Feedback De-excitation Research Articles

The negative feedback molecular mechanism which regulates excitation level in the plant photosynthetic complex LHCII: Towards identification of the energy dissipative state

Overexcitation of the photosynthetic apparatus is potentially dangerous because it can cause oxidative damage. Photoprotection realized via the feedback de-excitation in the pigment–protein light-harvesting complex LHCII, embedded in the chloroplast lipid environment, was studied with use of the steady-state and time-resolved fluorescence spectroscopy techniques. Illumination of LHCII results in the pronounced singlet excitation quenching, demonstrated by decreased quantum yield of the chlorophyll a fluorescence and shortening of the fluorescence lifetimes. Analysis of the 77K chlorophyll a fluorescence emission spectra reveals that the light-driven excitation quenching in LHCII is associated with the intensity increase of the spectral band in the region of 700nm, relative to the principal band at 680nm. The average chlorophyll a fluorescence lifetime at 700nm changes drastically upon temperature decrease: from 1.04ns at 300K to 3.63ns at 77K. The results of the experiments lead us to conclude that: (i) the 700nm band is associated with the inter-trimer interactions which result in the formation of the chlorophyll low-energy states acting as energy traps and non-radiative dissipation centers; (ii) the Arrhenius analysis, supported by the results of the FTIR measurements, suggests that the photo-reaction can be associated with breaking of hydrogen bonds. Possible involvement of photo-isomerization of neoxanthin, reported previously (Biochim. Biophys. Acta 1807 (2011) 1237-1243) in generation of the low-energy traps in LHCII is discussed.

Enhanced Photoprotection by Protein-Bound vs Free Xanthophyll Pools: A Comparative Analysis of Chlorophyll b and Xanthophyll Biosynthesis Mutants

When light absorbed by plants exceeds the capacity of photosynthesis, the xanthophyll violaxanthin is reversibly de-epoxidized to zeaxanthin in the so-called xanthophyll cycle. Zeaxanthin plays a key role in the protection of photosynthetic organisms against excess light, by promoting rapidly reversible (qE) and long-term (qI) quenching of excited chlorophylls, and preventing lipid oxidation. The photoprotective role of zeaxanthin, either free or bound to light-harvesting complexes (Lhcs), has been investigated by using mutants lacking Chl b (ch1) and/or specific xanthophyll species (npq, lut2). The ch1 mutation causes (1) the absence of Lhcb proteins; (2) strong reduction of the feedback de-excitation (qE); and (3) accumulation of xanthophylls as free pigments into thylakoids. Ch1 mutants showed extreme sensitivity to photo-oxidative stress in high light, due to higher singlet oxygen (¹O₂) release. The double mutant ch1npq1 was more sensitive to photo-oxidation than ch1, showing that zeaxanthin does protect lipids even when free in the membrane. Nevertheless, lack of zeaxanthin had a much stronger impact on the level of lipid peroxidation in Lhcs-containing plants (WT vs npq1) with respect to Lhc-less plants (ch1 vs ch1npq1), implying that its protective effect is enhanced by interaction with antenna proteins. It is proposed that the antioxidant capacity of zeaxanthin is empowered in the presence of PSII-LHCs-Zea complexes, while its effect on enhancement of qE only provides a minor contribution. Comparison of the sensitivity of WT vs npq1 plants to exogenous ¹O₂ suggests that besides the scavenging of ¹O₂, at least one additional mechanism is involved in chloroplast photoprotection.

Quantitative genetic analysis of thermal dissipation in Arabidopsis.

