Ribulose-1,5-bisphosphate carboxylase and fruit set or degeneration of unpollinated ovaries of Pisum sativum L.

The polypeptide patterns obtained by sodium dodecylsulphate-polyacrylamide gel electrophoresis of undigested and autodigested extracts from pea (Pisum sativum L.) ovaries at the early stages of development or degeneration have been studied. Development of unpollinated ovaries was stimulated by application of different plant growth regulators (gibberellic acid, 2,4-dichlorophenoxyacetic acid, and N⁶-benzyladenine) or by plant topping. Polypeptide bands of similar mobility to ribulose-1,5-bisphosphate carboxylase (RuBPCase) subunits (16 and 55 kDa) could be detected in all types of autodigested extracts from stimulated ovaries. However these bands were absent in electrophoretic patterns of autodigested extracts from unstimulated ovaries after 3 d post anthesis and in patterns of autodigested mixtures of these extracts with either those from stimulated ovaries or those from unstimulated ovaries before day 3. These observations indicate that a proteolytic activity which promotes the hydrolysis of RuBPCase appears in unstimulated ovaries about 3 d after anthesis. This event coincides with the loss of the capacity of unpollinated ovaries to develop in response to gibberellic acid and with the degeneration of the ovary wall.Abbreviations BA N⁶-benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - RuBPCase ribulose-1,5-bisphosphate carboxylase - SDS-PAGE sodium dodecylsulphate-polyacrylamide gel electrophoresis     

Identification,quantitation and distribution of gibberellins in fruits of Pisum sativum L. cv. Alaska during pod development

In addition to the previously-reported gibberellins: GA₁; GA₈, GA₂₀ and GA₂₉ (García-Martínez et al., 1987, Planta 170, 130–137), GA₃ and GA₁₉ were identified by combined gas chromatography-mass spectrometry in pods and ovules of 4-d-old pollinated pea (Pisum sativum cv. Alaska) ovaries. Pods contained additionally GA₁₇, GA₈₁ (2-hydroxy GA₂₀) and GA₂₉-catabolite. The concentrations of GA₁, GA₃, GA₈, GA₁₉, GA₂₀ and GA₂₉ were higher in the ovules than in the pod, although, with the exception of GA₃, the total content of these GAs in the pod exceeded that in the seeds. About 80% of the GA₃ content of the ovary was present in the seeds. The concentrations of GA₁₉ and GA₂₀ in pollinated ovaries remained fairly constant for the first 12 ds after an thesis, after which they increased sharply. In contrast, GA₁ and GA₃ concentrations were maximal at 7 d and 4–6 d, respectively, after anthesis, at about the time of maximum pod growth rate, and declined thereafter. Emasculated ovaries at anthesis contained GA₈, GA₁₉ and GA₂₀ at concentrations comparable with pollinated fruit, but they decreased rapidly. Gibberellins a₁ and A₃ were present in only trace amounts in emasculated ovaries at any stage. Parthenocarpic fruit, produced by decapitating plants immediately above an emasculated flower, or by treating such flowers with 2,4-dichlorophenoxyacetic acid or GA₇, contained GA₁₉ and GA₂₀ at similar concentrations to seeded fruit, but very low amounts of GA₁ and GA₃ Thus, it appears that the presence of fertilised ovules is necessary for the synthesis of these last two GAs. Mature leaves and leaf diffusates contained GA₁, GA₈, GA₁₉ and GA₂₀ as determined by combined gas chromatography-mass spectrometry using selected ion monitoring. This provides further evidence that vegetative tissues are a possible alternative source of GAs for fruit-set, particularly in decapitated plants.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - FW fresh weight - GA_n gibberellin A_n - GC-MS combined gas chromatography-mass spectrometry - HPLC high-performance liquid chromatography - KRI Kovats retention index - m/z mass to charge ratio We thank Mr M.J. Lewis for qualitative GC-MS analyses and Ms M.V. Cuthbert (LARS), R. Martinez Pardo and T. Sabater (IATA) for technical assistance. We are also grateful to Professor B.O. Phinney, University of California, Los Angeles, for gifts of [17-¹³C]GA₈ and -GA₂₉ and to Mr Paul Gaskin, University of Bristol, for the mass spectrum of GA₂₉-catabolite and for a sample of GA₈₁ The work in Spain was supported by Dirección General de Investigación Cientifica y Técnica (grant PB87-0402 to J.L.G.-M.). We also acknowledge the British Council and Ministerio de Educacion y Ciencia for travel grants through Accion Integrada Hispano-Britanica 56/142 (J.L.G.-M. and P.H.).     

