Pyrophosphate and fructose 2,6-bisphosphate effects on glycolysis in pea seed extracts

The participation of pyrophosphate-dependent phosphofructokinase (PPi-PFK) in plant glycolysis was examined using extracts from pea seeds (Pisum sativum L. cv Alaska). Glycolysis starting with fructose 6-phosphate was measured under aerobic conditions as the accumulation of pyruvate. Pyruvate accumulated in a medium containing PPi and adenosine diphosphate at about two-thirds of the rate in a medium containing adenosine diphosphate and adenosine triphosphate (ATP). The PPi-dependent pyruvate accumulation had the same reactant requirements and sensitivity to glycolysis inhibitors, sodium fluoride, and iodoacetamide, as the well-established ATP-dependent glycolysis. Added fructose 2,6-bisphosphate stimulated both the PPi-dependent pyruvate accumulation and PPi-PFK activity whereas this modulator had no effect on ATP-dependent glycolysis or ATP-PFK. Collectively these results demonstrate a PPi-dependent glycolytic pathway in plants which is responsive to fructose 2,6-bisphosphate.     

Essential role of pyrophosphate homeostasis mediated by the pyrophosphate-dependent phosphofructokinase in Toxoplasma gondii

Many biosynthetic pathways produce pyrophosphate (PPi) as a by-product, which is cytotoxic if accumulated at high levels. Pyrophosphatases play pivotal roles in PPi detoxification by converting PPi to inorganic phosphate. A number of apicomplexan parasites, including Toxoplasma gondii and Cryptosporidium parvum, express a PPi-dependent phosphofructokinase (PPi-PFK) that consumes PPi to power the phosphorylation of fructose-6-phosphate. However, the physiological roles of PPi-PFKs in these organisms are not known. Here, we report that Toxoplasma expresses both ATP- and PPi-dependent phosphofructokinases in the cytoplasm. Nonetheless, only PPi-PFK was indispensable for parasite growth, whereas the deletion of ATP-PFK did not affect parasite proliferation or virulence. The conditional depletion of PPi-PFK completely arrested parasite growth, but it did not affect the ATP level and only modestly reduced the flux of central carbon metabolism. However, PPi-PFK depletion caused a significant increase in cellular PPi and decreased the rates of nascent protein synthesis. The expression of a cytosolic pyrophosphatase in the PPi-PFK depletion mutant reduced its PPi level and increased the protein synthesis rate, therefore partially rescuing its growth. These results suggest that PPi-PFK has a major role in maintaining pyrophosphate homeostasis in T. gondii. This role may allow PPi-PFK to fine-tune the balance of catabolism and anabolism and maximize the utilization efficiency for carbon nutrients derived from host cells, increasing the success of parasitism. Moreover, PPi-PFK is essential for parasite propagation and virulence in vivo but it is not present in human hosts, making it a potential drug target to combat toxoplasmosis.     

Kinetics of the Vacuolar H-Pyrophosphatase : The Roles of Magnesium, Pyrophosphate, and their Complexes as Substrates, Activators, and Inhibitors

The responses of the vacuolar membrane (tonoplast) proton-pumping inorganic pyrophosphatase (H⁺-PPase) from oat (Avena sativa L.) roots to changes in Mg²⁺ and pyrophosphate (PPi) concentrations have been characterized. The kinetics were complex, and reaction kinetic models were used to determine which of the various PPi complexes were responsible for the observed responses. The results indicate that the substrate for the oat root vacuolar H⁺-PPase is Mg₂PPi and that this complex is also a non-competitive inhibitor. In addition, the enzyme is activated by free Mg²⁺ and competitively inhibited by free PPi. This conclusion differs from that reached in previous studies, in which it was proposed that MgPPi is the substrate for plant vacuolar H⁺-PPases. However, models incorporating MgPPi as a substrate were unable to describe the kinetics of the oat H⁺-PPase. It is demonstrated that models incorporating Mg₂PPi as the substrate can describe some of the published kinetics of the Kalanchoë daigremontiana vacuolar H⁺-PPase. Calculations of the likely concentrations of Mg₂PPi in plant cytoplasm suggest that the substrate binding site of the oat vacuolar H⁺-PPase would be about 70% saturated in vivo.     

