Winter down-regulation of intrinsic photosynthetic capacity coupled with up-regulation of Elip-like proteins and persistent energy dissipation in a subalpine forest

Overwintering, sun-exposed and photosynthetically inactive evergreens require powerful photoprotection. The goal of this study was to seasonally characterize photosynthesis and key proteins/components involved in electron transport and photoprotection. Maximal photosystem II (PSII) efficiency and photosynthetic capacity, amounts of zeaxanthin (Z), antheraxanthin (A), pheophytin and proteins (oxygen-evolving 33 kDa protein (OEC), PSII core protein D1 and subunit S (PsbS) protein, and members of the early light-inducible protein (Elip) family) were assessed in five conifer species at high altitude and in ponderosa pine (Pinus ponderosa) at moderate altitude during summer and winter. Relative to summer, winter down-regulation of photosynthetic capacity and loss of PSII efficiency at the high-altitude sites were paralleled by decreases in OEC, D1, and pheophytin; massive nocturnal retention of (Z + A) and up-regulation of two to four proteins cross-reactive with anti-Elip antibodies; and no change in PsbS amount. By contrast, ponderosa pine at moderate altitude exhibited no down-regulation of photosynthetic capacity, smaller depressions in PSII efficiency, and less up-regulation of Elip family members. These results support a function for members of the Elip family in the acclimation of sun-exposed needles that down-regulate photosynthesis during winter. A possible role in sustained photoprotection is considered.     

The relationship between maximum tolerated light intensity and photoprotective energy dissipation in the photosynthetic antenna: chloroplast gains and losses

The principle of quantifying the efficiency of protection of photosystem II (PSII) reaction centres against photoinhibition by non-photochemical energy dissipation (NPQ) has been recently introduced by Ruban & Murchie (2012 Biochim. Biophys. Acta 1817, 977–982 (doi:10.1016/j.bbabio.2012.03.026)). This is based upon the assessment of two key parameters: (i) the relationship between the PSII yield and NPQ, and (ii) the fraction of intact PSII reaction centres in the dark after illumination. In this paper, we have quantified the relationship between the amplitude of NPQ and the light intensity at which all PSII reaction centres remain intact for plants with different levels of PsbS protein, known to play a key role in the process. It was found that the same, nearly linear, relationship exists between the levels of the protective NPQ component (pNPQ) and the tolerated light intensity in all types of studied plants. This approach allowed for the quantification of the maximum tolerated light intensity, the light intensity at which all plant leaves become photoinhibited, the fraction of (most likely) unnecessary or ‘wasteful’ NPQ, and the fraction of photoinhibited PSII reaction centres under conditions of prolonged illumination by full sunlight. It was concluded that the governing factors in the photoprotection of PSII are the level and rate of protective pNPQ formation, which are often in discord with the amplitude of the conventional measure of photoprotection, the quickly reversible NPQ component, qE. Hence, we recommend pNPQ as a more informative and less ambiguous parameter than qE, as it reflects the effectiveness and limitations of the major photoprotective process of the photosynthetic membrane.     

Photoprotection of green plants: a mechanism of ultra-fast thermal energy dissipation in desiccated lichens

In order to survive sunlight in the absence of water, desiccation-tolerant green plants need to be protected against photooxidation. During drying of the chlorolichen Cladonia rangiformis and the cyanolichen Peltigera neckeri, chlorophyll fluorescence decreased and stable light-dependent charge separation in reaction centers of the photosynthetic apparatus was lost. The presence of light during desiccation increased loss of fluorescence in the chlorolichen more than that in the cyanolichen. Heating of desiccated Cladonia thalli, but not of Peltigera thalli, increased fluorescence emission more after the lichen had been dried in the light than after drying in darkness. Activation of zeaxanthin-dependent energy dissipation by protonation of the PsbS protein of thylakoid membranes was not responsible for the increased loss of chlorophyll fluorescence by the chlorolichen during drying in the light. Glutaraldehyde inhibited loss of chlorophyll fluorescence during drying. Desiccation-induced loss of chlorophyll fluorescence and of light-dependent charge separation are interpreted to indicate activation of a highly effective mechanism of photoprotection in the lichens. Activation is based on desiccation-induced conformational changes of a pigment-protein complex. Absorbed light energy is converted into heat within a picosecond or femtosecond time domain. When present during desiccation, light interacts with the structural changes of the protein providing increased photoprotection. Energy dissipation is inactivated and structural changes are reversed when water becomes available again. Reversibility of ultra-fast thermal dissipation of light energy avoids photo-damage in the absence of water and facilitates the use of light for photosynthesis almost as soon as water becomes available.     

