Transcription Factor ATAF1 in Arabidopsis Promotes ... Factor ATAF1 in Arabidopsis Promotes Senescence by Direct Regulation of Key Chloroplast Maintenance and Senescence Transcriptional

Transcription Factor ATAF1 in Arabidopsis PromotesSenescence by Direct Regulation of Key ChloroplastMaintenance and Senescence TranscriptionalCascades1[OPEN]

Prashanth Garapati, Gang-Ping Xue, Sergi Munné-Bosch, and Salma Balazadeh*

University of Potsdam, Institute of Biochemistry and Biology, 14476 Potsdam-Golm, Germany (P.G., S.B.);Plant Signaling Group, Max Planck Institute of Molecular Plant Physiology, 14476 Potsdam-Golm, Germany(P.G., S.B.); Commonwealth Scientific and Industrial Research Organization Plant Industry, St. Lucia,Queensland 4067, Australia (G.-P.X.); and Departament de Biologia Vegetal, Universitat de Barcelona, Facultatde Biologia, 08028 Barcelona, Spain (S.M.-B.)

ORCID IDs: 0000-0001-6523-6848 (S.M.-B.); 0000-0002-5789-4071 (S.B.).

Senescence represents a fundamental process of late leaf development. Transcription factors (TFs) play an important role forexpression reprogramming during senescence; however, the gene regulatory networks through which they exert their functions,and their physiological integration, are still largely unknown. Here, we identify the Arabidopsis (Arabidopsis thaliana) abscisicacid (ABA)- and hydrogen peroxide-activated TF Arabidopsis thaliana ACTIVATING FACTOR1 (ATAF1) as a novel upstream regulator ofsenescence. ATAF1 executes its physiological role by affecting both key chloroplast maintenance and senescence-promoting TFs,namely GOLDEN2-LIKE1 (GLK1) and ORESARA1 (ARABIDOPSIS NAC092), respectively. Notably, while ATAF1 activatesORESARA1, it represses GLK1 expression by directly binding to their promoters, thereby generating a transcriptional outputthat shifts the physiological balance toward the progression of senescence. We furthermore demonstrate a key role of ATAF1 forABA- and hydrogen peroxide-induced senescence, in accordance with a direct regulatory effect on ABA homeostasis genes,including NINE-CIS-EPOXYCAROTENOID DIOXYGENASE3 involved in ABA biosynthesis and ABC TRANSPORTER G FAMILYMEMBER40, encoding an ABA transport protein. Thus, ATAF1 serves as a core transcriptional activator of senescence by couplingstress-related signaling with photosynthesis- and senescence-related transcriptional cascades.

Leaf senescence involves the degradation of cellularcomponents and their remobilization to developingorgans such as young and immature leaves duringvegetative growth or seeds during the reproductivephase. It is assumed that the timely onset and pro-gression of senescence confers an ecological fitnessto the plant, as it contributes to optimizing the reuseof nitrogen and carbon during seed formation. Leafsenescence itself is an ordered physiological process,

and although it is principally mediated by an underly-ing genetic program, environmental factors have a largeimpact on the onset and rate of progression of senes-cence. Abiotic stresses such as drought, high canopytemperature, soil salinity, and nutrient deficiencieshasten senescence and thus affect plant productivity.

Senescence is accompanied by transcriptional repro-gramming of a large number of genes, including thoseencoding transcription factors (TFs), as shown byglobal transcriptome profiling in various plant species,including Arabidopsis (Arabidopsis thaliana; Buchanan-Wollaston et al., 2005; Balazadeh et al., 2008; Breezeet al., 2011), wheat (Triticum aestivum; Gregersen andHolm, 2007; Cantu et al., 2011), aspen (Populus tremula;Andersson et al., 2004), maize (Zea mays; Zhang et al.,2014), and others. Recently, the role of several NAC (forNOAPICALMERISTEM/ARABIDOPSIS ACTIVATIONFACTOR1 [ATAF1-2]/CUP-SHAPED COTYLEDON)TFs for senescence has been reported, and both positiveand negative regulators were identified. In Arabidopsis,positive senescence regulators include ORESARA1(ORE1; also called ARABIDOPSIS NAC092 [ANAC092]or AtNAC2; Kim et al., 2009; Balazadeh et al., 2010a;Rauf et al., 2013a), ORESARA1 SISTER1 (ORS1; ANAC059;Balazadeh et al., 2011), AtNAP (for Arabidopsis NAC-LIKE,ACTIVATED BY APETALA3/ PISTILLATA; ANAC029;

1 This work was supported by the Deutsche Forschungsgemein-schaft (FOR 948 grant no. BA4769/1–2 to S.B.), the Spanish Govern-ment (grant no. BFU2012–32057), and the International Max-PlanckResearch School Primary Metabolism and Plant Growth program(fellowship to P.G.).

* Address correspondence to [email protected] author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:Salma Balazadeh ([email protected]).

S.B. designed the research and supervised the group; S.M.-B. per-formed the hormone analysis; G.-P.X. performed the CELD experi-ment; P.G. and S.B. performed all other experiments; S.B. wrote thearticle with contributions from the other authors.

[OPEN] Articles can be viewed without a subscription.www.plantphysiol.org/cgi/doi/10.1104/pp.15.00567

1122 Plant Physiology�, July 2015, Vol. 168, pp. 1122–1139, www.plantphysiol.org � 2015 American Society of Plant Biologists. All Rights Reserved. www.plantphysiol.orgon July 2, 2018 - Published by Downloaded from

Copyright © 2015 American Society of Plant Biologists. All rights reserved.

http://orcid.org/0000-0001-6523-6848

http://orcid.org/0000-0002-5789-4071

mailto:[email protected]

http://www.plantphysiol.org

mailto:[email protected]

http://www.plantphysiol.org/cgi/doi/10.1104/pp.15.00567


Guo and Gan, 2006), and ANAC016 (Kim et al., 2013),while negative regulators include JUNGBRUNNEN1 (JUB1;ANAC042; Wu et al., 2012) and VND-INTERACTING2(ANAC083; Yang et al., 2011). Furthermore, down-stream target genes of some of the senescence-regulatedNAC TFs have been reported, including BIFUNCTIONALNUCLEASE I (BFN1), RIBONUCLEASE3, SEVEN-IN-ABSENTIA1 (SINA1), and SENESCENCE-ASSOCIATEDGENE29 (SAG29; SWEET15) for ORE1 (Balazadeh et al.,2010a; Matallana-Ramirez et al., 2013; Rauf et al., 2013a),DEHYDRATION-RESPONSIVE ELEMENT BINDINGPROTEIN2 for JUB1 (Wu et al., 2012), and SAG113 forAtNAP (Guo and Gan, 2006). These studies reveal firstinsights into the complex regulatory networks under-lying plant senescence.An important aspect of senescence is not only the up-

regulation of the above-mentioned TFs and their targetgenes but also the reduced expression of photosynthesis-associated genes (PAGs), ultimately resulting in a de-cline of photosynthetic activity. The loss of chloroplastmaintenance mechanisms and chlorophyll are impor-tant markers of senescence (Hensel et al., 1993). Twomembers of the GARP family of Myb TFs, namelyGOLDEN2-LIKE1 (GLK1) and GLK2, are known to playkey roles in chloroplast development and maintenancein Arabidopsis (Waters et al., 2008, 2009). Photosynthesis-related direct downstream targets of GLKs includinglight-harvesting chlorophyll a/b-binding (LHC) protein-encoding genes LHCB2.4, LHCB3, LHCA4, and LHCB4.2have been identified (Waters et al., 2009). Recently, wereported that the positive senescence regulator ORE1interacts at the protein level with the chloroplast main-tenance TFs GLK1 and GLK2 (Rauf et al., 2013a). Ourexperimental data showed that GLK target genes arerepressed when ORE1 expression is enhanced (mostlikely due to the formation of GLK-ORE1 heteromers),while ORE1 target genes are not affected. Based on ourfindings, we proposed the model that the onset of se-nescence is affected by the interaction of the two TFs(Rauf et al., 2013a).Phytohormones play an important role in the mod-

ulation of the timing and progression of senescence(Guo and Gan, 2005; Lim et al., 2007; Jibran et al.,2013; Mueller-Roeber and Balazadeh, 2014). Abscisicacid (ABA) is one of the most effective plant hormonesin terms of promoting leaf senescence apart from eth-ylene (Zacarias and Reid, 1990; Lee et al., 2011). ABAappears to initiate senescence, while ethylene appears toexert its effects at a later stage. An increase in endoge-nous ABA has been shown to coincide with the senes-cence of leaves, while genes involved in ABA signalingwere induced earlier during senescence (Breeze et al.,2011). However, the molecular elements of the cellularregulatory networks that trigger the ABA-dependentcontrol of senescence are largely unknown; however,recent experimental evidence indicates that AtNAP ac-tivates ABSCISIC ALDEHYDE OXIDASE3, which en-codes an enzyme responsible for the last step in ABAbiosynthesis (Yang et al., 2014). ABA itself influencesthe redox as well as antioxidant status of the cell, as it

involves hydrogen peroxide (H2O2) production via theactivation of NADPH oxidases and the expression ofantioxidant genes; thus, ABA helps balance both cellu-lar protection activities and the progression of senes-cence (Torres and Dangl, 2005; Lim et al., 2007).

The NAC transcription factor ATAF1 in Arabidopsisnegatively regulates the expression of stress-responsivegenes under drought stress and ABA treatment, andfunctions in abiotic and biotic stress responses havebeen reported (Lu et al., 2007; Jensen et al., 2008; Wuet al., 2009).

Drought induction and/or recovery experiments re-vealed an enhanced drought tolerance in ataf1 knockoutmutants (Lu et al., 2007; Jensen et al., 2008), indicating anegative regulatory role of ATAF1 in relation to droughttolerance. By contrast, in another report (Wu et al., 2009),ATAF1 overexpression led to increased tolerance todrought stress, demonstrating a positive correlationwith drought tolerance. These authors also observeda marked increase of the expression of several stress-responsive marker genes in ATAF1 overexpressors atthe later stress time points, in accordance with the en-hanced drought tolerance observed. At the protein level,ATAF1 is known to interact with the catalytic subunitsAKIN10 andAKIN11 of SUCROSENONFERMENTING1-RELATED SERINE/THREONINE-PROTEIN KINASE1(SnRK1; Kleinow et al., 2009). SnRK1 is a key integratorof transcription networks in response to stress and isimplicated in sugar and ABA signaling pathways (Baena-González et al., 2007; Jossier et al., 2009). Recently, ATAF1has been reported to directly regulate the expressionof NINE-CIS-EPOXYCAROTENOID DIOXYGENASE3(NCED3), which encodes a key enzyme of ABA bio-synthesis in Arabidopsis (Jensen et al., 2013). Accord-ingly, overexpression of ATAF1 led to elevated ABAlevels, and NCED3 transcript abundance was higher inATAF1 overexpressors but lower in ataf1 knockoutmutants compared with the wild type. The increaseddrought tolerance reported for ATAF1 overexpressorsby Wu et al. (2009) is in accordance with this finding,but currently it cannot explain the contrasting obser-vation by Jensen et al. (2008) that ataf1 mutant lineshave a higher drought tolerance.

We previously observed that the expression of ATAF1increases during developmental and salinity stress-induced leaf senescence (Balazadeh et al., 2008; Alluet al., 2014). Furthermore, ATAF1 is rapidly (within1 h) induced in plants treated with H2O2 (Balazadehet al., 2010b) and in seedlings treated with the catalaseinhibitor 3-aminotriazole, which triggers an intracellularrise of H2O2 level (expression data are summarized inSupplemental Fig. S1, A–C). In addition, the expressionof ATAF1 is enhanced in the catalase2-1 mutant (Gene-vestigator), which accumulates high levels of H2O2 underphotorespiratory conditions (Hu et al., 2010). Notably,however, the expression of ATAF1 is not induced whensenescence in induced by nitrogen starvation (Balazadehet al., 2014); thus, ATAF1 appears to be preferentiallyinvolved in senescence-triggering stresses that involveH2O2 as a physiological signal or metabolite.

