Exposure to (Z)-11-hexadecenal [(Z)-11-16:Ald] increases Brassica nigra susceptibility to subsequent herbivory

It is well established that plants emit, detect and respond to volatile organic compounds; however, knowledge on the ability of plants to detect and respond to volatiles emitted by non-plant organisms is limited. Recent studies indicated that plants detect insect-emitted volatiles that induce defence responses; however, the mechanisms underlying this detection and defence priming is unknown. Therefore, we explored if exposure to a main component of Plutella xylostella female sex pheromone namely (Z)-11-hexadecenal [(Z)-11-16:Ald] induced detectable early and late stage defence-related plant responses in Brassica nigra. Exposure to biologically relevant levels of vapourised (Z)-11-16:Ald released from a loaded septum induced a change in volatile emissions of receiver plants after herbivore attack and increased the leaf area consumed by P. xylostella larvae. Further experiments examining the effects of the (Z)-11-16:Ald on several stages of plant defence-related responses showed that exposure to 100 ppm of (Z)-11-16:Ald in liquid state induced depolarisation of the transmembrane potential (Vm), an increase in cytosolic calcium concentration [Ca2+]cyt, production of H2O2 and an increase in expression of reactive oxygen species (ROS)-mediated genes and ROS-scavenging enzyme activity. The results suggest that exposure to volatile (Z)-11-16:Ald increases the susceptibility of B. nigra to subsequent herbivory. This unexpected finding, suggest alternative ecological effects of detecting insect pheromone to those reported earlier. Experiments conducted in vitro showed that high doses of (Z)-11-16:Ald induced defence-related responses, but further experiments should assess how specific the response is to this particular aldehyde.


Results
Experiment 1: Effects of (Z)-11-16:Ald on Brassica nigra volatile emissions and feeding by Plutella xylostella larvae. To test whether (Z)-11-16:Ald induces detectable defence-related responses in B. nigra plants, treated plants were exposed for 24 h to (Z)-11-16:Ald released from septa loaded with 100 µl of a 100 ppm (Z)-11-16:Ald solution. Volatile emissions were collected from both control and (Z)-11-16:Aldexposed plants before and after P. xylostella larvae feeding on the plants for 24 h. Immediately after exposure, volatile emissions did not differ between the control and (Z)-11-16:Ald-exposed plants (P = 0.507) (Fig. 1). However, after 24 h of feeding by P. xylostella larvae, the volatile emissions were significantly different (P = 0.039) (Fig. 2). At the end of the feeding time, and after the collection of VOC, we measured the area consumed by larvae and found that P. xylostella larvae fed more on plants exposed to (Z)-11-16:Ald than plants exposed to clean air (P = 0.044) (Fig. 3).

Experiment 2: Effects of Plutella xylostella pheromone components on the transmembrane potential of Brassica nigra plants.
A change in transmembrane potential (Vm) is known to be the first step in plant detection of chemical cues 19 . We consequently studied whether the insect-emitted compounds could lead to a change in Vm of B. nigra leaves by applying 10 to 100 ppm aqueous solutions directly to leaf segments. Treatment with (Z)-11-16:Ald elicited Vm depolarisation at all concentrations tested except 10 ppm suggesting a threshold between 25 and 10 ppm (Fig. 4). In this experiment we also tested the effects of (Z)-11-16:Ac, another component of P. xylostella sex pheromone, on the transmembrane potential of B. nigra. Treatment with (Z)-11-16:Ac only elicited Vm depolarisation at 100 ppm (Table S1) showing that plants are more sensitive to (Z)-11-16:Ald exposure.  20,21 . Therefore, we determined [Ca 2+ ] cyt in plant leaves after 30 min of incubation with 50 and 100 ppm of (Z)-11-16:Ald in aqueous solution (Fig. 5). Exposure to both concentrations increased intracellular [Ca 2+ ], but the strongest green fluorescence was observed after exposure to 100 ppm of (Z)-11-16:Ald. Since an increased [Ca 2+ ] cyt is often associated with an increased production of ROS 22  www.nature.com/scientificreports/ and observed that plants treated with 100 ppm of (Z)-11-16:Ald, showed a strong production of H 2 O 2 in treated cells, with respect to controls (Fig. 6).

