Plastid Terminal Oxidase Research Articles

Implications of alternative electron sinks in increased resistance of PSII and PSI photochemistry to high light stress in cold-acclimated Arabidopsis thaliana

Exposure of control (non-hardened) Arabidopsis leaves to high light stress at 5°C resulted in a decrease of both photosystem II (PSII) (45%) and Photosystem I (PSI) (35%) photochemical efficiencies compared to non-treated plants. In contrast, cold-acclimated (CA) leaves exhibited only 35 and 22% decrease of PSII and PSI photochemistry, respectively, under the same conditions. This was accompanied by an accelerated rate of P700(+) re-reduction, indicating an up-regulation of PSI-dependent cyclic electron transport (CET). Interestingly, the expression of the NDH-H gene and the relative abundance of the Ndh-H polypeptide, representing the NDH-complex, decreased as a result of exposure to low temperatures. This indicates that the NDH-dependent CET pathway cannot be involved and the overall stimulation of CET in CA plants is due to up-regulation of the ferredoxin-plastoquinone reductase, antimycin A-sensitive CET pathway. The lower abundance of NDH complex also implies lower activity of the chlororespiratory pathway in CA plants, although the expression level and overall abundance of the other well-characterized component involved in chlororespiration, the plastid terminal oxidase (PTOX), was up-regulated at low temperatures. This suggests increased PTOX-mediated alternative electron flow to oxygen in plants exposed to low temperatures. Indeed, the estimated proportion of O(2)-dependent linear electron transport not utilized in carbon assimilation and not directed to photorespiration was twofold higher in CA Arabidopsis. The possible involvement of alternative electron transport pathways in inducing greater resistance of both PSII and PSI to high light stress in CA plants is discussed.

Investigating the Production of Foreign Membrane Proteins in Tobacco Chloroplasts: Expression of an Algal Plastid Terminal Oxidase

Chloroplast transformation provides an inexpensive, easily scalable production platform for expression of recombinant proteins in plants. However, this technology has been largely limited to the production of soluble proteins. Here we have tested the ability of tobacco chloroplasts to express a membrane protein, namely plastid terminal oxidase 1 from the green alga Chlamydomonas reinhardtii (Cr-PTOX1), which is predicted to function as a plastoquinol oxidase. A homoplastomic plant containing a codon-optimised version of the nuclear gene encoding PTOX1, driven by the 16S rRNA promoter and 5′UTR of gene 10 from phage T7, was generated using a particle delivery system. Accumulation of Cr-PTOX1 was shown by immunoblotting and expression in an enzymatically active form was confirmed by using chlorophyll fluorescence to measure changes in the redox state of the plastoquinone pool in leaves. Growth of Cr-PTOX1 expressing plants was, however, more sensitive to high light than WT. Overall our results confirm the feasibility of using plastid transformation as a means of expressing foreign membrane proteins in the chloroplast.

SUSCEPTIBILITY AND PROTECTIVE MECHANISMS OF MOTILE AND NON MOTILE CELLS OF HAEMATOCOCCUS PLUVIALIS (CHLOROPHYCEAE) TO PHOTOOXIDATIVE STRESS(1).

The life cycle of the unicellular green alga Haematococcus pluvialis consists of motile and nonmotile stages under typical growing conditions. In this study, we observed that motile cells were more susceptible than nonmotile cells to high light, resulting in a decrease in population density and photo-bleaching. Using two Haematococcus strains, CCAP 34/12 (a motile cell dominated strain) and SAG 34/1b (a nonmotile cell dominated strain), as model systems we investigated the cause of cell death and the protective mechanisms of the cells that survived high light. The death of motile cells under high light was attributed to the generation of excess reactive oxygen species (ROS), which caused severe damage to the photosynthetic components and the membrane system. Motile cells were able to dissipate excess light energy by nonphotochemical quenching and to relax ROS production by a partially up-regulated scavenging enzyme system. However, these strategies were not sufficient to protect the motile cells from high light stress. In contrast, nonmotile cells were able to cope with and survive under high light by (i) relaxing the over-reduced photosynthetic electron transport chain (PETC), thereby effectively utilizing PETC-generated NADPH to produce storage starch, neutral lipid, and astaxanthin, and thus preventing formation of excess ROS; (ii) down-regulating the linear electron transport by decreasing the level of cytochrome f; and (iii) consuming excess electrons produced by PSII via a significantly enhanced plastid terminal oxidase pathway.

