| The leaf is the main organ for photosynthesis, about 95% of rice yield are from leaf photosynthesis. Chlorophyll is an important pigment involved in photosynthesis in plant chloroplasts. Generally, the plant leaf is green; however, once the synthesis or degradation pathway of chlorophyll goes wrong, it will cause abnormal leaf color phenotype. In fact, leaf color mutants can be easily obtained natually. And the leaf color mutant is usually often been used as a donor of specific marker character in breeding, materials for understanding the chlorophyll biosynthesis and degradation pathway, chloroplast structure and function, mechanism of plant photosynthesis, rice functional genomics. Therefore, it is significant to explore and identify the genes associated with rice leaf color by characterizing the rice leaf color mutants.In the present study, a rice yellow-green leaf mutant ygl3 isolated from Shuhui 527, was physiologically and phenotypically characterized, and the YGL3 gene was cloned with the map-based cloning strategy. The main results are as follows:1. Phenotypic characteristics and the main agronomic characters of mutant ygl3. The ygl3 mutant has the typical yellow-green leaves during the whole growth period. The results of main agronomic traits in mature stage showed that the panicle length increased by 23.25%, the flag leaf length increased by 34.93%, and the number of tiller per plant decreased by 42.86%. The grain density of the mutant was 85.57% of that of wild type, the seed setting rate increased by 6.79%, the grain weight increased by 13.89%, but the yield of the mutant was significantly lower than that of wild type.2. The chlorophyll content evaluation and chloroplast structure analysis of the mutant ygl3. The chlorophyll content of wild type and mutant were evaluated at the seedling stage, the tillering stage and the heading stage, respectively, the results showed that, the chlorophyll a, chlorophyll b and total chlorophyll were significantly lower than those of wild type at all stages, suggesting that the decreased chlorophyll content may be reseason causing the the yellow-green leaf phenotype. Transmission electron microscope ananlysis showed that the shape of the chloroplast in ygl3 mutant was irregular and the number of chloroplast was reduced. Moreover, it was found that granum lamella in the ygl3 mutant contains only fewer thylakoid stacks, in addition, no matured starch granules were observed, and the osmiophilic bodies were outside. This indicated that the chloroplast development of mutant ygl3 was inhibited.3. The genetic analysis and gene location for ygl3. The mutant ygl3 was crossed to 9311, Fi individuals exhibited green leaves. In the F2 population, the ratio of the wild-type plants to the yellow-green leaf plants fits well with 3:1, indicating that the yellow-green mutant is controlled by a nuclear recessive gene. Furthermore, by using an F2 popultion derived from the cross of the mutant ygl3/9516, YGL3 was assigned to the interval governed by SSR-19 and NEW-85 on chromosome 3. In this interval, there are 16 opem reading frame (ORF) in total, and sequencing result showed that a single nucleotide substitution at position 235 in coding region was found in LOC Os03g22780 (OsDVR), resulting in a change from glycine into tryptophan. Therefore, OsDVR (LOC os03g22780) was considered as the putative candidate of YGL3.4. The chlorophyll component analysis. Bioinformatic analysis indicated that OsDVR (LOC_Os03g22780) encodes a divinyl reductase,which can reduce all kinds of chlorophyll intermediates 8-vinyl group into ethyl, the process is indispensable to the chlorophyll biosynthesis. So, the high performance liquid chromatography (HPLC) was employed to determine the the chlorophyll compositions of the wild type and the ygl3 mutant. The result showed that the content of chlorophyll a and chlorophyll b decreased significantly in mutant ygl3 plants compared to that of wild type, while the precussors of chlorophyll a significantly increased, only small amounts of single ethylene chlorophyll a was detected. We deduced that the decreased chlorophyll content resulted in the alteration of leaf color in the ygl3 mutant. Taken together, the results showed a single base substitution in OsDVR results in a defective divinyl reductase (OsDVR), consequently, leading to the alteration of the composition of chlororphyll in ihe ygl3 mutant. Thus, we considered that the candidate gene of YGL3 is OsDVR (LOC_Os03g22780).5. Transcriptional expression pattern analysis. We analyzed the expression pattern of OsDVR gene by real-time PCR, it was founded that the gene expressed in leaves with the highest level. And compared to the wild type, the expression of OsDVR gene in the leaf of mutant ygl3 was significantly reduced. |
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