Molecular study of drought response in the Mediterranean conifer Pinus pinaster Ait.: Differential transcriptomic profiling reveals constitutive water deficit-independent drought tolerance mechanisms.

Adaptation of long-living forest trees to respond to environmental changes is essential to secure their performance under adverse conditions. Water deficit is one of the most significant stress factors determining tree growth and survival. Maritime pine (Pinus pinaster Ait.), the main source of softwood in southwestern Europe, is subjected to recurrent drought periods which, according to climate change predictions for the years to come, will progressively increase in the Mediterranean region. The mechanisms regulating pine adaptive responses to environment are still largely unknown. The aim of this work was to go a step further in understanding the molecular mechanisms underlying maritime pine response to water stress and drought tolerance at the whole plant level. A global transcriptomic profiling of roots, stems, and needles was conducted to analyze the performance of siblings showing contrasted responses to water deficit from an ad hoc designed full-sib family. Although P. pinaster is considered a recalcitrant species for vegetative propagation in adult phase, the analysis was conducted using vegetatively propagated trees exposed to two treatments: well-watered and moderate water stress. The comparative analyses led us to identify organ-specific genes, constitutively expressed as well as differentially expressed when comparing control versus water stress conditions, in drought-sensitive and drought-tolerant genotypes. Different response strategies can point out, with tolerant individuals being pre-adapted for coping with drought by constitutively expressing stress-related genes that are detected only in latter stages on sensitive individuals subjected to drought.

Rising [CO2] effect on leaf drought-induced metabolome in Pinus pinaster Aiton: Ontogenetic- and genotypic-specific response exhibit different metabolic strategies.

Rising atmospheric CO2 concentrations ([CO2]) together with water deficit can influence ecological interactions of trees through an array of chemically driven changes in plant leaves. In four drought stressed Pinus pinaster genotypes, grown under two levels of atmospheric [CO2] (ambient (aCO2) and enriched (eCO2)) the metabolome of adult and juvenile needles was analyzed to know if the metabolic responses to this environmental situation could be genotype-dependent and vary according to the stage of needle ontogeny. Drought had the highest incidence, followed by needle ontogeny, being lower the eCO2 effect. The eCO2 reduced, eliminated or countered the 50 (adult needles) - 44% (juvenile) of the drought-induced changes, suggesting that CO2-enriched plants could perceived less oxidative stress under drought, and proving that together, these two abiotic factors triggered a metabolic response different from that under single factors. Genotype drought tolerance and ontogenetic stage determined the level of metabolite accumulation and the plasticity to eCO2 under drought, which was mainly reflected in antioxidant levels and tree chemical defense. At re-watering, previously water stressed plants showed both, reduced C and N metabolism, and a "drought memory effect", favoring antioxidants and osmolyte storage. This effect showed variations regarding genotype drought-tolerance, needle ontogeny and [CO2], with remarkable contribution of terpenoids. Chemical defense and drought tolerance were somehow linked, increasing chemical defense during recovery in the most drought-sensitive individuals. The better adaptation of trees to drought under eCO2, as well as their ability to recover better from water stress, are essential for the survival of forest trees.

Increased root investment can explain the higher survival of seedlings of 'mesic' Quercus suber than 'xeric' Quercus ilex in sandy soils during a summer drought.

In Mediterranean-type ecosystems, drought is considered the main ecological filter for seedling establishment. The evergreen oaks Quercus ilex L. and Quercus suber L. are two of the most abundant tree species in the Mediterranean Basin. Despite their shared evergreen leaf habit and ability to resist low soil water potentials, traditionally it has been suggested that Q. ilex is better suited to resist dry conditions than Q. suber. In this study, we examined how seedlings of Q. ilex and Q. suber grown in sandy soils responded to different levels of water availability using natural dry conditions and supplemental watering. Specifically, we estimated survival and water status of seedlings and explored the role of acorn mass and belowground biomass in seedling performance. To our surprise, Q. suber was better able to survive the summer drought in our experiment than Q. ilex. Nearly 55% of the Q. suber seedlings remained alive after a 2-month period without rain or supplemental water, which represents almost 20% higher survival than Q. ilex over the same period. At the end of the dry period, the surviving seedlings of Q. suber had strikingly higher water potential, potential maximum quantum yield of photosystem II (Fv/Fm) and stomatal conductance (gs) than those of Q. ilex. Acorn mass was associated with the probability of survival under dry conditions; however, it did not explain the differences in survival or water status between the species. In contrast, Q. suber had a higher root ratio and root:shoot ratio than Q. ilex and these traits were positively associated with predawn leaf water potential, Fv/Fm, gs and survival. Taken together, our results suggest that the higher relative investment in roots by Q. suber when growing in a sandy acidic substrate allowed this species to maintain better physiological status and overall condition than Q. ilex, increasing its probability of survival in dry conditions.

