Interaction of pea (Pisum sativum L.) lectins with rhizobial strains

Lectins from two varieties (PG-3 and LFP-48) of pea have been purified by affinity chromatography on Sephadex G-50. The specific activity increased by 23 and 25 folds, respectively. These lectins from both the varieties were found to be specific for mannose. The purified fluorescein isothiocyanate (FITC) – labelled lectins showed binding reaction with homologous as well as heterologous strains of Rhizobium spp. The results revealed that pea lectins are not highly specific to their respective rhizobia. Moreover, these lectins showed a greater stimulatory effect on homologous Rhizobium leguminosarum strains.     

Fabacyl acetate, a germination stimulant for root parasitic plants from Pisum sativum

A germination stimulant, fabacyl acetate, was purified from root exudates of pea (Pisum sativum L.) and its structure was determined as ent-2′-epi-4a,8a-epoxyorobanchyl acetate [(3aR,4R,4aR,8bS,E)-4a,8a-epoxy-8,8-dimethyl-3-(((R)-4-methyl-5-oxo-2,5-dihydrofuran-2-yloxy)methylene)-2-oxo-3,3a,4,5,6,7,8,8b-decahydro-2H-indeno[1,2-b]furan-4-yl acetate], by 1D and 2D NMR spectroscopic, ESI- and EI-MS spectrometric, X-ray crystallographic analyses, and by comparing the ¹H NMR spectroscopic data and relative retention times (RR_t) in LC-MS and GC-MS with those of synthetic standards prepared from (+)-orobanchol and (+)-2′-epiorobanchol. The ¹H NMR spectroscopic data and RR_t of fabacyl acetate were identical with those of an isomer prepared from (+)-2′-epiorobanchol except for the opposite sign in CD spectra. This is the first natural ent-strigolactone containing an epoxide group. Fabacyl acetate was previously detected in root exudates of other Fabaceae plants including faba bean (Vicia faba L.) and alfalfa (Medicago sativa L.).     

Sucrose metabolism in stele and cortex isolated from roots of Pisum sativum

Stele and cortex were separated from the region 6–24 mm from the tip of roots of seedlings of Pisum sativum L. that had been grown in the dark for 5 days. The activities of sucrose synthetase (E.C. 2.4.1.13) and sucrose phosphate synthetase (E.C. 2.4.1.14) in extracts of stele were 34 and 5·9 nmol product formed/min per mg protein, respectively. The corresponding figures for extracts of cortex were 17 and 5·2. Appreciable labelling of sucrose occurred when samples of either stele or cortex were incubated in [¹⁴C]glucose for 90 min. The labelling of sucrose after incubation of the tissues for 45 min in [¹⁴C]glucose followed by 45 min in glucose suggested some turnover of sucrose in the cortex but none in the stele. These results are discussed in relation to the control of sucrose metabolism in the root.     

Isolation and identification of lateral bud growth inhibitor,indole-3-aldehyde,involved in apical dominance of pea seedlings

A lateral bud growth inhibitor was isolated from etiolated pea seedlings and identified as indole-3-aldehyde. The indole-3-aldehyde content was significantly higher in the diffusates from explants with apical bud and indole-3-acetic acid treated decapitated explants, in which apical dominance is maintained, than in those from decapitated ones releasing apical dominance. When the indole-3-aldehyde was applied to the cut surface of etiolated decapitated plants or directly to the lateral buds, it inhibited outgrowth of the latter. These results suggest that indole-3-aldehyde plays an important role as a lateral bud growth inhibitor in apical dominance of pea seedlings.     

DNA polymerase activity and DNA synthesis in roots of pea (Pisum sativum) seedlings

Soluble DNA polymerase (DNA polymerase-α) and chromatin-bound DNA polymerase (DNA polymerase-β) have been assayed in serial sections cut from the roots of 5-day-old pea seedlings. The activity of DNA polymerase-α is high in regions of the root which exhibit high rates of DNA replication, and declines during cell differentiation and maturation. The activity of DNA polyrnerase-β is low in cells which show high rates of DNA replication, and increases during differentiation and maturation.     

Identification of enzyme activity that conjugates indole-3-acetic acid to aspartate in immature seeds of pea (Pisum sativum)

This study describes the first identification of plant enzyme activity catalyzing the conjugation of indole-3-acetic acid to amino acids. Enzymatic synthesis of indole-3-acetylaspartate (IAA-Asp) by a crude enzyme preparation from immature seeds of pea (Pisum sativum) was observed. The reaction yielded a product with the same Rf as IAA-Asp standard after thin layer chromatography. The identity of IAA-Asp was verified by HPLC analysis. IAA-Asp formation was dependent on ATP and Mg2+, and was linear during a 60 min period. The enzyme preparation obtained after poly(ethylene glycol) 6000 fractionation showed optimum activity at pH 8.0, and the temperature optimum for IAA-Asp synthesis was 30 degrees C.     

