Metabolic Integration of Spectral and Chemical Cues Mediating Plant Responses to Competitors and Herbivores

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Plant growth after far red light supplementation: Seeds of S. altissima were bulk collected in winter 2020 from plants around Bebe Lake, Ithaca, NY and then stored in a freezer at −20 °C. After a month, the seeds were put into LM1 germination mix soil (Lambert) for germination at Cornell University’s green-house with a photoperiod of 16:8 h light:dark. Once the plants had germinated, they were repotted into individual clear polyethylene terephthalate plastic cups of 500 mL capaci-ty, and an initial measurement of the length of the plant and the number of leaves was taken. All plants (n = 128) were grown under natural light supplemented with high-pressure sodium lamps that produce 200 µmol/m2/s of white light to complete a photoperiod of 16:8 h day:night. In addition, half of the plants were under supplemental far-red (FR) light, using a FR LED strip (Forever Green Indoors, λ = 730 nm) of 114 cm length with 32 LED bulbs. The FR lamps were covered with a blue filter (Roscolux, Su-pergel, Cinegel no. 83 Medium Blue) to remove residual red light following the protocol of [14]. The lamp was located at 15 cm to the side of the plant and 10 cm from the ground to simulate the angle of light and the intensity coming from a neighboring plant. After one week, the second measurement of height and number of leaves was recorded to as-sess the effect of increased ratios of FR light on plant growth.  [CSV file]   (5.396Kb)
Plant volatile organic compound emissions after herbivore damage and FR light supplementation: After plant growth measurements, two larvae of Spodoptera frugiperda in their third instar were added to each of 10 plants in each light treatment (FR and control), completing four groups of plants: Control, Damage, FR, and FR + Damage (Figure 1c). After another four days with the larvae actively feeding, 10 plants in the damage treatment and 10 plants in the control treatment were used as emitter plants in a plant VOC-exposure experiment. Their VOC emissions were pulled into receiver plant chambers that included either con-trol plants under normal light conditions or plants supplemented with FR light (Figure 1d). The chambers of both the emitter and receiver plants were connected through 0.7 cm diameter silicon tubing (BIO-RAD, Hercules, CA, USA), and the chamber of the receiver was connected to an active air sampling vacuum pump (IONTIK) pulling air at about 450 mL/min. The pumps generate a constant flow of air from the emitter to the receiver plants (Figure 1d). The pumps were changed twice a day, to ensure that there would be at least 22 h of flow per day. After four days of VOC exposure, we collected VOCs using adsorbent traps and leaf material to analyze non-volatile metabolites. Volatile samples of each plant were taken by enclosing the plant into 500 mL polyethylene cups that were connected to an ORBO-32 charcoal adsorbent tube (Supelco®, SIGMA-ALDRICH, Inc. St Louise, MO, USA). The air was pulled through the charcoal traps using an active air sampling vacu-um pump (IONTIK) pulling air at about 450 mL/min. Additionally, leaf samples were collected and flash-frozen in liquid nitrogen and later stored at −80 °C until further anal-ysis. To understand if the chemical response to damage is affected by the presence of a neighbor, we compared the volatile and non-volatile chemical profiles of the emitter plants (Control, Damage, FR, and FR + Damage). To understand if the perception of vola-tiles is affected by FR exposure, we compared the volatile and non-volatile metabolites produced by the receiver plants. For the analysis of VOCs emitted from the experimental plants, each of the ORBO-32 charcoal traps that were used in the collection was spiked with 5 µL of tetraline (90 ng/mL) as an internal standard. The charcoal traps were washed with 400 μL of di-chloromethane, which was then injected in a Varian CP-3800 gas chromatograph (GC) coupled with a Saturn 2200 mass spectrometer (MS) and equipped with a CP-8400 au-tosampler. The GC-MS was fitted with a DB-WAX column, (Agilent, J&W Scientific, Santa Clara, CA, USA) of 60 m × 0.25 mm id capillary column coated with polyethylene glycol (0.25 mm film thickness). The temperature program began with an injection tem-perature of 225 °C, heated from 45 °C to 130 °C at 10 °C/minute, then from 130 °C to 180 at 5 °C/min, and finally from 180° to 250 °C at 20 °C/minute with a 5 min hold at 230 and 250 °C. The samples were standardized by expressing signal intensity (peak area) of each peak relative to that of the internal standard. Compound and compound class identity were determined comparing mass spectra and retention time indices with NIST library records and previously published VOC data of S. altissima [27,59].  [CSV file]   (62.48Kb)
Non-volatile secondary metabolites in response to herbivory and FR light supplementation: For the high-performance liquid chromatography (HPLC) analysis of non-volatile compounds, leaf samples (150–250 mg/sample) were homogenized and extracted in 1 mL of 90% methanol using a FastPrep® tissue homogenizer (MP Biomedicals®, Irvine, CA, USA) at 6 m/s for 90 s using 0.9 g grinding beads (Zirconia/Silica 2.3 mm, Biospec®, Bartlesville, OK, USA). The samples were then centrifuged at 4 °C for 15 min at 14,000 rpm, and we analyzed 15 µL of the by HPLC on an Agilent® 1100 series HPLC. We used 99.9% acetonitrile and 0.25% H3PO4 as the mobile phase. The elution system consisted of aqueous 0.25% H3PO4 and acetonitrile (ACN), which were pumped through a Gemini C18 reverse-phase column (3 μm, 150 × 4.6 mm, Phenomenex, Torrance, CA, USA) at a rate of 0.7 mL/min with increasing concentrations of ACN: 0–5 min, 0–20% ACN; 5–35 min, 20–95% ACN; and 35–45 min, 95% ACN. The area of each peak was standardized by the mass of the leaf tissue extracted. The individual compound or class identity was determined based on the UV spectra and retention times of authentic standards.  [CSV file]   (109.5Kb)
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Abstract
Light quality and chemicals in a plant’s environment can provide crucial information about the presence and nature of antagonists, such as competitors and herbivores. Here, we evaluate the roles of three sources of information—shifts in the red:far red (R:FR) ratio of light reflected off of potentially competing neighbors, induced metabolic changes to damage by insect herbivores, and induced changes to volatile organic compounds emitted from herbivore-damaged neighboring plants—to affect metabolic responses in the tall goldenrod, Solidago altissima. We address the hy-pothesis that plants integrate the information available about competitors and herbivory to opti-mize metabolic responses to interacting stressors by exposing plants to the different types of en-vironmental information in isolation and combination. We found strong interactions between the exposure to decreased R:FR light ratios and damage on the induction of secondary metabolites (volatile and non-volatile) in plants. Similarly, the perception of VOCs emitted from neighboring plants was altered by the simultaneous exposure to spectral cues from neighbors. These results suggest that plants integrate spectral and chemical environmental cues to change the production and perception of volatile and non-volatile compounds and highlight the role of plant con-text-dependent metabolic responses in mediating population and community dynamics.
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These are the raw data associated with the publication "Metabolic Integration of Spectral and Chemical Cues Mediating Plant Responses to Competitors and Herbivores"., which was published in the journal "plants" in 2022.
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The research was funded by a grant to AC by Fundación CEIBA (Centro de Estudios Interdisci-plinarios Básicos y Aplicados) and a grant from NIFA Multistate NE-1501 (2021-22-194) to AK.
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2022
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Plants
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