Chlororespiration and cyclic electron flow around PSI during photosynthesis and plant stress response

Besides major photosynthetic complexes of oxygenic photosynthesis, new electron carriers have been identified in thylakoid membranes of higher plant chloroplasts. These minor components, located in the stroma lamellae, include a plastidial NAD(P)H dehydrogenase (NDH) complex and a plastid terminal plastoquinone oxidase (PTOX). The NDH complex, by reducing plastoquinones (PQs), participates in one of the two electron transfer pathways operating around photosystem I (PSI), the other likely involving a still uncharacterized ferredoxin-plastoquinone reductase (FQR) and the newly discovered PGR5. The existence of a complex network of mechanisms regulating expression and activity of the NDH complex, and the presence of higher amounts of NDH complex and PTOX in response to environmental stress conditions the phenotype of mutants, indicate that these components likely play a role in the acclimation of photosynthesis to changing environmental conditions. Based on recently published data, we propose that the NDH-dependent cyclic pathway around PSI participates to the ATP supply in conditions of high ATP demand (such as high temperature or water limitation) and together with PTOX regulates cyclic electron transfer activity by tuning the redox state of intersystem electron carriers. In response to severe stress conditions, PTOX associated to the NDH and/or the PGR5 pathway may also limit electron pressure on PSI acceptor and prevent PSI photoinhibition.     

Stimulation of chlororespiration by heat and high light intensity in oat plants

High irradiance and moderate heat inhibit the activity of the photosynthetic apparatus of oat (Avena sativa L.) leaves. The incubation of oat leaves under high light intensity in conjunction with high temperatures strongly decreased the maximal quantum yield of photosystem (PS) II, indicating the close synergistic effect of both stress factors on PS II inhibition and the subsequent irreversible damage to the photosynthetic apparatus. The PS I A/B protein levels remained similar to control values in leaves incubated under high light intensity or moderate heat, and decreased only when both stress factors were simultaneously applied. Immunoblot analysis of thylakoid membranes using specific antibodies raised against the NDH-K subunit of the thylakoidal NADH dehydrogenase complex (NADH DH) and against plastid terminal oxidase (PTOX) revealed an increase in the amount of both proteins in response to high light intensity and/or heat treatments. In addition, these stress treatments were seen to stimulate the activity of electron donation by NADPH and ferredoxin to plastoquinone, the PTOX activity in plastoquinone oxidation and the NADH DH activity in thylakoid membranes. Incubation with n-propyl gallate (an inhibitor of PTOX) inhibited the increase of NDH-K and PTOX levels under high light intensity and heat, and slightly stimulated the activity of electron donation by NADPH and ferredoxin to plastoquinone. Antimycin A (an inhibitor of cyclic electron flow) increased the NADH DH activity and preserved the levels of NDH-K and PTOX in thylakoid membranes from leaves incubated under high light intensity and heat. The up-regulation of the PTOX and the thylakoidal NADH DH complex under these stress conditions supports a role for chlororespiration in the protection against high irradiance and moderate heat.     

UDP-glucose:(6-methoxy)podophyllotoxin 7-O-glucosyltransferase from suspension cultures of Linum nodiflorum

Cell cultures of Linum species store 6-methoxypodophyllotoxin (MPTOX), podophyllotoxin (PTOX) and related lignans as O-glucosides. UDP-glucose:(M)PTOX 7-O-glucosyltransferase has been detected and characterised in protein preparations of suspension-cultured cells of Linum nodiflorum L. (Linaceae). The maximal lignan glucoside contents in the cells are preceded by a rapid increase of the specific glucosyltransferase activity on day six of the culture period. MPTOX glucoside is the major lignan with up to 1.18 mg g(-1) of the cell dry wt which is more than 30-fold of the PTOX glucoside content. Of the three aryltetralin lignans tested as substrates, PTOX and MPTOX display comparable apparent K(m) values of 4.7 and 5.4 microM, respectively. 5'-Demethoxy-6-methoxypodophyllotoxin is converted with the highest velocity of 25.2 pkat mg(-1) while also possessing a higher K(m) of 14.7 microM. Two-substrate test series indicate that all three compounds compete for the active site of a single protein. The structurally similar lignan beta-peltatin acts as competitive inhibitor as well. However, the 6-O-glucosidation is most likely catalysed by a separate enzyme. The (M)PTOX 7-O-glucosyltransferase works best at a pH around 9 and a temperature around 35 degrees C. A 15-30% increase of the reaction rate is effected by the addition of 0.9 mM Mn(2+).     

