There is increasing evidence that the circadian clock is a significant driver of photosynthesis that becomes apparent when environmental cues are experimentally held constant. We studied whether the composition of photosynthetic pigments is under circadian regulation, and whether pigment oscillations lead to rhythmic changes in photochemical efficiency. To address these questions, we maintained canopies of bean and cotton, after an entrainment phase, under constant (light or darkness) conditions for 30-48 h. Photosynthesis and quantum yield peaked at subjective noon, and non-photochemical quenching peaked at night. These oscillations were not associated with parallel changes in carbohydrate content or xanthophyll cycle activity. We observed robust oscillations of Chl a/b during constant light in both species, and also under constant darkness in bean, peaking when it would have been night during the entrainment (subjective nights). These oscillations could be attributed to the synthesis and/or degradation of trimeric light-harvesting complex II (reflected by the rhythmic changes in Chl a/b), with the antenna size minimal at night and maximal around subjective noon. Considering together the oscillations of pigments and photochemistry, the observed pattern of changes is counterintuitive if we assume that the plant strategy is to avoid photodamage, but consistent with a strategy where non-stressed plants maximize photosynthesis.

Winter evergreens living in mountainous areas have to withstand a harsh combination of high light levels and low temperatures in wintertime. In response, evergreens can activate a photoprotective process that consists of the downregulation of photosynthetic efficiency, referred to as winter photoinhibition (WPI). WPI has been studied mainly in woody evergreens and crops even when, in many instances, other functional groups such as lichens or bryophytes dominate in alpine and boreal habitats. Thus, we aimed to (1) assess the occurrence of WPI within overwintering evergreens comprising woody species, herbs, mosses and lichens, (2) compare the recovery kinetics among those groups and (3) clarify the role of thylakoid proteins and pigments in both processes: WPI and recovery. With this aim, WPI was analyzed in 50 species in the field and recovery kineticcs were studied in one model species from each functional group. Results showed that high levels of WPI are much more frequent among woody plants than in any other group, but are also present in some herbs, lichens and mosses. Winter conditions almost always led to the de‐epoxidation of the xanthophyll cycle. Nevertheless, changes in the de‐epoxidation level were not associated with the activation/deactivation of WPI in the field and did not match changes in photochemical efficiency during recovery treatments. Seasonal changes in thylakoid proteins [mainly D1 (photosystem II core complex protein) and PsbS (essential protein for thermal dissipation)] were dependent on the functional group. The results highlight the diversity of physiological solutions and suggest a physical–mechanical reason for the more conservative strategy of woody species compared with other groups.

Poplar species are economically important sources of timber and bioenergy and they also have a valuable application in phytoremediation and in forest restoration programmes. The characterization of their responses in the face of environmental constraints is essential to ascertain their adaptation capacity. Quantification of chloroplast pigments and antioxidant compounds, nutrients and carbon isotope discrimination (Δ13C) of a large set of samples in ecophysiological studies is important for determining the plant’s physiological responses. Δ13C and nitrogen are parameters of special interest for providing information on photosynthesis, water relations and nutritional characteristics in forest stands. Photoprotective and antioxidant compounds participate in the physiological defence of plants subjected to stressful environmental conditions. The measurement of these leaf components by traditional techniques is laborious and expensive. To evaluate them, a calibration model was obtained using Near-Infrared Reflectance Spectroscopy (NIRS) and chemical analyses of Populus spp. leaves sampled from different clones in different years under different physiological conditions. Predictive calibration equations for the concentration of chloroplast pigments (Chlorophylls a and b, lutein, neoxanthin, β-carotene (β-Car), VAZ (violaxanthin + antheraxanthin + zeaxanthin), epoxidation index (EI = (0.5·antheraxanthin + violaxanthin)/VAZ)), antioxidants (total phenolics (TPhe), ascorbate and α-tocopherol), Δ13C and N content were established using a partial least squares regression algorithm. Results showed a remarkable accuracy of the calibration equations for quantifying several antioxidative and photoprotective defence compounds such as neoxanthin (R2 = 0.844), β-Car (R2 = 0.827), VAZ (R2 = 0.846), EI (R2 = 0.831) and excellent accuracy for TPhe (R2 = 0.957), N (R2 = 0.963) and Δ13C (R2 = 0.922). As shown, results can be obtained on multiple physiological attributes with a single measurement, saving time and money and demonstrating the suitability of NIRS for ecophysiological purposes and bioremediation programs.

