Effects of Post-Anthesis High-Temperature Stress on Carbon Partitioning and Starch Biosynthesis in a Spring Wheat (Triticum aestivum L.) Adapted to Moderate Growth Temperatures.

This study investigates carbon partitioning in the developing endosperm of a European variety of spring wheat subjected to moderately elevated daytime temperatures (27°C/16°C d/night) from anthesis to grain maturity. Elevated daytime temperatures caused significant reductions in both fresh and dry weights and reduced the starch content of harvested grains compared to plants grown under a 20°C/16°C d/night regimen. Accelerated grain development caused by elevated temperatures was accounted for by representing plant development as thermal time (°C DPA). We examined the effects of high-temperature stress (HTS) on the uptake and partitioning of [U-14C]-sucrose supplied to isolated endosperms. HTS caused reduced sucrose uptake into developing endosperms from the second major grain-filling stage (approximately 260°C DPA) up to maturity. Enzymes involved in sucrose metabolism were unaffected by HTS, whereas key enzyme activities involved in endosperm starch deposition such as ADP-glucose pyrophosphorylase and soluble isoforms of starch synthase were sensitive to HTS throughout grain development. HTS caused a decrease in other major carbon sinks such as evolved CO2, ethanol-soluble material, cell walls and protein. Despite reductions in the labeling of carbon pools caused by HTS, the relative proportions of sucrose taken up by endosperm cells allocated to each cellular pool remain unchanged, except for evolved CO2, which increased under HTS and may reflect enhanced respiratory activity. The results of this study show that moderate temperature increases can cause significant yield reductions in some temperate wheat cultivars chiefly through three effects: reduced sucrose uptake by the endosperm, reduced starch synthesis and increased partitioning of carbon into evolved CO2.

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.

Isolation and characterisation of a full-length genomic clone encoding a plastidic glucose 6-phosphate dehydrogenase from Nicotiana tabacum.

We describe here the isolation and characterisation of the first full-length genomic clone encoding a plant glucose 6-phosphate dehydrogenase (G6PDH; EC 1.1.1.49) from Nicotiana tabacum L. cv Samsun. The gene was expressed in all tissues, including roots, leaves, stems and flowers. Comparison of the gene with other known plant G6PDH cDNAs grouped this sequence with plastidic isoforms. The protein, minus a putative plastidic transit sequence, was overexpressed in Escherichia coli as a glutathione S-transferase fusion protein. The resulting protein was shown to be immunologically related to the potato plastidic G6PDH. This suggests that the sequence described here codes for a plastidic isoform. Plastidic G6PDH mRNA was induced in both roots and leaves in response to KNO3, and the induction in roots was approximately 4 times the response seen in leaves. Sequence analysis of the 5'-untranslated region of the genomic clone indicated the presence of several NIT2 elements, which may contribute to the control of the expression of this gene. Plastidic G6PDH mRNA levels did not appear to respond to light.

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.

NONPHOTOSYNTHETIC METABOLISM IN PLASTIDS.

Nonphotosynthetic plastids are important sites for the biosynthesis of starch, fatty acids, and the assimilation of nitrogen into amino acids in a wide range of plant tissues. Unlike chloroplasts, all the metabolites for these processes have to be imported, or generated by oxidative metabolism within the organelle. The aim of this review is to summarize our present understanding of the anabolic pathways involved, the requirement for import of precursors from the cytosol, the provision of energy for biosynthesis, and the interaction between pathways that share common intermediates. We emphasize the temporal and developmental regulation of events, and the variation in mechanisms employed by different species that produce the same end products.

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.

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.

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.

Effects of the bisulphite ion on growth and photosynthesis in Sphagnum cuspidatum Hoffm.

Shoots of Sphagnum cuspidatum Hoffm. were collected from two sites: one, a relatively unpolluted site in N. Wales, remote from pollution sources and the other, a grossly polluted site in the South Pennines.* Material from both sites was grown in the laboratory and exposed to artificial rainwater solution with and without bisulphite (HSO3 ) amendment (0.1 mM). Effects of exposure to HSO3 for up to 21 days on growth, photosynthesis, chlorophyll a fluorescence and chlorophyll concentrations were studied in the two Sphagnum populations. Application of HSO3 produced significantly less than maximum growth in Sphagnum from both sites. This effect was far greater, however, in the material from the unpolluted Welsh site. Photosynthesis in the Welsh material treated with HSO3 decreased steadily with time; after 21 days of exposure, photosynthetic oxygen evolution had ceased. This decrease was accompanied by a decrease in fluorescence quenching (as [(P - T)/P]), suggesting a gradual loss of water-splitting activity. In contrast, HSO3 initially stimulated photosynthesis in Sphagnum from the polluted site. Chlorophyll concentration was decreased in Spliagmtm from both sites in the presence of HSO3 Possible mechanisms of tolerance to HSO3 are discussed.

