Differentially Expressed Genes in Smoke-Exposed Grapes
Sampling for gene expression analysis was conducted at 0, 2, 4, 6, 12, 24, and 36 hours following smoke exposure. Both pairwise and time course DE analyses were performed to better understand gene expression patterns in pre-veraison and post-veraison smoked grapes.
In pre-veraison smoke-exposed grapes, 1615 contigs were identified by pairwise expression analysis to be differentially expressed for at least one-time point; of these, 854 were mapped and functionally annotated with GO IDs (Supplementary File 2). The overall DE gene expression response for the pre-veraison grapes was characterized by a large number of downregulated DE genes 2 hours following smoke exposure, followed by an increasing number of upregulated DE genes with a peak at 6 hours post-smoke exposure (Fig. 1A). Post-veraison smoke-exposed grapes displayed a reduced number of DE genes in comparison with the pre-veraison grapes, with 548 DE genes identified in response to smoke for at least one-time point. Of these, 286 were assigned GO IDs and functional annotations. In contrast to the pre-veraison grapes, the DE gene expression of the post-veraison grapes was characterized by a peak in upregulated DE genes at 2 hours of smoke exposure, with relatively few genes displaying reduced expression in the smoked vs control grapes (Fig. 1B). The observation regarding the number of DE genes at the two developmental stages gives the impression that the response of pre-veraison grapes to smoke is more pronounced than that of the post-veraison grapes. However, analysis of the global gene expression levels over time for both stages indicate that the post-veraison grapes display a strong overall response to smoke that is not necessarily reflected in the DE analysis (Fig. 1C). A large degree of variation observed between replicates at the post-veraison developmental stage for both smoke-exposed and control berries could explain this, as it likely resulted in fewer genes being identified as significantly differentially expressed at the post-veraison stage, despite the strong global gene expression profile displayed by post-veraison grapes in response to smoke. A possible explanation for the high degree of inter-replicate variation in the post-veraison grapes is that at veraison the transcriptional program in different berries in a cluster proceeds at different rates24. This is expected to result in a different baseline transcriptomic response as well as potential differential responses to smoke across replicates after veraison. To address this finding in our analysis, we primarily focused on the genes that displayed shared expression patterns over time, per both the pairwise and time course DE analyses, between pre-veraison and post-veraison grapes.
Complementary to the pairwise DE analysis, time course analysis of transcriptomic responses of grapevines to smoke exposure revealed further insights into the plant's detoxification mechanisms against smoke-derived compounds over the course of the experiment (Fig. 2). Per this analysis, a total of 531 and 119 transcripts were identified in pre-veraison and post-veraison grapes, respectively, that displayed significantly different expression trends in the smoke-exposed vs control grape berries; 34 transcripts displayed significant expression trends in at both developmental stages (Supplementary File 2). The analysis of genes that were significantly differentially expressed at individual time points (pairwise DE analysis), as well as over time (time course DE analysis) in the smoke-exposed grape berries versus the control berries revealed notable upregulation of genes associated with UDP-glycosyltransferases (UGTs), with increasing expression over time in the smoke-exposed berries, particularly in the second half of the smoking time course (12–36 hours). UGTs attach sugar molecules to other molecules via a glycosylation step. In the context of smoke exposure, the molecules undergoing UGT conjugation are most likely VPs. The ability of plants to manage volatile organic compounds (VOCs), including VPs, extends beyond responses to exogenous environmental stressors like smoke. These compounds are crucial for plant defense and communication, and they also significantly influence the flavor and aroma profiles of plant-derived food and beverages. Plants have evolved mechanisms such as the storage of VOCs within cellular compartments and enhancing their water solubility through glycosylation, involving the activity of UGTs. UGTs act on volatile compounds, rendering them temporarily non-volatile and ensuring a balance that mitigates toxicity while mobilizing plant defenses. This glycosylation process also explains the phenomenon observed in wine production, where the fermentation process cleaves the glycosidic bonds of the VPs, altering the wine's aroma profile 18,25,26.
