MdVHA-A encodes an apple subunit A of vacuolar H+-ATPase and enhances drought tolerance in transgenic tobacco seedlings

Journal of Plant Physiology 170 (2013) 601–609

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Molecular Biology

MdVHA-A encodes an apple subunit A of vacuolar H+ -ATPase and enhances drought tolerance in transgenic tobacco seedlings Qing-Long Dong a,b , Chun-Rong Wang a , Dan-Dan Liu a , Da-Gang Hu a , Mou-Jing Fang a , Chun-Xiang You a , Yu-Xin Yao a,∗ , Yu-Jin Hao a,∗ a State Key Laboratory of Crop Biology, National Research Center for Apple Engineering and Technology, College of Horticulture Science and Engineering, Shandong Agricultural University, Tai’an, Shandong 271018, China b Shandong Institute of Pomology, Tai’an, Shandong 271000, China

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Article history: Received 9 April 2012 Received in revised form 29 November 2012 Accepted 4 December 2012 Available online 9 February 2013 Keywords: Apple (Malus domestica B.) Vacuolar H+ -ATPase gene Gene expression Lateral root Drought tolerance

s u m m a r y Vacuole H+ -ATPases (VHAs) are plant proton pumps, which play a crucial role in plant growth and stress tolerance. In the present study, we demonstrated that the apple vacuolar H+ -ATPase subunit A (MdVHAA) is highly conserved with subunit A of VHA (VHA-A) proteins from other plant species. MdVHA-A was expressed in vegetative and reproductive organs. In apple in vitro shoot cultures, expression was induced by polyethylene glycol (PEG)-mediated osmotic stress. We further verified that over-expression of MdVHA-A conferred transgenic tobacco seedlings with enhanced vacuole H+-ATPase (VHA) activity and improved drought tolerance. The enhanced PEG-mimic drought response of transgenic tobacco seedlings was related to an extended lateral root system (dependent on auxin translocation) and more efficient osmotic adjustment. Our results indicate that MdVHA-A is a candidate gene for improving drought tolerance in plants. © 2013 Elsevier GmbH. All rights reserved.

Introduction Vacuole H+ -ATPase (VHA) is one of plant proton pumps in the tonoplast (Marty, 1999; Ratajczak, 2000; Martinoia et al., 2007). VHAs are multi-subunit enzyme complexes with a molecular weight of 450–600 kDa, which are conserved in all eukaryotes and have been localized to vacuoles and other membranes of the plant secretory system (Sze et al., 2002). VHAs are composed of the cytosol ATP-hydrolyzing V1 sub-complex (which consists of 8 subunits [A–H]) and the membrane-bound proton-trans-locating V0 sub-complex (Nishi and Forgac, 2002). In Arabidopsis, most V1 subunits are encoded by a single gene, whereas V0 subunits are encoded by multiple genes (Sze et al., 2002). Subunit A of VHA (VHA-A) is one of the most highly conserved eukaryotic proteins, and is responsible for ATP hydrolysis (Gaxiola et al., 2007). In Arabidopsis VHA-A is encoded by a single gene (Sze et al., 2002). In

Abbreviations: CON, empty vector control; DW, dry weight; FW, fresh weight; MDA, malondialdehyde; Md, Malus domestica; ORF, open reading frame; PEG, polyethylene glycol; RT-PCR, reverse transcription-PCR; RWC, relative water content; TIBA, 2,3,5-triiodobenzoic acid; TW, total weight; UTR, untranslated region; VHA, vacuole H+ -ATPase; VHA-A, subunit A of VHA; VHP, vacuole H+ pyrophosphatase. ∗ Corresponding authors. Tel.: +86 0538 8246692; fax: +86 0538 8242364. E-mail addresses: [email protected] (Y.-X. Yao), [email protected] (Y.-J. Hao). 0176-1617/$ – see front matter © 2013 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.jplph.2012.12.014

