Maize ABP2 enhances tolerance to drought and salt stress in transgenic Arabidopsis

Journal of Integrative Agriculture 2018, 17(11): 2379–2393 Available online at www.sciencedirect.com

ScienceDirect

RESEARCH ARTICLE

Maize ABP2 enhances tolerance to drought and salt stress in transgenic Arabidopsis ZONG Na1*, LI Xing-juan1, 2*, WANG Lei1, WANG Ying1, WEN Hong-tao1, LI Ling2, ZHANG Xia1, FAN Yunliu1, ZHAO Jun1 1

National Key Facility for Crop Gene Resources and Genetic Improvement/Faculty of Maize Functional Genomics, Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, P.R.China 2 Guangdong Key Laboratory of Biotechnology for Plant Development/College of Life Science, South China Normal University, Guangzhou 510631, P.R.China

Abstract Abiotic stresses, especially drought and salt, severely affect maize production, which is one of the most important cereal crops in the world. Breeding stress-tolerant maize through biotechnology is urgently needed to maintain maize production. Therefore, it is important to identify new genes that can enhance both drought and salt stress tolerance for molecular breeding. In this study, we identified a maize ABA (abscisic acid)-responsive element (ABRE) binding protein from a 17day post-pollination (dpp) maize embryo cDNA library by yeast one-hybrid screen using the ABRE2 sequence of the maize Cat1 gene as bait. This protein, designated, ABRE binding protein 2 (ABP2), belongs to the bZIP transcription factor family. Endogenous expression of ABP2 in maize can be detected in different tissues at various development stages, and can be induced by drought, salt, reactive oxygen species (ROS)-generating agents, and ABA treatment. Constitutive expression of ABP2 in transgenic Arabidopsis plants enhanced tolerance to drought and salt stress, and increased sensitivity to ABA. In exploring the mechanism by which ABP2 can stimulate abiotic stress tolerance, we found that ROS levels were reduced and expression of stress-responsive and carbon metabolism-related genes was enhanced by constitutive ABP2 expression in transgenic plants. In short, we identified a maize bZIP transcription factor which can enhance both drought and salt tolerance of plants. Keywords: ABP2, maize, transcription factor, drought, salt, transgenic Arabidopsis

1. Introduction Received 2 November, 2017 Accepted 14 March, 2018 Correspondence ZHAO Jun, Tel/Fax: +86-10-82105320, E-mail: [email protected] * These authors contributed equally to this study. © 2018, CAAS. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http:// creativecommons.org/licenses/by-nc-nd/4.0/) doi: 10.1016/S2095-3119(18)61947-1

Maize (Zea mays L.) is one of the most important cereal crops because of its high yield potential and nutritional value as food, feed, and fuel. Although maize yield has increased over the past two decades, drought tolerance has decreased at the same time (Lobell et al. 2014). Maize experiences an approximately 39% yield reduction under reduced water conditions (approximately 40%) (Daryanto et al. 2016). Droughts are becoming increasingly more frequent with the

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increased warming of the climate worldwide, highlighting the water crisis that threatens maize productivity worldwide (Boyer et al. 2013; Yu 2011). According to a survey released by the SGS company in Geneva, China’s corn production dropped by 13% in 2009 (Yu 2011). The lowest production level in 4 years was reached because of drought in the main producing areas. With the exception of the United States, major producers of maize (e.g., China, Brazil, and France) have been estimated to have experienced declines of 3.8% in production due to climate variability from 1980 to 2008 (Lobell et al. 2011). Second only to drought, salt stress has become a serious threat to crop production in the arid and semi-arid regions of the world (Flowers and Yeo 1995; Munns 2002). Because of the low heritability of drought tolerance and high magnitude of environmental interactions, development of drought tolerant crops using a conventional breeding approach is greatly restricted (Pardo 2010; Hill et al. 2013; Liu et al. 2014). Breeding tolerant maize varieties that can survive and even maintain yields under complex abiotic stress conditions through biotechnology is becoming increasingly urgent to stabilize and increase global maize production (Joshi et al. 2016). Many researches have been done in the model plant Arabidopsis thaliana to elucidate the complex drought tolerance mechanisms and metabolic networks for adapting to environmental stresses (Shinozaki and YamaguchiShinozaki 2000). The plant phytohormone abscisic acid (ABA), which plays a major role in regulating plant growth and development, seed dormancy and germination, stomatal movement, seedling growth, and flowering, is the key hormone mediating plant adaptations to abiotic stresses, including drought, salt, and cold (Koornneef et al. 1998; Leung and Giraudat 1998; Finkelstein et al. 2002; Zhu et al. 2007). Under abiotic stress conditions, high levels of ABA are produced to regulate the expression of various stress-responsive genes. The functional dissection of promoters of such ABA-responsive genes has identified several cis-acting elements involved in ABA-induced gene expression. A G-box ACGT core motif designated as an ABA-responsive element (ABRE) is shared by promoters of some ABA-responsive genes, such as Rab16 and LEA from rice, and the Em LEA gene from wheat (Guiltinan et al. 1990; Skriver et al. 1991; Ono et al. 1996). Abiotic stresses also lead to the overproduction of reactive oxygen species (ROS) in plants, which are highly reactive, toxic, and cause damage to proteins, lipids, carbohydrates, and DNA, ultimately resulting in oxidative stress (Gill and Tuteja 2010). Excess ROS can cause oxidative damage to cells, while ROS at low concentrations function as signaling molecules to regulate plant protective stress responses, including ABA-induced activation of Ca2+ channels and stomatal closure and induction of defense gene expression