Despite being a small geographic extension, Mediterranean Basin is characterized by an exceptional plant biodiversity. Adaptive responses of this biocoenosis are delineated by an unusual temporal dissociation along the year between optimal temperature for growth and water availability. This fact generates the combination of two environmental stress factors: a period of summer drought, variable in length and intensity, and the occurrence of mild to cold winters. Both abiotic factors, trigger the generation of (photo) oxidative stress and plants orchestrate an arsenal of structural, physiological, biochemical, and molecular mechanisms to withstand such environmental injuries. In the last two decades an important effort has been made to characterize the adaptive morphological and ecophysiological traits behind plant survival strategies with an eye to predict how they will respond to future climatic changes. In the present work, we have compiled data from 89 studies following a meta-analytical approach with the aim of assessing the composition and plasticity of photosynthetic pigments and low-molecular-weight antioxidants (tocopherols, glutathione, and ascorbic acid) of wild Mediterranean plant species. The influence of internal plant and leaf factors on such composition together with the stress responsiveness, were also analyzed. This approach enabled to obtain data from 73 species of the Mediterranean flora, with the genus Quercus being the most frequently studied. Main highlights of present analysis are: (i) sort of photoprotective mechanisms do not differ between Mediterranean plants and other floras but they show higher plasticity indexes; (ii) a-tocopherol among the antioxidants and violaxanthin-cycle pigments show the highest responsiveness to environmental factors; (iii) both winter and drought stresses induce overnight retention of de-epoxidised violaxanthin-cycle pigments; (iv) this retention correlates with depressions of Fv/Fm; and (v) contrary to what could be expected, mature leaves showed higher accumulation of hydrophilic antioxidants than young leaves, and sclerophyllous leaves higher biochemical photoprotective demand than membranous leaves. In a global climatic change scenario,the plasticity of their photoprotective mechanisms will likely benefit Mediterranean species against oceanic ones. Nevertheless, deep research of ecoregions other than the Mediterranean Basin will be needed to fully understand photoprotection strategies of this extremely biodiverse floristic biome: the Mediterranean ecosystem.

Lichens and free-living terrestrial algae are widespread across many habitats and develop successfully in ecosystems where a cold winter limits survival. With the goal of comparing photoprotective responses in free-living and lichenized algae, the physiological responses to chilling and photochilling conditions were studied in three lichens and their isolated algal photobionts together as well as in a fourth free-living algal species. We specifically addressed the following questions: (i) Are there general patterns of acclimation in green algae under chilling and photochilling stresses? (ii) Do free-living algae exhibit a similar pattern of responses as their lichenized counterparts? (iii) Are these responses influenced by the selection pressure of environmental conditions or by the phylogenetic position of each species? To answer these questions, photosynthetic fluorescence measurements as well as pigment and low molecular weight carbohydrate pool analyses were performed under controlled laboratory conditions. In general, photochemical efficiency in all free-living algae decreased with increasing duration of the stress, while the majority of lichens maintained an unchanged photochemical activity. Nevertheless, these patterns cannot be generalized because the alga Trebouxia arboricola and the lichen Ramalina pollinaria (associated with Trebouxia photobionts) both showed a similar decrease in photochemical efficiency. In contrast, in the couple Elliptochloris bilobata-Baeomyces rufus, only the algal partner exhibited a broad physiological performance under stress. This study also highlights the importance of the xanthophyll cycle in response to the studied lichens and algae to photochilling stress, while the accumulation of sugars was not related to cold acclimation, except in the alga E. bilobata. The differences in response patterns detected among species can be mainly explained by their geographic origin, although the phylogenetic position should also be considered, especially in some species.

Carotenoids distribution and function in seeds have been very scarcely studied, notwithstanding their pivotal roles in plants that include photosynthesis and phytohormone synthesis, pigmentation, membrane stabilization and antioxidant activity. Their relationship with tocochromanols, whose critical role in maintaining seed viability has already been evidenced, and with chlorophylls, whose retention in mature seed is thought to have negative effects on storability, remain also unexplored. Here, we aimed at elucidating seed carotenoids relationship with tocochromanols and chlorophylls with regard to phylogenetic and ecological traits and at understanding their changes during germination. The composition and distribution of carotenoids were investigated in seeds of a wide range of wild species across the Fabaceae (the second-most economically important family after the Poaceae). Photosynthetic pigments and tocochromanols were analyzed by HPLC in mature dry seeds of 50 species representative of 5 subfamilies within the Fabaceae (including taxa that represent all continents, biomes and life forms within the family) and at key timepoints during seedling establishment in three species representative of distinct clades. Total-carotenoids content positively correlated with tocopherols in the basal subfamilies Detarioideae, Cercidoideae, and Dialioideae, and with chlorophylls in the Papilionoideae. Papilionoideae lacked tocotrienols and had the highest total-carotenoids, chlorophyll and γ-tocopherol contents. Interestingly, lutein epoxide was present in 72% of the species including several herbs from different subfamilies. Overall, species original from temperate biomes presented higher carotenoids and lower tocochromanols levels than those from tropical biomes. Also shrub species showed higher carotenoids content than herbs and trees. During germination, total content of photosynthetic pigments increased in parallel to changes in relative abundance of carotenoids: zeaxanthin and anteraxanthin decreased and β-carotene augmented. Notably, the highest contents of nutritionally valuable carotenoids were found in Papilionoideae subfamily to which all pulses of socio-economic importance belong. The major differences in carotenoids and tocochromanols composition across the Fabaceae are apparently related to phylogeny in conjunction with ecological traits such as biome and growth form.

