FAR5, a fatty acyl-coenzyme A reductase, is involved in primary alcohol biosynthesis of the leaf blade cuticular wax in wheat (Triticum aestivum L.)

Highlight

TaFAR5, a fatty acyl-coenzyme A reductase, catalyses the synthesis of primary alcohols in wheat cuticle and is linked to response to abiotic stress in an ABA-dependent manner.

Abstract

A waxy cuticle that serves as a protective barrier against non-stomatal water loss and environmental damage coats the aerial surfaces of land plants. It comprises a cutin polymer matrix and waxes. Cuticular waxes are complex mixtures of very long chain fatty acids (VLCFAs) and their derivatives. Results show that primary alcohols are the major components of bread wheat (Triticum aestivum L.) leaf blade cuticular waxes. Here, the characterization of TaFAR5 from wheat cv Xinong 2718, which is allelic to TAA1b, an anther-specific gene, is reported. Evidence is presented for a new function for TaFAR5 in the biosynthesis of primary alcohols of leaf blade cuticular wax in wheat. Expression of TaFAR5 cDNA in yeast (Saccharomyces cerevisiae) led to production of C22:0 primary alcohol. The transgenic expression of TaFAR5 in tomato (Solanum lycopersicum) cv MicroTom leaves resulted in the accumulation of C26:0, C28:0, and C30:0 primary alcohols. TaFAR5 encodes an alcohol-forming fatty acyl-coenzyme A reductase (FAR). Expression analysis revealed that TaFAR5 was expressed at high levels in the leaf blades, anthers, pistils, and seeds. Fully functional green fluorescent protein-tagged TaFAR5 protein was localized to the endoplasmic reticulum (ER), the site of primary alcohol biosynthesis. SDS–PAGE analysis indicated that the TaFAR5 protein possessed a molecular mass of 58.4kDa, and it was also shown that TaFAR5 transcript levels were regulated in response to drought, cold, and abscisic acid (ABA). Overall, these data suggest that TaFAR5 plays an important role in the synthesis of primary alcohols in wheat leaf blade.

Introduction

All of the primary aerial organs of land plants are covered with a cuticle that is essential for their protection and interaction with the environment. The cuticle comprises two main types of lipids: cutin, the core structural polymer composed of a three-dimensional polymer of mostly C16 and C18 hydroxy fatty acids cross-linked by ester bonds (Beisson et al., 2012); and cuticular waxes, which are the complex mixtures of very long chain fatty acids (VLCFAs) and their derivatives, including primary and secondary alcohols, aldehydes, alkanes, ketones, esters, triterpenes, sterols, and flavonoids (Bianchi et al., 1990; Kunst and Samuels, 2009), with chain lengths of >20 carbons. Wax composition varies according to species, organ, and developmental state (Samuels et al., 2008). Cuticular wax is believed to play important roles in limiting non-stomatal water loss, a feature contributing to drought tolerance (Hall and Jones, 1961; Premchandra et al., 1992; Kerstiens, 2006; Kosma and Jenks, 2007). It also protects the plant from UV radiation (Yeats and Rose, 2013), defends against bacterial and fungal pathogens (Jenks et al., 1994), and prevents inappropriate organ fusion during development (Sieber et al., 2000). The cuticular waxes of most plants are often present in the form of a film or microcrystals which are mainly classified into platelets, tubules, and rod-shaped structures and give the plant surface a glaucous or grey appearance (Post-Beittenmiller, 1996; Jenks and Ashworth, 1999; Barthlott et al., 1998; Jeffree, 2006).

The first step in wax biosynthesis is the elongation of C18:0 fatty acid produced in the plastid to generate VLCFA wax precursors by joining the C2 building blocks of acetyl-coenzyme A into chains of up to 34 carbons in length (Baker, 1982; Post-Beittenmiller, 1996; Samuels et al., 2008). VLCFAs are formed by a microsomal fatty acid elongation (FAE) system involving four consecutive enzymatic reactions that are catalysed in turn by a β-ketoacyl-CoA synthase (KCS), a β-ketoacyl-CoA reductase (KCR), a β-hydroxyacyl-CoA dehydratase (HCD), and an enoyl-CoA reductase (ECR). Subsequently, VLCFAs are converted through several biosynthetic pathways to the remaining wax components. In Arabidopsis, two principal wax biosynthetic pathways are found: (i) the acyl reduction pathway, which is initiated by the reduction of VLCFAs to aldehydes, followed by a further reduction of aldehydes by an aldehyde reductase to produce even-chained primary alcohols (Kolattukudy, 1971); and (ii) the decarbonylation pathway, leading to aldehydes, alkanes, secondary alcohols, and ketones, which account for 70–80% of total wax in Arabidopsis (Kolattukudy, 1971; Kunst and Samuels, 2003; Samuels et al., 2008).

