Inter-vine Signalling via Plant Volatiles

Abstract

With the help of an evolved innate immune system, the sessile plants have created mechanisms to withstand the background noise of their natural environment. One of these mechanisms is the ability of plants to release volatile organic compounds (VOCs) during episodes of environmental stress and subsequently triggering defence responses that might give them some level of tolerance against the stresses. In some instances, plants will “eavesdrop” these volatile cues from their stressed neighbours to adjust their phenotypes against imminent stress. The priming effect of stress-induced VOCs on non-stressed neighbouring plants for quicker and effective defence response to post stress challenge has been demonstrated in many studies involving biotic stress, with only a few studies being related to abiotic stress. There is an emerging knowledge of the mechanisms involved, and compelling discussions about the ecological significance of such interactions under various environmental stress conditions have been documented. To that end, this thesis begins by reviewing the significance of VOC-mediated inter-plant interactions under both biotic and abiotic stresses and highlights the potential to manipulate outcomes in agricultural systems for sustainable crop protection via enhanced defence (Chapter 1). The overarching aim of my PhD research was to investigate plant-plant volatile-mediated communication in grapevine under drought stress using physiological, transcriptome and volatilome analyses. The volatile-mediated interplay between stressed and non-stressed plants has been described in many studies involving both biotic and abiotic stresses as a one-way channel, where the focus is mainly on how inducible VOCs from stressed ‘emitter’ plants prime their non-stressed ‘receiver’ neighbours for defence against impending stress. In Chapter 2, an investigation of VOC-mediated interactions between stressed and non-stressed V. vinifera L. cv. Shiraz during drought and recovery was carried out. Under these experimental conditions, I was interested in exploring the role played by water stress on VOC emissions, specifically α-pinene, isoprene, methyl jasmonate, methyl salicylate, and (Z)-3-hexen-1-ol, as well as how these VOCs induce drought defence responses and stomatal closure in well-watered (WW) receiver vines. A synchronised decline in α-pinene concentration in colocated drought-stressed (DS) and WW receivers, as well as a differential increase in the isoprene levels in the DS emitter compared to the isolated DS vines was observed. An over-expression of a key gene in the jasmonic acid (JA) biosynthesis pathway, allene oxide synthase (AOS) was identified in only the DS emitter during peak drought. Transcript expression of key genes involved in SA and α-pinene biosynthesis, chorismate synthase and α-pinene synthase, respectively, showed similar trends to those of AOS. The results suggested that isoprene and α-pinene may be inter-plant signalling molecules used by grapevine during drought and the study presents the first report of a bidirectional interaction between the emitters and receivers under drought stress, mediated by the JA and terpenoid biosynthesis pathways. To confirm the involvement of isoprene and α-pinene in VOC-mediated signalling in grapevine, as well as other well-known VOC biomarkers, MeJA, MeSA, methanol, and (Z)-3-hexen-1-ol, experiments were carried using sensitive leaf hydraulic and gas exchange measurements to determine the individual VOCs’ effects on stomatal responses (Chapter 3). A modified Xyl'EM embolism meter in conjunction with a high sensitivity liquid mass flow meter was used to monitor the flow rate into single leaves detached from grapevines. Transient changes in the flow rate were observed in response to methanol and (Z)-3-hexen-1-ol volatiles. Grapevine leaves measured in vivo showed a decline in leaf gas exchange in response to methanol, but not with (Z)-3-hexen-1-ol. The hydraulics data from this study indicated that both methanol and (Z)-3-hexen-1-ol are signalling molecules in grapevine, which were hypothesised are elicitors of plant defence responses. Following on from this study, a comprehensive genome-wide transcriptome analysis using RNAseq, together with physiological and metabolite analyses were carried out in Chapter 4 to further explore the molecular basis of intervine signalling under drought stress. No differentially expressed genes (DEGs) were identified between WW control and co-located WW receivers at peak drought, therefore implying that the presence of the co-located DS emitter did not modulate the gene expression of the WW receiver. Contrary to what was observed in Chapter 2, VOCs collected from all the treatment vines showed no treatment effects. The gs of the receivers was also not affected by DS emitters. Overall, no evidence of VOC-mediated communication between the emitters and receivers was found, in contrast to findings from the previous study. It is likely that this difference may have been as a result of the variation in growth stages of each treatment in the previous study, where treatments were evaluated sequentially rather than concurrently, as was the case with the current study (Chapter 4). Nevertheless, from the RNAseq data from the study, drought was shown to induce the expression of DEGs related to the biosynthesis and signalling pathways of well-known VOC biomarkers, including JA, green leaf volatiles (GLVs), and isoprenoids. Although DEGs related to SA biosynthesis were not found, an up-regulation of SA-responsive pathogenesis-related genes and associated transcription factors were identified, suggesting that SA signalling pathway is also involved in grapevine drought defence responses. In summary, my PhD thesis has been devoted to advance the knowledge of the underlying mechanisms and signalling pathways involved in VOC-mediated inter-plant communication using grapevine as a model plant. Although inter-vine signalling could not be concluded from the results obtained in Chapter 4, the gene information from the study identified DEGs related to VOC biosynthesis and drought defence responses, which could be explored in future plant communication studies under drought stress. Using real-time monitoring of VOC-induced transpiration, the study was able to provide conclusive evidence of immediate grapevine responses to volatile cues. Future studies involving other putative VOC signals will benefit from this approach. Post challenge experiments following VOC exposure is also recommended for future investigations to determine the relevance of VOC-mediated priming in eliciting drought tolerance in grapevine and other plant species. The outcomes of this thesis offer opportunities for a wider application of VOCs in agricultural systems as an eco-sustainable plant protection strategy against biotic and abiotic stressors, such as using specific VOCs to enhance disease resistance and mitigate the effects of abiotic stressors on plant health.