Leaf anatomy affects optical properties and enhances photosynthetic performance under oblique light.

Photosynthesis under oblique illumination has not been studied extensively despite being the prevailing light regime under natural conditions. We studied how photosynthetic rate (An) is affected by the geometrical arrangement between leaf lamina and light rays, in conjunction with key anatomical features; studied plant species selected based on the absence (homobaric) or the occurrence of bundle sheath extensions (BSEs; heterobaric) and the arrangement of these structures, that is, parallel (monocots) or reticulated (dicots). The direction of light ray affected leaf absorptance (Abs) and An; both were maximal when the angle of incidence of light on leaf surface (polar angle, θ) was 90°. For any lower θ, both Abs and An were higher when the angle between the leaf axis and the light rays (azimuthal angle, φ) was zero. The dependence of Abs and An from φ was only evident in monocots and, especially, in heterobaric compared to homobaric leaves. In some species, An was substantially higher than predicted from calculated photon flux density of oblique light. The occurrence of BSEs, especially in monocots, significantly alters leaf optical properties, resulting in more efficient photosynthesis under oblique illumination conditions.

Acclimation of the Grapevine Vitis vinifera L. cv. Assyrtiko to Water Deficit: Coordination of Structural and Functional Leaf Traits and the Dynamic of Calcium Oxalate Crystals.

Grapevine leaves contain abundant CaOx crystals located either within the mesophyll in the form of raphides, or in the bundle sheaths as druses. CaOx crystals function as internal carbon pools providing CO2 for a baseline level of photosynthesis, named "alarm photosynthesis", despite closed stomata; thus, preventing the photoinhibition and the oxidative risk due to carbon starvation under adverse conditions. Structural and functional leaf traits of acclimated grapevine plants (Vitis vinifera L. cv. Assyrtiko) were investigated in response to water availability, in order to evaluate the dynamic functionality of CaOx. Leaf water potential, leaf area, leaf mass per area, stomatal properties, gas exchange parameters and performance index (PI) were decreased in leaves of vines acclimated to water deficit in comparison to the leaves of well-irrigated vines, although the chlorophyll fluorescence parameters showed that the operational efficiency of the photosystem II (PSII) photochemistry (Fv/Fm) did not change, indicating that the photosynthetic apparatus was not subjected to water stress. During the afternoon, more than half of the morning's existing druses disappeared in the drought-acclimated leaves. Also, the raphides' area of the drought-acclimated leaves was reduced more than that of the well-watered leaves. The substantial decomposition of druses under water deficit conditions compared to that of the raphides may have important implications for the maintenance of their different though overlapping roles. According to the results, it seems likely that, under water deficit conditions, a mechanism of "alarm photosynthesis" provides an additional tolerance trait in the leaves of Vitis vinifera cv. Assyrtiko; hence, leaf structure relates to function.

The Optical Properties of Leaf Structural Elements and Their Contribution to Photosynthetic Performance and Photoprotection.

Leaves have evolved to effectively harvest light, and, in parallel, to balance photosynthetic CO2 assimilation with water losses. At times, leaves must operate under light limiting conditions while at other instances (temporally distant or even within seconds), the same leaves must modulate light capture to avoid photoinhibition and achieve a uniform internal light gradient. The light-harvesting capacity and the photosynthetic performance of a given leaf are both determined by the organization and the properties of its structural elements, with some of these having evolved as adaptations to stressful environments. In this respect, the present review focuses on the optical roles of particular leaf structural elements (the light capture module) while integrating their involvement in other important functional modules. Superficial leaf tissues (epidermis including cuticle) and structures (epidermal appendages such as trichomes) play a crucial role against light interception. The epidermis, together with the cuticle, behaves as a reflector, as a selective UV filter and, in some cases, each epidermal cell acts as a lens focusing light to the interior. Non glandular trichomes reflect a considerable part of the solar radiation and absorb mainly in the UV spectral band. Mesophyll photosynthetic tissues and biominerals are involved in the efficient propagation of light within the mesophyll. Bundle sheath extensions and sclereids transfer light to internal layers of the mesophyll, particularly important in thick and compact leaves or in leaves with a flutter habit. All of the aforementioned structural elements have been typically optimized during evolution for multiple functions, thus offering adaptive advantages in challenging environments. Hence, each particular leaf design incorporates suitable optical traits advantageously and cost-effectively with the other fundamental functions of the leaf.

