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.

Expression of multiple forms of ferredoxin NADP+ oxidoreductase in wheat leaves.

In higher plants there are two forms of ferredoxin NADP(+) oxidoreductase (FNR), a photosynthetic pFNR primarily required for the photoreduction of NADP(+), and a heterotrophic hFNR which generates reduced ferredoxin by utilizing electrons from NADPH produced during carbohydrate oxidation. The aim of this study was to investigate the presence of multiple forms of FNR in wheat leaves and the capacity of FNR isoforms to respond to changes in reductant demand through varied expression and N-terminal processing. Two forms of pFNR mRNA (pFNRI and pFNRII) were expressed in a similar pattern along the 12 cm developing primary wheat leaf, with the highest levels observed in plants grown continuously in the dark in the presence (pFNRI) or absence (pFNRII) of nitrate respectively. pFNR protein increased from the leaf base to tip. hFNR mRNA and protein was in the basal part of the leaf in plants grown in the presence of nitrate. FNR activity in plants grown in a light/dark cycle without nitrate was mainly due to pFNR, whilst hFNR contributed significantly in nitrate-fed plants. The potential role of distinct forms of FNR in meeting the changing metabolic capacity and reductant demands along the linear gradient of developing cells of the leaf are discussed. Furthermore, evidence for alternative N-terminal cleavage sites of pFNR acting as a means of discriminating between ferredoxins and the implications of this in providing a more effective flow of electrons through a particular pathway in vivo is considered.

Compartmentation of metabolism within mitochondria and plastids.

An important component of metabolic regulation is compartmentation and specialization. Subcellular compartmentation and the role of individual organelles is well studied, though less consideration has been given to the extent to which organelles differ between cells. Organelles extracted from whole tissue homogenates will have generally originated from a range of cell types. This review describes and assesses the regulation of metabolic activity in plants at both the cellular and subcellular level by considering specialization of mitochondria and plastids.

Reconstitution of the hexose phosphate translocator from the envelope membranes of wheat endosperm amyloplasts.

Amyloplasts were isolated and purified from wheat endosperm and the envelope membranes reconstituted into liposomes. Envelope membranes were solubilized in n-octyl beta-D-glucopyranoside and mixed with liposomes supplemented with 5.6 mol% cholesterol to produce proteoliposomes of defined size, which showed negligible leakage of internal substrates. Transport experiments with proteoliposomes revealed a counter-exchange of glucose 1-phosphate (Glc1P), glucose 6-phosphate (Glc6P), inorganic phosphate (Pi), 3-phosphoglycerate and dihydroxyacetone phosphate. The Glc1P/Pi counter-exchange reaction exhibited an apparent K(m) for Glc1P of 0.4 mM. Glc6P was a competitive inhibitor of Glc1P transport (Ki 0.8 mM), and the two hexose phosphates could exchange with each other, indicating the operation of a single carrier protein. Glc1P/Pi antiport in proteoliposomes showed an exchange stoichiometry at pH 8.0 of 1 mol of phosphate transported per mol of sugar phosphate.

Nitrite reduction and carbohydrate metabolism in plastids purified from roots of Pisum sativum L.

Intact preparations of plastids from pea (Pisum sativum L.) roots have been used to investigate the metabolism of glucose-6-phosphate and reduction of inorganic nitrite within these organelles. The ability of hexose-phosphates to support nitrite reduction was dependent on the integrity of the preparation and was barely measurable in broken organelles. In intact plastids, nitrite was reduced most effectively in the presence of glucose-6-phosphate (Glc6P), fructose-6-phosphate and ribose-5-phosphate and to a lesser extent glucose-1-phosphate. The Km (Glc6P) of plastid-located Glc6P dehydrogenase (EC 1.1.1.49) and Glc6P-dependent nitrite reduction were virtually identical (0.68 and 0.66 mM respectively) and a similar relationship was observed between fructose-6-phosphate, hexose-phosphate isomerase (EC 5.3.1.9) and nitrite reduction. The pattern of release of CO2 from different carbon atoms of Glc6P supplied to root plastids, indicates the operation of both glycolysis and the oxidative pentose-phosphate pathway with some recycling in the latter. During nitrite reduction the evolution of CO2 from carbon atom 1 of Glc6P was stimulated but not from carbon atoms 2, 3, 4, or 6. The importance of these results with regard to the regulation of the pathways of carbohydrate oxidation and nitrogen assimilation within root plastids is discussed.

