Enhanced expression of EsWAX1 improves drought tolerance with increased accumulation of cuticular wax and ascorbic acid in transgenic Arabidopsis

Plant Physiology and Biochemistry 75 (2014) 24e35

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Research article

Enhanced expression of EsWAX1 improves drought tolerance with increased accumulation of cuticular wax and ascorbic acid in transgenic Arabidopsis Lin Zhu a, Jiansheng Guo a, Jian Zhu a, **, Cheng Zhou a, b, * a b

Department of Molecular and Cell Biology, School of Life Science and Technology, Tongji University, Shanghai 200092, China School of Life Science, Anhui Science and Technology University, Bengbu 233100, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 16 September 2013 Accepted 29 November 2013 Available online 11 December 2013

Drought can activate several stress responses in plants, such as stomatal closure, accumulation of cuticular wax and ascorbic acid (AsA), which have been correlated with improvement of drought tolerance. In this study, a novel MYB gene, designed as EsWAX1, was isolated and characterized from Eutrema salsugineum. EsWAX1 contained a full-length open reading frame (ORF) of 1068 bp, which encoding 355 amino acids. Transcript levels of EsWAX1 were quickly inducible by drought stress and ABA treatment, indicating that EsWAX1 may act as a positive regulator in response to drought stress. Ectopic expression of EsWAX1 increased accumulation of cuticular wax via modulating the expression of several wax-related genes, such as CER1, KCS2 and KCR1. Scanning electron microscopy further revealed higher densities of wax crystalline structures on the adaxial surfaces of leaves in transgenic Arabidopsis plants. In addition, the expression of several AsA biosynthetic genes (VTC1, GLDH and MIOX4) was significantly upregulated in EsWAX1-overexpressing lines and these transgenic plants have approximately 23e27% more total AsA content than WT plants. However, the high-level expression of EsWAX1 severely disrupted plant normal growth and development. To reduce negative effects of EsWAX1 over-expression on plant growth, we generated transgenic Arabidopsis plants expressing EsWAX1 driven by the stress-inducible RD29A promoter. Our data indicated the RD29A::EsWAX1 transgenic plants had greater tolerance to drought stress than wild-type plants. Taken together, the EsWAX1 gene is a potential regulator that may be utilized to improve plant drought tolerance by genetic manipulation. Ó 2013 Elsevier Masson SAS. All rights reserved.

Keywords: Eutrema salsugineum EsWAX1 Wax biosyhnthesis Ascorbic acid Drought tolerance Transgenic Arabidopsis

1. Introduction Drought is one of the most serious problems for sustainable agriculture worldwide. For productive and sustainable agriculture, it is important to improve drought stress tolerance by genetic engineering. Previous studies have shown that over-expression of a

Abbreviations: EsWAX1-OX, EsWAX1 over-expressing lines; TFs, transcription factors; WT, wild type; ABA, abscisic acid; IAA, indole-3-acetic acid; mJA, methyl jasmonate; KT, kinetin; ACC, 1-aminocyclopropane-1-carboxylic acid; AsA, ascorbate acid; SOD, superoxide dismutase; APX, ascorbate peroxidase; POD, peroxidase; CAT, catalase; qRT-PCR, quantitative reverse transcription polymerase chain reaction. * Corresponding author. Department of Molecular and Cell Biology, School of Life Science and Technology, Tongji University, Shanghai 200092, China. Tel.: þ86 21 65989464. ** Corresponding author. E-mail addresses: [email protected] (J. Zhu), [email protected] (C. Zhou). 0981-9428/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.plaphy.2013.11.028

few MYB transcription factors can improve tolerance to drought stress in plants. MYB transcription factors have a conserved MYB DNA-binding domain (MYB domain), which contain up to three imperfect repeats (named sequentially as R1, R2 and R3, respectively). According to the number of peptide repeats in the MYB domain, the MYB family is categorized into 1R-MYB, 2R-MYB, 3RMYB and 4R-MYB, respectively (Jin and Martin, 1999). Among the MYB proteins in plants, the MYB family with the two-repeat (R2R3) is the most common one, which have been demonstrated to involve in the regulation of secondary metabolism, control of cellular morphogenesis and plant stress responses (Abe et al., 2003; Ding et al., 2009; Lippold et al., 2009; Seo et al., 2011). In Arabidopsis, AtMYB2 can be apparently induced by drought stress and functions as a transcriptional activator in abscisic acid (ABA)-mediated gene expression (Abe et al., 2003). The expression of MYB15 is up-regulated by drought stress, and its over-expression has been shown to improve drought tolerance in transgenic plants by increasing the expression levels of the genes involved in ABA

