RESEARCH ARTICLE

The ARF2–ANT–COR15A gene cascade regulates ABA-signaling-mediated resistance of large seeds to drought in ArabidopsisLai-Sheng Meng1,*, Zhi-Bo Wang2, Shun-Qiao Yao1 and Aizhong Liu1,*

ABSTRACTSeedlings of large-seededplants are considered tobeable towithstandabiotic stresses efficiently. Themolecularmechanisms that underlie theinvolved signaling crosstalk between the large-seeded trait and abiotictolerance are, however, largely unknown. Here, we demonstrate themolecular link that integrates plant abscisic acid (ABA) responses todrought stress into the regulation of seed mass. Both loss-of-functionmutants of the Auxin Response Factor 2 (ARF2 encoding atranscription factor) and lines overexpressing AINTEGUMENTA(ANT; a transcription factor) under the 35S promoter exhibited largeseed and drought-tolerant phenotypes as a result of abnormal ABA–auxin crosstalk signaling pathways in Arabidopsis. The target geneCOLD-REGULATED15A (COR15a) was identified as participating inthe regulation of seed development with ABA signaling through anegative regulationmechanism that ismediated byANT. Themolecularand genetic evidencepresented indicate that ARF2, ANTandCOR15Aform an ABA-mediated signaling pathway to link modulation of seedmass with drought tolerance. These observations indicate that theARF2 transcription factor servesasamolecular link that integratesplantABA responses to drought stress into the regulation of seed mass.

KEY WORDS: Auxin response factor 2, ARF2, AINTEGUMENTA,ANT, COLD-REGULATED15A, COR15A, Seed mass, Droughttolerance, ABA signal

INTRODUCTIONSeed size in higher plants is an important trait with respect toecology and agriculture. Generally, larger seeds are less easilydispersed, but offer the germinating seedling a larger supply ofnutrients, thus increasing its competitiveness during seedlingestablishment and tolerance to adverse environmental stresses(Westoby et al., 1992). By contrast, smaller seeds are more easilycolonized, thus giving rise to population dispersal and spread. Asfor the mother plant, nutrient resources for producing seedsgenerally are limited, thus causing a trade-off between the numberand size of the seeds produced (Venable, 1992; Ohto et al., 2005;Coomes and Grubb, 2003; Orsi and Tanksley, 2009). In agriculture,increasing seed size has been a crucial contributor to the yieldincreases in crop plants during domestication (Shomura et al.,2008).The seed size depends on the development of the embryo,

endosperm and seed coat tissues, which are derived from distinct

cells of the ovule and have distinct complements of maternal andpaternal genomes. The embryo constitutes the major volume of amature seed in Arabidopsis, and the changes in seed mass arereflected in the size of the embryos. Thus, the mature seed size islargely affected by both the embryo cell number and cell size inArabidopsis. Furthermore, seed size is often able to alter in anintraspecific manner in response to environmental cues (Ohtoet al., 2005). Previous studies have found that the loss-of-functionmutants of apetala2 (ap2), auxin response factor2 (arf2), shorthypocotyl under blue1 (shb1) and da1-1 give an increased seed-mass phenotype in Arabidopsis through enlargement of theembryonic cell size or an increase in the embryonic cell number(Ohto et al., 2005; Schruff et al., 2006; Zhou et al., 2009;Li et al., 2008). By contrast, the mutants of miniseed3 and iku2exhibit a smaller seed mass owing to a decrease in cell number,but not cell size (Luo et al., 2005). However, relatively little isknown about the specific regulatory mechanisms that underliethese genes that mediate seed development in response toenvironmental inducers.

Phytohormone abscisic acid (ABA) is broadly involved indevelopmental regulation and various stress-related responses inhigher plants. The germinating seedlings at different developmentalstages exhibit different physiological responses to ABA signals inArabidopsis, and the ABA-deficient mutants have significantlyreduced cell vigor (Wang et al., 2011). Also, differentconcentrations of ABA can regulate the root and stem growth byboosting or suppressing cell proliferation and differentiation duringArabidopsis seedling development (Wang et al., 2011; Zhanget al., 2010). In addition, a number of genotypes with mutations inDNA replication machinery display a hypersensitive response toABA during seed germination and seedling growth (Yin et al.,2009), strongly implying that ABA signaling suppresses cellproliferation by regulating DNA-replication-related proteins. Inparticular, ABA is a key regulator of plant responses toenvironmental cues, such as drought, cold and salt stresses (Guoet al., 2004). Usually, these environmental stresses induce ABAaccumulation, thereby triggering many physiological actions inresponse to these stresses (Lim et al., 2007). For example, underwater-deprived conditions, ABA can induce stomatal closure,leading to a decrease of transpirational water loss (Assmann andWang, 2001).

The transcription factor Auxin Response Factor 2 (ARF2),originally identified as an ARF1-binding protein (thereforeformerly known as ARF1-BP), binds to the AuxREs (TGTCTC)cis-element in the promoter region of auxin-regulated genes(Ulmasov et al., 1997). ARF2 has been identified as a regulatorthat is involved in negatively regulating ABA-mediated seedgermination and primary root growth by binding to homeodomaingene HB33 (Wang et al., 2011). The lost-of-function mutant arf2exhibits a bigger seed size phenotype, compared to the wild type,because of the extra cell proliferation (Schruff et al., 2006). At theReceived 6 March 2015; Accepted 14 September 2015

1Key Laboratory of Economic Plants and Biotechnology, Kunming Institute ofBotany, Chinese Academy of Sciences, 132 Lanhei Road, Kunming 650201,People’s Republic of China. 2School of Bioengineering and Biotechnology,Tianshui Normal University, TianShui City 741001, People’s Republic of China.

*Authors for correspondence (menglaisheng@mail.kib.ac.cn;liuaizhong@mail.kib.ac.cn)

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same time, studies have revealed that the transcription factorAINTEGUMENTA (ANT), a member of the AP2-domain family, isinvolved in mediating cell proliferation and growth control (Klucheret al., 1996; Mizukami and Fischer, 2000; Nole-Wilson and Krizek,2000; Krizek, 2003). The overexpressing transformants of the ANTgene also display a phenotype of larger seed size, compared tocontrols (Mizukami and Fischer, 2000).Compounding evidence suggests that seedlings that have been

germinated from larger seeds have an increased tolerance to abioticstresses compared to seedlings from smaller seeds (Coomes andGrubb, 2003; Ohto et al., 2005; Moles et al., 2005; Orsi andTanksley, 2009). However, the molecular mechanisms underlyingwhy large-seeded seedlings give stronger tolerance to abioticstresses are not fully understood. In this study, we found that ARF2mediates signaling crosstalk between the drought stress responseand seed development by negatively regulating ANT. Furthermore,we identified that the target gene COR15A is directly regulated byANT. Combining the molecular and genetic evidence, we show forthe first time that the ARF2–ANT–COR15A pathway forms theABA-mediated signal cascade that regulates seed mass and droughttolerance. This study provides new evidence to enhanceunderstanding of why large-seeded seedlings exhibit strongertolerance to drought.

