| Rice(Oryza sativa L.), one of the most important food crops in the world, is also a very important model plant for the study of monocotyledons. In rice, photosynthesis that is mainly performed in chloroplasts in leaves synthesizes carbohydrate molecules which are transferred and stored finally in rice seeds. Leaf-color mutation is a common phenomenon that occurs widely in plants. In general, leaf-color mutation is involved in the change of photosynthetic pigment content in leaves, which often directly results in the change of color in leaf blades. Photosynthetic pigments play a crucial role in photosynthesis of photosynthetic organisms because of its vital functions to capture light energy and transfer electrons. Plenty of studies showed that leaf-color mutations are ideal materials to study photosynthetic pigment metabolism, chloroplast structure and function, chloroplast development, photosynthesis and photomorphogenesis in plants.In this study, using two yellow-green leaf mutants which were derived from indica restorer line Jinhui10 treated with EMS(ethyl methane sulfonate), two yellow-green leaf genes, YGL8 and YGL9, were cloned and functional complementation assays were performed. The organ-specific expression of these two genes in rice plants were analyzed and the expression levels of related genes in these two mutants were analyzed. The structures and functions of proteins encoded by these two genes were also analyzed. The main results were shown as follows. 1. The rice yellow-green leaf gene YGL9(1) Gene cloning of YGL9The rice ygl9 mutant exhibited a yellow-green leaf phenotype throughout its whole growth period, but its leaves became green slightly in an age dependent manner. DNA and cDNA sequencing results showed that a single base at position 900 of LOC_Os03g03990 was mutated(G to A) in ygl9, which caused a change of the 300 th amino acid from Try to stop codon. LOC_Os03g03990 encodes a probable chloroplast signal recognition particle 43 kDa protein(cpSRP43) in rice. Thus, we speculated that LOC_Os03g03990 is the candidate gene for YGL9.(2) Functional complementation assay of YGL9To confirm whether the mutation of LOC_Os03g03990 resulted in the yellow-green leaf phenotype of the ygl9 mutant, we cloned a 6812 bp wild-type genomic DNA fragment containing a 4373 bp upstream region, the entire 1167 bp coding region and a 1272 bp downstream region to construct the complementation vector of YGL9 and introduced it into the calli of the ygl9 mutant. The leaves of positive transgenic lines all recovered to a green phenotype and their chlorophyll and carotenoid contents approached that in wild-type plants respectively. These results demonstrated that LOC_Os03g03990 is the YGL9 gene.(3) Structure analysis of YGL9The phylogenetic tree showed that YGL9 belongs to a conserved cpSRP43 protein family which presents in almost all photosynthetic organisms. The results of subcellular localization indicated that YGL9 localizes in the chloroplast. The alignment of amino acid sequences showed that YGL9 are highly similar to the Arabidopsis cpSRP43, and the mature YGL9 protein may also be composed of three CDs(CD1-3) domains and four Anks(Ank1-4) domains and these domains are most probably involved in protein-protein interactions.(4) Organ-specific expression analysis of YGL9 and expression analysis of related genesRealtime quantitative PCR(qRT-PCR) was used to analyze the organ-specific expression of YGL9 and the results showed that YGL9 is highly expressed in green flag leaves and leaf sheaths, but lower expression levels in roots, culms, young panicles(white) and green panicles. qRT-PCR results showed that the transcription levels of some chlorophyll synthesis-related genes were distinctively down-regulated in ygl9 compared with that in wild-type at tillering stage. The transcription levels of carotenoid synthesis-related genes detected were distinctively down-regulated in ygl9 at tillering stage. The transcription levels of some light-harvesting chlorophyll a/b-binding protein(LHCP)-related genes were distinctively down-regulated in ygl9 at tillering stage. Additionally, the transcription levels of almost all the chloroplast development- and photosynthesis-related genes were distinctively down-regulated in ygl9 at tillering stage. However, it is notable that the transcription levels of chlorophyll and carotenoid synthesis-related genes were distinctively up-regulated in ygl9 compared with that in wild-type at heading stage. The transcription levels of all the LHCP-related genes were equivalent in ygl9 compared with that in wild-type at heading stage, even some were obviously up-regulated in ygl9. The transcription levels of most of the chloroplast development- and photosynthesis-related genes were also equivalent in ygl9 at heading stage, even some were obviously up-regulated in ygl9, but psaL and ndhA were obviously down-regulated in ygl9.