Please use this identifier to cite or link to this item:
Application of harpin proteins on alpine tea tree seedlings growing in the greenhouse condition
|關鍵字:||茶樹;耐旱;青心烏龍;tea;heat resistance;harpin||引用:||連橫 (1967) 台灣通史卷二十七〈農業志〉。台灣省文獻委員會。台灣南投。 徐英祥 (1995) 台灣日據時期茶業文獻譯集。台灣省茶業改良場。台灣桃園。 張清寬 (2003) 茶樹的起源及分類地位。遠足文化。臺灣台北。 臺灣區茶輸出業同業公會 (1965)《臺茶輸出百年簡史》。臺灣區茶輸出業同業公會。臺灣。 Acharya K, Pal AK, Gulati A, Kumar S, Singh AK and Ahuja PS (2013) Overexpression of Camellia sinensis thaumatin-like protein, CsTLP in potato confers enhanced resistance to Macrophomina phaseolina and Phytophthora infestans infection. Mol Biotechnol 2:609-622. Ahmed FE and Hall AE ( 1992) Heat Injury during Early Floral Bud Development in Cowpea. Crop sci 4:764-767. Ahmad M, Majerczak DR, Pike S, Hoyos ME, Novacky A, Coplin DL (2001) Biological activity of harpin produced by Pantoea stewartii subsp. stewartii. Mol Plant Microbe Interact 14:1223-1234. Arlat M, Van Gijsegem F, Huet JC, Pernollet JC, Boucher CA (1994) PopA1, a protein which induces a hypersensitivity-like response on specific Petunia genotypes, is secreted via the Hrp pathway of Pseudomonas solanacearum. EMBO J 13:543-553. Assmann SM and Shimazaki Ki (1999) The multisensory guard cell. stomatal responses to blue light and abscisic acid. Plant Physiol 3:809-816. Atkinson NJ and Urwin PE (2012) The interaction of plant biotic and abiotic stresses: from genes to the field. J Exp Bot 10:3523-3543. Babu PV and Liu, D (2008) Green tea catechins and cardiovascular health. Curr Med Chem 15:1840-50. Baker CJ, Atkinson MM and Collmer A (1987) Concurrent loss in Tn5 mutants of Pseudomonas syringae pv. syringae of the ability to induce the hypersensitive response and host plasma membrane K+ / H+ exchange in tobacco. J Physiol Biochem 77:1268-1272. Bent AF and Mackey D (2007) Elicitors, effectors, and R genes: the new paradigm and a lifetime supply of questions. Annu Rev Phytopathol 45:399-436. Boccara M, Schwartz W, Guiot E, Vidal G, De Paepe R, Dubois A and Boccara AC (2007) Early chloroplastic alterations analysed by optical coherence tomography during a harpin-induced hypersensitive response. Plant J 50:338-346. Bonkowski M and Clarholm M (2012) Stimulation of plant growth through interactions of bacteria and protozoa: testing the auxiliary microbial loop hypothesis. Acta Protozool 51:237-247. Bowler C and Fluhr R (2000) The role of calcium and activated oxygens as signals for controlling cross-tolerance. Trends Plant Sci 5:241-246. Cao W and Moss DN (1989) Temperature effect on leaf emergence and phyllochron in wheat and barley. Crop Science 29: 1018–1021. Carpentier SC, Witters E, Laukens K, Deckers P, Swennen R, Panis B. (2005). Preparation of protein extracts from recalcitrant plant tissues: an evaluation of different methods for two-dimensional gel electrophoresis analysis. Proteomics 5:2497-2507. Chan EWC, Soh, EY, Tie PP and Law YP (2011). Antioxidant and antibacterial properties of green, black, and herbal teas of Camellia sinensis. Pharmacognosy Res 3:266–272. Chan YJ (2010) Biochemical features and application of harpin protein on controlling bacterial disease from Pseudomonas syringae pv. averrhoi. Master Thesis, National Chung Hsing University. pp.50. Chandra S, Chakraborty N, Chakraborty A, Rai R, Bera B and Acharya K (2014) Abiotic Elicitor-Mediated Improvement of Innate Immunity in Camellia sinensis. J. Plant Growth Regul 33:849–859. Chang X and Nick P (2012) Defence signalling triggered by flg22 and harpin is integrated into a different stilbene output in Vitis