Evidence for a graft-transmissible substance which delays apical senescence in Pisum sativum L.

Apical senescence in an early flowering line of pea, G2, is greatly delayed by short days. This behavior is controlled by two dominant genes. Apical senescence of ungrafted, insensitive (I) lines is unaffected by photoperiod. When I-type scions with one of the two required genes were grafted onto G2, apical senescence of the I-type was delayed in short days, but not in long days. Flowering of the I-type was unaffected. The apex of the G2 stock was unaffected as well. Apical senescence of an I-type line lacking both photoperiod genes was not delayed when grafted on G2 in short days. It is concluded that G2 plants grown in short days produce a graft-transmissible factor which delays apical senescence of photoperiodically insensitive lines.     

Proteolytic activity in relationship to senescence and cotyledonary development in Pisum sativum L.

Changes in the weight and in the chlorophyll, free amino-acid and protein content of developing and senescing, vegetative and reproductive organs of Pisum sativum L. (cv. Burpeeana) were measured, and the proteolytic activity in extracts from the senescing leaf and the subtended pod was followed in relation to these changes. Protein content decreased in the ageing leaf and pod while it increased in the developing cotyledon. The proteolytic activity of the leaf did not increase as the leaf protein content decreased. In contrast, proteolytic activity in the subtended pod increased while the protein level decreased. The proteolytic activity in the extracts from the ageing organs was greater than the rates of protein loss. The proteolytic activity of leaf and pod extracts was greater on protein prepared from the respective organ than on non-physiological substrates. Proteolysis was increased by 2-mercaptoethanol and ethylenediaminetetraacetate but was not influenced by addition of ATP to the reaction mixture. The pH optimum was at 5.0. Free amino acids did not accumulate in the senescing leaf or pod when protein was degraded in each organ. It is suggested that these amino acids were quickly metabolized in situ or translocated to sink areas in the plant, especially to the developing seeds.     

Ethylene metabolism in Pisum sativum L.

The possible role of C₂H₄ metabolism in mediating the responses of plants to C₂H₄ is re-examined. It is demonstrated that (i) the effects of inhibitors upon C₂H₄ action do not correspond with their effects on metabolism, (ii) elicitors of C₂H₄ effects do not have appropriate effects on C₂H₄ metabolism, (iii) inhibitors of C₂H₄ metabolism do not affect the response of plants to C₂H₄. It is concluded that metabolism of C₂H₄ is not linked to the mode of action of the growth regulator.Abbreviations DTC sodium diethyldithiocarbamate - FW fresh weight     

Enzymes of serine and glycine metabolism in leaves and non-photosynthetic tissues of Pisum sativum L.

A comparative study is presented of the activities of enzymes of glycine and serine metabolism in leaves, germinated cotyledons and root apices of pea (Pisum sativum L.). Data are given for aminotransferase activities with glyoxylate, hydroxypyruvate and pyruvate, for enzymes associated with serine synthesis from 3-phosphoglycerate and for glycine decarboxylase and serine hydroxymethyltransferase. Aminotransferase activities differ between the tissues in that, firstly, appreciable transamination of serine, hydroxypyruvate and asparagine occurs only in leaf extracts and, secondly, glyoxylate is transaminated more actively than pyruvate in leaf extracts, whereas the converse is true of extracts of cotyledons and root apices. Alanine is the most active amino-group donor to both glyoxylate and hydroxypyruvate. 3-Phosphoglycerate dehydrogenase and glutamate: O-phosphohydroxypyruvate aminotransferase have comparable activities in all three tissues, except germinated cotyledons, in which the aminotransferase appears to be undetectable. Glycollate oxidase is virtually undetectable in the non-photosynthetic tissues and in these tissues the activity of glycerate dehydrogenase is much lower than that of 3-phosphoglycerate dehydrogenase. Glycine decarboxylase activity in leaves, measured in the presence of oxaloacetate, is equal to about 30–40% of the measured rate of CO₂ fixation and is therefore adequate to account for the expected rate of photorespiration. The activity of glycine decarboxylase in the non-photosynthetic tissues is calculated to be about 2–5% of the activity in leaves and has the characteristics of a pyridoxal-and tetrahydrofolate-dependent mitochondrial reaction; it is stimulated by oxaloacetate, although not by ADP. In leaves, the measured activity of serine hydroxymethyltransferase is somewhat lower than that of glycine decarboxylase, whereas in root apices it is substantially higher. Differential centrifugation of extracts of root apices suggests that an appreciable proportion of serine hydroxymethyltransferase activity is associated with the plastids.Abbreviation GOGAT l-Glutamine:2-oxoglutarate aminotransferase     

Photoperiod-induced changes in gibberellin metabolism in relation to apical growth and senescence in genetic lines of peas (Pisum sativum L.)

