Plant Biotechnology Journal (2015), pp. 1–13

doi: 10.1111/pbi.12358

Arabidopsis EDT1/HDG11 improves drought and salt tolerance in cotton and poplar and increases cotton yield in the field Lin-Hui Yu1,†, Shen-Jie Wu2,†, Yi-Shu Peng3,†, Rui-Na Liu4,†, Xi Chen1,†, Ping Zhao1, Ping Xu1, Jian-Bo Zhu4, Gai-Li Jiao2, Yan Pei3 and Cheng-Bin Xiang1,* 1

School of Life Sciences, University of Science and Technology of China, Hefei, Anhui Province, China

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Cotton Research Institute, Shanxi Academy of Agricultural Sciences, Yuncheng, Shanxi Province, China

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Biotechnology Research Center, Southwest University, Chongqing, China

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College of Life Sciences, Shihezi University, Shihezi, Xinjiang Province, China

Received 26 August 2014; revised 25 November 2014 and 29 January 2015; accepted 16 February 2015. *Correspondence (Tel 86-55163600429; fax 86-5513601443; emails [email protected]; [email protected]) † These authors are equally contributed.

Keywords: AtHDG11, drought stress, salt stress, transgenic cotton, transgenic poplar, cotton yield.

Summary Drought and salinity are two major environmental factors limiting crop production worldwide. Improvement of drought and salt tolerance of crops with transgenic approach is an effective strategy to meet the demand of the ever-growing world population. Arabidopsis ENHANCED DROUGHT TOLERANCE1/HOMEODOMAIN GLABROUS11 (AtEDT1/HDG11), a homeodomainSTART transcription factor, has been demonstrated to significantly improve drought tolerance in Arabidopsis, tobacco, tall fescue and rice. Here we report that AtHDG11 also confers drought and salt tolerance in upland cotton (Gossypium hirsutum) and woody plant poplar (Populus tomentosa Carr.). Our results showed that both the transgenic cotton and poplar exhibited significantly enhanced tolerance to drought and salt stress with well-developed root system. In the leaves of the transgenic cotton plants, proline content, soluble sugar content and activities of reactive oxygen species-scavenging enzymes were significantly increased after drought and salt stress compared with wild type. Leaf stomatal density was significantly reduced, whereas stomatal and leaf epidermal cell size were significantly increased in both the transgenic cotton and poplar plants. More importantly, the transgenic cotton showed significantly improved drought tolerance and better agronomic performance with higher cotton yield in the field both under normal and drought conditions. These results demonstrate that AtHDG11 is not only a promising candidate for crops improvement but also for woody plants.

Introduction A tremendous challenge that we face today is to satisfy the growing demand of the world population for food and clothing, which is compounded with the dramatic losing arable land because of increasingly severe soil destruction by pollution and abiotic stresses. Among these abiotic stresses, drought and salinity are the major limiting factors in agricultural productivity, affecting more than 10% of arable land and crop yield, on average, of more than 50% for most major crop plants (Bartels and Sunkar, 2005). Global climate change will likely make the situation more serious in the near future (Ahuja et al., 2010). Therefore, it is imperative for researchers to develop strategies and technologies to make crops more productive with enhanced stress tolerance. Although some success in improving abiotic stress tolerance has been achieved so far using traditional breeding (Ashraf, 2010; Varshney et al., 2011), this procedure is time-consuming, costand labour-intensive. Genetic engineering has opened up new possibilities in creating stress tolerant crops in a shorter time with

the rapid progress made in plant genomics and biotechnology research. In fact, there have been many efforts to improve drought and salinity tolerance in plants by genetic engineering (Bartels and Sunkar, 2005; Borsani et al., 2003). Numerous genes responsive to drought and salinity stress have been identified and used as candidate genes in genetic engineering, such as genes encoding metabolites or osmoprotectants (Kreps et al., 2002), genes related to ion homeostasis and hormones (Alvarez et al., 2008; Apse et al., 1999; Yang et al., 2001; Yue et al., 2012), genes in signal transduction and transcription factors (Liu et al., 2014; Umezawa et al., 2006; Yang et al., 2010). However, in most cases, the genetic modifications were carried out in the model plant Arabidopsis, only a small proportion were transferred into crop plants (Peleg et al., 2011). Cotton, which is grown commercially in the tropical and temperate regions of more than 50 countries, is the world’s most important textile fibre crop and a significant oilseed crop (Chen et al., 2007). Cotton is estimated as one of the major consumers of agricultural water among all crops, and about 53% of the global cotton field is irrigated, producing 73% of the global

Please cite this article as: Yu, L.-H., Wu, S.-J., Peng, Y.-S., Liu, R.-N., Chen, X., Zhao, P., Xu, P., Zhu, J.-B., Jiao, G.-L., Pei, Y. and Xiang, C.-B. (2015) Arabidopsis EDT1/HDG11 improves drought and salt tolerance in cotton and poplar and increases cotton yield in the field. Plant Biotechnol. J., doi: 10.1111/pbi.12358