cells. PLoS One 7:e40446. Charkowski AO, Alfano JR, Preston G, Yuan J, He SY, Collmer A (1998) The Pseudomonas syringae pv. tomato HrpW protein has domains similar to harpins and pectate lyases and can elicit the plant hypersensitive response and bind to pectate. J Bacteriol 180:5211-5217. Chen GH, Yang CY, Lee SJ, Wu CC, Tzen JTC (2014) Catechin content and the degree of its galloylation in oolong tea are inversely correlated with cultivation altitude. J Food Drug Anal. 22:303-309. Chisholm ST, Coaker G, Day B and Staskawicz BJ (2006) Host-microbe interactions: shaping the evolution of the plant immune response. Cell 124:803-14. Chou J and Huang Y (2010) Differential expression of thaumatin-like proteins in sorghum infested with greenbugs. Z Naturforsch C. 65:271-276. Choudhury FK, Rivero RM, Blumwald E and Mittler R (2017) Reactive oxygen species, abiotic stress and stress combination. Plant J 5:856-867. Clarke A, Mur LA, Darby RM, Kenton P (2005) Harpin modulates the accumulation of salicylic acid by Arabidopsis cells via apoplastic alkalization. J. Exp. Bot 56:3129-3136. Coleman-Derr D and Tringe SG (2014) Building the crops of tomorrow: advantages of symbiont-based approaches to improving abiotic stress tolerance. Front Microbiol 5:283. da Cunha L, Sreerekha MV and Mackey D (2007) Defense suppression by virulence effectors of bacterial phytopathogens. Curr Opin Plant Biol 10:349-57. Dixon RA (2001) Natural products and plant disease resistance. Nature 411:843–847. Dong H, Delaney TP, Bauer DW, Beer SV (2002) Harpin induces disease resistance in Arabidopsis through the systemic acquired resistance pathway mediated by salicylic acid and the NIM1 gene. Plant J 20:207-215. Dong HP, Yu H, Bao Z, Guo X, Peng J, Yao Z, Chen G, Qu S, Dong H (2005) The ABI2-dependent abscisic acid signalling controls HrpN-induced drought tolerance in Arabidopsis. Planta 221:313-27. Dou J, Lee VSY, Tzen JTC and Lee MR (2007) Identification and comparison of phenolic compounds in the preparation of oolong tea manufactured by semifermentation and drying processes. J Agric Food Chem 18:7462-7468. Eleftheriou EP, Adamakis ID, Panteris E and Fatsiou M (2015) Chromium-induced ultrastructural changes and oxidative stress in roots of Arabidopsis thaliana. Int J Mol Sci 16:15852-15871. Finkel T (1998) Oxygen radicals and signaling. Curr Opin Cell Biol 2:248-253. Fontanilla JM, Montes M,. De Prado R (2005) Effects of the foliar-applied protein harpin(Ea) (messenger) on tomatoes infected with Phytophthora infestans. Commun Agric Appl Biol Sci 3:41-45. Graham HN (1992) Green tea composition, consumption, and polyphenol chemistry. Prev Med 21:334–350 Goh CH, Veliz Vallejos DF, Nicotra AB and Mathesius U (2013) The impact of beneficial plant-associated microbes on plant phenotypic plasticity. J Chem Ecol 39:826-839. Gómez-Gómez L and Boller T (2000) FLS2: an LRR receptor-like kinase involved in the perception of the bacterial elicitor flagellin in Arabidopsis. Mol Cell 5:1003-1011. Grant SR, Fisher EJ, Chang JH, Mole BM and Dangl JL (2006) Subterfuge and manipulation: type III effector proteins of phytopathogenic bacteria. Annu Rev Microbiol 60:425-449. Gu L (2003) Comment on 'Climate and management contributions to recent trends in U.S. agricultural yields'. Science 300:1505. Guo YW (2013) Characterization of HrpY and HrpW proteins in Acidovorax avenae subsp. avenae CH12. Master Thesis, National Chung Hsing University. pp.69. He F, Pan QH, Shi Y, Duan CQ (2008) Biosynthesis and genetic regulation of proanthocyanidins in