Planta DOI 10.1007/s00425-015-2290-8

ORIGINAL ARTICLE

TraeALDH7B1-5A, encoding aldehyde dehydrogenase 7 in wheat, confers improved drought tolerance in Arabidopsis Jiamin Chen1,2 • Bo Wei1 • Guoliang Li1,3,4 • Renchun Fan1 • Yongda Zhong1,5 Xianping Wang1 • Xiangqi Zhang1

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Received: 23 September 2014 / Accepted: 9 March 2015 Ó Springer-Verlag Berlin Heidelberg 2015

Abstract Main conclusion TraeALDH7B1-5A, encoding aldehyde dehydrogenase 7 in wheat, conferred significant drought tolerance to Arabidopsis, supported by molecular biological and physiological experiments. Drought stress significantly affects wheat yields. Aldehyde dehydrogenase (ALDH) is a family of enzymes catalyzing the irreversible conversion of aldehydes into acids to decrease the damage caused by abiotic stresses. However, no wheat ALDH member has been functionally characterized

J. Chen and B. Wei contributed equally to this work.

Electronic supplementary material The online version of this article (doi:10.1007/s00425-015-2290-8) contains supplementary material, which is available to authorized users. & Xiangqi Zhang [email protected] 1

State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, No. 1 West Beichen Road, Chaoyang District, Beijing 100101, People’s Republic of China

2

Hefei No. 1 High School, No. 2356 Xizang Road, Lakeshore New District, Hefei, Anhui 230601, People’s Republic of China

3

Institute of Genetics and Physiology, Hebei Academy of Agriculture and Forestry Sciences, Shijiazhuang 050051, People’s Republic of China

4

University of Chinese Academy of Sciences, Beijing 100049, People’s Republic of China

5

The Key Laboratory of Horticultural Plant Genetic and Improvement of Jiangxi, Institute of Biology and Resources, Jiangxi Academy of Sciences, No. 7777 Changdong Road, Nanchang, Jiangxi 330096, People’s Republic of China

to date. Here, we obtained a differentially expressed EST encoding ALDH7 from a cDNA-AFLP library of wheat that was treated with polyethylene glycol 6000. The three full-length homologs of TraeALDH7B1 were isolated by searching the NCBI database and by homolog-based cloning method. Using nulli-tetrasomic lines we located them on wheat chromosomes 5A, 5B and 5D, and named them as TraeALDH7B1-5A, -5B and -5D, respectively. Gene expression profiles indicated that the expressions of all three genes were induced in roots, leaves, culms and spikelets under drought and salt stresses. Enzymatic activity analysis showed that TraeALDH7B1-5A had acetaldehyde dehydrogenase activity. For further functional analysis, we developed transgenic Arabidopsis lines overexpressing TraeALDH7B1-5A driven by the cauliflower mosaic virus 35S promoter. Compared with wild type Arabidopsis, 35S::TraeALDH7B1-5A plants significantly enhanced the tolerance to drought stress, which was demonstrated by up-regulation of stress responsive genes and physiological evidence of primary root length, maintenance of water retention and contents of chlorophyll and MDA. The combined results indicated that TraeALDH7B15A is an important drought responsive gene for genetic transformation to improve drought tolerance in crops. Keywords Aldehyde dehydrogenase  Drought tolerance  Common wheat  Arabidopsis Abbreviations AFLP Amplified fragment length polymorphism ALDH Aldehyde dehydrogenase DAS Days after sowing DAR Days after rewatering EST Expressed sequence tag NAD Nicotinamide-adenine dinucleotide

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NADP PEG WT

Nicotinamide-adenine dinucleotide phosphate Polyethylene glycol Wild type

Introduction Drought stress leads to both physiological and biochemical changes in plants, including stomatal closure, repression of cell growth and photosynthesis, and activation of respiration. Plants also respond and adapt to dehydration at both the cellular and molecular levels, for instance by the accumulation of osmotic agents and activating a range of genes with diverse functions (Bartels and Sunkar 2005; Shinozaki et al. 2003). In addition, drought induces the biosynthesis of the abscisic acid (ABA), which in turn causes expression changes of stress-related genes. Actually, previous studies demonstrate the presence of both ABAindependent and ABA-dependent regulatory systems controlling drought-inducible gene expression (YamaguchiShinozaki and Shinozaki 2005). Furthermore, drought stress also leads to accumulation of reactive oxygen species (ROS), which promote aldehyde production by a lipid peroxidation chain reaction (Bartels 2001; Zhu 2001). Because of their chemically reactive nature and toxic effect, excessive amounts of aldehydes threaten the plant growth (Bartels 2001; Jakoby and Ziegler 1990). Aldehyde dehydrogenases (ALDHs) are considered important mechanisms for the detoxification of aldehydes by oxidation to their corresponding carboxylic acids (Bartels 2001; Lindahl 1992; Vasiliou et al. 2000). ALDHs are widely distributed across all organisms from bacteria to humans and plants (Brocker et al. 2013; Kirch et al. 2004; Yoshida et al. 1998). Previous studies of the ALDH superfamilies throughout all taxa identified 24 families based on their protein sequence identities (Hou and Bartels 2014; Perozich et al. 1999; Skibbe et al. 2002; Sophos and Vasiliou 2003). These families are named based on the criteria from the ALDH Gene Nomenclature Committee (AGNC) (Vasiliou et al. 1999). In plant, there are 14 distinct families: ALDH2, ALDH3, ALDH5, ALDH6, ALDH7, ALDH10, ALDH11, ALDH12, ALDH18, ALDH19, ALDH21, ALDH22, ALDH23 and ALDH24. Among them, family ALDH10, ALDH12, ALDH19, ALDH21, ALDH22, ALDH23 and ALDH24 are plant-specific, whereas other families have their mammalian orthologues (Chen et al. 2002; Hou and Bartels 2014). In plants, many ALDH family genes respond to a wide variety of abiotic stresses including dehydration, high salinity, heat, oxidative stress and ultraviolet radiation (UVR) by regulating the plant homeostasis (Brocker et al. 2013; Chugh et al. 2011; Inostroza-Blancheteau et al.

