Hydroxyproline and Peroxidases in Cell Walls of Pisum sativum: Regulation by Ethylene

Purified cell-wall preparations from the epicotyl of etiolatedPisum sativum contain covalently bound peroxidases and hydroxyproline-richproteins. Towards the end of cell elongation there is a largerise in these wall components and thereafter a continuing slowrise which is associated with increasing age of tissue. Ethyleneat concentrations of 0.1 ppm or more increases both peroxidaseactivity and hydroxyproline levels in the walls, the greatestresponse occurring in immature tissue including the apical hook.Growth of these tissues is highly sensitive to ethylene whichcauses an inhibition of elongation in extending cells and anenhanced lateral cell expansion. We suggest that the effectsof ethylene on wall-bound peroxidase and hydroxyproline areimplicated in the ethylene regulation of cell growth. The covalently bound wall peroxidase was found to be extremelystable and to contain unique isoenzymes which do not occur ineither the cytoplasm or in the peroxidase which is ionicallybound to walls. Ethylene increases peroxidase activity in boththe cytoplasmic and the ionically bound wall fractions, butthere is little or no increase in their hydroxyproline content.The possible relationships between covalently bound wall peroxidaseand hydroxyproline are discussed and we speculate that thisperoxidase may be involved in the hydroxylation of proline inthe walls.     

Gibberellin translocation in Pisum sativum L.

The physiological and biochemical consequences of treating Le (tall) and le (dwarf) pea seedlings with varying quantities of the gibberellins [³H]GA₂₀ and GA₁ have been investigated. Although the percentage uptake of these compounds from the site of application on the 3 stipules was low and most of the applied GA remained unmetabolised in situ, the quantitative relationship between GA translocation and GA dosage was found to be linear for GA₁ but saturating for GA₂₀. The movement of the GAs and their subsequently produced metabolites was mainly acropetal. They accumulated in greatest quantity in the apical extremities of the shoot. Overall, the extent to which GA₂₀ was metabolished in le seedlings was considerably less than in Le pea seedlings. Although all le tissues contained significantly less [³H]GA₁ than their Le counterparts, phenotypic effects of the le mutation were apparent only on internode and tendril development. Increased tissue growth, consequent upon GA treatment, was also apparent only in the internodes and tendrils of le plants. For internodes, GA₁ content determined the mid-logarithmic-phase growth rate and, consequently, final length. For tendrils, GA₂₀ rather than GA₁ may be the primary stimulatory agent.Abbreviations GA gibberellin - HPLC high-performance liquid chromatography - 1–6 consecutive developmental numbering system for plant tissues/organs as shown in Fig. 1 The author gratefully acknowledges financial support from Imperial Chemical Industries, Plant Protection, Jealott's Hill, Bracknell, Berks., UK and the Science and Engineering Research Council.     

Gibberellin induces endopeptidase activity in detached cotyledons of Pisum sativum

Endopeptidase activity in cotyledons of 5-day seedlings of Pisum sativum increased rapidly during germination. However, the increase of the activity in detached cotyledons was depressed. We examined whether a growth regulator can be substituted for the embryonic axis on the development of endopeptidase activity. As monitored by an assay with azoalbumin, the development of endopeptidase activity from crude extracts of detached cotyledons appeared to be slightly accelerated by incubation with 10^–5 M GA₃. However, the pattern after gelatin-polyacrylamide gel suggested that the activity induced in detached cotyledons during a 5-d incubation at 10^–7 M GA₃ was the same as that in attached ones during germination for 5 days and an even greater increase in activity was obtained with 10^–5 M GA₃. These results suggest that GA₃ from the embryonic axis induces endopeptidase activity in attached cotyledons at the first stage of germination.Abbreviations ABA abscisic acid - IAA indole-3-acetic acid - GA gibberellic acid     

Cell Division Heterogeneity in the Primary Meristems of Pisum sativum Root Tips after Excision

