Identification of ononitol and O-methyl-scyllo-inositol in pea root nodules

Ononitol (4-O-methyl-myo-inositol) and O-methyl-scyllo-inositol were identified in pea (Pisum sativum L.) root nodules formed by twoRhizobium leguminosarum strains. Ononitol was the major soluble carbohydrate in nodules formed by strain 1045 while O-methyl-scyllo-inositol and two unidentified components were dominant in the carbohydrate pattern of the nodules formed by strain 1 a. The cyclitols were also present in the denodulated roots, but to a much smaller extent; in the above-ground plant parts only traces were found. The identification of ononitol and O-methyl-scyllo-inositol was established by gas chromatography and gas chromatography-mass spectrometry utilizing trimethylsilyl- and acetyl-derivatives.Abbreviations GC-MS gas chromatography-mass spectroscopy - TLC thin-layer chromatography     

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.Abbreviations GADPH glyceraldehyde 3-phosphate dehydrogenase - PFK phosphofructokinase - PFP pyrophosphate: fructose 6-phosphate 1-phosphotransferase Bronwen A. Trimming gratefully acknowledges the award of a studentship from the Science and Engineering Research Council     

Accumulation of 14C from exogenous labelled auxin in lateral root primordia of intact pea seedlings (Pisum sativum L.)

When [¹⁴C]indol-3yl-acetic acid was applied to the apical bud of 5-day old dwarf pea seedlings which possessed unbranched primary roots, a small amount of ¹⁴C was transported into the root system at a velocity of 11–14 mm h^-1. Most of the ¹⁴C which entered the primary root accumulated in the young lateral root primordia, including the smallest detectable (20–30 mm from the primary root tip). In older (8-d old) seedlings in which the primary root bore well-developed lateral roots, ¹⁴C also accumulated in the tertiary root primordia. In contrast, little ¹⁴C was detected in the apical region of the primary root or, in older plants, in the apices of the lateral roots.Abbreviations IAA indol-3yl-acetic acid     

The role of phosphofructokinase in glycolytic control in the facultative anaerobe Sipunculus nudus

Summary The involvement of phosphofructokinase (PFK) in glycolytic control was investigated in the marine peanut worm Sipunculus nudus. Different glycolytic rates prevailed at rest and during functional and environmental anaerobiosis: in active animals glycogen depletion was enhanced by a factor of 120; during hypoxic exposure the glycolytic flux increased only slightly. Determination of the mass action ratio (MAR) revealed PFK as a non-equilibrium enzyme in all three physiological situations. Duirng muscular activity the PFK reaction was shifted towards equilibrium; this might account for the observed increase in glycolytic rate under these conditions. PFK was purified from the body wall muscle of S. nudus. The enzyme was inhibited by physiological ATP concentrations and an acidic pH; adenosine monophosphate (AMP), inorganic phosphate (P_i), and fructose-2,6-bisphosphate (F-2,6-P₂) served as activators. PFK activity, determined under simulated cellular conditions of rest and muscular work, agreed well with the glycolytic flux in the respective situations. However, under hypoxia PFK activity surpassed the glycolytic rate, indicating that PFK may not be rate-limiting under these conditions. The results suggest that glycolytic rate in S. nudus is mainly regulated by PFK during rest and activity. Under hypoxic conditions the regulatory function of PFK is less pronounced.Abbreviations ATP, ADP, AMP adenosine tri-, di-, monophosphate - DTT dithiothreitol - EDTA ethylene diaminetetra-acetic acid - F-6-P fructose-6-phosphate - F-1,6-P₂ fructose-1,6-bisphosphate - F-2,6-P₂ fructose-2,6-bisphosphate; bwm, body wall muscle; fresh mass, total body weight - G-6-P glucose-6-phosphate - H enthalpy change - K _a activation constant - K _eq equilibrium constant - K _i inhibition constant - K _m Michaelis constant - MAR mass action ratio - NMR nuclear magnetic resonance - PFK phosphofructokinase - P_i inorganic phosphate - PLA phospho-l-arginine - SD standard deviation - TRIS, TRIS (hydroxymethyl) aminomethane - TRA triethanolamine hydrochloride - V _max maximal velocity     