Dynamic changes in cell wall composition of mature sunflower leaves under distinct water regimes affect photosynthesis.

In previous work, we identified that exposure to limited water availability induced changes in cell wall composition of mature Helianthus annuus L. leaves that affected mesophyll conductance to CO2 diffusion (gm). However, it is unclear on which timescale these changes in cell wall composition occurred. Here, we subjected H. annuus to control (i.e. water availability), different levels of short-term water deficit stress (ST), long-term water deficit stress (LT), and long-term water deficit stress followed by gradual recoveries addressed at different timescales (LT-Rec) to evaluate the dynamics of modifications in the main composition of cell wall (cellulose, hemicelluloses, pectins and lignins) affecting photosynthesis. During gradual ST treatments, pectins enhancement was associated with gm decline. However, during LT-Rec, pectins content decreased significantly after only 5 h, while hemicelluloses and lignins amounts changed after 24 h, all being uncoupled from gm. Surprisingly, lignins increased by around 200% compared with control and were related to stomatal conductance to gas diffusion (gs) during LT-Rec. Although we suspect that the accuracy of the protocols to determine cell wall composition should be re-evaluated, we demonstrate for the first time that a highly dynamic cell wall composition turnover differently affects photosynthesis in plants subjected to distinct water regimes.

Cell wall composition and thickness affect mesophyll conductance to CO2 diffusion in Helianthus annuus under water deprivation.

Water deprivation affects photosynthesis, leaf anatomy, and cell wall composition. Although the former effects have been widely studied, little is known regarding those changes in cell wall major (cellulose, hemicelluloses, pectin, and lignin) and minor (cell wall-bound phenolics) compounds in plants acclimated to short- and long-term water deprivation and during recovery. In particular, how these cell wall changes impact anatomy and/or photosynthesis, specifically mesophyll conductance to CO2 diffusion (gm), has been scarcely studied. To induce changes in photosynthesis, cell wall composition and anatomy, Helianthus annuus plants were studied under five conditions: (i) control (i.e. without stress) (CL); (ii) long-term water deficit stress (LT); (iii) long-term water deficit stress with recovery (LT-Rec); (iv) short-term water deficit stress (ST); and (v) short-term water deficit stress with recovery (ST-Rec), resulting in a wide photosynthetic range (from 3.80 ± 1.05 µmol CO2 m-2 s-1 to 24.53 ± 0.42 µmol CO2 m-2 s-1). Short- and long-term water deprivation and recovery induced distinctive responses of the examined traits, evidencing a cell wall dynamic turnover during plants acclimation to each condition. In particular, we demonstrated for the first time how gm correlated negatively with lignin and cell wall-bound phenolics and how the (cellulose+hemicelloses)/pectin ratio was linked to cell wall thickness (Tcw) variations.

New insights into the functions of carbon-calcium inclusions in plants.

Carbon-calcium inclusions (CCaI) either as calcium oxalate crystals (CaOx) or amorphous calcium carbonate cystoliths are spread among most photosynthetic organisms. They represent dynamic structures with a significant construction cost and their appearance during evolution indicates an ancient origin. Both types of inclusions share some similar functional characteristics providing adaptive advantages such as the regulation of Ca levels, and the release of CO2 and water molecules upon decomposition. The latter seems to be essential under drought conditions and explains the intense occurrence of these structures in plants thriving in dry climates. It seems, however, that for plants CaOx may represent a more prevalent storage system compared with CaCO3 due to the multifunctionality of oxalate. This compound participates in a number of important soil biogeochemical processes, creates endosymbiosis with beneficial bacteria and provides tolerance against a combination of abiotic (nutrient deprivation, metal toxicity) and biotic (pathogens, herbivores) stress factors. We suggest a re-evaluation of the roles of these fascinating plant structures under a new and holistic approach that could enhance our understanding of carbon sequestration at the whole plant level and provide future perspectives.

