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. 2011 Sep 27;157(3):1555–1567. doi: 10.1104/pp.111.183533

A New Insight into Application for Barley Chromosome Addition Lines of Common Wheat: Achievement of Stigmasterol Accumulation1,[W]

Jianwei Tang 1,2, Kiyoshi Ohyama 1,2,3, Kanako Kawaura 1, Hiromi Hashinokuchi 1, Yoko Kamiya 1, Masashi Suzuki 1, Toshiya Muranaka 1,4, Yasunari Ogihara 1,*
PMCID: PMC3252158  PMID: 21951468

Abstract

Barley (Hordeum vulgare) has a much higher content of bioactive substances than wheat (Triticum aestivum). In order to investigate additive and/or synergistic effect(s) on the phytosterol content of barley chromosomes, we used a series of barley chromosome addition lines of common wheat that were produced by normal crossing. In determining the plant sterol levels in 2-week-old seedlings and dry seeds, we found that the level of stigmasterol in the barley chromosome 3 addition (3H) line in the seedlings was 1.5-fold higher than that in the original wheat line and in the other barley chromosome addition lines, but not in the seeds. Simultaneously, we determined the overall expression pattern of genes related to plant sterol biosynthesis in the seedlings of wheat and each addition line to assess the relative expression of each gene in the sterol pathway. Since we elucidated the CYP710A8 (cytochrome P450 subfamily)-encoding sterol C-22 desaturase as a key characteristic for the higher level of stigmasterol, full-length cDNAs of wheat and barley CYP710A8 genes were isolated. These CYP710A8 genes were mapped on chromosome 3 in barley (3H) and wheat (3A, 3B, and 3D), and the expression of CYP710A8 genes increased in the 3H addition line, indicating that it is responsible for stigmasterol accumulation. Overexpression of the CYP710A8 genes in Arabidopsis increased the stigmasterol content but did not alter the total sterol level. Our results provide new insight into the accumulation of bioactive compounds in common wheat and a new approach for assessing plant metabolism profiles.

Wheat (Triticum aestivum) is one of the three major cereal crops. It has an advantage over barley (Hordeum vulgare) in that wheat flour is more suitable for processing for many kinds of foods, such as bread, noodles, cookies, and cakes. In contrast, barley is rich in health-beneficial bioactive compounds, such as γ-aminobutyrate, β-glucan, minerals, and vitamins (Kihara et al., 2007). If these advantages of barley could be incorporated into wheat, we could utilize wheat enriched in these bioactive compounds.

Barley chromosome addition lines of common wheat are a suitable genetic resource for our studies. To introduce valuable traits to a recipient species in plants, transgenic methods are an efficient technique, but there are technical difficulties with using these in wheat, and transgenic products are not well accepted by consumers. Genetic hybridization (cross-breeding) is a nontransgenic method widely applied to plant breeding. A set of barley chromosome addition lines of common wheat were developed through distant hybridization between hexaploid wheat (cv Chinese Spring; 2n = 6x = 42; AABBDD) and diploid barley (cv Betzes; 2n = 2x = 14; HH; Islam et al., 1975). This set of disomic addition lines of barley chromosomes in a genetic background of common wheat contains six lines with additions of chromosomes 2H to 7H; the 1H disomic addition line was not produced, because gene(s) on 1HL caused sterility in hybrids (Islam et al., 1981; Islam and Shepherd, 1990). Subsequently, a ditelosomic addition line of barley 1HS was bred (Islam et al., 1981; Islam, 1983; Islam and Shepherd, 1990, 2000). Barley chromosome addition lines of common wheat have been utilized for a variety of purposes. Usually, they have been used for cytological and genetic mapping of barley genes (Ashida et al., 2007; Kato et al., 2008; Sakai et al., 2009; Sakata et al., 2010). Transcripts of wheat-barley disomic addition lines and ditelosomic addition lines were profiled using the Affymetrix Barley1 GeneChip probe array. The expressed barley genes in each addition line were mapped onto chromosomes and chromosome arms (Cho et al., 2006; Bilgic et al., 2007). On the other hand, it is believed that barley chromosomes in a wheat genetic background produce bioactive compounds with an additive or synergistic action. However, little is known about the nutritional effect of barley chromosome addition lines of common wheat. Therefore, we aimed to identify and estimate overall bioactive compounds in each of the barley chromosome addition lines by integrated analysis of the metabolome and transcriptome.

In this paper, we focused on phytosterols. Phytosterols are well known for reducing the plasma cholesterol levels of humans when administered orally. Phytosterols have received U.S. Food and Drug Administration clearance as generally recognized as safe. Foods containing phytosterols can carry health claims indicating their cholesterol-reducing properties (Kamal-Eldin and Moazzami, 2009). Phytosterols have also been reported that have antiinflammatory properties, such as antiosteoarthritic properties (Gabay et al., 2010). Stigmasterol, campesterol, and sitosterol are the main molecular species of phytosterols. Stigmasterol was reported to inhibit hepatic cholesterol synthesis (Batta et al., 2006) and to have the highest affinity to the chondrocyte membrane (Gabay et al., 2010). These observations suggest that stigmasterol is the most valuable of the three main phytosterols.