The source of gibberellins in the parthenocarpic development of ovaries on topped pea plants

The role and source of gibberellins (GAs) involved in the development of parthenocarpic fruits of Pisum sativum L. has been investigated. Gibberellins applied to the leaf adjacent to an emasculated ovary induced parthenocarpic fruit development on intact plants. The application of gibberellic acid (GA₃) had to be done within 1 d of anthesis to be fully effective and the response was concentration-dependent. Gibberellin A₁ and GA₃ worked equally well and GA₂₀ was less efficient. [³H]Gibberellin A₁ applied to the leaf accumulated in the ovary and the accumulation was related to the growth response. These experiments show that GA applied to the leaf in high enough concentration is translocated to the ovary. Emasculated ovaries on decapitated pea plants develop without application of growth hormones. When [³H] GA₁ was applied to the leaf adjacent to the ovary a substantial amount of radioactivity accumulated in the growing shoot of intact plants. In decapitated plants, however, this radioactivity was mainly found in the ovary. There it caused growth proportional to the accumulation of CA₁. Application of LAB 150978, an inhibitor of GA biosynthesis, to decapitated plants inhibited parthenocarpic fruit development and this inhibition was counteracted by the application of GA₃ (either to the fruit, or the leaf adjacent to the ovary, or through the lower cut end of the stem). All evidence taken together supports the view that parthenocarpic pea fruit development on topped plants depends on the import of gibberellins or their precursors, probably from the vegetative aerial parts of the plant.Abbreviations FW flesh weight - GA_n gibberellin A_n - HPLC high-performance liquid chromatography     

The relationship between fruit growth and apical senescence in the G2 line of peas

In the G2 line of peas (Pisum sativum L.), senescence of the shoot apex (which precedes leaf senescence) only occurs in long days (LD) though flowering is independent of photoperiod. It has been suggested that the photoperiodic control of senescence in G2 is mediated through different rates of seed growth. In LD seed growth is more rapid than in short days (SD) and this places a greater nutrient drain on the plant. In addition, more flowers develop into fruits in LD than in SD: 32% of flower buds abort in SD while almost none abort in LD. Senescence is associated with early seed growth and does not occur in deflowered or deseeded plants. Seed development is completed in 30d in LD while it takes 40d in SD, though the seed weights are similar. The maximum rate of fresh-weight gain of all the growing seeds of eight fruits on a plant in SD (1,440 mg/d) does not reach the maximum rate of weight gain of a similar fruit complement in LD (1,720 mg/d). The appearance of senescence symptoms in the shoot apices of LD-grown G2 plants occurs, however, prior to the time of the greatest rate of seed-weight gain. In LD, four fruits with a combined maximum growth rate of 1,250 mg/d are sufficient to cause the appearance of senescence symptoms. This is a lower combined seed growth rate than in SD where senescence does not occur. The seeds in up to 12 fruits can be growing at any time in SD with a combined maximum seed-growth rate (1,660 mg/d), only slightly less than the maximum in LD, with no sign of senescence. It is concluded that the different rates of seed growth occasioned by different photoperiods bear no relation to senescence. However, photoperiod does alter the spatial relationship of the shoot apex and the filling fruits. In LD apical growth becomes slower as fruiting proceeds so that the distance between the filling fruits and the apex is decreased to only two nodes while in SD, because of the delayed fruit development compared to LD, the spatial separation between the fruits and the shoot apex is nine nodes. Even if the growth rate of the plant had remained constant in LD it is calculated that an equivalent fruit complement would still be located three nodes further from the apex in SD than in LD. This increased spatial separation of fruits and apex in SD compared to LD probably alters the source/sink distribution of photosynthate and leaf derived hormones so that larger amounts are available to the apex in SD than LD. Also any senescence factor exported from fruits is less likely to reach the apex in SD. In continuously deflorated plants of G2 the two uppermost expanded stipules enclose the apex in SD while in LD they open out. The effect is reversible. Thus photoperiod probably affects the apex and its growth, directly, i.e. independent of fruit development, and this is accentuated by the differing spatial relationships of the apex and fruits resulting from different fruit growth rates under the different photoperiodic conditions.Abbreviations LD long day(s) - SD short day(s)     

