Map-based Cloning And Functional Analysis Of AVB Genes In Rice (Oryza Sativa L. Ssp.indica)

Rice(Oryza sativa L.)is not only an important food crops in the world,but also a model plant for studing gene function in plant.Research of rice structure always play an important role in rice breeding.All aerial lateral organs are developed from the shoot apical meristem(SAM).At the periphery of the dome-like SAM,founder cells are recruited to form the primordia of lateral organs;the conversion of primordia into mature organs is usually described to consist of two partially overlapping phases: cell division and cell expansion.The cell proliferation rate in primordia,which determines the final number of cells,is an important factor contributing to the final organ size.During the development of primordia into mature organs,procambium cells are recruited in specific positions and differentiate into mature vascular bundles.Plant vascular bundles interconnect all parts of the plant body from the root tip to the aerial organs by their conductive function to enable the efficient long-distance transport of water,nutrients and small molecules and to provide mechanical support.Owing to our superficial understanding of the mechanisms that regulate cell proliferation and procambium establishment,more research is required to obtain a deeper understanding of the mechanisms involved.Here,we first confirmed that ABNORMAL VASCULAR BUNDLES(AVB)plays an important role in the maintenance of appropriate cell proliferation in meristems.Second,we showed that AVB participates in the establishment of the procambium in lateral organ primordia.Third,we found that AVB influences the proliferation of procambium cells.The main results as follows: 1.Phenotypic observation of avb mutantsCompared with WT plants,the avb plants exhibited visibly narrower aerial organs.Measurement of the leaf width throughout the growth period indicated that the avb mutation significantly affected the width of the leaf blades,and the narrow-leaf phenotype became increasingly pronounced over the course of plant development.At plant maturation,measurement of the internode width showed that the width of internodes was also significantly reduced in the avb mutant.In addition,the panicles of avb were reduced in size with a narrow rachis and fewer branches in comparison with those in WT.Despite the reduction in width of the aerial organs,the lengths of these organs were similar to those of WT.2.Histological analysis of avb mutantsTo clarify the reason for the narrow aerial organs,histological analysis of transverse sections of leaf blades,leaf sheaths,internodes and rachis was performed,which showed that the total cell numbers along the widths of these organs were reduced and the vascular bundles in these organs were also decreased in number compared with those in WT.Notably,all vascular bundles,whether BVs or SVs,in sheaths,internodes and rachis,became significantly larger than their WT counterparts and there were more cells between the two adjacent vascular bundles.However,there was no apparent change in the transverse sections of root,primary branches and secondary branches between the avb mutant and WT.3.Genetic analysis and molecule mapping of the AVB geneTo explore whether the avb mutant phenotypes were controlled by a single gene,the avb mutant was crossed with Nipponbare.All of the F1 generation exhibited a normal phenotype,the same as in Nipponbare.Genetic analysis of the F2 progeny showed that the segregation of WT and mutant individuals was a good fit to the 3 : 1 segregation ratio,indicating that the mutational traits were controlled by a single recessive nuclear gene.To clone the AVB gene,the F2 plants with the narrow-leaf phenotype were collected and SSR and In Del markers were used to map the AVB gene.Finally,the AVB gene was mapped to the middle of the long arm of chromosome 3 between SF2 and SR3,with a physical interval of 46 kb.4.Cloning and functional identification of the AVB geneEight annotated genes within this interval were sequenced and a single-nucleotide substitution from C in WT to T in avb within a gene with an unknown function(Os03g0308200)was identified.The C to T mutation caused the early termination of translation.This gene was thus predetermined as a candidate for the AVB gene.To confirm that it was indeed the AVB gene,WT genomic fragment of Os03g0308200 was transformed into avb.The mutant phenotype was completely rescued in transgenic plants.We have transformed RNAi vector into Zhonghua 11,and in transgenic plants,the expression levels of Os03g0308200 were down-regulating,with same-like phenotype as the avb mutants.These results confirmed that Os03g0308200 was the AVB gene.5.The expression pattern analysis of the AVB gene qRT-PCR showed that AVB was universally expressed in various tissues,including roots,internodes,leaves and panicles,especially at growing points.The