Feedback deexcitation is a photosynthetic regulatory mechanism that can protect plants from high light stress by harmlessly dissipating excess absorbed light energy as heat. To understand the genetic basis for intraspecies differences in thermal dissipation capacity, we investigated natural variation in Arabidopsis (Arabidopsis thaliana). We determined the variation in the amount of thermal dissipation by measuring nonphotochemical quenching (NPQ) of chlorophyll fluorescence in Arabidopsis accessions of diverse origins. Ll-1 and Sf-2 were selected as high NPQ Arabidopsis accessions, and Columbia-0 (Col-0) and Wassilewskija-2 were selected as relatively low NPQ accessions. In spite of significant differences in NPQ, previously identified NPQ factors were indistinguishable between the high and the low NPQ accessions. Intermediate levels of NPQ in Ll-1 x Col-0 F1 and Sf-2 x Col-0 F1 compared to NPQ levels in their parental lines and continuous distribution of NPQ in F2 indicated that the variation in NPQ is under the control of multiple nuclear factors. To identify genetic factors responsible for the NPQ variation, we developed a polymorphic molecular marker set for Sf-2 x Col-0 at approximately 10-centimorgan intervals. From quantitative trait locus (QTL) mapping with undistorted genotype data and NPQ measurements in an F2 mapping population, we identified two high NPQ QTLs, HQE1 (high qE 1, for high energy-dependent quenching 1) and HQE2, on chromosomes 1 and 2, and the phenotype of HQE2 was validated by analysis of near isogenic lines. Neither QTL maps to a gene that had been identified previously in extensive forward genetics screens using induced mutants, suggesting that quantitative genetics can be used to find new genes affecting thermal dissipation.

Properties of zeaxanthin and its radical cation bound to the minor light-harvesting complexes CP24, CP26 and CP29

Nonphotochemical quenching (NPQ) is a fundamental mechanism in photosynthesis by which plants protect themselves against excess excitation energy and which is thus of crucial importance for plant survival and fitness. Recently, carotenoid radical cation (Car •+) formation has been discovered to be a key step in the feedback deexcitation quenching component (qE) of NPQ, whose molecular mechanism and location remains elusive. A recent model for qE suggests that the replacement of violaxanthin (Vio) by zeaxanthin (Zea) in photosynthetic pigment binding pockets can in principle result in qE via the so-called “gear-shift” or electron transfer quenching mechanisms. We performed pump-probe measurements on individual antenna complexes of photosystem II (CP24, CP26 and CP29) upon excitation of the chlorophylls (Chl) into their first excited Q y state at 660 nm when either Vio or Zea was bound to those complexes. The Chl lifetime was then probed by measuring the decay kinetics of the Chl excited state absorption (ESA) at 900 nm. The charge-transfer quenching mechanism, which is characterized by a spectral signature of the transiently formed Zea radical cation (Zea •+) in the near-IR, has also been addressed, both in solution and in light-harvesting complexes of photosystem II (LHC-II). Applying resonant two-photon two-color ionization (R2P2CI) spectroscopy we show here the formation of β-Car •+ in solution, which occurs on a femtosecond time-scale by direct electron transfer to the solvent. The β-Car •+ maxima strongly depend on the solvent polarity. Moreover, our two-color two-photon spectroscopy on CP29 reveals the spectral position of Zea •+ in the near-IR region at 980 nm.

Improper excess light energy dissipation in Arabidopsis results in a metabolic reprogramming

BackgroundPlant performance is affected by the level of expression of PsbS, a key photoprotective protein involved in the process of feedback de-excitation (FDE), or the qE component of non-photochemical quenching, NPQ.ResultsIn studies presented here, under constant laboratory conditions the metabolite profiles of leaves of wild-type Arabidopsis thaliana and plants lacking or overexpressing PsbS were very similar, but under natural conditions their differences in levels of PsbS expression were associated with major changes in metabolite profiles. Some carbohydrates and amino acids differed ten-fold in abundance between PsbS-lacking mutants and over-expressers, with wild-type plants having intermediate amounts, showing that a metabolic shift had occurred. The transcriptomes of the genotypes also varied under field conditions, and the genes induced in plants lacking PsbS were similar to those reportedly induced in plants exposed to ozone stress or treated with methyl jasmonate (MeJA). Genes involved in the biosynthesis of JA were up-regulated, and enzymes involved in this pathway accumulated. JA levels in the undamaged leaves of field-grown plants did not differ between wild-type and PsbS-lacking mutants, but they were higher in the mutants when they were exposed to herbivory.ConclusionThese findings suggest that lack of FDE results in increased photooxidative stress in the chloroplasts of Arabidopsis plants grown in the field, which elicits a response at the transcriptome level, causing a redirection of metabolism from growth towards defence that resembles a MeJA/JA response.

Carotenoid radical cations as a probe for the molecular mechanism of nonphotochemical quenching in oxygenic photosynthesis.