(Pei et al. 2000; Desikan et al. 2001; Rizhsky et al. 2002). As ROS are both necessary and harmful, a mechanism to balance ROS production and scavenging is necessary in plants (Apel and Hirt 2004; Mittler et al. 2004). Thus, elucidating the ROS homeostasis and signaling pathway of plants in response to abiotic stress will be useful to generate abiotic stress-tolerant crops. Many transcription factors related to drought tolerance have been identified (Joshi et al. 2016), but only a few of these are from maize including ABP9, bZIP72, bZIP17, ZmNAC55, ZmDREB2A, and ZmZF1 (Qin et al. 2007; Huai et al. 2009; Jia et al. 2009; Zhang et al. 2011; Ying et al. 2012; Mao et al. 2016), etc. In addition, few have been proven to function in maize; recent genome-wide association studies (GWAS) indicated that genetic variation of ZmNAC111 and ZmVPP1 genes contributed to drought tolerance in maize seedlings, and high expression levels of these genes were shown to result in drought tolerance (Mao et al. 2015; Wang et al. 2016). A cis-acting elements analysis in maize identified ABRE2, a 9-bp G-box-containing DNA sequence (5´-CCACGTGGA-3´), which is responsible for ABA-induced expression of Cat1, the major enzymatic scavenger of ROS (Guan and Scandalios 1998; Guan et al. 2000). Previously, a cDNA library constructed using mRNA extracted from 17 days post-pollination (dpp) maize embryos was screened for proteins that interact specifically with the ABRE2 motif of the maize Cat1 gene, and a series of ABRE binding proteins (ABPs) were identified (Wang et al. 2002). In this study, we report the cloning and functional analysis of ABP2, a bZIP transcription factor that specifically interacts with the ABRE2 motif of the maize Cat1 gene. We analyzed the ABP2 expression profile under different treatments and characterized its role in stress tolerance. Our results showed that transgenic Arabidopsis plants overexpressing ABP2 exhibited normal growth and enhanced tolerance to drought and salt stresses by regulating ABA and ROS signaling.

2. Materials and methods 2.1. Plant materials and growth conditions All A. thaliana lines-used were in the Columbia-0 background. Surface-sterilized seeds were sown on Murashige and Skoog (MS) medium and kept for 3 days at 4°C in the dark to break dormancy, and then transferred to a growth chamber (22°C, 120 μmol photons m–2 s–1 under a 16-h light/8-h dark photocycle). Seedlings at 1- or 2-wk-old were transferred from plates into pots filled with compost soil. The maize (Zea mays L.) inbred line Qi319 was used in this study. To analyze the expression patterns of ABP2 in tissues and through development, the root, stem, leaves,

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and female and male inflorescence primordia were collected from field-grown plants at V6 (the 6th leaf unclasps from the stem), V10 (the 10th leaf unclasps from the stem), and the flowering stages. Maize plants grown in vermiculite were used for analysis of ABP2 expression patterns.

polyacrylamide gel electrophoresis (PAGE) in 0.5×Trisborate-EDTA buffer solution. After electrophoresis, gels were dried and visualized by autoradiography.

2.2. Cloning of full-length ABP2 cDNA

To construct the pIGA reporter plasmid, pIG46 (Ono et al. 1996), which contains the Cauliflower mosaic virus (CaMV) 35S minimal promoter (part of domain A of the CaMV 35S promoter, which contains the TATA box and extends from the −90 position to the transcription start site +1, was used as the “minimal promoter”) and glucuronidase (GUS) gene, was digested with XhoI, filled in using Klenow enzyme, and ligated with four copies of ABRE2. To prepare the effector plasmid, pBI221-ABP2, the full-length ABP2 cDNA was isolated from pGEM-TEasy-ABP2 by BamHI/SacI double digestion, and then cloned into pBI221 digested with BamHI/SacI. The effector plasmid, pBI221-ABP2, and reporter plasmid, pIGA, were co-transformed into maize Black Mexican Sweet (BMS) suspension cells using the biolistic particle delivery system. Briefly, the cells maintained in 50 mL of liquid MS medium (Sigma-Aldrich, USA) supplemented with 2.0 mg L–1 2,4-D and 3% sucrose were subcultured into fresh culture medium and treated with 3% PEG 8000 at 26–28°C in the dark on a shaker at 150 r min–1 overnight before bombardment. The plasmids were introduced into osmotically treated cells on glass microfiber filters (GF/A; Whatman) using a rupture pressure of 1 100 pounds per square inch (psi). After bombardment, the glass microfiber filters with the bombarded cells were transferred to MS agar medium and cultured at 26–28°C in the dark for 2 d. In situ assays of GUS activity were performed as described previously (Jefferson et al. 1987).

The methods for constructing the maize cDNA library and reporter plasmids, yeast transformation, and yeast one hybrid screening were described previously (Wang et al. 2002). By screening a maize cDNA library using the cis-element ABRE2 of the maize Cat1 promoter as bait, a clone was isolated and named ABP2. The fulllength ABP2 cDNA was cloned by RT-PCR amplification of transcripts from 17-dpp maize embryos using primers designed based on sequences of 5´-RACE fragments, and clones from the library were isolated. The 5´-RACE system used was from Gibco-BRL (USA). Total RNA was extracted from 17-dpp embryos. Template cDNA was synthesized with a cDNA-specific primer (ABP2 rv2: 5´-GCGACAACAGATGCGACGGT-3´) and general primer (G1: 5´-GGCACGCGTCGACTAGTACGGGGGGGGGG-3´). Two PCR primers were designed to test the RT-PCR product: ABP2 rv3: 5´-AGGAACAGAGTCTCCTCCTA-3´ and ABP2 FW2: 5´-GCGAGGAGCAGGACGCGCAA-3´. Then, AUAP 5´-GGCCACGCGTCGACTAGTAC-3´ and ABP2 rv3 were used to amplify the ABP2 cDNA. The target products were cloned into the pGEM-TEasy vector and sequenced.