same expression pattern of the AVB gene was detected by in situ hybridization.Strong signals were observed at the growing points.For a more detailed analysis of the expression pattern of AVB in growing points,the transverse and longitudinal sections of fixed tissues crossing SAM were examined for AVB signals.First,there were strong signals at the leaf primordia initiation site.When procambium cells formed at the primordia,AVB signals were concentrated at the procambium cells.When vascular bundles developed in mature leaves,the weak signals of AVB were primarily restricted to the whole of the vascular bundles,with no preference for the xylem or phloem.6.The AVB gene was involved in procambium establishment induced by auxinConsidering that the major site of AVB expression was shown to be procambium cells,mutation of the AVB gene impeded procambium establishment and polar auxin transport(PAT)played a unique role in procambium establishment,it was speculated that the AVB gene might crosstalk with auxin.Therefore,after treatment with indole-3-acetic(IAA)and 2,3,5-triiodobenzoic acid(TIBA)(inhibitor of polar auxin transport).On treatment with TIBA,expression of the AVB gene at the growing points was dramatically activated at 2 h.And there were three typical Aux REs in the promoter sequence of the AVB gene,named ?,? and ?.Electrophoretic mobility shift assay(EMSA)showed that the conserved DBD of Os ARF could directly combine with ?,?,? and the capacity for combining with ?,which was the closest one from the start code,was much stronger than that for?and ?.These results confirm that the AVB gene is involved in procambium establishment induced by the auxin signal.7.Protein function analysis of the AVBThere are 988 amino acid residues of AVB protein and there are five members belong to the same protein family with AVB in rice based on the information provided by Phytozome web site.Although AVB with no conservative domain is a unknown function protein based on the NCBI web site,AVB protein belonged to the Armadillo-type fold family according to the information provided by the global protein resource database.Armadillo-type fold is consist with two ?-helixes and has been implicated in mediating protein-protein interactions.Phylogenetic analysis showed that the AVB protein is conserved in land plants from moss and gymnosperms to monocots and dicots.In order to get the further information of the distribution of AVB protein in plant,western blot was adopt to distribution analysis and the result showed that AVB protein only existed in the growing point and there was no AVB protein in avb mutant.The subcellular localization of AVB was in the nucleus and cytoplasm.The reduced number of cells along the width of aerial organs indicated that cell proliferation might be impaired during lateral primordia development in avb.To confirm this possibility,flow cytometry was used to monitor the amount of DNA in the nuclear suspensions in the shoot apex of WT and avb.Compared with WT,the proportion of nuclei in the S phase of avb in the shoot apex was decreased by 30%.This showed that,in avb,the shoot apex,including SAM and lateral organ primordia,had lower cell division activity than that in WT.The reduction of cell division was further characterized by analysis of the in situ expression patterns of histone H4,which is expressed specifically in S-phase cells.The frequency of cells expressing histone H4 was reduced in leaf primordia in avb.These results confirmed that the decreased number of cells was caused by impaired cell proliferation during the early development of organ primordia.In contrast with the impaired cell division in early primordia in avb,the cell division capability increased in procambium cells in the late period of organ development in avb,which explains the enlarged vascular bundles in these organs,such as leaf sheaths,internodes and rachis,in avb.8.Transcriptome analysis of the avb mutant compared with the wild typeTo obtain deeper insights into the function of AVB,the transcriptomic profiles of shoot apexes in avb and WT grown under normal conditions were characterized.A total of 730 DEGs between avb and WT were identified,including 678 up-and 52 down-regulated DEGs.GO classification was used to assign the functional categories of DEGs.GO enrichment analysis results showed that the expression of genes involved in multicellular organismal development was changed dramatically.The expression of 44 of the 45 genes annotated within the organismal development term,containing 36 upand eight down-regulated genes,changed significantly,indicating that the development process was strongly affected by the AVB mutation.The significant differential expression of key cell cycle regulators in avb implied that the cell cycle was affected in avb,which was confirmed by ploidy analysis and the in situ expression patterns of histone H4,as mentioned earlier.