Effects of 2,4-dinitrophenol and anoxia on the inorganic-pyrophosphate content of the spadix of Arum maculatum and the root apices of Pisum sativum

This work was done to determine whether the inorganic-pyrophosphate (PPi) content of plant tissues changes when the rate of glycolysis is altered. Treatment of excised clubs of the spadix of Arum maculatum L. and root apices of Pisum sativum L. with 2,4-dinitrophenol increased the rates of respiration but had no detectable effects on PPi contents. When the two tissues were subjected to up to 60 min anoxia, no changes in PPi were detected. Anoxia was shown to lead to a fall in ATP and concomitant rises in ADP and AMP in pea roots. It is argued (i) that variation in the rate of glycolysis was not accompanied by detectable changes in PPi content, (ii) that this observation does not favour the view that pyrophosphate fructose 6-phosphate 1-phosphotransferase mediates appreciable entry into glycolysis, and (iii) that PPi content can be maintained when respiratory-chain phosphorylation is inhibited.Abbreviations FW fresh weight - PFK(PPi) pyrophosphate fructose 6-phosphate 1-phosphotransferase - PPi inorganic pyrophosphate     

Distinction between Cytosol and Chloroplast Fructose-Bisphosphate Aldolases from Pea, Wheat, and Corn Leaves

A reinvestigation of cytosol and chloroplast fructose-1,6-bisphosphate (FBP) aldolases from pea (Pisum sativum L.), wheat (Triticum aestivum L.) and corn leaves (Zea mays L.) revealed that the two isoenzymes can be separated by chromatography on diethylaminoethyl (DEAE)-cellulose although the separation was often less clear-cut than for the two aldolases from spinach leaves. Definite distinction was achieved by immunoprecipitation of the two isoenzymes with antisera raised against the respective isoenzymes from spinach leaves. The proportion of cytosol aldolase as part of total aldolase activity was 8, 9, 14, and 4.5% in spinach (Spinacia oleracea L.), pea, wheat, and corn leaves, respectively. For corn leaves we also obtained values of up to 15%. The K_m (FBP) values were about 5-fold lower for the cytosol (1.1-2.3 micromolar concentration) than for the chloroplast enzymes (8.0-10.5 micromolar concentration). The respective K_m (fructose-1-phosphate, F1P) values were about equal for the cytosol (1.0-2.3 millimolar concentration) and for the chloroplast aldolase (0.6-1.7 millimolar concentration). The ratio V (FIP)/V (FBP) was 0.20 to 0.27 for the cytosol and 0.07 to 0.145 for the chloroplast aldolase. Thus, cytosol and chloroplast aldolases from spinach, pea, wheat, and corn leaves differ quite considerably in the elution pattern from DEAE-cellulose, in immunoprecipitability with antisera against the respective isoenzymes from spinach leaves, and in the affinity to FBP.     

Occurrence of alternative respiratory capacity in soybean and pea

Capacity for the alternative respiratory pathway was assessed in leaf and root tissue of male-sterile and fertile soybean (Glycine max [L.] Merr.) plants and in leaf, embryonic axis, and epicotyl tissue as well as isolated mitochondria of pea (Pisum sativum L.) by measurement of oxygen uptake in the presence and absence of KCN and salicylhydroxamic acid. Male-sterile and fertile soybean tissues showed similar responses to the inhibitors, and both possessed a capacity for alternative respiration. We also found that tissue and isolated mitochondria from `Progress No. 9' pea possessed alternative respiratory capacity similar to that of `Alaska' pea.     

