A Rice NAC Transcription Factor Promotes LeafSenescence via ABA Biosynthesis1[OPEN]

Chanjuan Mao,2 Songchong Lu,2 Bo Lv, Bin Zhang, Jiabin Shen, Jianmei He, Liqiong Luo, Dandan Xi,Xu Chen, and Feng Ming3,4

State Key Laboratory of Genetic Engineering, Institute of Genetics, Institute of Plant Biology, School of LifeSciences, Fudan University, Shanghai 200433, China

ORCID ID: 0000-0001-8232-2334 (F.M.).

It is well known that abscisic acid (ABA)-induced leaf senescence and premature leaf senescence negatively affect the yield of rice(Oryza sativa). However, the molecular mechanism underlying this relationship, especially the upstream transcriptional networkthat modulates ABA level during leaf senescence, remains largely unknown. Here, we demonstrate a rice NAC transcriptionfactor, OsNAC2, that participates in ABA-induced leaf senescence. Overexpression of OsNAC2 dramatically accelerated leafsenescence, whereas its knockdown lines showed a delay in leaf senescence. Chromatin immunoprecipitation-quantitative PCR,dual-luciferase, and yeast one-hybrid assays demonstrated that OsNAC2 directly activates expression of chlorophylldegradation genes, OsSGR and OsNYC3. Moreover, ectopic expression of OsNAC2 leads to an increase in ABA levels viadirectly up-regulating expression of ABA biosynthetic genes (OsNCED3 and OsZEP1) as well as down-regulating the ABAcatabolic gene (OsABA8ox1). Interestingly, OsNAC2 is upregulated by a lower level of ABA but downregulated by a higher levelof ABA, indicating a feedback repression of OsNAC2 by ABA. Additionally, reduced OsNAC2 expression leads to about 10%increase in the grain yield of RNAi lines. The novel ABA-NAC-SAGs regulatory module might provide a new insight into themolecular action of ABA to enhance leaf senescence and elucidates the transcriptional network of ABA production during leafsenescence in rice.

Senescence is the last stage of leaf development.During this period, various changes occur at thephysiological, biochemical, and molecular levels. Forexample, macromolecules including lipids, proteins,and nucleic acids are hydrolyzed, which leads to dis-assembly of mitochondria and nuclei, and to cell death(Buchanan‐Wollaston et al., 2005; Ulker et al., 2007).Although senescence is an active process to salvagenutrients from old tissues, precocious senescence willshorten the growth stage of crops and be unfavorable toagronomic production (Woo et al., 2013).

The most distinguishing feature in leaf senescence isthe yellowing phenotype, which is a visible marker of

the degradation of macromolecules (Kim et al., 2006).The chlorophyll degradation pathway is one of themost characterized ones for macromolecule degrada-tion in plants (Hörtensteiner, 2006). OverexpressingNON-YELLOW COLORING1 (NYC1) or NYC1-likegenes in rice (Oryza sativa) can induce degradationof chlorophyll (Sato et al., 2009). A pph (encodingpheophytinase) mutant is abnormal in chlorophylldegradation during senescence and therefore ex-hibits a stay-green phenotype (Schelbert et al., 2009).Mutation of the PAO (Pheophorbide a oxygenase)gene leads to retention of chlorophyll in leaves dur-ing dark-induced senescence in Arabidopsis (Arabidopsisthaliana; Pruzinská et al., 2005). Recently, the highlyconserved STAY-GREEN (SGR) in higher plants hasbeen identified to be chloroplast-localized dechelatase(Shimoda et al., 2016).

The senescence process is highly regulated by a rangeof important factors. It has been demonstrated that thetranscriptional level of NAC (NAM, ATAF1, andCUC2) transcription factors are up-regulated and playa fundamental role in leaf senescence (Gregersen andHolm, 2007). The expression of senescence-associatedgenes (SAGs) is regulated by these senescence-relatedNAC transcription factors, such as ORESARA1 (ORE1)(Kim et al., 2009), Oresara1 sister 1 (ORS1) (Balazadehet al., 2011), Phytochrome-interacting factor 4 (PIF4)and PIF5 (Sakuraba et al., 2014), ANAC046 (Oda-Yamamizo et al., 2016), and AtNAP (Guo and Gan,2006). However, the molecular mechanism underlyingNAC transcription factor (TF)-regulated leaf senescence

1 This work was supported by the Ministry of Agriculture of thePeople’s Republic of China (grant no. 2016ZX08009-001-008) and TheNational Natural Science Foundation of China (grant no. 31471152).

2 These authors contributed equally to this work.3 Address correspondence to fming@fudan.edu.cn.4 Current address: 2005 Songhu Road, Yangpu District, Shanghai

200433, China.The 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:Feng Ming (fming@fudan.edu.cn).

F.M. conceived the project and designed the study; C.M. and S.L.performed the experiments; B.L., B.Z., J.S., and J.H. provided techni-cal assistance to C.M. and S.L.; C.M. wrote the article; C.M., F.M., andS.L. revised the article; all of the authors discussed the results andcommented on the manuscript.

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remains poorly understood in rice. NTL4 promotesdrought-induced senescence probably due to directregulation of reactive oxygen species (ROS) synthesis(Lee et al., 2012). The expression of both OsNAC5(Sperotto et al., 2009) and OsNAC6 (Nakashima et al.,2007) was increased during leaf senescence. NAC106plays an important role in regulation of the aging pro-cess, and its overexpression defers senescence in rice(Sakuraba et al., 2015). Additionally, OsNAP wasreported to control expression of genes involved inchlorophyll degradation by direct binding to thepromoter regions (Liang et al., 2014).

In higher plants, the level of ABA is finely controlledby the balance between the rates of ABA biosynthesisand catabolism (Nambara and Marion-Poll, 2005).Key enzymes controlling ABA production include 99-cis-epoxycarotenoid dioxygenases (NCEDs), which areinvolved in xanthophyll cleavage (Tan et al., 2003);zeaxanthin epoxidase (ZEP), which converts zeaxan-thin to violaxanthin via antheraxanthin (Oliver et al.,2007); andABSCISICALDEHYDEOXIDASE3 (AAO3),which is responsible for the final step in ABA biosyn-thesis (Yang et al., 2014). The oxidation of ABA tophaseic acid is catalyzed by ABA 89-hydroxylase,which is encoded probably by three genes (OsABA8ox1,2, and 3) in rice (Saika et al., 2007).

ABA has vital function in stress responses and reg-ulates various plant developments, e.g. seed matu-ration and dormancy, organ abscission, and leafsenescence (Chandler and Robertson, 1994; Cutler et al.,2010). It has long been known that ABA participates inleaf senescence (Becker and Apel, 1993). ABA can in-duce the expression of some SAGs, e.g. NYC1 (Kusabaet al., 2007), SGR (Park et al., 2007), PPH (Schelbert et al.,2009), and PaO (Pruzinská et al., 2005), and then pro-motes plant senescence. The expression of NAC TFs,including VNI2 (Yang et al., 2011), SNAC-As (Takasakiet al., 2015),ORE1 (Kim et al., 2011), andOsNAP (Lianget al., 2014), is significantly up-regulated after ABAtreatment by an unknown molecular mechanism.AtNAP could specifically bind to the promoter ofAAO3, one of the key enzymes controlling ABA pro-duction, and promote transcription of chlorophylldegradation genes (Yang et al., 2014). Although thebiosynthetic pathways that generate ABA and itsdownstream signaling mechanisms have been exten-sively studied, and ABA-induced leaf senescence haslong been observed, the upstream transcriptional net-work that modulates its level during leaf senescenceand connects its action to leaf senescence is less clear.