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2 Lin-Hui Yu et al. cotton. These irrigated cotton fields, mainly located in dry regions: Egypt, Uzbekistan and the Xinjiang Province of China, are entirely irrigated, whereas supplementary irrigation supplies most of crop water in Pakistan and the north of India (Soth et al., 1999). Moreover, due to the poor irrigation management and high evaporation, many irrigated agricultural areas have greatly threatened by salt accumulation in soils (Basal, 2010). Although cotton is considered as a drought-tolerant and medium saltresistant crop, its sensitivity varies greatly among genotypes (Ashraf, 2002; Iqbal et al., 2011; Ohkama-Ohtsu et al., 2007). Drought and salinity stress still greatly affected cotton in growth, yield and fibre quality (Dong, 2012). Therefore, it is urgent to enhance drought and salt stress tolerance of cotton to maintain productivity and quantity on marginal land under water-limited and saline conditions. The success story of herbicide-resistant and Bt (Bacillus thuringiensis)-containing cotton has shed light on genetic engineering for cotton breeding. During the past decades, hundreds of potential candidates for improving tolerance to abiotic stresses have been identified. However, progress in improving cotton drought and salinity tolerance through genetic engineering is limited. Increasing solute contents in plant vacuoles conferred enhanced drought and salt tolerance in plants. For example, expression of Arabidopsis vacuolar sodium/proton antiporter gene AtNHX1 (He et al., 2005), Arabidopsis vacuolar H+-pyrophosphatase gene AVP1 (Pasapula et al., 2011; Qin et al., 2013) and Thellungiella halophila H+-PPase gene TsVP (Lv et al., 2008, 2009) in cotton improved drought and salt tolerance. Increase of glycine betaine synthesis by overexpression of betA gene from Escherichia coli (E. coli) (Lv et al., 2007) or AhCMO gene from Atriplex hortensis (Zhang et al., 2009) also enhanced drought and salt tolerance in cotton. Water deficit-inducible expression of cytokinin biosynthetic gene IPT (isopentenyltransferase) increased cotton drought tolerance (Kuppu et al., 2013), and overexpression of rice transcription factor SNAC1 enhanced drought and salt tolerance in cotton (Liu et al., 2014), partly because of welldeveloped root system of these transgenic plants. The vast majority of these studies was conducted under artificial conditions in the laboratory or controlled greenhouse. However, gaps still exist between the results in the laboratory and the application of the techniques to crops in the field (Cominelli et al., 2013). Only a few genes have been showed enhancing plant stress tolerance and increasing cotton yields in the field (He et al., 2005; Pasapula et al., 2011). Our previous studies have demonstrated that Arabidopsis ENHANCED DROUGHT TOLERANCE1/HOMEODOMAIN GLABROUS11 (AtEDT1/HDG11), which encodes a homeodomainleucine zipper (HD-ZIP) transcription factor, is a good candidate for improving plant drought and salt tolerance. Overexpression of this gene enhanced tolerance to drought stress in Arabidopsis and tobacco (Yu et al., 2008), rice (Yu et al., 2013), and sweet potato (Ruan et al., 2012). Ectopic overexpression of AtHDG11 in tall fescue improved its drought and salt stress tolerance (Cao et al., 2009). In this study, we generated AtHDG11 transgenic cotton and tested its drought and salt tolerance both in laboratory and in the field. Our results showed that overexpression of AtHDG11 in cotton not only significantly improved drought and salt tolerance, but also increased cotton yield in the field. Moreover, to test whether AtHDG11-conferred stress tolerance phenotypes could be recapitulated in tree plants, we transferred AtHDG11 into poplar and found that this gene also enhanced drought and salt tolerance in poplar. These results

demonstrate again that AtHDG11 is a promising candidate not only for crop improvement with drought and salt tolerance but also for woody plants.

Results Generation of the transgenic cotton plants that express AtHDG11 To create AtHDG11-overexpressing transgenic cotton, AtHDG11 cDNA was cloned into the construct pCB3000 and pCB3000T, in which the AtHDG11 cDNA is under the control of CaMV 35S promoter (p35S) and Arabidopsis Tublin2 promoter (pTUB2), respectively (Figure 1a). These two constructs were used to transform upland cotton (Gossypium hirsutum) cultivar R15 and WC. Transgenic cotton lines were generated via Agrobacteriummediated transformation. 32 3H (transformed with pCB3000) and 40 3TH (transformed with pCB3000T) independent transgenic lines were produced, respectively. The shoot of primary transformed T0 plants were grafted onto wild-type cotton stock to get T1 seeds. The hygromycin-resistant T2 plants were prescreened for drought resistance, and most of the transformants showed improved drought tolerance. Six of droughtresistant T3 homozygous lines were selected for further analysis by RT-PCR. Figure 1b revealed that the expression of AtHDG11 was detected for all the transgenic lines but not for R15 and WC wild-type controls.

Overexpression of AtHDG11 in cotton enhances drought tolerance in laboratory condition The AtHDG11-overexpressing cotton plants were grown under controlled irrigation conditions to test the effects of AtHDG11 overexpression on drought tolerance. For drought tolerance test at the seedling stage, four transgenic lines were grown in greenhouse without watering after germination. As shown in Figure 2a, no obvious difference between the transgenic and wild-type plants on day 16 was observed; however, as drought (a)

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Figure 1 Overexpression vectors and expression analysis of AtHDG11 in the transgenic cotton plants. (a) Schematic representation of the T-DNA region of AtHDG11-overexpressing vector. LB, left border; RB, right border; 35S polyA, cauliflower mosaic virus (CaMV) 35S polyA; NPT, hygromycin phosphotransferase gene; p35S, CaMV 35S promoter; pTUB2, Arabidopsis Tublin2 promoter; Tnos, 30 -termination signal of nopaline synthase. (b) AtHDG11 transcript levels in the transgenic lines, as revealed by RT-PCR analysis. GhHis3 was used as internal control.

ª 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd, Plant Biotechnology Journal, 1–13

Overexpression of AtHDG11 enhances drought and salt tolerance in cotton and polar 3

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Figure 2 AtHDG11 significantly enhances drought tolerance of the transgenic cotton in greenhouse. (a) Drought tolerance of the transgenic plants improved at seedling stage. Seeds were germinated in 16 9 24 cm pots with well-watered soil. No irrigation was applied during the whole growth period. a, 16 days after germination; b, 28 days after germination; c, 30 days after germination; d, 34 days after germination; e, drought for 34 days and then recovery for 2 days. Bar = 10 cm. (b) Survival rate of the plants after 2 days of recovery. Values are means  standard deviations (SD) of three replicates each containing 10 plants per line (***P < 0.001). (c) Root of the transgenic and wild-type cotton plants. Plants after 2 days of recovery were carefully poured out with the soil as a whole from the pots. Soil was washed off from the root carefully with water. Bar = 8 cm. (d) Shoot dry biomass of the plants after 2 days of recovery. Values are means  SD of 15 plants (*P < 0.05). (e–f) Root length and root dry weight of the plants after 2 days of recovery. Values are means  SD of 15 plants (*P < 0.05, **P < 0.01, ***P < 0.001). (g) Drought tolerance of the transgenic plants improved at reproductive stage. Plants were normally grown in pots with 5 kg soil each for 35 days, and then watering was withheld for 40 days.

stress went on day 28 and 30, the transgenic plants showed much delayed leaf-wilting symptom compared with wild type. After 34 days drought treatment and subsequent recovering for 2 days, most of the transgenic plants recovered with a survival ratio from 83.3% to 93.3%, whereas the wild-type plants only had survival ratio from 6.67% to 10% (Figure 2b). We also found the transgenic plants had slightly increased dry biomass under the drought conditions (Figure 2d). Additionally, the transgenic plants developed more enlarged root system with longer roots and significantly increased root dry biomass (Figure 2 c, e and f). This better-developed root system would benefit for the plant growth and drought tolerance. For drought tolerance assay at reproductive phase, 35-day-old well-watered plants were used for drought stress treatment by withholding watering for 40 days. As shown in Figure 2g, the wildtype plants showed an obvious drought-stressed phenotype with withered leaves, while all the six transgenic lines displayed a relative normal phenotype. These results demonstrate that AtHDG11 can significantly increase the drought tolerance of cotton plants likely at different developmental stages.