Rapid changes in xanthophyll cycle-dependent energy dissipation and photosystem II efficiency in two vines, Stephania japonica and Smilax australis, growing in the understory of an open Eucalyptus forest

Leaves of Stephania japonica and Smilax australis were characterized in situ on the coast of north-eastern New South Wales, Australia, where they were growing naturally in three different light environments: deep shade, in the understory of an open Eucalyptus forest where they received frequent sunflecks of high intensity, and in an exposed site receiving full sunlight. In deep shade the xanthophyll cycle remained epoxidized during the day and the vast majority of absorbed light was utilized for photosynthesis. In the exposed site both deepoxidation and epoxidation of the xanthophyll cycle and changes in the level of xanthophyll-dependent thermal energy dissipation largely tracked the diurnal changes in photon flux density (PFD). In the understory the xanthophyll cycle became largely deepoxidized to zeaxanthin and antheraxanthin upon exposure of the leaves to the first high intensity sunfleck and this high level of deepoxidation was maintained throughout the day both during and between subsequent sunflecks. In contrast, thermal energy dissipation activity, and the efficiency of photosystem II, fluctuated rapidly in response to the changes in incident PFD. These findings suggest a fine level of control over the engagement of zeaxanthin and antheraxanthin in energy dissipation activity, presumably through rapid changes in thylakoid acidification, such that they became rapidly engaged for photoprotection during the sunflecks and rapidly disengaged upon return to low light when continued engagement might limit carbon gain.     

Photosynthetic capacity and light harvesting efficiency during the winter-to-spring transition in subalpine conifers

Some coniferous forest ecosystems undergo complete photosynthetic down-regulation in winter. The present study examined the influence of several environmental parameters on intrinsic, needle-level photosynthesis and photoprotection during the spring reactivation of photosynthesis in subalpine conifers. Maximal photosystem II (PSII) efficiency, photosynthetic capacity, and amounts of zeaxanthin and early light-inducible protein (Elip) family members were assessed in three subalpine conifer species over 3 years, and intensively during the 2003 winter-to-spring transition. During summers, maximal PSII efficiency remained high while intrinsic photosynthetic capacity varied depending on precipitation. During winters and the winter-to-spring transition, photosynthetic capacity and PSII efficiency were highly correlated and (during the spring transition) strongly influenced by air and soil temperature and liquid water availability. Decreases in the amount of Elip family members from winter through spring paralleled disengagement of sustained zeaxanthin-dependent photoprotection, although one of four anti-Elip antibody-reactive bands increased during spring. Intrinsic photosynthetic capacity and maximal PSII efficiency were highly responsive to day-to-day environmental changes during spring, indicating that multiple environmental signals are integrated to orchestrate the reactivation of photosynthesis from the inactive winter state to the active summer state.     

Nocturnally retained zeaxanthin does not remain engaged in a state primed for energy dissipation during the summer in two Yucca species growing in the Mojave Desert

Differently oriented leaves of Yucca schidigera and Yucca brevifolia were characterized in the Mojave Desert with respect to photosystem II and xanthophyll cycle activity during three different seasons, including the hot and dry summer, the relatively cold winter, and the mild spring season. Photosynthetic utilization of a high percentage of the light absorbed in PSII was observed in all leaves only during the spring, whereas very high levels of photoprotective, thermal energy dissipation were employed both in the summer and the winter season in all exposed leaves of both species. Both during the summer and the winter season, when energy dissipation levels were high diurnally, xanthophyll cycle pools (relative to either Chl or other carotenoids) were higher relative to the spring, and a nocturnal retention of high levels of zeaxanthin and antheraxanthin (Z + A) occurred in all exposed leaves of both species. Although this nocturnal retention of Z + A was associated with nocturnal maintenance of a low PSII efficiency (F_v/F_m) on a cold winter night, pre‐dawn F_v/F_m was high in (Z + A)‐retaining leaves following a warm summer night. This indicates nocturnal engagement of Z + A in a state primed for energy dissipation throughout the cold winter night – while high levels of retained Z + A were not engaged for energy dissipation prior to sunrise on a warm summer morning. Possible mechanisms for a lack of sustained engagement of retained Z + A for energy dissipation at elevated temperatures are discussed.     

