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.     

Phosphoenolpyruvate carboxykinase in developing pea seeds is associated with tissues involved in solute transport and is nitrogen-responsive

The aim of this work was to investigate the occurrence of phosphoenolpyruvate carboxykinase (PEPCK) in developing pea (Pisum sativum) seeds in relation to their nitrogen supply. PEPCK was present throughout development, with the peak of PEPCK protein and activity in the seed coat and cotyledons preceding protein accumulation in the cotyledons. It showed a different developmental pattern from enzymes involved in amino acid metabolism (phosphoenolpyruvate carboxylase, glutamine synthetase and glutamate dehydrogenase). Immunolocalization showed that PEPCK was present in parts of the developing seed that are involved in the transport and metabolism of assimilates. Early in development, it was associated with the inner integument of the ovule, the endospermic cytoplasm and the outer cells of the embryo. In the middle of development, around the peak of activity, PEPCK was abundant at the outer surface of the developing cotyledons, in the embryonic axis and in the vasculature of the seed coat. Later in development, PEPCK was associated with the embryonic leaf primordia and meristem and cortex of the radicle. PEPCK protein was strongly induced in vitro in the seed coat by nitrate, ammonium and asparagine, in the cotyledons by asparagine and in planta by the supply of nitrogen, which led to an increase in asparagine secretion by empty seed coats. It is suggested that PEPCK is involved in the metabolism of nitrogenous solutes in developing pea seeds.     

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.     

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.     

Major differences in isoform composition of starch synthase between leaves and embryos of pea (Pisum sativum L.)

Isoforms of starch synthase (EC 2.4.1.21) in pea (Pisum sativum L.) leaves have been identified and compared with those in developing pea embryos. Purification and immunoprecipitation experiments show that most of the soluble starch synthase activity of the leaf is contributed by a novel isoform (SSIII) that is antigenically related to the major soluble isoform of the potato tuber. The major soluble isoform of the embryo (SSII) is also present in the leaf, but contributes only 15% of the soluble activity. Study of the leaf starch of lam mutant peas, which lack the abundant granule-bound isoform responsible for amylose synthesis in the embryo (GBSSI), indicates that GBSSI is not responsible for the synthesis of amylose-like material in the leaf. Leaves appear to contain a novel granule-bound isoform, antigenically related to GBSSI. The implications of the results for understanding of the role of isoforms of starch synthase are discussed. Received: 13 March 1997 / Accepted: 13 May 1997     

Temporary storage compounds and sucrose-starch metabolism in seed coats during pea seed development (Pisum sativum)

Quantitative data for growth, carbohydrate, protein and free amino acid nitrogen content of pea ( Pisum sativum L. cv. Finale) seed coat were obtained during the main stage of seed development. These data allowed us to define the role of the seed coat storage compounds. High amounts of arginine were measured in the seed coat and this amino acid is hypothesized to be synthesized de novo in the seed coat cells. Starch appeared to be stored in a specific parenchyma layer of the seed coat. Starch storage was shown to occur from phloem-unloaded sucrose and high activities of some enzymes of sucrose-starch metabolism (sucrose synthase, EC 2.4.1.13 and ADP glucose pyrophosphorylase, EC 2.7.7.27) were measured. The contribution of seed storage compounds is discussed in terms of buffering embryo nutrition. The sink strength of the young pea seed may be located within the seed coat.     

Starch metabolism in developing embryos of oilseed rape

The aim of this work was to characterise the metabolism of starch in developing embryos of oilseed rape (Brassica napus L. cv. Topaz). The accumulation of starch in embryos in siliques which were darkened or had been exposed to the light was similar, suggesting that the starch is synthesised from imported sucrose rather than via photosynthesis in the embryo. Starch content and the activities of plastidial enzymes required for synthesis of starch from glucose 6-phosphate (Glc6P) both peaked during the early-mid stage of cotyledon development (i.e. during the early part of oil accumulation) and then declined. The mature embryo contained almost no starch. The starch-degrading enzymes α-(EC 3.2.1.1) and β-amylase (EC 3.2.1.2) and phosphorylase (EC 2.4.1.1) were present throughout development. Most of the activity of these three enzymes was extraplastidial and therefore unlikely to be involved in starch degradation, but there were distinct plastidial and extraplastidial isoforms of all three enzymes. Activity gels indicated that distinct plastidial isoforms increase during the change from net synthesis to net degradation of starch. Plastids isolated from embryos at stages both before and after the maximum starch content could convert Glc6P to starch although the rate was lower at the later stage. The results are consistent with the idea that starch synthesis and degradation occur simultaneously during embryo development. The possible roles of transient starch accumulation during embryo development are discussed. Received: 15 May 1997 / Accepted: 30 May 1997     

