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     

Growth Regulators Have Rapid Effects on Photosynthate Unloading from Seed Coats of Phaseolus vulgaris L

Of nine plant growth regulators (indoleacetic acid, 1-naphthalene acetic acid, gibberellic acid, giberellin 4/7, 6-benzylaminopurine, 6-furfurylaminopurine, abscisic acid, and 1-aminocyclopropane carboxylic acid) tested, only 6-benzylaminopurine and abscisic acid affected ¹⁴C-photosynthate unloading from excised seed coats of Phaseolus vulgaris L. Unloading, in the presence of KCl, was stimulated by 25 to 40%. Stimulation occurred immediately for 6-benzylaminopurine and for abscisic acid within 10 to 12 minutes of application.     

Effect of Potassium on Photosynthate Unloading from Seed Coats of Phaseolus vulgaris L. Specificity and Membrane Location

Rates of ¹⁴C-photosynthate unloading from excised seed-coathalves of Phaseolus vulgaris L. plants were stimulated by externalKCI concentrations in excess of 10 mM with an optimal responseat 100–150 mM KCI. The cellular pattern of ¹⁴C-photosynthatemetabolism was not altered by KCI but the treatment preferentiallystimulated the release of sucrose from the seed-coats. Photosynthateunloading was insensitive to Cl^– and was stimulated bya range of membrane-permeable cations (Na⁺, Mg²⁺ and tetraphenylphosphoniumion) in addition to K⁺. The K⁺ ionophore, valinomycin, abolishedthe K⁺ stimulation of ¹⁴C-photosynthate unloading. A switchto a wash solution containing K⁺ elicited a rapid burst of ¹⁴C-photosynthateunloading; the rate constant for the final phase of ¹⁴C-efflux(probably across the tonoplast) was unaffected by K⁺. The KCItreatment did not change the passive permeability of eitherthe plasmalemma or tonoplast. While sucrose influx across theplasmalemma was insensitive to K⁺, sucrose transfer to the vacuolewas slowed. The results obtained support the postulate thatK⁺ (and other membrane permeable cations) preferentially stimulatesucrose efflux across the plasmalemma of the unloading cellsby serving to carry positive charge in the opposite direction. Phaseolus vulgaris, bean, photosynthate unloading, potassium stimulation, seed-coat     

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.     

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.     

Quantitative Analysis of Photosynthate Unloading in Developing Seeds of Phaseolus vulgaris L. : I. The Use of Steady-State Labeling

The pathway and kinetics of photosynthate unloading in developing seeds of bean (Phaseolus vulgaris L.) were investigated using steady-state labeling with ¹⁴CO₂. The continuous assimilation of ¹⁴CO₂ at constant specific activity produced stable tracer fluxes that facilitated straightforward analyses of photosynthate import and unloading in developing seeds. The kinetics of tracer equilibration within intact seeds were compatible with a symplastic route of photosynthate unloading in the seed coat. The import and partitioning of tracer within seeds were partially disrupted by the surgical excision of the distal halves of seeds as practiced during the preparation of “empty” seed coats for perfusion.     

The Pathway of Radial Transfer of Photosynthate in Decapitated Stems of Phaseolus vulgaris L.

[¹⁴C]Sucrose was found to be the predominant component of the¹⁴C-photosynthates that accumulated in the free space of decapitatedstems of P. vulgaris plants. The ¹⁴C-photosynthates appearedto occupy the entire free-space volume of the stems at totalsugar concentrations in the range of 3–12 mM. The free-spacesugar levels were found to rapidly decline once photosynthatetransfer to the stems was halted. Moreover, it was found thatestimates of the rate of in vitro sucrose uptake by the stemscould account fully for the decline in free-space sugar levels.Overall, the evidence indicated that at least part of the radialpathway of photosynthate transfer in bean stems involved thestem apoplast. It is tentatively proposed that, based on celland tissue distribution of ¹⁴C-photosynthates, the apoplasticpathway extends from the membrane boundary of the sieve element/companion-cellcomplex to all other cells of the stem. Apoplast, Phaseolus vulgaris L., bean, phloem unloading, photosynthates, symplast     

Chromium VI Induced Structural and Ultrastructural Changes in Bush Bean Plants (Phaseolus vulgaris L.)

