Turgor-dependent unloading of assimilates from coats of developing legume seed. Assessment of the significance of the phenomenon in the whole plant

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.     

Osmotic regulation of assimilate unloading from seed coats of Vicia faba. Role of turgor and identification of turgor-dependent fluxes

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.     

Osmotic regulation of assimilate unloading from seed coats of Vicia faba. Assimilate partitioning to and within attached seed coats.

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 versus –0. 75 MPa). Sucrose efflux from the coats was elevated at the higher osmotic potential (high osmotic concentration) for the first 190 min 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 variative 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.     

Turgor-dependent efflux of assimilates from coats of developing seed of Phaseolus vulgaris L.: water relations of the cells involved in efflux

The turgor-homeostat model of assimilate efflux from coats of developing seed of Phaseolus vulgaris L. was further characterised. The turgor pressure (P), the volumetric elastic modulus () and hydraulic conductivity (L_p) of the seed coat cells responsible for assimilate efflux and cotyledon storage parenchyma cells were determined with a pressure probe. In addition, turgor of the seed coat and cotyledons was estimated by measuring the osmolalities of symplastic and apoplastic fluids extracted by centrifugation. Osmolality of symplastic and apoplastic saps collected from the seed coat declined significantly over the period of seed development from a cotyledon water content of 80% to 50%. However, the difference in osmolalities of the apoplastic and symplastic saps remained relatively constant. For cotyledons, osmolality of the apoplastic sap exhibited a significant decline during seed development, while the osmolality of symplastic sap did not change significantly. Hence cotyledon P increased as the water content dropped from 80% to 50%. For both detached and attached empty seed coats, a small decrease (ca. 40mOsmol·kg^–1) in the osmolality of the bathing solution, led to a rapid increase in P of cells involved in assimilate efflux (efflux cells) by about 0.07 MPa. Thereafter, cell P exhibited a rapid decline to the original value within some 20–30 min. When P of the efflux cells was reduced by increasing the osmolality of the bathing solution, P exhibited a comparable rate of recovery for attached empty seed coats but there was no P recovery to its original value in the case of detached seed coats. In contrast, the cotyledon storage parenchyma cells did not exhibit P regulation when the osmolality of the bathing solution was changed. The observations that the efflux cells of P. vulgaris seed coats can rapidly adjust their P homeostatically in response to small changes in apoplastic osmolality are consistent with the operation of a turgor-homeostat mechanism. The volumetric elastic modulus () of the seed coat efflux cells exhibited a mean value of 7.3±0.8 MPa at P=0.15 MPa and was found to be linearly dependent on cell P. The e of the cotyledon storage parenchyma cells was estimated to be 6.1±1.0 MPa at P=0.41 MPa. Hydraulic conductivity (L_p) of the seed coat cells and the cotyledon cells was (8.2±1.5) × 10^–8m·s^–1·MPa^–1and (12.8±1.0) × 10^–8 m·s^–1·MPa^–1, respectively. The relatively high , i.e., low elasticity, for the seed coat cell walls would ensure that small changes in water potential of the seed apoplast will be reflected in large changes in cell P. The high L_p values for both the seed coat and the cotyledon cells is consistent with the rapid changes in P in response to changes in water potential of the seed apoplast.Abbreviations LYCH Lucifer Yellow CH - volumetric elastic modulus - L_p hydraulic conductivity - P turgor pressure - osmotic pressure - t_1/2 half-time for water exchange The investigation was supported by funds from the Australian Research Council. We are grateful to Louise Hetherington for competent technical assistance and to Kevin Stokes for raising the plant material.     