Cystoliths of Parietaria judaica can serve as an internal source of CO2 for photosynthetic assimilation when stomata are closed.

The recently reported 'alarm photosynthesis' acts as a biochemical process that assimilates CO2 derived from the decomposition of calcium oxalate crystals. This study examined whether CaCO3 cystoliths could also serve as CO2 pools, fulfilling a similar role. Shoots of Parietaria judaica were subjected to carbon starvation, abscisic acid (ABA), or bicarbonate treatments, and the volume of cystoliths and the photochemical parameters of photosystem II (PSII) were determined. The size of cystoliths was reduced under carbon starvation or ABA treatments, whereas it was restored by xylem-provided bicarbonate. Under carbon starvation, ABA, or bicarbonate treatments, the photochemical efficiency of PSII was higher, while non-photochemical quenching, representing the safe dissipation of excess PSII energy due to lack of electron sinks, was lower in treated samples compared with controls. This observation suggests the involvement of ABA or other carbon starvation cues in the release of subsidiary CO2 for photosynthesis, inevitably from an internal source, which could be the cystoliths. Carbon remobilized from cystoliths can be photosynthetically assimilated, thus acting as a safety valve under stress. Together with alarm photosynthesis, these results show a tight link between leaf carbon deposits and photosynthesis.

Changes in the properties of calcium-carbon inclusions during leaf development and their possible relationship with leaf functional maturation in three inclusion-bearing species.

In many plant species, carbon-calcium inclusion (calcium oxalate crystals or cystoliths containing calcium carbonate) formation is a fundamental part of their physiology even necessary for normal growth and development. Despite the long-standing studies on carbon-calcium inclusions, the alterations in their properties during leaf development and their possible association with the maturation of the photosynthetic machinery have not been previously examined. In order to acquire more insights into this subject, we examined three of the most common species bearing abundant inclusions of different types, i.e., Amaranthus hybridus, Vitis vinifera, and Parietaria judaica. Results of our study showed that, irrespective of species and type of inclusion, similar patterns in the alterations of their properties are observed during leaf maturation, except for some differences in cell differentiation and distribution between raphides and druses in Vitis vinifera. As expected, inclusion formation has taken place at very early developmental stages and maximum density was observed in very young leaves. Inclusion properties are changing in a coordinated way with leaf area and these modifications are compatible with the concept that each idioblast or lithocyst "services" a finite number/area of adjacent cells. This tight coordination is also evident at the whole leaf level. Moreover, we observed an association of the properties of carbon-calcium inclusions and gas exchange, suggesting a possible implication of these structures in photosynthesis.

Changes in size and composition of pigweed (Amaranthus hybridus L.) calcium oxalate crystals under CO2 starvation conditions.

The functional role(s) of plant calcium oxalate (CaOx) crystals are still poorly understood. Recently, it was shown that crystals function as dynamic carbon pools whose decomposition could provide CO2 to photosynthesis when stomata are closed (e.g. under drought conditions) and CO2 starvation conditions may be created within the mesophyll. This biochemical process, named as 'alarm photosynthesis', can become crucial for plant survival under adverse conditions. Here, we study crystal decomposition under controlled CO2 starvation conditions (either in the shoot or in the root) to obtain a better insight into the process of crystal formation and function. Hydroponically grown pigweed plants were kept in CO2 -free air and/or CO2 -free nutrient medium for 9 days. Crystal volume was monitored daily, and carbon stable isotope composition (δ13 C) and Fourier transformation Raman spectra were obtained at the end of the experiment. A considerable reduction in the leaf crystal volume was observed in shoot-CO2 -starved plants at the end of the experiment. The smallest crystals were isolated from the plants in which carbon was excluded from both the shoot and the root and contained potassium nitrate. Crystal δ13 C of CO2 -starved plants was altered in a predicted way. Specifically, it depended on the average calculated isotope fractionation of all carbon fixation processes considered to be contributing in each experimental treatment. The results of the present study confirmed the correlation between CO2 starvation conditions and the CaOx crystal decomposition. Inorganic carbon fixed in the root may represent a major carbon source for CaOx formation.

Reevaluation of the plant "gemstones": Calcium oxalate crystals sustain photosynthesis under drought conditions.

