MicroRNA6443-mediated regulation of FERULATE 5-HYDROXYLASE gene alters lignin composition and enhances saccharification in Populus tomentosa.

Ferulate 5-hydroxylase (F5H) is a limiting enzyme involved in biosynthesizing sinapyl (S) monolignol in angiosperms. Genetic regulation of F5H can influence S monolignol synthesis and therefore improve saccharification efficiency and biofuel production. To date, little is known about whether F5H is post-transcriptionally regulated by endogenous microRNAs (miRNAs) in woody plants. Here, we report that a microRNA, miR6443, specifically regulates S lignin biosynthesis during stem development in Populus tomentosa. In situ hybridization showed that miR6443 is preferentially expressed in vascular tissues. We further identified that F5H2 is the direct target of miR6443. Overexpression of miR6443 decreased the transcript level of F5H2 in transgenic plants, resulting in a significant reduction in S lignin content. Conversely, reduced miR6443 expression by short tandem target mimics (STTM) elevated F5H2 transcripts, therefore increasing S lignin composition. Introduction of a miR6443-resistant form of F5H2 into miR6443-overexpression plants restored lignin ectopic composition, supporting that miR6443 specifically regulated S lignin biosynthesis by repressing F5H2 in P. tomentosa. Furthermore, saccharification assays revealed decreased hexose yields by 7.5-24.5% in miR6443-overexpression plants compared with the wild-type control, and increased hexoses yields by 13.2-14.6% in STTM6443-overexpression plants. Collectively, we demonstrate that miR6443 modulates S lignin biosynthesis by specially regulating F5H2 in P. tomentosa.

The R2R3 MYB transcription factor MYB189 negatively regulates secondary cell wall biosynthesis in Populus.

Secondary cell wall (SCW) biosynthesis during wood formation in trees is controlled by a multilevel regulatory network that coordinates the expression of substantial genes. However, few transcription factors involved in the negative regulation of secondary wall biosynthesis have been characterized in tree species. In this study, we isolated an R2R3 MYB transcription factor MYB189 from Populus trichocarpa, which is expressed predominantly in secondary vascular tissues, especially in the xylem. A novel repression motif was identified in the C-terminal region of MYB189, which indicates this factor was a transcriptional repressor. Overexpression (OE) of MYB189 in Arabidopsis and poplar resulted in a significant reduction in the contents of lignin, cellulose and hemicelluloses. Vascular development in stems of MYB189 OE lines was markedly inhibited, leading to a dramatic decrease in SCW thickness of xylem fibers. Gene expression analyses showed that most of the structural genes involved in the biosynthesis of lignin, cellulose and xylans were significantly downregulated in MYB189-overexpressing poplars compared with the wild-type control. Chromatin immunoprecipitation-quantitative real-time polymerase chain reaction and transient expression assays revealed that MYB189 could directly bind to the promoters of secondary wall biosynthetic genes to repress their expression. Together, these data suggest that MYB189 acts as a repressor to regulate SCW biosynthesis in poplar.

R2R3-MYB transcription factor MYB6 promotes anthocyanin and proanthocyanidin biosynthesis but inhibits secondary cell wall formation in Populus tomentosa.

The secondary cell wall is an important carbon sink in higher plants and its biosynthesis requires coordination of metabolic fluxes in the phenylpropanoid pathway. In Arabidopsis (Arabidopsis thaliana), MYB75 and the KNOX transcription factor KNAT7 form functional complexes to regulate secondary cell wall formation in the inflorescence stem. However, the molecular mechanism by which these transcription factors control different branches of the phenylpropanoid pathway remains poorly understood in woody species. We isolated an R2R3-MYB transcription factor MYB6 from Populus tomentosa and determined that it was expressed predominately in young leaves. Overexpression of MYB6 in transgenic poplar upregulated flavonoid biosynthetic gene expression, resulting in significantly increased accumulation of anthocyanin and proanthocyanidins. MYB6-overexpression plants showed reduced secondary cell wall deposition, accompanied by repressed expression of secondary cell wall biosynthetic genes. We further showed that MYB6 interacted physically with KNAT7 and formed functional complexes that acted to repress secondary cell wall development in poplar and Arabidopsis. The results provide an insight into the transcriptional mechanisms involved in the regulation of the metabolic fluxes between the flavonoid and lignin biosynthetic pathways in poplar.

Auxin-mediated Aux/IAA-ARF-HB signaling cascade regulates secondary xylem development in Populus.