Dependence of photosynthesis and energy dissipation activity upon growth form and light environment during the winter

Two very distinctive responses of photosynthesis to winter conditions have been identified. Mesophytic species that continue to exhibit growth during the winter typically exhibit higher maximal rates of photosynthesis during the winter or when grown at lower temperatures compared to individuals examined during the summer or when grown at warmer temperatures. In contrast, sclerophytic evergreen species growing in sun-exposed sites typically exhibit lower maximal rates of photosynthesis in the winter compared to the summer. On the other hand, shaded individuals of those same sclerophytic evergreen species exhibit similar or higher maximal rates of photosynthesis in the winter compared to the summer. Employment of the xanthophyll cycle in photoprotective energy dissipation exhibits similar characteristics in the two groups of plants (mesophytes and shade leaves of sclerophytic evergreens) that exhibit upregulation of photosynthesis during the winter. In both, zeaxanthin + antheraxanthin (Z + A) are retained and PS II remains primed for energy dissipation only on nights with subfreezing temperatures, and this becomes rapidly reversed upon exposure to increased temperatures. In contrast, Z + A are retained and PS II remains primed for energy dissipation over prolonged periods during the winter in sun leaves of sclerophytic evergreen species, and requires days of warming to become fully reversed. The rapid disengagement of this energy dissipation process in the mesophytes and shade sclerophytes apparently permits a rapid return to efficient photosynthesis and increased activity on warmer days during the winter. This may be associated with a decreasing opportunity for photosynthesis in source leaves relative to the demand for photosynthesis in the plant's sinks. In contrast, the sun-exposed sclerophytes – with a relatively high source to sink ratio – maintain PS II in a state primed for high levels of energy dissipation activity throughout much of the winter. Independent of whether photosynthesis was up- or downregulated, all species under all conditions exhibited higher levels of soluble carbohydrates during the winter compared to the summer. Thus downregulation of photosynthesis and of Photosystem II do not appear to limit carbohydrate accumulation under winter conditions. A possible signal communicating an altered source/sink balance, or that may be influencing the engagement of Z + A in energy dissipation, is phosphorylation of thylakoid proteins such as D1.This revised version was published online in October 2005 with corrections to the Cover Date.     

Correlation between persistent forms of zeaxanthin-dependent energy dissipation and thylakoid protein phosphorylation

High light stress induced not only a sustained form of xanthophyll cycle-dependent energy dissipation but also sustained thylakoid protein phosphorylation. The effect of protein phosphatase inhibitors (fluoride and molybdate ions) on recovery from a 1-h exposure to a high PFD was examined in leaf discs of Parthenocissus quinquefolia (Virginia creeper). Inhibition of protein dephosphorylation induced zeaxanthin retention and sustained energy dissipation (NPQ) upon return to low PFD for recovery, but had no significant effects on pigment and Chl fluorescence characteristics under high light exposure. In addition, whole plants of Monstera deliciosa and spinach grown at low to moderate PFDs were transferred to high PFDs, and thylakoid protein phosphorylation pattern (assessed with anti-phosphothreonine antibody) as well as pigment and Chl fluorescence characteristics were examined over several days. A correlation was obtained between dark-sustained D1/D2 phosphorylation and dark-sustained zeaxanthin retention and maintenance of PS II in a state primed for energy dissipation in both species. The degree of these dark-sustained phenomena was more pronounced in M. deliciosa compared with spinach. Moreover, M. deliciosa but not spinach plants showed unusual phosphorylation patterns of Lhcb proteins with pronounced dark-sustained Lhcb phosphorylation even under low PFD growth conditions. Subsequent to the transfer to a high PFD, dark-sustained Lhcb protein phosphorylation was further enhanced. Thus, phosphorylation patterns of D1/D2 and Lhcb proteins differed from each other as well as among plant species. The results presented here suggest an association between dark-sustained D1/D2 phosphorylation and sustained retention of zeaxanthin and energy dissipation (NPQ) in light-stressed, and particularly photoinhibited, leaves. Functional implications of these observations are discussed.This revised version was published online in October 2005 with corrections to the Cover Date.     

