Transcriptional Regulation of Expression of the Maize Aldehyde Dehydrogenase 7 Gene (ZmALDH7B6) in Response to Abiotic Stresses

Journal of Integrative Agriculture 2014, 13(9): 1900-1908

September 2014

RESEARCH ARTICLE

Transcriptional Regulation of Expression of the Maize Aldehyde Dehydrogenase 7 Gene (ZmALDH7B6) in Response to Abiotic Stresses AN Xia*, DUAN Feng-ying*, GUO Song, CHEN Fan-jun, YUAN Li-xing and GU Ri-liang Department of Plant Nutrition, Center for Resources, Environment and Food Security, China Agricultural University, Beijing 100193, P.R.China

Abstract Aldehyde dehydrogenases (ALDHs) represent a large protein family, which includes several members that catalyze the oxidation of an aldehyde to its corresponding carboxylic acid in plants. Genes encoding members of the ALDH7 subfamily have been suggested to play important roles in various stress adaptations in plants. In this study, quantitative RT-PCR analysis revealed that a maize ALDH7 subfamily member (ZmALDH7B6) was constitutively expressed in various organs, including roots, leaves, immature ears, tassels, and developing seeds. The abundance of ZmALDH7B6 mRNA transcripts in maize roots was increased by ammonium, NaCl, and mannitol treatments. To further analyze tissue-specific and stress-induced expression patterns, the 1.5-kb 5´-flanking ZmALDH7B6 promoter region was fused to the β-glucuronidase (GUS) reporter gene and introduced into maize plants. In roots of independent transgenic lines, there was significant induction of GUS activity in response to ammonium supply, confirming ammonium-dependent expression of ZmALDH7B6 at the transcript level. Histochemical staining showed that GUS activity driven by the ZmALDH7B6 promoter was mainly localized in the vascular tissues of maize roots. These results suggested that ZmALDH7B6 is induced by multiple environmental stresses in maize roots, and may play a role in detoxifying aldehydes, particularly in vascular tissue. Key words: abiotic stress, aldehyde dehydrogenase, gene expression, promoter, transgenic maize

INTRODUCTION Aldehydes are common intermediates and/or by-products of several fundamental metabolic pathways in plants, including the metabolisms of vitamins, amino acids, carbohydrates, and lipids (Yoshida et al. 1998). Aldehydes can also be produced under several environmental stresses, such as salinity, dehydration, desiccation, and cold and heat shock (Laszlo et al. 1998; Bartels 2001; Kirch et al. 2004; Kotchoni et al. 2006). Nevertheless, excess aldehydes can have detrimental effects on cel-

lular metabolism because their electrophilic carbonyl groups have strong chemical reactivity (Lindahl 1992; Schauenstein et al. 1997; Wacker et al. 2001). They can attack cellular nucleophiles (e.g., nucleic acids) and lead to chromosomal aberrations or DNA adducts. Some studies have suggested that certain aldehydes might also have signaling functions (Weber et al. 2004). Given their important biochemical and physiological functions, it is important that in vivo concentrations of aldehydes are tightly controlled. Aldehyde dehydrogenases (ALDHs) catalyze the oxidation of aldehydes to the corresponding carboxylic acid, and play roles in regulating cellular aldehyde

Received 15 March, 2013 Accepted 17 April, 2013 AN Xia, E-mail: [email protected]; Correspondence GU Ri-liang, Tel: +86-10-62734424, Fax: +86-10-62731016, E-mail: [email protected] * These authors contributed equally to this study.

© 2014, CAAS. All rights reserved. Published by Elsevier Ltd. doi: 10.1016/S2095-3119(13)60518-3

Transcriptional Regulation of Expression of the Maize Aldehyde Dehydrogenase 7 Gene (ZmALDH7B6) in Response to

RESULTS Organ-dependent transcription of ZmALDH7B6 To explore the putative function of ZmALDH7B6, we determined the abundance of ZmALDH7B6 transcripts in various organs of field-grown maize plants. In general, we detected relatively low transcript levels of ZmALDH7B6, about 1/100 to 1/10 that of the housekeeping gene ZmGAPDH. ZmALDH7B6 was constitutively expressed in all tested organs and tissues; the highest level (in seeds) was only about 5-fold of the lowest one (in ear leaves) (Fig. 1). 0.10

