Silvia Fornalé

ZmMYB31 directly represses maize lignin genes and redirects the phenylpropanoid metabolic flux.

Few regulators of phenylpropanoids have been identified in monocots having potential as biofuel crops. Here we demonstrate the role of the maize (Zea mays) R2R3-MYB factor ZmMYB31 in the control of the phenylpropanoid pathway. We determined its in vitro consensus DNA-binding sequence as ACC(T)/(A) ACC, and chromatin immunoprecipitation (ChIP) established that it interacts with two lignin gene promoters in vivo. To explore the potential of ZmMYB31 as a regulator of phenylpropanoids in other plants, its role in the regulation of the phenylpropanoid pathway was further investigated in Arabidopsis thaliana. ZmMYB31 downregulates several genes involved in the synthesis of monolignols and transgenic plants are dwarf and show a significantly reduced lignin content with unaltered polymer composition. We demonstrate that these changes increase cell wall degradability of the transgenic plants. In addition, ZmMYB31 represses the synthesis of sinapoylmalate, resulting in plants that are more sensitive to UV irradiation, and induces several stress-related proteins. Our results suggest that, as an indirect effect of repression of lignin biosynthesis, transgenic plants redirect carbon flux towards the biosynthesis of anthocyanins. Thus, ZmMYB31 can be considered a good candidate for the manipulation of lignin biosynthesis in biotechnological applications.

Down-regulation of the maize and Arabidopsis thaliana caffeic acid O -methyl-transferase genes by two new maize R2R3-MYB transcription factors

The maize (Zea mays L.) caffeic acid O-methyl-transferase (COMT) is a key enzyme in the biosynthesis of lignin. In this work we have characterized the involvement of COMT in the lignification process through the study of the molecular mechanisms involved in its regulation. The examination of the maize COMT gene promoter revealed a putative ACIII box, typically recognized by R2R3-MYB transcription factors. We used the sequence of known R2R3-MYB factors to isolate five maize R2R3-MYB factors (ZmMYB2, ZmMYB8, ZmMYB31, ZmMYB39, and ZmMYB42) and study their possible roles as regulators of the maize COMT gene. The factors ZmMYB8, ZmMY31, and ZmMYB42 belong to the subgroup 4 of the R2R3-MYB family along with other factors associated with lignin biosynthesis repression. In addition, the induction pattern of ZmMYB31 and ZmMYB42 gene expression on wounding is that expected for repressors of the maize COMT gene. Arabidopsisthaliana plants over-expressing ZmMYB31 and ZmMYB42 down-regulate both the A. thaliana and the maize COMT genes. Furthermore, the over-expression of ZmMYB31 and ZmMYB42 also affect the expression of other genes of the lignin pathway and produces a decrease in lignin content of the transgenic plants.

The maize ZmMYB42 represses the phenylpropanoid pathway and affects the cell wall structure, composition and degradability in Arabidopsis thaliana

The involvement of the maize ZmMYB42 R2R3-MYB factor in the phenylpropanoid pathway and cell wall structure and composition was investigated by overexpression in Arabidopsis thaliana. ZmMYB42 down-regulates several genes of the lignin pathway and this effect reduces the lignin content in all lignified tissues. In addition, ZmMYB42 plants generate a lignin polymer with a decreased S to G ratio through the enrichment in H and G subunits and depletion in S subunits. This transcription factor also regulates other genes involved in the synthesis of sinapate esters and flavonoids. Furthermore, ZmMYB42 affects the cell wall structure and degradability, and its polysaccharide composition. Together, these results suggest that ZmMYB42 may be part of the regulatory network controlling the phenylpropanoid biosynthetic pathway.

Altered lignin biosynthesis improves cellulosic bioethanol production in transgenic maize plants down-regulated for cinnamyl alcohol dehydrogenase.

