A novel wheat ASR gene, TaASR2D, enhances drought tolerance in Brachypodium distachyon

Plant Physiology and Biochemistry xxx (xxxx) xxx

Contents lists available at ScienceDirect

Plant Physiology and Biochemistry journal homepage: www.elsevier.com/locate/plaphy

Research article

A novel wheat ASR gene, TaASR2D, enhances drought tolerance in Brachypodium distachyon Jin Seok Yoon a, Jae Yoon Kim a, b, Dae Yeon Kim a, Yong Weon Seo a, * a b

Department of Plant Biotechnology, Korea University, Seongbuk-Gu, Seoul, 02841, Republic of Korea Department of Plant Resources, Kongju National University, Yesan, Chungnam, 32439, Republic of Korea

A R T I C L E I N F O

A B S T R A C T

Keywords: Wheat Drought Abscisic acid ABA-Induced protein Stress-induced protein Ripening-induced protein Stress-responsive genes Stomatal closure

Abscisic acid-, stress-, and ripening-induced (ASR) proteins play an important role in protecting plants against adverse environmental conditions. Here, we identified 24 ASR genes in the wheat genome and analyzed their characteristics. Among these, five ASR genes highly induced by abscisic acid (ABA) and polyethylene glycol were cloned and further characterized. The TaASR genes were expressed in response to different abiotic stresses and ABA and were found to be localized in the nucleus and plasma membrane of transformed tobacco cells. Bra­ chypodium distachyon transgenic plants overexpressing TaASR2D showed enhanced drought tolerance by regu­ lating leaf transpiration. The expression levels of stress-related and ABA-responsive genes were higher in transgenic plants than in wild-type plants under drought stress conditions. Moreover, overexpression of TaASR2D increased the levels of both endogenous ABA and hydrogen peroxide in response to drought stress, and these plants showed hypersensitivity to exogenous ABA at the germination stage. Furthermore, plants overexpressing TaASR2D showed increased stomatal closure. Further analysis revealed that TaASR2D interacts with ABA biosynthesis and stress-related proteins in yeast and tobacco plants. Collectively, these findings indicate that TaASR2D plays an important role in the response of plants to drought stress by regulating the ABA biosynthesis pathway and redox homeostasis system.

1. Introduction The growth and development of plants can be limited to varying extents by unfavorable environmental conditions, among which, drought is one of the major environmental stresses responsible for reduced crop productivity. Severe drought stress results in the inhibition of photosynthesis causing metabolic disorders. To cope with drought stress conditions, plants have developed a range of physiological and biochemical response mechanisms, including the control of water loss via the regulation of stomatal movement and enhancement of antioxi­ dant enzyme activity and redox homeostasis. Abscisic acid (ABA), a major plant hormone, plays pivotal roles in a diverse range of physiological processes, including stomatal movement, seed germination, and abiotic stress responses. ABA promotes stomatal closure under drought stress, thereby reducing transpiration and water loss from plant leaves (Acharya et al., 2009), and in this process, hydrogen peroxide (H2O2) plays an important role as a secondary messenger by increasing the calcium levels in guard cells through the activation of plasma membrane calcium channels (Bright et al., 2006;

Kwak et al., 2003; Wang et al., 2008). A previous study has revealed an ABA-independent stomatal closure mechanism in rice, in which a zinc finger transcription factor (DST) negatively regulates H2O2-induced stomatal closure by regulating the expression of genes related to H2O2 scavenging (Huang et al., 2009). Reactive oxygen species (ROS) play important signaling roles in acclimation processes, such as the growth and developmental responses of plants to abiotic stresses. Adverse environmental conditions typically result in increased ROS levels that can cause oxidative damage to membranes, proteins, and RNA and DNA molecules (Apel et al., 2004; Hancock et al., 2001). To mitigate such cellular damage, plants have evolved a complex ROS scavenging system that includes defense mechanisms that regulate ROS production to control cellular ROS con­ centrations (Bowler et al., 1992; Noctor and Foyer, 1998). Glutaredoxins (Grxs) are ubiquitous oxidoreductases in the thioredoxin (Trx) family that play vital roles in maintaining cellular redox homeostasis and regulating the redox-dependent signaling pathway. They utilize the reducing power of glutathione (GSH) to catalyze the reversible reduc­ tion of disulfide bonds of cognate target proteins (Rouhier et al., 2004),

* Corresponding author. E-mail address: [email protected] (Y.W. Seo). https://doi.org/10.1016/j.plaphy.2020.11.014 Received 24 August 2020; Accepted 13 November 2020 Available online 18 November 2020 0981-9428/© 2020 Elsevier Masson SAS. All rights reserved.

Please cite this article as: Jin Seok Yoon, Plant Physiology and Biochemistry, https://doi.org/10.1016/j.plaphy.2020.11.014

J.S. Yoon et al.

Plant Physiology and Biochemistry xxx (xxxx) xxx

and it has been found that the overexpression of OsGRX8 improves the salt and drought stress tolerance in Arabidopsis (Sharma et al., 2013). The abscisic acid-, stress-, ripening-induced (ASR) genes belong to a small gene family, the members of which are characterized by the presence of a conserved ABA/WDS domain (PF02469). Although this gene family has been identified in various plant species, including to­ mato, maize, and rice, ASR genes are conspicuously absent in the model plant Arabidopsis. All ASR proteins contain a zinc-binding domain at the N terminus and a putative nuclear targeting signal at the C terminus (Cakir et al., 2003). It has been reported that ASR genes are expressed in various organs and respond to abiotic stresses and ABA (Hu et al., 2013; Joo et al., 2013a; Zhang et al., 2015). The rice ASR genes OsASR1 and OsASR3 have been demonstrated to improve drought and salinity tolerance in transgenic rice plants (Joo et al., 2013b), and Brachypodium distachyon BdASR1 enhances drought tolerance through the activation of antioxidant systems, including ROS-scavenging enzymes and non-enzymatic antioxidants (Wang et al., 2016). Using the yeast one-hybrid approach, the grape ASR gene VvMSA has been shown to bind to the promoter of a hexose transporter gene, VvHT1, in carbohy­ drate and ABA signaling pathways (Cakir et al., 2003), and more recently, OsASR5 has been found to bind to the promoter of an ABC transporter gene, STAR1, which is required for aluminum tolerance (Arenhart et al., 2014). In addition, using the yeast two-hybrid approach, Saumonneau et al. (2008) demonstrated the interaction be­ tween the VvMSA gene and the VvDREB transcription factor. However, although ASR genes have been widely reported to respond to abiotic stresses, the underlying molecular mechanisms and the physiological role of ASR genes under stress conditions remain unclear. In this study, we identified ASR gene family members in wheat and characterized the responses of five selected TaASR genes to abiotic stresses and ABA. In addition, we analyzed the functions and molecular mechanisms of TaASR2D in transgenic plants exposed to drought stress.

displayed using the Gene Structure Display Server (http://gsds.cbi.pku. edu.cn/). Conserved motifs or domains of the TaASR proteins were predicted using the MEME suite web server (http://meme-suite.org/). 2.3. Plant materials, growth conditions and plant transformation For the purposes of this study, we used the Korean wheat variety Keumgang (IT 213100). Seeds were initially vernalized at 4 ◦ C for 4 weeks in a dark chamber and were subsequently grown in a Magenta plant culture boxes (6.5 × 6.5 × 20 cm; Greenpia Technology Inc., Yeoju, Korea) containing polypro mesh (polypropylene mesh opening 0.1 cm; Greenpia Technology Inc., Yeoju, Korea). The boxes filled with 180 mL of half-strength Hoagland nutrient solution no. 2 (H2395; Sigma-Aldrich, St Louis, MI, USA), which were then placed in a growth chamber set at 25/20 ◦ C (day/night), 16/8 h (day/night) photoperiod, and 65% humidity, with the Hoagland nutrient solution being exchanged each day. Three-week-old seedlings were subjected to treatment with 25% polyethylene glycol (PEG 6000) solution, 250 mM sodium chloride (NaCl), low temperature (4 ◦ C), 100 μM abscisic acid (ABA), or 100 mM hydrogen peroxide (H2O2), and the treated wheat seedling leaves were harvested at time intervals of 2, 6, 12, and 24 h. For tissue-specific expression analysis, we collected tissue samples from the leaves, stems, roots, peduncles, palea, glumes, lemma, anthers, stigmas, and ovaries of flowering plants. All harvested samples were stored at − 80 ◦ C until required for further expression analysis. To generate transgenic plants overexpressing the TaASR2D gene, the full-length coding sequence of TaASR2D was inserted into an expression cassette under the control of a maize ubiquitin promoter with the first intron and nos terminator, as described by Kim et al. (2017). Following digestion with PacI and BstBI and removal of the WRI expression cassette (Zale et al., 2016), the TaASR2D expression cassette was subcloned into pJK308NPTII immediately upstream of a selectable neomycin phos­ photransferase II (nptII) marker expression cassette (Supplementary Fig. S5A). The AGL1 strain of Agrobacterium tumefaciens harboring pJK308:TaASR2D was used to transform Brachypodium Bd21 plants following the procedure described by Yoon et al. (2019).

