Methyl jasmonate meja

To examine the effects of Methyl jasmonate (MeJA, Sigma Aldrich) on the expression of the four selected farnesene correlated genes, a 1 mM MeJA aqueous solution in 0.05% DMSO was sprayed every hour as a fine mist onto tea shoots, leaves were collected at 0, and 12 h respectively.

Sweet potato (Ipomoea batatas cv. Tainung 57) and tobacco (Nicotiana tabacum L. cv. W38) plants were grown in growth chambers (16 h/25 °C light and 8 h/22 °C dark; 70% humidity) under illumination of 30 µmol photons m^–2 s^–1 and 60 µmol photons m^–2 s^–1, respectively. Plants with 6–8 fully developed leaves were used in this study. The third fully expanded sweet potato leaves counted from the terminal bud were treated by wounding, 50 µM methyl jasmonate (MeJA; Sigma, St. Louis, MO, USA), or Spodoptera litura. In the wounding treatment, leaves except the primary veins were pressed by tweezers. In the S. litura feeding assay, the third fully expanded leaves were placed in plastic Petri dishes (90 mm) containing wet filter paper. The third-instar S. litura were individually placed on each leaf at 25 °C under a 16 h light/8 h dark photoperiod. All the analyses were performed in at least three independent biological replicates.

The tomato cultivar ‘Moneymaker’ (MM) and its near-isogenic line BC₅S₂ were used for the experiments. BC₅S₂ was generated from the initial cross S. lycopersicum cv. Moneymaker ×S. pimpinellifolium acc. TO-937 followed by five cycles of combined recurrent crosses toward ‘Moneymaker’ and subsequent selfing steps with selection for high type-IV trichome density and acylsugar production, plus two additional final selfing steps (Supplementary Figure S1). Seedlings were sown in plastic pots of 12 cm containing 15% plant-nutrient loaded zeolite and 85% coconut fiber substrate. Plants were grown within a glasshouse under natural lighting with loose temperature control (22–27°C day, 17–20°C night) and watered when needed. Two leaf growth-stage plantlets were sprayed with 7.5 mM methyl jasmonate (MeJA) (Sigma-Aldrich) in 0.8% ethanol aqueous solution until the point of run-off. Mock treatment with 0.8% ethanol aqueous solution was used as control. Multiple MeJA inductions were performed by three applications on days 0, 7, and 14.

Seeds of 30DPH were soaked in 70% (v/v) ethanol for 1 min followed by washing in 2.5% (v/v) sodium hypochlorite solution containing 0.1% (v/v) Tween 20 for 15 min and rinsed thoroughly with sterile distilled water. Surface sterilized seeds were grown in PhytoCon culture vessels (Phytotechnology Laboratories, Overland Park, KS, USA) containing half strength Murashige and Skoog (MS) medium for 14 days. Seedlings were kept in sucrose free liquid half strength MS medium for 24 h. Seedlings of 15DPG (days post germination) were transferred to PhytoCon culture vessels (Phytotechnology Laboratories) containing liquid half strength MS supplemented with 100 µM abscisic acid (ABA, Sigma, St. Louis, MO, USA), 50 µM brassinolide (Bra, Sigma), 50 µM gibberellic acid (GA, Sigma), 50 µM indole-3-acetic acid (IAA, Sigma), 100 µM methyl jasmonate (MeJa, Sigma), 100 µM salicylic acid (SA, Sigma), 100 µM Zeatin (Zea, Sigma) and incubated for 6 h. Leaves from a total of 72 samples from seven treatments in three biological replicates including one untreated control of three genotypes were harvested and immediately frozen as mentioned in the earlier section.

Two-week-old cv. Yuanfengzao seedlings were treated with varied abiotic stresses or hormones. Drought stress was applied by transferring the hydroponically cultivated seedlings to thee layered filter papers for fast dehydration. Salt stress treatment was achieved by drenching 150 mM NaCl (Sinopharm Chemical Reagents Co., Shanghai, China) solution to the rice seedlings. For cold stress, seedlings were transferred to a growth chamber at 4 °C. Heat stress was applied by transferring the seedlings to a chamber with temperature at 42 °C. Leaf samples were harvested for all four abiotic stress treatments and root samples were only collected for drought and salt treatments. For hormone treatment, seedlings were sprayed with 100 µM ABA, 100 µM methyl jasmonate (MeJA) (Sigma-Aldrich, St. Louis, MO, USA), 100 µM ACC (Sigma-Aldrich, St. Louis, MO, USA), 150 µM SA (Sigma-Aldrich, St. Louis, MO, USA) in a solution containing 0.1% ethanol (Sinopharm Chemical Reagents Co., Shanghai, China) and 0.02% Tween-20 (Sinopharm Chemical Reagents Co., Shanghai, China) or with the solution as controls. All samples were stored at −80 °C until use.

Before stress induction, cells from two-week-old cultures were washed by filtration through a 10 µm nylon net followed by suspension of cells in 100 mL of sterile deionized water. For stress induction 400 mL of fresh media was inoculated with 10 mL of water-suspended cells (0.6 g DW L⁻¹), cultures grown for 72 h, and independent cultures treated with 50, 100, and 200 mM NaCl (KARAL); 120, 240, and 360 mM NaHCO₃ (KARAL); 1.5, 3.0, and 6.0 mM salicylic acid (SA; Sigma-Aldrich); 10, 20, and 30 µM methyl jasmonate (MeJA; Sigma-Aldrich) and 1.09, 2.18, and 4.33 mM acetic acid (KARAL). Samples were taken at 10, 30, 60, and 120 min after treatment and the cultures were either used for ROS detection by fluorescent staining or the cells were harvested by filtration, frozen in liquid N₂, and stored at −80 °C for future use.

Age-dependent leaf senescence was assayed as described by Woo et al. (2001) (link). The photochemical efficiency of photosystem II (PSII) was deduced from the chlorophyll fluorescence (Oh et al., 1996 (link)) using an Imaging-PAM chlorophyll fluorometer (Heinz Walz GmbH, Germany). The ratio of the maximum variable fluorescence to the maximum yield of fluorescence, which corresponds to the potential quantum yield of the photochemical reactions of PSII, was used as a measure of the photochemical efficiency of PSII (John et al., 1995 ; Raggi, 1995 ; Oh et al., 1997 (link)). Chlorophyll was extracted from individual leaves by heating in 95% ethanol at 80 °C. The chlorophyll concentration per fresh weight of leaf tissue was calculated as described by Lichtenthaler (1987) . For dark-induced leaf senescence experiments, the third or fourth rosette leaves of wild-type or mutant plants at 12 d of leaf age were carefully detached and incubated in 3mM MES buffer (pH 5.7) at 22 °C in the dark for the designated time. For hormone treatment, leaves were floated on 3mM MES buffer (pH 5.7) in the presence or absence of 50 μM 1-aminocyclopropane-1-carboxylic acid (ACC; Sigma-Aldrich, USA) or 50 μM methyl jasmonate (MeJA; Sigma-Aldrich, USA) for 5 d. All hormonal treatments were performed at 22 °C under continuous light.