Quantitative Analysis of Photosynthate Unloading in Developing Seeds of Phaseolus vulgaris L. : II. Pathway and Turgor Sensitivity

Phloem import and unloading in perfused bean (Phaseolus vulgaris L.) seed coats were investigated using steady-state labeling. Though photosynthate import and unloading were significantly reduced by perfusion, measurements of photosynthate fluxes in perfused seed coats proved useful for the study of unloading mechanisms in vivo. Phloem import was stimulated by lowered seed coat cell turgor, as demonstrated by an increase in tracer and sucrose import to seed coats perfused with high concentrations of an osmoticum. The partitioning of photosynthates between retention in the seed coat and release to the perfusion solution also was turgor sensitive; increases in seed coat cell turgor stimulated photosynthate release to the apoplast at the expense of photosynthate retention within the seed coat. There was no evidence of a turgor-sensitive sucrose uptake mechanism in perfused seed coats. Thus, the turgor sensitivity of photosynthate partitioning within perfused seed coats was consistent with a turgor-sensitive efflux control mechanism. Measurements of tracer equilibration and sugar partitioning in perfused seed coats provided strong evidence for symplastic phloem unloading in seed coats.     

Changes in Photosynthate Unloading from Perfused Seed Coats of Phaseolus vulgaris L. Induced by Osmoticum and Ethylenediaminetetraacetate (EDTA)

Photosynthate unloading in Phaseolus vulgaris L. seed coatswas studied by treating perfused seed coats with differing concentrationsof an osmoticum and ethylenediaminetetraacetate (EDTA). Largechanges in osmoticum concentration typically produced rapidchanges in efflux of unlabelled sugar and steady-state-labelled¹⁴C-photosynthate. Osmoticum-induced changes in photosynthateefflux were caused by phloem import stimulation at low cellturgor and net efflux stimulation by high cell turgor. Eventhough rapid changes in sugar and tracer efflux were often inducedby osmoticum treatments, the specific activity of sugar releasedfrom seed coats was not greatly affected by these treatmentsand was similar to the specific activity of sugar remainingin the seed coat after perfusion. Thus, tracer was transportedfrom the phloem throughout the seed coat sugar pool before itwas released to the apoplast. This result is most consistentwith symplastic phloem unloading throughout perfused seed coats,because apoplastic transport between cells within the seed coatwas blocked by perfusion. Photosynthate efflux was stimulatedby simultaneous treatment of seed coats with EDTA and differentconcentrations of an osmoticum; loss of photosynthate from seedcoats did not appear to be tissue-specific. Key words: Phaseolus vulgaris, seed coat, photosynthate unloading, turgor, EDTA     

Cellular pathway of photosynthate transport in coats of developing seed of Vicia faba L. and Phaseolus vulgaris L. II. Principal cellular site(s) of efflux

The cells responsible for the photosynthate efflux from coatsof developing seed of Vicia faba L. and Phaseolus vulgaris L.were elucidated using known properties of the efflux mechanism.Sensitivity of sucrose efflux to NEM and high potassium concentrationswas retained by seed-coat halves of Phaseolus following pectinaseremoval of the branch parenchyma cell layer. In contrast, removalof the thin-walled parenchyma transfer cell layer from Viciaseed-coat halves abolished this sensitivity. The membrane-impermeantthiol-binding fluorochrome, qBBr, selectively stained the surfaceof the thin-walled parenchyma transfer cells. This phenomenonwas inhibited by the slowly permeable sul-phydryl agent, PCMBS,indicating that the plasma membranes of these cells are enrichedin sulphydryl groups characteristic of membrance porter proteins.On the basis that carrier-mediated sucrose efflux from seedcoats appears to be proton coupled, the putative plasma membraneH+-ATPase was used as a marker for the cells responsible forcarrier-mediated photosynthate efflux. When seed-coat halveswere exposed briefly at pH 8.5 to the weak acid fluorochrome,SRG, the ground parenchyma and thin-walled parenchyma transfercell layers selectively accumulated the dye. The apparent lowpH environment in the walls of these cells that renders SRGmembrane permeant appeared to be maintained by a VAN-sensitiveproton pump. The findings with SRG were corroborated by thecyto-chemical localization of plasma membrane ATPase activityto the ground parenchyma and thin-walled parenchyma transfercells using precipitation of cerium phosphate. Together, ourobservations provide qualified support for the conclusion thatcarrier-mediated photosynthate efflux from coats of Phaseolusand Vicia seed is primarily restricted to the ground parenchymaand thin-walled parenchyma transfer cell layers, respectively. Key words: Ground parenchyma, Phaseolus vulgaris L., photosynthate efflux, seed coat, transfer cell, Vicia faba L.     

