Mol Biol Rep DOI 10.1007/s11033-014-3695-3

Overexpression of AtDREB1D transcription factor improves drought tolerance in soybean Satish K. Guttikonda • Babu Valliyodan • Anjanasree K. Neelakandan • Lam-Son Phan Tran • Rajesh Kumar • Truyen N. Quach • Priyamvada Voothuluru Juan J. Gutierrez-Gonzalez • Donavan L. Aldrich • Stephen G. Pallardy • Robert E. Sharp • Tuan-Hua David Ho • Henry T. Nguyen



Received: 12 June 2014 / Accepted: 21 August 2014 Ó Springer Science+Business Media Dordrecht 2014

Abstract Drought is one of the major abiotic stresses that affect productivity in soybean (Glycine max L.) Several genes induced by drought stress include functional genes and regulatory transcription factors. The Arabidopsis thaliana DREB1D transcription factor driven by the constitutive and ABA-inducible promoters was introduced into soybean through Agrobacterium tumefaciens-mediated gene transfer. Several transgenic lines were generated and molecular analysis was performed to confirm transgene integration. Transgenic plants with an ABA-inducible promoter showed a 1.5- to two-fold increase of transgene expression under severe stress conditions. Under wellwatered conditions, transgenic plants with constitutive and ABA-inducible promoters showed reduced total leaf area and shoot biomass compared to non-transgenic plants. No significant differences in root length or root biomass were observed between transgenic and non-transgenic plants under non-stress conditions. When subjected to gradual S. K. Guttikonda  B. Valliyodan  T. N. Quach  P. Voothuluru  J. J. Gutierrez-Gonzalez  D. L. Aldrich  S. G. Pallardy  R. E. Sharp  H. T. Nguyen (&) National Center for Soybean Biotechnology and Division of Plant Sciences, University of Missouri, Columbia, MO 65211, USA e-mail: [email protected] S. K. Guttikonda Biotechnology Regulatory Sciences, Regulatory Sciences and Government Affairs, Dow AgroSciences, Indianapolis, IN, USA A. K. Neelakandan Department of Agronomy, Iowa State University, Ames, IA, USA L.-S. P. Tran Signaling Pathway Research Unit, RIKEN Center for Sustainable Resource Science, Yokohama, Kanagawa, Japan

water deficit, transgenic plants maintained higher relative water content because the transgenic lines used water more slowly as a result of reduced total leaf area. This caused them to wilt slower than non-transgenic plants. Transgenic plants showed differential drought tolerance responses with a significantly higher survival rate compared to nontransgenic plants when subjected to comparable severe water-deficit conditions. Moreover, the transgenic plants also showed improved drought tolerance by maintaining 17–24 % greater leaf cell membrane stability compared to non-transgenic plants. The results demonstrate the feasibility of engineering soybean for enhanced drought tolerance by expressing stress-responsive genes. Keywords DREB  Drought tolerance  Environmental stresses  Cell membrane stability  Soybean  Transcription factor

R. Kumar National Research Center on DNA Fingerprinting, National Bureau of Plant Genetic Resources, New Delhi 110012, India T. N. Quach The Center for Plant Science Innovation, University of Nebraska, Lincoln, NE, USA J. J. Gutierrez-Gonzalez USDA-ARS Plant Science Research Unit and Department of Agronomy and Plant Genetics, University of Minnesota, St Paul, MN, USA T.-H. D. Ho Department of Biology, Washington University, Saint Louis, MO, USA

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Introduction Abiotic stresses such as drought, high salinity, and low temperature are major environmental factors that drastically reduce plant growth and productivity, often causing average yield losses of more than 50 % in major crops [1]. Among various abiotic stresses, drought is the most prevalent factor that limits agricultural crop production. Plants respond to drought by a combination of morphological, physiological, cellular and biochemical changes. Droughtrelated environmental signals are first perceived by specific receptors that, upon activation, initiate cascades to transfer intercellular signals, and, activate transcription factors (TFs) to activate the expression of specific sets of genes. Subsequently, induced metabolic changes lead to enhanced stress tolerance in plants [2–7]. Expression of genes under abiotic stress conditions involves various abscisic acid (ABA)-dependent as well as ABA-independent regulatory mechanisms [8–13]. Among different dehydration-responsive elements binding members (DREB), transcription factors DREB1 and DREB2 that belong to the APETELA 2/Ethylene Responsive Element Binding Protein (AP2/EREBP) group bind to the cis-acting dehydration responsive element (DRE) in the promoter region of stress-inducible genes and regulate their expression in response to different abiotic stresses [14–16]. Expression of AtDREB1A, B and C is induced by low temperature and is not responsive to ABA, but AtDREB1D/ CBF4 (C-repeat binding factor) is induced during drought stress and is responsive to ABA treatment. Several studies have reported that the overexpression of stress-inducible DREB TF encoding genes activates the expression of many target genes having DRE elements in their promoters and the resulting transgenic plants showed improved stress tolerance [10, 14, 17, 18]. Transgenic Arabidopsis plants overexpressing AtDREB1D under the control of the CaMV35S (cauliflower mosaic virus) promoter showed strong tolerance to drought and freezing [19]. Northern analysis of transgenic plants indicated that under both normal and cold stress, the transcript levels of AtDREB1D were much greater than those observed in wild-type (WT) plants, suggesting that AtDREB1D plays a role in the signal transduction of drought adaptation in Arabidopsis plants [19]. However, constitutive overexpression of AtDREB1D caused severe growth retardation under normal growing conditions. Recently, overexpression of grape VrCBF1 and VrCBF4 genes showed reduced plant height and adverse growth effects and these transgenic lines showed high expression of these genes. Transgenic lines overexpressed with both these showed improved cold tolerance, whereas, increased drought tolerance was observed only in increased expression of VrCBF1 [20]. To overcome these negative impacts on plant growth, an inducible expression of transgene only under the stress

