Natural forest ecosystems of the Mediterranean Basin are dominated by holm oak trees (Quercus ilex L). This species, along with cork oak (Q. suber L), is also a vital component of the “dehesas” or “montados” agroecosystem in the Iberian Peninsula. Holm oak is mainly used to provide acorns for animal feeding and for black truffle production but, lately, acorns nutritional value, its high contents in phytochemical compounds with biological activity (such as antioxidant, anticarcinogenic, and cardioprotective properties), have raised the interest in integrating these fruits into the human diet (Vinha et al., 2016). In recent years, however, populations of many oak species, and especially those of holm oak, have dramatically declined in Portugal, Italy and Spain, where tree mortality has significantly increased (Català et al., 2017).

Holm oak decline is a complex syndrome that is manifested by gradual crown de-foliation, loss of tree vigour and, finally, death of the trees (Vivas et al., 2021). The etiology of the disease is multiple and usually results from the interaction of several factors (Moreno-Fernández et al., 2019), such as aging and low natural regeneration of trees (Ruiz-Gómez et al., 2019), and the impact of extreme climate events promoted by climate change, such as increased temperatures and altered precipitation regimes (Gea-Izquierdo et al., 2021; Maldonado-Alconada et al., 2022; Sena et al., 2018), and the appearance of new diseases and outbreaks of soil pathogens, mainly Phytophthora spp (de Sampaio e Paiva Camilo-Alves et al., 2013; Morcillo et al., 2020). Studies on oaks infection by Phytophthora reported that although trees have been infected 50 years ago, water stress episodes can act synergistically at different spatial-temporal scales to produce tree death (Gea-Izquierdo et al., 2021). However, tolerance of holm oak against Phytophthora cinnamomi root rot is related to specific hydric and photosynthetic mechanisms that differ from those associated with drought (Ruiz Gómez et al., 2018).

The economic and social values of the biodiversity in this forest ecosystem (Ramírez-Hernández et al., 2014) have raised many efforts to study causes of the symptoms of the oak decline syndrome, and also to develop strategies that help to adopt good management practices (Martínez et al., 2022). Key requirements for successful prediction, control and management of the holm oak decline have been recently reviewed (Ruiz-Gómez et al., 2019). These include a thorough knowledge of the cellular and molecular basis of the pathogen’s biology and pathogenicity (Hardham and Blackman, 2018), as well as methods for its rapid and sensitive detection and identification (Sena et al., 2018), and multifaceted control procedures including the use of beneficial microorganisms such as Trichoderma spp (Corcobado et al., 2014; Ruiz-Gómez et al., 2019). Multiomics and molecular approaches to identify markers associated to Quercus species tolerance to abiotic and biotic stresses have been also reviewed (Escandón et al., 2021; Maldonado-Alconada et al., 2022; Triviño et al., 2025; Alcaide et al., 2025). The tools and knowledge derived from the molecular approach might help to select tolerant genotypes that, after clonal propagation, can be used in reforestation, conservation, and improvement programs, therefore contributing to mitigate the effect of climate change (Escandón et al., 2021).

Among the available biotechnological tools, somatic embryogenesis may be a valuable option regarding the propagation and conservation of selected holm oak genotypes (Martínez et al., 2021; 2022). Also, somatic embryogenesis protocols have been proved to be an excellent system to study the use of elicitors in this forest tree species (Morcillo et al., 2020; 2022; Ruiz-Galea et al., 2023). Pre-exposure of plants, seeds or somatic embryos, to an eliciting factor enables primed somatic embryos or plants to be more tolerant to later biotic or abiotic stress events. Priming induced an “stress memory” that exists in both the present generation and the offspring. Thus, priming is suggested to be a promising strategy for plants to cope with the biotic and abiotic stresses under global change scenarios (Liu et al., 2022). Although most studies of transgenerational induction of defences to pests and pathogens in plants have focused on short-lived annuals, recent evidences support the fact that transgenerational plasticity also occurs in long-lived forest trees (Morcillo et al., 2020). For instance, external application of methyl jasmonate (MeJA) increased the production of resin-based defences in mature P. pinaster trees for at least two years after its application (Vázquez-González et al., 2022), and Camisón et al. (2019) demonstrated that chestnut (Castanea sativa) seedlings of ink-diseased mother trees showed increased tolerance to the P. cinnamomi the causing agent of this disease. Interestingly, this tolerance was not mediated by seed size, but probably as a consequence of seed priming during fruit development. Similarly, transgenerational-induced resistance to P. cinnamomi has been recently reported in Q. ilex seedlings (Vivas et al., 2021). Recently it was reported that oomycete protection in chesnut seedlings, induced by a preventive MeJA spray, lasted one-year (Dorado et al., 2025).

