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Molecular cloning and functional analysis of 4-Coumarate:CoA ligase 4(4CL-like 1)from Fraxinus mandshurica and its role in abiotic stress tolerance and cell wall synthesis.

ABSTRACT: BACKGROUND:Four-Coumarate:CoA ligase gene (4CL) plays multiple important roles in plant growth and development by catalyzing the formation of CoA ester. 4CL belongs to the plant phenylpropane derivative, which is related to the synthesis of flavonoids and lignin and is a key enzyme in the biosynthetic pathway. RESULTS:In this study, 12 4CL genes of Fraxinus mandschurica were identified and named Fm4CL1-Fm4CL12, respectively. The analysis of the expression pattern of Fm4CL genes indicate that Fm4CL-like 1 gene may play a role in the lignin synthesis pathway. Our study indicate that overexpression of Fm4CL-like 1 increases the lignin content of transgenic tobacco by 39.5% compared to WT, and the S/G ratio of transgenic tobacco increased by 19.7% compared with WT. The xylem cell layer of transgenic line is increased by 40% compared to WT, the xylem cell wall thickness increased by 21.6% compared to the WT. Under mannitol-simulated drought stress, the root length of transgenic tobacco is 64% longer than WT, and the seed germination rate of the transgenic lines is 47% higher than that of WT. In addition, the H2O2 content in the transgenic tobacco was 22% lower than that of WT, while the POD and SOD content was higher than WT by 30 and 24% respectively, which showed Fm4CL-like 1 affect the accumulation of reactive oxygen species (ROS). The MDA content and relative conductivity was 25 and 15% lower than WT, respectively. The water loss rate is 16.7% lower than that of WT. The relative expression levels of stress-related genes NtHAK, NtAPX, NtCAT, NtABF2, and NtZFP were higher than those of WT under stress treatment. The stomatal apertures of OE (Overexpression) were 30% smaller than those of WT, and the photosynthetic rate of OE was 48% higher than that of WT. These results showed that the overexpression line exhibited stronger adaptability to osmotic stress than WT. CONCLUSIONS:Our results indicate that Fm4CL-like 1 is involved in secondary cell wall development and lignin synthesis. Fm4CL-like 1 play an important role in osmotic stress by affecting cell wall and stomatal development.

SUBMITTER: Chen X

PROVIDER: S-EPMC6545724 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature

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Molecular cloning and functional analysis of 4-Coumarate:CoA ligase 4(4CL-like 1)from Fraxinus mandshurica and its role in abiotic stress tolerance and cell wall synthesis.

Chen Xiaohui X Wang Hengtao H Li Xiaoyi X Ma Kai K Zhan Yaguang Y Zeng Fansuo F

BMC plant biology 20190603 1

<h4>Background</h4>Four-Coumarate:CoA ligase gene (4CL) plays multiple important roles in plant growth and development by catalyzing the formation of CoA ester. 4CL belongs to the plant phenylpropane derivative, which is related to the synthesis of flavonoids and lignin and is a key enzyme in the biosynthetic pathway.<h4>Results</h4>In this study, 12 4CL genes of Fraxinus mandschurica were identified and named Fm4CL1-Fm4CL12, respectively. The analysis of the expression pattern of Fm4CL genes in ...[more]

PMID: 31159735

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Project description:4-Coumarate:CoA ligase (4CL, EC6.2.1.12), located at the end of the phenylpropanoid metabolic pathway, regulates the metabolic direction of phenylpropanoid derivatives and plays a pivotal role in the biosynthesis of flavonoids, lignin, and other secondary metabolites. In order to understand the molecular characteristics and potential biological functions of the 4CL gene family in the pomegranate, a bioinformatics analysis was carried out on the identified 4CLs. In this study, 12 Pg4CLs were identified in the pomegranate genome, which contained two conserved amino acid domains: AMP-binding domain Box I (SSGTTGLPKGV) and Box II (GEICIRG). During the identification, it was found that Pg4CL2 was missing Box II. The gene cloning and sequencing verified that this partial amino acid deletion was caused by genome sequencing and splicing errors, and the gene cloning results corrected the Pg4CL2 sequence information in the 'Taishanhong' genome. According to the phylogenetic tree, Pg4CLs were divided into three subfamilies, and each subfamily had 1, 1, and 10 members, respectively. Analysis of cis-acting elements found that all the upstream sequences of Pg4CLs contained at least one phytohormone response element. An RNA-seq and protein interaction network analysis suggested that Pg4CL5 was highly expressed in different tissues and may participate in lignin synthesis of pomegranate. The expression of Pg4CL in developing pomegranate fruits was analyzed by quantitative real-time PCR (qRT-PCR), and the expression level of Pg4CL2 demonstrated a decreasing trend, similar to the trend of flavonoid content, indicating Pg4CL2 may involve in flavonoid synthesis and pigment accumulation. Pg4CL3, Pg4CL7, Pg4CL8, and Pg4CL10 were almost not expressed or lowly expressed, the expression level of Pg4CL4 was higher in the later stage of fruit development, suggesting that Pg4CL4 played a crucial role in fruit ripening. The expression levels of 4CL genes were significantly different in various fruit development stages. The results laid the foundation for an in-depth analysis of pomegranate 4CL gene functions.

Dataset Information

Molecular cloning and functional analysis of 4-Coumarate:CoA ligase 4(4CL-like 1)from Fraxinus mandshurica and its role in abiotic stress tolerance and cell wall synthesis.

Publications

Molecular cloning and functional analysis of 4-Coumarate:CoA ligase 4(4CL-like 1)from Fraxinus mandshurica and its role in abiotic stress tolerance and cell wall synthesis.

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