Cloning And Functional Analysis Of T-DNA Insertional Rice Yellow Leaf Coloration Mutant Gene

The chloroplast is the cellular compartment in the mesophyll cells of plants. Although the chloroplast has its own germ plasm and translation system, its normal development is the results of the intercoordination of the nuclear genes and the chloroplast genes. The mutations of these genes would lead to unnormal chloroplast, the reduction of chlorophyll synthesis, leaf colortion mutants, and so on. Leaf coloration mutants are the excellent materials for studying the structures, functions and mechanisms of chloroplasts and the photosystem. In this study, the genetics model, photosynthetic characterization, mutagenzed gene and its functions of mutant vyl (virescent yellow leaf, vyl) were studied so as to understand its mechanism at the levels of the plant physiology and the molecule biology , which should be useful for its applications in studies on rice photosynthesis physiology, functional genomics and agricultural productions. In addition, Photosystem 2 photochemistry and pigment composition of mutant yl (yellow leaf, yl) under different light intensities were also studied so as to understand the effects of light intensities on its photosynthetic characterization , which should contribute to its in-depth study and applications in agricultural productions. The important results were as follows.1. The results of detection and genetic analysis of vyl mutant showed that vyl mutant was a novel rice yellow leaf coloration mutant, which was governed by a single recessive nuclear gene and contained a single T-DNA copy, unemerged leaves of vyl mutant showed pale yellow phenotype .After the leaves emerged , its leaves began to be virescent from the top of the leaf, but its entirely green leaves were much more yellow-green than those of wild-type plants. Furthermore, this phenotype of vyl mutant had no changes during the whole leaf development. Though vyl mutant was much shorter than wild-type, its photosynthetic growth and seed setting were normal.2. The results of photosynthetic characterization indicated that vyl mutant was a chlorophyll b less mutant. Compared with the wild-type, its contents of chlorophyll a, chlorophyll b,β-Carotenoid and total chlorophyll in the second leaves were significantly decreased, but its ratio of chlorophyll a and chlorophyll b was higher than that of wild-type during the whole leaf development. Its chloroplasts developed much more tardily than that of wild-type with the leaf development. The chloroplasts from 2-day wild-type second leaves (The first day of the emergence of the second heart leaf was assumed to be 0 day.) had a fully developed thylakiod membrane system composed of grana connected by stromal lamellae. In contrast, the chloroplasts from 2-day vyl mutant second leaves had few or no membrane stacks and only long, appressed areas among the long, parallel, unstacked membranes. Furthermore, even the chloroplast from 12-day vyl mutant second leaves only had intermediated amount of membrane stacks that were less than those of 2-day wild-type leaves. Although its maximal efficiency of PSⅡphotochemistry (F_v/F_m) was little different from wild-type, its actual photochemical efficiency of PSⅡ (ΦPSⅡ), the electron transport rate (ETR), photochemical quenching (qP) and Non-photochemical quenching (qN) were greatly decreased and its PSⅡphotochemistry was greatly inhibited at both donor and accept side in contrast to wild-type. Furthermore, Compared with wild-type, its PSⅡreaction center proteins CP43, CP47, D1, D2 and the LHCⅡproteins were greatly reduced and its pigment-protein complexes of PSⅡcombined with chlorophyll were less than those of wild-type. In contrast, its more chlorophylls were combined with pigment-protein complexes of PSⅠ, which suggested that there was a great reduction of thylakoid membrane formation in its leaves.3. The T-DNA insertional right-side sequence of vyl mutant was amplified by PCR-Walking method and its candidate gene VYL was identified based on the blast analysis and the phenotype of vyl mutant co-segregated with T-DNA insertion. VYL gene was locus on rice chromosome 9 and T-DNA was inserted at 64bp site behind the code ATG of its first exon. Furthermore, the full-length cDNA of VYL gene was amplified by RT-PCR method with a pair of primer from its ORF and EST sequences. The identity of VYL gene was subsequently confirmed by complementing the vyl mutant with a wild-type. VYL gene was a novel rice leaf coloration mutant gene and had a AIR1 (Arginine methyltransferase -interacting protein, contains RING Zn-finger),which mainly existed in the genes which encod Zn-finger protein, retrotransposon, retroelement-like protein and a hypothetical protein in rice and was important for posttranslational modification, regulating the expressions of nuclear genes and signal transduction. In addition, rice genes with AIR1 from different chromosomes are high homologous, which suggested that AIRlwas more conservative. VYL was lowly expressed in roots, highly expressed in leaves and moderately expressed in stems. Furthrmore, except a little lowly expressed in the 2d-unemerged second leaves(The first day of the emergence of the second heart leaf was assumed to be 0 day.), VYL gene was highly expressed in the 4d, 6d, 8d, 12d second leaves, respectively. The results of northern blot analysis illuminated that the expression of VYL gene was compositive. In order to understand its subcellular localization, VYL gene was fused in the frame to the N-terminal of GFP and the transient expression of GFP fusions was achieved by transforming the constructs into onion epidermis cell using PDS 1000/He Gene System(Bio-Rad). The result of the subcellular localization indicated that VYL-GFP was localized in the cytoplasm. This is in accordance with AIR1 functions of posttranslational modification.4. yl mutant was a Chl b deficient mutant and showed yellow color phenotype at the whole developmental stages. The thylakoid membranes in the chloroplasts from yl mutant leaves were much less than those of wild-type, especially in the grana lamella contents. At the weak light intensity (50μmol photons m^-2s^-1), its chlorophyll (Chl) contents, the actual PS2 efficiency (ΦPSⅡ), photochemical quenching (qP), and the efficiency of excitation capture by open PS2 centers (Fv'/Fm') were much lower than those of wild-type and its Chl a/b ratio were 3 folds as that of wild-type except no difference in the maximal efficiency of PS2 photochemistry (Fv/Fm). With progressing of light intensities (100 and 200μmol photons m^-2s^-1), there was a change in the photosynthetic pigment stoichiometry. The increase of its total chlorophyll contents and the decrease in Ch1 a/b ratio were observed. ItsΦPSⅡ, qP, and Fv'/Fm' of yl mutant were increased gradually along with the increase of light intensities but still much less than those of wild-type. Absorption peaks of yl mutant and wild-type chlorophyll were 432nm at Soret band and 663nm at Q band, but the absorbance of pigments per unit of yl mutant was higher than that of wild-type. Along with the increase of light intensity, the absorbance of yl mutant chlorophylls was decreased. At 200μmol (photon) m^-2s^-1 light intensity, it was nearly identical with that of wild-type. The increase of (Z+A)/(V+A+Z) ratio associate with Non-photochemical quenching (qN) was found in yl mutant suggested that yl mutant dissipated excess energy through the turnover of xanthophylls cycle pigments. No significant differences in pigment composition were observed in wild-type under different light intensities, except Ch1 a/b ratio also gradually decreased. These results suggested that the yl mutant developed much more tardily than that of wild-type, which caused by low light utilization efficiency independent with light intensity. The xanthophylls cycle played a main role in the dissipation of excess light energy in yl mutant as function of photoprotection.