The large vascular meristem of poplar trees with its highly organized secondary xylem enables the boundaries between different developmental zones to be easily distinguished. This property of wood-forming tissues allowed us to determine a unique tissuespecific transcript profile for a well defined developmental gradient. RNA was prepared from different developmental stages of xylogenesis for DNA microarray analysis by using a hybrid aspen unigene set consisting of 2,995 expressed sequence tags. The analysis revealed that the genes encoding lignin and cellulose biosynthetic enzymes, as well as a number of transcription factors and other potential regulators of xylogenesis, are under strict developmental stage-specific transcriptional regulation.T ranscript profiling has the potential to reveal transcriptional hierarchy during development for thousands of genes, as well as providing expression data for many genes of unknown function (1, 2). This is especially true when expression patterns can be obtained for well defined tissues at specific developmental stages. However, this is technically demanding and so far there are no reports demonstrating tissue-specific analysis on cell types from a single developmental sequence. We have studied the developing secondary xylem of poplar, which is highly organized with easily recognized and distinct boundaries between the different developmental stages. Wood formation is initiated in the vascular cambium. Cambial derivatives develop into xylem cells through the processes of division, expansion, secondary wall formation, lignification, and finally, programmed cell death. The large physical size of the vascular meristem in trees offers a unique possibility to obtain samples from defined developmental stages by tangential cryo sectioning (3). To determine the steady-state mRNA levels at specific stages during the ontogeny of wood formation in Populus tremula ϫ Populus tremuloides (hybrid aspen) we sampled 30-m-thick sections through the wood development region and subsequently analyzed the samples by using a spotted cDNA-microarray (4) consisting of 2,995 unique ESTs from hybrid aspen. Our study provides a unique global examination of gene expression patterns that encompasses a developmental gradient within a multicellular organism. Materials and MethodsThe Unigene set was selected from the expressed sequence tags (ESTs) presented in ref. 5, using cluster analysis. ESTs were transformed into Escherichia coli by using TSS competent cells (6), plasmids were prepared by using 96-well Multiscreen FB plates (Millipore), inserts were PCR amplified by using vectorspecific primers, and PCR products were purified on Multiscreen PCR filter plates (Millipore) and spotted in duplicate onto CMT GAPS slides (Corning) by using the GMS 417 Arrayer (Affymetrix, Santa Clara, CA) as described (7). All PCR products were checked on ethidium bromide-stained agarose gels. Nine clones giving double PCR bands were excluded from the analysis.A subset of 2,085 of the 2,995 PCR products in the Unigene set...

Two cDNA libraries were prepared, one from leaves of a field-grown aspen (Populus tremula) tree, harvested just before any visible sign of leaf senescence in the autumn, and one from young but fully expanded leaves of greenhouse-grown aspen (Populus tremula ϫ tremuloides). Expressed sequence tags (ESTs; 5,128 and 4,841, respectively) were obtained from the two libraries. A semiautomatic method of annotation and functional classification of the ESTs, according to a modified Munich Institute of Protein Sequences classification scheme, was developed, utilizing information from three different databases. The patterns of gene expression in the two libraries were strikingly different. In the autumn leaf library, ESTs encoding metallothionein, early light-inducible proteins, and cysteine proteases were most abundant. Clones encoding other proteases and proteins involved in respiration and breakdown of lipids and pigments, as well as stress-related genes, were also well represented. We identified homologs to many known senescence-associated genes, as well as seven different genes encoding cysteine proteases, two encoding aspartic proteases, five encoding metallothioneins, and 35 additional genes that were up-regulated in autumn leaves. We also indirectly estimated the rate of plastid protein synthesis in the autumn leaves to be less that 10% of that in young leaves.Leaf senescence is the final stage in leaf development, and understanding senescence is important not only for purely scientific reasons, but also for practical purposes. Premature senescence leads, for example, to decreased photosynthetic capacity, and consequently lower yield. Senescence is not simply the passive death of a leaf because of aging, but is a tightly controlled process during which cell components are degraded in a coordinated fashion and, when nutrients have been relocated to other parts of the plant body, the cell finally dies (Gan and Amasino, 1997;Nooden et al., 1997). Despite the resemblance with apoptosis of animal cells (Yen and Yang, 1998), a form of programmed cell death, only a few orthologs of genes regulating apoptosis have been found in plants, indicating that there are significant differences between the processes (Koonin and Aravind, 2002). Plant cells respond to some animal apoptosis regulators (e.g. Danon et al., 2000), so there must be common elements between the processes. However, it seems as if plants have developed a unique mode of cell death (Beers, 1997) that, if understood, may give insight into processes that are important for cell integrity and viability. However, very little is known about the details of plant leaf senescence.During the last decade, studies of leaf senescence, focusing especially on Arabidopsis, and other annual species to a lesser extent, have identified a number of senescence-associated genes (SAGs) and cellular mechanisms of senescence have begun to be elucidated, as reviewed by various authors (BuchananWollaston, 1997;Nam, 1997;Quirino et al., 2000). The most obvious visual phenotype of senescen...