Rapid wall relaxation in elongating tissues

Reported differences in the relaxation of cell walls in enlarging stem tissues of soybean (Glycine max [L.] Merr.) and pea (Pisum sativum L.) cause measurements of the yield threshold turgor, an important growth parameter, to be in doubt. Using the pressure probe and guillotine psychrometer, we investigated wall relaxation in these species by excising the elongating tissue in air to remove the water supply. We found that the rapid kinetics usually exhibited by soybean could be delayed and made similar to the slow kinetics previously reported for pea if slowly growing or mature tissue was left attached to the rapidly growing tissue when relaxation was initiated. The greater the amount of attached tissue, the slower the relaxation, suggesting that slowly growing tissue acted as a water source. Consistent with this concept was a lower water potential in the rapidly elongating tissue than in the slowly growing tissue. Previous reports of wall relaxation in pea included slowly growing tissue. If this tissue was removed from pea, relaxation became as rapid as usually exhibited by soybean. It is concluded that the true relaxation of cell walls to the yield threshold requires only a few minutes and that the yield threshold should be constant during so short a time, thus reflecting the yield threshold in the intact plant before excision. Under these conditions, the yield threshold was close to the turgor in the intact plant regardless of the species. The presence of slowly growing or mature tissue delays wall relaxation and should be avoided during such measurements. However, this delay can be used to advantage when turgor of intact growing tissues is being measured using excised tissues because turgor does not change for a considerable time after excision.     

Phosphofructokinase activities in photosynthetic organisms : the occurrence of pyrophosphate-dependent 6-phosphofructokinase in plants and algae

A pyrophosphate-dependent phosphofructokinase (PPi-PFK) activity is detectable in extracts of a wide variety of primitive and advanced plants, the Charalean algae, and in the photosynthetic bacterium, Rhodospirillum rubrum. Angiosperms with extractable PPi-PFK activities 4- to 70-fold higher than the respective ATP-PFK activities tend to be succulent and to exhibit CAM. Even though PPi-PFK activity is not detected in crude extracts of some well known CAM plants, e.g. plants in the Crassulaceae, gel filtration of the extract and/or inclusion of the PPi-PFK activator, fructose 2,6-bisphosphate, in the assay reveals that a PPi-PFK activity is present in these species. Fructose 2,6-bisphosphate likewise activates PPi-PFK activities in extracts of C₃ and C₄ plants. C₃ and C₄ plant PPi-PFK activities are roughly equivalent to ATP-PFK activities in the same species. PPi-PFK activity is also detected in some bryophytes, lower vascular plants, ferns, and gymnosperms. The Charophytes, advanced algae presumed to be similar to species ancestral to vascular plants, exhibit at least 4-fold higher PPi-PFK than ATP-PFK activities. R. rubrum also exhibits a much higher PPi-PFK activity than ATP-PFK activity. These data indicate that PPi-PFK may serve as an alternate enzyme to ATP-PFK in glycolysis in a wide range of photosynthetic organisms.     

Immunological Characterization of Plant Ornithine Transcarbamylases

Pea (Pisum sativum L.) ornithine transcarbamylase (OTC) antisera were used to investigate the immunological relatedness of several plant and animal OTC enzymes. The antisera immunoprecipitated OTC activity in all monocot and dicot species tested, and sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis of immunoprecipitated protein revealed monomeric proteins ranging from 35,200 to 36,800 daltons in size. Pea OTC antisera did not recognize mammalian OTC protein. OTC activity and protein levels detected on sodium dodecyl sulfate polyacrylamide gel electrophoresis immunoblots from homogenates of green leaf, etiolated epicotyl and cotyledon, and root tissues of pea were poorly correlated. This might result from differences in amounts of enzymatically active OTC protein in the homogenates. Alternatively, the antisera may fail to recognize different isozyme forms of OTC, which have been reported for some plant species. A putative cytosolic precursor OTC (pOTC) polypeptide exhibiting and M_r = 39,500 to 40,000 daltons was immunoprecipitated from in vitro translation mixtures of total pea leaf poly(A)⁺ RNA. The size of the pOTC polypeptide, as compared with mature OTC monomer (36,000 daltons), suggests that a 4 kilodalton N-terminal leader sequence, like that responsible for mitochondrial targeting of the mammalian enzyme, may be involved in organellar import of the plant enzyme.     

