Genotype-specific soluble auxin-binding in etiolated pea epicotyls

Using different independent procedures for assaying soluble auxin-binding in etiolated pea epicotyls, wo could prove the reliability of the (XH₄)₂SO₄-pelleting assay both for crude cytosols as well as for specific protein fractions obtained after chromatofocusing. Three distinct genotypes (two parent lines, one tall recombinant) investigated so far exhibit characteristic differences with respect to soluble auxin-binding kinetics in their cytosols.     

Autolysis of cell walls in pea epicotyls during growth. Enzymatic activities involved

Pisum sativum L. (cv. Lincoln) epicotyl cell walls show autohydrolysis and release into the incubation medium up to 120 μg of sugar per mg of cell wall dry weight in 30 h. Cell walls from younger epicotyls with high growth capacity showed higher auto-lytic capacity than older epicotyls. This suggests that both processes, growth and au-tolysis, are related. The proteins responsible for autolysis were extracted from the wall fraction with high saline solution (3 M LiCl) and enzymatic activities associated with the proteins were studied. The highest activity corresponded to α-galactosidase; lower activities were found for β-galactosidase, a-arabinosidase and exoglucanase. Changes in enzymatic activities and changes in the proportion of sugars released in autolysis by cell walls during the growth of epicotyls support the notion that α-galac-tosidase is one of the enzymes involved in the process of autolysis, and that the liberation of arabinose and galactose in this process occurs as arabinogalactan.     

Protochlorophyllide transformations and chlorophyll accumulation in epicotyls of pea (Pisum sativum)

Low-temperature fluorescence emission spectra of epicotyls of 6.5-day-old dark-grown seedlings of pea ( Pisum sativum L.) showed the dominance of short-wavelength protoch lorophyllide forms with emission maxima at 629 and 636 nm, respectively. The presence of long-wavelength protochlorophyllide with emission maxima around 650 nm was just detectable. Accordingly, irradiation with millisecond flashes gave a minute formation of chlorophyllide. The chlorophyll(ide) formation varied along the epicotyl. Irradiation with continuous light for 1.5 h resulted in an evident accumulation of chlorophyll(ide) in the upper part of the epicotyl. Only small amounts accumulated in the middle section. The conversion of protochlorophyllide to chlorophyllide was temperature dependent and almost arrested at 0°C. The chlorophyll(ide) formed had one dominating fluorescence peak at 681 nm. Irradiation for 24 h gave almost 100 times more chlorophyll in the upper part of the epicotyl than in the lower part. Electron micrographs from the upper part of the epicotyl irradiated for 6 h showed plastids with several developing thylakoids, while the plastids in the lower part of the epicotyl had only a few thylakoids. The dominance of short-wavelength protochlorophyllide forms indicated the presence of protochlorophyllide not bound to the active site of NADPH-protochlorophyllide oxidoreductase (EC 1.3.1.33). The inability of the short-wavelength form to transform into chlorophyllide with flash light denotes a dislocation from the active site. The time and temperature dependence of the chlorophyll(ide) formation in continuous light indicates that a relocation is required of the short-wavelength protochlorophyllide before chlorophyllide formation can occur.     

Protochlorophyllide forms in non-greening epicotyls of dark-grown pea (Pisum sativum)

Low-temperature fluorescence emission spectra of 6.5-day-old dark-grown epicotyls of pea ( Pisum sativum ) revealed the presence of protochlorophyll(ide). The upper part of the epicotyl contained 30% of the protochlorophyll(ide) content per fresh weight found in pea leaves, whereas the lower part contained 3%. Three discrete spectral forms of protochlorophyll(ide) were clearly distinguished after Gaussian deconvolution of fluorescence excitation and emission spectra. Adding the satellite bands of the Qy_(0-0) transitions (the emission vibrational (Emv) bands with correlated amplitudes, gave the following delineation: Ex439–Em629–Emv684, Ex447–Em636–Emv700 and Ex456–Em650–Emv728. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) followed by immunodetection of whole tissue extracts of the epicotyl indicated the presence of NADPH-protochlorophyllide oxidoreductase (EC 1.3.1.33). Electron micrographs showed prolamellar bodies in at most 11 % of the plastid profiles of the epicotyl cells. These prolamellar bodies were smaller, and many of them showed less regular structure than those of the leaves. Taken together, the results indicate that the protochlorophyll(ide) in epicotyls is arranged in a different way than in leaves.     

