Transcriptome profiling provides new insights into the formation of floral scent in Hedychium coronarium

BMC Genomics

Yanping Fan²  Rangcai Yu¹  Yuechong Yue² 

[1] College of Life Sciences, South China Agricultural University, Guangzhou 510642, China;The Research Center for Ornamental Plants, College of Forestry and Landscape Architecture, South China Agricultural University, Guangzhou 510642, China

关键词: Transcription factor;    Benzenoid;    Terpenoid;    Secondary metabolism;    Floral scent;    Transcriptome;    Zingiberaceae;    Hedychium coronarium;   

Others  :  1221252
DOI  :  10.1186/s12864-015-1653-7

 received in 2015-03-27, accepted in 2015-05-20,  发布年份 2015

【 摘 要 】

Background

Hedychium coronarium is a popular ornamental plant in tropical and subtropical regions because its flowers not only possess intense and inviting fragrance but also enjoy elegant shape. The fragrance results from volatile terpenes and benzenoids presented in the floral scent profile. However, in this species, even in monocots, little is known about the underlying molecular mechanism of floral scent production.

Results

Using Illumina platform, approximately 81 million high-quality reads were obtained from a pooled cDNA library. The de novo assembly resulted in a transcriptome with 65,591 unigenes, 50.90 % of which were annotated using public databases. Digital gene expression (DGE) profiling analysis revealed 7,796 differential expression genes (DEGs) during petal development. GO term classification and KEGG pathway analysis indicated that the levels of transcripts changed significantly in “metabolic process”, including “terpenoid biosynthetic process”. Through a systematic analysis, 35 and 33 candidate genes might be involved in the biosynthesis of floral volatile terpenes and benzenoids, respectively. Among them, flower-specific HcDXS2A, HcGPPS, HcTPSs, HcCNL and HcBCMT1 might play critical roles in regulating the formation of floral fragrance through DGE profiling coupled with floral volatile profiling analyses. In vitro characterization showed that HcTPS6 was capable of generating β-farnesene as its main product. In the transcriptome, 1,741 transcription factors (TFs) were identified and 474 TFs showed differential expression during petal development. It is supposed that two R2R3-MYBs with flower-specific and developmental expression might be involved in the scent production.

Conclusions

The novel transcriptome and DGE profiling provide an important resource for functional genomics studies and give us a dynamic view of biological process during petal development in H. coronarium. These data lay the basis for elucidating the molecular mechanism of floral scent formation and regulation in monocot. The results also provide the opportunities for genetic modification of floral scent profile in Hedychium.

【 授权许可】

   
2015 Yue at al.

【 图 表 】

Fig. 1.

Fig. 2.

Fig. 3.

Fig. 4.

Fig. 5.

Fig. 6.

Fig. 7.

Fig. 8.

Fig. 9.

Fig. 10.

Fig. 11.

Fig. 12.

Fig. 13.

