a new insight into application for barley chromosome addition

A New Insight into Application for Barley ChromosomeAddition Lines of Common Wheat: Achievement ofStigmasterol Accumulation1[W]

Jianwei Tang2, Kiyoshi Ohyama2,3, Kanako Kawaura, Hiromi Hashinokuchi, Yoko Kamiya, Masashi Suzuki,Toshiya Muranaka4, and Yasunari Ogihara*

Kihara Institute for Biological Research, Yokohama City University, Yokohama 244–0813, Japan (J.T., K.K.,Y.K., T.M., Y.O.); and RIKEN Plant Science Center, Yokohama 230–0045, Japan (K.O., H.H., M.S., T.M.)

Barley (Hordeum vulgare) has a much higher content of bioactive substances than wheat (Triticum aestivum). In order toinvestigate additive and/or synergistic effect(s) on the phytosterol content of barley chromosomes, we used a series of barleychromosome addition lines of common wheat that were produced by normal crossing. In determining the plant sterol levels in2-week-old seedlings and dry seeds, we found that the level of stigmasterol in the barley chromosome 3 addition (3H) line inthe seedlings was 1.5-fold higher than that in the original wheat line and in the other barley chromosome addition lines, butnot in the seeds. Simultaneously, we determined the overall expression pattern of genes related to plant sterol biosynthesis inthe seedlings of wheat and each addition line to assess the relative expression of each gene in the sterol pathway. Since weelucidated the CYP710A8 (cytochrome P450 subfamily)-encoding sterol C-22 desaturase as a key characteristic for the higherlevel of stigmasterol, full-length cDNAs of wheat and barley CYP710A8 genes were isolated. These CYP710A8 genes weremapped on chromosome 3 in barley (3H) and wheat (3A, 3B, and 3D), and the expression of CYP710A8 genes increased in the3H addition line, indicating that it is responsible for stigmasterol accumulation. Overexpression of the CYP710A8 genes inArabidopsis increased the stigmasterol content but did not alter the total sterol level. Our results provide new insight into theaccumulation of bioactive compounds in common wheat and a new approach for assessing plant metabolism profiles.

Wheat (Triticum aestivum) is one of the three ma-jor cereal crops. It has an advantage over barley(Hordeum vulgare) in that wheat flour is more suitablefor processing for many kinds of foods, such as bread,noodles, cookies, and cakes. In contrast, barley is richin health-beneficial bioactive compounds, such asg-aminobutyrate, b-glucan, minerals, and vitamins(Kihara et al., 2007). If these advantages of barley couldbe incorporated into wheat, we could utilize wheatenriched 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 inwheat, and transgenic products are not well acceptedby consumers. Genetic hybridization (cross-breeding)is a nontransgenic method widely applied to plantbreeding. A set of barley chromosome addition linesof common wheat were developed through distanthybridization between hexaploid wheat (cv ChineseSpring; 2n = 6x = 42; AABBDD) and diploid barley(cv Betzes; 2n = 2x = 14; HH; Islam et al., 1975). This setof disomic addition lines of barley chromosomes ina genetic background of common wheat contains sixlines with additions of chromosomes 2H to 7H; the 1Hdisomic addition line was not produced, because gene(s) on 1HL caused sterility in hybrids (Islam et al., 1981;Islam and Shepherd, 1990). Subsequently, a ditelosomicaddition line of barley 1HS was bred (Islam et al.,1981; Islam, 1983; Islam and Shepherd, 1990, 2000).Barley chromosome addition lines of common wheathave been utilized for a variety of purposes. Usually,they have been used for cytological and geneticmapping of barley genes (Ashida et al., 2007; Katoet al., 2008; Sakai et al., 2009; Sakata et al., 2010).Transcripts of wheat-barley disomic addition linesand ditelosomic addition lines were profiled usingthe Affymetrix Barley1 GeneChip probe array. Theexpressed barley genes in each addition line weremapped 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

1 This work was supported by the Research and DevelopmentProgram for New Bio-industry Initiatives and by Grants-in-Aid forScientific Research and Bilateral Program from the Japan Society forthe Promotion of Science. This paper is contribution no. 1005 from theKihara Institute for Biological Research, Yokohama City University.

2 These authors contributed equally to the article.3 Present address: Department of Chemistry and Materials Sci-

ence, Tokyo Institute of Technology, Tokyo 152–8551, Japan.4 Present address: Department of Biotechnology, Graduate School

of Engineering, Osaka University, Osaka 565–0871, Japan.* Corresponding author; e-mail [email protected] author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policydescribed in the Instructions for Authors (www.plantphysiol.org) is:Yasunari Ogihara ([email protected]).

[W] The online version of this article contains Web-only data.www.plantphysiol.org/cgi/doi/10.1104/pp.111.183533

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genetic background produce bioactive compoundswith an additive or synergistic action. However, littleis known about the nutritional effect of barley chro-mosome addition lines of common wheat. Therefore,we aimed to identify and estimate overall bioactivecompounds in each of the barley chromosome addi-tion lines by integrated analysis of the metabolomeand transcriptome.