Gene expression during two alternative pathways of ovary development in Pisum sativum: fruit development and ovary senescence

Pea ovaries are induced to enter a fruit development pathway involving physiological and morphological changes by pollination or application of plant growth regulators. In the absence of these stimuli, overies stop growing and enter an alternative pathway of senesecence that leads to their degeneration. We have used two dimensional polyacrylamide gel electrophoresis in search of molecular changes underlying fruit development and ovary senescence at the level of total accumulated proteins, newly synthesized proteins, and translatable, RNA populations. We have found changes in gene expression during the processes of ovary formation and ovary senescence. Stimuli that induce fruit set do not appreciably alter the overall patterns of synthesized proteins or translatable RNAs, indicating that fruit development is apparently a natural continuation of ovary formation. However, ovary senescence is an alternative pathway that involves the presence of new RNA messengers and proteins as well as the disappearance of others. These changes were detected earlier than any morphological or structural changes could be observed in the ovary.     

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)     

Gibberellins in developing fruits of Pisum sativum cv. Alaska: Studies on their role in pod growth and seed development

Gibberellins A₁, A₈, A₂₀ and A₂₉ were identified by capillary gas chromatography-mass spectrometry in the pods and seeds from 5-d-old pollinated ovaries of pea (Pisum sativum cv. Alaska). These gibberellins were also identified in 4-d-old non-developing, parthenocarpic and pollinated ovaries. The level of gibberellin A₁ within these ovary types was correlated with pod size. Gibberellin A₁, applied to emasculated ovaries cultured in vitro, was three to five times more active than gibberellin A₂₀. Using pollinated ovary explants cultured in vitro, the effects of inhibitors of gibberellin biosynthesis on pod growth and seed development were examined. The inhibitors retarded pod growth during the first 7 d after anthesis, and this inhibition was reversed by simultaneous application of gibberellin A₃. In contrast, the inhibitors, when supplied to 4-d-old pollinated ovaries for 16 d, had little effect on seed fresh weight although they reduced the levels of endogenous gibberellins A₂₀ and A₂₉ in the enlarging seeds to almost zero. Paclobutrazol, which was one of the inhibitors used, is xylem-mobile and it efficiently reduced the level of seed gibberellins without being taken up into the seed. In intact fruits the pod may therefore be a source of precursors for gibberellin biosynthesis in the seed. Overall, the results indicate that gibberellin A₁, present in parthenocarpic and pollinated fruits early in development, regulates pod growth. In contrast the high levels of gibberellins A₂₀ and A₂₉, which accumulate during seed enlargement, appear to be unnecessary for normal seed development or for subsequent germination.Abbreviations GA_(a) gibberellin A_n - GC-MS combined gas chromatography-mass spectrometry - HPLC high-performance liquid chromatography - PFK perfluorokerosene - PVP polyvinylpyrrolidone     

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.     

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.     

Effect of ovular receptivity on seed set and fruit development in cucumber (Cucumis sativus L.)

Summary Seed set and fruit development in cucumber (Cucumis sativus L.) were studied in relation to female flower receptivity from day — 2 before anthesis to day + 2 after anthesis. The female cucumber flower is protogynous. The pistil was receptive 2 days before anthesis. The iso-electric focusing (IEF) patterns of the stigma/style proteins, were identical from day -5 to day +2. In pollinated flowers in vivo germination and pollen-tube growth in the ovary were affected by pistil age from day -2 to day +2. In addition, differences in sectorial filling in full seeds were observed within the fruits. A negative correlation was observed between the frequency of fertilized ovules in the pedoncular part of the fruit and ovary length at the time of pollination. In the whole fruit, significant differences in the number of full seeds and fruit size at maturity were found, and these were observed to be correlated with the various stages of female flower maturation at pollination. The day -2 and day +2 stages yielded the smallest fruits with few full seeds compared to the day -1, day 0 and day +1 stages, which had the biggest fruits and a large number of full seeds. A strong positive correlation was found between total seed number (including full and empty seeds), fruit length and weight at maturity. All these results suggest that both seed set in the different parts of the fruit and fruit development are controlled by ovular receptivity rather than by stigma/style receptivity.     