In plants, most phytosterols are involved in membrane function, modulating membrane permeability and fluidity (Benveniste, 2004; Schaller, 2004). Phytosterols also serve as precursors for the biosynthesis of brassinosteroids, functioning as plant growth regulators, including cell division and expansion, responses to light and dark, morphogenesis, apical dominance, and gene expression (Schrick et al., 2002; Nemhauser and Chory, 2004). In plants, sterol synthesis mainly starts from the cytosolic mevalonate pathway (Newman and Chappell, 1999; Kasahara et al., 2002), leading to the C5 building blocks isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP). IPP and DMAPP are also synthesized in plastids through a 2-C-methyl-d-erythritol 4-phosphate pathway. Although metabolic cross talk between the mevalonate and 2-C-methyl-d-erythritol 4-phosphate pathways was reported (Kasahara et al., 2002; Nagata et al., 2002; Hemmerlin et al., 2003), the contribution of this cross talk is minor (Suzuki et al., 2009). Cytosolic IPP and DMAPP are condensed to generate the C30 precursor squalene and further converts into 2,3-oxidosqualene. Cytosolic IPP is the starting point of several metabolic branches leading to the synthesis of a variety of essential isoprenoids, including sterols, brassinosteroids, polyprenols, sesquiterpenes, and cytokinins (Fig. 1). Phytosterol biosynthesis is initiated by the enzymatic conversion of 2,3-oxidosqualene to cycloartenol, which is catalyzed by cycloartenol synthase 1 (CAS1). Although genes encoding lanosterol synthase (Kolesnikova et al., 2006; Sawai et al., 2006; Suzuki et al., 2006) and phytosterol biosynthesis via lanosterol in Arabidopsis (Ohyama et al., 2009) were reported recently, no genes encoding lanosterol synthase have been identified from monocotyledonous plants (Suzuki et al., 2006). Phytosterol biosynthesis, then, is divided into two branches from 24-methylene lophenol. One branch produces 24-methyl sterols inducing campesterol and 24-methyl-Δ22-sterols (a mixture of brassicasterol and crinosterol). The second branch produces 24-ethyl sterols including sitosterol and stigmasterol (Fig. 1). This key branch point is controlled by sterol-C24-methyltransferase 2 (SMT2). The orientation of sterol biosynthetic flux toward campesterol or sitosterol is achieved via SMT2 enzyme activity in plants (Schaeffer et al., 2001; Carland et al., 2002). CAS1 and SMT2 genes are plant specific. The precise regulation of these compounds is important for normal plant growth and development. Stigmasterol synthesis is the end step of the 24-ethyl sterol pathway. The reaction is catalyzed by a C-22 desaturase belonging to the cytochrome P450CYP710A family. Arabidopsis (Arabidopsis thaliana) CYP710A genes consist of four isogenes (AtCYP710A1, -A2, -A3, and -A4). Overexpression of CYP710A1, -A2, and -A4 in Arabidopsis contributed to stigmasterol synthesis (Morikawa et al., 2006a; Arnqvist et al., 2008). CYP710A2 is also the desaturase responsible for the production of 24-methyl-Δ22-sterols (Morikawa et al., 2006a). No drastic phenotypic alterations were observed in AtCYP710A-overexpressing plants (Morikawa et al., 2006a; Arnqvist et al., 2008).

Figure 1.

Figure 1.

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Schematic diagram of the plant sterol biosynthetic pathway. The plant sterol biosynthetic pathway is shown referencing the KEGG pathway database (http://www.kegg.jp/kegg/pathway.html), Suzuki et al. (2004), and Morikawa et al. (2006a). The nomenclature used in Arabidopsis referencing the KEGG pathway database was adopted. The genes encoding enzymes analyzed are numbered. FPP, Farnesyl diphosphate; GPP, geranyl diphosphate; MAV, mevalonate; MEP, 2-C-methyl-d-erythritol 4-phosphate.

In this paper, by systematic analysis of the overall sterol profiling in 2-week-old seedlings and dry seeds from a series of barley chromosome addition lines of common wheat having six of seven whole barley chromosomes (2H–7H) and a ditelosomic addition line (1HS), we found that only stigmasterol accumulated in the seedlings of the barley chromosome 3 (3H) addition line but not in the grains. Simultaneously, we integrated these sterol profiles with the transcriptome related to phytosterol biosynthesis. Furthermore, we isolated full-length cDNAs of wheat and barley CYP710A8 (cytochrome P450 subfamily) genes. These CYP710A8 genes were mapped on chromosome 3 in barley (3H) and wheat (3A, 3B, and 3D), and the expression of CYP710A8 genes increased in the 3H addition line. Overexpression of the CYP710A8 genes in seedlings of Arabidopsis increased the stigmasterol content but did not alter the total sterol level. Thus, we elucidated the CYP710A8 genes encoding sterol C-22 desaturase as a key characteristic for the higher level of stigmasterol in the 3H addition line. Our results provide new insight into the accumulation of bioactive compounds in common wheat and an approach for assessing plant metabolism profiles.

RESULTS

Barley Chromosome Added to Common Wheat Increased the Functional Sterol Level in Seedlings

Phytosterol profiles of leaves from 2-week-old seedlings of common wheat (cv Chinese Spring [CS]) and barley (cv Betzes) and barley chromosome addition lines (1HS and 2H–7H) were examined (Fig. 2). The total sterol levels averaged 191.58 μg 100 mg−1 dry weight in Betzes and 203.74 μg 100 mg−1 dry weight in CS based on three measurements. No significant difference in campesterol or total sterol levels was observed in Betzes, CS, or barley chromosome addition lines. Stigmasterol, sitosterol, and campesterol were the main phytosterols, and stigmasterol is considered the most functional sterol among them (Batta et al., 2006). In Betzes, the sterols consisted of 34.3% stigmasterol, 42.1% sitosterol, and 18.5% campesterol, and in CS, they consisted of 15.6% stigmasterol, 59.0% sitosterol, and 21.4% campesterol. The sterol compositions of addition lines, except for the 3H addition line, were similar to that of CS. In contrast, the 3H addition line consisted of 22.8% stigmasterol, 49.6% sitosterol, and 23.8% campesterol, a stigmasterol level that was 1.5-fold higher than that of CS and other barley chromosome addition lines, with a corresponding decrease in the level of sitosterol. Cholesterol, sitostanol, and campestanol levels were very low in all barley, wheat, and barley chromosome addition lines, contributing 1% to 2% of the total sterol. 24-Methyl-Δ22-sterol was only detected at trace levels in both barley and wheat seedlings (data not shown). Our results showed that only the 3H addition line had an altered phytosterol profile, which suggested that certain gene(s) of 3H are very important for stigmasterol synthesis.

