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

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.

Plastoquinone and Ubiquinone in Plants: Biosynthesis, Physiological Function and Metabolic Engineering.

Plastoquinone (PQ) and ubiquinone (UQ) are two important prenylquinones, functioning as electron transporters in the electron transport chain of oxygenic photosynthesis and the aerobic respiratory chain, respectively, and play indispensable roles in plant growth and development through participating in the biosynthesis and metabolism of important chemical compounds, acting as antioxidants, being involved in plant response to stress, and regulating gene expression and cell signal transduction. UQ, particularly UQ10, has also been widely used in people's life. It is effective in treating cardiovascular diseases, chronic gingivitis and periodontitis, and shows favorable impact on cancer treatment and human reproductive health. PQ and UQ are made up of an active benzoquinone ring attached to a polyisoprenoid side chain. Biosynthesis of PQ and UQ is very complicated with more than thirty five enzymes involved. Their synthetic pathways can be generally divided into two stages. The first stage leads to the biosynthesis of precursors of benzene quinone ring and prenyl side chain. The benzene quinone ring for UQ is synthesized from tyrosine or phenylalanine, whereas the ring for PQ is derived from tyrosine. The prenyl side chains of PQ and UQ are derived from glyceraldehyde 3-phosphate and pyruvate through the 2-C-methyl-D-erythritol 4-phosphate pathway and/or acetyl-CoA and acetoacetyl-CoA through the mevalonate pathway. The second stage includes the condensation of ring and side chain and subsequent modification. Homogentisate solanesyltransferase, 4-hydroxybenzoate polyprenyl diphosphate transferase and a series of benzene quinone ring modification enzymes are involved in this stage. PQ exists in plants, while UQ widely presents in plants, animals and microbes. Many enzymes and their encoding genes involved in PQ and UQ biosynthesis have been intensively studied recently. Metabolic engineering of UQ10 in plants, such as rice and tobacco, has also been tested. In this review, we summarize and discuss recent research progresses in the biosynthetic pathways of PQ and UQ and enzymes and their encoding genes involved in side chain elongation and in the second stage of PQ and UQ biosynthesis. Physiological functions of PQ and UQ played in plants as well as the practical application and metabolic engineering of PQ and UQ are also included.

Genetic modification of wood quality for second-generation biofuel production.

How the abundant tree biomass resources can be efficiently used for future biofuel production has attracted a great deal of interest and discussion in the past few years. Capable technologies are expected to be developed to realize the production of biofuel from wood biomass. A significant effort is put into the field of modifying wood properties of trees and simplifying the process of biomass-to-ethanol conversion, which includes mainly genetic engineering of lignin, cellulose and hemicellulose of woods. Current research in this field has achieved some promising results and opened up new opportunities to utilize wood biomass efficiently. This review will discuss the main developments in genetic modification of lignin, cellulose and hemicellulose biosynthesis in trees as well as other potential genetic technology of biofuel production from wood biomass.

Distinct roles of cinnamate 4-hydroxylase genes in Populus.

Cinnamate 4-hydroxylase (C4H) catalyzes the conversion of cinnamate into 4-hydroxy-cinnamate, a key reaction of the phenylpropanoid pathway which leads to the biosynthesis of several secondary metabolites. C4H genes exist as a multigene family in various plant species. In order to understand the roles of individual C4H members, four C4H cDNAs (PtreC4H) were isolated from Populus tremuloides and three C4H loci (PtriC4H) were identified in the P. trichocarpa genome. The ability of Populus C4H isoforms to convert trans-cinnamate into p-coumaric acid was verified by the examination of yeast recombinant PtreC4H proteins. Populus C4H genes were expressed in various tissues, including developing xylem, phloem and epidermis; however, the expression patterns of individual members were different from each other. Sequential analysis of C4H promoters showed that the differential expression of C4H genes was associated with cis-acting regulatory elements such as box L, box P and H box, suggesting that the divergent C4H isoforms played distinct roles in the production of secondary metabolites. The involvement of specific C4H isoforms in the biosynthesis of guaiacyl and syringyl monolignols is discussed.

Clarification of cinnamoyl co-enzyme A reductase catalysis in monolignol biosynthesis of Aspen.

Cinnamoyl co-enzyme A reductase (CCR), one of the key enzymes involved in the biosynthesis of monolignols, has been thought to catalyze the conversion of several cinnamoyl-CoA esters to their respective cinnamaldehydes. However, it is unclear which cinnamoyl-CoA ester is metabolized for monolignol biosynthesis. A xylem-specific CCR cDNA was cloned from aspen (Populus tremuloides) developing xylem tissue. The recombinant CCR protein was produced through an Escherichia coli expression system and purified to electrophoretic homogeneity. The biochemical properties of CCR were characterized through direct structural corroboration and quantitative analysis of the reaction products using a liquid chromatography-mass spectrometry system. The enzyme kinetics demonstrated that CCR selectively catalyzed the reduction of feruloyl-CoA from a mixture of five cinnamoyl CoA esters. Furthermore, feruloyl-CoA showed a strong competitive inhibition of the CCR catalysis of other cinnamoyl CoA esters. Importantly, when CCR was coupled with caffeoyl-CoA O-methyltransferase (CCoAOMT) to catalyze the substrate caffeoyl-CoA ester, coniferaldehyde was formed, suggesting that CCoAOMT and CCR are neighboring enzymes. However, the in vitro results also revealed that the reactions mediated by these two neighboring enzymes require different pH environments, indicating that compartmentalization is probably needed for CCR and CCoAOMT to function properly in vivo. Eight CCR homologous genes were identified in the P. trichocarpa genome and their expression profiling suggests that they may function differentially.