Plant Physiol. Vol. 168, 2015 1123

ATAF1 Regulates GLK1 and ORE1

www.plantphysiol.orgon July 2, 2018 - Published by Downloaded from Copyright © 2015 American Society of Plant Biologists. All rights reserved.

http://www.plantphysiol.org/cgi/content/full/pp.15.00567/DC1


Here, we identify ATAF1 as a novel positive regula-tor of senescence in Arabidopsis. We demonstrate thatATAF1 regulates senescence by activating expression ofthe positive senescence regulator ORE1 and by repres-sing expression of the chloroplast maintenance TFGLK1. Thus, ATAF1 regulates senescence by oppositelyacting on two downstream TF genes, which fully alignswith the previous finding that the interaction of ORE1with GLKs at the protein level balances leaf senescenceagainst maintenance by antagonizing the role of GLKs(Rauf et al., 2013a). Our data also reveal that ATAF1affects the progression of senescence by regulatingthe expression of at least two ABA biosynthesis- andtransport-related genes, namely NCED3 and ABCTRANSPORTER G FAMILY MEMBER40 (ABCG40).Furthermore, we show that H2O2-triggered expressionof ORE1, and H2O2-triggered repression of GLK1, re-quire the presence of ATAF1. Thus, our data provideevidence that ATAF1 integrates ABA- and H2O2-dependent signaling with senescence through the di-rect regulation of key photosynthesis and senescencetranscriptional cascades.

RESULTS

ATAF1 Positively Affects Senescence

As ATAF1 shows elevated expression during senes-cence (Fig. 1A; Balazadeh et al., 2008), we next tested itspotential involvement in the regulation of this process.To this end, we compared Columbia-0 (Col-0) plantswith transgenic plants overexpressing ATAF1 from thecauliflower mosaic virus (CaMV) 35S promoter andataf1 knockout mutants. We characterized three homo-zygous transfer DNA (T-DNA) insertion lines, namelyataf1-2 (SALK-057618; Lu et al., 2007), ataf1-3 (GK239B09),and ataf1-4 (GK565H08; Supplemental Fig. S2A). Thehomozygosity of T-DNA insertions was confirmed byPCR-based genotyping, and the location of T-DNA in-sertion position was verified by PCR (Supplemental Fig.S2B) and sequencing. Absence of ATAF1 expression inall three ataf1 mutants was demonstrated by end-pointPCR and quantitative real-time (qRT)-PCR (SupplementalFig. S2, C and D). In 35S:ATAF1 plants, elevated ATAF1transcript levels were confirmed by qRT-PCR in threeindependent transformants (Supplemental Fig. S2E).

To test the effect of altered ATAF1 expression onsenescence, we determined senescence-related parame-ters. Leaf yellowing due to chlorophyll loss becameevident at approximately 25 d after sowing (DAS) in35S:ATAF1 plants (OX-17 and OX-20), while loss ofchlorophyll was delayed in leaves of all three ataf1knockout mutants compared with Col-0 (Fig. 1, B–E).Even at 45 DAS, chlorophyll content and number ofleaves with 50% green blades were higher in thethree ataf1 null mutants than in 35S:ATAF1 and Col-0plants (Fig. 1, D and E). Ion leakage as an indicator ofmembrane damage was higher in leaves of 35S:ATAF1(OX-17) than wild-type plants, while ataf1-2 and ataf1-4

null mutants exhibited decreased ion leakage (Fig. 1F).As a further indicator for leaf senescence, we measuredthe transcript abundance of early-senescence (SAG13)and late-senescence (SAG12) marker genes. At 45 DAS,SAG13 expression in leaf 9 was lower in the ataf1-4mutant than in the wild type, while SAG12 expressionwas not detected; on the contrary, SAG12 expressionwas highly elevated in 35S:ATAF1 plants, clearly indi-cating a senescence-promoting effect of ATAF1 (Fig. 1G).Notably, bolting time was not significantly differentbetween the genotypes (days to inflorescence emer-gence, 26.6 6 1, 25.9 6 1.1, and 27.3 6 0.9, and numberof leaves at inflorescence emergence, 9.7 6 1.2, 10.4 61.6, and 9.5 6 1.3 for Col-0, 35S:ATAF1, and ataf1-4,respectively; n = 10 from two experiments).

We then expressed ATAF1 from an estradiol-induciblepromoter (Zuo et al., 2000). ATAF1-IOE lines were sownon one-half-strength Murashige and Skoog (MS) me-dium containing 1% (w/v) Suc and 20 mM estradiol(or 0.2% [v/v] ethanol in control experiments; 16 h of lightat 22°C/8 h of dark at 18°C). Similar to plants grownin soil, ATAF1-IOE plants grown in medium supple-mented with estradiol showed precocious senescencecompared with those grown in mock-supplementedmedium (Fig. 2A). Accordingly, expression of PAGssuch as LHCB2.4, LHCB3, LHCA4, and LHCB4.2 wasreduced in ATAF1-IOE seedlings (at 5 h after estradioltreatment) and in 27-d-old 35S:ATAF1 plants, whileexpression of the senescence marker SAG13 was ele-vated. On the contrary, expression of PAGs was en-hanced in the 27-d-old ataf1-4 mutant, while SAG13expression was reduced (Fig. 2B; Supplemental TableS1). Collectively, these data reveal a role of ATAF1 inthe control of developmental leaf senescence.

ATAF1 Is Involved in Dark-Induced Senescence

Although the molecular mechanisms underlyingdark-induced senescence are not known in detail, somesimilarities with developmental senescence have beenreported (van der Graaff et al., 2006). Microarray hy-bridization data obtained from Arabidopsis plantssubjected to prolonged darkness for different time pe-riods revealed a number of up- or down-regulatedgenes involved (Lin and Wu, 2004), including ATAF1,which was strongly up-regulated upon dark treatment(Fig. 3A). To test whether dark-induced senescence re-quires the presence of a functional ATAF1 gene, de-tached leaves from 3-week-old soil-grown Arabidopsisplants were incubated for 3 d in darkness on moist filterpaper. Upon dark incubation, leaves of OX-17 turnedyellowish faster than wild-type leaves, while leaf yel-lowing was slowed in ataf1-4 (Fig. 3B). In accordancewith this, we observed a more rapid decline in chloro-phyll level in leaves of OX-17 and OX-20 plants than inCol-0 and a slowed decline in leaves of all three ataf1mutants (Fig. 3C). Furthermore, expression of SAG13 andORE1 was enhanced, and expression of PAGs such asLHCA4, LHCB3, LHCB4.2, and PROTOCHLOROPHYLLIDE

1124 Plant Physiol. Vol. 168, 2015

Garapati et al.











OXIDOREDUCTASE B was significantly reduced indark-treated ATAF1 overexpression plants (Fig. 3D;Supplemental Table S1). Finally, we tested the expres-sion of PPDK, which encodes pyruvate orthophosphatedikinase and was previously shown to be inducedduring dark-induced senescence; the enzyme is thoughtto play a role in generating Asn to function as a nitrogencarrier for nitrogen remobilization during senescence(Lin and Wu, 2004). PPDK transcript abundance waselevated inOX-17 leaves (Fig. 3E), in accordance with themore rapid senescence observed during dark incubation.

In contrast, expression of PPDK was reduced in the ataf1-4mutant (Fig. 3E). These data suggest that, besides arole in developmental senescence, ATAF1 also consti-tutes an important regulatory element of the cellularnetworks controlling dark-induced senescence.

Identification of the Consensus Target Sequence of ATAF1

Identifying TF-binding sites represents an importantstep toward unraveling and predicting regulatory

Figure 1. ATAF1 controls leaf senescence. A, ATAF1 expression increases during leaf aging. Data from two independent bi-ological replicates, obtained from Affymetrix ATH1 microarray hybridizations, are shown. DAE, Days after emergence. B, Earlyonset of senescence in an OX-17 plant (arrows) compared with Col-0 and ataf1-4 plants grown under long-day condition (25DAS). C, Delayed leaf senescence in the ataf1-4 mutant compared with Col-0 and OX-17 plants. At top, leaves detached fromplants (35 DAS) were numbered from bottom (oldest leaf) to top (youngest leaf); cotyledons were not included in the leaf count.The middle and bottom parts show plants at 45 DAS. D, Chlorophyll content of leaf 9 at 35, 45, and 55 DAS. SPAD values weredetermined at the center of each leaf. Chlorophyll content decreases more rapidly in 35S:ATAF1 overexpression lines and moreslowly in different ataf1 knockout mutants compared with the Col-0 wild type (n = 6). E, Leaf yellowing. Shown are the per-centages of leaves that had at least approximately 50% yellow blades (inspected visually) at given DAS (n = 12 for each plantline and time point). F, Higher and lower ion leakage, respectively, in 35S:ATAF1 (OX-17) and ataf1 mutants compared withCol-0. Leaves 9 and 11 were combined and used for measurements. Data are means 6 SD of three independent biologicalreplicates. Asterisks indicate significant differences from Col-0 plants (Student’s t test, P # 0.05). G, Expression of senescencemarker genes (SAG12 and SAG13) in ATAF1 transgenics (45 DAS). Data are means 6 SD of three independent biologicalreplicates with two technical replicates each. Expression was normalized against the expression of ACTIN2.






networks. To identify DNA-binding sites of ATAF1,we performed a binding-site selection assay using thecellulase D (CELD) fusion method (Xue, 2005). Afterselection, 34 clones with high binding activity weresequenced. The sequence alignment of these clonesand base substitution or insertion analysis, togetherwith knowledge about the allowable spacer length ofhomologous NAC protein-binding sites (Xue et al.,2006; Matallana-Ramirez et al., 2013), revealed twotypes of bipartite sequences, namely VDHVnnYRR(5-6n)YACGnMWSK (BS1) and RRWnnCGTR(5-6n)DACGTMWHD (BS2), as ATAF1-binding sites (under-lined nucleotides represent 100% conservation in thesequenced clones; Supplemental Table S2). Recently,Jensen et al. (2013) used protein-binding microarrayscovering all possible 8-mer DNA sequences and reported

the monopartite 6-mer motifs TT[A,C,G]CGT andT(A,C,G)CGT(A,G) as the binding sites of ATAF1. Dueto technical constraints associated with protein-bindingmicroarrays, longer (and here, bipartite) binding sitescannot be detected with high probability using thismethod. Still, the Jensen et al. (2013) binding site forATAF1 is included in the spectrum of bipartite bindingsites we detected here. The consensus binding siteidentified for ATAF1 bears similarity to the core bind-ing motif (YACG or CGT) reported for a few other NACfactors: TCNNNNNNNACACG for the three highlyhomologous drought-responsive NAC factors ANAC019,ANAC055, andANAC072 (Tran et al., 2004), (A/G)G(A/T)NNCGT(A/G)NNNNN(C/T)ACGT(A/C)A(C/T)(C/T)for wheat TaNAC69 (Xue, 2005), (A/G)CGT(A/G)(4-5N)(A/G)(C/T)ACGCAA for ORS1 (Balazadeh et al., 2011),TGCCGT(7N)ACG and TGCCGT(7N)CCGC for JUB1(Wu et al., 2012), and VMGTR(5-6N)YACR for ORE1(Matallana-Ramirez et al., 2013).

ATAF1 Directly Regulates ORE1 and GLK1 Expressionin Vivo

The early-senescence phenotype observed in ATAF1-overexpressing plants and the delayed senescence in theataf1 mutant suggested a role for ATAF1 in regulatingleaf senescence. To better understand the underlyingmechanism of this regulation, we tested the expressionof 15 PAGs and 158 senescence-associated genes (SAGs)by qRT-PCR in 45-d-old OX-17, ataf1-4, and Col-0 plants.This experiment revealed that expression of eight PAGs(including GLK1 and GLK2) was down-regulated inATAF1 overexpression plants compared with Col-0;in contrast, expression of the same genes was induced inthe ataf1-4 mutant (Supplemental Fig. S3A; SupplementalTable S3). With respect to SAGs, expression of 59 genes(37%) and 91 genes (58%) was down-regulated by 2- to4-fold and by more than 4-fold, respectively, in ataf1-4compared with Col-0 (measured in leaf 9). In accor-dance with this, 38 genes (24%) and 24 genes (15%)of the SAGs were up-regulated by 2- to 4-fold and bymore than 4-fold, respectively, in 35S:ATAF1 plants(Supplemental Fig. S3B; Supplemental Table S3). Thefact that many PAGs and SAGs are affected at the ex-pression level in ATAF1-modified plants suggested thatsome of them might be direct targets of the NAC TF.