RESULTSLarge seed mass and drought tolerance of arf2 mutants arecaused by abnormal ABA signalingThe mature seeds of arf2 mutants were observed to be obviouslybigger in size than their wild-type counterparts (Fig. 1B,D;Fig. S1A,C), consistent with a previous report (Schruff et al.,2006). Cytological observations showed that the average areas ofthe cotyledon embryo and the cotyledon embryo cell in the arf2mutant were ∼1.6 and ∼1.3 times larger than those of the wild type,respectively (Fig. 1A,C; Fig. S1B,D); the corresponding ratio of thearea of the embryo to that of the cell was 1.23 (1.6/1.3=1.23),suggesting that the overall enlargement of arf2 mutant embryosmight result from both the augmentation of cell size and the increasein cell number. Furthermore, the abnormal size of the cotyledons ofthe arf2-6 mutant could be restored on Murashige and Skoog (MS)medium with 3.0 µm ABA (see Fig. 1E). And, cytologicalobservations showed that the average size of the cotyledon cellsin the arf2-6 mutant was smaller than that of wild type upontreatment with 3.0 µm ABA (see Fig. 1E; Fig. S2). Theseobservations strongly imply that the function of ARF2 in seeddevelopment might be involved in the ABA signaling pathway.Using the ELISA method for quantifying the endogenous ABAcontent in developing seeds of the arf2-6 mutant and wild type, we

Fig. 1. Several mutants show a change in seed size as a result of increased embryo size. (A) Representative mature embryos of the indicated seedgenotypes were isolated and observed. Panels in a–e are magnified in f–j. Scale bars: 100 μm (a–e); 10 μm (f–j). cor15a is SALK_054513. (B) Representativemature dry seeds of the indicated plants. Scale bar: 0.1 mm (a–f ). cor15a is SALK_054513. ant-KO is SALK_022770. (C) Bar graph exhibiting the difference in theembryo area and embryo cell area between the indicated seeds. The data from Col-0 are set as 1.0. Data are means±s.d. from at least five independentlypropagated Col-0 and mutant lines (n>10 for embryo area; n>40 for embryo cell area. **P<0.01; *P<0.05). (D) Bar graph exhibiting the difference in seed areabetween the indicated seeds. The data from Col-0 are set as 1.0. Data are means±s.d. from at least five independently propagated Col-0 and mutant lines (n>20;**P<0.01; *P<0.05). (E) Bar graph exhibiting the difference in cotyledon area and cotyledon cell area between 8-day-old indicated seedlings grown onMSmediumwith 3.0 µM ABA. The data from Col-0 are set as 1.0. Data are means±s.d. from at least five independently propagated Col-0 and mutant lines (n>10; **P<0.01;*P<0.05).

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found that the endogenous ABA content in the arf2-6 mutant wassignificantly higher (see Fig. 4D), suggesting that the large seeds ofthe arf2-6 mutant are likely to be the result of the abnormal ABAsignals.Generally, ABA signaling is one of main factors that induces

plant drought tolerance. To investigate the response of arf2mutantsto drought stress, the arf2 and wild-type plants were grown for twoweeks in soil and subsequently subjected to water deprivation forthe indicated number of days. Initial water-deprivation experimentsshowed that the leaves of both arf2 and wild-type plants remainedgreen (Fig. 2A; Fig. S3A). Under water deprivation for the givennumber of days, most wild-type plants dried up and died, whereasthe arf2 plants remained turgid and retained their green leaves (seeFig. 2A and B; Fig. S3A). Consistent with these results, the

detached leaves of the arf2 plants lost water more slowly than thoseof the wild-type plants (Fig. 2C). Furthermore, the enhanced water-deficit survival of arf2-6 plants was closely associated with theircapacity to maintain higher leaf relative water content (RWC) thanthe wild type at ∼16% soil water content (SWC) (Fig. S3B). Thestomatal closing of the leaf surfaces in the arf2 plants was obviouslyincreased under treatment with ABA (Fig. 2D). This finding wasconsistent with the expression of β-glucuronidase under the ARF2promoter (ProARF2:GUS) on the stomata (Fig. 3F), and seedgermination and seedling root growth responded in a more sensitivemanner to ABA in the arf2 plants (Wang et al., 2011). Uponexposure to 10% polyethylene glycol (PEG) 6000, commonly usedto mimic drought tolerance under controlled conditions, the ABAcontent became significantly higher in the arf2-6 mutant, as shown

Fig. 2. arf2-7mutant and 35S:ANT-L1 transgenic plants show drought tolerance. (A) The drought tolerance of the arf2-7mutant and 35S:ANT-L1 and wild-type plants under identical water deprivation and re-watering periods. The samples were photographed at the indicated time points. (B) Bar graph exhibitingthe survival rate in Col-0, arf2-7mutant and 35S:ANT-L1 plants. The survival rates of the plants were determined 4 days after re-watering, and the values are themean±s.d. of three independent experiments (n>40; ***P<0.001). (C) Bar graph exhibiting water loss in Col-0 and arf2-7 mutant and 35S:ANT-L1 leaves. Theleaves at the same developmental stages were excised and weighed at various time points. The values are the mean±s.d. of three independent experiments(n>10). (D) Bar graph exhibiting stomatal behavior in Col-0, arf2-6 and arf2-7 plants in response to ABA. (E) Bar graph exhibiting stomatal behavior in Col-0, 35S:ANT and ant-KO (SALK_022770) plants in response to ABA. Stomata were opened by exposing plants for 12 h to light and high humidity, and leaves wereincubated for 2 h in stomatal-opening solution containing 50.0 mM KCl, 10.0 mM CaCl2, and 10.0 mM Mes, pH 6.0. Stomatal apertures were measured 1 h afteradding 3.0 mM ABA. Data represent means±s.d. (n>80 stomata; *P<0.05; **P<0.01).

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in Fig. 4D. Thus, the more sensitive stomatal closure in the arf2mutant was most likely caused by abnormal ABA accumulation.However, the stomatal density and size were not significantlydifferent between the arf2 and wild-type plants (Fig. S3). Takentogether, these results indicate that ARF2 is involved in regulatingthe responsiveness of ABA and that the drought tolerance of arf2mutants is caused by the ABA-mediated stomatal closure.

