Amino Acid Release from the Seed Coat of Developing Seeds of Vicia faba and Pisum sativum

After removal of the embryo from developing ovules of Viciafaba L. and Pisum sativum L., seed-coat exudates were collectedand the amino acid fraction of the exudate was analyzed. InV. faba, alanine was the most important compound of the aminoacid fraction. In P. sativum, alanine and glutamine were thetwo most important components, whereas only small amounts ofasparagine were present. Comparison with published data suggeststhat seed-coat exudates may differ from phloem sap in the relativeimportance of these amino acids. Pisum sativum, pea, Vicia faba, broad bean, amino acid transport, amino acid unloading, seed-coat exudate, seed development     

Time Course and Polarity of Primary Phloem Fibre Differentiation in Pisum sativum

Primary phloem fibres of Pisum sativum deposit lignified cellwalls, 2 and 5 days after germination in root and stem, respectively.Fibre bundles reach their final size within 4–6 days.The differentiation of the bundles as a whole is faster in thestem compared with the root. In the special diverging bundlesof the lower internodes of the stem the peripheral portionsmature earlier than the inner portions. A wound in the rootas well as in the stem, interrrupts the differentiation of fibresdirectly below it. At least one bundle below a wound is disturbedand often a whole bundle is missing. The meaning of these findingsconcerning the control of cell size is discussed. Pisum sativum, differentiation, induction, phloem fibres, polarity, time course     

An Acidic Polysaccharide in Seeds of Pisum sativum L.

A phenanthrene-assimilating bacterium which belongs to the genus Aeromonas was isolated from soil. The cells which adapted to phenanthrene required a growth lag time on a naphthalene medium. The cells oxidized l-hydroxy-2-naphthoate (1H2NA), 2-carboxybenzaldehyde (2CBAL), o-phthalate (OPA) and protocatechuate (PCA) but did not oxidize salicylaldehyde (SAL), salicylate (SA) and catechol (CAT) which are intermediates in naphthalene catabolism. Using the cell-free extract, the same results were obtained in oxidative capacity. The intact cells metabolized 1H2NA and 2CBAL without the lag time, giving 2CBAL and PCA, respectively. The ammonium sulfate-treated extract prepared from the cells grown in phenanthrene medium, converted 1H2NA to 2CBAL and 2CBAL to OPA. It was suggested that the Aeromonas sp. degraded phenanthrene through OPA.     

Effect of Potassium on Sucrose and Amino Acid Release from the Seed Coat of Developing Seeds of Pisum sativum

After removal of the embryo from developing seeds of Pisum sativum,the ‘empty’ ovules (seed coats without enclosedembryo) were filled with a solution (pH 5.5) containing mannitol(usually 400 mM) to which various salts were added. A solutioncontaining two isotopes ((a) [²H]-sucrose/[–¹⁴C]aminoisobutyricacid (AIB) or (b) [³H]valine/[₁₄C]asparagine mixture) was administeredto the plant via the petiole subtending the fruiting node, and[²H]solute and [¹⁴C]solute unloading from the seed coat wasmeasured, in pulse-labelling experiments of about 5 h. The presenceof 25 or 50 mM K⁺ in the ‘empty’ ovule enhancedthe release of sucrose from the seed coat particularly duringthe first hours of the experiment, but the stimulating effectof K⁺ on the release of labelled solutes derived from aminoacids was much smaller. The presence of 25 mM CaCl₂ did notaffect the release of sucrose or amino acids from the seed coat.The effect of K⁺ on sucrose and amino acid release is explainedas an inhibition of sucrose and amino acid resorption from theseed coat apoplast into seed coat cells, after unloading fromthe seed coat unloading sites. It is suggested that amino acidrelease is much less affected by K⁺ than sucrose release, becausefar less resorption of amino acids by seed coat parenchyma cellstakes place during amino acid transport into the seed coat cavity. Pisum sativum, pea, assimilate transport, assimilate unloading, seed-coat exudate, seed development, sucrose resorption, surgical treatment     

Isozymes of beta-N-Acetylhexosaminidase from Pea Seeds (Pisum sativum L.)

