Indoleacetic acid

Two-year-old cutting seedlings of tea plant (C. sinensis cv. ‘Longjing43’) were planted in pots containing a mixture of perlite, vermiculite, and sphagnum (ratio, 1:2:3) in a climate-controlled growth chamber programmed with 70% ± 10% relative humidity, 16 h light (25 °C) with a light intensity of 300 μmol·m⁻²·s⁻¹ during the daytime, and 8 h darkness (16 °C) during the nighttime (Supplementary Fig. S1). The 1st, 2nd, 3rd, 4th, and older leaves at different levels of development were collected (Fig. 1). The 3rd leaves were selected as materials for using in hormonal treatments and untreated control to unify standards. Leaves were sprayed with 1 mM gibberellins (GA treatment), 1 mM 3-indoleacetic acid (IAA treatment), 1 mM salicylic acid (SA treatment), 1 mM methyl jasmonate (MeJA treatment), or 0.1 mM abscisic acid (ABA treatment) for 2 h46 47 . While GA, IAA, SA, and MeJA were dissolved in distilled water with 2% absolute ethanol, ABA was dissolved in distilled water only. Three biological experimental replicates were performed in different pots for each treatment. Tea plant leaf materials were collected, quickly immersed in liquid nitrogen and stored at –80 °C for RNA extraction.

All strains are derivatives of W303 (KWY165) with the following exceptions: KWY7227 and KWY7246 are derivatives of BY4741 (KWY1601). All strains are listed in Supplementary file 2. gcn2∆, CDC33-IAA7-3V5, FBA1-PP7sl, GFA1-PP7sl and PGK1-PP7sl were generated by standard PCR based methods (Longtine et al., 1998 (link)). RPL28(Q38K) was generated by plating wild-type cells on 3 mg/mL cycloheximide plates, selecting for suppressors, backcrossing the suppressors at least three times and confirming the mutation by sequencing. HIS3 was generated by PCR replacement of the his3-11,15 allele using HIS3 from pRS303. leu2-3,112∆::CG-LEU2::pGPD1-OsTIR1, his3-11,15∆::CG-HIS3::pGPD1-OsTIR1, trp1-1∆::CG-TRP1::pGPD1-LexA-EBD-B112, his3-11,15∆::CG-HIS3::pGPD1-LexA-EBD-B112 and SCO2::p4xLexOcyc1-3xGST-V5-24xPP7sl-tCYC1-NatNT2 were generated by transforming strains with plasmids pKW2830 (PmeI digested), pKW2874 (PmeI digested), pKW3908 (SwaI digested), pKW4073 (SwaI digested) and pKW4190 (NotI/AscI digested) respectively. Strains were grown in CSM-lowURA (7 g/L YNB, 2% dextrose, 20 mg/L adenine, 20 mg/L arginine, 20 mg/L histidine, 60 mg/L leucine, 30 mg/L lysine, 20 mg/L methionine, 50 mg/L phenylalanine, 200 mg/L threonine, 20 mg/L tryptophan, 30 mg/L tyrosine, 10 mg/L uracil) unless otherwise indicated. The following chemicals were obtained from the indicated sources: cycloheximide [Sigma], hippuristanol [a generous gift of Junichi Tanaka, University of the Ryukyus], β-estradiol [Sigma], sordarin [Sigma], 3-indoleacetic acid [Sigma], IP₆ [Sigma], 4-thiouracil (4TU) [Arcos], 3-amino-1,2,4-triazole (3AT) [Sigma].

Quantification of auxin produced by strain LB400 was performed as previously described (Bric et al., 1991 (link)). Briefly, strain LB400 was cultured on a shaker at 30°C for 72 h in LB medium supplemented with 500 µg/mL tryptophan. Optical density at 600 nm of the bacterial culture was measured, and 1 mL was harvested and centrifuged (10 min, 13,000 rpm) to collect the supernatant. 20 µL of orthophosphoric acid and 2 mL of Salkowski reagent (50 mL of 35% perchloric acid, 1 mL of 0.5 M FeCl₃ solution) were added to the supernatant and incubated at room temperature for 20 minutes. The color intensity was measured using a spectrophotometer (OD₅₃₀), and the quantity of auxin produced was obtained via interpolation with a standard curve obtained with indoleacetic acid (IAA) 10 µg/mL–100 µg/mL and normalized with the bacterial culture OD₆₀₀.

The production of Indole acetic acid was analyzed according to the method of Gordon and Weber [27 (link)]. A freshly grown bacterial culture was inoculated on sterile DF minimal medium supplemented with 0.5 mg/mL of tryptophan and incubated for 4 days at 30°C. Two mL Salkowski Chromogenic Agent was mixed with 10 mL of the cell suspension. The mixture was subsequently allowed to stand in the dark for 30 min at 25°C. The production of a pink colour from the reaction was a positive indicator for IAA production.

