Two molecular forms of pea ferredoxin in the electron transport chain of chloroplasts

The effects of two molecular forms of water-soluble ferredoxin (Fd I and Fd II) on the kinetics of electron transport in bean chloroplasts (class B) were studied. The light-induced redox transitions of the photosystem I reaction center P700 were measured by the intensity of the EPR signal I produced by P700+. Both forms of ferredoxin, Fd I and Fd II, when added to the chloroplasts in catalytic amounts, stimulate the light-induced electron transfer from P700 to NADP+. Nevertheless, Fd I is a better mediator of the back reactions from NADPH to P700+. This electron transfer pathway is sensitive to the cyclic electron transport inhibitor, antimycin A, and to DCMU inhibitor of electron transport between photosystem II and plastoquinone. It may be concluded that the two molecular forms of ferredoxin, Fd I and Fd II, differ in their ability to catalyze cyclic electron transport in photosystem I. The role of Fd I and Fd II in regulation of electron transport at the acceptor site of photosystem I is discussed.     

Two crystal forms of Azotobacter ferredoxin.

Two crystal forms of Azotobacter vinelandii (4Fe-4s)2 ferredoxin I (Fd I) have been grown which are suitable for high resolution x-ray diffraction studies. Tetragonal crystals grow as square bipyramids from ammonium sulfate and Tris buffer using a temperature gradient. The space group is P41212 (or P43212) with a = 55.3, c = 95.9 A and 1 molecule/asymmetric unit. Triclinic crystals grow as plates or laths from ammonium sulfate and phosphate buffer at constant temperature. The space group is P1 with a = 46.8, b = 58.7, c = 64.3 A, alpha = = 105 degrees 05 min, beta = 82 degrees 30 min, gamma = 110 degrees 30 min and 4 or 5 molecules/unit cell. Both crystal forms are stable to x-ray irradiation and diffract beyond 3.0 A resolution.     

Identification of N-terminal regions of wheat leaf ferredoxin NADP+ oxidoreductase important for interactions with ferredoxin

Wheat leaves contain two isoproteins of the photosynthetic ferredoxin:NADP(+) reductase (pFNRI and pFNRII). Truncated forms of both enzymes have been detected in vivo, but only pFNRII displays N-terminal length-dependent changes in activity. To investigate the impact of N-terminal truncation on interaction with ferredoxin (Fd), recombinant pFNRII proteins, differing by deletions of up to 25 amino acids, were generated. During purification of the isoproteins found in vivo, the longer forms of pFNRII bound more strongly to a Fd affinity column than did the shorter forms, pFNRII(ISKK) and pFNRII[N-2](KKQD). Further truncation of the N-termini resulted in a pFNRII protein which failed to bind to a Fd column. Similar k(cat) values (104-140 s(-1)) for cytochrome c reduction were measured for all but the most truncated pFNRII[N-5](DEGV), which had a k(cat) of 38 s(-1). Stopped-flow kinetic studies, examining the impact of truncation on electron flow between mutant pFNRII proteins and Fd, showed there was a variation in k(obs) from 76 to 265 s(-1) dependent on the pFNRII partner. To analyze the sites which contribute to Fd binding at the pFNRII N-terminal, three mutants were generated, in which a single or double lysine residue was changed to glutamine within the in vivo N-terminal truncation region. The mutations affected binding of pFNRII to the Fd column. Based on activity measurements, the double lysine residue change resulted in a pFNRII enzyme with decreased Fd affinity. The results highlight the importance of this flexible N-terminal region of the pFNRII protein in binding the Fd partner.     

Two sites of interaction of ferredoxin with thylakoids

The interaction of ferredoxin with thylakoids is shown to occur at two distinct sites: at the reducing end of photosystem I, and at the site where ferredoxin-NADP reductase (FNR) is located on the membrane. The evidence is based on the lack of inhibition of ferredoxin photoreduction by the extraction of FNR or its inactivation by an antibody, and on the difference between K_m values for ferredoxin in reactions requiring FNR as compared to those only requiring ferredoxin.     

Novel forms of ferredoxin and ferredoxin-NADP reductase from spinach roots

Ferredoxin and the enzyme catalyzing its reduction by NADPH, ferredoxin-NADP reductase (ferredoxin-NADP+ oxidoreductase or FNR), were found to be present in roots of spinach (Spinacia oleracea). Localization experiments with endosperm of germinating castor beans (Ricinus communis), a classical nonphotosynthetic tissue for cell fractionation studies, confirmed that ferredoxin and FNR are localized in the plastid fraction. Both proteins were purified from spinach roots and found to resemble their leaf counterparts in activity, spectral properties, and complex formation, but to differ in amino acid composition and amino terminal sequence. The results indicate that the primary structures of the FNR and ferredoxin of spinach roots differ from that of the corresponding leaf proteins. Together with earlier findings, the present results provide evidence that nonphotosynthetic plastids, including those of roots, are capable of reducing ferredoxin with heterotrophically generated NADPH.     

