Effect of KCN and p-chloromercuribenzenesulfonic acid on the release of sucrose and 2-amino(1-14C)isobutyric acid by the seed coat of Pisum sativum

The process of sugar and amino acid release by the seed coat of Pisum sativum L. cv. Marzia was studied. Prior to measuring the release of solutes by the seed coat of developing ovules, the embryo was removed from each ovule studied. After this surgical treatment, each "empty" seed coat was filled with the appropriate solution (pH 5.5) with or without inhibitor. Both KCN and p-chloromercuribenzenesulfonic acid (PCMBS) strongly inhibited the release of sucrose and p -aminoisobutyric acid (AIB) by the seed-coat. These data support the view that phloem unloading is an energy-dependent process sensitive to the sulfhydryl group modifier PCMBS. In pulse-labelling experiments, addition of high concentrations of unlabelled sucrose (200 m M ) and AIB (25 m M ) to the solution filling the seed coat cavity did not diminish the release of labelled solutes by the unloading sites of the seed coat. This observation presents evidence against the view that phloem unloading into a strong sink is related to low sugar concentrations in the apoplast.     

Effect of a pretreatment of seed coats with a low-osmolality solution on subsequent [14C] sucrose transport into attached empty ovules of pea

This paper discusses the question as to whether or not the seed coat tissues can‘adapt’to a treatment with a solution containing a low osmoticum concentration, representing an environment which is sub-optimal for assimilate transport into attached surgically modified ovules. Before the start of a pulse-labelling procedure, in experiments on [¹⁴C] sucrose transport into fruits of pea (Pisum sativum) with four empty ovules, two empty ovules were filled with a low-osmolality solution (a 200 mol m^?3 mannitol medium or a solution without mannitol) and the other two ovules were filled with a 400 mol m^?3 mannitol medium. Pretreatment with a low-osmolality medium, during a period of 2–3 h, enhanced subsequent transport of [¹⁴C] sucrose into empty ovules filled with a low-osmolality medium, in comparison with [¹⁴C] sucrose transport into empty ovules filled with a 400mol m^?3 mannitol medium during the pretreatment period. This partial recovery of sink strength of attached empty ovules can be explained as the result of a stimulation of solute efflux from seed coat cells at high cell turgor.     

Effect of the osmotic environment of the seed coat on sucrose and amino acid transport into developing seeds of Lunaria annua, Acer pseudoplatanus and Glycine max

In contrast to the data reported for developing seeds of pea and broad bean, assimilate transport into empty kernels of maize is not reduced by a low osmolality of the substitute medium. Therefore, additional data were collected from representatives of other taxonomical groups. In pulse-labelling experiments with Lunaria annua L. and Acer pseudoplatanus L., sucrose and amino acid transport into empty ovules was strongly reduced by a low osmolality of the medium filling an empty ovule, compared to that seen with high osmolality. In experiments of 8 h without radioiso-topes, a very low osmolality of the medium (about 0 m M ) reduced the rate of sugar and amino acid release from attached seed coats of soybean [ Glycine max (L.) Merr. cv. Fiskeby V], in comparison with a 300 m M mannitol medium. It can be concluded that in all dicotyledonous plants studied (five species), a low osmotic potential of the seed apoplast is one of the most important factors controlling the rate of assimilate transport into developing seeds. At this moment, the data reported for maize have an isolated position.     

Turgor-sensitive transport in developing seeds of legumes: the role of the stage of development and the use of excised vs. attached seed coats

Abstract After removal of the embryo from developing seeds of Vicia faba L. and Pisum sativum L., the ‘empty’ ovules were filled with a substitute medium (pH 5.5) and the effect of the osmolality of this solution on assimilate transport was exandned. In pulse-labelling experiments with a mixture of [³H]sucrose and [¹⁴C]α-andnoisobutyric acid (AIB), a solute concentration of 400 mol m^?3 (100 mol m^3? sucrose + 300 mol m^?3 mannitol) was too low to maintain sugar and andno acid transport into empty ovules of V. faba in a very early stage of development (embryo dry weight < 100 mg) on the same level as transport into intact ovules within the same fruit. A 550-mol m^?3 solution could maintain the normal rate of transport. In experiments with seeds in a more advanced stage of development (embryo dry weight > 250 mg), transport of labelled sucrose and AIB into empty ovules filled with a 400-mol m^?3 solution was practically equal to transport into intact ovules within the same fruit. Experiments without isotopes, on sugar and andno acid release from the seed coat, confirmed the important role of the osmotic environment. A very low osmolality of the solution (e.g. 50 mol m^?3 mannitol) enhanced net efflux of assimilates from excised seed coats and cotyledons, by inhibiting resorption from the apoplast.     