Distribution of calmodulin in pea seedlings: Immunocytochemical localization in plumules and root apices

Immunofluorescence techniques have been used to study the distribution of calmodulin in several tissues in young etiolated pea (Pisum sativum L.) seedlings. A fairly uniform staining was seen in the nucleoplasm and background cytoplasm of most cell types. Cell walls and nucleoli were not stained. In addition, patterned staining reactions were seen in many cells. In cells of the plumule, punctate staining of the cytoplasm was common, and in part this stain appeared to be associated with the plastids. A very distinctive staining of amyloplasts was seen in the columella of the root cap. Staining associated with cytoskeletal elements could be shown in division stages. By metaphase, staining of the spindle region was quite evident. In epidermal cells of the stem and along the underside of the leaf there was an intense staining of the vacuolar contents. Guard cells lacked this vacuolar stain. Vacuolar staining was sometimes seen in cells of the stele, but the most distinctive pattern in the stele was associated with young conducting cells of the xylem. These staining patterns are consistent with the idea that the interactions of plastids and the cytoskeletal system may be one of the Ca²⁺-mediated steps in the response of plants to environmental stimuli. Nuclear functions may also be controlled, at least in part, by Ca²⁺.     

Role of ATP in nitrite reduction in roots of wheat and pea

Excised wheat (Triticum aestivum L.) and field pea (Pisum arvense L.) roots, incubated under anaerobic conditions or in the presence of uncouplers of oxidative phosphorylation [2,4-dinitrophenol (DNP), carbonylcyanide-m-chlorophenylhydrazone, pentachlorophenol] accumulated nitrite as a result of an inhibition of nitrite reduction. In isolated root plastids, nitrite reduction was dependent on a supply of glucose-6-phosphate (G6P) and did not require ATP. The estimated K_m value for glucose 6-phosphate was 1.25 mM. Glucose and fructose-1,6-diphosphate were ineffective substrates for nitrate reduction. Anaerobic conditions and treatment with DNP, which would result in a cessation of ATP production by the mitochondria and a stimulation of glycolysis via the Pasteur effect, were shown to decrease the G6P content of excised roots of wheat and pea. A negative correlation was observed between the level of G6P and nitrite accumulation on root tissues. It is proposed that an interruption in the supply of G6P to the root plastid under these conditions would result in an inhibition of nitrite reduction leading to nitrite accumulation.Abbreviation G6P glucose-6-phosphate     

Interaction of gibberellic and indole-3-acetic acid on root formation in pea (Pisum sativum L.) epicotyl cuttings

Indole-3-acetic acid (IAA) strongly enhanced rooting of etiolated pea epicotyl cuttings while gibberellic acid (GA₃) enhanced rooting only slightly. The promoting effects of the hormones appeared not until 14 d after the onset of treatment. When GA₃ and IAA were applied together, the initiation of rooting started already after 6 d after onset of treatment. It is suggested that gibberellin plays an important role, in combination with auxin, in the initiation of root formation in Pisum cuttings.Abbreviations IAA Indole-3-acetic acid - GA₃ Gibberellic acid     

Calcium transport by pea root membranes

Calcium transport has been studied using purified endomembrane vesicles from dark-grown roots of Pisum sativum L. Membranes from a mixed microsomal (non-mitochondrial) fraction showed ATP-dependent calcium uptake which was released by the ionophore A 23187, had a pH optimum of 7.2 and required Mg²⁺ for uptake. Membranes were further purified using a rapid sucrosedensity-gradient technique yielding vesicles suitable for transport studies, and were identified using marker enzymes. Uptake by plasma membrane, tonoplast, endoplasmic reticulum and Golgi apparatus was indicated. Uptake by membranes of low density (predominantly tonoplast) had a pH optimum of 7.2–7.4 and nucleotide specificity ATP> guanosine 5-triphosphate>inosine 5-triphosphate>ADP>, while that by high-density membranes had a pH optimum of 7.5–7.9 and less specificity for ATP. The importance of regulating sucrose concentrations in calcium transport studies was demonstrated.Abbreviations ER endoplasmic reticulum - GTP guanosine 5-triphosphate - IDPase inosine diphosphatase - IIP inosine 5-triphosphate     

