TOPOGRAPHICAL DYNAMICS OF DAMAGE-RELATED VOLATILE EMISSION IN PHASEOLUS LUNATUS L.

As sessile organisms, plants cannot avoid attacks by biotrophs that use them as a food source; therefore, through evolution they developed efficient strategies to defend themselves (1). This led plants to produce chemical compounds ecologically useful to avoid or limit damages (2) caused by herbivores and pathogens. Besides those compounds that directly act against herbivores, plants produce Volatile Organic Compounds (VOCs) that attract natural enemies, parasitoids or predators of herbivores feeding on them (1, 3, 4). The plant response to biotic/abiotic wounding has always been studied on the whole plant emissions (5) with either head-space or disruptive methods, often by analyzing different organs or leaves separately (6). Despite the consistent amount of data, the dynamics of VOCs emission is still an open question as well as the relationship between damaged area and the biosynthesis of bioactive molecules. In order to identify compounds actively related to plant-insect interaction, we developed a non-invasive and innovative in vivo high concentration-capacity sampling technique to capture volatiles from Phaseolus lunatus (Lima bean) leaves upon wounding of different nature. Direct Contact-Sorptive Tape Extraction (DC-STE) (7, 8, 9) is an innovative and non-disruptive technique based on the direct contact of polydimethylsiloxane (PDMS) tapes (6×10 mm) on biological surfaces. In this case, we applied the technique to P. lunatus fresh leaves that were sampled at fixed distances from the wounded areas in time-course experiments. The sampled fraction was then recovered from the tape by thermal desorption and on-line analysis by gas-chromatography coupled to mass spectrometry (GC-MS). Upon herbivore wounding caused by the larvae of the Noctuid Spodoptera littoralis, mechanical damage by a pattern wheel and the combination of mechanical damage with the oral secretions of S. littoralis, we used DC-STE-GC-MS to analyze the topographical dynamics of VOC emission from leaves of P. lunatus. The statistical data treatment with multivariate analysis emphasized interesting differences depending on both the kind of wounding and the distance from wounding. The non-disruptive VOC sampling method used allowed us to run gene expression analysis of the key VOC-related enzymes on the same leaves previously analyzed. A perfect topographical correlation was found between VOC emission noticed with DC-STE-GC-MS and gene expression analyzed by Quantitative Real Time PCR, confirming the reliability of this new in vivo sampling technique in plant interactions studies. 1) A. Mithöfer, W. Boland (2012) Annu. Rev. Plant Biol., 63, 431–50 2) E. Pichersky, E. Lewinsohn (2011) Annu. Rev. Plant Biol., 62, 549–66 3) M. E. Maffei, J. Gertsch, G. Appendino (2011) Nat. Prod. Rep., 28, 1359–80 4) C. M. De Moraes, W. J. Lewis, P. W. Paré, H. T. Alborn, J. H. Tumlinson (1998) Nature, 393, 570–573 5) D. Tholl, W. Boland, A. Hansel, F. Loreto, U. S. R. Röse, J.-P. Schnitzler (2006) Plant J., 45, 540–60 6) T. G. Köllner, C. Lenk, C. Schnee, S. Köpke, P. Lindemann, J. Gershenzon, J. Degenhardt (2013) BMC Plant Biol., 13:15 7) C. Bicchi, C. Cagliero, P. Rubiolo (2011) Flavour Fragr. J., 26, 321–325 8) B. Sgorbini, M. R. Ruosi, C. Cordero, E. Liberto, P. Rubiolo, C. Bicchi (2010) J. Chromatogr. A, 1217, 2599–605 9) P. Sandra, S. Sisalli, A. Adao, M. Lebel, I. Le Fur (2006) LCGC Eur., 19, 33–39