Flexibility in photosynthetic electron transport: a newly identified chloroplast oxidase involved in chlororespiration.

Besides electron transfer reactions involved in the 'Z' scheme of photosynthesis, alternative electron transfer pathways have been characterized in chloroplasts. These include cyclic electron flow around photosystem I (PS I) or a respiratory chain called chlororespiration. Recent work has supplied new information concerning the molecular nature of the electron carriers involved in the non-photochemical reduction of the plastoquinone (PQ) pool. However, until now little is known concerning the nature of the electron carriers involved in PQ oxidation. By using mass spectrometric measurement of oxygen exchange performed in the presence of 18O-enriched O2 and Chlamydomonas mutants deficient in PS I, we show that electrons can be directed to a quinol oxidase sensitive to propyl gallate but insensitive to salicyl hydroxamic acid. This oxidase has immunological and pharmacological similarities with a plastid protein involved in carotenoid biosynthesis.

Targeted inactivation of the plastid ndhB gene in tobacco results in an enhanced sensitivity of photosynthesis to moderate stomatal closure.

The ndh genes encoding for the subunits of NAD(P)H dehydrogenase complex represent the largest family of plastid genes without a clearly defined function. Tobacco (Nicotiana tabacum) plastid transformants were produced in which the ndhB gene was inactivated by replacing it with a mutant version possessing translational stops in the coding region. Western-blot analysis indicated that no functional NAD(P)H dehydrogenase complex can be assembled in the plastid transformants. Chlorophyll fluorescence measurements showed that dark reduction of the plastoquinone pool by stromal reductants was impaired in ndhB-inactivated plants. Both the phenotype and photosynthetic performance of the plastid transformants was completely normal under favorable conditions. However, an enhanced growth retardation of ndhB-inactivated plants was revealed under humidity stress conditions causing a moderate decline in photosynthesis via stomatal closure. This distinctive phenotype was mimicked under normal humidity by spraying plants with abscisic acid. Measurements of CO(2) fixation demonstrated an enhanced decline in photosynthesis in the mutant plants under humidity stress, which could be restored to wild-type levels by elevating the external CO(2) concentration. These results suggest that the plastid NAD(P)H:plastoquinone oxidoreductase in tobacco performs a significant physiological role by facilitating photosynthesis at moderate CO(2) limitation.

Electron flow between photosystem II and oxygen in chloroplasts of photosystem I-deficient algae is mediated by a quinol oxidase involved in chlororespiration.

In Chlamydomonas reinhardtii mutants deficient in photosystem I because of inactivation of the chloroplast genes psaA or psaB, oxygen evolution from photosystem II occurs at significant rates and is coupled to a stimulation of oxygen uptake. Both activities can be simultaneously monitored by continuous mass spectrometry in the presence of (18)O(2). The light-driven O(2) exchange was shown to involve the plastoquinone pool as an electron carrier, but not cytochrome b(6)f. Photosystem II-dependent O(2) production and O(2) uptake were observed in isolated chloroplast fractions. Photosystem II-dependent oxygen exchange was insensitive to a variety of inhibitors (azide, carbon monoxide, cyanide, antimycin A, and salicylhydroxamic acid) and radical scavengers. It was, however, sensitive to propyl gallate. From inhibitors effects and electronic requirements of the O(2) uptake process, we conclude that an oxidase catalyzing oxidation of plastoquinol and reduction of oxygen to water is present in thylakoid membranes. From the sensitivity of flash-induced O(2) exchange to propyl gallate, we conclude that this oxidase is involved in chlororespiration. Clues to the identity of the protein implied in this process are given by pharmacological and immunological similarities with a protein (IMMUTANS) identified in Arabidopsis chloroplasts.

A novel thioredoxin-like protein located in the chloroplast is induced by water deficit in Solanum tuberosum L. plants.

By analysing two-dimensional patterns of chloroplastic proteins from Solanum tuberosum, the authors observed the accumulation of a 32-kDa polypeptide in the stroma of plants subjected to water deficit. N-terminus and internal peptides of the protein, named CDSP 32 for chloroplastic drought-induced stress protein, showed no obvious homology with known sequences. Using a serum raised against the protein N-terminus, a cDNA encoding CDSP 32 was cloned by screening an expression library. The deduced mature CDSP 32 protein is 243 amino acids long and displays typical features of thioredoxins in the C-terminal region (122 residues). In particular, CDSP 32 contains a CGPC motif corresponding to a thioredoxin active site and a number of amino acids conferring thioredoxin-type structure. The CDSP 32 C-terminal region was expressed as a fusion protein in Escherichia coli and was shown to possess thioredoxin activity based on reduction assay of insulin disulfide bridges. RNA blot analysis showed that CDSP 32 transcript does not accumulate upon mild water deficit conditions corresponding to leaf relative water contents (RWC) around 85%, but high levels of CDSP 32 transcripts were observed for more severe stress conditions (RWC around 70%). In vivo labelling and immunoprecipitation revealed a substantial increase in CDSP 32 synthesis upon similar stress conditions. Rewatering of wilted plants caused decreases in both transcript and protein abundances. In tomato wild-type plants and ABA-deficient mutants, a similar accumulation of a CDSP 32-related transcript was observed upon water deficit, most likely indicating no requirement for ABA in the regulation of CDSP 32 synthesis. Based on these results, it is proposed that CDSP 32 plays a role in preservation of the thiol: disulfide redox potential of chloroplastic proteins during water deficit.

