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The last stage of xylem development is programmed death of the cells, which is followed by complete autolysis of the cell contents. Two cell types predominate in the xylem, the vessel elements and the fibres, and my earlier research in Populus trees has revealed that both of these cell types display programmed cell death (PCD), but in a very different manner. I am interested in why the xylem fibres have to die and the underlying molecular mechanism. From the biotechnological point of view, the fibres should stay alive as long as possible, assince extending the lifetime of the fibres is expected to result in thicker cell walls and therefore higher biomass yields of wood.

Identification of key regulators of xylem cell death should enable modifications in the cell death process using transgenic approaches, in order to test how they affect wood properties. This is especially tractable in xylem fibres, which constitute the main bulk of biomass.
We have taken three different approaches to identify key genes in the regulation of xylem cell death: one is based on knowledge from other PCD processes, mainly in Arabidopsis thaliana; one on sequencing of ESTs from Populus stem tissues undergoing fibre cell death, and the third isone based on microarray analyses of transcripts from various vascular tissues of Populus stems and comparative genom- ics approaches.

A longitudinal section of Populus wood is shown here after staining with DAPI, which stains nuclear DNA. The contours of the longitudinal xylem fibers and the radially oriented xylem rays are revealed by unspecific staining of the cell walls. The nuclei of the xylem fibers are appressed against the cell wall due to the high pressure from the vacuole in the living cells. When the fibers die, the vacuole bursts and the remaining cellular contents are degraded by hydrolytic enzymes released from the vacuole.

We have studied xylem maturation in a cell culture system where xylem vessel like structures, called tracheary elements (TEs), differentiate in a semi-synchronous manner after hormonal stimulus. This cell culture system has allowed us to define a critical role for ethylene in xylem maturation by using various pharmacological agents. The figure shows three mature TEs with spiral-type secondary cell wall thickenings.

We have identified a metacaspase gene that is specifically expressed in the xylem elements of both /Populus /and Arabidopsis. The figure shows a confocal microscope image of green fluorescent protein (GFP) expression that is driven by the Arabidopsis /metacaspase 9/ promoter in the Arabidopsis root. The GFP signal is located in the nuclei of the vessel elements that can be recognised by the spiral cell wall thickenings.These analyses have revealed novel putative regulators of xylem PCD, as well as several homologues of known PCD regulators, such as vacuolar processing enzymes, autophagy-related genes, Bcl-2-associated athanogene (BAG) genes and a metacaspase gene. Three candidate genes were selected from these genes for detailed functional and molecular characterisation, encoding: a metacaspase, a bifunctional nuclease, and a polyamine biosynthetic ACL5. The functions of the metacaspase and bifunctional nuclease genes are currently being studied with reverse genetics approaches in both Arabidopsis plants and Populus trees.
The polyamine biosynthetic ACL5 gene has been found to be specifically expressed in the early developing vessels, and a mutation in ACL5 resulted in altered xylem development. Results of xylem morphology analysis and experiments with the xylogenic Zinnia elegans cell culture (see below) lead us to conclude that ACL5 prevents premature death of the developing xylem vessels to allow complete differentiation. This model is supported by the finding that transgenic Arabidopsis plants expressing the DT-A toxin gene under the control of the ACL5 promoter display similar alterations in xylem development to the acl5 mutant.
We have also studied the role of the plant hormone ethylene in xylem differentiation. In an in vitro tracheary element (TE) differentiation system of Zinnia elegans, we showed that ethylene has an important role in the control of lignification and cell death of TEs, since application of ethylene signalling inhibitors blocked both of these processes. We also created suppressive subtractive hybridisation (SSH) libraries in Zinnia elegans, in order to identify genes that were
1) active during the cell death phase of vessel differentiation and 2) that were dependent on the cell-death stimulatory effect of ethylene. One gene fulfilling these criteria was a PIRIN gene that also showed activity during the xylem cell death stage in Populus. There are four PIRIN genes in Arabidopsis, and we have started functional characterisation of this whole gene family.

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