A balanced PGR5 level is required for chloroplast development and optimum operation of cyclic electron transport around photosystem I

PSI cyclic electron transport contributes markedly to photosynthesis and photoprotection in flowering plants. Although the thylakoid protein PGR5 (Proton Gradient Regulation 5) has been shown to be essential for the main route of PSI cyclic electron transport, its exact function remains unclear. In transgenic Arabidopsis plants overaccumulating PGR5 in the thylakoid membrane, chloroplast development was delayed, especially in the cotyledons. Although photosynthetic electron transport was not affected during steady-state photosynthesis, a high level of non-photochemical quenching (NPQ) was transiently induced after a shift of light conditions. This phenotype was explained by elevated activity of PSI cyclic electron transport, which was monitored in an in vitro system using ruptured chloroplasts, and also in leaves. The effect of overaccumulation of PGR5 was specific to the antimycin A-sensitive pathway of PSI cyclic electron transport but not to the NAD(P)H dehydrogenase (NDH) pathway. We propose that a balanced PGR5 level is required for efficient regulation of the rate of antimycin A-sensitive PSI cyclic electron transport, although the rate of PSI cyclic electron transport is probably also regulated by other factors during steady-state photosynthesis.     

Ribulose 1,5-bisphosphate carboxylase/oxygenase large subunit translation is regulated in a small subunit-independent manner in the expanded leaves of tobacco

Ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is composed of small subunits (SSs) encoded by rbcS on the nuclear genome and large subunits (LSs) encoded by rbcL on the chloroplast genome, and it is localized in the chloroplast stroma. Constitutive knockdown of the rbcS gene reportedly causes a reduction in LS quantity and the level of translation in tobacco and the unicellular green alga Chlamydomonas. Constitutively knockdown of the rbcS gene also causes a reduction in photosynthesis, which influences the expression of photosynthetic genes, including the rbcL gene. Here, to investigate the influence of the knockdown of the rbcS gene on the expression of the rbcL gene under normal photosynthetic conditions, we generated transgenic tobacco plants in which the amount of endogenous rbcS mRNA can be reduced by inducible expression of antisense rbcS mRNA with dexamethasone (DEX) treatment at later stages of growth. In already expanded leaves, after DEX treatment, the level of photosynthesis, RuBisCO quantity and the chloroplast ultrastructure were normal, but the amount of rbcS mRNA was reduced. An in vivo pulse labeling experiment and polysome analysis showed that LSs were translated at the same rate as in wild-type leaves. On the other hand, in newly emerging leaves, the rbcS mRNA quantity, the level of photosynthesis and the quantity of RuBisCO were reduced, and chloroplasts failed to develop. In these leaves, the level of LS translation was inhibited, as previously described. These results suggest that LS translation is regulated in an SS-independent manner in expanded leaves under normal photosynthetic conditions.     

Redox regulation of chloroplast enzymes in Galdieria sulphuraria in view of eukaryotic evolution

Redox modulation is a general mechanism for enzyme regulation, particularly for the post-translational regulation of the Calvin cycle in chloroplasts of green plants. Although red algae and photosynthetic protists that harbor plastids of red algal origin contribute greatly to global carbon fixation, relatively little is known about post-translational regulation of chloroplast enzymes in this important group of photosynthetic eukaryotes. To address this question, we used biochemistry, phylogenetics and analysis of recently completed genome sequences. We studied the functionality of the chloroplast enzymes phosphoribulokinase (PRK, EC 2.7.1.19), NADP-dependent glyceraldehyde 3-phosphate dehydrogenase (NADP-GAPDH, GapA, EC 1.2.1.13), fructose 1,6-bisphosphatase (FBPase, EC 3.1.3.11) and glucose 6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49), as well as NADP-malate dehydrogenase (NADP-MDH, EC 1.1.1.37) in the unicellular red alga Galdieria sulphuraria (Galdieri) Merola. Despite high sequence similarity of G. sulphuraria proteins to those of other photosynthetic organisms, we found a number of distinct differences. Both PRK and GAPDH co-eluted with CP12 in a high molecular weight complex in the presence of oxidized glutathione, although Galdieria CP12 lacks the two cysteines essential for the formation of the N-terminal peptide loop present in higher plants. However, PRK inactivation upon complex formation turned out to be incomplete. G6PDH was redox modulated, but remained in its tetrameric form; FBPase was poorly redox regulated, despite conservation of the two redox-active cysteines. No indication for the presence of plastidic NADP-MDH (and other components of the malate valve) was found.     