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levels (Lindahl 1992; Bartels 2001). ALDHs are encoded by members of a large gene family with at least 22 subfamilies (Sophos and Vasiliou 2003). In plants, ALDH members have been assigned into 12 subfamilies based on their protein sequences (Kirch et al. 2004; Kotchoni et al. 2006; Rodrigues et al. 2006; Gao and Han 2009). Expression analyses have revealed that a large number of ALDH members are transcriptionally activated in response to various environmental stresses (Kirch et al. 2001; Bouche et al. 2003; Kotchoni et al. 2006; Rodrigues et al. 2006), and play roles in plant stress tolerance (Nakazono et al. 2000; Chen et al. 2002; Sunkar et al. 2003; Huang et al. 2008). Among stress-related ALDHs, the ALDH7 subfamily is represented in most plant species by a single gene with an ancient origin (Chen et al. 2002; Brocket et al. 2012). This pattern of evolution implies that there are conserved functions of ALDH7 orthologs in the plant kingdom. ALDH7 expression in Arabidopsis or rice has been detected in various plant organs (e.g., roots, leaves, and reproductive organs), and in response to a range of stresses (Kirch et al. 2004; Gao and Han 2009). The level of ALDH7 transcripts in other plant species has also been reported to be enhanced under conditions of dehydration, low temperature, heat shock, and high concentrations of abscisic acid (Rodrigues et al. 2006; Zhou et al. 2012). Its stress-inducible expression pattern implies that ALDH7 is probably involved in plant adaptation to environmental stresses. Maize (Zea mays L.) is one of the most widely cultivated crop plants, providing food for humans, feed for animals, and materials for industries. A maize ALDH7 gene (ZmALDH7B6B6) was identified after whole genome surveys of the ALDH gene superfamily (Jimenez-Lopez et al. 2010; Brocker et al. 2012). However, the expression patterns and functions of ZmALDH7B6 remained under investigation. In this work, the organ-specific and stress-dependent expressions of the ZmALDH7B6 gene were analyzed by quantitative RT-PCR. The 1.5-kb promoter region of ZmALDH7B6 was fused to the β-glucuronidase (GUS) reporter gene and introduced into maize. The promoter activities of ZmALDH7B6 were evaluated to clarify its tissue-specific and stress-responsive expression patterns. This work provides solid results on the expression of the ZmALDH7B6 gene, which will help to uncover its functions.

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Fig. 1 ZmALDH7B6 transcript levels in maize organs. RNA samples were extracted from various organs or tissues of field-grown maize plants at different developmental stages. Roots and shoots were harvested at the five-leaf stage (28 days after sowing), ear leaves, tassels, and immature ears were collected at the silking stage (90 days after sowing), and seeds were sampled 15 days after pollination. Transcript levels of ZmALDH7B6 were quantified by real-time RTPCR and normalized to that of the housekeeping gene ZmGAPDH. Values are means±SD (n=3).

Stress-responsive expression of ZmALDH7B6 To investigate the expression pattern of ZmALDH7B6 under stress conditions, total RNA was extracted from roots of maize seedlings that grown hydroponically under mannitol and salt stresses. The transcript level of ZmALDH7B6 in roots was markedly increased by both treatments (Fig. 2). In the NaCl treatment, ZmALDH7B6 transcripts started to accumulate after 3 h NaCl supply and peaked at 6 h. Mannitol treatment resulted in a more rapid increase, with ZmALDH7B6 transcript levels increasing within 1 h and peaking at 3 h (Fig. 2). Next, we investigated the transcript levels of ZmAL© 2014, CAAS. All rights reserved. Published by Elsevier Ltd.

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Fig. 2 ZmALDH7B6 transcription in maize roots in response to NaCl and mannitol treatments. Hydroponically pre-cultured maize plants (five-leaf stage) were subjected to salt-stress (150 mmol L-1 NaCl) and osmotic-stress (300 mmol L-1 mannitol) treatments from 0 to 24 h. RNA was extracted from maize roots and used to determine ZmALDH7B6 transcript levels. Results from real-time RT-PCR were normalized to the housekeeping gene ZmGAPDH, and the value of pre-treated plants was set to 1. Values are means±SD (n=3). Different letters indicate significant differences at P<0.05. The same as in Fig. 3.