Wood development is strictly regulated by various phytohormones and auxin plays a central regulatory role in this process. However, how the auxin signaling is transducted in developing secondary xylem during wood formation in tree species remains unclear. Here, we identified an Aux/INDOLE-3-ACETIC ACID 9 (IAA9)-AUXIN RESPONSE FACTOR 5 (ARF5) module in Populus tomentosa as a key mediator of auxin signaling to control early developing xylem development. PtoIAA9, a canonical Aux/IAA gene, is predominantly expressed in vascular cambium and developing secondary xylem and induced by exogenous auxin. Overexpression of PtoIAA9m encoding a stabilized IAA9 protein significantly represses secondary xylem development in transgenic poplar. We further showed that PtoIAA9 interacts with PtoARF5 homologs via the C-terminal III/IV domains. The truncated PtoARF5.1 protein without the III/IV domains rescued defective phenotypes caused by PtoIAA9m. Expression analysis showed that the PtoIAA9-PtoARF5 module regulated the expression of genes associated with secondary vascular development in PtoIAA9m- and PtoARF5.1-overexpressing plants. Furthermore, PtoARF5.1 could bind to the promoters of two Class III homeodomain-leucine zipper (HD-ZIP III) genes, PtoHB7 and PtoHB8, to modulate secondary xylem formation. Taken together, our results suggest that the Aux/IAA9-ARF5 module is required for auxin signaling to regulate wood formation via orchestrating the expression of HD-ZIP III transcription factors in poplar.

Genome-wide identification and characterization of the Populus WRKY transcription factor family and analysis of their expression in response to biotic and abiotic stresses.

WRKY proteins are a large family of regulators involved in various developmental and physiological processes, especially in coping with diverse biotic and abiotic stresses. In this study, 100 putative PtrWRKY genes encoded the proteins contained in the complete WRKY domain in Populus. Phylogenetic analysis revealed that the members of this superfamily among poplar, Arabidopsis, and other species were divided into three groups with several subgroups based on the structures of the WRKY protein sequences. Various cis-acting elements related to stress and defence responses were found in the promoter regions of PtrWRKY genes by promoter analysis. High-throughput transcriptomic analyses identified that 61 of the PtrWRKY genes were induced by biotic and abiotic treatments, such as Marssonina brunnea, salicylic acid (SA), methyl jasmonate (MeJA), wounding, cold, and salinity. Among these PtrWRKY genes, transcripts of 46 selected genes were observed in different tissues, including roots, stems, and leaves. Quantitative RT-PCR analysis further confirmed the induced expression of 18 PtrWRKY genes by one or more stress treatments. The overexpression of an SA-inducible gene, PtrWRKY89, accelerated expression of PR protein genes and improved resistance to pathogens in transgenic poplar, suggesting that PtrWRKY89 is a regulator of an SA-dependent defence-signalling pathway in poplar. Taken together, our results provided significant information for improving the resistance and stress tolerance of woody plants.

Functional characterization of the poplar R2R3-MYB transcription factor PtoMYB216 involved in the regulation of lignin biosynthesis during wood formation.

Because of the importance of wood in many industrial applications, tremendous studies have been performed on wood formation, especially in lignin biosynthesis. MYB transcription factors (TFs), which consist of a large family of plant TFs, have been reported to directly regulate lignin biosynthetic genes in a number of plants. In this study, we describe the cloning and functional characterization of PtoMYB216, a cDNA isolated from Chinese white poplar (Populus tomentosa Carr.). PtoMYB216 encodes a protein belonging to the R2R3-MYB family and displays significant similarity with other MYB factors shown to regulate lignin synthesis in Arabidopsis. Gene expression profiling studies showed that PtoMYB216 mRNA is specifically expressed during secondary wall formation in wood. The 1.8-kb promoter sequence of PtoMYB216 was fused to the GUS coding sequence and introduced into wild-type A. thaliana. GUS expression was shown to be restricted to tissues undergoing secondary cell wall formation. Overexpression of PtoMYB216 specifically activated the expression of the upstream genes in the lignin biosynthetic pathway and resulted in ectopic deposition of lignin in cells that are normally unligninified. These results suggest that PtoMYB216 is specific transcriptional activators of lignin biosynthesis and involved in the regulation of wood formation in poplar.

Isolation and characterization of cDNAs encoding leucoanthocyanidin reductase and anthocyanidin reductase from Populus trichocarpa.

