Influence of Organic-Phosphates on 3-Phosphoglycerate Dependent O2 Evolution in C3 and C4 Mesophyll Chloroplasts

In C₄ plants phosphoenolpyruvate (PEP) of the C₄ cycle may betransported on a chloroplast transporter which also transports3-phosphoglycerate (3-PGA) and triosephosphates. In C₃ plantsPEP is not considered to be effectively transported on the chloroplastphosphate translocator. The influences of certain organic phosphates,having a similar structure to either PEP or triose-phosphates,on 3-PGA dependent O₂ evolution by C₄ (Digitaria sanquinalisL. Scop.) and C₃ (Hordeum vulgare L.) mesophyll chloroplastswere investigated. In the C₄ mesophyll chloroplasts phosphoglycolatewas a competitive inhibitor (K_i = 2.1 mM) of 3-PGA dependentO₂ evolution, and was as effective as previously reported forPEP. 2-Phosphoglycerate was also a competitive inhibitor (K_t= 8.6 mM) of O₂ evolution in the C₄ mesophyll chloroplasts with3-PGA as substrate, while phospholactate was a weak inhibitorand glyphosate had no effect. Neither PEP, phosphoglycolatenor 2-phosphoglycerate were effective inhibitors of 3- PGA dependentO₂ evolution in the C₃ chloroplasts. Phosphohydroxypyruvatewas a competitive inhibitor of 3-PGA dependent O₂2 evolutionin both chloroplast types. The selectivity in inhibition ofO₂ evolution with 3-PGA as substrate suggests that the C₄ mesophyllchloroplasts can recognize certain organic phosphates with thephosphate in the C-2 or C-3 position but that the C₄ mesophyllchloroplasts can only effectively recognize certain organicphosphates with the phosphate in the C-3 position. The resultsalso support the view that 3-PGA and PEP are transported onthe same phosphate translocator in C₄ mesophyll chloroplasts. ¹ Current address: Department of Horticulture, 2001 Fyffe Court,The Ohio State University, Columbus, Ohio 43210-1096. (Received March 24, 1987; Accepted April 16, 1987)     

Japanese barberry seed predation by Rhagoletis meigenii fruit flies harboring Wolbachia endosymbionts

The phytophagous fruit fly Rhagoletis meigenii harbors the bacterium Wolbachia pipientis and, together with Japanese barberry, form a tri-partite symbiosis. R. meigenii is a seed predator of invasive Japanese barberry plants and is dependent on this insect-plant interaction for reproductive success. The endosymbiotic bacterium W. pipientis is a reproductive parasite known to alter the sex ratios of offspring and the fitness of infected host insects. We investigated Japanese barberry fruit for the degree of infestation by R. meigenii and characterized the Wolbachia strain infecting R. meigenii. Densities of R. meigenii in four naturalized stands of Japanese barberry revealed low numbers of fruit flies with high variability in the population densities observed among individual plants. Overall, R. meigenii infested roughly 10–20 % of the Japanese barberry fruits analyzed; fruit with two seeds (vs. one seed) were the most frequently infested. Approximately, 90 % of the R. meigenii tested positive for Wolbachia infection via PCR amplification of the Wolbachia surface protein (wsp) gene. No bacterial strain diversity was observed when comparing multi-locus sequence typing (MLST) profiles within or among five R. meigenii populations in Maine, although the MLST profile obtained from R. meigenii differed from three co-occurring Rhagoletis. The Wolbachia endosymbiont of R. meigenii is a member of the Wolbachia supergroup A and the ST-13 cluster complex.     

Anaerobiosis in Echinochloa crus-galli (Barnyard Grass) Seedlings : Intermediary Metabolism and Ethanol Tolerance

Tolerance to ethanol and the ability to metabolize key intermediary substrates under anaerobiosis were studied in Echinochloa crus-galli (L.) Beauv. var oryzicola seeds to further characterize the mechanisms which enable it to germinate and grow without O₂.

Our results indicate that E. crus-galli var oryzicola possesses an inherently high tolerance to ethanol and is able to metabolize low levels of ethanol in the absence of O₂. Concentrations of ethanol 45-fold greater than endogenous levels did not prove toxic to germinating seeds.

Five-day anaerobically grown seedlings of E. crus-galli var oryzicola metabolized added [¹⁴C]sucrose primarily to CO₂ and ethanol. Of the soluble compounds labeled, the phosphorylated intermediates of glycolysis and the oxidative pentose phosphate pathway predominated more under anaerobiosis than in air. In addition, organic acids and lipids were labeled from [¹⁴C]sucrose, the latter indicating that metabolism of carbohydrate via acetyl-CoA occurred in the absence of O₂. Lipids were also labeled when seeds were supplied with [¹⁴C]ethanol or [¹⁴C]acetate. Labeling experiments using the above compounds plus [¹⁴C]NaHCO₃, showed further labeling of organic acids; succinate and citrate being labeled under nitrogen, while fumarate was formed in air.