Root tips (1.0 cm) were excised, transferred to culture medium,sampled at intervals, embedded in paraffin and sectioned. Themitotic index in each primary meristem in the 2.0 mm tips wasreduced immediately after excision. After 8–12 h mitosisresumed. The position of mitotic figures in the protoderm, groundmeristem, procambium, and root cap was determined. The greatestnumber of mitotic figures occurred in the procambium, and uponexcision the smallest percent reduction also occurred in thisregion. The ground meristem showed the greatest reduction inmitotic figures. After excision, the relative contribution tothe total number of mitotic figures increased in the procambiumtissue and decreased in the ground meristem. Cell division inall primary meristems of the root tip is reduced after tip excision,but the ground meristem (cortical region) is the primary targettissue.     

Effects of Nutrients and Light on Growth and Root Formation in Pisum sativum Cuttings

Cuttings obtained from seedlings of Pisum sativum L. were rooted in water solution. Shoot growth continued after excision and shoot length increased considerably before roots emerged. Increase in dry weight was strongly dependent on light supply. Continued growth was dependent on supply of mineral nutrients to the rooting solution. Mineral nutrients had no or slight influence on the number of roots formed on cuttings from stock plants grown in fertilized soil, but the growth in length of the roots was dependent on the presence of calcium in the solution. Root formation was dependent on photosynthetic products formed after excision. No roots were formed on cuttings kept in the dark. The number of roots increased with increasing irradiance given to the leafy part of the cutting. At a low level of irradiance sucrose supply through the rooting medium increased the number of roots. Light given to the basal part of the cuttings had a strongly inhibitory effect on the number of roots formed. It is concluded that the carbohydrate level easily becomes a limiting factor for root formation in growing pea cuttings. Availability of mineral nutrients influences in the first place the growth of the shoots.     

Gibberellin biosynthesis in a cell-free system from immature seeds of Pisum sativum

A cell-free system from immature pea seeds converts ¹⁴C-labelled $\text{ent}$-kaurene to $\text{ent}$-kaurenol, $\text{ent}$-kaurenal, $\text{ent}$-kaurenoic acid, $\text{ent}$-7α-hydroxykaurenoic acid, and gibberellin A₁₂-aldehyde. The latter becomes converted further to 13-hydroxygibberellin A₁₂, gibberellin A44, gibberellin A₁₂-alcohol, and several unidentified products. Thus the biosynthesis of gibberellins $\text{via ent}$-kaurene is now established for a member of the $\text{Leguminosae}$. It is the first time that 13-hydroxylation of gibberellins has been observed in a cell-free system and that gibberellin A₁₂-alcohol has been obtained in any biological system.     

Ethylene-Mediated Regulation of Gibberellin Content and Growth in Helianthus annuus L

Elongation of hypocotyls of sunflower can be promoted by gibberellins (GAs) and inhibited by ethylene. The role of these hormones in regulating elongation was investigated by measuring changes in both endogenous GAs and in the metabolism of exogenous [³H]- and [²H₂]GA₂₀ in the hypocotyis of sunflower (Helianthus annuus L. cv Delgren 131) seedlings exposed to ethylene. The major biologically active GAs identified by gas chromatography-mass spectrometry were GA₁, GA₁₉, GA₂₀, and GA₄₄. In hypocotyls of seedlings exposed to ethylene, the concentration of GA₁, known to be directly active in regulating shoot elongation in a number of species, was reduced. Ethylene treatment reduced the metabolism of [³H]GA₂₀ and less [²H₂]GA₁ was found in the hypocotyls of those seedlings exposed to the higher ethylene concentrations. However, it is not known if the effect of ethylene on GA₂₀ metabolism was direct or indirect. In seedlings treated with exogenous GA₁ or GA₃, the hypocotyls elongated faster than those of controls, but the GA treatment only partially overcame the inhibitory effect of ethylene on elongation. We conclude that GA content is a factor which may limit elongation in hypocotyls of sunflower, and that while exposure to ethylene results in reduced concentration of GA₁ this is not sufficient per se to account for the inhibition of elongation caused by ethylene.     