tomato, 2 isoforms (VHA-A1 and VHA-A2) are present (Bageshwar et al., 2005). VHA and vacuole H+ -pyrophosphatase (VHP) create the proton gradient and membrane potential, which are used to energize the transport of ions and metabolites across the tonoplast for optimal growth and development (Marty, 1999; Ratajczak, 2000; Martinoia et al., 2007). Given the use of different energy sources, it is thought that the combined action of the 2 enzymes enables plants to maintain transport into the vacuole, even under stressful conditions (Krebs et al., 2010). With respect to salt resistance, the compartmentalization of Na+ into the vacuole comprises a plant multifarious adaptation strategy, which minimizes the deleterious effects of excess Na+ in the cytosol, and maintains osmotic balance by using Na+ as a cheap osmoregulatory substance. This process is mediated by the vacuolar Na+ /H+ antiporter, which is driven by the electrochemical gradient of protons across the tonoplast, generated by the vacuolar H+ -pumps, VHA and VHP (Blumwald and Gelli, 1997). In addition to their role in ion transportation, VHA and VHP energize the transport of solutes such as amino acids (proline), betaine, polyols, and sugars, across the tonoplast (Marty, 1999; Ratajczak, 2000; Chen and Murata, 2002). The cellular accumulation of compatible solutes is a common mechanism by which plants protect themselves from cold and osmotic stress-induced damage (Chen and Murata, 2002). A salt-induced increase in VHA-A transcription and VHA activity has been demonstrated in Mesembryanthemum crystallinum (Löw

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et al., 1996), Arabidopsis (Magnotta and Gogarten, 2002), and barley (Fukada et al., 2004). Similar salt-induced transcript up-regulation of VHA-c and VHA-B has been observed in Pennisetum glaucum (Tyagi et al., 2005) and wheat (Wang et al., 2011), respectively. An increase in VHA enzyme activities following salt treatment has been demonstrated in tomato (Binzel, 1995), Suaeda salsa (Wang et al., 2001), and Thellungiella halophila (Vera-Estrella et al., 2005). Over-expression of VHA-c from Limonium bicolor and VHA-B from wheat was shown to improve salt tolerance in transgenic tobacco and Arabidopsis, respectively (Wang et al., 2011; Xu et al., 2011). Conversely, studies on vha-a2 vha-a3 indicate that VHA activity is limiting for nutrient storage, but not for sodium tolerance, during vegetative and reproductive growth of Arabidopsis (Krebs et al., 2010). By contrast, over-expression of VHP genes has a wellrecognized role in enhancing salt and drought tolerance of various transgenic plants, including Arabidopsis (Gaxiola et al., 2001), cotton (Pasapula et al., 2011), and apple (Dong et al., 2011). VHP also plays an important role in root development, through facilitating auxin fluxes (Li et al., 2005; Park et al., 2005; Lv et al., 2009). In some plant species, more extensive root systems are associated with increased drought and salt resistance. A more extensive root system allows water to be absorbed from a greater soil volume during periods of drought, thus reducing plant dehydration (Sharp et al., 2004; Park et al., 2005). It also enables Na+ /K+ uptake into vacuoles to improve sodium sequestration during growth on highly saline soils, thereby reducing Na+ toxicity. Over-expression of VHP has been shown to enhance drought resistance of cotton (Lv et al., 2009), and drought and salt resistance of Arabidopsis (Gaxiola et al., 2001) and tomato (Park et al., 2005) by improving root development. To date, however, there are no reports on the role of VHA in drought tolerance, through its impact on root development. Here, we investigated the effect of over-expression of an apple VHA-A (MdVHA-A), cloned in a previous study (Yao et al., 2009), on the response to the drought stress. Using transgenic tobacco seedlings ectopically expressing MdVHA-A, we elucidated the biological function of the gene in drought tolerance. We discuss the potential application of MdVHA-A in genetically improving drought tolerance of plants.

Materials and methods Plant materials, growth conditions, and stress treatments Different tissues of the ‘Fuji’ apple (Malus domestica (Md) B.) were used for gene expression assays. In vitro shoot cultures of the ‘Orin’ apple (Md B.) were used for analyzing the expression of MdVHA-A in response to polyethylene glycol (PEG)-mimic drought. The shoot cultures were sub-cultured on MS (Murashige and Skoog, 1962) medium containing 87 mM sucrose, 0.2 mg l−1 of IAA, 0.8 mg l−1 of 6-Benzylaminopurine, 7 g l−1 of agar with a 14-h light/10-h dark photoperiod at approximately 600 mmol m−2 s−1 at 25 ◦ C. For PEG-mimic drought stress assay, 2-week-old in vitro shoot cultures were treated with 32.3 mM PEG-6000. Tobacco (Nicotiana tobaccum L. ‘NC89’) was used for genetic transformation and also for the drought stress and auxin response assays. Transgenic and empty vector control (CON) tobacco seedlings were cultured on B5 (Gamborg et al., 1968) medium at 25 ◦ C, using a 14-h light/10-h dark photoperiod, at approximately 600 mmol m−2 s−1 . For the auxin response assay under the normal and PEG conditions, 5-day-old seedlings were cultured for 13 days on B5 medium supplemented with IAA and/or auxin transport inhibitor 2,3,5-triiodobenzoic acid (TIBA) at the following concentrations: 30 ␮M IAA, 1 ␮M TIBA, 30 ␮M IAA + 1 ␮M TIBA, or 30 ␮M IAA + 5 ␮M TIBA.

