Arabidopsis myrosinases link the glucosinolate-myrosinase system and the cuticle

Transcription

1 Arabidopsis myrosinases link the glucosinolate-myrosinase system and the cuticle Ishita Ahuja 1,2,3, Ric C. H. de Vos 2, Jens Rohloff 1, Geert Stoopen 2, Kari K. Halle 4, Samina Jam Nazeer Ahmad 5, Linh Hoang 6, Robert D. Hall 2, 7, 8, Atle M. Bones 1 * 1. Department of Biology, Norwegian University of Science and Technology (NTNU), Realfagbygget, NO-7491 Trondheim, Norway 2. Plant Research International, P.O. Box 16, 6700 AA Wageningen, The Netherlands 3. Norwegian Institute for Nature Research (NINA), NO-7485 Trondheim, Norway 4. Department of Mathematical Sciences, NTNU, Trondheim, Norway. 5. Department of Botany, University of Agriculture Faisalabad (UAF), Pakistan 6. Cellular and Molecular Imaging Core Facility (CMIC), Laboratory for Electron Microscopy, NTNU, Trondheim, Norway 7. Laboratory of Plant Physiology, Wageningen University, P.O. Box 16, 6700 AA Wageningen, The Netherlands 8. Netherlands Metabolomics Centre, Einsteinweg 55, 2333 CC Leiden, The Netherlands These senior authors contributed equally. * To whom correspondence should be addressed. atle.m.bones@ntnu.no Phone: Fax: Date of submission: 25/10/16

2 Figure S1. Morphological appearance of 3-4 weeks old plants of wild-type and tgg single and double mutants. (Scale bar = 1 cm). Figure S2. The contents of FAs (nmol g-1 tissue wt.) (Fresh wt. basis) in rosette leaves of wild-type, tgg1, tgg2 single and tgg1 tgg2 double mutants from FAME analysis. Error bars represent S.E. Figure S3. The contents of FAs in rosette leaves of wild-type, tgg1, tgg2 single and tgg1 tgg2 double mutants from leaf cutin analysis. Error bars represent S.E. Table S1. Compounds detected in leaves of wild-type, tggl, tgg2 single and tggl tgg2 double mutants of Arabidopsis from FAME analysis. Table S2. Compounds detected in wild-type, tggl, tgg2 single and tggl tgg2 double mutants of Arabidopsis from leaf cutin analysis. Table S3. Compounds detected by LC-QTOF-MS (ESI negative mode) in leaves of wildtype, tggl, tgg2 single and tggl tgg2 double mutants of Arabidopsis through untargeted metabolic profiling. Table S4a. The list of structurally annotated/identified metabolites (obtained from FAME (F), leaf cutin (C) and untargeted metabolic profiling (L)) with values (log2 ratio) used for 2D principal component analysis (PCA) and Hierarchical cluster analysis (HCA). Table S4b. The list of structurally annotated/identified metabolites (obtained from FAME (F), leaf cutin (C) and untargeted metabolic profiling (L)) with peak intensity values which were used to quantify log2 ratios (Table S4a).