In an early-flowering line of pea (G2) apical senescence occurs only in long days (LD), while growth in short days (SD) is indeterminate. In SD, G2 plants are known to produce a graft-transmissible substance which delays apical senescence in related lines that are photoperiod-insensitive with regard to apical senescence. Gibberellic acid (GA₃) applied to the apical bud of G2 plants in LD delayed apical senescence indefinitely, while N⁶-benzyladenine and -naphthaleneacetic acid were ineffective. Of the gibberellins native to pea, GA₉ had no effect whereas GA₂₀ had a moderate senescence-delaying effect. [³H]GA₉ metabolism in intact leaves of G2 plants was inhibited by LD and was restored by placing the plants back in SD. Leaves of photoperiod-insensitive lines (I-types) metabolized GA₉ readily regardless of photoperiod, but the metabolites differed qualitatively from those in G2 leaves. A polar GA₉ metabolite, GA_E, was found only in G2 plants in SD. The level of GA-like substances in methanol extracts from G2 plants dropped about 10-fold after the plants were moved from SD to LD; it was restored by transferring the plants back to SD. A polar zone of these GA-like materials co-chromatographed with GA_E. It is suggested that a polar gibberellin is synthesized by G2 plants in SD; this gibberellin promotes shoot growth and meristematic activity in the shoot apex, preventing senescence.Abbreviations GA gibberellin - GA₃ gibberellic acid - SD short days - LD long days     

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.     

Intracellular location of ATP citrate lyase in leaves of Pisum sativum L.

The aim of this work was to discover if there is enough ATP citrate lyase (EC 4.1.3.8) in the cytosol of the leaves of Pisum sativum L. to catalyse the synthesis of the acetyl CoA needed for terpenoid synthesis. Estimates of the maximum catalytic activity of the enzyme in leaves of 7-d-old peas gave values of 113 nmol min^-1 g^-1 fresh weight. The rate of carotenoid accumulation in these leaves corresponded to a requirement for acetyl CoA of 0.7 nmol min^-1 g^-1 fresh weight. The distribution of marker enzymes during fractionation of homogenates of leaves from 7 to 10-d-old peas showed that differential centrifugation led to the isolation in reasonable yields of chloroplasts, mitochondria, peroxisomes and the endomembrane system. None of the above components of the leaf contained appreciable detectable activity of ATP citrate lyase, the distribution of which closely paralleled that of the cytosolic marker. It was concluded that in young leaves of pea most of the ATP citrate lyase is in the cytosol.     

Carnitine acyltransferases in chloroplasts of Pisum sativum L.

Carnitine-acetyltransferase (EC 2.3.1.7) and carnitine-palmitoyltransferase (EC 2.3.1.21) activities were shown to be present in chloroplasts of green pea leaves and possibly to occur in leaf mitochondrial and peroxisomal fractions. A role for the enzymes in the transfer of acyl groups across membranes is suggested.     

Capacity for fermentation in roots and Rhizobium nodules of Pisum sativum L.

The aim of this work was to compare the capacities for fermentation and synthesis of malate from phosphoenolpyruvate in roots and Rhizobium nodules of Pisum sativum. The nodules and the cortices and apices of roots had similar activities of glycolytic enzymes and enzymes of ethanolic and lactic fermentation when expressed on a protein basis. The activity of phosphoenolpyruvate carboxylase was similar in nodules and apices, and three to four fold lower in cortices. All three tissues had very high activities of malate dehydrogenase, significant activity of NADP-malic enzyme, and no detectable activity of phosphoenolpyruvate carboxykinase. These results do not support the belief that nodules have a substantially greater capacity to convert phosphoenolpyruvate to malate than roots, or that there are major qualitative differences in the pathways of fermentation of nodules and roots.Abbreviation PEP phosphoenolpyruvate     

Two routes for asparagine metabolism in Pisum sativum L.