【 参考文献 】

• [1]Pichersky E, Dudareva N. Scent engineering: toward the goal of controlling how flowers smell. Trends Biotechnol. 2007; 25(3):105-10.
• [2]Muhlemann JK, Klempien A, Dudareva N. Floral volatiles: from biosynthesis to function. Plant Cell Environ. 2014; 37(8SI):1936-1949.
• [3]Raguso RA. Wake up and smell the roses: the ecology and evolution of floral scent. Annu Rev Ecol Evol Syst. 2008; 39:549-69.
• [4]Pichersky E, Gershenzon J. The formation and function of plant volatiles: perfumes for pollinator attraction and defense. Curr Opin Plant Biol. 2002; 5(3):237-43.
• [5]Dudareva N, Klempien A, Muhlemann JK, Kaplan I. Biosynthesis, function and metabolic engineering of plant volatile organic compounds. New Phytol. 2013; 198(1):16-32.
• [6]Dudareva N, Pichersky E. Metabolic engineering of plant volatiles. Curr Opin Biotech. 2008; 19(2):181-9.
• [7]Knudsen JT, Eriksson R, Gershenzon J, Stahl B. Diversity and distribution of floral scent. Bot Rev. 2006; 72(1):1-120.
• [8]Vranova E, Coman D, Gruissem W. Network analysis of the MVA and MEP pathways for isoprenoid synthesis. Annu Rev Plant Biol. 2013; 64:665-700.
• [9]Tholl D. Terpene synthases and the regulation, diversity and biological roles of terpene metabolism. Curr Opin Plant Biol. 2006; 9(3):297-304.
• [10]Pichersky E, Raguso RA, Lewinsohn E, Croteau R. Floral scent production in Clarkia (Onagraceae) I Localization and developmental modulation of monoterpene emission and linalool synthase activity. Plant Physiol. 1994; 106(4):1533-40.
• [11]Pichersky E, Lewinsohn E, Croteau R. Purification and characterization of S-linalool synthase, an enzyme involved in the production of floral scent in Clarkia breweri. Arch Biochem Biophys. 1995; 316(2):803-7.
• [12]Chen F, Tholl D, D'Auria JC, Farooq A, Pichersky E, Gershenzon J. Biosynthesis and emission of terpenoid volatiles from Arabidopsis flowers. Plant Cell. 2003; 15(2):481-94.
• [13]Tholl D, Chen F, Petri J, Gershenzon J, Pichersky E. Two sesquiterpene synthases are responsible for the complex mixture of sesquiterpenes emitted from Arabidopsis flowers. Plant J. 2005; 42(5):757-71.
• [14]Guterman I, Shalit M, Menda N, Piestun D, Dafny-Yelin M, Shalev G et al.. Rose scent: Genomics approach to discovering novel floral fragrance-related genes. Plant Cell. 2002; 14(10):2325-38.
• [15]Dudareva N, Martin D, Kish CM, Kolosova N, Gorenstein N, Faldt J et al.. (E)-beta-ocimene and myrcene synthase genes of floral scent biosynthesis in snapdragon: function and expression of three terpene synthase genes of a new terpene synthase subfamily. Plant Cell. 2003; 15(5):1227-1241.
• [16]Nagegowda DA, Gutensohn M, Wilkerson CG, Dudareva N. Two nearly identical terpene synthases catalyze the formation of nerolidol and linalool in snapdragon flowers. Plant J. 2008; 55(2):224-39.
• [17]Roeder S, Hartmann A, Effmert U, Piechulla B. Regulation of simultaneous synthesis of floral scent terpenoids by the 1,8-cineole synthase of Nicotiana suaveolens. Plant Mol Biol. 2007; 65(1–2):107-24.
• [18]Hong G, Xue X, Mao Y, Wang L, Chen X. Arabidopsis MYC2 interacts with DELLA proteins in regulating sesquiterpene synthase gene expression. Plant Cell. 2012; 24(6):2635-48.
• [19]Zvi MM, Shklarman E, Masci T, Kalev H, Debener T, Shafir S et al.. PAP1 transcription factor enhances production of phenylpropanoid and terpenoid scent compounds in rose flowers. New Phytol. 2012; 195(2):335-45.
• [20]Maeda H, Dudareva N. The shikimate pathway and aromatic amino acid biosynthesis in plants. Annu Rev Plant Biol. 2012; 63:73-105.
• [21]Wildermuth MC. Variations on a theme: synthesis and modification of plant benzoic acids. Curr Opin Plant Biol. 2006; 9(3):288-96.