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6 Table S1. Compounds detected in leaves of wild-type, tggl, tgg2 single and tggl tgg2 double mutants of Arabidopsis from FAME analysis. Compounds with systematic Structure/MS Molecular Retention and common names unknowns structure Index (RI) (analyte) Fatty acyls Decanoic acid (Capric acid) 10:0 C10H20O Tetradecanoic acid 14:0 C14 H28 O (Myristic acid) 12-Methyltetradecanoic acid 12-Me-14:0 C15H30O (Sarcinic acid) Pentadecanoic acid 15:0 C15H30O Hexadecanoic acid 16:0 C16 H32 O (Palmitic acid) (Z)-9-Hexadecenoic acid 9c-16:1 C16H30O (Palmitoleic acid) (E)-9-Hexadecenoic acid 9t-16:1 C16H30O (Palmitelaidic acid) (Z)-7-Hexadecenoic acid 7c-16:1 C16H30O (Hypogeic acid) (7E,10E)-Hexadeca-7,10-dienoic acid 7t10t-16:2 C16H28O (7Z, 10Z,13Z)-Hexadeca-7,10,13- trienoic acid (Roughanic acid) 7c10c13c-16:3 C16H26O Octadecanoic acid (Stearic acid) 18:0 C18H36O (Oleic acid) (Z)-9-Octadecenoic acid 9c-18:1 C18H34O (E)-9-Octadecenoic acid 9t-18:1 C18H34O2 (Elaidic acid) ,8,12-Trimethyloctadecanoic acid 4,8,12-triMe-18:0 C21H42O (9Z,12Z)-Octadeca- 9,12-dienoic acid 9c12c-18:2 C18H32O (Linoleic acid) 2-Hydroxyhexadecanoic acid 2-OH-16:0 C16H32O (2-Hydroxypalmitic acid) (9E,12E,15E)-Octadeca-9,12,15- trienoic acid (Linolenelaidic acid) 9t12t15t-18:3 C18H30O (9Z,12Z,15Z)-Octadeca-9,12,15-9c12c15c-18:3 C18H30O trienoic acid (α-linolenic acid) Unknown fatty acid 1 79(100), 91(77), 67(59), (37), 105(25) Icosanoic acid (Arachidic acid) 20:0 C20H40O Docosanoic acid (Behenic acid) (Z)-13-Docosenoic acid (Erucic acid) Fatty alcohol 1-Dodecanol (Lauryl alcohol) 22:0 C22H44O c-22:1 C22H42O OH-12 C12H26O 1472 Fatty acid phytyl esters Fatty acid phytyl ester 1 123(100), 81(39), (21), 55(14), 278(12) Fatty acid phytyl ester 2 123(100), 81(35), (18), 55(14), 278(11) Fatty acid phytyl ester 3 95(100), 81(74), (61), 69(43), 57(31) Fatty acid phytyl ester 4 68(100), 95(89), 82(75), (45), 81(42), 55(41), 123(40) Fatty acid phytyl ester 5 123(100), 81(32),

7 95(17), 55(13), 278(12) Fatty acid phytyl ester 6 81(100), 95(90), 68(79), 57(62), 123(61), 55(13), 278(14) Fatty acid phytyl ester 7 123(100), 81(32), 95(16), 55(12), 278(12) Fatty acid phytyl ester 8 82(100), 95(59), 57(27), 67(23), 123(21), 81(21), 278(14) Fatty acid phytyl ester 9 82(100), 95(74), 57(27), 67(23), 123(22), 81(22), 278(17) Fatty acid phytyl ester 10 81(100), 123(36), 69(19), 85(19), 57(16), 278(4) Fatty acid phytyl ester 11 81(100), 123(30), 85(23), 57(20), 95(20), 69(17), 278(5) Indoles Indole 1 99(100), 191(80), 57(13), 206(10), 163(7)