Transgenic and CON tobacco seedlings were planted in 15cm-diameter plastic pots filled with a 2:1 (v/v) mixture of soil:vermiculite and grown at 25 ◦ C, using a 14-h light/10-h dark photoperiod, at approximately 600 mmol m−2 s−1 . For the drought tolerance assay, 10-week-old tobacco seedlings were stressed by withholding water for 18 days. Gene expression analysis with semi-quantitative reverse transcription (RT)-PCR and real-time quantitative reverse transcription-PCR (RT-PCR) Total RNA was extracted using a hot borate method, as described previously (Yao et al., 2007). For semi-quantitative RT-PCR, 1 ␮g of total RNA from each sample was used for the reverse transcription reaction. Subsequently, 1 ␮l of the reverse transcription product was used as a template for PCR amplification. The specific PCR primer pairs were designed according to the 3 untranslated regions (UTRs) of MdVHA-A, IAA4.2, IAA2.5, and NtActin (as the endogenous control) sequences (Appendix S1 in supporting information). The PCR products were examined on a 0.8% agarose gel stained with ethidium bromide. For real-time quantitative RTPCR, the specific primers of MdVHA-A, IAA4.2, IAA2.5, and Md18S, NtActin gene (as the endogenous control) were designed according to the 3 -UTR (Appendix S1 in supporting information). Real-time quantitative RT-PCR was performed using a Bio-Rad iQ5 instrument (Bio-Rad, Hercules, USA), as described previously (Dong et al., 2011). VHA activity assay Tobacco tonoplast vesicles were isolated for measurement of VHA activity, using sucrose density gradient ultracentrifugation as described by Carystinos et al. (1995), with a minor modification. Approximately 5 g of leaves were homogenized in 10 ml of ice-cold buffer containing 100 mM Tricine/Tris (pH 8.0), 300 mM mannitol, 3 mM MgSO4 , and 3 mM EDTA. Prior to use, 5 mM dithiothreitol and 1 mM phenylmethylsulfonyl fluoride were added to the buffer. After filtration and pre-centrifugation of the homogenate at 5000 × g for 10 min, the suspension was centrifuged at 100,000 × g for 35 min, and the pellet was re-suspended in a small volume of ice-cold buffer containing 100 mM Tricine/Tris (pH 8.0), 300 mM mannitol, 3 mM MgSO4 , 3 mM EDTA, and 5 mM dithiothreitol. The suspension containing the vesicles was layered over a 10–25% (w/w) discontinuous sucrose gradient solution containing 5 mM HEPES/Tris (pH 7.5) and 2 mM dithiothreitol. After centrifugation at 100,000 × g for 2 h, the vesicles at the interface between the 10–25% sucrose solutions were collected and diluted with 3-fold volumes of ice-cold buffer containing 10 mM HEPES/NaOH (pH 7.0), 3 mM MgSO4 , and 1 mM dithiothreitol. Membranes were collected by centrifugation at 100,000 × g for 35 min, resuspended in storage buffer with 10 mM HEPES/NaOH (pH 7.0), 40% (v/v) glycerol, 1 mM dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride, and stored at −70 ◦ C for further analysis. The purity of the vesicles was estimated with vanadate, nitrate and azide as inhibitors. The tonoplast H+ -ATPase is characterized by inhibition by nitrate, the H+ -ATPase in plasma membrane and mitochondria are inhibited by vanadate and azide, respectively. In the present study, nitrate inhibited the H+ -ATPase activity by more than 81%, but vanadate and azide reduced the enzyme activity by less than 3%, demonstrating that the vesicles were enriched in tonoplasts. VHA activity was measured as the release of inorganic phosphate from ATP during an incubation period of 30 min at 37 ◦ C; the mixture for the assay of enzyme activity contained 30 mM HepesTris (pH 7.5), 50 mM KCl, 3 mM MgSO4 , 0.1 mM ammonium molybdate, 0.25 mM Na3 VO4 , 2 mM ATP-Na2 , and 100 ␮l of tonoplast

