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... Division, Samuel Roberts Noble Foundation, Ardmore, Oklahoma 73401, U.S.A.; and yInstitute ... lar wax production in transgenic alfalfa (Medicago sativa L.).

Int. J. Plant Sci. 170(8):969–978. 2009. Ó 2009 by The University of Chicago. All rights reserved. 1058-5893/2009/17008-0001$15.00 DOI: 10.1086/600138

PHYSIOLOGICAL CHARACTERIZATION OF TRANSGENIC ALFALFA (MEDICAGO SATIVA) PLANTS FOR IMPROVED DROUGHT TOLERANCE Qingzhen Jiang,* Ji-Yi Zhang,* Xiulin Guo,*,y Maria J. Monteros,* and Zeng-Yu Wang1 ,* *Forage Improvement Division, Samuel Roberts Noble Foundation, Ardmore, Oklahoma 73401, U.S.A.; and yInstitute of Genetics and Physiology, Hebei Academy of Agricultural and Forestry Science, Shijiazhuang, Hebei 050051, China

WXP1 is an ethylene-responsive element-binding transcription factor that has been shown to lead to improved drought tolerance and increased wax accumulation when overexpressed in transgenic alfalfa under the control of the CaMV35S promoter. In this study, alfalfa was transformed with the WXP1 gene, driven by the epidermisspecific promoter CER6. The transgenic CER6::WXP1 alfalfa plants and a previously tested CaMV35S::WXP1 line were subjected to water stress for 3 d and then rewatered. Gas exchange, chlorophyll fluorescence, relative water content, and water potential were measured. Compared with the controls, the transgenic lines showed higher net photosynthetic rate, transpiration rate, and stomatal conductance and higher efficiency of photosystem II, quantum yield of photosystem II, coefficient of photochemical quenching, and apparent electron transport rate under water stress and after rewatering. The transgenic lines also showed higher relative water content and leaf water potential under water deficit conditions. The growth and development of CER6::WXP1 plants were normal, and the plants showed enhanced drought tolerance. Gas exchange and chlorophyll fluorescence data provided consistent evidence that the WXP1 transgenic lines experienced less damage under water stress conditions. This study revealed that increased wax accumulation did not have a negative impact on photosynthesis. The physiological analyses indicated that WXP1 is involved not only in wax biosynthesis but also in other physiological responses associated with enhanced drought tolerance that warrant further investigation. Keywords: alfalfa, chlorophyll fluorescence, drought tolerance, gas exchange, transgenic plant, water potential.

Introduction

WXP1 gene under the CaMV35S promoter activated cuticular wax production in transgenic alfalfa (Medicago sativa L.) and Arabidopsis and resulted in enhanced drought tolerance (Zhang et al. 2005, 2007). Taken together with the effects of the overexpression of another wax-related transcription factor gene, WIN1/SHN1, in Arabidopsis (Aharoni et al. 2004; Broun et al. 2004), the results confirmed the hypothesis that cuticular waxes of plants serve as a barrier to avoid water loss and play an important role in protecting aerial organs from damage caused by drought stress. However, in transgenic alfalfa expressing WXP1 under the CaMV35S promoter, increased wax accumulation was accompanied by moderately delayed growth (Zhang et al. 2005). Overexpression of transcription factors conferring drought tolerance can result in retarded plant growth (Kasuga et al. 1999; Broun et al. 2004). The possible negative impact of constitutive WXP1 expression on plant growth may be overcome by using epidermis-specific promoters such as that of the CER6 gene. CER6 is a condensing enzyme involved in Arabidopsis surface wax production (Millar et al. 1999; Fiebig et al. 2000; Hooker et al. 2002). The CER6 promoter from Arabidopsis was shown to direct high levels of gene expression in the epidermal cells, and the specificity of this promoter was retained in other species such as tobacco (Hooker et al. 2002). Under drought stress, many physiological and biochemical processes are affected, including the negative regulation of CO2 assimilation through stomatal closure and/or nonstomatal factors, including damage to the photosynthetic appara-