2.3. Electrophoretic mobility shift assays To prepare recombinant ABP2 protein, pGEM-TEasy-ABP2 and pGEX-4T-1 were cut with EcoRI and XhoI restriction enzymes, and the target fragments were purified and ligated to generate the pGEX-4T-1-ABP2 construct. pGEX-4T-1ABP2 was transformed into Escherichia coli BL21 cells to produce a GST fusion protein. The GST-ABP2 fusion protein was purified using MicroSpin-GST purification modules (GE Healthcare, USA). Oligos (19-bp) containing ABRE2 (5´-GAAGTCCACGTGGAGGTGG-3´) or its mutant form mABRE2 (5´-GAAGTaacatgttcGGTGG-3´) were annealed and labeled with [g-32P]-ATP using a DNA 5´ End-Labeling System (Promega, USA). Binding reactions containing 4 μg of purified ABP2 protein, labeled probe DNA (20 000– 50 000 counts min–1 (cpm)), 25 mmol L–1 HEPES-KOH (pH 8.0), 50 mmol L–1 KCl, 10% glycerol, and 1 mmol L–1 dithiothreitol in a volume of 20 μL were incubated on ice for 15 min. When performing competition assays, unlabeled probes were added 30 min prior to addition of the 32P-labeled probes. Binding reactions were separated by 5% non-denaturing

2.4. Trans-activation assay in maize cells

2.5. Generation of transgenic Arabidopsis plants The full-length ABP2 cDNA fragment was obtained from pGEM-T-ABP2 by BamHI and XhoI digestion, and blunt-ended with Klenow enzyme. The cDNA was then cloned into XhoIdigested and blunt-ended pCHF3 to generate the pCHF335S-ABP2 binary vector. For green fluorescent protein (GFP) reporter construction, the full-length coding sequence of ABP2 without a stop codon was amplified with the primers 5´-GGGAGCTCTAGGCGGGGAAGATGGAGATG-3´ and 5´-CGCGGATCCCCATGGGCCAGTCAGGGTG-3´, and cut with SacI and BamHI; GFP was amplified with the primers 5´-CGCGGATCCGATCCCATGAGTAAAGGAGAAG-3´, and 5´-CGGGGTACCGATTGGCGTCGACTTATTTGTA-3´, and then cut with BamHI and KpnI; these two fragments were cloned into the pGEM-9Zf vector and cut with SacI and KpnI resulting in pGEM-9Zf-ABP2:GFP. To obtain pCHF3-35S-ABP2:GFP, the ABP2:GFP fragment was

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obtained by cutting pGEM-9Zf-ABP2:GFP with SacI and KpnI and blunt-ended, then cloned into the XbaI-digested and blunt-ended pCHF3 vector. The 35S promoter is a very strong constitutive promoter, causing high levels of gene expression in the entire dicot plant (Benfey and Chua 1990). The constructs were transduced into the Agrobacterium tumefaciens strain GV3101 by electroporation and used to transform A. thaliana using the floral dip method (Bechtold and Pelletier 1998). The seeds collected from floral dip plants were named transformation 1 generations (T 1) seeds, and the T1 plants were selected by kanamycin resistance. The transgenic positive plants were green, and the non-transgenic plants were yellow on the kanamycincontaining plates. The green plants were transferred to soil to complete their life cycle. The T1 plants were further confirmed by PCR analysis using primers, as follows: ABP2Fw11: 5´-CTCGAGTAGGCGGGGAAGATGGAGATG-3´ and ABP2-Rv11: 5´-ATCGATCCATGGGCCAGTCAGGGTG-3´. The positive plants seeds were harvested per plant and named T2 seeds. The T2 seeds were plated on kanamycincontaining plates. The green plants of the lines that showed a ratio of green plants:yellow plants=3:1 were transferred to soil, and the seeds were harvested per plant and named T3 seeds. The T3 lines (>50 seeds/line) were plated on kanamycin-containing plates. The lines that showed all green on the kanamycin-containing plates were selected as the homozygous transgenic lines. Three representative homozygous lines with similar ABP2 expression levels were chosen for detailed analyses.

2.6. Subcellular localization assay The roots of 3–4-day-old post germination seedlings of transgenic Arabidopsis plants overexpressing ABP2:GFP were used to examine the subcellular localization of ABP2. Confocal laser microscopy (TCS SP2; Leica) was used to examine the fluorescent signal of ABP2-GFP fusion.

2.7. Organ-specific expression analysis, ABA and stress treatments, quantitative RT-PCR analysis, and primers Field grown maize was used for the organ-specific expression analysis. The V6 and V10 stages of the root, stem (below the highest node), leaf (fully stretched youngest leaf), ear primordium, and tassel primordium were used. The root, stem (below the highest node), juvenile leaf (fully stretched youngest leaf), ear seat leaf, adult leaf (base blade), silk, ear, and tassel were used at the anthesis stage. The maize seeds were imbibed and incubated on moist filter paper at 28°C in the dark for 48 h for the ABA and stress treatment and were sown in vermiculite and grown in a