Inter-genotypic differences in drought tolerance of maritime pine are modified by elevated [CO2].

Background and Aims: Despite the importance of growth [CO 2 ] and water availability for tree growth and survival, little information is available on how the interplay of these two factors can shape intraspecific patterns of functional variation in tree species, particularly for conifers. The main objective of the study was to test whether the range of realized drought tolerance within the species can be affected by elevated [CO 2 ]. Methods: Intraspecific variability in leaf gas exchange, growth rate and other leaf functional traits were studied in clones of maritime pine. A factorial experiment including water availability, growth [CO 2 ] and four different genotypes was conducted in growth rooms. A 'water deficit' treatment was imposed by applying a cycle of progressive soil water depletion and recovery at two levels of growth [CO 2 ]: 'ambient [CO 2 ]' (aCO 2 400 µmol mol -1 ) and 'elevated [CO 2 ]' (eCO 2 800 µmol mol -1 ). Key Results: eCO2 had a neutral effect on the impact of drought on growth and leaf gas exchange of the most drought-sensitive genotypes while it aggravated the impact of drought on the most drought-tolerant genotypes at aCO2. Thus, eCO2 attenuated genotypic differences in drought tolerance as compared with those observed at aCO2. Genotypic variation at both levels of growth [CO2] was found in specific leaf area and leaf nitrogen content but not in other physiological leaf traits such as intrinsic water use efficiency and leaf osmotic potential. eCO2 increased Δ 13 C but had no significant effect on δ 18 O. This effect did not interact with the impact of drought, which increased δ 18 O and decreased Δ 13 C. Nevertheless, correlations between Δ 13 C and δ 18 O indicated the non-stomatal component of water use efficiency in this species can be particularly sensitive to drought. Conclusions: Evidence from this study suggests elevated [CO 2 ] can modify current ranges of drought tolerance within tree species.

Intra-population variability in the drought response of a beech (Fagus sylvatica L.) population in the southwest of Europe.

Phenotypic variability within forest species populations is considered of special relevance for local adaptation under new environments, albeit it has been analyzed to a lesser extent than inter-population phenotypic variability. A common garden study was carried out to assess phenotypic variability in response to water stress in half-sibling families from a marginal population of Fagus sylvatica L. at its south-western range edge distribution in Europe. Two irrigation regimes were applied, well-watered (WW) seedlings and those submitted to weekly cycles of drying-rewatering of growth media. Seedling growth and their leaf functional traits were recorded during the last cycle of water stress. Most of the phenotypic changes were explained by phenotypic plasticity in response to water stress, but there was also a significant effect of family in the expression of some of the studied traits. The relationship of carbon isotope fractioning with gas exchange traits across families under WW conditions did not follow the same pattern as the phenotypic trends. The leaf net photosynthesis across families was modified by the nitrogen content on a leaf mass basis that was in turn correlated positively with leaf nitrogen isotope fractionation. The results point to an important role of leaf nitrogen in determining the intrinsic water-use efficiency (WUE) across families. Variation in WUE was ruled mainly by control of stomatal conductance to water vapor under water stress, but by leaf net photosynthesis under wet conditions. Relatively high inter-family phenotypic variability in growth and functional traits were observed. Within-population phenotypic variability, and the plasticity of some of the studied traits, is of fundamental importance to cope with the harsher environments beech will have to endure in the future at different points in its distribution range.

Organ-specific metabolic responses to drought in Pinus pinaster Ait.

