Functional characterization of poplar and Arabidopsis wood - related hydrolases by reverse genetics and overexpression in Arabidopsis : effects on hypocotyl elongation

Abstract: Trees and their wood present a very valuable and renewable resource (paper industry, raw materials and constructions). Consequently study of wood formation is an important research field. The wood or secondary xylem is formed from the vascular cambium where meristematic activity takes place. During differentiation of wood cells, there is radial expansion and intrusive tip growth phase followed by the secondary wall deposition and programmed cell death. Cell expansion and intrusive tip growth is regulated by the plasticity of the primary cell wall. This wall is composed of pectin, cellulose, hemicelluloses (xyloglucans in particular), and proteins including some wall residing enzymes that are important for regulating wall plasticity. To study the process of wood cell expansion, I focused on a two kinds of hydrolases expected to be involved in the cell wall plasticity/biosynthesis: xyloglucan endotransglycosylases and cellulases. Xyloglucan endotransglycosylases /hydrolases (XTHs or formerly XETs) are enzymes capable of mediating the endocleavage of a xyloglucan molecule and the re-ligation of the cut donor to the acceptor that is another xyloglucan molecule or, in some cases, the water. Cellulases on the other hand are ≤,1-4 endoglucanases and could hydrolyze cellulose or hemicellulose molecules. Both these enzymes exist as multigene families and it is a challenge to determine the function of individual members of these families. PttXET16A is a XTH that has been previously found as expressed in the wood forming tissues of poplar (Bourquin et al, 2002). Single insert, homozygotic lines of Arabidopsis expressing PttXET16A ectopically were obtained. I studied effects of the ectopic expression of PttXET16A on the elongation of the hypocotyls in the dark and in the light condition, and the effects on cell sizes inboth light conditions. In both light and dark conditions, I observed the inhibition of hypocotyl elongation and the reduction in the final size of cells in epidermis, cortex and endodermis in different PttXET16A transgenic lines. This indicates that PttXET16A enzyme reduces cell wall plasticity probably by incorporation of xyloglucan to the wall and by creation of more cross-links between cellulose microfibrils. Arabidopsis XTHs have been previously identified in the hypocotyls of the plants induced to form a wood (N. Nishikubo et al., unpublished). Single locus T-DNA insertions mutants of these genes were obtained. I studied the elongation of the mutant hypocotyls and cell sizes in the hypocotyl in the light and dark conditions. In six mutants studied: XTH4, XTH9, XTH16, XTH24 and two independent lines of XTH22, I observed more or less visible decrease of the hypocotyl elongation only in the light and minor effects on the cell sizes in both experimental conditions. I presume that the XTH genes play role in the hypocotyl elongation in the light causing more cell division but no cell elongation because cell size was not changed in the mutant. To test if XTH genes have redundant functions, double mutants were created and I isolated homozygotic double mutant lines of XTH9 x XTH22, XTH4 x XTH9 and XTH22 x XTH4. PttCEL9B is a cellulase expressed in the meristematic and expanding cells of the wood-forming zone of poplar. Its expression pattern suggests a function in xylem cell expansion. Single insert, homozygotic lines expressing PttCEL9B ectopically were studied for effects on the hypocotyl elongation in the light and dark condition. I observed a big increase of the hypocotyl length in the light but no differences in the dark. These results suggest that PttCEL9B activity is important for hypocotyl cell expansions only in light conditions. KORRIGAN is a cellulase implicated in the cellulose biosynthesis in Arabidopsis. Its weak mutant allele, irx2, has a collapsed vessel phenotype, and the poplar orthologue of this gene is up regulated during the secondary cell wall synthesis in developing wood, suggesting a function for KORRIGAN in the wood formation. I studied the hypocotyl elongation in three mutants: kor1-1, which is a strong mutation, irx2-1 and irx2-2 -less serve mutations. I observed the inhibition in the hypocotyl length in the light condition in irx2-2 and in the dark condition in kor1-1. This result confirms that KORRIGAN endoglucanase can weaken cell wall and its absence could increase wall rigidity. Different result was obtained for irx2-1 mutant, which had longer hypocotyls in light conditions than wild type plants. It could be explained by fact that studied lines come from different backgrounds. Present work focused the hypocotyls elongation and the growth of various cell layers in the hypocotyls on the early postembryonic growth. I demonstrated the growth effects or the lack of effects at this stage for the several overexpressing lines and mutants in two hydrolases, XTH and cellulase. The continued work should examine the secondary growth of these lines.