Alternative oxidases (AOX1a and AOX2) can functionally substitute for plastid terminal oxidase in Arabidopsis chloroplasts.

The immutans (im) variegation mutant of Arabidopsis thaliana is caused by an absence of PTOX, a plastid terminal oxidase bearing similarity to mitochondrial alternative oxidase (AOX). In an activation tagging screen for suppressors of im, we identified one suppression line caused by overexpression of AOX2. AOX2 rescued the im defect by replacing the activity of PTOX in the desaturation steps of carotenogenesis. Similar results were obtained when AOX1a was reengineered to target the plastid. Chloroplast-localized AOX2 formed monomers and dimers, reminiscent of AOX regulation in mitochondria. Both AOX2 and AOX1a were present in higher molecular weight complexes in plastid membranes. The presence of these proteins did not generally affect steady state photosynthesis, aside from causing enhanced nonphotochemical quenching in both lines. Because AOX2 was imported into chloroplasts using its own transpeptide, we propose that AOX2 is able to function in chloroplasts to supplement PTOX activity during early events in chloroplast biogenesis. We conclude that the ability of AOX1a and AOX2 to substitute for PTOX in the correct physiological and developmental contexts is a striking example of the capacity of a mitochondrial protein to replace the function of a chloroplast protein and illustrates the plasticity of the photosynthetic apparatus.

Plastid terminal oxidase 2 (PTOX2) is the major oxidase involved in chlororespiration in Chlamydomonas

By homology with the unique plastid terminal oxidase (PTOX) found in plants, two genes encoding oxidases have been found in the Chlamydomonas genome, PTOX1 and PTOX2. Here we report the identification of a knockout mutant of PTOX2. Its molecular and functional characterization demonstrates that it encodes the oxidase most predominantly involved in chlororespiration in this algal species. In this mutant, the plastoquinone pool is constitutively reduced under dark-aerobic conditions, resulting in the mobile light-harvesting complexes being mainly, but reversibly, associated with photosystem I. Accordingly, the ptox2 mutant shows lower fitness than wild type when grown under phototrophic conditions. Single and double mutants devoid of the cytochrome b(6)f complex and PTOX2 were used to measure the oxidation rates of plastoquinols via PTOX1 and PTOX2. Those lacking both the cytochrome b(6)f complex and PTOX2 were more sensitive to light than the single mutants lacking either the cytochrome b(6)f complex or PTOX2, which discloses the role of PTOX2 under extreme conditions where the plastoquinone pool is overreduced. A model for chlororespiration is proposed to relate the electron flow rate through these alternative pathways and the redox state of plastoquinones in the dark. This model suggests that, in green algae and plants, the redox poise results from the balanced accumulation of PTOX and NADPH dehydrogenase.

Physiological roles of plastid terminal oxidase in plant stress responses

The plastid terminal oxidase (PTOX) is a plastoquinol oxidase localized in the plastids of plants. It is able to transfer electrons from plastoquinone (PQ) to molecular oxygen with the formation of water. Recent studies have suggested that PTOX is beneficial for plants under environmental stresses, since it is involved in the synthesis of photoprotective carotenoids and chlororespiration, which could potentially protect the chloroplast electron transport chain (ETC) from over-reduction. The absence of PTOX in plants usually results in photo-bleached variegated leaves and impaired adaptation to environment alteration. Although PTOX level and activity has been found to increase under a wide range of stress conditions, the functions of plant PTOX in stress responses are still disputed now. In this paper, the possible physiological roles of PTOX in plant stress responses are discussed based on the recent progress.