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vesicles (Liu et al., 2004). Protein was quantified by a dye-binding assay, using bovine serum albumin as a standard (Bradford, 1976). Transformation of MdVHA-A into tobacco seedlings To assess the function of MdVHA-A in tobacco, a 1872-bp open reading frame (ORF) of the MdVHA-A gene was cloned from the ‘Orin’ apple (Md B.) using a pair of primers, and then inserted into vector pBI121 downstream of the CaMV 35S promoter. The resultant vector and CON were introduced into Agrobacterium tumefaciens strain LBA4404 for tobacco transformation. Tobacco was transformed with LBA4404 using the leaf disc method (Horsch et al., 1988) with a minor modification: the selection medium was changed to MS medium containing 0.2 mg l−1 of naphthyl acetic acid, 3 mg l−1 of 6-Benzylaminopurine, 100 mg l−1 of kanamycin, and 250 mg l−1 of carbenicillin. Tobacco genomic DNA was extracted from young leaves as described by Doyle and Doyle (1990). Different primers for the NPT II and MdVHA-A sequences were used in PCR, to determine whether MdVHA-A is integrated into the tobacco genome. The primers for the MdVHA-A sequence were the same as those used in the isolation of the MdVHA-A ORF. Each PCR reaction mixture contained 200 ng of DNA, 2.5 ␮l of 10× Taq buffer, 200 ␮mol of each dNTP, 10 pmol of each primer, and 0.5 U of Taq DNA polymerase, in a total volume of 25 ␮l. Transgenic tobacco seedlings containing pBI121 empty vector were used as controls. The un-segregated T2 generations of the transgenic tobacco plants were used for various assays. Procedures for IAA extraction and measurement IAA extraction and determination were performed according to Liao et al. (2008) with some modifications. The IAA content was determined by HPLC. For HPLC analysis, 20 ␮l of the purified IAA sample was injected into a Kromasil 100-5 C18 column (250 mm × 4.6 mm) and eluted using a mobile phase composed of 0.5% (v/v) acetic acid/methanol (55:45, v/v). The effluent was monitored at 254 nm through a dual ␭ absorbance detector of Waters 2487 in HPLC (Waters 510, Milford, MA, USA). The HPLC results were quantified using an IAA calibration curve, obtained over the range 0.67–670 ␮M and with a correlation co-efficient of 0.9984. The external standard (a mixture of 220 ml of 80% (v/v) cold methanol and 1 ml of 674 ␮M IAA solution) was used to estimate the recovery rate throughout the extraction/purification procedures. Determination of malondialdehyde (MDA) and proline content The MDA content was determined and calculated according to Hodges et al. (1999) Hodges et al.’s method (1999). The content of free proline was determined according to Bates et al. (1973). Measurement of relative water content (RWC) and solute potential Fresh weights (FWs) of tobacco leaves were recorded immediately after excision from seedlings. After soaking the leaves in deionised water at 4 ◦ C overnight, the turgid weights were determined. Dry weights (DWs) were obtained after oven drying the leaf samples at 70 ◦ C for 72 h. The RWC was calculated as follows: RWC (%) = (FW − DW)/(TW − DW) × 100%. The solute potential (s) was determined with a freezing point micro-osmometer (Fiske 210, Norwood, MA, USA). The solute potential (s) expressed in MPa was calculated using the formula: s = moles of solute (R × T), where R = 0.008314 and T = 298◦ K.

