Mediterranean-type ecosystems are recognized as critical hotspots for both biodiversity and climate change. Within these environments, plants often interact with diverse species, including holoparasitic plants, while simultaneously facing increasing episodes of precipitation shortages and rising temperatures. Here, we investigated the impact of Orobanche latisquama Reut. ex Boiss infestation on the Mediterranean shrub Salvia rosmarinus (L.) Spenn (rosemary) across three populations along an altitudinal gradient, focusing on its effects on host tolerance and resilience to severe summer drought in its natural habitat. Results showed no major physiological impact of the parasite on the host during spring but revealed an enhanced photo- and antioxidant-protective response during the summer drought in rosemary plants infested with O. latisquama. Infested plants showed elevated contents of α-tocopherol and a shift in the ascorbate ratio towards its oxidized state during the summer, particularly in upper and sun-exposed leaves. This was accompanied by elevated malondialdehyde content, indicating enhanced lipid peroxidation. However, despite the heightened photo-oxidative stress observed in leaves from infested plants, no damage to photosystem II was observed, indicating a good tolerance of rosemary to the interaction between parasitism and drought. By autumn, all plants displayed similar recovery patterns, and the differences between infested and non-infested plants disappeared, thus indicating a high resilience to the combination of these biotic and abiotic stresses. Overall, these findings underscore the great adaptive mechanisms S. rosmarinus plants have evolved to endure severe summer drought, even when challenged by holoparasitic plant infestation, and provide new insights into plant-parasite interactions in Mediterranean-type ecosystems.

1 INTRODUCTION

Mediterranean-type ecosystems, characterized by hot, dry summers and mild, wet winters, are globally recognized as biodiversity hotspots but are also among the most vulnerable zones to climate change (Cowling et al., 1996; Giorgi & Lionello, 2008). Rising temperatures and declining precipitation have already been observed in the Mediterranean basin (Cramer et al., 2018; Cos et al., 2022), with models predicting ongoing warming and more frequent extreme weather events by the end of the 21st century (Giorgi & Lionello, 2008; Carvalho et al., 2022). Additionally, as the second largest and the third richest biodiversity hotspot globally (Mittermeier et al., 1998; Perret et al., 2023), Mediterranean ecosystems are defined by complex interactions, including those with parasitic plants, which modulate plant community structure and productivity (Hatcher et al., 2012; Mellado & Zamora, 2017). However, climate change is expected to accelerate biodiversity loss here, threatening conservation efforts (Newbold et al., 2020). Consequently, research increasingly focuses on the effects of climate change effects within this framework of species interactions and ecosystem dynamics (Marske et al., 2023; Hernández-Lambraño et al., 2024; Liu et al., 2024; Wang et al., 2024). Despite this, parasitic plant-host interactions remain largely unexplored.

Parasitic plants, representing approximately 1.6% of angiosperm species, have evolved a unique strategy for extracting water and nutrients from their hosts through the haustorium, a specialized structure that penetrates host roots or shoots (Press et al., 1990; Twyford, 2018; Nickrent, 2020). Among these, holoparasitic species lack photosynthetic capacity and rely entirely on their host for survival. In agroecosystems, parasitic plants are known for their harmful impacts on the host growth and crop yield (Yoder & Scholes, 2010; Rubiales & Fernández-Aparicio, 2012; Fernández-Aparicio et al., 2020). Conversely, in natural ecosystems, they play a significant role in shaping ecosystem structure by altering habitat resources and influencing the distribution of other organisms (Gibson & Watkinson, 1991; Press & Phoenix, 2005; Bardgett et al., 2006; Fisher et al., 2013; Mellado & Zamora, 2017; Casadesús & Munné-Bosch, 2021). Interestingly, some studies have even revealed positive, compensatory effects of parasitic plants on their hosts, such as enhanced photosynthesis and nutrient uptake (Irving & Cameron, 2009; Těšitel et al., 2021; Casadesús & Munné-Bosch, 2023). However, host responses to parasitism often depend on environmental conditions, and parasitism can, in turn, influence host tolerance to other abiotic stresses. For instance, mistletoe species have been shown to exacerbate forest mortality in conifer species under warming and drought conditions (Galiano et al., 2010; Bell et al., 2020). Therefore, understanding both the direct and indirect effects of parasitism on hosts is essential for managing biodiversity and predicting climate change impacts on ecosystems.