Regeneration systems from immature embryos of Bulgarian pea genotypes

Ten genotypes from Pisum sativum and Pisum arvense were screened for their regeneration abilities. Most of them were created through experimental mutagenesis from Bulgarian varieties and have various valuable agronomic traits. Embryonic axes from immature embryos were plated on modified Murashige and Skoog medium, containing different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-d), α-naphthaleneacetic acid (NAA) and benzyladenine (BA). Two schemes for direct and indirect organogenesis were established. Callus and shoot formation were induced on media containing 0.2 mM 2,4-d or 5 mM BA, respectively. Embryonic axes formed buds directly when plated on medium with 10 mM BA and 1 mM NAA. Organogenesis and adventitious bud formation were maintained on medium supplemented with BA and NAA. Rhizogenesis was induced on Gamborgs' B5 medium. All screened genotypes were able to regenerate plants with a high efficiency (50–100%) although some differences in their organogenetic response were observed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Evidence that glucose 6-phosphate is imported as the substrate for starch synthesis by the plastids of developing pea embryos

The aim of this work was to determine in what form carbon destined for starch synthesis crosses the membranes of plastids in developing pea (Pisum sativum L.) embryos. Plastids were isolated mechanically and incubated in the presence of ATP with the following ¹⁴C-labelled substrates: glucose, fructose, glucose 6-phosphate, glucose 1-phosphate, fructose 6-phosphate, fructose 1,6-bisphosphate, dihydroxyacetone phosphate. Glucose 6-phosphate was the only substrate that supported physiologically relevant rates of starch synthesis. Incorporation of label from glucose 6-phosphate into starch was dependent upon the integrity of the plastids and the presence of ATP. The rate of incorporation approached saturation at a glucose 6-phosphate concentration of less than 1 mM. It is argued that glucose 6-phosphate is likely to enter the plastid as the source of carbon for starch synthesis in vivo.Abbreviations ADPG PPase ADP-glucose pyrophosphorylase - DHAP dihydroxyacetone phosphate     

Differences in release of endogenous sugars and amino acids from attached and detached seed coats of developing pea seeds

The effect of p -chloromercuribenzenesulfonic acid (PCMBS), carbonylcyanide- m -chlorophenylhydrazone (CCCP) and a high apoplastic pH (pH 7.5 compared with pH 5.5) on the release of sugars (sucrose and glucose) and amino acids from attached and detached seed coats of Pisum sativum L. cv. Marzia into a bathing solution was measured by means of the 'empty seed coat technique'. PCMBS reduced the release of sugars and amino acids from attached as well as from detached seed coats, suggesting that carrier-mediated transport might be involved. CCCP reduced sugar release from attached seed coats while amino acid release was hardly affected. In experiments with detached seed coats CCCP had no effect on release of either sugar or amino acids, suggesting that it is not energy-dependent. Raising the pH of the bathing solution from pH 5.5 to pH 7.5 slightly increased sugar release from both attached and detached seed coats while amino acid release was not affected. This might indicate a role of the apoplastic pH in regulating sugar release from the seed coat via a retrieval mechanism. The presented data indicate that there are important differences between sugars and amino acids with respect to transport processes in the seed coat. This is supported by the observation that the rate of amino acid release from the seed coat was higher than the rate of sugar release. The release data of detached seed coats were subjected to compartmental analysis in order to calculate rate constants for release from cell compartments. In the case of sugars, the half-times for emptying the cytoplasmic and vacuolar compartment were 0.8 h and 12.5 h. respectively. For amino acids the half-times were 0.5 h for emptying the cytoplasmic and 3.8 h for emptying the vacuolar compartment.     