Drought is an important driver of plant survival, growth, and distribution. Water deficit affects different pathways of metabolism, depending on plant organ. While previous studies have mainly focused on the metabolic drought response of a single organ, analysis of metabolic differences between organs is essential to achieve an integrated understanding of the whole plant response. In this work, untargeted metabolic profiling was used to examine the response of roots, stems, adult and juvenile needles from Pinus pinaster Ait. full-sib individuals, subjected to a moderate and long lasting drought period. Cyclitols content showed a significant alteration, in response to drought in all organs examined, but other metabolites increased or decreased differentially depending on the analyzed organ. While a high number of flavonoids were only detected in aerial organs, an induction of the glutathione pathway was mainly detected in roots. This result may reflect different antioxidant mechanisms activated in aerial organs and roots. Metabolic changes were more remarkable in roots than in the other organs, highlighting its prominent role in the response to water stress. Significant changes in flavonoids and ascorbate metabolism were also observed between adult and juvenile needles, consistent with previously proven differential functional responses between the two developmental stages. Genetic polymorphisms in candidate genes coding for a Myb1 transcription factor and a malate dehydrogenase (EC 1.1.1.37) were associated with different concentration of phenylalanine, phenylpropanoids and malate, respectively. The results obtained will support further research on metabolites and genes potentially involved in functional mechanisms related to drought tolerance in trees.

Non-targeted metabolomic profile of Fagus sylvatica L. leaves using liquid chromatography with mass spectrometry and gas chromatography with mass spectrometry.

INTRODUCTION: Fagus sylvatica L. is one of the most widely distributed broad-leaved tree species in central and western Europe, important to the forest sector and an accurate biomarker of climate change. OBJECTIVE: To profile the beech leaf metabolome for future studies in order to investigate deeper into the characterisation of its metabolic response. METHODS: Leaf extracts were analysed using LC-MS by electrospray ionisation in negative mode from m/z 100-1700 and GC-MS by electron ionisation in scan mode from m/z 35-800. RESULTS: The LC-MS profile resulted in 56 compounds, of which 43 were identified and/or structurally characterised, including hydroxycinnamic acid derivatives, flavan-3-ols and proanthocyanidins, and flavonols. From a second analysis based on GC-MS, a total of 111 compounds were identified, including carbohydrates, polyalcohols, amino acids, organic acids, fatty acids, phenolic compounds, terpenoids, sterols and other related compounds. Many of the compounds identified were primary metabolites involved in major plant metabolic pathways, however, some secondary metabolites were also detected. Some of them play roles as tolerance-response osmoregulators and osmoprotectors in abiotic stress, or as anti-oxidants that reduce the effect of reactive oxygen species and promote many protective functions in plants. CONCLUSIONS: This study provides a broad and relevant insight into the metabolic status of F. sylvatica leaves, and serves as a base for future studies on physiological and molecular mechanisms involved in biotic or abiotic stress.

Variation in photosynthetic performance and hydraulic architecture across European beech (Fagus sylvatica L.) populations supports the case for local adaptation to water stress.

The aim of this study was to provide new insights into how intraspecific variability in the response of key functional traits to drought dictates the interplay between gas-exchange parameters and the hydraulic architecture of European beech (Fagus sylvatica L.). Considering the relationships between hydraulic and leaf functional traits, we tested whether local adaptation to water stress occurs in this species. To address these objectives, we conducted a glasshouse experiment in which 2-year-old saplings from six beech populations were subjected to different watering treatments. These populations encompassed central and marginal areas of the range, with variation in macro- and microclimatic water availability. The results highlight subtle but significant differences among populations in their functional response to drought. Interpopulation differences in hydraulic traits suggest that vulnerability to cavitation is higher in populations with higher sensitivity to drought. However, there was no clear relationship between variables related to hydraulic efficiency, such as xylem-specific hydraulic conductivity or stomatal conductance, and those that reflect resistance to xylem cavitation (i.e., Ψ(12), the water potential corresponding to a 12% loss of stem hydraulic conductivity). The results suggest that while a trade-off between photosynthetic capacity at the leaf level and hydraulic function of xylem could be established across populations, it functions independently of the compromise between safety and efficiency of the hydraulic system with regard to water use at the interpopulation level.