Seasonal changes in xanthophyll cycle-dependent energy dissipation in Yucca glauca Nuttall

The evergreen species Yucca glauca was characterized at the end of September and following exposure to low temperatures at the end of November. In November the diurnal pattern of xanthophyll cycle-dependent energy dissipation was altered such that this thermal dissipation process was engaged at a high level throughout the day, whereas in September it only became engaged when leaves received direct sunlight. An analysis of the diurnal partitioning of the absorbed excitation energy into photochemistry versus thermal dissipation suggested that a smaller fraction of that energy was utilized in photochemistry and a greater fraction was dissipated thermally at the end of November compared to September. Lower ratios of Chl a / b and β -carotene/xanthophylls both suggested a decrease in the ratio of reaction centre plus core antenna proteins compared to light-harvesting proteins, and a lower leaf chlorophyll content suggested a decrease in light-harvesting capacity in November versus September. Thus adjustments to the photosynthetic apparatus occurred on several levels in response to the increase in excess excitation energy that Y. glauca experienced during the onset of winter.     

Multiple feedbacks between chloroplast and whole plant in the context of plant adaptation and acclimation to the environment

This review focuses on feedback pathways that serve to match plant energy acquisition with plant energy utilization, and thereby aid in the optimization of chloroplast and whole-plant function in a given environment. First, the role of source–sink signalling in adjusting photosynthetic capacity (light harvesting, photochemistry and carbon fixation) to meet whole-plant carbohydrate demand is briefly reviewed. Contrasting overall outcomes, i.e. increased plant growth versus plant growth arrest, are described and related to respective contrasting environments that either do or do not present opportunities for plant growth. Next, new insights into chloroplast-generated oxidative signals, and their modulation by specific components of the chloroplast''s photoprotective network, are reviewed with respect to their ability to block foliar phloem-loading complexes, and, thereby, affect both plant growth and plant biotic defences. Lastly, carbon export capacity is described as a newly identified tuning point that has been subjected to the evolution of differential responses in plant varieties (ecotypes) and species from different geographical origins with contrasting environmental challenges.     

Photoinhibition of Photosynthesis: Role of Carotenoids in Photoprotection of Chloroplast Constituents

Exposure of plants to irradiation, in excess to saturate photosynthesis, leads to reduction in photosynthetic capacity without any change in bulk pigment content. This effect is known as photoinhibition. Photoinhibition is followed by destruction of carotenoids (Cars), bleaching of chlorophylls (Chls), and increased lipid peroxidation due to formation of reactive oxygen species if the excess irradiance exposure continues. Photoinhibition of photosystem 2 (PS2) in vivo is often a photoprotective strategy rather than a damaging process. For sustainable maintenance of chloroplast function under high irradiance, the plants develop various photoprotective strategies. Cars perform essential photoprotective roles in chloroplasts by quenching the triplet Chl and scavenging singlet oxygen and other reactive oxygen species. Recently photoprotective role of xanthophylls (zeaxanthin) for dissipation of excess excitation energy under irradiance stress has been emphasised. The inter-conversion of violaxanthin (Vx) into zeaxanthin (Zx) in the light-harvesting complexes (LHC) serves to regulate photon harvesting and subsequent energy dissipation. De-epoxidation of Vx to Zx leads to changes in structure and properties of these xanthophylls which brings about significant structural changes in the LHC complex. This ultimately results in (1) direct quenching of Chl fluorescence by singlet-singlet energy transfer from Chl to Zx, (2) trans-thylakoid membrane mediated, pH-dependent indirect quenching of Chl fluorescence. Apart from these, other processes such as early light-inducible proteins, D1 turnover, and several enzymatic defence mechanisms, operate in the chloroplasts, either for tolerance or to neutralise the harmful effect of high irradiance.     

