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.
Project description:Laccases were proposed to catalyze the oxidative polymerization of monolignols. We identified 49 laccase gene models in Populus trichocarpa, of which 29 were predicted to be targets of ptr-miR397a. We overexpressed Ptr-MIR397a in transgenic P. trichocarpa. In each of all 9 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 approximately 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. Total twelve trees were used. Those include nine individual transgenic trees for overexpressing Ptr-miR397a, as nine biological replicates, and three wild-type trees.
Project description:Lignified stone cells substantially reduce fruit quality. Therefore, it is desirable to inhibit stone cell development using genetic technologies. However, the molecular mechanisms regulating lignification are poorly understood in fruit stone cells. In this study, we have shown that microRNA (miR) miR397a regulates fruit cell lignification by inhibiting laccase (LAC) genes that encode key lignin biosynthesis enzymes. Transient overexpression of PbrmiR397a, which is the miR397a of Chinese pear (Pyrus bretschneideri), and simultaneous silencing of three LAC genes reduced the lignin content and stone cell number in pear fruit. A single nucleotide polymorphism (SNP) identified in the promoter of the PbrmiR397a gene was found to associate with low levels of fruit lignin, after analysis of the genome sequences of sixty pear varieties. This SNP created a TCA element that responded to salicylic acid to induce gene expression as confirmed using a cell-based assay system. Furthermore, stable overexpression of PbrmiR397a in transgenic tobacco plants reduced the expression of target LAC genes and decreased the content of lignin but did not change the ratio of syringyl- and guaiacyl-lignin monomers. Consistent with reduction in lignin content, the transgenic plants showed fewer numbers of vessel elements and thinner secondary walls in the remaining elements compared to wild-type control plants. This study has advanced our understanding of the regulation of lignin biosynthesis and provided useful molecular genetic information for improving pear fruit quality.
Project description:Laccases are widely distributed in plant kingdom catalyzing the polymerization of lignin monolignols. Rosmarinic acid (RA) has a lignin monolignol-like structure and is converted into salvianolic acid B (SAB), which is a representatively effective hydrophilic compound of a well-known medicinal plant Salvia miltiorrhiza and also the final compound of phenolic acids metabolism pathway in the plant. But the roles of laccases in the biosynthesis of SAB are poorly understood. This work systematically characterizes S. miltiorrhiza laccase (SmLAC) gene family and identifies the SAB-specific candidates. Totally, 29 laccase candidates (SmLAC1-SmLAC29) are found to contain three signature Cu-oxidase domains. They present relatively low sequence identity and diverse intron-exon patterns. The phylogenetic clustering of laccases from S. miltiorrhiza and other ten plants indicates that the 29 SmLACs can be divided into seven groups, revealing potential distinct functions. Existence of diverse cis regulatory elements in the SmLACs promoters suggests putative interactions with transcription factors. Seven SmLACs are found to be potential targets of miR397. Putative glycosylation sites and phosphorylation sites are identified in SmLAC amino acid sequences. Moreover, the expression profile of SmLACs in different organs and tissues deciphers that 5 SmLACs (SmLAC7/8/20/27/28) are expressed preferentially in roots, adding the evidence that they may be involved in the phenylpropanoid metabolic pathway. Besides, silencing of SmLAC7, SmLAC20 and SmLAC28, and overexpression of SmLAC7 and SmLAC20 in the hairy roots of S. miltiorrhiza result in diversification of SAB, signifying that SmLAC7 and SmLAC20 take roles in SAB biosynthesis. The results of this study lay a foundation for further elucidation of laccase functions in S. miltiorrhiza, and add to the knowledge for SAB biosynthesis in S. miltiorrhiza.
Project description:Laccases (EC 126.96.36.199) are enzymes known for their ability to catalyse the oxidation of phenolic compounds using molecular oxygen as the final electron acceptor. Lignin is a natural phenylpropanoids biopolymer whose degradation in nature is thought to be aided by enzymatic oxidation by laccases. Laccase activity is often measured spectrophotometrically on compounds such as syringaldazine and ABTS which poorly relate to lignin. We employed natural phenolic hydroxycinnamates having different degree of methoxylations, p-coumaric, ferulic and sinapic acid, and a lignin model OH-dilignol compound as substrates to assess enzyme kinetics by HPLC-MS on two fungal laccases Trametes versicolor laccase, Tv and Ganoderma lucidum laccase, Gl. The method allowed accurate kinetic measurements and detailed insight into the product profiles of both laccases. Both Tv and Gl laccase are active on the hydroxycinnammates and show a preference for substrate with methoxylations. Product profiles were dominated by the presence of dimeric and trimeric species already after 10 minutes of reaction and similar profiles were obtained with the two laccases. This new HPLC-MS method is highly suitable and accurate as a new method for assaying laccase activity on genuine phenolic substrates, as well as a tool for examining laccase oxidation product profiles.
Project description:Plant laccases are thought to function in the oxidation of monolignols which leads to higher order lignin formation. Only a hand-full of laccases in plants have been functionally evaluated, and as such little is known about the breadth of their impact on cell wall chemistry or structure. Here, we describe a previously uncharacterized laccase from Populus, encoded by locus Potri.008G064000, whose reduced expression resulted in transgenic Populus trees with changes in syringyl/guaiacyl ratios as well as altered sugar release phenotypes. These phenotypes are consistent with plant biomass exhibiting reduced recalcitrance. Interestingly, the transgene effect on recalcitrance is dependent on a mild pretreatment prior to chemical extraction of sugars. Metabolite profiling suggests the transgene modulates phenolics that are associated with the cell wall structure. We propose that this particular laccase has a range of functions related to oxidation of phenolics and conjugation of flavonoids that interact with lignin in the cell wall.