Under environmental stressors, plants accumulate reactive oxygen species (ROS) within chloroplasts, where they act as signaling molecules to regulate gene expression in response to stress conditions (Suzuki et al., 2012; Li & Kim, 2022). However, elevated and sustained contents of ROS can lead to photoinhibition and oxidative cell damage if antioxidant defenses are insufficient (Takahashi & Murata, 2008; Foyer, 2018). Plants in natural ecosystems face multiple stressors that exacerbate oxidative stress (Pascual et al., 2022; Rillig et al., 2023). For example, high sunlight combined with other stresses can lead to the generation of ROS through photooxidation, where singlet oxygen (¹O₂) is the first ROS produced in the electron transport chain of chloroplasts, requiring rapid detoxification to prevent irreversible oxidative damage (Foyer, 2018; D'Alessandro et al., 2020; Li & Kim, 2022). Chloroplast antioxidants, especially α-tocopherol, are critical for maintaining redox balance and protecting photosystem II (PSII) from photo-oxidative damage (Krieger-Liszkay, 2006; Mesa & Munné-Bosch, 2023a; Tasaka et al., 2023). α-Tocopherol acts both as a physical quencher and a chemical scavenger of ¹O₂ in proximity to PSII while also reacting with peroxyl radicals in thylakoid membranes to inhibit lipid peroxidation (Munné-Bosch, 2005; Krieger-Liszkay, 2006; Kumar et al., 2021). Furthermore, it can be regenerated by interacting with ascorbate, ensuring sustained photoprotection and membrane stability (Foyer et al., 2008; Szarka et al., 2012).

Salvia rosmarinus (L.) Spenn (rosemary) is an evergreen shrub native to the Mediterranean region, known for its high resistance to summer drought (Munné-Bosch et al., 1999; Nogués et al., 2015; Abbaszadeh et al., 2020). The holoparasitic plant Orobanche latisquama Reut. ex Boiss, a member of the Orobanchaceae family, is a widespread root parasite from the western Mediterranean basin, specifically infesting the roots of Salvia rosmarinus and Salvia jordanii. In this study, we investigated the impact of O. latisquama infestation on rosemary in its natural habitat, focusing on seasonal responses to parasitism and drought. By analyzing antioxidant responses across spring, summer, and autumn, this study aims to enhance understanding of combined parasitic and drought stress effects on rosemary under Mediterranean field conditions.

2 MATERIALS AND METHODS

2.1 Study site, plant material, and sampling

The research was carried out at three locations along the Catalan coast (NE Spain), encompassing an altitudinal gradient where both the host plant, Salvia rosmarinus (L.) Spenn, and the holoparasite Orobranche latisquama Reut. ex Boiss co-occurred (Figure 1B). The selected populations included a population in “Olivella” (Population 1, 41°18′59.9” N, 1°48′23.3″E) at an altitude of 211 m above sea level (m.a.s.l), a second population in “El Bruc” within the Montserrat mountain Natural Park (Population 2, 41°35′42.5” N, 1°46′10.0″E) at 527 m.a.s.l., and a third population in “Granera” (Population 3, 41°43′ 10.2” N, 2°03′08.5″E) at 782 m.a.s.l. Annual climate data for temperature and precipitation in Catalonia (Figure 1B) were obtained from the Meteorological Service of Catalonia (Meteocat), alongside monthly data from the nearest weather stations to the study areas for the year 2023 (Figure S1). Specifically, data were collected from the weather stations located in Sant Llorenç Savall (~4 km from Population 1), Sant Dimes in Montserrat (~6 km from Population 2), and Sant Pere de Ribes (~4 km from Population 3).