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biosynthesis and signaling (Ding et al., 2009). The AtMYB41 transcripts can be rapidly increased under various abiotic stresses and AtMYB44 over-expression confers drought tolerance in Arabidopsis by reducing the expression of genes encoding PP2Cs, which have been described as negative regulators of ABA signaling (Lippold et al., 2009). It has recently been shown that over-expression of AtMYB96 confers drought resistance in transgenic plants via modulating cuticular wax biosynthesis (Seo et al., 2011). In higher plants, cuticular wax forms a hydrophobic layer covering aerial organs, which is deposited either outside of the cuticle (epicuticular wax), or within the cuticular matrix (intracuticular wax). In addition to repelling atmospheric water, epidermal wax provides a protective barrier between the plant and its environment, which functions as a barrier to water loss (Riederer and Schreiber, 2001; Kerstiens, 1996). It has been well documented that drought stress significantly increases cuticular wax accumulation in plants (Kosma et al., 2009). Seo et al. reported that cuticular wax accumulation of plants treated with drought stress showed 3e4 folds higher than that of plants grown under well water conditions (Seo et al., 2011). The similar phenomenon has also been observed in other plant species, such as Pinus pinaster and Eutrema salsugineum, suggesting that cuticular wax accumulation is closely associated with drought tolerance responses (LeProvost et al., 2013; Xu et al., 2013). Regulatory mechanisms of drought tolerance have been extensively described (Yamaguchi-Shinozaki and Shinozaki, 2006), including regulation of transcription, functional protection of proteins (such as dehydrins and heat shock proteins), accumulation of osmolytes (proline, glycine betaine, trehalose, mannitol, myo-inositol) and induction of ascorbic acid. Among these osmolytes, myo-inositol has been shown to be involved in the synthesis of ascorbic acid (AsA) and as a precursor of AsA biosynthesis. It has previously been reported that myo-inositol oxygenase (MIOX) is a key monooxygenase which catalyzes the conversion of myo-inositol into D-glucuronic acid (DGlcUA). MIOX proteins have highly conserved across almost all eukaryotes. In animals, MIOX degrades myo-inositol into D-GlcUA and then was reduced to L-gulonic acid and ring formation to gulonolactone which is finally oxidized to AsA (Brown et al., 2006). In plants, MIOX has also been shown to catalyze myo-inositol to D-GlcUA, which is involved in AsA biosynthesis (Lorence et al., 2004). Recently, E. salsugineum has became an important model plant for studying abiotic stress, which shares similar sequence identity with Arabidopsis thaliana (Inan et al., 2004). E. Salsugineum is more drought, and cold tolerance than A. thaliana. Under normal conditions, there exist significant differences of morphological characteristics between A. thaliana and E. Salsugineum and the latter exhibits smaller, narrower and waxy leaves. The higher wax level may play a critical role in limiting transpirational water loss across the plant surface. Previous studies have shown that genetic engineering with genes involved in wax biosynthesis can improve tolerance of plants to drought stress (Aharoni et al., 2004; Zhang et al., 2005). A novel R2R3-MYB transcription factor EsWAX1 showed higher-level expression in expressed sequence tags (ESTs) library generated from abotic stress-treated E. Salsugineum plants (Taji et al., 2008), however the researches related to the MYB transcription factors in E. Salsugineum were rarely available. Here, we reported the isolation of the EsWAX1 gene from E. Salsugineum. Ectopic expression of EsWAX1 significantly increased accumulation of cuticular wax and AsA content in transgenic Arabidopsis plants, however high-level expression of EsWAX1 severely affect plant normal growth and development. Our findings further shown that the EsWAX1 gene, under control of the Arabidopsis RD29A promoter, could be used to generate transgenic plants with improved drought tolerance without impacting plant growth and development.

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2. Materials and methods 2.1. Plant materials and growth conditions A. thaliana (Columbia ecotype) and E. salsugineum (Shandong ecotype) were used in this study. Plants were grown in greenhouse at 22  C with 16 h light/8 h dark photoperiod and a relative humidity at approximately 80%. To shorten the flowering time, 7week-old E. salsugineum plants were transferred from 22  C to a 4  C cold room for 6 weeks for vernalization. 2.2. Treatments with different exogenous hormones and drought stresses 3-week-old E. salsugineum plants grown on MS medium containing 0.8% agar were transferred to MS agar medium supplemented with various exogenous hormones, including 10 mM indole-3-acetic acid (IAA), 10 mM abscisic acid (ABA), 10 mM kinetin (KT), 20 mM methyl jasmonate (mJA), 20 mM 1-aminocyclopropane1-carboxylic acid (ACC) for the indicated time periods. For analyzing the effects of cold and drought stress on the expression of EsWAX1, 3-week-old E. salsugineum plants grown on MS agar medium were treated for the indicated time periods according to a previously described method by Seo et al. (2011). To assess drought tolerance of RD29A::EsWAX1 transgenic Arabidopsis plants, the drought stress treatments were performed. 5week-old wild type (WT) and RD29A::EsWAX1 transgenic plants were firstly grown in soil under well water conditions. These plants were deprived of water for 14 d and then rewatered once. The survival rate recorded 10 d after being rewatered. 2.3. Plasmid construction and plant transformation Total RNA was isolated from 5-week-old E. salsugineum plants using the TRIzol reagent (Invitrogen, USA). Residual genomic DNA was removed by RNase-free DNase I (Invitrogen, USA) treatment. A gene-specific primer pair was designed to amplify the coding region of EsWAX1 based on the sequence from E. salsugineum (GenBank accession no. BAJ34253). The coding region of the EsWAX1 gene from E. salsugineum cDNA was amplified with gene specific primers and was cloned into pGEM-T vector (Promega, USA) for sequencing to verify its integrity. The amplification condition was followed by 94  C for 5 min, 56  C for 1 min, 72  C for 7 min (35 cycles in total) and finally 72  C for 10 min. The PCR product was cloned into pGEM-T vector (Promega, USA) for sequencing. For the over-expression of EsWAX1, the complete coding sequence of EsWAX1 was digested with Xbal and SacI, and the digested fragment was inserted into plant binary vector containing a super-promoter consists of a trimer of the octopine synthase (OCS) transcriptional activating element affixed to the mannopine 2 synthase 20 (mas2’) transcriptional activating element (Lee et al., 2007). To create a construct containing RD29A::EsWAX1 cDNA, the sequence of RD29A promoter from Arabidopsis was firstly amplified. The PCR products were cloned into pGEM-T vector (Promega, USA) for sequencing and then digested with HindIII and NcoI. The digested fragment was inserted into plant binary vector pCMBIA1304. Secondly, the EsWAX1 cDNA was inserted in sense orientation into the NcoI site of the pCMBIA1304-RD29A vector. The constructed plasmids were transferred into Agrobacterium tumefaciens strain GV3101 and used to transform Arabidopsis plants by the floral dip method (Clough and Bent 1998). These seeds were collected and grown on MS agar medium containing 50 mg/l hygromycinto to screen transgenic plants. Transformants were identified as hygromycin-resistant and verified by PCR. The fragment of