To identify potential direct target genes of ATAF1, weanalyzed the promoters of the affected genes for the pres-ence of ATAF1-binding sites (Supplemental Table S2) andidentified GLK1 [VDHVNNYRR(6N)YACGNMWSK atpositions22,383 to22,360 bpupstreamof the transcriptionstart site] and ORE1 [VDHVNNYRR(5N)YACGNMWSKat 2345 to 2323 bp upstream] as candidates, which arekey regulators of chloroplast maintenance and senes-cence, respectively.

To substantiate the model that ATAF1 directly reg-ulates the transcription of GLK1 and ORE1, we nexttested their expression in ATAF1-IOE plants after es-tradiol induction. Expression of both genes was tested

Figure 2. Inducible ATAF1 expression triggers senescence. A, Inductionof ATAF1 expression in ATAF1-IOE plants by estradiol (ESTR) treatmentresults in precocious senescence. Ten-day-old seedlings were transferredto agar plates containing one-half-strength MS medium supplementedwith 20 mM estradiol and grown for another 8 d (16 h of light at 22˚C/8 hof dark at 18˚C). No leaf senescence is visible in estradiol-treated empty-vector (EV) control transformants or in mock-treated seedlings. B, Ele-vated expression of ATAF1 results in reduced expression of PAGs inATAF1-IOE (5 h after estradiol induction) and OX-17 plants, whileknocking out ATAF1 in the ataf1-4 mutant has the opposite effect (27DAS). On the contrary, expression of the early-senescence marker geneSAG13 is enhanced in ATAF1 overexpressors but reduced in ataf1-4.The heat map indicates differences in the expression (log2 fold change)of genes in estradiol-induced versus mock-treated ATAF1-IOE plants,and in OX-17 and ataf1-4 plants, respectively, compared with Col-0(corresponding data are given in Supplemental Table S1). Data representmeans of three independent biological replicates, each determined intwo technical replicates.


Garapati et al.











by qRT-PCR at different time points (1, 2, 5, 10, and 24 h)after estradiol (10 mM) treatment and compared withmock treatment. Interestingly, expression of ORE1 wasrapidly (alreadywithin 1–2 h after estradiol treatment) up-regulated, while GLK1 was down-regulated in ATAF1-IOE plants (Fig. 4A; Supplemental Table S1).To test the physical binding of recombinant ATAF1

transcription factor to the promoters of the two genes,we performed electrophoretic mobility shift assays(EMSAs). ATAF1 protein fused to CELD (see above)was incubated with 59-DY682-labeled 40-bp double-stranded DNA fragments containing the binding sitesof ATAF1. As shown in Figure 4B, ATAF1 binds to boththe ORE1 and GLK1 promoter fragments. Binding of

ATAF1 was significantly reduced when unlabeledpromoter fragments were added in excess (competitor).

Next, we examined whether ATAF1 binds to thepromoters of the two genes in vivo. To this end, weperformed chromatin immunoprecipitation (ChIP)-quantitative PCR (qPCR) using a transgenic line thatexpresses the ATAF1-GFP fusion protein under thecontrol of the CaMV 35S promoter. We observed thepresence of ATAF1-GFP protein in nuclei of transgenicplants (Fig. 4C) and elevated expression of ORE1 com-pared with Col-0, while expression ofGLK1was repressed(Fig. 4D), consistent with the model that ATAF1-GFPfusion protein faithfully functions as a transcriptionfactor. Using these plants, we observed a significant

Figure 3. Dark-induced senescence. A, ATAF1 expression increases during dark-induced senescence. The graph was repro-duced from transcriptomic data reported in supplemental table 1 of Lin and Wu (2004). The x axis indicates the dark treatmentperiod. Time point c6d represents samples from plants grown for 6 d without dark treatment, which were used as controls toverify the differential gene expression specific to dark treatment rather than to developmental alteration after 6 d of darkness. B,Leaves detached from whole rosettes of 3-week-old, long-day-grown Col-0, OX-17, and ataf1-4 plants were placed on moistfilter paper in petri dishes. Samples were kept in continuous darkness for 3 d. Senescence is retarded in ataf1-4 in comparisonwith Col-0 plants, whereas it is enhanced in the overexpression line. Leaves are numbered from 1 (oldest) to 10 (youngest); thetwo cotyledons are shown as well. C, Chlorophyll content of leaves 5 and 9 from long-day-grown plants before and after 3 d ofdark incubation (DDI). SPAD values were determined at the leaf tips. Note the higher and lower levels of chlorophyll content inthe older leaf (i.e. leaf 5) in all three ataf1mutants and 35S:ATAF1 (OX-17 andOX-20) plants, respectively, compared with Col-0 at 3d of dark incubation. In the younger leaf (i.e. leaf 9), less chlorophyll content in OX-17 and OX-20 than in Col-0 at 3 d of darkincubation supports the conclusion that senescence is promoted by ATAF1 overexpression. Data are means 6 SD of three inde-pendent biological replicates. Asterisks indicate significant differences from Col-0 plants (P # 0.05). D, Transcript abundance ofATAF1, PAGs, and SAGs after a 3-d dark incubation (leaf 5). The heat map indicates expression differences (log2 fold change) inATAF1 transgenics compared with Col-0 (for corresponding data, see Supplemental Table S1). E, Transcript abundance of PPDK indark-incubated leaves (3 d) of ATAF1 OX-17 and ataf1-4 compared with Col-0. Fch, Fold change. Data in D and E represent meansof two independent biological replicates, each determined in two technical replicates.








enrichment of the promoters of ORE1 and GLK1 (Fig.4E), supporting the conclusion that both are directdownstream targets of ATAF1.

ATAF1 Requires ORE1 for Full Control over Senescence

As ORE1 (ANAC092) is a central positive regulator ofsenescence and a direct target of ATAF1, we hypothe-sized that ATAF1 executes its role in developmentalsenescence through ORE1. To test this model, we gen-erated and analyzed plants overexpressing ATAF1 inthe anac092-1mutant, which is null for ORE1 (Balazadehet al., 2010a). We determined ATAF1 expression in 35S:ATAF1/anac092-1 transformants and selected two in-dependent lines (called L1 and L6; Fig. 5A) showingsimilar levels of ATAF1 overexpression to the 35S:ATAF1/Col-0 plants (Supplemental Fig. S2E). As seen in Figure 5B,leaf senescence developed more rapidly in 35S:ATAF1/Col-0 (line OX-17) than in 35S:ATAF1/anac092-1 plants.We assessed the progression of leaf senescence by

determining the number of leaves with at least 50%yellow blades at three different plant ages (35, 45,and 55 DAS) and membrane ion leakage at two timepoints (35 and 45 DAS). In accordance with previousfindings (Balazadeh et al., 2010a), senescence was sig-nificantly retarded in the anac092-1 mutant comparedwith the Col-0 wild type and was faster in 35S:ATAF1/Col-0 plants, while in 35S:ATAF1/anac092-1 plants (L1and L6), senescence was only partly promoted (Fig. 5, Cand D). Thus, although ATAF1 has the capacity to speedup senescence, it does require intactORE1 (ANAC092) tobe able to fully execute its role.

In addition, to analyze senescence at the molecularlevel, we checked the expression of SAG12 in leaf 9,which was approximately 4-fold less induced in 35-d-old 35S:ATAF1/anac092-1 than inOX-17 plants (Fig. 5E).We also tested expression of the 158 SAGs (see above) inthe anac092-1 mutant as well as the 35S:ATAF1/anac092-1and 35S:ATAF1/Col-0 plants at 45 DAS. Of all SAGstested in OX-17, 38 genes (24%) and 24 genes (15%)were up-regulated by 2- to 4-fold and more than 4-fold,

Figure 4. ORE1 and GLK1 are direct targets of ATAF1. A, Expression of ORE1 and GLK1 in 2-week-old ATAF1-IOE seedlingstreated with estradiol (ESTR) for the indicated times. The heat map indicates expression compared with mock treatment (log2fold change). Data represent means of three independent biological replicates each. Note the rapid down- and up-regulation ofGLK1 and ORE1, respectively (for corresponding data, see Supplemental Table S1). B, EMSA. Purified ATAF1-CELD proteinbinds specifically to the ATAF1-binding site within the promoters of ORE1 and GLK1. In vitro DNA-binding reactions wereperformed with 40-bp-long promoter fragments containing the respective ATAF1 motifs (59-GTTGCCTAACAATCTACGCATCT-39)and GLK1 (59-GACCAGTAGTCCTTTTACGAAAGT-39). C, Nuclear localization of ATAF1-GFP fusion protein in transgenicArabidopsis plants. D, Expression of ORE1 and GLK1 in 2-week-old 35S:ATAF1-GFP seedlings compared with expression inCol-0 plants. Numbers on the y axis indicate expression fold change (log2 Fch). Data are means 6 SD of three independentbiological replicates, each determined in two technical replicates. E, ChIP-qPCR. Shoots of 2-week-old 35S:ATAF1-GFPseedlings were harvested for the ChIP experiment. qPCR was used to quantify the enrichment of the ORE1 and GLK1 promoterregions. As a negative control (NC), qPCR was performed on a promoter (CLAVATA1; At1g75820) lacking ATAF1-binding sites.Data represent means 6 SD (two independent biological replicates, each with three technical replicates).


Garapati et al.





respectively, compared with the wild type, while in 35S:ATAF1/anac092-1 (line L6), only 24 genes (15%) and 18genes (11%) were up-regulated by 2- to 4-fold and morethan 4-fold, respectively (Fig. 5F; Supplemental TableS1). Notably, of the 82 SAGs repressed by at least 2-foldin the anac092-1mutant (in accordance with the delay insenescence), 25 genes returned to normal level (log2 foldchange difference from the wild type between20.5 and0.5) or were even induced as in OX-17. Importantly, asmaller fraction of the SAGs returned to normal levelwhen ATAF1 was overexpressed in anac092-1 than inthe Col-0 background (Fig. 5F; Supplemental Table S1).Collectively, our data clearly demonstrate that ORE1 is

an important genetic determinant through which ATAF1exerts its senescence regulatory function.

ORE1 has been shown previously to regulate theexpression of several known SAGs, of which BFN1,SWEET15/SAG29, and SINA1 were shown to be directtargets (Balazadeh et al., 2010a, 2010b; Matallana-Ramirez et al., 2013). As ATAF1 regulates ORE1, wehypothesized that modifying ATAF1 expression affectsORE1 target genes. To test this model, we analyzed theexpression of ORE1 targets in transgenic plants overex-pressingATAF1 in the Col-0 and anac092-1 backgroundsat 35 DAS. As shown in Figure 6A and SupplementalTable S1, expression of all tested ORE1 targets was

Figure 5. ATAF1 requires ORE1 for full control over senescence. A, Elevated expression of ATAF1 in anac092-1 mutant plantstransformed with the 35S:ATAF1 construct at 25 DAS. Data are means6 SD from three independent biological replications from T3generation plants. Asterisks indicate significant differences from anac092-1 (Student’s t test, P # 0.05). dCT, Delta cycle threshold.B, Comparison of ATAF1 overexpressors with Col-0 and the anac092-1mutant grown in long-day conditions. Note the earlier leafyellowing in OX-17 compared with 35S:ATAF1/anac092-1 plants. C, Leaf yellowing. Values on the y axis indicate the percentageof leaves that had at least 50% yellow blades (inspected visually) at a given DAS (n = 12 for each plant line and time point). D,Higher ion leakage of plants overexpressing ATAF1 in the Col-0 and anac092-1 backgrounds compared with Col-0 and theanac092-1 mutant, respectively. Leaves 9 and 11 were combined and used for measuring ion leakage. Data in C and D aremeans 6 SD of three biological replicates (asterisks indicate significant differences from Col-0; Student’s t test, P # 0.05). E, Lowerexpression of the senescence marker gene SAG12 in 35S:ATAF1/anac092-1 than in 35S:ATAF1/Col-0 (OX-17) plants at 35 DASindicates partial but not full recovery of the anac092-1 senescence phenotype. The expression levels of SAG12 in 35S:ATAF1/anac092-1 and 35S:ATAF1/Col-0 (OX-17) plants were normalized against ACTIN2 and compared with Col-0. Data are means6 SD ofthree independent biological experiments. The asterisk indicates a significant difference from OX-17 (Student’s t test, P # 0.05). Fch,Fold change. F, Percentage of up- and down-regulated SAGs in leaf 9 of 35S:ATAF1/Col-0 (OX-17) and 35S:ATAF1/anac092-1 L6 plantsas well as in the anac092-1 mutant compared with Col-0 (45 DAS). The corresponding gene expression data from three independentbiological replications, each determined in two technical replicates, are given in Supplemental Table S1.











enhanced in ATAF1 overexpressors, while their expres-sion, with the exception of SWEET15, remained low in35S:ATAF1/anac092-1 plants, similar to the situation inthe anac092-1 mutant itself. These results are in full accor-dance with the model that ORE1 is a direct target ofATAF1. The fact that the expression of SWEET15 returnedto normal in plants overexpressing ATAF1 in the anac092-1backgroundmay indicate that ATAF1 regulates SWEET15through other transcription factors besides ORE1.