plants. Molecules 10:2674-2703. He SY, Huang HC, Collmer A (1993) Pseudomonas syringae pv. syringae harpinPss: a protein that is secreted via the Hrp pathway and elicits the hypersensitive response in plants. Cell 73:1255-1266. Hsieh SK, Lo YH, Wu CC, Chung TY, Tzen JTC (2015) Identification of biosynthetic intermediates of teaghrelins and teaghrelin-like compounds in oolong teas, and their molecular docking to the ghrelin receptor. J Food Drug Anal 4:660-670. Hurley B, Subramaniam R, Guttman DS and Desveaux D (2014) Proteomics of effector-triggered immunity (ETI) in plants. Virulence. 5:752-60. Igarashi D, Tsuchida H, Miyao M and Ohsumi C (2006) Glutamate:glyoxylate aminotransferase modulates amino acid content during photorespiration. Plant Physiol 3:901-910. Innis MA, Gelfand DH, Sninsky JJ and White TJI (1990) PCR protocols. San Diego: Academic Press. pp 21-27. Jaleel CA, Gopi R, Manivannan P and Panneerselvam R (2007) Antioxidative potentials as a protective mechanism in Catharanthus roseus (L.) G. Don. plants under salinity stress. TURK J BOT 3:245-251. Jeffrey LC, Amutha SK, Eunsook P, Meenu SP, Kyle H, Shannon M, Kirk C and S. P. Dinesh-Kumar (2016) Chloroplast stromules function during innate immunity. Dev Cell. Jiang M and Zhang J (2002a) Involvement of plasma-membrane NADPH oxidase in abscisic acid- and water stress-induced antioxidant defense in leaves of maize seedlings. Planta 215:1022-1030. Jiang M and Zhang J (2002b) Water stress-induced abscisic acid accumulation triggers the increased generation of reactive oxygen species and up-regulates the activities of antioxidant enzymes in maize leaves. J. Exp. Bot. 53:2401-2410. Kato M and Shimizu S (1985) Chlorophyll metabolism in higher plants VI. Involvement of peroxidase in chlorophyll degradation. Plant & Cell Physiol 26:1291-1301. Kim JF and Beer SV (1998) HrpW of Erwinia amylovora, a new harpin that contains a domain homologous to pectate lyases of a distinct class. J Bacteriol 180:5203-5210. Kim JG, Jeon E, Oh J, Moon JS, Hwang I (2004) Mutational analysis of Xanthomonas harpin HpaG identifies a key functional region that elicits the hypersensitive response in nonhost plants. J Bacteriol 186:6239-6247. Knight H and Knight MR (2001) Abiotic stress signalling pathways: specificity and cross-talk. Trends Plant Sci 6:262-267. Kuhnert N, Clifford MN, Müller A (2010) Oxidative cascade reactions yielding polyhydroxy-theaflavins and theacitrins in the formation of black tea thearubigins: evidence by tandem LC-MS. Food Funct 2:180-199. Lee J, Klüsener B, Tsiamis G, Stevens C, Neyt C, Tampakaki AP, Panopoulos NJ, Nöller J, Weiler EW, Cornelis GR, Mansfield JW and Nürnberger T (2001) HrpZPsph from the plant pathogen Pseudomonas syringae pv. phaseolicola binds to lipid bilayers and forms an ion-conducting pore in vitro. Proc Natl Acad Sci U S A 98: 289-294. Lee S, Razqan GS, Kwon DH (2017) Antibacterial activity of epigallocatechin-3-gallate (EGCG) and its synergism with β-lactam antibiotics sensitizing carbapenem-associated multidrug resistant clinical isolates of Acinetobacter baumannii. Phytomedicine 24:49-55. Li FL (2015) Role of APX8, GR2 and GR3 in ABA-induced rice seedling senescence. Master Thesis, National Chung Hsing University. pp.122. Liu H, Wang Y, Zhou X, Wang C, Wang C, Fu J and Wei T (2016) Overexpression of a harpin-encoding gene popW from Ralstonia solanacearum primed antioxidant defenses with enhanced drought tolerance in tobacco plants. Plant Cell Rep 6:1333-1344. Liu JJ, Sturrock R and Ekramoddoullah AK (2010) The superfamily