(5) Prediction and analysis of the probable rice cpSRP54The probable YGL9-interacting proteins were predicted via STRING. Two probable chloroplast signal recognition particle 54 kDa proteins(cpSRP54), LOC_Os11g05552 and LOC_Os11g05556, were predicted. Sequencing results showed that the coding sequences of LOC_Os11g05552 and LOC_Os11g05556 are 1653 bp and 1674 bp respectively, and both of these two genes contain 15 exons and 14 introns. The alignment of amino acid sequences of LOC_Os11g05552 and LOC_Os11g05556 showed that they are highly similar.qRT-PCR results showed that the transcription levels of LOC_Os11g05552 and LOC_Os11g05556 were obviously down-regulated in ygl9, which indicated that these two genes were affected directly or indirectly by the mutation of YGL9. Subcellular localization indicated that both LOC_Os11g05552 and LOC_Os11g05556 localizes in the chloroplast. The phylogenetic tree showed that monocotyledons contain two groups of similar proteins, LOC_Os11g05552-like and LOC_Os11g05556-like proteins, and they are both clustered in the same branch with the cpSRP54 proteins of dicotyledons and green algae. The alignment of amino acid sequences showed that LOC_Os11g05552 and LOC_Os11g05556 are highly similar to the Arabidopsis cpSRP54. And the C-terminus of LOC_Os11g05552 contains the consistent key conserved sequences, APPGTARRKR, with the C-terminus of the Arabidopsis cpSRP54. The ARR sequences in APPGTARRKR play a crucial role in binding to the Arabidopsis cpSRP43. However, LOC_Os11g05556 doesn't contain these key conserved sequences. These results indicated that the LOC_Os11g05552-like protein could be the cpSRP54 protein of monocotyledons. Additionally, the alignment of amino acid sequences showed that LOC_Os11g05552 possibly also contains a NG-domain and a M-domain.(6) Yeast two-hybrid analysis between YGL9 and LOC_Os11g05552The results of the yeast two-hybrid assays indicated that YGL9 interacted with L18 of LHCP, and further assays indicated that only the Ank1-4 domain of YGL9 interacted with L18 of LHCP and other domains of YGL9 failed to interact with L18 of LHCP. The results of the yeast two-hybrid assays also indicated that YGL9 failed to interact with the full-length of mature LOC_Os11g05552. The possible reason is that the NG-domain of LOC_Os11g05552 contains a probable transmembrane region. Thus, in order to eliminate the influence of a probable transmembrane region, the M-domain of LOC_Os11g05552 alone was used to hybrid with YGL9. The results indicated that YGL9 interacted with the M-domain of LOC_Os11g05552 and further assays indicated that only the CD2 domain of YGL9 interacted with the M-domain of LOC_Os11g05552, and other domains of YGL9 failed to interact with the M-domain of LOC_Os11g05552. These results further indicated that YGL9 is the rice cpSRP43 and LOC_Os11g05552 is the rice cpSRP54. 2. The rice yellow-green leaf gene YGL8(1) Gene cloning of YGL8The rice ygl8 mutant exhibited a yellow-green leaf phenotype throughout its whole growth period, but its leaves became green slightly in an age dependent manner. DNA and cDNA sequencing results showed that a single base at position 671 of LOC_Os01g73450 was mutated(C to T) in ygl8, which caused a change of the 224 th amino acid from Ala to Val. LOC_Os01g73450 encodes a probable UMP kinase. Thus, we speculated that LOC_Os01g73450 is the candidate gene for YGL8.(2) Functional complementation assay of YGL8To confirm whether the mutation of LOC_Os01g73450 resulted in the yellow-green leaf phenotype of the ygl8 mutant, we cloned a 6478 bp wild-type genomic DNA fragment containing a 1570 bp upstream region, the entire 3844 bp coding region and a 1054 bp downstream region to construct the complementation vector of YGL8 and introduced it into the calli of the ygl8 mutant. The positive transgenic line recovered to a green phenotype and its total chlorophyll content approached that in wild-type plants. These results demonstrated that LOC_Os01g73450 is the YGL8 gene.In addition, we silenced the LOC_Os01g73450 gene in normal Zhonghua11 plants through the RNAi approach. The results showed that the positive transgenic lines all exhibited a yellow-green leaf phenotype and the transcription levels of LOC_Os01g73450 in these positive transgenic lines were obviously down-regulated compared with that in Zhonghua11. These results further demonstrated that LOC_Os01g73450 is the YGL8 gene.(3) Structure analysis of YGL8The results of subcellular localization indicated that YGL8 localizes in the chloroplast. The phylogenetic tree showed that the UMP kinases homologous to YGL8 in higher plants are clustered in the same branch with the UMP kinases in prokaryotic cyanobacteria. The alignment of amino acid sequences showed that the UMP kinases homologous to YGL8 in higher plants share significant conserved domains of UMP kinase with the UMP kinases in prokaryotic bacteria and cyanobacteria. These results indicated that the chloroplast UMP kinases in higher plants and the UMP kinases in prokaryotic bacteria and cyanobacteria possibly contain similar structures and their functions are also analogical.(4) Organ-specific expression analysis of YGL8 and expression analysis of related genesqRT-PCR was used to analyze the organ-specific expression of YGL8 and the results showed that YGL8 is highly expressed in leaves, but lower expression levels in roots, culms, sheaths and young panicles. qRT-PCR results also showed that the transcription levels of some chlorophyll synthesis-related genes were obviously down-regulated in ygl8 compared with that in wild-type at seedling stage. Additionally, the transcription levels of psaA, psaB in PSI and psbC in PSII were obviously down-regulated in ygl9, but ndhB encoding a subunit of NADH dehydrogenase was obviously up-regulated in ygl9. The abnormal expression of these genes might affect the chloroplast development in ygl9. |
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