Drought stress is one of the major limiting factors for crop production in many areas of the world. Because of the longterm effects of global warming, drought episodes will likely become more frequent in the future (Rivero et al. 2007). The adverse effects of drought stress on plant growth and production are obvious: retarded growth and lower yield (BhatnagarMathur et al. 2008). Mechanisms of plant response to drought stress are complicated, involving regulations at both molecular and physiological levels (Bray 1993; Shinozaki and Yamaguchi-Shinozaki 2000; Shinozaki et al. 2003). Many genes are involved, and their products may be classified into two categories: those that are directly involved in processes that protect cells from dehydration and those that regulate gene expression in the stress response, such as transcription factors (Ingram and Bartel 1996; Shinozaki and YamaguchiShinozaki 1997; Kasuga et al. 1999). The use of single transcription factor genes to improve quantitative traits may be ideal because they can simultaneously regulate many genes involved in stress response (Jaglo-Ottosen et al. 1998; Zhang 2003; Kasuga et al. 2004). WXP1 is an ethylene-responsive element-binding family transcription factor identified in the model legume Medicago truncatula (Zhang et al. 2005, 2007). Overexpression of the 1

Author for correspondence; e-mail: [email protected]

Manuscript received March 2009; revised manuscript received April 2009.

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tus. Stomatal responses are typically evaluated by gas exchange parameters (Jiang et al. 2006), while measurements of chlorophyll fluorescence have been used to evaluate the integrity of photosystem II (PSII) on exposure to stress (Shabala 2002). In the previous investigations of wax-related transcription factor genes (Aharoni et al. 2004; Broun et al. 2004; Zhang et al. 2005, 2007), stress tolerance of the transgenic plants overexpressing them was detected only by observation of the phenotypes, while no physiological evidence and quantitative data were provided. Physiological analyses would help not only to elucidate the mechanism of drought tolerance but also to address important issues such as the effect of increased surface wax accumulation on photosynthesis. Alfalfa is the fourth most widely grown crop in the United States, behind only corn, wheat, and soybeans. It is a highyielding and high-quality perennial species that requires little or no nitrogen fertilizer because of its ability to fix nitrogen. The greatest constraint on alfalfa productivity is insufficient water in many areas. For alfalfa, like many other crops, drought tolerance is an important target trait for its improvement. In this study, a WXP1 binary vector driven by the CER6 promoter was constructed and introduced into alfalfa. The objectives were to investigate drought responses of the transgenics and the associated physiological changes caused by the transgene. Gas exchange and chlorophyll fluorescence parameters were used to characterize drought tolerance of the transgenic and control lines after ceasing watering for 3 d and after rewatering of the plants.

Material and Methods Construction of the CER6::WXP1 Vector and Generation of Transgenic Alfalfa (Medicago sativa L.) Plants The CER6 promoter was obtained by polymerase chain reaction (PCR) amplification of Arabidopsis thaliana genomic DNA, using the following primers: 59-AAGCTTCGATATCGGTTGTTG-39 and 59-CCATGGTCGGAGAGTTTTAATG-39. To replace the CaMV35S promoter in the vector pC35S::WXP1 (Zhang et al. 2005), the amplified CER6 promoter sequences were digested with Hind III and Nco I and ligated with Hind III/Nco I–digested pC35S::WXP1. The resulting binary vector, pCER6::WXP1, was transferred into Agrobacterium tumefaciens strain AGL1, using the freezing/ heat shock method. Transgenic alfalfa plants were obtained by Agrobacterium-mediated transformation of the genotype Regen SY-4D, using the protocol described by Samac and Austin-Phillips (2006). Vegetatively propagated plants from the original Regen SY-4D clone were used as a wild-type control (WT). Because both pC35S::WXP1 and pCER6::WXP1 were derived from the binary vector pCAMBIA3301, alfalfa lines transformed with the original pCAMBIA3301 were used as an empty-vector control (CK).

PCR and Reverse-Transcription PCR (RT-PCR) Analyses of Transgenic Plants Plants regenerated after transformation and phosphinothricin selection were subjected to PCR screening. The forward primer (59-TTGCTCCCATCACTTGCTTTTGT-39) was specific to the CER promoter, and the reverse primer (59-GCT-