We next tested expression of the chloroplast mainte-nance TFs GLK1 and GLK2 and their target genes (Waterset al., 2009). As seen in Figure 6B and Supplemental TableS1, expression of the genes positively regulated by GLKswas significantly reduced in 35S:ATAF1/Col-0 (OX-17)plants, as expected (see above). Notably, expression ofthese genes was high in the anac092-1 mutant, consis-tent with the delayed senescence in this line and the factthat GLK targets require functional ORE1 protein forrepression during senescence, as reported (Rauf et al.,2013a). The repression of GLKs and their target geneswas largely restored when ATAF1 was overexpressedin the anac092-1 knockout mutant (Fig. 6B; SupplementalTable S1). Thus, collectively, our data are consistent withthe model that ATAF1 regulates senescence by bothinhibiting GLK1 expression and at the same time acti-vating ORE1 expression. However, besides controllingsenescence through the GLK/ORE1 transcriptionalcascades, ATAF1 affects senescence through ABA sig-naling, as shown in the following.

Control of Senescence by ABA Is Mediated by ATAF1

ABA is one of the most effective plant hormonesknown to promote senescence (Zacarias and Reid,1990; Lee et al., 2011; Finkelstein, 2013), and increases inendogenous ABA levels during senescence have beenreported in several plant species, including oat (Avenasativa; Gepstein and Thimann, 1980), rice (Oryza sativa;Philosophhadas et al., 1993), maize (He et al., 2005a),and Arabidopsis (Breeze et al., 2011). As expression ofATAF1 is well induced by ABA treatment (Lu et al.,2007; Wu et al., 2009), we tested whether ATAF1 plays arole in the hormonal control of senescence. To this end,we determined hormone levels in soil-grown plants.The levels of most hormones tested were not signifi-cantly altered in ataf1 null mutants compared with Col-0at 35 DAS, with the exception of ABA, which was re-duced significantly in ataf1 null mutants compared withCol-0 (Supplemental Table S4), similar to a previousreport by Jensen et al. (2008). In addition, overexpres-sion of ATAF1 was reported to lead to higher levels ofABA compared with wild-type plants (Jensen et al.,2013). Thus, the change in ABA levels (down in ataf1knockout and up in ATAF1 overexpressors) well con-forms to the change in senescence observed (delayedin ataf1 mutants and precocious in 35S:ATAF1 lines),suggesting that ABA is causally linked to senescenceinduction or progression.

Previous research has identified a role of ABA as atrigger of H2O2 accumulation under environmentalstress (Jiang and Zhang, 2001; Kwak et al., 2003; Huet al., 2005, 2006; Ye et al., 2011). In addition, H2O2levels are known to increase in senescing leaves, andexternal application of H2O2 promotes leaf senescence,possibly through the activation of senescence-associatedtranscriptional cascades (Leshem, 1988; Navabpouret al., 2003; Mittler et al., 2004; Zimmermann andZentgraf, 2005; Wu et al., 2012). Notably, ATAF1 is not

Figure 6. Gene expression in 35S:ATAF1/Col-0 and 35S:ATAF1/anac092-1plants. A, Expression of ORE1 target genes in plants overexpressing ATAF1in the Col-0 and anac092-1 backgrounds. Note the higher expression ofORE1 targets genes in OX-17 and the reduced expression in 35S:ATAF1/anac092-1 (line L6) plants except for SWEET15. B, Reduced expression ofGLK1 and GLK2 and their target genes in OX-17 and 35S:ATAF1/anac092-1 plants. The heat map indicates the expression difference (log2fold change) between transgenic plants and Col-0. Data represent meansof three independent biological replicates determined in two technicalreplicates each (corresponding data are given in Supplemental Table S1).Measurements were done at 35 DAS.


Garapati et al.









only induced by ABA (Lu et al., 2007; Wu et al., 2009)but also by external treatment of plants with H2O2(Balazadeh et al., 2010b) or intracellular elevation ofH2O2 level by catalase inhibition (Supplemental Fig. S1C),indicating that it may be positioned at a cross-talk nodeconnecting ABA and H2O2 signaling in plant senescence.Accordingly, we asked whether ATAF1 plays a role inABA-induced leaf senescence. We tested the effect ofexogenously applied ABA on the accumulation of H2O2and the induction of senescence in ATAF1 transgenics(Supplemental Fig. S4). We selected nonsenescent leaves4, 5, and 6 of 25-d-old plants for the experiment. After3 d of ABA (20 mM) treatment under light, leaves of35S:ATAF1/Col-0 and 35S:ATAF1/anac092-1 plantsaccumulated significantly higher H2O2 levels than Col-0;in contrast, leaves of the ataf1-4mutant accumulated lessH2O2 after ABA treatment, which was accompanied bya significantly better retention of chlorophyll comparedwith Col-0 (Supplemental Fig. S4, A–C).Recently, Jia et al. (2013) reported that PHOSPHOLIPASE

Dd (PLDd) plays an important role in ABA-promotedleaf senescence in Arabidopsis. The enzyme also func-tions in various environmental stresses, including theresponse to dehydration, salinity stress, and freezing,and plays a role in programmed cell death in response toH2O2 treatment. As shown by the authors, expression ofPLDd rises during ABA-stimulated senescence in de-tached leaves, and a pldd knockout mutant exhibiteddelayed senescence in the presence of ABA, while nodifference comparedwith the wild type was observed inthe absence of the hormone. Here, we used qRT-PCR totest the expression of PLDd, along with genes involvedin calcium signaling and chlorophyll degradation. Weincluded calcium signaling genes in our analysis, as Ca2+-mediated signal transduction plays a fundamental rolein many abiotic stress responses, including those in-volving changes in ABA and reactive oxygen species(ROS; H2O2) levels (Pei et al., 2000; Tuteja and Mahajan,2007; Wang and Song, 2008). We included chlorophylldegradation genes to monitor the transcriptional onsetof pigment degradation as a proxy for senescence. Asevident from the data shown in Supplemental Table S5,overexpression of ATAF1 significantly increased the ex-pression of PLDd and various calcium signaling-relatedgenes, irrespective of the genetic background (Col-0 oranac092-1). Similarly, chlorophyll degradation genes wereup-regulated upon ATAF1 overexpression (SupplementalTable S5). These results, together with the higher accu-mulation of H2O2 in ATAF1 overexpressors (see above),strongly support the model that ATAF1 affects senes-cence through ABA-mediated signaling and that thiscontributes to the partial restoration of the senescencesyndrome observed in 35S:ATAF1/anac092-1 plants.

ATAF1 Regulates ABA Homeostasis Genes

ATAF1 modulates ABA biosynthesis by directly regu-lating the expression of the ABA biosynthetic geneNCED3(Jensen et al., 2013). To test whether ATAF1 affects other

genes involved in ABA homeostasis, we examined theexpression of 13 genes involved in ABA signaling,ABA metabolism/transport, and ABA responses inthe ataf1-4 mutant as well as in 35S:ATAF1/Col-0 and35S:ATAF1/anac092-1 plants (Fig. 7; Supplemental TableS1). The expression of BCH2 (involved in the synthesis ofthe epoxycarotenoid biosynthetic precursor zeaxanthin),NCED3 (involved in the cleavage of 9-cis-epoxycarotenoidsto xanthoxin), and CYP707A1 (involved in ABA catab-olism) was significantly down-regulated in the ataf1-4mutant and up-regulated in 35S:ATAF1 plants. Also,the expression of genes involved in ABA signaling (suchas protein phosphatase 2Cs, PYR1-LIKE6, and OPENSTOMATA1) and ABA import (ABCG40) as well as ofABA-responsive genes such as WRKY40 and WRKY63was strongly down-regulated in the ataf1-4 mutant andup-regulated in ATAF1 overexpressors. Furthermore,expression of these genes was not significantly al-tered in the anac092-1mutant compared with the wildtype (Supplemental Table S1), while they were inducedin 35S:ATAF1/anac092-1 plants, indicating that theATAF1-dependent increase in the expression of thegenes is independent of ORE1/ANAC092.

Next, we analyzed the promoters of the differentiallyexpressed genes for potential ATAF1-binding sites andfound full motifs [VDHVnnYRR(6-5n)YACGnMWSK]in the NCED3 (640 bp upstream the transcription startsite) and ABCG40 (2,246 bp upstream) promoters,suggesting that both genes are direct downstream tar-gets of ATAF1. As a further confirmation of this model,

Figure 7. Expression of ABA signaling and metabolic genes. Elevatedexpression is shown for genes involved in ABA biosynthesis, transport,signaling, responses, and catabolism in OX-17 and 35S:ATAF1/anac092-1plants. The heat map indicates the expression difference (log2 fold change)between transgenic plants and Col-0. Data represent means of two inde-pendent biological experiments with two technical replicates each (corre-sponding data are given in Supplemental Table S1).















we tested the expression of both genes in ATAF1-IOElines after estradiol induction. Expression of ABCG40was rapidly (within 1 h) induced after estradiol treat-ment, while expression of NCED3 was up-regulated atlater time points (Fig. 8A; Supplemental Table S1). Us-ing EMSA, we confirmed the in vitro binding of ATAF1to 40-bp-long double-stranded oligonucleotides repre-senting the respective promoter fragments, includingthe ATAF1 binding motifs (Fig. 8B). Finally, usingChIP-qPCR, we examined whether ATAF1 binds to theABCG40 promoter in vivo. The expression of ABCG40was induced in 35S:ATAF1-GFP plants, indicating faith-ful regulation of ABCG40 by the ATAF1-GFP fusionprotein (Fig. 8C). Using these plants, we observed asignificant enrichment of the ABCG40 promoter (Fig.8D), supporting the conclusion that ABCG40 is a directdownstream target of ATAF1. Recently, Jensen et al.(2013) showed in vivo binding of ATAF1 to the pro-moter of NCED3.

ABA- and H2O2-Triggered Expression Changes AreModified by ATAF1

In various plant cells or tissues, ABA can cause thegeneration of ROS such as H2O2 (Pei et al., 2000; Jiangand Zhang, 2001; Lin and Kao, 2001; Laloi et al., 2004;Hu et al., 2005), and increased levels of H2O2 result inan oxidative burst and ultimately cell death (Gechevand Hille, 2005). As the expression of ATAF1 is inducedby treatment of plants with ABA (Lu et al., 2007; Wuet al., 2009) or H2O2 (Balazadeh et al., 2010b), we pro-posed an ABA- and H2O2-dependent regulation of itstwo photosynthesis- and senescence-related targetgenes (i.e. ORE1 and GLK1) through ATAF1. To testthis hypothesis, we analyzed the expression of ORE1and GLK1, as well as of their direct downstream targets,in 2-week-old Arabidopsis seedlings (Col-0, OX-17, andataf1-4) after a 4-h treatment with ABA (10 mM) or H2O2(10 mM). In both conditions, expression of GLK1 and its

Figure 8. NCED3 and ABCG40 are direct targets of ATAF1. A, Expression of NCED3 and ABCG40 in 2-week-old ATAF1-IOEseedlings after estradiol (ESTR) induction of ATAF1. The heat map indicates the difference in expression (log2 fold change) com-pared with mock-treated samples. Data represent means of three independent biological replicates, determined in two technicalreplicates each (corresponding data are given in Supplemental Table S1). B, EMSA. Purified ATAF1-CELD protein binds specificallyto the ATAF1-binding site within the promoters of NCED3 and ABCG40. In vitro DNA-binding reactions were performed with40-bp promoter fragments containing the respective ATAF1-binding motifs (NCED3, 59-GACACTAATATAAACTACGTACC-39; andABCG40, 59-AGATGCGTGGGAGGCTTGGGGAC-39). C, Expression levels of ATAF1 and ABCG40 in 2-week-old 35S:ATAF1-GFPseedlings compared with Col-0; ATAF1 expression in 35S:ATAF1-GFP seedlings represents the combined expression of the en-dogenous ATAF1 gene and the ATAF1-GFP transgene. Numbers on the y axis indicate expression fold change (log2 Fch) of geneexpression in 35S:ATAF1-GFP compared with Col-0. Data are means 6 SD of three independent biological replicates, each de-termined in two technical replicates. D, ChIP-qPCR. Shoots of 2-week-old 35S:ATAF1-GFP seedlings were harvested for the ChIPexperiment. qPCR was used to quantify enrichment of the ABCG40 promoter. As a negative control (NC), qPCRwas performed on apromoter (CLAVATA1; At1g75820) lacking ATAF1-binding sites. Data represent means6 SD (two independent biological replicates,each with three technical replicates).