Figure 2.

Figure 2.

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Sterol profiles of leaves from common wheat (CS), barley (Betzes), and barley chromosome addition lines. Leaves of 2-week-old seedlings were analyzed. Data are mean values ± sd from three independent leaf samples. Asterisks indicate significant differences between common wheat and other barley chromosome addition lines of common wheat (P < 0.01). DW, Dry weight.

Barley Chromosome Addition Lines of Common Wheat Did Not Increase Functional Sterol Levels in Grains

Phytosterol profiles of dry grains of common wheat (CS), barley (Betzes), and barley chromosome addition lines (1HS and 2H–7H) were examined (Fig. 3). In the grains, seed and fruit coats were contained. The total sterol levels averaged 61.39 μg 100 mg−1 dry weight in Betzes and 55.67 μg 100 mg−1 dry weight in CS based on three measurements. Total sterol levels in grains were one-third-fold lower than those in seedlings (Fig. 3). All sterol levels of the barley 2H chromosome addition line so far detected were significantly lower than those of all other lines, including wheat and barley parental lines. Levels of campesterol and sitosterol in Betzes were significantly higher than those of the other lines, while levels of campestanol and sitostanol were dramatically lower in Betzes. Levels of stigmasterol and cholesterol were very low in all lines (Fig. 3). Our results indicate that barley chromosome addition to common wheat did not bring about clear additional and/or synergetic effects to increase functional steroid levels in the wheat grains. Therefore, we focused on the phytosterol pathway(s) in seedlings of wheat and barley.

Figure 3.

Figure 3.

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Sterol profiles of grains from common wheat (CS), barley (Betzes), and barley chromosome addition lines. Dry seeds were analyzed. Data are mean values ± sd from three independent leaf samples. DW, Dry weight.

Expression of Genes Related to the Biosynthesis of Phytosterols in Seedlings

The expression of sterol pathway-related genes in the seedlings of wheat and barley was examined in relation to stigmasterol accumulation, because an imbalance of certain enzymes in the sterol biosynthetic pathways can lead to an accumulation of functional steroids. In Arabidopsis, overexpression of 3-hydroxy-3-methylglutaryl-CoA reductase isoform 1S (HMGR1S) increased the total sterol level and overexpression of farnesyl diphosphate synthase isoform 1S (FPS1S) increased the stigmasterol level (about 1.8-fold that of the wild type; Masferrer et al., 2002; Manzano et al., 2004). The hydra1 (hyd1) and hydra2 (fk) mutants showed lower levels of campesterol and sitosterol but a higher level of stigmasterol than the wild type (Topping et al., 1997; Souter et al., 2002). Hence, it is interesting to assess the gene expression pattern at each step of the sterol pathway. A custom wheat 38k oligo-DNA microarray (wheat 38k microarray; Agilent) was used to examine overall gene expression patterns in CS and in each barley chromosome addition line. Orthologous genes in wheat and barley shared high sequence homology. Thus, 60-mer probes on the wheat 38k microarray could hybridize to either wheat genes or most barley genes. The wheat 38k microarray could be utilized for screening of genes with expression levels specifically increased by an additive effect or synergistic action between wheat and barley chromosomes. In this manner, each of the wheat and barley HMGR genes, FPS1 genes, and genes related to the biosynthetic pathways from (S)-2,3-epoxysqualene to campesterol and sitosterol (Fig. 1) were identified, using the corresponding amino acid sequences from Arabidopsis or rice (Oryza sativa) as queries for a tBLASTn search against the KOMUGI database (http://www.shigen.nig.ac.jp/wheat/komugi/), Barley DB (http://www.shigen.nig.ac.jp/barley/), TriFLDB (http://trifldb.psc.riken.jp/index.pl), and HarvEST: Barley version 1.78 (http://www.harvest-web.org/). We adopted the gene nomenclature of Arabidopsis, with the letters “Ta” for wheat and “Hv” for barley. The corresponding original gene names in the wheat 38k microarray are listed in Supplemental Table S1. The expression patterns of genes were analyzed according to the conditions described in “Materials and Methods.” Data are shown as values relative to CS (Fig. 4). The expression of the HYD1 and DWARF5 (DWF5) genes was significantly increased in the 3H addition line. The expression level of HYD1, which corresponds to the probe from wheat0130Contig13954, was 1.6-fold higher (Fig. 4B), and the DWF5 gene, which corresponds to each of the probes rwhf14j19 and MUGEST2003_23lib_Contig7208, was 2.3- to 3.1-fold higher than CS (Fig. 4C). The expression of other genes of addition lines was similar to that of CS wheat. The expression patterns of HYD1, DWF5, and SMT2 genes were confirmed by real-time PCR using primers annealing to both barley and wheat genes (Supplemental Fig. S1A). These gene expressions were compared with that of SMT2, because SMT2 is located at the key position for phytosterol biosynthesis. The results were consistent with those from the 38k wheat microarray analysis. On the 38k wheat microarray, no probes corresponding to CYP710A genes were spotted.

Figure 4.

Figure 4.