of thaumatin-like proteins: its origin, evolution, and expression towards biological function. Plant Cell Rep. 5:419-436. Lo YH, Chen YJ, Chang CI, Lin YW, Chen CY, Lee MR, Lee VSY and Tzen JTC (2014) Teaghrelins, unique acylated flavonoid tetraglycosides in chin-shin oolong tea, are putative oral agonists of the ghrelin receptor. J Agric Food Chem 22:5085-5091. Lobell DB and Asner GP (2003) Climate and management contributions to recent trends in U.S. agricultural yields. Science 299:1032. Lucas JA, García-Cristobal J, Bonilla A, Ramos B and Gutierrez-Mañero J (2014) Beneficial rhizobacteria from rice rhizosphere confers high protection against biotic and abiotic stress inducing systemic resistance in rice seedlings. Plant Physiol Biochem 82:44-53. Mair, VH, Hoh E (2009) The true history of tea. Thames & Hudson. pp. 280. Marasco R, Rolli E, Vigani G, Borin S, Sorlini C, Ouzari H, Zocchi G and Daffonchio D (2013) Are drought-resistance promoting bacteria cross-compatible with different plant models? Plant Signal Behav 10:e26741. Maron LG, Piñeros MA, Kochian LV and McCouch SR (2016) Redefining 'stress resistance genes', and why it matters. J Exp Bot 19:5588–5591. Matsunaga A, Sano T, Hirono Y and Horie H (2016) Effect of various directly covered shading levels on chemical components in the new shoots of the first flush. Tea Res J 122:1–7 (in Japanese with English abstract) Mayak S, Tirosh T and Glick BR (2004) Plant growth-promoting bacteria that confer resistance to water stress in tomatoes and peppers. Plant Sci 166:525–530. Memelink J, Kijne JW, van der Heijden R, Verpoorte R (2001) Genetic modification of plant secondary metabolite pathways using transcriptional regulators. Adv Biochem Eng Biotechnol 72:103-25. Michael AB, Arthur FM, Stephen E, Walden R and Jon DR (1996) The crystal structure of the antifungal protein zeamatin, a member of the thaumatin-like, PR-5 protein family. Nat Struct Biol 3:19–22. Mika A and Lüthje S (2003) Properties of guaiacol peroxidase activities isolated from corn root plasma membranes. Plant Physiol 132:1489–1498. Misra RC, Sandeep, Kamthan M, Kumar S and Ghosh S (2016) A thaumatin-like protein of Ocimum basilicum confers tolerance to fungal pathogen and abiotic stress in transgenic Arabidopsis. Sci Rep 6: 25340. Miya A, Albert P, Shinya T, Desaki Y, Ichimura K, Shirasu K, Narusaka Y, Kawakami N, Kaku H and Shibuya N (2007) CERK1, a LysM receptor kinase, is essential for chitin elicitor signaling in Arabidopsis. Proc Natl Acad Sci U S A 104:19613-19618. Mueller UG and Sachs JL (2015) Engineering microbiomes to improve plant and animal health. Trends Microbiol. 23:606-617. Muoki RC, Paul A and Kumar S (2012) A shared response of thaumatin like protein, chitinase, and late embryogenesis abundant protein3 to environmental stresses in tea [Camellia sinensis (L.) O. Kuntze]. Funct Integr Genomics 3:565-571. Mysore KS, Ryu CM (2004) Nonhost resistance: how much do we know? Trends Plant Sci 2:97-104. Najeeb U, Sarwar M, Atwell BJ, Bange MP and Tan DKY (2017) Endogenous ethylene concentration is not a major determinant of fruit abscission in heat-stressed cotton (Gossypium hirsutum L.). Front Plant Sci 8:1615. Nakano Y and Asada K (1981) Hydrogen peroxide is scavenged by ascorbate -specific peroxidase in spinach chloroplasts. Plant & Cell Physiol 22: 867-880. Nissinen RM, Ytterberg AJ, Bogdanove AJ, VAN Wijk KJ and Beer SV (2007) Analyses of the secretomes of Erwinia amylovora and selected hrp mutants reveal novel type III secreted proteins and an effect of