Improved drought tolerance, agronomic performance and cotton yield of the AtHDG11 transgenic cotton in the field Besides drought tolerance assays in the greenhouse, we also evaluated the drought tolerance of the AtHDG11-overexpressing cotton plants in the fields at two different locations. Figure 3a and b showed that two transgenic lines 3H-R15-119 and 3THWC-44 displayed apparent tolerance to drought stress compared with the controls in the fields in Yuncheng, Shanxi Province, China. Other field trials were carried out from April to September in Shihezi, Xinjiang Province of China where rainfall is scarce during the entire growing season. Two transgenic lines (3H-R15122 and 3TH-WC-44) were chosen for further detailed analysis in the field in 2013 in Shihezi. The field trial consisted of three replica plots of about 22.5 m2 each for each line. Figure 3c showed the R15 control and transgenic line 3H-R15-122 grown side by side in a drought stress trial. The transgenic plants exhibited significant growth advantages and improved drought tolerance compared with the wild-type plants.

ª 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd, Plant Biotechnology Journal, 1–13

10 Lin-Hui Yu et al. results suggest that AtHDG11 and GhHOX3 may at best play minor roles in drought stress response. However, altered expression pattern of the AtHDG11 confers novel functions of this gene in drought tolerance (Yu et al., 2008). We speculate that overexpression of AtHDG11 in cotton may promote cotton fibre elongation or overexpression of GhHOX3 in cotton may enhance drought tolerance, which await further investigation. Taken together, our study showed that overexpression of AtHDG11 in cotton and poplar enhances drought and salt tolerance of the plants. The transgenic plants improved multiple characteristics related to drought tolerance, including welldeveloped root system, enlarged stomatal size and reduced stomatal density, improved WUE, increased SOD and CAT activities and higher proline and soluble sugar contents. More importantly, the transgenic cotton had enhanced drought tolerance as well as improved agronomic traits and cotton yield in the field. Taken the previous reports together, most if not all of the observed phenotypes in Arabidopsis edt1 mutant could also be recapitulated in other species, including tobacco (Yu et al., 2008), rice (Yu et al., 2013), tall fescue (Cao et al., 2009), sweet potato (Ruan et al., 2012), cotton and tree plant poplar. These studies reveal that the dicot AtHDG11-conferred drought tolerance and other phenotypes probably are conserved in other plant species, implicating that changing the expression pattern of AtHDG11 may be one way that drought tolerance can evolve in nature and this gene could be widely used for crop improvement.

Experimental procedures

used as internal control. All the primers used are shown in Table S2.

Drought and salt tolerance assay For drought tolerance assay of cotton seedlings, the transgenic and wild-type seeds were separately germinated in well-watered soil in 16 9 24 cm pots. Watering was withheld during the whole growth period. For drought tolerance assay of cotton plants at reproductive stage, seeds were germinated in pots with 5 kg soil each. The plants were irrigated with 700 mL water per pot every 10 days for 35 days until the plants developed 7–8 leaves, and then watering was withheld. After another 40 days growth in the greenhouse without watering when the reproductive stage plants exhibited obvious drought stress phenotypes, photographs were taken. For drought tolerance assay of poplar, rooted plantlets were acclimatized in water at 25 °C for 5 days, then transferred to pots filled with well-watered perlite. Watering was then withheld for drought stress treatment for 10 days. Then, the plants were rewatered for recovery. For salt treatment of cotton seedlings, 10-day-old seedlings were grew in 0.6% NaCl solution for a few days. For salt stress of cotton at reproductive stage, 35-day-old plants were irrigated with 500 mL 2% NaCl solution per pot every 2–3 days for 60 days. For salt tolerance assay of poplar, 24-day-old rooted plantlets were transferred to pots filled with perlite after 5 days acclimatization in water and then irrigated with 0.6% NaCl solution every 2 days.

Field trials

Vector construction and plant transformation The full length of AtHDG11 cDNA was cloned into the binary vector pCB3000 and pCB3000T, which contains CaMV 35S promoter and Arabidopsis Tublin2 promoter, respectively. The overexpression vectors (Figure 1a) were then used for Agrobacterium-mediated cotton transformation as previously described (Li et al., 2002). The hypocotyl segments of upland cotton (Gossypium hirsutum) cultivar R15 and WC were used as explants for transformation. After plantlet regenerated, the plantlets were transferred to pots for further growth. The shoot of primary transformed T0 plants were grafted into mature wild-type cotton plants to get T1 seeds. For poplar transformation, a leaf disc transformation method as described by Jia et al. (2010) was used with the overexpression vector pCB2004-AtHDG11 (Yu et al., 2008). Leaves of Chinese white poplar (P. tomentosa Carr.) were used as explants. After adventitious buds induced, regenerated shoots of 2–3 cm length were transferred to rooting medium to generate roots. Rooted plantlets were then acclimatized in water at 25 °C under a 16-h light period for a few days before further studies.

RT-PCR and quantitative real-time PCR analysis Total RNA was extracted from the plant materials using a Trizol reagent (Invitrogen, Carlsbad, CA). 1 lg of total RNA from each sample was used for the reverse transcription reactions. For AtHDG11 expression level analyses, 0.5 lL of cDNA template was used for RT-PCR with gene-specific primers in Table S2. Cotton GhHis3 (AF024716) and poplar Histone H4 (XM_006383060.1) were amplified as internal controls. Quantitative real-time PCR (qRT-PCR) was performed as described previously (Yu et al., 2013). The expression levels of GhHOX3 and GhDREB2B (GQ849094) were examined using specific primers. GhHIS3 was

To evaluate drought tolerance of transgenic cotton in the field, the homozygous transgenic cotton lines and wild-type controls were grew in the fields under water proof shed in Yuncheng, Shanxi Province, China. The experiment was laid out in completely randomized block design with one replicate. Each plot comprised one row of transgenic plant and one row of wild-type control. Cotton seeds were germinated and irrigated as usual. One month after germination when seedlings were established, no irrigation was applied through the rest of the growing season. To evaluate drought tolerance and agronomic traits of transgenic cotton lines in the field, field trials were carried out from April to September in Shihezi, Xinjiang Province, China, in 2013. This field trial consists of three replica plots of about 22.5 m2 each for each line. The inter-row spacing was 30 cm and interplant spacing was 12.5 cm. Each plot had about 600 plants. One month after germination when seedlings were established, watering was withheld and plants grew under natural drought conditions during the rest of the growing season. 50 plants in each plot were randomly selected for agronomic traits and cotton yields analysis.