Overexpressing transformants of ANT have enhanced seedmass and drought toleranceThe mature seeds of the 35S:ANT (ANT-overexpressing)transformants were observed to be obviously bigger in size,consistent with a previous report (Mizukami and Fischer, 2000),whereas ant-knockout (ant-KO) mutants exhibited smaller seeds,compared to wild type (see Fig. 1B,D; Fig. S1A,C). Cytologicalobservations showed that the average area of the cotyledon embryoand the cotyledon embryo cell in the 35S:ANT plants were ∼1.50

times and ∼1.28 times larger than the wild type, respectively (seeFig. 1A and C); the corresponding ratio of the area of the embryo tothat of the cell was 1.17 (1.5/1.28=1.17). Similarly, this observationmeant that the overall enlargement of the 35S:ANT embryos couldhave resulted from both an augmentation in cell size and an increasein cell number. The enlarged cotyledons of the 35S:ANTtransformants could also be restored on MS medium with 3.0 µMABA (see Figs 1E and 4A). Cytological observations showed thatthe average size of the cotyledon cells in the 35S:ANT transformantswas also smaller than that of wild type (see Fig. S4). Consistent withthis, the seed germination and seedling root growth of the 35S:ANTtransformants were more sensitive to ABA signals, whereas the ant-KO mutant was less sensitive, compared to the wild type (seeFig. 4A–C). When quantifying the endogenous ABA content indeveloping seeds of the 35S:ANT-transformant and ant-KO mutantlines, we found that the endogenous ABA content in the 35S:ANTtransformants and ant-KO mutant (SALK_022770) was

Fig. 3. ARF2 negatively regulatesANTexpression. (A) Bar graph exhibiting theARF2 andANTexpression difference between the 10-, 15- and 20-day-old wild-type (WT; Col-0) seedlings. Data are means±s.d. (n=3). (B,C) Bar graph exhibiting the ANT expression difference between young leaves (B), developing siliques(B) and old leaves (C) in wild-type (Col-0), arf2-6 and arf2-7 plants. (D) The expression of ProARF2:GUS and ProANT:GUS on the roots (n>20). Plants were fromthe same plate. Magnifications are the same. Scale bar: 100 μm (a–i). The screening method of seedlings is described in Materials and Methods. (E,G) Theintensity of GUS coloration was quantified by using Adobe Photoshop CS (Adobe Systems) software, as described previously byWang et al. (2011). Ten roots (E)and 10 leaf blades (G) were measured. Data are means±s.d. The expression intensity of the ProARF2:GUS in the wild-type background in root meristem,elongation and differentiation zones was set as 1.0 in B. The expression intensity of the ProARF2:GUS in the wild-type background was set as 1.0 in D. Alltransgenic plants expressing GUSwere subjected to staining of GUS for 8 h. (F) The expression ofProARF2:GUS andProANT:GUS on the leaves (n>20). Plantswere from the same plate. Magnifications are the same. Scale bar: 10 μm (a–c). The seedling screening method is described in Materials and Methods. (H) Bargraph exhibiting the interaction between ARF2 and ANT promoter. ChIP analysis was performed to analyze the in vivo interaction between ARF2 and the ANTpromoter and the coding sequence (CDS). The input shows chromatin before immunoprecipitation was performed. Anti-FLAG M2 antibody was used toprecipitate chromatin bound to 35S:ARF2:FLAG. An anti-GFP antibody was used as a negative control for the specificity of immunoprecipitation. The ANTpromoter region that bound to ARF2 was amplified by quantitative PCR using ANT promoter-specific primers against distinct regions.

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significantly higher and lower than the wild type, respectively (seeFig. 4D; Fig. S1E). These observations indicate that the function ofANT is also connected to the ABA signaling pathway and that thelarge seeds of 35S:ANT plants are most likely to be the result of theabnormal ABA signals.While testing the drought tolerance of the 35S:ANT and wild-type

plants, the physiological changes and survival status of leaves uponwater deprivation were observed. Similar to the phenotype of thearf2 mutants, the 35S:ANT plants displayed a stronger tolerance todrought than the wild type (Fig. 2A and B; Fig. S3A). Consistentwith this observation, the detached leaves from the 35S:ANT plantslost water more slowly (Fig. 2C); furthermore, the enhanced water-deficit survival of the 35S:ANT plants was closely associated withtheir capacity to maintain higher leaf RWC than the wild type at∼16% SWC (Fig. S3B). Further, the performance of stomata wasanalyzed, and the stomatal aperture was obviously reduced in the35S:ANT plants and enhanced in the ant-KO mutant plants upontreatment with ABA (Fig. 2E). The analysis of the expression of β-glucuronidase under the ANT promoter (ProANT:GUS) indicatedthe ANT was preferentially expressed in the stomata (Fig. 3F).Accordingly, seed germination and seedling root growth were more

responsive to ABA treatment in the 35S:ANT plants, but lesssensitive in the ant-KO plants, compared to the wild type(Fig. 4A–C). Upon exposure to 10% PEG 6000, the ABA contentbecame significantly higher in the 35S:ANT seedlings, as shown inFig. 4D. Thus, drought tolerance of the 35S:ANT plants was mostlikely caused by abnormal ABA signaling. Overall, these resultsindicate that ANT is involved in regulating ABA-mediated droughttolerance. Clearly, the 35S:ANT transformants exhibited aphenotype that is consistent with that of the arf2 mutant plants(Figs 1 and 2).

The expression of ANT is negatively regulated by ARF2As noted previously, the expression level of ANT was significantlyhigher in the arf2mutants compared to that in the wild type (Schruffet al., 2006). To further dissect the potential link between ARF2 andANT, we first investigated the relevance of the expression of ARF2and ANT in wild-type leaves. Using 10-, 15- and 20-day-old leavesand real-time PCR analyses, we found that the expression of ARF2gradually enhanced with increasing leaf age and, correspondingly,that the expression of ANT decreased (see Fig. 3A). Using youngand old leaves, and developing siliques as source material, we

Fig. 4. Effects of different ABA concentrations. (A) Representative sensitivity to ABA signaling of the ant-KO (SALK_022770), Col-0 and 35S:ANTseedlings grown on MSmedium without ABA and with 1.5 µM ABA or 3.0 µM ABA. Scale bars: 10 mm (a–c). (B) Bar graph showing different primary root lengthsin the indicated seedlings that are shown in A. Data aremeans±s.d. (n>15). (C) Bar graph exhibiting different germination rates in the indicated seedlings grown onMS medium without ABA and with 1, 2, 3 µM ABA. Data are means±s.d. (n>40). (D) ABA contents. Twelve-day-old seedlings of the indicated genotypes wereused to quantify ABA. ABAwas assayed by using ELISA. The values are the mean±s.d. of three independent experiments (n=3; **P<0.05; *P<0.01). FW, freshweight. For the ABA content assay protocol, see Yu et al. (2008). (E) Bar graph exhibiting the difference in ANT expression between the wild-type seedlingstreated with 40 µM ABA for 0, 3 and 6 h. Data are means±s.d. (n=3). (F) Bar graph showing the difference in ARF2 expression between the wild-type seedlings insufficient water and in drought, and aba2-1 in drought. Data are means±s.d. (n=3). (G) Bar graph exhibiting the difference in the expression of ANT between thewild-type seedlings in sufficient water and in drought, and aba2-1 seedlings in drought. Data are means±s.d. (n=3).