Four isozymes of β-N-acetylhexosaminidase (β-NAHA) from pea seeds (Pisum sativum L.) have been separated, with one, designated β-NAHA-II, purified to apparent homogeneity by means of an affinity column constructed by ligating p-aminophenyl-N-acetyl-β-d-thioglucosaminide to Affi-Gel 202. The other three isozymes have been separated and purified 500- to 1750-fold by chromatography on Concanavalin A-Sepharose, Zn²⁺ charged immobilized metal affinity chromatography, hydrophobic chromatography, and ion exchange chromatography on CM-Sephadex. All four isozymes are located in the protein bodies of the cotyledons. The molecular weight of each isozyme is 210,000. β-NAHA-II is composed of two heterogenous subunits. The subunits are not held together by disulfide bonds, but sulfhydryl groups are important for catalysis. All four isozymes release p-nitrophenol from both p-nitrophenyl-N-acetyl-β-d-glucosaminide and p-nitrophenyl-N-acetyl-β-d-galactosaminide. The ratio of activity for hydrolysis of the two substrates is pH dependent. The K_m value for the two substrates and pH optima of the isozymes are comparable to β-NAHAs from other plant sources.     

An Analysis of Seed Development in Pisum sativum L.

Growth analysis has been performed on developing seeds and seedcomponents of six contrasting genotypes of Pisum sativum. Seeddevelopment has been divided into three phases of high growthrate separated by two ‘lag’ phases, or phases oflow growth rate. It is suggested that the timing of these growthphases may not be determined by the developing seed, since thereappeared to be no consistent correlation with particular physiologicalstages of seed development. The relationship between the absolutegrowth rate of the embryo sac and of the embryo as determinedfrom changes in volume is reflected in the accumulation andabsorption of the endosperm. The relative growth rate of theembryo volume was invariably higher than that of the embryosac although the difference between these two relative ratesvaried with genotype and may account in part for the differencein seed phenotype. It is suggested that the testa and embryo are sinks which bothcompete for the endosperm which may act as a common source,and that this relationship accounts for variation in endospermvolume. It is concluded that seed development is dependent on threelevels of plant organization, the maternal parent, interactionsbetween the components of the seed and the genetic constitutionof the embryo. Pisum sativum L., garden pea, seed development, growth analysis     

Iron Transport to Developing Ovules of Pisum sativum (I. Seed Import Characteristics and Phloem Iron-Loading Capacity of Source Regions)

To understand the processes that control Fe transport to developing seeds, we have characterized seed growth and Fe accretion and have developed a radiotracer technique for quantifying phloem Fe loading in vegetative source regions of Pisum sativum. In hydroponically grown plants of cv Sparkle, developing ovules exhibited a seed-growth period of 22 d, with Fe import occurring throughout the 22-d period. Average Fe content of mature seeds was 19 [mu]g. Source tissues of intact plants were abraded and pulse labeled for 4 h with 100 [mu]M 59Fe(III)-citrate. Fe was successfully phloem loaded and transported to seeds from leaflets, stipules, and pod walls. Total export of 59Fe from labeled source regions was used to calculate tissue-loading rates of 36, 40, and 51 pmol of Fe cm-2 h-1 for the leaflet, stipule, and pod wall surfaces, respectively. By comparison, surface area measurements, along with seed-growth results, allowed us to calculate average theoretical influx values of 42 or 68 pmol of Fe cm-2 h-1 for vegetative tissues at nodes with one or two pods, respectively. Additional studies with the regulatory pea mutant, E107 (a single-gene mutant of cv Sparkle that can overaccumulate Fe), enabled us to increase Fe delivery endogenously to the vegetative tissues. A 36-fold increase in Fe content of E107 leaves, relative to Sparkle, resulted in no increase in Fe content of E107 seeds. Based on these findings, we hypothesized that Fe is phloem loaded in a chelated form, and the expression/synthesis of the endogenous chelator is an important factor in the control of Fe transport to the seeds.     