A Cotyledonary Inhibitor of Root Nodulation in Pisum sativum

Root nodule formation was studied in 11-day-old seedlings of Pisum sativum L. cv. Alaska, which were inoculated with Rhizobium leguminosarum at the time of planting. On intact plants an identical nodulation pattern was observed in the lateral roots attached to the two xylem strands connected to the cotyledons. When one cotyledon was removed before germination, however, a highly significant increase in nodultion was observed on the lateral roots attached to the xylem strand which no longer was connected to a cotyledon. Excision of one cotyledon also caused an alteration in the radial location of root nodules on the primary root. Under these conditions there was a distinct promotion of nodular proliferations in the root cortex opposite the primary phloem poles. The fact that removal of one cotyledon increased nodulation on the lateral roots but had no effect on the rhizobial infection of lateral root hairs suggested that a cotyledonary inhibitor acts at a step between the infection process and the appearance of a macroscopic nodule. The data were interpreted in terms of an inhibitor of cortical cell division which is translocated from the cotyledons to the developing root via the phloem.     

Blue-Light Regulation of Epicotyl Elongation in Pisum sativum

Blue light is known to induce suppression of stem elongation. To avoid the complication of blue-light-induced transformation of phytochrome we have adapted the procedure of measuring blue-light-induced suppression of stem elongation in Pisum sativum L. var Alaska grown under continuous red light. The resulting fluence-response curve for suppression of epicotyl elongation measured twenty-four hours after a blue-light treatment is bell-shaped, with the peak of suppression between 10⁰ and 10¹ micromoles per square meter, and no suppression at 10⁴ micromoles per square meter. Suppression is first observed 5 and 11 hours after the blue-light treatment for the fourth and third internodes, respectively. No significant differences in elongation rates were noted for the 10⁴ micromoles per square meter treated seedlings throughout the 24 hour period. Reciprocity holds for both third and fourth internodes in response to 10¹ and 10⁴ micromoles per square meter of blue light over the range of irradiation times tested (10⁰ to 10⁴ seconds, 10¹ micromoles per square meter; 10⁰ to 10³ seconds, 10⁴ micromoles per square meter). In contrast to the bell-shaped fluence-response obtained for epicotyl elongation, measurements of chlorophyll and carotenoid accumulation indicate increasing accumulation with increasing fluence.     

Axial Distribution of Glycosidases in Relation to Cellular Growth and Ageing in Pisum sativum Root

Tanimoto, E. 1985. Axial distribution of glycosidases in relationto cellular growth and ageing in Pisum sativum root.—J.exp. Bot. 36: 1267–1274. Eight glycosidase activities were measured in relation to cellulargrowth and ageing along the axis of pea roots. The highest activities of ß-D-fucosidase and ß-D-glucosidasewere in the apical 1.0 mm segment containing the root cap andmeristem. Activities of -L-arabinosidase, -D-mannosidase, ß-D-galactosidase, -D-glucosidase and ß-D-xylosidase werehighest in the segment between 1 and 2 mm from the tip and containingyoung elongating cells with high growth potential. The activityof -D-galactosidase was high between 1 and 2 mm from the roottip, decreasing to a minimum 4-5 mm from the tip and increasingagain up to the base of root. This distribution of enzyme activitiesrelative to the root tip remained unchanged during 28 h whilethe root length doubled. No -L--fucosidase, ß-L-fucosidaseand -D-xylosidase activities were detected. Key words: Pisum sativum, root growth, glycosidase, galactosidase, mannosidase     

Gibberellin A20 in seed of Pisum sativum L., cv. Alaska

Summary The main gibberellin in immature seed of Pisum sativum L., cv. Alaska, is identified as GA₂₀ by GC-MS. GA₉ may also be present.     