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the EsWAX1 gene was amplified using two specific primers. Primers used in all above experiments were shown in supplemental Table. 2.4. Bioinformatic analysis of EsWAX1 Amino acid sequence alignment was carried out using the ClustalW2 program (www.ebi.ac.uk/Tools/msa/clustalw2) and the phylogenetic relationship tree was constructed using the neighborjoining method of MEGA (version 4.0). Analysis of nucleotide and amino acid sequences were performed using the BLAST program at National Centre for Biotechnology Information (NCBI) server (http://www.ncbi.nlm.nih.gov/BLAST/). SMART program (http:// smart.embl-heidelberg.de) was used to explore conserved domains.

were as an internal control to normalize the RT mixtures, respectively. The PCR parameters were as follows: 95  C (4 min); 28 cycles of 95  C (30 s), 56  C (30 s), 72  C (30 s), and 72  C (5 min). Quantitative reverse transcription PCR (qRT-PCR) were carried out using the SYBR Green qPCR kit (Takara, Japan). The reactions were performed on a 7500 Real-time PCR machine (ABI; American) by an initial denaturation step at 95  C for 30 s and then 40 cycles (95  C for 15 s, 60  C for 30 s), followed by a subsequent standard dissociation protocol. EsActin2 or AtActin2 was used as internal standard. All calculations and analyses were performed using the 2DDCt method. RT-PCR and qRT-PCR reactions were performed in triplicate for each of the three independent samples. The primers used for RT-PCR or qRT-PCR analyses were listed in supplemental Table.

2.5. Expression analyses 2.6. Scanning electron microscopy Reverse transcription PCR (RT-PCR) was performed from total RNA, which extracted from plants using the TRIzol reagent (Invitrogen, USA) and treated with DNase I. First-strand cDNA was synthesized from 500 ng of total RNA using an oligo (dT) primer and reverse transcriptase ReverTraAce (Takara, Japan). E. salsugineum actin2 (EsActin2) and A. thaliana actin2 (AtActin2)

Leaf samples were cut from leaves of both WT and transgenic plants with 2.5% glutardialdehyde, dehydration in an ethanol dilution series from 50% to 100%, CO2 critical point drying, and sputter-coated with coated with approximately 20 nm of 60/40 GoldePalladium particles. The scanning electron microscopy (SEM)

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Fig. 1. Multiple alignment and phylogenetic analysis of EsWAX1 and selected R2R3-MYB proteins from other plant species (A) Alignment of deduced amino acid sequences of EsWAX1 and with its closest homologs from other plant species. Identical amino acid residues are shaded in black, similar in gray and the putative R2 and R3 domains shown refer to two repeat units of the MYB DNA-binding domain were underlined. (B) Phylogenetic tree of EsWAX1 and related R2R3-MYB proteins from other plant species using the neighborjoining method by the MEGA 4.0 software. The amino acid sequences of all R2R3-MYB proteins were retrieved from NCBI database and accession numbers are the following: EsWAX1 (BAJ34253), AtMYB96 (NP_201053), AtMYB94 (NP_190344), PsMYB1 (AGG69481), VvMYB30 (NP_001267946), SiMYB306 (XP_004236011), AtHSR1 (AAM64792), ZmMYB94 (NP_001147454) and GmMYBJ2 (AGN96216).

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For measurement of water loss rate, the experiments were performed in the dark greenhouse. The rosette leaves from WT and RD29A::EsWAX1 transgenic Arabidopsis plants were detached after 12 h exposure to dark and then soaked in distilled water to equalize the water content of the plants before the assays were conducted. The rosette leaves were then weighed and subject to dry at 20  C, 60% relative humidity. The weight of leaf samples was recorded at 1 h intervals and fresh weights of the detached leaves were measured as described by Zhang et al. (Zhang et al., 2005). Cuticular waxes were extracted from leaves of plants and analyzed according to the method described by Seo et al. (2011). The ascorbic acid (AsA) was measured as previously described (Hemavathi et al., 2010).

coding region of EsWAX1 consisted of 1068 nucleotides, encoding a peptide of 355 aa with an estimated molecular mass of 39.5 kDa and a theoretical pl of 5.61. Domain search analysis using SMART program revealed the EsWAX1 protein contained a conserved DNAbinding domain (MYB domain) of 102 amino acids with two imperfect repeat (one between residues 13 and 63, the other between residues 66 and 114) in the MYB domain. Multiple alignment analysis using the ClustalW2 program indicated EsWAX1 shared 52% amino acid similarity with VvMYB30, 54% with PsMYB1, 63% with AtMYB94 and especially 86% with AtMYB96 (Fig. 1A). To gain insight into the evolutionary relationship, a phylogenetic tree was constructed based on the amino acid sequences of EsWAX1 and MYB proteins from other plant species using MEG 4.0. And several other R2R3-type MYB proteins in plants were obtained from the National Center for Biotechnology Information (NCBI) database. The phylogenetic analysis showed that the EsWAX1 and AtMYB96 belong to a same subgroup (Fig. 1B), suggesting that EsWAX1 might be involved in plant response to drought stress.

2.8. Measurement of antioxidant enzyme activity

3.2. Expression pattern of EsWAX1

The superoxide dismutase (SOD) activity was determined by the method of Dhindsa et al. (1981). The ascorbate peroxidase (APX) activity was measured as described by Mittler and Zilinskas (1993). The peroxidase (POD) activity was assayed according to the methods described by Kwak et al. (1995). Catalase (CAT) activity was assayed according to the methods described by Aebi (1984).