Details are in the caption following the image — **FIGURE 1**
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Climatic and geographical characterization of the study zone. (A) Map of the Mediterranean region highlighting the Mediterranean-type climate zone in red. A black box zooms on the specific study area of this investigation. (B) Annual anomalies in temperature (upper panel) and precipitation (lower panel) relative to the 1961–1990 reference period (recommended by the World Meteorological Organization for assessing long-term changes). In the upper panel, red shading indicates years with temperatures above the reference period, while blue shading indicates years with temperatures below it. In the lower panel, green shading represents years with precipitation levels above the reference period, while orange shading represents years with precipitation levels below it. (C) Map of Catalonia (NE Spain) showing the three study populations labeled as Population 1, Population 2, and Population 3, along with their respective elevations above sea level. Orange dots indicate the distribution of *O. latisquama* in the region. (D) Characterization of the three sampled populations during the sampling days (full sunny day, on midday) in spring, summer, and autumn, including temperature (T), relative humidity (HR), maximum photosynthetically active radiation (PAR), and soil water content (SWC). (E) Summary diagram of the conditions and factors investigated in this research (holoparasitic plant flowering, drought, and recovery). Temperature and solar radiation are represented by an orange line, and precipitation is represented by a purple line during spring, summer, and autumn typical of the Mediterranean climate. The studied variables are indicated for each season: *O. latisquama* flowering in spring, drought in summer, and recovery in autumn. (F) Photograph of a rosemary shrub infested with *O. latisquama* during spring 2023 at Population 3, and a schematic representation showing the distinction between upper leaves and lower leaves sampled in the study, with the PAR conditions considered for each. Maps were created with Geographic Information System (GIS). Reference system ETRS89 spindle 31 N EPSG: 25831.

Sampling of rosemary was conducted in spring (May), summer (August), and autumn (November) at all three locations. Ambient temperature (T), relative humidity (HR), and photosynthetically active radiation (PAR) were measured at each site on the sampling days using a portable thermohygrometer (DO9847 Multifunction Meter, Delta Ohm). Soil samples were collected from five distinct points within the sampling area at each population during all sampling periods to determine soil water availability by measuring soil water content (SWC). SWC was determined by collecting fresh soil samples on sampling days, weighing them to obtain soil fresh weight (SFW), drying them in an oven at 80°C to a constant weight, and then recording the soil dry weight (SDW). The soil water content (SWC) was calculated using the formula SWC = 100 × (SFW − SDW).

To identify infested rosemary shrubs (n = 12 per population), careful excavation around the holoparasitic plant was performed until the haustorial connection with the host root was found. The roots were then traced back to the host plant. For the selection of non-infested plants (n = 12 per population), the maximum root length of rosemary shrubs (approximately 200 cm in depth and 150 cm in lateral spread) was considered. Non-infested individuals were chosen at a minimum distance of 300 cm from infested plants, ensuring no signs of parasitism within the specified perimeter and that all selected plants were within the same microclimatic conditions as the population. These individuals were monitored for several weeks post-selection to confirm the absence of parasitic emergence. In all cases, plants of similar heights and perimeters were chosen to eliminate size and age effects. The plants used in the study were, on average, 120 cm high and 20 cm in diameter. Leaves were collected from the upper, sun-exposed portion of the shrub (where PAR exceeded 1000 μmol m⁻² s⁻¹) and the bottom part under the canopy (where PAR was below 200 μmol m⁻² s⁻¹). Only fully-expanded leaves from each part of the canopy were sampled at solar midday on sunny days. Fresh samples were taken to assess leaf water status, leaf mass per area (LMA), and the maximum efficiency of PSII (F_v/F_m). Immediately following collection, samples were frozen in liquid nitrogen and stored at −80°C until further biochemical analyses were conducted.

2.2 Leaf water status

To evaluate the LMA of the leaves, ten leaves from each sample were weighed to determine their fresh weight (FW) and then scanned to analyse their green area using ImageJ (Schneider et al., 2012). Leaves were then hydrated in distilled water at 4°C for 24 h to obtain the turgid weight (TW). Subsequently, leaves were dried in an oven at 80°C until constant weight to obtain the dry weight (DW). The leaf relative water content (RWC) was calculated using the formula RWC = 100 × (FW − DW) / (TW − DW).

2.3 Chlorophyll quantification and fluorescence

The maximum efficiency of PSII (F_v/F_m) was determined in 2-h dark-acclimated rosemary leaves, placing five leaves to cover the entire area of the fluorometer clip (MINI-PAM-II Photosynthesis Yield Analyzer, Walz).

Total chlorophyll content (Chl a + b) and the Chl a/b ratio were estimated spectrophotometrically following the methodology outlined by Lichtenthaler (1987). Briefly, 100 mg of frozen rosemary leaves were ground into a fine powder using liquid nitrogen and then extracted with 1.2 mL of cold methanol containing 0.01% butylated hydroxytoluene (BHT) to prevent oxidation. The extracts were ultrasonicated (Branson 2510 ultrasonic cleaner, Bransonic) and vortexed for 30 min. Following this, the samples were centrifuged (PrismR, Labnet International Inc.) at 4°C for 10 min at 15,980 × g. The supernatant was carefully collected, and the pellet was reextracted twice to ensure complete recovery of the pigments. The supernatants from all extractions were pooled and diluted 1:8 in cold, pure methanol. Pigment analysis was performed using UV/visible double-beam spectrophotometry with a CE Aquatius UCE7400 spectrophotometer (Cecil Instruments Ltd). Absorbances were read at wavelengths of 470, 653, 666, and 750 nm. The total chlorophyll content and the Chl a/b ratio were calculated using the equations established by Lichtenthaler (1987).