In this paper, we focused on phytosterols. Phy-tosterols are well known for reducing the plasmacholesterol levels of humans when administered orally.Phytosterols have received U.S. Food and DrugAdministration clearance as generally recognized assafe. Foods containing phytosterols can carry healthclaims indicating their cholesterol-reducing properties(Kamal-Eldin and Moazzami, 2009). Phytosterols havealso been reported that have antiinflammatory prop-erties, such as antiosteoarthritic properties (Gabayet al., 2010). Stigmasterol, campesterol, and sitosterolare the main molecular species of phytosterols. Stig-masterol was reported to inhibit hepatic cholesterolsynthesis (Batta et al., 2006) and to have the highestaffinity to the chondrocyte membrane (Gabay et al.,2010). These observations suggest that stigmasterol isthe most valuable of the three main phytosterols.

In plants, most phytosterols are involved in mem-brane function, modulating membrane permeabilityand fluidity (Benveniste, 2004; Schaller, 2004). Phytos-terols also serve as precursors for the biosynthesis ofbrassinosteroids, functioning as plant growth regula-tors, including cell division and expansion, responsesto light and dark, morphogenesis, apical dominance,and gene expression (Schrick et al., 2002; Nemhauserand Chory, 2004). In plants, sterol synthesis mainlystarts from the cytosolicmevalonate pathway (Newmanand Chappell, 1999; Kasahara et al., 2002), leading tothe C5 building blocks isopentenyl diphosphate (IPP)and its isomer dimethylallyl diphosphate (DMAPP).IPP and DMAPP are also synthesized in plastidsthrough a 2-C-methyl-D-erythritol 4-phosphate path-way. Although metabolic cross talk between themevalonate and 2-C-methyl-D-erythritol 4-phosphatepathways was reported (Kasahara et al., 2002; Nagataet al., 2002; Hemmerlin et al., 2003), the contributionof this cross talk is minor (Suzuki et al., 2009). Cyto-solic IPP and DMAPP are condensed to generate theC30 precursor squalene and further converts into 2,3-oxidosqualene. Cytosolic IPP is the starting pointof several metabolic branches leading to the synthesisof a variety of essential isoprenoids, including sterols,brassinosteroids, polyprenols, sesquiterpenes, andcytokinins (Fig. 1). Phytosterol biosynthesis is initiatedby the enzymatic conversion of 2,3-oxidosqualene tocycloartenol, which is catalyzed by cycloartenol syn-thase 1 (CAS1). Although genes encoding lanosterolsynthase (Kolesnikova et al., 2006; Sawai et al., 2006;Suzuki et al., 2006) and phytosterol biosynthesis vialanosterol in Arabidopsis (Ohyama et al., 2009) werereported recently, no genes encoding lanosterol syn-thase have been identified from monocotyledonous

plants (Suzuki et al., 2006). Phytosterol biosynthesis,then, is divided into two branches from 24-methylenelophenol. One branch produces 24-methyl sterols in-ducing campesterol and 24-methyl-D22-sterols (a mix-ture of brassicasterol and crinosterol). The secondbranch produces 24-ethyl sterols including sitosteroland stigmasterol (Fig. 1). This key branch point iscontrolled by sterol-C24-methyltransferase 2 (SMT2).The orientation of sterol biosynthetic flux towardcampesterol or sitosterol is achieved via SMT2 enzymeactivity in plants (Schaeffer et al., 2001; Carland et al.,2002). CAS1 and SMT2 genes are plant specific. Theprecise regulation of these compounds is important fornormal plant growth and development. Stigmasterolsynthesis is the end step of the 24-ethyl sterol pathway.The reaction is catalyzed by a C-22 desaturase belong-ing to the cytochrome P450CYP710A family. Arabi-dopsis (Arabidopsis thaliana) CYP710A genes consist offour isogenes (AtCYP710A1, -A2, -A3, and -A4). Over-expression of CYP710A1, -A2, and -A4 in Arabidop-sis contributed to stigmasterol synthesis (Morikawaet al., 2006a; Arnqvist et al., 2008). CYP710A2 is alsothe desaturase responsible for the production of 24-methyl-D22-sterols (Morikawa et al., 2006a). No drasticphenotypic alterations were observed in AtCYP710A-overexpressing plants (Morikawa et al., 2006a; Arnqvistet al., 2008).