Previously, the transcription factor OsNAC2 wasreported to promote shoot branching (Mao et al., 2007),and our previous study showed that OsNAC2 affectsplant height and regulates abiotic stress tolerance in rice(Shen et al., 2017). Previous study of our lab showedthat overexpression of OsNAC2 endowed rice withpremature senility, which indicated OsNAC2 mightplay a role in the pathway of leaf senescence (Chenet al., 2015). However, the underlying molecularmechanisms of OsNAC2 regulating leaf senescence

were poorly understood. Here, we show that OsNAC2promotes leaf senescence in rice by induction of ABAbiosynthetic genes (OsNCED3 andOsZEP1) and down-regulating ABA catabolic gene (OsABA8ox1) by tar-geting to their promoters, which leads to increasedlevels of ABA. OsNAC2 also directly regulated chlo-rophyll degradation genes, OsSGR and OsNYC3. Ourdata elucidate the transcriptional network of ABAproduction during leaf senescence in rice. The novelABA-NAC-SAG regulatory module might provide anew insight into the molecular action of ABA to en-hance leaf senescence and the function of NAC TFs inpathway ABA induce leaf senescence.

RESULTS

OsNAC2 Is Highly Expressed during Leaf Senescence andPositively Regulates Leaf Senescence in an Age-Dependent Manner

To study the role of OsNAC2 in leaf senescence,we examined the senescence symptoms of transgeniclines, including 35S::OsNAC2 lines (OsNAC2-OX, ON7,and ON11) and knockdown lines (OsNAC2-RNAi18,RNAi25, and RNAi31), which were previously reported(Chen et al., 2015). The third leaves of 6-week-old seed-lings hydroponically cultivated were used for statisticsof yellowing rate. It was evident that older leaves be-came yellow earlier in OsNAC2-OX lines but much laterin RNAi lines than in the wild type (Fig. 1A). Consistentwith this visible symptom, the yellowing rate of leavesfromOsNAC2-OXwas obviously higher comparedwiththat in the wild type, whereas the OsNAC2-RNAi linehad few yellow leaves in the 4-week-old rice plant (Fig.1B). It is considered that stay-green phenotype in grain-filling stage leads to the increase of rice yield (Sakurabaet al., 2015). We subsequently examined the natural se-nescence phenotype of OsNAC2 transgenic lines dur-ing grain-filling stage and found that OsNAC2-RNAiexhibited a delayed-senescence phenotype, whileOsNAC2-OX exhibited a precocious-senescence phe-notype (Supplemental Fig. S1).

We then investigated temporal and spatial expres-sion patterns of OsNAC2 using transgenic plants(pOsNAC2-GUS) expressing b-glucuronidase (GUS)driven by the native promoter of OsNAC2, a 2-kb ge-nomic DNA fragment upstream of the transcriptionstart site. GUS staining analysis showed that GUS ac-tivity was obviously higher at the senescing area thanat the no senescing area in the same leaves. Moreover,GUS was expressed specifically in coleoptiles in thecourse of germination (from 1 to 10 d; Supplemental Fig.S2). The coleoptile is known to protect true leaves againstsoil pressures and other physical constraints and is thefirst organ of rice plants to senescence after germination(Kawai and Uchimiya, 2000). Therefore, these data sug-gest that OsNAC2 is involved in the aging process.

To further assess the transcriptional level of OsNAC2during leaf aging, we tested OsNAC2 expression inleaves at different developmental stages by qPCR. The

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OsNAC2 transcripts were higher in late-senescing andearly-senescing leaves than in young leaves and non-senescing leaves (Fig. 1C). Consistent with the resultsfrom GUS staining, OsNAC2 expression increasedgradually from the tip to the base of a fully expandedleaf (Supplemental Fig. S2). Taken together, our resultsindicate that OsNAC2 plays an important role in leafsenescence.To further analyze evolutionary relationships of

OsNAC2 in the NAC family, a total of 234 NAC pro-teins, comprising 99 from Arabidopsis (TAIR, http://www.Arabidopsis.org/), and 135 from rice (Rice Ge-nome Annotation Project, http://rice.plantbiology.msu.edu/), were used for construction of an phylo-genetic tree, byMEGA5.1 software using the maximumlikelihood method and the bootstrap test carried outwith 1,000 replications. As illustrated in SupplementalFigure S3, the phylogenetic analysis classified theNACs into 14 groups, designated as Roman numeralfamilies (NAC-I to NAC-XIV) based on tree topologies.The published senescence-associated NACs (Kim et al.,2016; Liang et al., 2014; Nakashima et al., 2007;Oda-Yamamizo et al., 2016; Sperotto et al., 2009) fall ingroups I to IV. In detail, ANAC053, ANC016, ANAC017,and ANAC096 located in NAC II, ANAC083, andONAC106 located in NAC III, while ANAC042,ONAC058 (OsNAP), AtNAP, ANAC047, ANAC019,AtNAC3, OsNAC5, ATAF1, and OsNAC5 located inNAC IV. OsNAC2 had high homology with AtNAC6(ANAC092), together with ANAC046 and ANAC059(ORS1), forming a distinct cluster NAC I (Supplemental

Fig. S3), which was consistent with the previous reports(You et al., 2015; Hu et al., 2010).

Overexpression of OsNAC2 Causes Early Senescence ofRice and Tobacco Leaves

Dark treatment is used frequently as an effectivemethod to simulate synchronous senescence (Kim et al.,2006; Kong et al., 2006). To gain a mechanistic under-standing of the potential role of OsNAC2 in leaf se-nescence, rice plants were treated by darkness withvarious times. After 5-d dark treatment, OsNAC2-OXlines exhibited a more severe leaf senescent phenotype,while RNAi transgenic lines displayed weaker leafsenescence, compared with the wild type (Fig. 2A).Similarly, an accelerated yellowing phenotype was alsoobserved in detached and 4-d dark-treated leaves fromOsNAC2-OX plants (Fig. 2B). AtSAG12, an Arabidopsisgene encoding a Cys protease, is considered a molec-ular marker the study of developmental senescence(Weaver et al., 1998). In rice, OsSAG12-1 is the closesthomolog of AtSAG12 and the expression of OsSAG12-1is induced during senescence (Singh et al., 2013). Thus,we determined the mRNA level of OsSAG12-1 in de-tached leaves of OsNAC2 transgenic plants incubated3 d in the darkness and found that OsSAG12-1 signifi-cantly upregulated in OsNAC2-OX plants (ON11 andON7; Fig. 2C). Conformably, chlorophyll content inOsNAC2-OX leaves treated with darkness for 5 d was0.2 mg/g fresh weight (FW), only 25% of that found in

Figure 1. OsNAC2 is upregulated during leaf senescence. A, Phenotype of the third leaves in 6-week-old wild-type andOsNAC2transgenic plants. B, Quantitative analysis of the yellowing rate of the leaves shown in A. C, qPCR analysis ofOsNAC2 expressionin various ages and regions of rice leaves. LS, ES, NS, and YL mean late-senescing, early-senescing, no senescing, and youngleaves, respectively. T, M, and B mean the top, middle, and base of the leaf. Relative mRNA level was calculated using the DDCT

method from triplicate data.OsActinwas used as an internal control to normalize the different samples with the same amount ofplant RNA. Data are mean 6 SE with three replicates. Asterisk indicates a significant difference between the wild type andOsNAC2 transgenic lines by t test: *P , 0.05.

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wild-type leaves (Fig. 2D). These results demonstratethat chlorophyll is degraded at a more rapid rate inOsNAC2-OX than in wild-type plants.

