Positional Cloning And Functional Study Of The Nitrogen Metabolism-related Gene OsARG In Rice

Rice is an important food crop and any genetic improvement on agronomic traits would playbeneficial roles on its yield increment. Nitrogen is considered as an essential major element forplant growth and development, and its reasonable application could bring the increase of grainyield. However, excessively applied nitrogen would decrease nitrogen use efficiency (NUE) andcause serious environmental pollution. From both view points of economic and environmentprotection, any measure on improving NUE at lower level of fertilizer supplement should beadopted. Through the study to an aberrant panicle mutant abp1, we detected out a mutationoccurred at the gene OsARG that responsible for nitrogen use efficiency, a single base pairmutation from G to T caused the abolishment of OsARG's functions and excessive argnineaccumulation in such organs as stem, leaf and panicle, and finally resulted in the abnormalphenotype of abp1. Results from our experiment are summarized as follows:1. Compared with wild type plant, the mutant abp1exhibited shorter plant height and paniclelength, thinner grains and lightened1000-grain weight, as well as degenerated upper part ofpanicles, lower pollen fertility and seed-setting rate. By the exogenous nitrogen supplementexperiments, the abp1phenotypic defects could nearly be recovered to be normal along with theconcentration increment of applied nitrogen. At ripen stage, the nitrogen remained in stem andleaf were significantly higher than those in wild type. It suggested that certain amount of nitrogenhad not be transported out of stems and leaves. All results mentioned above suggested that abp1was a nitrogen nutrition related mutant.2. The abp1locus was previously mapped to the rice chromosome4between markers In10and AL1-1, spanning76kb physical distance in which13predicted open reading frames werefound. Sequence comparison between the mutant and wild type revealed a single base substitutionof G to T, introducing a new stop codon (TGA) at ORF8of abp1mutant. ORF8was subsequentlyconsidered as the likely candidate gene of abp1. The complement test transformed either cDNAor genomic fragment of ORF8into the abp1could recover abp1phenotype to be normal,especially, the panicle development fully restored. In addition, one T-DNA insertion to ORF8producing a similar phenotype as abp1provided an extra evidence that ORF8was responsible forthe mutant phenotype.3. ORF8was annotated as arginase encoding the gene OsARG which had only one copy inrice genome. Analysis of quantitative real-time PCR revealed that OsARG expressedconstitutively in various organs including root, stem, leaf blade, leaf sheath and panicle, whichwas further verified by GUS-staining analysis. In abp1, the OsARG expression level wasdecreased significantly. In addition, the expression level of OsGS1;1was increased, indicatingthat the mutation in OsARG had affected the transcript of other genes involved in the nitrogenmetabolism pathway. According to the result of GFP expression positions visualized by confocal microcope, OsARG exhibited spottedly distribution in protoplasts, which was corresponding tothe subcellular localization of mitochondria.4. In abp1, the mutation in OsARG caused the abolishment of OsARG's functions andexcessive argnine accumulation in such organs as stem, leaf and panicle, which resulted in theabnormality of nitrogen metabolism, and finally resulted in the abnormal phenotype of abp1.5. In transgenic plants overexpressing OsARG, significant increases were observed in filledgrain number, seed-setting rate and the grain yield, and grain number was the yield componentthat was mostly responsible for the increase in grain yield with a strong relationship. Theseresults display a potential value for this gene utilization on improvement of nitrogen useefficiency.