Measurement of proline, soluble sugars and MDA content Leaves of similar developmental stages from stress-treated plants or normal control plants were used for proline and soluble sugar contents measurement. Proline was assayed as described by Bates et al. (1973). Soluble sugars were determined by anthrone reagent (Dubois et al., 1956). MDA content was determinate by thiobarbituric acid (TBA) test as previously described (Cao et al., 2009).

Determination of SOD and CAT activity About 0.5 g leaves were ground with liquid nitrogen and extracted with 5 mL of 50 mmol phosphate buffer (pH 7.8),

ª 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd, Plant Biotechnology Journal, 1–13

Overexpression of AtHDG11 enhances drought and salt tolerance in cotton and polar 5

Reduced leaf stomatal density and increased water-use efficiency in AtHDG11-expressing cotton Similar to the Arabidopsis edt1 mutant (Yu et al., 2008), the stomatal size was increased significantly in the AtHDG11-expressing cotton plants (Figure 5 a–c). Consequently, the average stomatal density of the line 3H-R15-122 was 32.2% less than that

of the wild type (Figure 5d). The reduced stomatal density of the transgenic line was mainly due to the enlarged size of epidermal cells, which led to a significant reduction in cell density compared to the wild type (Figure 5e). More importantly, we found the AtHDG11-expressing cotton had higher water-use efficiency (WUE) (Figure 5f), which benefited from the reduced stomatal density (Figure 5d) and stomatal conductance (Figure 5g).

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Figure 4 Overexpression of AtHDG11 significantly enhances salt tolerance of cotton plants. (a) Salt tolerance of the transgenic plants was increased at seedling stage. 10-day-old seedlings were grew in 0.6% NaCl solution for 7 days. (b) Salt tolerance of the transgenic plants was increased at germination and seedling emergence stage. About 45 seeds per line were germinated for 5 days in soil irrigated with water or 1% NaCl solution. (c) Germination rate after 5 days germination. Values are means  SD of three replicates (***P < 0.001). (d) Salt tolerance of the transgenic plants was increased at reproductive stage. 35-day-old plants were irrigated with 500 mL 2% NaCl solution per pot every 2–3 days for 60 days Each pot has 5 kg soil.

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Figure 5 Reduced stomatal density, enlarged stomatal and epidermal cell size, increased WUE, reduced stomatal conductance in the AtHDG11 transgenic cotton. (a–b) Comparison of adaxial epidermal imprint images of the wild-type and the transgenic plant. A surface imprint was used as described in Experimental procedures. Bar = 100 lm. (c) Stomatal size of the wild-type and transgenic plant. One leaf from the relative same position was sample for each plant, and 10 plants were sampled for both the transgenic lines and wild type for surface imprint. The stomata were counted and measured under HIROX’s KH-7700 microscope. Values are means  SD of 50 stomata (***P < 0.001). (d–e) Stomatal density (c) and epidermal cell number of the wild-type and transgenic plants. Values are means  SD of 50 images (**P < 0.01). (f–g) WUE (F) and stomatal conductance (G) of the wild-type and transgenic plants. Three measurements were applied for each plant using a portable photosynthesis system, and 10 plants were used for each line. Values are means  SD (**P < 0.01). ª 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd, Plant Biotechnology Journal, 1–13

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Soluble sugar content, proline content and activities of ROS-scavenging enzymes significantly increased in the transgenic cotton under stress conditions Malondialdehyde (MDA), a product of lipid peroxidation and an index of toxic oxygen species generation, was found accumulated more in the wild-type cotton compared with the transgenic plants after drought and salt stress treatment (Figure 6a and f), indicating the wild-type plants suffering more serious oxidative damage. During the drought and salt stress treatment, proline and soluble sugar contents were increased significantly in both the transgenic and wild-type plants, but with more significant increases in the transgenic plants compared with the wild-type controls (Figure 6 b, c, g, h). Moreover, activities of reactive oxygen species (ROS)-scavenging enzymes, such as superoxide dismutase (SOD) and catalase (CAT), were significantly increased

in the AtHDG11 transgenic plants compared with wild-type plants after stress treatments (Figure 6 d, e, i, j). Therefore, our data indicate that the transgenic plants are better protected from oxidative damage during the drought and salt stress.

Overexpression of AtHDG11 in poplar improves biomass and root development To test whether AtHDG11 confers stress tolerance in tree species, we developed AtHDG11-overexpressing poplar plants with plant binary vector pCB2004-AtHDG11 (Figure 7a) through Agrobacterium-mediated transformation. Regenerated shoots of 2–3 cm length were transferred to rooting medium to generate roots. The putative transgenic plants were confirmed by RT-PCR. Results showed that line H1 to line H7 had detectable expression of AtHDG11 (Figure 7b). As showed in Figure 7c, the AtHDG11overexpressing poplar plants were bigger in size than the wild-

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Figure 6 MDA contents, proline contents, soluble sugar contents, SOD and CAT activities of transgenic and wild-type plants with or without stress treatments. (a–d) MDA contents (a), proline contents (b), soluble sugar contents (c), SOD activities (d), CAT activities (e) in the leaves of 20-day-old plants with or without 10 days drought treatments. Values are means  SD of three replicates. (*P < 0.05, **P < 0.01, ***P < 0.001). (f–j) MDA contents (f), proline contents (g), soluble sugar contents (h), SOD activities (i), CAT activities (j) in the leaves of 15-day-old plants grown under normal conditions or irrigated with 2% NaCl solution for 10 days. Values are means  SD of three replicates. (*P < 0.05, **P < 0.01, ***P < 0.001). ª 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd, Plant Biotechnology Journal, 1–13

Overexpression of AtHDG11 enhances drought and salt tolerance in cotton and polar 7 (a) pCB2004-AtHDG11

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CK Figure 7 AtHDG11 conferred increased biomass and more extensive root system in poplar. (a) Schematic representation of the T-DNA region of pCB2004-AtHDG11 vector. (b) Expression level analyses of AtHDG11 in the transgenic plants using RT-PCR. Poplar Histone H4 gene was used as internal control. Water was used as negative control. H1 to H7 refers to the transgenic line. (c) Phenotypes of 15-day-old rooted transgenic and nontransgenic control (CK) plantlets. Regenerated shoots of 2–3 cm length were transferred to rooting medium for 15 days to generate roots. (d) Root phenotypes of 15-day-old rooted transgenic and nontransgenic control plantlets. (e–f) Root length and root number of the 15-day-old rooted plantlets. Values are means  SD of 15 plants (*P < 0.05, **P < 0.01).