Long-chain primary alcohols synthesized by the acyl reduction pathway are found throughout the biological world, and are widespread on the surfaces of plants and animals (Metz et al., 2000). In diverse plant species and organs, the important compounds are primary alcohols with a chain length preference of C26 or C28 and, in some systems, C30 or C32 (Baker, 1982). The biochemistry of primary alcohol formation has been examined in a variety of plants (Kolattukudy, 1971; Vioque and Kolattukudy, 1997; Metz et al., 2000). The partial purification of reducing activities from Brassica oleracea initially led to the proposal that primary alcohol production is a two-step process carried out by two separate enzymes, an NADH-dependent acyl-CoA reductase required to reduce VLCFAs to aldehydes and an NADPH-dependent aldehyde reductase required to reduce aldehydes further to primary alcohols (Kolattukudy, 1971; Kunst and Samuels, 2003). Biochemical support for the two-step process leading to alcohol formation comes from experiments in B. oleracea, in which an aldehyde intermediate was isolated (Kolattukudy, 1971). Subsequent studies have demonstrated that primary alcohols are not formed in two steps. Initially, the green alga Euglena gracilis was found to generate alcohols from acyl-CoA precursors without releasing aldehyde intermediates (Kolattukudy, 1970). Subsequent biochemical studies revealed that VLCFA precursors are reduced to primary alcohols by a single fatty acyl-coenzyme A reductase (FAR) in jojoba (Simmondsia chinensis) embryos (Pollard et al., 1979) and pea (Pisum sativum) leaves (Vioque and Kolattukudy, 1997) (Fig. 1). Furthermore, functional expression of FARs from jojoba (Metz et al., 2000), silkmoth (Bombyx mori) (Moto et al., 2003), mouse (Mus musculus), and human (Homo sapiens) cells (Cheng and Russell, 2004) in heterologous systems led to the formation of primary alcohols from fatty acid precursors. To date, eight FAR genes have been identified in Arabidopsis. MS2 (FAR2), a gene previously implicated in exine production, catalyses the conversion of fatty acids to fatty alcohols when expressed in Escherichia coli (Aarts et al., 1997; Doan et al., 2009; Dobritsa et al., 2009). CER4 (FAR3) is specifically involved in the production of C24 to C28 very long chain primary alcohols (Rowland et al., 2006). FAR1, FAR4, and FAR5 can generate the fatty alcohols found in root, seed coat, and wound-induced leaf tissue (Domergue et al., 2010). Ectopic expression of TAA1a from wheat resulted in the production of fatty alcohols in tobacco seeds and in E. coli (Wang et al., 2002).

Fig. 1.

Proposed biosynthetic pathway of very long chain primary alcohol in plants (cuticle and seed coat). FAE, FAR1, FAR4, and CER4 are the wax biosynthetic enzymes from Arabidopsis. ScFAR is the wax biosynthetic enzyme from jojoba. (This figure is available in colour at JXB online.)

Here the identification of TaFAR5 encoding an alcohol-forming FAR from wheat leaf blades, which is identical to TAA1b, previously reported as an anther-specific gene (Wang et al., 2002), is reported. Evidence is also presented for a new function for TaFAR5 in the primary alcohol biosynthesis of leaf blade cuticular wax in wheat. The expression pattern of TaFAR5 coincides with known sites of primary alcohol deposition. The TaFAR5 protein is localized to the endoplasmic reticulum (ER), the site of wax synthesis. Furthermore, TaFAR5 is regulated by drought and cold stresses in an abscisic acid (ABA)-independent manner.

Materials and methods

Plant materials and stress treatments

The hexaploid wheat cultivar Xinong 2718 was used for cDNA cloning, genomic PCR, quantitative real-time PCR (qRT-PCR), and cuticular waxes analysis. Chinese Spring nullisomic–tetrasomic lines were provided by Dr L.W. Shan and used to determine the chromosomal position of TaFAR5, and were grown at the experimental farm of Northwest A&F University in Yangling during the 2011 and 2012 wheat-growing seasons. Simultaneously, seeds of Xinong 2718 were also soil-grown in a glasshouse at 22 °C with 8h dark/16h light, after which 4-week-old seedlings were subjected to various stress treatments. For drought treatment, seedlings were dehydrated on filter paper and harvested at various time points. For polyethylene glycol (PEG) 6000 and ABA treatments, seedlings were transferred to solutions containing 20% (w/v) PEG 6000 and 100 μM ABA, respectively. For cold stress treatment, seedlings were maintained at 4 °C.

Wax extraction and chemical characterization

Wax load was determined on seedlings and flowering plants. Leaf blades were harvested and immediately immersed in chloroform for 1min at room temperature to extract cuticular waxes (Leide et al., 2007; Wang et al., 2011; Zhang et al., 2013). After extraction, n-tetracosane (C24 alkane) was added as an internal standard, and the solvent was completely evaporated under a stream of nitrogen. Samples were transferred to a GC autosampler vial, dried under nitrogen, and derivatized with 100 μl of bis-N, N-(trimethylsilyl)trifluoroacetamide (Sigma) and 100 μl of pyridine (Fluka) for 60min at 70 °C. Solvent was again evaporated under nitrogen gas and the waxes were resuspended in 500 μl of chloroform for gas chromatography–mass spectroscopy (GC-MS) analysis. The qualitative composition of the wax was analysed using a capillary gas chromatograph equipped with a Rxi-5ms column (length 30 m, id 0.25mm, film thickness 0.25 μm) and attached to a mass spectrometer (GCMS-QP2010, Shimadzu, Japan) using helium as the carrier gas. The initial temperature of 50 °C was held for 2min, increased at 20 °C min^–1 to 200 °C, held for 2min at 200 °C, increased again at 2 °C min^–1 to 320 °C, and held for 15min at 320 °C. Injector and detector temperatures were set at 250 °C. The molecular identities of individual wax compounds were identified by comparing their mass spectra with those of authentic standards and literature data. The compounds were quantified by comparing the peak areas with that of the internal standard. The total amount of cuticular wax was expressed per unit of leaf surface area. The leaf areas were determined using ImageJ software (http://rsb.info.nih.gov/ij/) based on digital images of the leaves and multiplying by 2.

Scanning electron microscopy (SEM) analysis

For epidermal wax crystal examination, leaf blades of wheat cv Xinong 2718 were collected from seedlings and flowering plants. The samples were air-dried for 2 d in a desiccator at room temperature and carefully dissected. Dried leaf blade pieces were mounted on specimen stubs using double-sided copper tape and sputter-coated with gold particles using 90 s bursts from a sputter coater. Coated surfaces were viewed using a Hitachi S4800 scanning electron microscope at an accelerating voltage of 10kV and at a working distance of 12mm.