To determine the tissue distribution and specificity of EsWAX1 in E. Salsugineum, we examined its expression in some major organs by qRT-PCR. Total RNAs were extracted from roots, cauline leaves, rosette leaves, stems, flowers and siliques, respectively. The results of qRT-PCR analyses indicated that the EsWAX1 transcript was detected in all tested organs, whereas it was expressed at different levels in these organs. The EsWAX1 gene was expressed at high levels in aerial organs including leaves, stems and siliques, while the lowest expression was occurred in the roots (Fig. 2A). In addition, the expression of EsWAX1 was investigated under drought, salinity and cold stress conditions. The results of qRT-PCR analyses indicated that the EsWAX1 gene was induced rapidly by drought stress. The expression of EsWAX1 peaked within 12 h and

observation was performed on an S-3400N (Hitachi) at an acceleration voltage of 15 kV. 2.7. Analysis of water loss rate, cuticular wax and ascorbic acid

3. Results 3.1. Sequence analysis of EsWAX1 A complete coding sequence of EsWAX1 from E. Salsugineum was obtained by RT-PCR methods. Sequence analysis revealed that the

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Fig. 2. qRT-PCR analysis of the EsWAX1 gene (A) Tissue-specific expression of EsWAX1. RO, roots; CL, cauline leaves; RL, rosette leaves; ST, stems; FL, flowers; SI, siliques; (B) and (C) Effects of exogenous hormones (B) and abiotic stress (C). 3-week-old plants of E. salsugineum cultivated on the MS agar medium were used for further treatments. ABA, IAA and KT were used at 10 mM, ACC and mJA were used at 20 mM, and NaCl was used at 150 mM. Data represent means and standard errors of three replicates, asterisk indicated significant differences compared to untreated control plants (time 0 h) as determined by Student’s t-test (P < 0.05).

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Fig. 3. Phenotypes of Arabidopsis plants over-expressing EsWAX1. (A) Construction of a physical map for over-expression of EsWAX1. The sense EsWAX1 cDNA was driven by the super-promoter. hph, encoding hgromycin resistance gene. RB right border, LB left border. (B) The transcript levels of the EsWAX1 gene in WT and several transgenic Arabidopsis plants (OX-5, 12, 13, 16, 18 and 26). (C) Phenotypes of transgenic plants with relatively high expression of EsWAX1 (OX5 and OX12). 5-week-old transgenic plants overexpressing EsWAX1 were photographed for comparison. The insets are amplified views of the EsWAX1-OX5 and EsWAX1-OX12 plants. (D) Morphological analysis of leaves of the EsWAX1-OX lines. The rosette leaves from WT and transgenic plants were photographed.

then gradually decreased (Fig. 2B). When treated with 150 mM NaCl, its expression level increased gradually until 24 h after the treatment (Fig. 2B). In addition, the EsWAX1 transcript was also induced by cold treatment. The expression of EsWAX1 was upregulated by cold treatment within 6 h and was continuously elevated for up to 12 h and decreased after 24 h (Fig. 2B). As shown in Fig. 2C, the EsWAX1 transcript was significantly elevated after exposure to ABA. The expression of EsWAX1 was slightly increased by IAA and mJA treatment, but was unaffected by KT and ACC. These

data suggested that EsWAX1 might play a crucial role in ABAmediated drought stress response. 3.3. Effects of EsWAX1 over-expression on plant growth and development In order to study the physiological roles of the EsWAX1 gene, we generated transgenic Arabidopsis plants over-expressing EsWAX1 (EsWAX1-OX) under control of a super-promoter (Fig. 3A]. Fifty-six

Fig. 4. Scanning electron microscopy (SEM) analysis of epicuticular wax deposition on the adaxial leaf surfaces of both WT and EsWAX1-OX plants. (A) and (B) Rosette leaves of 5week-old WT (A) and EsWAX1-OX (B) plants grown in soil were photographed; Bars ¼ 100 mm (C) and (D) Scanning electron microscopy images of epicuticular wax crystals on the adaxial surface of the leaves in both WT (C) and EsWAX1-OX (D) transgenic plants. Bars ¼ 10 mm.

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leaf surface of the EsWAX1-OX plants, while there were no visible wax crystals on that of WT plants (Fig. 4). Moreover, the content and composition of cuticular wax was measured by gas chromatography with flame ionization detection and gas chromatography with mass spectrometry, respectively. The results indicated that the total wax load was elevated approximately 11 folds in EsWAX1-OX leaves compared to WT (Fig. 5). It was also observed that the composition of cuticular wax was altered in EsWAX1-OX lines (Fig. 5BeE]. Increased accumulation of epidermal wax in EsWAX1-OX lines indicated that the expression of genes involved in wax biosynthesis might be changed in those plants. qRT-PCR was performed to detect the transcript levels of several genes involved in wax biosynthesis, including CER1, KCS2 and KCR1, etc. As shown in Fig. 6, the expression of these wax biosynthesis-related genes was significantly up-regulated in EsWAX1-OX lines compared with WT plants.

independent transgenic lines were screened on MS agar medium containing 50 mg/l hygromycin and confirmed by qRT-PCR. The results of qRT-PCR analyses showed that the transcript ion levels of EsWAX1 were over-expressed constitutively in several independent transgenic plants compared with WT plants (Fig. 3B). In contrast to WT, EsWAX1-OX transgenic lines exhibited morphological alterations on plant growth and development under well water conditions. Among these transgenic lines, approximately 65% transgenic lines were dwarfed with smaller leaves compared with WT plants (Figs. 3C and D]. Transgenic plants with relatively high-level expression of EsWAX1 (EsWAX1-OX5 and eOX12) shared similar phenotypes with plants grown under abiotic stress conditions, which suggesting that the EsWAX1 gene might be associated with abiotic stress responses. 3.4. EsWAX1 over-expression increases accumulation of cuticular wax and induction of wax biosynthesis-related genes

3.5. EsWAX1 over-expression promotes AsA biosynthesis In this study, we investigated the expression of cuticular waxbiosynthetic genes in WT and EsWAX1-OX lines and further analyzed deposition of epicuticular wax crystals on the adaxial leaf surface by scanning electron microscopy (SEM). Obviously, a large amount of epicuticular wax crystals was observed on the adaxial

To detect whether ectopic expression of EsWAX1 could promote the biosynthesis of AsA in transgenic Arabidopsis, we measured the AsA content of EsWAX1-OX lines (OX5 and OX12). The AsA content of leaves in 5-week-old transgenic lines was approximately 23e27%

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Fig. 5. Gas Chromatography (GC) - Mass Spectrometry (MS) analysis of cuticular wax in leaves of WT and EsWAX1-OX transgenic Arabidopsis lines (OX5 and OX12). Rosette leaves of 5-week-old WT and transgenic lines grown in soil were used for detecting cuticular wax loads (A) and composition (B). FA, long-chain fatty acid; alk, long-chain alkanes; alc, longchain alcohols; long-chain aldehydes; ket, ketones. Bars are the means SE of three replicates, asterisks indicated significant differences compared with WT plants as determined by Student’s t-test (P < 0.05).