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DH7B6 in response to different forms of nitrogen. The transcript abundance of ZmALDH7B6 did not change significantly under nitrogen starvation for up to 96 h (Fig. 3-A). Re-supplying 4 mmol L-1 (NH4)2SO4 to N-deficient roots resulted in a sharp increase in ZmALDH7B6 transcripts, but there was not a significant change at transcript levels when N-deficient roots were resupplied with 4 mmol L-1 KNO3 (Fig. 3-A). These results indicated that ZmALDH7B6 transcription in maize roots could be induced by (NH4)2SO4, but not by KNO3. To further role out the possible effect of ZmALDH7B6 expression by SO42- or K+, we conducted comparative analysis of the effects of (NH4)2SO4, K2SO4, KNO3, or KCl on ZmALDH7B6 transcription (Fig. 3-B). Treatment with (NH4)2SO4 increased transcript levels of ZmALDH7B6 in maize roots, but treatments with K2SO4, KNO3, or KCl did not. This result suggested that ZmALDH7B6 transcription in maize roots could be induced by ammonium ions (NH4+), but not by nitrate ions (NO3-).

Generation of ZmALDH7B6p-GUS transformed maize To investigate whether the accumulation of ZmALDH7B6 transcripts in response to various stresses was controlled at the transcriptional level, transgenic maize plants were generated to express the GUS reporter gene

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Fig. 3 ZmALDH7B6 transcription in maize roots in response to nitrogen. A, ZmALDH7B6 transcript levels in maize plants (10-dayold) pre-cultured hydroponically with 2 mmol L-1 NH4NO3, then kept in nitrogen-free nutrient solution for 48 and 96 h (-N), and then resupplied with 2 mmol L-1 (NH4)2SO4 or 4 mmol L-1 KNO3 from 1 to 24 h. B, ZmALDH7B6 transcript levels in maize plants starved of nitrogen for 96 h, then resupplied with 2 mmol L-1 (NH4)2SO4, 2 mmol L-1 K2SO4, 4 mmol L-1 KNO3, or 4 mmol L-1 KCl for 12 h. RNA samples were extracted from maize root and used to analyze transcript levels of ZmALDH7B6. Results from real-time RT-PCR were normalized to the housekeeping gene ZmGAPDH, and the value under continuous NH4NO3 supply was set to 1.

under the control of the 1 499-bp ZmALDH7B6 gene promoter (ZmALDH7B6p) (Fig. 4-A). In total, 12 PPT-resistant T0 plants were regenerated and further screened by PCR (Fig. 4-B). Five PCR-positive plants were used for Southern blot analysis. Four lines showed a visible positive band; two lines showed a single copy insertion (lines 2 and 5) and the other two showed multiple copy insertions (lines 6 and 10; Fig. 4-C). Each transgenic line showed a distinctive hybridization pattern indicating that each was an independent transformation event.

Activity of ZmALDH7B6 gene promoter in maize organs To confirm the organ-dependent expression of ZmALD© 2014, CAAS. All rights reserved. Published by Elsevier Ltd.

Transcriptional Regulation of Expression of the Maize Aldehyde Dehydrogenase 7 Gene (ZmALDH7B6) in Response to

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Fig. 4 Generation of ZmALDH7B6p-GUS transgenic maize. A, schematic representation of plasmid construct ZmALDH7B6p-GUS. GUS gene and 35S polyA terminator were under the control of the ZmALDH7B6 gene promoter. B, RT-PCR analysis of GUS gene expression in transformed maize. cDNA reverse-transcribed from total RNA extracted from maize leaves served as template for PCR with GUS-specific primers. W, water (negative control); WT, wild type maize plant (non-transgenic plant) as negative control; P, corresponding plasmid used for transformation; M, DNA marker. C, Southern blot analysis of BC3F2 transgenic maize plants. EcoR I-digested total DNA (100 mg) from maize leaf was fractionated on 0.7% agarose gel, blotted onto a nylon membrane, and probed with 32P-labeled Bar gene.