2.4 Lipid peroxidation determination

To estimate the extent of lipid peroxidation in rosemary leaves, both lipid hydroperoxides (LOOH) and malondialdehyde (MDA) contents were quantified using spectrophotometric methods. For LOOH quantification, the ferrous oxidation-xylenol orange (FOX) assay described by Bou et al. (2008) was used. In brief, 100 μL of methanolic extract was taken from the previous extraction protocol. This was diluted with an equal volume of cold methanol containing 0.01% BHT, and 50 μL of the diluted extract was incubated with 10 mM triphenylphosphine (TPP) for 30 min at room temperature (~20°C) in the dark. After, 10 μL of each mixture were then incubated in triplicate with FOX-2 reagent, which consists of a solution of 90% methanol (v/v) combined with 25 mM sulfuric acid (H₂SO₄), 4 mM BHT, 0.25 mM ammonium iron(II) sulfate ((NH₄)₂Fe(SO₄)₂), and 0.1 mM xylenol orange, in a 96-well plate for 45 min. Absorbances were measured at 560 and 800 nm using the same plate spectrophotometer. Calculations were made using absorbance differences from methanol-diluted and TPP-incubated samples, using the H₂O₂ calibration curve as a standard.

For MDA quantification, the thiobarbituric acid (TBA) reactive substances assay was utilized, following the methodology of Hodges et al. (1999). In summary, 50 mg of frozen rosemary leaves were ground into a fine powder using liquid nitrogen and extracted with 0.6 mL of 80% (v/v) ethanol containing 0.01% BHT. This extraction was performed through two rounds of sonication, initially for 30 min and subsequently for 15 min, with samples vortexed before and after each sonication. The samples were then centrifuged at 15,980 × g for 10 min at room temperature (~20°C). The supernatant was collected, and the extraction procedure was repeated twice more using the same protocol. Then, the −TBA and +TBA tubes were prepared with 20% trichloroacetic acid (w/v) plus 0.01% BHT (w/v) and with 20% trichloroacetic acid (w/v), 0.01% BHT (w/v), and 0.65% TBA (w/v), respectively. The pooled supernatants were incubated at 95°C for 25 min with the TBA+ and TBA− solutions. MDA content in the samples was analyzed by measuring absorbances at 440, 532, 600, and 750 nm using a plate spectrophotometer (xMark™ Microplate Absorbance Spectrophotometer, Bio-Rad), with quantification based on the equations provided by Hodges et al. (1999).

2.5 α-Tocopherol quantification

Quantification of tocopherols was performed by high-performance liquid chromatography (HPLC) according to the method described by Amaral et al. (2005). From the previous methanolic extracts detailed above (section of chlorophyll quantification), 250 μL were filtered using a PTFE filter with a pore size of 0.22 μm (Phenomenex) before injection into the HPLC system. The system comprised a Waters 600 controller pump, a Waters 717 plus auto-sampler, and a Jasco FP-1520 fluorescence detector. The mobile phase used for the separation of tocopherols consisted of n-hexane and 1,4-dioxane (95.5,4.5 v/v). Tocopherols were separated using an Inertsil 100 A column (5 μm, 30 × 250 mm, GL Sciences Inc.), with fluorescence emission detection as described by Amaral et al. (2005). Fluorescence detection was conducted at an excitation wavelength of 295 nm and an emission wavelength of 330 nm. The quantification of tocopherol compounds was achieved using a calibration curve generated from authentic standards (Extrasynthese).

2.6 Ascorbate and dehydroascorbate

The concentrations of ascorbic acid (AA) and its oxidized form, dehydroascorbate (DHA), in rosemary leaves were quantified using an enzymatic method adapted from Queval and Noctor (2007). Briefly, 50 mg of frozen rosemary leaves were ground to a fine powder in liquid nitrogen. The powdered samples were then extracted with cold 6% (w/v) meta-phosphoric acid and 0.2 mM diethylene triamine pentacetic acid (DTPA) as solvents. The samples underwent ultrasonic treatment for 30 min, followed by centrifugation at 15,980 × g for 10 min at 4°C to separate the soluble components from the particulate matter. The resulting pellet was re-extracted following the same protocol to maximize the recovery of the target compounds. The supernatants from both extraction steps were pooled to achieve a final volume of 300 μL. The levels of AA and DHA were determined spectrophotometrically using the xMark Microplate Spectrophotometer (Bio-Rad) with quartz microplates, measuring absorbance at 265 nm.