Experiment 4:
The effect of (Z)-11-16:Ald on ROS-scavenging enzyme activities and gene expression. In order to assess whether the ROS production observed with the confocal laser scanning microscope (CLSM) was associated with increased enzyme activity and transcription, we measured the activities of ROS scavenging enzymes catalase (CAT), superoxide dismutase (SOD), and peroxidase (POX) and their associated gene expression for control plants and plants treated for 30 min with 100 ppm of (Z)-11-16:Ald in aqueous solution. CAT gene transcript levels and enzyme activity were both significantly (P < 0.001, Fig. 7a, and P = 0.007, Fig. 7b) enhanced by 100 ppm (Z)-11-16:Ald treatment. SOD enzyme activity increased in response to (Z)-11-16:Ald treatment (P = 0.011, Fig. 7b), but there was no detectable change at the transcriptomic level (P = 0.647, Fig. 7a). For POX, (Z)-11-16:Ald increased the gene transcript level (P = 0.002, Fig. 7a) and tended to increase the enzyme activity (P = 0.056, Fig. 7b).

Discussion
To determine whether exposure to (Z)-11-16:Ald induced detectable defence-related responses in B. nigra, we investigated the amount of feeding damage to plants and the volatile emissions of plants exposed to volatilised pure compound. Contrary to our hypothesis, P. xylostella larvae fed more on plants previously exposed to (Z)-11-16:Ald at biologically relevant levels than on controls, suggesting that exposure to (Z)-11-16:Ald might increase the susceptibility of plants to future herbivory. Earlier work investigating the responses of plants to insect sex pheromone showed that exposure primed defences 14,16 , resulting in higher volatile emissions 13 , and lower feeding 12 . Our results are different to these findings and suggest alternative ecological effects of detecting insect pheromone to those reported earlier.
The greater feeding on (Z)-11-16:Ald-exposed plants compared to controls could relate to the differences in volatile emissions of the differently treated plants. Although there were no detectable differences in volatile emissions between exposed plants and controls immediately after (Z)-11-16:Ald-exposure, there were differences after a subsequent 24 h of feeding. The difference could be due to (Z)-11-16:Ald-induced changes in the plant directly affecting herbivore-induced volatile emission, or could be related to altered plant nutrition or defences leading to an increase in the leaf area consumed by herbivores and a consequent difference in induction of volatile emissions. Thus (Z)-11-16:Ald could potentially alter plant defences and in doing so increase the survival of eggs and future hatched larvae. It has earlier been shown in Arabidopsis thaliana that application of Pieris brassicae    www.nature.com/scientificreports/ or Spodoptera littoralis egg extract onto leaves reduced the induction of genes related to defence against insects after caterpillar feeding 23 . However, at this stage our data does not allow us to further explore these ecological hypotheses. Additional studies would need to test in greater detail the deposition of eggs, the hatching of eggs, and performance and survival of developing larvae of plants exposed to (Z)-11-16:Ald. The observation of changes in feeding amount and plant volatile responses prompted us to examine the early and late signaling events in response to (Z)-11-16:Ald exposure. The results showed that exposure to (Z)-11-16:Ald induced a transmembrane depolarisation by plants. The depolarisation of the plasma membrane potential is known to be the first detectable event in the detection of a biotic or abiotic stress 19 . We estimated the detection threshold of the pheromone to be a concentration between 25 and 10 ppm. A lower level of transmembrane depolarisation was observed when plants were exposed to (Z)-11-16:Ac, hence (Z)-11-16:Ald appears to be the most phytoactive component of the P. xylostella sex pheromone. However, it remains unclear how specific the plant response is to the (Z)-11-16:Ald, which should be further elucidated by comparison with aldehydes that have a more similar chemical structures.
The plasma membrane is the only cellular structure in direct contact with the environment, which makes it critical for sensing environmental stimuli and initiating a cascade of events that eventually leads to a specific response 21 . As demonstrated in Arabidopsis, Vm depolarisation depends on a cascade of events that include changes in [Ca 2+ ] cyt and the production of ROS 24 . We found that 50 ppm and 100 ppm of (Z)-11-16:Ald increased both the [Ca 2+ ] cyt and the production of ROS. Moreover, we demonstrated that the increased ROS detected by CLSM were associated with the increased expression of reactive oxygen species (ROS)-mediated genes and www.nature.com/scientificreports/ ROS-scavenging enzyme activity. ROS participate in cell oxidation, during which H 2 O 2 is produced and later regulated by ROS-scavenging enzymes involved in its degradation to protect cells from oxidative stress 25,26 . The production of H 2 O 2 is potentially harmful and can result in oxidation in the cells