Pea Cotyledons Research Articles

Osmosensitivity of Sucrose Uptake by Immature Pea Cotyledons Disappears during Development

Sucrose uptake was studied in isolated, immature pea cotyledons (Pisum sativum L. cv Marzia) in relation to their developmental stage. During the developmental period examined the water content of the cotyledons decreased from approximately 80% "stage 1" to approximately 55% "stage 2". When assayed in an isotonic medium (400 osmoles per cubic meter) the influx capacity per gram fresh weight for sucrose was almost constant during this developmental period. The influx could be analyzed into a saturable component (K(m) approximately 9 moles per cubic meter; V(max) approximately 150 nanomoles per minute per gram fresh weight) and an unsaturable component (k(i) approximately 0.5 nanomoles per minute per gram fresh weight [per mole per cubic meter]). Incubation in a hypotonic medium reduced the sucrose influx in stage 1 cotyledons, up to 80% reduction at 0 milliosmole (medium without mannitol), but had no effect on sucrose uptake by stage 2 cotyledons. Reduced uptake in a hypotonic medium (100 osmoles per cubic meter) could be attributed to a lowering of the V(max) from 150 to 36 nanomoles per minute per gram fresh weight. During incubation of stage 1 cotyledons and stage 2-cotyledons in a hypotonic medium (200 osmoles per cubic meter) their volume increased by 16% and 5.6%, respectively, while the calculated turgor pressure increased from 0.2 to 0.6 megapascal for cotyledons of both developmental stages. Reduced sucrose influx in hypotonic medium, therefore, seems to be related to cell swelling (membrane stretching) rather than to increased turgor pressure.

The identification and metabolism of GA9 and its metabolites in seed coats and shoots of pea, line G2

[3H]gibberellin A9 was applied to shoots or seed parts of G2 pea to produce radiolabeled metabolites. These were used as markers during purification for the recovery of endogenous GA9 and its naturally occurring metabolites. GA9 and its metabolites were purified by HPLC, derivatized and examined by GC-MS. Endogenous GA9, GA20, GA29 and GA51 were identified in pea shoots and seed coats. GA51-catabolite and GA29-catabolite were also detected in seed coats. GA70 was detected in seed coats following the application of 1 μg of GA9. Applied [3H]GA9 was metabolized through both the 13-hydroxylation and 2β-hydroxylation pathways. Labeled metabolites were tentatively identified on the basis of co-chromatography on HPLC with endogenous compounds identified by GC-MS. In shoots [3H]GA51 and [3H]GA51-catabolite were the predominant metabolites after 6 hrs, but by 24 hrs there was little of these metabolites remaining, while [3H]GA29-catabolite and an unidentified metabolite predominated. In seed coats [3H]GA51 was the initial product, later followed by [3H]GA51-catabolite and an unidentified metabolite (different from that in shoots), with lesser amounts of [3H]GA20, [3H]GA29 and [3H]GA29-catabolite. [3H]GA70 was a very minor product in both cases. [3H]GA9 was not metabolized by pea cotyledons.

Developmental patterns of fumarase in storage tissues of seeds following germination

Mitochondrial fumarase was purified from cucumber ( Cucumis sativus L., cv. Kaga-aonaga-jibae) cotyledons. The apparent molecular weight of the native enzyme was 100 000 as determined by gel filtration. The subunit molecular weight was 48 000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating that the enzyme is a dimer composed of two identical subunits. A polyclonal antibody against cucumber fumarase was prepared. The antibody cross-reacted with fumarase from cotyledons of pumpkin, pea, mung bean and cowpea, and from the endosperm of castor beam. The development of mitochondrial fumarase in cucumber, pumpkin, mung bean and pea cotyledons was followed by using the anti-cucumber fumarase antiserum. Two types of developmental patterns could be distinguished. In one type, the content of fumarase continued to increase markedly through 4–5 days after the start of imbibition; this type was observed in cucumber and pumpkin. In the other type, the enzyme remained at a more or less constant level during the same period; this type was observed in pea and mung bean. The significance of such marked difference in developmental patterns of fumarase is discussed.