2.7 Stress-related phytohormones

The endogenous content of stress-related phytohormones, including abscisic acid (ABA), 12-oxophytodienoic acid (OPDA), jasmonic acid (JA), jasmonoyl-isoleucine (JA-Ile), and salicylic acid (SA), was determined using ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-ESI-MS/MS), as outlined by Müller and Munné-Bosch (2011). In short, 200 μL of methanolic extracts containing deuterium-labeled internal standards were filtered with a PTFE filter with a pore size of 0.22 μm (Phenomenex) before injection into the UHPLC-ESI-MS/MS system, which consisted of an HPLC coupled to a triple quadrupole mass spectrometer (QTRAP 4000, AB Sciex). Hormones were quantified in the negative ion mode, considering the recovery rates of each sample based on the deuterium-labeled internal standards. Calibration curves for each analyte were constructed using the MultiQuant™ 3.0.1 software.

2.8 Statistical analysis

To assess the effects of “infestation”, “season”, and their interaction, mean values were tested by two-way analyses of variance (ANOVA). Multiple comparisons were tested using Duncan's post-hoc test, with statistical significance accepted at p < 0.05. The normality of the data was evaluated using Levene's test, and the homoscedasticity of residuals was assessed using the Shapiro–Wilk test. In cases where normality or homoscedasticity was rejected, data underwent a logarithmic transformation, followed by re-evaluation with ANOVA. All statistical analyses were performed using RStudio software, while graphs were generated using SigmaPlot (System Software). Figure 1A was created using Geographic Information System (GIS) software.

3 RESULTS

3.1 Physiological impact of O. latisquama infestation and drought on rosemary

The holoparasitic plant O. latisquama is exclusively distributed in the southwestern region of Europe, particularly concentrated along the Mediterranean coast of the Iberian Peninsula, where it infests the native shrub S. rosmarinus. Given its distribution across different elevations relative to sea level, we selected rosemary populations from three locations with an altitudinal gradient (Figure 1C). The flowering of O. latisquama in the study year 2023 occurred during the spring, specifically from April to May, across all three study populations without any temporal differences between them. Infestation by O. latisquama did not exert negative physiological impacts on the rosemary plants located in the two lowest populations (Populations 1 and 2, see Figure 1C) during the parasite's flowering period in spring. However, infested rosemary plants located at the highest elevation, Population 3 (Figure 1C), exhibited slightly reduced leaf water content and a 30% reduction in total chlorophyll content during the parasite's flowering compared to the non-infested plants (Figure 2).

The climatic conditions of the three study sites demonstrated typical Mediterranean climate dynamics, with minor ecosystemic differences among them (Figures 1D and S1). Rainfall data and maximum temperature records from the three sites during the study year (Figure S1), as well as the temperature and annual precipitation anomaly indices for Catalonia (Figure 1B), indicated the exceptional drought conditions and temperature increase in the region during 2023. Additionally, a historical record was set for forests affected by drought in Catalonia in 2023, with 66,500 hectares impacted (Vayreda & Banqué, 2023). During this season, all plants from the three locations were clearly affected by the harsh drought, leading to reduced leaf RWC (Figure 2). Furthermore, the reduction in chlorophyll content and F_v/F_m in all plants, reaching F_v/F_m values of around 0.4 in Population 2, indicated photoinhibition caused by the summer climatic conditions (Figure 2). The chlorophyll a/b ratio was not affected (Figure S3). The integrity of PSII remained unaffected by holoparasitic infestation during summer in the two lowest populations, indicating no additional photosynthetic damage. However, rosemary plants from Population 3, which had been previously negatively affected by O. latisquama infestation during spring, showed both higher leaf RWC and F_v/F_m values (Figure 2, right panel) in all leaves during summer and a reduced LMA in the bottom leaves (Figure S2) compared to non-infested plants.