of aerobic living organisms 27 . It is also an important component of the signalling network in plants 28,29 and takes part in plant defence in response to environmental stress 30,31 . Several plant species trigger localized cell death by producing ROS at oviposition sites on leaves, which has been shown to be associated with an increase in egg mortality or a reduction of larval survival rate 32 . Recently, Bittner and colleagues demonstrated that when plants are previously exposed to the female pine sawfly (D. pini) sex pheromone, they produce H 2 O 2 and induce defence-related genes faster after egg deposition on leaves, compared to plants that have not been exposed to the pheromone 15 . They suggested that the sex pheromone acted as an environmental cue indicating to plants that there would be future egg deposition on needles and subsequent herbivory. Plants then responded to it by producing H 2 O 2 , which formed necrotic tissue and reduced survival of eggs. Taken together with our observations, it is possible that the (Z)-11-16:Ald also primes B. nigra plant defences by producing H 2 O 2 as a defensive mechanism to limit future egg deposition. It was shown that B. nigra plants induce the necrosis of cells located at the oviposition site of Pieris rapae and Pieris napi 33 , which can support this hypothesis. Contrary to the hypothesis of defence induction, B. nigra plants exposed to (Z)-11-16:Ald received more herbivore-feeding damage than control plants, but further investigations are needed to determine whether the production of H 2 O 2 following exposure to (Z)-11-16:Ald would reduce subsequent egg deposition or hatching. Interestingly, early signalling events following the exposure to (Z)-11-16:Ald are analogous to the responses induced by biotic stress such as herbivore-wounding 20,22 , and in response to HIPVs 21 . For example, exposure to Error bars indicate se. * P < 0.05, ** P < 0.01, *** P < 0.001. ns indicates non-significant. n = 3 to 4 for each treatment. www.nature.com/scientificreports/ HIPVs resulted in a depolarisation of the plasma membrane (Vm) 34,35 due to the entrance of calcium (Ca 2+ ) into the cytosol of cells 34 . The detection of HIPVs results in transcriptional 36,37 , metabolic and physiological changes in plants 38 . Several studies have shown that plants perceiving HIPVs deploy faster and stronger chemical defences upon subsequent stress 8,9 , which can negatively affect herbivorous insects 39 . The observed action of (Z)-11-16:Ald is typical to that of insect and mite elicitors 21,40,41 . However, it is notable that to determine if (Z)-11-16:Ald induced detectable responses in B. nigra plants, whole plants were exposed to vapourised (Z)-11-16:Ald for 24 h, while to determine early and late signalling events following exposure to (Z)-11-16:Ald we applied 100 ppm in aqueous state for 30 min to 1 h (Table S2). While we used a biologically realistic scenario with a realistic concentration of pheromone for the whole plant responses the in vitro experiments focussed on mechanisms do not represent biologically accurate scenarios and utilised high concentrations of pheromone. Future studies should bridge this methodological gap by utilising more biologically realistic scenarios in mechanism elucidation. (Z)-11-16:Ald has been found to be a main constituent of pheromones in many moth species from the Noctuidae family including the corn earworm Helicoverpa zea 42 , which is the second-most important economic pest species in North America 43 . Many plants that have co-evolved with the Noctuidae could have the ability to detect (Z)-11-16:Ald. Prior to this study, the ability of plants to detect insect-emitted volatiles had been reported for two species: a perennial plant, S. altissima, and a tree, P. sylvestris. We can now tentatively add an annual plant, B. nigra, to the list. These studies suggest that the ability to detect insect-emitted volatiles has widely evolved in a large variety of plant families, and highlight the need to determine how widespread this trait is. Further studies should also determine the threshold and distance of detection and the ecological consequences of this detection.
In summary our results indicate for the first time that exposing B. nigra plants to volatile (Z)-11-16:Ald increases the susceptibility of plants to feeding by P. xylostella larvae and induces an alteration in herbivoreinduced volatile emissions. Further mechanistic experiments conducted in vitro using high doses of pheromone indicated that exposure to (Z)-11-16:Ald induces responses in receiver plants that are characterised by a depolarisation of Vm, an increase in [Ca 2+ ] cyt and production of H 2 O 2 leading to an increase in ROS-mediated gene expression and ROS scavenging-enzyme activity, which are typical responses to insect elicitors. This study supports recent findings showing that plants can detect insect-emitted volatiles. However, further research should be conducted to determine an accurate dose response of whole plants to volatile pheromone and the specificity of the response to this particular aldehyde.