Affinity purification and kinetics of pea cotyledon starch phosphorylases

Affinity purification of two phosphorylases from pea cotyledons has been achieved by chromatography on amylopectin bound to Concanavalin A—Sepharose. Cytoplasmic enzyme bound to amylopectin from normal-starch but the plastid enzyme required the amylopectin from high-amylose starch, with a longer average chain length. The V max/ K m values for a series of branched α-glucans with differing average chain lengths were linearly related to average external chain length. Rabbit-muscle phosphorylase limit-dextrin competitively inhibited the cytoplasmic enzyme with a K is of 13 μM non-reducing end-groups. Progress curves of phosphorolysis with both enzymes showed no product inhibition by d-glucose α-1-phosphate. A study of molecular models of the two reactions suggests that separate binding patterns of glucan may not be necessary for phosphorolysis and synthesis.

Calorimetric studies of the state of water in seed tissues

To understand the physical state of water in hydrating biological tissues, thermodynamic properties of water in cotyledons of pea and soybean with moisture contents ranging from 0.01 g H(2)O/g dw to 1.0 g H(2)O/g dw were studied using differential scanning calorimetry. The heat capacity of the tissues increased abruptly at moisture contents above 0.08 and 0.12 g H(2)O/g dw for soybean and pea cotyledons, respectively. Melting transitions of water were observed at moisture contents >0.23 and 0.26 g H(2)O/g dw for soybean and pea. However, freezing of water was not observed unless moisture contents exceeded 0.33-0.35 g H(2)O/g dw. In both seed tissues, the temperatures of the freezing and melting varied with moisture content and showed hysterisis. The energy of the transition also varied with moisture content and was similar to the heats of fusion and crystallization of pure water only at moisture contents >0.54 and 0.58 gH(2)O/g dw for soybean and pea seeds, respectively. The thermal properties of water change distinctly as seed moisture content changes: at least five states or water can be identified.

Purification of the subunits of pea mitochondrial F 1-ATPase by high-performance liquid chromatography

The subunits of the F 1-ATPase from pea cotyledon mitochondria were purified by reversed-phase chromatography. The resolution of the subunits was affected by several chromatographic parameters: a reversed-phase C 8 column was superior to the less hydrophobic Bio-Gel TSK Phenyl-5-PW column for the resolution of the subunits, acetonitrile was more suitable for good separation of the subunits than 2-propanol and the flow-rate had a significant effect on peak height but little effect on the column resolving power. Tandem chromatography on two reversed-phase chromatography columns with different hydrophobicities was used in an attempt to isolate F 1-ATPase subunits directly from soluble proteins extracted from submitochondrial particles.

Changing kinetics of l-valine uptake by immature pea cotyledons during development

The influx of L-[(14)C]valine was measured over a wide concentration range (1 μM to 100 mM) in immature, isolated pea (Pisum sativum L. cv. Marzia) cotyledons at two developmental stages, both in the absence and in the presence of 0.4 M mannitol. At an early developmental stage (water content of the cotyledons ≈80%) the valine influx was strictly proportional to the external amino-acid concentration over the whole concentration range (so-called linear component). This system renders the plasmalemma of the cotyledonary cells approximately 1000-fold more permeable to valine than the presumed basic permeability of the membrane for valine. At a later stage of development (water content of the cotyledons ∼55%) this transport pathway was supplemented by a saturable system (K m = 5mM; V max = 9.5 μmol · gFW(-1)). The saturable system emerged when the water content of the cotyledons was about 65%, and its activity increased steadily up to the latest developmental stage examined (water content of the cotyledons =50%). As a result, uptake rates of L-valine at low concentrations, expressed on a fresh-weight basis, increased about 15-fold in this phase of development. Low osmolarity of the bathing medium (0 mM mannitol) had no effect on the linear component, and reduced the uptake by the saturable component only slightly by increasing its K m from 3.8 mM to 5.6 mM. The possible role of the saturable system in the acquisition of amino acids by the developing embryo is discussed.

Effect of iso-osmotic levels of salts and PEG-6000 on saccharides, free proline and nitrogen content during early seedling growth of pea (Pisum sativum L.)