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further compared the expression differences of ANT between thearf2 mutants and the wild type; the level of expression of ANT inarf2mutants was significantly higher than that in the wild type (seeFig. 3B and C). In addition, we introduced the ProARF2:GUS andPro ANT:GUS constructs into the wild type. As shown in Fig. 3Dand E, the ProARF2:GUS construct was strongly expressed in theroot differentiation zone, but not in the meristem and elongationzones; whereas ProANT:GUS was strongly expressed in the rootmeristem, but not in the elongation and differentiation zones. Also,ProARF2:GUS was strongly expressed in the mature leaf abaxialepidermis, including the epidermis cells and stomata, whereasProANT:GUS was expressed at a very low level only in stomata(Fig. 3F and G). However, when ProANT:GUS was introduced intothe arf2mutants, we found that ANTwas strongly expressed in bothleaf epidermis cells (including stomata) and the root tissues intransformants (see Fig. 3D–G). These results strongly imply thatARF2 negatively regulates ANT expression.Inspecting the structural characteristics of the ANT promoter

region, two reverse AuxREs cis-elements (GAGACA) wereidentified – i.e. in positions −376 to −370 and −470 to −463,respectively (see Fig. S4A). To test whether ANT is directlyregulated by ARF2, we performed both an electrophoresis mobilityshift assay (EMSA) and chromatin immunoprecipitation (ChIP)assays. For the EMSA assay, as shown in Fig. S4, when the targetprotein (GST–ARF2N1-470) and the labeled P3-P4 (the regionfrom -601 to -276) DNA probes were added, a shifted DNA-bindingband was detected; when the unlabeled P3-P4 DNA probes wereadded to the reaction mixture, the DNA-binding band waseliminated (Fig. S4B). Furthermore, the target protein did notbind to mutated DNA probes (mP3-P4) (Fig. S4C), and the amountof bound protein was significantly reduced in the presence of excessunlabeled P3-P4 fragments (Fig. S4D). These results clearly revealthat the ARF2 DNA-binding domain directly and specifically bindsto the cis-element AuxREs in the promoter of ANT in vitro. For theChIP analysis, as shown in Fig. 3H, amplification of the DNAwithP3-P4 (-601 to -276), covering a region with two reverse conservedN-terminal DNA-binding sites (GAGACA), resulted in a greateramount of PCR product than amplification with primers 1 and 2(P1-P2; -1405 to -1081), covering a region that does not containreverse conserved N-terminal DNA-binding sites (GAGACA),

which also applied to the coding sequence (CDS). These resultsindicate that ARF2 directly binds to the ANT promoter in vivo.

ABA regulates the cotyledon elongation in arf2 and 35S:ANTplants by altering auxin distribution and/or auxin signalingInitially, ARF2 was identified as factor that is sensitive to auxinsignals. To further examine the potential differences in theresponses of ARF2 and ANT to ABA and auxin signals, thevector DR5:GUS, which allows identification of auxinaccumulation (Ulmasov et al., 1997), was transformed into thearf2-6, 35S:ANT and wild-type plants, and the signal of auxinaccumulation in different tissues was inspected. Generally, theauxin accumulation in young leaves (10–20% expanded) usuallyexhibited a gradient – i.e. a relatively higher level at the base and arelatively lower level at the tip, which regulates the cell proliferationand elongation patterns in leaf development (Chen et al., 2001;Benkova et al., 2003). Our experiments showed that on the tips ofthe 10-day-old arf2-6, 35S:ANT and wild-type leaves, the auxinaccumulation and/or auxin signals could not be detected in any ofthe samples tested (see Fig. 7). By contrast, when treated with1.5 µm ABA, the leaf tips of the arf2-6 and 35S:ANT transformantsexhibited more visible auxin signals than the wild type (see sub-panels a,c,e in Fig. 5A,B). Further, when treated with 3.0 µm ABA,the leaf tips of the arf2-6 and 35S:ANT transformants exhibitedstronger auxin signals than the wild type (see sub-panels b,d,f inFig. 5A and B). Correspondingly, the leaf sizes of the arf2-6 and35S:ANT transformants were dramatically reduced after thetreatment with ABA, compared to those of the wild type (see sub-panels a–f in Fig. 5A). These results clearly indicate that treatmentwith ABA significantly enhances auxin accumulation in the leafblades of the arf2-6 and 35S:ANT transformants, compared to that inthe wild type. All of the above findings suggest that abnormal ABAsignaling significantly induces auxin accumulation in cotyledons ofthe arf2-6 mutants and 35S:ANT transformants, and leads to thesuppression of elongation of their cotyledon.

ANT positively regulates COR15A expressionIt has previously been shown that the two AP2 domains of ANT canbind selectively to gCAC(A/G)N(A/T)TcCC(a/g)ANG(c/t) cis-elements that are involved in regulating the COR15A promoter

Fig. 5. DR5:GUS expression in cotyledons of arf2-6, 35S:ANT and wild type (Col-0). (A) Representative DR5:GUS expression in cotyledons of arf2-6 (a,b),35S:ANT (c,d) and wild type (Col-0; e,f ) grown on solid MS medium with 1.5 µM ABA or 3.0 µM ABA. Magnifications are the same. (B)The intensity of GUScoloration was quantified by using Adobe PhotoshopCS (AdobeSystems) software, as described byWang et al. (2011). Ten cotyledons weremeasured. Data aremeans±s.d. The expression intensity of DR5:GUS in the wild-type (Col-0) background was set as 1.0. All transgenic plants with GUS were subjected to GUSstaining for 8 h.

