Controlling expression of IPT, a gene encoding isopentenyltransferase (the enzyme that catalyzes the rate-limiting step in cytokinin biosynthesis), with a senescence-specific promoter results in the suppression of leaf senescence. Transgenic tobacco plants expressing this chimeric gene do not exhibit the developmental abnormalities usually associated with IPT expression because the system is autoregulatory. Because sufficient cytokinin is produced to retard senescence, the activity of the senescence-specific promoter is attenulated. Senescence-retarded leaves exhibit a prolonged, photosythetically active life-span. This result demonstrates that endogenously produced cytokinin can regulate senescence and provides a system to specifically manipulate the senescence program.

SummaryLeaf senescence is a unique developmental process that is characterized by massive programmed cell death and nutrient recycling. The underlying molecular regulatory mechanisms are not well understood. Here we report the functional analysis of AtNAP, a gene encoding a NAC family transcription factor. Expression of this gene is closely associated with the senescence process of Arabidopsis rosette leaves. Leaf senescence in two T-DNA insertion lines of this gene is significantly delayed. The T-DNA knockout plants are otherwise normal. The mutant phenotype can be restored to wild-type by the intact AtNAP, as well as by its homologs in rice and kidney bean plants that are also upregulated during leaf senescence. Furthermore, inducible overexpression of AtNAP causes precocious senescence. These data strongly suggest that AtNAP and its homologs play an important role in leaf senescence in Arabidopsis and possibly in other plant species.

Leaf senescence is a complex developmental process during which essential nutrients are recycled. In order to unravel the biochemical pathways and regulatory mechanisms that underlie this process, it would be valuable to examine the transcriptome associated with leaf senescence. Accordingly, an Arabidopsis thaliana leaf senescence cDNA library with approximately 10 4 recombinant clones was subjected to large-scale single-pass sequencing. Approximately 6200 expressed sequence tags (ESTs) were obtained, corresponding to 2491 unique genes. These included 134 genes encoding transcription factors and 182 genes whose products are components of signal transduction pathways, such as the mitogen-activated protein kinase (MAPK) cascades. A total of 116 of these genes are predicted to be involved in protein turnover, including 75 genes associated with the ubiquitin-proteasome pathway and 35 proteinases. Many of the genes are predicted to encode transporters for ions, amino acids and sugars, consistent with the substantial nutrient recycling during leaf senescence. In addition, this study revealed ESTs for 98 annotated genes for which ESTs did not previously exist and 46 novel transcribed units that have not previously been annotated in the Arabidopsis genome. Approximately one-third of the 2491 genes are predicted to encode proteins with unknown functions. The genes are distributed evenly on the five chromosomes.

In this work, the role of jasmonic acid (JA) in leaf senescence is examined. Exogenous application of JA caused premature senescence in attached and detached leaves in wild-type Arabidopsis but failed to induce precocious senescence of JA-insensitive mutant coi1 plants, suggesting that the JA-signaling pathway is required for JA to promote leaf senescence. JA levels in senescing leaves are 4-fold higher than in non-senescing ones. Concurrent with the increase in JA level in senescing leaves, genes encoding the enzymes that catalyze most of the reactions of the JA biosynthetic pathway are differentially activated during leaf senescence in Arabidopsis, except for allene oxide synthase, which is constitutively and highly expressed throughout leaf development. Arabidopsis lipoxygenase 1 (cytoplasmic) expression is greatly increased but lipoxygenase 2 (plastidial) expression is sharply reduced during leaf senescence. Similarly, AOC1 (allene oxide cyclase 1), AOC2, and AOC3 are all up-regulated, whereas AOC4 is down-regulated with the progression of leaf senescence. The transcript levels of 12-oxo-PDA reductase 1 and 12-oxo-PDA reductase 3 also increase in senescing leaves, as does PED1 (encoding a 3-keto-acyl-thiolase for ␤-oxidation). This represents the first report, to our knowledge, of an increase in JA levels and expression of oxylipin genes during leaf senescence, and indicates that JA may play a role in the senescence program.Originally identified as a major component of fragrant oils, methyl jasmonate (MeJA) and its precursor jasmonic acid (JA) were first demonstrated to promote senescence in detached oat (Avena sativa) leaves (Ueda and Kato, 1980), and were subsequently shown to be a class of plant growth regulator that plays pervasive roles in several other aspects of plant development, including seed germination and pollen development, responses to mechanical and insect wounding, pathogen infection, and drought stress (for review, see Hildebrand et al., 1998;Schaller, 2001). Recent molecular genetic studies have confirmed the involvement of JA both in developmental (Xie et al., 1998;Sanders et al., 2000;Stintzi and Browse, 2000) and defense-related processes (Vijayan et al., 1998;Xie et al., 1998;Ryan, 2000). The role of JA in leaf senescence is not clear. Exogenously applied JA and MeJA led to decreased expression of photosynthesis-related genes encoding, for example, the small subunit of Rubisco, reduced translation and increased degradation of Rubisco, and rapid loss of chlorophyll in barley (Hordeum vulgare) leaves (Weidhase et al., 1987;Parthier, 1990). However, many questions remain unanswered, such as whether JA levels change in leaves undergoing senescence and whether specific genes of JA biosynthesis are induced during senescence.The biosynthetic pathway of JA, starting with ␣-linolenic acid, has been elucidated ( Fig. 1; Vick and Zimmerman, 1984;Schaller, 2001). There may exist two pathways for JA biosynthesis in plant tissues, a chloroplast-localized and a cytoplasm-localized pathway (Creelman a...