2. Materials and methods 2.1. Identification of wheat ASR genes To isolate TaASR gene family members, all predicted protein se­ quences from wheat were downloaded from EnsemblPlants release 31 (http://plants.ensembl.org). A seed file of the ABA/WDS domain (PF02469) was obtained from the PFAM database (https://pfam.xfam. org/) and HMM profiles were compiled using the ABA/WDS domain seed file as a query to search and select those proteins containing ASR/ WDS domains from wheat protein sequences using the HMMER 3.0 program (Finn et al., 2011) with a predefined threshold of E < 1e-5. Finally, selected proteins containing putative ASR domains in their peptide sequence were confirmed using InterProScan (https://www.ebi. ac.uk/interpro/search/sequence-search). The theoretical isoelectric point (IP) and molecular weight (MW) of the proteins thus identified were determined using the pI/Mw computation tool of the ExPASy server (https://web.expasy.org/compute_pi/). The chromosomal local­ ization of ASR genes was established based on Ensembl Plants release 31 (http://oct2017-plants.ensembl.org/index.html) using a BLASTN search, after which, graphical maps using MapChart software (Voorrips, 2002).

2.4. Gene expression analysis using quantitative reverse transcriptionpolymerase chain reaction (qRT-PCR) Total RNA was extracted from each of the different tissues using TRIzoll reagent (Invitrogen, Carlsbad, CA, USA). One microgram of total RNA was reverse transcribed to cDNA using a Power cDNA Synthesis kit (iNtRON Biotechnology, Seongnam, Gyeonggi, Korea) following the manufacturer’s instructions. Diluted cDNAs obtained from the tissues of treated wheat plants were used as templates for quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis using the fluorescent DNA intercalating dye SYBR Green (Applied Biological Materials Inc., Richmond, BC, Canada). qRT-PCR was performed using a CFX Connect Real-Time PCR Detection System (Bio-Rad, USA). Each reaction was performed using three biological replicates, with the Actin gene being used as an endogenous control for template cDNA normali­ zation. Fold-changes were calculated using the 2− ΔΔCT method (Livak et al., 2001). To evaluate the expression of stress-related and ABA-associated genes in Brachypodium transgenic and wild-type (WT) plants, qRT-PCR analysis was also conducted using RNA samples isolated from the tis­ sues of control and treated plants. All specific-primers were designed using Primer-BLAST (NCBI), the detailed information of which is pro­ vided in Supplementary Table S1.

2.2. Sequence characteristic analysis of TaASR genes Multiple alignment of the TaASR protein sequences obtained in the present study with those from other plant species was performed using Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/), and a phylogenetic tree was subsequently constructed based on the neighborjoining (NJ) method with 1000 bootstrap replicates using MEGA 7 software (Kumar et al., 2016). The exon–intron organization and splicing phase of these predicted TaASR genes were also investigated based on the wheat genome database in Ensembl Plant and graphically

2.5. Yeast two-hybrid, subcellular localization and biomolecular fluorescence complementation assays A wheat cDNA library in a pGADT7-AD vector was generated from the mixed leaves of 21-day-old seedlings treated with PEG and ABA for 2

J.S. Yoon et al.

Plant Physiology and Biochemistry xxx (xxxx) xxx

6 h using the Make Your Own “Mate&Plate™” Library System (Clontech, Palo Alto, CA, USA). A full-length coding sequence of TaASR2D was amplified via PCR using specific primers, and was subsequently cloned into a pGBKT7-BD vector. Yeast two-hybrid screening was conducted according to the manufacturer’s instructions (Matchmaker™ Gold Yeast Two-Hybrid System and Yeastmaker™ Yeast Transformation System; Clontech, USA). We also cloned the full-length coding regions of the genes of three interacting proteins (Supplementary Table S3) in a pGADT7-AD vector and co-transformed this with pGBKT7-TaASR2D into the AH109 yeast strain. The co-transformants were selected on SD medium without Leu and Trp, and then transferred to an X-α-gal-con­ taining SD medium lacking Ade, His, Leu, and Trp. For subcellular localization assay, full-length coding sequences, without the stop codon of TaASRs and interacting proteins, were inserted into binary vector pMDC83, and the recombinant vectors were introduced into Agrobacterium tumefaciens strain GV3101 using the freeze–thaw method described by Chen et al. (1994). Following infil­ tration, the tobacco plants were placed in a growth chamber under normal growth conditions for 3 days, after which the fluorescence of GFP fusion proteins was observed using a confocal laser scanning mi­ croscope (LSM 700; Carl Zeiss, Don Mills, ON, CA). For the biomolecular fluorescence complementation (BiFC) assay, full-length sequences of TaASR2D and interacting proteins were inserted into the pGTQL1211-YN and pGTQL1221-YC vectors, respectively. The BiFC constructs were introduced into Agrobacterium tumefaciens strain GV3101, and infiltrated into 4-week-old tobacco leaves. After 3 days of infiltration, the leaves were observed using a laser confocal scanning microscope.

2.8. Stomatal aperture assay Leaves of 7-week-old WT plants and plants overexpressing TaASR2D that had been exposed to drought stress (without irrigation for 5 days) and normal conditions were detached and directly fixed using 2.5% glutaraldehyde for 24 h at 4 ◦ C. The specimens were then sequentially dehydrated using ascending concentrations of ethanol (50%, 70%, 80%, 90%, 95%, and 100%, for 10 min each). Dehydrated samples were dried using the critical point drying (CPD) method (Araujo et al., 2003) by flooding with liquid carbon dioxide at 5 ◦ C for 20 min and raising the temperature to the critical point (32 ◦ C). For scanning electron micro­ scopy, the samples were sputter-coated with gold, and images of the stomata were obtained using a JSM-6700F scanning electron microscope (JEOL, Peabody, MA, USA). The percentages of stomata that were completely open, partially open, or completely closed were determined from these images. Four fully expanded leaves were used to measure stomatal conductance using an SC-1 leaf porometer (Dekagon Devices, Pullman, WA, USA). The measurements were conducted under light conditions between 09:00 and 11:00. 2.9. Measurement of hydrogen peroxide and total glutathione Histochemical assays for H2O2 were performed using 3,3-diamino­ benzidine (DAB) staining as described by Lim et al. (2014). Four fully expanded leaves of 7-week-old transgenic TaASR2D-overexpressing and WT plants exposed to drought stress and normal conditions were collected and immersed in DAB solution for 8 h at 25 ◦ C in the dark. The leaf samples were then placed in a bleaching solution (ethanol:acetic acid:glycerol 3:1:1), boiled for 15 min, and photographed. To the measurement of H2O2 content, H2O2 was extracted from the leaves and quantified as described by Uchida et al. (2002). Total GSH and oxidized GSH (GSSG) were measured using the DTNB [5,5′ -dithiobis(2-nitrobenzoic acid)] recycling method (Rahman et al., 2006). For the GSSG assay, 2 μL of 2-vinylpyridine was added to the neutralized supernatant and left for 1 h at room temperature to mask GSH, and the samples were analyzed as described by Rahman et al. (2006). GSH levels were determined by subtracting the level of GSSG from the total level of glutathione.

2.6. Stress treatment and physiology assays To examine the effects of abiotic stresses on the TaASR2D gene in Brachypodium, we used 7-week-old plants overexpressing TaASR2D and WT plants grown in soil. For the drought treatment, T3 homologous transgenic and WT plants were grown for 10 days without irrigation. After re-watering for 15 days, the survival rate was determined ac­ cording to the vitality level. Samples obtained from treated and nontreated plants were used to measure physiological parameters. To measure relative water content (RWC) and rate of leaf water loss, the leaves from 7-week-old transgenic and WT plants under non-stress and drought conditions were used for RWC and rate of water loss. RWC was calculated according to a standard method (Barrs and Weatherley, 1962). The loss of leaf water content was measured as described by Lim et al. (2014). The excised leaves were placed on clean filter papers and allowed to dry for up to 360 min.

2.10. Statistical analysis Student’s t-tests were performed using SAS™ v9.3 to determine if mean values were obtained for wild-type and transgenic plants. Different letters indicate significant differences compared to the control (p < 0.05). 3. Results

2.7. Endogenous abscisic acid levels and an exogenous abscisic acid sensitivity assay

3.1. Identification of the TaASR gene family in wheat In order to identify TaASR genes, we used the HMMER, BLASTP, and InterProScan programs to search for genes in the wheat reference genome that contain the ABA/WDS domain (PF02464). We identified a total of 24 ASR genes within the wheat genome, which were located at eight, seven, and nine loci in the wheat A, B, and D sub-genomes, respectively (Supplementary Table S2). The relative positions of these loci on chromosomes were identified on genetic maps (Supplementary Fig. S1A), and the genes were renamed from TaASR1 to TaASR10 based on the order of chromosomal location. Given that TaASR1 on chromo­ some 4 has previously been reported, the newly identified TaASR genes on this chromosome were renamed starting from TaASR2, and the TaASR homologous genes were regarded as equivalent partners based on the closest matching genes of protein and DNA sequences (Supple­ mentary Table S2). All TaASR genes exhibited a common ASR structure containing two exons and one intron as well as conserved motifs (Sup­ plementary Figs. S1B–D). Amino acid sequence analysis of the 24

To determine endogenous ABA concentrations, 100 mg of the ground leaves of transgenic and WT plants that had been exposed to drought stress and normal conditions were homogenized in extraction buffer (80% methanol; 1 mM butylated hydroxytoluene) and shaken overnight at 4 ◦ C. The resulting extracts were then centrifuged at 4000×g for 20 min. The supernatant thus obtained was eluted through a Sep-Pak C18 cartridge and dried in a speed vacuum at 4 ◦ C, and the residues were dissolved in 0.01 M PBS buffer (10 mM phosphate, 137 mM NaCl, 2.7 mM KCl and pH 7.5). ABA content was determined based on an enzymelinked immunosorbent assay (ELISA) using a Phytodetek ABA test kit (Agdia, Elkhar, IN, USA) according to the manufacturer’s instructions. To assess the response to exogenous ABA at the germination stage, seeds of two independent TaASR2D-overexpressing lines and WT plants were germinated on MS medium containing 1 μM ABA at 24 ◦ C under a 16/8 h light/dark photoperiod in a growth chamber and the germination rates were determined for 10 days after initiation. 3

J.S. Yoon et al.

