The identification of leaf wax genes involved in stress tolerance is expected to have great potential for crop improvement. Here we report the characterization of a novel AP2 domain-containing putative transcription factor gene from the model legume Medicago truncatula. The gene, designated WXP1, is able to activate waxproduction and confer drought tolerance in alfalfa (Medicago sativa), the most important forage legume species in the world and a close relative of M. truncatula. The predicted protein of WXP1 has 371 aa; it is one of the longest peptides of all the single AP2 domain proteins in M. truncatula. WXP1 is distinctly different from the most studied genes in the AP2/ERF transcription factor family such as AP2s, CBF/DREB1s, DREB2s, WIN1/SHN1 and GL15. Transcript level of WXP1 is inducible by cold, abscisic acid and drought treatment mainly in shoot tissues in M. truncatula. Overexpression of WXP1 under the control of the CaMV35S promoter led to a significant increase in cuticular wax loading on leaves of transgenic alfalfa. Scanning electron microscopy revealed earlier accumulation of wax crystals on the adaxial surface of newly expanded leaves and higher densities of wax crystalline structures on both adaxial and abaxial surfaces of mature leaves. Gas chromatography–mass spectrometry analysis revealed that total leaf wax accumulation per surface area increased 29.6–37.7% in the transgenic lines, and the increase was mainly contributed by C30 primary alcohol. WXP1 overexpression induced a number of wax-related genes. Transgenic leaves showed reduced water loss and chlorophyll leaching. Transgenic alfalfa plants with increased cuticular waxes showed enhanced drought tolerance demonstrated by delayed wilting after watering was ceased and quicker and better recovery when the dehydrated plants were re-watered.
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A cuticle layer covers most primary aerial organs of vascular plants and forms the contact zone between the plant and the environment (Kerstiens, 1996). Cuticular waxes are the major constituents of the plant cuticle and play an important role in protecting aerial organs from damage caused by environmental stresses. Cuticular waxes are complex mixtures of very long-chain fatty acids, alkanes, primary and/or secondary alcohols, aldehydes, ketones, esters, triterpenes, sterols, and flavonoids. Wax compounds can be embedded within the cutin polymer framework and form intracuticular wax. In many plants, however, more waxes are loaded outside of the cuticle membrane and form an epicuticular wax layer that gives the plant surface a glaucous or gray appearance (Jenks and Ashworth, 1999; Post-Beittenmiller, 1996). Plant cuticular wax biosynthesis and its loading to the plant surface is a complicated but actively regulated process (Broun et al., 2004; Jenks et al., 2002). Mutant analysis has greatly contributed to the identification of the components and genes involved in wax deposition. Mutants with reduced wax accumulation or altered wax composition are, in general, characterized by a bright green phenotype which can be detected visually (Aarts et al., 1995). In Arabidopsis, 120 cuticular wax mutants representing a total of 31 recessive mutant loci have been identified, although the dominant wax gene mutations have not been reported (Jenks et al., 2002). Wax-deficient mutants have also been identified in other species, including maize, sorghum, barley, and rape (Kunst and Samuels, 2003). Studies of the eceriferum (cer) mutants and T-DNA insertional mutants in Arabidopsis and glossy (gl) mutants in maize led to the identification and isolation of a number of wax-related genes. To date, 12 genes associated with wax biosynthesis or regulation have been identified by molecular-genetic approaches. Among these genes, CER1, CER2, CER6/CUT1, 3-ketoacyl-CoA synthase (KCS1), FIDDLEHEAD (FDH), GL1, GL8, and WAX2 may encode metabolic enzymes or be involved in the transport of wax compounds (Aarts et al., 1995; Chen et al., 2003; Fiebig et al., 2000; Hansen et al., 1997; Millar et al., 1999; Negruk et al., 1996; Pruitt et al., 2000; St-Pierre et al., 1998; Todd et al., 1999; Xia et al., 1996, 1997; Xu et al., 1997), while CER3, GL2, GL15, and WIN1/SHN1 appear to encode regulatory proteins (Aharoni et al., 2004; Broun et al., 2004; Hannoufa et al., 1996; Moose and Sisco, 1996; Tacke et al., 1995). Mutations in most of these genes showed altered wax accumulation (Jenks et al., 2002). Cosuppression of some of the genes resulted in waxless stems in Arabidopsis (Millar et al., 1999; Todd et al., 1999) and overexpression of some of the genes in Arabidopsis mutant background complemented corresponding mutant phenotypes (Fiebig et al., 2000; Hannoufa et al., 1996). However, only limited information is available on the effects of overexpression of these genes in the wild-type background. Overexpression of the condensing enzyme gene CER6/CUT1 under the control of the CaMV35S promoter failed to promote wax deposition (Millar et al., 1999), while under the control of the epidermis-specific CER6 promoter, CER6/CUT1 overexpression led to increased wax load in stems of Arabidopsis (Hooker et al., 2002). The only report on increased wax accumulation in leaf tissues of Arabidopsis was by the overexpression of a transcriptional activator (Aharoni et al., 2004; Broun et al., 2004). Because multiple biochemical processes are involved in wax biosynthesis, transgenic expression of regulatory genes could be an effective way to manipulate wax accumulation in plants.