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Figure 8 Drought and salt tolerance were increased significantly in the transgenic poplar. (a–b) Drought tolerance assay (a) and salt tolerance assay (b) of the transgenic and nontransgenic poplar plants. Rooted plantlets were acclimatized in water at 25 °C for 5 days and then used for drought and salt tolerance assay as described in Experimental procedures.

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type controls after 15 days growth on the rooting medium. The transgenic plant also had well-developed root system (Figure 7c and d) with longer primary root and more lateral roots (Figure 7e and f). These results are consistent with the data obtained in edt1 mutant (Yu et al., 2008) and AtHDG11 transgenic rice (Yu et al., 2013).

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Improved drought and salt tolerance in AtHDG11overexpressing poplar Regenerated shoots of 2–3 cm length were grown on rooting medium for 12 days. The rooted plantlets were acclimatized in water at 25 °C for 5 days and then transferred to pots filled with

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Figure 9 Reduced stomatal density, enlarged stomatal and epidermal cell size in the AtHDG11 transgenic cotton. (a–d) Images of the lower epidermis (a and b) and upper epidermis (c and d) of the control and a representative transgenic plant. Bar = 50 lm. (e–f) Stomatal density (e) and stomatal size (f) of the control and transgenic plant. Ten microscopic sights were observed for each plant. Five plants were used for each line. Values are means  SD. Bar = 50 lm. (g–h) Upper epidermal cell density (g) and cell size (h) of the control and transgenic plant. Ten microscopic sights were observed for each plant. Five plants were used for each line. Values are means  SD (**P < 0.01, ***P < 0.001). Bar = 50 lm.

well-watered perlite. As shown in Figure 8a, after watering was withheld for 1 day, the transgenic plants and the wild-type control showed no obvious growth difference. However, after 5 days drought treatment, the wild-type plants started to wither, but the transgenic plants still grew normally. After 10 days withholding watering, the wild-type plants were all died,even after 10 days recovery, whereas all the transgenic plants survived. For salt stress treatment, 24-day-old rooted plantlets were acclimatized in water for 5 days and then transferred to perlitefilled pots. The plants were irrigated with 0.6% NaCl solution every 3 days. As showed in Figure 8b, there was no difference between the wild-type and transgenic plants on day 1 during the salt treatment. On day 8, old leaves of the wild-type plants started to die, indicating severe salt injury in these plants. However, no obvious salt toxic effects were observed in the transgenic plants. After 15 days NaCl treatment, all the wild-type plants died, while all the transgenic plants still alive. All these results demonstrate that AtHDG11 overexpression in poplar can enhance drought and salt tolerance of the plants.

The size of stomata and epidermal cells is increased in AtHDG11-overexpressing poplar To investigate whether AtHDG11 increases cell size in poplar as in cotton, we examine the size of leaf stomata in the abaxial epidermis and cells in upper epidermis, respectively. As expected, AtHDG11-expressing poplar plants had bigger stomata with decreased stomatal density in the abaxial epidermis (Figure 9a and b). Statistical analysis showed that stomatal density reduced by 38.9% in the transgenic plants compared with the wild-type plants (Figure 9e). Meanwhile, stomatal length and width of the transgenic plant were increased by 27.3% and 27.6%, respectively (Figure 9f). In the upper leaf epidermis of the transgenic plants, cell enlarged significantly as showed in Figure 9c and d. As a result, upper epidermal cell density decreased by 39.3% in the AtHDG11 overexpression in poplar (Figure 9g), with the cell length and width increasing by 38.7% and 35.1%, respectively (Figure 9h).

Discussion Environmental stresses such as drought and salinity greatly affect crop growth and productivity. As such, it is of the utmost importance and urgency to breed crop cultivars with improved drought and salt tolerance while increasing the yield. Transgenic approach has been proven as a faster track towards crop improvement. In our previous studies, we demonstrated that transgenic plants overexpressing AtHDG11 showed great promise in improving agricultural productivity under drought and salt conditions. AtHDG11 conferred drought tolerance and increased biomass in Arabidopsis edt1 mutant as well as AtHDG11overexpressing wild-type Arabidopsis and tobacco. The enhanced drought tolerance was contributed by developmental alterations in stomatal density and root system, improved tolerance to oxidative stress, increased ABA content and improved photosynthesis and WUE (Yu et al., 2008). AtHDG11 was also reported to improve drought tolerance and enhance photosynthesis in sweet potato (Ruan et al., 2012). When overexpressed in monocots tall fescue and rice, AtHDG11 also conferred drought-tolerant phenotypes (Cao et al., 2009; Yu et al., 2013). Moreover, salt tolerance was also enhanced in the transgenic tall fescue (Cao et al., 2009). Overexpression of AtHDG11 in rice increased biomass and grain yield under both normal and drought conditions in the field (Yu et al., 2013). In this study, we expressed AtHDG11 in cotton and woody plant poplar. The transgenic plants also had significantly enhanced drought and salt tolerance, well-developed root system, reduced stomatal density and increased stomatal size and leaf epidermal cell size. More importantly, AtHDG11 conferred drought tolerance, better agronomic performance and increased cotton yield in cotton plants under both normal and drought conditions in the field. Similar to the AtHDG11 overexpression in Arabidopsis and rice, the drought tolerance of AtHDG11-overexpressing cotton and poplar is contributed from a collection of beneficial changes at the morphological and physiological levels. At first, the transgenic

ª 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd, Plant Biotechnology Journal, 1–13