Plant Physiology and Biochemistry xxx (xxxx) xxx

TaASRs revealed that the length of the sequences ranged from 91 to 290, the molecular weight (MW) ranged from 10.1 to 31.5 kDa, the isoelectric point (pI) ranged from 4.9 to 10.1, and the grand average of hydro­ pathicity (GRAVY) values ranged from − 1.75 to − 0.91 (Supplementary Table S2). To investigate the phylogenetic relationships of the TaASR genes, we conducted a phylogenetic analysis of TaASRs and members of other ASR gene families from different plant species, and found that TaASR proteins showed close relationships with a range of other ASR family proteins (Fig. 1A). To examine the response of these genes to drought stress, we

performed qRT-PCR using specific primers (Supplementary Table S1). Among the 24 ASR genes, we found that five (TaASR1A, TaASR2D, TaASR5A, TaASR8D, and TaASR9D) showed transcript levels over 2.5fold higher than other ASR genes in response to ABA and PEG treat­ ments (Supplementary Fig. S2), and these genes were selected for further analysis. The amino acid sequences of the five TaASRs were aligned with the ASR amino acid sequences of other species using Clustal Omega. Multiple alignment of the sequences of TaASRs revealed the presence of a C-terminal nuclear localization site (NLS) (Fig. 1B).

Fig. 1. Phylogenetic relationships and multiple alignments of the deduced amino acid sequences of TaASR proteins with those of other ASR proteins. (A) The phylogenetic tree was constructed using the neighbor-joining algorithm with 1000 bootstraps. Red squares represent target genes. (B) Multiple alignments were analyzed using Clustal Omega. The red box indicates the ABA/WDS domain; the single underlines denote the nuclear localization site (NLS). The accession numbers of genes are as follows: Q08655 (SlASR1), P37219 (SlASR2), P372520 (SlASR3), AAY98032 (SlASR4), Os01g72900 (OsASR1), Os01g72910 (OsASR2), Is02g33820 (OsASR3), Os04g34600 (OsASR4), Os11g06720 (OsASR5), Os01g73250 (OsASR6), ACZ60138 (MaASR1), ACZ50739 (MaASR2), ACZ50754 (MaASR3), ACZ50736 (MaASR4), ACZ60133 (MbASR1), ACZ50744 (MnASR2), GRMZM2g136910 (ZmASR1), GRMZM5g854138 (ZmASR2), GRMZM2g044132 (ZmASR3), GRMZM2g168552 (ZmASR4), GRMZM2g052100 (ZmASR5), GRMZM2g057841 (ZmASR6), GRMZM2g014797 (ZmASR7-1), GRMZM2g314075 (ZmASR7-2), GRMZM2g383699 (ZmASR7-3), BAJ89693 (HvASR1), NAJ89693 (HvASR2), MLOC_60871 (HvASR1-1), MLOC_5645 (HvASR2-1), MLOC_10955 (HvASR3), XP_020198893 (AtASR1), EMS67162 (TuASR1), EMS68095 (TuASR2), AF281656 (VvMSA), Bradi2g61590 (BdASR1), Bradi2g61600 (BdASR2), Bradi2g61607 (BdASR3), Bradi4g24650 (BdASR4), Bradi5g10027 (BdASR5), and ADQ85915.1 (TaASR1). 4

J.S. Yoon et al.

Plant Physiology and Biochemistry xxx (xxxx) xxx

3.2. Expression profile and subcellular localization of TaASR genes

the exception of TaASR8D, these genes showed up-regulated expression in response to NaCl treatment. In plants exposed to cold treatment, we observed an increase in the transcript levels of TaASR5A, whereas expression levels of the other ASRs did not differ significantly. Following ABA treatment, the expression level of all TaASRs showed an upregulated pattern, whereas in plants exposed to H2O2, we observed in­ creases in the expression of TaASR1A, TaASR2D, and TaASR9D from 6 h to 12 h, which subsequently decreased at 24 h. In contrast, the expres­ sion levels of TaASR5A and TaASR8D showed no notable differences in response to H2O2 treatment. In order to examine the subcellular localization of TaASRs, we were transformed into tobacco epidermal cells. All TaASR-GFP fusion proteins were accordingly detected in the nucleus and plasma membranes of tobacco cells (Supplementary Fig. S4).

To characterize the expression profiles of TaASR, we examined the expression patterns of TaASR genes in different tissues based on qRTPCR analysis (Supplementary Fig. S3). We found that TaASRs were differentially expressed in all tissues assessed: leaf, stem, root, peduncle, palea, glume, lemma, anther, stigma, and ovary. TaASR1A and TaASR2D were found to have higher expression in the roots than in the other tissues, whereas TaASR9D was highly expressed in the peduncle and glum, and the transcript levels of TaASR5A were markedly increased in leaves. To determine the responses of TaASR genes to abiotic stresses and the phytohormone ABA, we examined the expression patterns of these TaASRs in plants exposed to different treatments, namely, PEG, NaCl, low temperature (4 ◦ C), ABA, and H2O2 (Fig. 2). All five selected TaASR genes showed up-regulated expression under PEG treatment, and with

Fig. 2. Expression patterns of TaASR genes under abiotic stress and abscisic acid (ABA) treatment. Three-week-old wheat seedlings were treated for different lengths of time (0, 2, 6, 12, and 24 h), and leaf samples were used for qRT-PCR. TaActin was used as an internal control. (A–E) Expression patterns of TaASR genes in plants subjected to 25% PEG6000, 100 μM ABA, 250 mM NaCl, 100 mM H2O2, and low temperature (4 ◦ C). Data represent the means ± SD calculated from three bio­ logical replicates. 5

J.S. Yoon et al.

Plant Physiology and Biochemistry xxx (xxxx) xxx

3.3. Overexpression of TaASR2D improves drought tolerance in transgenic brachypodium plants

stress, the RWC of the leaves of plants overexpressing TaASR2D was higher than that of WT plants (Fig. 3C). In contrast, the leaf water loss rate was found to be lower in plants overexpressing TaASR2D than in WT plants (Fig. 3D).

To investigate the potential functions of TaASR2D in response to drought and osmotic stress, we introduced a TaASR2D overexpression cassette into the Brachypodium callus via the Agrobacterium-mediated transformation method, and subsequently obtained seven independent overexpressing transgenic lines of TaASR2D. Among these, we selected two lines containing a single copy, which showed high transgene tran­ scription levels to functionally characterize TaASR2D (Supplementary Fig. S5). Seven-week-old seedlings of transgenic overexpression (OE) and wild-type Brachypodium plants grown in the soil were subjected to drought stress treatment, in which irrigation was withheld for 10 days; after this, the seedlings were re-watered for 15 days. We found that under normal conditions, transgenic (OE3 and OE5) and WT plants showed no notable differences in phenotypes, whereas after 10 days of drought stress, WT plants exhibited significant leaf rolling compared with the transgenic plants. Moreover, after 15 days of recovery, trans­ genic plants of OE3 and OE5 showed 63.3% and 70.0% survival, respectively, whereas only 30% of the WT plants survived (Fig. 3A and B). To evaluate physiological changes, we measured the RWC and leaf water loss rate of seedling leaves. RWC is one of the indicato rs routinely used to determine drought tolerance, and we found that under drought

3.4. Overexpression of TaASR2D regulates stress-related and ABAresponsive genes under drought stress conditions To determine the molecular mechanisms by which TaASR2D confers tolerance to drought stress, we examined the expression levels of selected stress-related (BdCBF1, BdCBF2, BdCBF3, BdDREB2A, BdRAB16, and BdWRKY36) and ABA-responsive (BdAAO3, BdNCED3, BdERD1, and BdRAB18) genes in transgenic and WT plants under normal and drought stress conditions (Fig. 4). Under normal conditions, we observed little difference in the expression levels of stress-related and ABA-responsive genes in transgenic and WT plants. The expression levels of stress-related, ABA-responsive, and ABA-biosynthesis genes were found to be higher in transgenic plants than in WT plants under drought stress conditions. The one exception in this regard was BdERD1, the expression levels of which were lower in OE plants than in WT plants.