The above metabolic characteristics would allow for the maintenance of an active alcoholic fermentation system which, along with high alcohol dehydrogenase activity, would continue to recycle NAD and result in continued energy production without O₂. In addition, Echinochloa's ability to metabolize carbohydrate intermediates and to synthesize lipids indicates that mechanisms exist for providing the carbon intermediates for biosynthesis, particularly membrane synthesis for growth, even in the absence of O₂.

     

Inhibition of 3-Phosphoglycerate-Dependent O(2) Evolution by Phosphoenolpyruvate in C(4) Mesophyll Chloroplasts of Digitaria sanguinalis (L.) Scop

The effects of phosphoenolpyruvate (PEP), inorganic phosphate (Pi), and ATP on 3-phosphoglycerate (PGA)-dependent O₂ evolution by chloroplasts of Digitaria sanguinalis (L.) Scop. (crabgrass) were evaluated relative to possible mechanisms of PEP transport by the C₄ mesophyll chloroplast. Crude and Percoll purified chloroplast preparations exhibited rates of PGA-dependent O₂ evolution in the range of 90 to 135 micromoles O₂ per milligram chlorophyll per hour, and up to 180 micromoles O₂ per milligram chlorophyll per hour at optimal Pi concentrations (approximately 0.2 millimolar at 9 millimolar PGA). Higher concentrations of Pi were inhibitory. PEP inhibited O₂ evolution (up to 70%) in both chloroplast preparations when the PEP to PGA ratio was high (i.e. 9 millimolar PEP to 0.36 millimolar PGA). Usually no inhibition was seen when the PEP to PGA ratio was less than 2. PEP acted as a competitive inhibitor and, at a concentration of 9 millimolar, increased the apparent K_m (PGA) from 0.15 to 0.53 millimolar in Percoll purified chloroplasts. A low concentration of PGA and high ratio of PEP to PGA, which are considered unphysiological, were required to detect any inhibition of O₂ evolution by PEP. Similar results were obtained from crude versus Percoll purified preparations. Neither the addition of Pi nor ATP could overcome PEP inhibition. As PEP inhibition was competitive with respect to PGA concentration, and as addition of ATP or Pi could not prevent PEP inhibition of PGA-dependent O₂ evolution, the inhibition was not due to PEP exchange of adenylates or Pi out of the chloroplast. Analysis of the effect of Pi and PEP, separately and in combination, on PGA-dependent O₂ evolution suggests interactions between PEP, Pi, and PGA on the same translocator in the C₄ mesophyll chloroplast. C₃ spinach chloroplasts were also found to be sensitive to PEP, but to a lesser extent than crabgrass chloroplasts. The apparent K_i values (PEP) were 3 and 21 millimolar for crabgrass and spinach, respectively.     

Mollusc/algal chloroplast symbiosis: how can isolated chloroplasts continue to function for months in the cytosol of a sea slug in the absence of an algal nucleus?

A marine sea slug, Elysia chlorotica, has acquired the ability to carry out photosynthesis as a result of forming an intracellular symbiotic association with chloroplasts of the chromophytic alga, Vaucheria litorea. The symbiont chloroplasts (kleptoplasts) are functional, i.e. they evolve oxygen and fix CO₂ and actively transcribe and translate proteins for several months in the sea slug cytosol. Considering the dependency of plastid function on nuclear genes, the level of kleptoplast activity observed in the animal cell is quite remarkable. Possible factors contributing to this long-lasting functional association that are considered here include: the presence of an algal nuclear genome in the sea slug, autonomous chloroplasts, unusual chloroplast/protein stability, re-directing of animal proteins to the kleptoplast, and lateral gene transfer. Based on our current understanding, the acquisition and incorporation of intact algal plastids by E. chlorotica is aided by the robustness of the plastids and the long-term functional activity of the kleptoplasts appears to be supported by both plastid and protein stability and contributions from the sea slug.     

Fluorescence microscopy and radiolabeling of C3 and C4 chloroplasts using diisothiocyanatostilbene disulfonic acid as a marker for the phosphate translocator

The usefulness of 4,4-diisothiocyanatostilbene-2,2-disulfonic acid (DIDS) for in-situ studies of the chloroplast phosphate translocator was evaluated by fluorescence microscopy and radiolabeling of spinach (Spinacia oleracea L.) (C₃ plant) and maize (Zea mays L.) (C₄ plant) chloroplasts. In maize mesophyll and bundle-sheath chloroplasts and in spinach chloroplasts that were either intact, broken or swollen, DIDS fluorescence was only associated with the chloroplast envelope. Intact chloroplasts often had fluorescent patches corresponding to concave regions of the chloroplast which we assume to be regions enriched in DIDS-binding sites.Incubation of intact or broken spinach chloroplasts or maize mesophyll chloroplasts with [³H₂]DIDS resulted in the labeling of a single polypeptide (relative molecular mass, M_r, 30 kDa) in the envelope fraction, in each case. Label in the stromal fraction was not detected when intact chloroplasts were incubated with [³H₂]DIDS. However, when broken chloroplasts were incubated with [³H₂]DIDS, several polypeptides of various molecular masses were labeled, but not the 30×31-kDa polypeptide. In thylakoid fractions from both broken and intact chloroplasts, a single 30×31-kDa polypeptide was labeled inconsistently. When a mixture of intact maize mesophyll and bundle-sheath chloroplasts was labeled with [³H₂]DIDS, extracts of whole chloroplasts displayed radioactivity only in the 30×31-kDa band.We conclude that DIDS is a valuable probe for the in-situ identification and characterization of the 30-kDa protein — the presumptive phosphate translocator — in C₃ and C₄ chloroplasts since DIDS (1) does not penetrate the inner membrane of the envelope of intact chloroplasts and, therefore, (2) does not bind internal sites in intact chloroplasts, and (3) only binds the 30-kDa protein in the inner membrane of the envelope.Abbreviations CBB Coomassie brilliant blue - DIC differential interference contrast optics - DIDS 4,4-diisothiocyanatostilbene-2,2-disulfonic acid - [³H₂]DIDS 1,2-ditritio-1,2-(2,2-disulfo-4,4-diisothiocyano)diphenylethane - kDa kilodalton - M_r relative molecular mass - PGA 3-phosphoglycerate - Pitranslocator phosphate translocator - SDS sodium dodecyl sulfate     

