Map-Based Cloning And Functional Analysis Of The Retarded Root Growth Gene In Arabidopsis

In plants, the formation and growth of all root organizations are derived from cell proliferation and differentiation of root meristem. Accurate regulation of meristem cell division is important for normal root growth. In recent years, a number of important major regulatory factors in cell division have been identified, but how cell division progression regulates the morphology and growth of root meristem is still not clear. Therefore, identification and characterization of mutants that show defects in cell division will be helpful to reveal how cell division regulates the development of root meristem. In our study, we screened an EMS-mutagenized Arabidopsis thaliana mutant library and identified a mutant that displayed retarded root growth-1(rrg-1). We identified the RRG gene using the approach of map-based cloning and characterized its function in regulating root growth. The main results are presented as follows:1. We eatablished an ethylmethane sulfonate (EMS)-mutagenized Columbia-0(Col-0) pool containing15000lines and identified a number of mutants. One of the mutants displayed retarded root growth and was named as rrg-1. rrg-1has short root, sparse and long root hairs, slowly growth and wrinkle seeds.2. Genetic analysis indicated that the rrg-1mutation is single recessive. Sixty homozygous mutant plants were selected from the F2population and analyzed with22simple sequence length polymorphism (SSLP) markers evenly distributed on the five Arabidopsis chromosomes. The RRG was firstly located between the markers nga280and ngalll on chromosome1. For fine mapping,721plants conferring the rrg phenotypes were identified from the F2population and genotyped with eight INDEL and SNP markers between nga280and ngalll. The RRG locus was delimited to a25.3-kb region flanked by markers CER473407and CER481032on BAC clones F23O10and F10D13, respectively. Sequencing of the genomic DNA amplified from this region revealed a single G-to-A base change in the rrg-1mutant background, which led to a missense mutation from glutamic (Glu) to lysine (Lys) at the346th amino acid of the encoded protein of the At1g69380gene.3. Complementation test was carried out and all transgenic plants obtained were phenotypically indistinguishable from WT plants, confirming that the root growth phenotype observed in rrg-1were caused by the disruption of Atlg69380. In agreement with this result, a T-DNA insertion mutant in which the T-DNA fragment is located in the seventh exon of Atlg69380showed a similar short root phenotype as rrg-1. We thus renamed Atlg69380as RRG and designated the T-DNA mutant rrg-2. Notably, reverse transcription (RT)-PCR analysis of mRNA transcript levels of RRG in rrg-1, rrg-2and WT plants revealed that RRG transcript was absent in rrg-2but still present in rrg-1, although lower than that in WT, suggesting that rrg-1is a knock-down allele whereas rrg-2is null for the RRG gene. No obvious phenotypic abnormality was observed in the embryonic RAM of both mutant alleles when compared with the WT control, indicating that the role of RRG is confined to post-embryonic root development.4. The RRG gene consists of seven exons and six introns, and encodes a novel protein with373amino acids. More detailed protein sequence analysis revealed a mitochondria signal sequence at the amino terminus of the RRG protein, a conserved DUF155domain and a carboxy terminus transmembrane domain. Sequence search against NCBI protein database with the RRG protein identified putative orthologs of RRG in plant and non-plant organisms such as bacteria, fungi and other eukaryotes. All of these orthologs contain the DUF155domain whose function is not yet known. Two putative yeast orthologs of RRG, Sad1-interacting factor2(SIF2) and Required for Meiotic Nuclear Division1(RMND1), have been associated with cell division. This suggests a role for RRG in meristematic cell division in the Arabidopsis root.5. To examine the subcellular localization of RRG in living Arabidopsis cells, we generated transgenic Arabidopsis plants with the35S::RRG-GFP construct and analyzed the expression of RRG-GFP in vivo with confocal laser scanning microscope. We found that RRG-GFP expression was confined to small punctuate structures in root cells, indicating that RRG is localized to certain organelles, possibly mitochondria as suggested by protein sequence analysis. Indeed, we confirmed this possibility by showing that the mitochondria-specific dye MitoTracker Orange and the RRG-GFP fusion protein colocalize in the mitochondria. To further reveal the nature of the structures marked by RRG-GFP, we performed in tobacco leaf epidermal cells co-localization experiments of RRG-GFP with mt-rk CD3-991, a well-known mitochondria marker generated with a red fluorescent protein mcherry. We observed that RRG-GFP co-localized with mt-rk CD3-991at mitochondria and thus conclude that RRG is localized to mitochondria.6. We investigated the expression pattern