Cloning of wheat TaFAR5 full-length cDNA

Total RNA was extracted from durum wheat cv Xinong 2718 leaves using Trizol reagent (Invitrogen). To remove contaminating genomic DNA, total RNA was treated with DNase I (Promega). First-strand cDNA was synthesized using PrimeScript™ reverse transcriptase (TaKaRa) according to the manufacturer’s directions for use as a template for PCR amplification. A pair of gene-specific primers were designed based on the sequence of TaFAR5 (TaFAR5F 5′-ATGGTGGGCACGCTGGATGAGG-3′ and TaFAR5R 5′-TCACTTGTGGACGTACTTCATG-3′) and used to amplify the coding region. The cDNA was amplified using the following protocol: denaturation for 5min at 94 °C, followed by 35 cycles of 30 s at 94 °C, 30 s at 55 °C, and 2min at 72 °C, with a final extension for 10min at 72 °C. The resulting PCR product was separated by agarose gel electrophoresis (1% agarose) and extracted using a Gel Extraction Kit (TIANGEN). A 1.6kb DNA band was recovered, cloned into the pMD™ 18-T vector (TaKaRa) using T4 DNA ligase (TaKaRa), and subsequently transformed into DH5α cells. Successful isolation of TaFAR5 cDNA was confirmed by sequencing. The genomic sequence of TaFAR5 was deposited in the GenBank database under the accession number KJ725345.

Heterologous expression in yeast

The coding sequence of TaFAR5 was amplified from wheat cv Xinong 2718 cDNA using a primer labelled as TaFAR5-YS (Supplementary Table S1 available at JXB online). The corresponding PCR fragment was cloned into the yeast expression vector pYES2 (Invitrogen) to yield pYES-TaFAR5 under the control of the GAL1 promoter. The construct pYES-TaFAR5 was transformed into DH5α cells. Sequencing of an individual clone confirmed that there were no errors in TaFAR5. The pYES-TaFAR5 vector and an empty vector were transformed into the mutant yeast strain INVSc1 according to Gietz and Woods (2002). Transgenic yeast cells were grown on synthetic complete (SC) selection medium without uracil. Three individual cell lines were selected from each transgenic strain. After galactose induction and 24h of incubation in 0.1M potassium phosphate-containing glucose and haemin, cells were collected, refluxed for 5min in 20% (w/v) KOH/50% (v/v) ethanol, and extracted twice with hexane. Following phase separation, the chloroform phase was transferred to a fresh tube. The samples were evaporated to dryness under a stream of nitrogen, and then derivatized with 100 μl of bis-N, N-(trimethylsilyl)trifluoroacetamide plus 100 μl of pyridine at 70 °C for 60min, and analysed by GC-MS as described above for the plant wax analysis.

Agrobacterium tumefaciens-mediated genetic transformation of tomato

The TaFAR5 coding region was amplified from wheat cv Xinong 2718 cDNA using the LA Taq DNA polymerase (TaKaRa). The corresponding PCR fragment was cloned into the binary vector pCXSN which had been digested with XcmI (NEB) restriction enzyme under the control of the 35S promoter. Positive clones were identified by colony PCR and sequencing. Construct pCXSN-TaFAR5 and the empty vector were individually introduced into Agrobacterium tumefaciens strain GV3101, and verified by colony PCR and restriction digestion. Tomato cv MicroTom plants were transformed as described by Dan et al. (2006) with minor modifications. The transgenic plants were screened via hygromycin selection and confirmed by PCR.

Protein alignment and phylogenetic analysis

Multiple sequences were aligned with the ClustalW 1.83 program (Thompson et al., 1997) using default parameters (http://www.ebi.ac.uk/clustalw). The aligned sequences were imported into BioEdit for manual editing (http://www.mbio.ncsu.edu/BioEdit/bioedit.html; Hall, 1999). A Neighbor–Joining tree was constructed using MEGA software (version 3.1; http://www.megasoftware.net/index; Kumar et al., 2004) with the following parameters: Poisson correction, pairwise deletion, and bootstrap (1000 replicates, random seed).

Quantitative RT-PCR

RNA samples from various organs, including leaf, leaf sheath, root, internode, young panicle, anther, awn, glume, filling seed, and seedlings, under stress treatments were extracted using the Trizol reagent (Invitrogen) according to the manufacturer’s protocol. An additional DNase I (Promega) treatment was included to eliminate any contaminating DNA. The RNA was quantified spectrophotometrically. Then, total RNA was used to synthesize the oligo(dT)₁₈-primed first-strand cDNA using PrimeScript™ reverse transcriptase (TaKaRa) following standard protocols. Quantitative real-time PCR (RT-PCR) was performed in a 25 μl volume using a SYBR^® Premix Ex Taq™ Kit (TaKaRa) on a CFX96 real-time PCR detection system (Bio-Rad) according to the manufacturer’s instructions. The data were analysed using Opticon monitor software (Bio-Rad). Three replicates were performed for each sample. Wheat TaActin gene was used as an internal control.

Subcellular localization of TaFAR5

To determine the localization of TaFAR5 protein in plant cells, the coding region of TaFAR5 without a termination codon was cloned into the pA7-GFP vector, yielding a TaFAR5–green fluorescent protein (GFP) fusion construct. The fusion construct and ER marker mCherry–HDEL (Nelson et al., 2007) were transiently co-expressed in rice leaf protoplasts by PEG according to the previously described protocol (Chen et al., 2006). The fluorescence was observed with a confocal laser scanning microscope (Leica TCS-SP4).

Results

Chemical composition and chain-length distribution of the cuticular wax in wheat leaf blade

The chemical composition of the wheat leaf surface was analysed by GC-MS. Three or four leaf blades were obtained from the different seedling and flowering plants of wheat cv Xinong 2718, which is derived from Shaanxi Province, China. During the seedling stage (~80 d old), the leaf blades exhibited 594.9±21.0 μg dm^–2 of total wax load. Primary alcohols were the major components of the wax extract, accounting for 78.4±0.5% of the total wax load, followed by alkanes (11.3±0.7%), fatty acids (1.6±0.2%), aldehydes (1.2±0.2%), and esters (0.9±0.1%) (Table 1). The chain lengths of primary alcohols ranged from C20 to C32, of which C28 was the most dominant. Chain length distributions of alkanes varied from C23 to C33, with C27 being the dominant chain length. The major fatty acid constituent was C26, and lesser amounts of C20, C22, C24, and C28 fatty acids were found (Fig. 2A).

