Functional and structural insight into the flexibility of cytochrome P450 reductases from Sorghum bicolor and its implications for lignin composition.

Plant NADPH-dependent cytochrome P450 reductase (CPR) is a multidomain enzyme that donates electrons for hydroxylation reactions catalyzed by class II cytochrome P450 monooxygenases involved in the synthesis of many primary and secondary metabolites. These P450 enzymes include trans-cinnamate-4-hydroxylase, p-coumarate-3'-hydroxylase, and ferulate-5-hydroxylase involved in monolignol biosynthesis. Because of its role in monolignol biosynthesis, alterations in CPR activity could change the composition and overall output of lignin. Therefore, to understand the structure and function of three CPR subunits from sorghum, recombinant subunits SbCPR2a, SbCPR2b, and SbCPR2c were subjected to X-ray crystallography and kinetic assays. Steady-state kinetic analyses demonstrated that all three CPR subunits supported the oxidation reactions catalyzed by SbC4H1 (CYP73A33) and SbC3'H (CYP98A1). Furthermore, comparing the SbCPR2b structure with the well-investigated CPRs from mammals enabled us to identify critical residues of functional importance and suggested that the plant flavin mononucleotide-binding domain might be more flexible than mammalian homologs. In addition, the elucidated structure of SbCPR2b included the first observation of NADP+ in a native CPR. Overall, we conclude that the connecting domain of SbCPR2, especially its hinge region, could serve as a target to alter biomass composition in bioenergy and forage sorghums through protein engineering.

Bm-miR172c-5p Regulates Lignin Biosynthesis and Secondary Xylem Thickness by Altering the Ferulate 5 Hydroxylase Gene in Bacopa monnieri.

MicroRNAs (miRNAs) are small non-coding, endogenous RNAs containing 20-24 nucleotides that regulate the expression of target genes involved in various plant processes. A total of 1,429 conserved miRNAs belonging to 95 conserved miRNA families and 12 novel miRNAs were identified from Bacopa monnieri using small RNA sequencing. The Bm-miRNA target transcripts related to the secondary metabolism were further selected for validation. The Bm-miRNA expression in shoot and root tissues was negatively correlated with their target transcripts. The Bm-miRNA cleavage sites were mapped within the coding or untranslated region as depicted by the modified RLM-RACE. In the present study, we validate three miRNA targets, including asparagine synthetase, cycloartenol synthase and ferulate 5 hydroxylase (F5H) and elucidate the regulatory role of Bm-miR172c-5p, which cleaves the F5H gene involved in the lignin biosynthesis. Overexpression (OE) of Bm-miR172c-5p precursor in B. monnieri suppresses F5H gene, leading to reduced lignification and secondary xylem thickness under control and drought stress. By contrast, OE of endogenous target mimics (eTMs) showed enhanced lignification and secondary xylem thickness leading to better physiological response under drought stress. Taken together, we suggest that Bm-miRNA172c-5p might be a key player in maintaining the native phenotype of B. monnieri under control and different environmental conditions.

Fiber Cell-Specific Expression of the VP16-Fused Ethylene Response Factor 41 Protein Increases Biomass Yield and Alters Lignin Composition.

Arabidopsis thaliana transcription factors belonging to the ERFIIId and ERFIIIe subclade (ERFIIId/e) of the APETALA 2/ethylene response factor (AP2/ERF) family enhance primary cell wall (PCW) formation. These transcription factors activate expression of genes encoding PCW-type cellulose synthase (CESA) subunits and other genes for PCW biosynthesis. In this study, we show that fiber-specific expression of ERF035-VP16 and ERF041-VP16, which are VP16-fused proteins of ERFIIId/e members, promote cell wall thickening in a wild-type background with a concomitant increase of alcohol insoluble residues (cell wall content) per fresh weight (FW) and monosaccharides related to the PCW without affecting plant growth. Furthermore, in the ERF041-VP16 lines, the total amount of lignin and the syringyl (S)/guaiacyl (G) ratio decreased, and the enzymatic saccharification yield of glucose from cellulose per fresh weight improved. In these lines, PCW-type CESA genes were upregulated and ferulate 5-hydropxylase1 (F5H1), which is necessary for production of the S unit lignin, was downregulated. In addition, various changes in the expression levels of transcription factors regulating secondary cell wall (SCW) formation were observed. In conclusion, fiber cell-specific ERF041-VP16 improves biomass yield, increases PCW components, and alters lignin composition and deposition and may be suitable for use in future molecular breeding programs of biomass crops.

Overexpression of ferulate 5-hydroxylase increases syringyl units in Sorghum bicolor.