Changes in mitochondrial DNA levels during development of pea (Pisum sativum L.)

The percentage of mitochondrial DNA (mtDNA) present in total DNA isolated from pea tissues was determined using labeled mtDNA in reassociation kinetics reactions. Embryos contained the highest level of mtDNA, equal to 1.5% of total DNA. This value decreased in light- and dark-grown shoots and leaves, and roots. The lowest value found was in dark-grown shoots; their total DNA contained only 0.3% mtDNA. This may be a reflection of increased nuclear ploidy levels without concomitant mtDNA synthesis. It was possible to compare the mtDNA values directly with previous estimates of the amount of chloroplast DNA (ctDNA) per cell because the same preparations of total DNA were used for both analyses. The embryo contained 1.5% of both mtDNA and ctDNA; this equals 410 copies of mtDNA and 1200 copies of ctDNA per diploid cell. Whereas mtDNA levels decreased to 260 copies in leaf cells of pea, the number of copies of ctDNA increased to 10300. In addition, the levels of ctDNA in first leaves of dark-grown and light-transferred pea were determined, and it was found that leaves of plants maintained in the dark had the same percentage of ctDNA as those transferred to the light.Abbreviations ctDNA chloroplast DNA - mtDNA mitochondrial DNA     

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.     

Correlation between infection by Rhizobium leguminosarum and lectin on the surface of Pisum sativum L. roots

The lectin on the surface of 4- and 5-dold pea roots was located by the use of indirect immunofluorescence. Specific antibodies raised in rabbits against pea seed isolectin 2, which crossreact with root lectins, were used as primary immunoglobulins and were visualized with fluorescein- or tetramethylrhodamine-isothiocyanate-labeled goat antirabbit immunoglobulin G. Lectin was observed on the tips of newly formed, growing root hairs and on epidermal cells located just below the young hairs. On both types of cells, lectin was concentrated in dense small patches rather than uniformly distributed. Lectin-positive young hairs were grouped opposite the (proto)xylematic poles. Older but still-elongating root hairs presented only traces of lectin or none at all. A similar pattern of distribution was found in different pea cultivars, as well as in a supernodulating and a non-nodulating pea mutant. Growth in a nitrate concentration which inhibits nodulation did not affect lectin distribution on the surface of pea roots of this age. We tested whether or not the root zones where lectin was observed were susceptible to infection by Rhizobium leguminosarum. When low inoculum doses (consisting of less than 10⁶ bacteria·ml^-1) were placed next to lectin-positive epidermal cells and on newly formed root hairs, nodules on the primary roots were formed in 73% and 90% of the plants, respectively. Only a few plants showed primary root nodulation when the inoculum was placed on the root zone where lectin was scarce or absent. These results show that lectin is present at those sites on the pea root that are susceptible to infection by the bacterial symbiont.Abbreviations FITC fluorescein isothiocyanate - TRIC tetramethylrhodamine isothiocyanate     

The partial purification and characterization of a gibberellin C-20 hydroxylase from immature Pisum sativum L. seeds

A gibberellin (GA) C-20 hydroxylase that catalyses the conversion of GA₅₃ to GA₄₄ was purified from developing pea embryos by ammonium-sulfate precipitation, gel filtration and anion-exchange column chromatography. The purification was about 270-fold and 15% of the enzymic activity was recovered. The relative molecular mass was 44000 by Sephadex G-200 gel filtration. The apparent Michaelis constant was 0.7 M and the isoelectric point was 5.6–5.9. The enzymic activity was optimal at pH 7.0 2-Oxoglutarate and ascorbate were required for activity. Low concentrations of Fe²⁺ stimulated the reaction, but externally added Fe²⁺ was not essential, even in the most purified preparation. Catalase and bovine serum albumin also stimulated. Dithiothreitol preserved the activity during purification but was not needed during incubation. In fact, the simultaneous presence of dithiothreitol and Fe²⁺ in the incubation mixture was inhibitory to the purified enzyme. The cofactor requirements are typical for those of 2-oxoglutarate-dependent dioxygenases.When the incubation time was long enough, GA₅₃ was converted to both GA₄₄ and GA₁₉. The proportions of these two products remained constant throughout the purification, but this does not necessarily mean that their formations is catalysed by a single enzyme. Sodium dodecyl sulfatepolyacrylamide gel electrophoresis showed that the final preparation contained several proteins. Although the most prominent protein band was located within the range expected for the enzyme on the grounds of its molecular weight, this band did not represent the enzyme, since it separated from the GA C-20 hydroxylase activity on ultrathin-layer isoeletric focusing.Abbreviation BSA bovine serum albumin - DEAE diethylaminoethyl - DTT dithiothreitol - EDTA ethylenediamine-tetraacetic acid - GA_n gibberellin A_n - HPLC high-performance liquid chromatography - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol     