The purification and properties of ferredoxin-NADP(+)-oxidoreductase from roots of Pisum sativum L.

A ferredoxin-NADP(+)-oxidoreductase (FNR) was purified to homogeneity from pea root plastids to a specific activity of 200 nkat.mg protein-1, following acetone precipitation and ferredoxin affinity chromatography. The molecular weight of the enzyme was estimated to be 36,000 and 33,800 by SDS-polyacrylamide gel electrophoresis and molecular exclusion chromatography, respectively. The absorption spectrum of the enzyme suggests it contains flavin as a prosthetic group. The enzyme requires NADPH and did not use NADH as an electron donor. The Km values for NADPH and ferredoxin were calculated to be 28 and 5 microM, respectively. The enzyme exhibited optimal activity at pH 8.0. Although resembling the leaf enzyme in most properties, amino terminal sequencing demonstrates clear differences between the leaf and root proteins and suggests closer homology of the pea root enzyme with the enzyme from spinach roots. A polyclonal antibody against the pea root plastid enzyme was raised by the immunization of rabbits. Judging by immunodiffusion only partial identity was observed between the root plastid and chloroplast FNR. The root plastid FNR enzyme activity was precipitated with increasing concentrations of the antibody, in contrast to the chloroplast enzyme which was not inhibited. The potential usefulness of these antibodies is discussed.

Effect of light/dark cycles on expression of nitrate assimilatory genes in maize shoots and roots.

The level of nitrate reductase (NR) and nitrite reductase (NiR) varied in both shoot and root tissue from nitrate-fed Zea mays L. grown under a 16-hour light/8-hour dark regime over a 10-day period postgermination, with peak activity occurring in days 5 to 6. To study the effect of different light regimes on NR and NiR enzyme activity and mRNA levels, 6-day-old plants were grown in the presence of continuous KNO(3) (10 millimolar). Both shoot NRA and mRNA varied considerably, peaking 4 to 8 hours into the light period. Upon transferring plants to continuous light, the amplitude of the peaks increased, and the peaks moved closer together. In continuous darkness, no NR mRNA or NR enzyme activity could be detected by 8 hours and 12 hours, respectively. In either a light/dark or continuous light regime, root NRA and mRNA did not vary substantially. However, when plants were placed in continuous darkness, both declined steadily in the roots, although some remained after 48 hours. Although there was no obvious cycling of NiR enzyme activity in shoot tissue, changes in mRNA mimicked those seen for NR mRNA. The expression of NR and NiR genes is affected by the light regime adopted, but light does not have a direct effect on the expression of these genes.

The cloning of two tomato lipoxygenase genes and their differential expression during fruit ripening.

A membrane-associated lipoxygenase from breaker-stage fruit of tomato (Lycopersicon esculentum Mill.) was purified and partially sequenced. Using degenerate oligonucleotides corresponding to portions of this sequence, a cDNA was amplified by PCR and used to screen a breaker fruit cDNA library. Two clones, tomloxA and tomloxB, were isolated and one of these (tomloxA) corresponded to the isolated protein. Genomic clones were isolated and sequence data from these were used to obtain the 5' ends of the cDNAs. The 2.8-kb cDNAs encode proteins that are similar in size and sequence to each other and to other plant lipoxygenases. DNA blot analysis indicated that tomato contains three or more genes that encode lipoxygenase. RNA blot analysis showed that tomloxA is expressed in germinating seeds as well as in ripening fruit, where it reached its peak during breaker stage. tomloxB appears to be fruit specific and is at its highest level in ripe fruit.

