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     

Enzymatic control of the accumulation of verbascose in pea seeds

Verbascose, the pentasaccharide of the raffinose family of oligosaccharides, consists of galactose units joined to sucrose. In pea (Pisum sativum) seeds, the content of verbascose is highly variable. In a previous study on a high‐verbascose pea cultivar, the present authors have demonstrated that verbascose is synthesized by a multifunctional stachyose synthase (EC 2.4.1.67), which utilizes raffinose as well as stachyose as a galactosyl acceptor. Herein the results of a study of the cloning and functional expression of stachyose synthase from the low‐verbascose genotype SD1 are reported and it is demonstrated that this line contains a protein with a reduced ability to synthesize verbascose. Analysis of seeds from seven pea lines revealed a positive correlation between verbascose synthase activity and verbascose content. Among these genotypes, only the SD1 line showed low verbascose synthase activity when the data were normalized to stachyose synthase activity. These results suggest that differences in the level of verbascose synthase activity could be caused by mutations in the stachyose synthase gene as well as by variation in the amount of the protein. The lines were also analysed for activity of α‐galactosidase, a catabolic enzyme that could limit the extent of verbascose accumulation. No relationship was found between α‐galactosidase activity and the amount of raffinose family oligosaccharides.     

Role of β-amylase in starch metabolism during soybean seed development and germination

Differences in starch metabolism during seed development and germination of two soybean [ Glycine max (L.) Merrill] genotypes with normal seed β-amylase activity ['Williams' ( Sp 1^b and 'Altona' ( Sp 1^b)] and two soybean genotypes with undetectable seed β-amylase activity ['Chestnut' ( Sp 1^au) and 'Altona' ( Sp 1)] were investigated. Starch and soluble sugar profiles were essentially the same during seed development and germination. Total amylase activity of Williams and Altona ( Sp 1^b) peaked just prior to seed maturity and then dropped off slowly; whereas, the total amylase activity of Chestnut and Altona ( sp 1) was very low throughout seed development and germination. The differences in amylase activity between Altona ( Sp 1 ^b) and Altona ( sp 1) was also seen in leaves. α-Amylase activity was similar in the four genotypes when β-amylase was inhibited with Hg²⁺ but was higher in the two genotypes with normal β-amylase activity when β-amylase was inhibited with heat plus Ca²⁺. Low levels of starch phosphorylase activity were detected throughout seed development and germination, and the activity was similar in three of the genotypes and higher in Altona ( sp 1).
The protein, oil and oligosaccharide contents of mature seeds of the four genotypes were similar. Altona ( sp 1 ^b) and ( sp 1), which appear to be near isogenic lines, were not different in any morphological character or yield.
Altona ( Sp 1 ^b) showed greater hydrolysis of soybean seed starch than Altona ( sp 1), but the evidence indicates that the mutation resulting in greatly reduced or missing β-amylase activity has no effect on starch metabolism of developing and germinating soybean seeds.     

Effects of apoplastic sucrose on carbohydrate pools and sucrose efflux of mesophyll protoplasts of pea (Pisum sativum)

The effects of the apoplastic, i.e. external, concentration of sucrose (0–30 m M ) on O₂ evolution, O₂ consumption, starch, sucrose, glucose and fructose content, and uptake and efflux of sucrose in mesophyll protoplasts of Pisum sativum L. cv. Fenomen were studied. Neither photosynthesis, dark respiration, sucrose nor starch content change with increased apoplastic sucrose concentration. The contents of glucose and fructose in the protoplasts increase with increased apoplastic sucrose concentration. The sucrose efflux increases with increased external sucrose concentrations between 1 and 5 m M , but above this range the efflux decreases with increased external sucrose concentrations. These findings indicate that although external sucrose does not enter the protoplasts, there is a relationship between the external sucrose pool and the internal pools of sugars in the mesophyll protoplasts. The results suggest an active sucrose efflux from the protoplasts at physiological concentrations of apoplastic sucrose (max 5 m M ) and a simple diffusion mechanism at higher concentrations.     

Ascorbic acid effect on the onset of cell proliferation in pea root

The ability of ascorbic acid to induce cell proliferation of non-cycling cells was investigated in quiescent embryo root of Pisum sativum L. cv. Lincoln, as well as in the active plantlet root meristem, where a minor portion of the cells is non-proliferating. Quiescent embryo cells speeded up the G₀–G₁ transition during germination in the presence of ascorbic acid. In addition, proliferating cells present in the root tip of 3-day-old plantlets, arrested at the G₁/S boundary by hydroxyurea, resumed the cycle earlier than the control, when treated with ascorbic acid. In contrast, ascorbic acid was unable to induce the proliferation of non-cycling cells present in the active meristem. Therefore, these data suggest that the ability of ascorbic acid lo induce cell proliferation depends on the physiological status of the cell. In particular the data indicate that ascorbic acid is involved in cell proliferation as a factor necessary to enable already competent cells to progress through the cell cycle phases, but not as a factor able to induce non-competent cells to overcome proliferation arrest.     