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2011; Sunkar et al. 2003), while some members further affect the development processes indirectly (Fait et al. 2008; Shin et al. 2009; Toyokura et al. 2011). Three ALDH3 isoforms-ALDH3F1, ALDH3H1 and ALDH3Idiffer in their expression profiles and responsive patterns under stress conditions. ALDH3I expression restricting in leaves is induced by exogenous ABA treatment, high salinity, dehydration, heavy metals, oxidants and pesticides (Kirch et al. 2001; Sunkar et al. 2003). ALDH3H1 was up-regulated by the stress in roots and constitutively expressed in leaves with weak signals. By contrast, the expression of ALDH3F1 was not changeable after the treatment with stresses mentioned above (Kirch et al. 2004). Mutations of ALDH5F1, a member of the succinicsemialdehyde dehydrogenase (SSADH) family in plants, enhance the level of H2O2, suggesting that the gene causes adaptation to stress by preventing the accumulation of reactive oxygen species (Bouche´ et al. 2003). Furthermore, SSA, the substrate of ALDH5F1, affects the robust leaf patterning and structure along the adaxial-abaxial axis (Toyokura et al. 2011). Microarray studies reveal that ALDH6B3 and ALDH6B7 are up-regulated by the longterm stresses of high salinity and water-deficit (Zhang et al. 2012). Additionally, two members of the ALDH10 family, ALDH10A8 and ALDH10A9, are induced by dehydration and high salt stresses and catalyze oxidation of betaine aldehyde to glycine betaine (Weretilnyk and Hanson 1990). The accumulation of glycine betaine is a universal osmoregulation process (Rhodes and Hanson 1993). ALDH11A3, encoding a non-phosphorylating GAPDH, functions in the cytosol of autotrophic eukaryotes and catalyzes one of the classic glycolytic ‘bypass’ reactions which would facilitate the development and acclimate to unavoidable environmental stresses such as anoxia and Pi starvation (Mertens 1991; Plaxton 1996; Sung et al. 1988). In Arabidopsis, ALDH12A1, encoding a mitochondrial D1-pyrroline-5-carboxylate dehydrogenase (P5CDH), was identified by complementing the yeast Dput2 mutant, and its transcript is induced by exogenous proline application and salinity (Deuschle et al. 2001; Hare and Cress 1997). Also, plant ALDH18 genes are strikingly up-regulated by dehydration, which are coupled with proline accumulation to relief the osmotic imbalances (Yoshiba et al. 1997). Recently, ALDH22A1 in maize is found to be induced by various abiotic stresses such as dehydration, high salinity and ABA treatment. The transgenic plants overexpressed ALDH22A1 confer the stress tolerance (Huang et al. 2008). Members of ALDH7 family are known as D1-piperideine-6-carboxylate dehydrogenases (P6CDH), aaminoadipic semialdehyde dehydrogenases or antiquitins. In both Arabidopsis thaliana and rice, their ALDH7 members are responsive to oxidative and abiotic stresses

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(Kirch et al. 2005; Shin et al. 2009). In soybean, an antiquitin-like ALDH7 gene confers tolerance to high salinity and drought when overexpressed in Arabidopsis and tobacco (Rodrigues et al. 2006). Interestingly, it is found that OsALDH7 protein can maintain seed viability by detoxifying the aldehydes generated by lipid peroxidatoin (Shin et al. 2009) and involve in lysine catabolism (Shen et al. 2012). Also, the antiquitin level is increased during apple fruit development (Yamada et al. 1999), which leads to the suggestion that the antiquitin may function in development. Despite that, there was still few physiological and molecular biological evidence reported during the drought stress-responsive processes participated by ALDH7 members. Here, we obtained ALDH7 gene from common wheat through a modified cDNA-AFLP strategy. Expression analysis showed that it was induced by polyethylene glycol (PEG) 6000 and salt stress. To further test its functions, we over-expressed it in Arabidopsis and found that TraeALDH7B1-5A transgenic plants under drought stress produced elongated primary roots, and had improved water retention and drought tolerance. Simultaneously, the expression profiles of many abiotic stress responsive genes were changed, providing partial molecular evidence for enhanced drought tolerance. Our work identifies TraeALDH7B1-5A as a potential gene resource to improve drought tolerance of crop plants by genetic transformation.

Materials and methods Plant materials Wheat (Triticum aestivum L.) cultivar Jihan 044 treated with PEG 6000 was firstly used to conduct cDNA-AFLP to obtain differentially expressed ESTs (Bachem et al. 1996; Korpelainen and Kostamo 2010). To further study the expression pattern of TraeALDH7B1-5A, 12 other wheat accessions were used. To determine the chromosomal locations of the three homoeologous TraeALDH7B1 genes, one accession each of the A genome (Triticum urartu, AA, 2n = 2x = 14), S genome (Aegilops speltoides, the putative B genome donor, SS, 2n = 2x = 14), D genome (Aegilops tauschii, DD, 2n = 2x = 14) donor, one hexaploid wheat variety Chinese Spring (AABBDD, 2n = 6x = 42) and 21 nulli-tetrasomic lines of Chinese Spring were used. Arabidopsis thaliana (Columbia-0), for genetic transformation was grown in a growth chamber at 22 °C with 16 h light (light intensity of 120 lmol/m2s) and 18 °C with 8 h darkness and 70 % relative humidity after breaking the seed dormancy for 2 days at 4 °C. Three T3 homozygous transgenic lines were selected for functional analyses. All