TTGTGGGCTTTGGAGGTA-39) targeted the WXP1 region. Genomic DNA was extracted from newly developed leaves of the regenerated plants and from the controls, using DNeasy Plant Mini Kit (Qiagen, Valencia, CA), following manufacturer’s instructions. Total RNA was extracted from newly developed leaves of the transgenic and control plants with MRC Tri-Reagent (Molecular Research Center, Cincinnati, OH). The concentration of total RNA was quantified by spectrophotometer. For RT-PCR analysis of transgene expression, 2 mg of total RNA was transcribed into cDNA, using Omniscript RT Kit and oligo (dT) primer (Qiagen). Two microliters of cDNA was used in each PCR amplification (25 mL). Primers for the WXP1 gene were 59-AATGGGTTGCTGAGATAAGACTAC-39 and 59-CAAGACCGGCAACAGGATT-39 (this primer is located in the nos terminator region). The actin gene was used as a reference gene; primers for the actin gene were 59-GATATGGAAAAGATCTGGCATCAC-39 and 59-TCATACTCGGCCTTGGAGATCCAC-39. PCR conditions were 95°C for 2 min, 25–32 cycles of 94°C for 1 min, 50°C for 1 min, and 72°C for 1 min, followed by 72°C for 10 min. Plants containing or expressing the WXP1 gene were identified by electrophoresis of amplification products in 1% (w/v) agarose/ ethidium bromide gels.

Cloning of a WXP1 Orthologue from Alfalfa On the basis of the sequence information of the WXP1 gene from Medicago truncatula L. (MtGI TC115856), primers were designed to amplify its alfalfa orthologue by RT-PCR. The following primers were used for the cloning of the alfalfa WXP1 gene: 59-CGCATGGATTTCTTCAACAATTC-39 and 59-AGCAGTGTCAAAAGTACCAAGCC-39.

Plants and Growth Conditions After the initial screening, three independent pCER6::WXP1 transgenic lines were used for further analyses. A previously obtained CaMV35::WXP1 transgenic line T-47 (Zhang et al. 2005) and WT and CK plants were also included in the analyses. All plants were propagated by shoot cuttings. When roots and seedlings were well established (2–3 wk), plants were transferred into 4.5-inch pots with Turface MVP clay (Profile Products, Buffalo Grove, IL) as soil. Plants were reared in a growth chamber under conditions previously described by Zhang et al. (2005). Briefly, the chamber was set at 23°/19°C, with a 16L : 8D photoperiod for normal plant growth and an 8L : 16D photoperiod when plants were drought stressed. After 2 wk of growth, similarly sized plants were grouped into at least five replications, and water stress was applied. Watering was withheld for 3 or up to 7 d before rewatering. The experiments were repeated at least three times for all measurements, except for the observation of the phenotypic differences between WT and transgenic lines (two replications).

Gas Exchange and Chlorophyll Fluorescence Measurement Leaf gas exchange and chlorophyll fluorescence were measured on intact center leaflets from fully developed upper-

Fig. 1 Alignment of WXP1 sequences obtained from Medicago truncatula (MtWXP1) and Medicago sativa (MsWXP1).

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most trifoliates, using the photosynthesis system LiCor-6400 coupled with the 6400-40 leaf chamber fluorometer (Li6400; Li-Cor, Lincoln, NE). The leaf chamber fluorometer was used as a light source. The light intensity was set at 500 mmol m2 s1, and blue light was set at 10% of the total. The flow rate was 400 mmol m2 s1, and ambient CO2 concentration was 400 mmol mol1. Ambient temperature and relative humidity were about 25°C and 50%, respectively, during all measurements. Data were manually logged when gas exchange and chlorophyll fluorescence parameters became stable. Measurements were conducted at well-watered (WW) conditions, water-stressed (WS) conditions (3 d after watering was withheld), and rewatered (RW) conditions (the next day after rewatering the plants).

Measurement of Leaf Relative Water Content (RWC) and Water Potential (cw) After we completed gas exchange measurements, leaflets were collected into ziplock bags and kept on ice before leaf area (LA) was measured. LA was recorded using an area meter (LI-3000; Li-Cor) to recalculate gas exchange data. Fresh weight (FW) was measured right after LA measurements, and leaflets were then placed in water for 24 h at 4°C to achieve full turgor. After weighing the samples for turgid weight (TW), samples were oven-dried at 65°C for 48 h, and dry weight (DW) was measured. Plant water status was evaluated by RWC, calculated as (FW  DW) / (TW  DW) 3 100 (Ritchie et al. 1990). Total leaf water potential was measured using a Wescor (Logan, UT) thermocouple psychrometer for 3 d after watering was ceased and 24 h after plants were rewatered. Center leaflets from the uppermost fully developed trifoliates were measured from at least five plants for each transgenic line and the controls.

Characterization of Transgenic Lines by PCR and RT-PCR PCR screening of the regenerants identified 18 positive transgenic plants (data not shown). Three transgenic lines were selected for further analyses on the basis of high gene expression levels and their initial response to water withdrawal. RT-PCR analysis revealed consistently high WXP1 gene expression in the selected transgenic alfalfa lines (fig. 2).