Responses to desiccation stress in bryophytes and an important role of dithiothreitol-insensitive non-photochemical quenching against photoinhibition in dehydrated states

The effects of air drying and hypertonic treatments in the dark on seven bryophytes, which had grown under different water environments, were studied. All the desiccation-tolerant species tested lost most of their PSII photochemical activity when photosynthetic electron transport was inhibited by air drying, while, in all the sensitive species, the PSII photochemical activity remained at a high level even when photosynthesis was totally inhibited. The PSI reaction center remained active under drying conditions in both sensitive and tolerant species, but the activity became non-detectable in the light only in tolerant species due to deactivation of the cyclic electron flow around PSI and of the back reaction in PSI. Light-induced non-photochemical quenching (NPQ) was found to be induced not only by the xanthophyll cycle but also by a DeltapH-induced, dithiothreitol-insensitive mechanism in both the desiccation-tolerant and -intolerant bryophytes. Both mechanisms are thought to have an important role in protecting desiccation-tolerant species from photoinhibition under drying conditions. Fluorescence emission spectra at 77K showed that dehydration-induced quenching of PSII fluorescence was observed only in tolerant species and was due to neither state 1-state 2 transition nor detachment of light-harvesting chlorophyll protein complexes from PSII core complexes.The presence of dehydration-induced quenching of PSI fluorescence was also suggested.     

Influence of chloroplastic photo-oxidative stress on mitochondrial alternative oxidase capacity and respiratory properties: a case study with Arabidopsis yellow variegated 2

Mitochondrial alternative oxidase (AOX), the unique respiratory terminal oxidase in plants, catalyzes the energy-wasteful cyanide (CN)-resistant respiration. Although it has been demonstrated that leaf AOX is up-regulated under high-light (HL) conditions, the in vivo mechanism of AOX up-regulation by light is still unknown. In the present study, we examined whether the photo-oxidative stress in the chloroplast modulates mitochondrial respiratory properties, especially the AOX capacity, using Arabidopsis leaf-variegated mutant yellow variegated 2 (var2) and exposing plants to HL. var2 mutants lack FtsH2 metalloprotease required for the repair of damaged PSII. Indeed, var2-1 suffered from photo-oxidative stress even before the HL treatments. While the activities of tricarboxylic acid cycle enzymes and cytochrome c oxidase in var2-1 were almost identical to those in the wild type, the amount of AOX protein and the CN-resistant respiration rate were higher in var2-1. Real-time PCR analysis revealed that HL treatment induced the expression of some energy-dissipating respiratory genes, including AOX1a, NDB2 and UCP5, more strongly in var2-1. Western blotting using var2-1 leaf extracts specific to green or white sectors, containing functional or non-functional photosynthetic apparatus, respectively, revealed that more AOX protein was induced in the green sectors by the HL treatment. These results indicate that photo-oxidative stress by excess light is involved in the regulation of respiratory gene expression and the modulation of respiratory properties, especially the AOX up-regulation.     

