Os01g0831900

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Annotated Information

Function

Nitrogen is a crucial nutrient for plant growth and development. Arginine is considered to be an important amino acid for nitrogen transport and storage, playing a crucial role during plant seedling development. However, little is known about the role of arginine in nitrogen remobilization at the reproductive stage. We isolated a rice mutant nglf-1 with reduced plant height, small panicle and grain size, and low seed-setting rate (10% in nglf-1 compared to 93% in wild-type). Map-based cloning revealed that the mutant phenotype was caused by loss of function of a gene (OsARG) encoding an arginine hydrolysis enzyme, which is consistent with arginine accumulation in the mutant. The phenotype was partially corrected supplying exogenous nitrogen, and fully corrected by expression of a wild-type OsARG transgene. Over-expression of OsARG in rice (cv. Kitaake) increased grain number per plant under nitrogen-limited conditions. OsARG was ubiquitously expressed in various organs, but most strongly in developing panicles. The OsARG protein was localized in the mitochondria, consistent with other arginases. Our results suggest that the arginase encoded by OsARG, a key enzyme in Arg catabolism, plays a critical role during panicle development, especially under conditions of insufficient exogenous nitrogen. OsARG is a potential target for crop improvement. Crossing nglf-1 with IRAT129, a japonica variety, to construct a genetically segregating population. Within the F2 population, nglf-1 exhibited normal Mendelian segregation (1632 normal:560 nglf-1; v2 = 0.322; P > 0.05), indicating the involvement of a single gene mutation. Bulked segregant analysis with SSR markers covering the whole rice genome found that the RM335 marker on the short arm of chromosome 4 was linked to the nglf-1 locus, which was subsequently delimited using SSR primers AL36-1 and AL1-1, and further mapped to a 72 kb interval, delimited by the two markers In10 and AL1-1, using 510 F2 mutant plants (Figure a). Based on the Rice Genome Annotation Project (http://rice.plantbiology.msu.edu/), the mapped region contains 13 recognizable open reading frames (ORFs). Sequence comparison revealed a single nucleotide substitution resulting in a stop codon (TGA) in ORF8 of nglf-1. ORF8, gene LOC_Os04g01590, encodes a protein homologous to arginase and consists of six exons, with a total size of 1382 bp, including a 1023 bp coding region, a 126 bp 5′ UTR, and a 233 bp 3′ UTR. Thus the substitution occurred in the arginase domain of nglf-1, resulting in premature termination of the protein (Figure b), and thus ORF8 was considered a candidate for putative gene Nglf. To verify that the mutation in LOC_Os04g01590 is responsible for the nglf-1 phenotype, we introduced a genomic DNA fragment of 5233 bp containing the entire LOC_Os04g01590 coding region, 937 bp upstream promoter sequence and 426 bp downstream sequence cloned from WT, into nglf-1. Of 26 regenerated plants (T0), 20 plants were confirmed as positive by PCR, exhibiting the same phenotype as WT. We grew five plants to the T1 generation, and found that all PCR-positive plants performed similarly to WT (Figure c–e). We also searched the publicly available rice T-DNA database (http://signal.salk.edu/cgi-bin/RiceGE/) and found a line in which Nglf was tagged by a T-DNA (3A-10962). This allelic mutant had a panicle-defective phenotype similar to nglf-1, and was named nglf-2 (F. Further analysis indicated that the insertion was in the first exon of Nglf , and that such an insertion completely disrupted transcription of Nglf, as we were unable to detect any transcripts of Nglf in nglf-2 by RT-PCR .Together, these results confirm that LOC_Os04g01590 is Nglf, and it was renamed OsARG. In the rice genome, OsARG is the only gene encoding arginase. A BLAST search with translated OsARG revealed that most plant arginases are encoded by one or two genes.OsARG has high identity with proteins from other organisms, including Arabidopsis thaliana (Flores et al., 2008), Solanum lycopersicum (Chen et al., 2004), Pinus taeda (Todd et al., 2001) and Glycine max (Goldraij and Polacco, 1999) . OsARG also has high identity with arginases from five genera of monocotyledonous plants, including the arginases in Zea mays, Sorghum bicolor, Brachypodium distachyon, Triticum aestivum and Hordeum vulgare, all of which are members of the same clade, distinguishing them from all dicotyledonous species analyzed. The shared identity may indicate a different role in monocotyledonous plants than in dicotyledonous species . The OsARG protein comprises 340 residues with a predicted molecular mass of 36.96 kDa and pI of 5.90. Sequencealignment indicated a conserved arginase domain, and all plant arginases contain two His and four Asp residues that bind the Mn2+ co-factor (Chen et al., 2004). A predicted mitochondrial targeting peptide is located at the N-terminal end, and this region showed the greatest diversity among various plant arginases Improving nitrogen use efficiency is an important objective of breeding programs. In maize over-expressing the Gln1-3 gene, an increase in kernel number was observed under either high-nitrogen or low-nitrogen growth conditions (Martin et al., 2006). A recent investigation of rice transformed with the cytosolic glutamine synthetase (GS1) gene indicated that, due to enhanced nitrogen use efficiency, lines over-expressing GS1 exhibited a 25–35% higher spikelet yield (Brauer et al., 2011). Our data indicate the possibility of increasing OsARG expression, highlighting the potential use of manipulation of OsARG expression to improve rice yield under sub-optimal nitrogen conditions. However, extensive field experiments are needed. OsARG not only functions in nitrogen catabolism, but also participates directly in the control of grain yield in rice.

