| Rice(Oryza sative L.) is one of the most important food crops worldwide, and grain yield in rice is closely related to leaf and flower development. In recent years, many genes associated with leaf and flower development have been isolated and applied in rice breeding to improve grain yield. However, the complex genetic regulation remains unclear. Therefore, further identification of the novel genes affecting leaf and flower development would provide new insights into the regulation mechanism of rice development, and provide a basis for molecular breeding in rice plant arichtecture and grian yield. In the present study, we identified NRL2 gene controlling leaf, anther and seed shape in rice through position cloning approach, and preliminarily analyzed its molecular function. The main results were as follows:We screened rice mutant populations that were generated by ethyl methanesulphonate mutagenesis and identified two allelic mutants, named(narrow and rolled leaf 2-1) nrl2-1 and nrl2-2, with extremely narrow and semi-rolled leaves, male semi-sterility, and slender seeds. Histological analysis showed that the nrl2 mutant displayed a reduction in the number of longitudinal veins, defective abaxial sclerenchymatous cell differentiation, abnormal tapetum degeneration, and microspore development compared with the wild type. Using F2 individuals derived from a cross between the nrl2-1 mutant and the japonica variety Zhonghua 17, we narrowed down the location of the NRL2 gene to a c.59-kb physical region between markers M5 and M4 on chromosome 3. By comparing the nucleotide sequences of fine-mapping region in the WT and the nrl2 mutants, we found that LOC_Os03g 19520 had a guanine (G) deletion (+2662) in the ninth exon of the nrl2-1 mutant, causing a frame shift mutation and premature translation stop; and a single nucleotide substitution from guanine (G) to adenine (A) at the fifteenth intron splicing site (3’acceptor site) in the nrl2-2 mutant, leading to the change of splice pattern and premature termination. Further transformation evidence demonstrated that LOC_Os03g19520 is equivalent to the NRL2 gene. Yeast two-hybrid and bimolecular fluorescence complementation assays showed that the NRL2 protein interacted with RL14 protein. Measurement of the abundance of the major constituents of secondary cell walls in the flag leaves found that the cellulose content was significantly higher and the lignin content was significantly lower in the nrl2-1 mutant compared with the WT, which was consistent with the changes observed in the rl14 mutant. Analysis of the phenylpropanoid metabolism products showed that the cinnamyl alcohol, myricetin, and dihydrokaempfrol contents were dramatically reduced in both the nrl2 and nrl2lrl14 double mutants. And the expression of genes involved in the phenylpropanoid metabolism pathway was also altered between nrl2 and wild type, suggesting the mutation of NRL2 affected phenylpropanoid metabolism pathway, changed the content of lignin and flavonoid, further resulted in both the semi-rolled leaves and reduced male fertility. In addition, we found that the mutation of NRL2 affected the expression of genes associated with cell division through mRNA in situ hybridization and qRT-PCR analysis. We also found that the outer parenchyma cell layer of hulls in the nrl2-1 mutant contained fewer cells than that of the WT, indicating that NRL2 gene might modulate both leaf and seed size by regulating cell proliferation.Taken together, NRL2 gene is an essential developmental regulator controlling fundamental developmental processes, serving as a potential breeding target for high-yielding rice cultivars. |
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