The mitochondrial external NADPH dehydrogenase modulates the leaf NADPH/NADP+ ratio in transgenic Nicotiana sylvestris

Plant mitochondria contain alternative external NAD(P)H dehydrogenases,which oxidize cytosolic NADH or NADPH and reduce ubiquinonewithout inherent linkage to proton pumping and ATP production.In potato, St-NDB1 is an external Ca²⁺-dependent NADPH dehydrogenase.The physiological function of this enzyme was investigated inhomozygous Nicotiana sylvestris lines overexpressing St-ndb1and co-suppressing St-ndb1 and an N. sylvestris ndb1. In leafmitochondria isolated from the overexpressor lines, higher activityof alternative oxidase (AOX) was detected. However, the AOXinduction was substantially weaker than in the complex I-deficientCMSII mutant, previously shown to contain elevated amounts ofNAD(P)H dehydrogenases and AOX. An aox1b and an aox2 gene wereup-regulated in CMSII, but only aox1b showed a response, albeitsmaller, in the transgenic lines, indicating differences inAOX activation between the genotypes. As in CMSII, the increaseof AOX in the overexpressing lines was not due to a generaloxidative stress. The lines overexpressing St-ndb1 had consistentlylowered leaf NADPH/NADP⁺ ratios in the light and variably decreasedlevels in darkness, but unchanged NADH/NAD⁺ ratios. CMSII insteadhad similar NADPH/NADP⁺ and lower NADH/NAD⁺ ratios than thewild type. These results demonstrate that St-NDB1 is able tomodulate the cellular balance of NADPH and NADP⁺ at least inthe day and that reduction of NADP(H) and NAD(H) is independentlycontrolled. Similar growth rates, chloroplast malate dehydrogenaseactivation and xanthophyll ratios indicate that the change inreduction does not communicate to the chloroplast, and thatthe cell tolerates significant changes in NADP(H) reductionwithout deleterious effects.     

Molecular design of photosynthesis-elevated chloroplasts for mass accumulation of a foreign protein

In order to increase production of a useful protein by the chloroplast transformation technique, it seems to be necessary to determine the upper limit for the accumulation of a biologically active foreign protein in chloroplasts and then improve photosynthetic capacity and plant productivity. Here we show that the stromal fractions of tobacco chloroplasts could accommodate an additional 200-260 mg ml(-1) of green fluorescent protein in the stroma without any inhibition of gas exchange under various light intensity and growth conditions. The minimum amount of fructose-1,6-/sedoheptulose-1,7-bisphosphatase (FBP/SBPase) limiting photosynthesis was then calculated. Analyses of the photosynthetic parameters and the metabolites of transformants into which FBP/SBPase was introduced with various types of promoter (PpsbA, Prrn, Prps2 and Prps12) indicated that a 2- to 3-fold increase in levels of FBPase and SBPase activity is sufficient to increase the final amount of dry matter by up to 1.8-fold relative to the wild-type plants. Their increases were equivalent to an increase of <1 mg ml(-1) of the FBP/SBPase protein in chloroplasts and were calculated to represent <1% of the protein accumulated via chloroplast transformation. Consequently, >99% of the additional 200-260 mg ml(-1) of protein expressed in the chloroplasts could be used for the production of useful proteins in the photosynthesis-elevated transplastomic plants having FBP/SBPase.     

ABC1K1/PGR6 kinase: a regulatory link between photosynthetic activity and chloroplast metabolism

Arabidopsis proton gradient regulation (pgr) mutants have high chlorophyll fluorescence and reduced non‐photochemical quenching (NPQ) caused by defects in photosynthetic electron transport. Here, we identify PGR6 as the chloroplast lipid droplet (plastoglobule, PG) kinase ABC1K1 (activity of bc1 complex kinase 1). The members of the ABC1/ADCK/UbiB family of atypical kinases regulate ubiquinone synthesis in bacteria and mitochondria, and impact various metabolic pathways in plant chloroplasts. Here, we demonstrate that abc1k1 has a unique photosynthetic and metabolic phenotype that is distinct from that of the abc1k3 homolog. The abc1k1/pgr6 single mutant is specifically deficient in the electron carrier plastoquinone, as well as in β–carotene and the xanthophyll lutein, and is defective in membrane antioxidant tocopherol metabolism. After 2 days of continuous high light stress, abc1k1/pgr6 plants suffer extensive photosynthetic and metabolic perturbations, strongly affecting carbohydrate metabolism. Remarkably, however, the mutant acclimates to high light after 7 days together with a recovery of carotenoid levels and a drastic alteration in the starch‐to‐sucrose ratio. Moreover, ABC1K1 behaves as an active kinase and phosphorylates VTE1, a key enzyme of tocopherol (vitamin E) metabolism in vitro. Our results indicate that the ABC1K1 kinase constitutes a new type of regulatory link between photosynthetic activity and chloroplast metabolism.     