8 Table S2. Compounds detected in wild-type, tggl, tgg2 single and tggl tgg2 double mutants of Arabidopsis from leaf cutin analysis. Compounds with systematic and common names Structure/ MS unknowns Molecular structure Retention Index (RI) (analyte) Fatty alcohol Tetracosanol (Lignocerol) 1-OH-24 C24H50O 2612 Aldehyde 2,2 - Dimethylpentanal - C7H14O 1341 Phenolics 4-Hydroxy-3-methoxy-benzaldehyde - C8H8O (Vanillin) 4-Hydroxy-3,5-dimethoxy-benzaldehyde - C9H10O (Syringaldehyde) 4-(2-Hydroxyethyl)-2-methoxyphenol - C9H12O (Homovanillyl alcohol) Hydroxycinnamic acids 3-(2-Hydroxyphenyl)-2-propenoic acid - C9H8O (o-coumaric acid) 3-(4-Hydroxyphenyl)-2-propenoic acid - C9H8O (p-coumaric acid) 2-Propenoic acid, 3-(4-hydroxy-3- methoxyphenyl)-, methyl ester (Methyl ferulate) (2,3,4-Trimethoxyphenyl) propenoic acid (2,3,4-Trimethoxycinnamic acid) - C12H14O (Methyl sinapate) 3-(3,4,5-Trimethoxyphenyl)-2 -propenoic acid - C12H14O (4-Hydroxy-3,5-dimethoxyphenyl)-2- propenoic acid (Sinapic acid) Fatty acids - C11H12O5 4-Oxopentanoic acid (Levulinic acid) 4-oxo-5:0 C5H8O Nonanedioic acid (Azelaic acid) 9:0-diacid C9H16O Tetradecanoic acid (Myristic acid) 14:0 C14H28O Unknown fatty acid 1 79(100), 91(66), 93(52), (38), 77(35), 41(33), 105(33) (7E,10E)-Hexadeca-7,10-dienoic acid 7t10t-16:2 C16H28O (7Z,10Z,13Z)-Hexadeca-7,10,13 -trienoic acid 7c10c13c-16:3 C16 H26 O (Roughanic acid) (Z)-9-Hexadecenoic acid (Palmitoleic acid) 9c-16:1 C16H30O Hexadecanoic acid (Palmitic acid) 16:0 C16H32O Unknown fatty acid 2 75(100), 117(84), 285(32), (19), 41(19), 71(17), 211(14) 9c12c15c-18:3 C18H30O (9Z,12Z,15Z)-Octadeca-9,12,15-trienoic acid (α-linolenic acid) (9Z,12Z)-Octadeca-9,12-dienoic acid 9c12c-18:2 C18H32O2 (Linoleic acid) 2102 (Z)-9-Octadecenoic acid (Oleic acid) 9c-18:1 C18H34O (E)-9-Octadecenoic acid (Elaidic acid) 9t-18:1 C18H34O Octadecanoic acid (Stearic acid) 18:0 C18H36O Hexadecanedioic acid (Thapsic acid) 16:0-diacid C16H30O Unknown fatty acid 3 43(100), 98(87), 55(86), 69(56), 83(50), 41(43), 79(36) Icosanoic acid (Arachidic acid) 20:0 C20H40O Unknown fatty acid 4 98(100), 43(90), 55(76), (59), 69(54), 83(50), 2260

