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.     

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.     

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.     

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.     

No direct linkage between proton pumping and photosynthate unloading from seed coats of Phaseolus vulgaris L.

The energization of the active sucrose release from bean seed-coat halves was investigated. For this purpose, seed coat tissues adjacent to the apoplastic space were exposed to a variety of treatments and proton and photosynthate release were measured. Fusicoccin (10^–5 moll^–1) stimulated proton pump activities. Orthovanadate (2×10^–4 moll^–1) and abscisic acid (10^–5 moll^–1) diminished the proton extrusion evoked by fusicoccin. Fusicoccin inhibited sucrose release, whereas orthovanadate and abscisic acid stimulated it. Addition of 100 mmoll^–1 K⁺ had a promotory effect on photosynthate unloading, fading away with time. This extra unloading was linearly related to an enhanced proton loss. It was concluded that the photosynthate unloading apparently is not a proton/sucrose antiport and that a pump-leak system for photosynthate release is unlikely. A tentative model for photosynthate/proton symport not directly linked to proton pumping is presented as the mechanism of unloading.Abbreviations ABA abscisic acid - CCCP carbonyl cyanide m-chlorophenyl hydrazone - DTE diethioerythritol - FC fusicoccin - MES 2-(N-morpholino) ethanesulfonic acid monohydrate - NEM n-ethylmaleimide - PCMBS p-chloromercuriphenylsulfonic acid - TRIS 2-amino-2-(hydroxymethyl) propane-1,3 diol - VAN sodium orthovanadate     

Proton extrusion in seed coats of Phaseolus vulgaris L.

Abstract. The present investigations were designed to identify proton pumps in seed coats of Phaseolus vulgaris L. Vacated seed-coat halves were exposed to bathing solutions with indicators for proton pump action and the pH changes in the media were measured. Fusicoccin increased the rate of proton extrusion from the seed coats. Orthovanadate and abscisic acid retarded the proton extrusion evoked by fusicoccin. Abolition of the proton extrusion by parachloromercuriphenylsulphonic acid was partially reversed by diethioerythritol. The extrusion was stimulated by high osmolarities (100 mol m⁻³ sorbitol), potassium ions (100 mol m⁻³ KCI) and light. Old seed coats reacted more rapidly to fusicoccin treatments than young ones. Proton pumping in seed coats and cotyledons showed differential responses to fusicoccin, K⁺ and sucrose. In contrast to seed coats, medium acidification by cotyledons was prohibited by addition of sucrose. The significance of proton pumps for photosynthate transfer in vivo is discussed.     

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.     

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

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

The micromorphologies of seed coats and petal trichomes of the taxa of Viola subsect. Boreali-Americanae (Violaceae) and their utility in discerning orthospecies from hybrids

Viola subsect.Boreali-Americanae, confined to North America including northern México, has long been considered one of the most taxonomically difficult temperate groups of the angiosperms. Hybridization, followed in some localities by introgression, has made it difficult for systematists to discern the “core” species by using only standard macromorphological characters. Analyses that employed scanning electron microscopy (SEM) of seed coats and petal trichomes generated new characters. SEM of the surface of seeds of each of the orthospecies revealed a suite of primary and secondary sculpture characters unique to each species that are correlated with a suite of macromorphological characters. In a number of species, SEM of petal trichomes provided additional unique characters. SEM of the surface of seed coats enabled, also, the identification of instances of hybridization by revealing blends of structures of the primary and secondary sculpture typical of different orthospecies and instances of putative introgression by revealing deviations in surface structures from those typical of orthospecies. A procedure based on iterative examinations of macromorphology and micromorphology, which was developed in this research, enable the discernment of hybrids and putative introgressants. Combining the micromorphological and macromorphological data with ecological and phytogeographical data enabled the delimitation of sixteen orthospecies.