The cellular pathway of sucrose transport in developing cotyledons ofVicia faba L. andPhaseolus vulgaris L.: a physiological assessment

The cellular pathway of sugar uptake in developing cotyledons of Vicia faba L. and Phaseolus vulgaris L. seed was evaluated using a physiological approach. The cotyledon interface with the seed coat is characterised by a specialised dermal cell complex. In the case of Vicia faba cotyledons, the epidermal component of the dermal cell complex is composed of transfer cells. Sucrose is the major sugar presented to the outer surface of both cotyledons and it is taken up from the apoplasm unaltered. Estimated sucrose concentrations within the apparent free space of Vicia and Phaseolus cotyledons were 105 and 113 mM respectively. Rates of in-vitro uptake of [¹⁴C]sucrose by cotyledon segments or by whole cotyledons following physical removal or porter inactivation of the outer cells demonstrated that, for both Vicia and Phaseolus cotyledons, the dermal cell complexes are the most intense sites of sucrose uptake. Accumulation of [¹⁴C]sucrose in the storage parenchyma of whole cotyledons was directly affected by experimental manipulation of uptake by the outer cell layers and plasmolytic disruption of the interconnecting plasmodesmata. These findings indicated that sucrose accumulated by the dermal cell complexes is transported symplasmically to the storage parenchyma. Overall, it is concluded that the dermal cell complexes of the developing legume embryo, irrespective of the presence or absence of wall ingrowths, are the major sites for the uptake of sucrose released from the maternal tissues to the seed apoplasm. Thereafter, the accumulated sucrose is transported radially inward through the symplast to the storage parenchyma.Abbreviations AFS apparent free space - CF 5-(6)-carboxyfluorescein - CFDA 5-(6)-carboxyfluorescein diacetate - Mes 2-(N-morpholino)ethanesulfonic acid - PCMBS p-chloromercuribenzenesulfonic acid - SRG sulphorhodamine G The investigation was supported by funds from the Research Management Committee, The University of Newcastle and the Australian Research Council. One of us, R. McDonald, gratefully acknowledges the support of an Australian Postgraduate Research Award. We are grateful to Stella Savoury for preparing the photomicrographs.     

Aqueous pathways dominate permeation of solutes across Pisum sativum seed coats and mediate solute transport via diffusion and bulk flow of water

The permeability of seed coats to solutes either of biological or anthropogenic origin plays a major role in germination, seedling growth and seed treatment by pesticides. An experimental set‐up was designed for investigating the mechanisms of seed coat permeation, which allows steady‐state experiments with isolated seed coats of Pisum sativum. Permeances were measured for a set of organic model compounds with different physicochemical properties and sizes. The results show that narrow aqueous pathways dominate the diffusion of solutes across pea seed coats, as indicated by a correlation of permeances with the molecular sizes of the compounds instead of their lipophilicity. Further indicators for an aqueous pathway are small size selectivity and a small effect of temperature on permeation. The application of an osmotic water potential gradient across isolated seed coats leads to an increase in solute transfer, indicating that the aqueous pathways form a water‐filled continuum across the seed coat allowing the bulk flow of water. Thus, the uptake of organic solutes across pea testae has two components: (1) by diffusion and (2) by bulk water inflow, which, however, is relevant only during imbibition.     

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.     

Cellular pathway of photosynthate transport in coats of developing seed of Vicia faba L. and Phaseolus vulgaris L. I. Extent of transport through the coat symplast