We have been interested in characterizing certain transcription factor genes from Medicago truncatula, a model plant for legume biological studies in view of its small diploid genome, self-fertile nature, and relatively short life cycle. A large number of expressed sequence tags (>190 000) have been sequenced from M. truncatula and its whole genome sequencing is in progress (May, 2004; VandenBosch and Stacey, 2003). Medicago truncatula is an omni-Mediterranean forage legume species and is closely related to the world's most important forage legume, alfalfa (Medicago sativa) (May, 2004). Forages are the backbone of sustainable agriculture; they are often grown in less favorable areas and thus require sophisticated protective mechanisms to withstand severe environmental conditions (Wang et al., 2001). Like many other crops, drought tolerance is an important target for improvement in alfalfa. As cuticular waxes play a pivotal role in limiting transpirational water loss across the plant surface, it is expected that genetic engineering of plant waxes may eventually increase environmental stress tolerance in crops of agronomic importance (Millar et al., 1999; Vogg et al., 2004).
Here we report the characterization and transgenic expression of a novel gene, designated WXP1, which activates waxproduction in the acyl-reduction pathway. It is a putative AP2 domain-containing transcription factor gene from the model legume M. truncatula. Overexpression of WXP1 under the control of the CaMV35S promoter led to increased cuticular wax loading on the leaf surfaces of transgenic alfalfa. Furthermore, we demonstrate that the transgenic plants had reduced water loss and enhanced drought tolerance.
Sequence analysis and expression pattern of WXP1
A number of putative AP2 domain-containing transcription factor genes were identified from M. truncatula and characterized by sequence alignment, Northern hybridization analysis, and transgenic overexpression. This study provides insights into the possible role of one of these genes, WXP1. The open reading frame (ORF) of WXP1 encodes a peptide of 371 aa with an estimated molecular mass of 41.3 kDa and a theoretical pl of 5.55. It is one of the longest peptides among the 80 AP2 domain-containing transcription factors identified in M. truncatula (J.-Y. Zhang and Z.-Y. Wang, unpublished data). The deduced amino acid sequence of this gene contains one conserved AP2 domain when analyzed by Pfam (Bateman et al., 2002). When aligned with well-characterized AP2 domain-containing transcription factors from Arabidopsis (At), tomato (Le) and tobacco (Nt), the AP2 domain of WXP1 shared high similarity with that of the other proteins (Figure 1). All of these AP2 domains have a 100% identical WLG motif in the middle and an extremely conserved YRG motif in the front, but they are clearly divided into three groups based on the diversification of the RAYD/LAYD/RAHD motif. WXP1 contains a LAYD motif that is also found in three ERFs (LePti4, NtERF1, and AtERF1), Mt77128 (TC77128), and AtRAP2.4 (At1g78080) (Figure 1). Sequences of Mt77128 and AtRAP2.4 were obtained by blast search with WXP1. Furthermore, the element differences in front and closely after the AP2 domains grouped WXP1 together with Mt77128 and AtRAP2.4 and separated it from WIN1, ERFs, and CBFs.
Comparison of WXP1 to predicted protein sequences of AP2 domain-containing transcription factors from different species revealed that Mt77128 from M. truncatula is the closest homolog with 53.4% identity to WXP1, and AtRAP2.4 from Arabidopsis is the closest ortholog with 48.8% identity to WXP1. When compared with amino acid sequences of other stress- or wax-related genes, WXP1 is only 19.4–22.8% identical to AtCBFs, 16.7% to AtDREB2A, 20.6% to AtDREB2B, 22.8–25.3% to the three ERFs, 28.6% to AtWIN1/SHN1 and 14.0% to ZmGlossy15. Phylogenetic analysis showed that WXP1 is distinct from most of the known AP2 domain-containing transcription factors based on analysis of their complete protein sequences (Figure 2).
The expression pattern of the WXP1 gene in M. truncatula was detected by Northern hybridization analysis. Drought treatment slightly induced its expression in shoot tissues but suppressed its expression in root tissues (Figure 3). WXP1 transcript in shoot was quickly induced to a high level after transferring plants to 4°C. However, no such change was observed in root after a cold treatment by pouring pre-cold water to the pots (Figure 3). Abscisic acid (ABA) treatment induced WXP1 expression in both shoot and root in a relatively short time (Figure 3). The induction or suppression of WXP1 in M. truncatula was reversible, as transcription levels returned to normal levels when the stimuli were withdrawn.
Growth and development of transgenic alfalfa plants overexpressing WXP1
The ORF of WXP1 was placed under the control of the CaMV 35S promoter by replacing the gusA gene of the binary vector pCAMBIA3301. The resulting vector, pC35S-WXP1, was introduced into alfalfa by Agrobacterium-mediated transformation and 60 independent transgenic plants were produced. PCR screening indicated that more than 98% of the regenerated plants contained the target gene and Northern hybridization analysis revealed that 60% of the plants expressed the transgene with various mRNA levels (data not shown). Preliminary greenhouse experiments involving 24 independent transgenics allowed us to identify five lines having obvious improvement in drought tolerance. To facilitate the analysis, we focused on three transgenic lines (18, 41, and 47) for agronomic and biochemical analysis. Lines 4 and 45 were added for gene regulation analysis because they had different transgene expression levels. Lines 4, 18, 41, 47, and 45 showed very low, low, medium, high, and extremely high levels of WXP1 expression, respectively (Figure 4a). All the lines were vegetatively propagated by cuttings of young shoots.