The effect of a growth reducing chromium VI concentration (9.6x 10^–5M Cr as Na₂CrO₄) on the structure and ultrastructureof different organs of bush bean plants (Phaseolus vulgarisL.) grown on perlite was studied using light, transmission electronand scanning electron microscopy. The structural and ultrastructuralalterations observed were quite different in the various organsanalysed and did not always stay in direct relation to the averageCr content of the whole organ. The primary toxicity effect seemedto be membrane damage, due to the high oxidation power of CrVI. It is suggested that chromium is retained in vacuoles andcell walls of roots, and that the chromium reaching the leavesmay be principally Cr III and present in cell walls. The alterationsobserved in the upper plant parts seemed principally due toindirect Cr effects on the content of essential mineral nutrients. Phaseolus vulgaris L., bush bean, chromium VI toxicity, structure, ultranstructure, light microscopy, transmission electron microscopy, scanning electron microscopy     

Cell-Wall Proteins Induced by Water Deficit in Bean (Phaseolus vulgaris L.) Seedlings

In the last few years, much attention has been given to the role of proteins that accumulate during water deficit. In this work, we analyzed the electrophoretic patterns of basic protein extracts, enriched for a number of cell-wall proteins, from bean (Phaseolus vulgaris L.) seedlings and 21-d-old plants subjected to water deficit. Three major basic proteins accumulated in bean seedlings exposed to low water potentials, with apparent molecular masses of 36, 33, and 22 kD, which we refer to as p36, p33, and p22, respectively. Leaves and roots of 21-d-old plants grown under low-water-availability conditions accumulated only p36 and p33 proteins. In 21-d-old plants subjected to a fast rate of water loss, both p33 and p36 accumulated to approximately the same levels, whereas if the plants were subjected to a gradual loss of water, p33 accumulated to higher levels. Both p36 and p33 were glycosylated and were found in the cell-wall fraction. In contrast, p22 was not glycosylated and was found in the soluble fraction. The accumulation of these proteins was also induced by abscisic acid (0.1-1.0 mM) treatment but not by wounding or by jasmonate treatment.     

Nuclear and chloroplast microsatellite diversity in Phaseolus vulgaris L. from Sardinia (Italy)

Studies of the level and the structure of the genetic diversity of local varieties of Phaseolus vulgaris are of fundamental importance, both for the management of genetic resources and to improve our understanding of the pathways of dissemination and the evolution of this species in Europe. We have here characterized 73 local bean populations from Sardinia (Italy) using seed traits and molecular markers (phaseolins, nuSSRs and cpSSRs). American landraces and commercial varieties were also included for comparison. We see that: (a) the Sardinian material is distinct from the commercial varieties considered; (b) the variation in the seed traits is high and it mostly occurs among populations (95%); (c) compared to the American sample and the commercial varieties, the Sardinian collection has a low level of diversity; (d) the majority (>95%) of the Sardinian individuals belong to the Andean gene pool; (e) the Sardinian material shows a strong genetic structure, both for cpSSRs and nuSSRs; (f) the nuSSRs and cpSSRs concur in differentiating between gene pools, but a lack of congruence between nuclear and chloroplast has been observed within gene pools; and (g) there are three putative hybrids between the Andean and Mesoamerican gene pools. Despite the relatively low level of diversity, which is probably due to a strong founder effect, the Sardinian landraces are worth being conserved and studied further because of their distinctiveness and because hybridization within and between the gene pools could generate variation that will be useful for breeding. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Effects of SO(2) and O(3) on Allocation of C-Labeled Photosynthate in Phaseolus vulgaris

A series of laboratory exposures of two varieties of bush bean (Phaseolus vulgaris L., var 274 and var 290) was conducted to determine the sensitivity of [¹⁴C]photosynthate allocation patterns to alteration by SO₂ and O₃. Experiments with the pollution-resistant 274 variety demonstrated short-term changes in both ¹⁴C and biomass allocation to roots of ¹⁴CO₂-labeled plants but no significant effect on yield by up to 40 hours of exposure to SO₂ at 0.50 microliters per liter or 4 hours of O₃ at 0.40 microliters per liter. Subsequent experiments with the more sensitive 290 variety demonstrated significant alteration of photosynthesis, translocation, and partitioning of photosynthate between plant parts including developing pods. Significant increases in foliar retention of photosynthate (+40%) occurred after 8 hours of exposure to SO₂ at 0.75 microliters per liter (6.0 microliters per liter-hour) and 11 hours of exposure to O₃ at 0.30 microliters per liter-hour (3.3 microliters-hours). Time series sampling of labeled tissues after ¹⁴CO₂ uptake showed that the disruption of translocation patterns was persistent for at least 1 week after exposures ceased. Subsequent longer-term exposures at lower concentrations of both O₃ (0.0, 0.10, 0.15, and 0.20 microliters per liter) and SO₂ (0.0, 0.20, and 0.40 microliters per liter) demonstrated that O₃ more effectively altered allocation than SO₂, that primary leaves were generally more sensitive than trifoliates, and that responses of trifoliate leaves varied with plant growth stage. Altered rates of allocation of photosynthate by leaves were generally associated with alterations of similar magnitude and opposite direction in developing pods. Collectively, these experiments suggest that allocation patterns can provide sensitive indices of incipient growth responses of pollution-stressed vegetation.     