Responses of psbA, hli and ptox genes to changes in irradiance in marine Synechococcus and Prochlorococcus

Expression of 3 gene families involved with photoacclimation — psbA (encoding the photosystem II reaction center protein D1), hli (encoding the high-light inducible proteins), and ptox (encoding the plastid terminal oxidase) — was compared in the marine cyanobacteria Synechococcus WH8102 and Prochlorococcus MED4 acclimated to either low or high light. These 2 strains, adapted for growth in oligotrophic marine environments, have distinct light-harvesting systems and respond differently to changes in irradiance. In response to growth at higher irradiance, Synechococcus WH8102 increased expression of the psbA multigene family (psbA1-4) 5-fold. Within this gene family, the expression of psbA2 increased 60-fold. Expression of 4 hli genes increased 2- to 5-fold, whereas expression of the ptox gene decreased 3-fold. In comparison, expression of the psbA gene increased 2-fold in Prochlorococcus MED4 cultures grown at higher irradiances. Expression of the Prochlorococcus MED4 hli6-9 and hli16-19 operons increased 11- to 14-fold, while ptox expression increased 3-fold. Using psbA induction as a standard for acclimation to changes in irradiance, we observed that the induction ratio of ptox:psbA1 and hli:psbA1 was 144 and 70 times greater, respec- tively, in Prochlorococcus MED4 compared with Synechococcus WH8102. These observations sug- gest that induction of ptox and hli may play a key role in the phototolerance of Prochlorococcus MED4. Conversely, the induction of psbA, and the synthesis of the PSII reaction center protein D1, may be critical for the acclimation of Synechococcus WH8102 to high irradiances.

Interplay between non-photochemical plastoquinone reduction and re-oxidation in pre-illuminated Chlamydomonas reinhardtii: a chlorophyll fluorescence study

In photosynthetic eukaryotes, the redox state of the plastoquinone (PQ) pool is an important sensor for mechanisms that regulate the photosynthetic electron transport. In higher plants, a multimeric nicotinamide adenine dinucleotide (phosphate) (NAD(P))H dehydrogenase (NDH) complex and a plastid terminal oxidase (PTOX) are involved in PQ redox homeostasis in the dark. We recently demonstrated that in the microalgae Chlamydomonas reinhardtii, which lacks the multimeric NDH complex of higher plants, non-photochemical PQ reduction is mediated by a monomeric type-II NDH (Nda2). In this study, we further explore the nature and the importance of non-photochemical PQ reduction and oxidation in relation to redox homeostasis in this alga by recording the 'dark' chlorophyll fluorescence transients of pre-illuminated algal samples. From the observation that this fluorescence transient is modified by addition of propyl gallate, a known inhibitor of PTOX, and in a Nda2-deficient strain we conclude that it reflects post-illumination changes in the redox state of PQ resulting from simultaneous PTOX and Nda2 activity. We show that the post-illumination fluorescence transient can be used to monitor changes in the relative rates of the non-photochemical PQ reduction and reoxidation in response to different physiological situations. We study this fluorescence transient in algae acclimated to high light and in a mutant deficient in mitochondrial respiration. Some of our observations indicate that the chlororespiratory pathway participates in redox homeostasis in C. reinhardtii.

