A genomic approach to investigate developmental cell death in woody tissues of Populus trees

Abstract

Background: Poplar (Populus sp.) has emerged as the main model system for molecular and genetic studies of forest trees. A Populus expressed sequence tag (EST) database (POPULUSDB) was previously created from 19 cDNA libraries each originating from different Populus tree tissues, and opened to the public in September 2004. We used this dataset for in silico transcript profiling of a particular process in the woody tissues of the Populus stem: the programmed death of xylem fibers.

Results: One EST library in POPULUSDB originates from woody tissues of the Populus stem where xylem fibers undergo cell death. Analysis of EST abundances and library distribution within the POPULUSDB revealed a large number of previously uncharacterized transcripts that were unique in this library and possibly related to the death of xylem fibers. The in silico analysis was complemented by a microarray analysis utilizing a novel Populus cDNA array with a unigene set of 25,000 sequences.

Conclusions: In silico analysis, combined with the microarray analysis, revealed the usefulness of non-normalized EST libraries in elucidating transcriptional regulation of previously uncharacterized physiological processes. The data suggested the involvement of two novel extracellular serine proteases, nodulin-like proteins and an Arabidopsis thaliana OPEN STOMATA 1 (AtOST1) homolog in signaling fiber-cell death, as well as mechanisms responsible for hormonal control, nutrient remobilization, regulation of vacuolar integrity and autolysis of the dying fibers.

Figure 1

Sampling of xylem tissues. A transverse section from the base of the stem showing xylem tissues sampled from a Populus tree for the microarray and the RT-PCR analysis. The bark was peeled off resulting in a fracture in the cambial zone. For RT-PCR analysis, the different xylem tissues were successively scraped from the surface of the exposed stem to the border with the dead wood. For microarray analysis, the tissues were pooled into two samples: A (early fiber development) and B (fiber-cell death). The fiber-cell death sample corresponded closely to the tissues collected for construction of the fiber death cDNA library [13]. V, dead vessel; Fs, developing fibers. Note that the development of vessels is completed within the region of cell expansion, and that the fibers develop at a much slower pace, visualized by the gradual loss of the cytoplasm of the fibers. The asterisks denote fibers close to the moment of death with barely detectable cytoplasm.

Figure 2

Assignment of gene ontologies to ESTs in the cDNA libraries representing fiber death, tension wood and leaf senescence in the POPULUSDB.

Figure 3

The most abundant transcripts in the fiber death library. The EST distribution in the different cDNA libraries of the POPULUSDB is shown for the 28 most abundant transcripts of the fiber death library. The transcripts are shown in descending order of EST abundance in the library. For a detailed description of the cDNA libraries, see [13]. The color code at the bottom of the figure indicates the number of ESTs in each library for each transcript.

Figure 4

RT-PCR analysis of gene expression in different parts of a Populus tree. Expression of glycosyl hydrolase family 1 protein (POPLAR.11628), proline-rich protein (POPLAR.11658), oligopeptide transporter (POPLAR.11639), an expressed protein (POPLAR.11646), an F-box protein (POPLAR.11624) and a protease inhibitor/seed storage/lipid transfer protein (POPLAR.9335) is shown in relation to 18S DNA expression. Sampling of the different tissue types is described in Materials and methods.

Figure 5

Gene expression in the 25K Populus cDNA array and in the POPULUSDB. The 50 most highly expressed transcripts in the microarray analysis of xylem fibers undergoing cell death are shown together with the corresponding library distribution in POPULUSDB. The transcripts are listed in descending order of expression ratio between the fiber-cell death sample and the early fiber development sample (see Figure 1). The color code at the bottom of the figure indicates the number of ESTs in each library for each transcript. The whole list is given in Additional data file 2.