Light inactivation of transaldolase in pea leaf chloroplasts

Transaldolase is inactivated when intact pea leaf chloroplasts are irradiated. This light inactivation is 3-(3,4-dichlorophenyl)-1,1-dimethyl urea, arsenite and sulfite-sensitive, implicating the Light Effect Mediator system in light inactivation of this oxidative pentose phosphate pathway enzyme.     

Light modulation of phosphofructokinase in pea leaf chloroplasts

Chloroplastic phosphofructokinase, phosphorylase, phosphoglucomutase, and phosphoglucoisomerase in peas are light inactivated. The effect of light on phosphofructokinase is mimicked by dithiothreitol. DCMU, arsenite, and sulfite inhibit light modulation of the enzyme. No effect of light inactivation on the K_m (fructose-6-P) or pH optima of phosphofructokinase was observed.     

Causes of different feeding rates of pea leaf weevil Sitona lineatus on three pea cultivars

The causes of different feeding rate in female pea leaf weevil (Sitona lineatus L.) were investigated with three cultivars, namely Gorkovskij, Lancet and Neuga. A negative correlation between leaf thickness and feeding rate was demonstrated, but by using artificial diet saturated with leaf sap, some effects were shown to be of a chemical nature.The effect of some compounds, the concentrations of which differed in the experimental cultivars, was verified by feeding tests. A strong inhibition of feeding was induced by the amino acid tyrosine. Among tested sugars saccharose was the most effective for stimulation of feeding.

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zusammenfassung In Freilandversuchen wurden drei Erbsensorten mit unterschiedlicher Frassintensität durch Sitona lineatus ausgewählt, die stark befallene Gorkovskij, die intermediäre Lancet und die wenig befallene Neuga. Die Ursachen der Befallsunterschiede wurden dann im Laboratorium mit weiblichen Käfern untersucht. Blattscheibentests mit frischen Blättern der drei Sorten bestätigten die Feldversuche. Versuche mit künstlichen Substraten, welche mit Blattsaft gesättigt waren, deuteten auf einen Einfluss physikalischer Faktoren, also des anatomischen Baus der Blätter, hin. Zwischen Frassstärke und Blattdicke ergab sich eine negative Korrelation.Bei den Tests mit künstlichen Substraten wurde der Einfluss der biochemischen Zusammensetzung des Blattsafts nachgewiesen. Aufgrund der Pflanzensaftanalyse wurden dann einige Stoffe in ihrer Wirkung auf den Käferfrass geprüft. Die stärkste Frasshemmung bewirkte bei den Aminosäuren Tyrosin, die stärkste Stimulation bei den Zuckern Saccharose.

     

Changes in chloroplast number during pea leaf development

Protoplasts were prepared from pea (Pisum sativum L.) leaves throughout development and their contents spread in a monolayer to determine the number of chloroplasts per cell. This approach permitted the rapid analysis of more than 100 cells at each stage of development. The average number of chloroplasts per cell increased from 24±10 to 64±20 during greening and expansion of the first true foliage leaves; all cells containing chloroplasts apparently increase their chloroplast number. A parallel increase in the amount of DNA per nucleus was not observed. As the leaves senesced the chloroplast number gradually decreased to 44±12. We have correlated these changes with our previous results on the percentage of chloroplast DNA per cell. Chloroplast multiplication resulted in a 2.7-fold dilution (from 272 to 102) of the number of copies of the chloroplast DNA molecule per plastid.     

Inactivation of pea leaf phosphofructokinase by light and dithiothreitol

When intact pea (Pisum sativum L.) plants are illuminated, the glycolytic enzyme phosphofructokinase is inactivated. In crude extracts the enzyme is inhibited by dithiothreitol. It would seem that this cytoplasmic enzyme, like glucose-6-P dehydrogenase, is light-inactivated when the enzymes of photosynthetic carbon metabolism are light-activated.     

Electron microscopy of glutamine synthetase from pea leaf chloroplasts

The structure of pea leaf chloroplast glutamine synthetase was studied by electron microscopy. The enzyme is shown to consist of eight elongated subunits which are arranged with point 42 symmetry at the vertices of two squares. These squares are twisted about the 4-fold axis at 40 degrees relative to each other.     

Some regulatory properties of pea leaf carbamoyl phosphate synthetase

Carbamoyl phosphate synthetase of pea shoots (Pisum sativum L.) was purified 101-fold. Its stability was greatly increased by the addition of substrates and activators. The enzyme was strongly inhibited by micromolar amounts of UMP (Ki less than 2 mum). UDP, UTP, TMP, and ADP were also inhibitory. AMP caused either slight activation (under certain conditions) or was inhibitory. Uridine nucleotides were competitive inhibitors, as was AMP, while ADP was a noncompetitive inhibitor. Enzyme activity was increased manyfold by the activator ornithine. Ornithine acted by increasing the affinity for Mg.ATP by a factor of 8 or more. Other activators were IMP, GMP, ITP, and GTP, IMP, like ornithine, increased the Michaelis constant for Mg.ATP. The activators ornithine, GMP, and IMP (but not GTP and ITP) completely reversed inhibition caused by pyrimidine nucleotides while increasing the inhibition caused by ADP and AMP.