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Italy is among the European countries with the greatest plant diversity due to both a great environmental heterogeneity and a long history of man–environment interactions. Trait-based approaches to ecological studies have developed greatly over recent decades worldwide, although several issues concerning the relationships between plant functional traits and the environment still lack sufficient empirical evaluation. To draw insights on the association between plant functional traits and direct and indirect human and natural pressures on the environmental drivers, this article summarizes the existing knowledge on this topic by reviewing the results of studies performed in Italy adopting a functional trait approach on vascular plants, bryophytes and lichens. Although we recorded trait measurements for 1418 taxa, our review highlighted some major gaps in plant traits knowledge: Mediterranean ecosystems are poorly represented; traits related to belowground organs are still overlooked; traits measurements for bryophytes and lichens are lacking. Finally, intraspecific variation has been little studied at community level so far. We conclude by highlighting the need for approaches evaluating trait–environment relationship at large spatial and temporal scales and the need of a more effective contribution to online databases to tie more firmly Italian researchers to international scientific networks on plant traits.

Long-duration manned Space missions require the realization of artificial environments, similar to the ecosystems on Earth, called Bioregenerative Life Support Systems (BLSSs). In these self-sufficient closed habitats, higher plants would have a fundamental role in air regeneration and food production, reducing the demand for resource supply from Earth. In this study, we exposed germinated seeds of soybean to different doses of X-rays (0.3, 10 and 20 Gy) and light quality regimes (W-White, R-Red and RB-Red-Blue light), to assess possible radiation-induced stimulation in the production of useful nutritional compounds in seedlings. Irradiated and control seedlings, grown under different light-quality treatments were analyzed in terms of growth (measuring stem, root elongation, fresh and dry weight), photosynthetic pigment production (chlorophylls, carotenoids and anthocyanins), antioxidants (total polyphenols), sugar and protein content, localization of phenolic compounds in the various organs. Our results indicated the occurrence of a significant interaction between light quality and ionizing radiation; in particular RB-light induced in irradiated seedlings a more compact (P

Anthropogenic impacts affect plants also in several protected areas of the Mediterranean basin. These impacts can also produce changes in vegetation dynamics. In Southern Italy, some areas of the Vesuvius National Park are particularly exposed to the impact of seasonal tourism. The frequent and intense presence of tourists, both hiking and moving by means of vehicles, is responsible for the formation of dust clouds which are mainly deposited on the vegetation close to the trails (Fig.1). Dust deposition on leaf lamina may negatively affect gas exchanges, compromising the plant health status. In this study, we analysed the effects of dust deposition on the leaf surface in Robinia pseudoacacia L., an invasive tree, native to north America, widespread in many areas of the Vesuvius National Park. Samplings were conducted in different sub-areas either located a) along the side of the roads, where a high deposition of dust (HD) was reported, or b) at least five meters far from the roads/trails, where plants receive a lower amount of dust (LD). For each site, the amount of dust on leaves was quantified in different periods of the year (springsummer-autumn), which have a different touristic pressure. On both HD and LD leaves, the effects of the dust deposition on anatomical and physiological traits were evaluated by performing eco-physiological measurements (e.g. chlorophyll fluorescence emission, photosynthetic pigment extraction and leaf functional traits) and light microscopy analysis (e.g. thickness of spongy and palisade tissues, incidence of intercellular spaces, stomatal size and density) (Fig. 2). Our results showed dust-induced changes in some of the anatomical and physiological traits, suggesting that dust deposition acted as a natural screen to protect the leaf surface especially in HD plantsIn HD plants, photochemistry was not affected by the presence of dust, being comparable to LD plants thus showing no sign of photoinhibition. HD plants also presented a higher amount of photosynthetic pigments, which could be an adaptation to the shielding phenomenon by which the plant increment chlorophylls and carotenoids in order to capture more light for photosynthesis especially during the summer, when the photosynthetic tissues are exposed to the excess of sunlight. These responses can be considered additional adaptive traits when the leaf heliotropic movements are not sufficient to avoid photo-inhibition. The leaf morphological and physiological plasticity of R. pseudoacacia in response to dust deposition is another critical trait which allows this species to be particularly competitive, and to spread in natural areas of the Vesuvius National Park subjected to anthropogenic impact.