Asparagine, a major transport compound, is metabolized in Pisum sativum by two enzymes, asparaginase (EC 3.5.1.1) and asparagine-pyruvate aminotransferase. The relative amount of the two enzymes varies between tissues. In developing seeds, there are very high levels of asparaginase but only trace amounts of the aminotransferase. Asparaginase is high in young leaves but falls rapidly during leaf growth; the aminotransferase remains high throughout development. Inhibitor studies with aminooxyacetate and methionine sulfoximine confirm that the aminotransferase is the main enzyme involved in asparagine utilisation in the leaf. Root tissue has low levels of asparaginase and only trace amounts of the aminotransferase. The asparaginase is potassium dependent, but is also partially activated by ammonium ions. The leaf aminotransferase has a lower K _m for asparagine (4.5 mM) than the leaf asparaginase (8 mM). The seed asparaginase has a lower K _m for asparagine (3 mM) than the leaf asparaginase.     

Distribution of glucose/mannose-specific isolectins in pea (Pisum sativum L.) seedlings

We report on the distribution and initial characterization of glucose/mannose-specific isolectins of 4- and 7-d-old pea (Pisum sativum L.) seedlings grown with or without nitrate supply. Particular attention was payed to root lectin, which probably functions as a determinant of host-plant specificity during the infection of pea roots by Rhizobium leguminosarum bv. viciae. A pair of seedling cotyledons yielded 545±49 g of affinity-purified lectin, approx. 25% more lectin than did dry seeds. Shoots and roots of 4-d-old seedlings contained 100-fold less lectin than cotyledons, whereas only traces of lectin could be found in shoots and roots from 7-d-old seedlings. Polypeptides with a subunit structure similar to the precursor of the pea seed lectin could be demonstrated in cotyledons, shoots and roots. Chromatofocusing and isoelectric focusing showed that seed and non-seed isolectin differ in composition. An isolectin with an isoelectric point at pH 7.2 appeared to be a typical pea seed isolectin, whereas an isolectin focusing at pH 6.1 was the major non-seed lectin. The latter isolectin was also found in root cell-wall extracts, detached root hairs and root-surface washings. All non-seed isolectins were cross-reactive with rabbit antiserum raised against the seed isolectin with an isolectric point at pH 6.1. A protein similar to this acidic glucose/mannose-specific seed isolectin possibly represents the major lectin to be encountered by Rhizobium leguminosarum bv. viciae in the pea rhizosphere and at the root surface. Growth of pea seedlings in a nitrate-rich medium neither affected the distribution of isolectins nor their hemagglutination activity; however, the yield of affinity-purified root lectin was significantly reduced whereas shoot lectin yield slightly increased. Agglutination-inhibition tests demonstrated an overall similar sugar-binding specificity for pea seed and non-seed lectin. However root lectin from seedlings grown with or without nitrate supplement, and shoot lectin from nitrate-supplied seedlings showed a slightly different spectrum of sugar binding. The absorption spectra obtained by circular dichroism of seed and root lectin in the presence of a hapten also differed. These data indicate that nutritional conditions may affect the sugar-binding activity of non-seed isolectin, and that despite their similarities, seed and non-seed isolectins have different properties that may reflect tissue-specialization.Abbreviations IEF isoelectric focusing - MW molecular weight - pI isoelectric point - Psl1, Psl2 and Psl3 pea isolectins - SDSPAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis The authors wish to thank Professors L. Kanarek and M. van Poucke for helpful discussions.     

Glutamate dehydrogenase from Pisum sativum L.