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To examine ZmALDH7B6 promoter activity in response to nitrogen, transgenic maize plants grown hydroponically were subjected to nitrogen starvation and nitrate/ ammonium resupply. A quantitative GUS activity assay showed that the activity of ZmALDH7B6p in the transgenic plant roots was significantly increased by (NH4)2SO4 resupply, while there was no significant response to nitrogen starvation or KNO 3 resupply (Fig. 6-A). Ammonium-induced ZmALDH7B6 promoter activity began at 3 h after treatment and peaked at 12 h. As well as treatment with (NH4)2SO4, transgenic maize plants were treated with K2SO4, KNO3, and KCl, but

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H7B6, GUS activities driven by the ZmALDH7B6 promoter were determined in various organs of transgenic lines 2 and 5. We observed GUS signals in both lines in all tested organs, including roots, leaves, tassels, immature ears, and seeds (Fig. 5-A). A quantitative GUS activity assay showed that line 5 had higher GUS activity than that of line 2 (Fig. 5-B). Both lines showed constitutive enzyme activity in all investigated organs. The highest enzyme activity (in tassels) was approximately 3-5 times greater than its lowest activity (in seedling shoots). This result was consistent with the ZmALDH7B6 mRNA levels detected in these organs (Fig. 1).

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Fig. 5 GUS activity of ZmALDH7B6p-GUS in various organs of field-grown maize plants. Plant organs were harvested at seedling stage (28 days after sowing), silking stage (90 days after sowing), and 15 days after pollination (DAP). A, histochemical localization of GUS activity in maize organs. B, quantitative GUS activity in various organs. Values are means±SD (n=4-6).

only the (NH4)2SO4 treatment significantly increased ZmALDH7B6 promoter activity (Fig. 6-B). This result

© 2014, CAAS. All rights reserved. Published by Elsevier Ltd.

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was consistent with the pattern of ZmALDH7B6 transcription (Fig. 3).

Tissue-specific activity of ZmALDH7B6 promoter in maize roots To determine the tissue localization of ZmALDH7B6 promoter activity, we conducted a histochemical analysis of ZmALDH7B6 promoter activity in roots of transgenic Lines 2 and 5. Both transgenic lines showed strong GUS signals in the root elongation zone, but not at the root tip (Fig. 7-A). Further histochemical analyses of cross- and longitudinal-sections indicated that the ZmALDH7B6 promoter drove GUS expression mainly in the root vascular cells (Fig. 7-B and C).

ous works have shown that ALDHs indirectly detoxify cellular reactive oxygen species (ROS) and reduce the effects of cellular toxicity under environmental stresses (Kotchoni et al. 2006; Wei et al. 2013). The generation of ROS is common to many abiotic stresses, including ammonium (Laloi et al. 2004; Reinehr et al. 2007). Ammonium ions might mimic the effect of some environmental stresses (e.g., salinity) and trigger the increase of H2O2, probably via common mechanisms. Thus, it is possible that ALDH has a similar role to that of ROS (H2O2)-scavengers during ammonium resupply. There is a second possibility with regards to the mechanism of ammonium-activated ALDHs expression. Besides reducing the level of reactive aldehydes, ALDH also produces the corresponding carboxylic acid. The carbon backbone released from the ALDH-catalyzed

DISCUSSION GUS activity (nmol L-1 4-MU min-1 mg-1 protein)

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ALDH7 enzymes play important roles in the detoxification process of aldehydes, which are generated in plants in response to abiotic stresses (Bartels 2001; Kirch et al. 2004; Kotchoni et al. 2006). ALDH7 genes can be transcriptionally activated by abiotic stresses (Skibbe et al. 2002; Kirch et al. 2004) and transgenic plants over-expressing ALDH7 displayed enhanced tolerance to stress conditions (Kotchoni et al. 2006; Rodrigues et al. 2006). In this study, expression of ZmALDH7B6 was induced under mannitol and salt stresses (Fig. 2), similar to the regulatory responses of other ALDH7 orthologs. Given that the ALDH7 subfamily has evolved throughout the plant kingdom, and a single member is present in most plant species (Brocker et al. 2012), it is expected that ALDH7 orthologs would have a conserved function in response to environmental stresses. Besides mannitol and salt stresses, in this work we also reported that ZmALDH7B6 expression was induced by ammonium ion re-supply (Fig. 3). Promoter-GUS analysis in transgenic maize confirmed this ammonium-inducible expression pattern and further indicated that up-regulation of ZmALDH7B6 transcription was controlled by its promoter (Fig. 6). In contrast, nitrate, as another important nitrogen source, did not trigger up-regulation of ZmALDH7B6. These results suggested that ZmALDH7B6 transcription is induced in response to ammonium ions, rather than nitrogen nutrition. Previ-

