Biological outliers: essential elements to understand the causes and consequences of reductions in maximum photochemical efficiency of PSII in plants.

MAIN CONCLUSION: By studying Cistus albidus shrubs in their natural habitat, we show that biological outliers can help us to understand the causes and consequences of maximum photochemical efficiency decreases in plants, thus reinforcing the importance of integrating these often-neglected data into scientific practice. Outliers are individuals with exceptional traits that are often excluded of data analysis. However, this may result in very important mistakes not accurately capturing the true trajectory of the population, thereby limiting our understanding of a given biological process. Here, we studied the role of biological outliers in understanding the causes and consequences of maximum photochemical efficiency decreases in plants, using the semi-deciduous shrub C. albidus growing in a Mediterranean-type ecosystem. We assessed interindividual variability in winter, spring and summer maximum PSII photochemical efficiency in a population of C. albidus growing under Mediterranean conditions. A strong correlation was observed between maximum PSII photochemical efficiency (Fv/Fm ratio) and leaf water desiccation. While decreases in maximum PSII photochemical efficiency did not result in any damage at the organ level during winter, reductions in the Fv/Fm ratio were associated to leaf mortality during summer. However, all plants could recover after rainfalls, thus maximum PSII photochemical efficiency decreases did not result in an increased mortality at the organism level, despite extreme water deficit and temperatures exceeding 40ºC during the summer. We conclude that, once methodological outliers are excluded, not only biological outliers must not be excluded from data analysis, but focusing on them is crucial to understand the causes and consequences of maximum PSII photochemical efficiency decreases in plants.

Parasitic plant-host interaction between the holoparasite Cytinus hypocistis and the shrub Cistus albidus in their natural Mediterranean habitat: local and systemic hormonal effects.

Mediterranean-type ecosystems provide a unique opportunity to study parasitic plant-host interactions, such as the relationship between the dominant shrub Cistus albidus L. and the root holoparasitic plant Cytinus hypocistis L. We examined this interaction (i) locally, by measuring the hormonal profiling of the interaction zone between the holoparasitic plant and the host, and (ii) systemically, by examining the hormonal profiling and physiological status of leaves from infested and uninfested plants. Furthermore, we explored how temporal variation (seasonal effects) and geographical location influenced the systemic hormonal and physiological response of leaves. Results shed light on tissue-related variations in hormones, suggesting the parasite exerted a sink effect, mainly influenced by cytokinins. Jasmonates triggered a defense response in leaves, far from the infestation point, and both jasmonates and abscisic acid (ABA) appeared to be involved in the tolerance to holoparasitism when plants were simultaneously challenged with summer drought. Parasitism did not have any major negative impact on the host, as indicated by physiological stress markers in leaves, thus indicating a high tolerance of the shrub C. albidus to the root holoparasitic plant C. hypocistis. Rather, parasitism seemed to exert a priming-like effect and some compensatory effects were observed (increased chlorophyll contents) in the host under mild climatic conditions. We conclude that (i) cytokinins, jasmonates and ABA play a role at the local and systemic levels in the response of C. albidus to the biotic stress caused by C. hypocistis, and that (ii) seasonal changes in environmental conditions and geographical location may impact holoparasitic plant-host interactions in the field, modulating the physiological response.

Substrate composition affects the development of water stress and subsequent recovery by inducing physiological changes in Cistus albidus plants.