Fig. 3. Overexpression of TaASR2D enhanced the drought tolerance of Brachypodium plants. (A) The phenotypes of transgenic and wild-type (WT) plants under drought stress. (B–D) A physiological drought stress tolerance assay of transgenic and WT plants under drought stress. (B) Survival of transgenic and WT plants was examined after 15 days of re-watering. (C) Relative water content, (D) water loss rate of transgenic and WT plants under normal conditions and after 10 days of drought stress. Data represent the means ± SD (n = 10) of three replicates. Asterisks indicate significant differences between the transgenic and WT plants (*P < 0.05, **P < 0.01). 6

J.S. Yoon et al.

Plant Physiology and Biochemistry xxx (xxxx) xxx

Fig. 4. Expression patterns of stress-related and abscisic acid (ABA)-responsive genes in transgenic and wild-type (WT) plants under normal and drought stress conditions. Data represent the means ± SD calculated from three biological replicates. Asterisks indicate significant differences between the transgenic and WT plants (*P < 0.05, **P < 0.01). The accession numbers of genes are as follows: Bradi1g56667 (BdAAO3), Bradi1g13760 (BdNCED3), Bradi5g08920 (BdERD1), Bra­ di4g19525 (BdRAB18), Bradi3g51630 (BdCBF1), Bradi1g49560 (BdCBF2), Bradi4g35650 (BdCBF3), Bradi2g04000 (BdDREB2A), Bradi2g47575 (BdRAB16), Bra­ di5g20290 (BdWRKY36), and Bradi4g00660 (BdUBC18).

3.5. Y2H assay, BiFC assay, subcellular localization, and expression profiles of TaASR-interacting proteins

response to NaCl and H2O2 treatment at 24 h, whereas the transcript levels of TaGrx increased in response to PEG, NaCl, and H2O2 treatments after 6 h. The transcripts of all interacting proteins were up-regulated in seedlings exposed to the ABA treatment. In addition, using qRT-PCR, we analyzed the Brachypodium genes BdASR1, BdGrx and BdSDR, which are highly homologous to TaASR9D, TaGrx and TaSDR, respectively, under normal and drought stress conditions in TaASR2D-overexpressing and WT plants (Fig. 6). The expression levels of BdASR1, BdGrx and BdSDR genes were not significantly different under normal condition, while their expression levels under drought stress conditions were higher in OE3 and OE5 than in WT plants.

In our analysis of proteins that interact with TaASR2D, we identified eight positive clones based on yeast two-hybrid screening, three of which were confirmed to be unique interacting proteins: abscisic stressripening protein 1 (TaASR9D), short-chain dehydrogenase/reductase (TaSDR), and glutaredoxin (TaGrx). TaASR2D and full-length sequences of the three interacting proteins were co-transformed into yeast cells and subsequently selected on DDO and QDO/X media. We observed that all selected proteins showed strong interactions with TaASR2D in yeast cells (Fig. 5A). To elucidate the function of TaASR2D and the interacting proteins, we performed a BiFC assay in tobacco epidermal cells (Fig. 5B). We found that the pGTQL1221-YC:TaASR9D and pGTQL1211-YN:TaASR2D constructs were localized in the nucleus and plasma membrane, whereas pGTQL1221-YC:TaSDR was detected in the plasma membrane; pGTQL1221-YC:TaGrx showed strong signals in the nucleus and plasma membrane along with TaASR2D. To investigate the subcellular locali­ zation of the interacting proteins, we conducted a transient expression assay in tobacco epidermal cells, and accordingly observed that TaSDR was localized in the plasma membrane, whereas TaGrx was localized in the nucleus and plasma membrane (Supplementary Fig. S6). To further examine the expression patterns of interacting proteins, we determined the transcript levels of these proteins in response to abiotic stresses and ABA based on qRT-PCR analysis (Supplementary Fig. S7). We found that the TaSDR transcripts showed high expression in

3.6. TaASR2D increased the endogenous ABA content and ABA sensitivity of transgenic brachypodium plants To evaluate the effect of overexpressing TaASR2D on the sensitivity of plants to exogenous ABA, we compared the responses of transgenic lines (OE3 and OE5) and WT plants to ABA treatment at the germination stage. Sterilized seeds of T3 transgenic and WT plants were placed on MS medium containing 0 and 1 μM ABA. We observed that whereas 10-dayold transgenic and WT plants showed similar phenotypes on MS medium lacking ABA, the transgenic seeds showed delayed germination compared with WT seeds on MS medium containing 1 μM ABA (Fig. 7A). After 5 days, approximately 61.1% of WT seeds, and 22.2% and 27.8% of OE3 and OE5 transgenic seeds, respectively, had germinated, whereas after 10 days, the germination percentages of WT, OE3, and OE5 seeds were 94.4%, 44.4%, and 55.6% respectively (Fig. 7B). 7

J.S. Yoon et al.

Plant Physiology and Biochemistry xxx (xxxx) xxx

Fig. 5. Identification of proteins interacting with the TaASR2D protein. (A) A yeast two-hybrid assay of TaASR2D-interacting proteins. The full-length TaASR2D and each of the interaction proteins (TaASR9D, TaSDR, and TaGrx) were cloned into the pGBKT7 and pGADT7 vectors, respectively. BD-TaASR2D was co-transformed with AD-interacting proteins in the AH109 yeast strain. (B) A biomolecular fluorescence complementation assay of TaASR2D with interacting proteins in tobacco epidermal cells. Each of the interacting proteins was cloned into pGTQL1221-YC and TaASR2D was cloned into pGTQL1211-YN. YN-TaASR2D was co-transfected with YC-interacting proteins into tobacco leaves.

Our analysis of endogenous ABA content in seedling leaves revealed no obvious differences between TaASR2D-overexpressing and WT plants under normal conditions, whereas under drought stress conditions, ABA content was notably higher in the transgenic plants (21.7–24.5 ng/g FW) than in WT plants (14.6 ng/g FW) (Fig. 7C).

3.7. TaASR2D increases hydrogen peroxide content under drought stress To examine the accumulation of H2O2 in response to drought stress, we measured the H2O2 content of TaASR2D-overexpressing and WT plants under drought stress and normal conditions. Under normal con­ ditions, H2O2 content in the leaves of transgenic plants did not differ 8

J.S. Yoon et al.

Plant Physiology and Biochemistry xxx (xxxx) xxx

Fig. 6. Expression levels of TaASR2D and orthologous interacting proteins in plants overexpressing TaASR2D and wild-type (WT) plants under normal and drought stress conditions. Data represent the means ± SD calculated from three biological replicates. Asterisks indicate a significant difference between the transgenic and WT plants (*P < 0.05, **P < 0.01). The accession numbers of genes are as follows: Bradi2g61590 (BdASR1) Bradi5g15220 (BdGrx) and Bradi1g37175 (BdSDR).

significantly from those in WT plants, as determined by DAB staining analysis. However, under drought stress conditions, we observed that transgenic plants showed a larger number of reddish-brown spots than did WT plants under drought stress conditions (Fig. 7D), and detected higher levels of H2O2 production in the leaves of plants overexpressing TaASR2D (Fig. 7E).

conditions, we measured total GSH content in the leaves of TaASR2Doverexpressing and WT plants. Although we did not detect significant differences in the total GSH content in transgenic and WT plants under normal conditions, plants overexpressing TaASR2D showed higher total GSH content under drought stress condition than WT plants did (Fig. 9A). Furthermore, in both transgenic and WT plants, we detected higher GSH/GSSG ratios in plants subjected to drought stress than in those exposed to normal conditions, with the ratios being higher in plants overexpressing TaASR2D (Fig. 9B).

3.8. Effect of TaASR2D overexpression on stomatal closure To investigate the responses of stomata under drought stress and normal conditions, we examined the leaf stomata of TaASR2D-over­ expressing and WT plants using scanning electron microscopy (Fig. 8A). Observations indicated that the number of stomata in the leaves of overexpressing plants did not differ significantly from those in WT plants (Fig. 8C). Similarly, the percentages of completely closed, partially open, and completely open stomata showed no obvious differences be­ tween the transgenic and WT plants under normal conditions. However, under drought stress conditions, 48.7% and 48.7% of stomata were completely and partially closed, respectively, in plants overexpressing TaASR2D, whereas in WT plants, 24% of stomata were completely closed and 69.3% were partially closed (Fig. 8B). Stomatal conductance was found to be lower in plants overexpressing TaASR2D than in WT plants under drought stress conditions (Fig. 8D).

4. Discussion Since the first discovery of the tomato ASR1 gene, ASR gene families have been identified in numerous plant types, including five in tomato, nine in maize, five in Brachypodium, and six in rice (Wang et al., 2016; Uchida et al., 2002; Philippe et al., 2010; Virlouvet et al., 2011). Several studies have subsequently revealed that ASR genes are involved in the response to abiotic stresses and hormones (Cakir et al., 2003; Kim et al., 2009; Park et al., 2020; Perez-Diaz et al., 2019; Shkolnik et al. 2008). To date, however, there have been no genome-wide studies of the ASR gene family in wheat, and little information is available regarding the role played by wheat ASR genes in mediating abiotic stress and hormone responses. In this study, we identified and characterized 24 TaASR genes in the wheat genome database for genome-wide analysis. In addition, we used B. distachyon, a monocot model plant that is closely related to major monocot cereal crops, such as wheat, barley, and oats, to generate transgenic plants.

