Summary: Leaf 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 10⁴ 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.

AtNAP is a NAC family transcription factor gene that plays a key role in leaf senescence but its underlying mechanisms are not known. SENESCENCE-ASSOCIATED GENE113 (SAG113), a gene encoding a Golgi-localized protein phosphatase 2C family protein phosphatase, mediates abscisic acid (ABA)-regulated stomatal movement and water loss specifically during leaf senescence. Here we report that SAG113 is a direct target gene of the AtNAP transcription factor. We found that both AtNAP and SAG113 were induced by leaf senescence and ABA. When AtNAP was chemically induced, SAG113 was also induced whereas when AtNAP was knocked out, the ABA- and senescence-induced expression of SAG113 was reduced. These data suggest that the expression of SAG113 is predominantly dependent on AtNAP. Functionally, overexpression of SAG113 restored the markedly delayed leaf senescence phenotype in atnap knockouts to wild type. Yeast (Saccharomyces cerevisiae) one-hybrid experiments and electrophoresis mobility shift assays showed that AtNAP could physically bind to the SAG113 promoter in vivo and in vitro, respectively. Site-directed mutagenesis revealed that AtNAP binds to a 9-bp core sequence of the SAG113 promoter, 5′CACGTAAGT3′. These results indicate that there is a unique regulatory chain, ABA-AtNAP-SAG113 protein phosphastase 2C, which controls stomatal movement and water loss during leaf senescence.

The plant hormone salicylic acid (SA) plays critical roles in plant defense, stress responses, and senescence. Although SA biosynthesis is well understood, the pathways by which SA is catabolized remain elusive. Here we report the identification and characterization of an SA 3-hydroxylase (S3H) involved in SA catabolism during leaf senescence. S3H is associated with senescence and is inducible by SA and is thus a key part of a negative feedback regulation system of SA levels during senescence. The enzyme converts SA (with a K _m of 58.29 µM) to both 2,3-dihydroxybenzoic acid (2,3-DHBA) and 2,5-DHBA in vitro but only 2,3-DHBA in vivo. The s3h knockout mutants fail to produce 2,3-DHBA sugar conjugates, accumulate very high levels of SA and its sugar conjugates, and exhibit a precocious senescence phenotype. Conversely, the gain-of-function lines contain high levels of 2,3-DHBA sugar conjugates and extremely low levels of SA and its sugar conjugates and display a significantly extended leaf longevity. This research reveals an elegant SA catabolic mechanism by which plants regulate SA levels by converting it to 2,3-DHBA to prevent SA overaccumulation. The research also provides strong molecular genetic evidence for an important role of SA in regulating the onset and rate of leaf senescence.

Small Auxin Up RNA genes (SAURs) are early auxin-responsive genes, but whether any of them are involved in leaf senescence is not known. Auxin, on the other hand, has been shown to have a role in leaf senescence. Some of the external application experiments indicated that auxin can inhibit leaf senescence, whereas other experiments indicated that auxin can promote leaf senescence. Here, we report the identification and characterization of an Arabidopsis (Arabidopsis thaliana) leaf senescence-associated gene named SAG201, which is highly up-regulated during leaf senescence and can be induced by 1-naphthaleneacetic acid, a synthetic auxin. It encodes a functionally uncharacterized SAUR that has been annotated as SAUR36. Leaf senescence in transfer DNA insertion saur36 knockout lines was delayed as revealed by analyses of chlorophyll content, F_v/F_m ratio (a parameter for photosystem II activity), ion leakage, and the expression of leaf senescence marker genes. In contrast, transgenic Arabidopsis plants overexpressing SAUR36 (without its 3′ untranslated region [UTR]) displayed an early leaf senescence phenotype. However, plants overexpressing SAUR36 with its 3′ UTR were normal and did not exhibit the early-senescence phenotype. These data suggest that SAUR36 is a positive regulator of leaf senescence and may mediate auxin-induced leaf senescence and that the 3′ UTR containing a highly conserved downstream destabilizes the SAUR36 transcripts in young leaves.