The effect of ionizing radiation on plants has been studied with several purposes. Ionizing radiation has been used to generate stable mutants in breeding programs, or as a substitute for conventional decontamination methods. However, the larget amount of data on the effects of ionizing radiation on plants was obtained from radioecology studies, as well as from Space exploration applications. Events as Chernobyl or Fukushima disasters created “natural experimental fields” where to study radiation effects in contaminated ecosystems. The direct effects of radionuclides on plants and indirect long-term effects due to the contamination of other components of the ecosystem have been widely explored. However, Space biology is currently one of the most active fields in which radiation effects on plants are studied (1). In the sight of long duration exploratory-class manned missions, like the one towards Mars or the deep space, the protection of organisms from cosmic radiation is considered one of the main challenges (2). In such missions, plant cultivation is mandatory, since plants not only have an important role in mitigating psychological stress due to isolation, but are also key organisms for regeneration in artificial ecosystem (e.g. Bioregenerative Life Support Systems). Plants regenerate oxigen through photosynthesis, allow water recovery through transpiration, participate in waste recycling, and produce fresh food (3). However, the Space is an extreme environment where ionizing radiation is known to influence deeply organisms’ growth at molecular, morpho-structural, physiological and biochemical levels, thus affecting the efficiency of plants as regenerators. Herein, we summarize the key results of many experiments performed on crop species (e.g. soybean, Glycine max (L.) Merr.; azuki bean, Vigna radiata (L.) R. Wilczek; dwarf bean, Phaseolus vulgaris L.; tomato, Solanum lycopersicum L.), where plants, during several phenological phases; have been exposed to ionizing radiation. Seeds and adult plants were exposed to different types of radiation (e.g. Low- and High-LET, Linear Energy Transfer), and at different doses. The combined effect of radiation and other environmental factors, such as light quality, has also been investigated. The consequence of irradiation was assessed through the analysis of morphological development, some molecular traits, anatomical features of leaves and fruits, photosynthesis, flowering phenology, fruit development, and nutritional traits. Depending on several factors, including species, cultivar, plant phenological stage and traits of the target tissue/organ, the exposure to ionizing radiation produces several effects, which range from detrimental outcomes at very high doses, harmful consequences at intermediate levels, and stimulatory effects at very low doses. A severe interaction with other factors has also been evidenced. The interaction between factors and the different radiosensitivity of plants should be taken into account in ecosystem recovery in case of radiation contamination, as well as in the development of artificial ecosystems in extreme environments like Space.

The possibility of growing plants in Space is everyday more realistic. The cultivation of edible plants in Space is fundamental to minimize the need for food supplying from Earth and so to extend the duration of manned missions. One of the main constraints for plant cultivation in Space is ionizing radiation, which affects several growth processes, sometimes inducing positive outcomes at low doses. In this study, we compared the capacity to cope with radiation in two possible candidate species for Space farming: soybean (Glycine max L. Merr. ‘PR91M10’) and the mung bean (Vigna radiata L.). Both species were irradiated at the stage of seedlings with three different doses of X-rays (0.3, 10 and 20 Gy) and cultivated in a growth chamber under controlled environmental conditions. We focused our attention principally to young plants’ response at leaf level, both in terms of structure and function. More specifically, we investigated eco-physiological parameters and leaf functional anatomical traits linked with the photosynthetic efficiency. We also analyzed the content of pigments, proteins and antioxidant compounds. Results showed that both plant species exhibit an intrinsic radio-resistance at the highest dose of X-rays. In addition, the exposure of plants to low-doses promoted the synthesis of chlorophylls, carotenoids and membrane phenolics. The overall results confirmed that the positive outcomes of radiation are specie-specifics and dependent on the phenological stage. This should be taken into account in the choice of the species to be grown on board and in the design of cultivation chambers.