Plant Phenotype No differences in size or development were observed among the lines under normal water regime (fig. 3A, WW). Three days after watering was ceased, CK and WT plants started showing wilted branches (fig. 3B, WS). The CaMV35S::WXP1 transgenic line (T) showed less wilting than the controls (fig. 3B, WS), while no wilting was observed in the three CER6::WXP1 transgenic lines. When all the plants were rewatered after a 5-d water stress, the WXP1 transgenic lines recovered better and quicker than CK and WT (fig. 3C, RW). In the control plants, some wilted leaves did not recover and died (fig. 3C, RW).

Gas Exchange

Identification of a WXP1 Orthologue in Alfalfa

Under WW conditions, the transgenic lines and the controls had similar net photosynthetic rates (fig. 4A, An, WW). Stomatal conductance (gs) and transpiration rate (E) were lower in the CK and the CaMV35S::WXP1 line (T), with respect to CER6::WXP1 lines 10 and 14 (fig. 4B, 4C, WW). WT had values similar to those of the transgenic lines under WW conditions (fig. 4B, 4C, WW). After watering was ceased for 3 d, all the transgenic lines (10, 14, 17, and T) had significantly higher net photosynthetic rates than the controls. Transgenic line 14 had the highest net photosynthetic rate (16.0 mmol m2 s1), more than threefold those of both controls (fig. 4A, WS). Thus, all lines containing the WXP1 gene showed improved net photosynthetic rate under water stress; gs and E showed a similar trend. All the CER6::WXP1 lines (10, 14, and 17) had significantly higher stomatal conductance and transpiration rates than the controls (fig. 4B, 4C, WS). Line 14 had the highest levels of stomatal conductance and transpiration rate, which were more than fourfold those of both controls (fig. 4B, 4C, WS). The values of the CaMV35S::WXP1 line (T) were in between those of the CER6::WXP1 lines and the controls. Although line 14 had significantly higher stomatal conductance and transpiration rate than line T, the differences between lines 10, 17, and T were not significant (fig. 4B, 4C, WS). The differences between line T and the controls were not significant (fig. 4B, 4C, WS).

Amplification of the Medicago sativa WXP1 gene resulted in a single RT-PCR amplicon; its sequence was found to be 93% identical to the Medicago truncatula WXP1 gene. The alfalfa orthologue was named MsWXP1. It coded for a 366– amino acid protein, compared with 371 amino acids encoded by M. truncatula WXP1. The deduced protein sequence of MsWXP1 shared 92% identity with that of M. truncatula WXP1 (fig. 1).

Fig. 2 WXP1 expression in leaves from transgenic lines and emptyvector control (CK) and wild-type control (WT) alfalfa. Transgenic lines used were 10, 14, 17 (CER6-WXP1), and T (CaMV35S-WXP1).

Statistical Analyses The experiments were conducted using a completely randomized design with at least five replications for each line, including WT and CK. Because the trends in individual experiments were similar, all experimental data for gas exchange, chlorophyll fluorescence, and RWC were combined and analyzed together. Differences among lines were analyzed by the GLM procedure (SAS Institute, Cary, NC), with P < 0:05 as a significance level according to a least significant difference test.

Results

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Fig. 3 Phenotype observation of transgenic lines and empty-vector control (CK) and wild-type control (WT) alfalfa under well-watered (WW), water-stressed (WS), and rewatered (RW) conditions. Transgenic lines used were 10, 14, 17 (CER6-WXP1), and T (CaMV35S-WXP1).

When plants were rewatered after the stress treatment, most of the transgenic lines recovered better than the controls in their net photosynthetic rate, stomatal conductance, and transpiration rate (fig. 4, RW). Lines 14, 17, and T were

significantly better than the controls. Statistical analyses revealed strong positive correlations between stomatal conductance and transpiration rate under both WS and RW conditions (r ¼ 0:98 and r ¼ 0:91, respectively).

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Fig. 4 Net photosynthetic rate (An), stomatal conductance (gs), and transpiration rate (E) of transgenic lines and empty-vector control (CK) and wild-type control (WT) alfalfa under well-watered (WW), water-stressed (WS), and rewatered (RW) conditions. Transgenic lines used were 10, 14, 17 (CER6-WXP1), and T (CaMV35S-WXP1). Error bars represent standard errors. Mean values with the same letter were not significantly different (P < 0:05).