Project description:The single-celled cotton fibers, produced from seed coat epidermal cells are the largest natural source of textile fibers. The economic value of cotton fiber lies in its length and quality. The multifunctional laccase enzymes play important roles in cell elongation, lignification and pigmentation in plants and could play crucial role in cotton fiber quality. Genome-wide analysis of cultivated allotetraploid (G. hirsutum) and its progenitor diploid (G. arboreum and G. raimondii) cotton species identified 84, 44 and 46 laccase genes, respectively. Analysis of chromosomal location, phylogeny, conserved domain and physical properties showed highly conserved nature of laccases across three cotton species. Gene expression, enzymatic activity and biochemical analysis of developing cotton fibers was performed using G. arboreum species. Of the total 44, 40 laccases showed expression during different stages of fiber development. The higher enzymatic activity of laccases correlated with higher lignin content at 25 DPA (Days Post Anthesis). Further, analysis of cotton fiber phenolic compounds showed an overall decrease at 25 DPA indicating possible incorporation of these substrates into lignin polymer during secondary cell wall biosynthesis. Overall data indicate significant roles of laccases in cotton fiber development, and presents an excellent opportunity for manipulation of fiber development and quality.
Project description:The edible white rot fungus Lentinula edodes possesses a variety of lignin degrading enzymes such as manganese peroxidases and laccases. Laccases belong to the multicopper oxidases, which have a wide range of catalytic activities including polyphenol degradation and synthesis, lignin degradation, and melanin formation. The exact number of laccases in L. edodes is unknown, as are their complete properties and biological functions. We analyzed the draft genome sequence of L. edodes D703PP-9 and identified 13 multicopper oxidase-encoding genes; 11 laccases in sensu stricto, of which three are new, and two ferroxidases. lcc8, a laccase previously reported in L. edodes, was not identified in D703PP-9 genome. Phylogenetic analysis showed that the 13 multicopper oxidases can be classified into laccase sensu stricto subfamily 1, laccase sensu stricto subfamily 2 and ferroxidases. From sequence similarities and expression patterns, laccase sensu stricto subfamily 1 can be divided into two subgroups. Laccase sensu stricto subfamily 1 group A members are mainly secreted from mycelia, while laccase sensu stricto subfamily 1 group B members are expressed mainly in fruiting bodies during growth or after harvesting but are lowly expressed in mycelia. Laccase sensu stricto subfamily 2 members are mainly expressed in mycelia, and two ferroxidases are mainly expressed in the fruiting body during growth or after harvesting, and are expressed at very low levels in mycelium. Our data suggests that L. edodes laccases in same group share expression patterns and would have common biological functions.
Project description:Lignin has enabled plants to colonize land, grow tall, transport water within their bodies, and protect themselves against various stresses. Consequently, this polyphenolic polymer, impregnating cellulosic plant cell walls, is the second most abundant polymer on Earth. Yet, despite its great physiological, ecological, and economical importance, our knowledge of lignin biosynthesis in vivo, especially the polymerization steps within the cell wall, remains vague-specifically, the respective roles of the two polymerizing enzymes classes, laccases and peroxidases. One reason for this lies in the very high numbers of laccases and peroxidases encoded by 17 and 73 homologous genes, respectively, in <i>Arabidopsis</i> Here, we have focused on a specific lignin structure, the ring-like Casparian strips (CSs) within the root endodermis. By reducing candidate numbers using cellular resolution expression and localization data and by boosting stacking of mutants using CRISPR-Cas9, we mutated the majority of laccases in <i>Arabidopsis</i> in a nonuple mutant-essentially abolishing laccases with detectable endodermal expression. Yet, we were unable to detect even slight defects in CS formation. By contrast, we were able to induce a complete absence of CS formation in a quintuple peroxidase mutant. Our findings are in stark contrast to the strong requirement of xylem vessels for laccase action and indicate that lignin in different cell types can be polymerized in very distinct ways. We speculate that cells lignify differently depending on whether lignin is localized or ubiquitous and whether cells stay alive during and after lignification, as well as the composition of the cell wall.
Project description:BACKGROUND:Understanding lignin biosynthesis and composition is of central importance for sustainable bioenergy and biomaterials production. Species of the genus Miscanthus have emerged as promising bioenergy crop due to their rapid growth and modest nutrient requirements. However, lignin polymerization in Miscanthus is poorly understood. It was previously shown that plant laccases are phenol oxidases that have multiple functions in plant, one of which is the polymerization of monolignols. Herein, we link a newly discovered Miscanthus laccase, MsLAC1, to cell wall lignification. Characterization of recombinant MsLAC1 and Arabidopsis transgenic plants expressing MsLAC1 were carried out to understand the function of MsLAC1 both in vitro and in vivo. RESULTS:Using a comprehensive suite of molecular, biochemical and histochemical analyses, we show that MsLAC1 localizes to cell walls and identify Miscanthus transcription factors capable of regulating MsLAC1 expression. In addition, MsLAC1 complements the Arabidopsis lac4-2 lac17 mutant and recombinant MsLAC1 is able to oxidize monolignol in vitro. Transgenic Arabidopsis plants over-expressing MsLAC1 show higher G-lignin content, although recombinant MsLAC1 seemed to prefer sinapyl alcohol as substrate. CONCLUSIONS:In summary, our results suggest that MsLAC1 is regulated by secondary cell wall MYB transcription factors and is involved in lignification of xylem fibers. This report identifies MsLAC1 as a promising breeding target in Miscanthus for biofuel and biomaterial applications.