RNRL1Mutation Rescues The Phenotype Of Osnaatl Root

It has been demonstrated that Fe（Ⅲ）-uptake ability and root growth of Osnaatl mutant is inhibited grown in a solution culture supplied with Fe（Ⅲ）, while the mutated traits can be recovered when the mutants are grown in a solution culture with Fe（Ⅱ） as iron source, or in a solution culture without phosphate （Fe（Ⅲ-P））. For further understanding of the mechanisms underlying the phenotypic changes of Osnaatl mutant under different growth conditions, we developed an ethyl methane sulfonate （EMS） mutagenized mutant library with a background of Osnaatl mutation. We isolated a double mutant, designated as Osnaatl/rnrll according to the result of gene cloning, which showed partial rescue of root growth in a solution culture with Fe（Ⅲ） as iron source. Studies on phenotypic, physiological and molecular traits of the double mutant providing the following results:1. Osnaatl/rnrll mutant showed leaf chlorosis under solution cultures with Fe（Ⅲ）, Fe（Ⅱ） and Fe（Ⅲ-P） treatments. The leaf vein showed severe necrosis in old leaves. The phenotypic trait is shown during the whole plant growth period. Osnaatl/rnrll showed partial rescue of root growth under a solution with Fe（Ⅲ）, and normal root growth in solution cultures with Fe（Ⅱ） and Fe（Ⅲ-P） conditions.2. Genetic analysis using a backcross population derived from a cross between the wild type （Nipponbare） and Osnaatl/rnrll mutant indicates that a single recessive gene is responsible for the chlorotic symposium of Osnaatl/rnrll. Map-based cloning using an F2population derived from a cross between an indica rice （MH63） and Osnaatl/rnrl1revealed that a point mutation was occurred at the eighth exon of the gene OsRNRL1（LOC₀6g07210） located on chromosome6between two molecular markers, s6-3346and s6-3504. The mutation results in a change of C to T, and consequently a change of Alanine to Valerian. Complementation test by transformation of the wild type OsRNRL1gene into the Osnaatl/rnrll confirmed the mapping result. OsRNRL1encodes Ribonucleotide Reductases Large Subunit1. It has been known that loss of function of OsRNRL1reduces the rate of deoxyribonucleotide production for DNA synthesis and repair.3. The single mutant Osrnrl1was isolated from the backcross plants by sequencing of OsRNRL1gene and NAAT1gene. The rnrl1mutant showed normal root phenotype as the wild type plant and leaf chlorosis as Osnaatl/rnrl1.4. RT-PCR analysis indicates that OsRNRL1is a constitutive expressed gene in all plant tissues with higher expression levels in roots and shoot base. The GUS（P-glucuronidase） staining analysis of the transgenic plants harboring GUS report gene driven by promoter of OsRNRL1is consistent with results form RT-PCR analysis.5. OsRNRL1p::OsRNRL1::GFP fusion protein is localized in endoplasmic reticulum of Onion epidermal cells.6. In a solution culture with Fe（Ⅲ）, the accumulation of ethylene and nicotianamine （NA） in roots of Osnaatl/rnrl1is similar as inOsnaat1mutant. Under phosphate starvation （-Pi） and exogenous supply of gibberellin （GA3） conditions, the accumulation of ethylene was reduced and root growth was resuced. The results suggest that the accumulation of ethylene in roots results in the root growth inhibition in Osnaatl mutant and Pi-starvation and exogenous GA may alleviate the the accumulation of ethylene effect.7. qRT-PCR analysis indicates that under Fe（Ⅲ） condition, Fe-starvation responsive genes and GA synthesis related genes were repressed in root and leave of Osnaat1/rnrl1double mutant, but antioxidant genes were increased compared with naat1mutant,same as rnrl1compared with wild type.Taken together, the function loss of OsRNRL1may increase GA synthesis and change the ethylene signaling in naatl, which partially rescue the root growth under Fe（Ⅲ） condition. This hypothesis required further investigation.