Map-based Cloning And Functional Study Of The Rice Mutant Gene Ygl1 Controlling Yellow-green Leaf

Rice is one of the most important food crops in the world. Half of global populationlives on rice. However, world population continues to increase at an explosive rate, ourarable land is deteriorating. At present, rice production can not serve for economicdevelopment and can not reach people's demand. How to improve rice yield will be onemain mission for rice breeding scientists. High photosynthetic efficiency breeding is one ofthe approaches for high yield. In the past, physiological research played an important rolein photosynthesis research. However, it can not make great progress in improvingphotosynthesis efficiency for hybrid breeding. Leaf coloration mutants are very importantmaterials for photosynthesis research. Although much work of gene mapping was done onrice leaf coloration mutants, few mutant genes have been cloned so far. In this study, weisolated a spontaneous mutant from indica rice cultivar, Zhenhui 249, ygll, which exhibitsyellow-green leaf phenotype. Functional analysis and map-based cloning of the mutantresulted in the identification of molecular mechanism for mutant phenotype, and somestudies were done in physiology and cell morphological. The results are as followed:The ygl1 mutant is different from Chlorina mutant, and exhibits delayed greeningduring photomorphogenesis because of slow rates of chlorophyll accumulation. Eventually,mutant plants accumulated substantial quantities of chlorophyll, nearing wild-type levelswith the maturation of leaves. To investigate its photosynthetic ability, we compared theapparent photosynthetic efficiency of the mutant with wild-type seedlings, using lowtemperature (77K) Chl fluorescence emission spectrometry. The results indicate that ygl1mutant have relatively more emission energy from PSâ… and PSâ…¡per unit of chlorophyll,and the electron transport activity increase from PSâ…¡to PSI in ygll mutant. Chlfluorescence kinetic parameters indicate that the functioning efficiencies of the PSâ…¡reaction centers in ygl1 mutant are nearly equivalent to that of wild type. The xanthophyll cycle analyses show that mutant accumulated high levels of anteraxanthin and zeaxanthinunder excess light, which indicates that ygll mutant is much better in standingphotoinhibition than wild type. TEM show that granal stackes in ygl1 mutant appear lessdense, and lack granal membranes compared with those of wild type in developing leaves.However, granal stackes increased when the leaf became mature in ygll mutant comparedwith that in wild type. This result appeared consistent with the observation that ygl1 mutantplants did turn green at later developmental stages. Although mutant plant biomass andweight per panicle decreased, there were increasement in effective panicles, and ygl1mutant still get high yield (6.75 t/hm²).For genetic analysis of the ygl1 mutant, the phenotype of yellow-green leaf in the ygl1mutant is controlled by a single recessive nuclear locus, ygll. To map the ygl1 locus, wegenerated F₂ mapping population derived from a cross of the ygl1 mutant and the cultivarPA64. We mapped the ygll to an interval between markers RM516 and RM164 onChromosome 5. Comparison of the chromosomal locations and leaf color phenotypesindicate that this gene is different from the previously identified genes related to leaf coloralteration. To narrow down the search for a candidate gene affected in ygl1, a larger F₂mapping population consisting of more than 12,000 plants, of which 2741 segregantsshowing the ygll mutant phenotype were used for fine-mapping. Between markers RM516and RM164, we further developed three SSR markers and seven cleaved amplifiedpolymorphic sequence (CAPs) markers. The ygll locus was finally located in a 11-kb DNAregion between the two cleaved amplified polymorphic sequence (CAPs) markers P23 andP8 on a single BAC, AC136221 (Fig. 2C). Within this region, two opening reading frames(ORF) were predicted, the first ORF encodes a putative chlorophyll synthetase that shows astrong similarity to Oat CHLG, a protein required for Chl a biosynthesis; the second ORF isOsem gene coding a protein similar to embryonic abundant protein. To define the molecularlesions of ygll mutant, the two ORFs corresponding to the putative ygll gene from wildtype and ygll mutant were amplified by RT-PCR and were sequenced. Comparison of thesequences revealed that only the 1st ORF carries a single nucleotide mutation in codon 592(T→C) in the eighth exon, which results in an amino acid change from Pro-198 to Ser (Fig.3A). Therefore, we tentatively designated the 1st ORF as the YGL1 gene.The identity of ygll was subsequently confirmed by genetic complementation experiments. The levels of chlorophyll and ratio of Chl a:b in several independenttransgenic lines transformed with the wild-type YGL1 gene reached same values as that ofthe wild-type plants. Therefore, the ygll mutation at this position was responsible for theabnormal phenotypes of the ygll mutant. Chlorophyll synthetase was believed to be boundto the thylakoid membranes and to catalyze prenylation of Chlides with GGPP or PhyPP,the last step of chlorophyll biosynthesis. This step is essential for the accumulation of Chl aand probably is essential for stable assemble of other components of the thylakoidmembranes. YGL1 appears to be a single copy gene, encoding a 1, 131-bp ORE The codingregion of YGL1 gene comprises 15 exons and encodes a 376 amino-acids protein with themolecular mass of about 41kDa. YGL1 harbors an apparent chloroplast-targeting sequenceof 47 amino acids at its N terminus. Multiple amino acids sequence alignments showed thatYGL1 has significant sequence similarity to CHLGs from higher plants and cyanobacteria.For example, YGL1 is 88.39ï¼…identical to Oat CHLG, 74.68ï¼…identical to the A. thalianaG4 gene product, and 53.94ï¼…identity to the Synechocystis sp. PCC 6803 enzyme.Moreover, YGL1 is also homologous to various bacteriochlorophyll synthetases (23～30ï¼…).We then analyzed the possible phylogenetic relationships between YGL1 and its relatedproteins. The result indicated that rice YGL1 is more closely related to chlorophyllsynthetase from oat (A. sativa) than that of other species. Not surprisingly, YGL1 isphylogenetically more related to CHLGs of the higher plant species than to cyanobacteriaproteins. Analysis of YGL1 derived amino acid sequence showed that Pro-198 is inproximity to a motif (WAGHDF-197) specifically found only in chlorophyll synthetases,but not in bacteriochlorophyll synthetases, which differ, in part, based on preference ofsubstrates, either Chlide (targeted by chlorophyll synthetases) or bacteriochlorphyllide (forbacteriochlorophyll synthetases). Therefore, the importance of the essential Pro-198 residuein YGL1 could be attributed to its location in or proximity to the binding site of Chlide. Ananalysis using the transmembrane calculation programs indicated that the ygl1 mutation siteoccur at or close to the end of a transmembrane helix. A missense mutation was found in ahighly conserved residue of YGL1 in the ygll mutant, resulting in about 70ï¼…reduction onthe mutant enzymatic activity. YGL1 is constitutively expressed in all tissues, and itsexpression is not significantly affected in the ygll mutant. Interestingly, the mRNAexpression of the cablR gene encoding the Chl a/b-binding protein is largely abolished inthe ygll mutant. Moreover, the expressions of several other genes associated withchlorophyll biosynthesis or chloroplast development are also affected in the ygl1 plants. These results indicate that the expressions of nuclear genes encoding various chloroplastenzymes are feedback regulated by chlorophyll precursors.