Subcellular distribution of carbonic anhydrase in Solanum tuberosum L. leaves: characterization of two compartment-specific isoforms.

The intracellular compartmentation of carbonic anhydrase (CA; EC 4.2.1.1), an enzyme that catalyses the reversible hydration of CO2 to bicarbonate, has been investigated in potato (Solanum tuberosum L.) leaves. Although enzyme activity was mainly located in chloroplasts (87% of total cellular activity), significant activity (13%) was also found in the cytosol. The corresponding CA isoforms were purified either from chloroplasts or crude leaf extracts, respectively. The cytosolic isoenzyme has a molecular mass of 255,000 and is composed of eight identical subunits with an estimated Mr of 30,000. The chloroplastic isoenzyme (Mr 220,000) is also an octamer composed of two different subunits with Mr estimated at 27,000 and 27,500, respectively. The N-terminal amino acid sequences of both chloroplastic CA subunits demonstrated that they were identical except that the Mr-27,000 subunit was three amino acids shorter than that of the Mr-27,500 subunit. Cytosolic and chloroplastic CA isoenzymes were found to be similarly inhibited by monovalent anions (Cl-, I-, N3- and NO3-) and by sulfonamides (ethoxyzolamide and acetozolamide). Both CA isoforms were found to be dependent on a reducing agent such as cysteine or dithiothreitol in order to retain the catalytic activity, but 2-mercaptoethanol was found to be a potent inhibitor. A polyclonal antibody directed against a synthetic peptide corresponding to the N-terminal amino acid sequence of the chloroplastic CA monomers also recognized the cytosolic CA isoform. This antibody was used for immunocytolocalization experiments which confirmed the intracellular compartmentation of CA: within chloroplasts, CA is restricted to the stroma and appears randomly distributed in the cytosol.

Isolation and characterization of two wound-regulated tomato extensin genes.

Extensins comprise a family of structural cell wall hydroxyproline-rich glycoproteins in plants. Two tomato genomic clones, Tom J-10 and Tom L-4, were isolated from a tomato genomic DNA library by in situ plaque hybridization with extensin DNA probes. Tom J-10 encoded an extensin with 388 amino acid residues and a predicted molecular mass of 43 kDa. The Tom J-10 encoded extensin lacked a typical signal peptide sequence, but contained two distinct protein domains consisting of 19 tandem repeats of Ser-Pro4-Ser-Pro-Lys-Tyr-Val-Tyr-Lys at the amino terminus which were directly followed by 8 tandem repeats of the consensus sequence Ser-Pro4-Tyr3-Lys-Ser-Pro4-Ser-Pro at the carboxy terminus. RNA blot hybridization analysis with the Tom J-10 extensin probe demonstrated the presence of a 4.0 kb tomato stem mRNA which accumulated markedly in response to wounding. Tom L-4 encoded an extensin with 322 amino acid residues and a predicted molecular mass of 35 kDa. The Tom L-4 encoded extensin contained a typical signal peptide sequence at the amino terminus and was followed by at least 3 distinct domains. These domains consisted of an amino terminal domain containing several Lys-Pro and Ser-Pro4 repeat units, a central domain with repeats of the consensus sequence Ser-Pro2-5-Thr-Pro-Ser-Tyr-Glu-His-Pro-Lys-Thr-Pro, and a carboxy terminal domain containing repeats of the consensus sequence Ser-Ser-Pro4-Ser-Pro-Ser-Pro4-Thr-Tyr1-3. RNA blot hybridization analysis with the Tom L-4 extensin probe demonstrated the presence of a 2.6 kb tomato stem mRNA which accumulated in response to wounding.

Tomato extensin and extensin-like cDNAs: structure and expression in response to wounding.