Table 1.

Amount and percentage of cuticular wax in leaf blades of seedling and flowering wheat cv Xinong 2718Mean values (μg dm^–2) of total wax loads and coverage of individual compound classes are given with the SD (n=3).

Wax composition	Measurement	Seedling	Flowering
Fatty acids	Amount	9.8±1.1	17.8±1.4
	Percentage	1.6±0.2	2.2±0.1
Aldehydes	Amount	7.5±0.8	9.7±1.7
	Percentage	1.2±0.2	1.2±0.1
Alkanes	Amount	66.9±3.6	145.4±9.8
	Percentage	11.3±0.7	18.3±0.8
β-Disketone	Amount	ND	260.7±25.8
	Percentage	ND	32.8±2.6
OH-β-Disketone	Amount	ND	5.7±1.3
	Percentage	ND	0.7±0.2
Primary Alcohols	Amount	466.1±17.2	277.7±13.9
	Percentage	78.4±0.5	35.0±1.5
Esters	Amount	5.5±0.5	6.2±0.2
	Percentage	0.9±0.1	0.8±0.1
Unidentified	Amount	42.4±7.3	39.2±5.5
	Percentage	7.1±1.0	4.9±0.6
Total Load	Amount	594.9±21.0	794.8±38.6

ND, not detected.

Fig. 2.

Cuticular wax composition in the leaf blade of wheat cv Xinong 2718. Wax coverage is expressed as μg dm^–2 of leaf blade surface area. (A) Composition of the leaf cuticular wax of seedling plants. (B) Composition of the leaf cuticular wax of flowering plants. Each wax constituent is designated by carbon chain length, and is labelled by chemical class along the x-axis. Each value is the mean of three independent measurements of individual plants. FA, fatty acids; Ald, aldehydes; Alk, alkanes; β-Dik, β-diketone; Alc, primary alcohols; OH-β-Dik, hydroxy-β-diketone; Est, esters; Uni, unindentified compounds. Error bars indicate the SD.

After 150 d, the total wax load on leaf blades of flowering Xinong 2718 increased to 794.8±238.6 μg dm^–2. It was mainly composed of primary alcohols (35.0±1.5%) and β-diketone (32.8±2.6%), followed by alkanes (18.3±0.8%), fatty acids (2.2±0.1%), aldehydes (1.2±0.1%), and esters (0.8±0.1%) (Table 1). It is interesting to note that although primary alcohols remained the major component of the cuticular waxes, the relative proportions of primary alcohols decreased 43.4% compared with seedling primary alcohols. The most drastic changes in wax composition occurred during the flowering stage: the percentages of β-diketone and OH-β-diketone increased significantly from 0 to 32.8±2.6% and from 0 to 0.7±0.2%, respectively (Table 1). The percentages of alkanes and fatty acids also increased relatively. The flowering leaf blades exhibited a similar chain length distribution of primary alcohols to that observed in the seedling leaf blades; even-numbered chain lengths ranging from C20 to C32 were observed, and C28 chains predominated (Fig. 2B). Furthermore, the chain length distribution of alkanes ranged from C23 to C33, and a shift in the dominant chain length towards C29 could be observed. This trend also occurred for fatty acids, the chain lengths of which ranged from C20 to C28, with a shift in the dominant chain length from C26 to C28. A single carbon chain length, C31, was detected for β-diketone and OH-β-diketone (Fig. 2B). The developmental changes in composition and chain length distribution confirm that wax biosynthesis occurs largely during leaf surface expansion.

Cuticular wax morphology on the leaf blades of wheat

To gain insight into the micromorphology of cuticular wax, the wax crystallites deposited on the adaxial and abaxial surfaces of seedling and flowering flag leaf blades were examined by SEM. The cuticular wax structure and density were similar on both the adaxial and abaxial sides of seedling leaf surfaces, which all formed platelets and were connected to a dense network (Fig. 3A–F). At the seedling stage, SEM showed no differences in wax morphology between the adaxial and abaxial leaf surfaces of the leaf blades. Strikingly, a clearly different morphology of wax deposition was observed between the adaxial and abaxial surfaces of flowering flag leaf blades. The adaxial cuticular wax layer was characterized by a covering of platelet-shaped wax crystals with irregular margins, some of which were connected to their neighbouring crystals. In contrast, the rod-shaped wax crystallites deposited on the abaxial surface densely covered the guard cells (Fig. 3G–L). Tulloch et al. (1980) detected a different morphology of wax deposition between adaxial and abaxial surfaces of the flag leaf in bread wheat. Wax morphology is determined by wax composition (Zhang et al., 2013). Since the amount of β-diketone significantly increased from 0 μg dm^–2 at the seedling stage to 277.7 μg dm^–2 at anthesis, it is inferred that the wax composition of rod-shaped wax crystallites may be comprised of β-diketone. These results indicate that the cuticular wax crystals on the leaf blades change with developmental stages in wheat.

Fig. 3.

SEM analysis of the wax crystals on the leaf surface of wheat cv Xinong 2718 at the seedling stage and flowering stage. (A–C) The adaxial sides of leaf blades at the seedling stage. (D–F) The abaxial sides of leaf blades at the seedling stage. (G–I) The adaxial sides of leaf blades at the flowering stage. (J–L) The abaxial sides of leaf blades at the flowering stage. A, D, G and J were detected by SEM at ×2000 magnification. B, E, H and K were detected by SEM at ×10 000 magnification. C, F, I and L were detected by SEM at ×30 000 magnification. Scale bars=4 μm (A, D, G, and J), 1 μm (B, E, H, and K), 0.2 μm (C, F, I, and L).