Organic residues (compost) can be used as growth medium but may contain phytotoxic ions that, combined with a water deficit may alter the behavior of plants. The experiment was carried out in a growth chamber with Cistus albidus in a commercial substrate, C (sphagnum peat, coconut fiber and perlite, 8:7:1) and a mixture of compost substrates, Cp (slurry compost, coconut fiber and perlite, 3:6:1). Plants were grown in pots under well-watered, maintaining values of Ψl around -0.9 MPa (WW) and water-stressed (WS) conditions, where the irrigation was removed until reached values of Ψl around -3.0 MPa (water stress period), after then, water was re-established in all plants (recovery period). Although, the well-watered plants had a leaf water potential (Ψl) around -0.9 MPa, stomatal conductance (gs) was 125 mmol m-2s-1 in the commercial substrate and 30 mmol m-2s-1 in compost. The time taken to reach the threshold value at which water stress occurs was 13 days in the commercial substrate and 53 days in compost. Water-stressed plants in the commercial substrate had significantly lower values of Ψl and gs than well-watered. Plants in compost maintained values of gs similar in both irrigation treatments (WW and WS) and accumulated less biomass than those that grown in commercial. The water stress in compost led an increase in the adaxial epidermis, parenchyma and mesophyll, whereas water stress in commercial the proportions of the different tissues decreased. Higher lipid peroxidation values were found in plants grown in both substrates under water stress. The recovery time of the plants, until manage Ψl values around -0.9 MPa, depended on the type of substrate. The restoration of irrigation in commercial substrate act as a new stress, as reflected in the photochemical mechanisms.

Fatty Acid Content and Composition of the Morphological Fractions of Cistus Ladanifer L. and Its Seasonal Variation.

Cistus ladanifer L. is a shrub from Cistaceae family, widespread in Mediterranean countries. Fatty acids (FA) have multiple roles in plants and are involved in adaption mechanisms to environmental conditions. This work evaluated the FA content and composition of each morphological fraction of C. ladanifer (leaves, stems, flower buds, flowers and seed heads) throughout a full year. Cistus ladanifer plants were collected in southern Portugal, during four consecutive seasons (18 plants/season), and the different morphological plant fractions (leaves, stems, flower buds, flowers and seed heads) were separated. Cistus ladanifer morphological fractions showed distinct FA compositions, being possible to discriminate three groups-the leaves that showed to be dominated by saturated FA (main 20:0) and contain branched-chain FA (iso-19:0 and iso-21:0); the stems that are composed mainly by SFA (main 22:0); and the reproductive organs that showed higher contents of polyunsaturated FA (PUFA) and the 16:0 as the main SFA. The FA composition of leaves changed over seasons, with replacement of the PUFA by monounsaturated FA and branched-chain FA during hot seasons. Regarding the other C. ladanifer morphological fractions, the FA composition was more stable over seasons, suggesting that leaves are more prone to adaptations to environmental changes.

Physiological response of Cistus salviifolius L. to high arsenic concentrations.

Arsenic is a trace element found in the environment which can be particularly toxic to living organisms. However, some plant species such as those of the genus Cistus are able to grow in soils with high As concentrations and could be used in the sustainable rehabilitation of mining areas through phytostabilization. In this work, the growth and the physiological response of Cistus salviifolius L. to As-induced oxidative stress at several concentrations (reaching 30 mg L-1) in an hydroponic system were evaluated for 30 days. Several growth parameters, chlorophyll content, chemical composition, one indicator of oxidative stress (H2O2) and two of the major antioxidative metabolites (ascorbate and glutathione) were analysed. The toxic effect of As was better perceived in the plants submitted to treatments with concentrations of 20 and 30 mg As L-1. Plants subjected to these treatments had higher concentration of As in roots and shoots. The concentrations of Ca, Mg, K and Fe in the plants as well as a large part of the evaluated growth parameters were also affected. Arsenic did not interfere with the ability of the plant to perform photosynthesis, as there were no significant differences in the contents of chlorophyll a, b and total between the different treatments. Plants from all treatments accumulated higher amount of As in roots than in shoots, and it was also in the roots that the concentrations of H2O2, AsA and GSH were higher. Cistus salviifolius showed high tolerance to As up to the concentration of 5 mg L-1, which makes it a species with high potential to be used in the phytostabilization of soils contaminated with As and presenting high concentrations of the element in the soil solution.

Contrasting patterns of hormonal and photoprotective isoprenoids in response to stress in Cistus albidus during a Mediterranean winter.