growth chamber (28°C, 12-h light/12-h dark photocycle) until the V2 (the second leaf unclasps the stem) stage. Dosage treatments tests were used to evaluate the appropriate treatment concentration of ABA, NaCl, and methyl viologen dichloride (MV). For the dosage treatment test, the roots were washed of vermiculite and immersed in various concentrations of ABA (0, 1.0, 10, or 100 μmol L–1), NaCl (0, 100, 200, or 300 mmol L–1), or MV (0, 100, 200, or 300 mmol L–1) solutions, and the leaves were sampled at 1 h. At least 10 plants were sampled for each dosage treatment, and the experiment was repeated three times. After the treatments, 10 μmol L–1 ABA, 100 mmol L–1 NaCl, and 1.0 μmol L–1 MV were chosen for a further time course treatment. V2 stage maize seedlings were immersed in 10 μmol L–1 ABA, 100 mmol L–1 NaCl, or 1.0 μmol L–1 MV solutions, and the entire plant was sampled after 0, 0.25, 0.5, 1, 3, 6, and 12 h for RNA preparation. At least 10 plants were sampled at each time point for each treatment, and the experiments were repeated three times. Total RNA was extracted using TRIzol Reagent (Galen, Beijing, China). First-strand cDNA was synthesized using SuperScript II reverse transcriptase (Promega, USA). The primers used to amplify ABP2 were: 5´-GGATGAGGTCCTGGAGCGAAT-3´ and 5´-CAACCGAACCTAAACAGCGAACT-3´. The primers used for amplification of the maize reference gene, actin1 (GenBank accession no. J01238), were: 5´-ACCTCACCGACCACCTAATG-3´ and reverse 5´-CTGAACCTTTCTGACCCAAT-3´. The primers used for the Arabidopsis reference gene actin2 were: forward 5´-CCAACAGAGAGAAGATGACT-3´ and reverse 5´-ATGTCTCTTACAATTTCCCG-3´. PCR amplification was performed in a 20-μL reaction volume containing primers, SYBR Premix, Ex Taq (TaKaRa, Japan), and diluted cDNA templates in a PTC-200 Thermo Cycler (BioRad, USA). The cycle threshold (CT) values, corresponding to the PCR cycle number at which fluorescence emission reached a threshold above baseline emission, were determined and the relative fold differences were calculated by the 2–ΔΔCT method using the actin gene as an endogenous reference and the stem tissue of maize was used for calibration (Livak and Schmittgen 2001). PCR was performed in triplicate in all cases. At least two experiments on independently grown plant materials were performed to confirm the reproducibility of the results. The primers used for verification of the microarray results are listed in Appendix A.

2.8. Stomatal movement assays To assay stomatal opening, detached whole rosette leaves of 4-wk-old plants were floated on buffer containing 20 mmol L–1 KCl, 5 mmol L–1 MES-KOH, and 1 mmol L–1 CaCl2

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(pH 6.15) (Pei et al. 2000), with or without 10 μmol L–1 ABA, under 120–150 μmol m–2 s–1 white light at 23°C. To assay stomatal closure, leaves were pre-floated on the above buffer for 2 h under 120–150 µmol m–2 s–1 white light at 23°C to open the stomata, and then transferred to the above buffer containing 10 µmol L–1 ABA for 2 h. The lower leaf epidermis was detached with forceps and the stomatal apertures were checked by Zeiss Axio Imager images were analyzed using Image J Software (http://rsbweb.nih.gov/ij/).

2.9. Arabidopsis drought stress tolerance test Arabidopsis drought stress tolerance tests were performed as reported previously (Zhang et al. 2011). Briefly, 10-dayold seedlings grown aseptically on 1/2 MS agar medium were transferred into pots containing equal amounts of compost soil and grown for another 1 or 2 weeks before application of drought stress treatments. For the drought stress tolerance assay, soil-grown plants were fully watered, and then irrigation was withheld for 3 weeks, followed by re-watering of the plants. Survival rates were scored 10 days after re-watering. At least three experiments on independently grown plant materials were performed to confirm the reproducibility of the results.

2.10. Salt tolerance test for plate-grown Arabidopsis plants Surface-sterilized seeds were sown on 1/2 MS medium and kept for 3 days at 4°C in the dark to break dormancy. The plates were then transferred to a growth chamber at 22°C, 120 μmol photons m–2 s–1, under a 16-h light/8-h dark photo cycle for 48 h, and then the seedlings were transferred to 1/2 MS with 200 or 225 mmol L–1 NaCl. Survival rates were recorded at 8 days after growth on salt plates. Fifty seedlings were used for each genotype in the experiment, and three independent biological replicates were performed.

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and 18 plants in each line were used to measure chlorophyll contents. The experiments were repeated at least three times. Values are the mean±SD.

2.12. Detection of ROS Superoxide anions and hydrogen peroxide were detected in situ using a histochemical staining procedure as described previously (Fryer et al. 2002). Arabidopsis plants grown in MS for 3–4 weeks were used for ROS detection. Rosette leaves sampled from the same position of seedlings were immersed in 50 mmol L–1 NaCl or 10 μmol L–1 ABA for 15 min, 30 min, or 1 h, and then washed three times with sterile water. Samples were carefully dried by blotting on paper, and the leaves were then infiltrated with nitroblue tetrazolium (NBT; Amresco, 1 mg mL–1 NBT in 10 mmol L–1 sodium azide and 10 mmol L–1 phosphate buffer, pH 7.8) or 3,3´-diaminobenzidine (DAB) solution (Sigma, 1 mg mL–1 DAB-HCl, pH 3.8). The stained leaves were imaged after removal of chlorophyll by boiling in 96% ethanol for 10 min. Three independent biological replicates were used in all assays.

2.13. Microarray assay Arabidopsis seeds were plated on 1/2 MS medium and grown in a growth chamber. After 4 weeks of growth, whole plants were sampled after immersion in 150 mmol L–1 NaCl for 0.25 or 6 h. Total RNA was isolated using an EASY Spin Kit (Galen, Beijing, China). Microarray analysis was performed by Bioss China, using Affymetrix GeneChip arrays for transcript profiling. Three independent biological replicates were used in the assay.