• [22]Boatright J, Negre F, Chen XL, Kish CM, Wood B, Peel G et al.. Understanding in vivo benzenoid metabolism in petunia petal tissue. Plant Physiol. 2004; 135(4):1993-2011.
• [23]Klempien A, Kaminaga Y, Qualley A, Nagegowda DA, Widhalm JR, Orlova I et al.. Contribution of CoA ligases to benzenoid biosynthesis in petunia flowers. Plant Cell. 2012; 24(5):2015-30.
• [24]Qualley AV, Widhalm JR, Adebesin F, Kish CM, Dudareva N. Completion of the core beta-oxidative pathway of benzoic acid biosynthesis in plants. P Natl Acad Sci Usa. 2012; 109(40):16383-8.
• [25]Van Moerkercke A, Schauvinhold I, Pichersky E, Haring MA, Schuurink RC. A plant thiolase involved in benzoic acid biosynthesis and volatile benzenoid production. Plant J. 2009; 60(2):292-302.
• [26]Colquhoun TA, Marciniak DM, Wedde AE, Kim JY, Schwieterman ML, Levin LA et al.. A peroxisomally localized acyl-activating enzyme is required for volatile benzenoid formation in a Petunia×hybrida cv. 'Mitchell Diploid' flower. J Exp Bot. 2012; 63(13):4821-4833.
• [27]Bussell JD, Reichelt M, Wiszniewski AAG, Gershenzon J, Smith SM. Peroxisomal ATP-binding cassette transporter COMATOSE and the multifunctional protein ABNORMAL INFLORESCENCE MERISTEM are required for the production of benzoylated metabolites in Arabidopsis seeds. Plant Physiol. 2014; 164(1):48-54.
• [28]Long MC, Nagegowda DA, Kaminaga Y, Ho KK, Kish CM, Schnepp J et al.. Involvement of snapdragon benzaldehyde dehydrogenase in benzoic acid biosynthesis. Plant J. 2009; 59(2):256-65.
• [29]Verdonk JC, Haring MA, van Tunen AJ, Schuurink RC. ODORANT1 regulates fragrance biosynthesis in petunia flowers. Plant Cell. 2005; 17(5):1612-24.
• [30]Spitzer-Rimon B, Marhevka E, Barkai O, Marton I, Edelbaum O, Masci T et al.. EOBII, a gene encoding a flower-specific regulator of phenylpropanoid volatiles’ biosynthesis in petunia. Plant Cell. 2010; 22(6):1961-76.
• [31]Van Moerkercke A, Haring MA, Schuurink RC. The transcription factor EMISSION OF BENZENOIDS II activates the MYB ODORANT1 promoter at a MYB binding site specific for fragrant petunias. Plant J. 2011; 67(5):917-28.
• [32]Spitzer-Rimon B, Farhi M, Albo B, Cna'Ani A, Ben ZM, Masci T et al.. The R2R3-MYB-like regulatory factor EOBI, acting downstream of EOBII, regulates scent production by activating ODO1 and structural scent-related genes in petunia. Plant Cell. 2012; 24(12):5089-105.
• [33]Colquhoun TA, Kim JY, Wedde AE, Levin LA, Schmitt KC, Schuurink RC et al.. PhMYB4 fine-tunes the floral volatile signature of Petunia x hybrida through PhC4H. J Exp Bot. 2011; 62(3):1133-43.
• [34]Wang Z, Gerstein M, Snyder M. RNA-Seq: a revolutionary tool for transcriptomics. Nat Rev Genet. 2009; 10(1):57-63.
• [35]Grabherr MG, Haas BJ, Yassour M, Levin JZ, Thompson DA, Amit I et al.. Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nat Biotechnol. 2011; 29(7):644-52.
• [36]Hao DC, Ge G, Xiao P, Zhang Y, Yang L. The first insight into the tissue specific taxus transcriptome via Illumina second generation sequencing. Plos One. 2011; 6(6):e21220.
• [37]Tang Q, Ma X, Mo C, Wilson IW, Song C, Zhao H et al.. An efficient approach to finding Siraitia grosvenorii triterpene biosynthetic genes by RNA-seq and digital gene expression analysis. BMC Genomics. 2011; 12:343. BioMed Central Full Text
• [38]Hyun TK, Rim Y, Jang HJ, Kim CH, Park J, Kumar R et al.. De novo transcriptome sequencing of Momordica cochinchinensis to identify genes involved in the carotenoid biosynthesis. Plant Mol Biol. 2012; 79(4–5):413-27.