Garapati et al.





target genes was repressed in Col-0; notably, how-ever, this repression was significantly stronger in OX-17seedlings but less prominent in ataf1-4 seedlings(Supplemental Table S6). With respect to ORE1, thesame stress treatments triggered a prominent increaseof its expression in OX-17 seedlings, which was weakerin Col-0 and lowest in the ataf1-4 mutant; ORE1 targetsshowed the same clear response (Supplemental TableS6). Importantly, all three ABA-related genes we tested(i.e. NCED3, ABCG40, and CYP707A1) also showedenhanced expression after ABA or H2O2 treatment inCol-0 seedlings, a response that was stimulated in OX-17but lowered in ataf1-4 lines (Supplemental Table S6).These data provide strong evidence for the model thatATAF1 regulates ABA- and H2O2-modulated senescencethrough GLK1 andORE1. In addition, they reveal a tightcross talk between ABA- and H2O2-mediated sig-naling through the action of ATAF1.Previously, delayed senescence in plants was ob-

served to be associated with an increased tolerance tooxidative stress (Woo et al., 2004; Wu et al., 2012), andATAF1 overexpressors were reported to be hypersen-sitive to oxidative stress imposed by treating seedlingswith Rose Bengal or paraquat, which trigger the for-mation of singlet oxygen or superoxide free radical,respectively; however, no effect of these chemicals wasobserved in ataf1 knockout mutants (Wu et al., 2009).However, considering our above-reported results onthe central role of ATAF1 in conveying the expres-sional effect of H2O2 through GLK1 and ORE1 on theirdownstream target genes, we wondered whether H2O2treatment would differentially affect the phenotype ofwild-type versus ATAF1-altered plants. To this end,2-week-old ataf1 mutant seedlings were incubated for2 d in liquid MS medium containing 10 mM H2O2 andthe chlorophyll level was determined. The decreasein chlorophyll content after H2O2 treatment was lessprominent in the three ataf1 mutants compared withthe wild type (Fig. 9, A and B). A less severe H2O2-triggered chlorophyll loss was also observed in de-tached ataf1-2 (Fig. 9C) and ataf1-4 leaves (data notshown). In accordance with this finding, ATAF1 over-expressors accumulated more, and ataf1-2 mutantsless, H2O2 than the Col-0 wild type, as shown by 3,39-diaminobenzidine (DAB) staining (Fig. 9D). Similarly,overexpression of ATAF1 from an estradiol-induciblepromoter (ATAF1-IOE line) stimulated chlorophyll lossin the presence of both estradiol and H2O2, confirmingthe important role of ATAF1 in this physiological re-sponse. Importantly also, estradiol treatment alonetriggered chlorophyll loss (albeit to a lesser extent; Fig.9, E and F), fully supporting the model of a close in-teraction between ABA- and H2O2-mediated signaling.

DISCUSSION

Senescence is a highly controlled process that re-quires the coordinated reprogramming of hundreds ofgenes. Previous work has identified a large number of

NAC TFs being up-regulated during senescence, andSAGs are enriched for NAC-binding sites in theirpromoters, establishing the NAC family as the largestgroup of plant-specific senescence-related transcriptionfactors (Balazadeh et al., 2008; Breeze et al., 2011).Several NAC TFs induced during senescence are alsoresponsive to H2O2 treatment and are considered toplay a role in ROS-mediated signaling networks thatmodulate plant longevity (Balazadeh et al., 2010b; Wuet al., 2012). Genes affected by senescence-associatedNAC TFs have been reported for several members ofthe family, and in some cases direct target genes wereidentified, such as for ORE1 (Balazadeh et al., 2010a;Matallana-Ramirez et al., 2013; Rauf et al., 2013a),AtNAP (Zhang and Gan, 2012b), and JUB1 (Wu et al.,2012). Besides regulating senescence, NAC TFs playimportant roles in various other physiological pro-cesses, including the responses to abiotic and bioticstresses and in hormone signaling (Wu et al., 2012;De Clercq et al., 2013; Jensen et al., 2013; Rauf et al.,2013b; Xu et al., 2013; for review, see Nuruzzaman et al.,2013). Here, we demonstrate the senescence-associatedrole of another NAC TF, the ABA- and H2O2-inducedfactor ATAF1.

As shown, overexpression of ATAF1 promotes de-velopmental as well as dark- and ABA-induced senes-cence, while knocking out the gene’s function delayssenescence (Figs. 1 and 3; Supplemental Fig. S4), indi-cating that ATAF1 positively affects senescence. In linewith this model, the expression of ATAF1 increases inleaves during developmental and dark-induced senes-cence (Lin and Wu, 2004; Balazadeh et al., 2008). Fur-thermore, a number of abiotic stresses, including salinity(Wu et al., 2009; Supplemental Fig. S1B), drought (Luet al., 2007), and oxidative stress (Balazadeh et al., 2010a;Supplemental Fig. S1C), as well as pathogen attack(Jensen et al., 2008) trigger ATAF1 expression, in ac-cordance with the widely known senescence-inducingeffect these stresses have (Lim et al., 2007; Gepsteinand Glick, 2013). An increase in ATAF1 expression isalso observed after ABA treatment (Wu et al., 2009;Genevestigator).

To unravel the molecular mechanism through whichATAF1 regulates senescence, we performed qRT-PCR-based expression profiling of 15 PAGs and 158 SAGsin ATAF1 overexpressor and ataf1 mutant lines andcompared the data with those obtained from the Col-0wild type. Among the differentially expressed geneswere GLK1 and ORE1, both of which contain bipartiteATAF1-binding sites in their 59 upstream regulatoryregions (promoters), revealing them as potential directtargets of ATAF1. Indeed, the transcript level of ORE1increases rapidly, while that of GLK1 decreases, uponestradiol-mediated induction of ATAF1 in ATAF1-IOEplants. In addition, ATAF1 binds in vivo to the pro-moters of ORE1 and GLK1, as shown by ChIP, allowingthe direct regulation of both genes in planta. How-ever, it remains unknown at present how the sameTF (i.e. ATAF1) can both repress (GLK1) and activate(ORE1) gene expression. One possible model is that












ATAF1 interacts with different other TFs to exert itsdifferent regulatory functions toward its target genes. Asimilar situation appears to exist for PHYTOCHROME-INTERACTING FACTOR4 (PIF4), which activates thechlorophyll degradation regulator NONYELLOWING1but represses the expression of GLK2 by direct bindingto their promoters, thereby triggering senescence (Songet al., 2014).

As shown by several laboratories independently,ORE1 is a key positive regulator of senescence in Arabi-dopsis, limiting the longevity of leaves, cotyledons, andinflorescences (Kim et al., 2009; Balazadeh et al., 2010a;Trivellini et al., 2012). In accordance with this, severalSAGs have been identified to be under direct or indirectcontrol of ORE1 (Balazadeh et al., 2010a; Breeze et al.,2011; Matallana-Ramirez et al., 2013; Rauf et al., 2013a),highlighting this NAC as one of the core control elementsof the transcriptome underlying the senescence syn-drome. In contrast, GLK genes exist as a pair of func-tionally redundant homologous genes, GLK1 and GLK2,in Arabidopsis, and they have been shown to directlyregulate the expression of photosynthesis apparatus-related genes during chloroplast development andmaintenance (Waters et al., 2009).

We have shown previously that ORE1 and GLKsinteract at the protein level to inhibit the transcriptionalactivation of GLK target genes, while the expression ofORE1 target genes is not affected (Rauf et al., 2013a).Thus, in developing and expanding leaves, when GLK1and GLK2 expression is high and ORE1 expression islow, ORE1 cannot disrupt the chloroplast maintenancefunction of GLKs. However, this situation changeswhen leaves age and the expression of ORE1 increasesand the formation of GLK-ORE1 heteromers becomesphysically possible. Thus, ORE1 balances leaf senes-cence against maintenance by antagonizing GLK-mediated transcription (Rauf et al., 2013a). However,although the age-dependent expression change of GLKs(reduced during senescence) andORE1 (induced duringsenescence) was known before, the molecular mecha-nism steering these opposing expressional changes wasnot. Here, we present convincing evidence that theNAC TF ATAF1 represents such an upstream regu-latory element. In addition to ATAF1, several otherTFs, namely PIF4 and PIF5, the basic Leu zippersABSCISIC ACID INSENSITIVE5 and ENHANCED EMLEVEL involved in ABA signaling, and ETHYLENE-INSENSITIVE3 (EIN3), a key transcription factor in

Figure 9. ATAF1 negatively regulatesH2O2 tolerance. A, Two-week-old wild-type (WT) and ataf1-2 seedlings wereincubated for 2 d in liquid MS mediumcontaining 10 mM H2O2. Note the bettersurvival of the ataf1-2 seedlings (right).B, Seedlings of all three ataf1 knockoutmutants retained chlorophyll significantlybetter than the wild type after H2O2

treatment. FW, Fresh weight. C, Ma-ture leaves treated with 20 mM H2O2

for 2 d. Note the reduced chlorosis ofataf1-2 leaves compared with leavesfrom wild-type or OX-17 plants afterH2O2 treatment. A similar result wasobtained for the ataf1-4 mutant (datanot shown). D, DAB staining of H2O2-treated and untreated leaves. Leavesof the ATAF1 overexpressor exhibitedstronger brown staining than leaves fromwild-type or ataf1-2 plants. E, Change inleaf pigmentation in ATAF1-IOE seed-lings grown for 1 week in liquid MSmedium containing 10 mM estradiol(ESTR) or 10 mM estradiol plus 10 mM

H2O2. Estradiol and H2O2 were omittedin mock treatments. An empty-vector(EV) line was used as a control. Beforetreatment, seedlings were grown for 2weeks on solid MS medium. F, De-creased chlorophyll retention in estradiol-treated ATAF1-IOE seedlings upon H2O2

treatment. Data in B and F representmeans of three independent biologi-cal replicates 6 SD. Asterisks indicatedata significantly different from controlplants: the wild type in B and the emptyvector in F (Student’s t test, P # 0.05).


