Michael J. Emes

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.

Starch synthesis and carbohydrate oxidation in amyloplasts from developing wheat endosperm

The rates of incorporation of various metabolites into starch by isolated amyloplasts from developing endosperm of spring wheat (Triticum aestivum L. cv. Axona) were examined. Of the metabolites tested that were likely to be present in the cytosol at concentrations sufficient to sustain starch synthesis, only glucose 1-phosphate (Glc1P) supported physiologically relevant rates of starch synthesis. Incorporation of Glc1P into starch was both dependent on the presence of ATP and intact organelles. The rate of incorporation of hexose into starch became saturated at a Glc1P concentration of less than 1 mol·m-3 in the presence of 1 mol·m-3 ATP. Starch synthesis from 5 mol · m-3 ADP-glucose supplied to the organelles occurred at rates 15-fold higher than from similar concentrations of Glc1P, but it is argued that this is probably of little physiological relevance. The net incorporation of hexose units into starch from GlclP was inhibited 50% by 100 mmol.m-3 carboxyatractyloside. Carbohydrate oxidation in the amyloplast was stimulated by the addition of 2-oxoglutarate and glutamine, and in such circumstances incorporation of14C-labelled metabolites into starch was reduced. Glucose 6-phosphate proved to be a better substrate for oxidative pathways than Glc1P. Our results suggest that Glc1P is the primary substrate for starch synthesis in developing wheat endosperm, and that ATP required for starch synthesis is imported via an adenylate translocator.

A rapid method for the isolation of purified amyloplasts from wheat endosperm

A rapid method for the isolation and purification of amyloplasts from the endosperm of developing grains of Triticum aestivum L. has been developed. Cell-free amyloplasts were mechanically isolated from plasmolysed tissue, and then purified by low-speed centrifugation through a single layer of Nycodenz sedimenting onto a cushion of agar. Recovery of amyloplasts was greater than 20% with less than 1% contamination by cytosol, 0.2% by mitochondria, 0.5% by endomembrane system and no contamination by microbodies. This method yields preparations which are routinely 55–65% intact up to 2 h after extraction. Amyloplast integrity was shown to depend upon the external sorbitol concentration, and amyloplastic enzymes in intact preparations were protected from digestion by trypsin.

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.

Metabolite pools during starch synthesis and carbohydrate oxidation in amyloplasts isolated from wheat endosperm

Abstract. Starch synthesis and CO2 evolution were determined after incubating intact and lysed wheat (Triticum aestivum L. cv. Axona) endosperm amyloplasts with 14C-labelled hexose-phosphates. Amyloplasts converted [U-14C]glucose 1-phosphate (Glc1P) but not [U-14C]glucose 6-phosphate (Glc6P) into starch in the presence of ATP. When the oxidative pentose-phosphate pathway (OPPP) was stimulated, both [U-14C]Glc1P and [U-14C]Glc6P were metabolized to CO2, but Glc6P was the better precursor for the OPPP, and Glc1P-mediated starch synthesis was reduced by 75%. In order to understand the basis for the partitioning of carbon between the two potentially competing metabolic pathways, metabolite pools were measured in purified amyloplasts under conditions which promote both starch synthesis and carbohydrate oxidation via the OPPP. Amyloplasts incubated with Glc1P or Glc6P alone showed little or no interconversion of these hexose-phosphates inside the organelle. When amyloplasts were synthesizing starch, the stromal concentrations of Glc1P and ADP-glucose were high. By contrast, when flux through the OPPP was highest, Glc1P and ADP-glucose inside the organelle were undetectable, and there was an increase in metabolites involved in carbohydrate oxidation. Measurements of the plastidial hexose-monophosphate pool during starch synthesis and carbohydrate oxidation indicate that the phosphoglucose isomerase reaction is at equilibrium whereas the reaction catalysed by phosphoglucomutase is significantly displaced from equilibrium.

Glycolytic enzymes in non-photosynthetic plastids of pea (Pisum sativum L.) roots

The presence of the glycolytic enzymes from hexokinase to pyruvate kinase in plastids of seedling pea (Pisum sativum L.) roots was investigated. The recoveries, latencies and specific activities of each enzyme in different fractions was compared with those of organelle marker enzymes. Tryptic-digestion experiments were performed on each enzyme to determine whether activities were bound within membranes. The results indicate that hexokinase (EC 2.7.1.2) and phosphoglyceromutase (EC 5.4.2.1) are absent from pea root plastids. The possible function of the remaining enzymes is considered.