Marine cyanophages: tinkering with the electron transport chain

Marine cyanophages: tinkering with the electron transport chain

Cell physiology of plants growing in cold environments

The life of plants growing in cold extreme environments has been well investigated in terms of morphological, anatomical, and ecophysiological adaptations. In contrast, long-term cellular or metabolic studies have been performed by only a few groups. Moreover, a number of single reports exist, which often represent just a glimpse of plant behavior. The review draws together the literature which has focused on tissue and cellular adaptations mainly to low temperatures and high light. Most studies have been done with European alpine plants; comparably well studied are only two phanerogams found in the coastal Antarctic. Plant adaptation in northern polar regions has always been of interest in terms of ecophysiology and plant propagation, but nowadays, this interest extends to the effects of global warming. More recently, metabolic and cellular investigations have included cold and UV resistance mechanisms. Low-temperature stress resistance in plants from cold environments reflects the climate conditions at the growth sites. It is now a matter of molecular analyses to find the induced genes and their products such as chaperones or dehydrins responsible for this resistance. Development of plants under snow or pollen tube growth at 0 degrees C shows that cell biology is needed to explain the stability and function of the cytoskeleton. Many results in this field are based on laboratory studies, but several publications show that it is not difficult to study cellular mechanisms with the plants adapted to a natural stress. Studies on high light and UV loads may be split in two parts. Many reports describe natural UV as harmful for the plants, but these studies were mainly conducted by shielding off natural UV (as controls). Other experiments apply additional UV in the field and have had practically no negative impact on metabolism. The latter group is supported by the observations that green overwintering plants increase their flavonoids under snow even in the absence of UV. Thus, their defense and antioxidant role dominates. Ultrastructural comparisons were unable to find special light adaptations in plants taken from polar regions vs. high alpine species. The only adaptation found at the subcellular level for most alpine and polar plants are protrusions of the chloroplast envelopes. They are seen as a demand for fast membrane transport requiring additional membrane surface area, whereby the increase in stroma volume may help to support carbohydrate formation. Plants forming such protrusions have to cope with a short vegetation time. These observations are connected to the question as to how photosynthesis works quite well even at or under zero temperatures. The interplay between plastids, mitochondria, and peroxisomes, known as photorespiration, seems to be more intense than in lowland plants. This organelle cooperation serves as a valve for a surplus in solar energy input under cold conditions. Additional metabolic acclimations are under investigation, such as the role of an alternative plastid terminal oxidase. Plants from cold environments may also be seen as ideal objects for studying the combined effects of high light plus cold resistance-from the molecular level to the whole plant adaptation. Modern instrumentation makes it possible to perform vital metabolic measurements under outdoor conditions, and research stations in remote polar and alpine areas provide support for scientists in the preparation of samples for later cellular studies in the home laboratory.

Physiological links among alternative electron transport pathways that reduce and oxidize plastoquinone in Arabidopsis

In addition to linear electron transport from water to NADP(+) , alternative electron transport pathways are believed to regulate photosynthesis. In the two routes of photosystem I (PSI) cyclic electron transport, electrons are recycled from the stromal reducing pool to plastoquinone (PQ), generating additional ΔpH (proton gradient across thylakoid membranes). Plastid terminal oxidase (PTOX) accepts electrons from PQ and transfers them to oxygen to produce water. Although both electron transport pathways share the PQ pool, it is unclear whether they interact in vivo. To investigate the physiological link between PSI cyclic electron transport-dependent PQ reduction and PTOX-dependent PQ oxidation, we characterized mutants defective in both functions. Impairment of PSI cyclic electron transport suppressed leaf variegation in the Arabidopsis immutans (im) mutant, which is defective in PTOX. The im variegation was more effectively suppressed in the pgr5 mutant, which is defective in the main pathway of PSI cyclic electron transport, than in the crr2-2 mutant, which is defective in the minor pathway. In contrast to this chloroplast development phenotype, the im defect alleviated the growth phenotype of the crr2-2 pgr5 double mutant. This was accompanied by partial suppression of stromal over-reduction and restricted linear electron transport. We discuss the function of the alternative electron transport pathways in both chloroplast development and photosynthesis in mature leaves.