3. Results

2.11. Salt tolerance test for soil-grown Arabidopsis plants and determining the pigment concentration

3.1. ABP2 is a bZIP transcription factor that binds specifically to the ABRE2 motif of the maize Cat1 gene and transactivates gene expression by specific interaction with ABRE2 in plant cells

After growth for 7 days in growth chambers, seedlings were transferred into pots filled with equal amounts of compost soil and grown for another 1 or 2 weeks before salt stress treatments. For salt tolerance assays, soil-grown plants were subjected to high-salt stress by irrigation with NaCl solution in increasing concentrations (50, 100, and 200 mmol L–1) at 3-day intervals and maintained at a concentration of 200 mmol L–1 for 14 days when chlorophyll contents in the aboveground parts of the plants were measured according to the method described previously (Lichtenthaler 1987). Forty-five plants of each line were used to check survival,

We constructed a cDNA library using mRNA extracted from 17 dpp maize embryos (Wang et al. 2002). Using the ABRE2 sequence of the Cat1 promoter as bait, a group of His+ isolates was obtained from a screen of 1.3×106 yeast transformants. The corresponding proteins were designated as ABPs (Wang et al. 2002). One positive isolate was designated as ABP2. To verify the specificity of protein-DNA interaction between ABP2 and the ABRE2 sequence motif of the maize Cat1 gene, yeast strains pRS315 HIS-ABRE2 and pRS315 HIS-mABRE2 were transformed with pPC86ABP2 plasmid DNA. As shown in Fig. 1-A, yeast strains with

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ABRE2 grew on His– SC dropout medium, while strains with mABRE2 did not, indicating that ABP2 binds ABRE2 in a sequence-dependent manner and mutations in the ABRE2 core sequence abolish the ABP2-ABRE2 interaction. To obtain the full-length ABP2 sequence, we performed 5´RACE and cloned a 1 056-bp cDNA encoding the fulllength 351 amino acid sequence of ABP2. We registered the ABP2 cDNA at GenBank under accession no.: MF596177. The deduced ABP2 amino acid sequence was used to search GenBank and perform multiple sequence alignment (Appendix B); the results showed that ABP2 protein belongs to the bZIP transcription factor family. We determined whether ABP2 binds specifically to ABRE2

A

ABP2 ABRE2 mABRE2

+ + –

+ – +

B

Trp–Leu–

in vitro by electrophoretic mobility shift assay (EMSA) using purified GST-ABP2 fusion protein and radiolabeled ABRE2 or mABRE2 as the probe. Additionally, to examine the core binding sequence of ABRE2 responsible for binding to ABP2, the core sequence of ABRE2, “CCACGTGGA”, was mutated to form a series of ABRE2 mutants, m1–m9. As shown in Fig. 1-B, migration of radiolabeled ABRE2 was retarded by ABP2, while migration of radiolabeled mABRE2 was not, indicating that ABP2 binds specifically to ABRE2. The EMSA results also showed that ABP2 binds strongly to ABRE2 m1, m2, and m9, but not to m3–m8, indicating that ABP2 binds to the “ACGTGG” sequence in the core of ABRE2. To examine whether ABP2 can activate gene expression

ABRE2 mABRE2 m1 m2 m3 m4 m5 m6 m7 m8 m9

GAATCT CCACGTGGA GGTGG GAATGT a a c a t g t t c GGTGG GAATGT aCACGTGGA GGTGG GAATGT CaACGTGGA GGTGG GAATGT CCcCGTGGA GGTGG GAATGT CCAaGTGGA GGTGG GAATGT CCAC t TGGA GGTGG GAATGT CCcCG gGGA GGTGG GAATGT CCcCGT t GA GGTGG GAATGT CCcCGTG t A GGTGG GAATGT CCcCGTGG c GGTGG

mABRE2 ABRE2 m1 m2 m3 m4 m5 m6 m7 m8 m9 Trp–Leu–His–

C

D Vector control

GFP

Bright ABP2+ ABRE2

Merged

80 μm

80 μm

80 μm

Fig. 1 ABRE2 (ABA-responsive element 2) binding specificity, transactivation activity, and subcellular localization of ABP2 (ABRE binding protein 2). A, pPC86-ABP2 was transformed into yeast strains yABRE2 or ymABRE2 and grew on Trp–Leu– drop-out plates (upper panel), or Trp–Leu–His– drop-out plates (lower pannel). B, purified ABP2 protein was added to reactions containing 32 P-labeled ABRE2 or mABRE2 probes. Arrow indicates the ABP2-ABRE2 binding complex. Arrowhead indicates free probe. C, histochemical assay of transactivation activity of ABP2 in maize cells in suspension. A GUS reporter construct containing ABRE2 in the promoter was co-transformed by bombardment into maize cells with ABP2 effector plasmid (lower panel) or with empty vector as a background control (upper panel) and stained with X-Gluc buffer. Insets within the panels show magnified pictures of stained cells. D, subcellular localization of ABP2-GFP in transgenic Arabidopsis plants. Root cells were imaged through GFP and bright-field filter channels. Bars=80 μm.

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Relative transctipt level

V6 stage

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To examine its involvement in abiotic stress regulation, we examined ABP2 expression patterns in maize in response to abiotic stresses and ABA using qRT-PCR. As shown in Fig. 3-A, ABP2 expression was upregulated by NaCl, ABA, and methyl viologen dichloride (MV) in maize leaves after treatment of seedlings for 1 h, and the strongest induction was seen in response to 100 mmol L–1 NaCl, 100 µmol L–1 ABA, and 10 µmol L–1 MV. Furthermore, we performed 12-h time course ABP2 expression studies under conditions of dehydration, 100 mmol L–1 NaCl, 10 µmol L–1 ABA, and 1.0 µmol L–1 MV treatment. The results indicated that ABP2 was strongly upregulated by dehydration, with the peak at 0.5 h, and the upregulation was sustained for 12 h (Fig. 3-B). NaCl also rapidly induced ABP2 expression at 0.25 h, with the highest level of induction at 1 h, but ABP2 expression was suppressed at 3 h. Similarly, ABA also rapidly induced ABP2 expression, with an expression peak at 0.25 h, followed by suppression at 6 h. The expression

20

Ju v

3.3. Expression of ABP2 in maize is inducible by salt, oxidative, drought stresses, and ABA

To investigate the function of ABP2 in plant responses to

R

To investigate its function in maize, we examined ABP2 expression in various tissues of maize at different development stages by qRT-PCR. As shown in Fig. 2, ABP2 expression was detected in all tissues sampled, but its expression was especially strong in young ears at the V6 stage; at the V10 stage, roots and young tassels showed especially high levels of ABP2 expression; at the anthesis stage, strong ABP2 expression was also observed in the silk and ears. These observations indicated that ABP2 is involved in many aspects of plant development.