Overexpression of AtHDG11 enhances drought and salt tolerance in cotton and polar 9 plants had well-developed root system (Figs 2c and 7 c–f), which could maximize water and nutrients uptake to deal with drought stress (Kavar et al., 2008; Lynch, 2007; Yu et al., 2013). One possible reason for the extensive root system of the AtHDG11overexpressing plant is that AtHDG11 can directly up-regulate several cell-wall-loosening protein genes, which is correlated with altered root system architecture (Xu et al., 2014). Additionally, stomtal density and conductance decreased in the transgenic cotton and poplar, resulting to better water conservation and higher WUE (Figs 5 and 9). As revealed in edt1 mutant, upregulated ERECTA and down-regulated KAT1 might affect the stomatal development and behaviour, thus improved the WUE (Yu et al., 2008). Moreover, the transgenic plants were better protected from oxidative and osmotic damages by increasing soluble sugar, proline, SOD and CAT. ROS was overproduced under various environmental stress, causing damages in membrane and biomolecules (Foyer and Noctor, 2005; Mittler, 2002). The levels of MDA, an convenient index of membrane lipid peroxidation, is therefore an indicator of ROS destructive effects under stress conditions (Sharma et al., 2012). The significantly reduced MDA levels detected in the transgenic cotton, under both drought and salt conditions, indicate that they are better protected from oxidative damages. Increased antioxidant enzyme activities can improve plant drought and salt tolerance (Gao et al., 2003; Xue et al., 2009; Young-Pyo et al., 2007; Zhang et al., 2014). In our study, SOD and CAT activities were much higher in the AtHDG11-overexpressing cotton, therefore protecting the plants from oxidative damage by enhancing ROSscavenging capability. Proline and soluble sugar, two common compatible osmolytes in higher plants, which playing important roles in the oxidative stress responses of plants (Couee et al., 2006; Hare et al., 1998), were accumulated significantly higher in the transgenic cotton plants after drought and salt stress. This is considered to be another important factor in reducing oxidative damage in the transgenic plants. Salinity stress was usually accompanied with water deficit, especially in the dry regions, where irrigation management was poor and evaporation was high (Basal, 2010). Although drought stress and salinity stress trigger plant responses individually, they are often studied as a whole because they regulate many common genes and trigger many common reactions in plants. Both stresses lead to cellular dehydration, resulting in osmotic stress, ion toxicity and oxidative burst (Golldack et al., 2014; Mittler, 2002; Zhu, 2002). Therefore, some improvements in drought tolerance may also make the plant more resistant to salt stress (Han et al., 2013; Hu et al., 2006; Liu et al., 2014; Xiong et al., 2014). In this study, we found drought-tolerant AtHDG11 transgenic cotton and poplar also had significantly improved salt tolerance. The transgenic cotton showed enhanced salt tolerance from young seedling stage to reproductive stage. More importantly, salt tolerance at germination and seedling emergence stage, which is the most sensitive stage to salt stress (Ahmad et al., 2002), also increased significantly in the transgenic cotton plants. This is very important for actual cotton cultivation because cotton production is greatly threaten by low seed germination and weak seedling emergence which result in death of plants at early stage under salinity conditions. However, once the cotton plant tides over the most difficult phase at germination and seedling emergence stage in saline soil, its salt tolerance would increased and it could survive on the saline land. One general mechanism for the improved salt tolerance of the transgenic cotton is the transgenic plants had higher SOD and CAT activities

to protect cells from oxidative damage and increased accumulation of osmolytes such as proline and soluble sugar to counter the osmotic stress caused by high salinity. Another possible reason of the salt tolerance of the AtHDG11-overexpressing plants is the transgenic plants had better ability to maintain Na+/K+ homeostasis under salt stress conditions. Previous study had reported that the AtHDG11 transgenic tall fescue can maintain a relatively stable Na+/K+ ratio in both the leaves and roots after salt treatment, thus enhancing the salt tolerance of the transgenic plants (Cao et al., 2009). During the past two decades, many genes involved in biotic and abiotic stresses were identified under artificial and environment-controlled conditions and exemplified by the large number of articles, sequences and patent applications (Vain, 2007). However, compared with the selection systems by the environment in the field, this indoor approach is not so reliable in actual agricultural application because great gap between the results in the laboratory and its actual outdoor application in crops still existed (Mittler, 2006). Many identified genes were proved to improve biotic or abiotic stress tolerance of the crops in the laboratory (Datta et al., 2012; Liu et al., 2014; Lv et al., 2007; Rong et al., 2014; Yang et al., 2012; Yue et al., 2012), with very fewer tested in the field with disappointing results (Rommens, 2010; Tian et al., 2003; Yamaguchi and Blumwald, 2005). Yield is usually the ultimate goal of crop breeding, and crop improvement should be achieved without growth and yield penalty (Cattivelli et al., 2008). However, constitutive expression of many stressrelated genes often results in abnormal development and thus a loss in productivity (Heidel et al., 2004; Nakashima et al., 2007; Priyanka et al., 2010). Our previous study showed that overexpression of AtHDG11 in Arabidopsis and rice had no apparent negative effects on plant development; in contrast, it conferred increased biomass and higher grain yield under both normal and drought conditions (Yu et al., 2008, 2013). In this research, we found similar results in the AtHDG11 transgenic cotton and poplar. The transgenic cotton had obvious growth advantage over the wild-type plants with increased plant height, fruit branch number and boll number per plant in the fields, thus increased seed cotton yield under both normal and drought conditions (Table 1 and S1). However, seed cotton yield of 3H-R15-122 and 3TH-WC-44 increased by 24.5% and 35% under drought conditions compared with 19.6% and 18.1% under normal drought conditions, respectively. These results revealed that overexpression of AtHDG11 in cotton not only improves its drought tolerance, but also benefits for the yield increase under both normal and drought stress conditions (Table 1). Under drought conditions, the relative cotton yield of the transgenic lines increased more sharply compared with that under normal conditions. Apparently, the transgene-conferred tolerance to drought benefits for the cotton growth and yield increase. All these data indicate that AtHDG11 is a good candidate for crop drought and salt tolerance improvement while maintaining or increasing crop yield. The HD-ZIP transcription factor, AtHDG11, was reported to function in trichome morphogenesis (Nakamura et al., 2006). Its homologue in cotton, GhHOX3, plays a key role in controlling cotton fibre elongation (Shan et al., 2014). The transcript of AtHDG11 was not detectable in response to osmatic stress (Yu et al., 2008). Moreover, we found that the expression of GhHOX3 was reduced significantly after 1-h drought stress in the air, and then the expression level remained relatively stable during the drought treatment for 1.5–5.5 h (Figure S3). These

ª 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd, Plant Biotechnology Journal, 1–13