In this paper, by systematic analysis of the overallsterol profiling in 2-week-old seedlings and dry seedsfrom a series of barley chromosome addition linesof common wheat having six of seven whole barleychromosomes (2H–7H) and a ditelosomic addition line(1HS), we found that only stigmasterol accumulated inthe seedlings of the barley chromosome 3 (3H) addi-tion line but not in the grains. Simultaneously, weintegrated these sterol profiles with the transcriptomerelated to phytosterol biosynthesis. Furthermore, weisolated full-length cDNAs of wheat and barleyCYP710A8 (cytochrome P450 subfamily) genes. TheseCYP710A8 genes were mapped on chromosome 3 inbarley (3H) and wheat (3A, 3B, and 3D), and theexpression of CYP710A8 genes increased in the 3Haddition line. Overexpression of the CYP710A8 genesin seedlings of Arabidopsis increased the stigmasterolcontent but did not alter the total sterol level. Thus, weelucidated the CYP710A8 genes encoding sterol C-22desaturase as a key characteristic for the higher level ofstigmasterol in the 3H addition line. Our results pro-vide new insight into the accumulation of bioactivecompounds in common wheat and an approach forassessing plant metabolism profiles.

RESULTS

Barley Chromosome Added to Common Wheat Increasedthe Functional Sterol Level in Seedlings

Phytosterol profiles of leaves from 2-week-old seed-lings of common wheat (cv Chinese Spring [CS]) and

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barley (cv Betzes) and barley chromosome additionlines (1HS and 2H–7H) were examined (Fig. 2). Thetotal sterol levels averaged 191.58 mg 100 mg21 dryweight in Betzes and 203.74 mg 100 mg21 dry weight inCS based on three measurements. No significant dif-ference in campesterol or total sterol levels was ob-

served in Betzes, CS, or barley chromosome additionlines. Stigmasterol, sitosterol, and campesterol werethe main phytosterols, and stigmasterol is consideredthe most functional sterol among them (Batta et al.,2006). In Betzes, the sterols consisted of 34.3% stig-masterol, 42.1% sitosterol, and 18.5% campesterol, and

Figure 1. Schematic diagram of the plant sterol biosynthetic pathway. The plant sterol biosynthetic pathway is shown referencingthe 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 ana-lyzed are numbered. FPP, Farnesyl diphosphate; GPP, geranyl diphosphate; MAV, mevalonate; MEP, 2-C-methyl-D-erythritol4-phosphate.

Phytosterol Synthesis in Wheat-Barley

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in CS, they consisted of 15.6% stigmasterol, 59.0%sitosterol, and 21.4% campesterol. The sterol compo-sitions of addition lines, except for the 3H additionline, were similar to that of CS. In contrast, the 3Haddition line consisted of 22.8% stigmasterol, 49.6%sitosterol, and 23.8% campesterol, a stigmasterol levelthat was 1.5-fold higher than that of CS and otherbarley chromosome addition lines, with a correspond-ing decrease in the level of sitosterol. Cholesterol,sitostanol, and campestanol levels were very low in allbarley, wheat, and barley chromosome addition lines,contributing 1% to 2% of the total sterol. 24-Methyl-D22-sterol was only detected at trace levels in bothbarley and wheat seedlings (data not shown). Ourresults showed that only the 3H addition line had analtered phytosterol profile, which suggested that cer-tain gene(s) of 3H are very important for stigmasterolsynthesis.

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

Phytosterol profiles of dry grains of common wheat(CS), barley (Betzes), and barley chromosome additionlines (1HS and 2H–7H) were examined (Fig. 3). In thegrains, seed and fruit coats were contained. The totalsterol levels averaged 61.39 mg 100 mg21 dry weightin Betzes and 55.67 mg 100 mg21 dry weight in CSbased on three measurements. Total sterol levels in

grains were one-third-fold lower than those in seed-lings (Fig. 3). All sterol levels of the barley 2H chromo-some addition line so far detected were significantlylower than those of all other lines, including wheatand barley parental lines. Levels of campesterol andsitosterol in Betzes were significantly higher than thoseof the other lines, while levels of campestanol andsitostanol were dramatically lower in Betzes. Levelsof stigmasterol and cholesterol were very low in alllines (Fig. 3). Our results indicate that barley chromo-some addition to common wheat did not bring aboutclear additional and/or synergetic effects to increasefunctional steroid levels in the wheat grains. There-fore, we focused on the phytosterol pathway(s) in seed-lings of wheat and barley.

Expression of Genes Related to the Biosynthesis ofPhytosterols in Seedlings

The expression of sterol pathway-related genes inthe seedlings of wheat and barley was examined inrelation to stigmasterol accumulation, because an im-balance of certain enzymes in the sterol biosyntheticpathways can lead to an accumulation of functionalsteroids. In Arabidopsis, overexpression of 3-hydroxy-3-methylglutaryl-CoA reductase isoform 1S (HMGR1S)increased the total sterol level and overexpression offarnesyl diphosphate synthase isoform 1S (FPS1S) in-creased the stigmasterol level (about 1.8-fold that of the

Figure 2. Sterol profiles of leaves from common wheat (CS), barley (Betzes), and barley chromosome addition lines. Leaves of2-week-old seedlings were analyzed. Data are mean values 6 SD from three independent leaf samples. Asterisks indicatesignificant differences between common wheat and other barley chromosome addition lines of common wheat (P, 0.01). DW,Dry weight.

