Arabidopsis thaliana NGATHA1 transcription factorinduces ABA biosynthesis by activating NCED3gene during dehydration stressHikaru Satoa,1, Hironori Takasakia,2, Fuminori Takahashia, Takamasa Suzukib, Satoshi Iuchic, Nobutaka Mitsudad,Masaru Ohme-Takagid,e, Miho Ikedad,e, Mitsunori Seof, Kazuko Yamaguchi-Shinozakig, and Kazuo Shinozakia,1

aGene Discovery Research Group, RIKEN Center for Sustainable Resource Science, Tsukuba, 305-0074 Ibaraki, Japan; bCollege of Bioscience andBiotechnology, Chubu University, Kasugai, 487-8501 Aichi, Japan; cExperimental Plant Division, BioResource Research Center, RIKEN, Tsukuba, 305-0074Ibaraki, Japan; dPlant Gene Regulation Research Group, Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology,Tsukuba, 305-8566 Ibaraki, Japan; eGraduate School of Science and Engineering, Saitama University, Saitama, 338-8570 Saitama, Japan; fDormancy andAdaptation Research Unit, RIKEN Center for Sustainable Resource Science, Yokohama, 230-0045 Kanagawa, Japan; and gLaboratory of Plant MolecularPhysiology, Graduate School of Agricultural and Life Sciences, University of Tokyo, 113-8657 Tokyo, Japan

Edited by Julian I. Schroeder, Cell and Developmental Biology Section, Division of Biological Sciences, University of California, San Diego, La Jolla, CA, andapproved October 1, 2018 (received for review July 10, 2018)

The plant hormone abscisic acid (ABA) is accumulated afterdrought stress and plays critical roles in the responses to droughtstress in plants, such as gene regulation, stomatal closure, seedmaturation, and dormancy. Although previous reports revealeddetailed molecular roles of ABA in stress responses, the factorsthat contribute to the drought-stress responses—in particular, reg-ulation of ABA accumulation—remain unclear. The enzyme NINE-CIS-EPOXYCAROTENOID DIOXYGENASE 3 (NCED3) is essential forABA biosynthesis during drought stress, and the NCED3 gene ishighly induced by drought stress. In the present study, we isolatedNGATHAs (NGAs) as candidate transcriptional regulators of NCED3through a screen of a plant library harboring the transcriptionfactors fused to a chimeric repressor domain, SRDX. The NGA pro-teins were directly bound to a cis-element NGA-binding element(NBE) in the 5′ untranslated region (5′ UTR) of the NCED3 promoterand were suggested to be transcriptional activators of NCED3.Among the single-knockout mutants of four NGA family genes,we found that the NGATHA1 (NGA1) knockout mutant was drought-stress-sensitive with a decreased expression level of NCED3 during de-hydration stress. These results suggested that NGA1 essentially func-tions as a transcriptional activator of NCED3 among the NGA familyproteins. Moreover, the NGA1 protein was degraded under non-stressed conditions, and dehydration stress enhanced the accu-mulation of NGA1 proteins, even in ABA-deficient mutant plants,indicating that there should be ABA-independent posttranslationalregulations. These findings emphasize the regulatory mechanisms ofABA biosynthesis during early drought stress.

drought stress | ABA biosynthesis | transcriptional regulation | NCED3 |NGA

Plants have developed various systems to survive adverse andfluctuating environmental conditions, such as drought, high

salt, and extreme temperature, as sessile organisms. Droughtstress has negative effects on the plant growth and crop yield (1)and often causes severe damage to the agricultural crops (2). Theplant hormone abscisic acid (ABA) is known as an essential factorthat positively regulates the plant drought-stress responses, such asstomatal closure, induction of drought-inducible genes, and re-pression of plant growth (3). Previous studies revealed the de-tailed molecular mechanisms by which ABA is transported (4),received (5) to activate the cellular signal cascades (6, 7), and theninduces drought-stress-responsive gene expression (8); however,the factors that regulate early drought-stress responses beforeABA accumulation need to be further explored.Among the various enzymatic proteins involved in several ABA bio-

synthetic pathways in plants (9), NINE-CIS-EPOXYCAROTENOIDDIOXYGENASE 3 (NCED3) is known as an important enzymefor ABA accumulation during drought stress because expression of

the NCED3 gene is highly induced in the vascular tissues bydrought stress, and its knockout mutants revealed decreased ABAaccumulation under drought-stress conditions and drought-stress-sensitive phenotypes (10, 11). The NCED3 promoter has a G-boxsequence as a long-range enhancer that is essential for gene in-duction in the vascular tissues during dehydration stress (12, 13).This G-box is located ∼2.3 kb upstream from its translational startsite; however, the detailed molecular mechanisms by which theunidentified transcription factors activate the NCED3 gene beforeABA accumulation have not been elucidated. In the present study,we identified a transcription factor, NGATHA1 (NGA1), thatactivates the NCED3 gene during dehydration stress and charac-terized a cis-acting sequence, NGA-binding element (NBE), in the5′ untranslated region (5′ UTR) of the NCED3 promoter that isnecessary for gene induction (in this work, we use “promoter”to refer to the region upstream from the translational start siteof a protein coding gene and include the 5′ UTR region). More-over, it also was suggested that ABA-independent posttranslational

Significance

The plant hormone abscisic acid (ABA) is essential for drought-stress responses in plants, and its functions have been wellstudied; however, the detailed molecular mechanisms of ABAbiosynthesis during early drought stress need to be furtherexplored. The present study identified a transcription factor,NGTHA1 (NGA1), which positively regulates ABA accumulationduring dehydration stress by activating the NCED3 gene encodinga key ABA biosynthetic enzyme. We also identified a cis-actingelement bound by NGA1 in the 5′ untranslated region (5′ UTR)of the NCED3 promoter. The NGA1 protein was degraded undernonstressed conditions, but it was stabilized during dehydrationstress in an ABA-independent pathway.

Author contributions: H.S., H.T., F.T., K.Y.-S., and K.S. designed research; H.S., H.T., T.S.,and M.S. performed research; H.S., S.I., N.M., M.O.-T., and M.I. contributed new reagents/analytic tools; H.S., H.T., and T.S. analyzed data; and H.S. and K.S. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Published under the PNAS license.

Data deposition: Transcriptome datasets have been deposited in the DNA Data Bank ofJapan (accession no. DRA006360).1To whom correspondence may be addressed. Email: hikaru.sato@riken.jp or kazuo.shinozaki@riken.jp.

2Present address: Graduate School of Science and Engineering, Saitama University, Sai-tama, 338-8570 Saitama, Japan.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1811491115/-/DCSupplemental.

