Starch phosphorylase enzymes in developing and germinating pea seeds

Phosphorylase has been fractionated during development and germination of seeds of smooth and wrinkled-seeded peas. The total phosphorylase levels have been compared. In addition, a number of other pea tissues and other legumes have been examined. Some kinetic properties of the two enzymes present have been measured. Both enzymes have been further purified by affinity chromatography on Sepharose 4B-starch columns and by sequential gel filtration in the absence and presence of amylopectin. The MW and sub-unit structures of the two enzymes have been examined and their possible roles discussed.     

Kinetic properties of two starch phosphorylases from pea seeds

Both of the starch phosphorylase fractions from Victory Freezer pea seeds, that can be separated by DEAE—cellulose chromatography and purified by Sepharose 4B-starch affinity chromatography, contain pyridoxal 5′-phosphate. The addition of further quantities of pyridoxal 5′-phosphate causes inactivation. Both enzymes showed similar bi-substrate kinetics with d-Glc-1-P and varying amounts of amylopectin and also with P_i and varying amounts of amylopectin. In the direction of glucan sythesis the K_m for amylopectin with phosphorylase II was much higher than with phosphorylase I. However, the two enzymes differed in their behaviour on glucan degradation at varying concentrations of P_i. With phosphorylase II the K_m for amylopectin was dependent on the concentration of P_i but that for phosphorylase I was constant. Phosphorylase II was strongly inhibited by ADPG in the direction of glucan degradation but only slightly in the direction of glucan synthesis by both ADPG and UDPG. Phosphorylase I was only slightly inhibited by ADPG in both directions and by UDPG in synthesis. UDPG inhibited both enzymes moderately in glucan degradation,     

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.     

Contribution of sucrose synthase, ADP-glucose pyrophosphorylase and starch synthase to starch synthesis in developing pea seeds

Using genetic variability existing amongst nine pea genotypes (Pisum sativum L.), the biochemical basis of sink strength in developing pea seeds was investigated. Sink strength was considered to be reflected by the rate of starch synthesis (RSS) in the embryo, and sink activity in the seed was reflected by the relative rate of starch synthesis (RRSS). These rates were compared to the activities of three enzymes of the starch biosynthetic pathway [sucrose synthase (Sus), ADP-glucose pyrophosphorylase and starch synthase] at three developmental stages during seed filling (25, 50 and 75% of the dry seed weight). Complete sets of data collected during seed filling for the nine genotypes showed that, for all enzyme activities (expressed on a protein basis), only Sus in the embryo and seed coat was linearly and significantly correlated to RRSS. The contribution of the three enzyme activities to the variability in RSS and RRSS was evaluated by multiple regression analysis for the first two developmental stages. Only Sus activity in the embryo could explain, at least in part, the significant variability observed for both the RSS and the RRSS at each developmental stage. We conclude that Sus activity is a reliable marker of sink activity in developing pea seeds.     

The granular structure of C-type pea starch and its role in gelatinization

We have used a combination of techniques to study the structure and properties of C-type starch from pea seeds. It was found that all C-type starch granules contain both types of polymorph; the B polymorphs are in the center of the granule and are surrounded by the A polymorphs. During heating in excess salt solution the A and B polymorphs within C-type granules melt independently, giving a double transition in heat capacity and a two-step swelling, compared with single transitions for A- and B-type starches. It was shown that B polymorphs gave a transition with a lower peak temperature than A. The disruption of crystallinity during gelatinization began from the hilum area and was propagated along the granule, accompanied by swelling of disrupted areas. It is proposed that the swelling of disrupted parts of the granule decreases the melting temperature of the neighboring crystallites resulting in the progressive disruption of crystalline areas. The gelatinization process is dependent on the arrangement of A and B polymorphs within the granule. © 1998 John Wiley & Sons, Inc. Biopoly 45: 323–332, 1998     

Two isoforms of the GBSSI class of granule-bound starch synthase are differentially expressed in the pea plant (Pisum sativum L.)