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wild type; Masferrer et al., 2002; Manzano et al., 2004).The hydra1 (hyd1) and hydra2 (fk) mutants showedlower levels of campesterol and sitosterol but a higherlevel of stigmasterol than the wild type (Topping et al.,1997; Souter et al., 2002). Hence, it is interesting toassess the gene expression pattern at each step ofthe sterol pathway. A custom wheat 38k oligo-DNAmicroarray (wheat 38k microarray; Agilent) was usedto examine overall gene expression patterns in CSand in each barley chromosome addition line. Orthol-ogous genes in wheat and barley shared high sequencehomology. Thus, 60-mer probes on the wheat 38k mi-croarray could hybridize to either wheat genes or mostbarley genes. The wheat 38k microarray could beutilized for screening of genes with expression levelsspecifically increased by an additive effect or syner-gistic action between wheat and barley chromosomes.In this manner, each of the wheat and barley HMGRgenes, FPS1 genes, and genes related to the biosyn-thetic pathways from (S)-2,3-epoxysqualene to cam-pesterol and sitosterol (Fig. 1) were identified, usingthe corresponding amino acid sequences from Arabi-dopsis or rice (Oryza sativa) as queries for a tBLASTnsearch 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 genenomenclature of Arabidopsis, with the letters “Ta” forwheat and “Hv” for barley. The corresponding origi-nal gene names in the wheat 38k microarray are listedin Supplemental Table S1. The expression patterns of

genes were analyzed according to the conditions de-scribed in “Materials and Methods.” Data are shownas values relative to CS (Fig. 4). The expression of theHYD1 and DWARF5 (DWF5) genes was significantlyincreased in the 3H addition line. The expression levelof HYD1, which corresponds to the probe fromwheat0130Contig13954, was 1.6-fold higher (Fig. 4B),and the DWF5 gene, which corresponds to each of theprobes rwhf14j19 andMUGEST2003_23lib_Contig7208,was 2.3- to 3.1-fold higher than CS (Fig. 4C). Theexpression of other genes of addition lines was similarto that of CS wheat. The expression patterns of HYD1,DWF5, and SMT2 genes were confirmed by real-timePCR using primers annealing to both barley andwheatgenes (Supplemental Fig. S1A). These gene expres-sions were compared with that of SMT2, because SMT2is located at the key position for phytosterol biosyn-thesis. The results were consistent with those from the38k wheat microarray analysis. On the 38k wheat mi-croarray, no probes corresponding to CYP710A geneswere spotted.

Orthologous barley genes were further charac-terized. With reverse transcription (RT)-PCR usingbarley-specific primers, the expression of HvHYD1,HvDWF5, and HvSMT2 genes was found only in 3H,3H, and 4H addition lines, respectively (SupplementalFig. S1B), suggesting that these genes are located onthese barley chromosomes, respectively. Moreover,other steroid biosynthesis-related genes were assignedto each of seven barley chromosomes by identifica-tion of the probe sets from the Affymetrix Barley1GeneChip (Cho et al., 2006; Bilgic et al., 2007). The

Figure 3. Sterol profiles of grains from common wheat (CS), barley (Betzes), and barley chromosome addition lines. Dry seedswere analyzed. Data are mean values 6 SD from three independent leaf samples. DW, Dry weight.





barley chromosomes to which the genes were assignedare presented in Figure 4D.

Molecular Characterization of Wheat and BarleyCYP710A Genes

In Arabidopsis and tomato (Solanum lycopersicum),stigmasterol is synthesized from sitosterol by cyto-chrome P450 CYP710Avia C22 desaturation (Morikawaet al., 2006a). Overexpression of these CYP710A genesin transgenic Arabidopsis resulted in stigmasterolaccumulation at the expense of the sitosterol level,which was consistent with the alteration of the sterolprofile in the 3H addition line. Therefore, the CYP710Agene may be key for regulation of the stigmasterolcontent in barley chromosome addition lines, althoughit cannot be completely ruled out that other gene(s)located on the 3H might control stigmasterol accu-mulation. To isolate full-length cDNAs from wheatand barley, the amino acid sequence of CYP710A1(At2g34500) was used as a query for a homologysearch using tBLASTn against the National Center for

Biotechnology Information database (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Several wheat and barleyEST clones covering the putative 5# untranslatedregion and partial coding regions of CYP710A geneswere identified. Wheat or barley cDNAs encodingthis entire region of CYP710A proteins were isolated.A full-length cDNA of the barley CYP710A gene andthree full-length cDNAs of wheat CYP710A geneswere obtained. According to the deduced amino acidsequences from these cDNAs, the P450 nomenclaturecommittee (under Dr. D.R. Nelson) named them asCYP710A8, 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 aminoacid sequences of wheat CYP710A8s and barleyCYP710A8 proteins were aligned with CYP710A1and CYP710A2 (Fig. 5A). The cDNAs showed about63% amino acid homology to CYP710A1 protein andabout 58% amino acid homology to CYP710A2.