Published online November 5, 2018.

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regulation should be involved in the stabilization of the NGA1protein. NGA proteins are B3-type transcription factors, and Ara-bidopsis thaliana has four NGA family proteins. Previous workrevealed that the NGA proteins regulate the developmental pro-cess in the reproductive organs or leaves (14–16). In the presentwork, we revealed functions of NGA1, which acts as a positiveregulator of ABA biosynthesis during the drought-stress responsesby directly activating the expression of NCED3.

ResultsNGA2–SRDX-Overexpressing Plants Reveal Repressed Expression ofNCED3. To identify the transcription factors that regulate ABAbiosynthesis during early drought responses, we screened a libraryof transgenic plants overexpressing the transcription factors fusedto the repressive domain SRDX (CRES-T; chimeric repressorgene silencing technology) (17). ABA-deficient mutants have beenreported to show sucrose-insensitive phenotypes during germina-tion (18). Previous studies reported that sucrose or other sugar-insensitive mutants often carried mutations in the genes involvedin ABA biosynthesis (19, 20). Therefore, we screened 1,795 CRES-T plant lines of the library on the basis of the germinationphenotypes on high sucrose-containing (300 mM) medium andfound four sucrose-insensitive lines. Among these, the line 1008-2 overexpressing NGA2–SRDX indicated strong insensitivity to ahigh concentration of sucrose during germination (Fig. 1 A–C).Line 1008-2 also exhibited suppressed expression ofNCED3 duringdehydration stress (Fig. 1D), as well as drought-sensitive pheno-types with decreased leaf temperature, which also were observed in

NCED3 knockout mutants (nc3-2) (Fig. 1 E and F and SI Ap-pendix, Fig. S1 A–C). (In this work, “dehydration” and “drought”stresses are defined as water-deficit stress treatments of plants onparafilms from agar plates and plants on soil without a watersupply, respectively.) ABA accumulation during dehydration stresswas significantly suppressed in line 1008-2 (Fig. 1G) in accord withthe repression of NCED3 (Fig. 1D). The low level of ABA accu-mulation in nc3-2 likely results from low induction of anotherNCED family gene in nc3-2 that was not induced in the wild-typeplant (21). Thus, we hypothesized that the NGA family proteinsare involved in the transcription of NCED3 during drought stress.NGA proteins are B3-type transcription factors, and Arabi-

dopsis thaliana has four NGA family proteins and three NGA-like (NGAL) family proteins (SI Appendix, Fig. S1D). RAVsubfamily proteins have the conserved B3 domain with highhomology to NGA; however, the protein structures are quitedifferent because they also have conserved AP2 domains. Anamino acid sequence, K/RLFGV, was found in all of the NGA,NGAL, and RAV subfamily proteins, which was reported as aputative repressor motif (22). More comprehensive and detailedphylogenetic analysis of the NGA family proteins in land plantswas reported in a previous study (23).

NGA Family Proteins Directly Bind to and Activate the NCED3Promoter. The NGA family proteins contain the putative re-pression domain RLFGV, but previous studies reported thatanother K/RLFGV domain-containing transcription factor fam-ily, HEAT SHOCK FACTOR B (HsfB), acts as both repressor

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Fig. 1. The 35S:NGA2-SRDX plants (line 1008-2) revealed decreased expression of NCED3 and were sensitive to drought stress. (A) Expression levels of NGA2in the identified line through a screen. The error bars indicate the SD from triplicate technical repeats. (B and C) Sucrose insensitivity of the 35S:NGA2-SRDXplants in germination. (B) Images of plants grown on MS medium with or without 300 mM sucrose for 2 wk. (Scale bars: 1 cm.) (C) Rates of plants forming trueleaves grown on medium with or without 300 mM sucrose. The error bars indicate the SD from four replicates (n = 20 each). Asterisks indicate significantdifferences from the wild-type plants. *P < 0.01 (Bonferroni-corrected Student’s t test). (D) Expression levels of NCED3 in the identified line through a screen.Plants grown on MS medium for 2 wk were treated with dehydration stress. The error bars indicate the SD from triplicate technical repeats. Asterisksindicate significant differences between the 35S:NGA2-SRDX and wild-type plants. *P < 0.01 (Bonferroni-corrected Student’s t test). (E and F ) Drought-stress tolerance test of the 35S:NGA2-SRDX and nc3-2 mutant plants. (E ) Images of plants before and after drought and after rewatering. Plants grown onMS medium for 2 wk were transferred to soil and grown for 2 d. Water was withheld for 11 d. (Scale bars: 1 cm.) (F) Survival rates of plants afterrewatering. The error bars indicate the SD from five replicates (n = 35 total). Asterisks indicate significant differences from the wild-type plants. *P < 0.01(Bonferroni-corrected Student’s t test). (G) ABA contents of the 35S:NGA2-SRDX and nc3-2 mutant plants during nonstress (Cont) and dehydration stress(Dry). Plants grown on MS medium for 2 wk were treated with dehydration stress for 4 h. The error bars indicate the SD from six replicate samples. Theletters above the bars indicate significant differences among plant lines under nonstress and dehydration-stress conditions (P < 0.05, according to Tukey’smultiple range test).

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and activator during heat stress (24, 25). To elucidate whetherthe NGA proteins function as activators or repressors, we per-formed transactivation assays using the NCED3 promoter as a re-porter in Arabidopsismesophyll protoplasts (Fig. 2A). Transfectionsof the NGA family proteins resulted in activation of the NCED33-kb promoter (Fig. 2B). The deletion series of the NCED3 pro-moter revealed that the nearby region of a translational start site(−1 to −161) was sufficient for the activation of NCED3 by NGAs(Fig. 2 B and C). These data indicate that the NGA family pro-teins function as transcriptional activators of NCED3. We alsoperformed similar transactivation assays using mutated NGA2 andNGA4 within their putative repressor motifs RLFGV to five ala-nines (AAAAA) as effectors. Interestingly, the mutated proteinsdid not activate theNCED3 3-kb promoter (SI Appendix, Fig. S2A),which suggested that this motif is necessary for NGA to activate theNCED3 promoter. We confirmed that NGA has transcriptionalactivating functions by detecting increased mRNA levels of theGUS reporter gene in protoplasts transfected with NGAs (SI Ap-pendix, Fig. S2B). The endogenousNCED3 gene in protoplasts alsowas activated by transfecting the NGA family proteins (SI Appen-dix, Fig. S2C). These data indicate that the NGA family proteinsfunction as a transcriptional activator of NCED3.We investigated whether the NGA proteins directly bound to