Epigenetic variability in the genetically uniform forest tree species Pinus pinea L.

There is an increasing interest in understanding the role of epigenetic variability in forest species and how it may contribute to their rapid adaptation to changing environments. In this study we have conducted a genome-wide analysis of cytosine methylation pattern in Pinus pinea, a species characterized by very low levels of genetic variation and a remarkable degree of phenotypic plasticity. DNA methylation profiles of different vegetatively propagated trees from representative natural Spanish populations of P. pinea were analyzed with the Methylation Sensitive Amplified Polymorphism (MSAP) technique. A high degree of cytosine methylation was detected (64.36% of all scored DNA fragments). Furthermore, high levels of epigenetic variation were observed among the studied individuals. This high epigenetic variation found in P. pinea contrasted with the lack of genetic variation based on Amplified Fragment Length Polymorphism (AFLP) data. In this manner, variable epigenetic markers clearly discriminate individuals and differentiates two well represented populations while the lack of genetic variation revealed with the AFLP markers fail to differentiate at both, individual or population levels. In addition, the use of different replicated trees allowed identifying common polymorphic methylation sensitive MSAP markers among replicates of a given propagated tree. This set of MSAPs allowed discrimination of the 70% of the analyzed trees.

Genetic control of functional traits related to photosynthesis and water use efficiency in Pinus pinaster Ait. drought response: integration of genome annotation, allele association and QTL detection for candidate gene identification.

BACKGROUND: Understanding molecular mechanisms that control photosynthesis and water use efficiency in response to drought is crucial for plant species from dry areas. This study aimed to identify QTL for these traits in a Mediterranean conifer and tested their stability under drought. RESULTS: High density linkage maps for Pinus pinaster were used in the detection of QTL for photosynthesis and water use efficiency at three water irrigation regimes. A total of 28 significant and 27 suggestive QTL were found. QTL detected for photochemical traits accounted for the higher percentage of phenotypic variance. Functional annotation of genes within the QTL suggested 58 candidate genes for the analyzed traits. Allele association analysis in selected candidate genes showed three SNPs located in a MYB transcription factor that were significantly associated with efficiency of energy capture by open PSII reaction centers and specific leaf area. CONCLUSIONS: The integration of QTL mapping of functional traits, genome annotation and allele association yielded several candidate genes involved with molecular control of photosynthesis and water use efficiency in response to drought in a conifer species. The results obtained highlight the importance of maintaining the integrity of the photochemical machinery in P. pinaster drought response.

Effects of drought on mesophyll conductance and photosynthetic limitations at different tree canopy layers.

In recent years, many studies have focused on the limiting role of mesophyll conductance (gm ) to photosynthesis (An ) under water stress, but no studies have examined the effect of drought on gm through the forest canopy. We investigated limitations to An on leaves at different heights in a mixed adult stand of sessile oak (Quercus petraea) and beech (Fagus sylvatica) trees during a moderately dry summer. Moderate drought decreased An of top and lowest beech canopy leaves much more than in leaves located in the mid canopy; whereas in oak, An of the lower canopy was decreased more than in sunlit leaves. The decrease of An was probably not due to leaf-level biochemistry given that VCmax was generally unaffected by drought. The reduction in An was instead associated with reduction in stomatal and mesophyll conductances. Drought-induced increases in stomatal limitations were largest in leaves from the top canopy, whereas drought-induced increases in mesophyll limitations were largest in leaves from the lowest canopy. Sensitivity analysis highlighted the need to decompose the canopy into different leaf layers and to incorporate the limitation imposed by gm when assessing the impact of drought on the gas exchange of tree canopies.

Annotated genetic linkage maps of Pinus pinaster Ait. from a Central Spain population using microsatellite and gene based markers.