seeds were harvested from the same batch. For analysis of expression pattern, seed germination and root growth experiments, seeds of wild type (WT) and transgenic lines were planted on Murashige and Skoog (MS) medium supplemented with different amounts of PEG 6000 or mannitol (Man) (Murashige and Skoog 1962). Sequence alignment and phylogenetic analysis To obtain homolgous protein sequences of ALDH7 in other species, the putative amino acid sequence of TraeALDH7B1-5A was used as query to perform BlastP search (NCBI, Basic Local Alignment Search Tool). Sequences with 85–100 % identities were collected for further analysis. The MegAlign program (DNAStar package) was used to perform sequence alignment and estimate sequence similarity under default settings (Fig. S1 and S2). A rooted neighbor-joining (NJ) tree was constructed using the MEGA 6.06 package (Saitou and Nei 1987). Tree nodes were evaluated by bootstrap analysis for 100 replicates (Tamura et al. 2013). Branches with less than 50 % bootstrap values were collapsed. Accession numbers and organism sources of all ALDH7 proteins are listed in Table S1. RT-PCR Total RNAs of wheat and Arabidopsis were extracted using TRIZOL reagent following the manufacturer’s instructions (Invitrogen, CA, USA). Reverse transcription reactions were performed as described previously (Wei et al. 2013). Semi-RT-PCR were carried out to detect the expression profiles of TraeALDH7B1-5A, -5B and -5D under normal growing conditions, and 18 % PEG 6000 and 250 mM NaCl treatments. PCR was carried out using a Veriti 96-well Thermal Cycler (Applied Biosystems) with following program: initial denaturation at 95 °C for 3 min; 30 cycles at 95 °C for 15 s, 55 °C for 15 s, 72 °C for 30 s, and a final extension at 72 °C for 10 min or adjusted accordingly to give the best results. Gene-specific primer sets for the three homoeologs were used to detect the expression signals, and TaTubulin gene was used as internal control. Real-time quantitative PCR were performed to detect the expressions of abiotic stress-responsive genes using an Applied Biosystems 7500 Real-Time PCR System. PCR were performed according to the instructions of SYBR Premix Ex Taq (Perfect real time) (Takara, Japan). Fluorescence signals were measured at each polymerization step. The relative expression of each gene was calculated according to the method of 2-DDCT (Livak and Schmittgen 2001). Ct values were averages of three independent biological replicates. Actin 2 was used as internal control. All of the primer sets for abiotic stress-related

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genes, described previously or designed in this study, were listed in Table S2. Acetaldehyde dehydrogenase activity assay With primer set in Table S3, the ORF of TraeALDH7B1-5A was amplified using TransStart FastPfu DNA Polymerase (Transgen, Bejing) according to the manufacturer’s introduction. The PCR fragment was ligated pET-30a vector (Novagen) after double digesting with EcoRI and Sal I. The construct was transformed into E. coli strain BL21 cell (DE3). Positive clone, verified by DNA sequencing, was incubated at 37 °C overnight, then transferred to 200 ml fresh LB liquid medium (1.0 % tryptone, 0.5 % yeast extract, and 1.0 % NaCl) and incubated continuously in a 37 °C shaker. Once the OD 600 value reached to 0.6–0.8, 0.5 mM isopropyl b-D-1-thiogalactopyranoside (IPTG) was added, and incubation was continued at 25 °C for another 5 h. To purify TraeALDH7B1-5A, cells were harvested at 8000 rpm for 5 min, resuspended in 15 ml of 1 9 Ni-NTA bind buffer (300 mM NaCl, 50 mM Na3PO4 and 10 mM imidazole, pH 8.0) with 100 ll PMSF (10 mM) (Amreasco) and 0.02 g lysozyme (MP, Biomedical), and incubated on ice at 150 rpm for 30 min, followed by adding 20 ll Triton X-100 and incubating on ice at 150 rpm for another 40 min. The solution was sonicated three times for 20 s with a 20 s stop using a Uibra cell with 40 % duty cycle. The lysate was centrifuged at 12,000 rpm for 10 min at 4 °C, and the supernatant was filtered with aperture at 0.22 lm (MILLEX). Filtered supernatant was transferred to a new triangular flask and incubated for 1 h at 4 °C after adding 1.0 ml Resin (Roche). Then, the 6 9 His-tagged TraeALDH7B1-5A proteins were purified on a Ni-NTA Spin column (Qiagen), followed by washing three times with 4 ml 1 9 Ni-NTA wash buffer (300 mM NaCl, 50 mM Na3PO4 and 20 mM imidazole, pH 8.0), and eluting four times with 0.5 ml 1 9 Ni-NTA elution buffer (300 mM NaCl, 50 mM Na3PO4 and 250 mM imidazole, pH 8.0). For our enzyme assay, 10-50 lg of purified TraeALDH7B1-5A recombinant protein was added to a 1 ml reaction system containing 1.5 mM NAD (Sigma), 0.1 M sodium pyrophosphate buffer (pH 8.5) and 1 mM acetaldehyde. The abosorbances at 340 nm were detected at the 20 s time points using a Multiskan spectrum (Thermo). Enzyme activities were calculated according to the previous reported (op den Camp and Kuhlemeier 1997). The values were averages of three replicates. Generation of transgenic plants The slightly modified binary vector pCambia 1300 was used to construct the overexpressed vector. The coding sequence of TraeALDH7B1-5A including BamHI (50 ) and