Even though the overexpression of WXP1 did not result in severe growth retardation, which was typical in AP2 domain-containing gene-overexpressed Arabidopsis (Aharoni et al., 2004; Broun et al., 2004; Gilmour et al., 2000; Jaglo-Ottosen et al., 1998; Liu et al., 1998), the alfalfa transgenic lines tended to grow relatively slowly (Figure 4b). When compared with wild type and empty vector control plants, flowering time of the transgenic plants was delayed 5–28 days (Figure S1a), height of the transgenic lines was 2–32% shorter at flowering time (Figure S1b), and trifoliates of the transgenic lines were smaller (Figure 5a). Even though the transgenic lines produced more branches, fresh and dry-matter production of the transgenic lines decreased 5.6–26.8% and 6.7–42.7%, respectively (Figure S1c,d).
The most striking phenotypic change in the WXP1-overexpressed alfalfa plants was the more glaucous appearance in the leaves (Figure 5a). Veins were also less obvious in the transgenic leaves (Figure 5c) compared with the control leaves (Figure 5b). Increased glaucousness occurred in most of the transgenic lines with low to extremely high WXP1 overexpression levels, represented by lines 18, 27, 41, 47, 33, and 45. However, when the WXP1 gene was highly expressed in alfalfa, such as in lines 33 and 45, the plants sometimes had difficulty recovering from the cutting-regrowth cycle in the greenhouse. These lines were therefore not included in further analyses.
Observation of leaf surfaces under the stereomicroscope with strong light indicated that glaucousness was increased on both sides of the WXP1-overexpressed alfalfa leaves. However, the change of glaucous appearance on the adaxial and abaxial surfaces of the transgenic lines was not equal. Leaflets excised from the same trifoliate showed less light reflection on the adaxial side and thus less glaucousness than the abaxial side in control leaflets (Figure 5b). However, the difference between the two sides was drastically reduced in transgenic leaflets (Figure 5c). Compared with control leaflets, transgenic leaflets showed more obvious increase in glaucousness on the adaxial surface than on the abaxial side (Figure 5b,c). The results indicate that the difference in glaucousness between transgenic and control lines was more prominent on the adaxial side than on the abaxial side.
The impact of WXP1 overexpression on cuticular wax production in transgenic alfalfa
To confirm that the increased glaucous appearance on the leaves was caused by alteration of epicuticular wax production, WXP1 transgenic alfalfa lines and empty vector control plants were examined by scanning electron microscopy (SEM). When the adaxial and abaxial surfaces of the same leaflet from a control plant were compared, the time of epicuticular wax loading, wax crystal type, crystal size, and crystal density were different between the two sides (Figure 6a,c,e,g). It is obvious that the abaxial surface started loading epicuticular wax earlier than the adaxial surface (Figure 6a,c). Comparison of leaf epicuticular wax crystalline patterns between control and transgenic lines showed that overexpression of WXP1 in alfalfa resulted in increased wax loading on both sides and earlier accumulation of epicuticular waxes on the adaxial surface. When the new leaf became fully expanded (top first), there were no visible wax crystals on the adaxial leaf surface of control plants (Figure 6a). In contrast, wax crystal structures were already developed on the adaxial leaf surface of transgenic plants (Figure 6b). Wax crystal density of newly expanded leaf showed differences on the abaxial side, with a better coverage of wax crystals in transgenics than in the control plant (Figure 6c,d). For the top second trifoliate, differences in wax crystal density on the adaxial leaf surfaces were apparent between the transgenic and control plants, with many more wax crystals present in the transgenic plant (Figure 6e,f). On the abaxial surface of the top second trifoliate, the long coiled crystalline structure in the transgenic line was reduced, but the loss of the long coiled crystals was complemented by the increase in density of tubular and plate-like wax crystals (Figure 6g,h). Thus the SEM examination is in agreement with the increased leaf surface glaucous appearance observed under the light microscope. Stem surfaces of both control and transgenic alfalfa lines did not show visible wax crystalline structures (Figure S2).
To further determine whether the crystal pattern alteration in transgenic alfalfa was the result of qualitative or quantitative changes in wax loading, leaf samples from the top four fully expanded trifoliates and stem samples from the top four internodes of wild type, empty vector control, and WXP1 transgenic plants were subjected to gas chromatography–mass spectrometry (GC–MS) analyses. As shown in Figure 7(a,b), primary alcohols are the major constituents of alfalfa leaf wax while alkanes are the most abundant wax components in stem. Total primary alcohol content per leaf area dramatically increased in the WXP1-overexpressing lines (Figure 7a), while only a slight increase in alkane content was observed in stems of the transgenics (Figure 7b). No new compound was identified in the transgenic lines.
Total wax accumulation per leaf area was significantly higher in the transgenic lines relative to the control plants (Figure 7c). The largest change in cuticular wax deposition was observed in transgenic line 47, which had an increase of 37.7% in its wax load. Lines 18 and 41 showed increases in their total wax load by 29.6 and 35.3%, respectively. The increase was mainly contributed by the C30 primary alcohol, which is the major component of alfalfa leaf wax (Figure 8a). C30 alcohol in transgenic lines 18, 41, and 47 was 34.5, 43.0, and 45.3% more than that of the control plants, respectively. Other carbon length alcohols did not show significant changes (Figure 8a). Alkanes were not significantly affected by the overexpression of WXP1, even though there was a minor increase in C31 alkane in the transgenic lines (Figure 8b). The abundance of unsaturated octadecenoic acid (C18:1) was apparently increased in the transgenic lines, while saturated fatty acid (C18:0) remained essentially the same, and the amount of C22 and C24 fatty acids decreased slightly (Figure 8c). In leaf cuticular wax extracts, C30 aldehyde was the only aldehyde component detected. The amount of this compound decreased in line 47, but no significant changes were found in other transgenic lines (Figure 8d).