Two forms of sustained xanthophyll cycle-dependent energy dissipation in overwintering Euonymus kiautschovicus

Seasonal differences in PSII efficiency (F_v/F_m), the conversion state of the xanthophyll cycle (Z + A)/ (V + A + Z), and leaf adenylate status were investigated in Euonymus kiautschovicus. On very cold days in winter, F_v/F_m assessed directly in the field remained low and Z + A high throughout day and night in both sun and shade leaves. Pre-dawn transfer of leaves from subfreezing temperatures in the field to room temperature revealed that recovery (increases in F_v/F_m and conversion of Z + A to violaxanthin) consisted of one, rapid phase in shade leaves, whereas in sun leaves a rapid phase was followed by a slow phase requiring days. The pre-dawn ATP/ADP ratio, as well as that determined at midday, was similar when comparing overwintering leaves with those sampled in the summer, although pre-dawn levels of ATP + ADP were elevated in all leaves during winter relative to summer. After a natural transition to warmer days during the winter, pre-dawn F_v/F_m and Z + A in shade leaves had returned to values typical for summer, whereas in sun leaves F_v/F_m and Z + A levels remained intermediate between the cold day in winter and the summer day. Thus two distinct forms of sustained (Z + A)-dependent energy dissipation were identified based upon their differing characteristics. The form that was sustained on cold days but relaxed rapidly upon warming occurred in all leaves and may result from maintenance of a low lumenal pH responsible for the nocturnal engagement of (Z + A)-dependent thermal dissipation exclusively on very cold days in the winter. The form that was sustained even upon warming and correlated with slow Z + A to violaxanthin conversion occurred only in sun leaves and may represent a sustained engagement of (Z + A)-dependent energy dissipation associated with an altered PSII protein composition. In the latter, warm-sustained form, uncoupler or cycloheximide infiltration had no effect on the slow phase of recovery, but lincomycin infiltration inhibited the slow increase in F_v/F_m and the conversion of Z + A to violaxanthin.     

Role of thermal dissipation in the photoprotection in cucumber plants after exposure to a chill stress

Experiments were carried out to investigate the changes in CO₂ assimilation, photon allocation, and photosynthetic electron flux in leaves of cucumber (Cucumis sativus L.) plants after chilling stress. Chilling significantly decreased CO₂ assimilation, the energy flux via linear electron transport (J _PS2) and non-constitutive thermal dissipation (J _NPQ) but increased fluorescence and constitutive thermal dissipation (J _f,D) in chilling-sensitive genotype Jinyan No. 4. In contrast, chilling had little effects on J _NPQ and J _f,D although CO₂ assimilation and J _PS2 were inhibited in chilling-tolerant genotype Jinchun No. 3. In parallel with the reduction in J _PS2, electron flux to oxygenation and carboxylation by ribulose-1,5-bisphosphate carboxylase/oxygenase all significantly decreased while electron flux to O₂ significantly increased, especially in chilling-sensitive genotype. Thermal and fluorescence dissipation were the main energy dissipation pathways whilst water-water cycle was an important electron sink when photosynthetic carbon reduction was suppressed after chilling. Chilling sensitivity of the photosynthetic apparatus was related to the operation of different photoprotection mechanisms.     

Photoprotection of reaction centers: thermal dissipation of absorbed light energy vs charge separation in lichens

During desiccation, fluorescence emission and stable light-dependent charge separation in the reaction centers (RCs) of photosystem II (PSII) declined strongly in three different lichens: in Parmelia sulcata with an alga as the photobiont, in Peltigera neckeri with a cyanobacterium and in the tripartite lichen Lobaria pulmonaria. Most of the decline of fluorescence was caused by a decrease in the quantum efficiency of fluorescence emission. It indicated the activation of photoprotective thermal energy dissipation. Photochemical activity of the RCs was retained even after complete desiccation. It led to light-dependent absorption changes and found expression in reversible increases in fluorescence or in fluorescence quenching. Lowering the temperature changed the direction of fluorescence responses in P. sulcata. The observations are interpreted to show that reversible light-induced increases in fluorescence emission in desiccated lichens indicate the functionality of the RCs of PSII. Photoprotection is achieved by the drainage of light energy to dissipating centers outside the RCs before stable charge separation can take place. Reversible quenching of fluorescence by strong illumination is suggested to indicate the conversion of the RCs from energy conserving to energy dissipating units. This permits them to avoid photoinactivation. On hydration, re-conversion occurs to energy-conserving RCs.     