We also investigated ion leakage rates and ROS indark-treated leaves. Consistently, OsNAC2-OX linesdisplayed significantly higher ion leakage (Fig. 2G),

indicating that overexpressing OsNAC2 results in cellmembrane damage in dark. In addition, ROS analysisshowed an increased level of H2O2 and O2

2 in ON7 andON11 lines (Fig. 2E). Same results were obtained in atransient expression assay in tobacco (Nicotiana ben-thamiana) leaves (Fig. 2, F and H). These results suggest

Figure 2. Overexpression ofOsNAC2 causes early senescence in rice and tobacco. A, Phenotypes of 4-week-old wild-type andOsNAC2-transgenic plants after 0, 3, and 5 d dark treatment. B, Phenotypes of detached leaves in 4-week-old wild-type andtransgenic plants after 0, 2, 3, and 4 d dark treatment. C, Expression ofOsSAG12-1 inOsNAC2-OX, the wild type, andOsNAC2-RNAi lines after 3 d dark treatment. D, Chlorophyll content of the leaves in OsNAC2 plants after 0, 3, and 5 d dark treatment.Values are means 6 SD of five replicates. E, DAB and NBT staining of wild-type and transgenic seedlings leaves after 4 d darktreatment. F, DAB and NBT staining in tobacco leaves by a transient expression of OsNAC2 after 3 d dark treatment. G, Ionleakage analysis of leaves in 4-week-old wild-type and transgenic rice plants after 0, 3, and 5 d dark treatment. Values aremeans6 SD of five replicates. H, Ion leakage analysis of leaves in tobacco leaveswithOsNAC2 transient expression after 3 d darktreatment. Values are means 6 SD of five replicates. Asterisks indicate a significant difference between wild-type and OsNAC2transgenic lines by t test: *P , 0.05 and **P , 0.01.

Table I. List of senescence-related genes changed in OsNAC2-OX line by microarray analysis

Annotation Probe Set_ID Gene Symbol Fold Change References

Ospse1 Os.35614.1.S1_at Os01g0546800 0.721459 Wu et al. (2013)SPL28 Os.38379.1.S1_at Os01g0703600 0.689033* Qiao et al. (2010)OsSAG12-1 Os.4181.1.S1_at Os01g0907600 0.799077 Singh et al. (2013)NYC1 Os.18818.1.S1_a_at Os01g0227100 0.637737* Kusaba et al. (2007)OsWRKY80 Os.20028.1.S1_at Os09g0481700 1.545195* Ricachenevsky et al. (2010)OsSGR Os.11666.1.S1_at Os09g0532000 2.434277** Park et al. (2007)OsRCCR1 Os.5164.1.S1_at Os10g0389200 0.884418 Tang et al. (2011)OsNAC5 Os.4385.1.S1_at Os11g0184900 0.826093 Sperotto et al. (2009)NYC3 Os.11724.2.S1_s_at Os06g0354700 1.090994 Morita et al. (2009)NOL Os.17490.1.A1_at Os03g0654600 1.124019 Sato et al. (2009)OsI85 Os.22277.1.S1_at Os07g0529000 1.240426* Pistelli et al. (1991)PHYB Os.4601.1.S1_a_at Os03g0309200 0.598971* Piao et al. (2015)cZOGT1 Os.46328.1.S1_at Os04g0556500 0.706998* Kudo et al. (2012)OsSWEET5 Os.6338.1.S1_at Os05g0588500 1.333925* Zhou et al. (2014)TDC2 Os.49621.1.S1_at Os07g0437500 1.518717* Kang et al. (2009)TDC1 Os.27876.1.S1_x_at Os08g0140300 0.25689** Kang et al. (2009)

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Figure 3. qPCR confirmation of expression of senescence-related genes in the 2-week-old wild type andOsNAC2-OX. RelativemRNA level was calculated using the DDCT method from triplicate data. OsActin was used as internal control to normalize thedifferent samples with the same amount of plant RNA. Data are mean 6 SE with three replicates. Asterisks indicate a significantdifference between wild-type and OsNAC2 transgenic lines by t test: *P , 0.05 and **P , 0.01.

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that OsNAC2 is positively regulatory factor for leafsenescence.

Overexpression of OsNAC2 Alters the Expression ofSenescence-Related Genes

Based on the leaf aging phenotype mentioned above,we predicted that the expression of senescence-relatedgeneswould be significantly different betweenOsNAC2-overexpressing and RNAi lines. To understand themolecular mechanism of OsNAC2-regulated chloro-phyll degradation, we profiled genome-wide gene ex-pression in 2-week-old OsNAC2-OX and wild-typeplants using ricemicroarray.Many differently expressedgenes were involved in leaf senescence, such as chloro-phyll breakdown genes OsNYC3, OsNOL, OsRCCR1,OsPAO, and OsSGR; serotonin biosynthesis gene,OsTDC1; glyoxylate cycle related gene, OsI85; and Galtransport gene, OsSWEET5 (Table I). The changes ofsome SAGs expression, e.g. OsSAG12-1, inOsNAC2-OXwere not so significant by t test, probably because2-week-old seedlings may be not in the procedure ofsenescence. However, there were still some SAGsshowed significant changes inOsNAC2-OX, e.g.OsSGR,

OsSWEET5, and OsTDC1, which gave information ofprobably candidate downstream targets of OsNAC2.Transcript levels of these senescence relative genes werefurther confirmed by qPCR. Significant increases intranscript levels of OsI85, OsNOL, OsSGR, OsRCCR1,OsSWEET5, andOsNYC3 anddecrease in that ofOsTDC1were found inOsNAC2-OX comparedwith the wild type(Fig. 3). These results indicate that OsNAC2 plays a reg-ulatory role, either direct or indirect, in expression ofchlorophyll degradation genes during leaf senescence.

OsNAC2 Directly Regulates Expression of OsNYC3 andOsSGR Genes

To further investigate whether OsNAC2 directlyregulates expression of chlorophyll degradation genes,yeast one-hybrid assays were used to test the interac-tion of OsNAC2 with DNA. The full-length cDNA ofOsNAC2 was fused in frame to the GAL4 activationdomain in the pGADT7 vector. The promoter sequenceregions ofOsI85,OsSGR, andOsNYC3were ligated intothe pHIS vector. The yeast one-hybrid assays showedthat OsNAC2 directly interacts with the promoter se-quences of OsSGR and OsNYC3 (Fig. 4A).

Figure 4. OsSGR and OsNYC3 are the direct target genes of OsNAC2. A, Interaction of OsNAC2 with the promoters of OsSGRand OsNYC3 by yeast one-hybrid assays. The yeast cells were grown on SD/-Leu/-Trp/-His/+30 mM 3-amino-1, 2,4-triazolemedium. B, Interaction of OsNAC2 with the promoters ofOsSGR andOsNYC3 by dual-luciferase promoter activation assays intobacco. C and D, ChIP-qPCR assays. Total protein extracted from 35S:OsNAC2-mGFP transgenic plants hydroponically culti-vated for 4 weeks were immunoprecipitated with an anti-GFPantibody. Fragmented genomic DNAwas eluted from the protein-DNA complexes and subjected to qPCR analysis. The long black bars represent promoter regions for which we designed primers.The numbers under the bar show the distance from ATG start codon. Short bars represents for the corresponding region of eachpair of primers on the promoter. Error bars are the SE for three biological repeats: *P , 0.05 and **P , 0.01.