Land plants face the perpetual dilemma of using atmospheric carbon dioxide for photosynthesis and losing water vapors, or saving water and reducing photosynthesis and thus growth. The reason behind this dilemma is that this simultaneous exchange of gases is accomplished through the same minute pores on leaf surfaces, called stomata. In a recent study we provided evidence that pigweed, an aggressive weed, attenuates this problem exploiting large crystals of calcium oxalate as dynamic carbon pools. This plant is able to photosynthesize even under drought conditions, when stomata are closed and water losses are limited, using carbon dioxide from crystal decomposition instead from the atmosphere. Abscisic acid, an alarm signal that causes stomatal closure seems to be implicated in this function and for this reason we named this path "alarm photosynthesis." The so-far "enigmatic," but highly conserved and widespread among plant species calcium oxalate crystals seem to play a crucial role in the survival of plants.

Alarm Photosynthesis: Calcium Oxalate Crystals as an Internal CO2 Source in Plants.

Calcium oxalate crystals are widespread among animals and plants. In land plants, crystals often reach high amounts, up to 80% of dry biomass. They are formed within specific cells, and their accumulation constitutes a normal activity rather than a pathological symptom, as occurs in animals. Despite their ubiquity, our knowledge on the formation and the possible role(s) of these crystals remains limited. We show that the mesophyll crystals of pigweed (Amaranthus hybridus) exhibit diurnal volume changes with a gradual decrease during daytime and a total recovery during the night. Moreover, stable carbon isotope composition indicated that crystals are of nonatmospheric origin. Stomatal closure (under drought conditions or exogenous application of abscisic acid) was accompanied by crystal decomposition and by increased activity of oxalate oxidase that converts oxalate into CO2 Similar results were also observed under drought stress in Dianthus chinensis, Pelargonium peltatum, and Portulacaria afra Moreover, in A. hybridus, despite closed stomata, the leaf metabolic profiles combined with chlorophyll fluorescence measurements indicated active photosynthetic metabolism. In combination, calcium oxalate crystals in leaves can act as a biochemical reservoir that collects nonatmospheric carbon, mainly during the night. During the day, crystal degradation provides subsidiary carbon for photosynthetic assimilation, especially under drought conditions. This new photosynthetic path, with the suggested name "alarm photosynthesis," seems to provide a number of adaptive advantages, such as water economy, limitation of carbon losses to the atmosphere, and a lower risk of photoinhibition, roles that justify its vast presence in plants.

"Carbon gain vs. water saving, growth vs. defence": two dilemmas with soluble phenolics as a joker.

Despite that phenolics are considered as a major weapon against herbivores and pathogens, the primal reason for their evolution may have been the imperative necessity for their UV-absorbing and antioxidant properties in order for plants to compensate for the adverse terrestrial conditions. In dry climates the choice concerning the first dilemma (carbon gain vs. water saving) needs the appropriate structural and metabolic modulations, which protect against stresses such as high UV and visible radiation or drought, but reduce photosynthesis and increase oxidative pressure. Thus, when water saving is chosen, priority is given to protection (including phenolic synthesis), instead of carbon gain and hence growth. At the global level, the different choices by the individual species are expressed by an interspecific negative relationship between total phenolics and photosynthesis. On the other hand, the accumulation of phenolics in water saving plants offers additional defensive functions because these multifunctional compounds can also act as pro-oxidant, antifeeding or toxic factors. Therefore phenolics, as biochemical jokers, can give the answer to both dilemmas: water saving involves high concentrations of phenolics which also offer high level of defence.

Cessation of photosynthesis in Lotus japonicus leaves leads to reprogramming of nodule metabolism.

