SUMMARYFew 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.

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.

Bean (Phaseolus vulgaris L.) cell suspensions were adapted for growth in 12 &mgr;M dichlobenil (2,6-dichlorobenzonitrile or DCB) by a stepwise increase in the concentration of the inhibitor in each subculture. Non-tolerant suspensions (I50 = 0.3 &mgr;M) gave rise to single cells or small clusters while tolerant cell suspensions (I50 = 30 &mgr;M) grown in DCB formed large clusters. The cells in these clusters were surrounded by a thick and irregular cell wall with a lamellate structure and lacking a differentiated middle lamella. Analysis of habituated cell walls by Fourier transform infrared spectroscopy and cell wall fractionation revealed: (1) a reduced amount of cellulose and hemicelluloses, mainly xyloglucan (2) qualitative and quantitative differences in pectin levels, and (3) a non-crystalline and soluble beta-1,4-glucan. When tolerant cells were returned to medium lacking DCB, the size of the cell clusters was reduced; the middle lamella was only partly formed, and the composition of the cell wall gradually reverted to that obtained with non-tolerant cells. However, dehabituated cells (I50 = 12 &mgr;M) were 40-fold more tolerant to DCB than non-tolerant cells and were only 2.5-fold more sensitive than tolerant cells.

Growth of maize (Zea mays L.) callus-culture cells was inhibited using dichlobenil (2,6 dichlorobenzonitrile, DCB) concentrations > or =1 microM; I (50) value for the effect on inhibited fresh weight gain was 1.5 microM. By increasing the DCB concentration in the culture medium, DCB-habituated cells became 13 times more tolerant of the inhibitor (I (50): 20 microM). In comparison with non-habituated calluses, DCB-habituated calluses grew slower, were less friable and were formed by irregularly shaped cells surrounded by a thicker cell wall. By using an extensive array of techniques, changes in type II cell wall composition and structure associated with DCB habituation were studied. Walls from DCB-habituated cells showed a reduction of up to 75% in cellulose content, which was compensated for by a net increase in arabinoxylan content. Arabinoxylans also showed a reduction in their extractability and a marked increase in their relative molecular mass. DCB habituation also involved a shift from ferulate to coumarate-rich cells walls, and enrichment in cell wall esterified hydroxycinnamates and dehydroferulates. The content of polymers such as mixed-glucan, xyloglucan, mannans, pectins or proteins did not vary or was reduced. These results prove that the architecture of type II cell walls is able to compensate for deficiencies in cellulose content with a more extensive and phenolic cross-linked network of arabinoxylans, without necessitating beta-glucan or other polymer enhancement. As a consequence of this modified architecture, walls from DCB-habituated cells showed a reduction in their swelling capacity and an increase both in pore size and in resistance to polysaccharide hydrolytic enzymes.