Nonphotochemical quenching (NPQ) is a fundamental mechanism in photosynthesis which protects plants against excess excitation energy and is of crucial importance for their survival and fitness. Recently, carotenoid radical cation (Car*+) formation has been discovered to be a key step for the feedback deexcitation quenching mechanism (qE), a component of NPQ, of which the molecular mechanism and location is still unknown. We have generated and characterized carotenoid radical cations by means of resonant two color, two photon ionization (R2C2PI) spectroscopy. The Car*+ bands have maxima located at 830 nm (violaxanthin), 880 nm (lutein), 900 nm (zeaxanthin), and 920 nm (beta-carotene). The positions of these maxima depend strongly on solution conditions, the number of conjugated C=C bonds, and molecular structure. Furthermore, R2C2PI measurements on the light-harvesting complex of photosystem II (LHC II) samples with or without zeaxanthin (Zea) reveal the violaxanthin (Vio) radical cation (Vio*+) band at 909 nm and the Zea*+ band at 983 nm. The replacement of Vio by Zea in the light-harvesting complex II (LHC II) has no influence on the Chl excitation lifetime, and by exciting the Chls lowest excited state, no additional rise and decay corresponding to the Car*+ signal observed previously during qE was detected in the spectral range investigated (800-1050 nm). On the basis of our findings, the mechanism of qE involving the simple replacement of Vio with Zea in LHC II needs to be reconsidered.

Photoprotection mutants of Arabidopsis thaliana acclimate to high light by increasing photosynthesis and specific antioxidants

Biochemical and physiological acclimation to different light environments is crucial for plant growth and survival. In high light (HL), feedback de-excitation (qE) is a well-known photoprotective mechanism that dissipates excess excitation energy in the light-harvesting antenna of photosystem II (PSII) and relieves excitation pressure in the photosynthetic electron transport chain. The xanthophylls zeaxanthin (Z) and lutein (L) function in qE, but also have roles as antioxidants. Although several studies have shown that qE is important during short-term fluctuations in light intensity, here we show that it is not required for the growth of Arabidopsis thaliana in prolonged HL conditions in the laboratory. Mutants that are deficient in qE alone, qE and Z synthesis, or in qE, Z synthesis and also L synthesis were able to grow at 1800 micromol photons m(-2) s(-1) and exhibited no major symptoms of photooxidative stress. The mutants (and wild type) acclimated to HL by increasing photosynthetic capacity and decreasing light harvesting, which together rendered qE less important for photoprotection. At a metabolite level, the HL-grown mutants appeared to compensate for their remaining qE deficit with increased alpha-tocopherol and ascorbate levels compared to the wild type. The specificity of this response provides insight into the relationship between qE and the antioxidant network in plants.

Functional flexibility and acclimation of the thylakoid membrane

Light is an elusive substrate for the function of photosynthetic light reactions of photosynthesis in the thylakoid membrane. Therefore structural and functional dynamics, which occur in the timescale from seconds to several days, are required both at low and high light conditions. The best characterized short-time regulation mechanism at low light is a rapid state transition, resulting in higher absorption cross section of PSI at the expense of PSII. If the low light conditions continue, activation of the lhcb-genes and synthesis of the light-harvesting proteins will occur to optimize the functions of PSII and PSI. At high light, the transition to state 2 is completely inhibited, but the feedback de-excitation of absorbed energy as heat, known as the energy-dependent quenching (q(E)), is rapidly set up. It requires, at least, the DeltapH-dependent activation of violaxanthin de-epoxidase and involvement of the PsbS protein. Another crucial mechanism for protection against the high light stress is the PSII repair cycle. Furthermore, the water-water cycle, cyclic electron transfer around PSI and chlororespiration are important means induced under high irradiation, functioning mainly to avoid an excess production of reactive oxygen species.

What leads to reduced fitness in non‐photochemical quenching mutants?

Feedback de‐excitation (FDE) is a process that protects photosystem II from damage during short periods of overexcitation. Arabidopsis thaliana mutants lacking this mechanism have reduced fitness in environments with variable light intensities. We have assayed the physiological consequences of mutations resulting in the lack of FDE and analysed the differences between field‐grown plants and plants grown under fluctuating light in the laboratory. We show that FDE is an important mechanism in short‐term responses to fluctuating light. Anthocyanin and carbohydrate levels indicated that the mutant plants were stressed to a higher degree than wild‐type (WT) plants. Field‐grown mutants were photo‐inactivated to a greater degree than WT, whereas mutant plants in the fluctuating light environment in the laboratory seemed to downregulate the photosynthetic quantum yield, thereby avoiding photo‐damage but resulting in impaired growth in the case of one mutant. Finally, we provide evidence that FDE is most important under conditions when photosynthesis limits plant growth, for example during flower and seed development.