Characteristics of plastids responsible for starch synthesis in developing pea embryos

The nature of the starch-synthesising plastids in developing pea (Pisum sativum L.) embryos has been investigated. Chlorophyll and starch were distributed throughout the cotyledon during development. Chlorophyll content increased initially, then showed little change up to the point of drying out of the embryo. Starch content per embryo increased dramatically throughout development. The chlorophyll content per unit volume was highest on the outer edge of the cotyledon, while the starch content was highest on inner face. Nycodenz gradients, which fractionated mechanically-prepared plastids according to their starch content, failed to achieve any significant separation of plastids rich in starch and ADP-glucose pyrophosphorylase from those rich in chlorophyll and a Calvin-cycle marker enzyme, NADP-glyceraldehyde-3-phosphate dehydrogenase. However, material that was not sufficiently dense to enter the gradients was enriched in activity of the Calvin-cycle marker enzyme relative to that of ADP-glucose pyrophosphorylase. Nomarski and epi-fluorescence microscopy showed that intact, isolated plastids, including those with very large starch grains, invariably contained chlorophyll in stromal structures peripheral to the starch grain. We suggest that the starch-storing plastids of developing pea embryos are derived directly from chloroplasts, and retain chloroplast-like characteristics throughout their development. Developing pea embryos also contain chloroplasts which store little or no starch. These are probably located primarily on the outer edge of the cotyledons where there is sufficient light for photosynthesis at some stages of development.     

Studies on the biosynthesis of tropane alkaloids by Datura stramonium L. transformed root cultures

Transformed root cultures of Datura stramonium, competent in tropane-alkaloid biosynthesis, have been treated with exogenous plant growth regulators. It was found that combinations of -naphthalene-acetic acid, kinetin (N⁶-furfurylaminopurine) and 2,4-dichlorophenoxyacetic acid induced de-differentiation, causing both the rooty phenotype and the hyoscyamine-biosynthetic capacity to be lost. Alkaloid biosynthesis disappeared rapidly and prior to the loss of morphological integrity. It was observed that the enzymes ornithine decarboxylase (EC 4.1.1.17), arginine decarboxylase (EC 4.1.1.19) and N-methylputrescine oxidase did not show the increase in level normally associated with subculturing the roots. The level of putrescine N-methyltransferase (EC 2.1.1.53) activity, the first enzyme fully committed to hyoscyamine biosynthesis, rapidly declined, about 80% being lost from the roots within 12h. This activity, although showing some temporary restoration, declined further after a few days, and was totally absent from fully dispersed cultures. N-Methylputrescine oxidase persisted at a low level. Following sub-culture of established de-differentiated lines to plant-growth-regulator-free medium, limited root regeneration occurred. The roots formed showed renewed competence in alkaloid biosynthesis and putrescine N-methyltransferase and N-methylputrescine oxidase activities were restored to their normal levels. The relationship between the morphological state and alkaloid-biosynthetic capacity of the cultures is discussed in relation to the overall control of alkaloid biosynthesis.Abbreviations ADC arginine decarboxylase - FW fresh weight - MPO N-methylputrescine oxidase - NAA -naphthalineacetic acid - ODC ornithine decarboxylase - pgr plant growth regulator - PMT putrescine N-methyltransferase We are most grateful to Abigael Peerless and Bridget Chapman for assistance with various part of this work.     