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Fig. 6 ZmALDH7B6 promoter activity in transgenic maize root in response to different nitrogen treatments. A, GUS activity in 10-dayold transgenic maize plants (line 2) pre-cultured hydroponically with 2 mmol L-1 NH4NO3, then kept in nitrogen-free nutrient solution for 96 h (-N), and then resupplied with 2 mmol L-1 (NH4)2SO4 or 4 mmol L-1 KNO3. B, GUS activity in maize plants starved of nitrogen for 96 h, and then resupplied with 2 mmol L-1 (NH4)2SO4, 2 mmol L-1 K2SO4, 4 mmol L-1 KNO3 or 4 mmol L-1 KCl for 12 h. Maize root were harvested for GUS activity analysis. Values are means±SD (n =4-6). Different letters indicate significant differences at P<0.05. © 2014, CAAS. All rights reserved. Published by Elsevier Ltd.

Transcriptional Regulation of Expression of the Maize Aldehyde Dehydrogenase 7 Gene (ZmALDH7B6) in Response to

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by several abiotic stresses. This work provides new insights into the expression of ZmALDH7B6, which can provide clues for gene functional annotation.

MATERIALS AND METHODS B

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Fig. 7 Histochemical localization of GUS gene expression driven by ZmALDH7B6 promoter in transgenic maize root. Lines 2 and 5 were germinated on paper. Plant roots were harvested at 12 days after germination. A, whole maize root. GUS signals were observed in root elongation zone but not in root tip. B, longitudinal section of young root. C, transverse section of young root. High GUS activity was detected in vascular tissue.

reaction could fuel metabolism to accelerate ammonium assimilation. This metabolic link between ammonium nutrition and stress adaption in plants has been supported by previous studies on tobacco and grape. In those studies, abiotic stresses induced expressions of glutamate dehydrogenase (GDH) and isocitrate dehydrogenase genes, whose products contribute to ammonium assimilation (Skopelitis et al. 2006). Transgenic studies using gain-of-function or loss-of-function approaches would uncover the functions of ALDH7 in ammonium assimilation.

CONCLUSION This work provided solid results on the expression patterns of the ZmALDH7B6 gene by monitoring its mRNA abundance and promoter activity in transgenic maize. Both methods gave the same results about its expression patterns. In general, ZmALDH7B6 was constitutively expressed in maize organs. In roots, ZmALDH7B6 was mainly expressed in root vascular tissue and was induced

Maize plants were grown in the field and corresponding tissues were collected for organ-specific gene expression and GUS activity analysis. Roots, shoots, and leaves of maize seedlings were harvested at the five-leaf stage (28 days after sowing). Ear leaves, tassels, and immature ears were collected at the silking stage (90 days after sowing). Immature seeds were sampled at 15 days after pollination (DAP). For NaCl and mannitol treatments, seedlings (five-leaf stage) grown in a hydroponic system were subjected to 150 mmol L-1 NaCl or 300 mmol L-1 mannitol, respectively. The composition of the hydroponic nutrient solution (in mmol L-1) was as follows: 2.0 Ca(NO3)2, 0.75 K2SO4, 0.1 KCl, 0.25 KH2PO4, 0.65 MgSO4 and 0.1 EDTA-Fe, and (in μmol L-1) 1.0 MnSO4, 1.0 ZnSO4, 0.1 CuSO4, and 0.005 (NH4)6Mo7O24. Roots were collected from seedlings from 0 to 24 h after treatment and frozen in liquid nitrogen for RNA extractions or GUS activity assays. For nitrogen treatments, maize seedlings were grown in the solution described above with 2 mmol L-1 NH4NO3 as nitrogen sources. The five-leaf stage plants were transferred to nitrogen-free solution for 4 days, and then re-supplied with 2 mmol L-1 (NH4)2SO4, 2 mmol L-1 K2SO4, 4 mmol L-1 KNO3, or 4 mmol L-1 KCl. Root tissues were collected and subsequently frozen in liquid nitrogen until analysis.

