Coniferaldehyde 5-hydroxylase Research Articles

Modification of lignin composition by ectopic expressing wheat TaF5H1 led to decreased salt tolerance in transgenic Arabidopsis plants

Lignin is an important cell wall component that provides plants with mechanical support and improved tolerance to pathogen attacks. Previous studies have shown that plants rich in S-lignin content or with a higher S/G ratio always exhibit higher efficiency in the utilization of lignocellulosic biomass. Ferulate 5-hydroxylase, or coniferaldehyde 5-hydroxylase (F5H, or CAld5H), is the critical enzyme in syringyl lignin biosynthesis. Some F5Hs have been characterized in several plant species, e.g., Arabidopsis, rice, and poplar. However, information about F5Hs in wheat remains unclear. In this study, a wheat F5H gene, TaF5H1, together with its native promoter (pTaF5H1), was functionally characterized in transgenic Arabidopsis. Gus staining results showed that TaF5H1 could be expressed predominantly in the highly lignified tissues in transgenic Arabidopsis plants carrying pTaF5H1:Gus. qRT-PCR results showed that TaF5H1 was significantly inhibited by NaCl treatment. Ectopic expression of TaF5H1 driven by pTaF5H1 (i.e., pTaF5H1:TaF5H1) could increase the biomass yield, S-lignin content, and S/G ratio in transgenic Arabidopsis plants, which could also restore the traces of S-lignin in fah1-2, the Arabidopsis F5H mutant, to an even higher level than the wild type (WT), suggesting that TaF5H1 is a critical enzyme in S lignin biosynthesis, and pTaF5H1:TaF5H1 module has potential in the manipulation of S-lignin composition without any compromise on the biomass yield. However, expression of pTaF5H1:TaF5H1 also led to decreased salt tolerance compared with the WT. RNA-seq analysis showed that many stress-responsive genes and genes responsible for the biosynthesis of cell walls were differentially expressed between the seedlings harboring pTaF5H1:TaF5H1 and the WT, hinting that manipulation of the cell wall components targeting F5H may also affect the stress adaptability of the modified plants due to the interference to the cell wall integrity. In summary, this study demonstrated that the wheat pTaF5H1: TaF5H1 cassette has the potential to modulate S-lignin composition without any compromise in biomass yield in future engineering practice. Still, its negative effect on stress adaptability to transgenic plants should also be considered.

BEL1-like Homeodomain Protein BLH6a Is a Negative Regulator of CAl5H2 in Sinapyl Alcohol Monolignol Biosynthesis in Poplar.

Lignin is one of the major components of xylem cell walls in tree stems. The lignin in the wood of most flowering plants (dicotyledonous angiosperms) is typically polymerized from three monolignol precursors, coniferyl alcohol, sinapyl alcohol, and p-coumaroyl alcohol, resulting in guaiacyl (G), syringyl (S), and hydroxyphenyl (H) subunits, respectively. In this study, we focus on the transcriptional regulation of a coniferaldehyde 5-hydroxylase (CAld5H2) gene, which encodes a key enzyme for sinapyl alcohol biosynthesis. We carried out a yeast one-hybrid (Y1H) screen to identify candidate upstream transcription factors (TFs) regulating CAld5H2. We obtained 12 upstream TFs as potential regulators of CAld5H2. One of these TF genes, BLH6a, encodes a BEL1-like homeodomain (BLH) protein and negatively regulated the CAld5H2 promoter activity. The direct regulation of CAld5H2 promoter by BLH6a was supported by chromatin immunoprecipitation–quantitative polymerase chain reaction (ChIP–qPCR) and dominant repression of BLH6a in transgenic plants. Luciferase complementation imaging analyses showed extensive protein–protein interactions among these 12 TFs. We propose that BLH6a is a negative regulator of CAld5H2, which acts through combinatorial regulation of multiple TFs for sinapyl alcohol (S monolignol) biosynthesis in poplar.