In addition to the GBSSI isoform of starch synthase described previously, the pea plant contains a second, granule-bound isoform, GBSSIb. GBSSI is abundant in pea embryos and Rhizobium root nodules, is present at low levels in pods and is absent from leaves. Mutations at the lam locus eliminate GBSSI from all of these organs. GBSSIb is present in pods, leaves and nodules and is unaffected by mutations at the lam locus. GBSSI and GBSSIb are very similar in molecular mass, primary sequence, activity and antigenic properties. GBSSIb, like GBSSI, can synthesize amylose in the presence of malto-oligosaccharides in isolated starch granules. However, its role in vivo is unclear. The lam mutation eliminates amylose from the starch of embryos but does not affect the relatively small amounts of amylose-like material in the starch of pods, leaves and nodules. The significance of these results for understanding of the regulation of amylose synthesis is discussed.     

Kinetin effect on ATP synthesis and on adenylate kinase activity in pea seeds

Pea seed powder incubated in the presence of AMP and phosphoenolpyruvate (PEP) accumulated relatively large amounts of ATP. The rate of accumulation in     

Carnitine content of pea seedling cotyledons

Using a radioactive assay for the determination of carnitine, the amount in pea cotyledons was shown to vary with age.     

Gelatinization and melting of maize and pea starches with normal and high-amylose genotypes

Normal and high-amylose maize and pea starches (gene ae and ra respectively) were studied during gelatinization by their swelling and solubility patterns, as well as by differential scanning calorimetry. High-amylose (> 60%) starches showed restricted swelling and solubility, compared to normal maize (25%) and pea (35%) genotypes. At low water volume fractions (ν₁ < 0.75), gelatinization occurred by two (pea and high-amylose maize) or three (normal maize) melting steps of crystallites, following the Flory equation. At high water volume fractions, melting of the crystallites and swelling are cooperative processes. On the basis of these experiments, explanations for the differences in behaviour between normal and high-amylose genotypes are discussed.     

Isolation and preliminary characterization of cryptic satellite DNA in pea

Optimum conditions have been established for isolation of ‘cryptic’ satellite DNA from the genome of pea (Pisum sativum), using gradients of CS₂SO₄ containing silver ions. At an Ag⁺ :DNA-P ratio (R) of 0.1, and at alkaline pH, four fractions are obtained: mainband (buoyant density 1.437 g cm³; 67% of total DNA), satellite I (buoyant density 1.582 g/cm³; 7% of total DNA), satellite II (buoyant density 1.520 g/cm³, 11% of total) and satellite III (buoyant density variable between 1.45 and 1.51 g/cm³; 15% of total). The reiterated DNA content of these four fractions has been investigated by reassociation experiments conducted over a Cot range of 1 × 10^?5 to 2.0. All four fractions contain at least two kinetic components; each fraction, including the mainband, consists at least partly of highly reiterated DNA. Ribosomal RNA hybridizes only to the mainband.     

Soluble starch synthases and starch branching enzymes from developing seeds of sorghum

Soluble starch synthases and branching enzymes have been partially purified from developing sorghum seeds. Two major fractions and one minor fraction of starch synthase were eluted on DEAE-cellulose chromatography. The minor enzyme eluted first and was similar to the early eluting major synthase in citrate-stimulated activity, faster reaction rates with glycogen primers than amylopectin primers, and in K_m for ADP-glucose (0.05 and 0.08 mM, respectively). The starch synthase peak eluted last had no citrate-stimulated activity, was equally active with glycogen and amylopectin primers, and had the highest K_m for ADP-glucose (0.10 mM). Four fractions of branching enzymes were recovered from DEAE-cellulose chromatography. One fraction eluted in the buffer wash; the other three co-eluted with the three starch synthases. All four fractions could branch amylose or amylopectin, and stimulated α-glucan synthesis catalysed by phosphorylase. Electrophoretic separation and activity staining for starch synthase of crude extracts and DEAE-cellulose fractions demonstrated complex banding patterns. The colour of the bands after iodine staining indicated that branching enzyme and starch synthase co-migrated during electrophoresis.     