the NCED3 promoter in vitro by electrophoretic mobility shiftassay (EMSA) using the −1- to −161-bp promoter region ofNCED3. This region was divided into three shorter parts (probesE1–E3). Due to the unsuccessful expression of the full-lengthNGA proteins in Escherichia coli, the N-terminal region ofNGA2 that includes the DNA-binding domain (SI Appendix, Fig.S2D) fused to GST (NGA2N-GST), was expressed. Two bandshifts were observed when the NGA2N-GST protein was in-cubated with the probe E3 (SI Appendix, Fig. S2E), which sug-gested the possibility that NGA2 directly bound to the NCED3promoter by forming a dimer. Weak band shifts were observed byusing the E1 and E2 probes, but there was a large difference in theintensity of band shifts from probe E3, which suggests thatE3 likely contains the NGA binding site. The probe E3 was di-vided into the two shorter parts (probes 3a and 3b), and sub-sequent EMSAs suggested that the NGA2N-GST protein directlybound to the probe 3b (SI Appendix, Fig. S2F). This region con-tains the 6-bp motif CACTTG that was reported as a directbinding motif of NGAL2 (SOD7) on the KLU promoter (26). Themutated probe (probe m1-5) indicated that replacement of theCACTTG motif led to a complete loss of binding (Fig. 2D). ThisCACTTG motif therefore appears to be a common binding se-quence of NGA and NGAL family proteins. The NGA1 proteinalso directly bound to the CACTTG motif (Fig. 2E). This sequenceCACTTG motif was named as the NBE. Additional transactivationassays in protoplasts confirmed that the NGA proteins could notactivate the 3-kb NCED3 promoter with NBE mutations, becausethere was no detection of increased enzymatic activity or mRNAaccumulation of the GUS reporter (Fig. 2C and SI Appendix, Fig.S2B). These results suggest that the NGA family proteins directlybind to NBE on the NCED3 promoter and induced gene expres-sion. Two band shifts were not observed in other EMSAs, pre-sumably due to the concentrations of polyacrylamide gel; 6% gelwas used for the experiment presented in SI Appendix, Figs. S2E,and 8% was used for the other figures.

The NBE Sequence Is Important for the NCED3 Promoter Activity inTransgenic Plants During Dehydration Stress.NBE was found to be acis-acting element present in the NCED3 promoter. We generatedtransgenic plants harboring the GUS reporter gene driven bymodified NCED3 promoters (Fig. 3A). As previous papersreported (12, 13), the NCED3 3-kb promoter could induce theexpression of the reporter gene, whereas the 2-kb promoter couldnot (Fig. 3B). The promoter of the −250- to −3,000-bp regionfused to the RD29A minimal TATA box (3-kbp Δ1–250 mTATA)

and the 3-kbp region with mutation in NBE to six adenines (3-kbpmNBE) revealed almost no expression of the reporter genes rel-ative to the expression level of the reporter gene in the 3 kbp:GUSplants (Fig. 3 B, Upper), when all transgenic lines were treated withsimilar dehydration stress (Fig. 3 B, Lower). In comparison withthe expression levels of the reporter genes under control condi-tions in each line, the 3-kb mNBE promoter still activated thereporter gene, but the reporter gene was significantly suppressedrelative to the 3-kbp promoter (Fig. 3C). In addition, reporteractivation by the 2- and 3-kbp Δ1–250 mTATA promoter wassuppressed more than the 3-kbp mNBE promoter (Fig. 3C). Theseresults suggest that the NBE sequence was necessary for both fullactivation of the NCED3 promoter during dehydration stress andbasal expression under control conditions. The GUS staining wasalso observed in only the 3 kbp:GUS plants during dehydrationstress among the transgenic lines (SI Appendix, Fig. S3 A and B).

NGA Genes Are Expressed in Various Tissues Including Vascular Onesand Reveal Several Stress-Mediated Expression Patterns. The 35S:NGA2-SRDX plants ectopically expressed the NGA2 protein fusedto the repressor motif, and the transactivation assays in protoplastssuggested the presence of redundant NGA functions in activatingNCED3 (Fig. 2 A and B). However, overexpression in plants andprotoplasts does not reflect the actual tissue expression patterns orintensity in wild-type plants. To elucidate how each NGA proteincontributed to the induction of NCED3 during drought stress, weanalyzed tissue-specific expression and stress-inducible expressionof NGAs. We generated transgenic plants harboring the GUS re-porter gene driven by an ∼3-kb-long promoter of each NGA gene.In the NGA1p:GUS plants, GUS activity was observed in thevascular tissues of aerial organs; in particular, strong activity wasobserved in the petioles and major veins of true leaves (Fig. 4 Aand B). Weak GUS staining was also observed in the lateral roots(Fig. 4C). In the NGA2p:GUS and NGA3p:GUS plants, GUS ac-tivity was observed in the whole leaves, including vascular tissues,shoot meristematic region, root–shoot junction, and lateral roots(Fig. 4 D–I). In the NGA4p:GUS plants, GUS activity was ob-served in the lateral roots, shoot meristematic region, and leaf tips(Fig. 4 J–M). Weak GUS staining was detected in the petioles oftrue leaves (Fig. 4K). The NCED3 gene was expressed in thevascular tissues (SI Appendix, Fig. S3B) (12), which suggested thatthe NGA family genes were coexpressed with NCED3. In partic-ular, the GUS staining patterns of the NGA1p:GUS plantsexhibited a similar pattern to the NCED3 3 kbp:GUS plants. Weanalyzed the expression levels of NGAs under dehydration-stressconditions. NGA1, NGA2, and NGA3 were highly expressed in theshoots under the nonstress condition (dehydration: 0 h) (Fig. 4N).During dehydration stress, NGA1, NG2, and NGA4 genes wereinduced by stress in the root tissues, and NGA3 and NGA4 weretransiently and continuously induced in the shoots, respectively(Fig. 4N). Meanwhile, the expression levels of NGA1 and NGA2were transiently suppressed by dehydration stress in the early phaseand were elevated again in the later phase of dehydration (Fig.4N). Dehydration stress increased the intensity of GUS staining inthe NGA3p:GUS and NGA4p:GUS plants in shoot and root tis-sues, respectively (SI Appendix, Fig. S4A). The NGA3p:GUS plantsshowed similar tissue-specific expression patterns during de-hydration stress, and theNGA4p:GUS plants showed GUS stainingover a wider range in lateral roots.We also examined expression levels of NCED3 and NGAs

during germination on medium with or without high sucrose. Theresults indicated that during germination, high concentration ofsucrose induced higher expression ofNCED3 andNGAs relative tocontrol medium (SI Appendix, Fig. S4B). In analyses of spatialexpression patterns of the NCED3 and NGAs during germination,the NCED3 3 kbp:GUS and NGAp:GUS plants demonstratedGUS staining in whole embryos on control medium (SI Appendix,Fig. S4C). On a high-sucrose medium, the NCED3 3 kbp:GUS and

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NGA1p:GUS plants showed stronger GUS staining patterns in thetip regions of cotyledons and radicles of embryos, as well as in theseed coats near the micropylar endosperm. Weak GUS stainingwas detected in seed coats in the NGA2p:GUS plants (SI Appendix,Fig. S4C). These expression patterns during germination suggestthat NGAs might activate the NCED3 gene to inhibit germinationon high-sucrose-containing medium.

