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Plant Signal Behav. 2010 June; 5(6): 733–735.
PMCID: PMC3001574

Salt-triggered expression of the ANAC092-dependent senescence regulon in Arabidopsis thaliana


The NAC domain transcription factor ANAC092 plays a central role in leaf senescence in Arabidopsis thaliana. We recently identified 170 genes whose expression increases upon activation of ANAC092 in a chemically (estradiol) controlled experimental set-up, 78 of which are known senescence-associated genes (SAGs). In accordance with the well-known phenomenon that salt stress promotes early leaf senescence in many plant species, we previously observed salt stress-enhanced expression of many SAGs of the ANAC092 regulon. Global expression profiling now revealed that 36 genes, representing 46% of all ANAC092 downstream SAGs, are induced by long-term (4 days) salt stress in shoots of Arabidopsis, whereas short-term stress (6 hours) only slightly affects gene expression. Expression analysis also showed that 14 of the 36 genes are induced by hydrogen peroxide (H2O2) treatment. Additionally, 15 senescence-associated NAC genes (senNACs), including ANAC092, respond to H2O2 exposure. Our data support the model that salt-triggered senescence is at least partly mediated through the ANAC092 gene regulatory network. Other senNACs most likely contribute to the coordination of this process, potentially in concert with H2O2-mediated signaling.

Key words: ANAC092, binding site, hydrogen peroxide, longevity, NAC transcription factor, ORE1, reactive oxygen species, regulon, salt stress, senescence

NAC transcription factor (TF) ANAC092 (At5g39610; also called AtNAC2 and ORE1)1,2 has recently been discovered as a central regulator of age-dependent and salt-promoted senescence in Arabidopsis thaliana.2,3 Besides ANAC092, NAC family member AtNAP (At1g69490; identical to ANAC029) also controls senescence in Arabidopsis.4 Blocking the function of these genes individually delays senescence,24 establishing both NAC TFs as positive regulators of senescence.

Under normal growth conditions, in the absence of particular stress, ANAC092 expression follows a leaf age-dependent expression pattern with low to moderate expression in young leaves, and high expression in mature and senescent leaves.2,3 ANAC092 transcript abundance during leaf development is in part controlled by miRNA164.2 Salt stress triggers expression of ANAC092 in various organs, including roots, leaves and flowers.1,3 Age-dependent and salt-promoted expression of ANAC092 is controlled at the promoter level as shown by reporter gene fusion.3 Chromatin immune-precipitation (ChIP) experiments revealed in vivo binding of the MADS transcription factor SEPALLATA3 (SEP3) to the ANAC092 promoter,5 but the biological significance of this interaction is currently unknown.

The delayed-senescence mutant ore-sara1 (ore1) lacks a functional ANAC092 gene;2 ore1 exhibits increased tolerance towards various types of oxidative stresses.6 Similarly, detached leaves of the anac092-1 mutant retained chlorophyll at a higher level than the wild-type control when incubated for several days in 150 mM NaCl,3 indicating improved salt tolerance in this experimental setup. Abiotic and biotic stress is often accompanied by the formation and accumulation of reactive oxygen species (ROS), such as hydrogen peroxide (H2O2), superoxide anion radical, and hydroxyl radical.7,8 Under normal growth conditions an intricate antioxidative defense system provides adequate guard against ROS, however environmental stress may cause ROS to rise to unfavorable levels causing irreparable cell damage. ROS also accumulate during agedependent leaf senescence, and many SAGs are known to be controlled by changes in cellular ROS levels9 (see Genevestigator at

Downstream Targets of ANAC092

To explore the downstream gene regulatory network supervised by ANAC092 we performed microarray-based expression profiling after induction of ANAC092 expression in estradiol-inducible overexpression lines.3 We found that 78 of the 170 genes upregulated upon ANAC092 induction are known senescence-associated genes (SAGs), establishing the candidate core senescence regulon of ANAC092 (Fig. 1). We also showed previously in plants of different developmental stages that expression of 19 out of 22 SAGs selected from the full SAG gene set (78 genes) is promoted by long-term salt stress, a major promoter of plant senescence, resembling the behavior of ANAC092 which itself is a salt-responsive gene.3

Figure 1
Role of ANAC092/AtNAC2/ORE1 transcription factor in salt-promoted senescence. Left: The currently known ANAC092 senescence regulon includes 78 genes of which 36 genes are induced by long-term salt stress. Twenty-six of the salt-induced SAGs contain an ...