Characteristics of Sugar, Amino Acid and Phosphate Release from the Seed Coat of Developing Seeds of Vicia faba and Pisum sativum

After removal of the embryo from developing seeds of Vicia fabaL. and Pisum sativum L., the ‘empty’ ovules werefilled with a standard solution (pH 5.5). Seed coat exudatesof both species were collected during relatively long experiments(up to about 12 h) and the concentration of sugar (mainly sucrose),amino acids and phosphate in the exudate measured. A discussionis presented on the amino acid/sugar ratio and the phosphate/sugarratio in the seed coat exudate. A pretreatment (15 min) withp-chloromercuribenzenesulphonic acid (PCMBS) reduced the releaseof sugar, amino acids and phosphate from broad bean seed coats.After excision of ‘empty’ ovules of Vicia faba andPisum sativum from the maternal plant, 2–4 h after thistreatment a strong difference became visible between sucroserelease from excised seed coats and sucrose release from attachedseed coats. Similarly, when the rate of phloem transport ofsucrose into an ‘empty’ ovule of Vicia faba or Pisumsativum was reduced by a sub-optimal mannitol concentrationin the solution, a reduced rate of sugar release from the seedcoat could be observed. Excision and treatment with a sub-optimalmannitol concentration reduced the release of amino acids toa lesser extent than for sucrose. These treatments did not reducethe rate of phosphate release from the seed coat. Key words: Seed development, Seed coat exudate, Phloem transport     

豌豆种子吸胀过程中脱水耐性变化的时间模式

研究了豌豆种子吸胀过程中脱水耐性的变化模式。种子在吸胀初期迅速吸收水分,然后缓慢吸收直到平台期。电解质渗漏速率在吸胀初期增加直到11h,然后随着吸胀下降。在吸胀过程中,种子的萌发率逐渐增加,种子和胚轴的脱水耐性逐渐丧失,10％和50％的种子和胚轴被脱水致死的含水量明显增加。赤霉素和脱落酸处理改变豌豆种子的萌发特性,提高胚轴的脱水耐性。研究结果表明,吸胀的豌豆种子脱水耐性的丧失是一种数量性状,正常性种子吸胀后脱水耐性的变化能够作为种子顽拗性研究的模式系统。     

Karyotype revised of Pisum sativum using chromosomal DNA amount

Pisum sativum was one of the first plants for which the mitotic karyotype was recognized and the karyogram assembled. These achievements were required owing to the physical mapping of P. sativum, providing data for evolutionary approaches and breeding programs. In spite of significant advances, precise morphometric characterization of chromosomes and karyogram assembly of P. sativum have become a topical problem. The present study proposes an unambiguous classification for the chromosomes of P. sativum, based on classical cytogenetic rules and chromosomal DNA amount. Cytogenetic procedure yielded mitotic cells showing morphologically preserved and stoichiometrically stained chromosomes. Twelve mitotic cells were selected, and the mean values for total, short- and long-arm lengths and DNA amount were measured for each chromosome. Chromosomal DNA amount fully correlated with total chromosome length, whose value proportionally decreases with the amount of DNA. Considering these data, all seven chromosomes could be unambiguously identified, yielding a new cytogenetic classification for P. sativum chromosomes. Moreover, the chromosome pairs were ordered according to the classical cytogenetic rule for assembly of karyograms. Since P. sativum is considered a model plant, it was possible to correlate the newly outlined karyotype with other cytogenetic data and linkage groups.     

Phloem exudate metabolic content reflects the response to water-deficit stress in pea plants (Pisum sativum L.)

Drought stress impacts the quality and yield of Pisum sativum. Here, we show how short periods of limited water availability during the vegetative stage of pea alters phloem sap content and how these changes are connected to strategies used by plants to cope with water deficit. We have investigated the metabolic content of phloem sap exudates and explored how this reflects P. sativum physiological and developmental responses to drought. Our data show that drought is accompanied by phloem-mediated redirection of the components that are necessary for cellular respiration and the proper maintenance of carbon/nitrogen balance during stress. The metabolic content of phloem sap reveals a shift from anabolic to catabolic processes as well as the developmental plasticity of P. sativum plants subjected to drought. Our study underlines the importance of phloem-mediated transport for plant adaptation to unfavourable environmental conditions. We also show that phloem exudate analysis can be used as a useful proxy to study stress responses in plants. We propose that the decrease in oleic acid content within phloem sap could be considered as a potential marker of early signalling events mediating drought response.     