MAIN CONCLUSION: Seasonal accumulation of hormonal and photoprotective isoprenoids, particularly α-tocopherol, carotenoids and abscisic acid, indicate their important role in protecting Cistus albidus plants from environmental stress during a Mediterranean winter. The high diurnal amounts of α-tocopherol and xanthophylls 3 h before maximum light intensity suggest a photoprotective response against the prevailing diurnal changes. The timing to modulate acclimatory/defense responses under changing environmental conditions is one of the most critical points for plant fitness and stress tolerance. Here, we report seasonal and diurnal changes in the contents of isoprenoids originated from the methylerythritol phosphate pathway, including chlorophylls, carotenoids, tocochromanols, and phytohormones (abscisic acid, cytokinins, and gibberellins) in C. albidus during a Mediterranean winter. Plants were subjected not only to typically low winter temperatures but also to drought, as shown by a mean plant water status of 54% during the experimental period. The maximum PSII efficiency, however, remained consistently high (Fv/Fm > 0.8), proving that C. albidus had efficient mechanisms to tolerate combined stress conditions during winter. While seasonal α-tocopherol contents remained high (200-300 µg/g DW) during the experimental period, carotenoid contents increased during winter attaining maximum levels in February (minimum air temperature ≤ 5 °C for 13 days). Following the initial transient increases of bioactive trans-zeatin (about fivefold) during winter, the increased abscisic acid contents proved its important role during abiotic stress tolerance. Diurnal amounts of α-tocopherol and xanthophylls, particularly lutein, zeaxanthin and neoxanthin including the de-epoxidation state, reached maximum levels as early as 2 h after dawn, when solar intensity was 68% lower than the maximum solar radiation at noon. It is concluded that (1) given their proven antioxidant properties, both α-tocopherol and carotenoids seem to play a crucial role protecting the photosynthetic apparatus under severe stress conditions; (2) high seasonal amounts of abscisic acid indicate its important role in abiotic stress tolerance within plant hormones, although under specific environmental conditions, accumulation of bioactive cytokinins appears to be involved to enhance stress tolerance; (3) the concerted diurnal adjustment of α-tocopherol and xanthophylls as early as 3 h before maximum light intensity suggests that plants anticipated the predictable diurnal changes in the environment to protect the photosynthetic apparatus.

Species-specific interference exerted by the shrub Cistus clusii Dunal in a semi-arid Mediterranean gypsum plant community.

BACKGROUND: The gypsovag shrub Cistus clusii is locally dominant in semi-arid gypsum plant communities of North-Eastern Spain. This species commonly grows in species-poor patches even though it has nurse potential, suggesting interference on neighbouring species. Other Cistus species exert a chemically mediated interference on plant communities, suggesting that it might be a common phenomenon in this genus. This study aimed investigating whether C. clusii exerts chemically mediated interference on neighbouring species in gypsum plant communities. We tested in a greenhouse whether aqueous extracts from C. clusii leaves (L), roots (R) and a mixture of both (RL) affected germination, seedling survival, and growth of nine native species of gypsum communities, including C. clusii itself. We further assessed in the field richness and abundance of plants under the canopy of C. clusii compared to Gypsophila struthium (shrub with a similar architecture having a nurse role) and in open patches. Finally, we specifically assessed in the field the influence of C. clusii on the presence of the species tested in the greenhouse experiment. RESULTS: Aqueous extracts from C. clusii (R and RL) negatively affected either germination or survival in four of nine species. In the field, richness and abundance of plants were lower under the canopy of C. clusii than under G. struthium, but higher than in open patches. Specifically, five of nine species were less frequent than expected under the canopy of C. clusii. CONCLUSIONS: Cistus clusii shows species-specific interference with neighbouring species in the community, which may be at least partially attributable to its phytotoxic activity. To our knowledge, this is the first report of species-specific interference by C. clusii.

Neglected Mediterranean plant species are valuable resources: the example of Cistus ladanifer.