Cinnamyl alcohol dehydrogenase (CAD) is a key enzyme involved in the last step of monolignol biosynthesis. The effect of CAD down-regulation on lignin production was investigated through a transgenic approach in maize. Transgenic CAD-RNAi plants show a different degree of enzymatic reduction depending on the analyzed tissue and show alterations in cell wall composition. Cell walls of CAD-RNAi stems contain a lignin polymer with a slight reduction in the S-to-G ratio without affecting the total lignin content. In addition, these cell walls accumulate higher levels of cellulose and arabinoxylans. In contrast, cell walls of CAD-RNAi midribs present a reduction in the total lignin content and of cell wall polysaccharides. In vitro degradability assays showed that, although to a different extent, the changes induced by the repression of CAD activity produced midribs and stems more degradable than wild-type plants. CAD-RNAi plants grown in the field presented a wild-type phenotype and produced higher amounts of dry biomass. Cellulosic bioethanol assays revealed that CAD-RNAi biomass produced higher levels of ethanol compared to wild-type, making CAD a good target to improve both the nutritional and energetic values of maize lignocellulosic biomass.

AtMYB7, a New Player in the Regulation of UV-Sunscreens in Arabidopsis thaliana

The phenylpropanoid metabolic pathway provides a wide variety of essential compounds for plants. Together with sinapate esters, in Brassicaceae species, flavonoids play an important role in protecting plants against UV irradiation. In this work we have characterized Arabidopsis thaliana AtMYB7, the closest homolog of AtMYB4 and AtMYB32, described as repressors of different branches of phenylpropanoid metabolism. The characterization of atmyb7 plants revealed an induction of several genes involved in flavonol biosynthesis and an increased amount of these compounds. In addition, AtMYB7 gene expression is repressed by AtMYB4. As a consequence, the atmyb4 mutant plants present a reduction of flavonol contents, indicating once more that AtMYB7 represses flavonol biosynthesis. Our results also show that AtMYB7 gene expression is induced by salt stress. Induction assays indicated that AtMYB7 represses several genes of the flavonoid pathway, DFR and UGT being early targets of this transcription factor. The results obtained indicate that AtMYB7 is a repressor of flavonol biosynthesis and also led us to propose AtMYB4 and AtMYB7 as part of the regulatory mechanism controlling the balance of the main A. thaliana UV-sunscreens.

ZmXTH1, a new xyloglucan endotransglucosylase/hydrolase in maize, affects cell wall structure and composition in Arabidopsis thaliana

Xyloglucan endotransglucosylase/hydrolases (XTHs; EC 2.4.1.207 and/or EC 3.2.1.151) are enzymes involved in the modification of cell wall structure by cleaving and, often, also re-joining xyloglucan molecules in primary plant cell walls. Using a pool of antibodies raised against an enriched cell wall protein fraction, a new XTH cDNA in maize, ZmXTH1, has been isolated from a cDNA expression library obtained from the elongation zone of the maize root. The predicted protein has a putative N-terminal signal peptide and possesses the typical domains of this enzyme family, such as a catalytic domain that is homologous to that of Bacillus macerans beta-glucanase, a putative N-glycosylation motif, and four cysteine residues in the central and C terminal regions of the ZmXTH1 protein. Phylogenetic analysis of ZmXTH1 reveals that it belongs to subgroup 4, so far only reported from Poaceae monocot species. ZmXTH1 has been expressed in Pichia pastoris (a methylotrophic yeast) and the recombinant enzyme showed xyloglucan endotransglucosylase but not xyloglucan endohydrolase activity, representing the first enzyme belonging to subgroup 4 characterized in maize so far. Expression data indicate that ZmXTH1 is expressed in elongating tissues, modulated by culture conditions, and induced by gibberellins. Transient expression assays in onion cells reveal that ZmXTH1 is directed to the cell wall, although weakly bound. Finally, Arabidopsis thaliana plants expressing ZmXTH1 show slightly increased xyloglucan endohydrolase activity and alterations in the cell wall structure and composition.

A MYB/ZML Complex Regulates Wound-Induced Lignin Genes in Maize

A methyl jasmonate-dependent MYB-ZML regulatory mechanism links wounding stress to the derepression of lignin genes in maize. Lignin is an essential polymer in vascular plants that plays key structural roles in vessels and fibers. Lignification is induced by external inputs such as wounding, but the molecular mechanisms that link this stress to lignification remain largely unknown. In this work, we provide evidence that three maize (Zea mays) lignin repressors, MYB11, MYB31, and MYB42, participate in wound-induced lignification by interacting with ZML2, a protein belonging to the TIFY family. We determined that the three R2R3-MYB factors and ZML2 bind in vivo to AC-rich and GAT(A/C) cis-elements, respectively, present in a set of lignin genes. In particular, we show that MYB11 and ZML2 bind simultaneously to the AC-rich and GAT(A/C) cis-elements present in the promoter of the caffeic acid O-methyl transferase (comt) gene. We show that, like the R2R3-MYB factors, ZML2 also acts as a transcriptional repressor. We found that upon wounding and methyl jasmonate treatments, MYB11 and ZML2 proteins are degraded and comt transcription is induced. Based on these results, we propose a molecular regulatory mechanism involving a MYB/ZML complex in which wound-induced lignification can be achieved by the derepression of a set of lignin genes.