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conditions is highly preferred, e.g. the use of ABA- and/or abiotic stress-inducible promoters [21–23]. The phytohormone ABA is known to play a key role in plant response to abiotic stresses, such as drought, salinity and cold [24]. ABA accumulation increases in response to drought conditions [25, 26]. In turn, elevated ABA levels have been shown to strongly regulate gene expression. For example, two types of ABRC (ABA-responsive complex) promoter elements showed strong induction in response to ABA: ABRC1, consisting of ABRE (ABA Response Element) elements A1 and CE1 from the barley HVA22 (Hordeum vulgare) gene, and ABRC3, composed of CE3 (coupling element 3) and A2 from the HVA1 gene. The 68-bp ABRC3 promoter element showed stronger induction than the 48-bp ABRCl element. One copy of the ABRC3 fragment led to 20-fold induction, twice as high as that acquired by the ABRC1 fragment when tested by the GUS (b-glucuronidase) assays in barley aleurone tissues. The activity of the ABRC3 fragment was more responsive to ABA than ABRC1 when tested in the vegetative tissue of 6-week-old barley [27, 28]. The rationale for using these promoters is that ABA accumulation under drought conditions will lead to the promoter activation. The ABRC1 promoter fused to the minimal promoter of rice Act1 and the HVA22 intron (I) in transgenic rice plants conferred GUS expression under ABA, water-deficit and salinity treatments [29]. The overexpression of trehalose biosynthesis genes under the control of ABRC1 promoter in rice induced enhanced drought and salinity tolerance in the transgenic plants. Trehalose accumulation in the transgenic plants was increased up to three-fold under water-deficit conditions [30]. Overexpression of AtDREB1C with three copies of ABRC1 resulted in increased tolerance to cold, drought, and salt stress when compared with untransformed plants. AtDREB1C expression was induced by cold, drought, and salt treatment in transgenic tomato plants [31]. Overexpression of the HVA1 gene using two copies of the ABRC3 promoter in creeping bentgrass revealed that transgenic lines maintained higher leaf water content and showed reduced leaf wilting under water-deficit conditions compared with non-transgenic control plants. The transgenic plants showed increase in HVA1 protein with increased water-deficit conditions when compared with a constitutive promoter [23]. Several studies have reported improved drought tolerance with DREB genes using different promoters in major crops such as rice, tobacco, peanut and wheat [22, 32–34]. Thus, it is likely that these genes might have general applications in improving the drought tolerance of crop plants. Soybean is the leading oilseed crop produced in the world, and processed soybeans are the world’s largest source of vegetable oil and protein feed. The most critical

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period in soybean yield determination for water-deficit conditions was reported to be the flowering stage and stages immediately following flowering. Drought increased rates of flower and pod abortion, thus causing reduction in final yield [35]. In soybean, drought causes yield reductions of up to 40 % [4, 8, 11]. Therefore, finding alternative ways to alleviate and/or ameliorate the effects of waterdeficit conditions is crucial. In comparison with traditional breeding and marker-assisted selection programs, the direct introduction of a gene(s) by genetic engineering could be a more effective and rapid approach to improving abiotic stress tolerance in soybean. Genetic engineering for drought tolerance using transcription factors and osmoprotectants is showing progress in the major food crops. However, in the case of soybean, it is still in its preliminary stages. The present study constitutes the report of transgenic soybean developed using the DREB TF. In this study, we sought to characterize the mechanism(s) involved in transgenic enhancement of drought tolerance by subjecting the plants to progressive soil drying with a proper control of soil moisture depletion, including plant water relations and physiological measurements, to ensure that plants are exposed to stress levels and kinetics of water deficits that closely resemble field conditions. To achieve this objective, we developed soybean transgenic plants by transformation with AtDREB1D gene driven by constitutive and ABA-inducible promoters and performed physiological evaluation of transgenic plants.

Materials and methods Plasmid constructs and transformation To study the function of AtDREB1D, two constructs were developed using CaMV35S and an ABA-inducible promoter. The full-length of the AtDREB1D was amplified from genomic DNA by PCR (Polymerase Chain Reaction) using forward primer 50 -CATGCCATGGATGAATCCATTTTAC TCTACATT-30 and reverse primer 50 -GCTCTAGATTAC TCGTCAAAACTCCAGAGT-30 . The amplified fragment was cloned into pGEM-Easy vector and sequenced. The AtDREB1D fragment (675 bp) was cloned into subcloning vector pRTL2:CaMV35S (obtained from Dr. Thomas Clemente, University of Nebraska) having the enhanced dual CaMV35S promoter with Nco1 and Xba1 restriction sites. The ABRC3 (493 bp) fragment was amplified from QS264 plasmid [27] and cloned into subcloning vector pRTL2: CaMV35S:AtDREB1D by replacing the CaMV35S promoter. Both cassettes, CaMV 35S:AtDREB1D and ABRC3:AtDREB1D, were cloned into binary vector pZY101.ASc with HindIII (Fig. 1a, b). Stable soybean transgenic plants were developed in the Maverick genotype using the