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Molecular analysis of genes related to the biosynthesis of plant sterols. A, Expression patterns of HMGR and FPS1 genes. B, Expression patterns of genes related to the pathway from (S)-2,3-epoxysqualene to 24-methylenelophenol. C, Expression patterns of genes related to the pathway from 24-methylenelophenol to campesterol and sitosterol. The expression patterns in A to C were analyzed using a custom wheat 38k oligo-DNA microarray. Data are relative to CS and are mean values ± sd from three independent leaf samples. D, The assigned barley chromosome for steroid biosynthesis-related genes. Genes were assigned to each of seven barley chromosomes according to data obtained using the Affymetrix Barley1 GeneChip probe array for wheat-barley addition lines from Cho et al. (2006) and Bilgic et al. (2007; http://www.barleybase.org/; accession nos. BB8 and BB55). * The barley chromosome location of these genes was also identified by RT-PCR with barley-specific primers (Supplemental Fig. S1B); ** the corresponding expression pattern with the wheat 38k array is not shown because values were lower than 50 in the raw data (see “Materials and Methods”).

Orthologous barley genes were further characterized. With reverse transcription (RT)-PCR using barley-specific primers, the expression of HvHYD1, HvDWF5, and HvSMT2 genes was found only in 3H, 3H, and 4H addition lines, respectively (Supplemental Fig. S1B), suggesting that these genes are located on these barley chromosomes, respectively. Moreover, other steroid biosynthesis-related genes were assigned to each of seven barley chromosomes by identification of the probe sets from the Affymetrix Barley1 GeneChip (Cho et al., 2006; Bilgic et al., 2007). The barley chromosomes to which the genes were assigned are presented in Figure 4D.

Molecular Characterization of Wheat and Barley CYP710A Genes

In Arabidopsis and tomato (Solanum lycopersicum), stigmasterol is synthesized from sitosterol by cytochrome P450 CYP710A via C22 desaturation (Morikawa et al., 2006a). Overexpression of these CYP710A genes in transgenic Arabidopsis resulted in stigmasterol accumulation at the expense of the sitosterol level, which was consistent with the alteration of the sterol profile in the 3H addition line. Therefore, the CYP710A gene may be key for regulation of the stigmasterol content in barley chromosome addition lines, although it cannot be completely ruled out that other gene(s) located on the 3H might control stigmasterol accumulation. To isolate full-length cDNAs from wheat and barley, the amino acid sequence of CYP710A1 (At2g34500) was used as a query for a homology search using tBLASTn against the National Center for Biotechnology Information database (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Several wheat and barley EST clones covering the putative 5′ untranslated region and partial coding regions of CYP710A genes were identified. Wheat or barley cDNAs encoding this entire region of CYP710A proteins were isolated. A full-length cDNA of the barley CYP710A gene and three full-length cDNAs of wheat CYP710A genes were obtained. According to the deduced amino acid sequences from these cDNAs, the P450 nomenclature committee (under Dr. D.R. Nelson) named them as CYP710A8, CYP710A8a, CYP710A8b, and CYP710A8d. In this study, the names of a barley CYP710A gene, CYP710A8(Hv), and three wheat CYP710A genes, CYP710A8(Ta-A), CYP710A8(Ta-B), and CYP710A8(Ta-D), corresponded to CYP710A8, CYP710A8a, CYP710A8b, and CYP710A8d, respectively. The deduced amino acid sequences of wheat CYP710A8s and barley CYP710A8 proteins were aligned with CYP710A1 and CYP710A2 (Fig. 5A). The cDNAs showed about 63% amino acid homology to CYP710A1 protein and about 58% amino acid homology to CYP710A2. CYP710A8(Hv) showed 93.5%, 95.6%, and 95.8% homology to CYP710A8(Ta-A), -(Ta-B), and -(Ta-D), respectively. Sequence comparison with CYP710A1 and CYP719A2 proteins showed that the conserved sequence FLFA(A/S)QDAS(T/S)S, which corresponds to a substrate recognition site (SRS4) of P450s (Gotoh, 1992), is present in all wheat and barley CYP710A8 proteins. The conserved Ala-299 of CYP710A1 (double underlined in SRS4), which is crucial for the introduction of the double bond in the sterol side chain (Morikawa et al., 2006b), was also present in wheat and barley CYP710A8, corresponding to Ala-315 of CYP710A8(Ta-A) and Ala-314 of CYP710A8(Ta-B) and -(Ta-D) and CYP710A8(Hv), indicating that these CYP710A8 proteins are likely responsible for the C-22 desaturation reaction. A phylogenetic tree of CYP710A proteins from wheat, barley, Arabidopsis, rice, and tomato is shown in Figure 5B. The wheat and barley CYP710A8 proteins formed a cluster located on the same branch as the rice CYP710A8 protein.

Figure 5.

Figure 5.

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Isolation of wheat and barley CYP710A genes. A, The deduced amino acid sequences of wheat CYP710A8s and barley CYP710A were aligned with CYP710A1 and CYP710A2 using ClustalW2 software. Residues conserved in all six proteins are indicated by asterisks. Residues having a similar, or very similar, physiochemical character are indicated by dots or colons, respectively. The characteristic SRS4 of P450s (Gotoh, 1992) is boxed. The heme ligand Cys residue is marked with the black arrow. The amino acid residues differing between CYP710A8(Hv)/CYP710A8(Ta-A) and CYP7108(Ta-B)/(Ta-D) are indicated with white arrows. B, Phylogenetic relationships among the CYP710A proteins. A phylogenetic tree was constructed using MEGA 5.05 software with CYP710A8(Hv), CYP710A8(Ta-A), -(Ta-B), and -(Ta-D), CYP710A1 (At2g34500), CYP710A2 (At2g34490), CYP710A3 (At2g28850), CYP710A4 (At2g28860), CYP710A5 (Loc_Os01g11270), CYP710A6 (Loc_Os01g11280), CYP710A7 (Loc_Os01g11300), CYP710A8 (Loc_Os01g11340), and CYP710A11 (BAE93156).