Figure 4. Molecular analysis ofgenes related to the biosynthesisof plant sterols. A, Expression pat-terns of HMGR and FPS1 genes. B,Expression patterns of genes relatedto the pathway from (S)-2,3-epoxy-squalene to 24-methylenelophenol.C, Expression patterns of genesrelated to the pathway from 24-methylenelophenol to campesteroland sitosterol. The expression pat-terns in A to C were analyzed usinga custom wheat 38k oligo-DNAmicroarray. Data are relative to CSand are mean values 6 SD fromthree independent leaf samples. D,The assigned barley chromosomefor steroid biosynthesis-relatedgenes. Genes were assigned toeach of seven barley chromosomesaccording to data obtained usingthe Affymetrix Barley1 GeneChipprobe array for wheat-barley addi-tion lines from Cho et al. (2006)and Bilgic et al. (2007; http://www.barleybase.org/; accession nos.BB8 and BB55). * The barley chro-mosome location of these geneswas also identified by RT-PCRwith barley-specific primers (Sup-plemental Fig. S1B); ** the corre-sponding expression pattern withthe wheat 38k array is not shownbecause values were lower than 50in the raw data (see “Materials andMethods”).

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CYP710A8(Hv) showed 93.5%, 95.6%, and 95.8% ho-mology to CYP710A8(Ta-A), -(Ta-B), and -(Ta-D), re-spectively. Sequence comparison with CYP710A1 andCYP719A2 proteins showed that the conserved se-quence FLFA(A/S)QDAS(T/S)S, which correspondsto a substrate recognition site (SRS4) of P450s (Gotoh,1992), is present in all wheat and barley CYP710A8proteins. The conserved Ala-299 of CYP710A1 (dou-ble underlined in SRS4), which is crucial for the intro-duction of the double bond in the sterol side chain(Morikawa et al., 2006b), was also present in wheatand barley CYP710A8, corresponding to Ala-315 ofCYP710A8(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-22desaturation reaction. A phylogenetic tree of CYP710Aproteins from wheat, barley, Arabidopsis, rice, andtomato is shown in Figure 5B. The wheat and barleyCYP710A8 proteins formed a cluster located on thesame branch as the rice CYP710A8 protein.

The chromosome location of the CYP710A8(Hv)gene was assigned. The expression profile of theCYP710A8(Hv) gene in barley chromosome additionlines was analyzed based on RT-PCR using barley-specific primers. The CYP710A8(Hv) gene was onlyexpressed in the 3H addition line and parental barleyline (Betzes) and, therefore, could be mapped to barley3H (Fig. 6A). The chromosome locations of wheat

Figure 5. Isolation of wheat and barley CYP710A genes. A, The deduced amino acid sequences of wheat CYP710A8s and barleyCYP710A were aligned with CYP710A1 and CYP710A2 using ClustalW2 software. Residues conserved in all six proteins areindicated by asterisks. Residues having a similar, or very similar, physiochemical character are indicated by dots or colons,respectively. The characteristic SRS4of P450s (Gotoh, 1992) is boxed. Theheme ligandCys residue ismarkedwith theblack arrow.The amino acid residues differing between CYP710A8(Hv)/CYP710A8(Ta-A) and CYP7108(Ta-B)/(Ta-D) are indicated with whitearrows. B, Phylogenetic relationships among the CYP710A proteins. A phylogenetic tree was constructed using MEGA 5.05software 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).





CYP710A8 genes were determined with nullisomic-tetrasomic lines of CS (Sears, 1966). With genomic DNAfrom nullisomic-tetrasomic lines as a template, PCRusing CYP710A8(Ta-A), -(Ta-B), and -(Ta-D) specificprimers showed that the band for CYP710A8(Ta-A)disappeared in nulli3A-tetra3B, the CYP710A8(Ta-B) band was deleted in nulli3B-tetra3A, and theCYP710A8(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).