Vitis vinifera L. is the most widely cultivated and economically important fruit crop in the world (1). Italy, one of the world’s leading wine-producing countries, shows vineyards distributed all over the mainland. In this scenario, Campania region, due to its peculiar climatic conditions and fertile soils, has always been considered one of the most appreciated districts for wine production (2). Additionally, the presence of several autochthonous grape varieties is a mainstay for this region. Italian legislation does not allow the irrigation of vineyards in order to preserve wine quality in most quality assurance labels. However, the ongoing climate changes are determining constraints in viticulture due to rising temperatures, changes in precipitation frequency and occurrence of extreme events, such as heat waves. Grapevine (Vitis vinifera L.) yield is expected to suffer from climate change also given that fruit development and ripening occur in the hottest and driest season (3) (4). In the last years, in the sight of climate change, the attention has been rise non wine production sustainability, particularly considering the possibility to adopt cultivation techniques aimed to enhance physiological adaptation to drought (5). In some volcanic areas of the Campania Region, grapevine cultivation is traditionally performed by avoiding the use of rootstocks thanks to the soil type that is inhospitable to Phylloxera pest. In this context, the aim of the study is to assess how the use of grafting on rootstocks could influence plant adaptation to drought and grape production. This study was conducted on Vitis vinifera L. ‘Piedirosso’ grapevine which is an autochthonous cultivar of the Campania Region. Plant growth, yield, photosynthetic performance and anatomy of water stem-leaf pathway were analysed in plants grown on their own-roots or grafted onto 420A rootstock (Vitis riparia x Vitis berlandieri). The selected vineyard Vigna Jossa was located within the farm Cantine degli Astroni, in the Campi Flegrei area at 200m above sea level (Naples, Southern Italy). Grafted and non-grafted plants were compared on the basis of several morphological and eco-physiological parameters including: plant architecture, leaf and wood stem anatomical traits, photosynthetic efficiency (by determination of quantum yield of PSII electron transport, maximal photochemical efficiency and pigment content), gas exchanges, etc. The overall results showed that the expected differences in growth performance and productivity in the two types of plants, were consequent to different eco-physiological and structural properties suggesting a different capability to modulate gas-exchanges and water flow efficiency/safety against embolism under different conditions of water availability. Hence, this study suggested the occurrence of higher plasticity of plant traits involved in the regulation of water flow in the grafted system. Such findings induce to better evaluate the opportunity to move from a traditional cultivation with own-rooted grapevines towards grafted models in the sight of increasing drought. As an alternative, the maintenance of the traditional own-rooted cultivation system should be accompanied by further cultivation trials aimed to optimize training system and plant management.

Plant cultivation in Space is a challenge for biology. In this context, preliminary studies with high energetic particles provide information about the capability of plants to withstand ionizing radiation in space. Non-lethal levels of high-LET ionizing radiation may stimulate physiological and morphological traits in plants, increasing their ability to be cultivated in extraterrestrial environments. In this study, we test the radioresistance of Dolichos melanophtalmus DC. (dwarf bean) irradiated at seed stage with low doses of heavy ions, namely C 1Gy, C 10 Gy, Ti 1 Gy and Ti 10 Gy. These doses, lethal for mammalian cells resulted not as dangerous for plants. After germination, alterations in growth were recorded by morphometric analysis; the photosynthetic efficiency was evaluated by chlorophyll a fluorescence emission measurements and determination of photosynthetic pigments’ content.The occurrence of oxidative stress and plant capability to withstand it were tested by measuring the intracellular H2O2 level, the ascorbic acid content and the poly(ADPribose) polymerase (PARP) activity. The irradiation of the seeds with different types of heavy ions and doses did not prevent either seed germination or the completion of plant life cycle; indeed irradiated plants were able to produce a new generation of seeds. Stimulatory effects on plant height were observed at Ti10 Gy, whereas C 10 Gy treatment determined a decrease in plant growth. The dose of C 10 Gy also reduced plant photochemistry and pigment content compared to other treatments, indicating the occurrence of damages at the photosystem level. However, in C 10 Gy treated plants the increase in intracellular H2O2 concentration, together with the rise in ascorbic acid and PARP activity, suggests the occurrence of a radio-induced oxidative stress mitigated by plant capability to activate the defense mechanisms successfully.

In the view of Space exploration, plants will have a key role in Bioregenerative Life Support Systems (BLSSs) to sustain human permanence in extraterrestrial environments. In Space, plant growth might be strictly inhibited by ionizing radiation of different quality and type. Within this framework, there is common agreement on the need to investigate plant response to ionizing radiation as one of the most limiting Space factors. This study aimed to test the effects of sub-lethal doses (1, 10 Gy) of C and Ti heavy ions, provided at dry-seed stage, on the development of dwarf bean plants (Dolichos melanophtalmus DC.) to assess whether low doses of heavy ions may induce stimulation of specific physiological traits rather than cause constraints in plant growth and photosynthesis. To reach this goal, the life cycle of plants from irradiated seeds was followed from seed-to-seed. Plant development was monitored by recording plant height, leaf area, number of leaves, flowers and pods. Photosynthesis was monitored by fluorescence measurements and photosynthetic pigment content amount. The occurrence of oxidative stress, as well as plant capability to counteract the radiation-induced changes, was assessed by the determination of endogenous hydrogen peroxide production, acid ascorbic amount and poly (ADP-ribose) polymerase (PARP) activity. Our results showed that all plants complete the life cycle, producing new seeds; however C ions at dose of 10 Gy induced constraints in plant growth, seed production and leaf lamina expansion, compared to control and other treatments. Photosynthetic activity was also reduced by 10 Gy C treatment. Both C and Ti ions at dose of 10 Gy determined a significant an increase in intracellular hydrogen peroxide, ascorbic acid amount and PARP activity. The overall results indicate that both C and Ti ions at the dose of 10 Gy stimulate defense mechanisms in dwarf bean plants, thus preventing harmful consequences that may compromise plant viability.