In contrast to leaves, cuticular wax accumulation in stems was not significantly altered in transgenic alfalfa plants (Figure 7c). The most abundant stem wax component, C31 alkane, increased gradually with the increased transgene expression level from transgenic line 18 to line 47. However, C29 alkane, the second most important stem wax component, was negatively affected by the level of transgene expression (Figure S3a). For the minor components of alfalfa stem wax, only C20 fatty acid and triterpenoids were reduced in the transgenics; no consistent and significant changes were observed for the other components (Figure S3b–d).
Water loss rate, chlorophyll extraction rate, and drought tolerance of transgenic alfalfa overexpressing WXP1
Detached alfalfa trifoliates were subjected to free leaf water loss rate assay. Both the top second and top third trifoliates from the transgenic lines showed lower water evaporation rate than that from the vector control (Figure 9 and Figure S4).
Chlorophyll efflux analysis revealed that the rate of chlorophyll extraction from the top first through top fourth leaves during a 6-h period was much slower in the transgenic plants than in the control plants (Figure 10a,c,e,g and Figure S5a,c,e,g), indicating reduced epidermal permeability in transgenic leaves. In stems, the difference in chlorophyll extraction rate between transgenic and control plants was relatively small, with no difference in the fourth internode (Figure 10b,d,f,h and Figure S5b,d,f,h). In addition, little difference in epidermal permeability was observed between leaves at different positions in the stem, even though they represent different developmental stages. However, different internodes showed substantial differences, with older internodes having much less chlorophyll efflux than younger internodes (Figure 10).
Control plants and the three transgenic lines (18, 41, and 47) identified from the preliminary greenhouse experiments were analyzed further regarding their drought stress tolerance in growth chambers and again in the greenhouse. In the growth chamber experiments, watering was suspended for three replicates of 20-day-old alfalfa plants and the chambers were dehumidified. Three days after watering was stopped, all the control plants became wilted while all the transgenic plants still kept their whole plant turgor (Figure 11a). The transgenics began to wilt 1 day later than the control plants. After one more week in the same chambers without watering, all the plants became dehydrated and dead-like. Upon resumption of the normal watering scheme, the transgenic plants recovered much faster and better than the control plants (Figure 11b). In the greenhouse experiments, it took longer time to completely dehydrate the plants because of the fluctuations in relative humidity. Nevertheless, after three cycles of drought stress-rewatering in a period of 4 weeks, all the independent transgenic plants survived while the control plants either failed to recover or recovered very slowly (Figure 11c).
Effect of WXP1 overexpression on other wax biosynthesis-related genes
Northern hybridization analysis was used to characterize the expression of a number of wax-related genes in the transgenic plants. Total RNA was isolated from leaves of transgenic lines 4, 18, 41, 47, and 45; these lines had increasing levels of WXP1 expression (Figure S6). A list of candidate genes (Table 1) was selected based on tblastn search against the TIGR Medicago truncatula Gene Index database (Quackenbush et al., 2000) with a set of query genes that were implicated in cuticular wax loading or cutin biosynthesis in Arabidopsis and maize. All of the DNA probes used for hybridization were selected from M. truncatula cDNA libraries.
Table 1. Changes in the expression of wax-related genes in transgenic alfalfa plants overexpressing WXP1
Because of the potential role of fatty acid elongase (FAE) in wax production, and the finding that a putative FAE gene was strongly downregulated by WIN1 overexpression in Arabidopsis (Broun et al., 2004), five M. truncatula cDNA clones predicted to encode different FAE-like proteins were tested. In total, seven bands were revealed by Northern hybridization with these five FAE-like genes. Transcript levels in three of the FAE-like genes, MtTC79579, MtTC80406 and MtTC87247, were enhanced in the transgenic lines with increasing levels of WXP1 expression (Table 1, Figure S6). This is contrary to the effect of WIN1 overexpression. No obvious changes were detected for the other FAE-like genes in the transgenic lines (Table 1).
KCS and CER6 have been implicated in the synthesis of very-long-chain fatty acids (VLCFA) precursors for wax production (Kunst and Samuels, 2003). No changes in the expression levels of KCS- or CER6-like genes (TC78487, TC82553 and TC81348) were observed in the alfalfa transgenic plants (Table 1).
The LACERATA (LCR) gene encodes a cytochrome P450 monooxygenase, which catalyzes ω-hydroxylation of fatty acids ranging from C12 to C18:1 (Wellesen et al., 2001). Overexpression of WXP1 in alfalfa upregulated two LCR-like genes, TC81689 and TC84740. Two bands were observed when another LCR-like gene (TC80834) was used as probe; the intensity of the upper band was increased, while the intensity of the lower band was decreased with the expression of WXP1 (Table 1 and Figure S6).