The extent of post-phloem solute transport through the coatsymplasts of developing seeds of Vicia faba L. and Phaseolusvulgaris L. was evaluated. For Vicia seed coats, the membrane-impermeantfluorochrome, CF, moved radially from the chalazal vein to reachthe chlorenchyma and thin-walled parenchyma transfer cell layers.Thereafter, the fluorochrome moved laterally in these two celllayers around the entire circumference of the seed coat. Transferof CF from the chalazal vein was inhibited by plasmolysis ofattached ‘empty’ seed coats. In contrast, the spreadof phloem imported CF was restricted to the ground parenchymaof Phaseolus seed coats. Fluorochrome loaded into the outermostground parenchyma cell layer was rendered immobile followingplasmolysis of excised seed-coat halves. Phloem-imported [¹⁴C]sucroseand the slowly membrane permeable sugar, L-[¹⁴C]glucose, werepartitioned identically between the vascular and non-vascularregions of intact Vicia seed coats. For ¹⁴C-photosynthates,these partitioning patterns in attached ‘empty’Vicia seed coats were unaffected by PCMBS, but inhibited byplasmolysis. Tissue autoradiographs of intact Phaseolus seedcoats demonstrated that a pulse of ¹⁴C-photosynthate moved fromthe veins to the grounds tissues. In excised Vicia seed coats,preloaded with ¹⁴C-photosynthates, the cellular distributionof residual ¹⁴C-label was unaffected by PCMBS. In contrast,PCMBS caused the ¹⁴C-photosynthate levels to be elevated inthe veins and ground parenchyma relative to the branch parenchymaof Phaseolusseed coat halves. Based on the above findings, itis concluded that the phloem of Vicia seed coats is interconnectedto two major symplastic domains; one comprises the chlorenchyma,the other the thin-walled parenchyma plus thin-walled parenchymatransfer cells. For Phaseolusseed coats, the phloem forms amajor symplastic domain with the ground parenchyma. Key words: Phaseolus vulgaris L, phloem unloading, photosynthate transport, seed coat, symplast, Vicia faba L     

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     

Turgor-Regulated Sugar Release from the Source Leaves of Sugarbeet (Beta vulgaris L.)

Under mild water stress conditions, a potential site of regulationfor distribution of sucrose between osmotic adjustment and exportmay be at the mesophyll plasmalemma and/or tonoplast. This possibilitywas examined in attached leaves of sugarbeet (Beta vulgarisL.), labeled with ¹⁴CO₂. Leaf discs were exposed to solutionscontaining 400 or 50 mM mannitol to generate "low" or "high"cellular turgor, respectively and release of labeled soluteswas monitored. Response to changes in cell turgor was rapidand reversible. High turgor increased solute efflux rates todouble those at low turgor conditions. Approximately 30% and55% of the released label was in the sugar (sucrose and hexose)fractions at low and high turgor, respectively. Paramercuribenzenesulfonic acid (PCMBS) had no effect on efflux, but N-ethylmaleimide(NEM) and carbonylcyanide-m-chlorophenyl hydrazone (CCCP) enhancedefflux, especially at high turgor. Presence of unlabeled sucrosegreatly enhanced efflux in a turgor-dependent manner; suggestinga sucrose exchange system. While influx across the plasmalemmais both turgor sensitive and carrier-mediated, turgor-regulatedplasmalemma efflux did not appear to involve a carrier. Boththe tonoplast and plasmalemma appeared to be involved in turgor-inducedsugar efflux. Turgor-regulated efflux of solutes from vacuole-containingcells (mesophyll), may contribute to the establishment of ahomeostatic turgor pressure in these cells. (Received June 9, 1989; Accepted September 5, 1989)     

Seed coat cell turgor responds rapidly to air humidity in chickpea and faba bean

The turgor pressure in cells of chickpea (Cicer arietinum L.) and faba bean (Vicia faba L.) seed coats was measured with a pressure probe. Measurements were made under in situ conditions by removing a section of wall from a pod, which remained attached to the plant, and exposing the intact seed. If the pod wall was removed and the turgor measurements made under ambient laboratory conditions of 50% to 70% relative humidity (RH), cell turgor pressure declined over time, typically reaching 0 MPa. If the pod wall was removed and the turgor measurements made under conditions of 100% RH, however, cell turgor pressure was stable over time, relatively uniform within the seed coat tissue, and was found to be 0.1-0.3 MPa for chickpea, and 0.1-0.2 MPa for faba bean. In both species there was a marked decline in cell turgor, beginning within about 60 s, when humidification was discontinued. The decline in cell turgor occurred regardless of the depth of the cell within the seed coat tissue, and this decline could be stopped, but not entirely reversed, when humidification was restored. An increase in cell turgor could also be caused by wetting of the seed. These responses indicate that a very rapid water exchange can occur within the seed coat tissue in situ. The rapid and, in some cases, relatively permanent loss of seed coat cell turgor in the absence of humidification raises serious concerns regarding desiccation artefacts which may be involved in the empty seed coat technique, often used to study seed carbon and water relations in grain legumes.     