10 Lin-Hui Yu et al. results suggest that AtHDG11 and GhHOX3 may at best play minor roles in drought stress response. However, altered expression pattern of the AtHDG11 confers novel functions of this gene in drought tolerance (Yu et al., 2008). We speculate that overexpression of AtHDG11 in cotton may promote cotton fibre elongation or overexpression of GhHOX3 in cotton may enhance drought tolerance, which await further investigation. Taken together, our study showed that overexpression of AtHDG11 in cotton and poplar enhances drought and salt tolerance of the plants. The transgenic plants improved multiple characteristics related to drought tolerance, including welldeveloped root system, enlarged stomatal size and reduced stomatal density, improved WUE, increased SOD and CAT activities and higher proline and soluble sugar contents. More importantly, the transgenic cotton had enhanced drought tolerance as well as improved agronomic traits and cotton yield in the field. Taken the previous reports together, most if not all of the observed phenotypes in Arabidopsis edt1 mutant could also be recapitulated in other species, including tobacco (Yu et al., 2008), rice (Yu et al., 2013), tall fescue (Cao et al., 2009), sweet potato (Ruan et al., 2012), cotton and tree plant poplar. These studies reveal that the dicot AtHDG11-conferred drought tolerance and other phenotypes probably are conserved in other plant species, implicating that changing the expression pattern of AtHDG11 may be one way that drought tolerance can evolve in nature and this gene could be widely used for crop improvement.

Experimental procedures

used as internal control. All the primers used are shown in Table S2.

Drought and salt tolerance assay For drought tolerance assay of cotton seedlings, the transgenic and wild-type seeds were separately germinated in well-watered soil in 16 9 24 cm pots. Watering was withheld during the whole growth period. For drought tolerance assay of cotton plants at reproductive stage, seeds were germinated in pots with 5 kg soil each. The plants were irrigated with 700 mL water per pot every 10 days for 35 days until the plants developed 7–8 leaves, and then watering was withheld. After another 40 days growth in the greenhouse without watering when the reproductive stage plants exhibited obvious drought stress phenotypes, photographs were taken. For drought tolerance assay of poplar, rooted plantlets were acclimatized in water at 25 °C for 5 days, then transferred to pots filled with well-watered perlite. Watering was then withheld for drought stress treatment for 10 days. Then, the plants were rewatered for recovery. For salt treatment of cotton seedlings, 10-day-old seedlings were grew in 0.6% NaCl solution for a few days. For salt stress of cotton at reproductive stage, 35-day-old plants were irrigated with 500 mL 2% NaCl solution per pot every 2–3 days for 60 days. For salt tolerance assay of poplar, 24-day-old rooted plantlets were transferred to pots filled with perlite after 5 days acclimatization in water and then irrigated with 0.6% NaCl solution every 2 days.

Field trials

Vector construction and plant transformation The full length of AtHDG11 cDNA was cloned into the binary vector pCB3000 and pCB3000T, which contains CaMV 35S promoter and Arabidopsis Tublin2 promoter, respectively. The overexpression vectors (Figure 1a) were then used for Agrobacterium-mediated cotton transformation as previously described (Li et al., 2002). The hypocotyl segments of upland cotton (Gossypium hirsutum) cultivar R15 and WC were used as explants for transformation. After plantlet regenerated, the plantlets were transferred to pots for further growth. The shoot of primary transformed T0 plants were grafted into mature wild-type cotton plants to get T1 seeds. For poplar transformation, a leaf disc transformation method as described by Jia et al. (2010) was used with the overexpression vector pCB2004-AtHDG11 (Yu et al., 2008). Leaves of Chinese white poplar (P. tomentosa Carr.) were used as explants. After adventitious buds induced, regenerated shoots of 2–3 cm length were transferred to rooting medium to generate roots. Rooted plantlets were then acclimatized in water at 25 °C under a 16-h light period for a few days before further studies.

RT-PCR and quantitative real-time PCR analysis Total RNA was extracted from the plant materials using a Trizol reagent (Invitrogen, Carlsbad, CA). 1 lg of total RNA from each sample was used for the reverse transcription reactions. For AtHDG11 expression level analyses, 0.5 lL of cDNA template was used for RT-PCR with gene-specific primers in Table S2. Cotton GhHis3 (AF024716) and poplar Histone H4 (XM_006383060.1) were amplified as internal controls. Quantitative real-time PCR (qRT-PCR) was performed as described previously (Yu et al., 2013). The expression levels of GhHOX3 and GhDREB2B (GQ849094) were examined using specific primers. GhHIS3 was

To evaluate drought tolerance of transgenic cotton in the field, the homozygous transgenic cotton lines and wild-type controls were grew in the fields under water proof shed in Yuncheng, Shanxi Province, China. The experiment was laid out in completely randomized block design with one replicate. Each plot comprised one row of transgenic plant and one row of wild-type control. Cotton seeds were germinated and irrigated as usual. One month after germination when seedlings were established, no irrigation was applied through the rest of the growing season. To evaluate drought tolerance and agronomic traits of transgenic cotton lines in the field, field trials were carried out from April to September in Shihezi, Xinjiang Province, China, in 2013. This field trial consists of three replica plots of about 22.5 m2 each for each line. The inter-row spacing was 30 cm and interplant spacing was 12.5 cm. Each plot had about 600 plants. One month after germination when seedlings were established, watering was withheld and plants grew under natural drought conditions during the rest of the growing season. 50 plants in each plot were randomly selected for agronomic traits and cotton yields analysis.

Measurement of proline, soluble sugars and MDA content Leaves of similar developmental stages from stress-treated plants or normal control plants were used for proline and soluble sugar contents measurement. Proline was assayed as described by Bates et al. (1973). Soluble sugars were determined by anthrone reagent (Dubois et al., 1956). MDA content was determinate by thiobarbituric acid (TBA) test as previously described (Cao et al., 2009).

Determination of SOD and CAT activity About 0.5 g leaves were ground with liquid nitrogen and extracted with 5 mL of 50 mmol phosphate buffer (pH 7.8),

ª 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd, Plant Biotechnology Journal, 1–13

Overexpression of AtHDG11 enhances drought and salt tolerance in cotton and polar 11 the extract was centrifuged at 12 000 g for 10 min, and the supernatant was used for SOD and CAT activities measurement as previously described (Ruan et al., 2012). Total protein content was measured using the Bradford protein assay kit (Sangon Biotech, Shanghai, China).