Arabidopsis WAX2 is predicted to have a metabolic function associated with both cuticle membrane and wax synthesis. Transcript level of one WAX2-like gene (TC82822) was negatively affected by the overexpression of WXP1, while the mRNA level of the other WAX2-like gene (TC87337) did not change (Table 1 and Figure S6). Maize GL8 functions as a β-ketoacyl-reductase (KAR) in wax production (Kunst and Samuels, 2003). The intensity of the KAR bands showed a negative correlation with expression of WXP1 (Table 1 and Figure S6).
We also analyzed the expression pattern of a number of other genes that were reportedly upregulated by WIN1 in Arabidopsis (Broun et al., 2004). Among these genes, the expression of the two glycerol-3-phosphate dehydrogenase genes was positively correlated with WXP1 expression, while the phospholipid/glycerol acyltransferase gene was negatively correlated with WXP1 expression. The transcript levels of enoyl-CoA hydratase/isomerase and long-chain acyl-CoA synthase (LACS) were not affected by the overexpression of WXP1 in alfalfa (Table 1 and Figure S6).
WXP1 is a novel transcription factor gene that activates wax production in leaves of transgenic alfalfa plants
The WXP1 gene cloned from M. truncatula encodes one of the longest peptides of all the predicted AP2 domain-containing transcription factors in M. truncatula. Sequence analysis revealed that WXP1 is very different from other well-characterized transcription factors related to abiotic stress or wax accumulation, for example, DREB/CBF from Arabidopsis (Jaglo-Ottosen et al., 1998; Liu et al., 1998), WIN1/SHN1 from Arabidopsis (Aharoni et al., 2004; Broun et al., 2004), and GL15 from maize (Moose and Sisco, 1996). Northern hybridization analysis showed that the expression of WXP1 is inducible by cold or drought stress, which is similar to some members of the DREB/CBF family. The response to environmental stress has not been reported for WIN1/SHN1 and GL15. As discussed below, functional characterization of WXP1 in transgenic alfalfa plants further proved its novelty.
Because of the unique characteristics and modes of action of transcription factors, the overexpression strategy is considered particularly effective in revealing transcription factor function (Zhang, 2003). Different from DREB/CBF genes, constitutive expression of the WXP1 gene in alfalfa resulted in a significant increase in wax accumulation on the leaf surfaces. Wax crystals were produced earlier on the adaxial side of newly expanded transgenic leaves than on the abaxial side; a higher density of wax crystals was found on both adaxial and abaxial sides of mature transgenic leaves. As pointed out by Jenks et al. (2002), alteration of wax accumulation in crop leaf tissues is important, because leaves are the primary photosynthetic organs, comprise the primary biomass of most agronomic crops, and are often severely affected by environmental stresses. Most of the Arabidopsis mutants have alterations in their stem waxes (Jenks et al., 1995; Koornneef et al., 1989). Visual screening of mutagenesis populations of Arabidopsis to find mutants having increased leaf glaucousness due to changes in cuticular waxes has had limited success (Jenks et al., 2002). Thus, overexpression of transcription factor genes could be an effective approach to turn on the wax biosynthetic pathway and lead to increased wax production in leaves.
WXP1 is likely involved in the acyl-reduction pathway of wax biosynthesis
In most plants, there are two principal wax biosynthetic pathways: an acyl-reduction pathway, which gives rise to primary alcohols and wax esters, and a decarbonylation pathway, leading to the formation of aldehydes, alkanes, secondary alcohols, and ketones (Kunst and Samuels, 2003). Although alfalfa naturally accumulates wax, only limited information is available about the composition of waxes in leaves and stems (Bergman et al., 1991). Our analysis of wax constituents revealed that primary alcohols are the major wax components in leaves, while alkanes are predominant in stems, indicating the existence of preferential pathways in alfalfa leaves and stems.
Increased wax production by WXP1 in transgenic alfalfa leaves was mainly due to the increases in primary alcohols; the changes of alkane and other major wax components in the transgenic leaves were not significant. Thus, WXP1 is mainly involved in the acyl-reduction pathway of wax biosynthesis; this is different from the Arabidopsis WIN1/SHN1 gene (Aharoni et al., 2004; Broun et al., 2004), which is mainly involved in the decarbonylation pathway. There was only a minor alteration of cuticular waxes in transgenic alfalfa stems; this result further supports the view that WXP1 is involved in the acyl-reduction pathway, because the decarbonylation pathway is the major pathway in alfalfa stems, in which the major components of waxes are alkanes.
WXP1 overexpression affected the expression of genes that are potentially related to wax or cutin biosynthesis
Among the candidate enzymatic genes analyzed, several exhibited expression patterns that associated with the expression of WXP1. In contrast to Arabidopsis WIN1, which downregulated the expression of an FAE gene, WXP1 upregulated three FAE genes. It is known that acyl chain extensions are carried out by several distinct elongases with unique substrate chain specificities (Kunst and Samuels, 2003). Thus WXP1 may positively impact the fatty acid elongation process for the production of VLCFA chains that are used for the production of aliphatic wax components. The transcript levels of genes coding for cytochrome P450 monooxygenase and glycerol-3-phosphate dehydrogenase were also upregulated in the WXP1 transgenics. Glycerol-3-phosphate dehydrogenase is essential for phospholipid synthesis through both the prokaryotic and the eukaryotic glycerolipid pathway (Wei et al., 2001). Cytochrome P450 monooxygenase functions as a fatty acid ω-hydroxylase that is required in the formation of cutin monomers (Wellesen et al., 2001). The results indicate that in addition to its function in wax biosynthesis, WXP1 may also play a role in the biosynthesis of cutin, which may act as a barrier that mechanically isolates epidermis cells of adjoining organs.