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(Nole-Wilson and Krizek, 2000; Krizek, 2003). The COR15A gene,considered to be a marker gene of the response to drought stress, isinvolved in response to environmental stresses through an ABA-dependent signaling pathway (Gilmour et al., 1998, 2004). We foundthat COR15Awas significantly downregulated in the ant-KO plants,compared to its expression in wild type (Fig. 6B and C), suggestingthat the positive regulation of ANT on COR15A occurs at thetranscriptional level. To determine whether COR15A is directlyregulated by ANT through binding to the promoter in vivo, a ChIPassay was performed. The results showed that the regions C1 and C2(containing consensus sequences) led to greater amounts of PCRproduct than the amount obtained by the region C3 (not containingconsensus sequences) (Fig. 6D). Furthermore, expression of β-glucuronidase under the COR15A promoter (ProCOR15A:GUS) wasweakon the leaves, hypocotyls and roots in thewild-type background,whereas expression of ProCOR15A:GUS was strong on thecorresponding organs in the 35S:ANT background (Fig. 6E). Thesefindings indicate that ANT plays a major role in activating theCOR15A promoter under non-stress conditions.

To dissect whether ANT and COR15A form a signalingtransduction pathway to regulate seed mass, firstly, the cor15a-knockout mutant (cor15a-KO) seed mass was further checked. Asshown in Fig. 1B,D and Fig. S1A,C, the mutant cor15a-KO hadsmaller seeds than the wild type. Cytological observation indicatedthat the average areas of the cotyledon embryo and the cotyledonembryo cells in the cor15a seeds were∼33% and∼24% smaller thanthe wild type, respectively (Fig. S1B,D), meaning that the decreasedsize of the cor15a embryo results from decreased embryo cell sizeand cell number. Further, the seed mass, embryo size and embryocell area of the 35S:ANTcor15a double mutants exhibited a similarphenotype to the cor15a plants (see Fig. 1A–D; Fig. S1A–D). Thesefindings indicate that mutation ofCOR15A suppresses the seed massof 35S:ANT plants, probably because ANT and COR15A form anANT–COR15A gene cascade to regulate seed mass.

To better demonstrate that the ARF2–ANT–COR15A genecascade regulates seed mass, we crossed the arf2-6 mutant with acor15a. Our findings indicated that the resulting arf2-6cor15a planthad a smaller seed mass than thewild type, suggesting that cor15a is

Fig. 6. ANT directly regulates COR15A expression. (A) Schematic diagram of the COR15A loci and three amplicons initiating from the ATG start codon ofCOR15A: C1, C2 and C3 used for ChIP analysis. (B) Representative expression ofCOR15A between 2-week-old ant-KO1(SALK_022770), ant-KO2 (CS483900)and Col-0 plants. TUB4 is used as control. (C) Bar graph exhibiting the COR15A expression between the 2-week-old wild-type and ant-KO seedlings. Data aremeans±s.d. (n=3; **P<0.01). (D) Bar graph exhibiting the interaction between ANT and the COR15A promoter. ChIP was performed to analyze the in vivointeraction between ANT and the COR15A promoter. The input was chromatin before immunoprecipitation. An anti-HA antibody was used for precipitatingchromatin associated with 35S:ANT:HA. GFP was used as a negative control for the specificity of immunoprecipitation. The COR15A promoter region thatassociated with ANT was amplified with quantitative PCR using COR15A-promoter-specific primers for distinct regions. (E) The spatial expression pattern ofCOR15A in the wild-type and the 35S:ANT seedlings. The COR15A-promoter–GUS construct was introduced into the wild-type (Col-0) and 35S:ANT seedlings,and histochemical GUS assays were performed using 1-week-old seedlings of each genotype (n>20). Representative images of GUS staining are shown. Themethod used to screen the seedlings is described in Materials and Methods. Scale bar: 1.0 cm (a–b).

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epistatic to arf2-6 (Fig. S1F,G). In conclusion, ANT acts upstream ofCOR15A to regulate seed mass.

DISCUSSIONAs mentioned above, large-seeded seedlings are generally morerobust and exhibit a stronger tolerance to environmental stresses,compared to small-seeded seedlings (Muller-Landau, 2010).Although ecologists have examined the survival advantage oflarge-seeded and small-seeded species at the macroscopic level, andhave explained the trade-off strategy between colonization andcompetition in which smaller-seeded species are superior atcolonizing and larger-seeded species are superior competitors(Coomes and Grubb, 2003; Westoby et al., 1992), little is knownabout the potential molecular mechanisms underlying why large-seeded seedlings exhibit a stronger tolerance to environmentalstresses than the small-seeded seedlings. Our current study hasdemonstrated that the ARF2 transcription factor serves as amolecular link that integrates the regulation of seed developmentinto plant responses to drought stresses and has provided novelinsights into understanding why large-seeded seedlings are moreefficient at withstanding abiotic stresses than smaller-seedseedlings.The transcription factor ARF2 was originally identified as a

repressor that is involved in negatively regulating ABA-mediatedseed germination and primary root growth (Wang et al., 2011).Usually, with ABA treatment, the expression of ARF2 issignificantly upregulated (Wang et al., 2011). Our current studydemonstrated that the ARF2 loss-of-function mutants accumulateABA, consequently resulting in extra cell proliferation or larger seedmass, and an increase of stomatal closing or drought tolerance. Itseems clear that ARF2 acts not only in response to ABA signals butalso as a regulator of ABA accumulation. As Wang et al. (2011)have proposed previously, the function of ARF2 in the network ofABA signaling might be twofold because it responds to differentABA inducers – i.e. ABI3, ABI4 and ABI5 act upstream of ARF2,resulting in the regulation of ARF2 expression, whereas ABI1 andABI2 act further downstream, to regulate ABA accumulation (seeFig. 8). Not surprisingly, the change in ABA accumulation triggers

consequent physiological actions in the regulation of growth anddevelopment and responses to environmental stresses (Lim et al.,2007; Assmann and Wang, 2001). Interestingly, evidence has beenpresented that ARF2 modulates the hypocotyl bending of thehookless1 mutant through auxin signaling in the apical hook (Liet al., 2004), meaning that ARF2 might be involved in auxinsignaling. Our current study confirms that ARF2 is able to mediateauxin signaling by changing the levels of ABA, which causescrosstalk between ABA and auxin.

The transcription factor ANT plays a pivotal role in controllingcell proliferation, which determines the overall size of the organ(Klucher et al., 1996; Mizukami and Fischer, 2000). This studydemonstrated that the expression of ANT was negatively regulatedby ARF2 through binding to the ANT promoter region and that theregulation of ANT modulates seed mass by promoting cellproliferation. In particular, the expression of ANT increasedsignificantly under drought conditions or upon ABA treatment(see Fig. 4E–G). Thus, the regulation of ANT by ARF2 might be, atleast partly, dependent on ABA signaling.