Cell wall modifications triggered by the down-regulation of Coumarate 3-hydroxylase-1 in maize

Coumarate 3-hydroxylase (C3H) catalyzes a key step of the synthesis of the two main lignin subunits, guaiacyl (G) and syringyl (S) in dicotyledonous species. As no functional data are available in regards to this enzyme in monocotyledonous species, we generated C3H1 knock-down maize plants. The results obtained indicate that C3H1 participates in lignin biosynthesis as its down-regulation redirects the phenylpropanoid flux: as a result, increased amounts of p-hydroxyphenyl (H) units, lignin-associated ferulates and the flavone tricin were detected in transgenic stems cell walls. Altogether, these changes make stem cell walls more degradable in the most C3H1-repressed plants, despite their unaltered polysaccharide content. The increase in H monomers is moderate compared to C3H deficient Arabidopsis and alfalfa plants. This could be due to the existence of a second maize C3H protein (C3H2) that can compensate the reduced levels of C3H1 in these C3H1-RNAi maize plants. The reduced expression of C3H1 alters the macroscopic phenotype of the plants, whose growth is inhibited proportionally to the extent of C3H1 repression. Finally, the down-regulation of C3H1 also increases the synthesis of flavonoids, leading to the accumulation of anthocyanins in transgenic leaves.

Changes in Cell Wall Polymers and Degradability in Maize Mutants Lacking 3′- and 5′-O-Methyltransferases Involved in Lignin Biosynthesis

Caffeoyl coenzyme A 3-O-methyltransferase (CCoAOMT) and caffeic acid-O-methyltransferase (COMT) are key enzymes in the biosynthesis of coniferyl and sinapyl alcohols, the precursors of guaiacyl (G) and syringyl (S) lignin subunits. The function of these enzymes was characterized in single and double mutant maize plants. In this work, we determined that the comt (brown-midrib 3) mutant plants display a reduction of the flavonolignin unit derived from tricin (a dimethylated flavone), demonstrating that COMT is a key enzyme involved in the synthesis of this compound. In contrast, the ccoaomt1 mutants display a wild-type amount of tricin, suggesting that CCoAOMT1 is not essential for the synthesis of this compound. Based on our data, we suggest that CCoAOMT1 is involved in lignin biosynthesis at least in midribs. The phenotype of ccoaomt1 mutant plants displays no alterations, and their lignin content and composition remain unchanged. On the other hand, the ccoaomt1 comt mutant displays phenotypic and lignin alterations similar to those already described for the comt mutant. Although stems from the three mutants display a similar increase of hemicelluloses, the effect on cell wall degradability varies, the cell walls of ccoaomt1 being the most degradable. This suggests that the positive effect of lignin reduction on cell wall degradability of comt and ccoaomt1 comt mutants is counteracted by changes occurring in lignin composition, such as the decreased S/G ratio. In addition, the role of the flavonolignin unit derived from tricin in cell wall degradability is also discussed.

Characterization of structural cell wall polysaccharides in cattail (Typha latifolia): Evaluation as potential biofuel feedstock

Second generation bioethanol produced from lignocellulosic biomass is attracting attention as an alternative energy source. In this study, a detailed knowledge of the composition and structure of common cattail (Typha latifolia L.) cell wall polysaccharides, obtained from stem or leaves, has been conducted using a wide set of techniques to evaluate this species as a potential bioethanol feedstock. Our results showed that common cattail cellulose content was high for plants in the order Poales and was accompanied by a small amount of cross-linked polysaccharides. A high degree of arabinose-substitution in xylans, a high syringyl/guaiacyl ratio in lignin and a low level of cell wall crystallinity could yield a good performance for lignocellulose saccharification. These results identify common cattail as a promising plant for use as potential bioethanol feedstock. To the best of our knowledge, this is the first in-depth analysis to be conducted of lignocellulosic material from common cattail.