Isolation of TaFAR5 from wheat

The study demonstrates that primary alcohols are the most important components of cuticular waxes on the surface of wheat leaves. In order to isolate the primary alcohol synthetic gene in wheat cuticle, a BLASTN search of the wheat GenBank database was performed using the open reading frame (ORF) nucleotide sequence of Arabidopsis CER4 (GenBank accession no. NP_567936), which encodes a FAR involved in primary alcohol formation (Rowland et al., 2006). Eight sequences in the wheat genome exhibited a significant similarity to CER4 over their entire length, one of which was TAA1b, a cDNA that has previously been found to be expressed specifically in sporophytic tapetum cells (Wang et al., 2002). However, the cuticular wax synthesis function of TAA1b remains unknown. TAA1b is referred to hereafter as TaFAR5. PCR amplification was performed with a degenerate primer designed according to identified cDNA and gDNA sequences derived from leaf blades of wheat cv Xinong 2718 (Fig. 4A), and then the products were sequenced. The full-length cDNA of TaFAR5 is 1949bp and contains a 1569bp coding region, a 73bp 5’-untranslated region, and a 308bp 3’-untranslated region. The full-length TaFAR5 spans 4237bp of genomic DNA and contains eight exons and seven introns. The exons range from 91bp to 413bp, and the introns range from 78bp to 1758bp (Fig. 4B).

Fig. 4.

Homology cloning of TaFAR5. (A) Amplification of TaFAR5 from wheat cv Xinong 2718. (B) Schematic representation of the TaFAR5 gene structure. The thick black bars represent exons with the predicted translation start site (ATG) and stop code (TGA); the thin lines represent the introns. White boxes indicate the 5’- and 3’-untranslated regions (UTRs). gDNA, genomic DNA; M, DNA ladder DL2000 (left) and DL5000 (right).

Wheat TaFAR5 expression in yeast and transgenic tomato leaves results in primary alcohol production

To characterize the catalytic function of TaFAR5, its coding region was cloned into a yeast expression vector pYES2 under the control of the GAL1-inducible promoter. Subsequently, the recombinant plasmid was transformed into the yeast mutant strain INVSc1 (Invitrogen), which is deficient in storage lipid biosynthesis, thereby serving as a good host system to examine the involvement of TaFAR5 in wax biosynthesis. The recombinant strains containing pYES2-TaFAR5 or the empty vector as a negative control were induced with galactose for 24h, and then lipophilic compounds were extracted with hexane. An analysis of the wax indicated that the yeast cells that were transformed with TaFAR5 produced C22:0 primary alcohol (based on the GC-MS characteristics); in contrast, no primary alcohols were detected when using the empty vector control (Fig. 5A). This result confirms that TaFAR5 protein possesses a FAR activity, directly catalysing the reduction of fatty acids to primary alcohols using an endogenous C22:0 fatty acid as a substrate.

Fig. 5.

Heterologous expression of TaFAR5 in yeast and tomato. (A) GC analysis of primary alcohols in transgenic yeast. Lipids were hexane extracted from yeast cells and analysed by GC-MS. In the empty vector control, no primary alcohols were detected. In contrast, the yeast strains expressing TaFAR5 were found to contain the C22:0 primary alcohol. (B) Primary alcohol accumulation in mature leaves of transgenic T₁ generation tomato cv MicroTom. (C) Quantitative analysis of total leaf wax load. CK is the empty pCXSN vector control. The other lines contain the TaFAR5 coding region under the control of the 35S promoter. Significant differences are marked with asterisks (t-test: *P<0.05; **P<0.01).

Furthermore, TaFAR5 coding region fragments were subcloned into the binary vector pCXSN (Chen et al., 2009), and transformed into tomato cv MicroTom under the transcriptional control of the Cauliflower mosaic virus (CaMV) 35S promoter via A. tumefaciens infiltration (Supplementary Fig. S1 at JXB online). The transgenic lines harbouring the empty vector were used as control. No significant morphological differences were found between T₁ transgenic lines carrying the TaFAR5 gene and control lines. Subsequently, the lipidic compositions of chloroform-extractable leaf cuticular waxes derived from the transgenic lines were analysed by GC-MS. As anticipated, the total primary alcohol content per leaf area was dramatically higher in five independent T₁ transgenic lines harbouring pCXSN-TaFAR5 (Fig. 5B), whereas the contents of n-alkanes, branched alkanes, and triterpenoids were only slightly affected in the transgenic leaves (Fig. 5C). No new compound was identified in the transgenic lines. The largest change in the primary alcohol deposition was observed in transgenic line 37-3, which exhibited an increase of 75.3% in the amount of total primary alcohol, with a 116.9% increase in C26:0 primary alcohol, a 73.6% increase in C28:0 primary alcohol, and a 57.0% increase in C30:0 primary alcohol. These primary alcohols are the major components of the primary alcohols present (Fig. 5B). In addition, to determine further whether the quantitative differences in the primary alcohol content in the transgenic lines were caused by an alteration of the crystal pattern, TaFAR5 transgenic lines and empty vector control plants were examined by SEM. A comparison of the leaf cuticular wax crystalline patterns showed no significant differences between the transgenic lines and the control plants, the waxes of which were deposited as a smooth film without any crystalline structures present (Supplementary Fig. S2A–D). It was inferred that the increased primary alcohol content was not sufficient to form wax crystals on the leaf surface of tomato. Similar to the results for transgenic tomato leaves, the total primary alcohols of fruits, including C28:0-OH, C30:0-OH, C32:0-OH, and C34:0-OH, showed a significant increase in three independent transgenic lines (data not shown). Taken together, these results suggest that TaFAR5 synthesizes primary alcohol of the plant cuticle.