HrpJ on extracellular harpin levels. Mol Plant Pathol 1:55-67. Paoletti F, Aldinucci D, Mocali A, Caparrini A (1986) A sensitive spectrophotometric method for the determination of superoxide dismutase activity in tissue extracts. Anal Biochem 154:536-541. Poorter H, Niinemets Ü, Poorter L, Wright IJ and Villar R (2009) Causes and consequences of variation in leaf mass per area (LMA): a meta-analysis. New Phytol 182:565–588 Reboutier D, Frankart C, Briand J, Biligui B, Rona JP, Haapalainen M, Barny MA and Bouteau F (2007) Antagonistic action of harpin proteins: HrpWea from Erwinia amylovora suppresses HrpNea-induced cell death in Arabidopsis thaliana. J Cell Sci 18:3271-3278. Reginato MA, Castagna A, Furlán A, Castro S, Ranieri A, Luna V (2014) Physiological responses of a halophytic shrub to salt stress by Na2SO4 and NaCl: oxidative damage and the role of polyphenols in antioxidant protection. AoB Plants 6:plu042. Sambrook JF and Russell DW (2001) Molecular cloning：a laboratory manual：2nd edit. CSHL press. Scharbert S, Holzmann N and Hofmann T (2004) Identification of the astringent taste compounds in black tea infusions by combining instrumental analysis and human bioresponse. J Agric Food Chem 11:3498–3508. Tamaki M, Kondo S, Itani T, Goto Y (2002) Temperature responses of leaf emergence and leaf growth in barley. J AGR SCI-Cambridge 138: 17–20. Tampakaki AP and Panopoulos NJ (2000) Elicitation of hypersensitive cell death by extracellularly targeted HrpZPsph produced in planta. Mol Plant Microbe Interact 13: 1366-1374. Todoroki Y, Narita K, Muramatsu T, Shimomura H, Ohnishi T, Mizutani M, Ueno K, Hirai N (2011) Synthesis and biological activity of amino acid conjugates of abscisic acid. Bioorg Med Chem 5:1743-1750. Tsunemi K, Taguchi F, Marutani M, Watanabe-Sugimoto M, Inagaki Y, Toyoda K, Shiraishi T and Ichinose Y (2011) Degeneration of hrpZ gene in Pseudomonas syringae pv. tabaci to evade tobacco defence: an arms race between tobacco and its bacterial pathogen. Mol Plant Pathol 12:709-714. van der Biezen EA and Jones JD (1998) Plant disease-resistance proteins and the gene-for-gene concept. Trends Biochem Sci 23:454-6. van Loon LC and van Strien EA (1999) The families of pathogenesis-related proteins, their activities, and comparative analysis of PR-1 type proteins. Physiol Mol Plant P 55:85–97. Velazhahan R, Datta SK, Muthukrishnan S (Eds.) (1999) Occurrence and properties of plant pathogenesis-related proteins, Pathogenesis-related Proteins in Plants, CRC Press, Boca Raton, pp 107–129 Wei ZM, Laby RJ, Zumoff CH, Bauer DW, He SY, Collmer A and Beer SV (1992) Harpin, elicitor of the hypersensitive response produced by the plant pathogen Erwinia amylovora. Science 257:85-88. Witkowski ETF and Lamont BB (1991) Leaf specific mass confounds leaf density and thickness. Oecologia 88:486–493 Wittmann C, Pfanz H, Loreto F, Centritto M, Pietrini F, Alessio G (2006) Stem CO2 release under illumination: corticular photosynthesis, photorespiration or inhibition of mitochondrial respiration? Plant Cell Environ 6:1149-58. Wu C, Cui K, Wang W, Li Q,Fahad S, Hu Q, Huang J,Nie L and Peng S (2016) Heat-induced phytohormone changes are associated with disrupted early reproductive development and reduced yield in rice. Sci Rep 6:34978. Wu X, Wu T, Long J, Yin Q, Zhang Y, Chen L, Liu R, Gao T and Dong H (2007) Productivity and biochemical properties of green tea in response to full-length and functional fragments of HpaGXooc, a harpin protein from the bacterial rice leaf streak pathogen Xanthomonas oryzae pv. oryzicola. J Biosci 32:1119-1131. Xie Z and Chen Z (2000) Harpin-induced hypersensitive cell death is associated with altered mitochondrial functions in tobacco cells. Mol Plant Microbe Interact 2:183-190. Ye N, Zhu G, Liu Y, Li Y and Zhang J (2011) ABA controls H2O2 accumulation through the induction of OsCATB in rice leaves under water stress. Plant Cell Physiol. 