The capacity of plastids from developing pea cotyledons to synthesise acetyl CoA

In order to determine whether the enzymes required to convert triose phosphate to acetyl CoA were present in pea (Pisum sativum L.) seed plastids, a rapid, mechanical technique was used to isolate plastids from developing cotyledons. The plastids were intact and the extraplastidial contamination was low. The following glycolytic enzymes, though predominantly cytosolic, were found to be present in plastids: glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.12), phosphoglycerate kinase (EC 2.7.2.3), and pyruvate kinase(EC 2.7.1.40). Evidence is presented which indicates that plastids also contained low activities of enolase (EC 4.2.1.11) and phosphoglycerate mutase (EC 2.7.5.3). Pyruvate dehydrogenase, although predominantly mitochondrial, was also present in plastids. The plastidial activities of the above enzymes were high enough to account for the rate of lipid synthesis observed in vivo.Abbreviations FPLC fast protein liquid chromatography - PPi pyrophosphate     

The role of ethylene in the control of cell division in cultured pea root tips: a mechanism to explain the excision effect

Summary A transitory cell division block, or excision effect, occurs in the meristem of roots after excision and transfer to culture medium. This block can be induced, in intact seedling roots, by exogenous treatment with ethylene gas. With continuous treatment, the block is longer and the recovery less than after a 4 hour pulse. In excised roots the excision effect can be eliminated by treatment with an inhibitor of ethylene synthesis (aminoethoxyvinylglycine) or action (silver thiosulfate). These experiments provide evidence to support the hypothesis that ethylene from the wounded end of an excised root is involved in a process resulting in a transitory block in cell cycle progression in the meristem. The implications of this hypothesis are discussed.     

Regeneration of vascular tissue in wounded pea roots

Severance of the stele of young main roots of pea (Pisum sativum L.) results in formation of a bridge of vascular tissue in the remaining cortex. Cell divisions occur close to the severed vascular tissues on both the proximal and distal sides of the cut within 24 h. Differentiation of new vascular strands subsequently begins in the same locations and progresses from both sides of the wound into the remaining cortex and also back along the original vascular strands. Most of the vascular tissue which forms the bridge through the cortex differentiates in the acropetal direction. Continuous strands composed of single sieve elements bypass the wound somewhat sooner than the first complete xylem strands; the latter in 60–70% of the cases, are present by 3 d. Cambial activity subsequently adds more xylem and phloem. Vascular regeneration is not affected by removal of the epicotyl or the root tip; it is greatly reduced but not prevented by removal of the cotyledons.     

The role of cytokinins in root development of pea seedlings

Endogenous cytokinin-like activity was detected in pea seedlings usingthe soybean callus bioassay. Higher levels of activity were found in two-day-oldseedling roots and in the root free zone of four-day-old seedlings compared tothe lateral root zone of four-day-old seedlings. By day six, the levels ofendogenous cytokinin-like activity was greatly reduced in both the lateral rootzone and root free zone. Decapitation of the root tip disrupted the spatialorganization of the root. Lateral roots were subsequently found along the entirelength of the root rather than in a discreet lateral root zone. Application of10^–3 M dihydrozeatin to decapitated root tipsovercame the effect of root tip removal and restored the situation to what isnormally found in intact roots. There was little mobility of dihydrozeatin inthe root, with applied ³H-DHZ not moving from the root free zone,even 24 h after application.     

