Fisica

The Plant Journal (2004) 39, 612–628

doi: 10.1111/j.1365-313X.2004.02160.x

Molecular events in senescing Arabidopsis leaves
Ji-Feng Lin and Shu-Hsing Wu* Institute of Botany, Academia Sinica, Taipei 11529, Taiwan
Received 30 March 2004; revised 15 May 2004; accepted 25 May 2004. * For correspondence (fax 886-2-2782-7954; e-mail shuwu@gate.sinica.edu.tw).

Summary Senescence is the finalstage of leaf development. Although it means the loss of vitality of leaf tissue, leaf senescence is tightly controlled by the development to increase the fitness of the whole plant. The molecular mechanisms regulating the induction and progression of leaf senescence are complex. We used a cDNA microarray, containing 11 500 Arabidopsis DNA elements, and the whole-genome Arabidopsis ATH1 GenomeArray to examine global gene expression in dark-induced leaf senescence. By monitoring the gene expression patterns at carefully chosen time points, with three biological replicates each time, we identified thousands of up- or down-regulated genes involved in dark-induced senescence. These genes were clustered and categorized according to their expression patterns and responsiveness to dark treatment.Genes with different expression kinetics were classified according to different biological processes. Genes showing significant alteration of expression patterns in all available biochemical pathways were plotted to envision the molecular events occurring in the processes examined. With the expression data, we postulated an innovative biochemical pathway involving pyruvate orthophosphate dikinase ingenerating asparagine for nitrogen remobilization in dark-treated leaves. We also surveyed the alteration in expression of Arabidopsis transcription factor genes and established an apparent association of GRAS, bZIP, WRKY, NAC, and C2H2 transcription factor families with leaf senescence. Keywords: Arabidopsis, senescence, microarray, pyruvate orthophosphate dikinase, transcription factor,biochemical pathway.

Introduction Unable to flee the habitat in which they grow, plants proceed with senescence in a highly coordinated manner (BuchananWollaston et al., 2003; Gan, 2003; Lim et al., 2003; Quirino et al., 2000; Yoshida, 2003). Many biotic and abiotic stresses also trigger senescence. Processes associated with leaf senescence include the disorganization of chloroplasts, shrinkage ofcytoplasmic volume and decrease in cellular metabolic activities. Significant metabolic changes are involved in the degeneration and remobilization of macromolecules that accumulate during growth and maturation. Research efforts to reveal the underlying molecular mechanism of plant senescence began with the collecting and analyzing of senescence-associated genes (SAGs). Sequence and/or functionalanalyses revealed that SAG-encoded proteins include proteases, nucleases, lipid-, carbohydrate- and nitrogen-metabolizing enzymes, stressresponsive proteins, and transcriptional regulators (for review see Buchanan-Wollaston et al., 2003). Previous studies have shown that SAGs have common and distinct
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expression profiles in response to various senescenceinducing factors (He et al., 2001), whichsuggests that plant cells have a complex fine-tuning mechanism to cope with various signals in senescence. The advances in the tools of molecular biology research have greatly helped the investigation of SAGs. Bhalerao et al. (2003) reported on the genes, preferentially expressed in autumn leaves, by sequencing expressed sequence tags (ESTs) of cDNA libraries made from field-grown aspen (Populustremula). A DNA microarray with 13 490 aspen ESTs was later created and used to study the leaf transcriptome of aspen leaves during natural autumn senescence (Andersson et al., 2004). Large-scale identification of SAGs via suppression substractive hybridization has added 70 new members to the current SAG collection in Arabidopsis (Gepstein et al., 2003). More recently, transcriptome associated with...