of RRG gene in the Arabidopsis root and found that the RRG gene is preferentially expressed in quiescent center (QC) cells, cortex/endodermis stem cells and their daughter cells, endodermal and stele cells in the root meristem, suggesting a role for RRG in these cells. Moreover, we found that the expression pattern of pRRG::GUS in lateral roots was identical to that observed in primary roots, and that GUS staining could also be seen in leaves and pollen.7. The sizes of root meristem of WT and rrg mutants were compared. The average size of the root meristem was significant reduced and the average cortex cell length in the differentiation zone, however, was unchanged between WT and rrg mutants, suggesting that RRG functions specifically in the root meristem and RRG positively regulates the root meristem size. We thus analyzed the average cortex cell number and cell length in the root meristem of WT and rrg mutants. The average cortex cell number in the root meristem of rrg were significantly reduced and the kinematic analysis also showed that the increasing of cortex cells in rrg root meristem was always slower than that in the WT control, suggesting that the rate of cell proliferation was decreased in rrg mutants. In contrast, the cortex cell length in root meristem of rrg mutants was significantly increased compared to the WT control, suggesting that cell elongation in the meristematic cells was accelerated in the rrg mutant background.8. We examined the number and structure of mitochondria in the root meristem cells of WT and mutant plants by using transmission electron microscope. We analyzed the section plane of mitochondria in multiple sections and found that the number of mitochondria per unit cell area in rrg-2was comparable with that of WT, but a vast majority of mitochondria (82%) in mutant cells displayed internal vacuolization compared to WT controls. The results suggest that RRG is required for the maintenance of mitochondrial structure.9. To further dissect the role of RRG on cell proliferation, we estimated the average cell cycle duration in the root meristem of rrg mutants and WT and found that the rrg mutations led to increased cell cycle duration in the root mersitem. We then examined the expression level of some cell cycle progression related genes in WT and rrg-2by qRT-PCR, the results showed that the expression of CYCD4;1and E2Fa, DPa and DELI did not change significantly, but HISTONE H4, CYCB1;1, CDKB1;1and WEE1were significantly down-regulated in rrg-2mutant compared to the WT controls. In agreement with this indication, we found that the number of root meristem cells at the G2-M phase, revealed by the CycB1;1::GFP marker, was strongly reduced in rrg mutants than in WT. The experiments indicated that cell cycle progression was impaired in rrg-2mutant. Because mutants affecting cell division always display altered endoreduplication levels, we compared the ploidy levels of WT and rrg mutant roots. We found that, at9days after germination, the proportions of root cells with nuclear contents of4C,8C and16C were lower in rrg mutants than in WT, indicating that endoreduplication was seriously compromised in rrg mutants. By contrast, the proportion of2C cells was increased in rrg-2null mutants, further confirming that cell division was impaired.10. We analyzed the expression levels and patterns of pSHR::SHR-GFP, pSCR::H2B-YFP and pWOX5::ERGFP in the rrg mutant background and found that the expression levels and patterns of these markers in the root meristem were not obviously affected by the rrg mutations, suggesting that RRG is not involved in the regulation of QC and proximal root stem cell identity and functions independently of the key patterning genes.11. To learn whether the phenotypes observed in rrg mutants were caused by changes in auxin level and response, we examined the expression of the auxin-responsive marker DR5rev::GFP in rrg mutants. No obvious differences between rrg mutants and WT seedlings were detected with regard to the expression level and pattern of DR5rev::GFP in the root tip, suggesting that the RRG mutations did not cause changes in auxin level and response. Consistently, we found that rrg-2mutants exhibited a WT-like root growth response to NAA, IAA, IBA, or2,4-D at all concentration examined, indicating that rrg-2mutants are not defective in auxin response or sensitivity.Taken together, RRG is preferentially expressed in the root meristem and required for the cell division activity of postembryonic root meristem in Arabidopsis. The function of RRG is independent of auxin signalling and root patterning. RRG encodes a mitochondrial protein, and the mitochondria in root meristem cells of rrg mutants display extensive vocalization, implicating plant mitochondria has an important role in the regulation of cell division. Our data reveal for the first time the role of the Arabidopsis RRG gene in regulating cell division in the root meristem, and provide a new evidence for the involvement of plant mitochondria in cell division.