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Abstract: 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 ≈47mM compared to the monophasic system where a Km of ≈74μM 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. [Copyright &y& Elsevier]

Plastids have retained from their cyanobacterial ancestor a fragment of the respiratory electron chain comprising an NADPH dehydrogenase and a diiron oxidase, which sustain the so-called chlororespiration pathway. Despite its very low turnover rates compared with photosynthetic electron flow, knocking out the plastid terminal oxidase (PTOX) in plants or microalgae leads to severe phenotypes that encompass developmental and growth defects together with increased photosensitivity. On the basis of a phylogenetic and structural analysis of the enzyme, we discuss its physiological contribution to chloroplast metabolism, with an emphasis on its critical function in setting the redox poise of the chloroplast stroma in darkness. The emerging picture of PTOX is that of an enzyme at the crossroads of a variety of metabolic processes, such as, among others, the regulation of cyclic electron transfer and carotenoid biosynthesis, which have in common their dependence on the redox state of the plastoquinone pool, set largely by the activity of PTOX in darkness. [ABSTRACT FROM AUTHOR]

The mitochondrial alternative oxidase is a diiron carboxylate quinol oxidase (Dox) found in plants and some fungi and protists, but not animals. The plastid terminal oxidase is distantly related to alternative oxidase and is most likely also a Dox protein. Database searches revealed that the α-proteobacterium Novosphingobium aromaticivorans and the cyanobacteria Nostoc sp. PCC7120, Synechococcus sp. WH8102 and Prochlorococcus marinus subsp. pastoris CCMP1378 each possess a Dox homolog. Each prokaryotic protein conforms to the current structural models of the Dox active site and phylogenetic analyses suggest that the eukaryotic Dox genes arose from an ancestral prokaryotic gene. [Copyright &y& Elsevier]

The significance of plastid terminal oxidase ( PTOX) in phytoene desaturation and chloroplast function has been demonstrated using PTOX-deficient mutants, particularly in Arabidopsis. However, studies on its role in monocots are lacking. Here, we report cloning and characterization of the rice ( Oryza sativa) PTOX1 gene., Using Ecotype Targeting Induced Local Lesions IN Genomes (Eco TILLING) and TILLING as forward genetic tools, we identified the causative mutation of an EMS mutant characterized by excessive tillering, semi-dwarfism and leaf variegation that corresponded to the PTOX1 gene., The tillering and semi-dwarf phenotypes of the ptox1 mutant are similar to phenotypes of known strigolactone ( SL)-related rice mutants, and both phenotypic traits could be rescued by application of the synthetic SL GR24. The ptox1 mutant accumulated phytoene in white leaf sectors with a corresponding deficiency in β-carotene, consistent with the expected function of PTOX1 in promoting phytoene desaturase activity. There was also no accumulation of the carotenoid-derived SL ent-2′- epi-5-deoxystrigol in root exudates. Elevated concentrations of auxin were detected in the mutant, supporting previous observations that SL interaction with auxin is important in shoot branching control., Our results demonstrate that PTOX1 is required for both carotenoid and SL synthesis resulting in SL-deficient phenotypes in rice. [ABSTRACT FROM AUTHOR]

Plastid terminal oxidase (PTOX) is a chloroplast enzyme that catalyzes oxidation of plastoquinol (PQH2) and reduction of molecular oxygen to water. Its function has been associated with carotenoid biosynthesis, chlororespiration and environmental stress responses in plants. In the majority of plant species, a single gene encodes the protein and little is known about events of PTOX gene duplication and their implication to plant metabolism. Previously, two putative PTOX (PTOX1 and 2) genes were identified in Glycine max, but the evolutionary origin and the specific function of each gene was not explored. Phylogenetic analyses revealed that this gene duplication occurred apparently during speciation involving the Glycine genus ancestor, an event absent in all other available plant leguminous genomes. Gene expression evaluated by RT-qPCR and RNA-seq data revealed that both PTOX genes are ubiquitously expressed in G. max tissues, but their mRNA levels varied during development and stress conditions. In development, PTOX1 was predominant in young tissues, while PTOX2 was more expressed in aged tissues. Under stress conditions, the PTOX transcripts varied according to stress severity, i.e., PTOX1 mRNA was prevalent under mild or moderate stresses while PTOX2 was predominant in drastic stresses. Despite the high identity between proteins (97%), molecular docking revealed that PTOX1 has higher affinity to substrate plastoquinol than PTOX2. Overall, our results indicate a functional relevance of this gene duplication in G. max metabolism, whereas PTOX1 could be associated with chloroplast effectiveness and PTOX2 to senescence and/or apoptosis. [ABSTRACT FROM AUTHOR]

