Turgor-dependent unloading of photosynthates from coats of developing seed of Phaseolus vulgaris and Vicia faba. Turgor homeostasis and set points

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 be 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 turgordependent 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.     

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. 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.     

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     

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)     

Differential effects of turgor on sucrose and potassium transport at the tonoplast and plasma membrane of sugar beet storage root tissue

When turgor was increased, by decreasing the concentration of mannitol bathing discs of sugar beet storage root tissue, the rates of sucrose and potassium uptake into the vacuole were decreased. At all external mannitol concentrations the rate of sucrose and potassium uptake across the plasma membrane was an order of magnitude greater than the rate of quasi-steady uptake into the vacuole, implying a very large efflux. Efflux of both sucrose and potassium was increased at high turgor. However, while increasing turgor decreased the rate of K⁺ uptake, the rate of sucrose uptake at the plasma membrane increased with time. Compartmental analysis of tracer exchange kinetics was used to determine unidirectional K⁺ fluxes. From these results, it was estimated that the increase in K⁺ efflux accompanying a 1.5 MPa increase in turgor could lead to a net increase of 140mol^?3h^?1 in the external potassium concentration. It is suggested that the turgor-imposed increase in solute efflux is a means of regulating intracellular osmotic pressure and/or turgor in sugar beet storage roots, but that sucrose is preferentially retrieved from the apoplast, even under conditions of excessively high turgor. However, much of this sucrose is probably lost from the cell, implying a ‘futile’ sucrose transport cycle at the plasma membrane. The turgor-stimulated leak of potassium could play a major role in the regulation of turgor pressure in sugar beet storage root tissue.     

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.     

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.     

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.     

The effect of cell turgor on sugar uptake in strawberry fruit cortex tissue

A reduction in cell turgor has been shown to stimulate sugar uptake in several plant sink tissues and it may regulate the import of assimilate into the sink apoplast, as well as maintain cell turgor. To determine whether cell turgor influences sugar uptake by strawberry (Fragaria x ananassa Duch. cv. Brighton) fruit cortex tissue, disks were cut from greenhouse-grown primary fruit at the green-white stage of development and placed in buffered incubation solutions containing either mannitol or ethylene glycol as an osmoticum. Cell turgor of fruit disks was calculated from the difference between the water potential of bathing solution and tissue solute potential after incubation at various osmolarities. Cell turgor increased when tissue disks were placed into mannitol incubation solutions more dilute than the water potential of fresh tissue (about 415 mOsmol kg^?1). The rate of uptake of [¹⁴C]-sucrose or [¹⁴C]-glucose decreased as osmolarity of the incubation solution increased, i.e. as cell turgor declined. Cell turgor and the rate of [¹⁴C]-sucrose uptake were unaffected when rapidly permeating ethylene glycol was used as an osmoticum. A decrease in cell turgor reduced both the V_max of the saturable (carrier mediated) kinetic component of sucrose uptake, and the slope of the linear (diffusional) component. The sulfhydryl binding reagent p-chloromercuibenzenesulfonic acid, an inhibitor of the plasma membrane sucrose carrier, strongly inhibited only the saturable component of sucrose uptake. Increased uptake of the nonmetabolizable sugar, O-methyl-glucose, at high turgor was similar to that of glucose, indicating that carrier activity was influenced by cell turgor, not cell metabolism. Turgor did not influence efflux of [¹⁴C]-sucrose from disks and had no effect on cell viability. Strawberry fruit cells do not possess a sugar uptake system that is stimulated by a reduction in turgor.     

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.     