Plastid Alternative Oxidase (PTOX) Promotes Oxidative Stress When Overexpressed in Tobacco

Photoinhibition and production of reactive oxygen species were studied in tobacco plants overexpressing the plastid terminal oxidase (PTOX). In high light, these plants was more susceptible to photoinhibition than wild-type plants. Also oxygen-evolving activity of isolated thylakoid membranes from the PTOX-overexpressing plants was more strongly inhibited in high light than in thylakoids from wild-type plants. In contrast in low light, in the PTOX overexpressor, the thylakoids were protected against photoinhibition while in wild type they were significantly damaged. The production of superoxide and hydroxyl radicals was shown by EPR spin-trapping techniques in the different samples. Superoxide and hydroxyl radical production was stimulated in the overexpressor. Two-thirds of the superoxide production was maintained in the presence of DNP-INT, an inhibitor of the cytochrome b(6)f complex. No increase of the SOD content was observed in the overexpressor compared with the wild type. We propose that superoxide is produced by PTOX in a side reaction and that PTOX can only act as a safety valve under stress conditions when the generated superoxide is detoxified by an efficient antioxidant system.

Conserved Active Site Sequences in Arabidopsis Plastid Terminal Oxidase (PTOX): IN VITRO AND IN PLANTA MUTAGENESIS STUDIES

The plastid terminal oxidase (PTOX) is distantly related to the mitochondrial alternative oxidase (AOX). Both are members of the diiron carboxylate quinol oxidase (DOX) class of proteins. PTOX and AOX contain 20 highly conserved amino acids, six of which are Fe-binding ligands. We have previously used in vitro and in planta activity assays to examine the functional importance of the Fe-binding sites. In this report, we conduct alanine-scanning mutagenesis on the 14 other conserved sites using our in vitro and in planta assay procedures. We found that the 14 sites fall into three classes: (i) Ala-139, Pro-142, Glu-171, Asn-174, Leu-179, Pro-216, Ala-230, Asp-287, and Arg-293 are dispensable for activity; (ii) Tyr-234 and Asp-295 are essential for activity; and (iii) Leu-135, His-151, and Tyr-212 are important but not essential for activity. Our data are consistent with the proposed role of some of these residues in active site conformation, substrate binding, and/or catalysis. Titration experiments showed that down-regulation of PTOX to approximately 3% of wild-type levels did not compromise plant growth, at least under ambient growth conditions. This suggests that PTOX is normally in excess, especially early in thylakoid membrane biogenesis.

Photosystem II and pigment dynamics among ecotypes of the green alga Ostreococcus.

We investigated the photophysiological responses of three ecotypes of the picophytoplankter Ostreococcus and a larger prasinophyte Pyramimonas obovata to a sudden increase in light irradiance. The deepwater Ostreococcus sp. RCC809 showed very high susceptibility to primary photoinactivation, likely a consequence of high oxidative stress, which may relate to the recently noted plastid terminal oxidase activity in this strain. The three Ostreococcus ecotypes were all capable of deploying modulation of the photosystem II repair cycle in order to cope with the light increase, but the effective clearance of photoinactivated D1 protein appeared to be slower in the deepwater Ostreococcus sp. RCC809, suggesting that this step is rate limiting in the photosystem II repair cycle in this strain. Moreover, the deepwater Ostreococcus accumulated lutein and showed substantial use of the xanthophyll cycle under light stress, demonstrating its high sensitivity to light fluctuations. The sustained component of the nonphotochemical quenching of fluorescence correlated well with the xanthophyll deepoxidation activity. Comparisons with the larger prasinophyte P. obovata suggest that the photophysiology of Ostreococcus ecotypes requires high photosystem II repair rates to counter a high susceptibility to photoinactivation, consistent with low pigment package effects in their minute-sized cells.

Effect of photon flux densities on regulation of carotenogenesis and cell viability of Haematococcus pluvialis (Chlorophyceae).