Abstract: Oat (Avena sativa L., cv. Prevision) plants grown under high irradiance and moderate heat showed a decrease in the quantum yield of photosystem (PS) II and the capacity of photosynthetic electron transport as consequence of the PS II inhibition. Acclimation to the same conditions increased the tolerance to subsequent stresses involving even higher heat and light levels, in young and mature leaves, but not in senescent leaves. PS I was more stable than PS II, and its activity was slightly higher in young and mature leaves from acclimated plants than in those from control plants, probably due to the protective role which PS I plays in several alternative PS I-driven routes of electron transport. 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 young and mature leaves from plants acclimated to high light intensity and moderate heat. In addition, these stresses were seen to stimulate PTOX activity in plastoquinone oxidation and the NADH DH activity in thylakoid membranes from young and mature leaves. However, in senescent leaves chlororespiratory activities and NDH-K and PTOX levels were similar in stressed and unstressed plants, although the PTOX levels were lower in senescent than in young and mature leaves; the level and activity of the NADH DH complex increased during senescence. The upregulation of the PTOX and the thylakoidal NADH DH complex under these stress conditions supports a role for chlororespiration in the acclimation of oat leaves to high irradiance and moderate heat during development. [Copyright &y& Elsevier]

The immutans ( im ) variegation mutant of Arabidopsis defines the gene for PTOX (plastid terminal oxidase), a versatile plastoquinol oxidase in chloroplast membranes. In this report we used im to gain insight into the function of PTOX in etioplasts of dark-grown seedlings. We discovered that PTOX helps control the redox state of the plastoquinone (PQ) pool in these organelles, and that it plays an essential role in etioplast metabolism by participating in the desaturation reactions of carotenogenesis and in one or more redox pathways mediated by PGR5 (PROTON GRADIENT REGULATION 5) and NDH (NAD(P)H dehydrogenase), both of which are central players in cyclic electron transport. We propose that these elements couple PTOX with electron flow from NAD(P)H to oxygen, and by analogy to chlororespiration (in chloroplasts) and chromorespiration (in chromoplasts), we suggest that they define a respiratory process in etioplasts that we have termed “etiorespiration”. We further show that the redox state of the PQ pool in etioplasts might control chlorophyll biosynthesis, perhaps by participating in mechanisms of retrograde (plastid-to-nucleus) signaling that coordinate biosynthetic and photoprotective activities required to poise the etioplast for light development. We conclude that PTOX is an important component of metabolism and redox sensing in etioplasts. [ABSTRACT FROM AUTHOR]

S pathiphyllum 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. [ABSTRACT FROM AUTHOR]

This work studies the effects of water deficit and heat, as well as the involvement of chlororespiration and the ferredoxin-mediated cyclic pathway, on the tolerance of photosynthesis to high light intensity in Hibiscus rosa-sinensis plants. Drought and heat resulted in the down--regulation of photosynthetic linear electron transport in the leaves, although only a slight decrease in variable fluorescence (Fv)/maximal fluorescence (Fm) was observed, indicating that the chloroplast was protected by mechanisms that dissipate excess excitation energy to prevent damage to the photosynthetic apparatus. The incubation of leaves from unstressed plants under high light intensity resulted in an increase of the activity of electron donation by nicotinamide adenine dinucleotide phosphate (NADPH) and ferredoxin to plastoquinone, but no increase was observed in plants exposed to water deficit, suggesting that cyclic electron transport was stimulated by high light only in control plants. In contrast, the activities of the chlororespiration enzymes (NADH dehydrogenase (NDH) complex and plastid terminal oxidase (PTOX)) increased after incubation under high light intensity in leaves of the water deficit plants, but not in control plants, suggesting that chlororespiration was stimulated in stressed plants. The results indicate that the relative importance of chlororespiration and the cyclic electron pathway in the tolerance of photosynthesis to high illumination differs under stress conditions. When plants were not subjected to stress, the contribution of chlororespiration to photosynthetic electron flow regulation was not relevant, and another pathway, such as the ferredoxin-mediated cyclic pathway, was more important. However, when plants were subjected to water deficit and heat, chlororespiration was probably essential. [ABSTRACT FROM AUTHOR]