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Statistical analysis All statistical analysis was performed by SAS (V8.0) software. The significant difference was determined by one-way analysis of variance (ANOVA) according to the Duncan’s multiple range test. Results Sequence characteristics of apple vacuole H+ -ATPase (VHA) subunit A (MdVHA-A) In a previous study, we cloned the open reading frame (ORF) and 3 -untranslated region (UTR) of apple VHA subunit A (EF128033) from the ‘Fuji’ apple (Malus domestica (Md) B.) (Yao et al., 2009). The genome sequence of MdVHA-A was obtained by blasting the MdVHA-A ORF in the apple genome database, and confirmed by PCR amplification and sequencing. The MdVHA-A genome sequence contained 20 exons and 19 introns in the ORF, and was identical to VHA-As from grapevine and Arabidopsis (Appendix S2 in supporting information). Additionally, MdVHA-A showed similar exon sequences to VHA-As from Vitis and Arabidopsis. However, most introns differed clearly in length among the 3 VHA-As (Appendix S2 in supporting information). The amino acid sequence of MdVHA-A (ABO33173) showed high homology (>83%) with those of other plant VHA-As. In the phylogenic tree constructed using online software MEGA4.1 (http://www.megasoftware.net/) (Tamura et al., 2007), MdVHA-A and other dicotyledon VHA-As were clustered into a single group, while monocotyledon VHA-As formed a separate group (Appendix S3 in supporting information). Thus, VHA-A ORFs and their putative amino acids are highly conserved among different plant species. Differences in genome sequences result from the genetic divergence in intron sequences. Expression analysis of MdVHA-A in different apple tissues and in response to polyethylene glycol (PEG)-mimic drought Real-time quantitative reverse transcription (RT)-PCR indicated that MdVHA-A was constitutively expressed in all of the tissues tested, including young roots, stems, leaves, flowers, mature fruits, and seeds. However, the expression level varied according to tissue, with strong expression in young roots, stems, leaves, and mature fruits, but weak expression in seeds and flowers (Fig. 1A). Under PEG-mimic drought conditions, MdVHA-A expression was increased 3.8-fold at 8 h in apple in vitro shoot cultures. By contrast, at other time points, expression was increased less than 2-fold, or was even decreased (Fig. 1B). Thus, in general, MdVHA-A expression was positively induced after the possible adaptive decline by PEGminic drought. Increase in the VHA activity of transgenic tobacco seedlings, through ectopic expression of MdVHA-A To further characterize the biological function of the MdVHA-A gene, the full-length ORF was cloned into the plant expression vector pBI121, and genetically introduced into tobacco seedlings using an Agrobacterium-mediated transformation method. PCR screening of the kanamycin-resistant transgenic lines was performed using genomic DNA as a template, with primers specific to the NPT II and MdVHA-A genes. We observed that the MdVHA-A gene was integrated into the genomes of kanamycin-resistant lines 3, 4, 5, 9, 10, 11 (Fig. 2A). Semi-quantitative reverse transcription-PCR (RT-PCR) analysis was performed to check whether MdVHA-A was ectopically expressed in any of the putative transgenic lines. We observed that MdVHA-A was expressed at different levels in transgenic lines 3, 4, 5, 9, 10, and 11 (Fig. 2B). Among them, transgenic lines 5, 9, and 10,

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Relative expression.

A

B

1000 100 10 1 0.1

Seeds Flower Fruit

Root Stem

leaf

0.01 0.001

Relative expression

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5 4

32.3 mM PEG-6000

3 2 1 0

0

2

4

6

8

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Treatment time (h) Fig. 1. Relative expression of MdVHA-A (A) in different tissues and (B) in response to PEG-mimic drought. The expression values were calculated relative to MdVHA-A expression in seeds (0.98 ± 0.16). Values represent the means ± SD of 3 replicates.

which exhibited different levels of MdVHA-A expression (Fig. 2B), were selected for further investigation. To examine the contribution of MdVHA-A to VHA activity, tonoplasts were isolated from 10-week-old empty vector control (CON) and selected transgenic tobacco seedlings. In the VHA activity assay, transgenic lines 5 and 9 exhibited significantly higher VHA activities than the CON (Fig. 2C). Furthermore, the VHA activities of the 3 transgenic lines were positively related to the MdVHA-A expression level (Fig. 2C), indicating that ectopic expression of MdVHA-A contributed to increased VHA activity. Improved drought tolerance of transgenic tobacco seedlings, by enhanced osmotic adjustment and extended lateral root growth, through ectopic expression of MdVHA-A To confirm the function of MdVHA-A in drought tolerance, 10week-old transgenic tobacco lines 5, 9, and 10 were used in a drought tolerance assay. We observed that, transgenic and CON seedlings wilted after 18 days of water-deficit stress. After rewatering, the transgenic lines recovered and grew, whereas the CON seedlings continued to wilt, and even died (Fig. 3A). Thus, transgenic seedlings were more tolerant to drought than were CON seedlings. To elucidate the mechanism of increased drought tolerance, we investigated the osmotic adjustment and lateral root growth of transgenic seedlings. After 9 days and 18 days of water-deficit stress, transgenic seedlings exhibited a more negative solute potential (s) than did CON seedlings, indicating enhanced potential to retain water (Fig. 3B). The improved water retention was verified by relative water content (RWC) assay. After 9 days of waterdeficit stress, the RWC decreased by 15% in CON seedlings, but