Apoplastic Phloem Unloading in the Stem of Bean

Sucrose has been found in the apoplast of bean stems at a concentrationof 25–60 mM with an axial concentration gradient in theappropriate direction for Munch translocation. Removal of theepidermis from a 50 mm length of stem enabled the washout oflabelled photosynthate from the apoplast. The rate of labelwashout was strongly dependent on temperature, and the rateincreased on blockage of phloem pathways to the main sink forthat assimilate. Washout did not reduce when the bathed tissuewas plasmolyzed. We propose that sucrose is unloaded from thephloem into the apoplast, and a sucrose concentration is maintainedthere by a balance of sucrose uptake into sink tissue or reloadinginto the phloem. It is proposed that the apoplastic pool ofphotosynthate can act to buffer sudden changes in phloem contentswhen there are rapid changes in source-sink configuration. Key words: Sucrose, Phaseolus vulgaris, Apoplast, Phloem unloading     

Control of Protein Hydrolysis in the Cotyledons of Germinating Pea (Pisum sativum L.) Seeds

The effects of removal of the shoot or whole axis on the levelsof total, protein, and TCA-soluble nitrogen and on proteaseactivity in cotyledons during germination of garden pea (Pisumsativum L ) seedlings grown in the light have been examined. Removal of the shoot 1 week after soaking the seed caused areduction in the rates of protein hydrolysis and of nitrogentransport from the cotyledons and an increase in the level ofsoluble nitrogen When the entire axis was excised after 4 or9 days there was a great reduction in protein hydrolysis whilethe level of soluble nitrogen remained the same as in de-shootedplants. In the intact plant, proteolytic activity of cotyledon extractsrose to a peak about 15 days after soaking of the seed and thenfell rapidly This fall coincided with a decrease in water contentand in oxygen consumption by the cotyledons. Removal of theshoot or entire axis led to a much smaller and more gradualincrease in protease activity and the subsequent decline inactivity of the enzyme and senescence of the cotyledons werealso delayed. It is concluded that control of protein hydrolysis in pea cotyledonsis not mediated through the level of protease enzymes, as indicatedby the proteolytic activity of tissue extracts, or by the amountof soluble nitrogen compounds accumulated. Protease activityseems to be controlled by the shoot and to be closely linkedto senescence of the cotyledons Protein hydrolysis and transportof nitrogen to the axis, on the other hand, are affected bythe presence of both shoot and root and the axis appears toexert independent control on each of these processes.     

Isolation of a Novel Auxin,Methyl 4-Chloroindoleacetate from Immature Seeds of Pisum sativum

Human casein was separated by gel filtration on a column of Sephadex G–200 with 0.1 m Tris buffer (pH 8.5) containing 1.0 m NaCl. The effluent which increased in turbidity at 25°C was centrifuged at 25,000 × g for 30 min and the precipitate was obtained as Fraction 6. After centrifugation, the effluent was separated into 5 elution fractions.

Disc gel electrophoretic patterns of each fraction showed occurrence of secondary bands other than major bands especially in Fractions 3, 4 and 5. The casein solutions unheated and heated at 100°C for 5 and 10 min were kept at 5°C for 5 days. No marked changes of electrophoretic pattern were observed among these casein solutions. However, when a casein solution heated at 100°C for 5 min was chroma to graphed under the same condition, secondary bands also appeared.     