Fig. 1 T-DNA regions of the binary plasmids used for Agrobacterium tumefaciens-mediated transformation. a Construct containing bar (herbicide selectable marker) driven by the two copies of 35S promoter and AtDREB1D under the control of a modified 35S promoter with 50 enhancer sequence of CaMV35S promoter (E35S) and a sequence from the tobacco etch virus (TEV). b Construct containing bar driven by CaMV35S promoter and ABRC3 driven AtDREB1D gene and TEV sequence. All the constructs had VSP (vegetative storage protein) and CaMV35 terminator sequences (35S T). LB, left border; RB, right border. c Components of the ABRC3 promoter containing the ABRC3 core element with Amy64 and HVA22 intron–exon-intron (I,E,I) sequences

Agrobacterium-mediated transformation with organogenesis technology developed at the University of Missouri Plant Transformation Core Facility [36]. Molecular analysis of transgenic plants Genomic DNA was isolated from young leaves of the WT and putative transgenic lines using an AutoGenPrep 960 plant DNA extraction kit (AutoGen Inc. Holliston, MA, U.S.A). An initial screening of the WT and transgenic lines for the absence and presence of AtDREB1D and for the promoter and gene fragments was performed using PCR. Southern hybridization analysis was performed as described previously [37]. Briefly, genomic DNA (25 lg) from each of the putative transgenic lines in the T0 generation (first transformants) was digested with EcoRI, and the fragments were resolved on to a 1.0 % agarose gel followed by transferred to Hybond-N? nylon membrane (Amersham Pharmacia Biotech, Freiburg, Germany). The blot was probed with a 32P dCTP-labeled gene and DNA fragment, which was a combination of promoter and gene fragment using the Prime-It II Random labeling kit (Strategene, La Jolla, CA, U.S.A). Positive transgenic lines were reconfirmed by another probe developed using the reporter gene, bar (basta resistance) fragment. Shoot and root growth measurements of transgenic plants Three different experiments were conducted to study the pattern of shoot and root growth of transgenic plants

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compared to WT plants (Glycine max cv. Maverick). In the first experiment, WT and two independent homozygous transgenic lines for each construct (CaMV35S:AtDREB1D and ABRC3:AtDREB1D) were grown in 3-gallon pots filled with Promix soil. The experiment was carried out under the greenhouse conditions with 60 %/80 % humidity during day/night with 16/8 h light/dark and 30/18 °C day/night temperatures, respectively, in a completely randomized statistical design with four replications. The plants were grown for 30 days from the day of sowing under wellwatered conditions. After 30 days the plant height and number of trifoliate leaves per plant were measured. The plants were then harvested to determine total leaf area per plant and shoot dry weight. Leaf area was measured for each plant with a LI-COR LI-3000 leaf area meter (LICOR, INC., Lincoln, NE, U. S. A.). The dry weights were measured after drying the plants at 72 °C for 4 days. Plant height, total leaf area and shoot dry weight measurements were repeated under greenhouse conditions. The experiment was repeated in the greenhouse to measure total leaf area and plant height. In another experiment, two CaMV35S:AtDREB1D independent transgenic (A1, A2) lines and WT plants were compared for shoot and root growth morphology under well-watered conditions in a growth chamber. The growth chamber conditions were 65 % humidity, 250 lmol m-2 s-1 photosynthetic photon flux density, 16/8 h day/night photoperiod, and 27/18 °C day/night temperatures. The experiment was conducted in a completely randomized design with nine replications per transgenic line under the controlled conditions. The plants were grown for 15 days from day of sowing in plastic cones of 36 cm height and 6.4 cm base diameter filled with turface and sand (1:1 v/v) mixture. Plant height, shoot dry weight, root length, and root dry weight measurements were taken after carefully harvesting the plants from the cones. Shoot and root dry weights were measured after drying the plants at 72 °C for 3 days. In a third experiment, WT and one of the ABRC3:AtDREB1D transgenic lines (B1) were compared for shoot and root growth morphology under well-watered conditions (-0.12 MPa soil water potential) and under three water-deficit treatments (initial soil water potentials of -0.25, -0.5 and -0.9 MPa under the growth chamber conditions). The WT and transgenic seeds were germinated on germination paper, and seedlings with 2–2.5 cm radicles were transplanted into tubes (76 cm long and 21 cm diameter). The tubes were filled with premixed turface and vermiculite medium (1:1 v/v) with desired soil water potential for the well-watered and water-deficit treatments described above [38]. The growth chamber conditions were maintained at 80 % humidity, 16/8 h day/night, 400 lmol m-2 s-1 photosynthetic photon flux density at the top of plant height, with 29 °C day/night temperatures. The transgenic line B1