MAIN CONCLUSION: The combination of genotypic selection, targeted and improved cultivation, and processing techniques for specific applications gives C. ladanifer the potential to be used as a valuable resource in Mediterranean areas with poor agronomic advantages. Cistus ladanifer (rockrose) is a perennial shrub, well adapted to the Mediterranean climate and possibly to upcoming environmental changes. As a sequence to a thorough review on taxonomic, morphological, chemical and competitive aspects of C. ladanifer, the research team focuses here on the economic potential of C. ladanifer: from production to applications, highlighting also known biological activities of extracts and their compounds. The use of this natural resource may be a viable solution for poor and contaminated soils with no need for large agricultural techniques, because this species is highly resistant to pests, diseases and extreme environmental factors. In addition, this species reveals interesting aptitudes that can be applied to food, pharmaceutical, phytochemical and biofuel industries. The final synthesis highlights research lines toward the exploitation of this neglected resource, such as selection of plant lines for specific applications and development of agronomic and processing techniques.

No genetic adaptation of the Mediterranean keystone shrub Cistus ladanifer in response to experimental fire and extreme drought.

In Mediterranean ecosystems, climate change is projected to increase fire danger and summer drought, thus reducing post-fire recruitment of obligate seeder species, and possibly affecting the population genetic structure. We performed a genome-wide genetic marker study, using AFLP markers, on individuals from one Central Spain population of the obligate post-fire seeder Cistus ladanifer L. that established after experimental fire and survived during four subsequent years under simulated drought implemented with a rainout shelter system. We explored the effects of the treatments on marker diversity, spatial genetic structure and presence of outlier loci suggestive of selection. We found no effect of fire or drought on any of the genetic diversity metrics. Analysis of Molecular Variance showed very low genetic differentiation among treatments. Neither fire nor drought altered the small-scale spatial genetic structure of the population. Only one locus was significantly associated with the fire treatment, but inconsistently across outlier detection methods. Neither fire nor drought are likely to affect the genetic makeup of emerging C. ladanifer, despite reduced recruitment caused by drought. The lack of genetic change suggests that reduced recruitment is a random, non-selective process with no genome-wide consequences on this keystone, drought- and fire tolerant Mediterranean species.

How do Mediterranean shrub species cope with shade? Ecophysiological response to different light intensities.

Under natural conditions, light exposure for Mediterranean shrubs can be highly variable, especially during cloudy days or under a canopy, and can interfere with other environmental factors such as temperature and water availability. With the aim of decoupling the effect of radiation and temperature from water availability, we conducted an experiment where two perennial and three summer semi-deciduous shrub species were subjected to different levels of irradiation. In order to follow plant responses to light exposure, we measured gas exchange, photosystem II photochemical efficiency, photosynthetic pigments and leaf mass area in spring and summer. Results showed that all study species presented a plastic response to different light conditions, and that light-related traits varied in a coordinated manner. Summer semi-deciduous species exhibited a more opportunistic response, with higher photosynthesis rates in full sun, but under shade conditions, the two strategies presented similar assimilation rates. Stomatal conductance did not show such a drastic response as photosynthetsis, being related to changes in WUE. Daily cycles of Fv /Fm revealed a slight photoinhibitory response during summer, mainly in perennial species. In all cases photosynthetic pigments adjusted to the radiation level; leaves had lower chlorophyll content, higher pool of xanthophylls and higher proportion of the de-epoxydaded state of xanthophylls under sun conditions. Lutein content increased in relation to the xanthophyll pool under shade conditions. Our results evidenced that radiation is an important driving factor controlling morphological and physiological status of Mediterranean shrub species, independently of water availability. Summer semi-deciduous species exhibit a set of traits with higher response variability, maximising their photosynthetic assimilation under different sun conditions.

Highlighting the differential role of leaf paraheliotropism in two Mediterranean Cistus species under drought stress and well-watered conditions.