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We also performed transient expression assays fortransactivation in N. benthamiana. The promoter se-quences of OsSGR and OsNYC3 were cloned intopGreenII 0800-LUC, and the open reading frame ofOsNAC2 was cloned into pGreenII 62-SK. The highestactivities were observed when OsNAC2was coinfiltratedwith the promoter of OsNYC3 and OsSGR (Fig. 4B).To test whether endogenous OsNAC2 specifically

binds to its target genes, chromatin immunoprecipita-tion (ChIP)-qPCR was performed using the anti-greenfluorescent protein (anti-GFP) antibody and specificprimers. Our results showed that specific fragments inthe promoter ofOsNYC3 andOsSGRwere significantlyenriched in the GFP antibody-immunoprecipitatedDNA (Fig. 4, C and D). Taken together, the data indi-cate that OsNAC2 is a transcription factor specificallybinding to its target genes, OsNYC3 and OsSGR.

ABA Participates in Leaf Senescence by ModulatingOsNAC2 Expression

Leaf senescence can be modulated by a variety ofphytohormones and environmental factors, and ABA

may be one of the most important phytohormonesregulating both leaf senescence and expression of NACTFs (Liang et al., 2014). To determine whether ABA-regulated senescence is mediated by OsNAC2, exoge-nously ABA was applied on different transgenic lines.Our results showed that in the presence of ABA (either2 or 4 mM), the shoot length of OsNAC2-OX lines wassignificantly shorter than that of the wild type, whereasshoot length of RNAi lines was longer than that of thewild type, indicating that ABA sensitivity of rice plantsis positively correlated to the level of OsNAC2 expres-sion (Fig. 5, A and B). Next, we examined the effect ofexogenously applied ABA on chlorophyll degradation.Consistent with the visible phenotypes, among ABA-treated leaves, those excised fromOsNAC2-OX showedthe fastest yellowing while those from OsNAC2-RNAiexhibited a stay-green phenotype, compared with thewild type (Fig. 5C). Chlorophyll concentrations inOsNAC2-RNAi, wild-type, and OsNAC2-OX leavestreated with ABA for 3 d were 1.6, 0.8, and 0.1 mg/mgFW, respectively (Fig. 5D); the severity of ion leakagewas themost inOsNAC2-OX, followed by thewild typeand OsNAC2-RNAi (Fig. 5E). Moreover, expression of

Figure 5. Effects of exogenous ABA on chloro-phyll content and SAG gene transcript levels inwild-type, OsNAC2-OX, and OsNAC2-RNAiplants. A, ABA effect on seedling growth on MSmedia. B, Relative shoot length of wild-type,OsNAC2-OX, and OsNAC2-RNAi plants in thepresence of different ABA concentrations. Thevalue of shoot length without ABA treatmentwas set to 1. C, Phenotype of detached leavesfrom wild-type, OsNAC2-OX, and OsNAC2-RNAi plants in the presence of 20 mM ABA. D,Chlorophyll content of the leaves in wild-type,OsNAC2-OX, andOsNAC2-RNAi plants treatedwith 20mMABA for 3 d. E, Ion leakage analysis ofleaves in 2-week-old wild-type, OsNAC2-OX,and OsNAC2-RNAi plants treated with 20 mM

ABA for 3 d. F and G, Relative expression ofOsSGR and OsNYC3 72 h after 20 mM ABAtreatment. HAT, hours after treatment. Data aremeans 6 SE with at least three biological repli-cates. Asterisks represent statistically significantdifferences between wild-type and transgenicplants: *P, 0.05, **P, 0.01, and ***P, 0.001.

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two OsNAC2-targeted SAGs, OsSGR and OsNYC3,was significantly upregulated 72 h after ABA treatment(Fig. 5, F and G). These results imply that OsNAC2mediates ABA-induced leaf senescence.

OsNAC2 Enhances ABA Concentration via RegulatingExpression of ABA Metabolism Genes

To test whether endogenous ABA production wasaffected by OsNAC2, we examined the ABA level in2-week-old leaves ofOsNAC2-OX,OsNAC2-RNAi, andwild-type plants. The ABA content ofOsNAC2-OXwas31.81 6 2.11 ng/g FW, which was significantly higherthan that of wild-type plants (22.626 0.88 ng/g FW; Fig.6A). Combined with our previously reported data, wehypothesized that the expression of ABA metabolism-

associated genes is affected by OsNAC2. qPCR analysisshowed that transcription levels of key ABA biosyntheticgenes, including OsNCED2 (Fig. 6B), OsNCED3 (Fig. 6C),OsNCED5 (Fig. 6D), and OsZEP1 (Fig. 6E), were signifi-cantly upregulated in OsNAC2-OX, whereas ABA catab-olism genes, including OsABA8ox1 (Fig. 6F), OsABA8ox2(Fig. 6G), andOsABA8ox3 (Fig. 6H), were down-regulated.Thus, it appears that OsNAC2 enhances the level of ABAvia up-regulation of ABA biosynthetic genes and down-regulation of ABA catabolism genes as well.

OsNAC2 Binds Directly to Promoters of ABA Metabolism-Related Genes

To investigate whether OsNAC2 directly regulatesthe expression of ABA metabolism-related genes, yeast

Figure 6. ABA content and expression of ABA me-tabolism-related genes in wild-type, OsNAC2-OX,and OsNAC2-RNAi plants. Data were means 6 SE

with at least three biological replicates. Asterisksrepresent statistically significant differences be-tween wild-type and transgenic plants: *P , 0.05and **P , 0.01.

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one-hybrid analysis was performed to test direct in-teraction between OsNAC2 and DNA. Our resultsshowed that the GAL4 transcriptional activationdomain fused with OsNAC2 activates the HIS3 re-porter gene driven by the promoter of OsNCED3,OsZEP1, or OsABA8ox1 (Fig. 7A). However, OsNAC2did not bind directly to the promoters of OsNCED2,OsNCED5, OsABA8ox2, and OsABA8ox3 (SupplementalFig. S4). ChIP-qPCR assays further confirmed thatOsNAC2 indeed bound to the promoters of OsNCED3(Fig. 7B),OsZEP1(Fig. 7C), andOsABA8ox1 (Fig. 7D).Thus, OsNAC2 is a transcription factor directlyregulating expression of ABA metabolism-relatedgenes.Previous work has defined the 4-bp core sequence

of the NAC binding motif as CACG (Olsen et al.,2005). Thus, we checked whether CACG sequencesexist in the promoters of these five target genes.The ChIP experiments identified the positive frag-ments, 2500 ; 2800, 2150 ; 2400, 2850 ; 21,000,2450 ; 2650, and 250 ; 2250 in the promoters oftarget genes, OsSGR, OsNYC3, OsNCED3, OsZEP1,and OsABA8ox1, respectively (Fig. 7, B–D). Sequencesearching by Clone Manage Software showed that allthe five positive fragments contained the core se-quence of NAC binding motif CACG (SupplementalFig. S5), which indicated CACG might also be thebinding motif of OsNAC2.