Exposure of plants to treatments.
A 100 ppm solution of synthetic (Z)-11-16:Ald diluted in dichloromethane was prepared and 100 µl of the solution was injected into a rubber septum (7 mm O.D; Sigma-Aldrich) and left for 30 min for the dichloromethane to evaporate. As a solvent control, 100 µl of dichloromethane was deposited on a rubber septum, and left to evaporate for 30 min. A second control, without the rubber septum and dichloromethane, was also set up. Either treatment or control septa were enclosed in 0.5 L glass jars connected with Teflon tubing to plastic bags (Polyethylene terephthalate; overall dimensions 28 × 35 cm; Look Isopussi Eskimo oy, Finland). Plants were enclosed in the PET bags, that were previously baked for 1 h at 120 °C. For 24 h, a clean air flow was passed into the treatment or control glass jars and then into the bags containing the plants (Fig. S1 and Table S2).

Volatile collections and feeding assays.
After 24 h of exposure to the treatments, jars containing the rubber septa were disconnected from the plants, and a first VOC sampling was made before adding 22 first and second instar P. xylostella larvae were added to each plant for 24 h. After 24 h of feeding, volatile compounds were collected by dynamic headspace sampling (Fig. S1). VOCs were trapped by pulling clean air at 0. www.nature.com/scientificreports/ 40 °C for 1 min and then ramped 5 °C min −1 to 210 °C, and then further ramped at 20 °C min −1 to 290 °C. The carrier gas was helium, the transfer line temperature to the MSD was 300 °C, the ionization energy was 70 eV, and the full scan range was 29-355 m/z. We identified volatiles by comparison with a series of analytical standards (Sigma-Aldrich, Germany) and by comparison of their mass spectra to those in the NIST and Wiley 275 mass spectral libraries. Compound quantification was based on Total Ion Chromatograms (TIC) and according to the responses of analytical standards. Emission rates (ER) were calculated with the formula ER = X*Ai/ Dw*t*Ao. ER was expressed in ng gDW −1 h −1 , X was the compound quantity (ng), Ai and Ao were the inlet and outlet air flows (mL min-1), respectively, t was the sampling time of one hour and Dw is the dry weight of the plant sampled (g). After the volatile collection, we placed leaves of plants on A4 paper for scaling and digitally photographed them. Plants were then dried in paper bags in an oven at 60 °C for 3 days. The leaf area consumed by larvae was calculated using the LeafAreaAnalyzer software (https:// github. com/ EmilS talvi nge/ LeafA reaAn alyser; emil-stalvinge@gmail.com).

