Montserrat Capellades

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.

MAPK phosphatase MKP2 mediates disease responses in Arabidopsis and functionally interacts with MPK3 and MPK6

Mitogen-activated protein kinase (MAPK) cascades have important functions in plant stress responses and development and are key players in reactive oxygen species (ROS) signalling and in innate immunity. In Arabidopsis, the transmission of ROS and pathogen signalling by MAPKs involves the coordinated activation of MPK6 and MPK3; however, the specificity of their negative regulation by phosphatases is not fully known. Here, we present genetic analyses showing that MAPK phosphatase 2 (MKP2) regulates oxidative stress and pathogen defence responses and functionally interacts with MPK3 and MPK6. We show that plants lacking a functional MKP2 gene exhibit delayed wilting symptoms in response to Ralstonia solanacearum and, by contrast, acceleration of disease progression during Botrytis cinerea infection, suggesting that this phosphatase plays differential functions in biotrophic versus necrotrophic pathogen-induced responses. MKP2 function appears to be linked to MPK3 and MPK6 regulation, as indicated by BiFC experiments showing that MKP2 associates with MPK3 and MPK6 in vivo and that in response to fungal elicitors MKP2 exerts differential affinity versus both kinases. We also found that MKP2 interacts with MPK6 in HR-like responses triggered by fungal elicitors, suggesting that MPK3 and MPK6 are subject to differential regulation by MKP2 in this process. We propose that MKP2 is a key regulator of MPK3 and MPK6 networks controlling both abiotic and specific pathogen responses in plants.

The maize caffeic acid O-methyltransferase gene promoter is active in transgenic tobacco and maize plant tissues

The pattern of expression directed by the promoter of the maize caffeic acid O-methyltransferase (COMT) gene was studied by histochemical and fluorometric β-glucuronidase (GUS) analysis in transgenic maize and tobacco plants. The COMT promoter directs GUS expression to the xylem and the other tissues undergoing lignification, and it responds to wounding and to elicitors. In transgenic maize plants, expression of GUS corresponds to the pattern of expression of the endogenous COMT gene as determined by northern analysis and in situ hybridization. The pattern in transgenic tobacco plants clearly shows that the maize promoter sequence is recognized by tobacco transcriptional factors, in spite of the anatomical differences and the evolutionary distance between these two species. The results suggest that the most significant promoter signals that induce the specific expression of the lignin COMT are conserved in different species.

Maize α-tubulin genes are expressed according to specific patterns of cell differentiation

In the past few years many α- and β-tubulin genes of different organisms have been cloned and studied, and in most systems studied they constitute multigene families. In plants, most studies have been done in Arabidopsis thaliana and Zea mays. In this paper, the study of mRNA accumulation by in situ hybridization and the activity of three maize α-tubulin gene promoters (tua1, tua2 and tua3) in transgenic tobacco plants are described. In maize, the expression of these three tubulin isotypes differ in the root and shoot apex and is associated with different groups of cells throughout the distinct stages of cell differentiation. In transgenic tobacco plants the promoters of the genes, fused to the uidA reporter gene (GUS), direct expression to the same tissues observed by in situ hybridization experiments. The tua1 promoter is mainly active in cortex-producing meristematic cells and in pollen, whereas tua3 is active in cells which are differentiating to form vascular bundles in the root and shoot apices. The accumulation of tua2 mRNA is detected by RNA blot in a similar form as tua1, but at a very much low level. In situ hybridization indicates that the tua2 mRNA specifically accumulates in the maize root epidermis. No GUS staining was detected in transgenic tobacco plants with the tua2 promoter. The difference in expression of the specific genes may be linked to processes where microtubules have different functions, suggesting that in plants, as in animals, there are differences in the function of the tubulin isotypes.

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.