Leaf chloroplast ultrastructure and photosynthetic properties of a chlorophyll-deficient mutant of rice

Leaf chloroplast ultrastructure and photosynthetic properties of a natural, yellow-green leaf mutant (ygl1) of rice were characterized. Our results showed that chloroplast development was significantly delayed in the mutant leaves compared with the wild-type rice (WT). As leaves matured, more grana stacks formed concurrently with increasing leaf chlorophyll (Chl) content. Except for the lower intercellular CO₂ concentration, the ygl1 plants had a higher leaf net photosynthetic rate, stomatal conductance, and transpiration rate than those of the WT plants. Under equal amounts of Chl, the excitation energy of PSI and PSII was much stronger in the mutant than that in the WT. The ygl1 plants showed higher nonphotochemical quenching and lower photochemical quenching. They also exhibited higher actual photochemical efficiency of PSII with a higher electron transport rate. Under the light of 200 μmol(photon) m^?2 s^?1, the ygl1 mutant showed lesser deepoxidation of violaxanthin in the xanthophyll cycle than WT, but it increased substantially under strong light conditions. In conclusion, the photosynthetic machinery of the ygl1 remained stable during leaf development. The plants were less sensitive to photoinhibition compared with WT due to the active xanthophyll cycle. The ygl1 plants were efficient in both light harvesting and conversion of solar energy.     

The bacterial stringent response, conserved in chloroplasts, controls plant fertilization

The chloroplast, an essential organelle for plants, performs a wide variety of metabolic processes for host cells, which include photosynthesis as well as amino acid and fatty acid biosynthesis. The organelle conserves many bacterial systems in its functions, implicating its origin from symbiosis of a photosynthetic bacterium. In bacterial cells, the stringent response acts as a global regulatory system for gene expression mediated by a small nucleotide, guanosine 5'-diphosphate 3'-diphosphate (ppGpp), that is necessary for cell adaptation to diverse environmental stimuli such as amino acid starvation. Recent studies indicated that proteins similar to the bacterial ppGpp synthase/hydrolyase are conserved in plants, although their precise roles are not known. Here we show that the stringent response in chloroplasts is crucial for normal plant fertilization. Specifically, one of the Arabidopsis ppGpp synthase homologs, CRSH (Ca(2+)-activated RelA/SpoT homolog), exhibits calcium-dependent ppGpp synthesis activity in vitro, and is localized in chloroplasts in vivo. A knockdown mutation of CRSH in Arabidopsis results in a significant reduction in silique size and seed production, indicating that plant reproduction is under the control of chloroplast function through a ppGpp-mediated stringent response.     

The assembly of the FtsZ ring at the mid-chloroplast division site depends on a balance between the activities of AtMinE1 and ARC11/AtMinD1