• [39]Lou Q, Liu Y, Qi Y, Jiao S, Tian F, Jiang L et al.. Transcriptome sequencing and metabolite analysis reveals the role of delphinidin metabolism in flower colour in grape hyacinth. J Exp Bot. 2014; 65(12):3157-64.
• [40]Wu Z, Raven P. Hedychium. In: Flora of China. volume 24. Saint Louis: Missouri Botanical Garden Press; 2000;370–377.
• [41]Van Valkenburg JLCH, Bunyapraphatsara N. Plant resources of south-east Asia. Medicinal and poisonous plants 2., vol. 12. Backhuys Publishers, Leiden; 2001.
• [42]Macedo JF. The genus Hedychium Koening (Zingiberaceae) in Minas Gerais State. Daphne Revista do Herbário PAMG da EPAMIG. 1997; 7(2):27-31.
• [43]Baez D, Pino JA, Morales D. Floral scent composition in Hedychium coronarium J Koenig analyzed by SPME. J Essent Oil Res. 2011; 23(3):64-7.
• [44]Lu Y, Zhong CX, Wang L, Lu C, Li XL, Wang PJ. Anti-inflammation activity and chemical composition of flower essential oil from Hedychium coronarium. Afr J Biotechnol. 2009; 8(20):5373-7.
• [45]Lan JB, Yu RC, Yu YY, Fan YP. Molecular cloning and expression of Hedychium coronarium farnesyl pyrophosphate synthase gene and its possible involvement in the biosynthesis of floral and wounding/herbivory induced leaf volatile sesquiterpenoids. Gene. 2013; 518(2):360-7.
• [46]Yue Y, Yu R, Fan Y. Characterization of two monoterpene synthases involved in floral scent formation in Hedychium coronarium. Planta. 2014; 240(4):745-62.
• [47]Miller JR, Koren S, Sutton G. Assembly algorithms for next-generation sequencing data. Genomics. 2010; 95(6):315-27.
• [48]Tatusov RLTN, Fedorova NDFN, Jackson JDJN, Jacobs ARJN, Kiryutin BKNN, Koonin EVKN et al.. The COG database: An updated version includes eukaryotes. BMC Bioinformatics. 2003; 4:41. BioMed Central Full Text
• [49]Kanehisa M, Goto S, Kawashima S, Okuno Y, Hattori M. The KEGG resource for deciphering the genome. Nucleic Acids Res. 2004; 32(Database issue):D277-80.
• [50]Mortazavi A, Williams BA, McCue K, Schaeffer L, Wold B. Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nat Methods. 2008; 5(7):621-8.
• [51]Zhang N, Zhang HJ, Zhao B, Sun QQ, Cao YY, Li R et al.. The RNA-seq approach to discriminate gene expression profiles in response to melatonin on cucumber lateral root formation. J Pineal Res. 2014; 56(1):39-50.
• [52]Phillips MA, Leon P, Boronat A, Rodriguez-Concepcion M. The plastidial MEP pathway: unified nomenclature and resources. Trends Plant Sci. 2008; 13(12):619-23.
• [53]Ganjewala D, Kumar S, Luthra R. An account of cloned genes of methyl-erythritol-4-phosphate pathway of isoprenoid biosynthesis in plants. Curr Issues Mol Biol. 2009; 11 Suppl. 1:i35-45.
• [54]Hsieh MH, Chang CY, Hsu SJ, Chen JJ. Chloroplast localization of methylerythritol 4-phosphate pathway enzymes and regulation of mitochondrial genes in ispD and ispE albino mutants in Arabidopsis. Plant Mol Biol. 2008; 66(6):663-73.
• [55]Estevez JM, Cantero A, Reindl A, Reichler S, Leon P: 1-Deoxy-D-xylulose-5-phosphate synthase, a limiting enzyme for plastidic isoprenoid biosynthesis in plants. J Biol Chem 2001;276(25):22901–22909.
• [56]Cordoba E, Salmi M, Leon P. Unravelling the regulatory mechanisms that modulate the MEP pathway in higher plants. J Exp Bot. 2009; 60(10):2933-43.
• [57]Walter MH, Hans J, Strack D. Two distantly related genes encoding 1-deoxy-D-xylulose 5-phosphate synthases: differential regulation in shoots and apocarotenoid-accumulating mycorrhizal roots. Plant J. 2002; 31(3):243-54.
• [58]Phillips MA, Walter MH, Ralph SG, Dabrowska P, Luck K, Uros EM et al.. Functional identification and differential expression of 1-deoxy-D-xylulose 5-phosphate synthase in induced terpenoid resin formation of Norway spruce (Picea abies). Plant Mol Biol. 2007; 65(3):243-57.