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

Abstract. 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.

Recycling of carbon in the oxidative pentose phosphate pathway in non-photosynthetic plastids

Recycling of carbon in the oxidative pentose phosphate pathway (OPPP) of intact pea root plastids has been studied. The synthesis of dihydroxyacetone phosphate (DHAP) and evolution of CO2 was followed in relation to nitrite reduction. A close coupling was observed between all three measured fluxes which were linear for up to 60 min and dependent upon the integrity of the plastids. However, the quantitative relationship between 1-14CO2 evolution from glucose 6-phosphate and nitrite reduction varied with available hexose phosphate concentration. When 10 mM glucose 6-phosphate was supplied to intact plastids a stoichiometry of 1.35 was observed between 14CO2 evolution and nitrite reduction. As exogenous glucose 6-phosphate was decreased this value fell, becoming 0.47 in the presence of 0.2 mM glucose 6-phosphate, indicative of considerable recycling of carbon. This conclusion was reinforced when using [2-14C]glucose-6-phosphate. The measured release of 2-14CO2 was consistent with the data for 1-14CO2, suggesting complete recycling of carbon in the OPPP. Ribose 5-phosphate was also able to support nitrite reduction and DHAP production. A stoichiometry of 2 NO2−reduced: 1 DHAP synthesised was observed at concentrations of 1 mM ribose 5-phosphate or less. At concentrations of ribose 5-phosphate greater than 1 mM this stoichiometry was lost as a result of enhanced DHAP synthesis without further increase in nitrite reduction. It is suggested that this decoupling from nitrite reduction is a function of excess substrate entering directly into the non-oxidative reactions of the OPPP, and may be useful when the demand for OPPP products is not linked to the demand for reductant. The significance of recycling in the OPPP is discussed in relation to the coordination of nitrate assimilation with carbohydrate oxidation in roots and with the utilisation of carbohydrate by other pathways within plastids.

Phosphoglucomutase Activity During Development of Wheat Grains

Summary Phosphoglucomutase (PGM) activity was measured during the development of endosperm of spring wheat ( Triticum aestivum L. cv. Axona) in crude extracts and in isolated amyloplasts. Results show a 60 % reduction in activity of plastidial PGM activity between 5 and 15 days post anthesis. Measurements of the concentrations of the substrates and co-factors of the PGM reaction in the tissue during this period indicate that the enzyme should be saturated in vivo , suggesting that the reduction in activity of PGM during endosperm development is a function of the amount of PGM protein. The implications of this for starch synthesis are discussed.

Nitrite Reduction in The Roots And Leaves of Pisum Sativum

The location of the nitrate/nitrite reducing mechanisms in plants is distributed between the root and the shoot in proportions which vary according to species. At one extreme, nitrate may be reduced almost entirely in the shoot, e.g. Beta and Chenopodium, while at the other extreme most reduction occurs in the root system as in some woody species. In the majority of species, however, including many important crop plants, both root and shoot participate in reduction. This subject has been discussed in some detail (Pate 1973). The mechanisms of function of both nitrate and nitrite reduction are better understood in leaves than roots. Root nitrogen assimilation has been reviewed by Oaks and Hirel (1985). Shortly after methods became available for the extraction and assay of nitrite reductase (Hageman et al. 1962), the enzyme was shown to be active in tomato roots (Sanderson and Cocking 1964). A few years previously an interesting study by Butt and Beevers (1961), approaching the subject from the direction of carbon-nitrogen relations had shown that glucose-6-phosphate could increase the rate of disappearance of nitrite in contact with preparations from maize roots. The association of both nitrite reductase and the enzymes of the pentosephosphate pathway (PPP) with root plastids, has been developed in a number of studies in the intervening years (see, e.g. Emes and Fowler 1979 a,b). The present paper describes a dual approach to the subject of nitrite reduction by roots, in which studies were made of the purified nitrite reductase (NIR) enzyme and of carbohydratenitrite relationships as found in root plastids. Pea was chosen for this work as a species in which nitrate was thought to be reduced in roots, and from which well authenticated root plastids can be extracted. A large number of labelling techniques was used in this study. Details of these may be found in Bowsher et al. (1988,1989).