Sodium Recirculation and Loss from Phaseolus vulgaris L.

In a split-root experiment, ²²Na was supplied to Phaseolus vulgarisL. roots emerging from the stem, 2.5 cm above the main roots.Sodium exported from these upper roots was translocated a shortdistance upward in the stem and downward to the main roots.Most of the ²²Na arriving in the main roots was lost to themedium. Sodium loss from P. vulgaris roots into KCI or NaCl was similarand was not affected by oligomycin. The results confirm a previous hypothesis regarding the mechanismof sodium exclusion from the tops of sodium non-accumulatorplants. Phaseolus vulgaris L., bean, sodium transport     

Seed Development in Phaseolus vulgaris L. cv Seminole: I. Developmental Independence of Seed Maturation

Phaseolus vulgaris L. cv Seminole pods removed from the plant continued their development when incubated in suitable conditions. Seeds continued to grow and develop and pods and seeds passed through an apparently normal developmental sequence to dryness. Seed growth was at the expense of pod dry weight (DW) reserves. Losses of pod DW paralleled DW gains by seeds in detached pods and in pod cylinders containing a seed. The transfer activity was apparent only within the period 10 to 30 days after anthesis (DAA) with maximal activity between 15 to 20 DAA. This period corresponds to maximum pod growth and the attainment of maximal DW. Seeds are in only the early phase of seed growth at this time. No DW transfer was observed at developmental stages beyond 30 to 35 DAA when normal senescence DW losses in pods became evident and seeds were in the later phase of seed fill. Pods or pod cylinders remained green and succulent over the transfer period, later passing through yellowing and drying phases characteristic of normal development. DW transfer was dependent on funicle integrity and was readily detectable in pod cylinders after 7 days incubation. The DW transfer activity may contribute to continuing nutrition of seeds under conditions where the normal assimilate supply to seeds becomes limiting. Defoliation and water stress treatments applied to Phaseolus plants reduced seed yields but allowed persistence of seed maturation processes such that all seeds developing to dryness were capable of germination.     

Hormonally Induced Changes in the Stem and Petiole Anatomy and Cellulase Enzyme Patterns in Phaseolus vulgaris L

Time course changes were observed in petiole and stem anatomy and cellulase enzyme patterns in bean (Phaseolus vulgaris L.) explants when 10⁻⁵ or 10⁻²m indoleacetic acid in lanolin paste was applied to acropetal cut surfaces in the presence or absence of ethylene. Auxin (10⁻²m) in the presence of ethylene stimulated rapid ordered cell division and dedifferentiation, with ensuing lateral root formation. Auxin (10⁻⁵m) caused moderate cortical swelling, pit formation in pith parenchyma, and chloroplast development in certain cortical cells. Exogenous ethylene reduced cell division activity and caused cortical cell swelling and separation. Removal of endogenously generated ethylene by mercuric perchlorate resulted in less ordered cell division patterns and no lateral root formation. Auxin treatments enhanced formation of an active acidic pI cellulase, exogenous ethylene-stimulated formation of an active basic pI cellulase. The absence of basic pI cellulase activity by the removal of endogenously generated ethylene suggests a close dependence of basic pI cellulase activity on ethylene.     