Analysis of ZmALDH7B6 transcript levels The transcription profile of the ZmALDH7B6 gene was examined by quantitative RT-PCR (qPCR). Total RNA was extracted using Trizol reagent (Invitrogen, Carlsbad, CA, USA). DNA contamination in RNA samples was removed using RNasefree DNase (Invitrogen). cDNA was reverse-transcribed with M-MLV reverse transcriptase (Invitrogen). qPCR reactions were performed in a 7500 Real-Time PCR System (Applied Biosystems, CA, USA) using SYBR Green dye (Applied Biosystems). Amplification was carried out in a two-step PCR procedure with 40 cycles of 15 s at 95°C for denaturation, 15 s at 60°C for annealing, and 60 s at 72°C for extension. Dissociation curves obtained by heating the amplicon from 60 to 98°C were analyzed to verify the specificity of the reaction. The transcript level of the ZmGAPDH gene (GenBank accession no.: NM_001111943.1) served as the internal control for organ-specific expression, and that of the Alpha

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tubulin4 gene (ZmTUB4, AJ420856.1) served as the control for stress-dependent expression (Czechowski et al. 2005). Three biological replications of qPCR were performed for each sample. The primers for the target or control genes were as follows: GAPDH-F: 5´-CTGGTTTCTACCGACTTCCTTG-3´, GAPDH-R: 5´-CGGCATACACAAGCAGCAAC-3´; TUB4-F: 5´-GCTATCCTGTGATCTGCCCTGA-3´; TUB4-R: 5´-CGCCAAACTTAATAACCCAGTA-3´; and ZmALDH7B6-F: 5´-CTTGAACTTAGCGGAAACA-3´; ZmALDH7B6-R: 5´-TGAAGAATCAGCCTACGAC-3´.

Cloning of ZmALDH7B6 gene promoter and construction of pZmALDH7B6p-GUS vector The 1 449-bp upstream sequence of the ZmALDH7B6 gene (maize sequence number: GRMZM2G130440, www. maizegdb.org) (Brocker et al. 2012) from the translation start codon ATG was amplified from maize genomic DNA by pfu DNA polymerase (Promega, Madison, WI, USA). The primers were as follows: ALDH7p-F: 5´-AAGCTTAAGGAGCAAAGCGACTCGGTTAC3´and ALDH7p-R: 5´-CCATGGTGTCGGCGTTGGTAG GTGATGCG-3´. Two restriction enzyme sites, Hind III and Nco I, were introduced into the forward and reverse primers, respectively. The amplified product was subsequently cloned into the pGEM T-easy vector (Promega, USA) for sequencing and then cloned into the Hind III and Nco I-digested binary vector pCAMBIA3301 (Gu et al. 2006) after digestion of the PGEM T-easy vector using these two enzymes. The CaMV35S promoter in front of the GUS reporter gene was replaced by the ZmALDH7B6 promoter, yielding pZmALDH7B6p-GUS.

Maize transformation The pZmALDH7B6p-GUS construct was introduced into the Agrobacterium tumefaciens strain EHA105 using the freezethaw method. Single colonies were selected to transform maize hybrid line Hi-II (HA×HB) using the method adapted from Duncan et al. (1985). Immature embryos (1.0-2.0 mm long) were isolated and dissected into liquid infection medium (D basal medium (Duncan et al. 1985) plus 68.5 g L-1 sucrose, 36.0 g L-1 glucose, 100 μmol L-1 acetosyringone, pH 5.2) and washed twice with this medium. Then, 1.5 mL A. tumefaciens suspension was added for infection. All embryos were submerged in the suspension and incubated for 5 min before being transferred onto the surface of co-cultivation medium (D basal medium plus 20 g L-1 sucrose, 10 g L-1 glucose, 0.85 mg L-1 silver nitrate, 100 μmol L-1 acetosyringone, 8 g L-1 agar, pH 5.8), oriented with the embryo-axis side embedded in the medium (scutellum side up) and then incubated in the dark at 25°C for 3 days. Embryos were then transferred to resting medium (D basal medium plus 20 g L-1 sucrose, 10 g L-1 glucose, 0.85 mg L-1 silver nitrate, 250 mg L-1 cefotaxime,

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8 g L-1 agar, pH 5.8) and kept in the dark at 28°C for 7 days. Then, embryos were transferred to selection medium (resting medium plus 1.38 g L-1 L-proline, 0.5 mL L-1 2,4-D, 5 mg L-1 phosphinothricin (PPT) (Shinyo Sangyo, Tokyo, Japan) and kept for 2 weeks in the dark at 28°C. Then selection was increased to 10 mg L-1 PPT for another three successive selections. PPT-resistant calli were placed on regeneration medium (the same as selection medium but without hormones and PPT). Once shoots reached about 2 to 3 cm in height, plantlets were transferred to half-strength Murashige and Skoog (MS) rooting medium and grown under light conditions at 28°C. Regenerated plantlets were grown in a growth chamber at 28°C under fluorescent white lights with a 16 h L:8 h D cycle. Because of the complexity of the hybrid maize genome, the regenerated Hi-II plants were cross-pollinated with the inbred line B73 and their BC3F2 lines were selected to analyze GUS activity.

