SUPPLEMENTALL INFORMATION
ABIOTIC INDUCTION AFFECTS THE COSTS AND BENEFITS
OF INDUCIBLE HERBIVORE DEFENSES IN Datura wrightii.
H. M. KRUIDHOF, JEREMY D. ALLISON, and J. DANIEL HARE
Induction of Pins by Lema daturaphila. Two groups of 10 plants, 10 - 15 cm tall but not yet differentiated according to adult trichome phenotype, were selected from the glasshouse. These plants were fourth4th-generation backcross progeny of the highly inducible MVV8 line (Hare 2007) formed by backcrossing heterozygous sticky progeny to their original homozygous recessive pollen parent for four 4 generations. After four 4 generations of backcrossing, progeny are expected to be at least 96.9% similar and express the adult trichome phenotype in a 1:1 ratio. Prior to treatment, a single young but full-sized leaf was removed from each plant and assayed for Pin production, and none of the plants was found to produce Pins. Two fourth 4th (last) instar larvae of L. daturaphila from a laboratory colony were placed on a single leaf per plant of one group of 10 plants and allowed to feed for 48 hr. Larvae did not move from the leaf on which they were placed. After 48 hr, larvae were removed, and plants were left in the glasshouse for an additional 24 hr for Pin production. Control plants were maintained under identical conditions but not damaged by L. daturaphila. Both groups of plants were sampled for Pins 72 hr after the beginning of the experiment. Data were analyzed by ANOVA after the log10 (X+1) transformation by PROC GLM of SAS. Feeding by L. daturaphila increased Pin production by nearly eight-fold (mean + SE = 1.126 + 0.145 nmoles mg-1 protein) compared to control plants (0.146 + 0.026, F2, 27 = 34.16, P < 0.001).
Potential for Induction of Pins by Acephate or 0.1% Tween 20. Four groups of 11 undifferentiated plants, as above, were selected from the glasshouse. Prior to treatment, a single young but full-sized leaf was removed from each plant and assayed for Pin production. Plants then were sprayed just to run-off either with 1) deionized water, 2) 0.1% Tween 20 (Sigma-Aldrich) in deionized water, 3) 8 mM MeJA in 0.1% Tween 20 in deionized water, or 4) 1.2 g l-1 acephate (Orthene 97, Valent Chemical Co., Walnut Creek, CA, USAalif.) in deionized water. Plants were segregated by treatment in the glasshouse with at least 2 m between groups and with the plants treated with MeJA being placed downwind and closest to the exhaust fan that was continuously in operation to avoid any possible induction of other plants from volatile MeJA. Plants were resampled for Pins 72 hr later.
Pin concentration before and after treatment was analyzed by a repeated-measures, restricted maximum likelihood ANOVA using PROC MIXED of SAS. The log10 (X+1) transformation was used to ensure normality of errors. Fixed effects included treatment group (water, Tween, MeJA and Tween, or acephate) and sampling time (pre- or post-treatment) and their interaction. Subjects were the individual plants within each treatment group. The interaction was decomposed into a series of single degree-of-freedom tests using the SLICE option of the LSMEANS of PROC MIXED.
Plants did not differ among treatments in Pin concentration prior to treatment (F3, 40 = 1.97, P = 0.13) but did so after treatment (F3, 40 = 24.72, P < 0.001, Table S1). This was entirely due to a nearly eight-fold increase in Pins in the plants treated with 8 mM MeJA (F1, 40 = 85.29, P < 0.001, Table 1), because Pin concentration in the other treatments were unaffected by treatment (all F1, 40 3.18, all P 0.08).
Table S1. Mean + SE Pin production (nmoles mg-1 protein) prior to, and 72 hr after spraying plants with water, 0.1% Tween 20 in water, 8 mM MeJA in 0.1% Tween 20 in water, or 1.12 g l-1 Acephate in water (N = 11 plants per treatment).