The differential degree by which paraheliotropism may counterbalance the deleterious impact of high irradiance between congeneric species in relation to different water availabilities has been poorly investigated. We followed the evolution of gas exchange, quenching analysis and OJIP parameters in restrained (R) and free (F) to move leaves of Cistus monspeliensis (CM) and Cistus salvifolius (CS) under drought stress (WS) and well-watered conditions (WW). Concerning gas exchange parameters, leaf restriction effect was overall not significant in CM except in apparent carboxylation efficiency (Ce) under WS, while CS showed a significant sensitivity of maximum net photosynthetic rate (Amax), stomatal conductance (gs) and Ce even under WW. The recovery analysis highlighted also a faster gs recovery in F leaves. Furthermore, in both the species, restriction affected photon allocation pathways especially in terms of light-regulated and light-independent constitutive non-photochemical energy dissipation under WW, ultimately affecting electron transport rate (ETR). Nevertheless, the OJIP analysis provided us evidences that CM was characterized by a down-regulation of ETR while an impairment occurs in CS. In CM this was due to its ability to modify a certain fraction of reaction centers thus resulting in a higher capability for dissipation of excess light energy under well-watered conditions, not affecting electron transport efficiency. This response was not observed in CS. Overall, we demonstrated that congeneric species, even mostly sharing the same physiological targets, differ in the degree by which leaf movements help to counterbalance the negative effect of the high irradiance in relation with the amount of water available.

Differences in functional and xylem anatomical features allow Cistus species to co-occur and cope differently with drought in the Mediterranean region.

A significant increase in drought events frequency is predicted for the next decades induced by climate change, potentially affecting plant species mortality rates and distributions worldwide. The main trigger of plant mortality is xylem hydraulic failure due to embolism and induced by the low pressures at which water is transported through xylem. As the Mediterranean basin will be severely affected by climate change, the aim of this study was to provide novel information about drought resistance and tolerance of one of its most widely distributed and common genera as a case study: the genus Cistus. Different functional and anatomical traits were evaluated in four co-occurring Cistus species in the Mediterranean Montado ecosystem. Soil water availability for each species was also assessed to evaluate if they show different ecological niches within the area. Results showed physiological and xylem anatomical differences between the four co-occurring species, as well as in the soil water availability of the sites they occupy. Despite the significant differences in embolism resistance across species, no trade-off between hydraulic safety and efficiency was observed. Interestingly, species with narrower vessels showed lower resistance to embolism than those with higher proportions of large conduits. No correlation, however, was observed between resistance to embolism and wood density. The four species showed different water-use and drought-tolerance strategies, occupying different ecological niches that would make them cope differently with drought. These results will allow us to improve the predictions about the expected changes in vegetation dynamics in this area due to ongoing climate change.

Germination responses to current and future temperatures of four seeder shrubs across a latitudinal gradient in western Iberia.

PREMISE OF THE STUDY: Species differ in their temperature germination niche. Populations of a species may similarly differ across the distribution range of the species. Anticipating the impacts of climate variability and change requires understanding the differential sensitivity to germination temperature among and within species. Here we studied the germination responses of four hard-seeded Cistaceae seeders to a range of current and future temperatures. METHODS: Seeds were collected at sites across the Iberian Peninsula and exposed or not exposed to a heat shock to break dormancy, then set to germinate under four temperature regimes. Temperatures were varied daily and seasonally, simulating the temperature range across the gradient, plus an increased temperature simulating future climate. Time to germination onset and cumulative germination at the end of each season were analyzed for the effects of temperature treatments, seasons, and local climate (temperature of the germination period, Tgp) at each site. KEY RESULTS: Tgp was a significant covariate of germination in all species but Cistus populifolius. Temperature treatments significantly affected Cistus ladanifer, C. salviifolius, and Halimium ocymoides. Germination occurred in simulated autumn conditions, with little germination occurring at later seasons, except in unheated seeds of H. ocymoides. Exposure to a heat shock changed the sensitivity to temperature treatments and the relationships with Tgp. CONCLUSIONS: Germination responses to temperature differ not only among species but also within species across their latitudinal range. The responses were idiosyncratic and related to the local climate of the population. This germination variability complicates generalizing the impacts of climate variability and climate change.