The chromosome location of the CYP710A8(Hv) gene was assigned. The expression profile of the CYP710A8(Hv) gene in barley chromosome addition lines was analyzed based on RT-PCR using barley-specific primers. The CYP710A8(Hv) gene was only expressed in the 3H addition line and parental barley line (Betzes) and, therefore, could be mapped to barley 3H (Fig. 6A). The chromosome locations of wheat CYP710A8 genes were determined with nullisomic-tetrasomic lines of CS (Sears, 1966). With genomic DNA from nullisomic-tetrasomic lines as a template, PCR using CYP710A8(Ta-A), -(Ta-B), and -(Ta-D) specific primers showed that the band for CYP710A8(Ta-A) disappeared in nulli3A-tetra3B, the CYP710A8(Ta-B) band was deleted in nulli3B-tetra3A, and the CYP710A8(Ta-D) band was not found in nulli3D-tetra3A (Fig. 6B). CYP710A8(Ta-A), -(Ta-B), and -(Ta-D), therefore, were assigned to wheat chromosomes 3A, 3B, and 3D, respectively (Fig. 6B).

Figure 6.

Figure 6.

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Molecular characterization of wheat and barley CYP710A8 genes. A, Expression profile of the CYP710A8(Hv) gene in barley chromosome addition lines based on RT-PCR. Specific CYP710A8(Hv) gene primers were used for identification of the gene’s chromosome location. B, Chromosome assignments of wheat CYP710A8 genes. N3AT3B, N3BT3D, and N3DT3B are abbreviations for nullisomic-tetrasomics of common wheat, namely nulli3A-tetra3B, nulli3B-tetra3D, and nulli3D-tetra3B, respectively. C, Northern-blot analysis of CYP710A8 genes in CS and barley chromosome addition lines. D, Relative expression levels of total CYP710A8 genes in CS and barley 3H were compared with real-time PCR analysis using primers annealing to both barley and wheat CYP710A8 genes. E, Relative expression levels of CYP710A8 genes in CS and barley 3H were compared with real-time PCR using their specific primers.

Expression patterns of CYP710A8 genes in CS and barley chromosome addition lines were examined by northern-blot analysis (Fig. 6C). The expression of CYP710A8 genes appeared to increase in the 3H addition line. Thereafter, relative expression levels of CYP710A8 genes were compared in CS and the 3H addition line. The overall expression level of CYP710A8 genes in the 3H addition line was increased 1.5 fold compared with CS (Fig. 6D). Furthermore, relative expression levels of CYP710A8(Ta-A), -(Ta-B), and -(Ta-D) in the 3H addition line were compared by real-time PCR using the respective specific primers. The relative expression levels of wheat CYP710A8 genes did not change drastically in the 3H addition line (Fig. 6E). Among the homeologous genes of CS, the gene from the A genome was more highly expressed than those from the other two genomes (4-fold expression rates) and that from the barley genome (2-fold rate). Since the stigmasterol content in the 3H addition line was increased 1.5 fold (Fig. 2), the expression of the barley gene contributed to its gain. These gene expression profiles of four CYP710A8 genes in the 3H addition line were confirmed by the RT-PCR products. Using the primers for the conserved region among the four genes, RT-PCR products were cloned into the plasmid vector and sequenced. Out of 39 clones identified, 22 clones (56.4%), one clone (2.6%), four clones (10.2%), and 12 clones (30.8%) corresponded to the genes from CYP710A8(Ta-A), CYP710A8(Ta-B), CYP710A8(Ta-D), and CYP710A8(Hv), respectively.

Overexpression of Wheat and Barley CYP710A in Arabidopsis Increases the Stigmasterol Level at the Expense of Sitosterol

The roles of CYP710A8 and DWF5 were further investigated by overexpression of these genes in Arabidopsis. Transgenic Arabidopsis plants were generated expressing the HvDWF5, TaDWF5, CYP710A8(Hv), CYP710A8(Ta-A), CYP710A8(Ta-B), and CYP710A8(Ta-D) cDNAs under the control of the 35S promoter. None showed phenotypes differing from the wild type. Two-week-old seedlings from T3 transgenic and wild-type Arabidopsis plants were used for characterization of the total sterol composition (Fig. 7A). No significant changes were observed in the total sterol level or in the campesterol level in wild-type plants or any transgenic line. The sterols of wild-type Arabidopsis were 17.4 ± 2.9 μg 100 mg−1 dry weight stigmasterol (6.1% of total sterol), 202.3 ± 13.1 μg 100 mg−1 dry weight sitosterol (70.8% of total sterol), and 53.4 ± 7.0 μg 100 mg−1 dry weight campesterol (18.7% of total sterol; Fig. 7A). All the CYP710A8 transgenic lines contained stigmasterol at higher levels, ranging from 140 to 272 μg 100 mg−1 dry weight (66.8%–78.9% of total sterol), corresponding to an approximately 10- to 20-fold increase compared with wild-type plants. In these transgenic lines, sitosterol levels decreased to 7.8 to 53.2 μg 100 mg−1 dry weight (2.4%–14.7% of total sterol). Expression of these transgenes was confirmed by RT-PCR, indicating that the alteration of sterol metabolism in Arabidopsis was due to the introduction of these alien genes (data not shown).

Figure 7.

Figure 7.

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Plant sterol profiles in Arabidopsis transgenic lines with CYP710A8 or DWF5 overexpression. Barley and wheat CYP710A8 and DWF5 genes were overexpressed in Arabidopsis under the control of the 35S promoter. Two-week-old seedlings were analyzed. Seven species of phytosterols are shown in A and B. Data are mean values ± sd from three independent samples. DW, Dry weight; WT, wild type.