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and WT plants were grown in tubes with well-watered and drought stress treatments for 14 days after transplanting. The plants were harvested to determine shoot length, shoot dry weight, root length, and root dry weight. Dry weights were measured after drying at 72 °C for 3 days. For shoot length and root length, six replications were used, and for root and shoot dry weight, three replications were used. Greenhouse drought evaluation of transgenic plants The WT and two independent lines of transgenic plants (35S:AtDREB1D and ABRC3:AtDREB1D) were planted in 3-gallon pots filled with Promix soil. The previously wellwatered 30-day-old plants were then subjected to waterdeficit conditions by completely withholding water until the leaf water potential of WT and transgenic plants reached -3.5 MPa. The experiment was conducted under greenhouse conditions with a 16/8 h day/night cycle, 60/80 % day/night humidity, and 28/20 °C day/night temperatures using a randomized complete block design of four blocks with two replications in each block. Each pot was completely covered with two layers of plastic to avoid loss of water by evaporation from the soil. Leaf water potential was estimated at midday using a pressure chamber (PMS Instrument Co. Albany, CA, U.S.A) for a leaflet from the topmost fully expanded leaf of each plant at 4 day intervals from the onset of drought stress to the end of the experiment. Stomatal conductance was determined using a leaf porometer (Decagon Devices Inc., Pullman, WA, U.S.A) before the start of drought stress treatment and during the stress treatment at six-day intervals. The stomatal conductance of the abaxial leaf surface was measured at 7 a.m., 11 a.m. and 3 p.m. during each data collection day. Stomatal conductance was measured on the topmost fully expanded, middle fully expanded and lower leaves of each plant per replication. The data were represented as average stomatal conductance per plant One of the shoot traits which may affect drought tolerance is the reduced wholeplant water use during stress period. As the soil moisture reduces, plants adapts and changes from water-saturated state to a state where water use is dependent on the available soil moisture. This change in phase is caused by a lower stomatal conductance. Stomatal conductance can change during the duration of day, stomata are closed in early morning and late evening, while they are open during mid day. Thus, to precisely test the effect of stomatal conductance of transgenic plants under well watered and drought stress conditions, stomatal conductance was measured at different time points. To elucidate the mechanisms involved in drought tolerance, leaf OA and leaf cell membrane stability were determined when the plants reached a leaf xylem water

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potential of -3.5 MPa using methods previously described [39, 40]. Briefly, the second fully expanded leaf was sampled, and osmotic potential for each sample was measured using a Wescor Osmometer (Wescor Inc., Logan, UT, U.S.A). Leaf OA was calculated as the difference in measured leaf osmotic potential between well-watered plants and fully rehydrated plants after water-deficit treatments. Cell membrane injury was determined using the electrolyte conductivity method following the standard protocol (www.plantstress.com). Briefly, 20 cm2 sections of leaves collected from well-watered and severely waterstressed plants were cut and placed in glass tubes, and 20 ml of deionized water was added, followed by incubation at 10 °C for 24 h. After incubation, conductance was determined with a conductivity meter (CDM210 conductivity meter, Hach Company, Loveland, CO, U.S.A). After the initial measurement, the vials were autoclaved for 15 min and release all the electrolytes. Both measurements were carried out for each sample from the well-watered and severe water-deficit plants subjected to the same leaf water potential. Cell membrane stability was calculated by the following formula: cell membrane stability % = [(1-(T1/ T2))/(1-(C1/C2))] 9 100, in which T and C refer to the water-deficit treated and control well-watered samples, respectively, and the 1 and 2 refer to the initial conductance before autoclaving and final conductance after autoclaving readings, respectively. Survival rate of transgenic plants Survival rate was determined in transgenic and WT plants subjected to water deficit under greenhouse conditions using a completely randomized design with three replications. The transgenic and WT plants were grown for 15 days under normal well-watered conditions in 7-gallon pots filled with Promix. A stress treatment was imposed by completely withholding water until the plant-pot system reached 3.3 kg total mass (30 % soil water content of initial pot-plant mass of 11 kg). All the pots were then rewatered to allow plants to recover from the water-deficit treatment, and the number of surviving plants (not completely wilted) was assessed after 2 days. Gene expression analysis Two independent lines of each construct of CaMV35S:AtDREB1D and ABRC3:AtDREB1D along with WT were grown under normal well-watered conditions for 30 days. The plants were grown in 3-gallon pots filled with Promix soil under greenhouse conditions. The plants were subjected to water-deficit conditions by completely withholding water, and the leaf samples were collected for each plant individually when it reached the desired stress level.

The stress treatments included well-watered, mild stress and severe stress (-0.5 MPa, -1.1 MPa and -2.6 MPa leaf water potential, respectively). The experiment was conducted under greenhouse conditions in a randomized complete block design with three blocks and two replications per block. The leaf samples collected were immediately and freezed in liquid nitrogen and stored at -80 °C until RNA isolation. Total RNA was extracted leaf tissue using the TRIZOL For each sample, 18 lg of total RNA was digested in a volume of 50 ll with Turbo DNA-free DNaseI to eliminate genomic DNA contamination. RNA was quantified with NanoDrop and first-strans cDNA synthesis was conducted using iScript cDNA Synthesis Kit. Using ProbeFinder Version 2.44, gene-specific primers were developed. Primer specificity was confirmed by BLAST-ing against Phytozome (http://www.phytozome. net/search.php?show=blast) with BLASTN program. Soybean Calcium-dependent protein kinase (CDPK) and ubiquitin genes were used as endogenous control for stressresponsive expression. Quantitative RT-PCR (qRT-PCR) reactions were performed as previously described [41]. Statistical analysis Analysis of variance (ANOVA) was conducted using PROC GLM in SAS (Statistical Analysis System) software (SAS Corporation, Cary, NC, U.S.A.) for all variables evaluated. Mean comparisons were made according to Fisher’s least significance difference (LSD) at the 0.05 significance level.