A 2–8-fold increase in the activity of glutamate dehydrogenase (GDH), accompanied by an alteration of the GDH isoenzyme pattern, was observed in detached pea shoots floated on tap water (preincubated shoots). Sugars supressed the process, whereas NH ₊ ⁴ and various metabolites as well as inhibitors of energy metabolism and protein synthesis were ineffective. The subcellular distribution pattern revealed evidence that the GDH isoenzymes are exclusively located in the mitochondrial matrix. The alterations in GDH activity occurring in preincubated shoots are restricted to the mitochondria.An experimental device suitable for studying the GDH function in isolated intact mitochondria has been established. Using [¹⁴C] citrate as the carbon source and hydrogen donor, the mitochondria synthesized considerable amounts of glutamate upon addition of NH ₊ ⁴ . The rates of glutamate formation in dependency of increasing NH ₊ ⁴ levels follow simple Michaelis-Menten kinetics. Half-saturation concentrations of NH ₊ ⁴ of 3.6±1.2 mM; 1.9±0.06 mM and 1.6±0.1 mM were calculated for the mitochondria isolated from pea shoots, roots, and preincubated shoots, respectively. The results are discussed in relation to the possible role of GDH in NH_+/4 assimilation at elevated intracellular NH_+/4 levels.Abbreviations GDH Glutamate dehydrogenase - MDH malate dehydrogenase - GOT aspartate aminotransferase - SDH succinate dehydrogenase - HEPES 4-(2-hydroxyethyl)-1-piperazineethan-sulfonic acid - BSA bovine serum albumin - TPP thiamine pyrophosphate - DNP 2,4-dinitrophenol - CCCP carbonyl cyanide m-chlorophenylhydrazone - DCPIP 2,6-dichlorophenolindophenol Dedicated to Professor Dr. Maximilian Steiner on the occasion of his 75th birthday     

The quantitative relationship between gibberellin A1 and internode growth in Pisum sativum L.

The metabolism and growth-promoting activity of gibberellin A₂₀ (GA₂₀) were compared in the internode-length genotypes of pea, na le and na Le. Gibberellin A₂₉ and GA₂₉-catabolite were the major metabolites of GA₂₀ in the genotype na le. However, low levels of GA₁, GA₈ and GA₈-catabolite were also identified as metabolites in this genotype, confirming that the le allele is a leaky mutation. Gibberellin A₂₀ was approximately 20 to 30 times as active in promoting internode growth of genotype na Le as of genotype na le. However, the levels of the 3-hydroxylated metabolite of GA₂₀, GA₈ (2-hydroxy GA₁), were similar for a given growth response in both genotypes. In each case a close linear relationship was observed between internode growth and the logarithm of GA₈ levels. A similar relationship was found on comparing GA₂₀ metabolism in the three genotypes le ^d, le and Le. The former mutation results in a more severe dwarf phenotype than the le allele (which has previously been shown to reduce the 3-hydroxylation of GA₂₀ to GA₁). These results indicate that GA₂₀ has negligible intrinsic activity and support the contention that GA₁ is the only GA active per se in promoting stem growth in pea.Abbreviations GA_n gibberellin A_n - GC-MS gas chromatography-mass spectrometry - HPLC high-pressure liquid chromatography     

Lectin-gene expression in pea (Pisum sativum L.) roots

The expression of a lectin gene in pea (Pisum sativum L.) roots has been investigated using the copy DNA of a pea seed lectin as a probe. An mRNA which has the same size as the seed mRNA but which is about 4000 times less abundant has been detected in 21-d-old roots. The probe detected lectin expression as early as 4 d after sowing, with the highest level being reached at 10 d, i.e. just before nodulation. In later stages (16-d- and 21-d-old roots), expression was substantially decreased. The correlation between infection by Rhizobium leguminosarum and lectin expression in pea roots has been investigated by comparing root lectin mRNA levels in inoculated plants and in plants grown under conditions preventing nodulation. Neither growth in a nitrate concentration which inhibited nodulation nor growth in the absence of Rhizobium appreciably affected lectin expression in roots.Abbreviation cDNA copy DNA - poly(A)⁺RNA polyadenylated RNA     

Components of auxin transport in stem segments of Pisum sativum L.