Inheritance and mapping of isoenzymes in pea (Pisum sativum L.)

Summary Three isoenzyme systems (amylase, esterase and glutamate oxaloacetate transaminase) were examined in seeds of pea (Pisum sativum L.) and shown to give clear variation in their band patterns on gel electrophoresis between different lines. The inheritance of these isoenzyme systems, and the location of their genes on the pea genome was investigated. Reciprocal crosses were made between lines, F2 seeds were analysed for segregation in the band patterns of the isoenzymes, and F2 plants were investigated to find linkage between the genes for these isoenzymes and genes for selected morphological markers. The results obtained showed that each of the investigated isoenzyme systems is genetically controlled by co-dominant alleles at a single locus. The gene for amylase was found to be on chromosome 2, linked to the loci k and wb (wb ... 9 ... k ... 25 ... Amy). The gene for esterase was found to be linked with the gene Br (chromosome 4) but the exact location is uncertain because of the lack of the morphological markers involved in the cross. The gene for glutamate oxaloacetate transaminase was found to be on chromosome 1 and linked with the loci a and d (a... 24... Got... 41 ... d).     

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

Summary 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 M_r. 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.Abbreviations CLC Clathrin light chain - CHC clathrin heavy chain - CV coated vesicle - DTT dithiothreitol - EGTA ethyleneglycol-bis-(-aminoethyl ether) N,N-tetraacetic acid - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - TBS Tris buffered saline     

Sulphate uptake and xylem loading of young pea (Pisum sativum L.) seedlings

Sulphate uptake and xylem loading was analysed in young pea (Pisum sativum) seedlings. The rate of sulphate uptake into intact 8-days-old pea seedlings (determined by a 1 h exposure to radiolabelled sulphate in the nutrient solution) was 585 nmol sulphate g^–1 root fresh weight h^–1. When the cotyledons were removed on day 6 the 8-days-old seedlings took up only 7% of the controls. Interruption of the phloem transport by steam girdling of the stem or the root (1 h before incubation with radiolabelled sulphate) diminished sulphate uptake by approximately 50%. The addition of sucrose to the nutrient solution during incubation did not restore sulphate uptake rates indicating that the decrease was not due to a lack of energy. Apparently, a signal from the shoot and/or the cotyledons is necessary to stimulate sulphate uptake into the roots of pea seedlings. Glutathione fed to the roots for 3 h prior to incubation with radiolabelled sulphate diminished sulphate uptake by approximately 50%. The relative proportion of the sulphate taken up that was loaded into the xylem remained unchanged (between 7 and 9% of total uptake), even when the stem was girdled above the cotyledons or when the seedlings were pre-exposed to glutathione. Only removal of the cotyledons or girdling of the root below the cotyledons increased the proportion of sulphate loaded into the xylem to 13–15% of total uptake upon exposure to glutathione. Apparently, a signal from the cotyledons represses xylem loading to some extent.     

First report of non-mycorrhizal plant mutants (Myc) obtained in pea (Pisum sativum L.) and fababean (Vicia faba L.)

Genetic resistance to vesicular-arbuscular (VA) mycorrhiza formation has been obtained in spontaneous or chemically induced mutants of two mycorrhiza-forming species (Pisum sativum L. and Vicia faba L.). The eight mutants, termed myc⁻, are characterized by aborted infections limited to one or two host cells. Expression of the myc⁻ character is associated with that of the nod⁻ character in both legumes, and is likewise under recessive genetic control. Preliminary analysis of the genetic behaviour of the myc⁻ mutants in diallel crosses has shown that at least three genes are involved in VA mycorrhiza infection.     

Mechanism of uptake and translocation of zinc by pea plants (Pisum sativum L.)

Summary Radiotracer studies revealed that the mechanism of zinc uptake was not purely non-metabolic. The over all uptake of Zn by intact plant ofPisum sativum L. (Var. T-163) occurred through an initial nonmetabolic phase followed by a metabolically mediated absorption. The translocation of zinc from plant roots to shoot was more sensitive to metabolic inhibitors and thus was a metabolic function.Research Publication No. 1541/116/0 through Experiment Station, G. B. Pant University, Pantnagar-263145, India.     

Coinoculation of Bacillus thuringeinsis-KR1 with Rhizobium leguminosarum enhances plant growth and nodulation of pea (Pisum sativum L.) and lentil (Lens culinaris L.)