Expression patterns of CYP710A8 genes in CS andbarley chromosome addition lines were examinedby northern-blot analysis (Fig. 6C). The expression ofCYP710A8 genes appeared to increase in the 3H ad-dition line. Thereafter, relative expression levels ofCYP710A8 genes were compared in CS and the 3H ad-dition line. The overall expression level of CYP710A8genes in the 3H addition line was increased 1.5 foldcompared with CS (Fig. 6D). Furthermore, relativeexpression levels of CYP710A8(Ta-A), -(Ta-B), and -(Ta-D) in the 3H addition line were compared by real-timePCR using the respective specific primers. The relativeexpression levels of wheat CYP710A8 genes did notchange drastically in the 3H addition line (Fig. 6E).Among the homeologous genes of CS, the gene fromthe 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). Sincethe stigmasterol content in the 3H addition line wasincreased 1.5 fold (Fig. 2), the expression of the barleygene contributed to its gain. These gene expressionprofiles of four CYP710A8 genes in the 3H additionline were confirmed by the RT-PCR products. Usingthe primers for the conserved region among the fourgenes, RT-PCR products were cloned into the plasmidvector and sequenced. Out of 39 clones identified, 22clones (56.4%), one clone (2.6%), four clones (10.2%),and 12 clones (30.8%) corresponded to the genes fromCYP710A8(Ta-A), CYP710A8(Ta-B), CYP710A8(Ta-D), andCYP710A8(Hv), respectively.

Overexpression of Wheat and Barley CYP710A inArabidopsis Increases the Stigmasterol Level at the

Expense of Sitosterol

The roles of CYP710A8 and DWF5 were further in-vestigated by overexpression of these genes in Arabi-dopsis. Transgenic Arabidopsis plants were generatedexpressing the HvDWF5, TaDWF5, CYP710A8(Hv),CYP710A8(Ta-A), CYP710A8(Ta-B), and CYP710A8(Ta-D) cDNAs under the control of the 35S promoter. Noneshowed phenotypes differing from the wild type. Two-

Figure 6. Molecular characterization of wheat and barley CYP710A8 genes. A, Expression profile of the CYP710A8(Hv) gene inbarley chromosome addition lines based on RT-PCR. Specific CYP710A8(Hv) gene primers were used for identification of thegene’s chromosome location. B, Chromosome assignments of wheat CYP710A8 genes. N3AT3B, N3BT3D, and N3DT3B areabbreviations 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 expressionlevels of total CYP710A8 genes in CS and barley 3Hwere compared with real-time PCR analysis using primers annealing to bothbarley and wheat CYP710A8 genes. E, Relative expression levels of CYP710A8 genes in CS and barley 3H were compared withreal-time PCR using their specific primers.

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week-old seedlings from T3 transgenic and wild-typeArabidopsis plants were used for characterization ofthe total sterol composition (Fig. 7A). No significantchanges were observed in the total sterol level or in thecampesterol level in wild-type plants or any transgenicline. The sterols of wild-type Arabidopsis were 17.4 62.9 mg 100 mg21 dry weight stigmasterol (6.1% of totalsterol), 202.3 6 13.1 mg 100 mg21 dry weight sitosterol(70.8% of total sterol), and 53.4 6 7.0 mg 100 mg21 dryweight campesterol (18.7% of total sterol; Fig. 7A). Allthe CYP710A8 transgenic lines contained stigmasterolat higher levels, ranging from 140 to 272 mg 100 mg21

dry weight (66.8%–78.9% of total sterol), correspondingto an approximately 10- to 20-fold increase comparedwith wild-type plants. In these transgenic lines, sitos-terol levels decreased to 7.8 to 53.2 mg 100 mg21 dryweight (2.4%–14.7% of total sterol). Expression of thesetransgenes was confirmed by RT-PCR, indicating thatthe alteration of sterol metabolism in Arabidopsis wasdue to the introduction of these alien genes (data notshown).Arabidopsis also contained cholesterol, 24-methyl-

D22-sterols, campestanol, and sitostanol as minor frac-tions (1.5%, 1.4%, 0.13%, and 0.83% of total sterol,respectively; Fig. 7B). The 24-methyl-D22-sterol con-tents of the CYP710A8(Hv) and CYP710A8(Ta-A) trans-

genic plants were increased about 1.6-fold over thewild type. Furthermore, the 24-methyl-D22-sterol levelsin the CYP710A8(Ta-B) and -(Ta-D) transgenic plantswere increased 2.5- to 4.5-fold over wild-type plants.These results suggested that CYP710A8(Ta-B) andCYP710A8(Ta-D) were involved in the desaturationto produce 24-methyl-D22-sterols. Overexpression ofDWF5 genes did not lead to any significant alterationin total sterol levels or the sterol composition (stig-masterol, sitosterol, campesterol, etc.). This meant thatincreasing the expression of wheat and barley DWF5genes did not affect the phytosterol profile of trans-genic Arabidopsis.