Determination of stomatal size and density Leaves of the same age from the same relative position were sampled from 40-day-old cotton plants or 15-day-old rooted poplar plantlets grown under the same condition. A leaf surface imprint method was used (Yu et al., 2008). The imprint on the glass slide was observed under HIROX’s KH-7700 digital microscope. Stomata were counted and measured using the software tools of the digital microscope.

Measurement of transpiration rate and WUE Transpiration rate and WUE were measured using a portable photosynthesis system (LI-COR LI-6400XT) as previously described (Yu et al., 2013). 40-day-old cotton plants grown under the same conditions were used for measurements. All measurements were taken at a constant air flow rate of 500 lmol/s. CO2 concentration was 400 lmol/mol, and temperature was 26  2 °C, and photosynthetic photon flux density was 1200 lmol (photon)/ m2 per s.

Statistical analysis Statistically significant differences (P < 0.05 or P < 0.01 or P < 0.001) were computed based on the Student’s t-tests. Data are the means  SD of three independent replicates.

Acknowledgements This work was supported by the Ministry of Science and Technology of China (Grant Nos. 2014ZX08005, 2012CB 114304), the National Nature Science Foundation of China (Grant No. 30830075) and the Chinese Academy of Science (Grant No. KSCX3–YW–N–007).

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Supporting information Additional Supporting information may be found in the online version of this article:

ª 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd, Plant Biotechnology Journal, 1–13

Plant Biotechnology Journal (2015), pp. 1–13

doi: 10.1111/pbi.12358

Arabidopsis EDT1/HDG11 improves drought and salt tolerance in cotton and poplar and increases cotton yield in the field Lin-Hui Yu1,†, Shen-Jie Wu2,†, Yi-Shu Peng3,†, Rui-Na Liu4,†, Xi Chen1,†, Ping Zhao1, Ping Xu1, Jian-Bo Zhu4, Gai-Li Jiao2, Yan Pei3 and Cheng-Bin Xiang1,* 1

School of Life Sciences, University of Science and Technology of China, Hefei, Anhui Province, China

2

Cotton Research Institute, Shanxi Academy of Agricultural Sciences, Yuncheng, Shanxi Province, China

3

Biotechnology Research Center, Southwest University, Chongqing, China

4

College of Life Sciences, Shihezi University, Shihezi, Xinjiang Province, China

Received 26 August 2014; revised 25 November 2014 and 29 January 2015; accepted 16 February 2015. *Correspondence (Tel 86-55163600429; fax 86-5513601443; emails [email protected]; [email protected]) † These authors are equally contributed.

Keywords: AtHDG11, drought stress, salt stress, transgenic cotton, transgenic poplar, cotton yield.

Summary Drought and salinity are two major environmental factors limiting crop production worldwide. Improvement of drought and salt tolerance of crops with transgenic approach is an effective strategy to meet the demand of the ever-growing world population. Arabidopsis ENHANCED DROUGHT TOLERANCE1/HOMEODOMAIN GLABROUS11 (AtEDT1/HDG11), a homeodomainSTART transcription factor, has been demonstrated to significantly improve drought tolerance in Arabidopsis, tobacco, tall fescue and rice. Here we report that AtHDG11 also confers drought and salt tolerance in upland cotton (Gossypium hirsutum) and woody plant poplar (Populus tomentosa Carr.). Our results showed that both the transgenic cotton and poplar exhibited significantly enhanced tolerance to drought and salt stress with well-developed root system. In the leaves of the transgenic cotton plants, proline content, soluble sugar content and activities of reactive oxygen species-scavenging enzymes were significantly increased after drought and salt stress compared with wild type. Leaf stomatal density was significantly reduced, whereas stomatal and leaf epidermal cell size were significantly increased in both the transgenic cotton and poplar plants. More importantly, the transgenic cotton showed significantly improved drought tolerance and better agronomic performance with higher cotton yield in the field both under normal and drought conditions. These results demonstrate that AtHDG11 is not only a promising candidate for crops improvement but also for woody plants.

Introduction A tremendous challenge that we face today is to satisfy the growing demand of the world population for food and clothing, which is compounded with the dramatic losing arable land because of increasingly severe soil destruction by pollution and abiotic stresses. Among these abiotic stresses, drought and salinity are the major limiting factors in agricultural productivity, affecting more than 10% of arable land and crop yield, on average, of more than 50% for most major crop plants (Bartels and Sunkar, 2005). Global climate change will likely make the situation more serious in the near future (Ahuja et al., 2010). Therefore, it is imperative for researchers to develop strategies and technologies to make crops more productive with enhanced stress tolerance. Although some success in improving abiotic stress tolerance has been achieved so far using traditional breeding (Ashraf, 2010; Varshney et al., 2011), this procedure is time-consuming, costand labour-intensive. Genetic engineering has opened up new possibilities in creating stress tolerant crops in a shorter time with

the rapid progress made in plant genomics and biotechnology research. In fact, there have been many efforts to improve drought and salinity tolerance in plants by genetic engineering (Bartels and Sunkar, 2005; Borsani et al., 2003). Numerous genes responsive to drought and salinity stress have been identified and used as candidate genes in genetic engineering, such as genes encoding metabolites or osmoprotectants (Kreps et al., 2002), genes related to ion homeostasis and hormones (Alvarez et al., 2008; Apse et al., 1999; Yang et al., 2001; Yue et al., 2012), genes in signal transduction and transcription factors (Liu et al., 2014; Umezawa et al., 2006; Yang et al., 2010). However, in most cases, the genetic modifications were carried out in the model plant Arabidopsis, only a small proportion were transferred into crop plants (Peleg et al., 2011). Cotton, which is grown commercially in the tropical and temperate regions of more than 50 countries, is the world’s most important textile fibre crop and a significant oilseed crop (Chen et al., 2007). Cotton is estimated as one of the major consumers of agricultural water among all crops, and about 53% of the global cotton field is irrigated, producing 73% of the global

Please cite this article as: Yu, L.-H., Wu, S.-J., Peng, Y.-S., Liu, R.-N., Chen, X., Zhao, P., Xu, P., Zhu, J.-B., Jiao, G.-L., Pei, Y. and Xiang, C.-B. (2015) Arabidopsis EDT1/HDG11 improves drought and salt tolerance in cotton and poplar and increases cotton yield in the field. Plant Biotechnol. J., doi: 10.1111/pbi.12358

ª 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd

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