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Fig. 6. qRT-PCR analysis of wax biosynthetic genes in EsWAX1-OX transgenic Arabidopsis lines (OX5 and OX12). Total RNAs were extracted from 5-week-old WT and transgenic lines grown in soil. Transcript levels of wax biosynthetic genes were investigated using qRT-PCR. Bars are the means SE of three replicates, asterisks indicated significant differences compared with WT plants as determined by Student’s t-test (P < 0.05).

higher in the EsWAX1-OX lines than that in the WT (Fig. 7A). Furthermore, the expression of genes involved in AsA biosynthesis was investigated using qRT-PCR. Our data indicated that the expression of key genes (VTC1 and GLDH) in the mannose/galactose (Man/Gal) route and the myo-inositol oxygenase gene (MIOX4) encoding an important enzyme involved in the myo-inositol pathway were greatly up-regulated in these EsWAX1-OX lines

(Fig. 7B). The transcript levels of several AsA recycling genes, which have been shown to play critical roles in the in AsAeglutathione (AsA-GSH) cycle (Chen et al., 2003; Stevens et al., 2008), were also detected in both WT and EsWAX1-OX lines. As shown in Fig. 7B, the expression of the AsA recycling genes monodehydroascorbate reductase 3 (MDAR3), chloroplast dehydroascorbate reductase (ChlDHAR), cytosolic dehydroascorbate reductase (CytDHAR) and

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Fig. 7. Analysis of ascorbic acid (AsA) biosynthesis in WT and EsWAX1-OX transgenic Arabidopsis lines (OX5 and OX12). (A) Content of AsA in the WT and EsWAX1-OX lines. (B) qRTPCR analysis of the expression of genes related to AsA biosynthesis in both WT and transgenic lines. Bars are the means SE of three replicates, asterisks indicated significant differences compared with WT plants as determined by Student’s t-test (P < 0.05).

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glutathione reductase 1 (GR1) was no obvious difference between WT and EsWAX1-OX lines. 3.6. RD29A-inducible expression of EsWAX1 enhances tolerance to drought stress in transgenic Arabidopsis plants To drive the expression of the EsWAX1 gene under control of the drought stress-inducible promoter RD29A and to minimize adverse effects of this gene on plant growth, we generated transgenic Arabidopsis plants expressing the coding region of EsWAX1 driven by the Arabidopsis RD29A promoter. In our experiments, twenty-six independent transgenic lines were selected on MS agar medium containing 50 mg/l hygromycin and the plant growth and development was further monitored. As shown in Fig. 8, the RD29A::EsWAX1 lines shared similar phenotype with WT plants and there is no obvious difference of shoot dry weight between WT and transgenic lines under well water conditions. In addition, we examined the expression patterns of the EsWAX1 gene in response to drought stress. The expression of EsWAX1 in two independent RD29A::EsWAX1 lines (RD29A::EsWAX1-6 and RD29A::EsWAX1-9) was determined under well-water and water deprivation (drought) stress conditions. The transcription levels of EsWAX1 were quantified by RT-PCR using the gene-specific primers

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(supplemental Table). As shown in Fig. 9A, the expression of EsWAX1 was barely detected in nearly all RD29A::EsWAX1 lines under well water conditions, while the expression of EsWAX1 was slightly increased after water deprivation for 3 d. After water deprivation of 10 d, the transcript levels of EsWAX1 significantly upregulated in these transgenic lines. Thus, these data suggested that the expression of EsWAX1 could be driven by the RD29A promoter under drought stress conditions. To determine whether stress-inducible expression of EsWAX1 could improve tolerance of transgenic plants to drought stress, WT and RD29A::EsWAX1 lines were grown under the same conditions. 5-week-old WT and RD29A::EsWAX1 lines were not watered for 14 d and then allowed to recover for 10 d. The leaves of WT exhibited severe withering, whereas only few of the leaves of RD29A::EsWAX1 lines wilted slightly (Fig. 9B. After re-watering, 78% (RD29A::EsWAX1-6) and 89% (RD29A::EsWAX1-9) of transgenic plants recovered, but only 15% WT plants did. Together, RD29A::EsWAX1 plants showed higher survival rates than WT under drought stress conditions, indicating that elevated expression of EsWAX1 improved more tolerance to drought stress in transgenic plants. Moreover, we compared water loss rates of these transgenic plants with those of WT under drought stress conditions. The detached leaves were subjected to free leaf water loss rate assay. WT plants lost about 36% of their water within 5 h, whereas the RD29A::EsWAX1 transgenic plants lost about 27% of their water content (Fig. 9C), suggesting that drought-induced expression of EsWAX1 in transgenic plants made the plant reduce water evaporation. 3.7. Analysis of antioxidant enzyme activity, content of AsA and wax levels in RD29A::EsWAX1 transgenic plants under drought stress To explore the physiological characterizations for the improved drought tolerance of the RD29A::EsWAX1 lines, the content of AsA and several major antioxidant enzyme activities, including SOD, APX, POD and CAT were measured. As shown in Figs. 10 and 11, there was no discernible difference in the ASA content and antioxidant enzyme activities between WT and RD29A::EsWAX1 lines under well water conditions. After 14 d exposure to drought stress, there were apparent increases in the ASA content and antioxidant enzyme activities of both WT and transgenic lines. However, the AsA content and antioxidant enzyme activities were significantly higher in the two transgenic lines than WT after deprivation of water for 14 d. We next examined whether the content of cuticular wax altered in these RD29A::EsWAX1 lines. As shown in Fig. 12, there was no apparent difference of cuticular wax accumulation in WT and transgenic plants. However, the total cuticular wax load and composition was significantly increased in the mature leaves of both WT and transgenic lines under drought stress conditions. Furthermore, leaf wax accumulation was up to 3e4 folds higher in these transgenic lines than in WT plants. Together, these data indicated that the elevated expression of EsWAX1 efficiently coped with oxidative stress and water loss evoked with drought stress by enhancing antioxidant enzyme activities and accumulating more cuticular wax.