3.9. Effects of TaASR2D on total glutathione content under drought stress To examine the redox status of GSH under drought stress and normal 9

J.S. Yoon et al.

Plant Physiology and Biochemistry xxx (xxxx) xxx

Fig. 7. Abscisic acid (ABA) sensitivity and accumulation of ABA and hydrogen peroxide (H2O2) in TaASR2D-overexpressing and wild-type (WT) plants. (A) The phenotypes of TaASR2D-overexpressing and WT plants in response to exogenous ABA. (B) Germination rates of the transgenic and WT plant seeds subjected to 1 μM ABA treatment for 10 days (n = 12). (C) ABA content of TaASR2D-overexpressing and WT plants under normal and drought stress conditions. (D) DAB staining for H2O2 in the leaves of TaASR2D-overexpressing and WT plants under normal and drought stress conditions. (E) H2O2 content of TaASR2D-overexpressing and WT plants under normal and drought stress conditions. Data represent the means ± SD calculated from three replicates. Asterisks indicate significant differences between the transgenic and WT plants (*P < 0.05, **P < 0.01).

The ASR genes in wheat comprise two exons and a single intron and contain a conserved ABA/WDS domain (Supplementary Fig. S1), which is consistent with the ASR gene family members characteristic of other plants (Liang et al., 2019; Huang et al., 2016), and the phylogenetic tree constructed in this study revealed that TaASRs show are closely related to the ASR genes of other species (Fig. 1A). Previous studies have indi­ cated that ASR genes are expressed in response to abiotic stresses and hormones and exhibit tissue-specific expression in plants (Joo et al., 2013b; Takasaki et al., 2008). In the present study, we examined the transcript patterns of five TaASR genes in different tissues and in response to abiotic stresses and ABA treatment, and accordingly observed different patterns of expression in different plant tissues

(Supplementary Fig. S3). This is similar to OsASRs that show specific expression patterns in different organs of rice (Perez-Diaz et al., 2014). We found that the transcript levels of all examined TaASR genes were up-regulated in response to treatments with PEG, H2O2, and ABA, whereas only TaASR5A expression increased after cold treatment (Fig. 2). These results are consistent with the findings of previous studies that have indicated that ASR genes may play a role in the responses to abiotic stress and hormones (Hu et al., 2014; Yang et al., 2005). Thus, there is reasonable evidence to indicate that TaASRs are expressed in a tissue-specific manner as well as in response to multiple abiotic stresses and ABA. Most ASR proteins have been shown to be localized in both the 10

J.S. Yoon et al.

Plant Physiology and Biochemistry xxx (xxxx) xxx

Fig. 8. Stomatal aperture status of TaASR2D-overexpressing and wild-type (WT) plants. (A) Scanning electron microscope images of three states of stomatal ap­ ertures (completely closed, partially open, and completely open). (B) Percentages of the three states of stomatal apertures in the leaves of TaASR2D-overexpressing and WT plants (n = 150 stomata for WT and OE3 under normal and drought stress conditions). (C) Number of stomata in the middle leaves of transgenic and WT plants (n = 3). (D) Stomatal conductance of the transgenic and WT plants under normal and drought stress conditions (n = 3). Data represent the means ± SD. Asterisks indicate significant differences between the transgenic and WT plants (*P < 0.05, **P < 0.01).

Fig. 9. Effect of drought stress on total glutathione content and the ratio of GSH to GSSG in TaASR2D-overexpressing and wild-type (WT) plants. (A) Total GSH content of transgenic and WT plant leaves under normal and drought stress conditions. (B) The GSH/GSSG ratio indicating the redox state in the leaves of transgenic and WT plants under drought stress. Total glutathione (GSH + GSSG) and GSSG were measured in transgenic and WT plant leaves under normal and drought stress conditions. GSH; reduced glutathione, GSSG; oxidized glutathione. Data represent the means ± SD calculated from three biological replicates. Asterisks indicate significant difference between the transgenic and WT plants (*P < 0.05, **P < 0.01).

nucleus and cytosol; in the present study, we detected TaASR proteins in the nucleus and plasma membrane (Supplementary Fig. S4). Given that the SiASR4 protein, a known transcription factor, is localized in both the nucleus and plasma membrane in tobacco epidermal cells, and that SiASR4 shows altered expression patterns in response to abiotic stress and ABA treatments (Li et al., 2017a), we suspect that TaASRs may also exhibit dual localization and regulate responses to multiple stresses. The ASR gene transcription factors SlASR1 and LLA23, which have similar structural features and harbor a putative nuclear targeting signal at the C terminus, have been found to enhance tolerance to salt and

drought stress by regulating the transcription of downstream genes (Yang et al., 2005; Kalifa et al., 2004). In the present study, we similarly identified a putative C-terminal nuclear targeting signal in the nuclearand plasma membrane-localized TaASR2D gene, which showed higher transcript levels in response to abiotic stresses and ABA treatment (Fig. 1B). Based on these observations, we speculate that TaASR2D may play multiple roles as a defense regulator or transcription factor in response to abiotic stress. To examine the functions of TaASR2D under abiotic stress condi­ tions, we generated transgenic Brachypodium plants overexpressing 11

J.S. Yoon et al.

Plant Physiology and Biochemistry xxx (xxxx) xxx

TaASR2D and found that two T3 homologous single-copy transgenic lines (OE3 and OE5) showed improved growth performance under drought stress conditions (Fig. 3A), as well as enhanced survival rates and water retention capacity (Fig. 3B–D). These findings indicate that plants overexpressing TaASR2D may confer a heightened resistance to drought stress-induced damage. To gain a better understanding of the molecular mechanisms un­ derlying the function of TaASR2D, we analyzed the expression of selected stress-related and ABA-responsive genes (Fig. 4). Accordingly, we found that the expression of the stress-related BdCBF, BdDREB2A, and BdWRKY36 genes was relatively higher in transgenic plants than in WT plants under drought stress conditions, which is consistent with the findings that BdCBF genes contribute to enhanced abiotic stress toler­ ance (including drought and cold stress) in transgenic plants (Ryu et al., 2014). Transcription factors, including DREB2A and WRKY36, enhance abiotic stress tolerance (Sun et al., 2015; Sakuma et al., 2006), and in the present study, we found that the expression of the ABA-responsive genes BdAAO3, BdNCED3 and BdRAB18 was higher in transgenic plants than in WT plants under drought stress conditions (Fig. 4). In contrast, expression levels of BdERD1 were lower in transgenic plants. ERD1 is induced not only by dehydration, but is also up-regulated during senescence (Simpson et al., 2003), and it has been demonstrated that NAC transcription factors bind to the promoter of ERD1 and affect the transcript levels of several stress-related genes in Arabidopsis under drought and salt stress (Tran et al., 2004). Thus, these observations indicate that the TaASR2D gene might play multiple roles as a regulator and/or transcription factor in the expression of stress-related and ABA-responsive genes under drought stress. ABA plays a vital role in the adaptive response of plants to abiotic stress, and one of the well-established physiological mechanisms asso­ ciated with the response to ABA is stomatal closure (Acharya et al., 2009). In maize, ZmASR3 regulates the expression of ABA-responsive genes and confers an improvement in tolerance to drought stress (Liang et al., 2019). Similarly, Brachypodium BdASR4 confers drought resistance and affects the expression of ABA-responsive genes, and also shows sensitivity to exogenous ABA in transgenic plants (Yoon et al., 2019). In the present study, we found that the overexpression of TaASR2D conferred hypersensitivity to exogenous ABA at the seed germination stage, and that endogenous ABA content in plants over­ expressing TaASR2D was higher than that in WT plants under drought stress conditions (Fig. 7A–C). Moreover, we found that TaASR2D regu­ lates the transcription of ABA biosynthesis genes (BdAAO3, BdNCED3, and BdSDR) in response to drought stress (Figs. 4 and 6) and interacts with the TaSDR gene in the plasma membrane of tobacco epidermal cells (Fig. 5). 9-Cis-epoxycarotenoid dioxygenase (NCED3) is a key enzyme involved in ABA biosynthesis, and in Arabidopsis, AtNCED3 is induced by drought stress and controls endogenous ABA levels under drought stress conditions (Seo et al., 2002). Overexpression of AtNCED3 not only in­ creases endogenous ABA levels but also the transcription of stress-related and ABA-responsive genes, thereby enhancing tolerance to dehydration stress in Arabidopsis (Iuchi et al., 2001). The short-chain dehydrogenase/reductases (SDRs) are a large family of enzymes, most of which are known to be NAD(P)(H)-dependent oxidoreductases. It has been reported that SDRs play catalytic roles in a wide range of reactions, including lipid, carbohydrates, amino acid, and hormone metabolism (Kavanagh et al., 2008; Persson et al., 1991, 2003). In the ABA biosynthesis pathway of Arabidopsis, the SDR gene AtABA2 catalyzes the conversion of xanthoxin to ABA aldehyde, which in turn is converted to ABA via the action of ABA-aldehyde oxidase (AAO3) (Cheng et al., 2002; Seo et al., 2004). Furthermore, it has been found that the exogenous application of ABA reduces H2O2 content in aba2-1 mutant plants sub­ jected to osmotic stress (Ozfidan et al., 2012). These results indicate that TaASR2D may play a role in the ABA biosynthesis pathway of wheat in response to drought stress. Recently, the molecular mechanisms underlying stomatal movement integrated by environmental signaling and ion channels have been