It waswell known that COR15A and otherCOR genes are involvedin responding to environmental stresses, particularly drought, coldand dehydration, and responses to these conditions are associatedwithABA signaling (Stockinger et al., 1997; Gilmour et al., 2004;Yamaguchi-Shinozaki and Shinozaki, 2006). Moreover, COR15A isinvolved in regulating plant growth and development, for example,leaf senescence (Yang et al., 2011). In this study, the cor15amutantsexhibited smaller seed size, indicating that the gene is involved incontrolling seed mass. The phenotype of the 35S:ANTcor15a doublemutant showed that mutation of COR15A can inhibit the large seedmass of the 35S:ANT plants (see Fig. 1). Particularly, the arf2, 35S:ANT and cor15a mutant plants all exhibited an altered seed massbecause of altered embryo sizes. These findings indicate that theARF2–ANT–COR15A signal cascade forms an ABA-mediatedsignal transduction pathway that regulates seed mass.

Studies have identified that the ABA2, ABI5 and SHB1 genes areinvolved in controlling seed size (Cheng et al., 2014). Specifically,ABA2 is positively regulated upon ABA accumulation, activatingABI5 in ABA signaling, consequently negatively modulating SHB1

Fig. 7. DR5:GUS expression incotyledons of arf2-6, 35S:ANT and wildtype (Col-0). (A–C) Representative DR5:GUS expression in cotyledons of wild type(Col-0) (A), arf2-6 (B), and 35S:ANT (C)grown on solid MS medium with 0.0 µMABA. Magnifications are the same. Scalebars: 0.25 mm. (D) The intensity of GUScoloration was quantified by using AdobePhotoshop CS (Adobe Systems)software, as described by Wang et al.(2011). Ten cotyledons were measured.Data are means±s.d. The expressionintensity of DR5:GUS in the wild-type(Col-0) background was set as 1.0. Alltransgenic plants with GUS weresubjected to GUS staining for 8 h.

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expression through the direct binding between ABI5 and the ABREcis-elements in the SHB1 promoter region (see Fig. 7). Further,SHB1 has been associated with both MINI3 and IKU2 in theregulation of seed development (Zhou et al., 2009). In this study,we identified that the ARF2–ANT–COR15A signal cascadeparticipates in controlling seed size within ABA-mediatedsignaling through ABA–auxin crosstalk, which causes an increasein cell proliferation. These studies indicate that the mechanisms tocontrol the size of seeds are complex and involve diverse networks.Clearly, our current study adds new evidence to help theunderstanding of the molecular mechanisms that control seed size.Based on our current study, a potential role of the ARF2–ANT–

COR15A signal cascade in integrating ABA signals into theregulation of seed mass and drought tolerance is proposed. Asshown in Fig. 7, ARF2 responds to ABA signals and mediates ABAaccumulation by triggering ABI1 and ABI2. ARF2 negativelyregulates the expression of ANT, consequently ANT directlyregulates the function of COR15A to regulate seed mass and stressresistance. The ARF2–ANT–COR15A–ABA-mediated signalcascade that we have identified in this study might be different fromthat which regulates seed development through the SHB1–ABA-mediated pathway, which has been proposed by Cheng et al. (2014).

MATERIALS AND METHODSPlant materials and growth conditionsThe arf2-6, arf2-7 (Okushima et al., 2005) and 35S:ARF2:FLAG (Wanget al., 2011), ant-8 (CS3944) (described by ABRC) and 35S:ANT transgenicplants (Mizukami and Fischer, 2000) on the Col-0 background have beendescribed previously. The arf2-6 and arf2-7 homozygous seeds have similarphenotypes (such as large seedmass, delayed flowering and leaf senescence,etc.), as have been described by Okushima et al. (2005).

cor15a (SALK_054513), cor15a (SALK_008461) and ant-KO (CS3944)seeds were obtained from the Arabidopsis Biological Resource Center(ABRC; Ohio State University). The arf2-6, arf2-7 and 35S:ARF2:FLAGhomozygous seeds were kindly provided by Professor Z. Z. Gong (ChinaAgricultural University, China). The ant-KO (SALK_022770) and 35S:ANT(hygromycin B; Ph2GW7) homozygous seeds were kindly provided byProfessor H. G. Nam (Daegu Gyeongbuk Institute of Science andTechnology, Korea).

Homozygous lines of cor15a (SALK_054513) and cor15a(SALK_008461) were obtained through herbicide selection for three ormore generations and analysis of segregation ratios. Absence of geneexpression in these mutants was verified by reverse transcriptase (RT)-PCRanalysis before use. The arf2 ant-KO/+ lines were obtained from F2seedlings of arf2-6×ant-KO (SALK_022770)/+ lines that did not show anydefect in apical hook formation when grown in the dark (Li et al., 2004).Then, the arf2 ant-KO homozygous lines were obtained from arf2 ant-KO/+lines through hygromycin selection for three or more generations andanalysis of segregation ratios. Absence of ARF2 and ANT expression in thearf2 ant-KO mutant was verified by RT-PCR analysis before use. The 35S:ANTcor15a homozygous lines were obtained from 35S:ANTcor15a/+through hygromycin and herbicide selection for three or more generationsand analysis of segregation ratios. Absence of COR15A gene expression in35S:ANTcor15a lines was verified by RT-PCR analysis before use;overexpression of the ANT gene in 35S:ANTcor15a lines was alsoverified by RT-PCR analysis before use.

We firstly generated (+/−) arf2-6cor15a double mutant (F1) seeds. (+/−)arf2-6cor15a double mutants (F1) seeds were sown, and (+/+, +/−, −/−)arf2-6cor15a double mutants (F2) seeds were obtained. Then, we selected(+/−, −/−) arf2-6cor15a seeds (F2), which are smaller size than those ofwild type, sowed them and gained (+/+, +/−, −/−) arf2-6cor15a seeds (F3).We randomly selected (+/−, −/−) a few arf2-6cor15a lines (F3) withsmaller seed size compared with those of wild type and their developedsmall seeds were identified by RT-PCR (data not shown). Our findingsindicated that ARF2 andCOR15A expression in these developed small seedscould be not detected, suggesting that these developed small seeds are (−/−)arf2-6cor15a double mutant (F3).