The green alga Haematococcus pluvialis produces large amounts of the pink carotenoid astaxanthin under high photon flux density (PFD) and other oxidative stress conditions. However, the regulation and physiological role of carotenogenesis leading to astaxanthin formation is not well understood. Comparative transcriptional expression of five carotenoid genes along with growth and pigment composition as a function of PFD was studied using a wild-type and an astaxanthin-overproduction mutant of H. pluvialis NIES144. The results indicate that astaxanthin biosynthesis was mainly under transcriptional control of the gene encoding carotenoid hydroxylase, and to a lesser extent, the genes encoding isopentenyl isomerase and phytoene desaturase, and to the least extent, the genes encoding phytoene synthase and carotenoid oxygenase. The expression of a plastid terminal oxidase (PTOX) gene ptox2 underwent transient up-regulation under elevated PFDs, suggesting that PTOX may be functionally coupled with phytoene desaturase through the plastoquinone pool and may play a role in reducing redox-potential-dependent and oxygen-concentration-dependent formation of reactive oxygen species in the chloroplast. Over-expression of both the carotenogenic and PTOX genes confers to the astaxanthin-overproduction mutant more effective photoprotective capability than that of the wild type under photooxidative stress.

Occurrence and environmental stress responses of two plastid terminal oxidases in Haematococcus pluvialis (Chlorophyceae)

The plastid terminal oxidase (PTOX) is a plastoquinol oxidase involved in carotenoid biosynthesis in higher plants, and may also represent the elusive oxidase in chlororespiration. Haematococcus pluvialis is a green alga that has the ability to synthesize and accumulate large amounts of the red carotenoid astaxanthin (ca. 2% of dry weight) under various stress conditions. However, the occurrence and function of PTOX in astaxanthin synthesis and the stress response in this organism is unknown. In this study, two ptox cDNAs were cloned and sequenced from H. pluvialis and were designated as ptox1 and ptox2. Genome sequence analysis and database searching revealed that duplication of PTOX gene occurred in certain eukaryotic algae, but not in cyanobacteria and higher plants. The physiological and biochemical evidence indicated that PTOX is involved in astaxanthin synthesis and plays a critical protective role against stress. Analysis of the transcriptional expression of the PTOXs and phytoene desaturase gene further suggests that it may be PTOX1 rather than PTOX2 that is co-regulated with astaxanthin synthesis. The fact that the changes in transcripts of ptoxs in response to high light and other stressors and the differential expression of ptox1 and ptox2, suggests that PTOX, coupled with astaxanthin synthesis pathway, exerts broad, yet undefined functions in addition to those identified in higher plants.

Contrasting Responses of Photosynthesis to Salt Stress in the Glycophyte Arabidopsis and the Halophyte Thellungiella: Role of the Plastid Terminal Oxidase as an Alternative Electron Sink

The effects of short-term salt stress on gas exchange and the regulation of photosynthetic electron transport were examined in Arabidopsis (Arabidopsis thaliana) and its salt-tolerant close relative Thellungiella (Thellungiella halophila). Plants cultivated on soil were challenged for 2 weeks with NaCl. Arabidopsis showed a much higher sensitivity to salt than Thellungiella; while Arabidopsis plants were unable to survive exposure to greater than 150 mM salt, Thellugiella could tolerate concentrations as high as 500 mM with only minimal effects on gas exchange. Exposure of Arabidopsis to sublethal salt concentrations resulted in stomatal closure and inhibition of CO2 fixation. This lead to an inhibition of electron transport though photosystem II (PSII), an increase in cyclic electron flow involving only PSI, and increased nonphotochemical quenching of chlorophyll fluorescence. In contrast, in Thellungiella, although gas exchange was marginally inhibited by high salt and PSI was unaffected, there was a large increase in electron flow involving PSII. This additional electron transport activity is oxygen dependent and sensitive to the alternative oxidase inhibitor n-propyl gallate. PSII electron transport in Thellungiella showed a reduced sensitivity to 2'-iodo-6-isopropyl-3-methyl-2',4,4'-trinitrodiphenylether, an inhibitor of the cytochrome b6f complex. At the same time, we observed a substantial up-regulation of a protein reacting with antibodies raised against the plastid terminal oxidase. No such up-regulation was seen in Arabidopsis. We conclude that in salt-stressed Thellungiella, plastid terminal oxidase acts as an alternative electron sink, accounting for up to 30% of total PSII electron flow.

