Beatriz Fernández-Marín

Physiology of the seasonal relationship between the photochemical reflectance index and photosynthetic light use efficiency

The photochemical reflectance index (PRI) is regarded as a promising proxy to track the dynamics of photosynthetic light use efficiency (LUE) via remote sensing. The implementation of this approach requires the relationship between PRI and LUE to scale not only in space but also in time. The short-term relationship between PRI and LUE is well known and is based on the regulative process of non-photochemical quenching (NPQ), but at the seasonal timescale the mechanisms behind the relationship remain unclear. We examined to what extent sustained forms of NPQ, photoinhibition of reaction centres, seasonal changes in leaf pigment concentrations, or adjustments in the capacity of alternative energy sinks affect the seasonal relationship between PRI and LUE during the year in needles of boreal Scots pine. PRI and NPQ were highly correlated during most of the year but decoupled in early spring when the foliage was deeply downregulated. This phenomenon was attributed to differences in the physiological mechanisms controlling the seasonal dynamics of PRI and NPQ. Seasonal adjustments in the pool size of the xanthophyll cycle pigments, on a chlorophyll basis, controlled the dynamics of PRI, whereas the xanthophyll de-epoxidation status and other xanthophyll-independent mechanisms controlled the dynamics of NPQ at the seasonal timescale. We conclude that the PRI leads to an underestimation of NPQ, and consequently overestimation of LUE, under conditions of severe stress in overwintering Scots pine, and most likely also in species experiencing severe drought. This severe stress-induced decoupling may challenge the implementation of the PRI approach.

Thermal energy dissipation and xanthophyll cycles beyond the Arabidopsis model

Thermal dissipation of excitation energy is a fundamental photoprotection mechanism in plants. Thermal energy dissipation is frequently estimated using the quenching of the chlorophyll fluorescence signal, termed non-photochemical quenching. Over the last two decades, great progress has been made in the understanding of the mechanism of thermal energy dissipation through the use of a few model plants, mainly Arabidopsis. Nonetheless, an emerging number of studies suggest that this model represents only one strategy among several different solutions for the environmental adjustment of thermal energy dissipation that have evolved among photosynthetic organisms in the course of evolution. In this review, a detailed analysis of three examples highlights the need to use models other than Arabidopsis: first, overwintering evergreens that develop a sustained form of thermal energy dissipation; second, desiccation tolerant plants that induce rapid thermal energy dissipation; and third, understorey plants in which a complementary lutein epoxide cycle modulates thermal energy dissipation. The three examples have in common a shift from a photosynthetically efficient state to a dissipative conformation, a strategy widely distributed among stress-tolerant evergreen perennials. Likewise, they show a distinct operation of the xanthophyll cycle. Expanding the list of model species beyond Arabidopsis will enhance our knowledge of these mechanisms and increase the synergy of the current studies now dispersed over a wide number of species.

Photoprotective implications of leaf variegation in E. dens-canis L. and P. officinalis L

Variegated leaves occur rarely in nature, but there are some species, primarily in the forest understory, that possess this characteristic. We recently studied two variegated plants: Erytronium dens-canis L., which is characterised by a pattern of red patches and Pulmonaria officinalis L., with light green spots. These non-green areas could attenuate light reaching mesophyll cells with respect to green sections. The aim of the study was to verify whether such red and light green parts are more photoprotected than green ones and if this trait could be of adaptive value. Red patches in E. dens-canis were due to a single layer of red cells in the upper parenchyma, which accumulated anthocyanins. Light green spots in P. officinalis were caused by the presence of loosely arranged cells instead of a well-established layer of packed cells in the palisade parenchyma. Chlorophyll fluorescence imaging was performed under light treatment, showing a greater decrease of photochemical efficiency in red and light green patches than in green sections. Differences in the extent of photochemical efficiency among patches were not attributable to different activation of the xanthophyll cycle. These observations failed to confirm our initial hypothesis, but they questioned the physiological reason for this higher sensitivity in red and light green patches of photosynthetic tissues. Chlorophyll fluorescence imaging was therefore performed in the field. The same pattern of photochemical efficiency was maintained only in E. dens-canis. The current results demonstrate that in both species the benefits of variegation, if any, are different from enhanced photosynthetic performance.

Distribution and evolutionary trends of photoprotective isoprenoids (xanthophylls and tocopherols) within the plant kingdom.