Some of the most widely utilized measurements in plant science research, such as total protein content or chlorophyll (chl) determination, are both straightforward and indispensable. The accuracy of the techniques employed for the quantification of these compounds is an important part of laboratory work, which will compromise the further determinations and even the conclusions of many of the articles published now and in the future. For the topic of this Letter, chl determination, a complete set of methodologies, from remote sensing to chromatographic analyses, are available (Blackburn & Ferwerda, 2008; Cortazar et al., 2015; Junker & Ensminger, 2016). Among this broad array of methodologies, one of the most (if not the most) successful protocol for the simultaneous quantification of chla and b is the one described by Arnon in the middle of last century (Arnon, 1949). Evidence of its success is the fact that it has received almost 14 000 citations up to now and it has been in the top 100 most cited papers of all time (Garfield, 1990) for decades, a position only recently lost (it ranks 139th, July 2017). Even so, Arnon's paper is frequently cited even now (actually, it is one of the four most‐cited papers ever in the area of Plant Science, according to the Web of Sciences, 2017). In fact, the highest number of citations was recorded in 2014, 2015 and 2016 (Fig. 1). Interestingly, chl determination was not the main achievement of this paper, which focused on copper metabolism in chloroplasts. However, the equations proposed for chl measurement became the reason for its success. In fact, his previous Nature paper on copper (Arnon, 1948) has only received 15 citations since its publication. Despite their great success in numeric terms and in spite of the fact that they enjoy such a huge following, Arnon's equations are based on old and inaccurate molar extinction coefficients calculated for 80% acetone as solvent.

Plants have been exposed to multiple environmental stressors on long-term (seasonal) and short-term (daily) basis since their appearance on land. During the last decades, however, plants have been frequently exposed to sudden changes in their environment (imposed by global change) which indeed involves the acclimation/adaptation syndrome of living organisms. The frequency of these unpredictable ‘stress’ events is expected to increase further in the near future. Such severe, even transient alterations in environmental stimuli (variables) represent new challenges to plants, which do not possess the ‘flight’ strategy usually displayed by other organisms.

Mining sites shelter a characteristic biodiversity with large potential for the phytoremediation of metal contaminated soils. Endophytic plant growth-promoting bacteria were isolated from two metal-(hyper)accumulator plant species growing in a metal contaminated mine soil. After characterizing their plant growth-promoting traits, consortia of putative endophytes were used to carry out an endophyte-assisted phytoextraction experiment using Noccaea caerulescens and Rumex acetosa (singly and in combination) under controlled conditions. We evaluated the influence of endophyte-inoculated plants on soil physicochemical and microbial properties, as well as plant physiological parameters and metal concentrations. Data interpretation through the grouping of soil properties within a set of ecosystem services was also carried out. When grown together, we observed a 41 and 16% increase in the growth of N. caerulescens and R. acetosa plants, respectively, as well as higher values of Zn phytoextraction and soil microbial biomass and functional diversity. Inoculation of the consortia of putative endophytes did not lead to higher values of plant metal uptake, but it improved the plants' physiological status, by increasing the content of chlorophylls and carotenoids by up to 28 and 36%, respectively, indicating a reduction in the stress level of plants. Endophyte-inoculation also stimulated soil microbial communities: higher values of acid phosphatase activity (related to the phosphate solubilising traits of the endophytes), bacterial and fungal abundance, and structural diversity. The positive effects of plant growth and endophyte inoculation on soil properties were reflected in an enhancement of some ecosystem services (biodiversity, nutrient cycling, water flow regulation, water purification and contamination control).

Aided phytostabilisation uses metal-tolerant plants, together with organic or inorganic amendments, to reduce metal bioavailability in soil while improving soil quality. The long-term effects of the following organic amendments were examined as part of an aided phytostabilisation field study in an abandoned Pb/Zn mining area: cow slurry, sheep manure and paper mill sludge mixed with poultry manure. In the mining area, two heavily contaminated vegetated sites, showing different levels of soil metal contamination (LESS and MORE contaminated site), were selected for this study. Five years after amendment application, metal bioavailability (CaCl2 extractability) along with a variety of indicators of soil microbial activity, biomass and diversity were analysed. Paper mill sludge mixed with poultry manure treatment resulted in the highest reduction of Cd, Pb and Zn bioavailability, as well as in stimulation of soil microbial activity and diversity, especially at the LESS contaminated site. In contrast, cow slurry was the least successful treatment. Our results emphasise the importance of the (i) long-term monitoring of soil quality at sites subjected to aided phytostabilisation and (ii) selection of the most efficient amendments and plants in terms of both reduction of metal bioavailability and improvement of soil quality.