Accumulation of Hydroxycinnamic Acid Amides Induced by Pathogen Infection and Identification

Accumulation of hydroxycinnamic acid amides induced by pathogen infection and identification of agmatine coumaroyltransferase in Arabidopsis thaliana

Atsushi Muroi, Atsushi Ishihara(), Chihiro Tanaka, Akihiro Ishizuka, Junji Takabayashi, Hideto Miyoshi, Takaaki Nishioka

Faculty of Agriculture, Tottori University, Tottori 680-8553, Japan

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Supplementary protocols

Chemicals

[1,4-13C2]Putrescine was synthesized according to the methods of Bottari et al. (2004) and Maruyoshi et al. (2004). p-Comaroyl- and feruloyl-CoAs were synthesized as described by Stockigt and Zenk (1975) by ester-exchange via the N-hydroxysuccinimide ester of p-coumaric and ferulic acids. p-Coumaroylagmatine (CouAgm), feruloylagmatine (FerAgm), p-coumaroylputrescine (CouPtr), and feruloylputrescine (FerPtr) were synthesized according to the method of Negrel et al. (1984). [8,9-13C2]p-Coumaric acid was synthesized from [13C3]malonic acid and 4-hydroxybenzylaldehyde as described by Ishihara et al. (1999).

N-Hydroxysuccinimide ester of [8,9-13C2]p-coumaric acid was synthesized according to Stökigt and Zenk (1975). [8,9-13C2]p-Coumaric acid (79.6 mg, 0.48 mmol) was dissolved in dry ethyl acetate (20 mL), and N-hydroxysuccinimide (55.2 mg, 0.48 mmol) and DCC (111 mg, 0.54 mmol) were added to the solution. The mixture was stirred for 24 h at room temperature. The precipitated dicyclohexylurea was filtered off and the filtrate was extracted with 1 M sodium bicarbonate. After the ethyl acetate layer was dried over sodium sulfate, the solvent was evaporated and the ester was purified by silica gel column chromatography [solvent: chloroform–methanol (20:1, v/v)].

[8,9-13C2]p-Coumaroyl-N-hydroxysuccinimide ester (94.8 mg).EI-MS m/z (relative intensity): 263 (4), 163 (3), 149 (100), 120 (18), 115 (13), 92 (15). 1H NMR (400 MHz, CD3OD) : 2.85 (4H, s), 6.53 (1H, ddd, 1JHC = 165.6 Hz, 3JHH = 15.9 Hz, 2JHC = 2.61 Hz), 6.84 (2H, d, J = 8.66 Hz), 7.57 (2H, d, J = 8.66 Hz), 7.87 (1H, ddd, 3JHH = 15.9 Hz, 3JHC = 7.17 Hz, 2JHC = 2.86 Hz). 13C NMR (100 MHz, CDCl3) : 26.5 (-CH2-CH2-), 108.5 (C-8, 1J8-9 = 79.0 Hz, 1J8-7 = 65.6 Hz), 117.0 (C-3, 5), 131.2 (C-1, 3J1-9 = 3.7 Hz), 132.1 (C- 2,6, 3J2, 6-8 = 4.9 Hz), 151.3 (C-7, 1J7-8 = 65.6 Hz),162.5 (C-4), 164.2 (C-9, 1J9-8 = 79.3 Hz), 172.1 (C=O, hydroxysuccinimide).

The N-hydroxysuccinimide ester was converted to N4-([8,9-13C2]p-coumaroyl)agmatine according to the method of Negrel and Smith (1984). Sodium bicarbonate (13.4 mg, 0.16 mmol) was added to an aqueous solution (20 mL) of agmatine sulfate (36.3 mg, 0.16 mmol). [8,9-13C2]p-Coumaroyl-N-hydroxysuccinimide ester (41.5 mg, 0.16 mmol) dissolved in acetone (20 mL) was added to the agmatine solution. The mixture was stirred for 24 h, and acidified with 0.5 mL of acetic acid. After removal of acetone by evaporation, the aqueous solution was extracted with ethyl acetate (20 mL x 3). The ethyl acetate layer was evaporated and subjected to preparative HPLC. HPLC conditions were as follows: column: Wakosil-II 5C18HG, 20 x 250 mm (Wako, Osaka, Japan); solvent: 0.1% TFA–methanol (67:33, v/v); flow rate: 7 mL min-1; detection: 280 nm. The peak corresponding to labeled CouAgm was collected and evaporated dry.