Localization of cytokinin biosynthetic sites in pea plants and carrot roots

The biosynthesis of cytokinins was examined in pea (Pisum sativum L.) plant organs and carrot (Daucus carota L.) root tissues. When pea roots, stems, and leaves were grown separately for three weeks on a culture medium containing [8-¹⁴C]adenine without an exogenous supply of cytokinin and auxin, radioactive cytokinins were synthesized by each of these organs. Incubation of carrot root cambium and noncambium tissues for three days in a liquid culture medium containing [8-¹⁴C]adenine without cytokinin demonstrates that radioactive cytokinins were synthesized in the cambium but not in the noncambium tissue preparation. The radioactive cytokinins extracted from each of these tissues were analyzed by Sephadex LH-20 columns, reverse phase high pressure liquid chromatography, paper chromatography in various solvent systems, and paper electrophoresis. The main species of cytokinins detectable by these methods are N⁶-(Δ²-isopentyl_adenine-5′-monophosphate, 6-(4-hydroxy-3-methyl-2-butenyl-amino)-9-β-ribofuranosylpurine-5′- monophosphate, N⁶-(Δ²-isopentenyl)adenosine, 6-(4-hydroxy-3-methyl-2-butenylamino)-9-β-ribofuranosylpurine, N⁶-(Δ²-isopentenyl)adenine, and 6-(4-hydroxy-3-methyl-2-butenylamino)purine. On the basis of the amounts of cytokinin synthesized per gram fresh tissues, these results indicate that the root is the major site, but not the only site, of cytokinin biosynthesis. Furthermore, cambium and possibly all actively dividing tissues are responsible for the synthesis of this group of plant hormones.     

Dependence of radiostrontium uptake by pea and lupin on the content of calcium in the nutrient solution

In lupin (Lupinus albus L.) and pea (Pisum sativum L., cv. Raman) it was shown that the uptake of⁸⁹Sr from Knop's nutrient solution is significantly increased from a solution with decreased calcium content (one tenth of the normal content) and is slightly decreased from a solution with higher calcium content (150% of the normal content). The calciphile pea absorbs approximately 50% more calcium than the calciphobe lupin, and accordingly 50% radiostrontium less. The pea plant more strongly blocks the translocation of radiostrontium from roots to overground parts, as is proved by the higher discrimination factor of pea (i.e. by the ratio of specific activities of mCi⁸⁹ Sr/g Ca of roots to overground parts). The presence of chlorine in the nutrient solution decreases the content of radiostrontium per gram of dry matter, both in pea and lupin. Radiostrontium is absorbed quickly by both species of plants and is autoradiographically detectable as early as 2 hours after the introduction of radiostrontium to the nutrient solution. *** DIRECT SUPPORT *** A01GP049 00004     

Differential competence for in vitro adventitous rooting of grass pea (Lathyrus sativus L.)

Grass pea (Lathyrus sativus L.) family leguminosae is cultivated as an important food and feed crop all over the world. It is very recalcitrant and difficult to regenerate and root under in vitro conditions. In this cotext, the study was carried out in three steps to find out the effects of three auxins [naphthalene acetic acid, indole 3 butyric acid, indole-3-acetic acid (IAA)], four sucrose concentrations and six types of substrate most suited for plant growth and helpful in acclimatisation of grass pea. The results showed that 2 mg L^?1 IAA, 3 % sucrose was most suitable for rooting of grass pea. When different concentrations of sucrose were supplied to optimum concentration of IAA in Murashige and Skoog medium, 4.5 % sucrose concentration induced maximum number of 13.70 roots per explants that had positive impact on root length, fresh and dry weight of roots, plant height and morphology of the growing plants. There was 92.66 % acclimatisation and survival rate of these plants using peat moss compared to five other substrates used in this study. The developing plants were vigorous, flowered and set seed contained in pods under glass house conditions. It is concluded that rooting is affected by type and concentration of plant growth regulators and type of substrate has direct bearing on acclimatisation, flowering, pod and seed set of grass pea. As such this paper reports an efficient rooting and acclimatisation system of grass pea that will be very useful in future genetic transformation and breeding for improved characteristics.     