Treatment / Pre-treatment / Post-treatmentWater / 0.17 + 0.12 / 0.59 + 0.26
Acephate / 0.78 + 0.46 / 0.64 +0.31
0.1% Tween 20 / 0.00 + 0.0 / 0.42 + 0.18
8 mM MeJA in 0.1% Tween 20 / 0.84 + 0.41 / 6.51 + 0.15
No Induction of Neighboring Plants by Drift of MeJA. Four groups of seven 7 potted plants, as above, were placed in a field devoid of vegetation at Agricultural Operations, University of California, Riverside, California. Each group of plants had one 1 focal plant that was treated, and a total of six 6 "sentinel" plants were arrayed in semicircles 1 m and 3 m from the focal plant. Each semicircle was arranged so that one plant was due east and downwind of the focal plant, and the other two were offset +/- 45° from the expected afternoon westerly winds. The focal plants were sprayed to runoff with 8 mM MeJA in 0.1% Tween 20 in the morning (ca. 09.00 h) and the plants were left in the field for 24 hr then returned to the glasshouse and segregated by treatment as above, left for an additional 48 hr for any Pin response to develop, then reassayed for Pins. Pin data were analyzed by ANOVA after the log10 (X+1) transformation by PROC GLM of SAS. The treatment effect was plant position (focal, 1 m, or 3-m away). Two plants were damaged during transport back to the glasshouse and were deleted from the analysis.
Plants differed in Pin production after treatment (F2, 23 = 21.46, P < 0.001). Focal plants produced a mean (+ SE) of 4.38 (+ 1.14, N = 4) nmoles of Pins mg-1 of protein after treatment whereas the plants 1 and 3 m away produced 0.10 + 0.10 (N = 10) and 0.13 + 0.13 (N = 12) nmoles of Pins mg-1 protein, respectively. Pin production of focal plants differed significantly from zero (P < 0.001) whereas Pin production of plants 1 m and 3 m away from focal plants did not (P > 0.12). Therefore, there was no induction of plants as close as 1 m away from MeJA-treated plants by drift.
Persistence of Pins after Induction. Twelve potted plants, as above were verified not to be producing Pins prior to the experiment. Six plants were sprayed just to run-off with 0.1% Tween 20 in water, and six 6 other plants were sprayed with 8 mM MeJA in 0.1% Tween 20. Plants were segregated in the glasshouse as above and sampled for Pin production 3, 17, 31, and 43 days post-treatment. Pin data were analyzed after applying the log10 (X+1) transformation by a repeated-measures, maximum likelihood ANOVA using PROC MIXED of SAS.
Pins were significantly induced in MeJA-treated plants three days3 d after treatment and remained significantly greater than in Tween-treated plants through day 31, although Pin concentration had begun to decline in MeJA-treated plants. By day 43, there were no significant differences in Pin production between treatments and two 2 of the Tween-treated plants expressed low levels of Pins by this time (Table S2). The experiment was terminated after day 43 because the plants were becoming root-bound and stressed by this time. Once induced, plants continue to show increased Pin concentration for at least 31 days following induction, but repeated sampling of plants for Pins through Day d 31 did not induce Pins in the plants treated only with 0.1% Tween.
Table S2. Mean (+ SE) nmoles Pin mg-1 total protein production in plants sprayed with 0.1% Tween 20 or 8 mM MeJA in Tween 20 when sampled from 3 – 43 days post-treatment. P; probability of significant difference between treatments (N = 6 plants per treatment).
Days post-treatment / 0.1% Tween 20 / 8 mM MeJA in 0.1% Tween 20 / P0 / 0.0 / 0.0 / --
3 / 0.03 + 0.03 / 1.76 + 0.94 / 0.008
17 / 0.0 / 1.78 + 0.49 / < 0.001
31 / 0.01 + 0.01 / 0.52 + 0.21 / 0.05
43 / 0.31 + 0.19 / 0.79 + 0.40 / 0.42
Light Transmittance of Polyester and Polyethylene Plastic Sheeting. Polyester and polyethylene plastic sheeting was obtained from McMaster-Carr, Elmhurst, IL, USAllinois, part numbers 8567K42 and 2008T41, respectively. Pieces were cut to fit between the carrier and a quartz flow cell for a UV-VIS spectrophotometer (Genesys10 UV, Thermo Fisher Scientific, Waltham MassMA, USA.) and transmission at selected wavelengths was determined relative to 100% transmittance through a "blank" quartz flow cell only.
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