3.2 Oxidative stress and photoprotective response of rosemary to O. latisquama infestation and drought

Under drought conditions during summer, rosemary plants from all populations increased α-tocopherol content, regardless of whether they were infested by the parasitic plant. However, infested plants exhibited a further increase in α-tocopherol content per unit of chlorophyll during this season, particularly in the leaves exposed to excess light (upper leaves, see Figures 1F and 3A). In contrast, leaves from the shaded parts of the plant (bottom leaves, see Figure 1F) were less affected by parasitism (Figure 3B). At Population 1, infested plants showed a 39% increase in α-tocopherol per chlorophyll content in upper leaves compared to non-infested plants. This increase was even more pronounced at Populations 2 and 3, where it reached 60% and 46%, respectively, in the upper leaves of infested shrubs during the summer (Figure 3A). In the more shaded bottom leaves, only those from infested rosemary at Population 2 displayed an increased tocopherol content during this period (Figure 3B). Additionally, an elevated DHA/Asc_t ratio was observed in the upper leaves of all infested plants during summer (Figure 3A). At the highest elevation, Population 3, where infested rosemary plants showed a compensatory response in summer, both α-tocopherol/chlorophyll and DHA/Asc_t ratios increased earlier in the upper leaves, already in spring, and remained higher during summer (Figure 3A). Infested rosemary shrubs exhibited higher levels of lipid peroxidation than non-infested plants, as indicated by greater MDA content (Figure 4A), except for those located at the lowest elevation and closest to the sea (Population 1).

Stress-related hormones exhibited complex patterns in rosemary plants in response to O. latisquama infestation and drought. The content of SA generally increased, while ABA content decreased systemically under drought conditions (Figure S4). Infestation by O. latisquama led to distinct hormonal responses depending on the geographical location. At Population 2, infested shrubs showed lower ABA content in both upper and bottom leaves, but SA levels remained unchanged. In contrast, at Populations 1 and 3, infested plants displayed lower SA content, particularly in exposed upper leaves during summer (Figure S4A, B). At Population 3, the highest elevation, not only decreased SA content in upper leaves from infested plants was observed during the drought period but also reduced ABA levels.

Jasmonates were largely unaffected by the conditions studied, with some population-specific variations (Figures S4A and S5). At Population 3, infested upper leaves showed a 138% increase in JA content compared to non-infested plants, but this was only evident during the holoparasite's flowering period in spring (Figure S5A). Variations in the jasmonate precursor OPDA were also population-dependent. At Population 2, infested plants had 133% more OPDA in upper leaves during summer compared to non-infested plants, while at Population 1, infested plants showed a 47% reduction in OPDA during spring (Figure S5A). No significant effects of parasitism were observed in the bottom leaves at any population (Figure S5B).

3.3 Rosemary recovery after sequential pressure interaction

After a period of rainfall in autumn (Figure S1), SWC at all three sites returned to levels comparable to those observed in spring (Figure 1D). Following the oxidative stress and antioxidant response triggered by the combined effects of O. latisquama infestation and severe summer drought, all rosemary plants recovered to pre-drought physiological levels across populations. For instance, leaf RWC, chlorophyll content, and F_v/F_m values in both the upper and bottom leaves of infested and non-infested plants increased, reaching levels indicating optimal physiological status (Figure 2). Therefore, by autumn, no physiological differences in the leaves of rosemary plants due to the infestation were observed.

Generally, the α-tocopherol content and the DHA/Asc_t ratio observed during summer returned to baseline levels following the rainy season, mirroring spring values. Besides, the observed differences due to parasitism also disappeared. α-Tocopherol content and DHA/Asc_t ratio became similar between infested and non-infested plants, except at Population 3, where infested plants maintained higher α-tocopherol levels in the upper leaves. In addition, the bottom leaves of infested rosemary at Population 2 —the only case where less exposed leaves showed elevated lipophilic antioxidant levels— continued maintaining increased α-tocopherol content during recovery (Figure 3B).

4 DISCUSSION

Holoparasitic plants act as an additional sink for their host plants, drawing essential resources like carbon, water, and nutrients, often weakening the host's growth and productivity, especially during the parasite's flowering period (Moreau et al., 2016; Pincovici et al., 2018). However, the extent of these impacts depends on the host's resilience, the parasite's aggressiveness, and the prevailing environmental factors. In our study, S. rosmarinus plants infested by O. latisquama did not exhibit significant physiological alterations during the spring flowering period in two out of the three populations studied (Figure 2). However, geographic location-related variations were observed. In the population located at higher altitudes, infested shrubs displayed reduced RWC and chlorophyll content, which aligns with previous observations in hosts infested by Orobanche species (Mauromicale et al., 2008; Jokinen & Irving, 2019). The higher altitude likely intensified environmental pressures, rendering rosemary plants in this population more vulnerable to parasitism.