The first evidence of transgenerational- induced resistance using somatic embryos in forest tree species was reported by Kvaalen and Johnsen (2008) demonstrating that temperature changes during somatic embryogenesis of Norway spruce affect phenological characteristic in their derived plants. More recently, our group demonstrated that applying high temperatures during somatic embryogenesis induction (Pérez-Oliver et al., 2021) or varying temperatures during maritime pine somatic embryo maturation (Sales et al., 2022) produced plants with better adaptation to heat stress. Furthermore, holm oak somatic embryos were used to study the potential of chemical elicitation treatments to induce defence responses to Phytophthora cinnamomi (Morcillo et al., 2020). Among several chemical elicitors, MeJA was proposed as an induced resistance (IR) stimulus (Morcillo et al., 2022) on the bases of the altered hormonal and phenolic profiles.

The regulatory mechanisms of plants in response to elicitation treatment and biotic stress can be studied by proteomic approaches (Lemaître-Guillier et al., 2017), that have great potential in plant biological research in general, and in the genus Quercus in particular, and that have been little exploited thus far. Several proteomic studies related to immune response of cork and holm oak to Phytophthora inoculation have been reported (Sghaier-Hammami et al., 2013; Coelho et al., 2021), while Escandón et al. (2021) reported that holm oak proteomic profile varies similarly after a biotic stress and after inoculation with the oomycete. Despite these recent findings, much is still unknown about the molecular mechanisms underpinning the establishment and maintenance of MeJA-IR in holm oak. Here we present and discuss changes in the proteomic profile after priming an embryogenic line with MeJA and further inoculation with P. cinnamomi.

Previously we reported that elicitation of holm oak embryogenic lines with 50 µM MeJA did not affect their further growth and development, while induced direct defence responses based on an increase in ABA and JA biosynthesis, and accumulation of phenolic compounds. In addition, after challenged against P. cinammomi, using a dual culture approach, elicited embryogenic material displayed a defence profile characterised by a further increase in JA, slightly inhibition of mycelia growth, and higher hydrogen peroxide production (Morcillo et al., 2020; 2022). To enlighten the mechanisms underlying this phenotype we studied the proteomic profile induced by MeJA before and after oomycete inoculation.

The proteomic response of holm oak (Quercus ilex) embryogenic lines to P. cinnamomi inoculation (INC) and/or methyl jasmonate (MeJA) elicitation revealed key insights into the defence-related metabolic reprogramming associated with biotic stress responses. Overall, P. cinnamomi inoculation led to a general down-regulation of protein abundance, consistent with previous studies in other plant species subjected to pathogenic stress (Sghaier-Hammami et al., 2021). This trend suggests suppression or redirection of core cellular metabolism during inoculation, possibly due to the pathogen’s manipulation of host physiological processes or the redirection to defence mechanisms like antioxidant compounds production. For example, our data pattern (Figure 2) is consistent with the expected effects of pathogen inoculation. Changes in metabolite levels often reflect either direct effects of inoculation or the host’s induced defence response. In general, decreases are interpreted as metabolic effects of pathogen activity or resource depletion, whereas increases are typically associated with host defence or priming responses (Bolton, 2009).

A key challenge in mitigating the effects of P. cinnamomi is the identification of molecular and physiological markers that differentiate resistant from susceptible genotypes. The constitutive expression of defence genes and the speed of defence activation are crucial indicators of resistance, as shown in Castanea crenata, which displays faster responses than the more susceptible C. sativa (Serrazina et al., 2015). Similarly, in avocado, early callose deposition at infection sites effectively limited P. cinnamomi penetration, underscoring the importance of early cellular defences in resistance (Coelho et al., 2021). Moreover, a recent multiomic approach (Triviño et al., 2025) reports on the identification of 29 candidate genes to be used as molecular markers to select holm oak genotypes with a better adaptation to drought and P. cinnamomi inoculation, both producing the species decline.