• [59]Cordoba E, Porta H, Arroyo A, Roman CS, Medina L, Rodriguez-Concepcion M et al.. Functional characterization of the three genes encoding 1-deoxy-D-xylulose 5-phosphate synthase in maize. J Exp Bot. 2011; 62(6):2023-38.
• [60]Vandermoten S, Haubruge E, Cusson M. New insights into short-chain prenyltransferases: structural features, evolutionary history and potential for selective inhibition. Cell Mol Life Sci. 2009; 66(23):3685-95.
• [61]Bohlmann J, Meyer-Gauen G, Croteau R. Plant terpenoid synthases: molecular biology and phylogenetic analysis. P Natl Acad Sci USA. 1998; 95(8):4126-33.
• [62]Degenhardt J, Kollner TG, Gershenzon J. Monoterpene and sesquiterpene synthases and the origin of terpene skeletal diversity in plants. Phytochemistry. 2009; 70(15–16):1621-37.
• [63]Tzin V, Galili G. New insights into the shikimate and aromatic amino acids biosynthesis pathways in plants. Mol Plant. 2010; 3(6):956-72.
• [64]Tzin V, Malitsky S, Ben Zvi MM, Bedair M, Sumner L, Aharoni A et al.. Expression of a bacterial feedback-insensitive 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase of the shikimate pathway in Arabidopsis elucidates potential metabolic bottlenecks between primary and secondary metabolism. New Phytol. 2012; 194(2):430-9.
• [65]Shockey J, Browse J. Genome-level and biochemical diversity of the acyl-activating enzyme superfamily in plants. Plant J. 2011; 66(1):143-60.
• [66]Murfitt LM, Kolosova N, Mann CJ, Dudareva N. Purification and characterization of S-adenosyl-L-methionine:benzoic acid carboxyl methyltransferase, the enzyme responsible for biosynthesis of the volatile ester methyl benzoate in flowers of Antirrhinum majus. Arch Biochem Biophys. 2000; 382(1):145-51.
• [67]Negre F, Kish CM, Boatright J, Underwood B, Shibuya K, Wagner C et al.. Regulation of methylbenzoate emission after pollination in snapdragon and petunia flowers. Plant Cell. 2003; 15(12):2992-3006.
• [68]Riechmann JL, Heard J, Martin G, Reuber L, Jiang CZ, Keddie J et al.. Arabidopsis transcription factors: Genome-wide comparative analysis among eukaryotes. Science. 2000; 290(5499):2105-10.
• [69]Yang CQ, Fang X, Wu XM, Mao YB, Wang LJ, Chen XY. Transcriptional regulation of plant secondary metabolism. J Integr Plant Biol. 2012; 54(10SI):703-712.
• [70]Perez-Rodriguez P, Riano-Pachon DM, Correa L, Rensing SA, Kersten B, Mueller-Roeber B. PInTFDB: updated content and new features of the plant transcription factor database. Nucleic Acids Res. 2010; 381(Database issue):D822-7.
• [71]Dubos C, Stracke R, Grotewold E, Weisshaar B, Martin C, Lepiniec L. MYB transcription factors in Arabidopsis. Trends Plant Sci. 2010; 15(10):573-81.
• [72]Annadurai RS, Jayakumar V, Mugasimangalam RC, Katta MA, Anand S, Gopinathan S et al.. Next generation sequencing and de novo transcriptome analysis of Costus pictus D. Don, a non-model plant with potent anti-diabetic properties. Bmc Genomics. 2012; 13:663. BioMed Central Full Text
• [73]Annadurai RS, Neethiraj R, Jayakumar V, Damodaran AC, Rao SN, Katta MA et al.. De Novo transcriptome assembly (NGS) of Curcuma longa L. rhizome reveals novel transcripts related to anticancer and antimalarial terpenoids. Plos One. 2013; 8(2):e56217.
• [74]Prasath D, Karthika R, Habeeba NT, Suraby EJ, Rosana OB, Shaji A et al.. Comparison of the transcriptomes of ginger (Zingiber officinale Rosc.) and mango ginger (Curcuma amada Roxb.) in response to the bacterial wilt infection. Plos One. 2014; 9(6):e99731.
• [75]Verdonk JC, Ric DVC, Verhoeven HA, Haring MA, van Tunen AJ, Schuurink RC. Regulation of floral scent production in petunia revealed by targeted metabolomics. Phytochemistry. 2003; 62(6):997-1008.