In Mediterranean ecosystems, the anthropogenic impact makes the preservation of plant biodiversity critical. Indeed, human pressure can be responsible for the depletion of vascular flora and the reduction in biomass production. Within the Campania region, some areas of the Vesuvius National Park are particularly exposed to the anthropogenic impact due to seasonal tourism. As a matter of fact, during spring and summer, a considerable amount of tourists pass through the Park areas, also with vehicles, thus causing the formation of dust clouds which are deposited over the vegetation close to the paths (Fig. 1). The main aim of this study is to analyse whether the deposition of dust on the leaf lamina of Centranthus ruber (L.) DC. subsp. ruber (Caprifoliaceae) plants can induce changes in leaf anatomy and in the efficiency of photosynthetic apparatus. The study was conducted on C. ruber plants growing at three sites, similar for environmental conditions and species composition. For each site two different sub-areas were considered: 1) the first, located along the side of the road, where a high deposition of dust (HD) was recorded on leaves, and 2) the second, placed at least five meters far from the path, where plants were subjected to a very low deposition of dust as control (LD). The amount of deposited dust was quantified at the study sites, in different periods of the year, characterized by different tourism access (spring-summer-autumn). Being C. ruber widespread throughout all the area of Vesuvius National park, it was chosen as an environmental indicator, making possible to evaluate the potential negative effects of the dust deposition on both anatomical and ecophysiological features. Functional traits in leaf lamina (e.g. stomatal size and frequency, percent of intercellular spaces in mesophyll) were quantified through light microscopy on fully-expanded leaves collected in summer (Fig. 2). Ecophysiological traits (chlorophyll a emission, photosynthetic pigment concentration and leaf traits) were carried out as well on fully expanded leaves, during spring and summer. Our results showed that HD leaves enhanced a pool of anatomical and photochemical traits allowing to overcome the effect of dust. Dust-induced changes in photosynthetic pigment content and stomata features were recorded. The overall analysis suggests that the greater amount of dust raised from the paths, shielded the leaf lamina: indeed leaves developing near the paths formed morphoanatomical traits as “shade leaves”. We infer that the phenomenon of dust deposition acts as a screen on leaves thus shading the photosynthetic apparatus from the light excess during summer. Although this phenomenon is positive for the optimization of C. ruber growth in these specific sites, the response of other species must be analysed to evaluate possible dust-induced vegetation dynamics consequent to possible species-specific adaptive capability.

Red leaf plants have aroused intriguing scientific debates over the last few years, not only to investigate their ecological role but especially to assess and improve their nutritional value as food plants. In most species, red colour in leaves is due to the accumulation of anthocyanins, the red pigment known for its antioxidant properties and therefore considered as an additional high nutritional value of red-leaf food plants. New research projects aimed at producing high quality food plants are supported by both Italian (ASI) and European Space Agencies (ESA) because plants represent a key element in bioregenerative life support systems, such as MELiSSA (Micro-Ecological Life Support System Alternative), not only for the water/air/waste recycling but also for the production of high quality plant food. Increasing attention is paid at such features in the selection of crop species aimed to become fresh food for astronauts in Space environment. Previous studies indicate that anthocyanins carry out several functions in plants, representing the nature’s Swiss army knife. A role in photoprotection is widely accepted, although this hypothesis is not always applicable and several exceptions are mostly explained ecologically by considering plant/herbivores interactions or various stress responses (1, 2). From a chemical and physical point of view, these watersoluble pigments, generally located within cellular vacuoles of epidermal and/or external mesophyll cells (Fig. 1), strongly absorb light in ultraviolet, blue and green wavelengths. The hypothesis was that highenergy wavelengths stimulate anthocyanins production to protect photosynthetic tissues, thus chloroplasts, from energizing wavelengths that commonly induce inhibition phenomena of the photosynthetic apparatus. Interestingly, green crop species grown under different light spectra are reported to modulate the synthesis of various compounds, including anthocyanins (3, 4). The general aim of our work was to apply the LED-based plant cultivation systems to maximise nutritional value of red-leaf plant food. More specifically, we grew green and red cultivars of Atriplex hortensis L. under different combination of light wavelengths (Fig. 2) to evaluate whether and how leaf anthocyanins content varies depending on light spectra. Our purpose was to understand plant’s response to different light wavelengths, especially in this valuable microgreens species intended for human diet. Our results showed that there is an increase in the foliar anthocyanins amount related to the amount of blue light within the incident light spectrum, especially in red leaf cultivar. Moreover, the combination of red and blue wavelengths stimulates biomass production and photochemical efficiency in both varieties of Atriplex hortensis L. These results provide useful informations to improve quality of plant food, without neglecting plant productivity, by using specific light spectra. Considering that anthocyanins content is significantly influenced by light quality, red leaf plants grown with adjustable lighting systems represent a promising biological system for high quality food production.