Fatty acids produced in the plastid from de novo synthesis are directed to at least three biosynthetic pathways that lead to the production of waxes, cutin/suberin, and glycerolipids, respectively (Post-Beittenmiller, 1996). This is accomplished by a partition occurring after their biosynthesis, which delivers C16:0 and unsaturated C18:1 fatty acids as precursors to produce glycerolipids or cutin/suberin and saturated C18:0 fatty acid as precursor to produce waxes (Post-Beittenmiller, 1996). In our transgenic WXP1 plants, no significant change was observed for the amount of saturated C18:0 fatty acid and total fatty acids, indicating the increase in wax accumulation, particularly the increase in primary alcohols in leaves, is most probably due to the regulation of genes that control fatty acid elongation and the acyl-reduction pathway. On the contrary, the amount of unsaturated C18:1 fatty acid in leaves was more than double that of controls. The increase in C18:1 fatty acid is consistent with the upregulation of cytochrome P450 monooxygenase, because the enzyme is directly involved in the biosynthesis of cutin (Wellesen et al., 2001). Further research with respect to WXP1 expression and cuticle composition is required to elucidate the potential role of WXP1 on cutin biosynthesis.
Although Northern hybridization analysis allowed the identification of several genes that are either upregulated or downregulated in transgenic plants, it is not clear what the direct target genes of WXP1 are. More information in this aspect could be obtained by further analysis using the Affymetrix oligonucleotide microarray, which is being developed in M. truncatula (G. May, The Noble Foundation, Ardmore, OK, USA, personal communication). In addition, large numbers of mutants are being generated in M. truncatula (May, 2004); the identification of wax mutants in this species may allow us to further elucidate wax biosynthetic pathways in legumes.
Overexpression of WXP1 confers drought tolerance in transgenic plants
Overexpression of transcription factor genes DREB/CBF in Arabidopsis activated C-repeat/DRE containing downstream genes that are involved in cold acclimation and drought adaptation. Although the transgenic Arabidopsis showed increased stress tolerance, no additional waxes were produced (Jaglo-Ottosen et al., 1998; Kasuga et al., 1999). Thus, the mechanism of DREB/CBF genes on drought tolerance improvement is different from that of WXP1. Overexpression of WXP1 activated wax production and led to the glaucous appearance of leaves in transgenic alfalfa. Glaucousness resulting from wax accumulation has been considered a beneficial trait for the adaptation of plants to water-limited environments (Jefferson, 1994). Under severe water-deficient conditions, plant stomata normally close. The survival of a plant will then depend largely on its ability to restrict water loss through the leaf epidermis (Rawson and Clarke, 1988). Transgenic alfalfa plants showed reduced water loss and decreased epidermal permeability, therefore the transgenic plants were much more drought-tolerant than the control plants. Genetic and mutant studies have suggested that wax accumulation is a potential drought adaptation trait (Jefferson, 1994). Our study using isogenic lines (wild-type control, empty vector control, and transgenic plants) clearly demonstrates the positive effects of cuticular waxes on drought tolerance. Despite the fact that both drought tolerance and wax accumulation are complicated traits that are under the control of multiple genes, our results demonstrate that overexpression of a single transcription factor gene, WXP1, could activate wax production and improve plant drought tolerance.
Alfalfa is the fourth most widely grown crop in the United States behind only corn, wheat, and soybeans. It contributes enormously to the world's dairy, beef, and wool production, although the contribution often goes unrecognized. As a perennial forage crop, alfalfa is a fairly hardy species and has a relatively high level of drought tolerance compared with many food crops. Even so, increased wax loading by overexpression of WXP1 on alfalfa leaves further enhances its drought and dehydration tolerance. Thus, manipulation of wax production by transgenic expression of WXP1 or its orthologs could have significant potential for the genetic improvement of other forage, food, or horticultural crops. Although transgenic alfalfa plants showed moderate slow growth due to increased wax accumulation, the problem may be overcome by the use of epidermis-specific promoters (Hooker et al., 2002) or drought-inducible promoters (Kasuga et al., 1999, 2004). The use of stress-inducible promoters minimizes the negative effects of DREB/CBF overexpression on plant growth (Kasuga et al., 1999, 2004).
We identified and characterized a novel AP2 domain-containing transcription factor gene by using an overexpression approach. Constitutive overexpression of the WXP1 gene led to a significant increase in cuticular wax production on leaves of transgenic alfalfa. The increase in wax production was mainly contributed by the increase in C30 primary alcohol. The accumulation of more cuticular waxes reduced water loss and increased plant drought tolerance. Therefore, genetic modification of leaf cuticular waxes has great potential for crop improvement. Further studies are needed to identify the downstream target genes of WXP1 to provide a better understanding of gene involvement in plant cuticular wax accumulation.