Purification and properties of nitrite reductase from roots of pea (Pisum sativum cv. Meteor).

Nitrite reductase (EC 1.6.6.4) prepared from pea roots was found to be immunologically indistinguishable from pea leaf nitrite reductase. Comparisons of the pea root enzyme with nitrite reductase from leaf sources showed a close similarity in inhibition properties, light absorption spectrum, and electron paramagnetic resonance signals. The resemblances indicate that the root nitrite reductase is a sirohaem enzyme and that it functions in the same manner as the leaf enzyme in spite of the difference in reductant supply implicit in its location in a non-photosynthetic tissue.

Protein phosphorylation in pea root plastids.

Protein phosphorylation has been investigated in non-photosynthetic plastids of pea roots. Intact and lysed preparations of plastids were incubated with [gamma-(32)P]ATP and three stromal proteins of sizes 41, 58 and 62 kDa were phosphorylated on a serine residue. No other proteins were significantly labelled under the conditions used. The 62 kDa protein is probably phosphoglucomutase and represents a phosphoenzyme catalytic intermediate. The protein kinase(s) and phosphatase(s) acting on the other proteins were not sensitive to exogenous calcium but were sensitive to magnesium. The protein phosphatase which acts on the 41 kDa protein is possibly of type 2C, whereas that acting on the 58 kDa phosphoprotein did not fall into any class defined by mammalian systems. Metabolism of exogenous glucose 6-phosphate by the oxidative pentose phosphate pathway in intact plastids abolished the phosphorylation of the 58 kDa protein. Dihydroxyacetone phosphate, phosphoenolpyruvate and 3-phosphoglycerate also inhibited phosphorylation of the 58 kDa protein and had a time-dependent effect on the phosphorylation of the 41 kDa protein. The significance of these results in relation to a possible role for protein phosphorylation in these plastids is considered.

Purification and partial characterization of a membrane-associated lipoxygenase in tomato fruit.

Membrane-associated lipoxygenase from green tomato (Lycopersicon esculentum L. cv Caruso) fruit has been purified 49-fold to a specific activity of 8.3 mumol.min(-1).mg(-1) of protein by solubilization of microsomal membranes with Triton X-100, followed by anion- exchange and size-exclusion chromatography. The apparent molecular mass of the enzyme was estimated to be 97 and 102 kD by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and size-exclusion chromatography, respectively. The purified membrane lipoxygenase preparation consisted of a single major band following sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which cross-reacts with immunoserum raised against soluble soybean lipoxygenase 1. It has a pH optimum of 6.5, an apparent K(m) of 6.2 mum, and V(max) of 103. mumol.min(-1).mg(-1) of protein with linoleic acid as substrate. Corresponding values for the partially purified soluble lipoxygenase from tomato are 3.8 mum and 1.3 mumol.min(-1).mg(-1) of protein, respectively. Thus, the membrane-associated enzyme is kinetically distinguishable from its soluble counterpart. Sucrose density gradient fractionation of the isolated membranes indicated that the membrane-associated lipoxygenase sediments with thylakoids. A lipoxygenase band with a corresponding apparent mol wt of 97,000 was identified immunologically in sodium dodecyl sulfate-polyacrylamide gel electrophoresis-resolved proteins of purified thylakoids prepared from intact chloroplasts isolated from tomato leaves and fruit.

Starch synthesis and carbon partitioning in developing endosperm.

The biosynthesis of starch is the major determinant of yield in cereal grains. In this short review, attention is focused on the synthesis of the soluble substrate for starch synthesis, ADPglucose (ADPG). Consideration is given to the pathway of ADPG production, its subcellular compartmentation, and the role of metabolite transporters in mediating its delivery to the site of starch synthesis. As ADPG is an activated sugar, the dependence of its production on respiration, changes which occur during development, and the constraints which ATP production may place on carbon partitioning into different end-products are discussed.