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Uptake of [U-14C]-sucrose (40 mM) by fresh and aged peeled leaf discs of broad bean (Vicia faba L. cv. Aguadulce) has been studied. In fresh discs, uptake was nearly insensitive to external pH, whereas the pH response of absorption in discs aged for 12 h was bell-shaped, with an optimum between pH 5 and 6. At this pH, uptake was nearly twice that in fresh tissue. The passive (insensitive to carbonyl cyanide m-chlorophenylhydrazone and to cold treatment) uptake was the same in fresh or aged discs. The development of pH sensitivity of absorption did not appear when ageing was performed in the presence of 10-4 M cycloheximide or 5.7 × 10-5 M actinomycin D. Similarly, when the tissues were treated with 10-3 M spermidine for 2 h after excision and then aged for 10 h, the development of the pH-sensitive uptake system was inhibited. Ca2+ (10-2 M) supplied together with spermidine prevented the inhibiting effect of spermidine. The appearance of the pH-sensitive system was also markedly reduced if ageing took place in the presence of 10-3 M aminoethoxyvinylglycine. Autoradiographs from fresh discs and from discs aged with or without the inhibitors suggest that pH sensitivity developed more intensively in the parenchyma than in the veins. The results suggest some caution when using excised leaf discs for studies on sucrose uptake and phloem loading. Development of pH sensitivity of uptake may require the synthesis of both DNA-dependent RNA and protein and could be related to ethylene metabolism. [ABSTRACT FROM AUTHOR]

The in vivo significance of turgor‐dependent unloading was evaluated by examining assimilate transport to and within intact developing seeds of Phaseolus vulgaris (cv. Redland Pioneer) and Vicia faba (cv. Coles Prolific). The osmotic potentials of the seed apoplast were low. As a result, the osmotic gradients to the seed coat symplast were relatively small (i.e. 0.1 to 0.3 MPa). Sap concentrations of sucrose and potassium in the seed apoplast and coat symplast accounted for some 45 to 60% of the osmotic potentials of these compartments. Estimated turnover times of potassium and sucrose in the seed apoplast of < 1 h were some 5 to 13 times faster than the respective turnover times in the coat symplast pools. The small osmotic gradient between the seed apoplast and coat symplast combined with the relatively rapid turnover of solutes in the apoplast pool, confers the potential for a small change in assimilate uptake by the cotyledons to be rapidly translated into an amplified shift in the cell turgor of the seed coat. Observed adjustments in the osmotic potentials of solutions infused between the coat and cotyledons of intact seed were consistent with the in vivo operation of turgor‐dependent unloading of solutes from the coat. Homeostatic regulation of turgor‐dependent unloading was indicated by the maintenance of apoplast osmotic potentials of intact seeds when assimilate balance was manipulated by partial defoliation or elevating pod temperature. In contrast, osmotic potentials of the coat symplast adjusted upward to new steady values over a 2 to 4 h period. The resultant downward shift in coat cell turgor could serve to integrate phloem import into the seed coat with the new rates of efflux to the seed apoplast. Circumstantial evidence for this linkage was suggested by the approximate coincidence of the turgor changes with those in stem levels of 32P used to monitor phloem transport. The results obtained provide qualified support for the in vivo operation of a turgor homeostat mechanism. It is proposed that the homeostat functions to integrate assimilate demand by the cotyledons with efflux from and phloem import into the coats of developing legume seed. The in vivo significance of turgor‐dependent unloading was evaluated by examining assimilate transport to and within intact developing seeds of Phaseolus vulgaris (cv. Redland Pioneer) and Vicia faba (cv. Coles Prolific). The osmotic potentials of the seed apoplast were low. As a result, the osmotic gradients to the seed coat symplast were relatively small (i.e. 0.1 to 0.3 MPa). Sap concentrations of sucrose and potassium in the seed apoplast and coat symplast accounted for some 45 to 60% of the osmotic potentials of these compartments. Estimated turnover times of potassium and sucrose in the seed apoplast of < 1 h were some 5 to 13 times faster than the respective turnover times in the coat symplast pools. The small osmotic gradient between the seed apoplast and coat symplast combined with the relatively rapid turnover of solutes in the apoplast pool, confers the potential for a small change in assimilate uptake by the cotyledons to be rapidly translated into an amplified shift in the cell turgor of the seed coat. Observed adjustments in the osmotic potentials of solutions infused between the coat and cotyledons of intact seed were consistent with the in vivo operation of turgor‐dependent unloading of solutes from the coat. Homeostatic regulation of turgor‐dependent unloading was indicated by the maintenance of apoplast osmotic potentials of intact seeds when assimilate balance was manipulated by partial defoliation or elevating pod temperature. In contrast, osmotic potentials of the coat symplast adjusted upward to new steady values over a 2 to 4 h period. The resultant downward shift in coat cell turgor could serve to integrate phloem import into the seed coat with the new rates of efflux to the seed apoplast. Circumstantial evidence for this linkage was suggested by the approximate coincidence of the turgor changes with those in stem levels of 32P used to monitor phloem transport. The results obtained provide qualified support for the in vivo operation of a turgor homeostat mechanism. It is proposed that the homeostat functions to integrate assimilate demand by the cotyledons with efflux from and phloem import into the coats of developing legume seed. [ABSTRACT FROM AUTHOR]