Knockout of NGA1 Results in Drought-Stress Susceptibility andSuppresses the Expression of the NCED3 Gene. To elucidate thecontribution of each NGA gene to drought-stress responses inseedlings, single-knockout mutants of each NGA gene wereisolated (nga1-, nga2-, nga3-, and nga4-1) (SI Appendix, Fig. S5A–C). A point mutation in the nga1-1 resulted in a truncatedNGA1 lacking the C-terminal region (SI Appendix, Fig. S5D).Among the NGA single-knockout mutants, only nga1-1 mutantsexhibited drought-stress sensitivity, and complementation of theNGA1 expression with the NGA1 genomic region (NGA1pro:NGA1) led to the recovery of drought-stress tolerance (Fig. 5 A–C and SI Appendix, Fig. S5E). The nga2-, nga3-, and nga4-1 didnot show any difference in drought-stress tolerance (SI Appendix,Fig. S5 F–K). The nga1-1mutants exhibited decreased expressionlevels of NCED3 and other ABA-responsive genes, RD22 andRD20 (Fig. 5D). Greater effects on RD22 and RD20 thanNCED3 might reflect amplification of ABA signaling duringdehydration stress, and the knockout of NGA1 has greater ef-fects on RD22 than RD20 because the induction of RD20 wassuppressed in nga1-1, while RD22 showed almost no inductionduring dehydration stress, as well as suppressed gene expressionunder nonstress conditions. The accumulation of ABA duringdehydration stress was also decreased in nga1-1 (Fig. 5E).Complemented expression of NGA1 recovered the expressionlevels of dehydration-inducible genes and ABA accumulation(Fig. 5 D and E). The expression levels of NCED3 during de-hydration stress were not significantly different from wild type innga2-, nga3-, and nga4-1 (SI Appendix, Fig. S5 L and M). Theseresults suggest that among the NGA family genes, NGA1 mainlycontributes to induction of NCED3 during drought stress. This isalso supported by the vascular-specific expression patterns of theNGA1 gene (Fig. 4 A–C), which are similar to those of NCED3(11). We also checked whether the truncated form of NGA1 innga1-1 (NGA1 mut) results in a dominant-negative effect withrepressive effects on NCED3 expression. In Arabidopsis meso-phyll protoplasts, transfection of the NGA1 mut did not affectthe endogenous NCED3 gene expression, and cotransfection ofthe NGA1 mut with the wild-type NGA1 (NGA1 wt) did notaffect induction of NCED3 by NGA1 wt (SI Appendix, Fig. S5N).These data suggest that the NGA1 mut in nga1-1 has no tran-scriptional activity or repressive effects on NCED3.

Transcriptomic Analysis Reveals That Dehydration-Inducible GenesAre Down-Regulated in the nga1-1 Mutants. We analyzed thetranscriptomic dynamics in nga1-1 under 1-h dehydration-stressconditions and nonstress conditions by RNA sequencing (Data-sets S1–S3). In the wild-type plants, 887 and 238 genes wereinduced and repressed, respectively, more than fourfold in re-sponse to dehydration stress [wild type (control) vs. wild type(dehydration)] (P < 0.05; Fig. 6 A and B). In the nga1-1 mutant,155 genes and 451 genes were down-regulated under nonstress

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Fig. 2. Transactivation assays and EMSAs revealed that the NGA familyproteins activated and directly bound to the NCED3 promoter in vitro. (A)Schematic diagram of the reporter and effector constructs. (B and C)Transactivation assays of the NGA family proteins using the various NCED3promoters as reporters. The error bars indicate the SD from three replicatesamples. Asterisks indicate statistically significant differences of reporteractivities from the vector control. *P < 0.05 (Bonferroni-corrected Student’st test). The 35S:ELUC plasmid was also cotransfected in each experiment asan internal control. (B) Reporters of 3, 2.4, and 1 kbp. (C) Reporters of 198-,

161-bp, and 3-kbp mNBE. (D and E) Schematic diagrams of the probes of theNCED3 promoters and EMSA using the recombinant NGA2 (D) and NGA1 (E)protein. The migration positions of the protein–DNA complexes are repre-sented by arrows. Mutated nucleotides in each probe are underlined, and anorange box indicates the position of the NBE. Incubation with 100- or 1,000-fold competitors was performed in the presence of the recombinantNGA1 proteins to confirm specific binding to the probes.

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[wild type (control) vs. nga1-1 (control)] or dehydration-stressconditions [wild type (dehydration) vs. nga1-1 (dehydration)],respectively, more than twofold of the level in the wild-typeplants (P < 0.05; Fig. 6A). A total of 248 (245 + 3) genesamong the 451 down-regulated genes in nga1-1 during de-hydration stress were dehydration-inducible genes, whereas10 (7 + 3) genes were dehydration-inducible genes under nonstressconditions (Fig. 6A). Among the 451 down-regulated genes, asignificant proportion of them were dehydration-inducible genes(55%; 248/451) relative to all Arabidopsis genes (3%; 887/27,417;P < 0.0001, Fisher’s exact test). In comparison with a previouspaper on the transcriptomic dynamics in nc3-2 subjected todrought stress (21), 79 genes among the 451 down-regulatedgenes in nga1-1 during dehydration stress were also down-

regulated in nc3-2 under drought stress. Among the 451 down-regulated genes, a significant proportion of them were NCED3-dependent genes (18%; 79/451) relative to all Arabidopsis genes(3%; 759/27,417; P < 0.001, Fisher’s exact test). These datasuggested that NGA1 is necessary for the dehydration-inducibletranscriptomic changes. Many dehydration-stress-repressedgenes (118 genes) were up-regulated in nga1-1 during de-hydration stress among all of the up-regulated genes duringdehydration stress (323 genes; Fig. 6B). This also suggested thatall of the dehydration-stress responses were attenuated in nga1-1,including stress-specific gene suppression. The expression levels ofthree down-regulated genes in nga1-1 during dehydration stress wereconfirmed by quantitative RT-PCR (qRT-PCR;MAPKKK18, ABR1,and At3g55940) (Fig. 6C). These three genes were dehydration-stress-inducible, were reported to be involved in drought-stress re-sponses (27–29), and have both NBE and G box motifs in theirpromoters (Dataset S3).A metaprofile analysis was performed by using a public tran-