Plants exhibiting the arf2-6 mutant phenotype [large rosette leaves andlarge seeds grown in white light (Schruff et al., 2006)] in the F2 populationswere screened for ProANT:GUS expression in roots. F3 seeds were collectedfrom those exhibiting expression, and lines expressing GUS in all F3 plantswere used for subsequent analysis, as described previously (Zgurski et al.,2005). Plants exhibiting the arf2-6 mutant and 35S:ANT phenotype [largerosette leaves and large seeds grown in white light (Schruff et al., 2006;Mizukami and Fischer, 2000)] in the F2 populations were screened for Dr5:GUS expression in roots. F3 seeds were collected from those exhibitingexpression, and lines expressing GUS in all F3 plants were used forsubsequent analysis. ProANT:GFP was introduced into the 35S:ARF2background by Agrobacterium-mediated transformation of 35S:ARF2homozygous plants. Transformants were selected on hygromycin B (Wanget al., 2011) for three or more generations and analyzed to determinesegregation ratios. Plants exhibiting the 35S:ANT phenotype [large rosetteleaves and large seeds grown inwhite light (Mizukami and Fischer, 2000)] inthe F2 populations were screened for ProCOR15A:GUS expression in roots.F3 seeds were collected from those exhibiting expression, and linesexpressing GUS in all F3 plants were used for subsequent analysis.Transgenic plants were generated using the Agrobacterium-tumefaciens-mediated floral dip method (Meng, 2015; Meng and Yao, 2015).

The seeds were subjected to 4°C for 3 days, and then sown onto solid MSmedium supplemented with 1% sucrose at pH 5.8 and 0.8% agar. The plantsgrown on agar were maintained in a growth room under 16–8 h light–darkcycles with cool white fluorescent light at 21±2°C. Plants grown in soil-lessmedium were maintained in a controlled environment growth room under16–8 h light–dark cycles with cool white fluorescent light at 21±2°C.

Quantitative PCRTotal RNAwas extracted from the tissues indicated in the figures using theTRIZOL reagent (Invitrogen), as has been described by Yu et al. (2008).SYBR green was used to monitor the kinetics of PCR product in real-time

Fig. 8. A proposedmodel to illustrate the relationship between seedmassand drought tolerance. Water-sufficient conditions result in a physiologicalconcentration of ABA, and through an ARF2–ANT–COR15A signal cascade,the ABA signal results in crosstalk with auxin to promote extra cell division,leading to an increase of seed mass, which results in a large seed mass.Conversely, water deficiency dramatically induces an ABA signal increaseand, through an ARF2–ANT signal cascade, this ABA signal results incrosstalk with auxin to inhibit cell division and to promote ABA-mediatedstomatal closing, reducing transpiration, which ultimately leads to resistance todrought stress. By contrast ARF2–ANT-mediated regulation of COR genescontributes to stress-resistance responses.

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RT-PCR, as has been described by Yu et al. (2008). Gene-specific PCRprimers are described below. For analyzing ARF2 expression in youngor old seedlings and developing siliques of wild type (Col-0), primers F5′-GAGTTTTGACTACCTCTGGTTAA-3′ and R 5′-GATAAAACCAC-CAATTTCACCTC-3′were used. For analyzing ANT expression in young orold seedlings and developing siliques of wild type (Col-0), primers F 5′-AGGTGGCAAGCACGGATTGGT-3′ and R 5′-AGGCAACGCGAAAA-TCGCCG-3′ were used. For analyzing COR15A expression in ant-KOseedlings, primers F 5′-AGCGGAGCCAAGCAGAGCAG-3′ and R5′-TGCCGCCTTGTTTGCGGCTT-3′ were used. These experimentswere repeated at least two times with similar results.

Cytological experimentsAverage seed weight was tested by weighing mature dry seeds in batches of100 using an electronic analytical balance (Mettler Toledo). The chlorophyllof young cotyledons was removed by using an ethanol gradient (30%, 50%,70%, 90% and 100%), for 30 min in each solution. These cotyledons werethen photographed using a HIROX three-dimensional video microscope,and the cotyledon cell size was measured by using ImageJ software.Measurements of cotyledon area were made through scanning these organsto form a digital image and then calculating the area using ImageJ software.Mature seeds were photographed at a relevant magnification using a HIROXthree-dimensional video microscope.

Mature dried seeds were imbibed for 60 to 100 min and dissected under amicroscope in order to isolate mature embryos. The embryos were incubatedovernight in buffer (30 mM sodium phosphate, pH 7.0, 10 mM EDTA, 1%Triton X-100 and 1% DMSO) at 37°C, fixed for 1 h in buffer (FAA with10% formalin, 5% acetic acid and 45% ethanol) and 0.01% Triton X-100,and dehydrated with an ethanol series, as described by Ohto et al. (2005).Then, the embryos were treated for 1 to 2 h in Hoyer’s buffer (3:0.8:0.4 ofchloral hydrate:water:glycerol). Using a HIROX three-dimensional videomicroscope, under relevant magnification, we observed the treated embryos.Using ImageJ software, cotyledon and hypocotyl area, and averageepidermal cell size in the central region of cotyledons and hypocotylswere measured, as described by Ohto et al. (2005).

Water loss measurementsFor water loss measurements, 6–8 leaves per individual mutant and wild-type plant that had been grown under normal conditions for 3 weeks wereexcised, and fresh weight was determined at the designated time intervals.Four replicates were performed for each line. Water loss was represented asthe percentage of initial fresh weight at each time point.

Stomatal aperture analysisStomata were opened by exposing plants for 12 h to light and high humidity,and leaves were incubated for 2 h in stomatal-opening solution containing50.0 mM KCl, 10.0 mM CaCl2 and 10.0 mM Mes, pH 6.0. Stomatalapertures were measured 1 h after adding 3.0 mM ABA. Data representmeans±s.d. (n>80 stomata; *P<0.05, **P<0.01). The stomatal aperture wasmeasured as previously described by Song et al. (2005). Subsequently, theepidermis was placed onto a slide and photographed under a Hirox three-dimensional video microscope. These experiments were repeated at leastthree times with similar results.