Photosynthate Unloading from Seed Coats of Phaseolus vulgaris L.--Nature and Cellular Location of Turgor-Sensitive Unloading

Patrick, J. W., Jacobs, E., Offler, C. E. and Cram, W. J. 1986.Photosynthate unloading from seed coats of Phaseolus vulgarisL.—Nature and cellular location of turgor-sensitive unloading—J.exp. Bot. 37: 1006–1019. Unloading rates of ¹⁴C-Photosynthates from excised seed-coathalves of Phaseolus vulgaris L. plants were sharply increasedat cell turgor potentials in excess of 5 ? 10⁵ Pa. Turgor-sensitiveunloading occurred in the absence of any change in the passivepermeability of, and active sucrose influx across, the plasmalemmaand tonoplast membranes. The proton ionophore CCCP, and lowtemperature significantly slowed turgor-sensitive unloadingwhile PCMBS, a non-permeating sulphydryl-modifying compound,was without effect. Turgor-sensitive unloading significantlydepressed the ¹⁴C-Photosynthate content of the ground and branchparenchyma, but had no effect on the ¹⁴C-Photosynthate levelsin the vascular tissues. Cycling of cell turgor potentials aboveand below 5 ? 10⁵ Pa elicited reproducible responses in theunloading rate of ¹⁴C-Photosynthates. Increasing turgor above5 ? 10⁵ Pa resulted in a burst of ¹⁴C-Photosynthate unloading.Reversal to turgors less than 5 ? 10⁵ Pa caused a rapid depressionin unloading rate. It is proposed that turgor-sensitive unloadingis facilitated by a specific turgor-sensitive porter locatedon the plasmalemma of the ground and/or branch parenchyma cellsof bean seed coats. Key words: Bean, seed coat, turgor-sensitive unloading, phloem     

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.     

Gradients of turgor, osmotic pressure, and water potential in the cortex of the hypocotyl of growing ricinus seedlings : effects of the supply of water from the xylem and of solutes from the Phloem

To evaluate the possible role of solute transport during extension growth, water and solute relations of cortex cells of the growing hypocotyl of 5-day-old castor bean seedlings (Ricinus communis L.) were determined using the cell pressure probe. Because the osmotic pressure of individual cells (πⁱ) was also determined, the water potential (ψ) could be evaluated as well at the cell level. In the rapidly growing part of the hypocotyl of well-watered plants, turgor increased from 0.37 megapascal in the outer to 1.04 megapascal in the inner cortex. Thus, there were steep gradients of turgor of up to 0.7 megapascal (7 bar) over a distance of only 470 micrometer. In the more basal and rather mature region, gradients were less pronounced. Because cell turgor ≈ πⁱ and ψ ≈ 0 across the cortex, there were also no gradients of ψ across the tissue. Gradients of cell turgor and πⁱ increased when the endosperm was removed from the cotyledons, allowing for a better water supply. They were reduced by increasing the osmotic pressure of the root medium or by cutting off the cotyledons or the entire hook. If the root was excised to interrupt the main source for water, effects became more pronounced. Gradients completely disappeared and turgor fell to 0.3 megapascal in all layers within 1.5 hours. When excised hypocotyls were infiltrated with 0.5 millimolar CaCl₂ solution under pressure via the cut surface, gradients in turgor could be restored or even increased. When turgor was measured in individual cortical cells while pressurizing the xylem, rapid responses were recorded and changes of turgor exceeded that of applied pressure. Gradients could also be reestablished in excised hypocotyls by abrading the cuticle, allowing for a water supply from the wet environment. The steep gradients of turgor and osmotic pressure suggest a considerable supply of osmotic solutes from the phloem to the growing tissue. On the basis of a new theoretical approach, the data are discussed in terms of a coupling between water and solute flows and of a compartmentation of water and solutes, both of which affect water status and extension growth.     