Consumption of oxygen by astaxanthin biosynthesis: A protective mechanism against oxidative stress in Haematococcus pluvialis (Chlorophyceae)

Haematococcus pluvialis, a unicellular green microalga, experiences photooxidative stress when exposed to excess photon flux density (PFD) relative to the capacity of photosynthesis, and particularly under other adverse environmental conditions (e.g., nutrient depletion, salinity, and excess heavy metals). Under stress, Haematococcus cells synthesize and accumulate large amounts of the secondary carotenoid astaxanthin stored in cytosolic lipid bodies. In this study, the transcriptional expression of five astaxanthin biosynthesis genes and two plastid terminal oxidase (PTOX) genes either in high PFD or in the presence of excessive sodium acetate and/or iron was determined by real-time reverse transcription PCR, and astaxanthin accumulation was measured by HPLC. Photosynthetic oxygen evolution, lipid peroxidation, and cell mortality were also investigated under these stress conditions. Our results indicate that the astaxanthin biosynthesis pathway may consume as much as 9.94% of the molecular oxygen evolved from photosynthesis under stress via at least two distinct routes: (1) extensive oxygen-dependent processes leading to astaxanthin formation, and (2) conversion of molecular oxygen into water using electrons derived from carotenogenic desaturation steps to PTOX via the photosynthetic plastoquinone (PQ) pool. Reduction of reactive oxygen species (ROS) production by reducing subcellular molecular oxygen substrates through the astaxanthin biosynthesis pathway may represent a novel protective mechanism to cope with oxidative stress. Reoxidation of the PQ pool by PTOX may further reduce photosynthetic electron transport chain-induced ROS formation.

Dual Role of the Plastid Terminal Oxidase in Tomato

The plastid terminal oxidase (PTOX) is a plastoquinol oxidase whose absence in tomato (Solanum lycopersicum) results in the ghost (gh) phenotype characterized by variegated leaves (with green and bleached sectors) and by carotenoid-deficient ripe fruit. We show that PTOX deficiency leads to photobleaching in cotyledons exposed to high light primarily as a consequence of reduced ability to synthesize carotenoids in the gh mutant, which is consistent with the known role of PTOX as a phytoene desaturase cofactor. In contrast, when entirely green adult leaves from gh were produced and submitted to photobleaching high light conditions, no evidence for a deficiency in carotenoid biosynthesis was obtained. Rather, consistent evidence indicates that the absence of PTOX renders the tomato leaf photosynthetic apparatus more sensitive to light via a disturbance of the plastoquinone redox status. Although gh fruit are normally bleached (most likely as a consequence of a deficiency in carotenoid biosynthesis at an early developmental stage), green adult fruit could be obtained and submitted to photobleaching high light conditions. Again, our data suggest a role of PTOX in the regulation of photosynthetic electron transport in adult green fruit, rather than a role principally devoted to carotenoid biosynthesis. In contrast, ripening fruit are primarily dependent on PTOX and on plastid integrity for carotenoid desaturation. In summary, our data show a dual role for PTOX. Its activity is necessary for efficient carotenoid desaturation in some organs at some developmental stages, but not all, suggesting the existence of a PTOX-independent pathway for plastoquinol reoxidation in association with phytoene desaturase. As a second role, PTOX is implicated in a chlororespiratory mechanism in green tissues.

Chlororespiration is involved in the adaptation of Brassica plants to heat and high light intensity

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.