Chlorophyll Fluorescence Most of the transgenic lines, excluding line 10, showed higher efficiency of PSII, quantum yield (FPSII), coefficient of photochemical quenching (qP), and apparent electron transport rate (ETR) than both controls under WS conditions (fig. 5, WS). Similar results were obtained after rewatering stressed plants (fig. 5, RW). The controls had more obvious reduction in their chlorophyll fluorescence parameters under WS and RW conditions when compared with the transgenic

plants under WW conditions. No difference was found between the transgenic and the control lines under WW conditions (fig. 5). This indicates that the PSII of these transgenic lines was less affected than that of the controls by drought stress. This is in agreement with the gas exchange parameters. In most of the cases, the lines in which WXP1 was driven by the CER6 promoter did not differ from CaMV35S::WXP1 line T, based on the efficiency of PSII (Fv9/Fm9), FPSII, qP, and ETR under WW (fig. 5, WW), WS (fig. 5, WS), and RW (fig. 5, RW) conditions.

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Fig. 5 Energy efficiency of photosystem II ([PSII] Fv9/Fm9), quantum yield (FPSII), coefficient of photochemical quenching (qP), and apparent electron transport rate (ETR) of transgenic lines and empty-vector control (CK) and wild-type control (WT) alfalfa under well-watered (WW), water-stressed (WS), and rewatered (RW) conditions. Transgenic lines used were 10, 14, 17 (CER6-WXP1), and T (CaMV35S-WXP1). Error bars represent standard errors. Mean values with the same letter were not significantly different (P < 0:05).

Leaf RWC and Water Potential under WS Conditions Compared with WT and CK, all the WXP1 transgenic alfalfa lines had significantly higher values of leaf RWC after watering was withheld for 3 d, indicating their improved

ability to withstand drought stress (fig. 6). Among these, line 14 showed the highest RWC (59.4%). On the first day after watering was suspended, the leaf water potential of most of the lines gradually decreased (fig. 7).

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INTERNATIONAL JOURNAL OF PLANT SCIENCES this time point, the transgenic WXP1 lines maintained higher leaf water potential (1.32 to 1.77 MPa) than the controls (2.07 and 2.18 MPa; fig. 7).

Discussion

Fig. 6 Relative water content (RWC, %) in leaves of transgenic lines and empty-vector control (CK) and wild-type control (WT) alfalfa after watering was ceased for 3 d. Error bars represent standard errors. Mean values with the same letter were not significantly different (P < 0:05).

On the second day, while the leaf water potential of the controls continued to decline, the changes in the transgenics were not obvious. However, on the third day, all the lines showed sharp declines in their leaf water potential (fig. 7). At

Plant drought tolerance is a quantitative trait controlled by multiple genes with complex regulation mechanisms that are still unclear (De Ronde et al. 2004; Bhatnagar-Mathur et al. 2008). Developing a crop plant with enhanced drought stress tolerance requires an essential understanding of the physiological, biochemical, and gene regulatory networks (Valliyodan and Nguyen 2006). Responses of plants to drought stress include stomatal closure, decreased photosynthetic activity, repressed cell growth, elevated respiration, and osmotic adjustment in the cells (Lawlor and Cornic 2002; Shinozaki and Yamaguchi-Shinozaki 2007). Previously, WXP1 and another wax-related gene, WIN1/SHN1, were shown to confer drought tolerance, probably by increasing wax accumulation and reducing water loss (Aharoni et al. 2004; Broun et al. 2004; Zhang et al. 2005). In this study, net photosynthetic rates were significantly higher in the transgenic lines compared with the controls under WS and RW conditions. However, E was also higher in the transgenic lines under unfavorable conditions. This is verified by the strong relationships between net photosynthetic rates and transpiration rates under WS

Fig. 7 Leaf water potential of transgenic lines and empty-vector control (CK) and wild-type control (WT) alfalfa after watering was suspended for 1, 2, and 3 d and also 24 h after rewatering.

