). † Both authors contributed equally to this work and should jointly be considered second authors. SUMMARYThe onset and progression of senescence are under genetic and environmental control. The Arabidopsis thaliana NAC transcription factor ANAC092 (also called AtNAC2 and ORE1) has recently been shown to control age-dependent senescence, but its mode of action has not been analysed yet. To explore the regulatory network administered by ANAC092 we performed microarray-based expression profiling using estradiolinducible ANAC092 overexpression lines. Approximately 46% of the 170 genes up-regulated upon ANAC092 induction are known senescence-associated genes, suggesting that the NAC factor exerts its role in senescence through a regulatory network that includes many of the genes previously reported to be senescence regulated. We selected 39 candidate genes and confirmed their time-dependent response to enhanced ANAC092 expression by quantitative RT-PCR. We also found that the majority of them (24 genes) are up-regulated by salt stress, a major promoter of plant senescence, in a manner similar to that of ANAC092, which itself is salt responsive. Furthermore, 24 genes like ANAC092 turned out to be stage-dependently expressed during seed growth with low expression at early and elevated expression at late stages of seed development. Disruption of ANAC092 increased the rate of seed germination under saline conditions, whereas the opposite occurred in respective overexpression plants. We also detected a delay of salinity-induced chlorophyll loss in detached anac092-1 mutant leaves. Promoter-reporter (GUS) studies revealed transcriptional control of ANAC092 expression during leaf and flower ageing and in response to salt stress. We conclude that ANAC092 exerts its functions during senescence and seed germination through partly overlapping target gene sets.

Leaf senescence is a key physiological process in all plants. Its onset is tightly controlled by transcription factors, of which NAC factor ORE1 (ANAC092) is crucial in Arabidopsis thaliana. Enhanced expression of ORE1 triggers early senescence by controlling a downstream gene network that includes various senescence-associated genes. Here, we report that unexpectedly ORE1 interacts with the G2-like transcription factors GLK1 and GLK2, which are important for chloroplast development and maintenance, and thereby for leaf maintenance. ORE1 antagonizes GLK transcriptional activity, shifting the balance from chloroplast maintenance towards deterioration. Our finding identifies a new mechanism important for the control of senescence by ORE1.Keywords: transcription factor; senescence; chloroplast; protein-protein interaction EMBO reports (2013EMBO reports ( ) 14, 382-388. doi:10.1038EMBO reports ( /embor.2013 INTRODUCTIONLeaf senescence is a developmentally controlled process that involves extensive reprogramming and modulation of gene expression to maximize plant fitness by remobilizing nutrients from deteriorating leaves to newly growing vegetative and reproductive organs. Early and noticeable features of leaf senescence are Rubisco and chlorophyll degradation, and a decline of photosynthetic activity owing to chloroplast dismantling [1,2]. Transcription factors (TFs) have important roles in coordinating the gene regulatory networks that underlie the senescence process [3,4]. One of the key senescence-control TF in A. thaliana is the NAC protein ORE1 (ANAC092; At5g39610). Overexpression of ORE1 in transgenic plants triggers early senescence, while its inhibition retards senescence [5,6]. ORE1 exerts its regulatory function by controlling the expression of various known senescence-associated genes (SAGs) [5]. Expression of ORE1 itself is controlled by at least two molecular mechanisms, one that involves currently unknown upstream TFs that determine leaf age-and abiotic stress-dependent ORE1 transcriptional activity [5], and a second one that leads to ORE1 messenger RNA degradation by transacting miR164 [6]. Both processes contribute to establishing a coordinated expression of ORE1, which is low in young, but high in aging leaves.In an aging leaf, the onset of senescence is counterbalanced by still vaguely defined chloroplast maintenance mechanisms. Key elements in this process are the Golden2-like TFs that act as nuclear regulators of chloroplast development and maintenance by coordinating the expression of genes-encoding proteins of the photosynthetic apparatus in various plant species, including A. thaliana, Zea mays and the moss Physcomitrella patens [7,8]. In Arabidopsis, GLK genes exist as a pair of homologous genes, GLK1 and GLK2, and they have been shown to be functionally redundant such that only glk1/glk2 double mutants show a clear phenotype [7,8].Herein, we report the unexpected finding that ORE1 interacts with GLK TFs at the protein level. Elevated expression of ORE1 in the presence of GLK expression str...

Senescence is a highly regulated process that involves the action of a large number of transcription factors. The NAC transcription factor ORE1 (ANAC092) has recently been shown to play a critical role in positively controlling senescence in Arabidopsis thaliana; however, no direct target gene through which it exerts its molecular function has been identified previously. Here, we report that BIFUNCTIONAL NUCLEASE1 (BFN1), a well-known senescence-enhanced gene, is directly regulated by ORE1. We detected elevated expression of BFN1 already 2 h after induction of ORE1 in estradiol-inducible ORE1 overexpression lines and 6 h after transfection of Arabidopsis mesophyll cell protoplasts with a 35S:ORE1 construct. ORE1 and BFN1 expression patterns largely overlap, as shown by promoter-reporter gene (GUS) fusions, while BFN1 expression in senescent leaves and the abscission zones of maturing flower organs was virtually absent in ore1 mutant background. In vitro binding site assays revealed a bipartite ORE1 binding site, similar to that of ORS1, a paralog of ORE1. A bipartite ORE1 binding site was identified in the BFN1 promoter; mutating the cis-element within the context of the full-length BFN1 promoter drastically reduced ORE1-mediated transactivation capacity in transiently transfected Arabidopsis mesophyll cell protoplasts. Furthermore, chromatin immunoprecipitation (ChIP) demonstrates in vivo binding of ORE1 to the BFN1 promoter. We also demonstrate binding of ORE1 in vivo to the promoters of two other senescence-associated genes, namely SAG29/SWEET15 and SINA1, supporting the central role of ORE1 during senescence.

The calcium-dependent protein kinase CPK1 regulates a pivotal developmental switch in senescence and leaf cell death through direct phosphorylation of transcription factor ORE1.