Quantitative changes in calmodulin and NAD kinase during early cell development in the root apex of Pisum sativum L.

Calmodulin and NAD kinase were extracted from serial developmental sections of the pea root apex. Highly purified samples of calmodulin were assayed by NAD-kinase activation, and whole-cell extracts were examined by two-dimensional polyacrylamide gel electrophoresis. Calmodulin was found to vary 17-fold in concentration over the apical 2 mm, being high in the region of the root cap and meristem, falling rapidly at the base of the meristem during early stages of rapid cell elongation. The rate of decline was different between stele and cortex. Except for a minor increase in concentration 2.5–5 mm from the apex, which coincides with the region of localised meristematic activity during initiation of lateral root primordia, the concentration of calmodulin remained at the lower level throughout the more basal sections of the apical 10 mm. In-vitro NAD-kinase activity was found to increase 17-fold per cell over the apical 30 mm, almost entirely as the result of an increase in calmodulin-dependent activity. Quantitative estimates of both calmodulin and NAD kinase were found to be highly dependent on extraction procedures.Abbreviation EGTA ethylene glycol-bis (-aminoethyl ether)-N,N,N,N-tetraacetic acid     

Determination for inflorescence development is a stable state,separable from determination for flower development in Pisum sativum L. buds

Terminal meristems of Pisum sativum (garden pea) transit from vegetative to inflorescence development, and begin producing floral axillary meristems. Determination for inflorescence development was assessed by culturing excised buds and meristems. The first node of floral initiation (NFI) for bud expiants developing in culture and for adventitious shoots forming on cultured meristems was compared with the NFI of intact control buds. When terminal buds having eight leaf primordia were excised from plants of different ages (i.e., number of unfolded leaves) and cultured on 6-benzylaminopurine and kinetin-supplemented medium, the NFI was a function of the age of the source plant. By age 3, all terminal buds were determined for inflorescence development. Determination occurred at least eight nodes before the first axillary flower was initiated. Thus, the axillary meristems contributing to the inflorescence had not formed at the time the bud was explanted. Similar results were obtained for cultured axillary buds. In addition, meristems excised without leaf primordia from axillary buds three nodes above the cotyledons of age-3 plants gave rise to adventitious buds with an NFI of 8.3 ±0.3 nodes. In contrast seed-derived plants had an NFI of 16.5 ±0.2. Thus cells within the meristem were determined for inflorescence development. These findings indicate that determination for inflorescence development in P. sativum is a stable developmental state, separable from determination for flower development, and occurring prior to initiation of the inflorescence at the level of meristems.     

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     

Biochemical and histochemical detection of endoproteolytic activities involved in ovary senescence or fruit development in Pisum sativum

The appearance of endoproteolytic activities related to the senescence of unpollinated pea ( Pisum sativum L. cv. Alaska) ovaries, or with fruit development induced by gibberellic acid (GA₃), was examined simultaneously by biochemical and histochemical techniques using gelatin as substrate. Biochemical detection was carried out by gelatin-containing sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Histochemical detection was performed using a gelatin film technique. No differences in endopeptidase activity were found in extracts from non-treated or developing ovaries during the two first days post-anthesis. After day 3 non-treated ovaries showed a marked increase in activity as well as two new bands with proteolytic activity, associated with the beginning of the senescence. At the same time a new activity was also located at the endocarp. In developing ovaries activity was only observed around vascular cells of the mesocarp at the end of the period studied (4–5 days post-anthesis). Activity detected in the ovules was essentially the same in both GA₃-treated and non-treated ovaries.