• [76]Muhlemann JK, Maeda H, Chang CY, San MP, Baxter I, Cooper B et al.. Developmental changes in the metabolic network of snapdragon flowers. Plos One. 2012; 7(7):e40381.
• [77]Hsiao Y, Jeng M, Tsai W, Chuang Y, Li C, Wu T et al.. A novel homodimeric geranyl diphosphate synthase from the orchid Phalaenopsis bellina lacking a DD(X) 2-4 D motif. Plant J. 2008; 55(5):719-33.
• [78]Chen F, Tholl D, Bohlmann J, Pichersky E. The family of terpene synthases in plants: a mid-size family of genes for specialized metabolism that is highly diversified throughout the kingdom. Plant J. 2011; 66(1):212-29.
• [79]Landmann C, Fink B, Festner M, Dregus M, Engel KH, Schwab W. Cloning and functional characterization of three terpene synthases from lavender (Lavandula angustifolia). Arch Biochem Biophys. 2007; 465(2):417-29.
• [80]Langer KM, Jones CR, Jaworski EA, Rushing GV, Kim JY, Clark DG et al.. PhDAHP1 is required for floral volatile benzenoid/phenylpropanoid biosynthesis in Petunia x hybrida cv ‘Mitchell Diploid’. Phytochemistry. 2014; 103:22-31.
• [81]Pott MB, Hippauf F, Saschenbrecker S, Chen F, Ross J, Kiefer I et al.. Biochemical and structural characterization of benzenoid carboxyl methyltransferases involved in floral scent production in Stephanotis floribunda and Nicotiana suaveolens. Plant Physiol. 2004; 135(4):1946-55.
• [82]Knudsen JT, Tollsten L. Trends in floral scent chemistry in pollination syndromes: floral scent composition in moth-pollinated taxa. Bot J Linn Soc. 1993; 113(3):263-84.
• [83]Kessler D, Diezel C, Clark DG, Colquhoun TA, Baldwin IT. Petunia flowers solve the defence/apparency dilemma of pollinator attraction by deploying complex floral blends. Ecol Lett. 2013; 16(3):299-306.
• [84]Huang M, Sanchez-Moreiras AM, Abel C, Sohrabi R, Lee S, Gershenzon J et al.. The major volatile organic compound emitted from Arabidopsis thaliana flowers, the sesquiterpene (E)-beta-caryophyllene, is a defense against a bacterial pathogen. New Phytol. 2012; 193(4):997-1008.
• [85]Junker RR, Bluthgen N. Floral scents repel facultative flower visitors, but attract obligate ones. Ann Bot-London. 2010; 105(5):777-82.
• [86]Junker RRRJ, Heidinger IMM, Bluethgen N. Floral scent terpenoids deter the facultative florivore Metrioptera bicolor (Ensifera, Tettigoniidae, Decticinae). J Orthopt Res. 2010; 19(1):69-74.
• [87]Finn RD, Clements J, Eddy SR. HMMER web server: interactive sequence similarity searching. Nucleic Acids Res. 2011; 39(Web Server issue):W29-37.
• [88]Gotz S, Garcia-Gomez JM, Terol J, Williams TD, Nagaraj SH, Nueda MJ et al.. High-throughput functional annotation and data mining with the Blast2GO suite. Nucleic Acids Res. 2008; 36(10):3420-35.
• [89]Li B, Dewey CN. RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome. BMC Bioinformatics. 2011; 12:323. BioMed Central Full Text
• [90]Wang L, Feng Z, Wang X, Wang X, Zhang X. DEGseq: an R package for identifying differentially expressed genes from RNA-seq data. Bioinformatics. 2010; 26(1):136-8.
• [91]Storey JD. The positive false discovery rate: A Bayesian interpretation and the q-value. Ann Stat. 2003; 31(6):2013-35.
• [92]Young MD, Wakefield MJ, Smyth GK, Oshlack A. Gene ontology analysis for RNA-seq: accounting for selection bias. Genome Biol. 2010; 11(2):R14. BioMed Central Full Text
• [93]Mao XZ, Cai T, Olyarchuk JG, Wei LP. Automated genome annotation and pathway identification using the KEGG Orthology (KO) as a controlled vocabulary. Bioinformatics. 2005; 21(19):3787-93.
• [94]Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2-DELTADELTACT method. Methods. 2001; 25(4):402-8.