ABSTRACT: ⢠Herbivore-induced plant volatiles (HIPVs), in addition to attracting natural enemies of herbivores, can serve a signaling function within plants by acting as wound signals that induce or prime defenses. However, particularly in woody plants, which compounds within HIPV blends are capable of acting as signaling molecules are largely unknown. ⢠Leaves of hybrid poplar (Populus deltoides x nigra) saplings were exposed in vivo to naturally wound-emitted concentrations of the green leaf volatile (GLV) cis-3-hexenyl acetate (z3HAC) and then subsequently fed upon by gypsy moth larvae (Lymantria dispar L.). Volatiles were collected throughout the experiments, and leaf tissue was collected to measure phytohormone levels and expression of defense-related genes. ⢠Relative to controls, z3HAC-exposed leaves had higher levels of jasmonic acid and ï¡-linolenic acid following gypsy moth feeding. Further, z3HAC primed transcripts of phytohormone signaling (lipoxygenase 1) and direct defense (a Kunitz proteinase inhibitor) genes. These qRT-PCR results were supported by microarray analysis using the AspenDB 7K EST microarray containing ~5400 unique gene models. Moreover, z3HAC also primed the release of herbivore-induced terpene volatiles. ⢠The widespread priming response suggests an adaptive benefit to detecting z3HAC as a wound signal. Thus, woody plants can detect and use z3HAC as a signaling cue to prime defenses before actually experiencing damage. GLVs may therefore have important ecological functions in arboreal ecosystems. Plants and Gypsy Moths Hybrid poplar (Populus deltoides x nigra, clone âOGYâ: Magnoliopsida; Malpighiales; Salicaceae; Aigeiros section of Populus) saplings were grown from cuttings in a walk-in growth chamber maintained at 25oC with a 16:8 photoperiod. Cuttings were planted in five-gallon pots in commercial potting soil (MetroMix 250, SunGro, Bellevue, WA, USA) and watered as necessary. Due to supplements in the soil, fertilization was not required and poplars grew to heights of ~1.5 m in 12-15 weeks. Newly molted 4th instar L. dispar larvae were used for all experiments. Egg masses were obtained from APHIS (Otis Plant Protection Lab, Cape Cod, MA, USA) and reared through 3rd instar on artificial diet. Newly molted 4th instar larvae cleared their gut contents prior to molting. Experimental Manipulations All experiments were conducted from March-April 2007 in a single walk-in growth chamber maintained as described above. Poplars were equilibrated to the chamber for 24 hours prior to manipulations. Two fully-expanded source leaves (leaf plastochron index LPI 8 and 9) were isolated per sapling using individual 15x15x1 cm Teflon/glass chambers (for photo see Appendix in Frost et al., 2007). These two leaves are non-orthostichous, which means that they do not readily share assimilates or wound signals (Davis et al., 1991). Thus, for the purposes of these experiments, treatment leaves did not interact with their respective control leaves. However, the leaves are of slightly different ages and, to control for this potential effect, we randomized which leaf would receive the z3HAC treatment. Flow into each chamber was set at 2.0 L/min, which was sufficient to avoid condensation within the chamber. The experiments were designed to have two consecutive albeit separate treatments: exposure to z3HAC and subsequent application of L. dispar larvae. We used commercially available z3HAC (Sigma-Aldrich), and prepared concentrations to infuse into each septum such that it delivered ~40 ng z3HAC per application. After a pretreatment volatile collection (Day 1 and night starting Day 2), z3HAC-infused rubber septa were placed into the leaf chambers with each treatment leaf (morning of Day 2). Control leaves each received a septum infused with just the dichloromethane solvent. Septa were replaced twice, once before the afternoon collection on Day 2, and before the morning collection on Day 3. All septa were removed during the night of Day 3, since most volatile