Construction of pC35S-WXP1 vector and alfalfa transformation
The coding sequence of WXP1 from M. truncatula (genotype Jemalong A17) was PCR-amplified using primers 5′-GGTACCATGGATTTCTTCAACA-3′ (forward) and 5′-AACCGGTCACCAAATTCATCCA-3′ (reverse) and digested by NcoI and BstE II. The fragment was inserted into the pCAMBIA 3301 vector by replacing the gusA gene. The resulting binary vector, pC35S-WXP1, was transferred into Agrobacterium tumefaciens strain C58C1 using freezing/heat shock method.
An alfalfa genotype, Regen SY-4D, was used for Agrobacterium-mediated transformation to generate transgenic plants (Austin et al., 1995). Vegetatively propagated plants from the original Regen SY-4D clone were used as wild-type control. Alfalfa lines transformed with the original pCAMBIA3301 vector were used as empty vector control.
Growth and treatments of M. truncatula
Medicago truncatula (genotype Jemalong A17) seeds were pre-germinated and planted in 4.5 in pots filled with Turface MVP clay (Profile Products LLC, Buffallo Grove, IL, USA). Plants were grown in greenhouse at 24/22°C with 16/8 h photoperiod and a relative humidity at approximately 70–80%. Four-week-old plants were used for drought, cold, and ABA treatment. For drought stress treatment, watering was suspended for 1–4 days, which represented very mild, mild, moderate, and severe drought stresses, respectively. Additional severely stressed plants were re-watered and tissue was sampled 24 h later as recovering treatment. Cold treatment was performed by transferring plants to a 4°C cold room, and tissues were collected at different time points. Samples from recovered tissues were also collected 24 h after transferring the plants back to greenhouse. For ABA treatment, 100 μm ABA (mixed isomers) in 0.02% Tween-20 water solution was sprayed on leaves and also poured into the pots. Controls were sprayed and watered with the same solution without ABA. Leaf and root tissues were sampled at different time points.
Growth and treatments of alfalfa
The transgenic and control plants were propagated using shoot cuttings. Root system was developed and seedlings established 2–3 weeks after transferring the shoot cuttings to Oasis® Rootcubes® Growing Medium (Smithers-Oasis U.S.A., Kent, OH, USA). Seedlings were transplanted to 4.5 in pots filled with BM-7 bark mix (Berger, Saint-Modeste, Quebec, Canada). For the analyses of wax content, chlorophyll leaching and leaf water loss rate, samples were taken from plants with six to seven fully expanded trifoliates on the major stem and four to six trifoliates on the branches. All plants were grown in a greenhouse at 23/19°C with 14/10 h photoperiod and relative humidity at approximately 50%. Flowering time was measured as the days from transplanting to the emergence of first flower. Plant height and fresh weight were measured when all the plants flowered after 80 days of growth. Dry matter data were obtained by drying individual plants for 48 h at 60°C.
For drought tolerance study, well-established seedlings were transplanted to 4.5 in pots filled with Turface MVP clay as soil. In the greenhouse experiments, the transgenic and control plants were grown at 24/22°C with 16/8 h photoperiod and subjected to three cycles of the drought-recovery treatments. For each cycle of the drought-recovery treatment, plants were drought-stressed for 7 days and watering resumed for 2 days. The plants were kept in normal growth conditions for 2 weeks before the recovery status was recorded. Five transgenic lines with improved drought tolerance were identified in a preliminary experiment involving 24 independent transgenics. Greenhouse drought stress experiment was repeated three times with three selected transgenic lines (18, 41, 47) that were used for other agronomic and biochemical analyses. All greenhouse experiments had three replications of plants.
Drought tolerance of the three transgenic lines was also tested in growth chambers at 23/19°C with 16/8 h photoperiod and relative humidity at 60%. After 20 days of growth, similar-sized plants in three replicates were drought-stressed by stopping watering. In the mean time, humidity was reset to 20% and photoperiod was reset to 8/16 h in order to keep the stomata closed most of the time. The drought-tolerant phenotype was recorded 3–4 days later. When the control plants became totally dried or dead-like (approximately 10 days after watering was withheld), all the pots were re-watered and humidity was reset to 60%. Plant recovery was recorded 2 weeks later. The growth chamber experiment was repeated another time.
RNA gel blotting and hybridization
Total RNA was extracted with MRC Tri-Reagent® (Molecular Research Center, Inc., Cincinnati, OH, USA). Twenty micrograms of RNA was loaded in each lane of 1.2% agarose gels with formaldehyde. WXP1 cDNA and other wax biosynthesis-related genes from M. truncatula were 32P-labeled using the RanPrime DNA Labeling System (Invitrogen, Carlsbad, CA, USA) as instructed by the manufacturer. Northern hybridization was conducted using High Efficiency Hybridization System and Washing/Pre-Hyb solution (Molecular Research Center) following the manufacturer's instructions.
The top first and the second trifoliates and the top two internodes from the major stem were harvested and air-dried at room temperature. The middle section between the leaf edge and the major vein of the leaflets or the middle of the internodes were mounted on stubs and coated with approximately 20 nm of 60/40 Gold-Palladium particles using a Hummer VI sputtering system (Anatech Ltd, Springfield, VA, USA). Coated surfaces were viewed using a JEOL JSM-840 scanning electron microscope at 15 kV (JEOL, Peabody, MA, USA).