Metabolic links between the biosynthesis of pyrrolizidine alkaloids and polyamines in root cultures of Senecio vulgaris

Isotope feeding and inhibitor experiments were performed in order to elucidate the pathway common to polyamine and alkaloid biosynthesis in root cultures of Senecio vulgaris L. -Difluoromethylarginine, a specific inhibitor of arginine decarboxylase, prevented completely the incorporation of radioactivity from [¹⁴C]arginine and [¹⁴C]ornithine into spermidine and the pyrrolizidine alkaloid senecionine N-oxide. In contrast, -difluoromethylornithine, a specific ornithine-decarboxylase inhibitor, had no effect on the flow of radioactivity from labelled ornithine and arginine into polyamines and alkaloids. Thus, putrescine, the common precursor of polyamines and pyrrolizidine alkaloids, is exclusively derived via the arginine-agmatine route. Ornithine is rapidly transformed into arginine. Recycling of the guanido moiety of agmatine back to ornithine can be excluded. Putrescine and spermidine were found to be reversibly interconvertable and to excist in a highly dynamic state. In contrast, senecionine N-oxide did not show any turnover but accumulated as a stable metabolic product. In-vivo evidence is presented that the carbon flow from arginine into the polyamine/alkaloid pathway may be controlled by spermidine. The possible importance of the metabolic coupling of pyrrolizidine-alkaloid biosynthesis to polyamine metabolism is discussed.Abbreviations DFMA D,l--difluoromethylarginine - DFMO D,l--difluoromethylornithine - FW fresh weight     

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 [1-¹⁴C]-and [6-¹⁴C]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.     

ent-Kaurene biosynthesis in a cell-free system from wheat (Triticum aestivum L.) seedlings and the localisation of ent-kaurene synthetase in plastids of three species

A cell-free system capable of converting [¹⁴C]geranylgeranyl diphosphate to ent-[¹⁴C]kaurene and to an unidentified acid-hydrolysable compound was obtained from the basal portions of 5-d-old shoots of wheat seedlings (Triticum aestivum L.). By means of marker enzyme activities, the synthesis of ent-kaurene and the unknown compound could be quantitatively assigned to a plastid fraction obtained by Percoll-gradient centrifugation of the homogenate. The enzyme activities were located within the plastids, probably in the stroma, because they withstood trypsin treatment of the intact plastids, and the plastids had to be broken to release the activity, which was then obtained in soluble form. Plastid membranes had no activity. Plastid stroma preparations obtained from pea (Pisum sativum L.) shoot tips and pumpkin (Cucurbita maxima L.) endosperm also yielded ent-kaurene synthetase activity, but did not form the unknown compound. The exact nature of the active plastids was not ascertained, but the use of methods for proplastid isolation was essential for full activity, and the active tissues are all known to contain high proportions of proplastids, developing chloroplasts or leucoplasts. We therefore believe that ent-kaurene synthesis may be limited to these categories. Mature chloroplasts from the wheat leaves did not contain ent-kaurene synthetase activity and did not yield the unknown component. Incorporation of [¹⁴C]geranylgeranyl diphosphate into ent-[¹⁴C]kaurene and the unknown component was assayed by high-performance liquid chromatography with on-line radiocounting. ent-[¹⁴C]Kaurene was identified by Kovats retention index and full mass spectra obtained by combined gas chromatography-mass spectrometry. The unknown component was first believed to be copalyl diphosphate, because it yielded a compound on acid hydrolysis, which migrated like copalol on high-performance liquid chromatography and gave a mass spectrum very similar to that of authentic copalol. However, differences in the mass spectrum and in retention time on capillary gas chromatography excluded identity with copalol. Furthermore, the unhydrolysed compound was not converted to ent-kaurene by a cell-free system from C. maxima endosperm as copalyl diphosphate would have been.Abbreviations ADH alcohol dehydrogenase - AMO 1618 2isopropyl-4-(trimethylammoniumchloride)-5-methylphenyl piperi-dine-1-carboxylate - BSA bovine serum albumin - DTT dithioth-reitol - GA_n gibberellin A_n - GAPDH NADP⁺-glyceraldehyde 3-phosphate dehydrogenase - GC-MS combined gas chromatography-mass spectrometry - GGPP all trans-isomer of geranyl-geranyl diphosphate - KS ent-kaurene synthetase - MDH malate dehydrogenase - MAA mevalonate activating activity - SOR shikimate oxidoreductase We thank Mrs. Gudrun Bodtke and Mrs. Dorothee Dasbach for able technical assistance, Prof. L.N. Mander (Australian National University, Canberra, Australia) for ent-[²H₂]kaurene and Dr. Yuji Kamiya (RIKEN, Saitama, Japan) for geranylgeraniol and copalol. The work was supported by the Deutsche Forschungsgemeinschaft.     