Structure of the developing pea seed coat and the post-phloem transport pathway of nutrients

An important function of the seed coat is to deliver nutrients to the embryo. To relate this function to anatomical characteristics, the developing seed coat of pea (Pisum sativum L.) was examined by light- and cryo-scanning electron microscopy (cryo-SEM) from the late pre-storage phase until the end of seed filling. During this time the apparently undifferentiated seed coat tissues evolve into the epidermal macrosclereids, the hypodermal hourglass cells, chlorenchyma, ground parenchyma and branched parenchyma. Using the fluorescent symplast tracer 8-hydroxypyrene-1,3,6-trisulfonic acid, it could be demonstrated that solutes imported by the phloem move into the chlorenchyma and ground parenchyma, but not into the branched parenchyma. From a comparison with literature data of common bean (Phaseolus vulgaris L.) and broad bean (Vicia faba L.), it is concluded that in the three species different parenchyma layers, but not the branched parenchyma, may be involved in the post-phloem symplasmic transport of nutrients in the seed coat. In pea, the branched parenchyma dies during the storage phase, and its cell wall remnants then form the boundary layer between the living seed coat parenchyma cells and the cotyledons. Using cryo-SEM, clear images were obtained of this boundary layer which showed that many intracellular spaces in the seed coat parenchyma are filled with an aqueous solution. This is suggested to facilitate the diffusion of nutrients from the site of unloading towards the cotyledons.     

Gibberellin-enhanced sugar accumulation in growing subhooks of etiolated Pisum sativum seedlings. Effects of gibberellic acid, indoleacetic acid and cycloheximide on invertase activity, sugar accumulation and growth

The possible involvement of invertase in the action of gibberellic acid (GA) on stimulating sugar accumulation in growing subhooks of Alaska pea ( Pisum sativum L. cv. Alaska) was studied. GA and indoleacetic acid (IAA) stimulated elongation growth to a similar extent. GA, in contrast to IAA, increased the amount of soluble sugars in the subhook. GA substantially increased invertase activity whereas IAA did not. These results suggest that the mode of action of GA and IAA differs, although both stimulate pea subhook growth.
Cycloheximide (CH) inhibited the effect of GA on invertase activity, accumulation of soluble sugars, and elongation growth. Good correlations were found between invertase activity, the amount of soluble sugars and growth. The results suggest that GA-induced enhancement of sugar accumulation in the subhook cells is dependent on increased invertase activity. The sugar accumulated in the subhook may be involved in growth promotion by GA.     

Effect of gibberellic acid on epicotyl growth and carbohydrate distribution in derooted Pisum sativum cuttings with or without cotyledons

The effect of gibberellic acid (GA) on subhook growth in derooted cuttings of pea ( Pisum sativum L. cv. Alaska) grown in the dark was studied in relation to the distribution of sugar-related compounds in the epicotyl and cotyledons. GA stimulated subhook growth of cuttings with or without cotyledons. In cuttings with cotyledons, the net inflow of sugar-related compounds (soluble sugars, starch, cell wall polysaccharides and sugars consumed by respiration) to the epicoiyl balanced with the net outflow from the cotyledons. GA stimulated the net inflow of sugar-related compounds to the epicotyl and the net outflow from cotyledons. Among these compounds, GA substantially increased the amount of soluble sugars, starch and cell wall polysaccharides in the subhook. In cuttings without cotyledons, on the other hand, the net inflow of sugar-related compounds to the subhook almost balanced with the net outflow from the epicotyl below the subhook. GA stimulated the net inflow of sugar-related compounds to the subhook and the net outflow from the epicotyl below the subhook. Among these compounds, GA substantially increased the amount of soluble sugars and cell wall polysaccharides in the subhook. These results suggest that GA stimulates an increase in the net inflow of sugar-related compounds to the subhook, thereby preventing an increase in osmotic potential and stimulating cell wall polysaccharide synthesis, when pea subhook growth is stimulated.     