Regulation of the phosphorylation of mitochondrial pyruvate dehydrogenase complex in situ: effects of respiratory substrates and calcium

The activity of the pyruvate dehydrogenase complex (PDC), as controlled by reversible phosphorylation, was studied in situ with mitochondria oxidizing dfifferent substrates. PDCs from both plant and animal tissues were inactivated when pyruvate became limiting. The PDC did not inactivate in the presence of saturating levels of pyruvate. Calcium stimulated reactivation of PDC in chicken heart but not pea (Pisum sativum L.) leaf mitochondria. With pea leaf mitochondria oxidizing malate, inactivation of PDC was pH dependent corresponding to the production of pyruvate via malic enzyme. When pea leaf mitochondria oxidized succinate or glycine, PDC was inactivated. This inactivation was reversed by the addition of pyruvate. Reactivation by pyruvate was enhanced by the addition of thiamine pyrophosphate, as previously observed with nonrespiring mitochondria. These results indicate a major role for pyruvate in regulating the covalent modification of the PDC.     

Phosphoribosyl pyrophosphate and the measurement on inorganic pyrophosphate in plant tissues

This work provides further evidence that plants contain appreciable amounts of inorganic pyrophosphate (PPi), and that breakdown of phosphoribosyl pyrophosphate (PPRibP) does not contribute significantly to the PPi detected in plant extracts. Inorganic pyrophosphate in extracts of the roots of Pisum sativum L., clubs of the spadices of Arum maculatum L., and the developing endosperm of Zea mays L. was assayed with pyrophosphate fructose 6-phosphate 1-phosphotransferase (EC 2.7.1.90), and with sulphate adenyltransferase (EC 2.7.7.4). The two different assays gave the same value for PPi content, and for recovery of added PPi. It was shown that PPRibP is converted to PPi during the extraction of PPi. However, the amounts of PPRibP in clubs of A. maculatum and the developing endosperm of Z. mays were negligible in comparison with the contents of PPi.Abbreviations EDTA ethylenediaminetetraacetic acid - PFK(PPi) pyrophosphate fructose 6-phosphate 1-phosphotransferase - PPi inorganic pyrophosphate - PPRibP phosphoribosyl pyrophosphate     

The proliferation of fungal hyphae in soils supporting mycorrhizal and non-mycorrhizal plants

This study investigated the impact of mycorrhizal plants, non-mycorrhizal plants and soil organic matter on the relative abundance of soil hyphae perceived to belong to indigenous arbuscular mycorrhizal (AM) plants. The mycorrhizal plants corn (Zea mays L.) and barley (Hordeum vulgare L.) and a non-mycorrhizal plant, canola (Brassica napus L.), were grown in unsterilized soil in pots inoculated with mycorrhizal corn root fragments. The abundance of hyphae was measured after 5 weeks and the response of fungal growth to the addition of corn residues in the absence of plants was assessed. The abundance of hyphae was higher in the presence of the mycorrhizal plants than in the other treatments. AM hyphae present under mycorrhizal plants accounted for more than 83% of the measured hyphae. The levels of root colonization of 32% in corn and 27% in barley confirmed the mycorrhizal status of the experimental plants. Only a few points of entry were observed in canola, the non-host plant. The percentage of mycorrhizal colonization was positively related (R ²?=?0.85) to the abundance of soil hyphae, indicating that AM hyphae were the major component of the soil hyphae in the presence of mycorrhizal plants in this study.     

Role of source-to-sink transport of methionine in establishing seed protein quantity and quality in legumes

Grain legumes such as pea (Pisum sativum L.) are highly valued as a staple source of protein for human and animal nutrition. However, their seeds often contain limited amounts of high-quality, sulfur (S) rich proteins, caused by a shortage of the S-amino acids cysteine and methionine. It was hypothesized that legume seed quality is directly linked to the amount of organic S transported from leaves to seeds, and imported into the growing embryo. We expressed a high-affinity yeast (Saccharomyces cerevisiae) methionine/cysteine transporter (Methionine UPtake 1) in both the pea leaf phloem and seed cotyledons and found source-to-sink transport of methionine but not cysteine increased. Changes in methionine phloem loading triggered improvements in S uptake and assimilation and long-distance transport of the S compounds, S-methylmethionine and glutathione. In addition, nitrogen and carbon assimilation and source-to-sink allocation were upregulated, together resulting in increased plant biomass and seed yield. Further, methionine and amino acid delivery to individual seeds and uptake by the cotyledons improved, leading to increased accumulation of storage proteins by up to 23%, due to both higher levels of S-poor and, most importantly, S-rich proteins. Sulfate delivery to the embryo and S assimilation in the cotyledons were also upregulated, further contributing to the improved S-rich storage protein pools and seed quality. Overall, this work demonstrates that methionine transporter function in source and sink tissues presents a bottleneck in S allocation to seeds and that its targeted manipulation is essential for overcoming limitations in the accumulation of high-quality seed storage proteins.

