dvb1 mutant.Mic10 in yeast has been reported to form polymers through the core G*G*G*G sequence to fold the mitochondrial inner membrane as cristae.Mutation of this sequence would cause Mic10to fail forming polymers and the mitochondrial inner membrane could not be folded.Aiming at the question whether DVB1 and DVB1^G90E could form polymers,we found both DVB1 and DVB1^G90E have their own interactions and can interact with each other through yeast two-hybrid.Subsequently,by western blotting of native-PAGE,both DVB1 and DVB1^G90E can form dimers and a small number of polymers.By RNA-Seq analysis,2,533 up-regulated genes and 2,500 down-regulated genes were found in the dvb1 mutant.GO enrichment analysis of these differential genes are concentrated in some pathways such as hormone signal transduction,amino acid metabolism and secondary metabolism.Considering that the phenotype of the dvb1mutant exhibits an abnormal auxin phenotype,then the auxin in the wild type and dvb1mutant at the seedling and tillering stages was measured,and it was found that the auxin content in the dvb1 mutant was significantly increased,and the increase at the tillering stage was greater than that at the seedling stage.Some amino acids are precursors of auxin synthesis,the amino acid content of wild type and dvb1 mutant in leaves at the tillering stage were tested.The amino acids of auxin precursors such as serine,tryptophan,phenylalanine,and tyrosine,respectively,increased by 105.3%,22.8%,28.6%,and 10.63%.Relative expression analysis of several genes related Trp to auxin synthesis in wild type and dvb1 mutant revealed that the expression of FIB,TAR1,TDD1,COW1 was higher in dvb1 mutant.The above results demonstrate that the abnormal mitochondria of the dvb1 mutant may lead to the disorder of amino acid metabolism,which affects the synthesis of auxin,causing the auxin to increase and exhibiting a phenotype of growth inhibition.