FAIR-CT96-5050 Molecular genetic analysis and tagging of specific genes controlling wood quality in walnut (Juglans spp)

Contacts




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FAIR Area 1.3 - Forestry-Wood Chain : FAIR Marie Curie Research Training Grants : Solid Wood Products

Objectives:

The main objective of this project is to tag genes controlling wood quality in walnut (Juglans spp). Wood quality is an important trait and the formation of heartwood is a key process as it affects colour and durability of wood. While some biochemical mechanisms of wood colouring are already described, no information is available on gene expression during heartwood formation in walnut. In this work, genes involved in heartwood formation will be tagged as genes potentially related to wood quality. This strategy is based on two approaches:

• RT-PCR and gene expression study for candidate genes controlling phenolics metabolism;
• Construction and screening of CDNA libraries. The selected CDNA clones will provide homologous probes and sequence information to be used in mapping programmes, as well as in wood physiology studies.

Summary of achievements:

The formation of heartwood has a strong influence on the quality of walnut wood. Nevertheless, very little information is available on gene expression during heartwood formation. The research activity has focused primarily on gaining a deeper knowledge of the molecular basis of this process by cloning genes expressed in aged sapwood (transition zone) transforming into heartwood.

Activities and Results:

The application of molecular analyses to aged wood tissues has required a special approach for the set up of suitable techniques. Taking into account the chemical composition of wood extractives, the key enzymes of phenylpropanoid pathway and flavonoids biosynthesis have been targeted as candidate genes, that could potentially control wood quality. The expression of specific genes was investigated by Reverse Transcriptase-PCR (RT-PCR) in xylem tissues of black walnut (Juglans nigra L.) trees. By this approach it was possible to detect the expression of genes encoding 4-Coumarate:CoA ligase (4CL), Chalconc synthase (CHS), Flavanone 3-hydroxylase (F3H), and Dlhydroflavonol-4-reductase (DFR) in the transition zone of Juglins nigra.

The RT-PCR products were cloned and sequenced and the obtained sequences confirmed the identity of the cloned CDNA fragments. These results represent a technical breakthrough as far as this particular tissue is concerned. This is the first report showing the activation of phenylpropanoid metabolism at the transcription level in the transition zone.

In order to describe the temporal dynamics of heartwood formation, walnut trees were cut and collected for analyses every 6 weeks along the year. According to the results of chemical analyses and anatomy observations, the study of gene expression was focused on Juglans nigra trees collected in July and January. Indeed, these periods should represent highest and lowest peaks in the accumulation of wood extractives. Flavonoids compounds play an important role as wood extractives. Thus the expression of genes specific of flavonoids biosynthesis (CHS, F3H, DFR) is being studied by RT-PCR in young and aged wood tissues. The expression of these genes was detected in all tissues analysed. By differential cleaving of RT-PCR products with restriction enzymes it was possible to show that only two members of CHS multigene family are expressed in walnut wood tissues. These analyses are now extended to F3H and DFR. A quantitative analysis of gene expression is being carried out on the same samples by Northern Blotting hybridisation. A severe limitation for this type of analysis is the very low yield of RNA extracted from aged wood tissues (1500 to 3000 folds lower than in young tissues).

These results may be a starting point for promising perspectives. The cloned CDNA fragments provide sequence information and homologous CDNA probes. Future evolution of the present work will be the construction of a CDNA library from transition zone. This library will be screened to tag genes differentially expressed in this tissue. Selected CDNA clones will be used as Expressed Sequence Tags (EST) in studies of genetic variability, in mapping programmes, as well as in wood physiology studies.

Keywords: Heartwood, walnut, Juglans spp

Contacts

Scientific Supervisor

• INRA Unité Amélioration, Génétique et Physiologie Forestière Physiology of Tree Development Team / FRANCE