production stops at night in this hybrid (Fig 5a). On the morning of Day 4, two newly molted 3rd or 4th instar gypsy moth larvae were added to each leaf, regardless of whether the leaf received the treatment or control septa on Days 2 and 3. The herbivores remained on the leaves for ~24 hours. On the day of collection, each leaf was excised at the petiole, photographed, and then placed in a labeled coin envelope and submerged directly into liquid N2. Each photograph also contained a standard of known area, from which leaf area and % damage calculations were determined (Sigma Scan 500, Systat Software Inc.). Larvae consumed similar amounts on the treatment and control leaves (24 hours: z3HAC-treated leaves: 4.7 ± 1.8 cm2; controls: 4.3 ± 1.7 cm2). The snap-frozen leaves were stored at -80oC until grinding. Each leaf was ground in an individual mortar and pestle under liquid N2. Ground tissue was weighed into vials for RNA extraction and JA analysis (see below). Care was taken to not allow the ground tissue to thaw at any point prior to extraction. This method therefore allowed us to measure gene expression and JA from the same tissue. Microarray Analysis To explore a transcriptome-level response of poplar leaves to z3HAC and herbivory, we conducted microarray analysis of the samples collected after 24 hours of herbivore damage. This allowed a comparison of transcriptome-level responses to herbivory in z3HAC-treated versus untreated leaves (i.e., the set of genes that were primed by z3HAC exposure). Total RNA was labeled using Ambion's Amino Allyl Message Amp II aRNA Amplification Kit (#AM1753) according to the manufacturer's instructions. Briefly, 1 ug of each sample was reversed transcribed using T7-oligo(dT) priming and subsequently second strand cDNA synthesis was performed. The resulting double stranded cDNA was purified and then used as a template for in vitro transcription during which amino allyl-modified UTP was incorporated. The resulting amino allyl-modified cRNA was purified and coupled with Cy3 or Cy5 (GE Amersham, #RPN5661) as appropriate. We used dye swapping to account for potential dye effects. Dye coupled cRNA was purified and 4 ug was fragmented and subsequently dissolved in 60ul of hybridization solution. We used an aspen 7K EST array containing replicate subarrays of 6,705 elements in a 4Ã4 grid with a spot diameter of ~150 µm and a spacing of 200 µm (Ranjan et al., 2004). The elements represented ~5,400 unique JGI Populus gene models, and included positive and negative controls (Lucidea Universal ScoreCard, Amersham) to monitor target labeling and hybridization efficiency, as well as many genes of known function. Clone information can be downloaded from the aspenDB website (www.aspenDB.mtu.edu). Arrays were hybridized following previously published methods (Xiang et al., 2002; Hughes et al., 2001). Chang S, Puryear J, Cairney J. 1993. A simple and efficient method for isolating RNA from pine trees. Plant Molecular Biology Reporter 11: 113-116. Davis JM, Gordon MP, Smit BA. 1991. Assimilate movement dictates remote sites of wound-induced gene expression in poplar leaves. Proceedings of the National Academy of Sciences (USA) 88: 2393-2396. Frost CJ, Appel HM, Carlson JE, De Moraes CM, Mescher MC, Schultz JC. 2007. Within-plant signalling via volatiles overcomes vascular constraints on systemic signalling and primes responses against herbivores. Ecology Letters 10: 490-498. Hughes TR, Mao M, Jones AR, Burchard J, Marton MJ, Shannon KW, Lefkowitz SM, Ziman M, Schelter JM, Meyer MR, Kobayashi S, Davis C, Dai HY, He YDD, Stephaniants SB, Cavet G, Walker WL, West A, Coffey E, Shoemaker DD, Stoughton R, Blanchard AP, Friend SH, Linsley PS. 2001. Expression profiling using microarrays fabricated by an ink-jet oligonucleotide synthesizer. Nature Biotechnology 19: 342-347. Xiang CC, Kozhich OA, Chen M, Inman JM, Phan QN, Chen YD, Brownstein MJ. 2002. Amine-modified random primers to label probes for DNA microarrays. Nature Biotechnology 20: 738-742.