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Plants have evolved an extraordinary capacity to adapt their external environments by means of morphological, physiological, and biochemical adaptations (Zhu, 2002; Xiong et al., 2002). Drought is one of most severe environmental culprits that greatly restrict crop production and plant distribution worldwide. Thus, understanding the genes underlying adaptation of drought stress is needed for both theoretical and applied researches. Recently, the

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Fig. 9. Stress-inducible expression of EsWAX1 enhanced tolerances to drought stress in RD29A:EsWAX1 transgenic Arabidopsis lines. (A) RT-PCR analysis of the EsWAX1 transcripts in two RD29A:EsWAX1 T3 transgenic lines. 5-week-old plants of E. salsugineum grown in soil were deprived of water for 0 d, 5 d, 7 d, and 10 d. The total RNA samples from both WT and transgenic lines were extracted for RT-PCR analysis and the Arabidopsis Actin2 (AtActin2) gene was used as an internal control. The number of the PCR cycles is listed on the right side. (B) Dehydration treatments of WT and transgenic lines. 5-week-old WT and T3 transgenic lines were withholding irrigation for 14 d and then were allowed to a 10-d recovery period. (C) Comparison of water loss rate of detached leaves from WT and RD29A::EsWAX1 lines with drought stress treatment. Fresh weight was measured at the indicated times. Amount of water loss expressed in percentage of the initial fresh weight. Data are the means SE of three replicates, with a minimum of 15 samples (n > 15) for each replicate.

study of stress-responsive transcription factors has been one of the main foci on drought stress tolerance. Over-expression of transcription factor (TF) improve tolerance of plants is a promising strategy because of the ability of a TF to regulate an entire suite of important genes in a stress-response pathway (Hu et al., 2006; Xiao et al., 2009). 4.1. EsWAX1 involves in regulatory pathways of abiotic stress response In this study, a novel MYB gene, EsWAX1, from E. Salsugineum was isolated and characterized. Based on the analysis of predicted

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amino acid sequence, EsWAX1 showed high homology to AtMYB96. Phylogenetic analysis further showed that EsWAX1 and AtMYB96 cluster in the same sub-group, indicating EsWAX1 might be involved in abiotic stress response. In addition, the EsWAX1 gene was mainly expressed in leaves stems, flowers and siliques, however there was weak expression in roots. The results indicated that EsWAX1 might play a certain role during the development of several aerial organs in E. Salsugineum. Moreover, the EsWAX1 gene was significantly up-regulated under drought stress, high-salinity and cold stress conditions. Previous studies have shown that exogenous application of ABA induces the expression of a number of genes that respond to drought stress, including the MYB TFs (Nakashima et al., 2009). In E. Salsugineum, the expression of EsWAX1 gene was also found to be induced rapidly by exogenous ABA. Thus, our data suggested that the EsWAX1 gene positively correlated with ABA signaling pathways and might modulate ABA-mediated genes expression in response to abiotic stress.

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Fig. 10. Stress-inducible expression of EsWAX1 promotes the biosynthesis of AsA in RD29A::EsWAX1 transgenic Arabidopsis lines under drought stress conditions. The AsA content from both WT and transgenic lines was detected under well water and drought stress conditions. Bars indicate SE of the mean, the bars with different letters indicate significant differences among three genotypes (WT, RD29A::EsWAX1-6 and RD29A::EsWAX1-9) within well water and drought stress treatments using a Dunnett test at P < 0.05. FW, fresh weight.

It has been documented that cuticular wax on the leaf surface provides primary protection against environmental stresses to reduce water loss (Kerstiens, 1996). Previous studies have reported that over-expression of WXP1 from Medicago truncatula leads to increased leaf wax accumulation in transgenic alfalfa and Arabidopsis plants, contributes to drought resistance (Zhang et al., 2005). The similar effects have been observed in transgenic plants overexpressing the Arabidopsis WIN1, an ethylene response factor-type transcription factor. And over-expression of WIN1 up-regulates the expression of several wax biosynthetic genes, including CER1, KCS1 and CER2, to promote leaf epidermal wax accumulation, which improves tolerance to drought stress in transgenic plants (Broun et al., 2004). Recently, Seo et al. (2011) have shown that ectopic expression of AtMYB96 improves tolerance of plants to

L. Zhu et al. / Plant Physiology and Biochemistry 75 (2014) 24e35

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Fig. 11. Stress-inducible expression of EsWAX1 enhances antioxidant enzyme activities in RD29A::EsWAX1 transgenic Arabidopsis lines under drought stress conditions. The activity of antioxidant enzymes between WT and transgenic lines was measured under well water and drought stress conditions. (A) SOD. (B) POD. (C) CAT. (D) APX. Data are the means SE of three replicates, the bars with different letters indicate significant differences among three genotypes (WT, RD29A::EsWAX1-6 and RD29A::EsWAX1-9) within well water and drought stress treatments using a Dunnett test at P < 0.05.