Effects of iso-osmotic levels of salts (NaCl, CaCl2, Na2So2) and PEG-6000 on saccharides, free proline and nitrogen contents were studied in cotyledons of pea. Saccharide (total, reducing andnon-reducing) and nitrogen contents decreased with increasing the salt and water stress as compared to control at all the stages of seedling growth. PEG-induced stress had more deleterious effect on total saccharide content and non-reducing saccharide (NRS) content, while salt stress was more harmful to reducing saccharide (RS) and nitrogen content. Free proline content of cotyledons increased with increasing the salt and especially PEG-induced stress at all the stages of seedling growth.

Développement d'axes embryonnaires de pois pourvus ou dépourvus de réserves

Growth of axes attached to, or excised from, cotyledons of pea (Pisum sativum) L. cv. Douce Provence) was measured during 6 days of germination or incubation on aseptic agar nutritive medium (mineral nutrients, sucrose). Growth of attached peas starts from the first hour on germination medium, whereas growth of excised axes starts after a lag period of 4 days. During this lag period, carbohydrate and nitrogen metabolism of the axis is dependent on reserve compounds in the cotyledons: on the one hand, the soluble sugar content of the excised axis decreases as a function of incubation time while no dilution due to water uptake occurs, whereas, on the other hand, the high proportion of mineral nitrogen in excised axes contrasts with their high glutamine synthetase and glutamate synthase activities. These observations emphasize the role of utilisation of sugars as respiratory substrate and of polypeptide synthesis involved in the growth process.

Characterization of α-Amylase from Shoots and Cotyledons of Pea (Pisum sativum L.) Seedlings

The most abundant alpha-amylase (EC 3.2.1.1) in shoots and cotyledons from pea (Pisum sativum L.) seedlings was purified 6700-and 850-fold, respectively, utilizing affinity (amylose and cycloheptaamylose) and gel filtration chromatography and ultrafiltration. This alpha-amylase contributed at least 79 and 15% of the total amylolytic activity in seedling cotyledons and shoots, respectively. The enzyme was identified as an alpha-amylase by polarimetry, substrate specificity, and end product analyses. The purified alpha-amylases from shoots and cotyledons appear identical. Both are 43.5 kilodalton monomers with pls of 4.5, broad pH activity optima from 5.5 to 6.5, and nearly identical substrate specificities. They produce identical one-dimensional peptide fingerprints following partial proteolysis in the presence of SDS. Calcium is required for activity and thermal stability of this amylase. The enzyme cannot attack maltodextrins with degrees of polymerization below that of maltotetraose, and hydrolysis of intact starch granules was detected only after prolonged incubation. It best utilizes soluble starch as substrate. Glucose and maltose are the major end products of the enzyme with amylose as substrate. This alpha-amylase appears to be secreted, in that it is at least partially localized in the apoplast of shoots. The native enzyme exhibits a high degree of resistance to degradation by proteinase K, trypsin/chymostrypsin, thermolysin, and Staphylococcus aureus V8 protease. It does not appear to be a high-mannose-type glycoprotein. Common cell wall constituents (e.g. beta-glucan) are not substrates of the enzyme. A very low amount of this alpha-amylase appears to be associated with chloroplasts; however, it is unclear whether this activity is contamination or alpha-amylase which is integrally associated with the chloroplast.

Crosslinking of microsomal proteins identifies P-9000, a protein that is co-transported with phaseolin and phytohemagglutinin in bean cotyledons

Developing cotyledons of the common bean, Phaseolus vulgaris L., transport within their secretory system (endoplasmic reticulum and Golgi apparatus) the abundant vacuolar proteins, phaseolin and phytohemagglutinin. To identify proteins that may play a role in vacuolar targeting, we treated cotyledon microsomal fractions with a bifunctional crosslinking reagent, dithiobis(succinimidyl propionate), isolated protein complexes with antibodies to phaseolin and phytohemagglutinin, and analysed the polypeptides by sodium dodecylsulfate polyacrylamide gel electrophoresis. This allowed us to identify a protein of Mr=9000 (P-9000) that was crosslinked to both phaseolin and phytohemagglutinin. P-900 is abundantly present in the endoplasmic reticulum. The aminoterminus of P-9000 shows extensive sequence identity with the amino-terminus of PA1 (Mr=11 000), a cysteine-rich albumin whose processing products accumulate in the vacuoles of pea (Pisum sativum L.) cotyledons. Like PA1, P-9000 is synthesized as a pre-proprotein that is posttranslationally processed into smaller polypeptides. The possible functions of P-9000 are discussed.