The relationship between extracellular metal accumulation and bisulphite tolerance in Sphagnum cuspidatum Hoffm.

Sphagnum cuspidatum Hoffm. was collected from a remote site in N. Wales, and a polluted site in the S. Pennines. When added to artificial rainwater solution, HSO3 - was oxidized to SO4 2- . The rate at which this oxidation occurred was modified differentially by the mosses from the two sites. S. cuspidatum from the S. Pennines promoted a rapid oxidation rate and disappearance of HSO3 - was complete in 6 h. S. cuspidatum from N. Wales, on the other hand, achieved a very slow oxidation rate and HSO3 - persisted in solution for more than 24 h. Prolonged exposure to HSO3 - in the Welsh material caused damage to, and eventual death of, this material but not of the S. Pennine moss. The rates of HSO3 - oxidation promoted by the mosses from the two sites appear to be related to the concentration of the transition metal ions, Fe(III), Mn(II), and Cu(II), present on the cell-wall cation-exchange sites. These metals, particularly Fe, present on the surface of the S. Pennine material catalysed a rapid chemical oxidation of HSO3 - to SO4 2- . The increased levels of transition metals associated with the S. Pennine moss originate in the peat as a legacy of past pollution events at this site. Levels of Fe were approximately 100 times greater than those for Mn or Cu and 5-10 times higher on the S. Pennine moss than on that from N. Wales. Removal of these metal ions (using EDTA) from the surface of the S. Pennine material removed the HSO3 - oxidizing ability of the moss, leading eventually to cell death. The ability to withstand high levels of HSO3 - was conferred upon the Welsh moss by supplying Fe(m) in artificial rainwater solution under laboratory conditions. Transplanting Sphagnum from the Welsh to the S. Pennine site gave rise to a similar response. Nomenclature of mosses follows Smith (1978).

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.

Purification of plastids from higher-plant roots.

A procedure is described for the purification of plastids from the roots of Pisum sativum L. The preparations obtained are appreciably free of contamination by other particles as judged by the distribution of organelle-specific marker enzymes and by electron microscopy. Latency of glutamate synthase (EC 2.6.1.53) within these preparations indicates that the plastids obtained are 90-95% intact, whilst the resistance of this enzyme, and glucose-6-phosphogluconate dehydrogenase (EC 1.1.1.43) to tryptic digestion in unlysed organelles indicates that they are at least 70-85% intact and may be suitable for studies of metabolite transport.

The supply of reducing power for nitrite reduction in plastids of seedling pea roots (Pisum sativum L.).

Plastids were separated from extracts of pea (Pisum sativum L.) roots by sucrose-density-gradient centrifugation. The incubation of roots of intact pea seedlings in solutions containing 10 mM KNO3 resulted in increased plastid activity of nitrite reductase and to a lesser extent glutamine synthetase. There were also substantial increases in the activity of glucose-6-phosphate and 6-phosphogluconate dehydrogenases. No other plastid-located enzymes of nitrate assimilation or carbohydrate oxidation showed evidence of increased activity in response to the induction of nitrate assimilation. Studies with [1-(14)C]-and [6-(14)C]glucose indicated that there was an increased flow of carbon through the plastid-located pentose-phosphate pathway concurrent with the induction of nitrate assimilation. It is suggested that there is a close interaction through the supply and demand for reductant between the pathway of nitrite assimilation and the pentose-phosphate pathway located in the plastid.

Intracellular interactions between the pathways of carbohydrate oxidation and nitrate assimilation in plant roots.

Using density gradient techniques we have shown that in addition to a location within the cytoplasm all the enzymes of the pentose phosphate pathway are also present within the plastids of apical cells of pea roots. The data are discussed in relation to the hypothesis that the pentose phosphate pathway provides the NADPH for nitrite assimilation, the enzymes of which pathway have previously been shown to be located within the plastids of apical cells of pea roots.

The intracellular location of the enzymes of nitrate assimilation in the apices of seedling pea roots.

The intracellular distribution of the enzymes of nitrate and ammonia assimilation in apical cells of pea (Pisum sativum L.) roots is described. Nitrate reductase (EC 1.6.6.2) was found to have no organelle association, and is considered to be located in the cytosol or possibly loosely bound to the outside of an organelle. Nitrite reductase and glutamate synthase (EC 2.6.1.53) are plastid located, as is glutamine synthetase (EC 6.3.1.2) although this enzyme also has activity in the cytosol. Glutamate dehydrogenase (EC 1.4.1.3) was found only in the mitochondrion.