drought stress with increased accumulation of cuticular wax. Furthermore, AtMYB96 can regulate directly the cuticular wax biosynthetic genes, such as KCS1, CER1, CER3, LTP3 and WSD1, via binding to the MYB binding consensus sequence (BS) in the promoters of these wax biosynthetic genes. Therefore, increased accumulation of cuticular wax is an important stress adaptation strategy that minimizes cellular and organismal dehydration in plants under drought stress conditions. In the present study, overexpression of EsWAX1 was sufficient to activate the expression of several wax biosynthetic genes and greatly elevated cuticular wax accumulation. Our experimental results therefore indicated that the EsWAX1 gene might play important roles in the regulation of cuticular wax biosynthesis. 4.3. EsWAX1 involves in AsA biosynthesis under drought stress conditions Previous studies have indicated that AsA is a major redox buffer, which involved in the regulation of photosynthesis, hormone biosynthesis, and regenerating other antioxidants. Recently, several biosynthetic routes to AsA have been well described. Some attempts have been made to increase AsA content by over-expression of critical genes of AsA biosynthetic pathways (Chen et al., 2003; Hemavathi et al., 2009). In potato, transgenic plants overexpressing GalUR promote the AsA biosynthesis in tubers compared to the control plants (Hemavathi et al., 2009). The similar result has been observed previously in maize, the over-expression of DHAR resulted in a significant increase in AsA content in nonphotosynthetic kernels (Chen et al., 2003). In addition, some studies have shown that the increase in resistance to abiotic stresses is associated with the antioxidant activity of AsA. Overexpression of DHAR (Kwon et al., 2003) and GALDH (Tokunaga et al., 2005) enhanced tolerance of transgenic plants with increased AsA content to salt and oxidative stresses, indicating that AsA recycling also plays an important role in the regulation of AsA levels in plants. Tóth et al. (2011) have reported that high AsA

content of leaves in transgenic Arabidopsis plants over-expressing MIOX4 contributes significantly to the ability of plants to withstand heat-stress conditions. Previous studies have indicated that light or various abiotic stresses such as salt and drought stress can result in significant increases in AsA content in plants (Ioannidi et al., 2009). However, there is little information on molecular mechanisms involved in light or stress-mediated regulation of AsA biosynthesis in plants. A recent study has reported that the photomorphogenic factor CSN5B interact with VTC1, encoding the enzyme GDP Man pyrophosphorylase, involve in the regulation of light-related AsA biosynthsis (Wang et al., 2013). In Arabidopsis, AtERF98 can be quickly induced by NaCl or H2O2 and regulate AsA biosynthesis occurs through the D-Man/L-Gal pathway, which contributes to salinity tolerance in transgenic Arabidopsis plants (Zhang et al., 2012). In the present study, we found that the elevated expression of EsWAX1 led to a significant increase in ASA content in transgenic plants. It was observed that the expression of VTC1, GLDH and MIOX4 and the AsA levels were significantly increased in EsWAX1-OX lines compared to WT. By contrast, there is no discernible difference in the expression of genes involved in the AsA-GSH cycle such as MDAR3, ChlDHAR, CytDHAR and GR1. It allowed us to speculate that EsWAX1 regulated the biosynthesis of AsA through modulating the expression of the mannose/galactose (Man/Gal) route (VTC1 and GLDH) and the myoinositol oxygenase gene (MIOX4). 4.4. RD29A::EsWAX1 transgenic plants exhibited improved drought-stress tolerance Previous studies have shown that abiotic stresses (cold, drought, and salt) induce the expression of genes driven by the promoter of RD29A and RD29B gene (Msanne et al., 2011). The promoter of RD29A is rapidly responded to drought and cold stresses, whereas the promoter of RD29B is highly responsive to salt stress. Thus, candidate genes driven by the promoter of RD29A and RD29B gene sequences has potential to confer abiotic stress resistance in

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Fig. 12. Stress-inducible expression of EsWAX1 increased cuticular wax accumulation in RD29A::EsWAX1 transgenic Arabidopsis lines under drought stress conditions. (A) Accumulation of cuticular wax of both WT and transgenic lines were determined. (BeE) Rosette leaves of 7-week-old WT and transgenic lines grown in soil were used for analysis of cuticular wax composition and loads under well water and drought stress conditions. Data are the means SE of three replicates, the bars with different letters indicate significant differences among three genotypes (WT, RD29A::EsWAX1-6 and RD29A::EsWAX1-9) within well water and drought stress treatments using a Dunnett test at P < 0.05. FW, fresh weight; DR, drought stress treatment.

transgenic plants grown in arid and semi-arid regions. It has recently shown that the stress-inducible RD29A promoter is useful for generating transgenic plants with enhanced tolerance to water stress without affecting on growth and development (Li et al., 2013). In the present study, over-expression of EsWAX1 led to increased accumulation of cuticular wax and the AsA content, however most transgenic plants exhibited phenotypic abnormalities such as curly and small leaves. Thus, we used the RD29A promoter instead of the constitutive CaMV 35S promoter for the over-expression of EsWAX1 in transgenic Arabidopsis plants. And the EsWAX1 transcript was hardly detected in transgenic plants under well water conditions. However, the expression of EsWAX1 was significantly increased in RD29A::EsWAX1 lines after exposure to drought stress. Similar results were observed in antioxidant enzyme activities, which showed significantly higher in RD29A::EsWAX1 lines compared to WT plants under drought stress conditions. In addition, accumulation of cuticular wax was much higher in RD29A::EsWAX1 lines than WT plants. Water loss rate assay further showed that the leaves of RD29A::EsWAX1 lines were lower water loss than that of

WT plants under drought stress conditions. Together, these data indicated that drought stress could be sufficient to induce the expression of EsWAX1 to improve tolerance to drought stress in transgenic plants. In conclusion, we here reported the cloning of a cDNA encoding a novel MYB transcription factor EsWAX1 from E. salsugineum. The EsWAX1 transcript was rapidly induced by ABA and drought stress. Ectopic expression of EsWAX1 in transgenic Arabidopsis plants promoted cuticular wax biosynthesis and increased the AsA content, while its over-expression caused negative effects on plant growth under normal conditions. Furthermore, the use of the RD29A stress-inducible promoter, rather than the 35S promoter, to control EsWAX1 expression improves drought tolerance of transgenic plants without impacting plant normal growth. Author contributions Conceived and designed the experiments: Jian Zhu and Cheng Zhou. Performed the experiments: Lin Zhu and Jiansheng Guo. Analyzed the data: Cheng Zhou. Wrote the manuscript: Lin Zhu.