Key physiological characteristics of turgor-dependent efflux of photosynthates were examined using excised coats and cotyledons of developing Phaseolus vulgaris (cv. Redland Poineer) and Vicia faba (cv. Coles Prolific) seed during the linear phase of seed fill. Exposure to solutions of high osmotic potential inhibited net uptake of [14C]sucrose by cotyledons at developmental stages less than 60% of their final dry weight. The effect could not he fully reversed by transferring cotyledons to solutions set at lower osmotic potentials. The inhibition became apparent at osmotic potentials that were higher than those that caused stimulation of efflux from seed coats. Net [14C]sucrose uptake by cotyledons at more advanced stages of development was unaffected by external osmotic potential. Specified tissue layers were removed from seed coats by pretreatment with pectinase. Efflux studies with the pectinase-modified coats of Phaseolus and Vicia seed demonstrated that the cellular site of turgor-dependent efflux was the ground parenchyma and thin-wall parenchyma transfer cells, respectively. Coats subjected to long-term (hours) incubations, under hypo-osmotic conditions, exhibited the capacity for turgor regulation. This was mediated by turgor- dependent efflux of solutes. The solutes exchanged were of nutritional significance to the developing embryo. The relationship between efflux and coat turgor was characterised by a turgor-independent phase at low turgors. Once turgor exceeded a minimal value (set point), efflux increased in proportion to the magnitude of the turgor deviation (error signal) from the set point. For coats of sink-limited seed of Vicia and Phaseolus, efflux exhibited apparent saturation at turgors above 0.25 and 0.5 MPa respectively. The putative turgor set point and slope of the turgor-dependent component of efflux varied with seed development, the prevailing source/sink ratio and genetic differences in seed growth rate. The nature of these kinetic variations was compatible with the competitive ability of the seed. A turgor homeostat model is proposed that incorporates the observed functional attributes of turgor-dependent efflux. Operationally, the model provides a mechanistic basis for the integration of assimilate demand by the cotyledons with assimilate import into and unloading from the seed coat. [ABSTRACT FROM AUTHOR]

Osmotic regulation of assimilate efflux from excised coats of developing Vicia faba (cv. Coles Prolific) seed was examined by exposing these to bathing solutions (adjusted to –0. 02 to –0. 75 MPa with sorbitol) introduced into the cavity vacated by the embryo. 14C photosynthate efflux was found to be independent of solution osmotic potentials below –0. 63 MPa. At higher osmotic potentials, efflux was stimulated and exhibited a biphasic response to osmotic potential with apparent saturation being reached at –0. 37 MPa. Efflux could be repeatedly stimulated and slowed by exposing seed coats to solutions of high and low osmotic potentials, respectively. Manipulation of components of tissue water potential, with slowly‐ and rapidly‐permeating osmotica, demonstrated that turgor functioned as the signal regulating 14C photosynthate efflux. Com‐partmental analysis of 14C photosynthate preloaded seed coats was consistent with exchange from 4 kinetically‐distinct compartments. The kinetics of turgor‐dependent efflux exhibited characteristics consistent with the transport mechanism residing in the plasma membranes of the unloading cells. These characteristics included the rapidity (<2 min) of the efflux response to turgor increases, similar rate constants for efflux from the putative turgor‐sensitive and cytoplasmic compartments and the apparent small pool size from which turgor‐dependent efflux could repeatedly occur. In contrast, influx of [14C] sucrose across the plasma and tonoplast membranes was found to be insensitive to turgor. The plasma membrane [14C] sucrose influx was unaffected by p‐chloromercuribenzenesulfonic acid and erythrosin B and exhibited a linear dependence on the external sucrose concentration. This behaviour suggested that influx across the plasma membrane occurs by passive diffusion. Preloading excised seed coats with a range of solutes demonstrated that turgor‐dependent efflux exhibited partial solute selectivity. Based on these findings, it is proposed that turgor controls assimilate exchange from the seed coat by regulating an efflux mechanism located in the plasma membranes of the unloading cells. [ABSTRACT FROM AUTHOR]

