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.     

Turgor-sensitive sucrose and amino acid transport into developing seeds of Pisum sativum. Effect of a high sucrose or mannitol concentration in experiments with empty ovules

Sugar and amino acid transport into empty ovules of Pisum sativum L. cv. Marzia was examined. In fruits containing 4–6 developing seeds, the embryo was removed from four ovules. After this surgical treatment, each empty seed coat was filled with a solution (pH 5.5) containing a low (0, 50 or 200 m M ), medium (350, 400 or 500 m M ) or high (0.7 or 1 M ) concentration of sucrose and/or mannitol. In pulse-labelling experiments with sucrose and α-aminoisobutyric acid (AIB), transport of sucrose and AIB into an empty ovule filled with a solution containing a high sucrose concentration was the same as transport into an ovule filled with a mannitol solution of similar osmolarity, demonstrating that a high sucrose concentration in the seed coat apoplast affects phloem transport of sucrose and AIB into the seed coat only by the osmotic effect. The osmolarity of a given solution filling the seed coat cavity appeared to be important for phloem transport of sucrose and AIB into empty ovules.
In our experiments, 350 m M appeared to be the optimal concentration for sucrose and AIB transport into the cavity within an empty ovule, giving results comparable with transport into intact ovules. A lower osmolarity of the solution induced less transport. Very high sucrose or mannitol concentrations caused a strong inhibition of sucrose and AIB unloading from the seed coat, so that transport into the empty ovules was inhibited. A low (strongly negative) but not too low osmotic potential of the solution in the seed coat apoplast seems necessary to maintain a normal rate of phloem transport into developing seeds. Apparently, the "sink strength" of developing seeds is turgor-sensitive.     

Effect of the Osmotic Environment on the Balance between Uptake and Release of Sucrose and Amino Acids by the Seed Coat and Cotyledons of Developing Seeds of Pisum sativum

Excised seed-coat halves and cotyledons of developing seedsof Pisum sativum L. were incubated in a bathing medium (pH 5·5),in order to measure the release or uptake of sucrose and aminoacids. Net efflux of sucrose and amino acids was reduced bya 250 mol m ^–3 mannitol solution and a 400 mol m ^–3solution, in comparison with a 100 mol m^–3 control. Thiseffect could not be observed in the case of the amino acid analogue-aminoisobutyric acid (AIB). Net uptake of labelled sucroseor valine by cotyledons and seed coats was enhanced by a highosmolality of the bathing medium. The data on AIB and the datafrom uptake experiments support the view that net efflux ofassimilates is reduced by a high solute concentration in theapoplast (e.g. 400 mol m^–3 mannitol), via a stimulationof carrier-mediated sucrose and amino acid uptake into cotyledonaryand seed coat tissues. In experiments with attached empty ovulesof pea in a very early stage of development, sugar release fromthe seed coat was enhanced by a low osmolality of the apoplastsolution (e.g. 100 mol m^–3 mannitol, in comparison witha 400 mol m ^–3 control). This paradoxical effect may beobserved when the stimulatory effect on net assimilate effluxfrom seed coat tissues is exceeding the inhibitory effect onassimilate import into the seed coat. Key words: Seed development, turgor-sensitive transport, assimilate transport     

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.     

Effect of Potassium on Sucrose and Amino Acid Release from the Seed Coat of Developing Seeds of Pisum sativum

After removal of the embryo from developing seeds of Pisum sativum,the ‘empty’ ovules (seed coats without enclosedembryo) were filled with a solution (pH 5.5) containing mannitol(usually 400 mM) to which various salts were added. A solutioncontaining two isotopes ((a) [²H]-sucrose/[–¹⁴C]aminoisobutyricacid (AIB) or (b) [³H]valine/[₁₄C]asparagine mixture) was administeredto the plant via the petiole subtending the fruiting node, and[²H]solute and [¹⁴C]solute unloading from the seed coat wasmeasured, in pulse-labelling experiments of about 5 h. The presenceof 25 or 50 mM K⁺ in the ‘empty’ ovule enhancedthe release of sucrose from the seed coat particularly duringthe first hours of the experiment, but the stimulating effectof K⁺ on the release of labelled solutes derived from aminoacids was much smaller. The presence of 25 mM CaCl₂ did notaffect the release of sucrose or amino acids from the seed coat.The effect of K⁺ on sucrose and amino acid release is explainedas an inhibition of sucrose and amino acid resorption from theseed coat apoplast into seed coat cells, after unloading fromthe seed coat unloading sites. It is suggested that amino acidrelease is much less affected by K⁺ than sucrose release, becausefar less resorption of amino acids by seed coat parenchyma cellstakes place during amino acid transport into the seed coat cavity. Pisum sativum, pea, assimilate transport, assimilate unloading, seed-coat exudate, seed development, sucrose resorption, surgical treatment     

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.     