ABA Regulates OsNAC2 and Senescence-Related GeneExpression in a Dose-Dependent Manner But Temporally

We then investigated whether ABA affects expres-sion of OsNAC2. As shown in Figure 8D, the level ofOsNAC2 transcripts increased up to 8- and 16-fold ofthe ABA-untreated seedlings at 2 and 4 h after 20 mM

ABA treatment, respectively. Consistently, expressionof OsNCED3 and OsZEP1 obviously increased 2 h after20 mM ABA treatment and was generally higher inOsNAC2-OX plants than the wild type (Fig. 8, A and B),whereas OsABA8ox1 significantly downregulated inOsNAC2-OX, compared with the wild type (Fig. 8C).Thus, it seems that ABA-promoted expression ofOsNCED3 and OsZEP1, and ABA-suppressed expres-sion of OsABA8ox1 are mediated by OsNAC2. How-ever, OsNAC2 transcription decreased rapidly 8 h afterABA treatment (Fig. 8D). We also examined the kineticexpression ofOsNAC2 4 h after different concentrationsof ABA. OsNAC2 was induced exclusively by 20 mM

ABA (Fig. 8E). In contrast, ABA concentrations over40 mM had an inhibitory action on the expression ofOsNAC2 (Fig. 8E). Thus, ABA regulates expression ofOsNAC2 either in a positive or a negative manner de-pendent on the dose of ABA. Interestingly, at 2 h ofABA treatment, the transcript level of OsABA8ox1 in-creased in the wild type, while that of OsZEP1 de-creased in OsNAC2-OX (Fig. 8, B and C). ABA was thefinal production of OsZEP1 and the substrate of

Figure 7. Interactions between OsNAC2 and the promoters ofOsNCED3,OsZEP1, andOsABA8ox1. A, Interaction of OsNAC2with the promoters ofOsNCED3,OsZEP1, andOsABA8ox1 by yeast one-hybrid assays. The yeast cells were grown on SD/-Leu/-Trp/-His/+30 mM 3-amino-1, 2,4-triazole medium. B to D, ChIP-qPCR assays of OsNAC2 binding to the promoters ofOsNCED3(B), OsZEP1 (C), and OsABA8ox1 (D). Total protein extracted from 35S:OsNAC2-mGFP transgenic plants hydroponically cul-tivated for 4 weeks was immunoprecipitated with an anti-GFPantibody. Fragmented genomic DNAwas eluted from the protein-DNA complexes and subjected to qPCR analysis. The long black bars represent for promoter region for which we designedprimers. The numbers under the bar show the distance fromATG start codon. Short bars represents for the corresponding region ofeach pair of primers on the promoter. Error bars are the SE for three biological repeats: *P, 0.05, **P, 0.01, and ***P, 0.001.

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OsABA8ox1 (Oliver et al., 2007; Zhu et al., 2009).Therefore, the accumulation of ABA might feedbackregulates expression of OsZEP1 and OsABA8ox1.

To gain a mechanistic understanding of the effect ofABA on rice senescence, transcript levels of some SAGswere measured by qPCR. There was no obvious change

in the expression ofOsSGR andOsNYC3 4 h after 20 mM

ABA treatment. Then, significant up-regulation ofOsSGR andOsNYC3 in OsNAC2-OX line was observedat 8 and 12 h of ABA treatment (Fig. 8, F andG). Instead,the expression of ABAmetabolism-related genes varied2 or 4 h after ABA treatment (Fig. 8, A–C), which

Figure 8. Expression of ABA metabolism and senescence-related genes after different concentration of ABA treatments. A to Dand F to H, Treatments with 20 mM ABA. I to K, Treatments with 80 mM ABA. Relative mRNA level was calculated using the DDCT

method from triplicate data.OsActinwas used as an internal control to normalize the different samples with the same amount ofplant RNA. Data are mean6 SE with three replicates. Asterisks indicate a significant difference between wild-type andOsNAC2transgenic lines by t test: *P , 0.05, **P , 0.01, and ***P , 0.001.

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suggested the temporality of OsNAC2 regulating ABAmetabolism-related genes and SAGs. Treatment ofleaves with ABA resulted in no significant change ofOsPAO transcript levels between OsNAC2-OX and thewild type at all three time points: 4, 8, and 12 h (Fig. 8H),which suggested that ABAmodulated the expression ofOsPAO in an OsNAC2-independent manner.On the contrary, 80 mM ABA led to significant in-

crease in transcript levels of both target (OsSGR andOsNYC3) and nontarget (OsPAO) genes inOsNAC2-OXline 4 h after treatment, compared with the wild type.In detail, expression of OsSGR and OsNYC3 increased7.6- and 1.72-fold, respectively, obviously lower thanthat of OsPAO (14.1-fold; Fig. 8, I–K), which was con-sistent with the pervious finding that high dose ofABA had a feedback regulation of OsNAC2 and theninhibited the expression of target-genes (Fig. 8E). So, it’sinteresting that high dose of ABA might mediate SAGsexpression probably in both OsNAC2-dependent andOsNAC2-independent pathway.

nced3 and aba8ox1 Mutants Exhibit a Stay-Green andSenescence Phenotype, Respectively

To test whether OsNCED3 and OsABA8ox1 areinvolved in leaf senescence, we obtained the tillingmutants of OsNCED3 (nced3-1,-2) and OsABA8ox1(aba8ox1-1,-2; Supplemental Fig. S6). After 5 d of darktreatment, nced3-1,-2 leaves showed a stay-green phe-notype similar to that of OsNAC2-RNAi plants, whileaba8ox1-1,-2 showed a senescent phenotype similar tothat of OsNAC2-OX plants (Fig. 9A). Consistent withthis, ion leakage rates, ROS levels, and chlorophyllconcentrations were significantly higher in aba8ox1 -1,-2leaves but lower in nced3-1,-2 leaves than in wild-typeleaves after dark treatment (Fig. 9, B–D). Takentogether, these data indicate that OsNCED3 andOsABA8ox1 are involved in OsNAC2-regulated plantsenescence.

Down-Regulation of OsNAC2 Leads to IncreasedGrain Yield

Our RNAi transgenic lines displayed distinctlydelayed leaf senescence (Fig. 10A), which was consis-tent with the observed decline in OsNAC2 expression(Fig. 10B). So, we next investigated whether grain yieldis improved when OsNAC2-RNAi lines were grown inthe field. As expected, the root length ofOsNAC2-RNAilines was significantly longer than that of the wild type(Fig. 10, C and D). After 150 mM NaCl treatment,OsNAC2-RNAi lines showed a lower withered ratecompared with the wild type (Fig. 10E). Furthermore,an examination of the agronomic traits related to grainyield in twoOsNAC2-RNAi lines, RNAi25 and RNAi31,showed a 16.1% and 10.9% increase in seed-setting ratioand a 3.6% and 4.6% increase in 1,000-grain weight,respectively, compared with nontransgenic controls(Fig. 10, F and G). These changes resulted in increasedgrain yields of 11.6% and 10.6% in the RNAi25 andRNAi31 lines, respectively (Fig. 10H). Thus, regulatingthe expression of OsNAC2 should be a useful strategyfor improving rice yield in the future.

DISCUSSION

Leaf senescence is a complex and highly pro-grammed process, including down-regulation of pho-tosynthesis, disintegration of chloroplasts, degradationof nucleic acid, protein, and lipid, and recycling ofnutrients. Although significant progress has beenmade toward our understanding of leaf senescence inArabidopsis, it remains largely unknown how leaf se-nescence is regulated in rice, where chlorophyll deg-radation is the most characterized, e.g. RLS1 (Jiao et al.,2012), OsDOS (Kong et al., 2006), and NYC1 (Sato et al.,2009). The NAC family is one of the most importanttranscriptional factors involved in leaf senescence(Gregersen and Holm, 2007). Previous reports have

Figure 9. Effect of darkness on de-tached leaves of 8-week-old wild-type, nced3-1,2, and aba8ox1-1,2plants. A, Phenotype of wild-type,nced3-1,2, and aba8ox1-1,2 leavesafter 5 d dark treatment. B, DAB andNBT staining of 6-week-old wild-type, nced3-1,2, and aba8ox1-1,2leaves treated with dark for 5 d. C,Ion leakage analysis of leaves in6-week-old wild-type, nced3-1,2,and aba8ox1-1,2 leaves after 5 ddark treatment. D, Chlorophyllcontent in leaves prior to and afterdark treatment. Values are means6SD of five measurements. Asterisksindicate a significant differencebetween wild-type and OsNAC2transgenic lines by t test: *P, 0.05,**P , 0.01, and ***P , 0.001.