Osmotic dependence of the transmembrane potential difference of broadbean mesocarp cells

Pod walls of broadbean (Vicia faba L. cv Aguadulce) were harvested at the import (S₁), at the transition (S₂) or at the export (S₃) phase for assimilate transport. Measurements of the transmembrane potential difference (PD) of mesocarp cells were made under various osmotic conditions. Internal osmotic potentials and cell turgor were calculated from osmolality measurements of cell saps recovered by freeze-thawing, after correction for the contribution of the free-space solution. Changes in the mannitol concentration of the medium altered the PD within a few minutes, and new stable values of PD were reached within 20 minutes after the osmotic change. With mannitol as the osmoticum, the most negative PD was measured at an external osmotic potential of -0.70 megapascals (MPa) for S₁ and S₂, while the most negative was at -0.40 MPa for S₃. Ethylene glycol, a permeant osmoticum, had little effect on PD, showing that the PD was sensitive to turgor, not to solute potential per se. For S₁ and S₂, the PD was less negative for turgor potentials lower than 0.1 MPa or greater than 0.3 MPa. S₃ samples exhibited a different turgor dependence, with a sharp optimum of the negativity of the PD at 0.3 MPa. The data are consistent with the proposal that the proton pump acts as a transducer of the osmotic conditions. They show that the osmotic sensitivity of the PD of mesocarp cells of broadbean changes with the stage of development of the pod.     

Cell specific expression of three genes involved in plasma membrane sucrose transport in developingVicia faba seed

Summary In developing seeds ofVicia faba, transfer cells line the inner surface of the seed coat and the juxtaposed epidermal surface of the cotyledons. Circumstantial evidence, derived from anatomical and physiological studies, indicates that these cells are the likely sites of sucrose efflux to, and influx from, the seed apoplasm, respectively. In this study, expression of an H⁺/sucrose symporter-gene was found to be localised to the epidermal-transfer cell complexes of the cotyledons. The sucrose binding protein (SBP) gene was expressed in these cells as well as in the thin-walled parenchyma transfer cells of the seed coat. SBP was immunolocalised exclusively to the plasma membranes located in the wall ingrowth regions of the transfer cells. In addition, a plasma membrane H⁺-ATPase was most abundant in the wall ingrowth regions with decreasing levels of expression at increasing distance from the transfer cell layers. The observed co-localisation of high densities of a plasma membrane H⁺-ATPase and sucrose transport proteins to the wall ingrowths of the seed coat and cotyledon transfer cells provides strong evidence that these regions are the principal sites of facilitated membrane transport of sucrose to and from the seed apoplasm.Abbreviations BCIP 5-bromo-4-chloro-3-indolyl phosphate - DIG digoxigenin - H⁺-ATPase plasma membrane H⁺-translocating adenosine triphosphatase - Ig immunoglobulin - LeSUT1 tomato H⁺/sucrose symporter - SBP sucrose binding protein     

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.     

Sucrose Release into the Endosperm Cavity of Wheat Grains Apparently Occurs by Facilitated Diffusion across the Nucellar Cell Membranes

Nutrients required for the growth of the embryo and endosperm of developing wheat (Triticum aestivum L.) grains are released into the endosperm cavity from the maternal tissues across the nucellar cell plasma membranes. We followed the uptake and efflux of sugars into and out of the nucellus by slicing grains longitudinally through the endosperm cavity to expose the nucellar surface to experimental solutions. Sucrose uptake and efflux are passive processes. Neither was sensitive to metabolic inhibitors, pH, or potassium concentration. p-Chloromercuribenzene sulfonate, however, strongly inhibited both uptake and efflux, although not equally. Except for p-chloromercuribenzene sensitivity, these characteristics of efflux and the insensitivity of Suc movement to turgor pressure are similar to those of sucrose release from maize pedicels, but they contrast with legume seed coats. Although the evidence is incomplete, movement appears to be carrier mediated rather than channel mediated. In vitro rates of sucrose efflux were similar to or somewhat less than in vivo rates, suggesting that transport across the nucellar cell membranes could be a factor in the control of assimilate import into the grain.     