GUN4 is a regulatory subunit of Mg-chelatase involved in the control of tetrapyrrole synthesis in plants and cyanobacteria. Here, we report the first characterization of a gun4 insertion mutant of the unicellular green alga Chlamydomonas reinhardtii. The mutant contains 50% of chlorophyll as compared to wild-type and accumulates ProtoIX. In contrast to the increase in LHC transcription, the accumulation of most LHC proteins is drastically diminished, implying posttranscriptional down-regulation in the absence of transcriptional coordination. We found that 803 genes change their expression level in gun4 as compared to wild-type, by RNA-Seq, and this wide-ranging effect on transcription is apparent under physiological conditions. Besides LHCs, we identified transcripts encoding enzymes of the tetrapyrrole pathway and factors involved in signal transduction, transcription, and chromatin remodeling. Moreover, we observe perturbations in electron transport with a strongly decreased PSI-to-PSII ratio. This is accompanied by an enhanced activity of the plastid terminal oxidase (PTOX) that could have a physiological role in decreasing photosystem II excitation pressure. [ABSTRACT FROM AUTHOR]

Two species of Brassica were used to study their acclimation to heat and high illumination during the first stages of development. One, Brassica fruticulosa, is a wild species from south-east Spain and is adapted to both heat and high light intensity in its natural habitat, while the other, Brassica oleracea, is an agricultural species that is widely cultivated throughout the world. Growing Brassica plants under high irradiance and moderate heat was seen to affect the growth parameters and the functioning of the photosynthetic apparatus. The photosystem II (PSII) quantum yields and the capacity of photosynthetic electron transport, which were lower in B. fruticulosa than in B. oleracea, decreased in B. oleracea plants when grown under stress conditions, indicating inhibition of PSII. However, in B. fruticulosa, the values of these parameters were similar to the values of control plants. Photosystem I (PSI) activity was higher in B. fruticulosa than in B. oleracea, and in both species this activity increased in plants exposed to heat and high illumination. 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 a higher amount of both proteins in B. fruticulosa than in B. oleracea. In addition, PTOX activity in plastoquinone oxidation, and NADH DH activity in thylakoid membranes were higher in the wild species ( B. fruticulosa) than in the agricultural species ( B. oleracea). The results indicate that tolerance to high illumination and heat of the photosynthetic activity was higher in the wild species than in the agricultural species, suggesting that plant adaptation to these stresses in natural conditions favours subsequent acclimation, and that the chlororespiration process is involved in adaptation to heat and high illumination in Brassica. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Plants of Chrysanthemum morifolium (sun species) and Spathiphyllum lanceifolium (shade species) were used to study the effects of chilling stems under high illumination. The stress conditions resulted in a greater accumulation of H 2 O 2 in C. morifolium than in S. lanceifolium , and in the down–regulation of photosynthetic linear electron transport in both species. However, only a slight decrease in the maximal quantum yield of PSII was observed under unfavorable conditions in both species, suggesting that mechanisms exist in the chloroplasts that dissipate excess excitation energy and prevent damage to the photosynthetic apparatus. Additionally, changes were observed in the PGR5 polypeptide involved in cyclic electron flow around PSI and in chlororespiratory enzymes (plastidial NDH complex and PTOX). The level of PGR5 increased significantly only in chilled plants of C. morifolium, whereas the levels of the PTOX and NDH-H polypeptide of the plastidial NDH complex and the NDH activity increased significantly only in chilled plants of S. lanceifolium . These findings suggest that the cyclic electron flow involving PGR5 is more active in C. morifolium , while in S. lanceifolium , other mechanisms involving chlororespiratory enzymes are stimulated in response to chilling and high light, resulting in less H 2 O 2 being accumulated in leaves. [ABSTRACT FROM AUTHOR]