The effect of different light quality on photosynthetic performance, leaf anatomy and isoprenoid emission was studied in two different plant systems: a fast-growing crop, tomato (Solanum lycopersicum L.), and a fast-growing tree, oriental plane (Platanus orientalis L.). Both species were subjected to three different light quality regimes: RGB, RB and white light (WL), considered as control. Plants were analyzed for growth, leaf anatomical traits, photosynthesis and emission of key volatile compounds, in order to evaluate the capacity of the two plant models to regulate gas exchanges and optimize functional metabolites and biomass production under different radiation quality. In both species, the growth under RGB and RB light induced a more compact plant size, i.e. the reduction in plant height, biomass and leaf area. In tomato, the RGB and RB growth determined higher values of CO2 assimilation rate (A), stomatal (gs) and mesophyll (gm) conductance than WL; the trend was similar in oriental plane, but only gs significantly increased under RB. The anatomical leaf traits were strongly affected by light quality: in tomato, leaf lamina thickness was significantly reduced in RGB and RB leaves compared to WL ones; in oriental plane the opposite trend was observed. In both species, RB leaves showed higher stomata size than WL and RGB leaves. Light quality also influenced photosynthesis-dependent volatile isoprenoids. In tomato, β-phellandrene was lower under RB and RGB compared to WL. However, RGB and RB treatments stimulated α-pinene, carene and α-terpinene emissions. In oriental planes the isoprene emission was reduced under RGB and more so under RB. Briefly, photosynthesis, leaf anatomy, biomass production, and volatile isoprenoid emission are affected by light quality differently in the two plant model. In both cases, light quality control may have important applications to modulate plant productivity and biosynthesis of useful biochemical compounds.

In Space exploration, plants will play a crucial role, not only for resource regeneration, but also as food resource. Ionizing radiation represents a key constraint for plant growth and photosynthesis in space interacting with many developmental processes. At very low doses, ionizing radiation may induce some positive effects related on plant physiological and morphological traits. Among studies focused on the effect of ionizing radiation on plants, the challenge to improve conditions for plant growth in order to induce specific nutritional properties still remains. This study is aimed to analyse the effects of four doses of X rays (0, 0.3, 10 and 20 Gy) on soybean seedlings growing in a climatic chamber under three different light quality regimes: White (W), Red (R) and Red-Blue (RB) light. The aim of the study is to assess if specific light quality treatments, during plant growth may reduce and/or enhance the production, in irradiated plants, of metabolites useful in human diet. At this purpose, irradiated and control seedlings were analyzed in terms of growth (measuring stem, root elongation and fresh weight) and phytochemicals production (chlorophylls, carotenoids polyphenols and anthocyanins). Our results showed that R-light induces a significant stem elongation in control and X-ray irradiated seedlings compared to W and RB. At the highest dose of X-rays, 20 Gy, conversely to W and RB, the R seedlings maintain higher values of fresh biomass. The RB-light treatment induced higher content of chlorophylls and carotenoids than W and R treatments. The R and RB light treatments determined in irradiated seedlings a significant increase in polyphenols and a significant decrease in anthocyanins compared to W and not-irradiated samples. The overall results indicated that there is significant interaction between the two factors suggesting that it is possible to induce specific traits in irradiated plants by opportunely manipulating light quality during plant growth.