52:689-698. Zhang L, Xiao S, Li W, Feng W, Li J, Wu Z, Gao X, Liu F and Shao M (2011) Overexpression of a Harpin-encoding gene hrf1 in rice enhances drought tolerance. J Exp Bot 12:4229-4238.||摘要:||
茶是世界上最廣泛飲用的飲料之一。隨著茶的需求日益增高，台灣高山茶園的產量已供給不足，而茶樹 (Camellia sinensis) 適合生長於高海拔山區，但山區開墾面積受限，因此發展平地栽種茶有其必要性。高溫低濕造成的熱傷害是平地栽種茶樹的主要問題之一，會引起茶樹脫水造成葉緣褐變，新芽灼傷枯萎，因此增加茶樹耐熱性是一項重要課題。harpin蛋白藉由細菌第三型分泌系統 (type III secretion system, T3SS) 分泌至細胞間隙，會引發植物產生過敏性反應 (hypersensitive response, HR)。許多的文獻中指出harpin蛋白可以促進植物生長，提高生物性 (biotic) 及非生物性 (abiotic) 逆境抗性，誘導抗病 (pathogenesis-related, PR) 基因表現。本研究利用大腸桿菌大量表現植物病原細菌之 harpin 蛋白群包含 HrpWAaa、HrpYAaa、 HrpZPav 及HopAK1Pav，再以煮沸破菌萃取 harpin 蛋白以不同劑量噴灑於溫室種植的茶樹苗葉片上，結果發現經 harpin 蛋白噴灑後之茶樹PR-5 家族成員Thaumatin-like protein (TLP) 基因表現量增加，茶苗存活率增加、抗氧化系統活性提高，然而也觀察到 harpin 蛋白誘導葉片細胞死亡。植物二次代謝物有助於抵抗環境逆境，且茶樹二次代謝物如兒茶素 (Catechin) 是影響風味品質的主要成分之一，本研究透過高效液相層析儀 (HPLC-UV) 分析茶葉中酚類化合物成分，結果顯示噴灑 harpin 蛋白於茶苗葉片的沒食子酸 (gallic acid, GA)、咖啡因 (caffeine)、酯化型兒茶素(epicatechin-3-gallate, ECG及epigallocatechin-3-gallate, EGCG)、茶飢素 (teaghrelin) 等成分含量均有上升趨勢。綜合以上結果，推測 harpin 蛋白可能誘導高山茶苗HR、抗性基因表現及增強對於高溫環境之耐受性，且影響了茶苗的次級代謝物累積。
Tea is one of the most widely consumed beverage in the world. As the growing demand for tea in Taiwan, alpine tea plantations production is insufficient supply. Tea tree (Camellia sinensis) is suitable for growing in high-altitude mountainous areas. Due to limited area of mountain reclamation, to grow alpine tea tree in lowland areas will be a promising project, but the heat damage will cause the browning of the leaf margin and dehydration of the tea tree, it is one of the main problems of grow alpine tea tree in lowland areas. The harpin proteins produced by phytobacteria are secreted into plant apoplast via type III secretion system (T3SS) and thereby elicit the hypersensitive response (HR). Several studies showed that harpin proteins can promote plant growth, enhance stress tolerance, pathogen resistance and induces pathogenesis-related (PR) gene expression. In this study, harpin proteins HrpWAaa, HrpYAaa, HrpZPav and HopAK1Pav were overexpressed in Escherichia coli using T7 RNA-polymerase- dependent system and then were partial purified by boiling. Subsequently, the tea tree seedlings growing on lowland were sprayed with different concentrations of harpin proteins. Interestingly, the tea tree seedlings treated with harpin proteins showed higher survival rate, antioxidant system activity, Thaumatin-like protein (TLP) gene expression and more cell death in the inoculated leaf. In addition, based on the high performance liquid chromatography (HPLC) analysis, the phenolic compounds including gallic acid (GA), caffeine, epicatechin-3-gallate (ECG), epigallocatechin-3-gallate (EGCG) and teaghrelin (TG) in the leaves were elevated when the tea tree seedlings were treated with harpin proteins. Taken together, the results revealed that the harpin proteins are able to enhance resistance to heat for tea tree seedlings growing in the lowland environment and affect the production of secondary metabolites.
|Appears in Collections:||生物科技學研究所|
Show full item record
Files in This Item:
TAIR Related Article
Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.