The possibility that the herbicide glyphosate (N-phophoaomethylglycine) may be taken up in plant cells via a phosphate transporter of the plasma membrane was investigated using protoplasts of broad bean leaves (Viccia faba L.). Phosphonoformic acid, a powerful inhibitor of phosphate transport in animal cells, was first demonstrated to be a competitive inhibitor of phosphate uptake in broad bean protoplasis. Glyphosate was able to inhibit phosphate uptake into the protoplasts, and to protect partially the phosphate transporter from inhibition by phosphonoformic acid. Concentration dependence studies showed that glyphosate uptake exhibited a saturable phase at low glyphosate concentrations (0.5 to 3 μM), superimposed by a linear uptake at higher concentrations (up to 100 μM). inhibition of glyphosate uptake by para-chloromercuribenzene sulphonic acid, sodium azide and carbonyl-cyanide-in- chlorophenylhydrazone was much stronger at 1 than at 100 μM gIyphosate. Kinetics indicated that the saturable component of glyphosate transport was competitively inhibited by either phosphate or phosphonoformic acid. It is concluded that glyphosate can be absorbed via a phosphate transporter of the plasma membrane. [ABSTRACT FROM AUTHOR]

The significance of the osmotic potential of the seed apoplast sap as a regulator of assimilate transfer to and within coats of developing seed of Vicia faba (cv. Coles Prolific) was assessed using attached empty seed coats and intact developing seed. Following surgical removal of the embryos, through windows cut in the pod walls and underlying seed coats, the resulting attached "empty" seed coats were filled with solutions of known osmotic potentials (-0.02 venus -0.75 MPa). Sucrose efflux from the coats was elevated at the higher osmotic potential (low osmotic concentration) for the first 190 mill of exchange. Thereafter, this efflux was depressed relative to efflux from coats exposed to the low osmotic potential (high osmotic concentration) solution. This subsequent reversal in efflux was attributable to an enhanced diminution of the coat sucrose pools at the high external osmotic potential. Indeed, when expressed as a proportion of the current sucrose pool size, relative efflux remained elevated for coats exposed to the high osmotic potential solution. Measurement of potassium and sucrose fluxes to and from their respective pools in the coat tissues demonstrated that the principal fluxes, sensitive to variation in the external osmotic potential, were phloem import into and efflux from the "empty" coats. Phloem import, consistent with a pressure-driven phloem transport mechanism, responded inversely with changes in the external osmotic potential. In contrast, sucrose and potassium efflux from the coats exhibited a positive dependence on the osmotic potential. Growth rates of whole seed were approximately doubled by enclosing selected pods in water jackets held at temperatures of 25°C compared to 15°C. The osmotic potential of sap collected from the seed apoplast remained constant and independent of the temperature-induced changes in seed growth rates and hence phloem import. Based on these findings, it is proposed that control of phloem import by changes in the external osmotic potential observed with "empty" seed coats has no significance as a regulator of assimilate import by intact seed. Rather, maintenance of the seed apoplast osmotic potential, independent of seed growth rate, suggests that the observed osmotic regulation of efflux from the coats may play a key role in integrating assimilate demand by the embryo with phloem import. [ABSTRACT FROM AUTHOR]

The short‐term effects of p‐chloromercuribenzenesulphonic acid (PCMBS) on the transmembrane potential difference (PD) of broad bean (Vicia faba L. cv. Aguadulce) cotyledon cells and on sugar beet (Beta vulgaris L. cv. Klein E.) leaf cells were studied by the electrophysiological method. These effects were compared with that of the permeant thiol reagents N‐ethylmaleimide and HgCl2. N‐Ethylmaleimide and HgCl2 markedly and rapidly depolarised the PD of all the material studied, while PCMBS caused either a slight depolarisation (cotyledon cells) or no depolarisation (leaf cells) during the first 30 min of treatment. In cotyledons, PCMBS markedly inhibited sucrose uptake (89%) and the sucrose‐induced depolarisation associated with the proton‐sucrose symport (67%), while it decreased the proton‐motive force only marginally (7%). It is concluded that during short treatments (30 min or less). PCMBS inhibits sucrose uptake directly by blocking the sucrose carrier, and not the proton pump. For longer treatments, indirect effects cannot be excluded. [ABSTRACT FROM AUTHOR]

Sugar export by broadbean (Vicia faba L. cv. Aguadulce) was blocked by a cold jacket (1 cm‐width, 1°C) applied on the petiole of a mature leaf or by heat‐girdling the petiole. A time course study was made on the effects of these treatments on apoplastic and intracellular soluble sugars of the leaf in relation to phloem loading and photosynthesis. Blocking of export by heat‐girdling induced an inhibition of phloem loading within 10 min, an accumulation of starch within 30 min and a rise in apoplastic sucrose within 60 min. By contrast, apoplastic hexoses and photosynthesis were not affected by this treatment within 8 h and intracellular sugars were not affected within 2 h. The cold jacket also increased the sucrose content of the apoplast. The increase in apoplastic sucrose induced by the cold barrier is reversed upon rewarming and less marked when the sink/source ratio is increased by defoliating all but the leaves studied. The results are discussed in terms of sink/source relationships. They show that the increase in apoplastic sucrose resulting from inhibition of loading is not part of the events leading from blocking of transport to change in carbon partitioning. [ABSTRACT FROM AUTHOR]