Arabidopsis also contained cholesterol, 24-methyl-Δ22-sterols, campestanol, and sitostanol as minor fractions (1.5%, 1.4%, 0.13%, and 0.83% of total sterol, respectively; Fig. 7B). The 24-methyl-Δ22-sterol contents of the CYP710A8(Hv) and CYP710A8(Ta-A) transgenic plants were increased about 1.6-fold over the wild type. Furthermore, the 24-methyl-Δ22-sterol levels in the CYP710A8(Ta-B) and -(Ta-D) transgenic plants were increased 2.5- to 4.5-fold over wild-type plants. These results suggested that CYP710A8(Ta-B) and CYP710A8(Ta-D) were involved in the desaturation to produce 24-methyl-Δ22-sterols. Overexpression of DWF5 genes did not lead to any significant alteration in total sterol levels or the sterol composition (stigmasterol, sitosterol, campesterol, etc.). This meant that increasing the expression of wheat and barley DWF5 genes did not affect the phytosterol profile of transgenic Arabidopsis.

DISCUSSION

Phytosterol Profiles in Common Wheat, Barley, and Barley Chromosome Addition Lines of Common Wheat

We measured the phytosterol contents in the seedlings and grains of wheat, barley, and barley chromosome addition lines. Overall levels of phytosterols in seedling leaves were approximately 3-fold higher than those in the grains (Figs. 2 and 3). The relative amount of stigmasterol in the grain was dramatically lower, while those of campestarol and sitostanol in the grains were higher than those in the seedlings. It is notable that campestanol and sitostanol in barley grains were scarcely at detectable levels (Fig. 3). Additionally, the phytosterol levels of the 2H addition line of CS were lower than the levels in the grain. These lines of evidence suggest that the biosynthesis system(s) for phytosterol revealed tissue and organ specificity in wheat and barley. Hence, gene manipulation to promote gene expression related to phytosterol biosynthesis could be applied to improve the functional phytosterol in wheat grains.

Systematic Analysis of Sterol Biosynthesis-Related Genes in Barley Chromosome Addition Lines of Common Wheat

Sterols maintain membrane integrity and fluidity and regulate membrane permeability. Sterols also serve as the precursors for a variety of steroidal hormones. In Arabidopsis, mutation of genes such as cas1 (Babiychuk et al., 2008), smt1 (Diener et al., 2000), fk (Jang et al., 2000), hyd1 (Souter et al., 2002), smt2 (Schaeffer et al., 2001; Carland et al., 2002), ste1/dwarf7 (Choe et al., 1999), and dwf5 (Choe et al., 2000) changed the composition of sterols and led to distinct phenotypes in embryogenesis and development at specific stages. On the other hand, overexpression of sterol biosynthesis-related genes also altered the total amount and composition of sterols, causing severe impairment in development. For example, an SMT2-overexpressing plant displayed reduced stature and growth, because the amount of campesterol decreased and the amount of sitosterol increased concomitantly (Schaeffer et al., 2001). Although it remains unknown how the modified ratio between the two phytosterols affected plant growth, it is plausible that balance between campesterol and sitosterol is important for normal plant growth and development.

To our knowledge, this paper is the first report on systematic analysis of the phytosterol biosynthetic pathway-related genes in barley chromosome addition lines of common wheat. The expression of genes related to sterol biosynthesis in barley chromosome addition lines of common wheat was systematically characterized. Although orthologous barley genes were located on their respective barley chromosomes (Fig. 4D; Supplemental Fig. S1B), the 38k wheat microarray analysis did not show these genes as having a high expression level in the corresponding addition lines, except for the barley chromosome 3 addition line of hexaploid wheat (Fig. 4, A–C). In the 3H addition line, HYD1, DWF5, and CYP710A8 displayed higher expression levels (Figs. 4, B and C, and 6, C and D). HvHYD1, HvDWF5, and CYP710A8(Hv) mapped to barley chromosome 3 (3H) and were expressed in the 3H addition line. These results also agree with phytosterol profiles in common wheat, barley, and barley chromosome addition lines of common wheat, in which an alteration of phytosterol composition was observed only in the 3H addition line. The sterol profile in the 3H addition line was characterized by an increased amount of stigmasterol at the expense of sitosterol. The amounts of campesterol and total sterol were not changed (Fig. 2). Additive effects of barley chromosome additions to the hexaploid wheat background on gene expression were only found in the 3H addition line, and only the amount of stigmasterol amount increased. This suggests that phytosterol composition is precisely controlled in the biopathway, except for sitgmasterol; that is, in plants, the ratio of 24-ethyl sterols (sitosterol and stigmasterol) to 24-methyl sterols (campesterol) might be precisely regulated, but the ratio of sitosterol to stigmasterol is not as strictly regulated.

Function of CYP710A8(Ta) and CYP710A8(Hv) in the Biosynthesis of Phytosterols

Additional expression of the barley CYP710A8 gene in the 3H addition line of CS wheat (Fig. 2) and overexpression of wheat and barley CYP710A8 in Arabidopsis (Fig. 7) increased the stigmasterol level at the expense of sitosterol but did not significantly alter the levels of campesterol, cholesterol, campestanol, or total sterol. These results demonstrate that wheat CYP710A8 and barley CYP710A8 catalyze the C-22 desaturase reaction converting sitosterol to stigmasterol. Although no significant difference in converting sitosterol to stigmasterol was observed among CYP710A8(Ta-A), -(Ta-B), -(Ta-D), and CYP710A8(Hv), production activities of 24-methyl-Δ22-sterols were different between CYP710A8(Hv)-CYP710A8(Ta-A) and CYP710A8(Ta-B)-CYP710A8(Ta-D) groups (Fig. 7). Since there were found some differences of amino acid residues (Fig. 5), these amino acids might be candidate(s) for enzyme activity. Overexpression of wheat and barley CYP710A8 in Arabidopsis reversed the ratio of stigmasterol to sitosterol but did not affect the phenotype of transgenic Arabidopsis. This indicates that accumulation of Δ22-sterols at high levels did not influence brassinosteroid (Fig. 1) biosynthesis or other developmental processes.