In this population, the physiological impacts during O. latisquama flowering were marked by increased levels of JA (Figures 4A and 5A) and activation of the antioxidant system associated with tocopherol, as evidenced by elevated α-tocopherol concentrations and an increased DHA/Asc_t ratio (Figure 3A). Since JA enhances antioxidant mechanisms, including the synthesis of vitamin E under various plant stress conditions via the induction of the enzyme tyrosine aminotransferase (TAT) (Sandorf & Holländer-Czytko, 2002; Sirhindi et al., 2015; Bali et al., 2018; Casadesús & Munné-Bosch, 2021), it was likely contributing to the antioxidant defense and photoprotection of infested rosemary plants in this population. Furthermore, infested shrubs here showed a compensatory mechanism, showing an improved physiological state during the post-summer period, as evidenced by higher RWC, greater F_v/F_m (Figure 2), and lower LMA (Figure 2). Similar to observations in Cistus albidus infested by the holoparasite Cytinus hypocistis (Casadesús & Munné-Bosch, 2023), this suggests a priming effect induced by infestation, potentially enhancing drought tolerance through cross-tolerance, as also described for aphid-infested plants (Foyer et al., 2016). Thus, we used the term “infestation” for this facultative ecological interaction between related angiosperms, which we believe is more appropriate here than “infection”.

The Mediterranean basin has experienced pronounced reductions in precipitation and a rise in temperature over recent decades, with rates exceeding the global average (Cramer et al., 2018; Cos et al., 2022; Feng et al., 2022; Zittis et al., 2022; Urdiales-Flores et al., 2023). These climatic shifts have intensified droughts, as evidenced by the severe drought in Catalonia (NE Spain) in 2023 (Toreti et al., 2022; Essa et al., 2023) (Figure 1B). Drought conditions severely impact Mediterranean vegetation, contributing to forest dieback and biodiversity loss (Senf et al., 2020; Feng et al., 2021), especially when combined with other stressors (Desprez-Loustau et al., 2006; Huang et al., 2020). In our study, the summer drought during 2023 led to decreased leaf water content and impaired photosynthetic machinery with reduced chlorophyll content and lowered F_v/F_m in S. rosmarinus shrubs (Figures 2 and 5). Despite its renowned drought resistance (Munné-Bosch et al., 1999; Müller et al., 2006), S. rosmarinus is susceptible to oxidative damage under severe drought conditions, particularly when its antioxidant defences are insufficient to counteract the resultant oxidative stress, leading to photosynthetic damage (Munné-Bosch & Alegre, 2000; Nogués & Baker, 2000; Sánchez-Blanco et al., 2004). Notably, our results revealed elevated α-tocopherol levels and increased DHA/Asc_t ratio, particularly in sun-exposed upper leaves (Figures 3A and 4). These results, together with the reduced contents of MDA observed during summer, supports the role of α-tocopherol and ascorbate in mitigating photo-oxidative stress and lipid peroxidation (Foyer et al., 2008). However, the antioxidant response of S. rosmarinus under the cumulative drought conditions of summer 2023 was probably insufficient to fully counteract the physiological impacts observed in the shrub.

An even more pronounced photo-oxidative stress was observed in infested plants during the summer, evidenced by elevated levels of α-tocopherol, DHA/Asc_t ratio, and MDA (Figures 3A, 4A, and 5). The combination of drought stress and biotic infection in plants can have both positive and negative effects, which often depend on the severity of these stresses (Ramegowda & Senthil-Kumar, 2015). For instance, Prasch & Sonnewald (2013) reported that severe drought combined with Turnip Mosaic Virus (TuMV) infection in Arabidopsis significantly reduced plant growth compared to either stress individually. In our study, the combined effects of infestation by the holoparasitic plant O. latisquama and the intense drought during the summer of 2023 exacerbated oxidative stress and consequently enhanced the antioxidant response in rosemary plants.