In addition to UGTs, a number of genes associated with glutathione S-transferases (GSTs) displayed increasing expression over time in the smoke-exposed grapes compared to control grapes. Like UGTs, GSTs play an important role in cellular detoxification processes by facilitating the conjugation of toxic molecules into less reactive forms. However, GSTs attach glutathione (GSH) instead of sugar to stress-inducing, xenobiotic, and/or phytotoxic compounds. This conjugation process renders the VPs more water-soluble, thereby reducing their toxicity and facilitating their sequestration into cellular compartments, such as vacuoles, for storage. The timing of the upregulation of UGT/GST genes indicates that the grapes’ response to smoke exposure involves not only immediate defense reactions but also a sustained effort to manage and detoxify smoke-derived compounds over time. By enhancing the conjugation and storage of VPs, grapevines mitigate the detrimental impacts of these compounds on the plant. This adaptive response highlights the crucial role of UGT and GST genes in the plant's metabolic adjustments to environmental stress, while also lending insight into a potential target for early detection of smoke taint. Specifically, the levels of UGT and GST expression in the grape berries during smoke exposure is likely directly related to the levels of toxic compounds (VPs and other smoke-derived xenobiotics) that are taken up and conjugated into non-toxic forms by the plant; this, in turn, is correlated with the amount of VPs and smoke-derived compounds that are released when the bonds of their conjugated forms during the fermentation process, leading to smoke taint. This is consistent with the findings of multiple metabolic studies investigating the content of smoke-taint-associated VPs and thiophenols, as well as their conjugated forms, before, during, and after the winemaking process 11,21,27.
In addition to genes associated with targeted inactivation of toxic compounds, we observed increased activity of several gene families with various other roles in plant stress responses. Notably, mannitol dehydrogenase (MDH) displayed increasing expression over time in the smoke-exposed vs control grape berries, which was strongly correlated with the expression pattern displayed by the GST/UGT genes. MDH, which converts mannitol to fructose, serves as a reactive oxygen species (ROS) scavenger, as well as a strong osmotic regulator during stress responses, directly engaging in the mitigation of oxidative damage through the scavenging of hydroxyl radicals28. Moreover, while it may seem counterintuitive to increase the activity of an enzyme that converts mannitol—given the role of mannitol as an antioxidant—the shift likely represents a nuanced strategy to utilize mannitol reserves more effectively in the context of reduced photosynthetically active radiation under smoke and broader stress response mechanisms, enabling the mobilization of mannitol reserves to fuel energy-demanding processes essential for repair, growth, and the synthesis of additional antioxidants and detoxification agents.
Several additional genes that were upregulated in response to smoke include those associated with vacuolar function and cell wall structural components. The expression of serine carboxypeptidase, a vacuolar marker, was also notably increased in the smoke-exposed grapes. This enzyme is commonly associated with the breakdown of proteins in the vacuoles and suggests an upregulation of vacuolar activity29. This activity may be related to the transport and sequestration of glycosylated VPs within the vacuoles, preventing their potential toxic effects within the cytosol and aiding in their eventual processing. Furthermore, the upregulation of genes encoding glycine-rich proteins, which are integral components of the cell wall and are associated with lignin-biosynthetic activity30, suggests a potential reinforcement of the cell wall structure. The activity of these proteins could indicate that lignin biosynthesis occurs as a reactive mechanism to smoke exposure. This biosynthesis could enhance the cell wall's barrier properties, mitigating the entry of additional volatile phenols and other xenobiotics into the cells. These responses seem to underline the grapevine's strategy to fortify its metabolic and structural defenses in response to environmental stressors. Overall, the observed upregulation of detoxification and structural-associated genes indicates a targeted response to smoke characterized by activation of multiple biochemical pathways to balance the immediate benefits of direct ROS scavenging and toxic compound inactivation and storage with the necessity to support the plant's overall health and recovery.
Gene Ontology Enrichment Analysis
GO enrichment analysis performed using the lists of DE genes at each time point identified 151 and 120 terms that were overrepresented in the pre-veraison and post-veraison smoke-exposed grapes, respectively (Supplementary File 3). Of these, 61 were enriched at both developmental stages during smoke exposure. The temporal dynamics of gene expression changes we observed in grapevines exposed to smoke reveal a dynamic response to smoke exposure over time that is characterized by initial activation of hormone signaling pathways, followed by metabolic adjustments for energy and osmotic regulation, and culminating in detoxification and structural reinforcement to mitigate damage and promote longer-term reactive compound mitigation strategies (Fig. 3). Initially, the enrichment of hormone-activated signaling pathways associated GOs suggests that, at the berry level, grape plants rapidly perceive and respond to smoke exposure. The early upregulation of "cytokinin-activated signaling" and "regulation of jasmonic acid mediated signaling" is particularly noteworthy. Cytokinins protect the plants from a novel type of stress induced by altered photoperiod, an addition to other abiotic stresses31. Similarly, Jasmonic acid is implicated in abiotic stress response including light stress32. These pathways are crucial for orchestrating plant stress-response mechanisms, and their early activation is likely instrumental to the activation of additional cellular processes for the detoxification and repair mechanisms, including those described in the remainder of this section.