9 236(17), 255(17) Octadecadienedioic acid 18:2-diacid C18H30O Unknown fatty acid 5 55(100), 81(78), 67(66), (54), 95(52), 276(47), 309(23) Octadecanedioic acid 18:0-diacid C18H32O Unknown fatty acid (100), 79(95), 43(85), 106(78), 67(64), 119(51), 133(35), 290(13) Unknown fatty acid 7 67(100), 79(91), 43(82), (71), 121(58), 55(57), 320(14), 292(12) 9,10-Dihydroxyoctadecanoic acid (9,10-9,10-diOH-18:0 C18H36O Dihydroxystearic acid) (11E,14E)-Icosa-11,14-dienoic acid 11t14t-20:2 C20H36O Docosanoic acid (Behenic acid) 22:0 C22H44O (Z)-15-Tetracosenoic acid (Nervonic acid) 15c-24:1 C24H46O Tetracosanoic acid (Lignoceric acid) 24:0 C24H48O Pentacosanoic acid (Hyenic acid) 25:0 C25H50O Hexacosanoic acid (Cerotic acid) 26:0 C26H52O (Z)-13-Docosenoic acid (Erucic acid) 13c-22:1 C22H42O Octacosanoic acid (Montanic acid) 28:0 C28H56O Fatty acid ester (10Z, 12Z)-9-Methyl- 10,12-hexadecadien-1-9Me- 10c12c-16:2-acet C19H34O yl acetate (9-Methyl-Z,Z- 10,12-hexadecadien- 1-ol acetate) Monoglycerides Hexadecanoic acid, 2,3-dihydroxypropyl ester MG(16:0/0:0/0:0) C19H38O (1-Monopalmitin) MG(0:0/18:0/0:0) C21H42O Octadecanoic acid, 2-hydroxy-1- (hydroxymethyl) ethyl ester (2-Monostearin) Indoles 1H-Indol-3-ylacetonitrile (3 - C11H9N 1819 Indoleacetonitrile) C10H9NO H-Indol-3-ylacetic acid (Indole-3-acetic acid) 1H-Indole-3-carboxylic acid (3- C9H7NO Indolecarboxylic acid) 2-(5-Methoxy-1H-indol-3-yl) acetic acid (5- Methoxyindol-3-ylacetic acid) C11H11NO (4-Methoxy-1H-indol-3-yl) acetonitrile C11H10N2O (Arvelexin) 2026 Unknown indole 1 160(100), 219(53), 130(39), (9), 89(9), 103(6) Unknown indole 2 130(100), 204(88), 118(51), 189(42), 145(21), 171(16) (5-Methoxy-1H-indol-3-yl) ethanol (5 - Methoxytryptophol) - C11H13NO Unknown indole 3 160(100), 234(85), 175(58), (29), 219(21), 117(14) Polyol 1,2,3-Propanetriol (Glycerol) C3H8O Carbohydrate 2,6 -Anhydro-D-fructofurano se (2,6- C6H10O Anhydrofructose) Diterpene alcohol (2E,7R,11R)-3,7,11,15-Tetramethyl-2- hexadecen-1-ol (Phytol) C20H40O 2126

10 Table S3. Compounds detected by LC-QTOF-MS (ESI negative mode) in leaves of wild- type, tggl, tgg2 single and tggl tgg2 double mutants of Arabidopsis through untargeted metabolic profiling. Compounds Measured mass (m/z) Calculated mass (m/z) Difference measured vs. calculated (ppm) Molecular structure Retention time (RT) min Glucosinolates (GSLs) Glucoiberin C11H21NO10S (3-Methylsulfinylpropyl GSL) Glucoraphanin (4-Methylsulfinylbutyl GSL) C12 H23NO10S Glucoalyssin C13H25NO10S (5-Methylsulfinylpentyl GSL) Glucoibarin (7-Methylsulfinylheptyl GSL) C15H29NO10S Glucoerucin C12H23NO9S (4-Methylthiobutyl GSL) Glucohirsutin C16H31NO10S (8-Methylsulfinyloctyl GSL) Hexyl glucosinolatet C13H25NO9S Glucobrassicin C16H20N2O9S (Indol-3-ylmethyl GSL) Neoglucobrassicin (1-Methoxy-indol-3-yl-methyl GSL) C17H22N2O10 S Kaempferol 3-O-rhaglu 7-O-rha (Kaempferol 3-O-rhamnosyl- glucoside 7- O-rhamnoside) Kaempferol-3 -glu-7-rha (Kaempferol-3 -O-glucoside-7-Orhamnoside) Flavonol glycosides C39H50O C 27 H 30 O C H O Kaempferol-soph-glu-coumaroyl (Kaempferol-3 -O-(pcoumaroyl)sophoroside 7-O- glucoside) Quercetin 7-O-rha-3-rhaglu C H O (Quercetin 7-O-rhamnoside 3-Orhamnosylglucoside) Quercetin 3-O-rha 7-O-glu C27H30O (Quercetin 3-O-rhamnoside 7-Oglucoside) Sinapoyl esters Sinapoylglucose C17H22O Sinapoylmalate C15H16O Sinapoyl-caffeoylquinic acid methyl C27H28O ester Sinapoyl-caffeoylquinic acid methyl ester (isomer) C 27 H28O t Hexyl glucosinolate identified on basis of elemental composition and from mass as reported before (Beekwilder et al and Matsuda et al. 2010).