Proanthocyanidins (PAs) contribute to poplar defense mechanisms against biotic and abiotic stresses. Transcripts of PA biosynthetic genes accumulated rapidly in response to infection by the fungus Marssonina brunnea f.sp. multigermtubi, treatments of salicylic acid (SA) and wounding, resulting in PA accumulation in poplar leaves. Anthocyanidin reductase (ANR) and leucoanthocyanidin reductase (LAR) are two key enzymes of the PA biosynthesis that produce the main subunits: (+)-catechin and (-)-epicatechin required for formation of PA polymers. In Populus, ANR and LAR are encoded by at least two and three highly related genes, respectively. In this study, we isolated and functionally characterized genes PtrANR1 and PtrLAR1 from P. trichocarpa. Phylogenetic analysis shows that Populus ANR1 and LAR1 occurr in two distinct phylogenetic lineages, but both genes have little difference in their tissue distribution, preferentially expressed in roots. Overexpression of PtrANR1 in poplar resulted in a significant increase in PA levels but no impact on catechin levels. Antisense down-regulation of PtrANR1 showed reduced PA accumulation in transgenic lines, but increased levels of anthocyanin content. Ectopic expression of PtrLAR1 in poplar positively regulated the biosynthesis of PAs, whereas the accumulation of anthocyanin and flavonol was significantly reduced (P<0.05) in all transgenic plants compared to the control plants. These results suggest that both PtrANR1 and PtrLAR1 contribute to PA biosynthesis in Populus.

The role of C-terminal amino acid residues of a Δ6-fatty acid desaturase from blackcurrant.

Δ6-fatty acid desaturase is an important enzyme in the catalytic synthesis of polyunsaturated fatty acids. Using domain swapping and a site-directed mutagenesis strategy, we found that the region of the C-terminal 67 amino acid residues of Δ6-fatty acid desaturase RnD6C from blackcurrant was essential for its catalytic activity and that seven different residues between RnD6C and RnD8A in that region were involved in the desaturase activity. Compared with RnD6C, the activity of the following mutations, V394A, K395I, F411L, S436P, VK3945AI and IS4356VP, was significantly decreased, whereas the activity of I417T was significantly increased. The amino acids N, T and Y in the last four residues also play a certain role in the desaturase activity.

Highly efficient phosphorescent organic light emitting diodes based on iridium(III) complex with bulky substituent spacers.

We developed highly efficient phosphorescent organic light emitting diodes (PHOLEDs) using iridium(III) complex, fac-tris[4-methyl-2-2(4'-trimethylsilylphenyl)pyridine] [Ir(msippy)3]. PHOLEDs based on Ir(msippy)3 complex exhibit the yellowish-green emission with CIE color coordinates of (0.31,0.64). These device performances were compared with those of the green emitting Ir(ppy)3-based devices. The higher external quantum efficiency (EQE) of 25.6% and the current efficiency of 84.4 cd/A were achieved for Ir(msippy)3-based device. The results show that the complete energy and/or charge transfer from the host to Ir(msippy)3 dopant in the emitting layer (EML) of the device resulted in the higher device efficiencies compared with those of Ir(ppy)3-based devices.

Identification and functional analysis of the genes encoding Delta6-desaturase from Ribes nigrum.

Gamma-linolenic acid (gamma-linolenic acid, GLA; C18:3 Delta(6, 9, 12)) belongs to the omega-6 family and exists primarily in several plant oils, such as evening primrose oil, blackcurrant oil, and borage oil. Delta(6)-desaturase is a key enzyme involved in the synthesis of GLA. There have been no previous reports on the genes encoding Delta(6)-desaturase in blackcurrant (Ribes nigrum L.). In this research, five nearly identical copies of Delta(6)-desaturase gene-like sequences, named RnD8A, RnD8B, RnD6C, RnD6D, and RnD6E, were isolated from blackcurrant. Heterologous expression in Saccharomyces cerevisiae and/or Arabidopsis thaliana confirmed that RnD6C/D/E were Delta(6)-desaturases that could use both alpha-linolenic acids (ALA; C18:3 Delta(9,12,15)) and linoleic acid (LA; C18:2 Delta(9,12)) precursors in vivo, whereas RnD8A/B were Delta(8)-sphingolipid desaturases. Expression of GFP tagged with RnD6C/D/E showed that blackcurrant Delta(6)-desaturases were located in the mitochondrion (MIT) in yeast and the endoplasmic reticulum (ER) in tobacco. GC-MS results showed that blackcurrant accumulated GLA and octadecatetraenoic acids (OTA; C18:4 Delta(6,9,12,15)) mainly in seeds and a little in other organs and tissues. RT-PCR results showed that RnD6C and RnD6E were expressed in all the tissues at a low level, whereas RnD6D was expressed at a high level only in seeds, leading to the accumulation of GLA and OTA in seeds. This research provides new insights to our understanding of GLA synthesis and accumulation in plants and the evolutionary relationship of this class of desaturases, and new clues as to the amino acid determinants which define precise enzyme activity.