Auxins Induce α-Amylase Activity in Pea Cotyledons

The report documents how the development of alpha-amylase activity in detached cotyledons of Pisum sativum cv Alaska is accelerated 2- to 12-fold during incubation with 1 micromolar to 10 micromolar 2,4-dichlorophenoxyacetic acid, 2,4,5-trichlorophenoxyacetic acid, or with 4-chloroindoleacetic acid, an endogenous auxin from Pisum sativum. It seems probable that auxin from the embryonic axis induces alpha-amylase in the attached cotyledons during germination.

Plant mitochondrial F1-ATPase. The presence of oligomycin-sensitivity-conferring protein (OSCP)

Purified pea (Pisum sativum) cotyledon F1-ATPase contains six subunits rather than the five usually reported for F1-ATPases. The additional 26.5 kDa (delta) subunit is shown by immunoblotting and N-terminal amino acid sequencing to be similar to bovine oligomycin-sensitivity-conferring protein (OSCP). It is concluded that the delta subunit of plant mitochondrial F1-ATPase is the plant OSCP. This OSCP subunit occurs in all mono- and di-cotyledonous species of plants tested (maize, oats, peas, potatoes, sweet potatoes and turnips).

Reconstitution of Oxidative Phosphorylation and of Oligomycin-Sensitive ATPase by Five- and Six-Subunit Forms of Pea Mitochondrial F1-ATPase

Five- and six-subunit forms of F(1)-ATPase were purified from pea (Pisum sativum L. cv Homesteader) cotyledon submitochondrial particles. Apart from the usual complement of five subunits, the six-subunit enzyme contained an additional 26,500-dalton protein. Both forms of the F(1)-ATPase were used to reconstitute oxidative phosphorylation in F(1)-depleted (ASU) as well as in F(1) and oligomycin-sensitivity conferring protein (OSCP)-depleted (ASUA) bovine mitochondrial membranes. The six-subunit enzyme was considerably more efficient in reconstituting the ATP synthesis than the five-subunit enzyme. Both forms of the enzyme were also able to reconstitute the ATPase activity in ASU- as well as in ASUA-particles. There were substantial differences, however, in the oligomycin sensitivity of the ATPase bound to the ASUA-particles: 20 and 60% inhibition by oligomycin was obtained in the case of the five-subunit and six-subunit enzyme, respectively. We conclude, that the 26,500-dalton protein present in the six-subunit F(1)-ATPase is responsible for the increase in oligomycin sensitivity of the bound enzyme and functions, therefore, as the plant OSCP.

Isolation and partial characterization of clathrin-coated vesicles from pea (Pisum sativum L.) cotyledons

Clathrin-coated vesicles have been isolated from cotyledons of both developing and germinating pea seeds using differential centrifugation, ribonuclease treatment, discontinuous sucrose gradients, and isopycnic centrifugation on a linear D2O-Ficoll gradient. The yield of coated vesicles from developing pea cotyledons was exceptional, being 1.6 × higher than the yield from hog and bovine brain, 5.3 × higher than the yield from carrot suspension cultures, and 13 × the yield from cotyledons of germinating pea seeds. The pea coated vesicles are similar to other plant coated vesicles in size (approximately 80 nm in diameter) and in having a clathrin heavy chain of 190,000 Mr. The lipid phosphorus to protein ratio, 190–250 nmol P per mg protein, of the coated vesicles from plants is comparable to that reported for highly purified coated vesicles from animals. The nondenatured pea clathrin reacted weakly with an antiserum to bovine brain clathrin, but pea clathrin denatured by sodium dodecyl sulfate did not.