Energy charge and emergence of the coleoptile and radicle at varying oxygen levels in Echinochloa crus-galli

The role of protein synthesis in senescence and in the inhibition of senescence by light and kinetin was studied in barley ( Hordeum vulgare L. cv. Hassan) leaves with different inhibitors of protein synthesis. A comparison of the actions of D- and L-chloramphenicol was made to compensate for the effects of D-chloramphenicol not mediated by inhibition of protein synthesis. The involvement of phytochrome was also studied. The results suggest that: 1) cytoplasmic protein synthesis is required for senescence in the light and in the dark; 2) chloroplasts, in the dark, synthesize protein which accelerates senescence; 3) kinetin inhibits the synthesis by chloroplasts of senescence-accelerating protein; 4) light changes the type of protein synthesized by chloroplasts from those accelerating to those retarding senescence; and 5) lightretar-dation of senescence is mediated by phytochrome and, probably, by photophos-phorylation.     

Molecular Characterization of the Calvin Cycle Enzyme Phosphoribulokinase in the Stramenopile Alga Vaucheria litorea and the Plastid Hosting Mollusc Elysia chlorotica

Phosphoribulokinase （PRK）, a nuclear-encoded plastid-localized enzyme unique to the photosynthetic carbon reduction （Calvin） cycle, was cloned and characterized from the stramenopile alga Vaucheria litorea. This alga is the source of plastids for the mollusc （sea slug） Elysia chlorotica which enable the animal to survive for months solely by photoautotrophic CO2 fixation. The 1633-bp V. litorea prk gene was cloned and the coding region, found to be interrupted by four introns, encodes a 405-amino acid protein. This protein contains the typical bipartite target sequence expected of nuclearencoded proteins that are directed to complex （i.e. four membrane-bound） algal plastids. De novo synthesis of PRK and enzyme activity were detected in E. chlorotica in spite of having been starved of V. litorea for several months. Unlike the algal enzyme, PRK in the sea slug did not exhibit redox regulation. Two copies of partial PRK-encoding genes were isolated from both sea slug and aposymbiotic sea slug egg DNA using PCR. Each copy contains the nucleotide region spanning exon 1 and part of exon 2 of V litorea prk, including the bipartite targeting peptide. However, the larger prk fragment also includes intron 1. The exon and intron sequences of prk in E. chlorotica and V/itorea are nearly identical. These data suggest that PRK is differentially regulated in V. litorea and E. chlorotica and at least a portion of the V. litorea nuclear PRK gene is present in sea slugs that have been starved for several months.     

Effect of Aerobic Priming on the Response of Echinochloa crus-pavonis to Anaerobic Stress (Protein Synthesis and Phosphorylation)

Echinochloa species differ in their ability to germinate and grow in the absence of oxygen. Seeds of Echinochloa crus-pavonis (H.B.K.) Schult do not germinate under anoxia but remain viable for extended periods (at least 30 d) when incubated in an anaerobic environment. E. crus-pavonis can be induced to germinate and grow in an anaerobic environment if the seeds are first subjected to a short (1-18 h) exposure to aerobic conditions (aerobic priming). Changes in polypeptide patterns (constitutive and de novo synthesized) and protein phosphorylation induced by aerobic priming were investigated. In the absence of aerobic priming protein degradation was not evident under anaerobic conditions, although synthesis of a 20-kD polypeptide was induced. During aerobic priming, however, synthesis of 37- and 55-kD polypeptides was induced and persisted upon return of the seeds to anoxia. Furthermore, phosphorylation of two 18-kD polypeptides was observed only in those seeds that were labeled with 32PO4 during the aerobic priming period. Subsequent chasing in an anaerobic environment resulted in a decrease in phosphorylation of these polypeptides. Likewise, phosphorylation of the 18-kD polypeptides was not observed if the seeds were labeled in an anaerobic atmosphere. These results suggest that the regulated induction of the 20-, 37-, and 55- kD polypeptides may be important for anaerobic germination and growth of E. crus-pavonis and that the specific phosphorylation of the 18-kD polypeptides may be a factor in regulating this induction.