Cloning And Functional Analysis Of ARL1 Gene Required For Adventitious Root Formation In Rice

Rice, one of the most important food crops worldwide, has a fibrous root system, which consists of one primary root and a mass of adventitious roots. Adventitious roots are shoot-borne roots that are the most important for anchorage, nutrient acquisition, and water uptake in rice. Adventitious root is a post-embryonic organogenesis event that is formed on the nodes and gives rise to most of the underground parts of rice. The developmental stages of adventitious root can be divided into the initiation of the root primodium and its subsequent outgrowth. Although the morphology and physiology of adventitious root have been well described, Little is known about the genes that control adventitious root development in rice. Through screening for mutants of adventitious root morphogenesis, we have got a mutant defective in adventitious root (arl1). The phenotypic, physiological and genetic characterization of the arl1mutant were investigated, and the ARL1 gene was cloned by map-based cloning. The results are summarized as follows:1.Phenotypic analysisThe arl1 (adventitious rootless 1) mutant seedlings showed a complete defect in adventitious roots, while the length of lateral roots on the primary root were enhanced. No significant difference was observed in shoot morphology between the wild type and the mutant at the seedling stage. The tiller primordia was developed earlier in the arl1 mutants than in the wild-type plants. No normal tillers were observed in arl1 mutant plants because the plants died due to lack of adventitious roots.2. Cytogenesis analysisCross-sections at the base of the stem of 4d-old seedlings revealed that the formation of adventitious primordium is impaired in the arl1 mutant. Adventitious root primordia were induced at the fourth node of 8-week-old wild-type plants by submergence for 24 hr, but not induced in the arl1 mutant.3.Hormone analysisTreating with different concentrations IAA, 6BA and ethephon could not rescue the defect in aril.4. Genetic analysisGenetic analysis with reciprocal crosses between aril and wild-type plants indicated that aril possesses a recessive mutation in a single nuclear locus.5. Gene mapping and cloningThe ARll gene was located on the short arm of chromosome 3 near STS9. Homology analysis demonstrated that ARD1 is a member of the plant-specific LBD gene family proteins. The binary plasmid carrying a 5.4-kb wild-type genomic fragment containing the entire ORF plus 3.7-kb upstream region was able to rescue the adventitious root defect phenotype of the ardl mutant.6. Gene expression patternSemi-quantity of RT-PCR analysis suggested that ARL1 is mainly expressed in adventitious roots and basal stem.7. ARL1 analysisThe 35S promoter-driven ARLl-mGFP4 fusion protein transiently expressed in onion epidermal cells. The ARL1 -GFP green fluorescent signal was detected mainly in nuclei, suggesting that ARL1 is a nuclear protein. Yeast two-hybrid experiments suggest that ARL1 may act as a transcription factor to regulate the expression of a certain gene(s) as a homodimer or heterodimer and thereby control the initiation of adventitious root primordia.