Characterization of the predicted TaFAR5 protein

Sequence analysis indicated that TaFAR5 encodes a polypeptide of 522 amino acids. Protein domain searches against the Pfam database revealed that the deduced protein TaFAR5 contains an NAD_binding_4 (NADB) domain and a Sterile domain (Fig. 6A). Previous reports revealed that EgFAR (Euglena gracilis) and DPW (rice) both contain an NADB domain, and a Male Sterile 2 (MS2) domain encodes a FAR; therefore, it is possible that TaFAR5 encodes a FAR protein that is involved in the synthesis of primary alcohol. Based on TMHMM program analysis, TaFAR5 appears to be an integral membrane protein possessing a highly conserved transmembrane-spanning domain at its C-terminus (data not shown). To determine the molecular mass of TaFAR5, the coding region was cloned into the pET28a vector (Novagen) and expressed in E. coli BL21 (DE3). Recombinant strains containing pET-TaFAR5 or the empty vector as a control were induced with 0.5mM isopropyl-β-d-1-thiogalactopyranoside for 48h and shaken slowly at 20 °C to improve expression of TaFAR5. In agreement with the predicted size, the molecular mass of TaFAR5 protein was estimated to be 58.4kDa by SDS–PAGE analysis (Fig. 6B). Multiple alignments were performed for the eight plant proteins with high similarity to the TaFAR5 protein. TaFAR5 exhibits 74% identity with TAA1a and 74% identity with TAA1c, which are the most similar orthologues to TaFAR5, and ~40% identity with five Arabidopsis homologues CER4, FAR1, FAR4, FAR5, and FAR8 (Fig. 6C). Phylogenetic analysis of the 18 proteins with high similarities to TaFAR5 showed that these proteins can be grouped into two clades. TaFAR5, TAA1a, TAA1c, and two rice proteins were grouped into the first clade. Five Arabidopsis proteins (CER4, FAR1, FAR4, FAR5, and FAR8) belong to the second clade. Interestingly, the monocot proteins form one clade, and the dicot proteins form the second clade (Fig. 6D). These results suggest that TaFAR5 and its homologues play key roles in primary alcohol biosynthesis among divergent monocot and dicot plants.

Fig. 6.

A schematic diagram illustrating the functional domains of TaFAR5, and multiple alignments and phylogenic analysis of TaFAR5-related proteins. (A) Protein structure of TaFAR5. (B) SDS–PAGE of TaFAR5 protein. Arrows indicate the HIS-TaFAR5 protein. (C) Multiple alignment of wheat TaFAR5 with seven related proteins with the ClustalX 1.83 program using default parameters. Identical and similar amino acid residues are shown on black and grey backgrounds, respectively. Gaps required for optimal alignment are indicated by dashes. NAD_binding_4 and Sterile domains are indicated by stars and grey lines under the sequences, respectively. (D) Phylogenetic tree of the TaFAR5-related proteins constructed by MEGA 3.1 with the Neighbor–Joining method. The number for each interior branch shows the percentage of the bootstrap value (1000 replicates), and only values >50% are shown. Homologous genes have the following accession numbers: Triticum aestivum Ta TAA1a (AJ459249), Ta TAA1c (AJ459253), Ta MSF_1 (CBI75514), and Ta MSF_2 (CBI75517); Brachypodium distachyon Bd LOC100827426 (XP_003578677) and Bd LOC100824003 (XP_003574925); Oryza sativa Os09g0567500 (NP_001063962) and Os08g0557800 (NP_001062488); Sorghum bicolor Sb02g011920 (XP_002459832) and Sb07g024240 (XP_002445686); Zea mays Zm LOC100501369 (NP_001183038); Arabidopsis thaliana At CER4 (NP_567936), At FAR1 (NP_197642), At FAR4 (NP_190040), At FAR5 (NP_190041), and At FAR8 (NP_190042); Artemisia annua Aa GFAR1 (ADK66305).

Expression pattern of TaFAR5 and the subcellular localization of the TaFAR5 protein

In order to gain a better understanding of the function of TaFAR5, its expression pattern was examined by quantitative RT-PCR analysis. Total RNA was isolated from various vegetative and reproductive organs of wheat cv Xinong 2718. As shown in Fig. 7A, a high level of TaFAR5 transcript was detected in leaf blades, anthers, pistils, and seeds of the wheat, and modest transcript levels were observed in internodes, leaf sheaths, young panicles, and glumes. Meanwhile, a low level of TaFAR5 transcript was detected in the roots. It is possible that TaFAR5 catalyses the production of primary alcohols in leaf blades, where primary alcohols were detected.

Fig. 7.

Expression analysis of TaFAR5 and subcellular localization of the TaFAR5 protein. (A) Quantitative RT-PCR analysis showing the expression of TaFAR5. LB, young leaf blades; LS, leaf sheaths; R, roots; IN, internodes; YP, young panicles; AN, anthers; PI, pistils; G, glumes; S, seeds. Wheat TaActin was used as reference. Error bars indicate the SE of three independent samples. (B–E) Subcellular localization of TaFAR5 in the rice protoplast. (B) GFP fluorescence of the protoplast cell expressing 35S:TaFAR5–GFP. (C) ER marker mCherry–HDEL. (D) Bright-field image of the protoplast cell. (E) A merged image of B, C, and D. Scale bars=5 μm. (F) Chromosomal localization of TaFAR5. RT-PCR was performed on 11 nullisomic–etrasomic (N-T) lines of wheat cultivar Chinese Spring with two pairs of primers, TaFAR5a and TaFAR5b, which specifically amplify different chromosomal assignment of TaFAR5, respectively. (This figure is available in colour at JXB online.)

Most of the known wax synthesis-related enzymes are located in the ER (Xu et al., 2002; Zheng et al., 2005; Rowland et al., 2006; Greer et al., 2007; Haslam et al., 2012; Mao et al., 2012; Rajangam et al., 2013). To investigate the subcellular localization of TaFAR5, TaFAR5 was transiently expressed in rice leaf protoplasts. The C-terminus of the TaFAR5 coding sequence was fused with GFP under the control of the CaMV 35S promoter, and the TaFAR5–GFP construct was transferred into rice leaf protoplasts by the PEG-mediated method. To mark the ER, a red fluorescent protein (RFP) mCherry–HDEL was co-transformed into the protoplasts (Nelson et al., 2007; Mao et al., 2012). As expected, overlap of the GFP signal generated by TaFAR5–GFP and the RFP signal generated by HDEL–RFP results in an orange fluorescent signal (Fig. 7B–E). Fluorescence microscopy revealed that the ER marker fluorescence and the TaFAR5–GFP fluorescence completely overlapped, indicating that TaFAR5 is located in the ER.