Hevea brasiliensis coniferaldehyde-5-hydroxylase (HbCAld5H) regulates xylogenesis, structure and lignin chemistry of xylem cell wall in Nicotiana tabacum

Key messageThe HbCAld5H1 gene cloned from Hevea brasiliensis regulates the cambial activity, xylem differentiation, syringyl–guaiacyl ratio, secondary wall structure, lignification pattern and xylan distribution in xylem fibres of transgenic tobacco plants.Molecular characterization of lignin biosynthesis gene coniferaldehyde-5-hydroxylase (CAld5H) from Hevea brasiliensis and its functional validation was performed. Both sense and antisense constructs of HbCAld5H1 gene were introduced into tobacco through Agrobacterium-mediated genetic transformation for over expression and down-regulation of this key enzyme to understand its role affecting structural and cell wall chemistry. The anatomical studies of transgenic tobacco plants revealed the increase of cambial activity leading to xylogenesis in sense lines and considerable reduction in antisense lines. The ultra-structural studies showed that the thickness of secondary wall (S2 layer) of fibre had been decreased with non-homogenous lignin distribution in antisense lines, while sense lines showed an increase in S2 layer thickness. Maule color reaction revealed that syringyl lignin distribution in the xylem elements was increased in sense and decreased in antisense lines. The immunoelectron microscopy revealed a reduction in LM 10 and LM 11 labelling in the secondary wall of antisense tobacco lines. Biochemical studies showed a radical increase in syringyl lignin in sense lines without any significant change in total lignin content, while S/G ratio decreased considerably in antisense lines. Our results suggest that CAld5H gene plays an important role in xylogenesis stages such as cambial cell division, secondary wall thickness, xylan and syringyl lignin distribution in tobacco. Therefore, CAld5H gene could be considered as a promising target for lignin modification essential for timber quality improvement in rubber.

Lignin characterization of rice CONIFERALDEHYDE 5-HYDROXYLASE loss-of-function mutants generated with the CRISPR/Cas9 system.

The aromatic composition of lignin is an important trait that greatly affects the usability of lignocellulosic biomass. We previously identified a rice (Oryza sativa) gene encoding coniferaldehyde 5-hydroxylase (OsCAld5H1), which was effective in modulating syringyl (S)/guaiacyl (G) lignin composition ratio in rice, a model grass species. Previously characterized OsCAld5H1-knockdown rice lines, which were produced via an RNA-interference approach, showed augmented G lignin units yet contained considerable amounts of residual S lignin units. In this study, to further investigate the effect of suppression of OsCAld5H1 on rice lignin structure, we generated loss-of-function mutants of OsCAld5H1 using the CRISPR/Cas9-mediated genome editing system. Homozygous OsCAld5H1-knockout lines harboring anticipated frame-shift mutations in OsCAld5H1 were successfully obtained. A series of wet-chemical and two-dimensional NMR analyses on cell walls demonstrated that although lignins in the mutant were predictably enriched in G units all the tested mutant lines produced considerable numbers of S units. Intriguingly, lignin γ-p-coumaroylation analysis by the derivatization followed by reductive cleavage method revealed that enrichment of G units in lignins of the mutants was limited to the non-γ-p-coumaroylated units, whereas grass-specific γ-p-coumaroylated lignin units were almost unaffected. Gene expression analysis indicated that no homologous genes of OsCAld5H1 were overexpressed in the mutants. These data suggested that CAld5H is mainly involved in the production of non-γ-p-coumaroylated S lignin units, common in both eudicots and grasses, but not in the production of grass-specific γ-p-coumaroylated S units in rice.

Regulation of CONIFERALDEHYDE 5-HYDROXYLASE expression to modulate cell wall lignin structure in rice.