Garapati et al.



ethylene signaling, were found to bind to the ORE1promoter to regulate its expression (Sakuraba et al.,2014). In addition, ORE1 is negatively regulated bymicroRNA164 (miR164), whose abundance is high indeveloping leaves but decreases in leaves undergoingsenescence (Kim et al., 2009). Recently, EIN3 was shownto repress miR164 transcription by binding to its pro-moter, thereby up-regulating transcript levels of ORE1,leading to early senescence (Li et al., 2013). Thus, eth-ylene is an important trigger of ORE1 gene expression,as reported earlier (He et al., 2005b; Du et al., 2014).ATAF1, shown here to function as a positive up-

stream regulator of ORE1, is induced by treatment withABA or H2O2. Similarly, ORE1 expression is induced byboth environmental signal mediators, while the ex-pression of GLK1 shows the opposite response (i.e.repression by ABA or H2O2 treatment or conditions thatlead to intracellular changes in H2O2 concentration;Supplemental Table S6; Supplemental Fig. S5, A and B),suggesting that the stress-dependent transcriptional re-sponse of ORE1 and GLK1 is conveyed by ATAF1.Indeed, as we show here, ABA-/H2O2-dependent ex-pression changes are impaired in ataf1 mutants butstimulated in ATAF1 overexpressors (SupplementalTable S6). Thus, our data extend the existing molec-ular model of ORE1 regulation (Kim et al., 2009; Wooet al., 2013) by adding a further layer of control con-necting it, via ATAF1, to ABA- and H2O2-dependentstress signaling (Fig. 9).Lower and higher basal ABA levels in ataf1 mutants

(Jensen et al., 2008; Supplemental Table S4) and ATAF1overexpressors (Jensen et al., 2013), respectively, sug-gest a role of ATAF1 in ABA-induced physiologicalchanges. Notably, exogenously applied ABA can actas an inducer of H2O2 generation in plant cells or tissues(Kwak et al., 2003; Hu et al., 2005; Xing et al., 2008; Yeet al., 2011), and increased levels of H2O2 trigger se-nescence through the activation of senescence-associatedtranscriptional cascades (Wu et al., 2012; Wang et al.,2013). As shown here, the ataf1-4 mutant exhibits adelayed progression of senescence, while ATAF1 over-expressors exhibit precocious senescence in responseto exogenously applied ABA. Additionally, the expres-sion of PLDd, which is an important mediator in ABA-promoted senescence (Jia et al., 2013), and genes involvedin calcium signaling along with chlorophyll degrada-tion is enhanced in ATAF1 overexpressors, highlightingthe role of ATAF1 as a transcriptional regulator of ABAuptake and/or an amplifier of the signaling cascade thatresults in ABA-promoted senescence.The synchronized expression of genes involved in

ABA signaling as well as ABA metabolism and trans-port help to maintain ABA homeostasis in crops suchas rice, wheat, and barley (Hordeum vulgare; Govindet al., 2011; Ji et al., 2011). The expression of genesinvolved in ABA signaling, ABA metabolism/import,and ABA responses was up-regulated in ATAF1 over-expressors. Considering the fact that ATAF1 overex-pressors show higher sensitivity to ABA treatment andprecocious senescence, we propose an overlap between

ATAF1 regulatory pathways in response to altered ABAhomeostasis and developmental senescence. Moreover,NCED3, which is an ABA biosynthetic gene, was foundto be a direct target of ATAF1 (Jensen et al., 2013). Here,we confirmed the activation of NCED3 by ATAF1 anddemonstrated in vitro binding of ATAF1 protein to itsbipartite binding site within the NCED3 promoter (Fig.8B), which, however, differed from the monopartitebinding site identified by Jensen et al. (2013). Thus,ATAF1 might regulate NCED3 by binding to at leasttwo 59-upstream binding sites. We additionally foundthat ABCG40 (encoding an ABA importer) harbors thebinding site in its promoter to which ATAF1 binds invitro and in vivo (Fig. 8, B and D).

Taking the experimental data together, we concludethat ATAF1 regulates senescence through a gene reg-ulatory network that involves various target genes thataffect aging-induced cell death (ORE1), photosynthesis(GLK1), and ABA biosynthesis and import (NCED3and ABCG40; Fig. 10). Our data thus provide a mecha-nistic explanation for the long-known stimulatory effect

Figure 10. Model for the integration of ATAF1 function during devel-opmental and stress-induced leaf senescence in Arabidopsis. ATAF1activatesORE1 and repressesGLK1 expression by directly binding to thepromoters of both genes (ATAF1-binding sites are indicated by blackboxes). Consequently, the expression of GLK target genes is impaired,resulting in an age-dependent decline in the expression of PAGs (GLKtargets), while the expression of ORE1 target genes is enhanced, leadingto the onset and subsequent progression of senescence. ATAF1 alsoactivates the expression of genes involved in ABA biosynthesis andtransport (NCED3 and ABCG40) and through this contributes to ABA-induced senescence. ATAF1 expression is regulated by unknown up-stream TFs, allowing it to respond to ABA and H2O2 in a stress-relatedmanner. Arrow-ending lines and T-ending lines indicate positive andnegative interactions, respectively. The inhibitory effect of the ORE1protein on GLK transcription factors was reported by Rauf et al. (2013a).










that ABA (and abiotic stresses promoting a rise in cellularABA level, such as drought) has on chloroplast deteri-oration, the down-regulation of photosynthesis, andsenescence (Noodén, 1988; Lim et al., 2007).

Another senescence regulatory NAC TF in Arabidopsis,namely AtNAP, has recently been shown to regulateABA- and developmental senescence-dependent ex-pression of SAG113 by directly binding to its promoter(Zhang and Gan, 2012a). SAG113 encodes a Golgi-localized protein phosphatase of the protein phospha-tase 2C family; the protein is involved in the control ofwater loss from leaves (through stomata) during se-nescence (Zhang et al., 2012). In addition, recent dataindicate that AtNAP also directly activates ABSCISICALDEHYDE OXIDASE3, which encodes an enzymeresponsible for the last step in ABA biosynthesis (Yanget al., 2014). In rice, the functional ortholog of AtNAP(i.e. OsNAP) promotes ABA-induced senescence byactivating SAGs, including those that encode chloro-phyll degradation enzymes (Liang et al., 2014). In ad-dition, a plasma membrane-anchored NAC TF, namelyNAC WITH TRANSMEMBRANE MOTIF1-LIKE4(ANAC053), has been reported to promote the pro-duction of ROS (H2O2 and superoxide radicals) duringdrought-stimulated senescence by activating the expres-sion of RESPIRATORY BURST OXIDASE HOMOLOGgenes leading to increased ROS formation; expressionof NTL4 is induced by drought and ABA treatment aswell as during developmental senescence (Lee et al.,2012). Thus, plants appear to have recruited variousNAC factors to integrate different cellular activities intoa coherent physiological process during stress-promotedleaf senescence. Further studies employing genetic andbiochemical approaches will help to further unravel theintricate regulatory networks connecting developmen-tal with stress-triggered senescence.

MATERIALS AND METHODS

General

Standard molecular techniques were performed as described (Sambrooket al., 2001). Oligonucleotide sequences are given in Supplemental Table S7.Unless indicated otherwise, chemicals and reagents were obtained from Merck,Invitrogen, Sigma-Aldrich, and Fluka. Molecular biological kits were obtainedfrom Qiagen and Macherey-Nagel. qRT-PCR primers were designed usingQuantPrime (www.quantprime.de). Genevestigator (www.genevestigator.com)and the eFP browser (www.bar.utoronto.ca/efp/cgi-bin/efpWeb.cgi) were usedfor gene expression pattern analysis.

Biological Material and Growth Conditions

All experiments were performed using Arabidopsis (Arabidopsis thaliana)accession Col-0 (Institut National de la Recherche Agronomique; http://dbsgap.versailles.inra.fr/publiclines). Seeds were germinated in soil (Einheitserde GS90;Gebrüder Patzer) in a climate-controlled chamber with a 16-h daylength providedby fluorescent light at approximately 100 mmol m22 s21, day/night temperatureof 20°C/16°C, and relative humidity of 60%/75%. After 2 weeks, seedlings weretransferred to a growth chamber with a 16-h day (80 or 120 mmol m22 s21), day/night temperature of 22°C/16°C, and 60%/75% relative humidity. For growth onplates, Arabidopsis seeds were surface sterilized using 10% (v/v) sodium hy-pochlorite solution and sown on nutrient agar medium (13MS medium and 1%[w/v] Suc, pH 5.8). The plates were stored for 2 d under vernalization conditions

(16 h of light at 22°C and 8 h of dark at 4°C) and then transferred to long-daygrowth conditions (16 h of light at 22°C and 8 h of dark at 18°C). Seed stocks ofATAF1T-DNA insertion lines (Supplemental Fig. S2) were obtained from the NottinghamArabidopsis Stock Centre (http://arabidopsis.info). The anac092-1 T-DNA in-sertion mutant was reported previously (Balazadeh et al., 2010a).

DNA Constructs

Constructs were generated employing PCR. Strategies for construct gen-eration and primer sequences are listed in Supplemental Table S7. Ampliconsgenerated by PCR were checked for correctness by DNA sequence analysis(Eurofins MWG Operon). Constructs were transformed into Arabidopsis viaAgrobacterium tumefaciens GV3101 (pMP90)-mediated transformation.

Gene Expression Analysis

Total RNA extraction, synthesis of complementary DNA, and qRT-PCRusing SYBR Green were performed as described (Caldana et al., 2007; Balazadehet al., 2008). PCR was performed using an ABI PRISM 7900HT sequence de-tection system (Applied Biosystems). PCR experiments were generally per-formed on plants from two or three independent growth experiments, asindicated in the figure legends, and each sample was measured in two technicalreplicates. Expression levels were normalized against the expression level ofACTIN2. Statistical significance was estimated using Student’s t test.

DNA-Binding Site Selection

In vitro binding site selection was performed using the CELD systemwiththe pTacATAF1-CELD6XHis construct, employing a biotin-labeled double-stranded oligonucleotide, which contained a 30-nucleotide random sequence(Xue, 2005). ATAF1-selected oligonucleotides were cloned and sequenced.The DNA-binding activity of ATAF1-CELD was measured using methyl-umbelliferyl b-D-cellobioside as substrate (Xue, 2002). In control experiments,DNA-binding assays were performed with a biotin-labeled single-stranded oli-gonucleotide or a biotin-labeled double-stranded oligonucleotide lacking a targetbinding site.

EMSA

Recombinant ATAF1-CELD fusion protein was expressed in Escherichia colistrain BL21 (DE3) pLysS (Agilent Technologies) as described (Dortay et al.,2011). Protein was purified using Protino Ni-IDA 150 packed columns(Macherey-Nagel). EMSA was performed as described (Wu et al., 2012; Raufet al., 2013b) using the Odyssey Infrared EMSA kit (Li-Cor). 59-DY682-labeledDNA fragments were purchased from Eurofins MWG Operon.

ChIP-qPCR

ChIP-qPCR was performed with 2-week-old Arabidopsis seedlings ex-pressing GFP-tagged ATAF1 protein from the CaMV 35S promoter (35S:ATAF1-GFP). Col-0 plants served as negative controls. ChIP was performed asdescribed (Kaufmann et al., 2010) employing anti-GFP antibody to immuno-precipitate protein-DNA complexes. The ChIP experiment was run in two bio-logical replications with three technical replications per assay. Primers used forqPCR flanked the ATAF1-binding sites within the promoter regions of ORE1,GLK1, and ABCG40. Primers annealing to promoter regions of an Arabidopsisgene (CLAVATA1; At1g75820) lacking an ATAF1-binding site were employed innegative-control experiments. ChIP-qPCR data were analyzed as described(Wu et al., 2012).

Treatments

For estradiol induction, 12-d-old seedlings were incubated in liquid MSmedium containing 10 mM estradiol (control treatment; 0.1% [v/v] ethanol).The seedlings were kept on a rotary shaker for 1, 2, 5, or 10 h, harvested, andimmediately frozen in liquid nitrogen. Gene expression levels were deter-mined by qRT-PCR. For estradiol induction on plates, MS medium was sup-plemented with 20 mM estradiol (control; 0.2% [v/v] ethanol). For ABA treatment,fully expanded green and nonsenescent leaves were detached from the ro-settes of 25-d-old plants (Col-0, anac092-1, and ATAF1 transgenics) grown on


Garapati et al.



http://www.quantprime.de

http://www.genevestigator.com

http://www.bar.utoronto.ca/efp/cgi-bin/efpWeb.cgi

http://dbsgap.versailles.inra.fr/publiclines

http://dbsgap.versailles.inra.fr/publiclines


http://arabidopsis.info



soil at 22°C under a 16-h-light/8-h-dark photoperiod in 50% relative humidityand incubated for 3 d in water with or without ABA (20 mM) under white lightat room temperature. For ABA or H2O2 treatment, 12-d-old seedlings wereincubated for 4 h on a rotary shaker (90 rpm) in liquid MS medium containing10 mM ABA or 10 mM H2O2, respectively.

Determination of H2O2 Level

Quantitative measurement of H2O2 level was performed using the AmplexRed Hydrogen Peroxide/Peroxidase Assay kit (Molecular Probes) as de-scribed by Wu et al. (2012). For DAB staining, detached leaves were vacuuminfiltrated in DAB solution (0.5 mg L21) for 30 min and then incubated for 4 hat room temperature. Chlorophyll was removed by extraction with 80% (v/v)hot ethanol.