Using Affymetrix ATH1 arrays we now analyzed global salt-dependent expression patterns in stage 1 plants of the experimental set-up described previously3 (results will be published in detail elsewhere). We found that 57 (34%) of the 170 ANAC092 downstream targets, representing 36 genes (46%) of the senescence regulon of ANAC092, are significantly induced by long-term salt stress (4 days), but not or only weakly after short-term (6 h) stress (150 mM NaCl). An exception was At2g02990 that was slightly higher expressed at short-term stress in the Affymetrix micro-arrays (Fig. 1; Suppl. Table 1), but not when we determined its expression by quantitative RT-PCR.3 Induction of ANAC092 expression was already detectable after shortterm salt stress and increased further upon extended stress treatment. Of the 36 saltinduced SAGs in stage 1 plants, 13 were already known to be salt-regulated.3 Thus, with the global expression profiling experiment performed here we discovered 23 new salt-induced SAGs of the ANAC092 senescence regulon.

Twenty-six of the 36 salt-responsive SAGs (i.e., 72%; Fig. 1) harbor the known binding site of the ANAC092 transcription factor, i.e., T[TAG][GA]CGT[GA] [TCA][TAG],10 in their 1-kb upstream promoter sequence (Suppl. Table 1), strongly supporting the conclusion that salt-dependent enhancement of their expression is controlled by this transcription factor.

H2O2-Responsive senNACs

Generally, expression of many senescence-regulated NACs (senNACs) is strongly upregulated by salinity stress and to a lesser extent by drought.11 As abiotic stress affects intracellular ROS levels (including that of H2O2),7,8,12 we analyzed the effect of H2O2 on gene expression using Affymetrix ATH1 micro-arrays (details will be published elsewhere). Treatment of two-week-old Arabidopsis seedlings for 5 h with 10 mM H2O2 enhanced ANAC092 expression approximately two-fold, whereas no induction was observed after 1 h. We also analyzed the expression of all other known senNACs and found that 15 of them (including ANAC092) are H2O2-responsive (Suppl. Table 1). Induction of some genes was also observed after 1 h of treatment with a higher concentration of H2O2 (20 mM).13 The most strongly responding genes in our experiment were AtNAP (18-fold after 5 h of treatment) as well as ANAC032 and ANAC042 (both ~25-fold), whereas all other senNACs were induced by 2- to 7-fold after 5 h of H2O2 exposure (Suppl. Table 1). Various other treatments known to trigger accumulation of intracellular ROS levels lead to upregulation of senNAC gene expression. For example expression of ANAC019, ANAC032, ANAC072 (RD26), and ANAC0102 is strongly induced by ozone and methyl viologen, and ANAC042 is induced by ozone14 and treatment with 3-aminotriazole, which blocks catalase leading to an intracellular rise of H2O2 level.15

H2O2-Responsive ANAC092 Downstream Targets

Next we analyzed how many of the 36 salt-responsive SAGs of the ANAC092 regulon are affected by H2O2 and found that 14 of them are induced by 2-fold or more upon H2O2 treatment (Suppl. Table 1), representing ~40% of the ANAC092 salt-dependent senescence regulon. However, five genes were repressed by H2O2-treatment, suggesting complex signaling interplay. To untie the relationship between H2O2 accumulation, expression of ANAC092 targets and development of senescence symptoms it might therefore be important to perform longer-term H2O2 stress experiments (with lower concentrations of external H2O2) and include H2O2 signaling mutants in such studies.

In addition to regulation at the transcript level various senescence-regulated NAC transcription factors have been shown to be targets of protein kinases in vitro. Notably, five senNACs, i.e., ANAC029 (AtNAP), ANAC032, ANAC074, ANAC084 and ANAC092, are phosphorylated by MAP kinases.16,17 Another senNAC, ANAC002 (ATAF1), physically interacts with protein kinase SnRK1 α-subunits AKIN10 and AKIN11,18 but phosphorylation of ATAF1 by SnRK1 has not been demonstrated yet. So far, functional proof for a physiologically relevant role of NAC transcription factor phosphorylation in relation to senescence is lacking.


Global expression profiling disclosed 36 salt-enhanced genes representing a large proportion (46%) of the currently known 78 genes of the ANAC092 senescence regulon, suggesting that it constitutes an important element of the network that controls salt-promoted senescence. Lack of ANAC092 function in the ore1 mutant extends plant longevity under oxidative stress conditions.6 Some of the salt- and H2O2-responsive genes identified here (Fig. 1; Suppl. Table 1) might play an important role in this process. Further work is clearly needed to unravel the precise make-up of the ANAC092 senescence regulon and its integration into other signaling networks that control plant longevity.


We are grateful to Annapurna Devi Allu for performing expression profiling experiments on salt-treated Arabidopsis plants, the results of which will be published in detail elsewhere. We thank the Deutsche Forschungsgemeinschaft (FOR 948-Nitrogen uptake, metabolism and remobilization in leaves during plant senescence; MU 1199/14-1) for funding.


Addendum to: Balazadeh S, Siddiqui H, Allu AD, Matallana-Ramirez LP, Caldana C, Mehrnia M, et al. Gene regulatory network controlled by NAC transcription factor ANAC092/AtNAC2/ORE1 during salt-promoted senescencePlant J2010In press doi: 10.1111/j.1365-313X.2010.04151.x.


Supplementary Material

Supplementary Material:


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