Results Construction of CaMV35S:AtDRED1D and ABRC3:AtDREB1D transgenic plants To examine the role of AtDREB1D in transgenic soybean plants, two constructs were used for soybean transformation: CaMV35S:AtDREB1D and ABRC3:AtDREB1D (Fig. 1a, b) Seventeen and twenty independent transgenic lines for each construct CaMV35S:AtDREB1D and ABRC3:AtDREB1D, respectively, were obtained in the first generation immediately after transformation using the Agrobacterium-mediated method [36]. PCR analysis was performed to identify positive transgenic plants. Presence of transgene and copy number in all transgenic plants was determined by genomic Southern blot hybridization in T0 (first transformants). Southern blot analysis identified at least three independent lines for CaMV35S:AtDREB1D and ABRC3:AtDREB1D with single copy number (data not shown).

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Fig. 2 AtDREB1D expression of transgenic plants. Expression in leaf tissue of 30-day-old transgenic and WT plants subjected to waterdeficit treatments by withholding water. The leaf tissue was collected from plants subjected to three treatments, well-watered (-0.5 MPa), mild stress (-1.1 MPa), and severe stress (-2.6 MPa). A1 and A2 represent two independent transgenic lines of the 35S:AtDREB1D construct, while B1 and B2 represent two independent lines of the ABRC3:AtDREB1D construct

Expression of AtDREB1D in transgenic soybean plants Total RNA was isolated from leaf tissues of non-stress (-0.5 MPa), mild stress (-1.1 MPa), and severe stress (-2.6 MPa) 30-day-old WT and T2 homozygous transgenic plants. The AtDREB1D transcript was detected at different levels for all transgenic lines tested. Of the two independent lines of the 35S:AtDREB1D, one A1-independent line showed 2.5 times stronger expression than the other independent A2 line under both stress and non-stress conditions (Fig. 2). Two independent lines of ABRC3:AtDREB1D showed a similar expression pattern, which was also similar to the expression of the A2 line of the 35S:AtDREB1D plants under non-stress conditions. However, under severe water-stress conditions, the ABRC3:AtDREB1D lines showed 1.5- to 2-fold induction when compared to well-watered conditions; the expression level was also slightly higher in the A2 line. Under normal conditions, the transgene expression level varies to a small degree among different lines using both constitutive and stress-inducible promoters, which may cause the differences in growth parameters like plant height, biomass (A1 transgenic line). The differences in expression may be used of strong dicot constitutive promoter (2 9 35S). Shoot growth and root growth of transgenic plants Previous reports indicated severe growth retardation in Arabidopsis plants in which AtDREB1D or AtDREB1A was overexpressed [19, 21, 42]. Shoot growth and morphology

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of CaMV35S:AtDREB1D and ABRC3:AtDREB1D transgenic and WT plants were measured under well-watered conditions at 30 days after sowing (Fig. 3). One of the CaMV35:AtDREB1D transgenic lines, A1, showed significant reduction in plant height, trifoliate number, total leaf area, and shoot dry weight compared to WT plants. The other transgenic line, A2, showed greater plant height and a similar number of trifoliate leaves compared to WT. The total leaf area and shoot dry weight of A2 plants were significantly reduced compared with WT, but overall, there was no severe growth retardation (Fig. 3a–e). One independent line of ABRC3:AtDREB1D plants, B1, showed similar plant height compared to the WT. The transgenic lines B1 of ABRC3 and A2 of CaMV35S constructs showed a similar shoot growth pattern, with similar plant height, number of trifoliate leaves, total leaf area per plant and shoot dry weight (Fig. 3a–d). The other transgenic line, B2, showed a significant reduction in plant height, fewer trifoliate leaves, and significantly reduced total leaf area and shoot dry weight. However, it was not as severely affected as the 35S A1 line (Fig. 3e, f). Measurements of total leaf area per plant and plant height were replicated in another separate greenhouse experiment and similar trends were observed (data not shown). To investigate the role of the AtDREB1D gene in root growth and development, the primary root length and root dry weight were determined for 14-day-old 35S:AtDREB1D transgenic plants grown under non-stress conditions. Plants of the transgenic lines A1 and A2 exhibited similar root length and root dry weight compared to WT plants grown under well-watered conditions (Fig. 4a, b). The shoot and root growth parameters of one independent line of ABRC3:AtDREB1D, B1, was also examined under non-stress and stress conditions. The transgenic and WT plants were grown for 14 days in tubes filled with premixed soil with known soil water potential (non-stress -0.12 and stress treatments -0.25, -0.5, and -0.9 MPa). The shoot length showed severe growth inhibition with increased stress, whereas root length was less affected with an increase of stress. The transgenic line B1 exhibited similar primary root length, root dry weight, shoot length, and shoot dry weight compared to WT grown under non-stress and different stress conditions (Fig. 4c–f). Drought stress tolerance in transgenic soybean plants To investigate whether expression of AtDREB1D was correlated with stress tolerance in transgenic plants, 30-day-old WT and two independent lines of 35S:AtDREB1D and ABRC3:AtDREB1D T2 homozygous transgenic soybean plants were subjected to slow drying by completely withholding water. The transgenic plants maintained higher soil water content and exhibited less