Ferulate 5-hydroxylase (F5H) of the monolignol pathway catalyzes the hydroxylation of coniferyl alcohol, coniferaldehyde and ferulic acid to produce 5-hydroxyconiferyl moieties, which lead to the formation of sinapic acid and syringyl (S) lignin monomers. In contrast, guaiacyl (G) lignin, the other major type of lignin monomer, is derived from polymerization of coniferyl alcohol. In this study, the effects of manipulating S-lignin biosynthesis in sorghum (Sorghum bicolor) were evaluated. Overexpression of sorghum F5H (SbF5H), under the control of the CaMV 35S promoter, increased both S-lignin levels and the ratio of S/G lignin, while plant growth and development remained relatively unaffected. Maüle staining of stalk and leaf midrib sections from SbF5H overexpression lines indicated that the lignin composition was altered. Ectopic expression of SbF5H did not affect the gene expression of other monolignol pathway genes. In addition, brown midrib 12-ref (bmr12-ref), a nonsense mutation in the sorghum caffeic acid O-methyltransferase (COMT) was combined with 35S::SbF5H through cross-pollination to examine effects on lignin synthesis. The stover composition from bmr12 35S::SbF5H plants more closely resembled bmr12 stover than 35S::SbF5H or wild-type (WT) stover; S-lignin and total lignin concentrations were decreased relative to WT or 35S::SbF5H. Likewise, expression of upstream monolignol biosynthetic genes was increased in both bmr12 and bmr12 35S::SbF5H relative to WT or 35S::SbF5H. Overall, these results indicated that overexpression of SbF5H did not compensate for the loss of COMT activity. KEY MESSAGE: Overexpression of F5H in sorghum increases S-lignin without increasing total lignin content or affecting plant growth, but it cannot compensate for the loss of COMT activity in monolignol synthesis.

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.

PdWND3A, a wood-associated NAC domain-containing protein, affects lignin biosynthesis and composition in Populus.

BACKGROUND: Plant secondary cell wall is a renewable feedstock for biofuels and biomaterials production. Arabidopsis VASCULAR-RELATED NAC DOMAIN (VND) has been demonstrated to be a key transcription factor regulating secondary cell wall biosynthesis. However, less is known about its role in the woody species. RESULTS: Here we report the functional characterization of Populus deltoides WOOD-ASSOCIATED NAC DOMAIN protein 3 (PdWND3A), a sequence homolog of Arabidopsis VND4 and VND5 that are members of transcription factor networks regulating secondary cell wall biosynthesis. PdWND3A was expressed at higher level in the xylem than in other tissues. The stem tissues of transgenic P. deltoides overexpressing PdWND3A (OXPdWND3A) contained more vessel cells than that of wild-type plants. Furthermore, lignin content and lignin monomer syringyl and guaiacyl (S/G) ratio were higher in OXPdWND3A transgenic plants than in wild-type plants. Consistent with these observations, the expression of FERULATE 5-HYDROXYLASE1 (F5H1), encoding an enzyme involved in the biosynthesis of sinapyl alcohol (S unit monolignol), was elevated in OXPdWND3A transgenic plants. Saccharification analysis indicated that the rate of sugar release was reduced in the transgenic plants. In addition, OXPdWND3A transgenic plants produced lower amounts of biomass than wild-type plants. CONCLUSIONS: PdWND3A affects lignin biosynthesis and composition and negatively impacts sugar release and biomass production.

MYB103 is required for FERULATE-5-HYDROXYLASE expression and syringyl lignin biosynthesis in Arabidopsis stems.

The transcription factor MYB103 was previously identified as a member of the transcriptional network regulating secondary wall biosynthesis in xylem tissues of Arabidopsis, and was proposed to act on cellulose biosynthesis. It is a direct transcriptional target of the transcription factor SECONDARY WALL ASSOCIATED NAC DOMAIN PROTEIN 1 (SND1), and 35S-driven dominant repression or over-expression of MYB103 modifies secondary wall thickness. We identified two myb103 T-DNA insertion mutants and chemically characterized their lignocellulose by pyrolysis/GC/MS, 2D NMR, FT-IR microspectroscopy and wet chemistry. The mutants developed normally but exhibited a 70-75% decrease in syringyl (S) lignin. The level of guaiacyl (G) lignin was co-ordinately increased, so that total Klason lignin was not affected. The transcript abundance of FERULATE-5-HYDROXYLASE (F5H), the key gene in biosynthesis of S lignin, was strongly decreased in the myb103 mutants, and the metabolomes of the myb103 mutant and an F5H null mutant were very similar. Other than modification of the lignin S to G ratio, there were only very minor changes in the composition of secondary cell-wall polymers in the inflorescence stem. In conclusion, we demonstrate that F5H expression and hence biosynthesis of S lignin are dependent on MYB103.

Parallels in lignin biosynthesis: A study in Selaginella moellendorffii reveals convergence across 400 million years of evolution.

A hallmark of vascular plants is the development of a complex water-conducting system, which is physically reinforced by the heterogeneous aromatic polymer lignin. Syringyl lignin, a major building block of lignin, is often thought to be uniquely characteristic of angiosperms; however, it was demonstrated over fifty years ago that that syringyl lignin is found in another group of plants, known as the lycophytes, the ancestors of which diverged from all the other vascular plant lineages 400 million years ago.1 To determine the biochemical basis for this common biosynthetic ability, we isolated and characterized cytochrome P450-dependent monooxygenases (P450s) from the lycophyte Selaginella moellendorffii and compared them to the enzyme that is required for syringyl lignin synthesis in angiosperms. Our results showed that one of these P450s encodes an enzyme that is functionally analogous to but phylogenetically independent from its angiosperm counterpart. Here, we discuss the evolution of lignin biosynthesis in vascular plants and the role of Selaginella moellendorffii in plant comparative biology and genomics.