BACKGROUND: Pinus pinaster Ait. is a major resin producing species in Spain. Genetic linkage mapping can facilitate marker-assisted selection (MAS) through the identification of Quantitative Trait Loci and selection of allelic variants of interest in breeding populations. In this study, we report annotated genetic linkage maps for two individuals (C14 and C15) belonging to a breeding program aiming to increase resin production. We use different types of DNA markers, including last-generation molecular markers. RESULTS: We obtained 13 and 14 linkage groups for C14 and C15 maps, respectively. A total of 211 and 215 markers were positioned on each map and estimated genome length was between 1,870 and 2,166 cM respectively, which represents near 65% of genome coverage. Comparative mapping with previously developed genetic linkage maps for P. pinaster based on about 60 common markers enabled aligning linkage groups to this reference map. The comparison of our annotated linkage maps and linkage maps reporting QTL information revealed 11 annotated SNPs in candidate genes that co-localized with previously reported QTLs for wood properties and water use efficiency. CONCLUSIONS: This study provides genetic linkage maps from a Spanish population that shows high levels of genetic divergence with French populations from which segregating progenies have been previously mapped. These genetic maps will be of interest to construct a reliable consensus linkage map for the species. The importance of developing functional genetic linkage maps is highlighted, especially when working with breeding populations for its future application in MAS for traits of interest.

Functional and genetic characterization of gas exchange and intrinsic water use efficiency in a full-sib family of Pinus pinaster Ait. in response to drought.

Drought is an important environmental factor in Mediterranean ecosystems affecting seedling recruitment, productivity or susceptibility to fires and pathogens. Studying water use efficiency in these environments is crucial due to its adaptive value allowing trees to cope with low water availability. We studied the phenotypic variability and genetic control of intrinsic water use efficiency (WUE(i)) and related traits in a full-sib family of Pinus pinaster under drought imposition. We detected significant differences in WUE(i) between clones of the same family and moderate heritability estimates that indicate some degree of genetic control over this trait. Stomatal conductance to water vapor was the trait most affected by drought imposition and it showed the strongest influence in WUE(i). Stomatal conductance to water vapor and specific leaf area (SLA) were the traits with highest heritabilities and they showed a significant genetic correlation with WUE(i), suggesting that selection of needles with low SLA values will improve WUE(i) in this species by reducing water losses through stomatal control.

Phenotypic plasticity and local adaptation in leaf ecophysiological traits of 13 contrasting cork oak populations under different water availabilities.

Plants distributed across a wide range of environmental conditions are submitted to differential selective pressures. Long-term selection can lead to the development of adaptations to the local environment, generating ecotypic differentiation. Additionally, plant species can cope with this environmental variability by phenotypic plasticity. In this study, we examine the importance of both processes in coping with environmental heterogeneity in the Mediterranean sclerophyllous cork oak Quercus suber. For this purpose, we measured growth and key functional traits at the leaf level in 9-year-old plants across 2 years of contrasting precipitation (2005 and 2006) in a common garden. Plants were grown from acorns originated from 13 populations spanning a wide range of climates along the distribution range of the species. The traits measured were: leaf size (LS), specific leaf area (SLA), carbon isotope discrimination (Delta(13)C) and leaf nitrogen content per unit mass (N(mass)). Inter-population differences in LS, SLA and Delta(13)C were found. These differences were associated with rainfall and temperature at the sites of origin, suggesting local adaptation in response to diverging climates. Additionally, SLA and LS exhibited positive responses to the increase in annual rainfall. Year effect explained 28% of the total phenotypic variance in LS and 2.7% in SLA. There was a significant genotype x environment interaction for shoot growth and a phenotypic correlation between the difference in shoot growth among years and the annual mean temperature at origin. This suggests that populations originating from warm sites can benefit more from wet conditions than populations from cool sites. Finally, we investigated the relationships between functional traits and aboveground growth by several regression models. Our results showed that plants with lower SLA presented larger aboveground growth in a dry year and plants with larger leaf sizes displayed larger growth rates in both years. Overall, the study supports the adaptive value of SLA and LS for cork oak under a Mediterranean climate and their potentially important role for dealing with varying temperature and rainfall regimes through both local adaptation and phenotypic plasticity.

Summer drought impedes beech seedling performance more in a sub-Mediterranean forest understory than in small gaps.