Drought responses, phenotypic plasticity and survival of Mediterranean species in two different microclimatic sites.

Climate models predict a further drying of the Mediterranean summer. One way for plant species to persist during such climate changes is through acclimation. Here, we determine the extent to which trait plasticity in response to drought differs between species and between sites, and address the question whether there is a trade-off between drought survival and phenotypic plasticity. Throughout the summer we measured physiological traits (photosynthesis - Amax , stomatal conductance - gs , transpiration - E, leaf water potential - ψl) and structural traits (specific leaf area - SLA, leaf density - LD, leaf dry matter content - LDMC, leaf relative water content - LRWC) of leaves of eight woody species in two sites with slightly different microclimate (north- versus south-facing slopes) in southern Spain. Plant recovery and survival was estimated after the summer drought period. We found high trait variability between species. In most variables, phenotypic plasticity was lower in the drier site. Phenotypic plasticity of SLA and LDMC correlated negatively with drought survival, which suggests a trade-off between them. On the other hand, high phenotypic plasticity of SLA and LDMC was positively related to traits associated with rapid recovery and growth after the drought period. Although phenotypic plasticity is generally seen as favourable during stress conditions, here it seemed beneficial for favourable conditions. We propose that in environments with fluctuating drought periods there can be a trade-off between drought survival and growth during favourable conditions. When climate become drier, species with high drought survival but low phenotypic plasticity might be selected for.

Germination sensitivity to water stress in four shrubby species across the Mediterranean Basin.

Mediterranean shrublands are generally water-limited and fire-driven ecosystems. Seed-based post-fire regeneration may be affected by varying rainfall patterns, depending on species sensitivity to germinate under water stress. In our study, we considered the germination response to water stress in four species from several sites across the Mediterranean Basin. Seeds of species with a hard coat (Cistus monspeliensis, C. salviifolius, Cistaceae, Calicotome villosa, Fabaceae) or soft coat (Erica arborea, Ericaceae), which were exposed or not to a heat shock and smoke (fire cues), were made to germinate under water stress. Final germination percentage, germination speed and viability of seeds were recorded. Germination was modelled using hydrotime analysis and correlated to the water balance characteristics of seed provenance. Water stress was found to decrease final germination in the three hard-seeded species, as well as reduce germination speed. Moreover, an interaction between fire cues and water stress was found, whereby fire cues increased sensitivity to water stress. Seed viability after germination under water stress also declined in two hard-seeded species. Conversely, E. arborea showed little sensitivity to water stress, independent of fire cues. Germination responses varied among populations of all species, and hydrotime parameters were not correlated to site water balance, except in E. arborea when not exposed to fire cues. In conclusion, the species studied differed in germination sensitivity to water stress; furthermore, fire cues increased this sensitivity in the three hard-seeded species, but not in E. arborea. Moreover, populations within species consistently differed among themselves, but these differences could only be related to the provenance locality in E. arborea in seeds not exposed to fire cues.

Mycorrhization between Cistus ladanifer L. and Boletus edulis Bull is enhanced by the mycorrhiza helper bacteria Pseudomonas fluorescens Migula.

Boletus edulis Bull. is one of the most economically and gastronomically valuable fungi worldwide. Sporocarp production normally occurs when symbiotically associated with a number of tree species in stands over 40 years old, but it has also been reported in 3-year-old Cistus ladanifer L. shrubs. Efforts toward the domestication of B. edulis have thus focused on successfully generating C. ladanifer seedlings associated with B. edulis under controlled conditions. Microorganisms have an important role mediating mycorrhizal symbiosis, such as some bacteria species which enhance mycorrhiza formation (mycorrhiza helper bacteria). Thus, in this study, we explored the effect that mycorrhiza helper bacteria have on the efficiency and intensity of the ectomycorrhizal symbiosis between C. ladanifer and B. edulis. The aim of this work was to optimize an in vitro protocol for the mycorrhizal synthesis of B. edulis with C. ladanifer by testing the effects of fungal culture time and coinoculation with the helper bacteria Pseudomonas fluorescens Migula. The results confirmed successful mycorrhizal synthesis between C. ladanifer and B. edulis. Coinoculation of B. edulis with P. fluorescens doubled within-plant mycorrhization levels although it did not result in an increased number of seedlings colonized with B. edulis mycorrhizae. B. edulis mycelium culture time also increased mycorrhization levels but not the presence of mycorrhizae. These findings bring us closer to controlled B. edulis sporocarp production in plantations.