Effect of apex excision and replacement by 1-naphthylacetic acid on cytokinin concentration and apical dominance in pea plants

As known from literature lateral buds from pea ( Pisum sativum ) plants are released from apical dominance when repeatedly treated with exogenous cytokinins. Little is known, however, about the endogenous role of cytokinins in this process and whether they interact with basipolar transported IAA, generally regarded as the main signal controlling apical dominance. This paper presents evidence that such an interaction exists.
The excision of the apex of pea plants resulted in the release of inhibited lateral buds from apical dominance (AD). This could be entirely prevented by applying 1-naphthylacetic acid (NAA) to the cut end of the shoot. Removal of the apex also resulted in a rapid and rather large increase in the endogenous concentrations of zeatin riboside (ZR), isopentenyladenosine (iAdo) and an as yet unidentified polar zeatin derivative in the node and internode below the point of decapitation. This accumulation of ZR and iAdo, was strongly reduced by the application of NAA. The observed increase in cytokinin concentration preceded the elongation of the lateral buds, suggesting that endogenous cytokinins play a significant role in the release of lateral buds from AD. However, the effect of NAA on the concentration of cytokinins clearly demonstrated the dominant role of the polar basipetally transported auxin in AD. The results suggest a mutual interaction between the basipolar IAA transport system and cytokinins obviously produced in the roots and transported via the xylem into the stem of the pea plants.     

Beneficial interaction between photosynthesis and respiration in mesophyll protoplasts of pea during short light-dark cycles

The respiratory uptake or photosynthetic evolution of oxygen by mesophyll protoplasts of pea ( Pisum sativum L. cv. Arkel) were monitored during successive short. (3–5 min) cycles of darkness and illumination. The rate of respiration was nearly doubled after 3–4 short periods of illumination while there was a 15–20% enhancement in photosynthesis with cycles of illumination and darkness preceding illumination. Such interaction between photosynthesis and respiration was statistically significant when bicarbonate was present in the reaction medium. The inhibitors of photosynthesis [3(3,4–dichlorophenyl)-l,l-dimethylurea (DCMU), glyceraldehyde] decreased respiration after periods of illumination, whereas inhibitors of respiratory electron transport (Rotenone, antimycin A, NaN₃) suppressed photosynthesis, as well. We suggest that a rapid beneficial interaction exists between photosynthesis and respiration in protoplasts, even during short cycles of light and darkness.     

Interaction of 4-chloroindole-3-acetic acid and gibberellins in early pea fruit development

In pea, normal pod (pericarp) growth requires the presence of seeds; and in the absence of seeds, gibberellins (GAs) and/or auxins can stimulate pericarp growth. To further characterize the function of naturally occurring pea GAs and the auxin, 4-chloroindole-3-acetic acid (4-Cl-IAA), on pea fruit development, profiles of the biological activities of GA3, GA1, and 4-Cl-IAA on pericarp growth were determined separately and in combination on pollinated deseeded ovaries (split-pericarp assay) and nonpollinated ovaries. Nonpollinated ovaries (pericarps) responded differently to exogenous GAs and 4-Cl-IAA than pollinated deseeded pericarps. In nonpollinated pericarps, both GA3 and 4-Cl-IAA stimulated pericarp growth, but GA3 was significantly more active in stimulating all measured parameters of pericarp growth than 4-Cl-IAA. 4-Cl-IAA, GA1, and GA3 were observed to stimulate pericarp growth similarly in pollinated deseeded pericarps. In addition, the synergistic effect of simultaneous application of 4-Cl-IAA and GAs on pollinated deseeded pericarp growth supports the hypothesis that GAs and 4-Cl-IAA are involved in the growth and development of pollinated ovaries.     

Crystalline arrays of ribosomes in isolated pea chloroplasts

Abstract Transmission electron microscopy of chloroplasts isolated by osmotic lysis of pea leaf protoplasts has revealed crystalline arrays of ribosomal particles associated with the thylakoid membranes. Optical diffraction techniques have established the crystallinity of the arrays and an image-enhancement technique has given an indication of ribosomal macrostructure. A model of crystal-packing is presented. This apparently artefactual induction of ribosome crystals should provide a valuable approach towards the elucidation of the details of the structure of chloroplast ribosomes.     

The presence of a p53-like protein during pea seed maturation and germination

ABSTRACT

The mechanisms that allow monitoring of DNA damage and the activation of repair systems in plants are poorly known. In mammalian cells the tumor suppressor protein p53 plays an important role in the checkpoint pathway induced by DNA damage. In this work, we investigated the presence and distribution of the p53-like protein in pea root tip nuclei and its role during early germination in relation to DNA damage. In pea seed, PFGE and TdT assays show that DNA fragmentation occurs during maturation and dry seed storage, and that this DNA fragmentation is repaired at the beginning of germination before the onset of proliferation. In the same seeds, the p53-like protein was found during maturation and germination. Immunoblotting characterization of this protein led to the identification of a single specific protein of about 94 kDa, more abundant at the beginning of the hydration process than in actively cycling cells. Furthermore, the p53-like protein revealed different nuclear distribution patterns, probably in relation to the formation of DNA fragments in dry seeds, and to the reactivation of repair mechanisms during early germination. These data suggest that the presence of a p53-like protein in quiescent or proliferating pea embryo cells is related to DNA damage, and serves for the maintenance of genetic information and the development of normal seedlings.     