The earliest land photosynthesis would have increased the risk of photo-oxidations and the demand of anti-oxidative protection. In this work, we aimed to determine the evolutionary trends in photoprotection across a wide representation of the plant kingdom and to verify whether the non-ubiquitous lutein-epoxide (Lx) cycle is a polyphyletic or an ancient character. Carotenoids and alpha-tocopherol (alpha-toc) were analysed by HPLC in 266 species. Phylogenetic analyses of the presence of photoprotective compounds and zeaxanthin-epoxidase (ZE) sequences were performed. Violaxanthin-cycle pigments (VAZ) and alpha-toc were taxonomically ubiquitous. Ancient groups showed higher contents of VAZ than vascular plants, while alpha-toc showed the opposite pattern. Lutein-epoxide was present in 45% of the species. It showed a remarkable variation across groups but with a clear increasing trend from algae to basal angiosperms. Lutein-epoxide was also related to woody trait and leaf longevity. No correlation between the presence of Lx and recurrent mutations in ZE sequences, including the duplications, was found. Thus, there is an evolutionary trend to increase the content of alpha-toc and to decrease the total amount of VAZ pigments. Absence of Lx in algae discards an ancestral origin. Present results are also inconsistent with a polyphyletic origin of Lx in angiosperms.

Genome-wide association mapping and biochemical markers reveal that seed ageing and longevity are intricately affected by genetic background and developmental and environmental conditions in barley

Globally, over 7.4 million accessions of crop seeds are stored in gene banks, and conservation of genotypic variation is pivotal for breeding. We combined genetic and biochemical approaches to obtain a broad overview of factors that influence seed storability and ageing in barley (Hordeum vulgare). Seeds from a germplasm collection of 175 genotypes from four continents grown in field plots with different nutrient supply were subjected to two artificial ageing regimes. Genome-wide association mapping revealed 107 marker trait associations, and hence, genotypic effects on seed ageing. Abiotic and biotic stresses were found to affect seed longevity. To address aspects of abiotic, including oxidative, stress, two major antioxidant groups were analysed. No correlation was found between seed deterioration and the lipid-soluble tocochromanols, nor with oil, starch and protein contents. Conversely, the water-soluble glutathione and related thiols were converted to disulphides, indicating a strong shift towards more oxidizing intracellular conditions, in seeds subjected to long-term dry storage at two temperatures or to two artificial ageing treatments. The data suggest that intracellular pH and (bio)chemical processes leading to seed deterioration were influenced by the type of ageing or storage. Moreover, seed response to ageing or storage treatment appears to be significantly influenced by both maternal environment and genetic background.

Unravelling the roles of desiccation-induced xanthophyll cycle activity in darkness: a case study in Lobaria pulmonaria.

Desiccation-tolerance ability in photosynthetic organisms is largely based on a battery of photoprotective mechanisms. Xanthophyll cycle operation induced by desiccation in the absence of light has been previously proven in the desiccation-tolerant fern Ceterach officinarum. To understand the physiological function of xanthophyll cycle induction in darkness and its implication in the desiccation tolerance in more detail, we studied its triggering factors and its photochemical effects in the lichen Lobaria pulmonaria. We found that both the drying rate and the degree of desiccation play a crucial role in the violaxanthin de-epoxidase activation. De-epoxidation of violaxanthin to zeaxanthin (Z) occurs when the tissue has lost most of its water and only after slow dehydration, suggesting that a minimum period of time is required for the enzyme activity induction. Fluorescence analysis showed that Z, synthesised during tissue dehydration in the absence of light, prevents photoinhibition when rewatered tissues are illuminated. This is probably due to Z implication in both non-photochemical quenching and/or antioxidative responses.

Evidence for the absence of enzymatic reactions in the glassy state. A case study of xanthophyll cycle pigments in the desiccation-tolerant moss Syntrichia ruralis

Desiccation-tolerant plants are able to withstand dehydration and resume normal metabolic functions upon rehydration. These plants can be dehydrated until their cytoplasm enters a ‘glassy state’ in which molecular mobility is severely reduced. In desiccation-tolerant seeds, longevity can be enhanced by drying and lowering storage temperature. In these conditions, they still deteriorate slowly, but it is not known if deteriorative processes include enzyme activity. The storage stability of photosynthetic organisms is less studied, and no reports are available on the glassy state in photosynthetic tissues. Here, the desiccation-tolerant moss Syntrichia ruralis was dehydrated at either 75% or <5% relative humidity, resulting in slow (SD) or rapid desiccation (RD), respectively, and different residual water content of the desiccated tissues. The molecular mobility within dry mosses was assessed through dynamic mechanical thermal analysis, showing that at room temperature only rapidly desiccated samples entered the glassy state, whereas slowly desiccated samples were in a ‘rubbery’ state. Violaxanthin cycle activity, accumulation of plastoglobules, and reorganization of thylakoids were observed upon SD, but not upon RD. Violaxanthin cycle activity critically depends on the activity of violaxanthin de-epoxidase (VDE). Hence, it is proposed that enzymatic activity occurred in the rubbery state (after SD), and that in the glassy state (after RD) no VDE activity was possible. Furthermore, evidence is provided that zeaxanthin has some role in recovery apparently independent of its role in non-photochemical quenching of chlorophyll fluorescence.