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SalI (30 ) was amplified using the primers shown in Table S3. Both the PCR product and pCambia 1300 plasmid were double digested with BamHI and SalI. Then, the PCR product was cloned into binary vector pCambia 1300 under control of the cauliflower mosaic virus 35S promoter in the sense orientation. The entire construct was transformed into Arabidopsis plants using the floral-dip method according to previously described procedures (MartinezTrujillo et al. 2004) using Agrobacterium tumefaciens strain GV3101 (Koncz and Schell 1986). Transgenic Arabidopsis plants were screened on solid MS medium containing 25 mg/l hygromycin. Seed germination assays For seed germination assays, seeds from TraeALDH7B15A transgenic and WT plants were treated in 100 % ethanol for 1 min, surface-sterilized in 10 % (v/v) NaClO with 0.2 % (v/v) Triton X-100 for 10 min and followed by six times washing with sterilized distilled water. Thirty seeds, in three replicates, for each transgenic and the WT were sown on solid MS medium plates with 100 mM mannitol. The percentages of germinated seeds were calculated based on the number of seedlings that reached the cotyledon stage at 2 weeks (Saleki et al. 1993). Root growth experiments Surface-sterilized seeds of transgenic and WT plants were separately sown on MS medium plates and moved to normal growing conditions after vernalization at 4 °C for 2 days. Then, three-day-old well-developed seedlings were chosen to grow vertically on MS medium with 200 mM mannitol. The root lengths of 10 plants in each line were measured when the seedlings had grown for nine or 10 days under normal culture conditions. Morphological characterization of transgenic plants Twelve uniform seven-day-old seedlings of 35S::TraeALDH7B1-5A transgenic lines and WT plants were transferred to well-watered soil from MS medium. The plants were grown without further watering until evidence of drought stress. At 47 days after planting they were rewatered. Survival rates were calculated by dividing the total numbers of transplants by the numbers of surviving plants. Three replicates were conducted. Measurement of malondialdehyde and chlorophyll content For measurements of malondialdehyde (MDA) contents, leaves of seedlings were grinded in 2.0 ml tubes with

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0.5 M phosphate buffer (pH 7.8), then centrifuged at 4500 rpm, 4 °C for 10 min. The supernatants (0.6 ml) were transferred to 2.0 ml fresh tubes with 0.9 ml of 0.5 % (w/v) TBA (Thiobarbituric acid) in 5 % (w/v) TCA (Trichloracetic acid). The supernatants were boiled for 20 min, cooled quickly on ice, and centrifuged at 4500 rpm, 4 °C for 10 min. Absorbances at 532 and 600 nm were measured using a Multiskan spectrum (Thermo). The MDA contents were calculated as described previously (Hodges et al. 1999). The values were averages of five biological replicates. Chlorophyll was extracted from detached leaves with 95 % ethanol. Chlorophyll a and b contents were calculated from absorption values at 663 and 645 nm according to a previously published report (Lichtenthaler 1987). The values were averages of 10 leaves from each tested line.

fresh diploid cells were plated on SD medium lacking Trp, Leu, His, and Ade. Plates were incubated for up to 7 days at 30 °C for detecting interactions that activate both the HIS and ADE reporter genes. All assays were repeated three times with fresh transformants. ABA content measurement For ABA content measurement, the seeds from TraeALDH7B1-5A transgenic and WT plants were sown on the MS medium plants. The 10-day old seedlings were subjected to PEG 6000 treatment for 3 h and harvested for measurements. The ABA content measurement was performed as described (Fu et al. 2012).

Results Yeast two hybrid assays Entire wheat seedlings treated with PEG 6000 and 200 mM NaCl were harvested to prepare a protein expression library. mRNA was extracted using a FastTrackÒ 2.0 Kit (Life Technology), then double-strand cDNA was synthesized according the manufacturer’s instructions. A yeasttwo-hybrid library (Prey) was developed by recombination of double-strand cDNA and the pGADT7-Rec2 vector (Clontech). The bait vector was constructed by cloning the coding sequence of TraeALDH7B1-5A into the pGBKT7 vector (Clontech). The bait vector and prey plasmid (library) were co-transformed into yeast strain AH109. Interaction signals appeared on SD solid medium lacking Trp, Leu, His, and Ade in 3–7 days. All positive signals were confirmed through DNA sequencing. To confirm protein interactions, the coding sequences of putative interactive proteins (AD-1, -2, -3 -4, -5 and -6) were isolated and cloned into pAD-Gal4-2.1 (prey), and coding sequence of TraeALDH7B1-5A was cloned into pBD-Gal4Cam (bait) (Stratagene, La Jolla, CA, USA). Using the LiAc method (Clontech, Mountain View, CA, USA), bait and prey constructs were transformed into MATa (PJ69-4A) and MATa (PJ69-4A), respectively (James et al. 1996). A pair-wise scheme was used to detect interactions. Interactions between the bait protein (BDTraeALDH7B1-5A) and empty AD, prey proteins and empty BD, empty BD and empty AD provided negative controls. Yeast cells containing bait or prey vectors were grown overnight (30 °C, 250 rpm). Mating was conducted by dropping 5 ll of each culture serially onto solid YPD (Clontech). Cells were grown at 30 °C overnight and transferred to SD medium lacking Trp and Leu to screen diploids containing prey and bait vectors. After 2 days,