Immunolocalization of a thiol-protease induced during the senescence of unpollinated pea ovaries

A thiol-endoprotease induced during the senescence of unpollinated ovaries of Pisum sativum L. cv. Alaska has been localized at both cellular and subcellular levels using purified antibodies. Immunoblot analysis showed a single band of 30 kDa in extracts from senescent ovaries 3 and 4 days post-anthesis. Immunolocalization showed the accumulation of the protease within the exocarp and in the outer cell layers of the mesocarp of the senescent ovaries, although with an asymmetric distribution as illustrated in transverse sections. Ultrastructural localization indicates that the protease is associated with the tonoplast and with electron dense materials within the vacuole, where lysis of cell components occurs in senescent ovaries.     

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.     

The nature of protein differentiation in the growing pea root

Abstract Sections of the growing root of pea (Pisum sativum) have been microdissected into stele, cortex and epidermis. Using labelled amino acids, two dimensional separations employing non-equilibrium pH gel electrophoresis (NEPHGE) and isoelectric focusing (IEF), and silver staining, the complexity of protein differences between the cortex and the stele has been assessed. Analyses commenced as cells in these two tissues appear in the meristem (0.7—1 mm from the tip) and continued up to 30 mm from the tip as they subsequently mature. From the earliest stages at which the cortex and stele can be distinguished and dissected apart the protein patterns differ substantially. However these tissue differences, involving over one third of the detected protein species, are almost all quantitative. Very few qualitative (i.e. tissue specific) proteins were detected. Many proteins also show quantitative stage-specific variation, detected using successively older root segments. In vitro translation studies involving isolated mRNA showed only a very limited stage-specific variation in translated proteins. This supports the notion that translational controls may contribute significantly to the development of these two tissue types.     

Maximum axial root growth pressure in pea seedlings: effects of measurement techniques and cultivars

Values of the maximum axial growth pressure (σ_max) of seedling pea (Pisum sativum L.) roots reported in the literature, obtained using different apparatuses and cultivars, range from 0.3 MPa to 1.3 MPa. To investigate possible reasons for this large range, we studied the effect of apparatus and cultivar on measurements of σ_max in peas. We describe four types of apparatus which can be used to measure axial root growth force and hence σ_max, and used them to measure σ_max in seedling pea roots using cultivar Meteor. Two of these apparatuses were also used to compare σ_max for three pea cultivars (Helka, Meteor and Greenfeast). Both cultivar and apparatus significantly affected σ_max , but there were greater differences between apparatuses than between the three cultivars. Estimating root cross-sectional area from the diameter of cross-sections, rather than from in situ measurements (i.e. measurements made with the root still in place in the apparatus) may explain these differences. This revised version was published online in June 2006 with corrections to the Cover Date.     

Competitive inhibition of the auxin-induced elongation by α-D-oligogalacturonides in pea stem segments

Branca, C, De Lorenzo, G. and Cervone, F. 1988. Competitive inhibition of the auxin-induced elongation by α-D-oligogalacturonides in pea stem segments. - Physiol. Plant. 72: 499–504.
α-D-galacturonide oligomers (OG) were prepared by partial hydrolysis of sodium polypectate with an homogeneous Aspergillus niger endopolygalacturonase (EC 3.2.1.15). OG, obtained after digestion for 10, 20, 30, 60, 120 min and 24 h, were assayed for their ability to interfere with the IAA-induced elongation of pea ( Pisum sativum L. cv. Alaska) stems. Maximum inhibiting activity was exhibited by oligomers with an approximate degree of polymerization higher than 8. Inhibition by longer OG was much lower, and the products of the 24 h digestion and the unhydrolysed polypectate were ineffective. The addition of OG to pea stems caused a parallel shift to the right of the IAA dose-effect curve. The shift depended on the amount of OG used, showing that oligogalacturonides behave as competitive antagonists of IAA. The presence of OG caused the disappearance of the second maximum of the elongation rate and reduced the first maximum. OG were also tested for their ability to inhibit IAA-induced ethylene evolution of pea stem segments. Maximal inhibition was obtained with OG of the same size as those that interfered with IAA-induced elongation. Inhibition of the auxin action seemed to be specific as OG did not interfere with the activity of gibberellic acid (GA₃) or kinetin. It was concluded that oligogalacturonides strongly interfere with the activity of IAA, although they are by themselves incapable to influence the elongation of pea stem segments directly.