scriptome database (Genevestigator) with the top 300 down- orup-regulated genes in nga1-1 during dehydration stress. Manygenes down-regulated in nga1-1 were suggested to be induced byABA and dehydration stress treatment (SI Appendix, Fig. S6A).The genes that were up-regulated in nga1-1 during dehydrationstress exhibited almost the opposite trend, and especially manyof the up-regulated genes in nga1-1 were salt-stress-repressedgenes (SI Appendix, Fig. S6B). To characterize the genes whoseexpression levels were affected by the knockout of NGA1, wecompared the frequency of the hexamer sequences in the pro-moters of the top 300 genes that were up- or down-regulated innga1-1 during dehydration stress with their normalized fre-quencies in the promoters of the entire Arabidopsis genome. TheZ-scores of all hexamer sequences are presented in the scatterplots. The results revealed that the hexamers related to theABRE sequences that contain the G-box core sequence (ACGT)were highly enriched on the promoters of the down-regulatedgenes in nga1-1 (SI Appendix, Fig. S6C). This is consistent withthe suppressed expression of NCED3 and other ABA-responsivegenes in nga1-1. Meanwhile, these hexamer sequences were notabundant on the promoters of the up-regulated genes in nga1-1(SI Appendix, Fig. S6D). The NBE sequence was not enriched onthe promoters of the down-regulated genes (CACTTG; Zscore = 1.44, P = 0.075), presumably because the large effect ofthe suppressed dehydration-stress responses in ABA signalingmight mask the enrichment of fewer down-regulated genes thatcontain the NBE on their promoters. These suggested thatNGA1 should affect the whole transcriptome by regulating thelimited numbers of its target genes including NCED3. We per-formed Gene Ontology (GO) analyses using the public GO da-tabase agriGO (Version 2.0) (30). The top 300 genes that weredown- or up-regulated in nga1-1 during dehydration stress wereanalyzed compared with the entire whole genome. Genesencoding proteins involved in ABA (P < 0.005) and reproductivestructure development (P < 0.00005) were overrepresentedamong the down-regulated genes in nga1-1 (SI Appendix, Fig. S6E and G). This result is consistent with the function of NGA1 asan activator of NCED3 during dehydration stress and in theregulation of reproductive growth as reported (15). Among theup-regulated genes in nga1-1, genes encoding proteins involvedin growth related-hormone; auxin, cytokinin, and gibberellinwere overrepresented (SI Appendix, Fig. S6F). These data sug-gest that NGA1 inhibited plant growth during dehydration stressby activating ABA biosynthesis.

NGA1 Is Posttranslationally Regulated Through ABA-IndependentPathways During Dehydration Stress. To analyze the binding ac-tivity of the NGA protein in planta by chromatin immunopre-cipitation (ChIP) assays, the transgenic plants overexpressingNGA1 fused to GFP were generated (35S:GFP-NGA1-a, -b, and -c).

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GFP-NGA1 was localized in the nuclei (SI Appendix, Fig. S7A).We found that the binding activity of NGA1 to the NCED3promoter region was enhanced by the dehydration stress (Fig.7A). In these transgenic plants, protein accumulation of GFP-NGA1 increased due to dehydration stress (Fig. 7 B and C), butmRNA levels were not affected (SI Appendix, Fig. S7B). Treat-ment of MG132, a 26S proteasome inhibitor, also led to accu-mulation of GFP-NGA1 (Fig. 7 D and E). Importantly, theaccumulation of GFP-NGA1 was observed in the ABA-deficientmutant aba2-2 during dehydration stress (Fig. 7 F and G), andABA treatment did not affect protein accumulation (SI Appen-dix, Fig. S7C). These results suggest that NGA1 was degraded bya 26S proteasome under control conditions and that NGA1 wasposttranslationally stabilized in an ABA-independent manner.The NGA proteins have been shown to be modified by bothphosphorylation and O-linked N-acetylglucosaminylation (O-GlcNAcylation) (31), and NGA1 is believed to have six phos-phorylation sites (T80, T83, S160, S289, S290, and S291) andthree possible O-GlcNAcylation sites (T3, S6, or T8) (31). Wefound that modification of these sites is conserved among all orpart of the other NGA family proteins (SI Appendix, Fig. S7D).To determine whether these modifications affect the transcrip-tional activity or protein stability of NGA1, transactivation assaysusing Arabidopsis mesophyll protoplasts were performed. Amongthe phosphorylated amino acids, a S160D phospho-mimic mu-tation significantly increased endogenous NCED3 expression(Fig. 7H), and additional T80D and S83D phospho-mimic mu-tations further enhanced expression (Fig. 7H). However, alaninemutations at T80, S83, and S160 suppressed the NCED3 ex-pression (Fig. 7I). These data suggest that the phosphorylation ofthese three amino acids can enhance the transcriptional activityof NGA1 for NCED3 during dehydration stress. However, nodifferences in protein accumulation were found for the mutatedNGA1 proteins (SI Appendix, Fig. S7E), implying that there aredifferent activating mechanisms of NGA1 involved in regulation

of protein stability and transcriptional activity. Any combination,including phospho-mimic or alanine mutations of the C-terminalregions (S289, S290, and S291), repressed the transcriptionalactivity of NGA1 (Fig. 7 H and I). Mutations at the possible O-GlcNAcylated amino acids also caused negative effects on thetranscriptional activity of NGA1 (SI Appendix, Fig. S7F). Theseresults suggest that three serine amino acids at positions 289,290, and 291 and O-GlcNAcylation are involved in regulatingtranscriptional activity of NGA1 during dehydration stress andthat complex regulatory systems of NGA1 protein stability andtranscriptional activity likely exist.