The metabolic profiles obtained indicate distinct and complementary effects of MeJA elicitation and P. cinnamomi inoculation. MeJA alone promoted an extensive metabolic reprogramming, reinforcing energy-producing pathways and JA-dependent defence mechanisms, consistent with the establishment of a primed defensive state (Bertini et al., 2019; Mageroy et al., 2020). In contrast, inoculation by P. cinnamomi caused a general metabolic downshift, possibly reflecting a pathogen-induced restriction of host metabolic activity and resource allocation as described in Cai et al. (2011). When both stimuli were combined, MeJA priming attenuated the inoculation-induced suppression by maintaining the activation of key defence-related routes, particularly phenylpropanoid and glutathione metabolism as described by Ji et al. (2021) and Liu et al. (2023). These findings suggest that MeJA preconditioning enables holm oak cells to prioritise antioxidant and phenolic defences during pathogen challenge, partially compensating for the broad repression of primary metabolism caused by inoculation.

In our experimental setting, MeJA elicitation promoted the accumulation of a higher number of proteins, particularly those related to phenylpropanoid biosynthesis (e.g., cinnamate-4-hydroxylase and caffeoyl-CoA O-methyltransferase), stress signalling (e.g., Stress-induced-phosphoprotein 1, Heat Shock Protein 90), pathogen related (e.g., thaumatin-like proteins, basic chitinase type I), and redox homeostasis (e.g., calreticulin, glutathione-related enzymes). These changes reflect the central role of jasmonate signalling in orchestrating induced defences and secondary metabolite pathways that contribute to pathogen resistance (Wasternack and Song, 2017). The accumulation of cinnamate-4-hydroxylase, that catalyses the second step of the phenylpropanoid pathway to generate p-coumaric acid, the precursor of numerous phenolic compounds (Khatri et al., 2023), might account for the higher content in phenolic acids previously reported for the MeJA-elicited holm oak embryogenic lines (Morcillo et al., 2022). In turn, S-adenosylmethionine synthase and caffeoyl-CoA O-methyltransferase are responsible for methylation of the hydroxy groups of flavonoids (Liu et al., 2023).

Interestingly, the MeJA+INC group did not synergistically amplify this protein accumulation and, in some cases, showed a diminished response compared to MeJA alone (e. g., chitinase and thaumatin-like protein). This suggests that while MeJA pre-conditioning primes the cells for defence, the pathogen may interfere with or override certain aspects of this priming upon inoculation. Notably, two PR identified proteins were over-accumulated in the MeJA+INC samples, the basic chitinase type I, protein that target and degrade chitin, which is the main structural part of cell walls of the pathogenic fungi (Durand et al., 2021), and thaumatin-like protein that confers enhanced resistance to a broad spectrum of phytopathogenic fungi (Hejgaard et al., 1991; Grenier et al., 1999) because of their ability to change the permeability of cell membranes and inhibit spore germination and mycelial growth of the target fungi (Jiao et al., 2018; Zhao et al., 2023). Despite the lack of chitin in oomycetes, there are some studies that characterised these genes in Phytophthora spp (Hinkel and Ospina-Giraldo, 2017). Note, however, that some isoforms of both proteins were under accumulated (Table 2). It is worth noting that in bottom-up proteomics, protein identification often relies on conserved peptide regions, which can match multiple isoforms or members of multigene families, even if they differ in expression or substrate specificity (Dupree et al., 2020; Miller et al., 2022). The observation that some defence protein isoforms were over−accumulated while others were under−accumulated following methyl jasmonate (MeJA) treatment can be explained by both biological regulation and technical factors. Many defence proteins, such as chitinases (PR-3 family) and thaumatin-like proteins (P-5 family), are encoded by large multigene families whose members are differentially regulated by hormonal signalling pathways; thus, MeJA induces specific chitinase isoforms involved in jasmonate-mediated defence responses whereas other PR proteins can be constitutively expressed or more responsive to salicylic acid (SA) signalling (Sabater-Jara et al., 2011; van Loon et al., 2006). The antagonistic crosstalk between JA and SA pathways is well documented in Arabidopsis, where SA strongly suppresses expression of many jasmonate-responsive genes, providing a mechanistic basis for why some defence isoforms may be repressed even under JA/MeJA treatment (Pieterse et al., 2012). From a technical perspective, bottom-up proteomics often infers protein identities based on shared peptide sequences among closely related paralogs, which can mask isoform-specific abundance changes and contribute to apparently contradictory results.