Expression

We examined the pattern of OsARG expression in various organs using quantitative real-time PCR. OsARG was expressed ubiquitously in all organs, including the root, stem, leaf blade, leaf sheath and panicle. Compared to WT, the expression in nglf-1 was reduced (Figure f). For a detailed evaluation of OsARG expression, we generated transgenic plants expressing the GUS reporter gene driven by the native promoter of OsARG. The GUS signal was found in the root, stem, leaf and panicle, with the strongest signal in the spikelets, consistent with the RT-PCR results (Figure g).

Evolution

You can also add sub-section(s) at will. The rice narrow-grain and low-fertility mutant nglf-1 was derived from an anther culture of autotetraploid indica/ japonica hybrid H3774 (H2088 9 H891) (Cheng et al., 2005;Qin et al., 2005). During the vegetative growth stage, the mutant and its wild-type (WT) were indistinguishable. At the ripening stage, the apparent characteristics of the mutant included a small and erect panicle accompanied by low fertility (10% in nglf-1 compared to 93% in WT) and lower kernel weight under growth conditions in Beijing in summer (39° 54′N, temperate climate) or Sanya, Hainan Province in winter (18° 16′N, tropical climate). The abnormal phenotype was more severe in plants without exogenous nitrogen application, and included lack of seed set on the panicle. Compared to WT, the mutant displayed reduced plant height but an increase in the number of small, non-seed-setting tillers ,which resulted in dry weight differences in stem, leaf sheath and panicle. However, no significant differences in total dry weight were found between the mutant and WT.

Labs working on this gene

National Key Facility for Crop Gene Resources and Genetic Improvement, Institute of Crop Science, Chinese Academy of Agricultural Sciences, Beijing, 100081, China.

Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing, 100081, China.

National Key Laboratory for Crop Genetics and Germplasm Enhancement, Jiangsu Plant Gene Engineering Research Center, Nanjing Agricultural University, Nanjing, 210095, China.

References

Xuefeng Ma， Zhijun Cheng， Ruizhen Qin et al. OsARG encodes an arginase that plays critical roles in panicle development and grain production in rice. The Plant Journal ，2013（ 73）：190–200

Flores, T., Todd, C.D., Tovar-Mendez, A., Dhanoa, P.K., Correa-Aragunde, N., Hoyos, M.E., Brownfield, D.M., Mullen, R.T., Lamattina, L. and Polacco, J.C. (2008) Arginase-negative mutants of Arabidopsis exhibit increased nitric oxide signaling in root development. Plant Physiol. 147,1936–1946.

Chen, H., McCaig, B.C., Melotto, M., He, S.Y. and Howe, G.A. (2004) Regulation of plant arginase by wounding, jasmonate, and the phytotoxin coronatine. J. Biol. Chem. 279, 45998–46007.

Todd, C.D., Cooke, J.E.K., Mullen, R.T. and Gifford, D.J. (2001) Regulation of loblolly pine (Pinus taeda L.) arginase in developing seedling tissue during germination and post-germinative growth. Plant Mol. Biol. 45, 555–565.

Goldraij, A. and Polacco, J.C. (1999) Arginase is inoperative in developing soybean embryos. Plant Physiol. 119, 297–304.

Martin, A., Lee, J., Kichey, T. et al. (2006) Two cytosolic glutamine synthetase isoforms of maize are specifically involved in the control of grain production. Plant Cell, 18, 3252–3274.

Brauer, E.K., Rochon, A., Bi, Y.-M., Bozzo, G.G., Rothstein, S.J. and Barry, J.S. (2011) Reappraisal of nitrogen use efficiency in rice overexpressing glutamine synthetase1. Physiol. Plant. 141, 361–372.

Cheng, Z.J., Qin, R.Z., Zhang, X., Lei, C.L., Guo, X.P. and Wan, J.M. (2005) Molecular mechanism for phenotypic mutation arisen from polyploidization in plant. Acta Agron. Sin. 31, 940–943.