ROS-scavenging enzyme activities and soluble protein determination. Leaves were collected
immediately after 30 min of exposure to either 100 ppm of (Z)-11-16:Ald or control (Table S2). Intact leaves of two pooled plants were frozen in liquid N 2 and stored at -80ºC before enzyme extraction. Frozen leaves were used for extraction of ROS scavenger enzymes following the method described in Maffei et al. 22 . All steps were carried out at 4 °C. Plant material was ground with a mortar and pestle under liquid nitrogen in cold 50 mM sodium phosphate buffer, pH 7.5, containing 250 mM sucrose, 1.0 mM EDTA, 10 mM KCl, 1 mM MgCl 2 , 0.5 mM phenylmethylsulfonyl fluoride (PMSF), 0.1 mM dithiothreitol (DTT), and 1% (w/v) polyvinylpolypyrrolidone (PVPP) in a 1:10 proportion (weight of plant material to buffer volume). The homogenate was then centrifuged at 25,000 g for 20 min at 4 °C and the supernatant was used directly for measurement of enzyme activity. The soluble protein concentration was measured using the method established by Bradford 46 using bovine serum albumin as a standard.
Catalase (CAT) activity was assayed spectrophotometrically by monitoring the absorbance change at 240 nm due to the decreased absorption of H 2 O 2 (ɛ = 39.4 mM −1 cm −1 ). The reaction mixture in 1 mL final volume contained 50 mM Na-P, pH 7.0, 15 mM H 2 O 2 , and the enzyme extract. The reaction was initiated by addition of H 2 O 2 .
Peroxidase (POX) activity was measured by detecting the oxidation of guaiacol (ɛ = 26.6 mM −1 cm −1 ) in the presence of H 2 O 2 . The reaction mixture contained 50 mM Na-P, pH 7.0, 0.33 mM guaiacol, 0.27 mM H 2 O 2 , and the enzyme extract in a 1.0 mL final volume. The reaction was started by addition of guaiacol and measured spectrophotometrically at 470 nm.
Superoxide dismutase (SOD) activity was measured by reduction of nitro blue tetrazolium due to a photochemically generated superoxide anion. One ml of assay mixture consisted of 50 mM Na-P buffer, pH 7.8, 13 mM methionine, 75 μM nitro blue tetrazolium (NBT), 2 μM riboflavin, 0.1 mM EDTA, and the enzyme extract. Riboflavin was added as the last reagent. Samples were placed 30 cm below a light source (60 µmol m −1 s −1 ), and www.nature.com/scientificreports/ the reaction was allowed to run for 15 min. The reaction was stopped by switching off the light. A non irradiated reaction mixture, which was run in parallel, did not develop colour and served as a control. The absorbance was read at 560 nm.

Quantitative gene expression analysis by Real-time PCR.
Total RNA was isolated from control or treatment leaf tissues using RNA Isolation mini Kit (Machery-Nagel, Germany), and RNase-Free DNase according to the manufacturer's protocols. The quality of RNA was checked in 1% agarose gel and the final yield was checked with a Spectrophotometer (Pharmacia Biotech Ultrospec 3000, United States). The cDNA synthesis was performed starting from 1 µg RNA using the High Capacity cDNA Reverse Transcription kit (Applied Biosystem, United States). Primers for real-time PCR were designed using the Primer 3.0 software 47 and the relative sequences are listed in Supplementary Table S3. The real-time PCR was performed on an Mx3000P Real-Time System (Agilent Technologies, United States) using SYBR green I with the dye ROX as an internal loading standard. The reaction mixture was 10 µl in volume and comprised 5 µl of 2 × Maxima SYBR Green qPCR Master Mix (Thermo Fisher Scientific), 0.5 ml of cDNA, and 100 nM of primers (Integrated DNA Technologies, United States). The thermal conditions were as follows: 10 min at 95 °C, 40 cycles of 15 s at 95 °C, 20 s at 57 °C, and 30 s at 72 °C. Fluorescence was read after each annealing and extension phase. All runs were followed by a melting curve analysis from 55 to 95 °C. Two reference genes, ACT1 and eEF1Balpha2, were used to normalize the results. The sequences of the primers used in this work for CAT1, CuZnSOD1, PER4, ACT1 and eEF1Balpha2 are reported in Table S3. All amplification plots were analyzed with the MX3000P software (Agilent Technologies, United States) to obtain Ct values. Real-time PCR data are expressed as fold change of the treatment with respect to the control.
Statistical analysis. Area consumed by larvae, Vm measurements, enzyme activity and gene expression data were analysed using SPSS 25 software (IBM Corp. Armonk, USA). The normality of data and homogeneity of variances were checked and log transformed when the data did not meet assumptions for parametric analyses. Because we observed no significant differences between the control and the solvent control, we directly compared the control with (Z)-11-16:Ald for all analyses. Differences between treatments were analysed using T-tests for the fed area, gene expression and enzyme activity. Volatile emission rates were log transformed and auto-scaled (mean-centered and divided by the standard deviation of each variable). Partial Least Squares -Discriminant Analysis (PLS-DA) was performed on emission rates with the R software (v. 3.4.3) with the package vegan and RVAideMemoire with cross validation based on 50 submodels (fivefold outer loop and fourfold inner loop). A pairwise test was performed, based on PLS-DA with 999 permutations, to highlight the differences between treatments. The PLS-DA graphics were done with metaboanalyst (https:// www. metab oanal yst. ca/).

Data availability
The datasets generated during the current study are available from the corresponding author on reasonable request.