GC–MS analysis of cuticular wax composition
Leaf cuticular wax samples were collected from the top four fully expanded trifoliates excised from the major stems. The stem cuticular wax samples were collected from the top four internodes. One leaflet was excised from each trifoliate and the four leaflets were combined as one leaf sample. Four internodes were combined as one stem sample. Each sample was inserted into a 20-ml glass tube, and 10 ml (for leaves) or 5 ml (for stem) of hexane (Sigma-Aldrich, Inc., St Louis, MO, USA) was added. Tissues were agitated for 2 min on a rotator at 50 rpm, and the solvent was decanted into new glass tubes. Tissues and tubes were given a 10-sec rinse with the same amount of hexane, and both solutions were combined in a new tube. Hexane-soluble wax extracts were evaporated to a small volume (approximately 1 ml) under a nitrogen stream and then transferred into 2-ml autosampler vials. After complete evaporation in the 2-ml vials, the extracts were resuspended and derivatized in 15.0 μl of 70% pyridine and 30% MSTFA solution (with 0.01 μg μl−1 of cholesterol as internal standard) for every 1 cm2 leaf section or 1 cm2 stem surface area. Derivatization was performed for 60 min at 50°C. One microliter of the solution was injected onto an Agilent 6890 gas chromatograph (Agilent, Palo Alto, CA, USA) using a splitless injection. The injector was held at 280°C and the transfer arm held at 250°C. Separation was achieved using an oven programmed at an initial temperature of 120°C (2 min), ramped linearly to 315°C at 5°C min−1, and held at 315°C for 8 min. The GC was coupled to an Agilent 5973 MSD using electron impact ionization while scanning 50–650 m/z. Duplicate injections were performed for each sample, and the average value of the two injections was used for statistical analysis. Chromatographic peak areas were extracted using Agilent Chemstation integration of the total ion chromatogram.
Quantification was based on peak areas and normalization based on the internal standard as described by Jenks et al. (1995) and Bergman et al. (1991) with modifications. Dose–response curves for correction were developed for each class of wax components: heptadecanoic acid for free fatty acids, tridecanal for adehydes, hentriacontane for alkanes, docosanol for primary alcohols, and cholesterol for sterols, triterpenes and other unknown peaks. The amount of each cuticular wax component and total wax composition was expressed per unit of leaf or stem surface area. Leaf areas were determined using computer digitization of the leaf images by scanning (NIH ImageJ 1.31t). Stem surface areas were calculated as the surfaces of right circular cylinders for every internode (Chen et al., 2003). All values represent averages of six replicate plant samples ± SE.
Quantification of epidermal traits
To quantify leaf water loss rate, five to six trifoliates from the top second and top third internodes were detached from 20-day-old alfalfa after the plants were kept in constant dark for 10 h. Dehydration and measurement was performed in a dark room at 23°C. Humidity was around 50% in the room. Four replicates were included for each sample in the experiment.
Epidermal permeability was measured using a chlorophyll extraction assay. Three trifoliates or stem internode segments from the same stem position were collected from 20-day-old alfalfa plants, and immersed in 50 ml tubes with 15 ml (for trifoliates) or 10 ml (for internodes) of 80% ethanol. Tubes were agitated gently on a rotator platform at 50 rpm. Aliquots of 1000 μl were taken out for chlorophyll quantification and poured back to the same tube at every time point. The amount of chlorophyll extracted into the solution was quantified using a U-60 spectrophotometer (Beckman, Fullerton, CA, USA) and calculated from light absorption at wavelength of 647 and 664 nm as described by Lolle et al. (1997). Chlorophyll extracted at each time point was expressed as a percentage of total chlorophyll extracted after 48 h of immersion. Data were obtained from two independent experiments, each of which was replicated thrice.
We thank Mr William F. Chissoe III from the Noble Electron Microscopy Laboratory, University of Oklahoma, for assistance with SEM. This research was supported by the Samuel Roberts Noble Foundation.
Figure S1. Growth and development of transgenic alfalfa plants overexpressing WXP1. (a) Days to flowering after transplanting. (b) Plant height. (c) Plant fresh weight. (d) Plant dry weight. WT, wild type; VCTR, empty vector control. All values are mean ± SE, n = 3 replicates.
Figure S2. Scanning electron microscopy of stem surfaces of top first and top second internodes of vector control (a, c) and transgenic line T-47 (b, d). Images were taken at ×3,000 magnification.
Figure S3. Stem cuticular wax profile of transgenic alfalfa plants. WT, wild type; VCTR, empty vector control; T, transgenic lines overexpressing WXP1. All values are mean ± SE, n = 6 replicates.
Figure S4. Water loss rate of detached leaves from transgenic line 41 and empty vector control (VCTR). (a) Top second trifoliates. (b) Top third trifoliate. Each point is mean ± SE, n = 4 replicates.
Figure S5. Chlorophyll leaching from leaves and stems of transgenic line 41 and empty vector control (VCTR). Data represent the mean ± SE of two independent experiments (n = 3 for each experiment).
Figure S6. Northern hybridization analysis of the expression of wax-related genes in WXP1 transgenic alfalfa plants. FAE, fatty acid elongase; CYP450, cytochrome P450 monooxygenase; LTP, lipid transfer protein; G3PD, glycerol-3-phosphate dehydrogenase; KAR, β-keto acyl reductase; ECH/ECI, Enoyl-CoA hydratase/isomerase; VCTR, empty vector control; T, transgenic lines overexpressing WXP1.