Chlorophyll Measurement

Chlorophyll content was determined using a SPAD analyzer (N-tester;Hydro Agri). Alternatively, frozen Arabidopsis leaves were ground in liquidnitrogen, resuspended in 5mL of 80% (v/v) acetone inwater, and homogenizedfor 1min. Chlorophyll content was determinedwith a spectrophotometer at 663and 646 nm as described by Arnon (1949).

Electrolyte Leakage Measurements

Ion leakage in pooled leaves 9 and 11was determined by immersing them in10 mL of deionized water and shaking at room temperature for 30 min. Initialelectrical conductivity was measured at 25°C using a conductometer (Schott).Then, samples were boiled for 15 min and cooled down to 25°C, and totalconductivity was measured again. Conductivity was expressed as the per-centage of the initial conductivity versus the total conductivity.

Other Methods

Hormone levels were determined as reported by Müller and Munné-Bosch(2011). The presence of ATAF1-GFP fusion protein in transgenic plants wasanalyzed by confocal fluorescence microscopy (Eclipse E600 microscope; Nikon).Student’s t test embedded in Microsoft Excel was used for testing statisticalsignificance; only the return of P # 0.05 was regarded as statistically significant.

Sequence data from this article can be found in the Arabidopsis GenomeInitiative or GenBank/EMBL databases under the following codes: ABCG40(At1g15520),ACTIN2 (At3g18780),ATAF1/ANAC002 (At1g01720),GLK1 (At2g20570),ORE1/ANAC092 (At5g39610), NCED3 (At3g14440), and PLDd (At4g35790).Additional codes are given in Supplemental Tables S1, S3, S5, S6, and S7.

Supplemental Data

The following supplemental materials are available.

Supplemental Figure S1. Senescence- and stress-dependent expressionof ATAF1.

Supplemental Figure S2. T-DNA insertions and ATAF1 expression.

Supplemental Figure S3. Expression of PAGs and SAGs.

Supplemental Figure S4. ABA-induced senescence.

Supplemental Figure S5. Hydrogen peroxide effects on gene expression.

Supplemental Table S1. Data of figures.

Supplemental Table S2. DNA-binding site selection.

Supplemental Table S3. Data of supplemental figures.

Supplemental Table S4. Hormone concentrations.

Supplemental Table S5. Expression of genes involved in calcium signalingand chlorophyll degradation in leaves during ABA-promoted senescence.

Supplemental Table S6. Expression of ATAF1 target genes in H2O2- andABA-treated seedlings.

Supplemental Table S7. Constructs and primer sequences.

ACKNOWLEDGMENTS

We thank Karin Koehl and the Max Planck Institute of Molecular PlantPhysiology for plant care, Maren Müller (University of Barcelona) for tech-nical assistance in hormone analyses, and the University of Potsdam andthe Max Planck Institute of Molecular Plant Physiology for supporting theresearch.

Received April 17, 2015; accepted May 5, 2015; published May 7, 2015.

LITERATURE CITED

Allu AD, Soja AM, Wu A, Szymanski J, Balazadeh S (2014) Salt stress andsenescence: identification of cross-talk regulatory components. J Exp Bot65: 3993–4008

Andersson A, Keskitalo J, Sjödin A, Bhalerao R, Sterky F, Wissel K,Tandre K, Aspeborg H, Moyle R, Ohmiya Y, et al (2004) A transcrip-tional timetable of autumn senescence. Genome Biol 5: R24

Arnon DI (1949) Copper enzymes in chloroplasts: polyphenoloxidases inBeta Vulgaris. Plant Physiol 32: 691–697

Baena-González E, Rolland F, Thevelein JM, Sheen J (2007) A centralintegrator of transcription networks in plant stress and energy signal-ling. Nature 448: 938–942

Balazadeh S, Kwasniewski M, Caldana C, Mehrnia M, Zanor MI, Xue GP,Mueller-Roeber B (2011) ORS1, an H2O2-responsive NAC transcriptionfactor, controls senescence in Arabidopsis thaliana. Mol Plant 4: 346–360

Balazadeh S, Riaño-Pachón DM, Mueller-Roeber B (2008) Transcriptionfactors regulating leaf senescence in Arabidopsis thaliana. Plant Biol(Stuttg) (Suppl 1) 10: 63–75

Balazadeh S, Schildhauer J, Araújo WL, Munné-Bosch S, Fernie AR,Proost S, Humbeck K, Mueller-Roeber B (2014) Reversal of senescenceby N resupply to N-starved Arabidopsis thaliana: transcriptomic andmetabolomic consequences. J Exp Bot 65: 3975–3992

Balazadeh S, Siddiqui H, Allu AD, Matallana-Ramirez LP, Caldana C,Mehrnia M, Zanor MI, Köhler B, Mueller-Roeber B (2010a) A generegulatory network controlled by the NAC transcription factorANAC092/AtNAC2/ORE1 during salt-promoted senescence. Plant J 62:250–264

Balazadeh S, Wu A, Mueller-Roeber B (2010b) Salt-triggered expression ofthe ANAC092-dependent senescence regulon in Arabidopsis thaliana.Plant Signal Behav 5: 733–735

Breeze E, Harrison E, McHattie S, Hughes L, Hickman R, Hill C, Kiddle S,Kim YS, Penfold CA, Jenkins D, et al (2011) High-resolution temporalprofiling of transcripts during Arabidopsis leaf senescence reveals a distinctchronology of processes and regulation. Plant Cell 23: 873–894

Buchanan-Wollaston V, Page T, Harrison E, Breeze E, Lim PO, Nam HG,Lin JF, Wu SH, Swidzinski J, Ishizaki K, et al (2005) Comparativetranscriptome analysis reveals significant differences in gene expressionand signalling pathways between developmental and dark/starvation-induced senescence in Arabidopsis. Plant J 42: 567–585

Caldana C, Scheible WR, Mueller-Roeber B, Ruzicic S (2007) A quanti-tative RT-PCR platform for high-throughput expression profiling of2500 rice transcription factors. Plant Methods 3: 7

Cantu D, Pearce SP, Distelfeld A, Christiansen MW, Uauy C, Akhunov E,Fahima T, Dubcovsky J (2011) Effect of the down-regulation of the highgrain protein content (GPC) genes on the wheat transcriptome duringmonocarpic senescence. BMC Genomics 12: 492

De Clercq I, Vermeirssen V, Van Aken O, Vandepoele K, Murcha MW,Law SR, Inzé A, Ng S, Ivanova A, Rombaut D, et al 2013) Themembrane-bound NAC transcription factor ANAC013 functions in mi-tochondrial retrograde regulation of the oxidative stress response inArabidopsis. Plant Cell 25: 3472–3490

Dortay H, Schmöckel SM, Fettke J, Mueller-Roeber B (2011) Expression ofhuman c-reactive protein in different systems and its purification fromLeishmania tarentolae. Protein Expr Purif 78: 55–60

Du J, Li M, Kong D, Wang L, Lv Q, Wang J, Bao F, Gong Q, Xia J, He Y(2014) Nitric oxide induces cotyledon senescence involving co-operationof the NES1/MAD1 and EIN2-associated ORE1 signalling pathways inArabidopsis. J Exp Bot 65: 4051–4063

Finkelstein R (2013) Abscisic acid synthesis and response. The ArabidopsisBook 11: e0166, doi/10.1199/tab.0166

Gechev TS, Hille J (2005) Hydrogen peroxide as a signal controlling plantprogrammed cell death. J Cell Biol 168: 17–20


















Gepstein S, Glick BR (2013) Strategies to ameliorate abiotic stress-inducedplant senescence. Plant Mol Biol 82: 623–633

Gepstein S, Thimann KV (1980) The effect of light on the production ofethylene from 1-aminocyclopropane-1-carboxylic acid by leaves. Planta149: 196–199

Govind G, Seiler C, Wobus U, Sreenivasulu N (2011) Importance of ABAhomeostasis under terminal drought stress in regulating grain fillingevents. Plant Signal Behav 6: 1228–1231

Gregersen PL, Holm PB (2007) Transcriptome analysis of senescence in theflag leaf of wheat (Triticum aestivum L.). Plant Biotechnol J 5: 192–206

Guo Y, Gan S (2005) Leaf senescence: signals, execution, and regulation.Curr Top Dev Biol 71: 83–112

Guo Y, Gan S (2006) AtNAP, a NAC family transcription factor, has animportant role in leaf senescence. Plant J 46: 601–612

He P, Osaki M, Takebe M, Shinano T, Wasaki J (2005a) Endogenoushormones and expression of senescence-related genes in different se-nescent types of maize. J Exp Bot 56: 1117–1128

He XJ, Mu RL, Cao WH, Zhang ZG, Zhang JS, Chen SY (2005b) AtNAC2,a transcription factor downstream of ethylene and auxin signalingpathways, is involved in salt stress response and lateral root develop-ment. Plant J 44: 903–916

Hensel LL, Grbi�c V, Baumgarten DA, Bleecker AB (1993) Developmentaland age-related processes that influence the longevity and senescence ofphotosynthetic tissues in Arabidopsis. Plant Cell 5: 553–564

Hu X, Jiang M, Zhang A, Lu J (2005) Abscisic acid-induced apoplastic H2O2

accumulation up-regulates the activities of chloroplastic and cytosolicantioxidant enzymes in maize leaves. Planta 223: 57–68

Hu X, Zhang A, Zhang J, Jiang M (2006) Abscisic acid is a key inducer ofhydrogen peroxide production in leaves of maize plants exposed towater stress. Plant Cell Physiol 47: 1484–1495

Hu YQ, Liu S, Yuan HM, Li J, Yan DW, Zhang JF, Lu YT (2010) Functionalcomparison of catalase genes in the elimination of photorespiratory H2O2

using promoter- and 39-untranslated region exchange experiments in theArabidopsis cat2 photorespiratory mutant. Plant Cell Environ 33: 1656–1670

Jensen MK, Hagedorn PH, de Torres-Zabala M, Grant MR, Rung JH,Collinge DB, Lyngkjaer MF (2008) Transcriptional regulation by anNAC (NAM-ATAF1,2-CUC2) transcription factor attenuates ABA sig-nalling for efficient basal defence towards Blumeria graminis f. sp. hordeiin Arabidopsis. Plant J 56: 867–880

Jensen MK, Lindemose S, de Masi F, Reimer JJ, Nielsen M, Perera V,Workman CT, Turck F, Grant MR, Mundy J, et al (2013) ATAF1transcription factor directly regulates abscisic acid biosynthetic geneNCED3 in Arabidopsis thaliana. FEBS Open Bio 3: 321–327

Ji X, Dong B, Shiran B, Talbot MJ, Edlington JE, Hughes T, White RG,Gubler F, Dolferus R (2011) Control of abscisic acid catabolism andabscisic acid homeostasis is important for reproductive stage stresstolerance in cereals. Plant Physiol 156: 647–662

Jia Y, Tao F, Li W (2013) Lipid profiling demonstrates that suppressingArabidopsis phospholipase Dd retards ABA-promoted leaf senescenceby attenuating lipid degradation. PLoS ONE 8: e65687

Jiang M, Zhang J (2001) Effect of abscisic acid on active oxygen species,antioxidative defence system and oxidative damage in leaves of maizeseedlings. Plant Cell Physiol 42: 1265–1273

Jibran R, Hunter DA, Dijkwel PP (2013) Hormonal regulation of leaf se-nescence through integration of developmental and stress signals. PlantMol Biol 82: 547–561

Jossier M, Bouly JP, Meimoun P, Arjmand A, Lessard P, Hawley S, Hardie DG,Thomas M (2009) SnRK1 (SNF1-related kinase 1) has a central role in sugarand ABA signalling in Arabidopsis thaliana. Plant J 59: 316–328

Kaufmann K, Muiño JM, Østerås M, Farinelli L, Krajewski P, Angenent GC(2010) Chromatin immunoprecipitation (ChIP) of plant transcription factorsfollowed by sequencing (ChIP-SEQ) or hybridization to whole genome arrays(ChIP-CHIP). Nat Protoc 5: 457–472

Kim JH, Woo HR, Kim J, Lim PO, Lee IC, Choi SH, Hwang D, Nam HG(2009) Trifurcate feed-forward regulation of age-dependent cell deathinvolving miR164 in Arabidopsis. Science 323: 1053–1057

Kim YS, Sakuraba Y, Han SH, Yoo SC, Paek NC (2013) Mutation of theArabidopsis NAC016 transcription factor delays leaf senescence. PlantCell Physiol 54: 1660–1672