Molecular identification of transgenic maize plants Total RNA was extracted from leaves of different transformed T 0 maize plants using Trizol reagent (Invitrogen) and reverse-transcribed into cDNA by M-MLV reverse transcriptase (Invitrogen). The GUS transcript was amplified using the following GUS gene primers: (GUS-F: 5´-CAGGAAGTGATGGAGCATCAG-3´ and GUS-R: 5´-TCGTGCACCATCAGCACGTTA-3´). Maize tubulin cDNA was also amplified as a control with the primers TUB-F: 5´-GCTATCCTGTGATCTGCCCTGA-3´ and TUB-R: 5´-CGCCAAACTTAATAACCCAGTA-3´. Southern blot analyses were carried out to analyze integration of the GUS gene in transgenic plants by standard procedures as described by Sambrook and Russell (2001). DNA was isolated from maize leaves by the sodium dodecyl sulfate method and then purified and quantified. DNA (approx. 100 μg) was digested by EcoR I (Promega), fractionated on 0.7% agarose gels, and transferred to a nylon membrane (Amersham Biosciences, Buckinghamshire, UK). The membrane was probed with the α-32P dCTP labeled Bar gene using the Primera-GeneTM Labeling System (Promega). The Bar gene was amplified by PCR with the primers Bar-F: 5´-ATGAGCCCAGAACGACGCCCGGCCG-3´ and Bar-R: 5´-TCAAATCTCGGTGACGGGCAGGAC-3´. Blots were exposed to X-ray film (Kodak, Tokyo, Japan) for imaging.

GUS activity assay and GUS staining Plant samples were harvested and immediately homogenized by grinding in 0.7 mL protein extraction buffer (0.05 mol L-1 Na2HPO4-NaH2PO4, pH 7.0, 0.1% sodium dodecyl sulfate, 10 mmol L -1 ethylenediamine tetraacetic acid (EDTA), 20% methanol, 10 mmol L-1 β-mercaptoethanol, and Triton

© 2014, CAAS. All rights reserved. Published by Elsevier Ltd.

Transcriptional Regulation of Expression of the Maize Aldehyde Dehydrogenase 7 Gene (ZmALDH7B6) in Response to

X-100). After centrifugation at 13 000×g for 15 min at 4°C, 50 μL supernatant was transferred into a microcentrifuge tube containing 250 μL GUS reaction buffer (protein extraction buffer containing 2 mmol L-1 4-methylumbelliferyl-β-Dglucuronide; MUG) pre-heated to 37°C, and then mixed. Then, 50 μL of the solution was immediately mixed with 950 μL GUS stop buffer (0.2 mol L-1 Na2CO3) to serve as the control. The remaining solution was incubated at 37°C. Aliquots (50 μL) were collected at 10, 20, 30, 40, and 60 min and mixed with 950 μL GUS stop buffer. Activity assays were performed in a Microfluor fluorometer (Model 450) with emission at 455 nm and excitation at 365 nm. Protein concentrations in samples were determined by the Bradford method. GUS enzyme activity is expressed as nmol 4-MU produced per minute per mg protein. For GUS histochemical staining, samples were immersed in staining solution (50 mmol L-1 NaPO4, pH 7.0/10 mmol L-1 EDTA/2 mmol L-1 5-bromo-4-chloro-3-indoyl glucuronide/ 1 mmol L -1 potassium ferrocyanide) at 37°C overnight after being fixed in 42.5% ethanol/5% acetic acid/3.7% formaldehyde. The samples were cleared of chlorophyll using 70% ethanol and used for imaging.

Acknowledgements

We thank Mr. Ayaz Ali Soomro (China Agricultural University, China) for English editing. This work was financially supported by the National 863 Program of China (2012AA100306), the National 973 Program of China (2011CB100305), the National Natural Science Foundation of China (30971863, 31121062), and the Ministry of Agriculture of China (2011ZX08003-005).

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