Mol Biol Rep Fig. 3 Shoot growth of transgenic plants under wellwatered conditions. a Plant height of 30-day-old WT and transgenic plants. b Number of trifoliate leaves per plant. c Total leaf area per plant. d Shoot dry weight. e, f Visual observations of 30-day-old WT and transgenic plants. A1 and A2 represent two independent transgenic lines of the 35S:AtDREB1D construct, while B1 and B2 represent two independent lines of the ABRC3:AtDREB1D construct. Means with statistical significance at p \ 0.05 are shown by different alphabetical letters

daily water loss compared to the WT plants. During the first 5 days of water-deficit stress, the WT plants showed a significantly greater reduction in soil water content and significantly higher water loss compared to transgenic plants (Fig. 5a, b). The WT soybean plants reached -3.5 MPa leaf water potential after 10 days of stress, whereas the transgenic plants reached same water potential after 11–12 days of stress. The transgenic plants maintained higher leaf water potential during the drying period compared to the WT plants (Fig. 5c). The WT and transgenic plants showed similar stomatal conductance measured at three different time points of a single day under well-watered conditions before onset of stress treatments (Fig. 5d). For both WT and transgenic plants, stomatal conductance under stress conditions decreased progressively with increased stress; there was no significant difference in this parameter between WT and transgenic plants (data not shown).

All WT and transgenic plants were eventually stressed to the same leaf water potential of -3.5 MPa, irrespective of the time required (Fig. 5c), but there were differences in the timing of plant responses. The WT plants exhibited wilting symptoms, lower midday water potentials, and drought-induced rolling of young leaves at 10 days of stress, whereas the transgenic plants showed no or only slight symptoms of leaf drooping at 10 days (Fig. 5e, f). However, soil water extraction data (Fig. 5a) indicated that the smaller transgenic plants were depleting soil water more gradually and that the initial differential morphological and water relations responses were likely attributable to differences in plant size, not inherent drought tolerance differences. When all plants had been allowed to dry to a targeted low water potential (-3.5 MPa), then reirrigated and allowed to recover in moist soil for 2 days, transgenic plants did show apparent enhanced drought tolerance, as indicated by better recovery determined by

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Fig. 4 Shoot and root growth under well-watered and water-deficit conditions. Shoot and root growth of 15-day-old WT and transgenic plants. a Root and shoot length of WT and 35S transgenic plants under well-watered conditions. b Root and shoot dry weight of WT and 35S transgenic plants under well-watered conditions. c, d Primary

root length and root dry weight of WT and B1 (ABRC3:AtDREB1D transgenic) plants under different stress conditions. e, f Shoot length and shoot dry weight of WT and B1 under different stress conditions. Means with no statistical significance at p \ 0.05 are shown by same alphabetical letters

visual observation, compared to the WT plants, although both were stressed to the same low leaf water potential (Fig. 6). Leaf osmotic adjustment (OA) and cell membrane stability were measured with the goal of understanding potential physiological traits that contribute to the enhanced stress tolerance of transgenic plants. After the plants reached -3.5 MPa leaf xylem water potential

under water-deficit conditions, leaf samples were collected to measure OA and cell membrane stability before rewatering the plants. The transgenic plants did not show any increase in OA compared to WT plants measured at the same leaf water potential (Table 1). In contrast, transgenic plants had significantly and substantially greater values of the cell membrane stability index than WT plants (Table 1). The leaf cell membrane stability

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Fig. 5 Response of transgenic plants to drought. Performance of WT and transgenic plants during water-deficit conditions. a, b, c, d Daily pot weight, daily pot water loss and leaf water potential of WT and transgenic plants during soil water depletion. e Leaf abaxial stomatal conductance of WT and transgenic plants under well-watered

conditions. f, g WT and transgenic plants at 6 and 10 days after stress. Means with statistical significance at p \ 0.05 are shown by asterisk. A1 and A2 are two independent transgenic lines of the 35S:AtDREB1D construct, while B1 and B2 are two independent lines of the ABRC3:AtDREB1D construct

experiment was repeated and similar results were obtained (data not shown). These results support the hypothesis that transgenic plants have increased tolerance to drought

stress; further, they indicate that overexpression of Arabidopsis AtDREB1D in soybean confers increased tolerance to drought stress.

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Fig. 6 Transgenic plants’ recovery after drought stress. Visual appearance of transgenic and WT plants after 2 days of recovery after stress. Note the greater number of necrotic leaves and smaller leaf/leaflet size of the WT plants

Table 1 Leaf osmotic adjustment (OA) of transgenic plants under water-deficit conditions Plants

Osmotic adjustment (MPa)

Cell membrane stability (%)

WT

1.11a

66.71c

A1

0.94a

89.53a

A2

0.98a

83.59b

B1

0.95a

90.41a

B2

0.96a

86.60ab

A1 and A2 are two independent lines of 35S:AtDREB1D construct, while B1 and B2 are two independent lines of ABRC3: AtDREB1D construct. The measurements were taken from WT and transgenic plants subjected to water-deficit conditions with -3.5 MPa leaf water potential. LSD (0.05) of leaf OA = 0.1814 and cell membrane stability = 5.497. In each column, values followed by a different letter are significantly different

Survival rate of transgenic plants To compare the drought tolerance of the soybean transgenic plants with the WT plants, 15-day-old plants were grown in soil. Stress treatment was induced by withholding water, followed subsequently by rewatering when all plants had reached 30 % soil water content. The survival rate was observed 2 days after rewatering. Only 21 % of the WT plants survived stress treatment, but survival rate for all transgenic plants was much higher, determined by better recovery of transgenic plants compared to WT plants based on visual observation and the number of leaves recovered. The survival rate for 35S:AtDREB1D plants was 87.5 % for transgenic line A1 and 75.3 % for A2, and the survival

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rate for ABRC3:AtDREB1D plants was 83.5 % for B1 and 91.6 % for B2 (Table 2). These results demonstrate that the overexpression of AtDREB1D resulted in transgenic plants with improved drought tolerance compared to the WT controls.