Spathiphyllum wallisii plants were sensitive to temperature stress under high illumination, although the susceptibility of leaves to stress may be modified by root temperature. Leaves showed higher tolerance to high illumination, in both cold and heat conditions, when the roots were cooled, probably because the chloroplast were protected by excess excitation energy dissipation mechanisms such as cyclic electron transport. When the roots were cooled both the activity of electron donation by NADPH and ferredoxin to plastoquinone and the amount of PGR5 polypeptide, an essential component of cyclic electron flow around PSI, increased. However, when the stems were heated or cooled under high illumination, but the roots were heated, the quantum yield of PSII decreased considerably and neither the electron donation activity by NADPH and ferredoxin to plastoquinone nor the amount of PGR5 polypeptide increased. In such conditions, the cyclic electron flow cannot be enhanced by high light and PSII is damaged as a result of insufficient dissipation of excess light energy. Additionally, the damage to PSII induced the increase in both chlororespiratory enzymes, NDH complex and PTOX. [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: Sun (Chrysanthemum morifolium) and shade (Spathiphyllum wallisii) plants were used to study the effects of drought, heat and high illumination. The stress conditions caused a greater accumulation of hydrogen peroxide in Chrysanthemum morifolium than in Spathiphyllum wallisii leaves. They also resulted in down-regulation of linear electron transport in the leaves of both species, as indicated by a gradual reduction in the photochemistry efficiency of PS II, which was associated with an increase in the non-photochemical quenching of fluorescence. Only a slight decrease in F v/F m was observed under stress conditions in either plant species, suggesting that the chloroplast is protected by mechanisms that dissipate excess excitation energy to prevent damage to the photosynthetic apparatus. In addition to the effects on photosynthetic activity, changes were also observed by immunoblot analysis in the plastidial NADH DH complex, PTOX and PGR5. The quantities of the PTOX and NDH–H subunit of the thylakoidal NADH DH complex, and the NADH DH activity in the thylakoid membranes were similar in control plants of both species and increased in stressed plants, particularly in Spathiphyllum wallisii. The level of PGR5 polypeptide was higher in Chrysanthemum morifolium than in Spathiphyllum wallisii control plants, while after stress, the quantity of PGR5 increased significantly in Chrysanthemum morifolium and remained constant in Spathiphyllum wallisii. These results indicate that the relative importance of chlororespiration and the cyclic electron pathways in the tolerance to drought, heat and high illumination differs in sun and shade plants, indicating different adaptive mechanisms to the environment. In the conditions studied, the PGR5-dependent cyclic pathway is more active in Chrysanthemum morifolium, a sun species, whereas in Spathiphyllum wallisii, a shade species, other ways involving the NADH DH complex and PTOX are stimulated in response to stress, which results in lower levels of ROS accumulation in the leaves. [Copyright &y& Elsevier]

Abstract: Sun (Chrysanthemum morifolium) and shade (Spathiphyllum wallisii) plants were used to study the effects of the inhibitors antimycin A and n-propyl gallate on photosynthesis, using chlorophyll fluorescence imaging. Photochemical efficiency in sun and shade plants was not inhibited by antimycin A under low light intensity, but the effective PS II quantum yield was inhibited under high illumination and heat, more so in sun than in shade species. The inhibitor n-propyl gallate had no or little effect on photochemical efficiency under low or high illumination and heat in control plants of both species. However, in stressed plants the effect of n-propyl gallate under high illumination and heat increased, strongly inhibiting the effective PS II quantum yield in S. wallisii. In addition to the effects produced by the inhibitors, changes were also observed by immunoblot analysis in the plastidial NADH DH complex, PTOX and PGR5. The quantities of PTOX and NDH-H subunit of the thylakoidal NADH DH complex and the NADH DH activity in the thylakoid membranes were similar in control plants of both species and increased in stressed plants, particularly in S. wallisii. However, the level of PGR5 polypeptide increased in C. morifolium but remained similar to control in S. wallisii. The results indicate that under high illumination and heat the cyclic electron flow around the PSI antimycin A-sensitive is important for the photosynthetic function in the sun species while another pathway involving the NADH DH complex and PTOX might be more important in the shade species. [Copyright &y& Elsevier]