DISCUSSION

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

We measured the phytosterol contents in the seed-lings and grains of wheat, barley, and barley chromo-some addition lines. Overall levels of phytosterols inseedling leaves were approximately 3-fold higher thanthose in the grains (Figs. 2 and 3). The relative amountof stigmasterol in the grain was dramatically lower,while those of campestarol and sitostanol in the grains

Figure 7. Plant sterol profiles in Arabidopsis transgenic lines with CYP710A8 or DWF5 overexpression. Barley and wheatCYP710A8 andDWF5 genes were overexpressed in Arabidopsis under the control of the 35S promoter. Two-week-old seedlingswere analyzed. Seven species of phytosterols are shown in A and B. Data are mean values6 SD from three independent samples.DW, Dry weight; WT, wild type.





were higher than those in the seedlings. It is notablethat campestanol and sitostanol in barley grains werescarcely at detectable levels (Fig. 3). Additionally, thephytosterol levels of the 2H addition line of CS werelower than the levels in the grain. These lines of evi-dence suggest that the biosynthesis system(s) for phy-tosterol revealed tissue and organ specificity in wheatand barley. Hence, gene manipulation to promote geneexpression related to phytosterol biosynthesis couldbe applied to improve the functional phytosterol inwheat grains.

Systematic Analysis of Sterol Biosynthesis-RelatedGenes in Barley Chromosome Addition Lines of

Common Wheat

Sterolsmaintainmembrane integrity and fluidity andregulate membrane permeability. Sterols also serve asthe precursors for a variety of steroidal hormones. InArabidopsis, mutation of genes such as cas1 (Babiychuket 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 com-position of sterols and led to distinct phenotypes inembryogenesis and development at specific stages. Onthe other hand, overexpression of sterol biosynthesis-related genes also altered the total amount and com-position of sterols, causing severe impairment indevelopment. For example, an SMT2-overexpressingplant displayed reduced stature and growth, becausethe amount of campesterol decreased and the amountof sitosterol increased concomitantly (Schaeffer et al.,2001). Although it remains unknown how the modi-fied ratio between the two phytosterols affected plantgrowth, it is plausible that balance between campes-terol and sitosterol is important for normal plantgrowth and development.

To our knowledge, this paper is the first report onsystematic analysis of the phytosterol biosyntheticpathway-related genes in barley chromosome additionlines of common wheat. The expression of genesrelated to sterol biosynthesis in barley chromosomeaddition lines of common wheat was systematicallycharacterized. Although orthologous barley geneswere located on their respective barley chromosomes(Fig. 4D; Supplemental Fig. S1B), the 38k wheat micro-array analysis did not show these genes as having ahigh expression level in the corresponding additionlines, except for the barley chromosome 3 addition lineof hexaploid wheat (Fig. 4, A–C). In the 3H additionline, HYD1, DWF5, and CYP710A8 displayed higherexpression levels (Figs. 4, B and C, and 6, C and D).HvHYD1, HvDWF5, and CYP710A8(Hv) mapped tobarley chromosome 3 (3H) and were expressed in the3H addition line. These results also agree with phyto-sterol profiles in common wheat, barley, and barleychromosome addition lines of common wheat, inwhich an alteration of phytosterol composition wasobserved only in the 3H addition line. The sterol

profile in the 3H addition line was characterized byan increased amount of stigmasterol at the expense ofsitosterol. The amounts of campesterol and total sterolwere not changed (Fig. 2). Additive effects of barleychromosome additions to the hexaploid wheat back-ground on gene expression were only found in the 3Haddition line, and only the amount of stigmasterolamount increased. This suggests that phytosterol com-position is precisely controlled in the biopathway,except for sitgmasterol; that is, in plants, the ratio of24-ethyl sterols (sitosterol and stigmasterol) to 24-methyl sterols (campesterol) might be precisely regu-lated, but the ratio of sitosterol to stigmasterol is not asstrictly regulated.

Function of CYP710A8(Ta) and CYP710A8(Hv) in theBiosynthesis of Phytosterols

Additional expression of the barley CYP710A8 genein the 3H addition line of CS wheat (Fig. 2) and over-expression of wheat and barley CYP710A8 in Arabi-dopsis (Fig. 7) increased the stigmasterol level at theexpense of sitosterol but did not significantly alterthe levels of campesterol, cholesterol, campestanol,or total sterol. These results demonstrate that wheatCYP710A8 and barley CYP710A8 catalyze the C-22desaturase reaction converting sitosterol to stigmas-terol. Although no significant difference in convert-ing sitosterol to stigmasterol was observed amongCYP710A8(Ta-A), -(Ta-B), -(Ta-D), and CYP710A8(Hv),production activities of 24-methyl-D22-sterols were dif-ferent between CYP710A8(Hv)-CYP710A8(Ta-A) andCYP710A8(Ta-B)-CYP710A8(Ta-D) groups (Fig. 7). Sincethere were found some differences of amino acid res-idues (Fig. 5), these amino acids might be candidate(s)for enzyme activity. Overexpression of wheat andbarley CYP710A8 in Arabidopsis reversed the ratioof stigmasterol to sitosterol but did not affect thephenotype of transgenic Arabidopsis. This indicatesthat accumulation of D22-sterols at high levels did notinfluence brassinosteroid (Fig. 1) biosynthesis or otherdevelopmental processes.