Legumin antibodies recognize polypeptides in coated vesicles isolated from developing pea cotyledons

A highly enriched coated vesicle fraction has been isolated from cotyledons of developing pea seeds. This, and coated vesicles isolated from bovine brain as well as from bean leaves were subjected to SDS-PAGE followed by Western blotting with legumin antibodies. A distinct cross reaction with two polypeptides at around 60 kDa was seen, but only with the coated vesicles isolated from peas. Since legumin is synthesized as a 60 kDa precursor, but occurs as 40 and 20 kDa polypeptides in the protein body, we interpret our results as giving support to the idea that reserve proteins, like lysosomal proteins, are transported via coated vesicles.

Extrusion of Protoplasm and Protein Bodies through Pores in Cell Walls of Pea, Bean, and Faha Bean Cotyledons during Imbibition

Several processes contribute to leakage of intracellular substances from legume seeds during imbibition. This study was conducted to determine whether protoplasm and protein bodies also leak from cotyledonary cells by a process analogous to pressure‐driven extrusion from multicellular blisters. The effects of soaking, and air‐drying or fixing excised cotyledons and intact seeds of field‐grown bean (Phaseolus vulgaris L.), pea (Pisum sativum L.), and faba bean (Vicia faba L.) were investigated using scanning electron microscopy. When dissolution and dispersion of intracellular substances were minimized by removing cotyledons from water, cells of excised ‘OSU 605’ pea cotyledons provided direct evidence for cellular pressure‐driven extrusion. Streams of intracellular substances from OSU 605 ranged from 0.2 to 1 μm in diameter. Similar extrusion streams were not found in situ on cotyledons of other genera, cultivars, or treatments; however, projections from protoplast surfaces, pores through cell walls, and orifices and protein bodies in intercellular spaces were consistent with cellular extrusion. The path for cellular extrusion probably was pores through contact cell walls (regions of cell wall in contact with adjacent cells), and then channels along the middle lamella to orifices at intercellular spaces or cotyledonary surfaces.

Sequences responsible for the tissue specific promoter activity of a pea legumin gene in tobacco.

Maturing pea cotyledons accumulate large quantities of storage proteins at a specific time in seed development. To examine the sequences responsible for this regulated expression, a series of deletion mutants of the legA major seed storage protein gene were made and transferred to tobacco using the Bin19 disarmed Agrobacterium vector system. A promoter sequence of 97 bp including the CAAT and TATA boxes was insufficient for expression. Expression was first detected in a construct with 549 bp of upstream flanking sequence which contained the the leg box element, a 28 bp conserved sequence found in the legumin-type genes of several legume species. Constructs containing -833 and -1203 bp of promoter sequence significantly increased levels of expression. All expressing constructs preserved seed specificity and temporal regulation. The results indicate that promoter sequences between positions -97 and -549 bp are responsible for promoter activity, seed specificity, and temporal regulation of the pea legA gene. Sequences between positions -549 and -1203 bp appear to function as enhancer-like elements, to increase expression.

Terminal N-Acetylglucosamine Containing Proteins and N-Acetylglucosamine Binding Proteins from Organelles and Membranes of Developing Pea ( Pisum sativum L.) Cotyledons

Summary Endoplasmic reticulum, Golgi apparatus, and transit vesicles, the organelles involved in intracellular transport of storage proteins, were isolated from developing pea cotyledons. After organelle lysis and washing of the membranes with salt to remove the bulk of the matrix proteins, additional proteins were eluted from all three membrane fractions with N-acetylglucosamine. A N-acetylglucosamine-binding, 67,000 Mr integral membrane protein was released from all three membrane fractions, as well as protein body membranes, by treatment with Triton X-100 and partially purified by affinity chromatography.

Diamine Oxidase in Cotyledons of Pisum sativum Develops as a Result of the Supply of Oxygen through the Embryonic Axis during Germination

The activity of diamine oxidase (EC 1.4.3.6.) in pea, Pisum sativum cv Alaska, cotyledons was studied. The rapid hydration caused by soaking seeds in water, the excision of the embryonic axis, and the suppression of the elongation of the embryonic axis by indoleacetic acid generate anaerobic conditions in these cotyledons that suppress diamine oxidase activity. These results show that oxygen is essential for the induction of diamine oxidase activity in pea cotyledons. During germination cotyledonary diamine oxidase develops as a result of the supply of oxygen through the embryonic axis of the intact pea seedling.