Role of electron-transfer quenching of chlorophyll fluorescence by carotenoids in non-photochemical quenching of green plants

NPQ (non-photochemical quenching) is a fundamental photosynthetic mechanism by which plants protect themselves against excess excitation energy and the resulting photodamage. A discussed molecular mechanism of the so-called feedback de-excitation component (qE) of NPQ involves the formation of a quenching complex. Recently, we have studied the influence of formation of a zeaxanthin-chlorophyll complex on the excited states of the pigments using high-level quantum chemical methodology. In the case of complex formation, electron-transfer quenching of chlorophyll-excited states by carotenoids is a relevant quenching mechanism. Furthermore, additionally occurring charge-transfer excited states can be exploited experimentally to prove the existence of the quenching complex during NPQ.

Carotenoid Cation Formation and the Regulation of Photosynthetic Light Harvesting

Photosynthetic light harvesting in excess light is regulated by a process known as feedback deexcitation. Femtosecond transient absorption measurements on thylakoid membranes show selective formation of a carotenoid radical cation upon excitation of chlorophyll under conditions of maximum, steady-state feedback deexcitation. Studies on transgenic Arabidopsis thaliana plants confirmed that this carotenoid radical cation formation is correlated with feedback deexcitation and requires the presence of zeaxanthin, the specific carotenoid synthesized during high light exposure. These results indicate that energy transfer from chlorophyll molecules to a chlorophyllzeaxanthin heterodimer, which then undergoes charge separation, is the mechanism for excess energy dissipation during feedback deexcitation.

Carotenoid-chlorophyll complexes: Ready-to-harvest

Abstract The fundamental interactions between naturally occurring pigments in light-harvesting systems are responsible for the high efficiency of the photosynthetic apparatus. We describe the role of carotenoids (Cars) in light-harvesting systems, including our work elucidating the mechanism of energy transfer from the optically dark Car singlet excited state (S1) to chlorophyll (Chl) and calculations on the electronic structure of Cars by means of time-dependent density functional theory (TDDFT). We highlight new studies on the charge-transfer state of the Car, peridinin (Per), which enhances the light-harvesting efficiency of the Car by increasing the electronic coupling to Chl. The role of another Car, zeaxanthin (Zea), is discussed with respect to its role in the mechanism of the feedback deexcitation quenching in green plants, a vital regulation process under light conditions which exceed photosynthetic capacity. Lastly, we provide insight on how the 96 Chls in Photosystem I are optimized to generate a pigment-protein complex which utilizes solar energy with near unit efficiency.

Toward an understanding of the mechanism of nonphotochemical quenching in green plants.

Oxygenic photosynthesis in plants involves highly reactive intermediates and byproducts that can damage the photosynthetic apparatus and other chloroplast constituents. The potential for damage is exacerbated when the amount of absorbed light exceeds the capacity for light energy utilization in photosynthesis, a condition that can lead to decreases in photosynthetic efficiency. A feedback de-excitation mechanism (qE), measured as a component of nonphotochemical quenching of chlorophyll fluorescence, regulates photosynthetic light harvesting in excess light in response to a change in thylakoid lumen pH. qE involves de-excitation of the singlet excited state of chlorophyll in the light-harvesting antenna of photosystem II, thereby minimizing the deleterious effects of high light via thermal dissipation of excess excitation energy. While the physiological importance of qE has been recognized for many years, a description of its physical mechanism remains elusive. We summarize recent biochemical and spectroscopic results that have brought us closer to the goal of a mechanistic understanding of this fundamental photosynthetic regulatory process.

Acclimation of Arabidopsis thaliana to the light environment: the relationship between photosynthetic function and chloroplast composition.