by only 1%, 4%, and 6% in transgenic lines 5, 9, and 10, respectively. Similarly, after 18 days of water-deficit stress, the RWC declined by 51% in CON seedlings, but by only 37%, 38%, and 41% in transgenic lines 5, 9, and 10, respectively (Fig. 3B). In general, transgenic seedlings accumulated more proline and generated less malondialdehyde (MDA) than did CON seedlings (Fig. 3B), indicating enhanced osmotic adjustment and reduced membrane injury, respectively. Taken together, these results suggest that ectopic expression of MdVHA-A in transgenic tobacco seedlings may trigger an enhanced capacity for osmotic adjustment, resulting in greater water retention and reduced membrane injury during water-deficit stress. In comparison with CON seedlings, transgenic tobacco seedlings produced more extensive lateral root systems under normal conditions, and especially after PEG-mimic drought treatment (Fig. 4A and B). This suggests that MdVHA-A can influence root growth, especially under conditions of drought stress. To further investigate the role of MdVHA-A in root growth, we compared 10-week-old CON and transgenic tobacco seedlings after drought treatment. Under normal and drought-stress conditions, the transgenic seedlings produced bushier root systems (Fig. 5A). To confirm the visual observation, we compared fresh weight (FW) and dry weight (DW) of the root systems. We recorded higher FW and DW for the root systems of transgenic seedlings (Fig. 5B). After 9 days of drought treatment, the root systems of transgenic seedlings exhibited >0.77-fold higher FW and DW than did those of CON seedlings (Fig. 5B). Taken together, these results indicate that ectopic expression of MdVHA-A in transgenic tobacco seedlings improves tolerance to drought, at least partly by enhancing osmotic adjustment and promoting root growth.

Fig. 2. (A) PCR identification, (B) MdVHA-A expression, and (C) VHA activity determination of transgenic tobacco seedlings. (A) NPT II and MdVHA-A indicate that the primers used to identify the transgenic lines were designed according to the 2 genes. (B) Semi-quantitative RT-PCR determination of MdVHA-A expression (RT-MdVHA-A) in transgenic seedlings; NtActin was used as the control (RT-NtActin). (C) VHA activity in the functional leaves of transgenic and CON seedlings. M, DNA marker; NC, negative control (H2 O); PC, positive control (plasmid DNA of 35S::MdVHA-A pBI121 vectors); CON, empty vector control. Data are presented as means ± SD (n = 3). *, Significant difference, p < 0.05.

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Fig. 3. Assessment of drought tolerance in control and transgenic tobacco seedlings. (A) Phenotypes of CON and transgenic seedlings under conditions of drought stress. Photographs were taken after 0, 9, and 18 days of drought, respectively. Pots were irrigated to field capacity at day 18, and photographs were taken on day 2 after rewatering. (B) The solute potential, leaf RWC, and proline and MDA content in CON and transgenic seedlings at 0, 9, and 18 days under conditions drought stress. Data are presented as means ± SD (n = 3). *, Significant difference, p < 0.05; **, highly significant difference, p < 0.01.