DNA Topoisomerase in Nuclei Purified from Root Meristems of Pisum sativum

DNA topoisomerase is present in nuclei purified from the rootmeristems of Pisum sativum seedlings. The DNA topoisomeraseis solubilized from nuclei by 500 mol m^–3 NaCl and relaxessupercoiled pBR322 DNA forming a series of DNA topoisomers whichmigrate electrophoretically between the supercoiled and opencircular forms. The presence of ATP in the incubation mixtureincreases the number of DNA topoisomers migrating electrophoreticallyin the region with slightly greater mobility than the open circularform. The formation of topoisomers with different linking numbersmight be the result of the activation of a different DNA topoisomerasewhich has a peculiar relaxing activity or introduces supercoilsinto the open circular form of pBR322 DNA. A low unknottingactivity with knotted P4 DNA is also present in the same nuclearpreparation. The hypothesis is made that both DNA topoisomerase I and IImight be present contemporaneously in these nuclei. The DNArelaxing activity seems to be stable and is activated by KC1.Partial purification by ion-exchange chromatography is not sufficientto separate these two DNA topoisomerases. Key words: Pisum sativum, pea, DNA topoisomerase, nuclei, cell proliferation     

Genetic Regulation of Flowering in Grafts on Pisum sativum L

Lf, E, Sn, and Hr are major loci that condition the flowering and photoperiod responses of Pisum sativum L. Genetic lines containing the dominant alleles of these loci are characterized by flowering in long days, but having a prolonged (>50 node) vegetative phase in short days. A representative of this class, response type G, was used as a receptor in short days for donors of other flowering response types. The qualitative and quantitative flowering response of G receptors depended on the genotype of the donor. Donors containing sn hr induced the earliest development, followed by sn Hr and Sn hr donors. The Lf and E loci in foliar donors apparently did not affect flowering of G. Five-leaved > single-leaved > cotyledonary donors in effecting a flowering response in G, in part due to the longer life of the foliar donors. The responses of G to the various donors were generally consistent with the proposed roles of Lf, E, and Sn, but the role of Hr in these grafts was unclear.     

Gibberellin Substitution for the Requirement of the Cotyledons in Stem Elongation in Pisum sativum Seedlings

The removal of the cotyledons from 8-day-old light-grown Pisum sativum cv. Alaska seedlings caused a reduction in the rate of stem elongation to 50% of the intact control value. Gibberellic acid restored the stem elongation rate of decotylized plants to the level of the intact controls. The effect of decotylization was to lower both the rate of node formation and the rate of internode elongation. The steady state rate of internode elongation was reduced to 50% of the control rate by decotylization. Applied gibberellic acid did not restore the normal rate of node formation nor the lag in internode elongation caused by decotylization, but gibberellic acid did restore the normal steady state rate of internode elongation. Analysis of variance demonstrated an interaction between the cotyledons and applied gibberellic acid. 2-Isopropyl-4-dimethylamino-5-methyl phenyl-1-piperidine carboxylate methyl chloride inhibited internode elongation to the same extent in both intact and decotylized plants. The results indicate that the cotyledons are an effective source of gibberellin for the young pea seedling.     

Stomata on the Primary Root of Pisum sativum L.

The first record of stomata on a non-specialized root was obtainedby scanning electron microscopy of 4-d-old Pisum sativum L.In some cases subsidiary cells were trichoblasts. Stomata andthe root triarch vascular structure were simultaneously presentin transverse sections through the root. Pisum sativum, pea, root stomata, guard cells, trichoblasts     