DiscussionRecent studies of ABA signaling have revealed the detailedmolecular mechanisms in which the varieties of proteins (trans-porter, receptor, signaling regulator, transcription factor, etc.)function to activate ABA responses in plants during droughtstress (4–8, 32); however, the factors that are involved in theearly response to dehydration stress to activate NCED3 for ABAaccumulation need to be further explored. The present studyidentified a transcription factor, NGA1, which activated NCED3during dehydration stress, through a unique screening method(Fig. 1). Among the NGA transcription factors, single-knockoutmutants of NGA1 exhibited not only suppressed expression ofNCED3 during dehydration stress (Fig. 5F), but also reducedaccumulation of ABA (Fig. 5G). This NGA1 transcription factorexhibited a positive effect on the reporter activity driven by theNCED3 promoter in Arabidopsis mesophyll protoplasts (Fig. 2 Aand B) and directly bound to the NCED3 promoter in vitro (Fig.2E) and in vivo (Fig. 7A). These results clearly suggest thatNGA1 is a transcriptional activator for the induction of NCED3under dehydration-stress conditions. Moreover, the transcriptomicchanges during dehydration stress (Fig. 6A) and decreased drought-stress tolerance (Fig. 5B) in nga1-1 revealed that NGA1 plays certain

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critical roles in the dehydration-stress responses and survivalduring drought stress in plants.The present study identifies posttranslational activating

mechanisms of NGA1 during dehydration stress. The NGA1proteins appear to be degraded through the 26S proteasomepathways under nonstress conditions (Fig. 7D), but they arestabilized by dehydration stress (Fig. 7B). Importantly, proteinstabilization of NGA1 during dehydration stress was detectedeven in the ABA-deficient mutant (Fig. 7F). These results sug-gest that the NGA1 protein is posttranslationally regulatedthrough protein stabilization in an ABA-independent pathway inthe early responses to drought stress (33) (SI Appendix, Fig. S8).Moreover, the transactivation assays using the mutated NGA1suggest the presence of complex mechanisms of regulatingtranscriptional activity of NGA1. Increased transcriptional ac-tivity of the phospho-mimic mutations of NGA1 and the adverseeffects of the alanine mutations at positions T80, S83, and 160Ssuggest that phosphorylation of these amino acids is involved inactivating mechanisms of NGA1 during drought stress. However,there was no effect of these amino acid mutations on proteinaccumulation in protoplasts (SI Appendix, Fig. S7E), which im-plies that the increase in transcriptional activity by phosphory-lation at position T80, S83, and 160S might be due to differentactivating mechanisms from those required for protein stabili-zation of NGA1. Decreased transcriptional activation due tomutations at possible O-GlcNAcylated amino acids suggests thatO-GlcNAcylation also might be involved in the activation ofNGA1 (SI Appendix, Fig. S7F).In the present study, an important cis-acting element NBE

(CACTTG) in the 5′ UTR region of the NCED3 promoter wasisolated. The NGA proteins bound to the NBE in vitro (Fig. 2 Dand E), and activated the NCED3 promoter through NBE inprotoplasts (Fig. 2 B and C). Mutation of NBE in the 3-kb pro-moter of NCED3 decreased the reporter activity in the transgenicplants (Fig. 3). These findings strongly suggest that the NBE se-

quence is necessary for activation of NCED3 during dehydrationstress by NGA. Interestingly, the NBE is located in the 5′ UTRregion of NCED3. Recently, several reports have demonstratedthat genomic regions other than promoters (intron or 3′-nontranscribed intergenic region) play important roles in thetranscriptional activation of genes (34, 35); however, few papershave indicated the importance of cis-acting elements in the 5′UTR region for transcriptional activation (36). It was alsoreported that a G-box sequence located ∼2.3 kb upstream fromthe translational start site was necessary for the induction ofNCED3 (Fig. 3) (12, 13), implying that any transcription factorsbinding to the G-box coordinately activate NCED3 collectivelywith NGA1 (SI Appendix, Fig. S8). Analysis of the transcriptionalmechanisms of NCED3 is important not only for elucidating theearly drought responses in plants, but also for studying and pro-posing an intriguing model of unique transcriptional mechanisms.Moreover, future studies will be necessary to identify the noveltranscription factors that bind to the long-range enhancer G-box.Previous reports revealed that NGAs also are involved in plant

growth. Marginal growth of leaves is regulated by NGAs and theTCP transcription factors by restricting meristem activity in leafdistal regions (16). This study revealed that NGA and TCP havenegative effects on the expression of KANADI, which encodes atranscription factor involved in the regulation of adaxial–abaxialpolarity (16). Other studies reported that KANADI has negativeeffects on expression of genes involved in ABA signaling (37) andthat one of the KANADI-regulating genes encoded a transcriptionfactor ABIG1 (ABA INSENSITIVE GROWTH 1) that was nec-essary for ABA-mediated growth inhibition (38). GO analysis in thepresent study suggested that NGA1 is involved in growth inhibitionduring dehydration stress (SI Appendix, Fig. S6F). Our findings thuspresent a possibility that the NGA proteins control plant growth ororgan polarity during drought stress through ABA biosynthesis.In conclusion, the present study revealed that: (i) NGA1 is a

transcriptional activator of NCED3; (ii) NGA1 regulates NCED3

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Fig. 5. The nga1-1 single mutants revealed de-creased expression of NCED and were sensitive todrought stress. (A) Expression levels ofNGA1 in nga1-1and complemented lines. The error bars indicate theSD from triplicate technical repeats. (B and C) Drought-stress tolerance test of nga1-1 and complemented lines.(B) Images of plants before and after drought and afterrewatering. Plants grown onMS medium for 2 wk weretransferred to soil and grown for 2 d. Water waswithheld for 15 d. (Scale bars: 1 cm.) (C) Survival ratesof plants after rewatering. The error bars indicate theSD from five replicates (n = 35 in total). Asterisk indi-cates significant differences from the wild-type plants.*P < 0.01 (Bonferroni-corrected Student’s t test).(D) Expression levels of NCED3 and other dehydration-inducible genes during dehydration stress in nga1-1and complemented lines. Plants grown on MS me-dium for 2 wk were treated with dehydration stress.The error bars indicate the SD from triplicate technicalrepeats. The letters above the bars indicate significantdifferences between the plant lines at each time point(P < 0.05, according to Tukey’s multiple range test).(E) ABA contents of nga1-1 and complemented linesduring nonstress (Control) and dehydration stress(Dry). Plants grown on MS medium for 2 wk weretreated with dehydration stress for 3 h. The error barsindicate the SD from at least four replicate samples.Asterisk indicates significant differences from thewild-type plants. *P < 0.01 (Bonferroni-correctedStudent’s t test).