Improved root growth of transgenic tobacco seedlings, dependent on auxin translocation, through ectopic expression of MdVHA-A The enhanced lateral root initiation and growth of transgenic tobacco seedlings suggest the involvement of the phyto-hormone auxin. To determine whether lateral root formation was dependent on auxin synthesis and/or translocation, we compared the levels of IAA in the shoot and root tips of transgenic and CON seedlings (Fig. 6). Under normal conditions, transgenic seedlings accumulated slightly less IAA in their shoot tips, but more IAA in their root tips (Fig. 6), suggesting that ectopic expression of MdVHA-A promoted auxin translocation but not synthesis. Following PEGmimic drought treatment, transgenic seedlings accumulated ∼10% less IAA in their shoot tips, but 32% more IAA in their root tips (p < 0.05), than did CON seedling (Fig. 6), further confirming the role of MdVHA-A in auxin translocation but not synthesis. To further elucidate the role of auxin translocation in lateral root formation, we cultured 5-day-old CON and transgenic tobacco seedlings for 13 days on B5 medium supplemented with IAA and/or 2,3,5-triiodobenzoic acid (TIBA) (Fig. 4). Under normal and PEG-mimic drought conditions, IAA supplementation significantly reduced tap root length, but increased lateral root numbers, in CON and transgenic tobacco seedlings. Furthermore, supplementation of B5 medium with 1 ␮M TIBA, or with 1 ␮M TIBA and 30 ␮M IAA, completely inhibited lateral root formation in CON and transgenic seedlings. Following supplementation of MS medium with 32.2 mM PEG, 30 ␮M IAA, and 1 ␮M TIBA, CON seedlings failed to generate lateral roots. By contrast, transgenic seedlings continued to produce more extensive lateral root systems, indicating that ectopic expression of MdVHA-A suppressed the negative effect of TIBA on auxin transportation. The addition of 5 ␮M TIBA to MS medium containing 32.2 mM PEG and 30 ␮M IAA completely inhibited lateral root formation in transgenic seedlings (Fig. 4A and B).

To determine whether the auxin response pathway is activated in transgenic tobacco seedlings ectopically expressing MdVHA-A, we analyzed the expression of the early auxin responsive genes IAA4.2 and IAA2.5, which are selectively induced as a primary response to auxin and prior to the initiation of cell growth (Abel and Theologis, 1996; Shukla et al., 2006). We revealed that transgenic tobacco seedlings produced much higher levels of IAA4.2 and IAA2.5 transcripts than did CON seedlings (Fig. 7A and B). Taken together, our results indicate that ectopic expression of MdVHAA improves lateral root formation in transgenic tobacco seedlings, by increasing auxin transportation and activating auxin responses, and thereby positively contributes to enhanced drought tolerance. Discussion VHA-A is one of the most highly conserved eukaryotic proteins (Appendix S3 in supporting information; Gaxiola et al., 2007). The VHA-A gene is constitutively expressed in different tissues of apple (Fig. 1A), Arabidopsis (Magnotta and Gogarten, 2002), and tomato (Bageshwar et al., 2005), suggesting that it may be involved in multiple aspects of plant growth and development (Ratajczak, 2000). VHA-A is expressed differentially in response to plant abiotic stresses. For example, VHA-A expression was shown to be induced by salt treatment in tobacco (Narasimhan et al., 1991), sugar beet (Kirsch et al., 1996), barley (Fukada et al., 2004) and tomato (Bageshwar et al., 2005), and by cold treatment in Arabidopsis (Magnotta and Gogarten, 2002). In the present study, VHA-A expression in apple in vitro shoot cultures was induced by PEG-mediated osmotic stress (Fig. 1B). Ectopic expression of MdVHA-A in transgenic tobacco seedlings improved drought tolerance and enhanced VHA activity (Figs. 2C and 3). Thus, we propose that MdVHA-A are involved in tolerance to abiotic stresses, probably by regulating VHA activity.

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Fig. 4. Effect of MdVHA-A over-expression on lateral root growth of tobacco seedlings, through alteration of IAA synthesis and/or translocation, under normal and PEG conditions. (A) Morphology of CON and transgenic seedlings on B5 medium supplemented with IAA and/or TIBA at day 13 under normal and PEG conditions. (B) Number and length of lateral roots of CON and transgenic seedlings on MS medium supplemented with IAA and/or TIBA at day 13 under normal and PEG conditions. See legend of Fig. 3 for definitions.

The improved drought tolerance of transgenic tobacco seedlings was also derived from increased accumulation of solutes. In principle, enhanced expression of vacuolar proton pumps increases vacuolar solute accumulation, by increasing the availability of protons (Gaxiola et al., 2001; Bao et al., 2009). Previously, we reported that over-expression of MdVHP1 in transgenic apple calluses promoted tolerance to drought, by increasing the proline content (Dong et al., 2011). Proline is an important organic solute, and has been demonstrated to accumulate in plants under conditions of drought, salinity, and cold stress (Claussen, 2005; Yamada et al., 2005; Li et al., 2010). In the present study, we observed a similar situation in MdVHA-A-over-expressing tobacco seedlings (Fig. 3B). The increase in solute concentration leads to enhanced osmotic adjustment (Gaxiola et al., 2001). In the present study, droughtstressed transgenic tobacco seedlings exhibited a more negative solute potential, and a higher leaf RWC, than did CON seedlings (Fig. 3B). Moreover, under conditions of drought stress, transgenic seedlings suffered reduced cell damage, as indicated by a lower MDA level (Fig. 3B), which, as an end product of free radical chain reactions and lipid peroxidation in bio-membranes, can reflect the