Isolation and molecular characterization of homoeologous ALDH7 genes in common wheat Modified cDNA-AFLP was used to detect differentially expressed ESTs in wheat cultivar Jihan 044 when grown under normal conditions and PEG 6000 stress. A gene fragment (21-1-1) was significantly up-regulated by PEG 6000 treatment (Fig. S3). Nucleotide blast analysis revealed that the sequence of the fragment was highly similar to genes family 7 ALDHs in monocots, such as TrurALDH7B1 in Triticum urartu (KD107258.1), AetaALDH7B1 in Aegilops tauschii (KD510307.1) and BrdiALDH7B1 Brachypodium distachyon (XM_003578133.2) (Fig. S1 and S2). It was therefore suggested to be ALDH7 family member genes and named TraeALDH7B1. Moreover, all three homoeologous ALDH7 genes were isolated using the homologous cloning method. The open reading frames of the three TraeALDH7B1 genes were 1530, 1473 and 1530 bp, putatively encoding polypeptides with 510 aa (amino acid residues), 492 and 510 aa, respectively (Fig. S1). Multiple sequence alignment showed that the ALDH7 family proteins in various species were highly conserved, especially members from the subfamily Pooideae, sharing at least 95 % similarities (Fig. S2). The three wheat homologs showed about 90 % similarities with counterparts in maize, sorghum and foxtail millet, 77–79 % with ALDH7B4 (NP_175812), 75–77 % with GlmaALDH7B1 (XP_003546512) in soybean and about 58 % with HosaALDH7B1 (P49419) in humans (Fig. S2). A phylogenetic tree constructed for the wheat proteins and their homologs from other plants revealed a monocots-specific clade including TrurALDH7B1, AespALDH7B1, AetaALDH7B1, HovuALDH7B1 and BrdiALDH7B1 (Fig. 1).

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Planta Fig. 1 Phylogenetic tree of three homoeologous TraeALDH7B1 proteins and orthologs from other plant species. The TraeALDH7B1 proteins are marked with solid triangles. Accession numbers and species sources were provided in Table S1

Chromosome localizations of three homoeologous TraeALDH7B1 genes in wheat

To investigate the expression patterns of the three homoeologous TraeALDH7B1 genes, we designed genome-

specific primer sets and examined their expression profiles in roots, culms, leaves and spikelets under normal growing condition and two abiotic stresses, drought (18 % PEG 6000) and salt (250 mM NaCl) using a semi-quantitative RT-PCR method. TraeALDH7B1-5A, -5B and -5D displayed similar expression patterns under both normal and stress conditions (Fig. 3), while the expressions were rather weak under normal growing condition. However, expression signals were dramatically increased following treatment with 18 % PEG 6000 (Fig. 3). In addition, the three genes were up-regulated in leaves following exposure to drought (18 % PEG 6000) and salt (250 mM NaCl). The highly similarities (98.6 %) among the three TraeALDH7B1 protein and similar expression patterns (Fig. 3) among the three genes revealed that they might play similar roles in drought stress tolerance. Therefore, in this study, the TraeALDH7B1-5A gene was selected for the further functional analyses. Further investigation showed that TraeALDH7B1-5A was strongly up-regulated by the PEG 6000 in 12 other wheat accessions, but

Fig. 2 Chromosomal localizations of three homoeologous TraeALDH7B1 genes in wheat. CS represents the Hexaploid wheat accession Chinese Spring; AA, BB and DD represent three diploid

relative wheat accessions with AA, BB and DD genome, respectively; 1A–7D represent nulli-tetrasomic lines deleting specific corresponding chromosome

To determine the chromosome localizations of three TraeALDH7B1 genes, we detected them in hexaploid wheat cultivar Chinese Spring, three diploid related species accessions and a series of nulli-tetrasomic lines using allele-specific primer pairs. As Fig. 2 shown, three homoeologous TraeALDH7B1 genes were not amplified in 5A, 5B and 5D nulli-tetrasomic lines, which indicated that they were assigned to chromosomes 5A, 5B and 5D, respectively (Fig. 2). Furthermore, they were named as TraeALDH7B1-5A, TraeALDH7B1-5B and TraeALDH7B1-5D, respectively. Responses of TraeALDH7B1 genes to PEG 6000 and NaCl stresses

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Fig. 3 Expression profile analysis of TraeALDH7B1-5A, -5B and 5D. C and P in the first lane represent control and 18 % PEG 6000 treatment, respectively. The second and third lanes show the

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Fig. 4 Enzymatic assay of acetaldehyde dehydrogenase activity of TraeALDH7B1-5A protein. a Purification of TraeALDH7B1-5A recombinant protein. The purified protein contains a His tag consisting of six histidine residues. 1 Marker, 2 the noninduced protein, 3 the induced protein, 4 the purified recombinant protein. b Enzymatic assay of TraeALDH7B1-5A recombinant protein

expression patterns of three homoeologous TraeALDH7B1-5A genes at the testing time points under 18 % PEG 6000 and 250 mM NaCl stresses, respectively

       



     

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reaching highest expression levels at different time points (Fig. S4). TraeALDH7B1-5A had acetaldehyde dehydrogenase activity Multiple sequence alignment of TraeALDH7B1-5A and its counterparts from other species revealed that TraeALDH7B1-5A shared typical functional residues represented in aldehyde dehydrogenases (Fig. S1). As Fig. S1 shown, Gly254, Gly259, Glu432 and Phe434 were integral for binding the nicotinamide ring of NAD (Ni et al. 1997; Ohta et al. 2005; Wymore et al. 2004), whereas Cys331 was the catalytic cysteine and Gly328 an essential residue for the positioning of the catalytic nucleophile (Liu et al. 1997; Wymore et al. 2004). In addition, it also had the GxxxG conserved NAD-binding motif (Liu et al. 1997). To detect whether TraeALDH7B1-5A protein possessed the acetaldehyde dehydrogenase activity, we expressed it in E. coli cell, and purified it. As Fig. 4a showed, the molecular weight of the tested purified protein is about 61 kD, which was consistent with that of the 6 9 His tagged TraeALDH7B1-5A recombinant protein. Aldehyde dehydrogenases are a group of enzymes catalyzing the conversion of aldehydes to the corresponding acids by means

of an NAD(P)?-dependent virtually irreversible reaction (Yoshida et al. 1998). In our study, the amount of NADH was increased after adding substrate acetaldehyde into TraeALDH7B1-5A protein enzymatic assay system (Fig. 4b). The result suggested that TraeALDH7B1-5A had the acetaldehyde dehydrogenase activity. Seed germination was delayed in TraeALDH7B1-5A transgenic Arabidopsis plants under mannitolinduced drought stress Biochemical result showed that TraeALDH7B1-5A had acetaldehyde dehydrogenase activity, to further test its possible roles in planta, we over-expressed TraeALDH7B1-5A in wild type Arabidopsis (ecotype Colombia-0). The construct consisted of the TraeALDH7B1-5A open reading frame driven by the cauliflower mosaic virus 35S promoter. A total of 45 positive transgenic plants were obtained. Three T3 homozygous lines were selected for further functional analyses. As shown in Fig. 5a, under normal growing condition (MS medium), the three 35S::TraeALDH7B1-5A lines and WT had similar germination rates. However, after 24 h of culturing on 100 mM mannitol, germination rates of the transgenic lines (50 %) were significantly lower than