Cistus creticus subsp. eriocephalus as a Model for Studying Plant Physiological and Metabolic Responses to Environmental Stress Factors.

Variations in physiology and metabolic products of Cistus creticus subsp. eriocephalus along an altitudinal gradient (350-750 m.a.s.l.) within the Monti Lucretili Regional Natural Park (central Italy) were studied. The results showed that the phenol production was in relationship with the net photosynthetic rates and the chlorophyll content. In particular, the increasing caffeic acid (CA) content with altitude suggested its role in providing an additional photo-protection mechanism, by its ability to consume photochemical reducing power and acting as an alternative C-atom sink under high light conditions. The metabolic production was tested by high performance thin layer chromatography (HPTLC) fingerprint analysis, highlighting the potential of this technique in biologic studies.

Redox proteomics and physiological responses in Cistus albidus shrubs subjected to long-term summer drought followed by recovery.

MAIN CONCLUSION: The interaction between enzymatic and non-enzymatic antioxidants, endogenous levels of ABA and ABA-GE, the rapid recuperation of photosynthetic proteins under re-watering as well the high level of antioxidant proteins in previously drought-stressed plants under re-watering conditions, will contribute to drought resistance in plants subjected to a long-term drought stress under Mediterranean field conditions. This work provides an overview of the mechanisms of Cistus albidus acclimation to long-term summer drought followed by re-watering in Mediterranean field conditions. To better understand the molecular mechanisms of drought resistance in these plants, a proteomic study using 2-DE and MALDI-TOF/TOF MS/MS was performed on leaves from these shrubs. The analysis identified 57 differentially expressed proteins in water-stressed plants when contrasted to well watered. Water-stressed plants showed an increase, both qualitatively and quantitatively, in HSPs, and downregulation of photosynthesis and carbon metabolism enzymes. Under drought conditions, there was considerable upregulation of enzymes related to redox homeostasis, DHA reductase, Glyoxalase, SOD and isoflavone reductase. However, upregulation of catalase was not observed until after re-watering was carried out. Drought treatment caused an enhancement in antioxidant defense responses that can be modulated by ABA, and its catabolites, ABA-GE, as well as JA. Furthermore, quantification of protein carbonylation was shown to be a useful marker of the relationship between water and oxidative stress, and showed that there was only moderate oxidative stress in C. albidus plants subjected to water stress. After re-watering plants recovered although the levels of ABA-GE and antioxidant enzymes still remain higher than in well-watered plants. We expect that our results will provide new data on summer acclimation to drought stress in Mediterranean shrubs.

Zeatin modulates flower bud development and tocopherol levels in Cistus albidus (L.) plants as they age.

In a previous study we showed that Cistus albidus (L.) experiences an age-dependent decay in flower vigour correlated with a decline in trans-zeatin (tZ) levels. In the present study we aimed to establish a causal relationship between these two phenomena. Exogenous tZ applied to plants grown under semi-controlled conditions did not rescue flower vigour; however, it accelerated flower development, but only in younger individuals. Older plants showed lower tocopherol levels in flower buds, which were restored by exogenous tZ, suggesting that a loss of antioxidant defences may underlie the age-dependent decay in flower vigour. We conclude that declining tZ levels may not be directly responsible for the age-associated loss of floral vigour; that tZ modulates the speed of flower development as plants age; and that flower buds alter their sensitivity to tZ as plants age.