Symbiotic nitrogen fixation (SNF) involves global changes in gene expression and metabolite accumulation in both rhizobia and the host plant. In order to study the metabolic changes mediated by leaf-root interaction, photosynthesis was limited in leaves by exposure of plants to darkness, and subsequently gene expression was profiled by real-time reverse transcription-PCR (RT-PCR) and metabolite levels by gas chromatography-mass spectrometry in the nodules of the model legume Lotus japonicus. Photosynthetic carbon deficiency caused by prolonged darkness affected many metabolic processes in L. japonicus nodules. Most of the metabolic genes analysed were down-regulated during the extended dark period. In addition to that, the levels of most metabolites decreased or remained unaltered, although accumulation of amino acids was observed. Reduced glycolysis and carbon fixation resulted in lower organic acid levels, especially of malate, the primary source of carbon for bacteroid metabolism and SNF. The high amino acid concentrations together with a reduction in total protein concentration indicate possible protein degradation in nodules under these conditions. Interestingly, comparisons between amino acid and protein content in various organs indicated systemic changes in response to prolonged darkness between nodulated and non-nodulated plants, rendering the nodule a source organ for both C and N under these conditions.

Photosynthetic capacity is negatively correlated with the concentration of leaf phenolic compounds across a range of different species.

BACKGROUND AND AIMS: Phenolic compounds are the most commonly studied of all secondary metabolites because of their significant protective-defensive roles and their significant concentration in plant tissues. However, there has been little study on relationships between gas exchange parameters and the concentration of leaf phenolic compounds (total phenolics (TP) and condensed tannins (CT)) across a range of species. Therefore, we addressed the question: is there any correlation between photosynthetic capacity (A(max)) and TP and CT across species from different ecosystems in different continents? METHODOLOGY: A plethora of functional and structural parameters were measured in 49 plant species following different growth strategies from five sampling sites located in Greece and Australia. The relationships between several leaf traits were analysed by means of regression and principal component analysis. PRINCIPAL RESULTS: The results revealed a negative relationship between TP and CT and A(max) among the different plant species, growth strategies and sampling sites, irrespective of expression (with respect to mass, area or nitrogen content). Principal component analysis showed that high concentrations of TP and CT are associated with thick, dense leaves with low nitrogen. This leaf type is characterized by low growth, A(max) and transpiration rates, and is common in environments with low water and nutrient availability, high temperatures and high light intensities. Therefore, the high TP and CT in such leaves are compatible with the protective and defensive functions ascribed to them. CONCLUSIONS: Our results indicate a functional integration between carbon gain and the concentration of leaf phenolic compounds that reflects the trade-off between growth and defence/protection demands, depending on the growth strategy adopted by each species.

The winter-red-leaf syndrome in Pistacia lentiscus: evidence that the anthocyanic phenotype suffers from nitrogen deficiency, low carboxylation efficiency and high risk of photoinhibition.

Recent evidence indicates that winter-red leaf phenotypes in the mastic tree (Pistacia lentiscus) are more vulnerable to chronic photoinhibition during the cold season relative to winter-green phenotypes occurring in the same high light environment. This was judged by limitations in the maximum quantum yield of photosystem II (PSII), found in previous studies. In this investigation, we asked whether corresponding limitations in leaf gas exchange and carboxylation reactions could also be manifested. During the cold ("red") season, net CO2 assimilation rates (A) and stomatal conductances (g(s)) in the red phenotype were considerably lower than in the green phenotype, while leaf internal CO2 concentration (Ci) was higher. The differences were abolished in the "green" period of the year, the dry summer included. Analysis of A versus Ci curves indicated that CO2 assimilation during winter in the red phenotype was limited by Rubisco content and/or activity rather than stomatal conductance. Leaf nitrogen levels in the red phenotype were considerably lower during the red-leaf period. Consequently, we suggest that the inherently low leaf nitrogen levels are linked to the low net photosynthetic rates of the red plants through a decrease in Rubisco content. Accordingly, the reduced capacity of the carboxylation reactions to act as photosynthetic electron sinks may explain the corresponding loss of PSII photon trapping efficiency, which cannot be fully alleviated by the screening effect of the accumulated anthocyanins.

Leaf anatomy affects the extraction of photosynthetic pigments by DMSO.