Evidence for alternative electron sinks to photosynthetic carbon assimilation in the high mountain plant species Ranunculus glacialis

The high mountain plant species Ranunculus glacialis has a low antioxidative scavenging capacity and a low activity of thermal dissipation of excess light energy despite its growth under conditions of frequent light and cold stress. In order to examine whether this species is protected from over-reduction by matching photosystem II (PSII) electron transport (ETR) and carbon assimilation, both were analysed simultaneously at various temperatures and light intensities using infrared gas absorption coupled with chlorophyll fluorescence. ETR exceeded electron consumption by carbon assimilation at higher light intensities and at all temperatures tested, necessitating alternative electron sinks. As photorespiration might consume the majority of excess electrons, photorespiration was inhibited by either high internal leaf CO₂ molar ratio (C_i), low oxygen partial pressure (0.5% oxygen), or both. At 0.5% oxygen ETR was significantly lower than at 21% oxygen. At 21% oxygen, however, ETR still exceeded carbon assimilation at high C_i, suggesting that excess electrons are transferred to another oxygen consuming reaction when photorespiration is blocked. Nevertheless, photorespiration does contribute to electron consumption. While the activity of the water –water cycle to electron consumption is not known in leaves of R. glacialis, indirect evidence such as the high sensitivity to oxidative stress and the low initial NADP-malate dehydrogenase (NADP-MDH) activity suggests only a minor contribution as an alternative electron sink. Alternatively, the plastid terminal oxidase (PTOX) may transfer excess electrons to oxygen. This enzyme is highly abundant in R. glacialis leaves and exceeds the PTOX content of every other plant species so far examined, including those of transgenic tomato leaves overexpressing the PTOX protein. Finally, PTOX contents strongly declined during deacclimation of R. glacialis plants, suggesting their important role in photoprotection. Ranunculus glacialis is the first reported plant species with such a high PTOX protein content.     

Modulation of photosynthetic electron transport in the absence of terminal electron acceptors: Characterization of the rbcL deletion mutant of tobacco

Tobacco rbcL deletion mutant, which lacks the key enzyme Rubisco for photosynthetic carbon assimilation, was characterized with respect to thylakoid functional properties and protein composition. The ΔrbcL plants showed an enhanced capacity for dissipation of light energy by non-photochemical quenching which was accompanied by low photochemical quenching and low overall photosynthetic electron transport rate. Flash-induced fluorescence relaxation and thermoluminescence measurements revealed a slow electron transfer and decreased redox gap between Q_A and Q_B, whereas the donor side function of the Photosystem II (PSII) complex was not affected. The 77 K fluorescence emission spectrum of ΔrbcL plant thylakoids implied a presence of free light harvesting complexes. Mutant plants also had a low amount of photooxidisible P700 and an increased ratio of PSII to Photosystem I (PSI). On the other hand, an elevated level of plastid terminal oxidase and the lack of F₀ ‘dark rise’ in fluorescence measurements suggest an enhanced plastid terminal oxidase-mediated electron flow to O₂ in ΔrbcL thylakoids. Modified electron transfer routes together with flexible dissipation of excitation energy through PSII probably have a crucial role in protection of PSI from irreversible protein damage in the ΔrbcL mutant under growth conditions. This protective capacity was rapidly exceeded in ΔrbcL mutant when the light level was elevated resulting in severe degradation of PSI complexes.     

Stimulation of chlororespiration by drought under heat and high illumination in Rosa meillandina

Rosa meillandina plants were used to study the effects of water deficit on photosynthesis and chlororespiration. Plants showed high tolerance to heat and high illumination in controlled conditions that ensured that there was no water deficit. However, when heat and high illumination were accompanied by low watering photosynthetic linear electron transport was down regulated, as indicated by the reduced photochemistry efficiency of PS II, which was associated with an increase in the non-photochemical quenching of fluorescence. In addition to the effects on the photosynthetic activity, changes were also observed in the plastidial NDH complex, PTOX and PGR5. In plants exposed to heat and high illumination without water deficit, the activities and amounts of the chlororespiration enzymes, NDH complex and PTOX, remained similar to the control and only increased in response to drought, high light and heat stress, applied together. In contrast, both the PS I activity and the amount of PGR5 polypeptide were higher in plants exposed to heat and high illumination without water deficit than in those with water deficit. The results indicated that in the conditions studied, the contribution of chlororespiration to regulating photosynthetic electron flow is not relevant when there is no water deficit, and another pathway, such as cyclic electron flow involving PGR5 polypeptide, may be more important. However, when PS II activity is inhibited by drought, chlororespiration, together with other routes of electron input to the electron transfer chain, is probably essential.     