Chloroplast division comprises a sequence of events that facilitatesymmetric binary fission and that involve prokaryotic-like stromaldivision factors such as tubulin-like GTPase FtsZ and the divisionsite regulator MinD. In Arabidopsis, a nuclear-encoded prokaryoticMinE homolog, AtMinE1, has been characterized in terms of itseffects on a dividing or terminal chloroplast state in a limitedseries of leaf tissues. However, the relationship between AtMinE1expression and chloroplast phenotype remains to be fully elucidated.Here, we demonstrate that a T-DNA insertion mutation in AtMinE1results in a severe inhibition of chloroplast division, producingmotile dots and short filaments of FtsZ. In AtMinE1 sense (overexpressor)plants, dividing chloroplasts possess either single or multipleFtsZ rings located at random intervals and showing constrictiondepth, mainly along the chloroplast polarity axis. The AtMinE1sense plants displayed equivalent chloroplast phenotypes toarc11, a loss-of-function mutant of AtMinD1 which forms replicatingmini-chloroplasts. Furthermore, a certain population of FtsZrings formed within developing chloroplasts failed to initiateor progress the membrane constriction of chloroplasts and consequentiallyto complete chloroplast fission in both AtMinE1 sense and arc11/atminD1plants. Our present data thus demonstrate that the chloroplastdivision site placement involves a balance between the opposingactivities of AtMinE1 and AtMinD1, which acts to prevent FtsZring formation anywhere outside of the mid-chloroplast. In addition,the imbalance caused by an AtMinE1 dominance causes multiple,non-synchronous division events at the single chloroplast level,as well as division arrest, which becomes apparent as the chloroplastsmature, in spite of the presence of FtsZ rings.     

Digalactosyldiacylglycerol is required for better photosynthetic growth of Synechocystis sp. PCC6803 under phosphate limitation

Digalactosyldiacylglycerol (DGDG) is a typical membrane lipid of oxygenic photosynthetic organisms. Although DGDG synthase genes have been isolated from plants, no homologous gene has been annotated in the genomes of cyanobacteria and the unicellular red alga Cyanidioschyzon merolae. Here we used a comparative genomics approach and identified a non-plant-type DGDG synthase gene (designated dgdA) in Synechocystis sp. PCC6803. The enzyme produced DGDG in Escherichia coli when co-expressed with a cucumber monogalactosyldiacylglycerol synthase. A DeltadgdA knock-out mutant showed no obvious phenotype other than loss of DGDG when grown in a BG11 medium, indicating that DGDG is dispensable under optimal conditions. However, the mutant showed reduced growth under phosphate-limited conditions, suggesting that DGDG may be required under phosphate-limited conditions, such as those in natural niches of cyanobacteria.     

NADK2, an <Emphasis Type="Italic">Arabidopsis</Emphasis> Chloroplastic NAD Kinase,Plays a Vital Role in Both Chlorophyll Synthesis and Chloroplast Protection

As one of terminal electron acceptors in photosynthetic electron transport chain, NADP receives electron and H⁺ to synthesize NADPH, an important reducing energy in chlorophyll synthesis and Calvin cycle. NAD kinase (NADK), the catalyzing enzyme for the de novo synthesis of NADP from substrates NAD and ATP, may play an important role in the synthesis of NADPH. NADK activity has been observed in different sub-cellular fractions of mitochondria, chloroplast, and cytoplasm. Recently, two distinct NADK isoforms (NADK1 and NADK2) have been identified in Arabidopsis. However, the physiological roles of NADKs remain unclear. In present study, we investigated the physiological role of Arabidiposis NADK2. Sub-cellular localization of the NADK2–GFP fusion protein indicated that the NADK2 protein was localized in the chloroplast. The NADK2 knock out mutant (nadk2) showed obvious growth inhibition and smaller rosette leaves with a pale yellow color. Parallel to the reduced chlorophyll content, the expression levels of two POR genes, encoding key enzymes in chlorophyll synthesis, were down regulated in the nadk2 plants. The nadk2 plants also displayed hypersensitivity to environmental stresses provoking oxidative stress, such as UVB, drought, heat shock and salinity. These results suggest that NADK2 may be a chloroplast NAD kinase and play a vital role in chlorophyll synthesis and chloroplast protection against oxidative damage.     