Discussion In this study, the AtDREB1A gene was overexpressed by the CaMV35S promoter for constitutive expression and by the ABA-inducible promoter for stress-inducible expression. Two independent T2 homozygous lines were selected for each construct for all further molecular and physiological analysis. The expression of AtDREB1D was studied in the leaves of 30-day-old 35S:AtDREB1D and ABRC3:AtDREB1D transgenic plants that were subsequently subjected to water-deficit conditions by withholding water for 12 days. Transgene expression was similar in well-watered and water deficit-treated 35S:AtDREB1D plants and expression in the A1 line expression was twice as high as in the A2 line. Expression of AtDREB1D gene is twice high in A1 line compare to A2 line when using the same constitutive promoter due the random insertion of the T-DNA by Agrobacterium transformation. The difference in expression lines may due to the insertion of T-DNA at different locations in genome. The ABRC3:AtDREB1D lines showed basal expression in well-watered conditions (-0.5 MPa). With progressively greater stress, there was a 1.5- to two-fold increase in induction of expression under

Mol Biol Rep Table 2 Survival rate of transgenic plants Plants

No. of plants survived/ Total plants tested

Survival %

WT

5/24

20.8a

A1

23/24

91.6c

A2

18/24

75.3b

B1

20/24

83.5bc

B2

23/24

91.6c

A1 and A2 are two independent lines of 35S:AtDREB1D construct, while B1 and B2 are two independent lines of ABRC3: AtDREB1D construct. The alphabetical letters indicate significant difference between WT and transgenic lines at Least Significant Difference (0.05) = 15.3

severe water-deficit conditions. It is well known from previous reports that ABA accumulation is increased in shoots and roots of water-stressed plants compared to wellwatered plants [25, 43]. In this study, ABRC3:AtDREB1D transgenic plants under non-stress conditions showed basal expression, as a result of the endogenous presence of ABA in the leaves. When subjected to prolonged water-deficit conditions, higher accumulation of ABA in the plant might have caused a stronger induction of transgene under the influence of the ABRC3 promoter. In basic agreement with these results, the ABRC3 promoter was sufficient to confer ABA induction when fused to a minimal promoter (Amy64) with the transient expression when barley aleurone tissue was subjected to 20 lM ABA for 24 h [27, 44]. In transgenic rice plants, the presence of one copy of the ABRC1 showed a six-fold induction of GUS activity when the plants were subjected to eight days of stress [29]. In another study, overexpression of HVA1 using two copies of ABRC3 promoter in creeping bentgrass showed that transgenic lines had higher levels of the HVA1 protein after 9 days of drought stress. The authors also tested the promoter efficiency of ABRC3 by fusing with GFP in callus, and found that one copy of ABRC3 expressed very weakly compared with the two-copy ABRC3 promoter [23]. Some previous studies suggest that a single ABAinducible promoter might not be sufficient for the very strong induction (tenfold or higher) during water-deficit conditions, compared with well-watered conditions. In this study, one copy of ABRC3 promoter was used for the expression of the transgene, and under stress conditions, we observed only a two-fold induction. The efficiency of the ABA-inducible promoter depends on both the number of the promoter element and on the threshold level of ABA accumulation in the plants that were subjected to stress and well-watered conditions. This needs to be investigated further, particularly in soybean. Shoot and root morphology should be examined in detail to compare non-transgenic control plants and transgenic plants because water loss is a function of leaf transpiration,

and apparently drought tolerance in transgenic plants can be mimicked by the slower water loss of plants with reduced leaf area. Observations of shoot growth in this study demonstrated that all the transgenic lines showed a significant reduction in total leaf area and in shoot dry weight. Transgenic lines B1 and A2 showed similar plant heights and the same number of trifoliate leaves. One transgenic line of 35S:AtDREB1A (A1) plants exhibited severe reduction in growth under well-watered conditions, compared with other transgenic lines and the WT plants. The expression of AtDREB1A by constitutive promoter in Arabidopsis transgenic plants resulted in severe growth retardation [21]. Similarly, the overexpression of AtDREB1D in Arabidopsis using 35S promoter induces growth retardation in transgenic plants. The degree of growth retardation correlated with the level of AtDREB1D expression. The high level of transgene expression resulted in a dwarf phenotype. The transgenic plants with growth retardation exhibited improved survival rates, which might be simply due to difference in shoot growth (and resultant reduction in the rate of soil water depletion) compared with WT plants [19]. In this study, 35S transgenic lines showed similar root length and root dry weight compared to WT plants (Fig. 4a, b). To understand whether AtDREB1D interacts with other stress-inducible proteins, and therefore plays a role in root growth and development under water-deficit conditions, one independent line from the ABRC3:AtDREB1D (B1) transgenic lines was grown for 14 days to monitor shoot and root growth under different stress conditions (Fig. 4c– f). As expected, shoot growth was severely affected with increase in stress conditions, but root growth was less severely affected [26]. There was no significant difference in root length or in root dry weight of transgenic and WT plants at 14 days after transplant of the seedlings. These results indicate that expression of AtDREB1D is not involved in root morphology-related traits that contribute to drought tolerance. To drive the AtDREB1D expression, use of abiotic stress-induced promoter in the soybean transgenic plants can lead to increased stress tolerance associated with less negative effects on growth, since stress-induced promoter drives the expression only under stress conditions. Use of constitutive promoter for the expression of AtDREB1D in soybean transgenic plants can lead to high level stress tolerance but could lead to negative or abnormal growth effects due to expression at all time. Expression of CBF/DREB genes using stress-inducible promoters showed undetectable expression of these genes under normal conditions. The transgenic plants did not show any abnormal effects of growth parameters [45]. To study the response of transgenic plants to water-deficit conditions, 30-day-old plants were subjected to slow drying by withholding water. Transgenic plants in the initial