Regulation of a gene encoding coniferaldehyde 5-hydroxylase leads to substantial alterations in lignin structure in rice cell walls, identifying a promising genetic engineering target for improving grass biomass utilization. The aromatic composition of lignin greatly affects utilization characteristics of lignocellulosic biomass and, therefore, has been one of the primary targets of cell wall engineering studies. Limited information is, however, available regarding lignin modifications in monocotyledonous grasses, despite the fact that grass lignocelluloses have a great potential for feedstocks of biofuel production and various biorefinery applications. Here, we report that manipulation of a gene encoding coniferaldehyde 5-hydroxylase (CAld5H, or ferulate 5-hydroxylase, F5H) leads to substantial alterations in syringyl (S)/guaiacyl (G) lignin aromatic composition in rice (Oryza sativa), a major model grass and commercially important crop. Among three CAld5H genes identified in rice, OsCAld5H1 (CYP84A5) appeared to be predominantly expressed in lignin-producing rice vegetative tissues. Down-regulation of OsCAld5H1 produced altered lignins largely enriched in G units, whereas up-regulation of OsCAld5H1 resulted in lignins enriched in S units, as revealed by a series of wet-chemical and NMR structural analyses. Our data collectively demonstrate that OsCAld5H1 expression is a major factor controlling S/G lignin composition in rice cell walls. Given that S/G lignin composition affects various biomass properties, we contemplate that manipulation of CAld5H gene expression represents a promising strategy to upgrade grass biomass for biorefinery applications.

Rescue of syringyl lignin and sinapate ester biosynthesis in Arabidopsis thaliana by a coniferaldehyde 5-hydroxylase from Eucalyptus globulus.

The gene coding for F5H from Eucalyptus globulus was cloned and used to transform an f5h -mutant of Arabidopsis thaliana , which was complemented, thus verifying the identity of the cloned gene. Coniferaldehyde 5-hydroxylase (F5H; EC 1.14.13) is a cytochrome P450-dependent monooxygenase that catalyzes the 5-hydroxylation step required for the production of syringyl units in lignin biosynthesis. The Eucalyptus globulus enzyme was characterized in vitro, and results showed that the preferred substrates were coniferaldehyde and coniferyl alcohol. Complementation experiments demonstrated that both cDNA and genomic constructs derived from F5H from E. globulus under the control of the cinnamate 4-hydroxylase promoter from Arabidopsis thaliana, or a partial F5H promoter from E. globulus, can rescue the inability of the A. thaliana fah1-2 mutant to accumulate sinapate esters and syringyl lignin. E. globulus is a species widely used to obtain products that require lignin removal, and the results suggest that EglF5H is a good candidate for engineering efforts aimed at increasing the lignin syringyl unit content, either for kraft pulping or biofuel production.

Functional redundancy of the two 5-hydroxylases in monolignol biosynthesis of Populus trichocarpa: LC–MS/MS based protein quantification and metabolic flux analysis

Flowering plants have syringyl and guaiacyl subunits in lignin in contrast to the guaiacyl lignin in gymnosperms. The biosynthesis of syringyl subunits is initiated by coniferaldehyde 5-hydroxylase (CAld5H). In Populus trichocarpa there are two closely related CAld5H enzymes (PtrCAld5H1 and PtrCAld5H2) associated with lignin biosynthesis during wood formation. We used yeast recombinant PtrCAld5H1 and PtrCAld5H2 proteins to carry out Michaelis-Menten and inhibition kinetics with LC-MS/MS based absolute protein quantification. CAld5H, a monooxygenase, requires a cytochrome P450 reductase (CPR) as an electron donor. We cloned and expressed three P. trichocarpa CPRs in yeast and show that all are active with both CAld5Hs. The kinetic analysis shows both CAld5Hs have essentially the same biochemical functions. When both CAld5Hs are coexpressed in the same yeast membranes, the resulting enzyme activities are additive, suggesting functional redundancy and independence of these two enzymes. Simulated reaction flux based on Michaelis-Menten kinetics and inhibition kinetics confirmed the redundancy and independence. Subcellular localization of both CAld5Hs as sGFP fusion proteins expressed in P. trichocarpa differentiating xylem protoplasts indicate that they are endoplasmic reticulum resident proteins. These results imply that during wood formation, 5-hydroxylation in monolignol biosynthesis of P. trichocarpa requires the combined metabolic flux of these two CAld5Hs to maintain adequate biosynthesis of syringyl lignin. The combination of genetic analysis, absolute protein quantitation-based enzyme kinetics, homologous CPR specificity, SNP characterization, and ER localization provides a more rigorous basis for a comprehensive systems understanding of 5-hydroxylation in lignin biosynthesis.