Changes in carotenoids, tocopherols and diterpenes during drought and recovery, and the biological significance of chlorophyll loss in Rosmarinus officinalis plants

Two-year-old rosemary (Rosmarinus officinalis L.) plants were subjected to severe stress by exposure to prolonged drought during a Mediterranean summer. Severely stressed plants recovered completely after the autumn rainfalls although the relative water content remained below 35% for 3 months and the chlorophyll content of leaves was reduced by up to 85% during the drought. In severe stress: (i) α-tocopherol increased 9-fold per g dry weight and 20-fold per unit of chlorophyll; (ii) lutein and β-carotene contents decreased on a dry-weight basis, but an 80% increase in lutein and constant levels of β-carotene were observed on a chlorophyll basis; (iii) there were transient and sustained increases in the de-epoxidation state of the xanthophyll cycle; and (iv) the highly oxidised abietane diterpene isorosmanol increased 8-fold as a result of the oxidation of carnosic acid. With the autumn rainfalls, water status, α-tocopherol and violaxanthin recovered first and the levels of photosynthetic pigments and abietane diterpenes increased later. The photoprotection conferred by the xanthophyll cycle and the antioxidant function of tocopherols, lutein and diterpenes may help to avoid irreversible damage in severe drought, making possible the recovery of functional membranes after the autumn rainfalls. Besides, chlorophyll loss reduces the amount of photons absorbed by leaves, which enhances the photoprotective and antioxidant capacity of leaves per amount of photons absorbed, since the ratios of xanthophylls, α-tocopherol and abietane diterpenes to chlorophyll increase. Received: 12 July 1999 / Accepted: 25 November 1999     

Phosphatidylglycerol Depletion Induces an Increase in Myxoxanthophyll Biosynthetic Activity in Synechocystis PCC6803 Cells

Phosphatidylglycerol (PG) depletion suppressed the oxygen-evolvingactivity of Synechocystis PCC6803 pgsA mutant cells. Shortageof PG led to decreased photosynthetic activity, which, similarto the effect of high light exposure, is likely to generatethe production of reactive oxygen species (ROS) or free radicals.Protection of the PG-depleted cells against light-induced damageincreased the echinenone and myxoxanthophyll content of thecells. The increased carotenoid content was localized in a solublefraction of the cells as well as in isolated thylakoid and cytoplasmicmembranes. The soluble carotenoid fraction contained carotenederivatives, which may bind to proteins. These carotene–proteincomplexes are similar to orange carotenoid protein that is involvedin yielding protection against free radicals and ROS. An increasein the content of myxoxanthophyll and echinenone upon PG depletionsuggests that PG depletion regulates the biosynthetic pathwayof specific carotenoids.     

Heat stress: an inducer of programmed cell death in Chlorella saccharophila

Programmed cell death (PCD) has been recognized as a fundamental cellular process conserved in metazoans, plants and yeast. However, the cellular mechanisms leading to PCD have not been fully elucidated in unicellular organisms. Evidence is presented that heat stress induces PCD in Chlorella saccharophila cells. Our results demonstrate that heat shock triggers a PCD pathway occurring with characteristics features such as chromatin condensation, DNA fragmentation, cell shrinkage and detachment of the plasma membrane from the cell wall, and suggest the presence of caspase 3-like activity. The caspase 3 inhibitor Ac-DEVD-CHO gave significant protection against heat shock-induced cell death. Moreover, a reduction in photosynthetic pigment contents associated with alteration of chloroplast morphology and a fairly rapid disappearance of the ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit and the light-harvesting complex of PSII have been observed. The timing of events in the signaling cascade associated with the C. saccharophila heat shock PCD response is discussed. Insights into this field may have general implications for understanding the pathway of cell death in unicellular green algae.     