Functional and molecular characterization of plastid terminal oxidase from rice (Oryza sativa)

The plastid terminal oxidase (PTOX) is a plastohydroquinone:oxygen oxidoreductase that shares structural similarities with alternative oxidases (AOX). Multiple roles have been attributed to PTOX, such as involvement in carotene desaturation, a safety valve function, participation in the processes of chlororespiration and setting the redox poise for cyclic electron transport. We have investigated a homogenously pure MBP fusion of PTOX. The protein forms a homo-tetrameric complex containing 2 Fe per monomer and is very specific for the plastoquinone head-group. The reaction kinetics were investigated in a soluble monophasic system using chemically reduced decyl-plastoquinone (DPQ) as the model substrate and, in addition, in a biphasic (liposomal) system in which DPQ was reduced with DT-diaphorase. While PTOX did not detectably produce reactive oxygen species in the monophasic system, their formation was observed by room temperature EPR in the biphasic system in a [DPQH₂] and pH-dependent manner. This is probably the result of the higher concentration of DPQ achieved within the partial volume of the lipid bilayer and a higher Km observed with PTOX-membrane associates which is ≈ 47 mM compared to the monophasic system where a Km of ≈ 74 μM was determined. With liposomes and at the basic stromal pH of photosynthetically active chloroplasts, PTOX was antioxidant at low [DPQH₂] gaining prooxidant properties with increasing quinol concentrations. It is concluded that in vivo, PTOX can act as a safety valve when the steady state [PQH₂] is low while a certain amount of ROS is formed at high light intensities.     

Effect of constitutive expression of bacterial phytoene desaturase CRTI on photosynthetic electron transport in Arabidopsis thaliana

The constitutive expression of the bacterial carotene desaturase (CRTI) in Arabidopsis thaliana leads to increased susceptibility of leaves to light-induced damage. Changes in the photosynthetic electron transport chain rather than alterations of the carotenoid composition in the antenna were responsible for the increased photoinhibition. A much higher level of superoxide/hydrogen peroxide was generated in the light in thylakoid membranes from the CRTI expressing lines than in wild-type while the level of singlet oxygen generation remained unchanged. The increase in reactive oxygen species was related to the activity of plastid terminal oxidase (PTOX) since their generation was inhibited by the PTOX-inhibitor octyl gallate, and since the protein level of PTOX was increased in the CRTI-expressing lines. Furthermore, cyclic electron flow was suppressed in these lines. We propose that PTOX competes efficiently with cyclic electron flow for plastoquinol in the CRTI-expressing lines and that it plays a crucial role in the control of the reduction state of the plastoquinone pool.     

Involvement of chlororespiration in chilling stress in the tropical species Spathiphyllum wallisii

Spathiphyllum wallisii plants were used to study the effect of chilling stress under high illumination on photosynthesis and chlororespiration. Leaves showed different responses that depended on root temperature. When stem, but not root, was chilled, photosystem II (PSII) was strongly photoinhibited. However, when the whole plant was chilled, the maximal quantum yield of PSII decreased only slightly below the normal values and cyclic electron transport was stimulated. Changes were also observed in the chlororespiration enzymes and PGR5. In whole plants chilled under high illumination, the amounts of NADH dehydrogenase (NDH) complex and plastid terminal oxidase (PTOX) remained similar to control and increased when only stem was chilled. In contrast, the amount of PGR5 polypeptide was higher in plants when both root and stem were chilled than in plants in which only stem was chilled. The results indicated that the contribution of chlororespiration to regulating photosynthetic electron flow is not relevant when the whole plant is chilled under high light, and that another pathway, such as cyclic electron flow involving PGR5 polypeptide, may be more important. However, when PSII activity is strongly photoinhibited in plants in which only stem is chilled, chlororespiration, together with other routes of electron input to the electron transfer chain, is probably essential.     

Effect of Chlamydomonas plastid terminal oxidase 1 expressed in tobacco on photosynthetic electron transfer

The plastid terminal oxidase PTOX is a plastohydroquinone:oxygen oxidoreductase that is important for carotenoid biosynthesis and plastid development. Its role in photosynthesis is controversially discussed. Under a number of abiotic stress conditions, the protein level of PTOX increases. PTOX is thought to act as a safety valve under high light protecting the photosynthetic apparatus against photodamage. However, transformants with high PTOX level were reported to suffer from photoinhibition. To analyze the effect of PTOX on the photosynthetic electron transport, tobacco expressing PTOX‐1 from Chlamydomonas reinhardtii (Cr‐PTOX1) was studied by chlorophyll fluorescence, thermoluminescence, P700 absorption kinetics and CO₂ assimilation. Cr‐PTOX1 was shown to compete very efficiently with the photosynthetic electron transport for PQH₂. High pressure liquid chromatography (HPLC) analysis confirmed that the PQ pool was highly oxidized in the transformant. Immunoblots showed that, in the wild‐type, PTOX was associated with the thylakoid membrane only at a relatively alkaline pH value while it was detached from the membrane at neutral pH. We present a model proposing that PTOX associates with the membrane and oxidizes PQH₂ only when the oxidation of PQH₂ by the cytochrome b₆f complex is limiting forward electron transport due to a high proton gradient across the thylakoid membrane.