JIANG ET AL.—PHYSIOLOGICAL CHARACTERIZATION OF TRANSGENIC ALFALFA (r ¼ 0:92, P < 0:0001) and RW (r ¼ 0:69, P < 0:0001) conditions. One possible reason is that stomata are the major routes for gas exchange, with only a fraction of total transpiration occurring through the rest of the leaf surface (Chaerle and Van Der Straeten 2007). Therefore, enhanced wax production may have a limited contribution to the reduced water loss during stomata opening. Higher photosynthetic and transpiration rates may indicate that the transgenics were less affected by drought stress and still maintained certain levels of metabolic activities. Consistent with previous results, the transgenic alfalfa plants indeed showed higher leaf water content and improved capacity to withstand water withdrawal than did the controls. However, the mechanism behind drought tolerance improvement in WXP1-overexpressing alfalfa is more complicated than previously thought. Plants possess multiple mechanisms to cope with low water availability. Upon water stress, leaf water potential decreases. In the first 2 d of water stress, the changes in leaf water potential were not drastic. Sharp decreases in leaf water potential occurred 3 d after water was withheld. In this phase, the transgenic lines had higher leaf water potential than the controls. One explanation is that cuticular wax may play a significant role when stomata are closed in response to drought stress. Studies have shown that wax deposition on the plant surface increased under drought stress conditions (Kim et al. 2007). Less reduction in water potential may be helpful in maintaining higher levels of photosynthesis in the transgenics. Drought stress may inhibit photosynthesis through photochemical and energy-dependent quenching (De Ronde et al. 2004). It is believed that PSII plays a key role in response to environmental perturbations (Baker 1991; Lal et al. 2008). Chlorophyll fluorescence parameters such as Fv9/Fm9, qP, and ETR indicate efficiencies of PSII. As pointed out by Lal et al. (2008), when the rate of damage of PSII is higher than the rate of repair in chloroplasts, photoinhibition will occur. The defense system of drought-sensitive plants may be damaged because of the irreversible photoinhibition related to PSII. The drought-tolerant alfalfa lines could maintain relatively high photosynthetic activity because of less damage to the PSII, as demonstrated by a smaller decline in Fv9/Fm9. Our data on gas exchange and chlorophyll fluorescence suggest that the WXP1 transgenic lines experienced less damage to the photosynthetic apparatus under stress conditions when compared with the controls. Previous work on WXP1 used the CaMV35S promoter to drive the expression of the gene in alfalfa (Zhang et al. 2005). In this study, an epidermis-specific promoter was employed to direct the expression of the WXP1 gene. The growth and development of transgenic alfalfa overexpressing the WXP1 gene under control of the CER6 promoter were

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normal, and the plants showed enhanced drought tolerance with higher net photosynthetic rates. This further confirmed the function of the WXP1 gene in stress tolerance, in addition to its role in wax production. Analysis of total wax content did not show significant difference between transgenic lines driven by the CER6 promoter and those driven by the CaMV35S promoter (data not shown). In particular, CER6::WXP1 line 14 showed increased wax accumulation and consistently improved physiological parameters under water stress conditions. Unexpectedly, the CER6 promoter did not offer a significant advantage in driving WXP1 regarding gas exchange parameters under RW conditions and RWC under stress conditions. A possible reason is reduced activity of the CER6 promoter in alfalfa compared with its activity in Arabidopsis, from which the promoter was isolated. This view is supported by our observation that transgenic alfalfa plants possessing the CER6::GUS construct had weaker staining of GUS activity in leaves and stems compared with CaMV35S::GUS plants (data not shown). The weak GUS staining from cross section analysis made it difficult to conclude whether the CER6 promoter led to tissue-specific expression in alfalfa. The results support our conclusion that WXP1 is important in drought stress response and the use of a weak promoter could still lead to improved drought tolerance. A commonly asked question regarding altered wax production is whether such increased cuticular wax accumulation affects photosynthesis. This study showed that under our experimental conditions, increased wax accumulation had no negative impact on photosynthesis in the transgenic plants studied. However, as discussed above, this physiological study also revealed that the role of WXP1 is more complex than simply altering wax biosynthesis. Further investigations of the relevant network and downstream genes of this important transcription factor will clarify its function and may lead to more effective ways to use relevant genes in enhancing abiotic stress tolerance. Alfalfa has much higher natural stress tolerance than model species such as Arabidopsis. Therefore, it may be more challenging to observe the impact of introduced stress tolerance transgenes. To date, WXP1 is the only gene that was shown to improve drought tolerance in alfalfa in laboratory tests. Because alfalfa is an outcrossing species, future work should involve crossing the transgenics with commercial cultivars and evaluating the progenies under field conditions.

Acknowledgment The work was supported by the Samuel Roberts Noble Foundation.

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