Photoprotective responses of Mediterranean and Atlantic trees to the extreme heat-wave of summer 2003 in Southwestern Europe

Summer 2003 was extremely hot in Europe. High light in combination with heat and drought exacerbates the generation of photo-oxidative stress. Under these conditions photoprotective responses can be critical for plant survival. Photoprotection was analysed in 2003 in several Mediterranean and Atlantic woody species. These data were compared with previous summers (1998, 1999 and 2001) to evaluate the potential acclimation for each species. A pattern of changes consisting on a decrease in chlorophyll, ascorbate and Fv/Fm and an increase in tocopherol, xanthophyll cycle pigments (VAZ) and de-epoxidation index was regularly observed. Acclimation potential was measured by the use of the plasticity index for each parameter. Mediterranean species were more plastic than Atlantic ones. The latter were unable to increase antioxidant pools to the same extent or to down-regulate the efficiency of light energy conversion. These results indicate that most Mediterranean species are able to perform an efficient acclimation to heat stress, whilst Atlantic species will be more affected by climate warming.

Dark induction of the photoprotective xanthophyll cycle in response to dehydration.

Some plants tolerate tissue dehydration. Dehydration conditions suppress photosynthesis, exacerbating photooxidative stress. In this study, fern samples were collected from the field, desiccated in darkness, and subsequently re-watered. During dark dehydration, zeaxanthin (Z) was formed and maximal photochemical efficiency of PS II was strongly reduced. Rehydration in the dark reversed these effects. Violaxanthin de-epoxidase was responsible for the dark formation of Z as illustrated by its complete inhibition by DTT. Nonetheless, its activity was not affected by nigericin, indicating that Z formation in the dark could be a process independent of the transmembrane pH-gradient into the thylakoids. Synthesis de novo of Z was rejected after blocking carotenogenesis with norfluorazon. Dark formation of Z was also observed in dehydrating leaves of desiccation-intolerant plants, which seems to indicate that this is a phenomenon scattered among different taxa within the plant kingdom. Plants may trigger this mechanism during dehydration, for chlorophyll protection during desiccation, and for faster acclimation when rehydrating conditions return. Violaxanthin de-epoxidation to form Z is typically a light-dependent process, but the formation induced solely by dehydration might represent an anticipatory mechanism for preventing early morning photodamage in desiccation-tolerant plants such as the fern Ceterach officinarum.

Carotenoid composition in Rhodophyta: insights into xanthophyll regulation in Corallina elongata

Macroalgae possess several photoprotection mechanisms, including xanthophyll cycles. Among these cycles, the VAZ (violaxanthin–anteraxanthin–zeaxanthin) cycle controls the interconversion of epoxidated xanthophyll (V) to de-epoxidated forms anteraxanthin and zeaxanthin. This conversion modulates the amount of excess energy that it is dissipated as heat. Presence of functional xanthophyll cycles in Rhodophyta is controversial. In this work we investigated the presence of xanthophyll cycles in red algae. Carotenoid composition of 13 red macroalgae collected in northern Spain was studied by HPLC. This screening showed that carotenoid composition is a conservative trade in rhodophytes, with very few exceptions to the general pattern formed by β-carotene and one to three xanthophylls: anteraxanthin, zeaxanthin or lutein. Corallina elongata and Jania rubens were the only algae containing anteraxanthin as the main xanthophyll. The first species was selected to study whether any truncated xanthophyll cycle between anteraxanthin and zeaxanthin could operate under photoinhibitory conditions. Upon illumination, xanthophyll composition remained stable, without any short-term light-induced de-epoxidation of anteraxanthin to zeaxanthin. However, an inverse relation between both xanthophylls was found. In addition, a seasonal trend of changes in anteraxanthin and zeaxanthin was observed when pigment composition was studied in field samples over the course of a year. We concluded that these variations were more likely due to differential rates of synthesis and degradation of xanthophylls than to the operation of a xanthophyll cycle. The great amount of anteraxanthin in C. elongata may play an structural stability role on light-harvesting complexes.