N4-([8,9-13C2]p-Coumaroyl)agmatine (31.8 mg). EI-MS m/z (relative intensity):278 (1, [M]+), 261 (4), 178 (4), 164 (5), 149 (30), 87 (50), 69 (100). 1H NMR (400 MHz, CDCl3) : 1.65 (4H, m), 3.30 (2H, m), 3.31 (2H, m) 6.41 (1H, ddd, 1JHC = 156.6 Hz, 3JHH = 15.7 Hz, 2JHC = 3.94 Hz), 6.79 (2H, d, J = 8.64 Hz), 7.40 (2H, d, J = 8.64 Hz), 7.45 (1H, ddd, 3JHH = 15.7 Hz, 3JHC = 6.47 Hz, 2JHC = 2.81 Hz). 13C NMR (100 MHz, CDCl3) : 27.1 (C-2 or 3), 27.8 (C-2 or 3), 39.6 (C-1), 42.0 (C-4), 116.7 (C-3', 5'), 117.9 (C-8', 1J8'-9' = 64.7Hz, 1J8'-7' = 72.2 Hz),127.6 (C-1', 3J1'-9' = 6.5 Hz), 130.5 (C- 2',6', 3J2', 6'-8' = 4.7 Hz), 142.0 (C-7', 1J7'-8' = 72.2 Hz), 158.6 (C-4'), 160.6 (guanidino), 169.4 (C-9', 1J9'-8' = 64.9 Hz).

Preparation of radiolabeled CouAgm

N4-([U-14C]p-Coumaroyl)agmatine was prepared by feeding barley seedlings with [U-14C]L-phenylalanine (Moravek Biochemicals, Brea, CA). Thirty seedlings of barley (2-d-old) were transplanted to a 1 mM L-phenylalanine solution (15 mL, 0.555 MBq). After a 24-h incubation at 25°C, the seedlings were extracted with methanol (15 mL). Distilled water (4 mL) was added to the extract, and the mixture was passed through a Sep-Pak Plus C18 cartridge (Waters, Boston, MA) that had been equilibrated with methanol–water (8:2, v/v). The cartridge was eluted with methanol–water (8:2, v/v, 5 mL). The column-through fraction and 80% methanol fraction were combined and concentrated to approximately 500 µL by evaporation. The solution was applied to a Sep-Pak Plus C18 cartridge equilibrated with methanol–water (2:8, v/v), and the cartridge was eluted with the same solution. The column-through fraction and 20% methanol fraction were combined, and evaporated to approximately 150 µL. This solution was subjected to preparative HPLC to give radiolabeled CouAgm (0.722 µmol, 5.19 Bqnmol-1). HPLC conditions were as follows: column: Mightysil RP-18 GP 3 µm, 4.6 x 150 mm (Kanto Chemical, Tokyo, Japan); solvent: 0.1% TFA–acetonitrile (88:12, v/v); flow rate: 0.8 mL min-1; temperature: 40°C; detection: 280 nm.

RNA isolation

Total RNA was extracted from A. thaliana leaves using an RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the instructions with some modifications. Leaves were ground with liquid nitrogen and lysed in 450 µL of Buffer RLT. The lysate was transferred to a QIAshredder spin column, and centrifuged at 18,500g for 2 min. The supernatant was transferred to an RNeasy spin column after mixing with a half volume of ethanol, and centrifuged at 8,000g for 15 sec. Buffer RW1 was added to the column, and centrifuged at 8,000g for 15 sec. Genomic DNA was digested with DNase I (Qiagen, Hilden, Germany) for 30 min at 25°C. Buffer RW1 was added to the column, and centrifuged at 8,000g for 15 sec. After two washes with 500 mL of Buffer RPE, total RNA was eluted with 30 mL of nuclease-free water by centrifugation at 8,000g for 1min. cDNA was prepared from 1 µg of total RNA, which was reverse transcribed with an RNA PCR KIT (Takara, Kyoto, Japan) using the oligo(dT) primer in a reaction volume of 10 µL.