TaFAR5 is located on wheat chromosome 4D

To determine the chromosomal location of TaFAR5, a query of the wheat genome database (http://wheat-urgi.versailles.inra.fr/Seq-Repository/BLAST) with the genomic sequence of TaFAR5 using BLAST search programs revealed that the entire 4237bp of TaFAR5 sequence completely matches the sequence of the wheat chromosome 4DS contig, without nucleotide mutation, indicating that TaFAR5 is located on wheat chromosome 4DS. In addition, a set of 11 wheat cv Chinese Spring nullisomic–tetrasomic substitution lines were used as templates for cDNA amplification using two pairs of TaFAR5-specific primers, named TaFAR5a and TaFAR5b, respectively. As shown in Fig. 7F, no PCR product was detected in N4DT4A which lacked the whole of chromosome 4A and had the whole of chromosome 4D added; in contrast, the PCR product was obtained from all of the other 10 Chinese Spring nullisomic–tetrasomic substitution lines. Consistent with the result of the BLAST search, TaFAR5 was mapped to wheat chromosome 4DS.

Transcriptional regulation of TaFAR5 under abiotic stress and ABA treatments

Previous investigations have established that Arabidopsis wax synthesis is induced by water deficit, sodium chloride, and ABA treatments (Kosma et al., 2009). To examine how the level of TaFAR5 transcript is induced, the abundance of TaFAR5 under stress conditions was analysed. Quantitative RT-PCR was performed to measure the abundance of TaFAR5 transcripts in 4-week-old chamber-grown seedlings of wheat cv Xinong 2718 subjected to various stress treatments at six different times (0, 2, 4, 6, 12, and 24h). When seedlings were exposed to the air, dehydration led to an increase of the relative abundance of TaFAR5 transcripts by 2.8-fold for up to 2h compared with 0h, and then dropped to normal levels by 4h (Fig. 8A). Consistent with the dehydration induction, TaFAR5 transcripts increased 2.6- and 2.2-fold within 4h under 20% PEG 6000 and cold treatments, respectively (Fig. 8B, C). Based on these observations, it is concluded that transcriptional control plays a major role in the regulation of TaFAR5 during drought and cold stress. ABA is an important signal in abiotic stress responses. Previous studies have shown that some wax genes are induced in ABA-dependent stress signalling. To explore this notion further, the ABA-induced expression of TaFAR5 was analysed in 4-week-old wheat seedlings exposed to 100 μM ABA treatments. As expected, TaFAR5 expression exhibited a continual rise up to at least 4h after induction by ABA, thereby demonstrating that TaFAR5 is induced by drought and cold stresses in an ABA-dependent manner (Fig. 8D).

Fig. 8.

Expression analysis of TaFAR5 in wheat cv Xinong 2718 seedlings under abiotic stress and ABA treatment. Four-week-old soil-grown plants were exposed to various stress treatments. Relative transcript levels were determined by quantitative RT-PCR using the TaActin gene as an internal control. Error bars represent the SE of three independent experiments. Drought treatment, plants were removed from the soil and allowed to dry on filter paper; PEG treatment, 20% (w/v) PEG 6000; cold treatment, 4 °C; ABA treatment, 100 μM ABA.

Discussion

Analysis of primary alcohols in cuticular wax from wheat leaf blades has shown that they are all saturated with C20–C32 carbons, and C28 carbons are the most frequent species (Bianchi and Figini, 1986; Koch et al., 2006; Adamski et al., 2013; Zhang et al., 2013). Very little is known about the importance of FAR enzymes in governing the composition of the primary alcohols in wheat cuticle. The principal goal of this study was to identify and characterize a primary alcohol biosynthesis-related gene that is involved in the acyl reduction pathway. Here, a new function for TaFAR5, which is identical to TAA1b, an anther-specific gene (Wang et al., 2002), is shown, namely its involvement in the primary alcohol biosynthesis of leaf blade cuticular wax in wheat.

The present study shows that the components, amount, and micromorphology of cuticular waxes vary considerably during various stages of growth, and primary alcohols are major components of cuticular waxes in the leaf blades of wheat. These results are consistent with those of Tulloch (1973), who revealed that primary alcohols are major components of wax from leaf blades of spring wheat varieties Selkirk and Manitou. Before sheaths and flag leaf are completely developed, the major component is octacosanol. When sheath development is complete, β-diketone content is greatest. Additional studies in other species have also supported the present view. Cuticular wax of leaves in Hedera helix contained high percentages of alkanes, aldehydes, primary alcohols, and fatty acids early on, and increasing amounts of alkyl esters during seasonal development (Hauke and Schreiber, 1998). In fact, primary alcohols are always the major components of cuticular waxes on wheat leaf blades; when β-diketone is present, the primary alcohol content is correspondingly lower. Based on the observed differences in the component and micromorphology of wheat leaf blades, it is proposed that platelet-shaped wax crystals are associated with primary alcohols, and rod-shaped wax crystallites are characteristic of β-diketones. This view is supported by the findings that lobed plates of cuticular wax on Quercus robur are associated with primary alcohols (Gülz, 1994), and long thin tubes are characteristic of β-diketones on Hordeum vulgare (barley) lemma (von Wettstein-Knowles, 1995).