Seed Vigour in Bean (Phaseolus vulgaris L. cv. Apollo) as Influenced by Temperature and Water Regime during Development and Maturation

Bean plants grown in a controlled temperature glasshouse athigh temperatures (33/28, 30/25, or 27/22 °C) during theperiod of seed development and maturation matured early andproduced small seeds. The seeds were of lower vigour than thosegrown at 21/16 or 18/13 °C. The detrimental effect of highmaturation temperatures was observed even on plants bearingwell-developed seeds (yellow, fleshy-pod stage). Seeds maturedat high temperatures were also more susceptible to deteriorationwith delay in harvest, and to mechanical damage. Heavy wateringof plants with seed ready to harvest caused a reduction in seedvigour. For optimum quality bean seed, it appears essentialthat the seed develops and matures at cool temperatures, ina dry environment.     

Partial Purification of a cis-trans-Isomerase of Zeatin from Immature Seed of Phaseolus vulgaris L

Investigation of the conversion of exogenous cis-zeatin to trans-zeatin in immature seeds of Phaseolus vulgaris L. led to the isolation of a cis-trans-isomerase from the endosperm. The enzyme was purified more than 2000-fold by chromatography on a series of fast protein liquid chromatography (anion exchange, gel filtration, and hydrophobic interaction) and concanavalin A columns. The enzymic reaction favors conversion from the cis to the trans form and requires flavin, light, and dithiothreitol. cis-Zeatin riboside is also a substrate for the enzyme. Retention on the concanavalin A column indicated that the enzyme is a glycoprotein. The enzyme was stable for at least 8 weeks when stored at -80[deg] C. The occurrence of cis-trans-isomerization suggests that cis-zeatin and cis-zeatin riboside formed by tRNA degradation could be precursors of biologically active cytokinins.     

Sugar Efflux from Attached Seed Coats of Glycine max (L.) Men.

Ellis, E. C. and Spanswick, R. M. 1987. Sugar efflux from attachedseed coats of Glycine max (L.) Merr.—J. exp. Bot. 38:1470—1483. Sugar efflux (sucrose + glucose) from attached seed coats ofGlycine max (L.) Merr. was measured at high sampling rates toimprove the kinetic characterization of seed coat exudation.This study confirms that sugar efflux in seed coats has at leasttwo components, and demonstrates that the concentration of mannitolosmoticum bathing the seed coat may influence one or both ofthese components. High leaf irradiance increased sugar effluxrelative to a low leaf irradiance at the same mannitol concentration.A high concentration of mannitol (500 mol m³) enhanced sugarefflux relative to a medium concentration (100 mol m³) underboth high and low leaf irradiance. A low mannitol concentration(10 mol m³) stimulated sugar efflux (relative to 100 mol m³)to a greater extent when leaf irradiance was high. Rapid changesin mannitol concentration produced immediate stimulations ofsugar efflux. Effects of osmoticum on sugar efflux are explainedby simultaneous turgor-mediated effects on import of sucroseby the phloem and retrieval of apoplastic sucrose, presumablyby seed coat parenchyma.     

Dynamics of Imbibition in Phaseolus vulgaris L. in Relation to Initial Seed Moisture Content

The seed moisture level marking the onset of imbibitional injury (breakpoint) was determined for two cultivars of Phaseolus vulgaris L. cvs `Tendercrop' (TC) and `Kinghorn Wax' (KW). At 20°C the breakpoints were 0.15 gram H₂O/gram dry weight (gram per gram) for TC and 0.11 gram per gram for KW. When seeds were imbibed at 5°C, the breakpoints were 0.19 gram per gram (TC) and 0.16 gram per gram (KW). Below the breakpoint germination changed 4.6%/0.01 gram per gram for all treatments. Imbibition rates were maximal at 0.07 gram per gram and 0.33 gram per gram after 20 minutes imbibition. Rates of electrolyte leakage were correlated with the imbibition rate maximum at 0.07 gram per gram but were unaffected by the maximum at 0.33 gram per gram. The transition from tightly bound to semibound water occurred at 0.09 gram per gram and 0.11 gram per gram for KW and TC, respectively. T1 values increased exponentially as seed moisture decreased from 0.47 gram per gram to 0.05 gram per gram. ¹³C-NMR sugar signals increased at moisture levels above 0.14 gram per gram and plateaued at approximately 0.33 gram per gram seed moisture. These results suggest that the breakpoint moisture level for imbibitional damage is a function of temperature while the injury process is similar at both 5 and 20°C. Imbibition and leakage rate maxima reflect transitions in the states of seed water. NMR data support the application of the Water Replacement Hypothesis to seeds. Thus, imbibitional injury may be related to specific, temperature dependent moisture levels that are determined by water binding characteristics in the seed tissue.