Kleinow T, Himbert S, Krenz B, Jeske H, Koncz C (2009) NAC domaintranscription factor ATAF1 interacts with SNF1-related kinases and si-lencing of its subfamily causes severe developmental defects in Arabi-dopsis. Plant Sci 177: 360–370

Kwak JM, Mori IC, Pei ZM, Leonhardt N, Torres MA, Dangl JL, Bloom RE,Bodde S, Jones JD, Schroeder JI (2003) NADPH oxidase AtrbohD andAtrbohF genes function in ROS-dependent ABA signaling in Arabidopsis.EMBO J 22: 2623–2633

Laloi C, Apel K, Danon A (2004) Reactive oxygen signalling: the latestnews. Curr Opin Plant Biol 7: 323–328

Lee IC, Hong SW, Whang SS, Lim PO, Nam HG, Koo JC (2011) Age-dependent action of an ABA-inducible receptor kinase, RPK1, as a pos-itive regulator of senescence in Arabidopsis leaves. Plant Cell Physiol 52:651–662

Lee S, Seo PJ, Lee HJ, Park CM (2012) A NAC transcription factor NTL4promotes reactive oxygen species production during drought-inducedleaf senescence in Arabidopsis. Plant J 70: 831–844

Leshem YY (1988) Plant senescence processes and free radicals. Free RadicBiol Med 5: 39–49

Li Z, Peng J, Wen X, Guo H (2013) Ethylene-insensitive3 is a senescence-associated gene that accelerates age-dependent leaf senescence by directlyrepressing miR164 transcription in Arabidopsis. Plant Cell 25: 3311–3328

Liang C, Wang Y, Zhu Y, Tang J, Hu B, Liu L, Ou S, Wu H, Sun X, Chu J,et al (2014) OsNAP connects abscisic acid and leaf senescence by fine-tuning abscisic acid biosynthesis and directly targeting senescence-associated genes in rice. Proc Natl Acad Sci USA 111: 10013–10018

Lim PO, Kim HJ, Nam HG (2007) Leaf senescence. Annu Rev Plant Biol 58:115–136

Lin CC, Kao CH (2001) Abscisic acid induced changes in cell wall peroxi-dase activity and hydrogen peroxide level in roots of rice seedlings.Plant Sci 160: 323–329

Lin JF, Wu SH (2004) Molecular events in senescing Arabidopsis leaves.Plant J 39: 612–628

Lu PL, Chen NZ, An R, Su Z, Qi BS, Ren F, Chen J, Wang XC (2007) Anovel drought-inducible gene, ATAF1, encodes a NAC family proteinthat negatively regulates the expression of stress-responsive genes inArabidopsis. Plant Mol Biol 63: 289–305

Matallana-Ramirez LP, Rauf M, Farage-Barhom S, Dortay H, Xue GP,Dröge-Laser W, Lers A, Balazadeh S, Mueller-Roeber B (2013) NACtranscription factor ORE1 and senescence-induced BIFUNCTIONALNUCLEASE1 (BFN1) constitute a regulatory cascade in Arabidopsis. MolPlant 6: 1438–1452

Mittler R, Vanderauwera S, Gollery M, Van Breusegem F (2004) Reactiveoxygen gene network of plants. Trends Plant Sci 9: 490–498

Mueller-Roeber B, Balazadeh S (2014) Auxin and its role in plant senes-cence. J Plant Growth Regul 33: 21–33

Müller M, Munne-Bosch S (2011) Rapid and sensitive hormonal profilingof complex plant samples by liquid chromatography coupled to elec-trospray ionization tandem mass spectrometry. Plant Methods 7: 37

Navabpour S, Morris K, Allen R, Harrison E, A-H-Mackerness S,Buchanan-Wollaston V (2003) Expression of senescence-enhanced genes inresponse to oxidative stress. J Exp Bot 54: 2285–2292

Noodén LD (1988) The phenomena of senescence and aging. In LD Noodén,AC Leopold, eds, Senescence and Aging in Plants. Academic Press, Inc.,San Diego, pp 1–50

Nuruzzaman M, Sharoni AM, Kikuchi S (2013) Roles of NAC transcrip-tion factors in the regulation of biotic and abiotic stress responses inplants. Front Microbiol 4: 248

Pei ZM, Murata Y, Benning G, Thomine S, Klüsener B, Allen GJ, Grill E,Schroeder JI (2000) Calcium channels activated by hydrogen peroxidemediate abscisic acid signalling in guard cells. Nature 406: 731–734

Philosophhadas S, Hadas E, Aharoni N (1993) Characterization and use inELISA of a new monoclonal antibody for quantitation of abscisic acid insenescing rice leaves. Plant Growth Regul 12: 71–78

Rauf M, Arif M, Dortay H, Matallana-Ramírez LP, Waters MT, Nam HG,Lim PO, Mueller-Roeber B, Balazadeh S (2013a) ORE1 balances leafsenescence against maintenance by antagonizing G2-like-mediatedtranscription. EMBO Rep 14: 382–388

Rauf M, Arif M, Fisahn J, Xue GP, Balazadeh S, Mueller-Roeber B(2013b) NAC transcription factor SPEEDY HYPONASTIC GROWTHregulates flooding-induced leaf movement in Arabidopsis. Plant Cell 25:4941–4955

Sakuraba Y, Jeong J, Kang MY, Kim J, Paek NC, Choi G (2014)Phytochrome-interacting transcription factors PIF4 and PIF5 induce leafsenescence in Arabidopsis. Nat Commun 5: 4636

Sambrook J, Russell DW (2001) Molecular Cloning: A Laboratory Manual.Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY


Garapati et al.



Song Y, Yang C, Gao S, Zhang W, Li L, Kuai B (2014) Age-triggered anddark-induced leaf senescence require the bHLH transcription factorsPIF3, 4, and 5. Mol Plant 7: 1776–1787

Torres MA, Dangl JL (2005) Functions of the respiratory burst oxidase inbiotic interactions, abiotic stress and development. Curr Opin Plant Biol8: 397–403

Tran LS, Nakashima K, Sakuma Y, Simpson SD, Fujita Y, Maruyama K,Fujita M, Seki M, Shinozaki K, Yamaguchi-Shinozaki K (2004) Isola-tion and functional analysis of Arabidopsis stress-inducible NAC tran-scription factors that bind to a drought-responsive cis-element in theearly responsive to dehydration stress 1 promoter. Plant Cell 16: 2481–2498

Trivellini A, Jibran R, Watson LM, O’Donoghue EM, Ferrante A, SullivanKL, Dijkwel PP, Hunter DA (2012) Carbon deprivation-driven tran-scriptome reprogramming in detached developmentally arresting Ara-bidopsis inflorescences. Plant Physiol 160: 1357–1372

Tuteja N, Mahajan S (2007) Calcium signaling network in plants: anoverview. Plant Signal Behav 2: 79–85

van der Graaff E, Schwacke R, Schneider A, Desimone M, Flügge UI,Kunze R (2006) Transcription analysis of Arabidopsis membranetransporters and hormone pathways during developmental and inducedleaf senescence. Plant Physiol 141: 776–792

Wang P, Song CP (2008) Guard-cell signalling for hydrogen peroxide andabscisic acid. New Phytol 178: 703–718

Wang T, Mao X, Li H, Qiao S, Xu A, Wang J, Lei S, Liu Z, Ng KF, Wong GT,et al (2013) N-Acetylcysteine and allopurinol up-regulated the Jak/STAT3and PI3K/Akt pathways via adiponectin and attenuated myocardial post-ischemic injury in diabetes. Free Radic Biol Med 63: 291–303

Waters MT, Moylan EC, Langdale JA (2008) GLK transcription factors regulatechloroplast development in a cell-autonomous manner. Plant J 56: 432–444

Waters MT, Wang P, Korkaric M, Capper RG, Saunders NJ, Langdale JA(2009) GLK transcription factors coordinate expression of the photo-synthetic apparatus in Arabidopsis. Plant Cell 21: 1109–1128

Woo HR, Kim JH, Nam HG, Lim PO (2004) The delayed leaf senescencemutants of Arabidopsis, ore1, ore3, and ore9 are tolerant to oxidativestress. Plant Cell Physiol 45: 923–932

Woo HR, Kim HJ, Nam HG, Lim PO (2013) Plant leaf senescence anddeath: regulation by multiple layers of control and implications for ag-ing in general. J Cell Sci 126: 4823–4833

WuA, Allu AD, Garapati P, Siddiqui H, Dortay H, ZanorMI, Asensi-FabadoMA,Munné-Bosch S, Antonio C, Tohge T, et al (2012) JUNGBRUNNEN1, a reactiveoxygen species-responsive NAC transcription factor, regulates longevity inArabidopsis. Plant Cell 24: 482–506

Wu Y, Deng Z, Lai J, Zhang Y, Yang C, Yin B, Zhao Q, Zhang L, Li Y, Yang C,et al (2009) Dual function of Arabidopsis ATAF1 in abiotic and biotic stressresponses. Cell Res 19: 1279–1290

Xing Y, Jia W, Zhang J (2008) AtMKK1 mediates ABA-induced CAT1 ex-pression and H2O2 production via AtMPK6-coupled signaling in Ara-bidopsis. Plant J 54: 440–451

Xu ZY, Kim SY, Hyeon Y, Kim DH, Dong T, Park Y, Jin JB, Joo SH, Kim SK,Hong JC, et al (2013) The Arabidopsis NAC transcription factor ANAC096cooperates with bZIP-type transcription factors in dehydration and osmoticstress responses. Plant Cell 25: 4708–4724

Xue GP (2002) Characterisation of the DNA-binding profile of barley HvCBF1using an enzymatic method for rapid, quantitative and high-throughputanalysis of the DNA-binding activity. Nucleic Acids Res 30: e77

Xue GP (2005) A CELD-fusion method for rapid determination of the DNA-binding sequence specificity of novel plant DNA-binding proteins. PlantJ 41: 638–649

Xue GP, Bower NI, McIntyre CL, Riding GA, Kazan K, Shorter R (2006)TaNAC69 from the NAC superfamily of transcription factors is up-regulated by abiotic stresses in wheat and recognises two consensusDNA-binding sequences. Funct Plant Biol 33: 43–57

Yang J, Worley E, Udvardi M (2014) A NAP-AAO3 regulatory modulepromotes chlorophyll degradation via ABA biosynthesis in Arabidopsisleaves. Plant Cell 26: 4862–4874

Yang SD, Seo PJ, Yoon HK, Park CM (2011) The Arabidopsis NAC tran-scription factor VNI2 integrates abscisic acid signals into leaf senescencevia the COR/RD genes. Plant Cell 23: 2155–2168

Ye N, Zhu G, Liu Y, Li Y, Zhang J (2011) ABA controls H2O2 accumulationthrough the induction of OsCATB in rice leaves under water stress. PlantCell Physiol 52: 689–698

Zacarias L, Reid MS (1990) Role of growth regulators in the senescence ofArabidopsis thaliana leaves. Physiol Plant 80: 549–554

Zhang H, Gan J (2012a) A reproducing kernel-based spatial model inPoisson regressions. Int J Biostat 8: 1–26

Zhang K, Gan SS (2012b) An abscisic acid-AtNAP transcription factor-SAG113 protein phosphatase 2C regulatory chain for controlling dehy-dration in senescing Arabidopsis leaves. Plant Physiol 158: 961–969

Zhang K, Xia X, Zhang Y, Gan SS (2012) An ABA-regulated and Golgi-localized protein phosphatase controls water loss during leaf senescencein Arabidopsis. Plant J 69: 667–678

Zhang WY, Xu YC, Li WL, Yang L, Yue X, Zhang XS, Zhao XY (2014)Transcriptional analyses of natural leaf senescence in maize. PLoS ONE9: e115617

Zimmermann P, Zentgraf U (2005) The correlation between oxidativestress and leaf senescence during plant development. Cell Mol Biol Lett10: 515–534

Zuo J, Niu QW, Chua NH (2000) An estrogen receptor-based transactivatorXVE mediates highly inducible gene expression in transgenic plants.Plant J 24: 265–273





Documents

Transcription Factor ATAF1 in Arabidopsis Promotes ... Factor ATAF1 in Arabidopsis Promotes Senescence by Direct Regulation of Key Chloroplast Maintenance and Senescence Transcriptional