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through a cis-element NBE in the 5′ UTR in the promoter;and (iii) NGA1 is posttranslationally stabilized in an ABA-independent manner (SI Appendix, Fig. S8). These findings pro-vide mechanisms for the early plant response during droughtstress before ABA accumulation. However, additional experi-ments are necessary to elucidate the detailed molecular mecha-nisms, especially with respect to the posttranslational activationmechanisms of NGA1. We revealed that mutations in putativemodified amino acids (31) had positive and negative effects ontranscriptional activity of NGA1 (Fig. 7 H and I). Future inves-tigations should determine how these modifications affect themolecular function of NGA1 and what factors regulate themodification and protein stabilization of NGA1 under control anddehydration-stress conditions. A recent study reported that a smallpeptide CLAVATA3/EMBRYO-SURROUNDING REGION-RELATED 25 (CLE25) induced the NCED3 expression duringdehydration stress (39). It is also an intriguing question whetherand how the CLE25-dependent signaling cascade is involved inthe activation mechanisms of NGA1.

Materials and MethodsPlant Materials and Growth Conditions. A. thaliana ecotype Columbia (Col-0),Landsberg erecta (Ler-0), or Nossen (Nos-0) plants were grown on germi-nation medium (GM) agar plates and on soil at 22 °C under a 16-h photo-period at a photon flux density of 40 μmol m−2·s−1 as described (40).Knockout mutants of the NGA family genes were provided by John PaulAlvarez, Monash University, Clayton, VIC, Australia (14) (nga2-1, CS110052;nga3-1, CS872330; and nga4-1, N16752). Knockout mutants of NCED3 (nc3-2)and ABA2 (aba2-2) were described in previous studies (21, 41). Transgenicplants were generated through the floral dip method by using Agro-bacterium tumefaciens GV3101 (pMP90) cells as described (42). More thantwo independent transgenic lines were analyzed for each experiment.

Screen for Sucrose-Insensitive Lines in a Plant Library. Transgenic plants harboringthe transcription factors fused to a chimeric repressor SRDX were reported (17).Sterilized seeds of the transgenic plants were sown on Murashige and Skoog(MS) medium (0.8% agar) supplemented with 300 mM sucrose. The plantswere grown under control conditions for 2 wk, and phenotypic differenceswere analyzed.

Stress and Chemical Treatment. For the dehydration-stress treatment, 14-d-old plants grown on solid GM agar plates were transferred on parafilm.For the drought-stress treatment, 14-d-old plants grown on solid GM agarplates were transferred to soil and grown for 2 d under control conditions.Water supply was withheld until plants withered. During periods ofdrought-stress treatment, pot weights were measured, and relative watercontent (RWC) was calculated from the dried pot weight and adjustedamong plant lines to ensure equal intensity of drought stress. RWC wascalculated as {(pot weight during stress) − (dried pot weight)}/{(initial potweight) − (dried pot weight)} × 100. Survival rates of plants were calculated7 d after rewatering under control conditions. For RNA extraction and GUSstaining using germinating seeds, sterilized seeds were sown in one layer offilter paper containing 2 mL of liquid GM with or without 300 mM sucrose in60-mm plastic Petri dishes. The seeds were cold stratified at 4 °C for 3 d in thedark, and then seeds were transferred to the light at 22 °C. For ABA orMG132 treatment, 14-d-old plants were transferred to a Petri dish filled withdistilled water containing 100 μM ABA (Sigma-Aldrich) or 100 μM MG132(Calbiochem).

RNA Preparation and qRT-PCR. The total RNA from seedlings was isolated withRNAiso plus (TaKaRa) according to the supplier’s instructions. Total RNA fromseeds was isolated as described (43). The cDNA was synthesized by using Su-perScript IV VILO (Thermo Fisher Scientific), and qRT-PCR was performed byusing an Applied Biosystems 7500 Fast Real-Time PCR system and Fast SYBRGreen (Thermo Fisher Scientific), as described in the supplier’s instructions.The obtained values were normalized according to the amounts of ACT2.

Measurement of Leaf Temperature and ABA Content. Leaf temperature wasmeasured by using a FLIR T640 infrared camera and FLIR tool software. Thetemperature of three spots in each plant was calculated by using seven plantsof each line. Extraction of ABA from plants was performed as described (44).Parameters for liquid chromatography tandem mass spectrometry analysisto measure the endogenous ABA and internal control D6-ABA (Icon Isotepes)by Triple TOF 5600+ (SCIEX) are described in SI Appendix, Tables S4 and S5.

Sequence Alignment and Phylogenetic Analysis. The alignment of the peptidesequences and the construction of phylogenetic trees were performed asdescribed (45). The peptide sequences of B3-type transcription factors wereobtained through Phytozome 12 (https://phytozome.jgi.doe.gov/pz/portal.html) using PFAM ID PF02362.

Transient Expression with Arabidopsis Mesophyll Protoplasts and TransactivationAssays. Isolation and transient transformation of Arabidopsis mesophyll pro-toplasts was performed as described (46), and transactivation assays were per-formed as described (45) by using Perkin-Elmer Multimode Plate Reader EnSpirefor measurement of the luciferase luminescence and methylumbelliferonefluorescence. For transfection, 3, 2, and 5 μg of the reporter, internal control,and effector plasmids were used.

GFP Fluorescence Observation and Histochemical GUS Staining. GFP fluores-cence was observed by using a confocal laser scanning microscope (OlympusFLUOVIEWFV1000).HistochemicalGUS stainingwasperformedasdescribed (45).

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Fig. 6. Transcriptome analysis revealed the essential roles of NGA1 duringdehydration stress. (A) Venn diagram comparing the dehydration-induciblegenes in the wild-type plants and down-regulated genes in nga1-1 undernonstress and dehydration-stress conditions. The total numbers of each setof genes are presented in parentheses. (B) Venn diagram comparing thedehydration-stress-repressed genes in the wild-type plants and up-regulatedgenes in nga1-1 under nonstress and dehydration-stress conditions. The totalnumbers of each set of genes are given in parentheses. (C) Expression levelsof the dehydration-inducible genes identified by RNA-seq in nga1-1 andcomplemented lines during dehydration stress. The error bars indicate theSD from triplicate technical repeats. The letters above the bars indicatesignificant differences between the plant lines at each time point (P < 0.05,according to Tukey’s multiple range test).

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EMSA and ChIP Assay. EMSA was performed as described (47) with minormodifications. A mixture of 10,000 dpm of 32P-labeled probe and 1 μg wasincubated in binding buffer [25 mM Hepes–KOH (pH 7.9), 40 mM KCl, 1 mMDTT, 1 mM ETDA, 10% glycerol, 1% BSA, and 1 mg of poly(dI-dC)] for30 min at room temperature. The reaction mixtures were resolved byelectrophoresis through a 6% or 8% polyacrylamide gel in 0.5× Tris–bo-rate–EDTA buffer at 100 V for 50 min. ChIP assays were performed by usingan EpiQuik Plant ChIP kit (Epigenetek) according to the user guide. Onegram of plants with or without stress treatment was collected. To immu-noprecipitate the DNA–protein complex, the polyclonal anti-GFP antibody(48) was used.