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Planta Fig. 5 Seed germination, primary root growth and water retention levels in TraeALDH7B1-5A transgenic and wild type Arabidopsis plants. a Seeds of three 35S::TraeALDH7B1-5A lines and WT were sown on MS medium to measure seed germination rates at 24, 48 and 72 h. b Germination rates of three 35S::TraeALDH7B1-5A lines and WT on MS plates supplemented with 100 mM mannitol. c Comparison of primary root lengths of 35S::TraeALDH7B1-5A and WT on MS plates supplemented with 200 mM mannitol. d Statistical comparison of primary root lengths of 35S::TraeALDH7B1-5A and WT under 200 mM mannitol treatment. At least 10 plants for each line were measured. *P \ 0.05, **P \ 0.01. e Comparisons of water loss rates from detached tissue sections between transgenic and WT plants. Five separate plants for each line were measured. Tests were replicated three times. Values are mean ± SD

that of WT (70 %) (Fig. 5b). At 48 h, nearly all seeds had germinated on both MS and 100 mM mannitol (90–100 %) (Fig. 5a, b). These results indicated that overexpression of TraeALDH7B1-5A delayed the early stages of seed germination when subjected to mannitol-induced drought stress.

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Primary root elongation was promoted in TraeALDH7B1-5A transgenic plants under mannitol-induced drought stress Root growth is highly sensitive to osmotic stress induced by drought and salt stresses (Hsiao and Xu 2000). In this

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study, we found that the primary roots of TraeALDH7B15A transgenic lines were significantly longer than those of WT when treated with 200 mM mannitol (Fig. 5c, d). TraeALDH7B1-5A over-expression in Arabidopsis improved water retention Water retention abilities were assessed on three TraeALDH7B1-5A transgenic lines and WT. As shown in Fig. 5e, water loss rates of three 35S::TraeALDH7B1-5A transgenic lines were significantly lower than those of WT, indicating that 35S::TraeALDH7B1-5A plants may be able to reduce transpiration and save water to enhance the drought tolerance. Drought tolerance in TraeALDH7B1-5A overexpressing plants

phenotypes, and most of them maintained normal greenness (Fig. 6a). Chlorophyll (a and b) contents in the three transgenic lines were much higher than that in WT Arabidopsis (Fig. 6b, c), and MDA contents were lower in transgenic lines than that in WT (Fig. 6d), which were consistent with their drought tolerance. Before rewatering at 47 DAS the frequent dead leaves on WT plants indicated a severe degree of drought stress. At 7 days after rewatering, 52–87 % of 35::TraeALDH7B1-5A plants survived, whereas the survival rate of WT plants was less than 10 % (Fig. 6e). Thus, overexpression of TraeALDH7B1-5A can enhance the drought tolerance in Arabidopsis. Expression changes of abiotic stress related-genes in TraeALDH7B1-5A transgenic Arabidopsis plants

To determine whether the TraeALDH7B1-5A gene improves drought tolerance we observed the comparative morphological changes in three 35S::TraeALDH7B1-5A lines and WT under water deficit condition. Twelve plants for each line were cultured under the normal growing conditions after 2 days of vernalization (Fig. 6a). At 42 DAS (days after sowing) leaves of WT plants were obviously withered and even whitened, whereas only some individuals of the transgenic lines showed severe stress

Morphological analyses indicated that TraeALDH7B1-5A transgenic plants enhanced tolerance to drought stress. To uncover the underlying molecular mechanisms, transgenic lines A7A-32-8 and A7A-35-1 were selected for expression pattern assays under normal and PEG 6000 stress conditions (Fig. 7). A total of 20 genes were detected including five ABA synthesis or responsive genes and 15 abiotic stress-related genes involved in various pathways associated with drought stress. Compared to normal growing conditions, expression signals of 17 genes were significantly increased when

Fig. 6 Drought tolerance was improved in Transgenic TraeALDH7B1-5A Arabidopsis lines. a Morphological changes in three transgenic lines and WT under water-deficit conditions. Twelve plants were transplanted for each line, and five replicates were

conducted. DAS days after sowing, DAR days after rewatering. b, c Chlorophyll a and b contents in 42-day-old plants. Data are means of 10 plants per line. d MDA contents in transgenic lines and WT. e Survival rates of transgenic lines and WT