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     

Characterization of the uptake of sucrose and glucose by isolated seed coat halves of developing pea seeds. Evidence that a sugar facilitator with diffusional kinetics is involved in seed coat unloading

Uptake of ¹⁴C-labelled sucrose and glucose by isolated seed coat halves of pea (Pisum sativum L. cv. Marzia) seeds was measured in the concentration range <0.1 μM to 100 mM. The initial influx of sucrose was strictly proportional to the external concentration, with a coefficient of proportionality (k) of 6.2 μmol·(g FW)^?1·min^?1·M^?1. Sucrose influx was not affected by 10 μM carbonylcyanide m-chlorophenylhydrazone (CCCP), but it was inhibited by 40% in the presence of 2.5 mM p-chloromercuribenzenesulfonic acid (PCMBS). Influx with diffusional kinetics was also observed for glucose (k = 4.8 μmol·(g FW)^?1·min ^?1·M ^?1) and mannitol (k = 5.1 μmol·(g FW)^?1·min^?1·M^?1). For glucose an additional saturable system was found (K_m = 0.26 mM, V _max = 4.2 nmol·(g FW)^?1·min^?1), which appeared to be completely inhibited by CCCP and partly by PCMBS. In contrast to the diffusional pathway, uptake by this saturable system was slightly pH-dependent, with an optimum at pH 5.5. The influx of sucrose appears to be by the same pathway as the efflux of endogenous sucrose, which was inhibited by 36% in the presence of 2.5 mM PCMBS (De Jong A, Wolswinkel P, 1995, Physiol Plant 94: 78–86). It is argued that passive transport may be the only mechanism for sucrose transport through the plasma membrane of seed coat parenchyma cells. The estimated permeability coefficient of the plasma membrane for sucrose (P = 3.5·10^?7 cm·s^?1) is more than 1 × 10⁶-fold higher than that reported for artificial lipid membranes. This relatively high permeability is hypothesized to result from pore-forming proteins that allow the diffusion of sucrose. Furthermore, it is shown that a sucrose gradient across the plasma membrane of the seed coat parenchyma of only 22 mM will suffice to result in the net efflux of sucrose which is required to feed the embryo.     

The role of gibberellins A1 and A3 in fruit growth of Pisum sativum L. and the identification of gibberellins A4 and A7 in young seeds

Gibberellins A₁ and A₃ are the major physiologically active gibberellins (GAs) present in young fruit of pea (Pisum sativum L.). The relative importance of these GAs in controlling fruit growth and their biosynthetic origins were investigated in cv. Alaska. In addition, the non-13-hydroxylated active GAs, GA₄ and GA₇, were identified for the first time in young seeds harvested 4 d after anthesis, although they are minor components and are not expected to play major physiological roles. The GA₁ content is maximal in seeds and pods at 6 d after anthesis, the time of highest growth-rate of the pod (Garcia-Martinez et al. 1991, Planta 184: 53–60), whereas gibberellic acid (GA₃), which is present at high levels in seeds 4–8 d after anthesis, has very low abundance in pods. Gibberellins A₁₉, A₂₀ and A₂₉ are most concentrated in seeds at, or shortly after, anthesis and their abundance declines rapidly with development, concomitant with the sharp increase in GA₁ and GA₃ content. Application of GA₁ or GA₃ to the leaf subtending an emasculated flower stimulated parthenocarpic fruit development. Measurement of the GA content of the pods at 4 d after anthesis indicated that only 0.002–0.5% of the applied GA was transported to the fruit, depending on dose. There was a linear relationship between GA₁ content and pod weight up to about 2 ng · (g FW)⁻¹, whereas no such correlation existed for GA₃ content. The concentration of endogenous GA₁ in pods from pollinated ovaries is just sufficient to give the maximum growth response. It is concluded that GA₁, but not GA₃, controls pod growth in pea; GA₃ may be involved in early seed development. The distribution of GAs within the seeds at 4 d post anthesis was also investigated. Most of the GA₁, GA₈, GA₁₉, GA₂₀ and GA₂₉ was present in the testa, whereas GA₃ was distributed equally between testa and endosperm and GA₄ was localised mainly in the endosperm. Of the GAs analysed, only GA₃ and GA₂₀ were detected in the embryo. Metabolism experiments with intact tissues and cell-free fractions indicated compartmentation of GA biosynthesis within the seed. Using ¹⁴C-labelled GA₁₂, GA₉, 2,3-didehydroGA₉ and GA₂₀ as substrates, the testa was shown to contain 13-hydroxylase and 20-oxidase activities, the endosperm, 3β-hydroxylase and 20-oxidase activities. Both tissues also produced 16,17-dihydrodiols. However, GA₁ and GA₃ were not obtained as products and it is unlikely that they are formed via the early 13-hydroxylation pathway. [¹⁴C]gibberellin A₁₂, applied to the inside surface of pods in situ, was metabolised to GA₁₉, GA₂₀, GA₂₉, GA₂₉-catabolite, GA₈₁ and GA₉₇, but GA₁ was not detected. Gibberellin A₂₀ was metabolised by this tissue to GA₂₉ and GA₂₉-catabolite. Received: 23 July 1996 / Accepted: 2 September 1996