Regulation of Glutathione Synthesis by Cadmium in Pisum sativum L

In roots and shoots of pea plants (Pisum sativum L.) cultivated with CdCl₂ concentrations up to 50 micromolar, growth, the content of total acid soluble thiols, and the activity of glutathione synthetase (EC 6.3.2.3) and of adenosine 5′-phosphosulfate sulfotransferase were measured. In addition, the occurrence of Cd-binding peptides (phytochelatins) and the contents of glutathione and cysteine were determined in roots of plants exposed to 20 micromolar Cd and/or 1 millimolar buthionine sulfoximine, an inhibitor of glutathione synthesis. An appreciable increase in activity of glutathione synthetase at 20 and 50 micromolar Cd and of adenosine 5′-phosphosulfate sulfotransferase at 5 micromolar and higher Cd concentrations was detected in the roots. Most of the additional thiols formed due to Cd treatment were eluted from a gel filtration HPLC column together with Cd, indicating the presence of phytochelatins. In plants treated with buthionine sulfoximine and Cd, no phytochelatins could be detected but the cysteine content increased 21-fold. Additionally, a larger increase in both enzyme activities occurred than with Cd alone. Taken together, our results are consistent with the hypothesis that glutathione is a precursor for phytochelatin synthesis.     

The Response to Gibberellin in Pisum sativum Grown under Alternating Day and Night Temperature

The application of gibberellins (GA) reduces the difference in stem elongation observed under a low day (DT) and high night temperature (NT) combination (negative DIF) compared with the opposite regime, a high DT/low NT (positive DIF). The aim of this work was to investigate possible thermoperiodic effects on GA metabolism and tissue sensitivity to GA by comparing the response to exogenously applied GA (in particular, GA₁ and GA₃) in pea plants (Pisum sativum cv. Torsdag) grown under contrasting DIF. Control plants not treated with growth inhibitors or additional GA were 38% shorter under negative (DT/NT 13/21°C) than positive DIF (DT/NT 21/13°C) because of shorter internodes. Additional GA₁ or GA₃ decreased the difference between positive and negative DIF. In pea plants dwarfed with paclobutrazol, which inhibits GA biosynthesis at an early step, the response to GA₁ was reduced more strongly by negative compared with positive DIF than the response to GA₃. The induced stem elongation by GA₁₉ and GA₂₀ did not deviate significantly from the response to GA₁. Plants treated with prohexadione-calcium, an inhibitor of both the production and the inactivation of GA₁, grew equally tall under the two temperature regimes in response to both GA₁ and GA₃. We hypothesize that the reduced response to GA₁ compared with GA₃ in paclobutrazol-treated plants grown under negative DIF is caused by a higher rate of 2β-hydroxylation of GA₁ into GA₈ under negative than positive DIF. This contributes to lower levels of GA₁ and consequently shorter stems and internodes in pea plants grown under negative than positive DIF. Differences in tissue sensitivity to GA alone cannot account for this specific thermoperiodic effect on stem elongation. Received May 28, 1998; accepted May 29, 1998     

Evidence for Phytochrome Regulation of Gibberellin A(20) 3beta-Hydroxylation in Shoots of Dwarf (lele) Pisum sativum L

The effect of light on the dwarfing allele, le, in Pisum sativum L. was tested as the growth response to gibberellins prior to or beyond the presumed block in the gibberellin biosynthetic pathway. The response to the substrate (GA₂₀), the product (GA₁), and a nonendogenous early precursor (steviol) was compared in plants bearing the normal Le and the deficient lele genotypes in plants made low in gibberellin content genetically (nana lines) or by paclobutrazol treatment to tall (cv Alaska) and dwarf (cv Progress) peas. Both genotypes responded to GA₁ under red irradiation and in darkness. The lele plants grew in response to GA₂₀ and steviol in darkness but showed a much smaller response when red irradiated. The Le plants responded to GA₂₀ and steviol in both light and darkness. The red effects on lele plants were largely reversible by far-red irradiation. It is concluded that the deficiency in 3β-hydroxylation of GA₂₀ to GA₁ in genotype lele is due to a Pfr-induced blockage in the expression of that activity.