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stage of water-deficit conditions maintained high water potential compared to WT plants and showed no significant difference in stomatal conductance. The WT plants reached -3.5 MPa leaf xylem water potential sooner and showed wilting symptoms earlier than did transgenic plants, but these responses were associated with reduced total leaf area in the transgenic plants, and hence, slower pot-water consumption compared to WT plants. The transgenic and WT plants eventually reached the same leaf xylem water potential (-3.5 MPa) and were then re-watered. After 2 days of recovery, by visual observation the transgenic plants showed better recovery and growth than WT plants. To understand physiological traits involved in tolerance, the leaf OA and leaf cell membrane stability were determined in all of the plants when they reached -3.5 MPa leaf xylem water potential. The transgenic plants showed similar leaf OA compared with WT plants. The accumulation of osmolytes might not be enough to contribute to OA [30, 46, 47]. Significantly, transgenic plants showed 17–24 % higher values of a cell membrane stability index than did the WT plants. Previous reports had shown that transgenic Arabidopsis plants overexpressed with AtDREB1D exhibited less electrolyte leakage compared to WT plants [19]. Similarly, transgenic tomato plants overexpressed with AtDREB1B using the ABRC1 promoter showed 65–70 % reduction in electrolyte leakage compared to WT plants [31]. Recently transgenic soybean lines developed by overexpression of the DREB1A gene under drought conditions in greenhouse conditions showed higher survival rates due to reduced water use by lower transpiration under normal conditions [48]. These results suggest that improved drought tolerance observed in the transgenic soybean lines (as evidenced by superior survival under severe water stress) was mainly through enhanced cell membrane stability. The structural integrity of all cellular membranes is a desirable property that is necessary for the maintenance of cellular functions. Therefore, plant capacity to maintain membrane integrity and stability is critical for survival under severe waterdeficit conditions [46, 49]. Under severe water-deficit conditions, specific osmolytes and LEA proteins might prevent protein denaturation and maintain structural integrity by preferential exclusion associated with the membrane macromolecules. In a severe air-dry state, the hydroxyl groups substitute for water to maintain hydrophyllic interactions with membrane lipids and proteins. This concept has been termed the water replacement hypothesis [50]. The accumulation and role of osmolytes and/or LEA proteins in maintaining membrane integrity by preferential exclusion should be a focus of future research. In conclusion, we have developed transgenic lines of soybean with the AtDREB1D transcription factor encoding gene overexpressed with constitutive CaMV35S and ABA-

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inducible ABRC3 promoters. All of the independent transgenic lines tested showed reduced total leaf area and shoot biomass compared to non-transgenic plants. Transgenic lines overexpressed by a constitutive promoter showed the highest expression of the gene, which correlated with reduced shoot growth. Under water-deficit conditions, transgenic plants maintained higher relative water content compared to the WT plants, as a result of slower pot-water depletion, which was in turn caused by reduced total leaf area per plant. When the WT and transgenic plants were subjected to -3.5 MPa leaf xylem water potential, the transgenic plants showed improved survival and greater apparent drought tolerance by maintaining cell membrane stability better than WT plants. No difference in leaf OA was observed between transgenic and WT plants, which might be because the accumulation was not sufficient to maintain turgor under stress conditions. Evaluation of performance of transgenic plants under natural drought conditions in the field is in progress. Future research should involve the identification of downstream target genes of AtDREB1D to understand the genes involved in drought tolerance. The accumulation and role of specific osmolytes and of LEA proteins up-regulated by the AtDREB1D in maintaining structural integrity need to be investigated. This approach of engineering for abiotic stress tolerance with stress-inducible transcription factors has the potential to develop more drought-tolerant genotypes of soybean in the near future. Acknowledgments We thank Dr. Zhanyuan Zhang, Plant Transformation Core Facility, University of Missouri for the soybean transformation and Dr. Thomas Clemente, University of Nebraska for providing sub-cloning vectors. This work was supported by the United Soybean Board and the Missouri Soybean Merchandising Council funding to HTN.

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Overexpression of AtDREB1D transcription factor improves drought tolerance in soybean.

Drought is one of the major abiotic stresses that affect productivity in soybean (Glycine max L.) Several genes induced by drought stress include func...
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