Transcript abundances of LIM transcription factor, 4CL, CAld5H and CesAs affect wood properties in Eucalyptus globulus

Abstract Eucalyptus globulus is the main hardwood species grown in pulpwood plantations in temperate regions of the world. We have cloned six genes influencing wood quality including the LIM domain transcription factor (LIM), 4-coumarate-CoA ligase (4CL), coniferaldehyde 5-hydroxylase (CAld5H) and the three catalytic units of cellulose synthase (CesA), from E. globulus. The transcript abundances of LIM in basal stems of ten independent E. globulus lines showed similar patterns to those of 4CL. We investigated the correlation between gene transcript abundances and wood qualities such as Klason lignin (KL) content, syringaldehyde/vanillin (S/V) ratio and holocellulose (HC) content. Expression of the LIM and 4CL were positively correlated with KL content. A highly significant positive correlation was observed between CAld5H expression and S/V ratio. Furthermore, a ratio of the sum of the transcript abundances of three CesA1, CesA2 and CesA3 to 4CL showed a positive correlation with a ratio of HC/KL content that positively correlated with the chemically extracted fiber content in this woody plant.

Wood quality-related gene expressions of Eucalyptus globulusgrown in a greenhouse

Eucalyptus species constitute the most widely planted hardwood trees in temperate and subtropical regions. Their wood is used as a raw material for the production of cellulose. Eucalyptus species have fast growth rates and the ability to adapt to a broad range of geographic locations. Most importantly, Eucalyptus has been listed as one of the candidate biomass energy crops [1]. Eucalyptus globulus is the main hardwood species grown in pulpwood plantations in temperate regions of the world. In this study, we focused on four kinds of key genes, 4-coumarate-CoA ligase (4CL), LIM domain transcription factor (LIM) [2], coniferaldehyde 5-hydroxylase (CAld5H) and the three catalytic units of cellulose synthase (CesA1, CesA2 and CesA3) influencing wood quality . We investigated correlation between relative expression levels of these genes and wood qualities. We have cloned the genes encoding LIM, 4CL, CAld5H and the cellulose synthase (CesA1, CesA2 and CesA3) from E. globulus by the method of cDNA library screening (Fig.1). A cDNA library was constructed using mRNA purified from stems of four-month old E. globulus grown in the greenhouse. The expression levels of LIM in basal stems of ten independent E. globulus lines showed similar patterns to those of 4CL, indicating that the LIM may control 4CL expression. We investigated the correlation between gene expression levels and wood qualities such as Klason lignin (KL) content, syringyl/guaiacyl (S/G) ratio and holocellulose (HC) content.Expression of the LIM and 4CL were positively correlated with KL content. A highly significant positive correlation was observed between CAld5H expression and S/G ratio. Furthermore, a ratio of the sum of the expression levels of three CesA1, CesA2 and CesA3 to 4CL showed positive correlation with a ratio of HC/KL content that positively correlated to the chemically extracted fiber content in this woody plant. Overall, our results provide a strong foundation for manipulating candidate genes such as LIM, 4CL, CAld5H and CesA towards the production of desirable wood qualities in an extremely important biomass plant, the Eucalyptus species.