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DREB2A

RAB18

RD29A

RD29B

COR15A

1C 2C WTC 1P 2P WTP

1C 2C WTC 1P 2P WTP

1C 2C WTC 1P 2P WTP

MYB2

MYC2

1C 2C WTC 1P 2P WTP

1C 2C WTC 1P 2P WTP

1C 2C WTC 1P 2P WTP

AREB2

1C 2C WTC 1P 2P WTP

1C 2C WTC 1P 2P WTP

AREB1

1C 2C WTC 1P 2P WTP

1C 2C WTC 1P 2P WTP

RD22 1.2 1 0.8 0.6 0.4 0.2 0

ABA2

WRKY54

1C & & 2C :7& WTC 3 1P 3 2P :73 WTP

DREB1A

1.2 1 0.8 0.6 0.4 0.2 0

1C 2C WTC 1P 2P WTP

COR47

1C 2C WTC 1P 2P WTP

CBF2

1C 2C WTC 1P 2P WTP

ABI5

1C 2C WTC 1P 2P WTP

CBF1

& 1C & 2C :7& WTC 3 1P 3 2P :73 WTP

ABI2

1C 2C WTC 1P 2P WTP

ABI1

& 1C & 2C :7& WTC 3 1P 3 2P :73 WTP

ABA1

1C 2C WTC 1P 2P WTP

TraeALDH7B1-5A

1C 2C WTC 1P 2P WTP

1.2 1 0.8 0.6 0.4 0.2 0

1C 2C WTC 1P 2P WTP

Planta

Fig. 7 Relative expression of stress-responsive genes in TraeALDH7B1-5A transgenic plants and WT under normal and PEG 6000 stress conditions. Two transgenic lines were selected, and three independent biological replicates for each line were performed

for quantitative RT-PCR. Values are mean ± SE. C, normal growing conditions (control); P, PEG 6000 treatment; 1 and 2, transgenic lines A7A-32-8 and A7A-35-1, respectively

plants were subjected to PEG 6000 treatment (Fig. 7). With the exceptions of ABI5, CBF1, COR15A and RD29A that showed similar levels of expression in 35S::TraeALDH7B1-5A plants and WT, the other 13 genes, ABI1, ABI2, CBF2, DREB1A, DREB2A, COR47, RAB18, RD29B, RD22, AREB1, AREB2, MYB2 and MYC2, were significantly up-regulated in transgenic plants when compared with WT (Fig. 7). ABA1 and ABA2 expressed equally in both transgenic and wild type plants under normal conditions; however, in 35S::TraeALDH7B1-5A plants they showed much higher levels of expression than in WT when subjected to PEG 6000 treatment. Interestingly, WRKY54, a negative regulator of stomatal closure (Li et al. 2013), was expressed at a much higher level under normal conditions than under PEG 6000 stress, and transgenic plants showed a lower transcription signal than WT in both normal and PEG 6000 stress conditions. Thus, improvements in drought tolerance of 35S::TraeALDH7B1-5A plants probably resulted from expression changes of

drought stress-related genes, including those detected in this research.

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TraeALDH7B1-5A protein interaction detection In humans catalytically active forms of ALDH3 and 4 are homodimers (Yoshida et al. 1998). Also, human antiquitin is enzymatically active as homotetramers (Tang et al. 2008). However, no interaction proteins of ALDH7 family members are reported in plants. To investigate the possible interaction partners of TraeALDH7B1-5A protein in wheat, we developed a TraeALDH7B1-5A expression vector with pGBDK and expressed TraeALDH7B1-5A in AH109 yeast cells and screened the protein expression library (pGADT-Rec2). A total of six putative interaction proteins were identified (Table S4). To further confirm the result, we expressed TraeALDH7B1-5A in pBD-Gal4Cam and six putative interacted proteins in pAD-Gal4-2.1, and transformed them into yeast MATa (PJ69-4A) and MATa

Planta Fig. 8 Protein interaction assays of TraeALDH7B1-5A protein. BDe and ADe, empty BD and AD vectors were used as negative controls; SD-TL, synthetic dropout nutrient medium lacking Trp and Leu; SD-TLHA, SD solid medium lacking Trp, Leu, His, and Ade

(PJ69-4A) cells. Only ALDH7B was confirmed as an interaction protein. Therefore, TraeALDH7B1-5A may interact with itself in common wheat (Fig. 8).

Discussion Functions of ALDH7 family genes in plants In this study, we characterized a PEG 6000-induced gene TraeALDH7B1-5A, which encodes aldehyde dehydrogenase 7 in common wheat. Enzymatic activity assay showed that TraeALDH7B1-5A had the aldehyde dehydrogenase activity in mean of conversion NAD? into NADH? (Fig. 4). Transgenic TraeALDH7B1-5A Arabidopsis had enhanced drought tolerance. Furthermore, we provided physiological and molecular biological evidence to explain its drought tolerance. We found the lengths of primary roots were significantly longer than that of WT, which could make TraeALDH7B1-5A-overexpressing plants absorb more water than WT from soil (Fig. 5c, d). Furthermore, stronger water retaining capacity reduced more transpiration and saved more water to increase the drought tolerance (Fig. 5e). Additionally, in transgenic plants, many stress related genes were also triggered which urged us to consider that the enhancement of drought tolerance of transgenic lines were resulted from contributions of multiple abiotic responsive pathways (Figs. 7, 9). ALDH7 family genes are highly conserved in animals and plants (Shen et al. 2012). In this study, we showed that TraeALDH7B1-5A shared 91.8 % identity with its counterpart in rice (Fig. S1 and S2). In spite of that, slight different expression patterns were observed between TraeALDH7B1-5A and OsALDH7. Unlike TraeALDH7B15A showing the most abundant expression signals in leaves (Fig. 3), OsALDH7 expressed the highest level of transcript in matured seeds (Shen et al. 2012), which was consistent with its roles on seed maturation, seed viability and accumulation of oryzamutaic acid A (Shen et al. 2012; Shin