L. Zhu et al. / Plant Physiology and Biochemistry 75 (2014) 24e35

Acknowledgments We thank Song Rentao, professors of Shanghai University, generous donation of Eutrema salsugineum (Shandong ecotype) seeds, and for their valuable suggestions. This work was supported in part by the Natural Science Foundation of China (Grant No. 30970169). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.plaphy.2013.11.028 References Abe, H., Urao, T., Ito, T., Seki, M., Shinozaki, K., Yamaguchi-Shinozaki, K., 2003. Arabidopsis AtMYC2 (bHLH) and AtMYB2 (MYB) function as transcriptional activators in abscisic acid signaling. Plant Cell 15, 63e78. Aebi, H., 1984. Catalase in vitro. Meth. Enzymol. 105, 121e126. Aharoni, A., Dixit, S., Jetter, R., Thoenes, E., van Arkel, G., Pereira, A., 2004. The SHINE clade of AP2 domain transcription factors activates wax biosynthesis, alters cuticle properties, and confers drought tolerance when overexpressed in Arabidopsis. Plant Cell 16, 2463e2480. Broun, P., Poindexter, P., Osborne, E., Jiang, C.Z., Riechmann, J.L., 2004. WIN1, a transcriptional activator of epidermal wax accumulation in Arabidopsis. Proc. Natl. Acad. Sci. U. S. A. 101, 4706e4711. Brown, P.M., Caradoc-Davies, T.T., Dickson, J.M., Cooper, G.J., Loomes, K.M., Baker, E.N., 2006. Crystal structure of a substrate complex of myo-inositol oxygenase, a di-iron oxygenase with a key role in inositol metabolism. PNAS 103, 15032e15037. Chen, Z., Young, T.E., Ling, J., Chang, S.C., Gallie, D.R., 2003. Increasing vitamin C content of plants through enhanced ascorbate recycling. Proc. Natl. Acad. Sci. U. S. A. 100, 3525e3530. Dhindsa, R.A., Plumb-Dhindsa, P., Thorpe, T.A., 1981. Leaf senescence: correlated with increased permeability and lipid peroxidation and decreased levels of superoxide dismutase and catalase. J. Expt. Bot 32, 93e101. Ding, Z., Li, S., An, X., Liu, X., Qin, H., Wang, D., 2009. Transgenic expression of MYB15 confers enhanced sensitivity to abscisic acid and improved drought tolerance in Arabidopsis thaliana. J Genet. Genom. 36, 17e29. Hemavathi, Upadhyay, C.P., Ko, E.Y., Nookaraju, A., Kim, H.S., Heung, J.J., Oh, M.O., Reddy, A.C., Chun, S.C., Kim, D.H., Park, S.W., 2009. Over-expression of strawberry d-galacturonic acid reductase in potato leads to accumulation of vitamin C with enhanced abiotic stress tolerance. Plant Sci. 177, 659e667. Hemavathi, Upadhyaya, C.P., Akula, N., Young, K.E., Chun, S.C., Kim, D.H., Park, S.W., 2010. Enhanced ascorbic acid accumulation in transgenic potato confers tolerance to various abiotic stresses. Biotechnol. Lett. 32, 321e330. Hu, H., Dai, M., Yao, J., Xiao, B., Li, X., Zhang, Q., Xiong, L., 2006. Overexpressing a NAM, ATAF, and CUC (NAC) transcription factor enhances drought resistance and salt tolerance in rice. Proc. Natl Acad. Sci. U. S. A. 103, 12987e12992. Inan, G., Zhang, Q., Li, P., Wang, Z., Cao, Z., Zhang, H., Zhang, C., Quist, T.M., Goodwin, S.M., Zhu, J., Shi, H., Damsz, B., Charbaji, T., Gong, Q., Ma, S., Fredricksen, M., Galbraith, D.W., Jenks, M.A., Rhodes, D., Hasegawa, P.M., Bohnert, H.J., Joly, R.J., Bressan, R.A., Zhu, J.K., 2004. Salt cress. A halophyte and cryophyte Arabidopsis relative model system and its applicability to molecular genetic analyses of growth and development of extremophiles. Plant Physiol. 135, 1718e1737. Ioannidi, E., Kalamaki, M.S., Engineer, C., Pateraki, I., Alexandrou, D., Mellidou, I., Giovannonni, J., Kanellis, A.K., 2009. Expression profiling of ascorbic acidrelated genes during tomato fruit development and ripening and in response to stress conditions. J. Exp. Bot. 60, 663e678. Jin, H., Martin, C., 1999. Multifunctionality and diversity within the plant MYB-gene family. Plant Mol. Biol. 41, 577e585. Kerstiens, G., 1996. Cuticular water permeability and its physiological significance. J. Exp. Bot. 47, 1813e1832. Kosma, D.K., Bourdenx, B., Bernard, A., Parsons, E.P., Lü, S., Joubès, J., Jenks, M.A., 2009. The impact of water deficiency on leaf cuticle lipids of Arabidopsis. Plant Physiol. 151, 1918e1929. Kwak, S.S., Kim, S.K., Lee, M.S., Jung, K.H., Park, I.H., Liu, J.R., 1995. Acidic peroxidase from suspension cultures of sweet potato. Phytochemistry 39, 981e984.

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