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shown that NAC transcription factors, NTL4 (Leeet al., 2012), OsNAC5 (Sperotto et al., 2009), OsNAC6(Nakashima et al., 2007), OsORE1 (Kim et al., 2014),ONAC106 (Sakuraba et al., 2015), and OsNAP (Lianget al., 2014), participated in aging process of rice.Among them, OsNAP was identified to directly con-trol expression of SAGs, OsSGR, OsNYC1, OsNYC3,OsRCCR1 and OsI57, which function in chlorophylldegradation and glyoxylate cycle (Liang et al., 2014).Additionally, ONAC106 also directly controls the ex-pression ofOsSGR andOsNYC1 (Sakuraba et al., 2015).In this study, we identified and characterized OsNAC2,which acts as an important regulator in leaf senescence.OsNAC2 is upregulated during leaf senescence (Fig. 1),and overexpression ofOsNAC2 can alter the expressionof SAGs (Fig. 3). Furthermore, OsNAC2 can directlyinteract with the promoter sequences of OsSGR andOsNYC3 (Fig. 4). AlthoughOsNAP andOsNAC2 play a

similar role in modulating expression of SAGs, wefound that expression of OsNAC2 and OsNAP was notaffected in OsNAP-OX and OsNAC transgenic plants,respectively (Supplemental Fig. S7). These results sug-gest that OsNAC2 accelerates leaf senescence in anOsNAP-independent manner.

ABA is generally considered as a senescence initiator(Cutler et al., 2010). Recently, OsNAP, a transcriptionalactivator of senescence in rice, is found to repress ABAbiosynthesis. ABA concentration is lower in rice leavesof a gain-of-function ps1-D mutant while higher inthose of OsNAP-RNAi plants, compared with the wildtype. ABA biosynthetic genes, such as OsNCED1,OsNCED3, OsNCED4, and OsZEP, appeared to bedownregulated in the ps1-Dmutant (Liang et al., 2014).However, how OsNAP regulates ABA biosyntheticgenes, and how ABA accelerates senescence remainsunclear in rice. In this study, we found that ABA

Figure 10. Agronomic traits of OsNAC2-RNAi transgenic lines. A, Morphology of 4-month-old mature OsNAC2-RNAi linesgrown in field conditions. B, Expression of OsNAC2 in RNAi plants. C, Root phenotype of 2-week-old wild-type and OsNAC2-RNAi lines. D, Root length of 2-week-oldOsNAC2-RNAi plants comparedwith thewild type. E, Relativewithered rate of 2-week-old wild-type andOsNAC2-RNAi line 3 d after 150 mM NaCl treatment. F to H, Seed-setting rate (F), 1,000-grain weight (G), andgrain yield per plant (H) in the RNAi lines. Values are means6 SD of 10 measurements. Asterisks indicate a significant differencebetween wild-type and OsNAC2 transgenic lines by t test: *P , 0.05, **P , 0.01, and ***P , 0.001.

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content was significantly higher in OsNAC2-OX trans-genic plants than in the wild-type plants (Fig. 6A).Four ABA biosynthetic genes were upregulated, whilethree ABA degradation genes were downregulatedin OsNAC2-OX plants (Fig. 6). These findings indicatethat OsNAC2 regulates rice leaf senescence in a dif-ferent way from OsNAP. Further analysis revealedthat OsNAC2 can directly bind to the promoter ofOsNCED3, OsZEP1, and OsABA8ox1 (Fig. 7). In Ara-bidopsis leaves, NAP was reported to promote chlo-rophyll degradation by directly binding the promoterof ABA biosynthetic gene, AAO3, which enhances thetranscription of AAO3 and leads to increased levels ofABA (Yang et al., 2014). The enzymatic steps of ABAbiosynthesis, from zeaxanthin to active ABA, and ca-tabolism have been elucidated to be potential points ofcontrol to regulate endogenous ABA concentrations(Seo et al., 2009). Our work provided evidence to sup-port that OsNCED3, OsZEP1, and OsABA8ox1 are keypoints to control ABA levels. OsNCED3 is one of thefive NCED genes encoding 9-cis epoxy-carotenoid di-oxygenase, which is involved in xanthophyll cleavage(Hwang et al., 2010), while OsZEP1 encodes a zeaxan-thin epoxidase responsible for the first step in ABA bi-osynthesis (Oliver et al., 2007).OsABA8ox1 is a key genein ABA catabolism and has ABA 89-hydroxylase activ-ity in rice (Zhu et al., 2009). Importantly, nced3 leavesshowed a stay-green phenotype similar to that ofOsNAC2-RNAi plants, while aba8ox1 showed prema-ture senility similar to that of OsNAC2-OX plants (Fig.9A). It was probably that OsNAC2 might not activate

the expression of downstream genes without target-ing OsNCED3 and OsABA8ox1, which indicated thatOsNCED3 andOsABA8ox1were the targets of OsNAC2directly. The hybridization of nced3 or aba8ox1 withOsNAC2-OX line or OsNAC2 RNAi line should be fur-ther conducted for genetic analysis. These data indicatethat OsNCED3, OsZEP1, and OsABA8ox1 act down-stream of OsNAC2, consistent with the conclusion thatOsNAC2 accelerates senescence via induction of ABAbiosynthesis in rice.

Based on the above results, we proposed a followingmodel for the role of OsNAC2 in leaf senescence (Fig.11). The plant hormone ABA, together with others in-cluding ethylene, jasmonic acid, and salicylic acid, isthe major regulator for senescence. In our research,lower concentrations of ABA induce the transcriptionof OsNAC2 (Fig. 8E), which activates expression ofOsNCED3 and OsZEP1, but inhibits expression ofOsABA8ox1 by binding to a specific sequence in theirpromoters (Figs. 6 and 7). The subsequent increasein OsNCED3 and OsZEP1 activity and decrease inOsABA8ox1 activity enhance ABA accumulation in riceleaves (Fig. 6A). Furthermore, OsNAC2 can also di-rectly bind to the promoters of OsSGR and OsNYC3,and increase their expression (Figs. 3 and 4), resulting inchlorophyll degradation and leaf senescence (Figs. 2and 3). In contrast, although OsNAC2 increases its ownexpression by induction of ABAbiosynthesis, it appearsthat high level of ABA may have a feedback repressionof the expression of OsNAC2 (Fig. 8E) and then slowdown ABA synthesis, which might be one of the

Figure 11. Proposed model of OsNAC2 role inplant leaf senescence of rice.

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mechanisms to finely regulate plant growth and de-velopment. The resulting also increases the transcrip-tion of chlorophyll degradation genes, OsPAO, OsSGR,and OsNYC3, via unknown intermediates and leads toleaf senescence. Thus, the NAC transcription factorOsNAC2 acts as a key regulator linking ABA and leafsenescence processes, which might provide a new vi-sion of NAC TFs function in plant development.

