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...