1. The uptake of indol-3-yl acetic acid ([1-¹⁴C]IAA, 0–2.0 M) into light-grown pea stem segments was measured under various conditions to investigate the extent to which mechanisms of auxin transport in crown gall suspension culture cells (Rubery and Sheldrake, Planta 118, 101–121, 1974) are also found in a tissue capable of polar auxin transport. — 2. IAA uptake increased as the external pH was lowered. IAA uptake was less than that of benzoic acid (BA), naphthylacetic acid (NAA) or 2,4 dichlorophenoxyacetic acid (2,4D) under equivalent conditions. TIBA enhanced net IAA uptake through inhibition of efflux, and to a lesser extent, also increased uptake of NAA and 2,4D while it had no effect on BA uptake. — 3. Both DNP and, at higher concentrations, BA, reduced IAA uptake probably because of a reduction of cytoplasmic pH. However, low concentrations of both BA and DNP caused a slight enhancement of IAA net uptake, possibly through a reduction of carrier-mediated IAA efflux. In the presence of TIBA, the inhibitory effects of DNP and BA were more severe and there was no enhancement of uptake at low concentrations. — 4. Non-radioactive IAA (10 M) reduced uptake of labelled IAA but further increases in concentration up to 1.0 mM produced first an inhibition (0–10 min) of labelled IAA uptake, followed by a stimulation at later times. Non-radioactive 2,4 D decreased, but was not observed to stimulate, uptake of labelled IAA. In the presence of TIBA labelled IAA uptake was inhibited by non-radioactive IAA regardless of its concentration. — 5. Sulphydryl reagents PCMB and PCMBS promoted or inhibited IAA uptake depending, respectively, on whether they penetrated or were excluded from the cells. The penetrant PCMB also reduced the promotion of labelled IAA uptake by TIBA or by high concentrations of added non-labelled IAA. — 6. Our findings are interpreted as being consistent with the diffusive entry of unionised IAA into cells together with some carrier-mediated uptake. Auxin efflux from the cells also appears to have a carrier-mediated contribution, at least part of which is inhibited by TIBA, and which has a capacity at least as great as that of the uptake carrier. The data indicate that pea stem segments contain cells whose mechanisms of trans-membrane auxin transport fit the model of polar auxin transport proposed from experiments with crown gall suspension cells, although differences, particularly of carrier specificity, are apparent between the two systems.Abbreviations IAA indol-3-yl acetic acid - BA benzoic acid - NAA 1-naphthylacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - TIBA 2,3,5-triiodobenzoic acid - DNP 2,4-dinitrophenol - PCMB p-chloromercuribenzoic acid - PCMBS p-chloromercuribenzene sulphonic acid This work was performed in Cambridge during the tenure of a sabbatical leave by P.J.D. Supported by a grant for supplies from the American Philosophical Society to P.J.D.     

Regeneration of whole plants from callus culture of diverse genetic lines of Pisum sativum L.

Sixteen genetic lines of peas were screened for their ability to regenerate whole plants from callus cultures. Epicotyl sections from germinating seeds were placed on callus-inducing medium; the resulting callus was subcultured monthly and was tested every other month for its regeneration ability. Six lines were found that would regenerate after 2 months' growth as callus. Four of these continued to regenerate after 4 months and, of these, two after 6 months. The cultivars Frosty and Alaska were among the lines that would not regenerate at all.Michigan Agricultural Experiment Station Journal Article No. 8932     

Changes in legumin messenger RNAs throughout seed development in Pisum sativum L.

The patterns of accumulation of three classes of legumin mRNA from Pisum sativum have been followed through seed development by cell-free translation and hybridization to complementary DNAs. Maximum amounts of mRNA were found at 19 days after flowering (DAF) for two classes and at 24 DAF for the third class. The proportions of the three classes varied through development: the RNA species which was 40% of the total legumin mRNA at 14 DAF represented 15–20% of the total at 25–27 DAF, whereas a second mRNA species represented approx. 30% and 70% at the same stages, respectively. Based on these results, some deductions about the possible contribution of individual genes within classes are made.Abbreviations cDNA complementary DNA - DAF days after flowering - IgG immunoglobulin G - SSC standard saline citrate (0.15 M NaCl, 0.015 M trisodium citrate)     

Plasmatubules in transfer cells of pea (Pisum sativum L.)

Plasmatubules are tubular evaginations of the plasmalemma. They have previously been found at sites where high solute flux between apoplast and symplast occurs for a short period and where wall proliferations of the transfer cell type have not been developed (Harris et al. 1982, Planta 156, 461–465). In this paper we describe the distribution of plasmatubules in transfer cells of the leaf minor veins of Pisum sativum L. Transfer cells are found in these veins associated both with phloem sieve elements and with xylem vessels. Plasmatubules were found, in both types of transfer cell and it is suggested that the specific distribution of the plasmatubules may reflect further membrane amplification within the transfer cell for uptake of solute from apoplast into symplast.