The CUC subfamily has provided information aboutthe roles in plant development, such as the formation ofthe cotyledon boundary, the shoot apical meristem(Vroemen et al., 2003), gynoecium, and ovule (Ishidaet al., 2000). NAC proteins, which belong to theCUC subfamily, have been demonstrated to play apivotal part in plant development (Olsen et al., 2005),stress responses (Puranik et al., 2012), and senes-cence (Gregersen and Holm, 2007). ANAC096 (Xuet al., 2013), ANAC019 (Jensen et al., 2010), ANAC055,ANAC072/RD26, ANAC002/ATAF1, ANAC081/ATAF2, ANAC102, and ANAC032 (Takasaki et al.,2015) function as positive regulators of ABA signaling.ORE1/ANAC092/NAC2 (Balazadeh et al., 2010), ORS1(Balazadeh et al., 2011), andAtNAP (Guo andGan, 2006)are positive regulators of senescence. ANAC016 also hasan important role in ABA-inducible leaf senescence bydirectly promoting AtNAP (Kim et al., 2013). Tran-scription factors act as not only activators but also re-pressors in plants. For instance, ATAF1 functions as anegative regulator in drought signaling pathwaysthrough modulation of osmotic stress-responsive geneexpression (Lu et al., 2007). ATAF2, in the same cladewith ATAF1, and ANAC019/ANAC055 were recentlyreported to negatively regulate the expression ofpathogenesis-related genes (Bu et al., 2008). JUB1 is anegative regulator of senescence (Wu et al., 2012). Al-though, in general, we proposed OsNAC2 acceleratesleaf senescence in rice, it is novel thatOsNAC2positivelyregulated OsSGR, OsNYC3, OsNCED3, and OsZEP1,and negatively regulated OsABA8ox1 by directly bind-ing to their promoters (Figs. 4 and 7). Similarly, Chenet al. (2015) reported that OsNAC2 repressed the ex-pression of GA biosynthetic genes, OsKO2, and en-hanced the expression of OsEATB, which encodesa repressor of GA biosynthesis (Chen et al., 2015). Ad-ditionally, MYC2, a basic helix-loop-helix domain-containing TF, acts as both activator and repressor tomodulate the expression jasmonic acid-responsive genesin Arabidopsis (Dombrecht et al., 2007). The recognitionsite of MYC2 appears to function as both positive andnegative regulator of the expression of the MYC2promoter-GUS fusion gene (Abe et al., 1997). Recently,CONSTANS, the eponymous member of the family ofCONSTANS-like transcriptional regulators, mediatesphotoperiodic flowering by physically interacting withmicroproteins, miP1a and miP1b (Graeff et al., 2016).Thus, TFs play a multiple and complicated role in de-velopment and stress responses of plants. Future workon the interaction of OsNAC2with other proteins mighthelp us better understand the regulatory mechanism oftranscriptional activation in TFs.

MATERIALS AND METHODS

Plant Materials and Treatments

Plant material used in this study was Nipponbare (Oryza sativa japonica cvNipponbare). The vector in which 1,500-bp full-length promoter of OsNAC2 isconnectedwith GUSwas as described by Shen et al. (2017). Overexpression andRNAi transgenic lines were constructed before and the details are described byChen et al. (2015). The seeds of OsNAC2-OX, wild-type, and OsNAC2-RNAiplants were germinated in petri dishes with wetted filter paper at 37°C underdark condition. After 48 h incubation, uniformly germinated seeds were se-lected and cultivated in a beaker containing half-strength Kimura B solution asdescribed previously (Chu and Lee, 1990). The hydroponically cultivatedseedlings were grown in a phytotron in a 16/8-h light/dark cycle, 28°C, 300 W/m2

light intensity, and 40% humidity regime. Leaves were used in experiments and allexperiments were repeated three times independently.

For dark-induced leaf senescence, 4-week-old seedlings (cultivated as de-scribed above) were treated with 0, 3, and 5 d of darkness to observe theirphenotypic characters. The leaveswere alsoused for chlorophyll and ion leakagerates measurements. The detached leaves were treated with 0, 2, 3, and 4 d ofdarkness to obtain the phenotype in vitro. ForABA-induced leaf senescence, riceseeds were germinated on Murashige and Skoog medium containing 0, 2, and4 mM ABA, and the sensitivity index of seed germination and shoot length toABA was evaluated. Detached leaves (2 weeks old) were placed in a petri dishwetted with 20 mM ABA and kept under dim light (40 mmol/s/m2). Chloro-phyll and ion leakage rates were also measured. The 20 and 80 mM ABA-treatedleaves were collected at designated time intervals for total RNA extraction.Additionally, treatment of ABA at 0, 20, 40, 80, and 160 mM was conducted forOsNAC2 expression pattern.

GUS Staining

The native promoter ofOsNAC2, a 2-kb genomicDNA fragment upstreamofthe transcription start site, was cloned and transcriptionally fused to the up-stream of the GUS-coding sequence, and theOsNAC2 promoter::GUS construct(pOsNAC2-GUS) was transformed into wild-type rice plants (Nipponbare)plants. Seeds of transgenic rice (T1) harboring an OsNAC2 Pro::GUS constructwas used for GUS staining from 1 to 10 d. Primers for amplifying OsNAC2promoter are shown in Supplemental Table S1. b-GUS staining was performedas described previously (Woo et al., 2001). Images were taken directly or with astereomicroscope (Leica ZOOM 2000).

Histochemical Staining

O22 andH2O2 were determined using nitro blue tetrazolium (NBT) and 3,39-

diaminobenzidine (DAB) histochemical staining, respectively. Leaves afterdifferent treatment were detached from different lines and submerged in NBTsolution (1 mg/mL NBT plus 10 mM NaN3 solution in 10 mM potassium phos-phate buffer, pH 7.8) or DAB solution (1 mg/mL, pH 5.5). After leaves werestained for 40 min (NBT) or 2 h (DAB), leaves were boiled in 95% ethanol for15 min to remove the chlorophyll completely and stored in 60% glycerol forobservation and imaging.

Chlorophyll Measurements

Chlorophyll was extracted from 50mg of leaf tissuewith different treatment,and its content was determined by measuring the A663 and 645 nm using avisspectro photometer [Jinghua (Shanghai) Co.] as described previously (Kimet al., 2006): total Chl (mg/L) = Chla + Chlb = 20.2A645 + 8.02A663.

Measurement of Ion Leakage Rates

Ion leakage rates were measured as described previously (Lee et al., 2015).The conductivity of the solution was measured using an FG3-ELK conductivitymeter (Mettler Toledo Instruments). The rate of ion leakage is expressed as thepercentage of initial conductivity divided by the total conductivity.

Rice Microarray Analysis

Formicroarray analysis, rice seedlings were grown in basal nutrient solutionunder the condition of a 16-h-light/8-h-dark photoperiod at 28°C. Total RNA

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was extracted from the leaves of 2-week-old wild-type and OsNAC2-OX(ON11) plants according to the manufacturer’s instructions, and biotin-labeledcRNA was obtained by GeneChip 39IVT Express Kit (Affymetrix). Array hy-bridization and wash were then conducted with the GeneChip Hybridization,Wash and Stain Kit (Affymetrix) in Hybridization Oven 645 (Affymetrix) andFluidics Station 450 (Affymetrix). Slides were scanned by GeneChip Scanner3000 and Command Console software 3.1 (Affymetrix) with default settings.Three independent biological replicates were conducted for the wild-type andOsNAC2-OX (ON11) plants. Raw data were normalized by the robust multi-array average algorithm, and fold change and pfp values were calculated byrank product method using R (3.0.0). The differently expressed genes in wild-type and OsNAC2-OX plants were classified functionally using the biologicalprocess category of Rice Gene Ontology (www.geneontology.com). Significantinteractors were determined using a two-sample analysis (t test).

Endogenous ABA Determination

Two-week-old leaves of OsNAC2-OX, wild type, and OsNAC2-RNAi lineswere collected, weighed, and immediately frozen in liquid nitrogen. Frozenleaves were pulverized and ABA was extracted as described previously (Chenet al., 2012). Quantitative determination of endogenous ABAwas performed ona UPLC-MS/MS system consisting of an AB SCIEX 4500 triple quadrupolemass spectrometer and a Shimadzu LC-30ADUPLC system.Three independentbiological repeats were performed.

qPCR Analysis

Untreated and treated leaf tissue obtained from different lines about 2weeksoldwas used as a source of total RNA. Total RNAswere extractedwith RNAisoreagent (TaKaRa code no. 9752A). The total RNAswere purified and reversed tocDNAwith the PrimeScript RT reagent kit with gDNAEraser (TaKaRa code no.RR047A). The qPCR analysis was performed using SYBR Premix EX Taq(TaKaRa code no. RR420A) on a MyiQ2 real-time PCR detection system(Bio-Rad) according to the manufacturer’s instructions. PCR efficiency (95–105%)was verified. Transcript levels of target genes were normalized to that of thehousekeeping gene OsActin using the equation of 22△△CT, where CT is thethreshold cycle for each gene in every sample. Primer sequences are listed inSupplemental Table S1.