Identification of pH-sensing Sites in the Light Harvesting Complex Stress-related 3 Protein Essential for Triggering Non-photochemical Quenching in Chlamydomonas reinhardtii

Light harvesting complex stress-related 3 (LHCSR3) is the protein essential for photoprotective excess energy dissipation (non-photochemical quenching, NPQ) in the model green alga Chlamydomonas reinhardtii. Activation of NPQ requires low pH in the thylakoid lumen, which is induced in excess light conditions and sensed by lumen-exposed acidic residues. In this work we have used site-specific mutagenesis in vivo and in vitro for identification of the residues in LHCSR3 that are responsible for sensing lumen pH. Lumen-exposed protonatable residues, aspartate and glutamate, were mutated to asparagine and glutamine, respectively. By expression in a mutant lacking all LHCSR isoforms, residues Asp¹¹⁷, Glu²²¹, and Glu²²⁴ were shown to be essential for LHCSR3-dependent NPQ induction in C. reinhardtii. Analysis of recombinant proteins carrying the same mutations refolded in vitro with pigments showed that the capacity of responding to low pH by decreasing the fluorescence lifetime, present in the wild-type protein, was lost. Consistent with a role in pH sensing, the mutations led to a substantial reduction in binding the NPQ inhibitor dicyclohexylcarbodiimide.     

A few molecules of zeaxanthin per reaction centre of photosystem II permit effective thermal dissipation of light energy in photosystem II of a poikilohydric moss

The relationship between thermal dissipation of light energy (as indicated by the quenching of chlorophyll fluorescence), zeaxanthin availability and protonation reactions was investigated in the moss Rhytidiadelphus squarrosus (Hedw.) Warnst. In the absence of zeaxanthin and actinic illumination, acidification by 20% CO₂ in air was incapable of quenching basal, so-called F ₀ fluorescence either in the moss or in spinach (Spinacia oleracea L.) leaves. However, 1-s light pulses given either every 40, 60 or 200 s increased thermal dissipation as indicated by F ₀ and F _m quenching in the presence of 20% CO₂ in air in the moss, but not in spinach while reaction centres of photosystem II (PSII) were photochemically open. In the moss, a few short light pulses, which were separated by prolonged dark times, were sufficient to raise zeaxanthin levels in the presence of 20% CO₂ in air. Simultaneously, quantum efficiency of charge separation in PSII was decreased. Increasing the CO₂ concentration beyond 20% further decreased quantum efficiency even in the absence of short light pulses. Under conditions optimal for fluorescence quenching, one molecule of zeaxanthin per reaction centre of PSII was sufficient to decrease quantum efficiency of charge separation in PSII by 50%. Thus, in combination with a protonation reaction, one molecule of zeaxanthin was as efficient at capturing excitation energy as a photochemically open reaction centre. The data are discussed in relation to the interaction between zeaxanthin and thylakoid protonation, which enables effective thermal dissipation of light energy in the antennae of PSII in the moss but not in higher plants when actinic illumination is absent. Received: 8 April 2000 / Accepted: 31 August 2000     

依赖叶黄素循环的非辐射能量耗散在防御珊瑚树叶片光抑制破坏中的作用

使用脉冲调制荧光仪观测了珊瑚树叶片光合作用的光抑制发生与恢复过程中几个主要荧光参数(初始荧光F_0,可变荧光与最大荧光之比F_v/F_M和非光化学荧光猝灭q_E及其快组分 q_(E—fast)、慢组分 q(E—slow))的变化,以探讨非光化学荧光猝灭不同组分的作用。 强光(约 1500μmol photons m~(-2) s~(-1))照射叶片使F_0、F_V/F_M和q_(E—fast)降低.q_(E—slow)和q_E增高。NH_4Cl处理使 F_V/F_M降低的幅度和q_E提高的幅度都增加。DTT处理使q_E水平和q_(E—slow)增加的幅度降低,而F_0和稳态荧光水平增加,强光下降低了的F_V/F_M在弱光下不易恢复。NaF处理对这些荧光参数都没有明显的影响。