Plants respond to growth under different environmental conditions by adjusting the composition of the photosynthetic apparatus. To investigate the consequences of the acclimation strategies adopted by Arabidopsis thaliana, we have assessed the functioning of the photosynthetic apparatus in plants with very different chloroplast compositions. Using chlorophyll fluorescence analysis, we have determined the efficiency of, and capacity for, electron transport, assessed the ability to undergo state transitions, and measured non-photochemical quenching over a range of actinic irradiances followed by its resolution into fast- and slow-relaxing components; parallel measurements of leaf carotenoid composition were also carried out. The data clearly show that acclimation serves to maintain the electron transport chain in an oxidised state, ensuring efficient photochemistry. Furthermore, plants grown in high light have a greater capacity for energy-dependent feedback de-excitation, but this is not correlated with xanthophyll cycle pigment levels or de-epoxidation state. Surprisingly, even plants with very low levels of light-harvesting complexes were able to undergo state transitions. We also show that apparent discrepancies between chloroplast composition and photosynthetic function can be attributed to varying degrees of light penetration through the leaf. Thus, leaf chlorophyll content is an important factor influencing acclimation within the leaf.

Zeaxanthin deficiency enhances the high light sensitivity of an ascorbate-deficient mutant of Arabidopsis.

The ascorbate content of plants is usually increased in high light (HL), implying a function for ascorbate in the acclimation of plants to HL. Nevertheless, the importance of ascorbate in HL acclimation has not yet been tested directly. Here, we report on the acclimation process of an ascorbate-deficient Arabidopsis mutant to HL. The mutant vtc2 has only 10% to 30% of wild-type levels of ascorbate, and it is also slightly deficient in feedback de-excitation (qE), a photoprotective mechanism that causes the dissipation of excess light as heat. The vtc2 mutant was unable to acclimate to HL, when transferred from low light to HL. Its mature leaves bleached, and it showed an increased degree of lipid peroxidation and photoinhibition. In parallel, we tested the photosensitivity of an ascorbate-deficient xanthophyll cycle mutant, vtc2npq1, which also lacks zeaxanthin and nearly all qE. The double mutant bleached sooner and had higher degrees of lipid peroxidation and photoinhibition than the vtc2 mutant. This was in contrast to the npq1 single mutant that showed only slight deviations from the wild-type phenotype under the conditions used. These results demonstrate the antioxidant role of ascorbate in the acclimation process to HL and point to the relative importance of ascorbate in comparison with other photoprotective processes, such as specific xanthophylls or feedback de-excitation. The results also provide further support for the proposed role of zeaxanthin as an antioxidant and lipid stabilizer.

PsbS-dependent enhancement of feedback de-excitation protects photosystem II from photoinhibition.

Feedback de-excitation (qE) regulates light harvesting in plants to prevent inhibition of photosynthesis when light absorption exceeds photosynthetic capacity. Although the mechanism of qE is not completely understood, it is known to require a low thylakoid lumen pH, de-epoxidized xanthophylls, and the photosystem II protein PsbS. During a short-term 4-h exposure to excess light, three PsbS- and qE-deficient Arabidopsis thaliana mutants that differed in xanthophyll composition were more photoinhibited than the wild type. The extent of photoinhibition was the same in all of the mutants, suggesting that qE capacity rather than xanthophyll composition is critical for photoprotection in short-term high light, in contrast to longer-term high light conditions (days) when additional antioxidant roles of specific xanthophylls are evident. Plants with a 2-fold increase in qE capacity were generated by overexpression of PsbS, demonstrating that the level of PsbS limits the qE capacity in wild-type Arabidopsis. These results are consistent with the idea that variations in PsbS expression are responsible for species-specific and environmentally induced differences in qE capacity observed in nature. Furthermore, plants with higher qE capacity were more resistant to photoinhibition than the wild type. Increased qE was associated with decreased photosystem II excitation pressure and changes in the fractional areas of chlorophyll a fluorescence lifetime distributions, but not the lifetime centers, suggesting that qE protects from photoinhibition by preventing overreduction of photosystem II electron acceptors. Engineering of qE capacity by PsbS overexpression could potentially yield crop plants that are more resistant to environmental stress.

Rapid regulation of light harvesting and plant fitness in the field.

We used Arabidopsis thaliana mutants to examine how a photosynthetic regulatory process, the qE-type or DeltapH-dependent nonphotochemical quenching, hereafter named feedback de-excitation, influences plant fitness in different light environments. We show that the feedback de-excitation is important for plant fitness in the field and in fluctuating light in a controlled environment but that it does not affect plant performance under constant light conditions. Our findings demonstrate that the feedback de-excitation confers a strong fitness advantage under field conditions and suggest that this advantage is due to the increase in plant tolerance to variation in light intensity rather than tolerance to high-intensity light itself.