SDS-PAGE and western blot analysis

SDS-PAGE was carried out in a 10% acrylamide gel. The running buffer contained 25 mM Tris, 200 mM glycine, and 0.1% SDS. Separated proteins were transferred to a PVDF membrane. After blocking of the membrane for 1 h with 5% blocking agent (GE Healthcare, Buckinghamshire, UK) in PBS-T, the membrane was incubated for 1 h in PBS-T containing anti-His antibody (GE Healthcare, Buckinghamshire, UK). After a wash, the membrane was incubated for 1 h in PBS-T containing anti-mouse antibody linked with AP (NEB, MA). After another wash, the secondary antibody was detected with an Alkaline Phosphatase Conjugate Substrate Kit (BIO-RAD,Hercules, CA).

References

Bottari P, Aebersold R, Turecek F, Gelb MH (2004) Design and synthesis of visible isotope-coded affinity tags for the absolute quantification of specific proteins in complex mixtures. Bioconjugate Chem 15:380-388.

Ishihara A, Ohtsu Y, Iwamura H (1999) Induction of biosynthetic enzymes for avenathramides in elicitor-treated oat leaves. Planta 208:512-518.

Negrel J, Smith T (1984) Oxidation of p-coumaroylagmatine in barley seedling extracts in the presence of hydrogen peroxide or thiols. Phytochemistry 23:739-741.

Stöckigt J, Zenk MH (1975) Chemical syntheses and properties of hydorxycinnamoyl-coenzyme A derivatives. Z Naturforsch 30c:352-358.

Supplementary figures

Supplementary Fig.1 Expression of AtACT in E. coli.(a) SDS-PAGE analysis with coomassie stain. (b) Western blot analysis of soluble proteins. Proteins were transferred to a PVDF membrane after SDS-PAGE. The His-Tag in the fusion protein was detected with anti-HisTag antibody.

Supplementary Fig. 2 A phylogenic tree of the plant BAHD family of acyltransferases including AtACT. Protein sequences were analyzed by the neighbor-joining method and a bootstrap analysis with CLUSTAL W ( I – V represent the clades categorized by D'Auria et al. (2006). ACT, H. vulgare agmatine coumaroyltransferase; AtHCT, A. thaliana hydroxycinnamoyl-CoA:shikimate/quinate hydroxycinnamoyltransferase; AsHHT1, A. sativa hydroxycinnamoyl-CoA:hydroxyanthranilate N-hydroxycinnamoyltransferase; BanAAT, banana alcohol acyltransferase; CbBEAT, C. breweri benzylalcohol O-acetyltransferase; CbBEBT, C. breweri benzoyl-CoA:benzylalcohol O-benzoyltransferase; CER2, A. thaliana unknown; CHAT, (Z)-3-hexen-1-ol O-acetyltransferase; CmAAT4, C. melo alcohol acyltransferase; DAT, deacetylvindoline 4-O-acetyltransferase; Dv3MAT, D. variabilis malonyl-CoA:anthocyanidin 3-O-glucoside-6 ”-O-malonyltransferase; Glossy2, Z. mays unknown; GmIF7MaT, G. max malonyl-CoA:isoflavone 7-O-glucoside-6 ”-O-malonyltransferase; Gt5AT, G. triflora anthocyanine 5-aromatic acyltransferase; HCBT, anthranilate N-hydroxycinnamoyl/benzoyltransferase; HQT, N. tabacum hydroxycinnamoyl-CoA quinate hyroxycinnamoyl transferase; MAT, minovincinine-19-hydroxy-O-acetyltransferase; NtBEBT, N. tabacum benzoyl-CoA: benzylalcohol O-benzoyltransferase; NtHCT, N. tabacum hydroxycinnamoyl-CoA: shikimate/quinate hydroxycinnamoyltransferase; NtMAT1, N. tabacum malonyl-CoA flavonoid/napthol glucoside acyltransferase; Pun1, C. annum unknown; SalAT, salutaridinol 7-O-acetyltransferase; RhAAT1, R. hybrida alcohol acetyltransferase; SAAT, strawberry alcohol acyltransferase; Sc3MaT, Senecia curentus malonyl-CoA:anthocyanidin 3-O-glucoside 6 ”-O-malonyltransferase; SHT, A. thaliana spermidine hydroxycinnamoyl transferase; Ss5MaT1/Ss5MaT2, S. splendens malonyl-CoA:anthocyanin 5-O-glucoside-6 ”-O-malonyltransferase; VAAT, Fragaria vesca alcohol acyltransferase; Vh3MAT1, V. hybrida malonyl-CoA:flavonol 3-O-glucoside-6 ”-O-malonyltransferase; Vinorine, R. serpentine vinorine synthase.

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