Methionine transporter function in pea phloem and embryo affects sulfur, nitrogen, and carbon acquisition, metabolism, and partitioning, resulting in increased seed yield, protein levels, and quality.     

Relationships between shoot to root ratio, growth and leaf soluble protein concentration of Pisum sativum, Phaseolus vulgaris and Triticum aestivum under different nutrient deficiencies

Relations between shoot to root dry weight ratio (S : R), total plant dry weight (DW), shoot and plant N concentration and leaf soluble protein concentration were examined for pea ( Pisum sativum L.), common bean ( Phaseolus vulgaris L.) and wheat ( Triticum aestivum L.) under different nutrient deficiencies. A regression model incorporating leaf soluble protein concentration and plant DW could explain greater than 80% of the variation in S : R within and between treatments for pea supplied different concentrations of NO₃^– or NH₄⁺ in solid substrate; pea and bean supplied different concentrations of N, P, K and Mg in liquid culture; and wheat supplied different concentrations of N, P, K, Mg, Ca and S in liquid culture. Addition of shoot or plant N concentration to the model explained little more of the variation in S : R. It is concluded that results are consistent with the proposal that macronutrient effects on S : R are primarily mediated through their effects on protein synthesis and growth.     

Concentration of Indole-3-acetic Acid and Its Derivatives in Plants

Seeds of oat, coconut, soybean, sunflower, rice, millet, kidney bean, buckwheat, wheat, and corn and vegetative tissue of oat, pea, and corn were assayed for free indole-3-acetic acid (IAA), esterified IAA, and peptidyl IAA. Three conclusions were drawn: (a) all plant tissues examined contained most of their IAA as derivatives, either esterified or as a peptide; (b) the cereal grains examined contained mainly ester IAA; (c) the legume seeds examined contained mainly peptidyl IAA. Errors in analysis of free and bound IAA are discussed.     

The ectopic expression of the wheat Puroindoline genes increase germ size and seed oil content in transgenic corn

Plant oil content and composition improvement is a major goal of plant breeding and biotechnology. The Puroindoline a and b (PINA and PINB) proteins together control whether wheat seeds are soft or hard textured and share a similar structure to that of plant non-specific lipid-transfer proteins. Here we transformed corn (Zea mays L.) with the wheat (Triticum aestivum L.) puroindoline genes (Pina and Pinb) to assess their effects upon seed oil content and quality. Pina and Pinb coding sequences were introduced into corn under the control of a corn Ubiquitin promoter. Three Pina/Pinb expression positive transgenic events were evaluated over two growing seasons. The results showed that Pin expression increased germ size significantly without negatively impacting seed size. Germ yield increased 33.8% while total seed oil content was increased by 25.23%. Seed oil content increases were primarily the result of increased germ size. This work indicates that higher oil content corn hybrids having increased food or feed value could be produced via puroindoline expression.     

Superoxide Dismutases: II. Purification and Quantitative Relationship with Water-soluble Protein in Seedlings

Superoxide dismutase was purified from pea (Pisum sativum L., cv. Wando) seeds and corn (Zea mays L., cv. Michigan 500) seedlings. The purified pea enzyme eluting as a single peak from gel exclusion chromatography columns contained the three electrophoretically distinct bands of superoxide dismutase characterizing the crude extract. The purified corn enzyme eluted as the same peak as the pea enzyme, and contained five of the seven active bands found in the crude extract. The similar molecular weights and the cyanide sensitivities of these bands indicated that they are probably isozymes of a cupro-zinc superoxide dismutase. One of the remaining corn bands was shown to be a peroxidase.