FAIR-CT97-3972 Wood formation processes: the key to improvement of the raw material

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FAIR Area 1.3 - Forestry-Wood Chain : Plant Genetics : Wood (Lignocellulose)

	Contract No:	FAIR-CT97-3972
	Date Prepared:	April 2001
	Source:	Second Annual Progress Report Abstract

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Second Annual Progress Report Abstract

Objectives

The main objective of the project is to provide the generic scientific information needed to guide strategies for genetic engineering of wood so that its properties can be tailored for the pulp industry. This will be achieved by:

• characterising key regulatory processes during commitment of cambial cells. Examining the subsequent differentiation of cambial cell derivatives at the molecular, biochemical, ultrastructural and anatomical levels.
• quantification of the role of endogenous auxin in the control of these processes and the verification of the hypothesis that different endogenous levels of this growth substance influence wood structure and properties.

Activities

The objectives will be achieved through a study of cellular differentiation processes occurring during the formation of wood cells from the vascular cambium in hybrid aspen (Populus tremula x tremuloides). Wild-type plants, and plants genetically modified to have altered endogenous auxin levels have been produced and are being studied to understand fundamental processes as the molecular, biochemical, ultrastructural and anatomical levels. This work may be summarised as follows:

• At the anatomical level: the effect of different nitrogen levels in the soil, and altered endogenous auxin levels on the rate and pattern of cell production by the cambium, and on wood anatomy is being investigated by light microscopy and histochemistry.
• At the ultrastructural level: the role of the cytoskeleton and intercellular communication in cell commitment to differentiate along a particular pathway.
• At the biochemical level: the role of different iso-forms of the enzyme pectin methylesterase on the pathway of differentiation, coupled with a study of the structure of the different iso-forms in an attempt to discover how this affects their activity.
• At the molecular level: The genes responsible for coding for the pectin methylesterase iso-forms have been sequenced, and will be used to produce anti-sense genes to down- regulate the enzyme in a second generation of transformed plants to obtain more information on its function.

Progress

Most of the work as set out above has been completed, although a few items are still outstanding. These include completion of the study of the association of actin with plasmodesmata, some remaining cell modelling work, and examination of plants modified with antisense PME. Work on the cytoskeleton has been completed and the changes in differentiating cells described. Of particular interest has been the observation of actin and myosin associated with developing bordered pits in vessel elements of the xylem. It has become clear that there is a continuum of cytoskeletal elements through the long-lived parenchyma cells of the symplastic system. Work on the distribution of plasmodesmata and their de novo formation in the cambium and differentiating xylem cells has been completed. No evidence has been found to suggest de novo formation of plasmodesmata in differentiating cells, showing that all secondary formation of plasmodesmata must be occurring in the cambial zone.

IAA levels have been measured in tension wood, and surprisingly, found to be higher than levels in normal wood. Cell counts to study patterns of wood formation have shown that the number of cells produced is dependent on temperature as well as time, with a linear relationship existing between number of degree days and number of xylem cells produced by the cambium. Vessel diameters have been found to be smaller in transformed trees as compared with wild-type trees, although fibre diameters remained unchanged.

Work to determine the structure of pectin methylesterase was delayed owing to a lack of sufficient protein for satisfactory crystallisation. As a result, this work was pre-empted by the publication of the structure of a bacterial PME by another group of workers. This was entirely in agreement with the structure predicted on the basis of sequencing work carried out early in this project. In transformed plants, acidic forms of PNE were shifted towards the differentiating xylem, while in the wild-types, they were predominantly associated with the cambium.

Achievements

• IAA measurements in tension wood carried out.
• Propagation of hybrid aspen tissue with sense and antisense PME genes.
• Completion of analysis of PME distribution in the cambium and differentiating tissues.
• Mapping of the distribution of cytoskeletal elements within the parenchyma system in the wood.
• Description of the association of actin and myosin with developing pit borders.
• Completion of study of de novo formation of plasmodesmata.
• Identification of degree-days as a major factor controlling xylem cell production.
• Completion of anatomical study of plants grown in 1998, 1999 and 2000.

Future activities

To be carried out:

• Continuation of investigations of the role of actin in association with plasmodesmata.
• Completion of the study of antisense PNE plants, and a study of the structure and anatomy of wood they produce.
• Further data analysis on cell production rates.