et al. 2009). In addition, our study also showed that the seed germination of TraeALDH7B1-5A transgenic lines was slower than WT on MS medium with 100 mM mannitol (Fig. 5b), which was different from the results in soybean (Rodrigues et al. 2006). This functional divergence of ALDH7 family members could be real since there is only 77.3 % identity at the protein level between TraeALDH7B1-5A and GlmaALDH7B1 (AAP02957.1) (Fig. S2). The possible mechanisms of drought tolerance in TraeALDH7B1-5A transgenic plants TraeALDH7B1-5A was significantly up-regulated under drought and salt stress, and TraeALDH7B1-5A transgenic Arabidopsis lines had enhanced drought tolerance. This was partially consistent with previous works on homologs in rice and Arabidopsis (Rodrigues et al. 2006; Shen et al. 2012). Moreover, we found that many abiotic stress responsive genes were triggered in TraeALDH7B1-5A transgenic plants when they were subjected to PEG 6000 treatment (Figs. 7, 9). The fact that two ABA biosynthesis-related genes ABA1 and ABA2 were up-regulated by PEG 6000 stress in TraeALDH7B1-5A transgenic plants indicated that ABA might be responsive in transgenic plants (Le´on-Kloosterziel et al. 1996; Xiong et al. 2001). The ABA response stimulates ABA-dependent pathways and enhances integrative tolerance to multiple abiotic stresses (Roychoudhury et al. 2013). As Fig. 9 shown, two stress responsive genes MYB2 and MYC2, and their targeting gene RD22, were up-regulated (Abe et al. 1997, 2003); similarly, AREB1 and AREB2, and their regulating gene RD29B were also induced in transgenic plants (Figs. 7, 9) (Uno et al. 2000). Additionally, the ABA-independent pathways involving DREB2A, CBF2/DREBC, DREB1A, and their target gene COR47 were also activated in TraeALDH7B1-5A transgenic plants (Figs. 7, 9) (Gilmour et al. 1998; JagloOttosen et al. 1998; Liu et al. 1998). In contrast,

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Planta Fig. 9 Putative genetic pathways up-regulated in TraeALDH7B1-5A transgenic plants under PEG 6000 stress conditions. Dotted lines represent deductions, whereas the solid lines are previously known pathways. MYBRS, MYB-recognition sequence; MYCRS, MYC-recognition sequence; ABRE, ABAresponsive element; DRE, dehydration-responsive element. MYBRS, MYCRS, ABRE and DRE, cis-elements in the promoter regions of RD22, RD29B and COR47, respectively

transcriptions of some well-known stress responsive genes, such as ABI5, CBF1, COR15A and RD29A, were not changed, indicating only certain pathways were probably triggered in 35S::TraeALDH7B1-5A plants under PEG 6000 stress (Fig. 7). WRKY54 functions as a negative regulator of stomatal closure and consequently enhances osmotic stress tolerance in Arabidopsis (Li et al. 2013). It was coincided with the results that expression of WRKY54 was significantly decreased under PEG 6000 treatment compared with normal growing condition (Fig. 7). Additionally, to some extent, expression change of WRKY54 could explain why TraeALDH7B1-5A transgenic plants loss more water than WT during the same time intervals (Figs. 5e, 7). Interestingly, in this study, we also found the ABA contents in WT Arabidopsis plants were significantly higher than those in transgenic lines (Fig. 10), which was likely to be explained by the findings that reactive oxygen species (ROS) may regulate the ABA signaling (Xiong et al. 2002) and ABA biosynthesis (Zhao et al. 2001). As revealed by the recent studies, ROS, which is considered as a signaling, may trigger the ROS scavenging system and even talk crossly with ABA to protect the plants from damage by environmental stresses (Kar 2011; Kwak et al. 2003). Aldehyde dehydrogenases (ALDHs) detoxify aldehydes promoted by the ROS during the abiotic stress tolerance (Bartels 2001; Lindahl 1992; Vasiliou et al. 2000). In this study, compared with the WT plants, the 35S::TraeALDH7B1-5A lines may deoxidize more aldehyde, thereby decrease the levels of ROS content, which was supported by the result that malonyldialdehyde (MDA)

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Fig. 10 Comparisons of ABA contents in WT and 35S::TraeALDH7B1-5A plants after PEG 6000 treatment. At least twenty separate plants for each line were mixed and sampled. Measurements were replicated three times. Values are mean ± SD

contents in transgenic plants were significantly lower than that in WT plants (Fig. 6d). Furthermore, the relative low level of ROS in 35S::TraeALDH7B1-5A lines might regulate the ABA content. Actually, it is very interesting that what is the relationship among drought stress, ABA and ROX and how does the plant balance the two signals to minimize the damage caused by drought stress in TraeALDH7B1-5A transgenic plants. Identification and characterization of stress-responsive genes are necessary for the improvement of stress tolerance of crops using transgenic approaches. In this study, we cloned a drought-induced gene, TraeALDH7B1-5A in common wheat. Transgenic Arabidopsis system revealed

Planta

its potential function in water-deficit stress tolerance. These results provided important clues for thoroughly understanding the functions and mechanisms of TraeALDH7B15A. However, we cannot ignore the differences between model plant Arabidopsis and common wheat. Therefore, a further functional analysis of TraeALDH7B1-5A in drought tolerance would need to be conducted using transgenic modified wheat lines. Thus, the ultimate potential value of this gene in wheat improvement can be evaluated. This research reported a drought induced gene TraeALDH7B1-5A in common wheat. The gene conferred significant drought tolerance of Arabidopsis, which was supported by molecular biological and physiological experiments in this research. Our study laid an important molecular foundation for further utilizing it to improve the drought tolerance of wheat cultivars. Author contribution XZ conceived and designed research. JC, BW, GL and YZ conducted experiments. RF and XW contributed new reagents or analytical tools. AM and GR analyzed data. BW wrote the manuscript. All authors read and approved the manuscript. Acknowledgments We thank the expertise of Miss Shuang Fang and Dr. Jinfang Chu (National Centre for Plant Gene Research (Beijing), Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China) in determining the ABA contents of the plant materials. We thank Professor Robert A. McIntosh (Plant Breeding Institute, University of Sydney, NSW, Australia) for revising the manuscript and constructive advice. The project was sponsored by National Natural Science Foundation of China (31101141) and National Key Technology R & D Program (2011BAD07B00). Conflict of interest declare.

The authors have no conflicts of interest to

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