Lipid peroxidation in and photo-damage to a light-sensitive chlorescence pea mutant

Leaves of 10- to 12-day-old chlorescence lethal Pisum sativum L. mutant are similar to control plants with respect to the content of chlorophylls, carotenoids, fatty acids and α-tocopherol. Subsequent development of the mutant under high irradiation resulted in th destruction of the photosynthetic pigments, polyunsaturated fatty acids, α-tocopherol, and also in the accumulation of liposoluble fluorescent products. No increase in the level of malondialdehyde was observed. In chloroplasts isolated from mutant plants the contents of chlorophyll a and β-carotene were decreased to a greater extent than the more oxidized pigments (xanthophylls and chlorophyll b ). The data obtained are discussed with special reference to the role of lipid peroxidation in the injury of plant cells under the action of visible light and to the antioxidative mechanisms stabilizing photosynthetic membranes.     

Physiological studies on pea tendrils

When tendrils which have been dark adapted for 3 days are mechanically stimulated, they will only coil appreciably if they are irradiated with light. A spectral response curve suggests that this is a blue light effect. Red or far red light do not modulate this response, but a high intensity flash of blue white light given before the actinic blue light blocks coiling for 30 min. The dark-adapted tendril, which has not been previously rubbed, has the ability to store the tactile sensory information for about 60 min, but loses it if it is not irradiated within 120 min after mechanical perturbation.     

Cyclic AMP-dependent phosphorylation of pea proteins induced by forskolin

An increase in the level of phosphorylation of low-molecular-weight polypeptides in pea (Pisum sativum L.) leaves and changes in the content of some polypeptides after 10-min forskolin action in situ were demonstrated. A total level of protein phosphorylation in the homogenate of forskolin-treated leaves diminished after 25 min of its incubation in vitro. Using a highly specific inhibitor of cAMP-dependent protein kinases and cAMP treatments, we established that forskolin-induced change in the phosphorylation level of some polypeptides was cAMP-dependent.Translated from Fiziologiya Rastenii, Vol. 52, No. 1, 2005, pp. 27–35.Original Russian Text Copyright © 2005 by Karimova, Tyrykina, Zakharova.     

Isolation,characterisation and expression of a cDNA for pea cholinephosphate cytidylyltransferase

In plants, phosphatidylcholine is the major phospholipid in extra-plastid membranes and is synthesised mainly by the CDP-choline pathway. Evidence from studies in animals, as well as in plants, suggests that the intermediate step catalysed by cholinephosphate cytidylyltransferase (CPCT) has a major control in carbon flux to this lipid. We have isolated a full-length CPCT cDNA (designated PCT2) from Pisum sativum cv. Feltham First using an Arabidopsis probe and the polymerase chain reaction (PCR). The deduced amino acid of PCT2 is 48%, 43% and 76% identical to the rat, yeast and Brassica napus amino acid sequences, respectively. Expression of the CPCT protein in Escherichia coli confirmed the activity of the enzyme. Expression of the PCT2 mRNA in pea roots and stems was increased by treatment with 0.1 µM indole-3-acetic acid.     

Effect of brassinolide on tyrosine phosphorylation of pea leaf proteins

Brassinosteroid-induced phosphorylation of tyrosine residues in proteins was studied. Proteins of crude extract of pea leaves were analyzed by one- and two-dimensional electrophoresis followed by Western blotting with monoclonal antibodies PY20 to phosphotyrosine proteins. One- and two-dimensional electrophoresis revealed 7 and 13 tyrosine-phosphorylated proteins, respectively. Brassinolide increased the phosphorylation level of most of these proteins. With inhibitors of tyrosine protein phosphatases, such as phenylarsine oxide and orthovanadate, the level of tyrosine phosphorylation of these proteins increased.     

Protoplast regeneration and organogenesis from pea protoplasts

Summary Protoplasts of young Pisum Sativum L. seedlings from 7 different genotypes were isolated and regenerated to the callus stage. Germinating embryos were cultivated with cotyledons removed, thus avoiding intracellular starch accumulation in donor tissue. The first lateral shoots provided a source of homogenous meristematic cells which gave rise to sustained protoplast division and resulted in callus formation within 4, weeks. Root formation occurred on hormone-free medium and shoots developed on medium containing kinetin, 2iP or zeatin in the third subculture, when subcultured in monthly intervals. This work was made possible by a joint GFP/BMFT—project to H.-J. Jacobsen (grant A 24/87-ZF).     

Isolation and properties of a lectin from the roots of Pisum sativum (Garden pea)

Abstract The roots of pea (Pisum sativum L. ev. Feltham First) seedlings contained haemagglutinating activity and a protein which reacted with antibodies directed against pea seed lectin. This protein was shown to be present on the surface of root hairs and in the root cortical cells by immunofluorescence. Lectin (haemagglutinin) was purified from pea seedling roots by both immunoaffinity chromatography and affinity chromatography on Sephadex G-100. The pea root lectin was similar to the seed lectin when analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, and was antigenically identical: however, the isoelectric focussing band patterns of the proteins differed. The sugar specificity of the root lectin differed from that of the seed lectin, and the haemagglutinating activity of the root lectin was less than the seed lectin. These results are discussed with reference to the hypothesis that lectins mediate in the symbiotic association of legume and Rhizobium through their carbohydrate-binding properties.     