The TaFAR5 gene cloned from wheat leaf blade encodes a FAR with a predicted NADB domain and a Sterile domain. SDS–PAGE confirmed that TaFAR5 has a molecular mass of 58.4kDa, which is similar to the molecular masses of Arabidopis CER4 (Rowland et al., 2006), jojoba FAR (Metz et al., 2000), and E. gracilis EgFAR (Teerawanichpan and Qiu, 2010). Sequence analysis revealed that TaFAR5 shares ~40% identity with five Arabidopsis homologues, CER4, FAR1, FAR4, FAR5, and FAR8. Therefore, it is most likely that TaFAR5 enzyme has a similar function in catalysing primary alcohol synthesis to Arabidopis FARs, jojoba FAR, and E. gracilis EgFAR. Indeed, two pieces of evidence clearly indicate that TaFAR5 functions as a FAR enzyme in primary alcohol biosynthesis for the production of cuticular waxes. First, functional analysis of TaFAR5 in yeast indicated that TaFAR5 effectively converts C22:0 fatty acid to the corresponding primary alcohol. Secondly, the expression of TaFAR5 in transgenic tomato results in the accumulation of C26:0, C28:0, and C30:0 primary alcohols in leaves. Previous studies have demonstrated that expression of CER4 in yeast results in the production of C24:0-OH and C26:0-OH (Rowland et al., 2006; Domergue et al., 2010). Moreover, in yeast, recombinant FAR1, FAR4, and FAR5 are able to form alcohols using distinct but overlapping substrates with a chain length ranging from C18:0 to C24:0 (Domergue et al., 2010). Expression of E. gracilis EgFAR in yeast led to the accumulation of C14:0 and C16:0 primary alcohols (Teerawanichpan and Qiu, 2010), and expression of jojoba FAR in E. coli resulted in accumulation of C16:0-OH and C18:1-OH (Metz et al., 2000). TaFAR5 possesses a narrower substrate range than other biochemically characterized FARs and only uses saturated fatty acid with a C22 carbon chain as substrate, with the C22:0 fatty acid preferred. Interestingly, it was found that TaFAR5 has very distinct substrate specificities between yeast and tomato, with C22:0 fatty acid preferred in yeast, but C26:0, C28:0, and C30:0 fatty acids preferred in tomato. Previous studies have established that wheat TAA1a can produce C16:0 and C18:1-OH when expressed in E. coli, but that transgenic tobacco accumulates C18:1, C24:0, and C26:0 primary alcohols (Wang et al., 2002). Expression of CER4 in yeast results in the production of C24:0-OH and C26:0-OH, but molecular complementation of cer4-1 produced C24:0, C26:0, C28:0, and C30:0 primary alcohols (Rowland et al., 2006). This raises the questions of to what extent the specificity of the FAR governs the composition of the primary alcohol produced in the plant, and to what extent this specificity is governed by FAR in yeast cells.

As anticipated, TaFAR5 expression was detected most prominently in leaf blades, consistent with a role for this gene in cuticular wax production. Surprisingly, TaFAR5 expression was not restricted to the epidermis of aerial parts. Its expression was also detected in anthers, pistils, and seeds, and this result led the authors to suggest that TaFAR5 is implicated not only in wax metabolism but also in the anther cutin biosynthesis pathway. Indeed, TaFAR5 has previously been reported as an anther-specific gene (Wang et al., 2002). Moreover, this is not the first study in which anther expression of genes associated with wax metabolism has been reported. Other genes involved in cuticle formation are also expressed in anthers, namely CER1 (Aarts et al., 1995), CER2 (Xia et al., 1996), CER3 (Hannoufa et al., 1996), WAX2 (Chen et al., 2003), WIN1 (Broun et al., 2004), and CER8 (Lü et al., 2009). Additional RNA interference experiments in wheat are required to determine whether TaFAR5 is implicated in anther cutin biosynthesis. Enzymes that catalyse the initial steps of wax synthesis, the formation of VLCFA wax precursors, are associated with the ER in all plant species investigated to date (Samuels et al., 2008). It was likewise anticipated that if TaFAR5 were involved in primary alcohol synthesis, this protein would localize to the ER. Visualization of the functional TaFAR5–GFP fusion protein in rice leaf protoplasts revealed that the wheat TaFAR5 is also localized to the ER. In Arabidopsis, the midchain hydroxylase MAH1 that catalyses the final step of the decarbonylation pathway, the formation of secondary alcohols and ketones, was also confined to the ER of stem epidermal cells (Greer et al., 2007). The diacylglycerol acyltransferase WSD1 which catalyses the formation of final alkyl esters of the acyl reduction pathway of cuticular wax biosynthesis, resides in the ER membranes (Li et al., 2008). Accordingly, it is proposed that the ER is the subcellular compartment in which most of the cuticular wax components are deposited. These wax components are then delivered from this compartment to the plasma membrane for export toward the cuticle (Samuels et al., 2008).

Cuticular waxes are known to play an important role in plant resistance to environmental stresses, such as water-limited environments (Kosma and Jenks, 2007). In Arabidopsis, cuticle composition and gene expression indicate that wax synthesis is induced by water deficit, sodium chloride, and ABA treatments (Kosma et al., 2009). This suggests that TaFAR5 plays a vital role in stress response, including drought and cold stresses, as its expression was induced under these conditions. Like TaFAR5, several genes encoding enzymes involved in wax biosynthesis are induced by osmotic stresses. CER1, with a function in alkane synthesis, was highly induced by water deficit, NaCl, and ABA (Kosma et al., 2009; Bourdenx et al., 2011). CER6, a VLCFA-condensing enzyme, was induced by osmotic stress and the presence of ABA (Hooker et al., 2002). DSO is also induced by multiple stresses, including ABA, high salt, and glucose (Alvarado et al., 2004; Panikashvili et al., 2007). Nevertheless, the molecular events implicated in these regulations have not yet been established.

In conclusion, this study presents strong evidence that TaFAR5 plays an important role in leaf surface wax synthesis in the monocot wheat. Furthermore, TaFAR5 is linked to responses to drought and cold stresses in an ABA-dependent manner.

Supplementary data

Supplementary data are available at JXB online.

Figure S1. Genetic transformation of TaFAR5 in tomato cv MicroTom driven by the CaMV 35S promoter.

Figure S2. SEM images of cuticular wax crystal patterns on tomato leaves.

Table S1. Sequences of the primers used for genomic PCR, qRT-PCR, yeast expression, and subcellular localization experiments in this study.

Glossary

Abbreviations:

ER

endoplasmic reticulum

FAR

fatty acyl-coenzyme A reductase

GC-MS

gas chromatography–mass spectroscopy

SEM

scanning electron microscopy

VLCFA

very long chain fatty acid.