14C sucrose efflux from the perimedulla of growing potato tubers

Abstract An experimental system has been developed for studying efflux of ¹⁴C assimilates in growing potato tubers. Small wells are cut into the phloem-rich perimedulla and filled with trap solutions of varying composition which inhibit or promote assimilate efflux. One well on each tuber acts as the treatment while a second well acts as the control. Movement of ¹⁴C into wells occurred at comparable rates to that found in intact tissue, harvested from importing tubers in the form of microcores. Sucrose was the predominant translocated sugar in the stolon and was not hydrolysed in either the wells or the microcores following unloading. Efflux into wells containing agar traps was stimulated 40-fold relative to buffer controls by the addition of 20 mol m^?3 EGTA to the agar. This was interpreted as passive efflux to the apoplast due to increased membrane permeability in the pathway between the sieve elements and the collecting wells. The EGTA stimulation was reversed by addition of Ca²⁺. ¹⁴C efflux into buffered solutions was inhibited significantly by both DNP and PCMBS, suggesting the involvement of active and carrier-mediated transport components. However, it was not possible to determine whether these compounds acted at the site of unloading only, or on the short-distance transfer step between phloem and collecting wells. The rate of tracer efflux was not significantly different when 1 mol m^?3 and 300 mol m^?3 sucrose were applied to the wells, indicating insensitivity of solute movement to low apoplastic solute concentrations. However, raising the solute concentration to 800 mol m^?3 caused a severe inhibition of tracer efflux. These results were duplicated with mannitol as the osmoticum. It is suggested that plasmolysis prevented further efflux by disruption of a predominantly symplastic transport pathway between the phloem and collecting wells.     

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.     

Enhancement of seed germination in high salinity by engineering mannitol expression in Arabidopsis thaliana

The bacterial gene mtlD, which encodes mannitol 1-phosphate dehydrogenase (E. C. 1. 1. 1. 17), was transformed into Arabidopsis thaliana and expressed under control of the CaMV 35S promoter. MtlD-transformants accumulated mannitol, a sugar alcohol that is not normally found in Arabidopsis. Amounts of soluble carbohydrates, sucrose, glucose, fructose, myo-inositol and mannitol were determined in different tissues of wild-type and transgenic plants. We estimated that less than 1& of the carbon assimilated was converted into mannitol by the transgenic plants. The establishment of individual transformed lines (after self-crossing three times) resulted in high and low mannitol-producing lines which were stably maintained. The presence of mannitol did not alter plant appearance or growth habit. When MtlD-expressing seeds and control seeds (T3 generation) were imbibed with solutions containing NaCl (range 0 to 400 mol m^?3), transgenic seeds containing mannitol germinated in medium supplemented with up to 400 mol m^?3 NaCl, while control seeds ceased germination at 100 mol m^?3 NaCl. It is doubtful whether the ability to germinate in high salt was a result of an osmotic effect exerted by elevated levels of mannitol, considering that mannitol concentrations were in the mol m^?3 range in seeds. A specific effect of polyols, for example on the integrity of subcellular membranes or enzymes, cannot be excluded.     

Membrane Transport in Isolated Vesicles from Sugarbeet Taproot : II. Evidence for a Sucrose/H-Antiport

The process of sucrose transport was investigated in sealed putative tonoplast vesicles isolated from sugarbeet (Beta vulgaris L.) taproot. If the vesicles were allowed to develop a steady state pH gradient by the associated transport ATPase and 10 millimolar sucrose was added, a transient flux of protons out of the vesicles was observed. The presence of an ATPase produced pH gradient allowed [¹⁴C]sucrose transport into the vesicles to occur at a rate 10-fold higher than the rate observed in the absence of an imposed pH gradient. Labeled sucrose accumulated into the sealed vesicles could be released back to the external medium if the pH gradient was dissipated with carbonylcyanide-m-chlorophenyl hydrazone (CCCP). When the kinetics of ATP dependent [¹⁴C]sucrose uptake were examined, the kinetic profile followed the simple Michaelis-Menten relationship and a Michaelis constant of 12.1 millimolar was found. When a transient, inwardly directed sucrose gradient was imposed on the vesicles in the absence of charge compensating ions, a transient interior negative membrane potential was observed. This membrane potential could be prevented by the addition of CCCP prior to sucrose or dissipated by the addition of CCCP after sucrose was added. These results suggest that an electrogenic H⁺/sucrose antiport may be operating on the vesicle membrane.