3.4. Constitutive expression of ABP2 in Arabidopsis leads to enhanced tolerance to abiotic stress and sensitivity to ABA

Relative transctipt level

3.2. ABP2 expression can be detected in various tissues of maize

pattern of ABP2 in response to 1.0 µmol L–1 MV showed two peaks at 1 and 12 h. Taken together, these results suggested that ABP2 is involved in plant responses to abiotic stress and ABA.

Relative transctipt level

through binding to ABRE2 in vivo, the GUS coding sequence of pBI221 was substituted by ABP2 to form pBI221-35SABP2 effector plasmid, and ABRE2 was inserted upstream of the 35S mini-promoter of pIG46 to obtain the pIGA46 reporter plasmid. pBI221-35S-ABP2 and pIGA46 were bombarded into maize cells in suspension culture. Detection of GUS protein expression by histochemical staining showed that the GUS reporter gene was activated in maize cells by ABP2 via interaction with the ABRE2 motif in the chimeric promoter (Fig. 1-C). These observations strongly suggested that ABP2 acts as a transcriptional activator in plant cells. To determine its subcellular localization, ABP2 was fused in-frame to the N-terminus of GFP and expressed under the control of the CaMV 35S promoter. Confocal imaging of GFP fluorescence in transgenic plants indicated that the ABP2GFP fusion protein was present in the nucleus (Fig. 1-D).

Fig. 2 ABRE binding protein 2 (ABP2) expression in various tissues at different developmental stages in maize. qRT-PCR analysis of ABP2 expression in maize at the V6 (the 6th leaf unclasps from the stem), V10 (the 10th leaf unclasps from the stem), and anthesis stages. Expression levels were normalized to the endogenous control (maize actin1) relative to the calibrator (stem tissue). The expression level in the stem tissue of each development stage was defined as 1. Data represent mean±SD of three replicates.

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1 10 ABA (μmol L –1)

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Fig. 3 ABRE binding protein 2 (ABP2) expression is upregulated by abscisic acid (ABA) and abiotic stress. A, qRT-PCR analysis of ABP2 expression under conditions of ABA, NaCl, or methyl viologen dichloride (MV) treatment for 1 h at various dosage. B, time course of ABP2 expression under treatment with 10 μmol L–1 ABA, 100 mmol L–1 NaCl, 10 μmol L–1 MV, or dehydration. Maize seedlings were pretreated with ABA, NaCl, and MV as described in the Materials and methods section. Expression levels were normalized to the endogenous control (maize actin1) relative to the control plants. The expression level in the control plants was defined as 1. Data represent mean±SD of three replicates.

abiotic stress and ABA, we generated transgenic Arabidopsis plants overexpressing the ABP2 cDNA under the control of the CaMV 35S promoter. Three representative lines with similar ABP2 expression levels, 24-2, 194-4, and 229-5, were selected for the study. First, drought stress tolerance of transgenic plants was compared to that of wild-type controls. Under normal conditions, the growth condition of the ABP2overexpressing and wild-type plants were not different at the vegetative or developmental stages (Appendix C). However, as shown in Fig. 4-A, after exposure to drought stress for 3 weeks, the transgenic plants were still green and vigorous, while most of the wild-type plants had become withered and bleached. After resuming watering, the survival rate of wild-type plants was 30%, while that of the transgenic plants was about 65%. These observations indicated that ABP2 expression in transgenic plants significantly increased drought tolerance. Next, as ABP2 expression was induced by ABA, we compared the ABA sensitivity of stomatal closure in ABP2-

overexpressing transgenic plants with wild-type controls. As shown in Fig. 4-B, the stomatal aperture of transgenic plants was smaller than that of wild-type plants in the absence of exogenously applied ABA. When treated with 10 µmol L–1 ABA, the stomatal apertures of transgenic lines 24-2, 194-4, and 229-5 were reduced by 46, 53, and 54% compared to untreated plants, respectively, while that of wild-type plants was reduced by 31%. These results suggest that overexpression of ABP2 reduced the stomatal aperture of plants and also increased the sensitivity of ABA-induced stomatal closure. We further investigated the response of seedling growth to NaCl by transferring 2-day-old seedlings from NaCl-free medium to medium containing NaCl. As shown in Fig. 4-C, there were no differences in growth status among the seedlings of ABP2-overexpressing and wild-type plants in the NaCl-free medium. However, with NaCl at 225 mmol L–1, most wild-type plants showed bleaching, while fewer bleached seedlings were found in ABP2-overexpressing

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Fig. 4 Constitutive expression of ABRE binding protein 2 (ABP2) in Arabidopsis thaliana leads to enhanced tolerance to abiotic stress and sensitivity to abscisic acid (ABA). A, drought tolerance in 35S-ABP2 Arabidopsis plants. Survival rates were scored 10 days after resuming watering. Col-0, all A. thaliana lines used were in the Columbia-0 background. 24-2, 194-4, and 229-5, three representative lines with similar ABP2 expression levels. The data are shown as mean±SD of three replicates with 45 plants per line each. B, stomatal aperture measurement of wild-type and 35S-ABP2 transgenic plants in response to 0 and 10 μmol L–1 ABA. Bars=2 μm. Values are mean±SD of three repeats with at least 75 stomata each. C, salt tolerance test of plate-grown plants. Survival rates were recorded at 8 days after growth on salt plates. The data are shown as mean±SD of three replicates with 50 plants per line each, three independent biological replicates were performed. D and E, salt tolerance test of soil-grown plants. The aboveground parts of plants were used to measure chlorophyll contents. Values in D are mean±SD of three repeats with at least 45 plants each; values in E are mean±SD of three repeats with at least 18 plants each. * and **, significant difference at P<0.05 and P<0.01, respectively (Student t-test).