Refugia of mixed beech forest persist in the central mountains of the Iberian Peninsula at the south-western limit of European beech (Fagus sylvatica L.) distribution. The lack of beech regeneration is a concern in this region that has experienced reduced rainfall and higher temperatures over the past 30 years. Beech is considered especially susceptible to climate change because of its conservative shade-tolerant growth strategy; hence seedling responses to drought stress in gaps and in the understory are of particular interest. During the summer of 2007, a watering treatment raised the soil water content by up to 5% in gap and understory plots of beech seedlings in a mixed beech forest. Root-collar diameter was increased by our watering treatment in understory seedlings. Neither drought-avoidance through stomatal closure nor physiological drought-tolerance mechanisms were able to mitigate the effects of water stress in the understory seedlings, whereas osmotic adjustment enhanced the ability of the gap seedlings to tolerate water stress. Overall, high photosynthetic rates in the gaps, despite the photoinhibitory effects of high radiation, allowed gap seedlings to survive and grow better than the understory seedlings irrespective of water availability. Our results indicate that further intensification of summer drought, predicted for the Iberian Peninsula, will hinder the establishment of a beech seedling bank in the understory because of the conflicting seedling trait responses to simultaneously withstand water stress and to tolerate shade.

Is shade beneficial for mediterranean shrubs experiencing periods of extreme drought and late-winter frosts?

BACKGROUND AND AIMS: Plants are naturally exposed to multiple, frequently interactive stress factors, most of which are becoming more severe due to global change. Established plants have been reported to facilitate the establishment of juvenile plants, but net effects of plant-plant interactions are difficult to assess due to complex interactions among environmental factors. An investigation was carried out in order to determine how two dominant evergreen shrubs (Quercus ilex and Arctostaphylos uva-ursi) co-occurring in continental, Mediterranean habitats respond to multiple abiotic stresses and whether the shaded understorey conditions ameliorate the negative effects of drought and winter frosts on the physiology of leaves. METHODS: Microclimate and ecophysiology of sun and shade plants were studied at a continental plateau in central Spain during 2004-2005, with 2005 being one of the driest and hottest years on record; several late-winter frosts also occurred in 2005. KEY RESULTS: Daytime air temperature and vapour pressure deficit were lower in the shade than in the sun, but soil moisture was also lower in the shade during the spring and summer of 2005, and night-time temperatures were higher in the shade. Water potential, photochemical efficiency, light-saturated photosynthesis, stomatal conductance and leaf 13C composition differed between sun and shade individuals throughout the seasons, but differences were species specific. Shade was beneficial for leaf-level physiology in Q. ilex during winter, detrimental during spring for both species, and of little consequence in summer. CONCLUSIONS: The results suggest that beneficial effects of shade can be eclipsed by reduced soil moisture during dry years, which are expected to be more frequent in the most likely climate change scenarios for the Mediterranean region.

Leaf palmate venation and vascular redundancy confer tolerance of hydraulic disruption.

Leaf venation is a showcase of plant diversity, ranging from the grid-like network in grasses, to a wide variety of dendritic systems in other angiosperms. A principal function of the venation is to deliver water; however, a hydraulic significance has never been demonstrated for contrasting major venation architectures, including the most basic dichotomy, "pinnate" and "palmate" systems. We hypothesized that vascular redundancy confers tolerance of vein breakage such as would occur during mechanical or insect damage. We subjected leaves of woody angiosperms of contrasting venation architecture to severing treatments in vivo, and, after wounds healed, made detailed measurements of physiological performance relative to control leaves. When the midrib was severed near the leaf base, the pinnately veined leaves declined strongly in leaf hydraulic conductance, stomatal conductance, and photosynthetic rate, whereas palmately veined leaves were minimally affected. Across all of the species examined, a higher density of primary veins predicted tolerance of midrib damage. This benefit for palmate venation is consistent with its repeated evolution and its biogeographic and habitat distribution. All leaves tested showed complete tolerance of damage to second- and higher-order veins, demonstrating that the parallel flow paths provided by the redundant, reticulate minor vein network protect the leaf from the impact of hydraulic disruption. These findings point to a hydraulic explanation for the diversification of low-order vein architecture and the commonness of reticulate, hierarchical leaf venation. These structures suggest roles for both economic constraints and risk tolerance in shaping leaf morphology during 130 million years of flowering plant evolution.

Does growth irradiance affect temperature dependence and thermal acclimation of leaf respiration? Insights from a Mediterranean tree with long-lived leaves.