An Analysis of Seed Development in Pisum sativum: VI. ABSCISIC ACID ACCUMULATION

The abscisic acid (ABA) content of wrinkled (rr) pea seed tissueshas been quantified during development using multiple-ion-monitoringcombined gas chromatography-mass spectrometry and a deuteratedinternal standard. The level of ABA in the embryo generallyincreased with increasing cotyledon fresh weight while thatin the testa showed a distinct maximum at the time of maximumendosperm volume and the slowing in the growth of the testa.Pods contained relatively little ABA on a fresh weight basis.The total seed ABA content showed a biphasic distribution, thefirst maximum following the maximum growth rate of the testaand the second that of the embryo. The biphasic distributionof ABA in the pea seed was confirmed using a second pea genotype,near-isogenic to the first except for the r locus, and by theanalysis of individual seeds using a radioimmunoassay for ABA.The first maximum was composed mainly of a testa component andthe second mainly of an embryo component. When plants were grownin different environments, wrinkled seeds were found to containslightly more ABA than round (RR) but this was only significantlate in development. Immature seeds were capable of metabolizing17'-deoxy ABA to ABA, as determined by incorporation of either³H or ²H, and the metabolite was present mainly in the testa.The production of ABA in pea seeds is discussed in relationto the development of the different seed tissues. Key words: Abscisic acid, peas, seed development     

Transformation of peas (Pisum sativum L.) using immature cotyledons

A reliable Agrobacterium tumefaciens-mediated transformation method has been developed for peas (Pisum sativum) using immature cotyledons as the explant source. Transgenic plants were recovered from the four cultivars tested: Bolero, Trounce, Bohatyr and Huka. The method takes approximately 7 months from explant to seed-bearing primary regenerant. The binary vector used carried genes for kanamycin and phosphinothricin resistance. Transformed pea plants were selected on 10 mg/l phosphinothricin. The nptII and bar genes were shown to be stably inherited through the first sexual generation of transformed plants. Expression of the phosphinothricin-resistance gene in the transformed plants was demonstrated using the Buster (=Basta) leaf-paint test and the phosphinothricin acetyl transferase enzyme assay.Abbreviations BA 6-benzylaminopurine     

Transport of Materials from the Cotyledons during Germination of Seeds of the Garden Pea (Pisum sativum L.)

After the first week of germination the relationship betweenthe amounts of total dry matter, nitrogen, phosphorus, sulphur,and potassium transferred to the axis from the cotyledons inthe intact plant remained approximately constant irrespectiveof the conditions of growth. It is proposed that the ratio inwhich the individual elements are transported is determinedby the proportions in which they are released by the storagecells. Deviation from this ratio during the first week of germination,and over a longer period in deshooted plants is attributed tocompetition for the available nutrients between actively metabolizingcells in the cotyledons and axis. It is demonstrated by steam-girdling that movement of materialsfrom the cotyledons into the shoot probably occurs via the phloem.Calcium is mobile in the phloem during the early stages of germination,possibly because the amount of free calcium in the cotyledonsis high.     

Effect of the Osmotic Environment on the Balance between Uptake and Release of Sucrose and Amino Acids by the Seed Coat and Cotyledons of Developing Seeds of Pisum sativum

Excised seed-coat halves and cotyledons of developing seedsof Pisum sativum L. were incubated in a bathing medium (pH 5·5),in order to measure the release or uptake of sucrose and aminoacids. Net efflux of sucrose and amino acids was reduced bya 250 mol m ^–3 mannitol solution and a 400 mol m ^–3solution, in comparison with a 100 mol m^–3 control. Thiseffect could not be observed in the case of the amino acid analogue-aminoisobutyric acid (AIB). Net uptake of labelled sucroseor valine by cotyledons and seed coats was enhanced by a highosmolality of the bathing medium. The data on AIB and the datafrom uptake experiments support the view that net efflux ofassimilates is reduced by a high solute concentration in theapoplast (e.g. 400 mol m^–3 mannitol), via a stimulationof carrier-mediated sucrose and amino acid uptake into cotyledonaryand seed coat tissues. In experiments with attached empty ovulesof pea in a very early stage of development, sugar release fromthe seed coat was enhanced by a low osmolality of the apoplastsolution (e.g. 100 mol m^–3 mannitol, in comparison witha 400 mol m ^–3 control). This paradoxical effect may beobserved when the stimulatory effect on net assimilate effluxfrom seed coat tissues is exceeding the inhibitory effect onassimilate import into the seed coat. Key words: Seed development, turgor-sensitive transport, assimilate transport