At 4- and 6-hours post-exposure, the nature of the enriched GOs shifted towards those associated with energy metabolism and osmotic regulation, as well as with early detoxification efforts. The enrichment of numerous carbohydrate metabolism and response-associated ontologies ("response to carbohydrate," "pyruvate kinase activity," "glycolytic process," and "carbohydrate phosphorylation") suggests the grapevine's metabolic adjustment to meet the energetic demands of the smoke-induced stress response. This phase likely also involves an increase in carbohydrate-associated osmotic adjustments as a strategy for maintaining cellular homeostasis during smoke exposure. Complementary to these processes, the upregulation of ion binding-associated GO terms (“potassium ion binding” and “calcium ion binding”) could indicate increased vacuolar activity essential for the sequestration of conjugated volatile phenols and maintenance of ionic balance within the cell. Finally, alongside terms associated with carbohydrate metabolism, osmotic and ionic regulation, and vacuolar function, we observed an increase in "thiol oxidase activity", which indicates the onset of the detoxification response, with thiol oxidases playing a role in the conversion of thiols to their less reactive disulfide forms, potentially as a protective mechanism against smoke-associated oxidative damage33. These responses lay the foundations for the longer detoxification process that follows.
The period from 12–36 hours was characterized by enrichment of GOs associated with detoxification and mitigation of entry of toxins into the cell, including GOs associated with glutathione and glycosylation conjugation of compounds, as well as GOs associated with cell wall fortification. Considering the role of glutathione in detoxification of ROS and xenobiotics34, the enrichment of glutathione-related ontologies (“glutathione metabolic process”, “protein glutathionylation”, and “glutathione transferase activity”) lends further support to its utility in mitigating the oxidative stress in the context of smoke exposure. In parallel to enriched glutathione GOs, the enrichment of glycosylation processes, particularly through UGTs, emphasizes the plant's strategy to render volatile phenolic compounds less toxic and more amenable to sequestration. These processes not only reduce the immediate harmful effects of smoke-derived toxins but also aid in their eventual processing and elimination. Finally, in addition to pathways responsible for detoxification, we observed enrichment of the lignin biosynthetic pathway (“lignin biosynthetic process”) from 12–36 hours. Increased lignin biosynthesis is indicative of structural modifications at the cellular level. This response to smoke may serve multiple purposes, including reinforcing the cell wall against further xenobiotic penetration and adjusting the phenolic content of the fruit.
Overall, the enrichment of these pathways illustrates a dynamic defense mechanism of grapevines to smoke exposure. The integrated response ensures both the immediate mitigation of stress impacts and the long-term resilience of the grape berries as toxic compounds are rendered inert and stored in the vacuole.
Proposed Mode of Action for Detoxification of Smoke-Derived Compounds in Grapes
Based on the findings of the DE and GO enrichment analyses, we propose a hypothetical mode of action for the processing of smoke-derived compounds in grapes. Due to the relatively small size and lipophilic nature of VPs from smoke, these compounds are able to diffuse across the berry cuticle and membrane. Once smoke-derived compounds reach the cytosol, the grape berry initiates a series of coordinated responses to mitigate the potential damage caused by reactive VPs. This includes the activation of key enzymes, such as GSTs and UGTs, which begin the detoxification process. GST enzymes deactivate electrophilic phenols through the addition of a GSH group, forming GS-O conjugates that are more water-soluble and less reactive35. UGT enzymes, on the other hand, process VPs through glycosylation, attaching sugar molecules to render them less toxic26. The modified phenols, including thiophenols that may form due to the plant's further metabolism of the phenols 4, are then transported to the vacuole via active transporters. The vacuole serves as a storage compartment to avoid the toxicity of these compounds, effectively sequestering them away from critical cellular processes (Fig. 4).