The plant cell wall is a plastic structure of variable composition that constitutes the first line of defence against environmental challenges. Lodging and drought are two stressful conditions that severely impact maize yield. In a previous work, we characterised the cell walls of two maize inbreds, EA2024 (susceptible) and B73 (resistant) to stalk lodging. Here, we show that drought induces distinct phenotypical, physiological, cell wall, and transcriptional changes in the two inbreds, with B73 exhibiting lower tolerance to this stress than EA2024. In control conditions, EA2024 stalks had higher levels of cellulose, uronic acids and p-coumarate than B73. However, upon drought EA2024 displayed increased levels of arabinose-enriched polymers, such as pectin-arabinans and arabinogalactan proteins, and a decreased lignin content. By contrast, B73 displayed a deeper rearrangement of cell walls upon drought, including modifications in lignin composition (increased S subunits and S/G ratio; decreased H subunits) and an increase of uronic acids. Drought induced more substantial changes in gene expression in B73 compared to EA2024, particularly in cell wall-related genes, that were modulated in an inbred-specific manner. Transcription factor enrichment assays unveiled inbred-specific regulatory networks coordinating cell wall genes expression. Altogether, these findings reveal that B73 and EA2024 inbreds, with opposite stalk-lodging phenotypes, undertake different cell wall modification strategies in response to drought. We propose that the specific cell wall composition conferring lodging resistance to B73, compromises its cell wall plasticity, and renders this inbred more susceptible to drought.

Lodging is one of the causes of maize (Zea mays L.) production losses worldwide and, at least, the resistance to stalk lodging has been positively correlated with stalk strength. In order to elucidate the putative relationship between cell wall, stalk strength and lodging resistance, twelve maize inbreds varying in rind penetration strength and lodging resistance were characterized for cell wall composition and structure. Stepwise multiple regression indicates that H lignin subunits confer a greater rind penetration strength. Besides, the predictive model for lodging showed that a high ferulic acid content increases the resistance to lodging, whereas those of diferulates decrease it. These outcomes highlight that the strength and lodging susceptibility of maize stems may be conditioned by structural features of cell wall rather than by the net amount of cellulose, hemicelluloses and lignin. The results presented here provide biotechnological targets in breeding programs aimed at improving lodging in maize.

Trabajo presentado al Internal Seminar of the Centre de Recerca Agrigenomica (CRAG), celebrado el 12 de julio de 2019.

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.

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.

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 characterised 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 displays 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 the ones 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, being the cell walls of the ccoaomt1 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.

Comunicacion presentada en el 4th Symposium on Biotechnology applied to Lignocelluloses, June 19-22, Madrid (2016)

Comunicacion oral presentada en la XIV Reunion de la Red Tematica Biotecnologia de Materiales Lignocelulosicos (2015)

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.

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.

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.

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.

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.

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.

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.

Plant laccases are enzymes that have been proposed to participate in the last step of lignin biosynthesis. The Polymerisation event remains still much unknown, implicating other enzymes such as peroxidases. To gain more insight in how this polymerisation process takes place in maize, we isolated by differential screening of an elongation maize root cDNA library four cDNA clones encoding a family of laccases. Three of them (ZmLac2, ZmLac4, ZmLac5) were basic enzymes, while one of them (ZmLac3) was an acidic enzyme. Southern analysis indicates that laccases belong to a multigene family in maize. Phylogenetic analysis reveals that ZmLac2, ZmLac4. and ZmLac5 are closely related enzymes, whereas ZmLac3 is a slightly different enzyme. The pattern of mRNA accumulation of ZmLac2, ZmLac3. ZmLac4, and ZmLac5 genes correlates with the maize regions undergoing lignification. Moreover, ZmLac3 is induced by wounding, whereas ZmLac2 and ZmLac5 are repressed and ZmLac4 gene expression is not affected.Taken together, our results suggest that the acidic ZmLac3 enzyme could be involved in the polymerisation of phenolic compounds in maize. Instead, and in agreement with the idea that laccases are enzymes involved in a wide range of physiological processes, results obtained with ZmLac2, ZmLac4, and ZmLac5 lead us to exclude a direct role of these laccases in lignin polymerisation. (c) 2006 Elsevier Ireland Ltd. All rights reserved.

In the present study we analysed polyamine metabolism in Arabidopsis thaliana (ecotype Columbia) rosette leaves collected at vegetative and reproductive stages from plants germinated and grown under increasing salt stress (0-75 mM NaCl) conditions. The expression level of the different isoforms of polyamine biosynthetic enzymes was analysed by reverse transcriptase-polymerase chain reaction (RT-PCR) and the polyamine biosynthetic enzyme activities were determined both in supernatant and pellet fractions. Free and perchloric acid (PCA)-conjugated (soluble and insoluble) polyamines, were measured. At vegetative stage, plants were able to adapt up to 50 mM NaCl, showing a significant growth inhibition only at 75 mM NaCl. At this growth stage and NaCl concentration there was an up-regulation of spermine biosynthesis. At reproductive stage, plants were able to flower up to 50 mM NaCl, even if with a delay of 7 days. On the contrary, at 75 mM NaCl two different phenotypes were isolated: 75/01 (salt sensitive) and 75/02 (salt tolerant). The sensitive plants (75/01) showed a severely stressed phenotype, compared to the tolerant ones (75/02), and the polyamine metabolism was up-regulated, with the increase of free putrescine and spermine.

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. Arabidopsis thaliana 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.