The possibility to cultivate plants in Space represents an ongoing challenge because in extraterrestrial environments, plant growth is controlled by factors, some of which are also acting on Earth (e.g. temperature, relative humidity, light) while others are peculiar such as altered gravity in addition to a profoundly different ionizing radiation (IR) field (De Micco et al., 2011). In particular, exposure to Space IR may determine several outcomes depending on the dose, radiation quality (high vs. low LET), exposure rate (acute vs. chronic), but also by the intrinsic characteristics of the organism, such as species, cultivars, developmental stage, structure of organs and tissues and genetic traits (De Micco et al., 2011). Generally, plant response to IR is manifestly dose-dependent with irreparable damage at high doses, sublethal consequences at intermediate levels and stimulatory effects at low dose (De Micco et al., 2011; Arena et al., 2014). In this study we explore the possibility that low doses of heavy ions, namely C ions at dose of 25 Gy delivered at seed stage, may exert beneficial effects on Solanum Lycopersicum L. cv ‘Micro-Tom’ on physiological, biochemical and anatomical traits, promoting the fruit ripening and quality. This would have important consequences in the view of its utilization on board of the Bioregenerative Life Support Systems (BLSSs) as food for crew. For this purpose, the plant life cycle from germination to fruit harvesting was monitored in terms of plant growth, photosynthetic efficiency, leaf anatomical traits and antioxidant production in leaf and fruits. The irradiation did not affect plant germination. Plants from irradiated seeds showed reduced height and a more compact size. The PSII quantum yield as well as the electron transport rate was promoted in irradiated compared to control plants. These data are consistent with a high level of D1 protein and photosynthetic pigment content in the leaves. As regards fruit, plants from irradiated seeds showed a significantly higher content of ascorbic acid, carotenoids and anthocyanins.

In the view of long-term manned missions for Space exploration, the knowledge of mechanisms of plant resistance to IR is an open challenge for biologists. The selection of the best plant species to be cultivated in closed ecosystems on board space platforms, should consider not only the plant role in regenerating resources (e.g. oxygen and air), but also their importance as source of edible biomass for astronaut diet. In order to reach efficient plant growth, it is important to modulate the lighting during cultivation, also considering its possible interaction with space factors, such as IR. In this study, we analyse the behaviour of mung bean (Vigna radiata L.) seedlings grown at different light quality regimes (W-White, R-Red and RB-Red-Blue light) after the exposure of germinated seeds at three doses of X-rays (0.3, 10 and 20 Gy) along with the non-irradiated control. Plant growth was monitored by measuring stem elongation, dry biomass, and total leaf area. Fluorescence a emission measurements, the amount of chlorophyll and carotenoids, as well as the Rubisco level, were analysed to evaluate the integrity of the photosynthetic machinery. More specifically, we evaluated the possibility that different light qualities would affect plant capability to withstand IR. Our results showed that the growth under R wavelengths promoted stem elongation compared to W and RB, independently from applied X-ray dose; the different doses did not cause changes in root length and root/shoot ratio. The RB treatment was responsible for the lowest production of chlorophylls and carotenoids at all doses of X-rays. Photochemistry was affected by neither different light regimes nor X-rays doses. R light induced a higher stability of protein expression level that was insensitive to the delivered dose of X-rays. Overall results indicate that the growth under R light helps seedlings to buffer the effects of different X-ray doses.

Erica arborea (L) is a widespread Mediterranean species, able to cope with water stress and colonize semiarid environments. The eco-physiological plasticity of this species was evaluated by studying plants growing at two sites with different soil moistures on the island of Elba (Italy), through dendrochronological, wood-anatomical analyses and stable isotopes measurements. Intra-annual density fluctuations (IADFs) were abundant in tree rings, and were identified as the key parameter to understand site-specific plant responses to water stress. Our findings showed that the formation of IADFs is mainly related to the high temperature, precipitation patterns and probably to soil water availability, which differs at the selected study sites. The recorded increase in the 13C-derived intrinsic water use efficiency at the IADFs level was linked to reduced water loss rather than to increasing C assimilation. The variation in vessel size and the different absolute values of δ18O among trees growing at the two study sites underlined possible differences in stomatal control of water loss and possible differences in sources of water uptake. This approach not only helped monitor seasonal environmental differences through tree-ring width, but also added valuable information on E.arborea responses to drought and their ecological implications for Mediterranean vegetation dynamics. © 2013 John Wiley & Sons Ltd.