不同含水量的豌豆种子萌发时物质动员及代谢研究

不同含水量的豌豆种子在饱和水蒸气中保持7d过程中,含水量低于萌动临界含水量时,子叶中贮藏蛋白质和淀粉的动员不能启动;含水量达到或超过萌动临界含水量,贮藏物质的动员被启动,豌启种子萌动后,子叶中蛋白质和淀动员程度与种子含水量呈正相关,前3d物质动员的程度比后4d强烈得多,因此,含水量是豌豆种子萌发时物质动员的启动因子和调节因子,同时,豌豆种子的含水量直接影响胚轴的生长状况。     

Starch synthesis in developing wheat grain

The effect of light on the in-vivo rate of starch synthesis in the endosperm of developing wheat (Triticum aestivum cv. Mardler) grain was studied. Individual grains from spikelets grown on the same spike either in darkness or bright light showed no difference in their ability to accumulate radioactivity or to convert this to starch over a 14-h period. Similarly, there was no difference in final grain dry weight between spikes which had been kept in either darkness or normal light from 10 d post anthesis. In contrast, when half-grains (grain which had been bisected longitudinally along the crease region) were incubated by being submerged in culture solution (in vitro) the incorporation of [¹⁴C]sucrose into starch was stimulated by increased irradiance. Further experiments showed that the in-vitro dependence on light could be linked to the availability of oxygen. We suggest that in vitro the diffusion of oxygen into the endosperm cells combined with an increased rate of respiration of the tissue during the incubation causes this limitation. Thus the dependence of starch synthesis on light is an artefact of the in-vitro incubation system. The photosynthetic ability of the green pericarp tissue can be used to prevent the development of anoxia in the endosperm tissue of half-grains incubated in vitro. In conclusion, we propose that starch synthesis in vivo is not dependent on oxygen production by photosynthesis in the green layer of the pericarp.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - dpa days post anthesis - PCA perchloric acid     

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     

Soluble auxin-binding proteins in pea epicotyls

Auxin-binding proteins, have been identified in the soluble cytoplasrnic protein fraction of etiolated pea epicotyls, Pisum sativum L., cv. "Dippes Gelbe Victoria". The binding is specific for the auxins NAA, IAA and 2,4-D with a K_D in the range of 0.1–0.4 μ M . Moreover, the binding is competitive, sensitive to digestion by proteinase and shows linearity with the protein content of the assay mixture. The binding proteins appear to be very labile, since repeated freezing and thawing destroys specific binding. No clear pH-optimum could be detected in the physiological pH-range 5.5–8.0, but the binding was doubled at pH 8.0 compared to pH 5.5–7.0.     

The purification and characterisation of the two forms of soluble starch synthase from developing pea embryos

Soluble starch synthase was purified 10000-fold from developing embryos of pea (Pisum sativum L.). The activity was resolved into two forms which together account for most if not all of the soluble starchsynthase activity in the embryo. The two isoforms differ in their molecular weights but are similar in many other respects. Their kinetic properties are similar, neither isoform is active in the absence of primer, and both are unstable at high temperatures, the activity being abolished by a 20-min incubation at 45° C. Both isoforms are recognised by antibodies raised to the granule-bound starch synthase of pea. Isoform II, which has the same molecular weight (77 kDa) as the granulebound enzyme, is recognised more strongly than Isoform I.     

Development of fertilized ovules and their role in the growth of the pea pod

The histological development of fertilized ovules during fruit-set and development in pea ( Pisum sativum L. cv. Alaska) has been investigated. Killing the ovules on day 0 (anthesis) or day 1 prevented fruit-set and resulted in ovary degeneration. When the ovules were destroyed at later stages the ovaries developed, though the rate of growth of the pod was reduced significantly. Pollination in pea occurs normally the day before anthesis, and fertilization of the egg cell 32 to 48 h later. The first divisions of the zygote and endosperm nuclei started simultaneously (ca 48 h after pollination) but the endosperm developed more rapidly than the embryo; the embryo sac cavity was lined with free endosperm nuclei at the time of beginning suspensor elongation. Extracts of endosperm and ovule coats from ovules at day 7 after anthesis showed fruit-set activity in pea, the latter material having about 3 times more activity than the former per ovule basis. These results indicate that fertilization of the ovule is necessary for fruit-set in pea, and that compounds which induce fruit-set are probably synthesized in the ovules following fertilization.     

Multivariate analyses of polypeptide synthesis in developing maize embryos

Summary Variation in polypeptide synthesis was examined in developing maize embryos of two inbred and two hybrid genotypes. Multivariate analyses were used to evaluate the variation among two-dimensional, electrophoretic separations of polypeptides. Several features of the data set were revealed. Similar developmental patterns were exhibited by all genotypes and no evidence was obtained for differential rates of development for inbreds and hybrids. The differential synthesis of two subsets of polypeptides during embryo development was observed. The multivariate methods employed in this study were a valuable aid in interpreting the results from a large and complex data set.