Phototropic Response of the Bean Pulvinus: Movement of Water and Ions

Segmental analysis of the laminar pulvinus of Phaseolus vulgaris L. showed that its phototropic curvature is accompanied by efflux of inorganic ions and water from its contracting sector and a comparable influx into its expanding one. All the major ions, except Na⁺, contributed to this transport, suggesting that the response to light involves changes in the driving force, or conductivity of a wide range of solutes. During the curvature, K⁺ and CI^? made the greatest and equivalent contributions to efflux, but only Cl^? exhibited a matching influx into the expanding sector, while K⁺ influx was much less. Use of the cell pressure probe showed that, as the laminar angle of elevation changed between ?40° to +40°, turgor pressure in the expanding motor cells increased by 0.48 MPa and decreased in the contracting cells by 0.32 MPa. Picoliter osmometry of single-cell samples showed that during this movement vacuolar osmotic pressure remained constant. Thus, changes in turgor pressure resulted from changes in apoplastic, rather than the protoplastic osmotic pressure. Volumetric modulus of elasticity of pulvinar motor cells is very low, showing that their walls are very elastic. These properties increase the effectiveness of converting osmotic work into the large-scale, reversible volume changes responsible for leaf movements.     

Abscisic Acid and its relationship to seed filling in soybeans

The effect of exogenous abscisic acid (ABA) on the rate of sucrose uptake by soybean (Glycine max L. Merr.) embryos was evaluated in an in vitro system. In addition, the concentrations of endogenous ABA in seeds of three soybean Plant Introduction (PI) lines, differing in seed size, were commpared to their seed growth rates. ABA (10⁻⁷ molar) stimulated in vitro sucrose uptake in soybean (cv `Clay') embryos removed from plants grown in a controlled environment chamber, but not in embryos removed from field-grown plants of the three PI lines. However, the concentration of ABA in seeds of the three field-grown PI lines correlated well with their in situ seed growth rates and in vitro [¹⁴C] sucrose uptake rates.

Across genotypes, the concentration of ABA in seeds peaked at 8.5 micrograms per gram fresh weight, corresponding to the time of most rapid seed growth rate, and declined to 1.2 micrograms per gram at physiological maturity. Seeds of the large-seeded genotype maintained an ABA concentration at least 50% greater than that of the small-seeded genotype throughout the latter half of seed filling. A higher concentration of ABA was found in seed coats and cotyledons than in embryonic axes. Seed coats of the large-seeded genotype always had a higher concentration of ABA than seed coats of the small-seeded line. It is suggested that this higher concentration of ABA in seed coats of the large-seeded genotype stimulates sucrose unloading into the seed coat apoplast and that ABA in cotyledons may enhance sucrose uptake by the cotyledons.

     

Effluxes of solutes from developing seed coats of Phaseolus vulgaris L. and Vicia faba l.: locating the effect of turgor in a coupled chemiosmotic system

Cells lining the developing seed coats of legumes efflux photosynthates (mostly sucrose) and salts (mostly of potassium) into the apoplast for uptake by the developing embryo. These effluxes increase transiently in response to an increase in turgor in the effluxing cells. Detached coats of developing seed of P. haseolus vulgaris and Vicia faba were used to study the effects of turgor on the rates of efflux, on the membrane potential difference and on the membrane pH difference, using a number of inhibitors and agents which might affect signal cascades involving cytoplasmic calcium concentration. Effluxes were measured by measuring the concentrations of solutes of interest in solution samples placed in halves of detached seed coats, the paired halves serving as control and treated sample where appropriate. It is shown that a number of substances affect sucrose and potassium effluxes differently, and that hypo-osmotic shock depolarizes the efflux cells and acidifies the cytoplasm (in P. vulgaris). It is concluded that sucrose and potassium effluxes, although both are increased by an increase in turgor, are affected by different signal pathways. Further, it is also concluded that the signal that increases the rates of both sucrose efflux (via sucrose-proton antiport) and proton pump acts directly on the antiporter rather than on the pump. There are interesting parallels and contrasts between these processes and those in plants such as the charophyte Lamprothamnium after hypo-osmotic shock.