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plants, and the survival rate of wild-type controls was also significantly lower than that of ABP2 transgenic plants. Similar results were obtained with seedlings on plates containing 200 mmol L–1 NaCl. To estimate the salt stress tolerance of transgenic plants, salt stress was imposed by watering with solutions containing progressively increasing concentrations of salt. After salt treatment, the wild-type plants showed leaf chlorosis and could neither flower nor fruit, while the transgenic plants showed green leaves, upright stems, and could successfully flower and fruit, and the survival rate of the wild-type controls was also significantly lower than that of the ABP2 transgenic plants (Fig. 4-D). Analysis of pigment contents indicated that the chlorophyll a/b and carotenoid contents of wild-type and transgenic plants did not differ significantly under normal growth conditions. However, after salt treatment, the chlorophyll a/b and carotenoid contents of transgenic plants were significantly higher than those of wild-type plants (Fig. 4-E). These results confirmed that ABP2 enhanced the salt tolerance of transgenic plants.

3.5. Constitutive expression of ABP2 in Arabidopsis leads to reduced ROS levels Since the ROS-generating agent MV upregulated ABP2 expression, we examined whether ABP2 is involved in control of ROS levels under normal growth conditions and in response to salt stress or ABA. For this purpose, we compared O2-· and H2O2 levels in 35S-ABP2 transgenic and wild-type plants under normal growth conditions and under salt stress/ABA treatment. The O2-· and H2O2 levels of plants under normal growth conditions and salt stress/ABA treatment were detected by staining. As shown in Fig. 5, under normal growth

10 μmol L–1 ABA 0

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conditions, the O2-· and H2O2 levels, visualized as dark blue and deep brown products, respectively, were lower in the three ABP2-overexpressing transgenic lines than in wildtype plants. After treatment with either 50 mmol L–1 NaCl or exposure to 10 µmol L–1 ABA for 0.25, 0.5, or 1 h, the levels of O2-· and H2O2 accumulation were also markedly lower in transgenic plants than in wild-type plants. These results indicated that ABP2 can influence ROS accumulation in plants under normal conditions and under salt stress/ ABA treatment.

3.6. Constitutive expression of ABP2 enhances stress-responsive and carbon metabolism-related gene expression To explore the molecular mechanism by which ABP2 regulates ABA signaling and ROS accumulation, and thus salt stress tolerance, we performed microarray analysis to examine global changes in gene expression in ABP2overexpressing transgenic plants in comparison to wild-type controls using the Arabidopsis Affymetrix GeneChip (USA). The chip assay showed that, after treatment with 150 mmol L–1 NaCl for 0.5 h, 20 genes exhibited expression level changes greater than twofold over wild-type plants in the 24-2 and 229-5 transgenic lines (Appendix D). To verify the microarray results, we analyzed the expression levels of these 20 upregulated genes by qRTPCR. As shown in Fig. 6, under normal conditions, the expression levels of these genes were elevated in transgenic plants in comparison with wild-type controls; in particular, the stress-responsive genes At5g52300 (RD29B), At1g52690 (LEA7), and At5g16980 (putative NADP-dependent oxidoreductase) showed 2–3 folds increases in expression in transgenic lines relative to the wild-type controls. All

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Fig. 5 Cellular levels of H2O2 and O2 · in leaves of wild-type and 35S-ABP2 plants in response to 10 μmol L–1 abscisic acid (ABA) and 50 mmol L–1 NaCl. Detached leaves from wild-type (Col-0) and transgenic plants (24-2, 194-4, and 229-5) pretreated with 50 mmol L–1 NaCl or 10 µmol L–1 ABA were stained with 3´-diaminobenzidine (DAB) and nitroblue tetrazolium (NBT) to visualize H2O2 and O2 ·, respectively. All experiments were repeated at least 3 times, and about 15 leaves collected from multiple seedlings (3–4-week-old) were inspected in each experiment.

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4

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Relative transcript level RA LF AT 3G L9 01 24 0 PG AT 3G A4 AT 012 3G 70 28 75 SK 0 S1 RD 3 29 B AG P2 3 AT AG 2G P6 AT 470 3G 50 07 82 0 P AT G A 1G 3 AT 107 5G 70 50 03 0 AT LEA 5G 7 19 AT 5 5G 80 50 36 0 VG D1 AC AT 5G A3 16 98 0

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Fig. 6 Constitutive ABRE binding protein 2 (ABP2) expression enhanced expression of stress-responsive and carbon metabolismrelated genes. Seedlings grown on agar plates were untreated or treated with 150 mmol L–1 NaCl for 0.25 and 6 h. Transcript levels of each gene detected by qRT-PCR, normalized to the endogenous control (actin2: At3g18780) relative to untreated plants as determined by the 2–ΔΔCT method. For each gene, the expression level in the wild-type plants was defined as 1. Data represent mean±SD of three replicates.

of the test genes showed upregulation after treatment with 150 mmol L–1 NaCl for 0.25 h; At1g02790 (PGA4), At3g07820 (PGA3), At2g47040 (pectin esterase, putative), and At3g01270 (pectate lyase family protein, putative) showed the highest levels of induction (>20-fold). The genes At3g01240 (polygalacturonase, putative), At5g52300 (RD29B), and At5g45880 (Pollen Ole e 1 allergen and

extensin family protein) showed >14-fold induction. After treatment with 150 mmol L–1 NaCl for 6 h, the expression of stress-responsive genes showed the highest levels of induction: At5g52300 (RD29B), >95-fold; At1g52690 (LEA7), >25-fold induction; and At5g16980 (putative NADPdependent oxidoreductase), >5-fold induction. A gene of unknown function, At5g50360, also showed high (15-fold)

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induction, while all of the other genes showed comparatively weak induction.