Nodulation and the subsequent nitrogen fixation are important factors that determine the productivity of legumes. The beneficial effects of nodulation can be enhanced when rhizobial inoculation is combined with plant-growth-promoting bacteria (PGPB). The PGPB strain Bacillus thuringiensis-KR1, originally isolated from the nodules of Kudzu vine (Pueraria thunbergiana), was found to promote plant growth of field pea (Pisum sativum L.) and lentil (Lens culinaris L.) under Jensen’s tube, growth pouch and non-sterile soil, respectively, when co-inoculated with Rhizobium leguminosarum-PR1. Coinoculation with B. thuringiensis-KR1 (at a cell density of 10⁶ c.f.u. ml⁻¹) provided the highest and most consistent increase in nodule number, shoot weight, root weight, and total biomass, over rhizobial inoculation alone. The enhancement in nodulation due to coinoculation was 84.6 and 73.3% in pea and lentil respectively compared to R. leguminosarum-PR1 treatment alone. The shoot dry-weight gains on coinoculation with variable cell populations of B. thuringiensis-KR1 varied from 1.04 to 1.15 times and 1.03 to 1.06 times in pea and lentil respectively, while root dry weight ratios of coinoculated treatments varied from 0.98 to 1.14 times and 1.08 to 1.33 times in pea and lentil respectively, those of R. leguminosarum-PR1 inoculated treatment at 42 days of plant growth. While cell densities higher than 10⁶ c.f.u. ml⁻¹ had an inhibitory effect on nodulation and plant growth, lower inoculum levels resulted in decreased cell recovery and plant growth performance. The results of this study indicate the potential of harnessing endophytic bacteria of wild legumes for improving the nodulation and growth of cultivated legumes.     

Inhibitory effect of essential oils of Allium sativum and Piper longum on spontaneous muscular activity of liver fluke, Fasciola gigantica

Effects of essential oil of Allium sativum (garlic) and Piper longum (Indian long pepper) were evaluated on muscular activity of whole Fasciola gigantica and its strip preparation. The whole flukes and longitudinal strip preparations of the flukes were isometrically mounted to record the spontaneous muscular activity (SMA) and to evaluate effects of cumulative doses (0.1, 0.3, 1.0 and 3.0 mg/ml) of the plant essential oils. Whole flukes and the strip preparations exhibited continuous SMA without any significant difference in its baseline tension, frequency and amplitude for 2 h. Essential oil of A. sativum produced significant reduction in the frequency and the amplitude of the SMA of whole fluke at 1 and 3 mg/ml concentrations. It caused complete paralysis of the fluke after 15 min of administration of 3 mg/ml concentration. Similar to whole fluke, essential oil of A. sativum (3 mg/ml) also produced flaccid paralysis in the strip preparations of the flukes. Essential oil of P. longum firstly induced marked excitatory effect and then there was flaccid paralysis of the whole fluke following 15 min exposure at 3 mg/ml concentration. Complete flaccid paralysis of the strip preparation was also ensued after 15 min of administration of 3 mg/ml concentration of P. longum. In both the essential oils, the whole fluke and strip preparations did not recover from paralysis following 2-3 washes. In conclusion, the observations demonstrated irreversible paralytic effect of essential oils of A. sativum and P. longum on F. giganticain vitro which might possibly help to developing herbal-based anthelmintic.     

The organisation and expression of the genes encoding the mitochondrial glycine decarboxylase complex and serine hydroxymethyltransferase in pea (Pisum sativum)

Summary Restriction fragment length polymorphisms have been used to determine the chromosomal location of the genes encoding the glycine decarboxylase complex (GDC) and serine hydroxymethyltransferase (SHMT) of pea leaf mitochondria. The genes encoding the H subunit of GDC and the genes encoding SHMT both show linkage to the classical group I marker i. In addition, the genes for the P protein of GDC show linkage to the classic group I marker a. The genes for the L and T proteins of GDC are linked to one another and are probably situated on the satellite of chromosome 7. The mRNAs encoding the five polypeptides that make up GDC and SHMT are strongly induced when dark-grown etiolated pea seedlings are placed in the light. Similarly, when mature plants are placed in the dark for 48 h, the levels of both GDC protein and SHMT mRNAs decline dramatically and then are induced strongly when these plants are returned to the light. During both treatments a similar pattern of mRNA induction is observed, with the mRNA encoding the P protein of GDC being the most rapidly induced and the mRNA for the H protein the slowest. Whereas during the greening of etiolated seedlings the polypeptides of GDC and SHMT show patterns of accumulation similar to those of the corresponding mRNAs, very little change in the level of the polypeptides is seen when mature plants are placed in the dark and then re-exposed to the light.