Profiling of phenylpropanoid monomers in developing xylem tissue of transgenic aspen (Populus tremuloides)

Here we describe alterations in the cinnamate/monolignol pathway in three transgenic aspen lines: one with downregulated expression of 4-coumarate:CoA ligase (4CL), one with upregulated expression of coniferaldehyde 5-hydroxylase (CAld5H), and a 4CL downregulated/CAld5H upregulated line. Compared with the wild type, the 4CL downregulated line showed significantly increased levels of p-hydroxycinnamic acids such as p-coumaric, ferulic, and sinapic acids. In contrast, the CAld5H upregulated line had increased content of p-coumaryl and 5-hydroxyconiferyl alcohols. In the 4CL downregulated line, it was likely that most hydroxycinnamic acids were glycosylated. These results strongly suggest that the downregulation of 4CL and upregulation of CAld5H disrupt the metabolic flow through the cinnamate/monolignol pathway and thus alter the amount and structure of its final product, lignin.

Cytochromes P450 in phenolic metabolism

Three independent cytochrome P450 enzyme families catalyze the three rate-limiting hydroxylation steps in the phenylpropanoid pathway leading to the biosynthesis of lignin and numerous other phenolic compounds in plants. Their characterization at the molecular and enzymatic level has revealed an unexpected complexity of phenolic metabolism as the major route involves shikimate/quinate esters and alcohol/aldehyde intermediates. Engineering expression of CYP73s (encoding cinnamate 4-hydroxylase), CYP98s (encoding 4-coumaroylshikimate 3′-hydroxylase) or CYP84s (encoding coniferaldehyde 5-hydroxylase) leads to modified lignin and seed phenolic composition. In particular CYP73s and CYP98s also play essential roles in plant growth and development, while CYP84 constitutes a check-point for the synthesis of syringyl lignin and sinapate esters. Although recent data shed new light on the main path for lignin synthesis, they also raised new questions. Mutants and engineered plants revealed the existence of (an) alternative pathway(s), which most likely involve(s) different precursors and oxygenases. On the other hand, phylogenetic analysis of plant genomes show the existence of P450 gene duplications in each family, which may have led to the acquisition of novel or additional physiological functions in planta. In addition to the main lignin pathway, P450s contribute to the biosynthesis of many bioactive phenolic derivatives, with potential applications in medicine and plant defense, including lignans, phenylethanoids, benzoic acids, xanthones or quinoid compounds. A very small proportion of these P450s have been characterized so far, and rarely at a molecular level. The possible involvement of P450s in salicylic acid is discussed.

Monolignol biosynthesis and genetic engineering of lignin in trees, a review

This paper summarizes our previous and current research on genetic engineering of lignin biosynthesis for the purposes of improving wood pulping and bleaching efficiency. For these purposes, our targets were to produce transgenic trees with low content of lignin that is also chemically reactive (high lignin S/G ratio). Using aspen as a model species, we have characterized the biochemical functions of various genes and the kinetic properties of these gene products involved in monolignol biosynthetic pathway. The results of these characterizations proved strong evidence for a principle phenolic flux leading to the formation of monolignols. Biochemical evidence further demonstrated that, in this principle flux, 4CL could be the enzyme limiting total lignin accumulation, whereas CAld5H might control the lignin S/G ratio. These propositions were fully supported by the in vivo functions of these enzymes. Transgenic trees with inhibited 4CL enzyme activity exhibited 5–45% reduction in lignin contents. The chemical structure of the resulting lignin remained essentially unchanged. More importantly, the lignin reduction was compensated for by a concomitant increase in cellulose content. When antisense 4CL and sense coniferaldehyde 5-hydroxylase (CAld5H) genes were simultaneously transferred into aspen via Agrobacterium, transgenic trees expressing each one and both of the transgenes were produced. Lignin reductions up to 55% were achieved in antisense 4CL plants and up to 3-fold S/G increases were observed in sense CAld5H plants. These effects were independent but additive, with plants expressing both transgenes having less lignin and higher S/G ratio. Consistent with our previous results, lignin reduction has always resulted in an increase in cellulose content. These transgenics could be potentially valuable for pulp production. But more importantly, these benchmark transgenics are rich sources of information for functional genomics and metabolic engineering, allowing the generation of the ultimate raw materials for wood pulp production.