4. Discussion 4.1. ABP2 enhances plant drought and salt stress tolerance by modulating ROS balance, stomatal aperture, and cell damage protection In this study, using the ABRE2 sequence from the promoter of the ROS-scavenging gene Cat1 as bait in a yeast onehybrid assay, we identified the native maize ABRE2-binding protein, ABP2, from a 17-dpp maize embryo cDNA library. ABP2 was shown to belong to the bZIP transcription factor family, and its constitutive expression in Arabidopsis led to enhanced drought and salt stress tolerance by affecting ABA-mediated stomatal aperture closing (Fig. 4), ROS accumulation (Fig. 5), and defense gene expression (Fig. 6). In addition, under normal conditions, the growth of transgenic plants did not differ from that of wild-type plants, suggesting that ABP2 may be a good drought and salt tolerance candidate gene for molecular breeding. Various environment stresses can lead to ROS accumulation, which is a major cause of loss of crop productivity worldwide (Mittler et al. 2004; Neri et al. 2010; Wilson et al. 2014). ROS are highly reactive and toxic, and cause damage to proteins, lipids, carbohydrates, and DNA, ultimately resulting in cell death (Mittler et al. 2004). Thus, the ability to rapidly remove excess ROS is very important for plants to survive under conditions of stress. Similar to results previously shown for ABP9, which can regulate ROS levels to strengthen multi-stress tolerance of plants (Zhang et al. 2011), the O2 · and H2O2 accumulation levels of ABP2 transgenic plants under normal conditions or with 10 µmol L–1 ABA or 50 mmol L–1 NaCl treatment were all lower than those in wild-type plants, indicating that ABP2 also regulates production or scavenging of ROS to reduce injury to plants by stress and thus enhances their abiotic tolerance. We also found that the stomatal aperture was smaller in transgenic plants than in wild-type controls in the absence or presence of exogenously applied ABA (Fig. 4-B). Stomata, which function as epidermal valves, play central roles in gas exchange and water loss in plants (Qu et al. 2017). Under conditions of water stress, plants close their stomata as a defense response to minimize the loss of water (Singh et al. 2017). Many drought tolerance genes, such as ATDIF1, have been shown to interact with stomatal aperture (Gao et al. 2017). The results showed that chlorophyll a/b and carotenoid contents of the transgenic plants were significantly higher after the salt treatment than those of the wild-

type plants (Fig. 4-E). Many studies have reported that the loss of chlorophyll under saline conditions is related to photoinhibition or ROS formation and reduced O2 · absorption (Kato and Shimizu 1985; GarcıA ́ -Sánchez et al. 2002). Thus, transgenic plants protect chlorophyll a/b and carotenoids to maintain photosynthetic capacity under the salt treatment, meaning that they are more tolerant. In addition, to adjust the ROS balance and stomatal aperture, ABP2 upregulates expression of some stress tolerance proteins. Stress-responsive proteins, such as LEAs, chlorophyll a/b and carotenoid have been implicated in detoxification and alleviation of cellular damage during dehydration. LEA proteins may also function as chaperone-like protective molecules to combat cellular damage (Umezawa et al. 2006). It was suggested that LEA7 protein stabilizes enzymes during drying and freezing (Popova et al. 2011). The RD29A and RD29B genes are highly responsive to multiple abiotic stressors, including cold, drought, and salt (Yamaguchi-Shinozaki and Shinozaki 1993). Expression of RD29A and RD29B through ABA-dependent or -independent mechanisms confers tolerance to stresses (Jia et al. 2012). A. thaliana NADPH oxidoreductase homolog plays a distinct role as an antioxidant in plants (Babiychuk et al. 1995). Our data showed that expression of ABP2 in Arabidopsis leads to upregulation of stress-responsive genes; after treatment with 150 mmol L–1 NaCl for 6 h, the expression levels of the stress-responsive genes RD29B and LEA7 were highly induced. In addition, one gene putatively encoding an NADP-dependent oxidoreductase was also highly induced in ABP2 transgenic plants under normal or salt stress conditions. ABP2 enhances plant drought and salt stress tolerance by modulating ABA-mediated ROS balance, stomatal aperture, and cell damage protection.

4.2. ABP2 induces expression of a series of pectin esterase-related proteins The microarray results also showed that two exopolygalacturonases (PGA4 and PGA3), one pectin esterase putative protein, and one pectate lyase family putative protein were highly induced in ABP2-overexpressing plants. Exopolygalacturonase expression usually helps fruit to ripen, and is detected in many kinds of fruit (Downs and Brady 1990). Pectin esterase is an enzyme responsible for the demethylation of galacturonyl residues in high molecular weight pectin, and is believed to play an important role in cell wall metabolism (Phan et al. 2007). This enzyme has been reported in many plant tissues and is involved in many developmental processes, including cellular adhesion, stem elongation (Micheli 2001), pollen tube development (Bosch and Hepler 2005), abscission, and fruit ripening (Bosch

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and Hepler 2005). However, no study has investigated the relationships of these cell wall metabolism-related enzymes to abiotic stresses. We speculate that the high expression levels of these genes suggest that ABP2 is involved in some other developmental pathways in plants or these pectin esterase-related proteins may have functions under abiotic stress conditions. Further studies are required to examine these possibilities.

5. Conclusion In this study, the maize ABRE binding bZIP transcription factor ABP2 was identified by a yeast one-hybrid from a maize seed 17 dpp library using the ABRE2 motif of the maize Cat1 gene. The expression analysis showed that drought, salt, ROS-generating agents, and ABA treatmentinduced ABP2 expression, and constitutive expression of ABP2 in transgenic Arabidopsis plants led to enhanced tolerance to drought and salt stress and increased sensitivity to ABA.

Acknowledgements This work was supported by the National Natural Science Foundation of China (30870202), the National Key Research and Development Program of China (2016YFD0101002), and the National Special Program for Genetically Modified Organism (GMO) Development of China (2016ZX08003004). Appendices associated with this paper can be available on http://www.ChinaAgriSci.com/V2/En/appendix.htm

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