Two tomato cDNA libraries were synthesized from poly(A)+ RNAs isolated from unwounded and wounded tomato stems. These cDNA libraries were packaged in lambda gt10 and screened by in situ plaque hybridization with a tomato extensin gene clone (pTom 5.10). Several cDNA clones were identified and isolated from both libraries in this manner and subjected to restriction enzyme digestion. Southern gel blot hybridization, RNA gel blot hybridization, and DNA sequence analyses. From these analyses, the various cDNA clones were found to fall into one of five distinct classes (classes I-V). Class I clones hybridized to a 4.0 kb mRNA which accumulated markedly after wounding and encoded an extensin characterized largely by Ser-(Pro)4-Ser-Pro-Ser-(Pro)4-(Tyr)3-Lys repeats. Class II clones hybridized to a 2.6 kb mRNA which showed no accumulation following wounding and encoded an extensin containing Ser-(Pro)4-Ser-Pro-Ser-(Pro)4-Thr-(Tyr)1-3-Ser repeats. Class III clones hybridized to a 0.6 kb mRNA which greatly accumulated in response to wounding and encoded a glycine-rich protein (GRP) with (Gly)2-6-Tyr-Pro and (Gly)2-6-Arg repeats. Class IV clones contained both class I and class III DNA sequences and consequently hybridized to both the 4.0 kb and the 0.6 kb wound-accumulating mRNAs; these clones encoded a portion of a GRP sequence on one DNA strand and encoded a portion of an extensin sequence on the other DNA strand. Class V clones hybridized to a 2.3 kb mRNA which decreased following wounding and encoded a GRP sequence characterized by (Gly)2-5-Arg repeats.

Extensin and Phenylalanine Ammonia-Lyase Gene Expression Altered in Potato Tubers in Response to Wounding, Hypoxia, and Erwinia carotovora Infection.

Potato (Solanum tuberosum L.) tubers are susceptible to infection by Erwinia carotovora, causal agent of bacterial soft rot, when wounded and subjected to wet, hypoxic environments. The expression of two putative plant defense genes, extensin and phenylalanine ammonia-lyase (PAL), was examined by monitoring their respective mRNA levels and cell wall hydroxyproline levels in tuber tissues under various conditions leading to susceptibility or resistance and after inoculation with E. carotovora in order to assess the possible roles of these genes and their products in this plant-pathogen interaction. Extensin and PAL mRNA levels as well as cell wall hydroxyproline levels accumulated markedly in response to wounding and subsequent aerobic incubation. Extensin and PAL mRNA levels as well as cell wall hydroxyproline levels decreased in response to wounding and subsequent anaerobic incubation; these changes were correlated with high susceptibility of tuber tissue to E. carotovora infection. Inoculation of wound sites with E. carotovora caused some additional accumulation of the wound-regulated extensin and PAL mRNAs under certain aerobic conditions, but never under anaerobic conditions.

Two different families of hydroxyproline-rich glycoproteins in melon callus: biochemical and immunochemical studies.

Two different families of hydroxyproline-rich glycoproteins, HRGP(1) and HRGP(2), have been isolated from melon callus and separated by ion exchange chromatography on CM-sepharose. HRGP(1) corresponds to an arabinogalactan protein. The sugar portion of HRGP(1) accounts for 94% of the molecule and contains galactose (66%) and arabinose (34%); these residues are present as polysaccharide side chains attached to hydroxyproline. Hydroxyproline is the main amino acid residue (46%) of the protein moiety. The arabinogalactan protein nature of HRGP(1) has been checked by its ability to positively react with the beta-glucosyl Yariv antigen; the (3)H-labeled deglycosylated HRGP(1) also called HRP(1) migrates upon electrophoresis as a single band of molecular weight 76,000. HRGP(2) was fractionated by affinity chromatography on heparin-Ultrogel into three different glycoproteins, HRGP(2a,2b) and (2c). Two of these glycoproteins behave as polycations (HRGP(2b) and (2c)) and are chemically distinct from HRGP(2a). HRGP(2b) is the most abundant component and contains 41% protein and 50% sugar. Hydroxyproline, lysine, tyrosine, and arabinose are the most prominent residues of their respective moiety. The glycosylation pattern of hydroxyproline indicates that HRGP(2b) is related to and possibly a precursor of the wall HRGP; as in melon cell wall HRGP, Hyp-Ara(3) predominates, and small amounts of a putative Hyp-Ara(5) a hitherto unreported hyp-arabinoside, are recorded. The molecular weight of HRP(2b), the protein portion of HRGP(2b) is 55,000 +/- 5,000, as estimated after deglycosylation of the molecule with trifluoromethane sulfonic acid. Antibodies have been raised against HRGP(2b) and HRP(2b). Immunodiffusion shows that each antigen (HRGP(2b) or HRP(2b)) reacts with its own IgG, and cross-reacts with the heterologous IgG, thereby indicating the presence of common (unglycosylated) and specific (glycosylated and deglycosylated) epitopes. The arabinogalactan protein HRGP(1) is not recognized by either antibody and HRGP(2b) does not react with the Yariv antigen. Immunoprecipitation of (3)H-labeled HRP(1) and HRP(2b) in the presence of goat antirabbit IgG, followed by gel electrophoresis, allows to recover HRP(2b) only. Again, HRP(2b) is immunoprecipitated by the two antisera.