The 24-methyl-D22-sterol levels increased signifi-cantly in all CYP710A8-overexpressing lines, butthe levels in CYP710A8(Ta-B)- and CYP710A8(Ta-D)-overexpressing lines were much higher than those inCYP710A8(Hv) and CYP710A8(Ta-A) lines. These re-sults indicate that all wheat and barley CYP710A8proteins are able to produce 24-methyl-D22-sterols butthat the CYP710A8(Ta-B) and -(Ta-D) proteins havehigher activity than CYP710A8(Ta-A) and CYP710A8(Hv). In Arabidopsis, CYP710A2 can catalyze theproduction of 24-methyl-D22-sterols, but CYP710A1does 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 therewere four candidate positions: both CYP710A8(Ta-A)and CYP710A8(Hv) have the same amino acid resi-dues at positions 9, 31, 381, and 453 (Fig. 5), while

Tang et al.




CYP710A8(Ta-B) and CYP710A8(Ta-D) have distinctamino acid residues from those of CYP710A8(Ta-A)and CYP710A8(Hv) (Fig. 5A, white arrows). Amongthese amino acid residue alterations, the change of Proto Thr at position 381 (Fig. 5A) might be the key to thedifferences of brassicasterol production, because thisamino 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 residuedifference is not observed between CYP710A1 andCYP710A2 in Arabidopsis. This hypothesis should beconfirmed by further experiments.In wheat, barley, and barley chromosome addition

lines of common wheat, the 24-methyl-D22-sterol levelswere almost nothing. Since activities of CYP710A fromwheat and barley per se were functional, there is apossibility that the biosynthesis of 24-epi-campesterolshould be weak, probably because of enzymatic activ-ity for DWF1 (CYP85A1; Fig. 1) in wheat and barley.Since overexpression of both wheat and barley DWF5genes in Arabidopsis did not change the profile ofphytosterol in Arabidopsis, at least in the plants so farexamined, we can conclude that the key protein forproducing stigmasterol is CYP710A. The AtDWF5gene was reported to be involved in the biosyntheticsteps that convert D7-sterol intermediates into cam-pesterol and sitosterol (Choe et al., 2000). The brassi-nosteroid profile was not examined in this study, sowe do not know if the brassinosteroid profiles werechanged by overexpression of these DWF5 genes.

Accumulation of Stigmasterol in Common Wheat HasPossible Applications

Of the main phytosterols, stigmasterol would be themost beneficial for health. The barley 3H addition lineof common wheat and Arabidopsis transgenic lineswith wheat and barley CYP710A8 genes accumulatedstigmasterol with a concurrent decrease in the sitos-terol level, but they did not display any obviousphenotypic alterations. That means that accumulationof stigmasterol in common wheat is possible. Ourstudy demonstrated that CYP710A proteins were thekey enzymes controlling the accumulation of stigmas-terol, and Manzano et al. (2004) reported that over-expression of HMGR could increase total sterol. Theseresults suggest that increased expression of CYP710Agenes, singly or combined with theHMGR gene, mightlead to an increased amount of stigmasterol. In thisstudy, 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 newbarley-wheat addition lines harboring two pairs ofbarley chromosomes. Therefore, the double chromo-some addition lines could further increase the stig-masterol content in wheat, and we are studying theamount of stigmasterol in the double barley chromo-some addition lines. Consequently, barley chromo-some addition lines are greatly useful for the study ofthe biosynthesis pathway of plant substrates and

research into the key gene(s) for increasing the effec-tive level of plant substrates. Hence, our system pro-vides a new insight into the accumulation of bioactivecompounds in common wheat and a new approach forassessing 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 mg 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, PCRwas

performed using KOD FX polymerase (Toyobo). Real-time PCR was per-

formed 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 compar-

ative threshold cycle method and normalized to wheat Tubulin. SYBR Premix

Ex Taq (TaKaRa) was used for theDWF5,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 [a-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 mg 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 PCRwith 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 con-





taining 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 ofTransgenic 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 trans-

formation into Arabidopsis (Arabidopsis thaliana) Columbia plants using the

floral dip method (Clough and Bent, 1998). T1 seeds were screened on agar

plates of 13 Murashige and Skoog medium (Duchefa Biochemie) containing

25 mg mL21 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 13Murashige 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 Gene-

Spring 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 inde-

pendent samples.

Sterol Extraction and Quantification

For wheat, barley, and each addition sample, leaves from about 25 seed-

lings 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.

Supplemental Figure S1. Confirmation of the location of the genes by PCR

analysis.

Supplemental Table S1. List of the phytosterol biosynthesis-related gene

probes found in the wheat oligo-DNA microarray or the barley DNA

chip.

Supplemental Table S2. List of PCR primers used in this study

Supplemental Table S3. List of PCR primers used for wheat and barley

CYP710A8 cDNA cloning

Received July 18, 2011; accepted September 16, 2011; published September 27,

2011.

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a new insight into application for barley chromosome addition

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