Changes in concentration of endogenous growth inhibitors during growth recovery of dwarf pea seedlings

In order to clarify the role of endogenous growth inhibitors A-2α and A-2β in a dwarf pea plant, red light (emission peak 657 nm) treated, 9-d-old seedlings of dwarf pea (Pisum sativum L. cv. Progress No. 9) were transferred to darkness, and the resulting changes in growth rate and concentrations of A-2α and A-2β were monitored. The growth rate of the epicotyls increased, and the concentration of the inhibitors in the epicotyls decreased, according to sigmoidal time courses. The relationship between the logarithms of the concentration of the inhibitors and the corresponding growth rate was linear. These results suggest that A-2α and A-2β, may play an important role in the growth recovery process of the dwarf pea cultivar after termination of red light irradiation. This revised version was published online in July 2006 with corrections to the Cover Date.     

Low temperature spectrophotometry of the phototransformation of Pfr to Pr in pelletable pea phytochrome

Manabe, K. 1987. Low temperature spectrophotometry of the phototransformation of P_fr to P_r, in pelletable pea phytochrome.
Low temperature spectrophotometry was used to study the phototransformation of P_fr to P_r in 1000–7000 g pelletable fractions extracted from dark grown pea ( Pisum sativum L. cv. Alaska) epicotyls which had been irradiated with red and then far-red light. At -170°C, far-red irradiation of the pelletable phytochrome which had been pre-irradiated with saturating fluence of red light before freezing caused formation of an intermediate (named I₆₆₀), the difference spectrum of which showed a marked ab-sorbance decrease at 740 nm and a concomitant small increase at about 660 nm. The inermediate I₆₆₀ was converted to another intermediate (I₆₆₀) when it was warmed above -80°C. The difference spectrum of this intermediate showed a positive peak at 670 nm. This intermediate was photoconverted to P_fr by red irradiation and also underwent dark reversion to P_fr at -60°C. I₆₆₀ formed P_r if the temperature was above -10°C. The basic features of the phytochrome intermediates resemble those obtained in vivo and in degraded purified phytochrome.     

Indole-3-acetaldehyde oxidase of pea seedlings

Indole-3-acetaldehyde oxidase was partially purified from the epicotyl of Pisum satiyum seedlings by column chromatography using CM-Sephadex and Sephadex G-150. The enzyme was only active in the presence of molecular oxygen. The activity was maximal at pH 8.0, and the Km value for indole-3-acetaldehyde was 1.4 × 10⁻³ M . The enzyme was inhibited strongly by p -hydroxymercuribenzoate, cyanide and hydroxylamine, suggesting that it contains sulfhydryl group(s) and a metal component such as iron.     

Regeneration of shoots from pea (Pisum sativum) hypocotyl explants

A sequential indole-3-acetic acid (IAA)-zeatin treatment was applied to Pisum sativum hypocotyl explants, resulting in shoot formation from 50% of the explants. Shoots were easily rooted and transplantable plants could be obtained in 3 months. The method has been applicable to the 5 cultivars tested. Histological examination of explants suggests the shoots to be of de novo origin, which would make the system suitable for transformation experiments.     

Somatic embryogenesis in pea (Pisum sativum L.): effect of explant, genotype and culture conditions

In this study 16 cultivars of pea (Pisum sativum L.) were screened in vitro for the formation of somatic embryos which were dependent on the genotype, culture conditions and explant source used. The cultivars Stehgolt, Maro and Progreta showed the highest tendency to form somatic embryos (c. 25%) while Alaska, Rondo and Ascona did not show any embryo production. Using the cultivar Belman, the highest embryo production was achieved by using nodal explants of shoots (10.6%) and a cotyledon-free embryo as explant source (14.1%) in the light (15.8%) compared to using apices as explants (1.8%) and a seedling as the explant source (9.4%) in the dark (3.3%). Media containing picloram (0.75 mg/litre) followed by BA (1 mg/litre) or kinetin (1 mg/litre), each for four weeks gave the highest somatic embryo production. The development of embryos to whole plants was unreliable and some 90% of the embryos induced did not develop any further, died, recallused or formed secondary embryos. The size of the embryo at separation and the timing of the separation from the original callus were important factors determining success for complete development to whole plant. Regeneration of 184 plants was achieved with ensuing flowering, pod formation and viable seed production from the techniques described.     

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.