| Phenylpropanoid metabolism is a major metabolic pathway in plants. 4-Coumarate:coenzyme A ligase (4CL) is a key enzyme of the general phenylpropanoid metabolism that provides the precursors for numerous secondary metabolites, including flavonoids, lignin and wall-bound phenolics.4CL genes often appear as a small gene family, with two to five members. Enzymatic properties, expression characteristics and evolutionary relationships of 4CL and their genes have been extensively studied extensively in Arabidopsis, polar and soybean. In dicots,4CLs can be divided into two distinct groups. One group of 4CLs participate in lignin formation, while 4CLs in other group impact flavonoid metabolism.Monocotyledons contain different monolignol compositions and different wall-bound phenolic compounds compared to dicots. Rice is a model species for monocots. Enzymatic properties, expression characteristics and evolutionary relationships of rice 4CLs were characterized in this study. We also analysed nucleotide divergence in the 4CL genes from 16 species and used likelihood methods with various evolutionary models to investigate potential patterns of positive selection.The main results are as follows:1. Enzymatic properties of rice 4CLThe recombinant Os4CLl, Os4CL2, Os4CL3, Os4CL4, and Os4CL5 exhibited highest relative Vmax/Km values in single-substrate assays with coumarate and ferulic acid. Caffeic acid was a relatively poor substrate for recombinant rice 4CLs. It should be noted that SA was not accepted as a substrate under experimental conditions. The rank in order of turnover rate for different 4CL was Os4CL5>Os4CL4>Os4CLl>Os4CL3>Os4CL2. In mixed-substrate assays, the PA:FA:CA product ratio for Os4CL1, Os4CL2, Os4CL3, Os4CL4, and Os4CL5 was 1.00:0.72:0.06,1.00:0.00:0.09,1.00:0.43:0.07,1.00:0.45:0.07, and 1.00:0.48:0.02, respectively. The data indicated that PA-CoA was the predominant product of rice 4CLs. PA and FA strongly inhibited rice 4CL utilization of CA.2. Expression characteristics of rice 4CL genesThe 4CLs were expressed at a range of levels in a variety of tissues. Os4CL3 was expressed at relatively high levels in most tissues, especially in the hulls where relatively large amounts of lignin accumulated. The Os4CL1 transcript was the least abundant in all organs. A different expression pattern was observed for Os4CL2. The highest relative transcript levels of Os4CL2 were detected in anther, which were 10 and 30-fold above hull and stem, respectively, and 20-fold above other 4CLs in anther. All rice 4CL genes were strongly expressed at the onset of lignin deposition in the plumules and radicles. Os4CL3, Os4CL4, and Os4CL5 transcript accumulation was significantly upregulated by wounding, with levels of 6,3, and 7-fold above the untreated control within 4,1, and 6 hours after wounding, respectively. Os4CL2 expression was induced by up to 16-fold at 12 h after the onset of irradiation, and remained high at 24 h after treatment.3. Function of rice 4CLsBased on the enzymatic properties, expression characteristics, and evolutionary relationships, Os4CL2 was identified as the key 4CL isoenzyme involved in flavonoid biosynthesis, whereas Os4CL1, Os4CL3, Os4CL4, and Os4CL5 were involved in lignin formation. 4CL conversion through PA intermediate was found to be a main pathway by which lignin was synthesized in rice. These findings are consistent with the hypothesis that PA is a main substrate of 4CL for lignin biosynthesis in dicots. 4CL conversion through FA intermediate was also a pathway for lignin formation.4. Phylogenetic analysis of 4CLs in angiospermsBased on the phylogenetic analysis,4CLs were categorized into two major groups. One group contains the 4CLs of the dicots involved in this study, while the other group contains the 4CLs of the monocots, suggesting that function divergence occurred after monocot-dicot split.5. Evolution of the 4CL genes in angiosperms4CLs may share a common ancestor as a result of a duplication event. The evolution of 4CLs involved in flavonoid biosynthesis is constrained by purifying selection and maintains the ancestral role of the protein in response to biotic and abiotic factors. Lignin-biosynthetic 4CLs are under positive selection, suggesting that positive selection drives adaptive diversification of the 4CL genes in angiosperms. Four and six candidate sites for positive selection were identified using the BEB method in dicots and monocots, respectively. The majority of the positively selected sites are located in the substrate-binding pocket and the catalytic centre, indicating that nonsynonymous substitutions might contribute to the functional evolution of 4CLs for lignin biosynthesis.Celliilosic ethanol provide a potenial route to avoiding the global energy crisis.Cellulosic biomass is both abundant and renewable. However, the celluloses and pretreatment processes involved are very expensive. Genetically engineering plants to produce cellulases, and to reduce the need for pretreatment processes through lignin modification are promising paths th solving this problem,together with other strategies, such as increasing plant polysaccharide content.The main contents in the paper are as follows:① We expressed the Trichodenna reesei endoglucanase Ⅱ in rice to investigate the feasibility of producing this enzyme in plants.② Downregulation of lignin biosynthesis enzyme Os4CL4 was perforaied using RNA interference for modifying lignin composition and content.③ We expressed the Arabidopsis cellulose synthase AtCESA8 in rice for modifying cell wall composition. The main Results are as follows:1.Codon optimization of EGⅡ Eight codons of EGⅡ from Trichoderma reesei were used rarely in rice(less than15%). These codons were optimized according to the frequency of codon usage in rice by PCR.2.Construction of expression vector containing EGⅡ① Promoter: rice Rbcs promoter and XVE inducible system② 5’UTR: tobacco mosaic virus Ω translational enhancer③ Subcellular targeting: targeting the protein to the chloroplast or apoplast④ The full cDNA of EGⅡ or its catalytic domain was expressed in rice.3.Construction of RNAi vector The RNAi vector pTCK303-RNAi-Os4CL4 was constructed for the gene Os4CL4 silencing, which is the key enzyme for lignin biosynthesis.4.Construction of expression vector containing AtCESA8Arabidopsis cellulose synthase AtCESA8, which is required for the biosynthesis of the secondary walls, are transcribed under the control ofpromoter in rice.5.Identify of transgenic ricesPlants from 19 vectos were positive by gene specific PGR.6. EGⅡ antigen preparation The variant region of EGⅡ was selected th prepare antigen in E.coli... |
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