Leaf-level analysis across canopy layers provided insights into the stress response, which was accentuated in the uppermost leaves exposed to higher light conditions, with PAR levels reaching 2,000 μmol m⁻² s⁻¹ during summer. Despite this, PSII integrity was not compromised (Figure 2A), suggesting that rosemary shrubs infested by O. latisquama efficiently enhanced their α-tocopherol-mediated antioxidant response to withstand the photo-oxidative stress from combined parasitism and severe drought (Figure 5). This observed increase in α-tocopherol likely contributed to scavenging ROS and protecting PSII, while simultaneously preventing lipid peroxidation and safeguarding thylakoid membrane lipids. Ascorbate may regenerate tocopherol from tocopheroxyl radicals; thus, the observed increase in the oxidized-to-reduced ascorbate ratio might indicate a high regeneration rate of α-tocopherol (Figure 3C) (Foyer et al., 2008; Mesa & Munné-Bosch, 2023b). Additionally, all plants exhibited full physiological recovery by autumn, highlighting the remarkable resilience of rosemary to both parasitism and drought (Figure 2).

Stress-related phytohormones are low-molecular-weight compounds synthesized in small quantities, yet they play a crucial role in activating signaling pathways that induce physiological changes in plants. These hormones are integral to the plant's response to both abiotic and biotic stressors. The accumulation of ABA generally increases as drought progresses in most plants and remains elevated under these conditions (Zeevaart, 1980; Brodribb et al., 2014). However, during prolonged droughts, ABA may follow a divergent dynamic when plants are dehydrated to approximately −4 MPa, known as a “peaking-type” (p-type) ABA dynamic (Brodribb & McAdam, 2013; Mcadam & Brodribb, 2015; Hasan et al., 2021). In our study, summer ABA reductions in rosemary may reflect such dynamics under the severe drought conditions of 2023 (Figure S4). On the other hand, since SA is synthesized in response to the detection of phytopathogens at the infection site, triggering a systemic defense response in distal tissues to protect undamaged areas, we expected an increase in this hormone in the leaves of plants infested by O. latisquama. However, paradoxically, the endogenous levels of SA increased during the summer (Figure S4). This increase may have contributed to drought tolerance in rosemary plants, as seen in other studies (Okuma et al., 2014; Abbaszadeh et al., 2020). Notably, parasitism combined with drought led to reduced ABA and SA in photoinhibitory conditions (Figure S4), resembling drought-tolerant responses observed in virus-infected plants (González et al., 2021). However, further research is needed to better understand the hormonal regulation in infested plants under drought.

5 CONCLUSION

In conclusion, this study reveals an adaptive and resilient strategy of the Mediterranean shrub S. rosmarinus to the combined stress from infestation by O. latisquama and severe summer drought in its natural Mediterranean environment. Despite experiencing significant photoinhibitory conditions, rosemary plants demonstrated a high stress tolerance and full recovery of key physiological traits, including leaf relative water content, chlorophyll content, and PSII efficiency, by autumn. Our findings suggest that an enhanced α-tocopherol-mediated antioxidant response appears integral in protecting the photosynthetic machinery under these combined stressors. Additionally, the antioxidant response and physiological status of the host were modulated by microclimatic-related effects associated with geographical differences, suggesting that the infestation may even trigger a compensatory priming response that enhances the physiological status of the host under severe water stress in some climatic contexts. This study provides valuable insights into the effects of holoparasitic plants in natural Mediterranean-type ecosystems and their interactions with drought, which can ultimately help us better manage the biodiversity of these unique ecological communities.

AUTHOR CONTRIBUTIONS

L.J. and S.M.B. conceived the idea and designed the experiments. L.J. performed the experiments with the help of M.M.R. L.J. prepared the first draft of the manuscript with the help of S.M.B. All authors read and approved the final manuscript.

FUNDING INFORMATION

The research was funded by the Catalan Government through an ICREA Academia award and the 2021SGR00640 grant given to S.M.B.

AKNOWLEDGMENTS

We are very grateful to the Experimental Fields Service of the University of Barcelona for their equipment and technical assistance. We also thank Sabina Villadangos, Tania Mesa, Paula Muñoz, and Alba Arabia (all of them from the University of Barcelona) for their methodological assistance during sampling.

Open Research

DATA AVAILABILITY STATEMENT

The data that supports the findings of this study are available in the article and in the supplementary materials of this article.

Supporting Information

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Citing Literature

Volume176, Issue6

November/December 2024

e14652

Interactive effects of Orobanche latisquama parasitism and drought stress in Salvia rosmarinus plants growing under Mediterranean field conditions

Abstract

1 INTRODUCTION

2 MATERIALS AND METHODS