The 24-methyl-Δ22-sterol levels increased significantly in all CYP710A8-overexpressing lines, but the levels in CYP710A8(Ta-B)- and CYP710A8(Ta-D)-overexpressing lines were much higher than those in CYP710A8(Hv) and CYP710A8(Ta-A) lines. These results indicate that all wheat and barley CYP710A8 proteins are able to produce 24-methyl-Δ22-sterols but that the CYP710A8(Ta-B) and -(Ta-D) proteins have higher activity than CYP710A8(Ta-A) and CYP710A8(Hv). In Arabidopsis, CYP710A2 can catalyze the production of 24-methyl-Δ22-sterols, but CYP710A1 does not have this ability (Morikawa et al., 2006a). CYP710A8(Ta-A), -(Ta-B), -(Ta-D), and CYP710A8(Hv) shared more than 93.5% amino acid homology (Fig. 5). Alignment of these sequences made it clear that there were four candidate positions: both CYP710A8(Ta-A) and CYP710A8(Hv) have the same amino acid residues at positions 9, 31, 381, and 453 (Fig. 5), while CYP710A8(Ta-B) and CYP710A8(Ta-D) have distinct amino acid residues from those of CYP710A8(Ta-A) and CYP710A8(Hv) (Fig. 5A, white arrows). Among these amino acid residue alterations, the change of Pro to Thr at position 381 (Fig. 5A) might be the key to the differences of brassicasterol production, because this amino acid change is located between SRS4 (Gotoh, 1992) and the heme ligand C residue of the enzyme (Meunier et al., 2004; Fig. 5A). This amino acid residue difference is not observed between CYP710A1 and CYP710A2 in Arabidopsis. This hypothesis should be confirmed by further experiments.

In wheat, barley, and barley chromosome addition lines of common wheat, the 24-methyl-Δ22-sterol levels were almost nothing. Since activities of CYP710A from wheat and barley per se were functional, there is a possibility that the biosynthesis of 24-epi-campesterol should be weak, probably because of enzymatic activity for DWF1 (CYP85A1; Fig. 1) in wheat and barley. Since overexpression of both wheat and barley DWF5 genes in Arabidopsis did not change the profile of phytosterol in Arabidopsis, at least in the plants so far examined, we can conclude that the key protein for producing stigmasterol is CYP710A. The AtDWF5 gene was reported to be involved in the biosynthetic steps that convert Δ7-sterol intermediates into campesterol and sitosterol (Choe et al., 2000). The brassinosteroid profile was not examined in this study, so we do not know if the brassinosteroid profiles were changed by overexpression of these DWF5 genes.

Accumulation of Stigmasterol in Common Wheat Has Possible Applications

Of the main phytosterols, stigmasterol would be the most beneficial for health. The barley 3H addition line of common wheat and Arabidopsis transgenic lines with wheat and barley CYP710A8 genes accumulated stigmasterol with a concurrent decrease in the sitosterol level, but they did not display any obvious phenotypic alterations. That means that accumulation of stigmasterol in common wheat is possible. Our study demonstrated that CYP710A proteins were the key enzymes controlling the accumulation of stigmasterol, and Manzano et al. (2004) reported that overexpression of HMGR could increase total sterol. These results suggest that increased expression of CYP710A genes, singly or combined with the HMGR gene, might lead to an increased amount of stigmasterol. In this study, we mapped the CYP710A8(Hv) gene to 3H (Fig. 6D) and HvHMGR genes to 5H and 7H (Fig. 4D). Cross-hybridization makes it possible to produce new barley-wheat addition lines harboring two pairs of barley chromosomes. Therefore, the double chromosome addition lines could further increase the stigmasterol content in wheat, and we are studying the amount of stigmasterol in the double barley chromosome addition lines. Consequently, barley chromosome addition lines are greatly useful for the study of the biosynthesis pathway of plant substrates and research into the key gene(s) for increasing the effective level of plant substrates. Hence, our system provides a new insight into the accumulation of bioactive compounds in common wheat and a new approach for assessing plant metabolism profiles.

MATERIALS AND METHODS

Plant Growth Conditions

Common wheat (Triticum aestivum ‘Chinese Spring’), barley (Hordeum vulgare ‘Betzes’), six barley chromosome disomic addition lines (2H–7H), and a ditelosomic addition line (1HS) were used (Islam et al., 1981; Islam and Shepherd, 2000). Seeds were germinated and grown on soil at 22°C under 16-h-light and 8-h-dark conditions. Leaves and mature dry seeds from 2-week-old seedlings were used for sterol determination, and leaves were supplied for RNA extraction.

RNA Extraction, RT-PCR, and Real-Time PCR

Total RNA was extracted using an RNeasy Plant Mini Kit (Qiagen). Total RNA quality was checked with an Agilent 2100 Bioanalyzer. For first-strand cDNA synthesis, 1 μg of total RNA was treated with RNase-free DNase (Invitrogen), then first-strand synthesis was carried out using Rever Tra Ace (Toyobo) according to the manufacturer’s instructions. To examine gene expression, PCR was performed for 40 cycles using KOD plus polymerase (Toyobo). To amplify the entire coding sequence of genes for cloning, PCR was performed using KOD FX polymerase (Toyobo). Real-time PCR was performed on a Thermal Cycler Dice Real Time System TP800 (TaKaRa). SYBR Premix Ex Taq II (TaKaRa) was used for the CYP710A8 genes. Four replicates were performed. The relative quantification was calculated with the comparative threshold cycle method and normalized to wheat Tubulin. SYBR Premix Ex Taq (TaKaRa) was used for the DWF5, HYP1, and SMT2 genes. The relative quantification was calculated with a standard method and normalized to wheat Tubulin. Primers used in this study are listed in Supplemental Table S2.