Regarding the response to inoculation of elicited plant material (MeJA+INC), we also observed that protein categories involved in hormone signalling, particularly jasmonate-associated regulators, were more abundant in MeJA+INC samples, indicating partial retention of elicitor-triggered defence signalling under pathogen challenge. This result can be associated to the elevated jasmonic acid content found in MEJA+INC lines in our previous work (Morcillo et al., 2022). Gene ontology and protein functional analyses further supported these findings, highlighting the enrichment of proteins involved in cell wall biogenesis, amino acid metabolism, and secondary metabolism detected in the MeJA treatment. Conversely, the infected group (INC) was characterised by a significant depletion of these functional categories, emphasising the pathogen’s impact on the host’s structural and metabolic defences. This is consistent with reports that P. cinnamomi targets host cell wall integrity and metabolism to facilitate penetration and colonisation (Coelho et al., 2021).

The estimation of protein fold changes across the 3,205 identified proteins confirmed a higher number of up-regulated proteins in MeJA-elicited lines than in those only infected, reinforcing the role of jasmonate in pre-activating protective pathways. These results align with findings in other woody species, where jasmonate signalling enhanced resistance through pre-emptive activation of defence genes and proteins (García-Pineda et al., 2010). From a broader perspective, our results highlight the potential of MeJA elicitation as a strategy to prime holm oak tissues against P. cinnamomi. Identifying proteins that show significant accumulation upon elicitation or depletion upon inoculation can contribute to the development of molecular markers for resistance screening. For example, consistent over-accumulation of specific secondary metabolism enzymes or redox regulators could serve as early indicators of a primed state. Specifically, the overlap between MeJA elicitation and pathogen inoculation (Figure 4) highlights a conserved defence signature, particularly the activation of phenylpropanoid metabolism, which is central to the synthesis of lignin, flavonoids, and other antimicrobial compounds known to fortify structural barriers and restrict pathogen spread (Dixon et al., 2002; Vogt, 2010).

The simultaneous upregulation of purine and thiamine metabolism, together with the glyoxylate cycle, suggests a reprogramming of primary metabolism to support defence-related demands, including nucleotide biosynthesis and energy balance under stress conditions (Bolton, 2009). Interestingly, the consistent induction of glycolysis under both conditions may reflect the need to provide carbon skeletons for secondary metabolite biosynthesis, such as ellagic acid precursors, which have been reported to contribute to antioxidant and antimicrobial defence (Landete, 2011). These shared responses reinforce the idea that elicitor-induced priming and pathogen-triggered immunity converge on core metabolic pathways that underpin effective resistance.

This study contributes to the ongoing search for physiological and molecular markers capable of distinguishing resistant from susceptible genotypes in holm oak (Quercus ilex), a keystone Mediterranean species of ecological and agronomic value. The proteomic adjustments observed under MeJA elicitation and Phytophthora cinnamomi inoculation underscore the crucial role of hormone signalling, redox regulation, and secondary metabolism in the plant’s defence repertoire. The over-accumulation of pathogenesis-related (PR) proteins, particularly basic chitinase type I and thaumatin-like proteins, reinforces their established antifungal functions through chitin degradation and membrane disruption, mechanisms that collectively inhibit fungal spore germination and mycelial growth (Durand et al., 2021; Zhao et al., 2023; Grenier et al., 1999; Li et al., 2023). Significant up-regulation of chitinase during P. cinnamomi root inoculation have been also reported in Q. suber (Ebadzad and Cravador, 2014) what suggests the importance of this enzyme in plant defence against the pathogen attack and constitutes probably, an effective response to lessen P. cinnamomi damage. These findings align with previous studies in other woody crops, where constitutive defence gene expression and rapid activation of defence responses were critical determinants of resistance, as shown in chestnut (Serrazina et al., 2015); and avocado (Coelho et al., 2021).

Beyond their physiological relevance, these insights carry practical implications for the sustainability of forest and nut crops. Holm oak is not only essential to Mediterranean ecosystems but also supports the acorn-based agri-food sector, where acorns are gaining renewed attention as a nutrient-rich, gluten-free flour alternative with potential for functional food applications. Enhancing resistance to P. cinnamomi in holm oak through molecular breeding or biotechnological approaches could therefore preserve genetic resources, safeguard acorn yields, and support the diversification of sustainable plant-based food sources. Altogether, the molecular signatures identified in this work provide valuable insight into the mechanisms of induced resistance in holm oak, and represent promising targets for selecting tolerant genotypes and guiding future breeding and biotechnological strategies that integrate both forest conservation and food innovation goals. In line with integrated pest management strategies, MeJA-induced defence reprogramming represents a preventive approach that can be implemented at early developmental stages in nurseries, complementing genetic and biotechnological interventions aimed at improving long-term resistance.