Understanding the response of leaf respiration (R) to changes in irradiance and temperature is a prerequisite for predicting the impacts of climate change on plant function and future atmospheric CO2 concentrations. Little is known, however, about the interactive effects of irradiance and temperature on leaf R. We investigated whether growth irradiance affects the temperature response of leaf R in darkness (Rdark) and in light (Rlight) in seedlings of a broad-leaved evergreen species, Quercus ilex. Two hypotheses concerning Rdark were tested: (1) the Q10 (i.e. the proportional increase in R per 10 degrees C rise in temperature) of leaf Rdark is lower in shaded plants than in high-light-grown plants, and (2) shade-grown plants exhibit a lower degree of thermal acclimation of Rdark than plants exposed to higher growth irradiance. We also assessed whether light inhibition of Rlight differs between leaves exposed to contrasting temperatures and growth irradiances, and whether the degree of thermal acclimation of Rlight is dependent on growth irradiance. We showed that while growth irradiance did impact on photosynthesis, it had no effect on the Q10 of leaf Rdark. Growth irradiance had little impact on thermal acclimation when fully expanded, pre-existing leaves were exposed to contrasting temperatures for several weeks. When Rlight was measured at a common irradiance, Rlight/Rdark ratios were higher in shaded plants due to homeostasis of Rlight between growth irradiance treatments and to the lower Rdark in shaded leaves. We also showed that Rlight does not acclimate to the same degree as Rdark, and that Rlight/Rdark decreases with increasing measuring and growth temperatures, irrespective of the growth irradiance. Collectively, we raised the possibility that predictive carbon cycle models can assume that growth irradiance and photosynthesis do not affect the temperature sensitivity of leaf Rdark of long-lived evergreen leaves, thus simplifying incorporation of leaf R into such models.

Functional traits and plasticity in response to light in seedlings of four Iberian forest tree species.

We investigated the differential roles of physiological and morphological features on seedling survivorship along an experimental irradiance gradient in four dominant species of cool temperate-Mediterranean forests (Quercus robur L., Quercus pyrenaica Willd., Pinus sylvestris L. and Pinus pinaster Ait.). The lowest photochemical efficiency (F(v)/F(m) in dark-adapted leaves) was reached in deep shade (1% of full sunlight) in all species except Q. robur, which had the lowest photochemical efficiency in both deep shade and 100% of full sunlight. Species differed significantly in their survival in 1% of full sunlight but exhibited similar survivorship in 6, 20 and 100% of full sunlight. Shade-tolerant oaks had lower leaf area ratios, shoot to root ratios, foliage allocation ratios and higher rates of allocation to structural biomass (stem plus thick roots) than shade-intolerant pines. Overall phenotypic plasticity for each species, estimated as the difference between the minimum and the maximum mean values of the ecophysiological variables studied at the various irradiances divided by the maximum mean value of those variables, was inversely correlated with shade tolerance. Observed morphology, allocation and plasticity conformed to a conservative resource-use strategy, although observed differences in specific leaf area, which was higher in shade-tolerant species, supported a carbon gain maximization strategy. Lack of a congruent suite of traits underlying shade tolerance in the studied species provides evidence of adaptation to multiple selective forces. Although the study was based on only four species, the importance of ecophysiological variables as determinants of interspecific differences in survival in limiting light was demonstrated.

Performance of seedlings of Mediterranean woody species under experimental gradients of irradiance and water availability: trade-offs and evidence for niche differentiation.

The aim of the study was to assess the potential importance for Mediterranean plants of trade-offs in the response to irradiance and water availability at the regeneration stage. Survival and growth patterns across an experimentally imposed irradiance gradient (1, 6, 20 and 100% sunlight) were studied in seedlings of eight Mediterranean woody species, together with the impact of a simulated summer drought. We found evidence of some of the trade-offs previously reported for non-Mediterranean plant communities, such as between survival in the shade and relative growth rate (RGR) at high light, but no evidence for others, such as between shade and drought tolerances. The impact of drought on survival and RGR was stronger in high light than in deep shade. The observed species-specific differences in performance provide a mechanistic basis for niche differentiation at the regeneration stage, contributing to possible explanations of species coexistence in Mediterranean ecosystems.