Dye-sensitized photo-oxidation of acid ribonuclease from pea cotyledons

In crude extracts, pea cotyledon acid ribonuclease is not inactivated by photo-oxidation, but after 150-fold purification, it is markedly inactivated when illuminated in the presence of methylene blue at pH 7.2. It is, however, still resistant to methylene blue-sensitized photo-oxidation at pH 5.4. These data suggest that photo-oxidation of methionine, cysteine and tryptophan has no effect on enzyme activity, whereas photo-oxidation of histidine markedly reduces catalytic acitivity. It is thus likely that the mode of action of acid ribonuclease from pea cotyledons is similar to that of pancreatic ribonuclease.     

The effect of light on lipoxygenase activity in dwarf pea seedlings

Continuous illumination of 10-day-old etiolated dwarf pea seedlings caused an increase in lipoxygenase activity. At the same time the activity in both stem and leaf tissue decreased. The lipoxygenase isoenzymes of the whole seedling and separated leaf and stem tissue were affected differently by light. It is concluded that lipoxygenase is not involved directly in photosynthesis or chloroplast development.     

Enzymes involved in starch synthesis in the developing mung bean seed

The levels of free sugars, starch and enzymes involved in starch metabolism—sucrose synthetase, UDP and ADP glucose pyrophosphorylase, phosphorylase and starch synthetase—were assayed during seed development of three cultivars of mung bean (Vigna radiata). Free sugars and starch increased with increasing seed weight. Changes in levels of sucrose synthetase, UDP- and ADP-glucose pyrophosphorylases, and phosphorylase were paralleled by changes in starch accumulation. After the maximum activity levels of these enzymes had been reached, maximum activities of soluble starch synthetase and starch granule-bound starch synthetase occurred. There were high activities of sucrose synthetase and phosphorylase at maximum rates of starch accumulation. Thus, starch could be synthesized via the ADP glucose pathway in mung bean seeds. However, phosphorylase may account for the starch accumulation in the early stages of mung bean seed development.     

Purification of ctp: cholinephosphate cytidylyl-transferase from pea stems

CTP:cholinephosphate cytidylyltransferase (EC 2.7.7.15) was purified from pea (Pisum sativum) stems. The purification involved ammonium sulphate fractionation, ion exchange chromatography, removal of proteases with α₂-macroglobulin and gel filtration. The purified enzyme had K_m values for phosphorylcholine and CTP of 2.1 mM and 0.55 mM respectively. It was found to have a pH optimum of 7.5, a requirement for Mg²⁺ and an M_r of 56000. It could not utilize phosphorylethanolamine and its activity was not stimulated by added phospholipids.     

Vicilin from cajanus cajan seeds

Vicilin from pigeon pea (Cajanus cajan) seeds was purified and characterised. It has a M, of ca 180 000 and consists of two types of subunits having M,s of ca 72 000 and 57 000. The subunits are not linked by disulphide bonds. The vicilin of pigeon pea differs from that of Pisum or Vicia in the absence of small M, subunits.     

Biochemical changes in developing seeds of pigeonpea (Cajanus cajan)

Soluble sugars, starch, soluble nitrogen and protein nitrogen were studied in developing seeds of 3 cultivars of pigeonpea. When expressed on a per seed basis soluble sugars increased up to 35 days after flowering and then declined slightly. Rapid starch accumulation was observed between 14 and 28 days after flowering. The levels of soluble nitrogen and protein nitrogen underwent rapid changes during the same period. Amino-acid composition of seed protein was also studied at different stages of maturation. Sodium dodecyl sulphate polyacrylamide gel electrophoresis of salt-soluble proteins revealed that seed storage globulins are formed after 14 days of flowering and do not change much during later stages of maturation.     

Effect of pisatin inducing fungi on pea polyamines

The spermine and spermidine content of pea pod tissue is not significantly altered by inoculation with the pisatin-inducing fungi, Fusarium solani. Although these polyamines induce pisatin, it appears that they do not accumulate in levels sufficient to serve as internal mediators of pisatin production in infected tissues.