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Ptr-miR397a is a negative regulator of laccase genes affecting lignin content in Populus trichocarpa.

ABSTRACT: Laccases, as early as 1959, were proposed to catalyze the oxidative polymerization of monolignols. Genetic evidence in support of this hypothesis has been elusive due to functional redundancy of laccase genes. An Arabidopsis double mutant demonstrated the involvement of laccases in lignin biosynthesis. We previously identified a subset of laccase genes to be targets of a microRNA (miRNA) ptr-miR397a in Populus trichocarpa. To elucidate the roles of ptr-miR397a and its targets, we characterized the laccase gene family and identified 49 laccase gene models, of which 29 were predicted to be targets of ptr-miR397a. We overexpressed Ptr-MIR397a in transgenic P. trichocarpa. In each of all nine transgenic lines tested, 17 PtrLACs were down-regulated as analyzed by RNA-seq. Transgenic lines with severe reduction in the expression of these laccase genes resulted in an ?40% decrease in the total laccase activity. Overexpression of Ptr-MIR397a in these transgenic lines also reduced lignin content, whereas levels of all monolignol biosynthetic gene transcripts remained unchanged. A hierarchical genetic regulatory network (GRN) built by a bottom-up graphic Gaussian model algorithm provides additional support for a role of ptr-miR397a as a negative regulator of laccases for lignin biosynthesis. Full transcriptome-based differential gene expression in the overexpressed transgenics and protein domain analyses implicate previously unidentified transcription factors and their targets in an extended hierarchical GRN including ptr-miR397a and laccases that coregulate lignin biosynthesis in wood formation. Ptr-miR397a, laccases, and other regulatory components of this network may provide additional strategies for genetic manipulation of lignin content.

SUBMITTER: Lu S

PROVIDER: S-EPMC3696765 | biostudies-literature | 2013 Jun

REPOSITORIES: biostudies-literature

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Ptr-miR397a is a negative regulator of laccase genes affecting lignin content in Populus trichocarpa.

Lu Shanfa S Li Quanzi Q Wei Hairong H Chang Mao-Ju MJ Tunlaya-Anukit Sermsawat S Kim Hoon H Liu Jie J Song Jingyuan J Sun Ying-Hsuan YH Yuan Lichai L Yeh Ting-Feng TF Peszlen Ilona I Ralph John J Sederoff Ronald R RR Chiang Vincent L VL

Proceedings of the National Academy of Sciences of the United States of America 20130610 26

Laccases, as early as 1959, were proposed to catalyze the oxidative polymerization of monolignols. Genetic evidence in support of this hypothesis has been elusive due to functional redundancy of laccase genes. An Arabidopsis double mutant demonstrated the involvement of laccases in lignin biosynthesis. We previously identified a subset of laccase genes to be targets of a microRNA (miRNA) ptr-miR397a in Populus trichocarpa. To elucidate the roles of ptr-miR397a and its targets, we characterized t ...[more]

PMID: 23754401

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Project description:IntroductionBiofuel is a kind of sustainable, renewable and environment friendly energy. Lignocellulose from the stems of woody plants is the main raw material for "second generation biofuels". Lignin content limits fermentation yield and is therefore a major obstacle in biofuel production. Plant laccase plays an important role in the final step of lignin formation, which provides a new strategy for us to obtain ideal biofuels by regulating the expression of laccase genes to directly gain the desired lignin content or change the composition of lignin.MethodsMultiple sequence alignment and phylogenetic analysis were used to classify PtrLAC genes; sequence features of PtrLACs were revealed by gene structure and motif composition analysis; gene duplication, interspecific collinearity and Ka/Ks analysis were conducted to identify ancient PtrLACs; expression levels of PtrLAC genes were measured by RNA-Seq data and qRT-PCR; domain analysis combine with cis-acting elements prediction together showed the potential function of PtrLACs. Furthermore, Alphafold2 was used to simulate laccase 3D structures, proLAC23::LAC23-eGFP transgenic Populus stem transects were applied to fluorescence observation.ResultsA comprehensive analysis of the P. trichocarpa laccase gene (PtLAC) family was performed. Some ancient PtrLAC genes such as PtrLAC25, PtrLAC19 and PtrLAC41 were identified. Gene structure and distribution of conserved motifs clearly showed sequence characteristics of each PtrLAC. Combining published RNA-Seq data and qRT-PCR analysis, we revealed the expression pattern of PtrLAC gene family. Prediction results of cis-acting elements show that PtrLAC gene regulation was closely related to light. Through above analyses, we selected 5 laccases and used Alphafold2 to simulate protein 3D structures, results showed that PtrLAC23 may be closely related to the lignification. Fluorescence observation of proLAC23::LAC23-eGFP transgenic Populus stem transects and qRT-PCR results confirmed our hypothesis again.DiscussionIn this study, we fully analyzed the Populus trichocarpa laccase gene family and identified key laccase genes related to lignification. These findings not only provide new insights into the characteristics and functions of Populus laccase, but also give a new understanding of the broad prospects of plant laccase in lignocellulosic biofuel production.

Project description:Forest trees generally show high levels of local adaptation and efforts focusing on understanding adaptation to climate will be crucial for species survival and management. Here, we address fundamental questions regarding the molecular basis of adaptation in undomesticated forest tree populations to past climatic environments by employing an integrative quantitative genetics and landscape genomics approach. Using this comprehensive approach, we studied the molecular basis of climate adaptation in 433 Populus trichocarpa (black cottonwood) genotypes originating across western North America. Variation in 74 field-assessed traits (growth, ecophysiology, phenology, leaf stomata, wood, and disease resistance) was investigated for signatures of selection (comparing QST-FST) using clustering of individuals by climate of origin (temperature and precipitation). 29,354 SNPs were investigated employing three different outlier detection methods and marker-inferred relatedness was estimated to obtain the narrow-sense estimate of population differentiation in wild populations. In addition, we compared our results with previously assessed selection of candidate SNPs using the 25 topographical units (drainages) across the P. trichocarpa sampling range as population groupings. Narrow-sense QST for 53% of distinct field traits was significantly divergent from expectations of neutrality (indicating adaptive trait variation); 2,855 SNPs showed signals of diversifying selection and of these, 118 SNPs (within 81 genes) were associated with adaptive traits (based on significant QST). Many SNPs were putatively pleiotropic for functionally uncorrelated adaptive traits, such as autumn phenology, height, and disease resistance. Evolutionary quantitative genomics in P. trichocarpa provides an enhanced understanding regarding the molecular basis of climate-driven selection in forest trees and we highlight that important loci underlying adaptive trait variation also show relationship to climate of origin. We consider our approach the most comprehensive, as it uncovers the molecular mechanisms of adaptation using multiple methods and tests. We also provide a detailed outline of the required analyses for studying adaptation to the environment in a population genomics context to better understand the species' potential adaptive capacity to future climatic scenarios.

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Ptr-miR397a is a negative regulator of laccase genes affecting lignin content in Populus trichocarpa.

Publications

Ptr-miR397a is a negative regulator of laccase genes affecting lignin content in Populus trichocarpa.

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