Project leaders: Björn Sundberg,
Geoffrey Daniel,Tuula Teeri
Tel.: +46 90 7865800 
Fax: +46 90 7865901, 78690092


Biosynthesis of the Cell Wall

Enzymes Determining Wood Fibre Composition and Ultrastructure

Aim: This project strives to elucidate the mechanisms and enzymes responsible for the physico-chemical properties of wood fibres. It will focus on the carbon skeleton of the fiber, i.e. cellulose and hemicellulose.

Background: Wood fibers are composite structures consisting of a network of cellulose and hemicellulose encrusted with lignin and structural proteins. These components form predetermined patterns resulting in characteristic fiber ultrastructure. The fiber morphology, ultrastructure and chemical composition differ between types of fibers, e.g. compression wood fibers, juvenile fibers and early wood/late wood fibers, giving them their specific physico-chemical properties. These properties are dictated by strictly controlled expression of a number of different enzyme systems acting in developing xylem cells. Despite their importance, the enzymes involved in the wood fiber formation are not very well understood, partially since they were not easily isolated using traditional biochemical techniques. High through-put sequencing of genes expressed in the wood-forming tissues of a model tree species, the hybrid aspen, created an opportunity to identify enzymes involved in fiber formation and to elucidate their role. This in turn opens the prospects for the development of new biotechnical applications in fiber modification.

Methods: The project can be divided into three major tasks.
Task 1. Identification and characterization of enzymes potentially involved in fiber formation In this task we will take advantage of the EST-sequence database of the wood forming tissues of poplar (www.biochem.kth.se/PopulusDB/) in order to identify and characterize the enzymes involved in the different steps of cell wall synthesis and assembly. Based on sequence similarities with enzymes in other organisms, we have identified several different enzyme families, which may play an important role in the fiber biosynthesis, particularly in the formation of carbohydrate components of wood fibers. These include e.g. cellulose synthases, cellulases, xyloglucan endo transglycosylases (XET), expansins and a putative xylan endohydrolase. At KTH, a number of such clones are currently being full-length sequenced followed by detailed expression analysis using the microchip technology.

Task 2. Localization of the enzymes active during fiber formation Localization of enzymes in the wood forming tissues can be done using antibodies specific of each different enzyme. Using a robust expression system for several of the target enzymes in E. coli, we have generated antibodies in egg yolk. The antibodies will specifically recognize each enzyme in the tissue extract or a section allowing us to follow its expression during the process of xylem cell development. We are currently performing immuno localization experiments for XET - an enzyme involved in the assembly of hemicellulose-cellulose network in developing fibers. An example of immunolocalisation of XET in the cambial region of hybrid aspen using confocal laser microscopy is shown below.

A
   B
A. Immunlocalisation of XET in the cambial region of poplar using egg yolk anti- XET antibodies. Label (green) is evident in the peripheral cytoplasm of sieve tubes cells, cambium and developing xylem cells.
B shows the same region labeled with control pre-immune egg yolk proteins.

Task 3. Investigation of the role of enzyme function for cell wall ultrastructure
Transgenic techniques allow us to up- and down regulate the activity of any enzyme in planta. Careful phenotypic analysis of the clones allows us to determine the role of each enzyme and elucidate mechanisms behind assembly of the cell wall components into a functional structure. We are currently producing hybrid aspen trees with altered expression of selected cell wall enzymes. The ultrastructure and the chemical composition of the fiber in these trees will be characterized by light and electron microscopy as well as by chemical analysis.

Participants: The project is a part of a research network of Swedish groups in the fields of molecular biology and enzymology (KTH, Stockholm), wood biology (SLU, Umeň) as well as wood structural analysis and utilization in the pulp and paper industries (SLU, Uppsala).

Project group:
Bj÷rn Sundberg, SLU Umeå
Ewa Mellerowicz, SLU, Umeň
Geoffrey Daniel, SLU
Tuula Teeri, KTH
Kristina Blomqvist, KTH
Harry Brumer, KTH