3-indoleacetic acid (1a, 0.2 mmol), nitriles (2, 0.1 mmol), K₂CO₃ (1 equiv.) and fac-Ir(ppy)₃ (2 mol%) were successively added to a dried reaction tube (10 mL) with a magnetic stirring bar. Air was then withdrawn and backfilled with argon 3 times. Subsequently, degassed DMSO (1 mL) was injected into the tube by syringe. Then, the resulting reaction mixture was performed at room temperature under blue LED (6 W) irradiation for 3 h. The reaction progress was monitored by TLC. After the reactions were completed, the reaction mixture was diluted with water (10 mL) and washed with EA (3 × 10 mL). The combined organic layers were dried over anhydrous Na₂SO₄ and concentrated under reduced pressure. The residue was purified by column chromatography to afford the desired compounds 4 (ethylacetate/n-Hexane = 1:3 to 1:10).
4-((1H-indol-3-yl)methyl)-2-methylbenzonitrile (4a) and 4-((1H-indol-3-yl)methyl)-3-methylbenzonitrile (4a’): The desired pure product was obtained in 80% yield as a white solid (rr = 4:5). ¹H NMR (400 MHz, CDCl₃) δ 8.07 (s, 1H), 7.53–7.39 (m, 2H), 7.36 (d, J = 3.6 Hz, 1H), 7.26–7.11 (m, 3H), 7.11–7.02 (m, 1H), 6.94 (s, 0.4H), 6.75 (s, 0.5H), 4.10 (d, J = 6.7 Hz, 2H), 2.46 (s, 1.3H), 2.35 (s, 1.7H). ¹³C NMR (151 MHz, CDCl₃) δ 146.9, 145.1, 141.9, 137.9, 136.5, 133.4, 132.5, 130.5, 129.9, 129.8, 127.2, 127.2, 126.6, 122.6, 122.3, 122.3, 119.6, 119.4, 118.9, 118.8, 118.5, 114.0, 113.2, 111.3, 111.3, 110.1, 109.8, 31.7, 29.5, 20.5, 19.4. HRMS (ESI) exact mass calcd for C₁₇H₁₅N₂ [M+H]⁺ m/z 247.1235, found 247.1240.
3-(pyridin-4-ylmethyl)-1H-indole (4b): The desired pure product was obtained in 72% yield as a white solid. ¹H NMR (400 MHz, CDCl₃) δ 8.85 (s, 1H), 8.47 (d, J = 5.0 Hz, 2H), 7.43 (d, J = 7.8 Hz, 1H), 7.34 (d, J = 8.1 Hz, 1H), 7.18 (t, J = 6.7 Hz, 3H), 7.07 (t, J = 7.4 Hz, 1H), 6.96 (s, 1H), 4.10 (s, 2H). ¹³C NMR (151 MHz, CDCl₃) δ 150.8, 149.4, 136.6, 127.2, 124.2, 122.9, 122.2, 119.5, 118.8, 113.0, 111.4, 31.1. HRMS (ESI) exact mass calcd for C₁₄H₁₃N₂ [M+H]⁺ m/z 209.1079, found 209.1082.
3-((2-fluoropyridin-4-yl)methyl)-1H-indole (4c): The desired pure product was obtained in 71% yield as a white solid. ¹H NMR (400 MHz, CDCl₃) δ 1H NMR (400 MHz, CDCl3) δ 8.18 (s, 1H), 8.08 (d, J = 5.1 Hz, 1H), 7.46–7.31 (m, 2H), 7.24–7.19 (m, 1H), 7.14–7.06 (m, 2H), 7.02 (s, 1H), 6.80 (s, 1H), 4.14 (s, 2H). ¹³C NMR (151 MHz, CDCl₃) δ 164.2 (d, J = 238.2 Hz), 156.6 (d, J = 7.6 Hz), 147.2 (d, J = 15.1 Hz), 136.4, 127.0, 122.7, 122.5, 121.7 (d, J = 3.9 Hz), 119.8, 118.7, 112.6, 111.3, 109.2 (d, J = 37.0 Hz), 30.9, 30.9. HRMS (ESI) exact mass calcd for C₁₄H₁₂FN₂ [M+H]⁺ m/z 227.0985, found 227.0989.
3-((2-chloropyridin-4-yl)methyl)-1H-indole (4d): The desired pure product was obtained in 74% yield as a white solid. ¹H NMR (400 MHz, CDCl₃) δ 8.31 (s, 1H), 8.24 (d, J = 5.1 Hz, 1H), 7.47–7.36 (m, 2H), 7.22 (t, J = 7.5 Hz, 2H), 7.15–7.06 (m, 2H), 7.00 (s, 1H), 4.09 (s, 2H). ¹³C NMR (151 MHz, CDCl₃) δ 154.0, 151.6, 149.4, 136.4, 126.9, 124.2, 122.8, 122.8, 122. 5, 119.8, 118.7, 112.5, 111.3, 30.8. HRMS (ESI) exact mass calcd for C₁₄H₁₂ClN₂ [M+H]⁺ m/z 243.0689, found 243.0683.
3-((3-chloropyridin-4-yl)methyl)-1H-indole (4e): The desired pure product was obtained in 79% yield as a white solid. ¹H NMR (400 MHz, CDCl₃) δ 8.59 (s, 1H), 8.55 (d, J = 2.3 Hz, 1H), 8.30 (d, J = 5.0 Hz, 1H), 7.48 (d, J = 7.9 Hz, 1H), 7.39 (d, J = 8.1 Hz, 1H), 7.22 (d, J = 7.2 Hz, 1H), 7.16–7.10 (m, 1H), 7.08 (d, J = 5.0 Hz, 1H), 7.03 (d, J = 2.1 Hz, 1H), 4.23 (s, 2H). ¹³C NMR (151 MHz, CDCl₃) δ 148.9, 148.0, 147.5, 136.4, 132.1, 127.1, 125.0, 123.2, 122.4, 119.7, 118.8, 111.4, 111.4, 28.5. HRMS (ESI) exact mass calcd for C₁₄H₁₂ClN₂ [M+H]⁺ m/z 243.0689, found 243.0692.