RNA Sequencing and Data Analysis. CDNA libraries were constructed by usingthe TruSeq RNA Sample Preparation Kit (Version 2; Illumina), and the li-braries were sequenced by NextSeq 500 (Illumina). The produced bcl fileswere converted to fastq files by bcl2fastq (Illumina). The reads were ana-lyzed as described (49). The data were deposited into the DNA Data Bank ofJapan (accession no. DRA006360). Metaprofile analysis was performed byusing the public transcriptome database Genevestigator (https://geneves-tigator.com/gv/) (50), and overrepresentation analysis of hexamers on thepromoters of sets of genes by RNA-sequencing (RNA-seq) data were per-formed as described (51) using 1-kb upstream sequences from the trans-lational start sties obtained through Phytozome (Version 12; https://phytozome.jgi.doe.gov/pz/portal.html). GO analysis was performed by us-ing the public GO database agriGO (Version 2.0; systemsbiology.cau.edu.

cn/agriGOv2/) (30) and comparing to the TAIR10 whole-genome sequenceas a reference.

Protein Immunoblot Analysis. Total protein was extracted from seedlings andprotoplasts by using protein extraction buffer (8 M urea, 5% 2-mercaptoe-thanol, 2 mM EDTA, 1% SDS, and50 mM Tris·HCl, pH 6.8) and centrifuged at20,000 × g for 10 min at room temperature. The supernatant was boiled at95 °C for 3 min. The resultant extracts, which corresponded to a fresh weight of3 mg of seedling or 1 × 104 protoplasts, were separated by SDS-polyacrylamidegel electrophoresis. Immunoblotting was performed by using a polyclonalanti-GFP antibody (48) as the primary antibody and goat anti-Rabbit IgGperoxidase-conjugate (Thermo Fisher Scientific) as a secondary antibody. Thesignals were developed by SuperSignal West Dura Extended Duration Substrate(Thermo Fisher Scientific) according to the manufacturer’s protocol and de-tected with an image analyzer (ChemiDoc MP; Bio-Rad). Ponceau S (APROSCIENCE) staining was performed according to the manufacturer’s instructions.Band intensity was measured by using Image Lab Software (Bio-Rad) andnormalized to the intensity of the rubisco large subunit (rbcL).

ACKNOWLEDGMENTS. We thank Dr. John Paul Alvarez and Dr. Yuval Eshed forproviding materials for the nga knockout mutants; and Ms. Fuyuko Shimoda,Ms. SahoMizukado, Ms. Saya Kikuchi, Ms. Hiroko Kobayashi, Ms. KumikoMatsuo,Ms. Michie Etoh, Ms. Ayami Furuta, and Ms. Tomomi Shinagawa for their excel-lent technical assistance. This work was supported by Grant-in-Aid for ScientificResearch for Young Scientists (B) 16K21626 (to H.S.) and Grants-in-Aid for Scien-tific Research on Innovative Areas JP16H01475 and JP18H04792 (to F.T.).

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35S:GFP-NGA1-a (cont)35S:GFP-NGA1-a (dry)35S:GFP (cont)35S:GFP (dry)

Promoter CDS

Promoter CDS 500 bp

NCED3 CDS5’ UTR 3’ UTR

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35S:GFP-NGA1a b c

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35S:GFP-NGA1/aba2-2

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(S289, S290, S291)A

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NGA1 wt

Vector control

(T80, S83, S160)A

all-A

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*

Relative expression of NCED3

Moc

kM

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GM

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Ponceau S

35S:GFP-NGA1a b c

D

0 5 10 15 20Relative expression

of NCED3

(S289, S290, S291)DS160D

(T80, S83)DNGA1 wt

Vector control

(T80, S83, S160)D(S160, S289, S290, S291)D

(T80, S83, S289, S290, S291)Dall-D

*

****

****

**

**H

Fig. 7. NGA1 is posttranslationally regulated during dehydration stress. (A) ChIP assays of the NGA1 proteins. A schematic diagram displays the amplifiedregion of the NCED3 genome by qRT-PCR. Plants grown on MS medium for 2 wk were treated with dehydration stress for 1 h. Yellow and closed boxesindicate the coding sequence (CDS) regions and UTRs of NCED3, respectively. Error bars indicate the SD from six technical replicates. Asterisks indicate sig-nificant differences from control plants. *P < 0.01 (Bonferroni-corrected Student’s t test). (B–G) Accumulation levels of the GFP-NGA1 protein during de-hydration stress (B, C, F, and G) and with treatment of MG132, a 26S proteasome inhibitor (D and E) in the 35S:GFP-NGA1 (lines a, b, and c) (B–E) and 35S:GFP-NGA1/aba2-2 (lines a and b) (F and G). Immunoblotting (B, D, and F) and relative band intensity of GFP-NGA1 (C, E, and G) are shown. Plants grown on MSmedium for 2 wk were treated with dehydration stress for 1 h (Dry) or 100 μM MG132 for 4 h (MG). (B, D, and F) Total extracted proteins were analyzed byimmunoblotting with an anti-GFP antibody (Upper), and the rubisco large subunits (rbcL) were detected by Ponceau S staining (Lower). Relative band in-tensity of the GFP-NGA1 proteins from the transgenic line a under control condition was set to 1. The error bars indicate the SD from three replicate samples.Asterisks indicate significant differences between conditions. *P < 0.01 (Bonferroni-corrected Student’s t test). (H and I) Relative expression levels of theendogenous NCED3 in Arabidopsis mesophyll protoplasts transfected with wild-type and mutated NGA1. The effects of mutations to aspartic acid (H) andalanine (I) are shown. The serine and threonine in the brackets show the mutated positions, and “all-D” and “all-A” indicate mutations in the six amino acids(T80, S83, S160, S289, S290, and S291). Relative expression levels of NCED3 among protoplasts transfected with the wild-type and mutated NGA1 werenormalized to expression levels in the wild-type and mutated NGA1. The error bars indicate the SD from three replicate samples. Asterisks indicate significantdifferences in relative expression from protoplasts transfected with the wild-type NGA1. *P < 0.05; **P < 0.01 (Bonferroni-corrected Student’s t test).

E11186 | www.pnas.org/cgi/doi/10.1073/pnas.1811491115 Sato et al.

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