studied in plants (Daszkowska-Golec et al., 2013; Desikan et al., 2004; Pei et al., 2000), including the role of H2O2 as a second messenger in ABA-mediated stomatal closure (Bright et al., 2006; Zhang et al., 2001). Hydrogen peroxide generation is dependent on ABA content and is essential for ABA-induced stomatal movement in plants (Kwak et al., 2003; Wang et al., 2008), and in the present study, we observed the accumulation of H2O2 in plants overexpressing TaASR2D, as detected by DAB staining, as well as increased H2O2 and ABA levels under drought stress conditions (Fig. 7C–E). Furthermore, these plants exhibited increased stomatal closure and decreased stomatal conductance in response to drought stress (Fig. 8). These findings thus tend to indicate that TaASR2D is involved in H2O2-mediated stomatal closure via an ABA-dependent pathway under drought stress conditions. In a previous study, the rice genes OsASR1 and OsASR5 were observed to act in concert and complementarily to regulate gene expression in response to aluminum (Arenhart et al., 2016). Similarly, we detected an interaction between TaASR2D and TaASR9D in the nu­ cleus and plasma membrane of tobacco epidermal cells (Fig. 5B). Moreover, the expression profiles of TaASR2D and TaASR9D showed complementary expression patterns in response to PEG and NaCl treat­ ments (Fig. 2). In response to PEG treatment, the expression of TaASR2D increased at 6 h and subsequently decreased, whereas TaASR9D tran­ scripts increased at 12–24 h. Likewise, in plants exposed to NaCl, TaASR2D transcripts increased at 6–12 h and subsequently decreased, whereas TaASR9D transcripts increased at 24 h. These results indicate that TaASR2D and TaASR9D may play beneficial roles in tolerance to drought and salt stress. Glutathione plays important roles in the maintenance of redox ho­ meostasis, detoxification of heavy metals, and regulation of cellular signaling in response to oxidative stress. Glutathione can exist in both reduced glutathione (GSH) and oxidized glutathione (GSSG) in which two molecules are linked via a disulfide bond (Rouhier et al., 2008). Glutaredoxins (Grxs), also known as thio-disulfide oxidoreductases, have diverse functions in plant development, signal transduction, and stress responses by playing roles in mitigating abiotic stress, either via direct ROS scavenging or redox regulation of target proteins (Rouhier et al., 2004; Cheng et al., 2011; Sundaram et al., 2010; Wu et al., 2017). Grxs can regulate protein activity by reversible glutathionylation or reduction of disulfide bonds of target proteins to maintain cellular redox homeostasis (Rouhier et al., 2004). Recently, overexpression of SlGRX1 in Arabidopsis has been shown to promote tolerance to oxidative, drought, and salt stresses; it also modulates the expression of stress-related genes (Guo et al., 2010). Overexpression of OsGrxs in Arabidopsis shows drought tolerance, compared to WT plants, by enhancing the antioxidant enzyme defense system, as well as molecules including GSH content GSH/GSSG ratio and GST (Kumar et al., 2020). In the present study, we found that TaASR2D interacted with TaGrx in the plasma membrane and nucleus in tobacco epidermal cells (Fig. 5B). We observed up-regulation of TaGrx transcripts in response to PEG, NaCl, H2O2, and ABA. Orthologous BdGrx transcripts were higher in plants overexpressing TaASR2D than in WT plants under drought stress (Sup­ plementary Fig. S7 and Fig. 6). Furthermore, overexpression of TaASR2D was found to increase the total GSH content and GSH/GSSG ratios of plants exposed to drought stress (Fig. 9). These results imply that TaASR2D may also be involved in the cellular redox homeostasis system by regulating or interacting with Grx. On the other hand, over­ expressing TaASR2D plants has been shown to increase H2O2 content compared to WT plants. Recently, Li et al. (2017b) reported that OsASR5 overexpressing plants showed increased H2O2 accumulation via decreased APX activity, and OsARS5 overexpression affected the decrease in expression levels of DST, a negative regulator of genes related to H2O2 homeostasis, compare with WT plants, under drought stress conditions. Based on the different strands of evidence obtained in the present study, TaASR2D might play multiple roles in the response of plants to drought stress, as depicted in Fig. 10.

12

J.S. Yoon et al.

Plant Physiology and Biochemistry xxx (xxxx) xxx

Fig. 10. A proposed model depicting the function of TaASR2D in drought stress tolerance. Under drought stress conditions, overexpression of TaASR2D pro­ motes the up-regulated expression of abscisic acid (ABA) biosynthesis and stress-related genes such as BdNECD3, BdSDR, and BdGrx. Furthermore, TaASR2D interacts with the ABA biosynthesis gene TaSDR, leading to ABA and H2O2 accumulation. ABA and H2O2 accumulation induce stomatal closure, resulting in the conservation of leaf water content and thus enhanced drought tolerance. Drought stress causes oxidative damage via the excessive production of reactive oxygen species (ROS), the accumulation of which stimulates the synthesis of glutathione (GSH), which regulates cell redox homeostasis. The accu­ mulation of oxidized GSH (GSSG) does not disturb the redox potential if it is compensated by an increase in the total concentration of GSH. TaASR2D interacts with TaGrx in response to drought stress, resulting in an increase in both GSH content and the GSH/GSSG ratio. TaASR2D thus appears to play a defensive role in the redox homeostasis system by maintaining cellular redox potential and enhancing drought tolerance. Furthermore, TaASR2D and the interacting TaASR9D show up-regulated expression under drought stress conditions. Collectively, the findings of this study indicate that TaASR2D might play impor­ tant roles in the tolerance of wheat plants to drought stress.

5. Conclusions

that TaASR2D plays multiple roles in the regulation of drought stress tolerance via an ABA-dependent pathway and the redox homeostasis system. Although the precise mechanisms underlying the functions of TaASR2D and the interacting proteins in drought stress tolerance require further study, our results provide new evidence for the role of TaASR2D in mediating the modulation of stomatal closure and regulation of the cell redox system in plants exposed to drought stress.

In this study, we identified 24 TaASR genes in wheat, and investi­ gated the characteristics of five genes selected from among them. The function of one of these (TaASR2D) was examined in detail for its role in responding to drought and salt stress. Compared with WT plants, transgenic Brachypodium plants overexpressing TaASR2D showed enhanced tolerance to drought and salt stress. TaASR2D promoted tolerance to drought stress by upregulating the expression levels of stress-related and ABA-responsive genes. Furthermore, plants over­ expressing TaASR2D were found to be sensitive to exogenous ABA, which was associated with increases in endogenous ABA content and H2O2 accumulation in plants subjected to drought stress. Hydrogen peroxide accumulation due to increased ABA content resulted in increased stomatal closure, thereby reducing transpiration and conserving leaf water, which ultimately enhanced drought stress toler­ ance. In addition, our observations indicate that TaAsR2D may play a role in an ABA-dependent pathway and the redox homeostasis system, given that it interacts with ABA biosynthesis and redox homeostasisrelated genes, including TaSDR and TaGRX. These findings indicate

Author contribution statement JSY conceived and designed the experiments. JSY performed ex­ periments, analyzed data, and wrote the manuscript with support from JYK and YWS. JYK carried out vector construction and Southern blot analysis. DYK carried out wheat genome data analysis. YWS contributed to valuable discussions. All authors have discussed the results and approved the final manuscript. Declaration of competing interest The authors declare that they have no known competing financial 13

J.S. Yoon et al.