Rapid modulation of nitrate reductase in pea roots

The regulatory properties of nitrate reductase (NR; EC 1.6.6.1) in root extracts from hydroponically grown pea (Pisum sativum L. cv. Kleine Rheinländerin) plants were examined and compared with known properties of NR from spinach and pea leaves. Nitrate-reductase activity (NRA) extracted from pea roots decreased slowly when plants were kept in the dark, or when illuminated plants were detopped, with a half-time of about 4 h (= slow modulation in vivo). In contrast, the half-time for the dark-inactivation of NR from pea leaves was only 10 min. However, when root tip segments were transferred from aerobic to anaerobic conditions or vice versa, changes in NRA were as rapid as in leaves (= rapid modulation in vivo). Nitrate-reductase activity was low when extracted from roots kept in solutions flushed with air or pure oxygen, and high in nitrogen. Okadaic acid, a specific inhibitor of type-1 and type-2A protein phosphatases, totally prevented the in vivo activation by anaerobiosis of NR, indicating that rapid activation of root NR involved protein dephosphorylation. Under aerobic conditions, the low NRA in roots was also rapidly increased by incubating the roots with either uncouplers or mannose. Under these conditions, and also under anaerobiosis, ATP levels in roots were much lower than in aerated control roots. Thus, whenever ATP levels in roots were artificially decreased, NRA increased rapidly. The highly active NR extracted from anaerobic roots could be partially inactivated in vitro by preincubation of desalted root extracts with MgATP (2 mM), with a half-time of about 20 min. It was reactivated by subsequently incubating the extracts with excess AMP (2 mM). Thus, pea root NR shares many of the previously described properties of NR from spinach leaves, suggesting that the root enzyme, like the leaf enzyme, can be rapidly modulated, probably by reversible protein phosphorylation/ dephosphorylation.     

Pyruvate dehydrogenase complex and acetyl-CoA carboxylase in pea root plastids: their characterization and role in modulating glycolytic carbon flow to fatty acid biosynthesis

The pyruvate dehydrogenase complex (PDC) and acetyl-CoA carboxylase(ACC, EC 6.4.1.2 [EC] ) have been characterized in pea root plastids.PDC activity was optimum in the presence of 1.0 mM pyruvate,1.5 mM NAD⁺ 0.1 mM CoA, 0.1 mM TPP, 5 mM MgCl₂, 3.0 mM cysteine-HCl,and 0.1 M Tricine (pH 8.0) and represents approximately 47%of the total cellular activity. ACC activity was greatest inthe presence of 1.0 mM acetyl-CoA, 4 mM NaHCO₃ mM ATP, 10 mMMgCl₂, 2.5 mM dithiothreitol, and 100 mM Tricine (pH 8.0). Bothenzymes were stimulated by reduced sulphydryl reagents and inhibitedby sulphydryl inhibitors. ACC was also inhibited by malonyl-CoAwhile PDC was inhibited by both malonyl-CoA and NADH. Both enzymeswere stimulated by DHAP and UDP-galactose while ACC was alsostimulated by PEP and F₁,6P. Palmitic acid and oleic acid bothinhibited ACC, but had essentially no effect on PDC. Palmitoyl-CoAinhibited both enzymes while PA and Lyso-PA inhibited PDC, butstimulated ACC. The results presented support the hypothesisthat PDC and ACC function in a co-ordinated fashion to promoteglycolytic carbon flow to fatty acid biosynthesis in pea rootplastids. Key words: Pisum sativum L., pyruvate dehydrogenase complex, acetyl-CoA carboxylase, roots, non-photosynthetic plastids