level of lipid peroxidation in bio-membranes, and also indirectly reflects the extent of membrane injury (Fang and Liu, 2006). Thus, MdVHA-A improves the drought resistance of transgenic tobacco seedlings, at least partly by enhancing osmotic adjustment. The extended root system of transgenic tobacco seedlings may provide an additional morphological and/or physiological basis for enhanced performance under drought stress (Figs. 4A and 5A). Deeper and/or more extensive root systems are known to be associated with increased drought resistance (Pinheiro et al., 2005). A more robust root system facilitates water uptake, especially when plants are exposed to low soil water conditions (Lv et al., 2009). In the present study, the improved root system of transgenic tobacco seedlings was mainly attributed to more extensive lateral root formation (Figs. 4A and 5A). In higher plants, auxins play a crucial role in lateral root formation. The auxins are endogenously produced in the shoot apical meristem, and are then rapidly transported down to the roots via the phloem (Malamy and Ryan, 2001; Bhalerao et al., 2002; Zandonadi et al., 2007). Some studies have demonstrated that auxins stimulate the hydrolytic activity of vacuole pumps (Ozolina et al., 1996; Salyaev et al., 1999; Fukuda and Tanaka, 2006). In

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Fig. 5. Root growth of CON and transgenic tobacco seedlings under conditions of drought stress. (A) Root phenotypes of CON and transgenic seedlings under conditions of drought stress. (B) Root FW and DW of CON and transgenic seedlings under conditions of drought stress. See legend of Fig. 3 for definitions.

comparison with VHA, VHP appears to be much more sensitive to auxin (Zandonadi et al., 2007). Auxins (IAA and humic acids) improve root growth through the proliferation of lateral roots, together with the differential activation of the plasmalemma, and also vacuolar VHA and VHP. This suggests that concerted activation of the plasmalemma and tonoplast proton pumps plays a key role in auxin-induced root growth (Zandonadi et al., 2007). Thus, it is possible that the plasmalemma and tonoplast proton pumps are activated to differing extents by auxins and therefore respond to different physiological and stress conditions. On the other hand, VHA is required to support root growth of Arabidopsis under normal nutrient conditions and under mild salt stress (Padmanaban et al., 2004). Furthermore, over-expression of the vacuole proton pump gene (H+ -PPase) increases root growth, and thereby facilitates improved water-deficit recovery in Arabidopsis (Li et al., 2005), tomato (Park et al., 2005), turf grass (Li et al., 2010), and cotton

B

1000 100 10 1 0.1 0.01 0.001

Seeds Flower Fruit

Root Stem

leaf

Relative expression

Relative expression.

A

(Lv et al., 2009; Pasapula et al., 2011). Characterization of gain-offunction and loss-of-function mutants reveals that AVP1 plays an important role in root development, through facilitating the auxin fluxes that regulate organogenesis (Li et al., 2005). In the present study, improved lateral root growth (dependent on auxin transportation) was observed in MdVHA-A transgenic tobacco seedlings (Fig. 4). We propose that a VHP-like mechanism may underlie this improved lateral root formation. Further studies are required to confirm this hypothesis. Taken together, our findings indicate that ectopic expression of MdVHA-A in transgenic tobacco seedlings confers drought tolerance. Furthermore, the role of MdVHA-A in tolerance to drought stress resembles that of MdVHP1 (Dong et al., 2011). The functional relationship between, and relative contributions of, the 2 vacuole proton pumps remain to be elucidated. We believe that this is the first report to verify the role of VHA-A over-expression in drought

5 4

32.3 mM PEG-6000

3 2 1 0

0

2

4

6

8

12

Treatment time (h) Fig. 6. IAA content in the shoot and root tips of transgenic and CON seedlings under normal and PEG conditions.

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Fig. 7. Semi-quantitative RT-PCR (A) and real-time RT-PCR (B) analysis of transcript levels of auxin response genes in transgenic and CON seedlings under normal conditions.

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