Nicotine Production by Tissue Cultures of Tobacco as Influenced by Various Culture Parameters

Assimilate efflux from vacuum-infiltrated leaf slices (spinach, barley) into a buffered solution was examined in relation to Ca^{+ +} -activity and osmotic conditions. Efflux from isolated mesophyll protoplasts and from a unicellular green alga (Eremosphaera viridis de Bary) was also measured.In the presence of Ca^{+ +}, assimilate efflux from leaf slices was small (1 to 5 % of the total carbon fixation rate, depending on osmotic conditions). Efflux was drastically stimulated by addition of Ca^{+ +} -chelators. If expressed as µmol carbon mg^-1 chlorophyll h^-1, it reached 50 % of the assimilation rate. Efflux from protoplasts or algae was slow and insensitive to Ca^{+ +} chelators at concentrations which caused fast efflux from leaf slices.Assimilate efflux from leaf slices was rather unspecific. Both in the tissue and the surrounding medium, sucrose was the most abundantly labelled compound (70 to 80 % of total soluble labelled material).A 50 % decrease of efflux was observed when turgor pressure was lowered by addition of sorbitol (200 to 300 mosmol kg^-1). At extremely high sorbitol concentrations (> 1500 mosmol kg^-1) efflux increased again and was relatively less stimulated by EDTA.It is suggested that assimilate efflux from leaf slices is mainly diffusion through open veins and/or plasmodesmata. When these symplastic connections are closed by addition of Ca^{+ +}, the remaining transmembrane flux into the apoplast is small. Thus, assimilate movement from the mesophyll to the phloem appears to be symplastic, not apoplastic as suggested in the literature.     

Mechanism of Photosynthate Efflux from Vicia faba L. Seed Coats

In order to develop a tentative model of the mechanism of photosynthateefflux from the vascular region of Vicia faba L. seed coats,wash-out experiments were performed after removal of the embryo. The sulphydryl group modifiers, pCMBS and NEM, reduced ¹⁴C-photosynthateefflux by 40% and 50%, respectively. Their inhibitory effectcould be prevented or reduced (in the latter case) by includingDTT in the bathing solution. Maltose competed with sucrose forefflux; a concentration of 300 mol m^–3 inhibited ₁₄C-photosynthaterelease by 35%. The cations K⁺ , Na⁺ Mg²⁺ and TPP⁺ enhancedefflux significantly, whereas the countenon Cl^– had noeffect. The presence of the protonophore CCCP (0·1 molm^–3) led to a reduction of efflux by 50% net proton extrusiondropped by 34%. To a lesser extent, an efflux inhibition wasalso achieved by decreasing the cytoplasmic pH with the weakacid DM0. In contrast, alterations in the external pH causedonly a feeble response. The ATPase inhibitor, EB, decreasedphotosynthate efflux and H⁺ extrusion. DES reduced efflux slightly,presumably by affecting ATPase activity as well as energy metabolism. Based on these findings, it is proposed that a sucrose/protonantiport mechanism could be responsible for photosynthate effluxfrom Vicia faba seed coats. Key words: Photosynthate efflux, proton extrusion, proton/sucrose antiport, seed coat, Vicia faba L.     

Gradients in water potential and turgor pressure along the translocation pathway during grain filling in normally watered and water-stressed wheat plants

The water relations parameters involved in assimilate flow into developing wheat (Triticum aestivum L.) grains were measured at several points from the flag leaf to the endosperm cavity in normally watered (Psi approximately -0.3 MPa) and water-stressed plants (Psi approximately -2 MPa). These included direct measurement of sieve tube turgor and several independent approaches to the measurement or calculation of water potentials in the peduncle, grain pericarp, and endosperm cavity. Sieve tube turgor measurements, osmotic concentrations, and Psi measurements using dextran microdrops showed good internal consistency (i.e. Psi = Psi(s) + Psi(p)) from 0 to -4 MPa. In normally watered plants, crease pericarp Psi and sieve tube turgor were almost 1 MPa lower than in the peduncle. This suggests a high hydraulic resistance in the sieve tubes connecting the two. However, observations concerning exudation rates indicated a low resistance. In water-stressed plants, peduncle Psi and crease pericarp Psi were similar. In both treatments, there was a variable, approximately 1-MPa drop in turgor pressure between the grain sieve tubes and vascular parenchyma cells. There was little between-treatment difference in endosperm cavity sucrose or osmotic concentrations or in the crease pericarp sucrose pool size. Our results re-emphasize the importance of the sieve tube unloading step in the control of assimilate import.