Dimethylsulfoxide (DMSO) is a widely used solvent for the extraction of chlorophylls (Chls) from leaves of higher plants. The method is preferred because the time-consuming steps of grinding and centrifuging are not required and the extracts are stable for a long time period. However, the extraction efficiency of this solvent is not comparable among plant species, whereas the particular leaf anatomical characteristics responsible for this unevenness remain unknown. In order to examine the influence of leaf anatomy on the extraction efficiency of DMSO (i.e. the concentration of Chls extracted with DMSO as % of the concentration of Chls extracted with 80% acetone), leaves of 19 plant species with different anatomical characteristics were incubated for 40min in DMSO at 65 degrees C. Under these conditions, heterobaric leaves, which are characterized by the occurrence of bundle sheath extensions in the mesophyll, showed lower extraction efficiency of DMSO compared to homobaric leaves and conifer needles. Microscopical observations of DMSO incubated leaf tissues showed that bundle sheath extensions behave as anatomical barriers which prevent the diffusion of DMSO within heterobaric leaves, even after prolonged incubation with the solvent. The effect was stronger in heterobaric leaves possessing thick bundle sheath extensions. The extraction efficiency of DMSO in these leaves was improved by vacuum infiltration of the samples in the presence of warm (65 degrees C) solvent.

The photoprotective role of epidermal anthocyanins and surface pubescence in young leaves of grapevine (Vitis vinifera).

BACKGROUND AND AIMS: Depending on cultivar, surfaces of young leaves of Vitis vinifera may be glabrous-green ('Soultanina') or transiently have anthocyanins ('Siriki') or pubescence ('Athiri'). A test is made of the hypothesis that anthocyanins and pubescence act as light screens affording a photoprotective advantage to the corresponding leaves, and an assessment is made of the magnitude of their effect. METHODS: Measurements were made on young leaves of the three cultivars in spring under field conditions. Photosynthetic gas-exchange and in vivo chlorophyll fluorescence were measured. Photosynthetic and photoprotective pigments were analysed by HPLC. KEY RESULTS: Compared with glabrous-green leaves, both anthocyanic and pubescent leaves had greater dark-adapted PSII photochemical efficiency and net photosynthesis. In leaves possessing either anthocyanins or pubescence, the ratio of xanthophyll cycle components to total chlorophyll, and mid-day de-epoxidation state of the xanthophyll cycle were considerably smaller, than in glabrous-green leaves. These differences were more evident in pubescent leaves, probably indicating that trichomes were more effective in decreasing light stress than anthocyanins in the epidermis. CONCLUSIONS: Light screens, especially in the form of pubescence, decrease the risk of photoinhibition whilst allowing leaves to maintain a smaller content of xanthophyll cycle components and depend less on xanthophyll cycle energy dissipation. This combination of photoprotective features, i.e. decreased photon flux to the photosynthetic apparatus and lower xanthophyll cycle utilization rates may be particularly advantageous under stressful conditions.

The relationship between anatomy and photosynthetic performance of heterobaric leaves.

Heterobaric leaves show heterogeneous pigmentation due to the occurrence of a network of transparent areas that are created from the bundle sheaths extensions (BSEs). Image analysis showed that the percentage of photosynthetically active leaf area (Ap) of the heterobaric leaves of 31 plant species was species dependent, ranging from 91% in Malva sylvestris to only 48% in Gynerium sp. Although a significant portion of the leaf surface does not correspond to photosynthetic tissue, the photosynthetic capacity of these leaves, expressed per unit of projected area (Pmax), was not considerably affected by the size of their transparent leaf area (At). This means that the photosynthetic capacity expressed per Ap (P*max) should increase with At. Moreover, the expression of P*max could be allowing the interpretation of the photosynthetic performance in relation to some critical anatomical traits. The P*max, irrespective of plant species, correlated with the specific leaf transparent volume (lambda(t)), as well as with the transparent leaf area complexity factor ((CF)A(t)), parameters indicating the volume per unit leaf area and length/density of the transparent tissues, respectively. Moreover, both parameters increased exponentially with leaf thickness, suggesting an essential functional role of BSEs mainly in thick leaves. The results of the present study suggest that although the Ap of an heterobaric leaf is reduced, the photosynthetic performance of each areole is increased, possibly due to the light transferring capacity of BSEs. This mechanism may allow a significant increase in leaf thickness and a consequent increase of the photosynthetic capacity per unit (projected) area, offering adaptive advantages in xerothermic environments.