Combinatorial modification of multiple lignin traits in trees through multigene cotransformation.

Lignin quantity and reactivity [which is associated with its syringyl/guaiacyl (S/G) constituent ratio] are two major barriers to wood-pulp production. To verify our contention that these traits are regulated by distinct monolignol biosynthesis genes, encoding 4-coumarate-CoA ligase (4CL) and coniferaldehyde 5-hydroxylase (CAld5H), we used Agrobacterium to cotransfer antisense 4CL and sense CAld5H genes into aspen (Populus tremuloides). Trees expressing each one and both of the transgenes were produced with high efficiency. Lignin reduction by as much as 40% with 14% cellulose augmentation was achieved in antisense 4CL plants; S/G-ratio increases as much as 3-fold were observed without lignin quantity change in sense CAld5H plants. Consistent with our contention, these effects were independent but additive, with plants expressing both transgenes having up to 52% less lignin, a 64% higher S/G ratio, and 30% more cellulose. An S/G-ratio increase also accelerated cell maturation in stem secondary xylem, pointing to a role for syringyl lignin moieties in coordinating xylem secondary wall biosynthesis. The results suggest that this multigene cotransfer system should be broadly useful for plant genetic engineering and functional genomics.

Expression of a poplar cDNA encoding a ferulate-5-hydroxylase/coniferaldehyde 5-hydroxylase increases S lignin deposition in Arabidopsis thaliana

Ferulate-5-hydroxylase or coniferaldehyde 5-hydroxylase (F5H or Cald5H; CYP84A1) is the enzyme responsible for the last hydroxylation of the syringyl-type lignin precursors. A cDNA clone highly homologous to the Arabidopsis thaliana F5H/ Cald5H cDNA was identified during the random sequencing of a poplar ( Populus trichocarpa) differentiating xylem cDNA library. Present as a multigenic family in poplar, this gene ( PopF5H) was found to be highly expressed in lignified tissues supporting its role in lignin biosynthesis. When placed under the control of the CaMV 35S promoter, the poplar F5H was able to complement the A. thaliana fah1-2 mutation. Overexpression in wild-type A. thaliana substantially increased the level of syringyl units in lignins. These transgenic lines were also typified by substantial levels of S-units in root lignins, whereas these units are present in low amounts in wild-type A. thaliana. These results support that PopF5H is a functional homolog of the A. thaliana F5H gene.

Monolignol biosynthesis in microsomal preparations from lignifying stems of alfalfa ( Medicago sativa L.)

Microsomal preparations from lignifying stems of alfalfa ( Medicago sativa L.) contained coniferaldehyde 5-hydroxylase activity and immunodetectable caffeic acid 3- O-methyltransferase (COMT), and catalyzed the S-adenosyl l-methionine (SAM) dependent methylation of caffeic acid, caffeyl aldehyde and caffeyl alcohol. When supplied with NADPH and SAM, the microsomes converted caffeyl aldehyde to coniferaldehyde, 5-hydroxyconiferaldehyde, and traces of sinapaldehyde. Coniferaldehyde was a better precursor of sinapaldehyde than was 5-hydroxyconiferaldehyde. The alfalfa microsomes could not metabolize 4-coumaric acid, 4-coumaraldehyde, 4-coumaroyl CoA, or ferulic acid. No metabolism of monolignol precursors was observed in microsomal preparations from transgenic alfalfa down-regulated in COMT expression. In most microsomal preparations, the level of the metabolic conversions was independent of added recombinant COMT. Taken together, the data provide only limited support for the concept of metabolic channeling in the biosynthesis of S monolignols via coniferaldehyde.