Plant Physiology and Biochemistry xxx (xxxx) xxx

interests or personal relationships that could have appeared to influence the work reported in this paper.

tolerance by gene manipulation of 9-cis-epoxycarotenoid dioxygenase, a key enzyme in abscisic acid biosynthesis in Arabidopsis. Plant J. 27, 325–333. Joo, J., Lee, Y.H., Choi, D.H., Cheong, J.J., Kim, Y.K., Song, S.L., 2013a. Rice ASR1 has function in abiotic stress tolerance during early growth stages of rice. J. Korean Soc. Appl. Biol. Chem. 56, 349–352. Joo, J., Lee, Y.H., Kim, Y.K., Nahm, B.H., Song, S.L., 2013b. Abiotic stress responsive rice ASR1 and ASR3 exhibit different tissue-dependent sugar and hormone-sensitivities. Mol. Cell 35, 421–435. Kalifa, Y., Perlson, E., Gilad, A., Konrad, Z., Scolnik, P.A., Bar-Zvi, D., 2004. Overexpression of the water and salt stress-regulated Asr1 gene confers an increased salt tolerance. Plant Cell Environ. 27, 1459–1468. Kavanagh, K.L., Jornvall, H., Persson, B., Oppermann, U., 2008. Medium- and shortchain dehydrogenase/reductase gene and protein families : the SDR superfamily: functional and structural diversity within a family of metabolic and regulatory enzymes. Cell. Mol. Life Sci. 65, 3895–3906. Kumar, A., Dubey, A.K., Kumar, V., Ansari, M.A., Narayan, S., Meenakshi, Kumar, S., Pandey, V., Shirke, P.A., Pande, V., Sanyal, I., 2020. Overexpression of rice glutaredoxin genes LOC_Os02g40500 and LOC_Os01g27140 regulate plant responses to drought stress. Ecotoxicol. Environ. Saf. 200, 110721. Kim, S.J., Lee, S.C., Hong, S.K., An, K., An, G., Kim, S.R., 2009. Ectopic expression of a cold-responsive OsAsr1 cDNA gives enhanced cold tolerance in transgenic rice plants. Mol. Cell 27, 449–458. Kim, J.Y., Nong, G., Rice, J.D., Gallo, M., Preston, J.F., Altpeter, F., 2017. In planta production and characterization of a hyperthermostable GH10 xylanase in transgenic sugarcane. Plant Mol. Biol. 93, 465–478. Kumar, S., Stecher, G., Tamura, K., 2016. MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol. Biol. Evol. 33, 1870–1874. Kwak, J.M., Mori, I.C., Pei, Z.M., Leonhardt, N., Torres, M.A., Dangl, J.L., Bloom, R.E., Bodde, S., Jones, J.D., Schroeder, J.I., 2003. NADPH oxidase AtrbohD and AtrbohF genes function in ROS-dependent ABA signaling in Arabidopsis. EMBO J. 22, 2623–2633. Li, J., Dong, Y., Li, C., Pan, Y., Yu, J., 2017a. SiASR4, the target gene of SiARDP from Setaria italica, improves abiotic stress adaption in plants. Front. Plant Sci. 7, 2053. Li, J., Li, Y., Yin, J., Jiang, J., Zhang, M., Guo, X., Ye, Z., Zhao, Y., Xiong, H., Zhang, Z., Shao, Y., Jiang, C., Zhang, H., An, G., Paek, N.C., Ali, J., Li, Z., 2017b. OsASR5 enhances drought tolerance through a stomatal closure pathway associated with ABA and H2O2 signalling in rice. Plant Biotechnol. J. 15, 183–196. Liang, Y., Jiang, Y., Du, M., Li, B., Chen, L., Chen, M., Jin, D., Wu, J., 2019. ZmASR3 from the maize ASR gene family positively regulates drought tolerance in transgenic Arabidopsis. Int. J. Mol. Sci. 20, 2278. Lim, S.D., Lee, C., Jang, C.S., 2014. The rice RING E3 ligase, OsCTR1, inhibits trafficking to the chloroplasts of OsCP12 and OsRP1, and its overexpression confers drought tolerance in Arabidopsis. Plant Cell Environ. 37, 1097–1113. Livak, K.J., Schmittgen, T.D., 2001. Analysis of relative gene expression data using realtime quantitative PCR and the 2− ΔΔCT method. Methods 25, 402–408. Noctor, G., Foyer, C.H., 1998. Ascorbate and glutathione: keeping active oxygen under control. Annu. Rev. Plant Physiol. Plant Mol. Biol. 49, 249–279. Ozfidan, C., Turkan, I., Sekmen, A.H., Seckin, B., 2012. Abscisic acid-regulated responses of aba2-1 under osmotic stress: the abscisic acid-inducible antioxidant defense system and reactive oxygen species production. Plant Biol. 14, 337–346. Park, S.I., Kim, J.J., Shin, S.Y., Kim, S.Y., Yoon, H.S., 2020. ASR enhances environmental stress tolerance and improves grain yield by modulating stomatal closure in rice. Front. Plant Sci. 10, 1752. Pei, Z.M., Murata, Y., Benning, G., Thomine, S., Klusener, B., Allen, G.A., Grill, E., Schroeder, J.I., 2000. Calcium channels activated by hydrogen peroxide mediate abscisic acid signalling in guard cells. Nature 406, 731–734. Perez-Diaz, J., Wu, T.M., Perez-Diaz, R., Ruiz-Lara, S., Hong, C.Y., Casaretto, J.A., 2014. Organ- and stress-specific expression of the ASR genes in rice. Plant Cell Rep. 33, 61–73. Perez-Diaz, J., Perez-Diaz, J.R., Medeiros, D.B., Zuther, E., Hong, C.Y., Nunes-Nesi, A., Hincha, D.K., Ruiz-Lara, S., Casaretto, J.A., 2019. Transcriptome analysis reveals potential roles of a barley ASR gene that confers stress tolerance in transgenic rice. J. Plant Physiol. 238, 29–39. Persson, B., Krook, M., Jornvall, H., 1991. Characteristics of short-chain alcohol dehydrogenases and related enzymes. Eur. J. Biochem. 200, 537–543. Persson, B., Kallberg, Y., Oppermann, U., Jornvall, H., 2003. Coenzyme-based functional assignments of short-chain dehydrogenases/reductases (SDRs). Chem. Biol. Interact. 143–144, 271–278. Philippe, R., Courtois, N., McNally, K.L., Mournet, P., El-Malki, R., Le Paslier, M.C., Fabre, D., Billot, C., Brunel, D., Glaszmann, J.C., This, D., 2010. Structure, allelic diversity and selection of Asr genes, candidate for drought tolerance. In: Oryza Sativa L. And Wild Relatives. Theor. Appl. Genet, vol. 121, pp. 769–787. Rahman, I., Kode, A., Biswas, S.K., 2006. Assay for quantitative determination of glutathione and glutathione disulfide levels using enzymatic recycling method. Nat. Protoc. 1, 3159–3165. Rouhier, N., Gelhaye, E., Jacquot, J.P., 2004. Plant glutaredoxins: still mysterious reducing systems. Cell. Mol. Life Sci. 61, 1266–1277. Rouhier, N., Lemaire, S.D., Jacquot, J.P., 2008. The role of glutathione in photosynthetic organisms: emerging functions for glutaredoxins and glutathionylation. Annu. Rev. Plant Biol. 59, 143–166. Ryu, J.Y., Hong, S.Y., Jo, S.H., Woo, H.C., Lee, S., Park, C.M., 2014. Molecular and functional characterization of cold-responsive C-repeat binding factors from Brachypodium distachyon. BMC Plant Biol. 14, 15. Sakuma, Y., Maruyama, K., Qin, F., Osakabe, Y., Shinozaki, K., Yamaguchi-Shinozaki, K., 2006. Dual function of an Arabidopsis transcription factor DREB2A in water-stress-