Plasmid constructsForARF2, ANT andCOR15A promoter analysis, promoter–GUS constructs ofAt5g62000, At4g37750 and At2g42540 were created by inserting ∼2.0 kb,∼1.0 kb and ∼1.1 kb promoter fragments, respectively, which were amplifiedusing a primer into pCB308R, as previously described by Lei et al. (2007). Foramplifying ARF2 promoter fragments, primers F 5′-ggggacaagtttgtacaaaaaa-gcaggctTTTCTCGTCCTTTTCCTCTCAA-3′ and R 5′-ggggaccactttgtacaa-gaaagctgggtAGCTTCAATCATTTCAACCGC-3′ were used. For amplifyingANT promoter fragments, primers F 5′-ggggacaagtttgtacaaaaaagcaggctAGC-TTATAATGTGACAAAAGTTA-3′ and R 5′-ggggaccactttgtacaagaaagctgg-gtCTAATAATTAGGTTTCTTGTCACTT-3′ were used. For amplifyingCOR15A promoter fragments, primers F 5′-ggggacaagtttgtacaaaaaagcaggct-CTTCGGAACAACAACAAGAGTT-3′ and R 5′-ggggaccactttgtacaagaaag-ctgggtTGTAATCATATTTGTGGTTTTCAG-3′ were used. To generate the

ANT:HA plasmid, the primers F 5′-GTTTGTACAAAAAAGCAGGCTAT-GCAACAGCACCTGATGCAGAT-3′ and R 5′-CTTTGTACAAGAAAG-CTGGGTTCAATTCCCATCATCTGATGATTTC-3′ were used. Foramplifying ANT promoter fragments, primers F 5′-ggggacaagtttgtacaaaaaa-gcaggctAGCTTATAATGTGACAAAAGTTA-3′ and R 5′-ggggaccactttgt-acaagaaagctgggtCTAATAATTAGGTTTCTTGTCACTT-3′ were used. Inthis section, lowercase letters denote sequences from the connector primers,whereas capital letters denote the sequences of targeted genes.

GUS stainingUsing a mix buffer [1 mM X-gluc, 60 mM NaPO4 buffer, 0.4 mM ofK3Fe(CN)6/K4Fe(CN)6 and 0.1% (v/v) Triton X-100], samples (transgenicplants harboring and expressing ProARF2:GUS, Pro ANT:GUS, ProCOR15A:GUS DR5:GUS) were stained, and then incubated at 37°C for 8 h.After staining of GUS, chlorophyll was removed using a 30, 50, 70, 90 and100% ethanol series, with an incubation of 30 min at each concentration.GUS staining was performed as previously described byMeng et al. (2015).

ChIP assayLeaf tissues of 2-week-old transgenic lines overexpressing 35S:ARF2:FALG and 35S:ANT:HA were used in this assay. ChIP was performed, asdescribed previously (Meng, 2015; Meng and Yao, 2015; Meng and Liu,2015). A FLAG-M2-tag-specific monoclonal antibody was used for ChIPanalysis in overexpressing 35S:ARF2:FALG lines. A hemagglutinin (HA)-tag-specific monoclonal antibody was used for ChIP analysis inoverexpressing 35S:ANT:HA lines. Green fluorescent protein (GFP)-tag-specific monoclonal antibody was used as a control in the aboveexperiments. The ChIP DNA products were analyzed by usingquantitative PCR with primers that had been synthesized to amplify∼300-bp DNA fragments in the promoter region of ANT and COR15Ain the ChIP analysis. The primer sequences used for ARF2 ChIP analysiswere: ANT-1 (P1 5′-AGCTTATAATGTGACAAAAGTTATT-3′; P2 5′-T-GTCTTGGGTTATTTTGTGGTG-3′); ANT-2 (P3 5′-TAGATACAGTAT-AAACTAACTTTAA-3′; P4 5′-CTAATAATTAGGTTTCTTGTCACTT-3′). The primer sequences used for ANTChIP analysis were: COR15A-1 (P15′-CTTCGGAACAACAACAAGAGTT-3′; P2 5′-TTAAATTTTTACAA-AATTAAATT-3′); COR15A-2 (P3 5′-AGGAGATGTTACTGTCCGTC-AG-3′; P4 5′-ATGAGTTGAAACCACAAACCATT-3′) and COR15A-3(P3 5′-GGCTTTTGGTAGATTTGGGCTTG-3′; P4 5′-ACGTGTAATCA-TATTTGTGGTTT-3′).

Protein expression and purificationThe plasmid pGEX-5X-1 for ARF2 was used in this experiment. The codingsequence of ARF2 was amplified by using the primer pair (5′-GGATCC-ATGGCGAGTTCGGA-3′ and 5′-GAATTCAGATTGCCAGACAAC-3′),and then cloned into the BamHI and EcoRI restriction sites of the pET28aplasmid to generate the final plasmid. Recombinant glutathione S-transferase binding protein (GST)-tagged ARF2 was extracted fromtransformed Escherichia coli (Rosetta2) after 10 h of incubation at 16°Cfollowing induction with 10 μM isopropylβ-D-1-thiogalactopyranoside.These recombinant proteins were purified using GST-agarose affinity.

Electrophoretic mobility shift assayThe EMSA protocol has been described previously (Meng et al., 2015). Thebiotin-labeled ANT DNA fragments (5′-GAAAAAGAGACAAAAGGA-GGGAATTTAGAAATGAGGTGGTGAAGGTATGTTGGATTGTTGTG-GAACGATATGGTCAATAAAGCATATCGCATTATTGGAGAGACATT-ACAT-3′) and mutated ANT DNA fragments (5′-GAAAAAGTGTCAA-AAGGAGGGAATTTAGAAATGGTGAAGGTATGTTGGATTGTTGT-GGAACGATATGGTCAATAAAGCATATCGCATTATTGGAGTGTCAT-TACAT-3′) were synthesized, annealed and used as probes, and the biotinunlabeled same DNA fragments were used as competitors in this assay. Theprobes were incubated with the ARF2 fusion protein at room temperature for20 min in a binding buffer [50 mM HEPES-KOH (pH 7.5), 375 mM KCl,6.25 mM MgCl, 1 mM DTT, 0.5 mg/ml BSA, glycerol 25%].

AcknowledgementsWe thank Professors Hong-Gil Nam (DGIST, Korea) and Cheng-Bin Xiang(University of Science and Technology of China, China) for their support during the

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initial stages of this work. We extend our thanks to Zi-Qing Miao (University ofScience and Technology of China, China) for completing the ANT:HA plasmid andgaining ANT:HA seeds.

Competing interestsThe authors declare no competing or financial interests.

Author contributionsL.-S.M. designed experiments. L.-S.M. performed the experiments. L.-S.M., S.-Q.Y.and Z.-B.W. completed statistical analysis of data. L.-S.M. and A.L. wrote, edited andrevised this manuscript.

FundingThis study was financially supported by National Key Basic Research Program ofChina [grant number 2014CB954100]; and National Key Technology R&D Program[grant number 2015BAD15B02].

Supplementary informationSupplementary information available online athttp://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.171207/-/DC1

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RESEARCH ARTICLE Journal of Cell Science (2015) 128, 3922-3932 doi:10.1242/jcs.171207

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