Xanthophyll cycle--a mechanism protecting plants against oxidative stress

Six different xanthophyll cycles have been described in photosynthetic organisms. All of them protect the photosynthetic apparatus from photodamage caused by light-induced oxidative stress. Overexcitation conditions lead, in the chloroplast, to the over-reduction of the NADP pool and production of superoxide, which can subsequently be metabolized to hydrogen peroxide or a hydroxyl radical, other reactive oxygen species (ROS). On the other hand, overexcitation of photosystems leads to an increased lifetime of the chlorophyll excited state, increasing the probability of chlorophyll triplet formation which reacts with triplet oxygen forming single oxygen, another ROS. The products of the light-dependent phase of xanthophyll cycles play an important role in the protection against oxidative stress generated not only by an excess of light but also by other ROS-generating factors such as drought, chilling, heat, senescence, or salinity stress. Four, mainly hypothetical, mechanisms explaining the protective role of xanthophyll cycles in oxidative stress are presented. One of them is the direct quenching of overexcitation by products of the light phase of xanthophyll cycles and three others are based on the indirect participation of xanthophyll cycle carotenoids in the process of photoprotection. They include: (1) indirect quenching of overexcitation by aggregation-dependent light-harvesting complexes (LHCII) quenching; (2) light-driven mechanisms in LHCII; and (3) a model based on charge transfer quenching between Chl a and Zx. Moreover, results of the studies on the antioxidant properties of xanthophyll cycle pigments in model systems are also presented.     

Role of the reversible xanthophyll cycle in the photosystem II damage and repair cycle in Dunaliella salina

The Dunaliella salina photosynthetic apparatus organization and function was investigated in wild type (WT) and a mutant (zea1) lacking all beta,beta-epoxycarotenoids derived from zeaxanthin (Z). The zea1 mutant lacked antheraxanthin, violaxanthin, and neoxanthin from its thylakoid membranes but constitutively accumulated Z instead. It also lacked the so-called xanthophyll cycle, which, upon irradiance stress, reversibly converts violaxanthin to Z via a de-epoxidation reaction. Despite the pronounced difference observed in the composition of beta,beta-epoxycarotenoids between WT and zea1, no discernible difference could be observed between the two strains in terms of growth, photosynthesis, organization of the photosynthetic apparatus, photo-acclimation, sensitivity to photodamage, or recovery from photo-inhibition. WT and zea1 were probed for the above parameters over a broad range of growth irradiance and upon light shift experiments (low light to high light shift and vice versa). A constitutive accumulation of Z in the zea1 strain did not affect the acclimation of the photosynthetic apparatus to irradiance, as evidenced by indistinguishable irradiance-dependent adjustments in the chlorophyll antenna size and photosystem content of WT and zea1 strain. In addition, a constitutive accumulation of Z in the zea1 strain did not affect rates of photodamage or the recovery of the photosynthetic apparatus from photo-inhibition. However, Z in the WT accumulated in parallel with the accumulation of photodamaged PSII centers in the chloroplast thylakoids and decayed in tandem with a chloroplast recovery from photo-inhibition. These results suggest a role for Z in the protection of photodamaged and disassembled PSII reaction centers, apparently needed while PSII is in the process of degradation and replacement of the D1/32-kD reaction center protein.     

Disruption of a gene encoding C4-dicarboxylate transporter-like protein increases ozone sensitivity through deregulation of the stomatal response in Arabidopsis thaliana

To understand better the plant response to ozone, we isolated and characterized an ozone-sensitive (ozs1) mutant strain from a set of T-DNA-tagged Arabidopsis thaliana ecotype Columbia. The mutant plants show enhanced sensitivity to ozone, desiccation and sulfur dioxide, but have normal sensitivity to hydrogen peroxide, low temperature and high light levels. The T-DNA was inserted at a single locus which is linked to ozone sensitivity. Identification of the genomic sequences flanking the T-DNA insertion revealed disruption of a gene encoding a transporter-like protein of the tellurite resistance/C(4)-dicarboxylate transporter family. Plants with either of two different T-DNA insertions in this gene were also sensitive to ozone, and these plants failed to complement ozs1. Transpiration levels, stomatal conductance levels and the size of stomatal apertures were greater in ozs1 mutant plants than in the wild type. The stomatal apertures of ozs1 mutant plants responded to light fluctuations but were always larger than those of the wild-type plants under the same conditions. The stomata of the mutant and wild-type plants responded similarly to stimuli such as light, abscisic acid, high concentrations of carbon dioxide and ozone. These results suggest that OZS1 helps to close stomata, being not involved in the responses to these signals.