Acknowledgments This work was carried out with the support of the “Next-Generation BioGreen21 Program for Agriculture & Technology Development (Project No. PJ01103501)” Rural Development Administration, Re­ public of Korea. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.plaphy.2020.11.014. References Acharya, B.R., Assmann, S.M., 2009. Hormone interactions in stomatal function. Plant Mol. Biol. 69, 451–462. Apel, K., Hirt, H., 2004. Reactive oxygen species: metabolism, oxidative stress, and signal transduction. Annu. Rev. Plant Biol. 55, 373–399. Araujo, J.C., Teran, F.C., Oliveira, R.A., Nour, E.A., Montenegro, M.A., Campos, J.R., Vazoller, R.F., 2003. Comparison of hexamethyldisilazane and critical point drying treatments for SEM analysis of anaerobic biofilms and granular sludge. J. Electron. Microsc. 52, 429–433. Arenhart, R.A., Bai, Y., de Oliveira, L.F.V., Neto, L.B., Schunemann, M., Maraschin, F.D., Mariath, J., Silverio, A., Sachetto-Martins, G., Margis, R., Wang, Z.Y., MargisPinheiro, M., 2014. New insights into aluminum tolerance in rice: the ASR5 protein binds the STAR1 promoter and other aluminum-responsive genes. Mol. Plant 7, 709–721. Arenhart, R.A., Schunemann, M., Bucker-Neto, L., Margis, R., Wang, Z.Y., MargisPinheiro, M., 2016. Rice ASR1 and ASR5 are complementary transcription factors regulating aluminium responsive genes. Plant Cell Environ. 39, 645–651. Barrs, H., Weatherley, P., 1962. A re-examination of the relative turgidity technique for estimating water deficits in leaves. Aust. J. Biol. Sci. 15, 413–428. Bowler, C., Vanmontagu, M., Inze, D., 1992. Superoxide-dismutase and stress tolerance. Annu. Rev. Plant Physiol. Plant Mol. Biol. 43, 83–116. Bright, J., Desikan, R., Hancock, J.T., Weir, I.S., Neill, S.J., 2006. ABA-induced NO generation and stomatal closure in Arabidopsis are dependent on H2O2 synthesis. Plant J. 45, 113–122. Cakir, B., Agasse, A., Gaillard, C., Saumonneau, A., Delrot, S., Atanassova, R., 2003. A grape ASR protein involved in sugar and abscisic acid signaling. Plant Cell 15, 2165–2180. Chen, H., Nelson, R.S., Sherwood, J.L., 1994. Enhanced recovery of transformants of Agrobacterium tumefaciens after freeze-thaw transformation and drug selection. Biotechniques 16, 664–668, 670. Cheng, N.H., Liu, J.Z., Liu, X., Wu, Q.Y., Thompson, S.M., Lin, J., Chang, J., Whitham, S. A., Park, S., Cohen, J.D., Hirschi, K.D., 2011. Arabidopsis monothiol glutaredoxin, AtGRXS17, is critical for temperature-dependent postembryonic growth and development via modulating auxin response. J. Biol. Chem. 286, 20398–20406. Cheng, W.H., Endo, A., Zhou, L., Penney, J., Chen, H.C., Arroyo, A., Leon, P., Nambara, E., Asami, T., Seo, M., Koshiba, T., Sheen, J., 2002. A unique short-chain dehydrogenase/reductase in Arabidopsis glucose signaling and abscisic acid biosynthesis and functions. Plant Cell 14, 2723–2743. Daszkowska-Golec, A., Szarejko, J., 2013. Open or close the gate–stomata action under the control of phytohormones in drought stress conditions. Front. Plant Sci. 4, 138. Desikan, R., Cheung, M.K., Bright, J., Henson, D., Hancock, J.T., Neill, S.J., 2004. ABA, hydrogen peroxide and nitric oxide signalling in stomatal guard cells. J. Exp. Bot. 55, 205–212. Finn, R.D., Clements, J., Eddy, S.R., 2011. HMMER web server: interactive sequence similarity searching. Nucleic Acids Res. 39, W29–W37. Guo, Y., Huang, C., Xie, Y., Song, F., Zhou, X., 2010. A tomato glutaredoxin gene SlGRX1 regulates plant responses to oxidative, drought and salt stresses. Planta 232, 1499–1509. Hancock, J., Desikan, R., Neill, S., 2001. Role of reactive oxygen species in cell signalling pathways. Biochem. Soc. Trans. 29, 345–350. Hu, W., Huang, C., Deng, X., Zhou, S., Chen, L., Li, Y., Wang, C., Ma, Z., Yuan, Q., Wang, Y., Cai, R., Liang, X., Yang, G., He, G., 2013. TaASR1, a transcription factor gene in wheat, confers drought stress tolerance in transgenic tobacco. Plant Cell Environ. 36, 1449–1464. Hu, Y.X., Yang, X., Li, X.L., Yu, X.D., Li, Q.L., 2014. The SlASR gene cloned from the extreme halophyte Suaeda liaotungensis K. enhances abiotic stress tolerance in transgenic Arabidopsis thaliana. Gene 2, 243–251. Huang, K., Zhong, Y., Li, Y., Zheng, D., Cheng, Z.M., 2016. Genome-wide identification and expression analysis of the apple ASR gene family in response to Alternaria alternata f. sp. Mali. Genome 59, 866–878. Huang, X.Y., Chao, D.Y., Gao, J.P., Zhu, M.Z., Shi, M., Lin, H.X., 2009. A previously unknown zinc finger protein, DST, regulates drought and salt tolerance in rice via stomatal aperture control. Genes Dev. 23, 1805–1817. Iuchi, S., Kobayashi, M., Taji, T., Naramoto, M., Seki, M., Kato, T., Tabata, S., Kakubari, Y., Yamaguchi-Shinozaki, K., Shinozaki, K., 2001. Regulation of drought

14

J.S. Yoon et al.

Plant Physiology and Biochemistry xxx (xxxx) xxx Uchida, A., Jagendorf, A.T., Hibino, T., Takabe, T., Takabe, T., 2002. Effects of hydrogen peroxide and nitric oxide on both salt and heat stress tolerance in rice. Plant Sci. 163, 515–523. Virlouvet, L., Jacquemot, M.P., Gerentes, D., Corti, H., Bouton, S., Gilard, F., Valot, B., Trouverie, J., Tcherkez, G., Falque, M., Damerval, C., Rogowsky, P., Perez, P., Noctor, G., Zivy, M., Coursol, S., 2011. The ZmASR1 protein influences branchedchain amino acid biosynthesis and maintains kernel yield in maize under waterlimited conditions. Plant Physiol 157, 917–936. Voorrips, R.E., 2002. MapChart: software for the graphical presentation of linkage maps and QTLs. J. Hered. 93, 77–78. Wang, L., Hu, W., Feng, J., Yang, X., Huang, Q., Xiao, J., Liu, Y., Yang, G., He, G., 2016. Identification of the ASR gene family from Brachypodium distachyon and functional characterization of BdASR1 in response to drought stress. Plant Cell Rep. 35, 1221–1234. Wang, P., Song, C.P., 2008. Guard-cell signalling for hydrogen peroxide and abscisic acid. New Phytol. 178, 703–718. Wu, Q., Yang, J., Cheng, N., Hirschi, K.D., White, F.F., Park, S., 2017. Glutaredoxins in plant development, abiotic stress response, and iron homeostasis: from model organisms to crops. Environ. Exp. Bot. 139, 91–98. Yang, C.Y., Chen, Y.C., Jauh, G.Y., Wang, C.S., 2005. A Lily ASR protein involves abscisic acid signaling and confers drought and salt resistance in Arabidopsis. Plant Physiol 139, 836–846. Yoon, J.S., Kim, J.Y., Lee, M.B., Seo, Y.W., 2019. Over-expression of the Brachypodium ASR gene, BdASR4, enhances drought tolerance in Brachypodium distachyon. Plant Cell Rep. 38, 1109–1125. Zale, J., Jung, J.H., Kim, J.K., Pathak, B., Karan, R., Liu, H., Chen, X., Wu, H., Candreva, J., Zhai, Z., Shanklin, J., Altpeter, F., 2016. Metabolic engineering of sugarcane to accumulate energy-dense triacylglycerols in vegetative biomass. Plant Biotechnol. J. 14, 661–669. Zhang, L.L., Hu, W., Wang, Y., Feng, R.J., Zhang, Y.D., Liu, J.H., Jia, C.H., Miao, H.X., Zhang, J.B., Xu, B.Y., Jin, Z.Q., 2015. The MaASR gene as a crucial component in multiple drought stress response pathways in Arabidopsis. Funct. Integr. Genom. 15, 247–260. Zhang, X., Zhang, L., Dong, F.C., Gao, J.F., Galbraith, D.W., Song, C.P., 2001. Hydrogen peroxide is involved in abscisic acid-induced stomatal closure in Vicia faba. Plant Physiol 126, 1438–1448.

responsive and heat-stress-responsive gene expression. Proc. Natl. Acad. Sci. U.S.A. 103, 18822–18827. Saumonneau, A., Agasse, A., Bidoyen, M.T., Lallemand, M., Cantereau, A., Medici, A., Laloi, M., Atanassova, R., 2008. Interaction of grape ASR proteins with a DREB transcription factor in the nucleus. FEBS Lett. 582, 3281–3287. Seo, M., Koshiba, T., 2002. Complex regulation of ABA biosynthesis in plants. Trends Plant Sci. 7, 41–48. Seo, M., Aoki, H., Koiwai, H., Kamiya, Y., Nambara, E., Koshiba, T., 2004. Comparative studies on the Arabidopsis aldehyde oxidase (AAO) gene family revealed a major role of AAO3 in ABA biosynthesis in seeds. Plant Cell Physiol. 45, 1694–1703. Sharma, R., Priya, P., Jain, M., 2013. Modified expression of an auxin-responsive rice CCtype glutaredoxin gene affects multiple abiotic stress responses. Planta 238, 871–884. Shkolnik, D., Bar-Zvi, D., 2008. Tomato ASR1 abrogates the response to abscisic acid and glucose in Arabidopsis by competing with ABI4 for DNA binding. Plant Biotechnol. J. 6, 368–378. Simpson, S.D., Nakashima, K., Narusaka, Y., Seki, M., Shinozaki, K., YamaguchiShinozaki, K., 2003. Two different novel cis-acting elements of erd1, a clpA homologous Arabidopsis gene function in induction by dehydration stress and darkinduced senescence. Plant J. 33, 259–270. Sundaram, S., Rathinasabapathi, B., 2010. Transgenic expression of fern Pteris vittata glutaredoxin PvGrx5 in Arabidopsis thaliana increases plant tolerance to high temperature stress and reduces oxidative damage to proteins. Planta 231, 361–369. Sun, J., Hu, W., Zhou, R., Wang, L., Wang, X., Wang, Q., Feng, Z., Li, Y., Qiu, D., He, G., Yang, G., 2015. The Brachypodium distachyon BdWRKY36 gene confers tolerance to drought stress in transgenic tobacco plants. Plant Cell Rep. 34, 23–35. Takasaki, H., Mahmood, T., Matsuoka, M., Matsumoto, H., Komatsu, S., 2008. Identification and characterization of a gibberellin-regulated protein, which is ASR5, in the basal region of rice leaf sheaths. Mol. Genet. Genom. 279, 359–370. Tran, L.S.P., Nakashima, K., Sakuma, Y., Simpson, S.D., Fujita, Y., Maruyama, K., Fujita, M., Seki, M., Shinozaki, K., Yamaguchi-Shinozaki, K., 2004. Isolation and functional analysis of Arabidopsis stress-inducible NAC transcription factors that bind to a drought-responsive cis- element in the early responsive to dehydration stress 1 promoter. Plant Cell 16, 2481–2498.

15