Northern-Blot Analysis

Ten micrograms of total RNA was separated on agarose-formaldehyde gels, blotted to Hybond-N+ membranes (GE Healthcare), and hybridized according to the manufacturer’s instructions. The DNA probe specific for both wheat and barley CYP710A8 was amplified by PCR using the primers listed in Supplemental Table S2 and labeled with [α-32P]dCTP using the BcaBEST labeling kit (TaKaRa).

Isolation of CYP710A8 Genes and DWF5 Genes from CS and Betzes

For the isolation of CYP710A8 genes, we used the amino acid sequence of CYP710A1 (At2g34500) as a query for a tBLASTn search against the National Center for Biotechnology Information database (http://blast.ncbi.nlm.nih.gov/Blast.cgi) and identified several wheat EST clones covering the putative 5′ untranslated region and partial coding regions of CYP710A8 genes. Then, 3′-RACE PCR was performed using 1 μg of total RNA prepared from seedlings of CS or Betzes. First-strand cDNA was synthesized using a Rever Tra Ace kit (Toyobo). Primer 2 in Supplemental Table S3 (for wheat) or primer 1 (for barley) and 3′-RACE coding sequence primer A from the SMART RACE cDNA amplification kit (TaKaRa) were used for initial PCR, and primer 3 and nested universal primer A from the SMART RACE cDNA amplification kit were used for nested PCR. Nested PCR products were cloned into the pGEM-T vector (Promega) and sequenced. Using the 3′ sequence acquired, primer 5 (for wheat) and primer 4 (for barley) were designed to obtain the entire coding region. Fragments containing the untranslated regions of CYP710A8 genes were amplified by PCR with primers 2 and 5 for wheat CYP710A8 and primers 1 and 4 for barley CYP710A8, and their products were cloned into pENTR/D-TOPO (Invitrogen) and sequenced.

For the isolation of DWF5 genes, we used the amino acid sequence of AtDWF5 (At1g50430) as a query for a tBLASTn search against TriFLDB (http://trifldb.psc.riken.jp/index.pl) and identified two cDNA clones containing the putative entire coding region for wheat DWF5 (AK333433) and barley DWF5 (AK249569). The entire coding sequences of wheat DWF5 and barley DWF5 genes were thus amplified by PCR using the cDNA from CS or Betzes as a template. The PCR products were then cloned into pENTR/D-TOPO (Invitrogen) and sequenced.

Construction of 35S Promoter:CYP710A8 and 35S Promoter:DWF5 Fusion Genes and Generation of Transgenic Plants

The pENTR-CYP710A8(Ta-A), -(Ta-B), -(Ta-D), CYP710A8 (Hv), TaDWF5, and HvDWF5 obtained above were integrated into the binary vector pBCR-79 (Seki et al., 2008) using the Gateway system. The resultant construct was transferred into Agrobacterium tumefaciens strain GV3101, followed by transformation into Arabidopsis (Arabidopsis thaliana) Columbia plants using the floral dip method (Clough and Bent, 1998). T1 seeds were screened on agar plates of 1× Murashige and Skoog medium (Duchefa Biochemie) containing 25 mg mL−1 kanamycin, and resistant seedlings were transferred to soil and allowed to set seed. 35S:CYP710A8(Ta-A), -(Ta-B), -(Ta-D), and 35S:CYP710A8(Hv) homozygous lines were selected by examining the kanamycin resistance of T3 seedlings. Arabidopsis Columbia, CYP710A8(Ta-A), -(Ta-B), -(Ta-D), CYP710A8(Hv), TaDWF5, and HvDWF5 transgenic plants were germinated and grown on agar plates of 1× Murashige and Skoog medium containing 3% Suc. After stratification for 3 d at 4°C, plates were incubated for 2 weeks at 23°C under a photoperiodic cycle of 16 h of light/8 h of dark.

Microarray Analysis

We used a custom wheat 38k oligo-DNA microarray (Agilent; Kawaura et al., 2008) consisting of 37,826 probes (http://www.shigen.nig.ac.jp/wheat/komugi/). A Cy3-labeled copy RNA probe was prepared using a Low RNA Input Linear Amp Kit (Agilent), and microarray hybridization was performed following the recommended protocol. Data were acquired by an Agilent G2565BA microarray scanner. Microarray data were analyzed using GeneSpring GX software (Agilent). The normalization and baseline transformation were performed as follows: (1) threshold raw signals to 1.0; (2) normalization algorithm: shift to 50th percentile; (3) baseline to median of control samples (CS). The genes in this study had values greater than 50 in the raw data for at least three samples (belonging to the same addition line). Leaves from two seedlings of CS and each addition line were sampled for each experiment. The experiments were replicated three times for each addition line using independent samples.

Sterol Extraction and Quantification

For wheat, barley, and each addition sample, leaves from about 25 seedlings of each genotype were harvested and lyophilized for each experiment. Twenty grains of each genotype were supplied for phytosterol extraction. For Arabidopsis samples, about 50 seedlings were harvested for each experiment. Experiments were replicated three times using independent samples. Sterols were extracted and quantified using gas chromatography-mass spectrometry according to a method described previously (Suzuki et al., 2004).

Sequence data from this report are available from the DNA Data Bank of Japan under the following accession numbers: CYP710A8(Ta-A), AB620024; CYP710A8(Ta-B), AB620025; CYP710A8(Ta-D), AB620026; and CYP710A8(Hv), AB620027. Microarray data can be found in the Gene Expression Omnibus under accession number GSE28023.

Supplemental Data

The following materials are available in the online version of the article.

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