Constructions for Yeast One-Hybrid Assay

For yeast one-hybrid assays, the coding sequence ofOsNAC2was amplifiedusing thermostable DNA polymerase from Thermococcus kodakarensis (KOD)polymerase (Toyobo) and was subcloned into the EcoRI and XhoI sites ofpGADT7 vector. To generate OsSGRp::HIS3, OsI85p::HIS3, OsNYC3p::HIS3,OsNCED2p::HIS3, OsNCED3p::HIS3, OsNCED5p::HIS3, OsZEP1p::HIS3,OsABA8ox1p::HIS3, OsABA8ox2p::HIS3, and OsABA8ox3p::HIS3 reporterconstructs, the promoter fragments were amplified by PCR and then clonedinto pHIS2.1 vector through EcoRI-SpeI, EcoRI- SpeI, SacI-SpeI, EcoRI-MluI,EcoRI-MluI, MluI- SpeI, EcoRI-MluI, EcoRI-MluI, EcoRI-MluI, and EcoRI-MluIsites, respectively. All primers used for cloning these constructs are listed inSupplemental Table S1. These vectors and empty vector were transformed intoyeast strain AH109 by the PEG/LiAc method, and yeast cells were plated ontoSD/–His/–Trp/–Leu + 30 mM 3-amino-1, 2,4-triazole medium for stringentscreening of the possible interactions, according to the protocol of MatchmakerGAL4 One-Hybrid System (Clontech).

Dual-Luciferase Assay of Transiently TransformedTobacco Leaves

To generate the construct of LUC reporter for the dual-luciferase assays, thecoding sequence of OsNAC2 was cloned into pGreenII 62-SK vector. The pro-moter sequences of OsSGR and OsNYC3 were amplified from rice genome andinserted into pGreenII0800-LUC through KpnI-NcoI sites, respectively. Primersare listed in Supplemental Table S1.

The Nicotiana benthamiana leaves were used for dual-luciferase assays asdescribed previously (Hellens et al., 2005). The effector constructs (p35S-OsNAC2 or p35S) and the above reporter constructs harboring the luciferase(LUC) gene were introduced into Agrobacterium tumefaciens strain GV3101, re-spectively. After cotransformation, the tobacco plants were placed under 23°Cfor 3 d. The firefly LUC and Renilla luciferase (REN) activities were measured

using the Dual-Luciferase Reporter Assay System (Promega).The LUC activitywas normalized to the REN activity.

ChIP

ChIP was performed based on a previous report (Gendrel and Colot, 2005)with OsNAC2-OX transgenic seedlings, in which a GFP coding sequence wasfused in frame to the 39end of the OsNAC2 gene. About 4 g of 4-week-oldOsNAC2-OX seedlings was treated and fixed with 1.0% formaldehyde for10 min subsequently. Antibodies against mGFP (Santa Cruz Biotechnology)were used for immunoprecipitation. Protein-A-agarose beads were blockedwith salmon spermDNA and used to pull down the protein-DNA complex. TheDNA fragments of the ChIP were used for qPCR. Primers were selected in thepromoter regions of each selected gene. The ChIP experiments were repeatedthree times with the similar data. Primer pairs for qPCR were listed inSupplemental Table S1.

Field Cultivation of Rice

The rice plants were grown in a standard paddy field at the ExperimentalStationof FudanUniversity inTaicang Jiangshuprovince. Thefieldplantingwaswith three replicates according to a randomized complete-block design; thespacing of the transplanted plants was 20 3 20 cm. The area per plot wasdesigned as 4 m2. These plants were then grown under the conditions of con-ventional cultivation. For agronomic traits measurement, at least 10 plantsrandomly obtained from the center of the plot were used to avoid any irregu-larities at the margins of the plot.

Tilling Mutant and Verification

The tilling mutant of OsABA8ox1 and OsNCED3 bought from Liu Lab at KeyLaboratory of Plant Molecular Physiology, CAS, was germinated and hydro-ponically cultivated as described previously (Chu and Lee, 1990). These tillingmutants were planted in the paddy field at Fudan University. For mutant veri-fication, 2-week-old leaf tissue of each mutant line was used for DNA extractingby Plant Genomic DNA Kit (Tiangen; code DP305). The primers, aba8ox1-F (59-CTAACTCCTCTTCCCACTCTGCTT-39), aba8ox1-R (59-ATTTCAGTTAAG-GAACCAGTCTGC-39), nced3-F (59-TCTCTCTCTCGGCAGAAACACACC-39),and nced3-R (59-CGGGTCGAGGAGGCCGCAGGCGGC-39), were provided byLiuLab atKey Laboratory of PlantMolecular Physiology,CAS, for amplifying thefragments ofOsABA8ox1 andOsNCED3. PCRwas carried out using PrimerSTARMaxDNAPolymerase (TaKaRa code no. R045A) according to themanufacturer’sinstruction. The obtained fragments were sequenced by Sangon Biotech (Shang-hai) Co.Additionally, leaves of themutants collected from the heading stageweretreated with 5 d of darkness to observe their phenotypes. Chlorophyll and ionleakage rates were also measured.

Accession Numbers

Sequence data from this article can be found in GenBank/EMBL databasesunder the following accession numbers: OsNAC2 (Os04g0460600), OsNAP(Os03g0327800), OsSGR (Os09g0532000), OsNYC3 (Os06g0354700), OsI85(Os07g0529000),OsRCCR1 (Os10g0389300),OsPaO (Os03g0805900),OsNCED2(Os12g0435200), OsNCED3 (Os03g0645900), OsNCED5 (Os12g0617400),OsABA8ox1 (Os02g0703600), OsABA8ox2 (Os08g0472800), OsABA8ox3(Os09g0457100),OsZEP1 (Os04g0448900),OsActin (Os10g0510000),OsNOL(Os03g0654600), OsSWEET5 (Os05g0588500), OsTDC1 (Os08g0140300),and OsNAP (Os03g0327800).

Supplemental Data

The following supplemental materials are available.

Supplemental Figure S1. Natural senescence phenotype of OsNAC2 trans-genic lines during grain-filling stage in field condition.

Supplemental Figure S2. Histochemical staining of a transgenic lineexpressing pOsNAC2-GUS in 6-week-old wild-type leaf and seeds 1 to10 d after germination.

Supplemental Figure S3. Phylogenetic tree among NACs in Arabidopsisand rice.

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Supplemental Figure S4. Yeast one-hybrid assays between OsNAC2 andthe promoter of senescence and ABA metabolism-related genes.

Supplemental Figure S5. NAC binding motif CACG searching.

Supplemental Figure S6. Sequence alignment of mutant aba8ox1 and nced3with the wild type.

Supplemental Figure S7. Expression of OsNAC2 in OsNAP-OX (OX11 andOX17) and OsNAP-SRDX (SRDX4, SRDX6, and SRDX7) lines andOsNAP in OsNAC2-OX and OsNAC2-RNAi lines.

Supplemental Table S1. Primers used for the sequencing of different genesin rice.

Received April 21, 2017; accepted May 9, 2017; published May 12, 2017.

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