Sugar uptake by the dermal transfer cells of developing cotyledons of Vicia faba L.

Two experimental systems were developed to study the uptake of sucrose by the dermal transfer cells of developing cotyledons of Vicia faba L. First, the in-vivo state was approximated by short-term (10 min) incubation of whole cotyledons in [¹⁴C]sucrose solutions. Under these conditions, a minimum of 67% of the ¹⁴C label entered the dermal transfer cell complex. Of this, at least 40% crossed the plasma membranes of the epidermal transfer cells. Second, a protocol was developed to enzymatically isolate and purify dermal transfer cell protoplasts. The yields of the transfer cell protoplasts were relatively low and their preparation incurred a significant loss of plasma membrane. However, the protoplasts remained viable up to 24 h following purification and proved to be a suitable system to verify transport properties observed with whole cotyledons. Using these two experimental systems, it was established that [¹⁴C]sucrose uptake by the dermal transfer cells exhibited features consistent with mediated energy-dependent transport. This included saturation kinetics, competition for uptake between structurally similar molecules, and inhibition of uptake by p-chloromercuribenzenesulfonic acid and several other metabolic inhibitors. For comparative purposes, sugar uptake by the storage parenchyma of the Vicia cotyledons was also examined. In contrast to the dermal transfer cell complex, sucrose uptake by the storage parenchyma displayed characteristics consistent with simple diffusion.Abbreviations CCCP carbonylcyanide m-chlorophenylhydrazone - DNP 2,4-dinitrophenol - NEM N-ethylmaleimide - PCMBS p-chloromercuribenzenesulfonic acid 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 indebted to Stella Savory for preparing the ultrathin sections for electron microscopy.     

Sugar uptake by the dermal transfer cells of developing cotyledons of Vicia faba L.

The mechanism of carrier-mediated sucrose uptake by the dermal transfer cells of developing Vicia faba L. cotyledons was studied using excised cotyledons and isolated transfer cell protoplasts. Addition of sucrose resulted in a transitory alkalinization of the bathing solution whereas additions of glucose, fructose or raffinose had no effect. Dissipating the proton motive force by exposing cotyledons and isolated transfer cell protoplasts to an alkaline pH, carbonylcyanide m-chlorophenylhydrazone, weak acids (propionic acid and 5,5-dimethyl-oxazolidine-2,4-dione) or tetraphenylphos-phonium ion resulted in a significant reduction of sucrose uptake. The ATPase inhibitors, erythrosin B (EB), diethylstilbestrol (DES) and N,N-dicyclohexylcarbodiimide (DCCD) were found to abolish the sucrose-induced medium alkanization as well as reduce sucrose uptake. Cytochemical localization of the ATPase, based on lead precipitation, demonstrated that the highest activity was present in the plasma membranes located in wall ingrowth regions of the dermal transfer cells. The presence of a transplasma-membrane redox system was detected by the extracellular reduction of the electron acceptor, hexacyanoferrate III. The reduction of the ferric ion was coupled to a release of protons. The redox-induced proton extrusion was abolished by the ATPase inhibitors EB, DES and DCCD suggesting that proton extrusion was solely through the H⁺-ATPase. Based on these findings, it is postulated that cotyledonary dermal transfer cells take up sucrose by a proton symport mechanism with the proton motive force being generated by a H ⁺ -ATPase. Sucrose uptake by the storage parenchyma and inner epidermal cells of the cotyledons did not exhibit characteristics consistent with sucrose-proton symport.Abbreviations CCCP carbonylcyanide m-chlorophenylhydrazone - DCCD N,N-dicyclohexylcarbodiimide - DES diethylstilbestrol - EB erythrosin B - Em membrane potential - FC fusicoccin - HCF II hexacyanoferrate II - HCF III hexacyanoferrate III - Mes 2-(N-morpholino)ethanesulfonic acid - pmf proton motive force - TPP⁺ tetraphenylphosphonium ion 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 indebted to Stella Savory for preparing the ultrathin sections for electron microscopy.     

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     

Transfer cell induction in cotyledons ofVicia faba L.

Summary Immediately prior to seed fill, a dermal transfer cell complex, comprised of epidermal and subepidermal cells, differentiates on the abaxial surface of the cotyledons in seed ofVicia faba. Over the period of differentiation of this complex in vivo, the principal sugars of the seed apoplasmic sap change from hexoses, glucose and fructose, to sucrose. Cotyledons were removed from seeds before differentiation of the transfer cell complex and cultured for 6 days on an agar-based medium in the dark with their abaxial surface in contact with a medium containing either 100 mM hexoses (glucose and fructose in equimolar concentrations) or 100 mM sucrose. On both media, cotyledon growth rate was maintained throughout the culture period at, or above, that of in vivo grown cotyledons of equivalent developmental age. When cotyledons were cultured on a medium containing glucose and fructose, epidermal cells of both the ab- and adaxial surfaces developed wall ingrowths on their outer periclinal walls and their cytoplasm became dense, vesicular, and rich in mitochondria. Extensive ingrowth deposition also occurred on walls of the subepidermal cells and several rows of underlying storage cells where they abutted intercellular spaces. This latter ingrowth development was apparent on both cotyledon surfaces, but extended into more of the underlying cell layers on the abaxial surface at the funicular end of the cotyledon. In in vivo grown cotyledons, such ingrowth development is restricted to the subepidermal cells of the abaxial surface. Ingrowth morphology was commensurate with that of transfer cells of in vivo grown cotyledons. In contrast to the observed induction on a medium containing glucose and fructose, cotyledons cultured with sucrose as the sole sugar source exhibited no ingrowth deposition or small wall ingrowths in some abaxial epidermal cells. While the potential sugar signalling mechanism is unknown, this culture system offers an exciting opportunity to explore the molecular biology of transfer cell development.Abbreviations DAA days after anthesis - GC-MS gas chromatography and mass spectrometry - PAR photosynthetically active radiation - RGR relative growth rate - SCM standard culture medium     

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.     

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.     

Cell-type specific,coordinate expression of two ADP-glucose pyrophosphorylase genes in relation to starch biosynthesis during seed development of Vicia faba L.

Several cDNA clones encoding two different ADP-glucose pyrophosphorylase (AGPase, EC 2.7.7.27) polypeptides denoted VfAGPC and VfAGPP were isolated from a cotyledonary library of Vicia faba L. Both sequences are closely related to AGPase small-subunit sequences from other plants. Whereas mRNA levels of VfAGPP were equally high in developing cotyledons and leaves, the mRNA of VfAGPC was present in considerable amounts only in cotyledons. During development of cotyledons, both mRNAs accumulated until the beginning of the desiccation phase and disappeared afterwards. The increase of AGPase activity in cotyledons during the phase of storage-product synthesis was closely followed by the accumulation of starch. The AGPase activity in crude extracts of cotyledons was insensitive to 3-phosphoglycerate whereas the activity from leaves could be activated more than five-fold. Inorganic phosphate inhibited the enzyme from both tissues but was slightly more effective on the leaf enzyme. There was a correlation at the cellular level between the distribution of VfAGPP and VfAGPC mRNAs and the accumulation of starch, as studied by in-situ hybridisation and by histochemical staining in parallel tissue sections of developing seeds, respectively. During the early phase of seed development (12–15 days after fertilization) VfAGPase mRNA and accumulation of starch were detected transiently in the hypodermal, chlorenchymal and outer parenchymal cell layers of the seed coat but not in the embryo. At 25 days after fertilization both synthesis of VfAGPase mRNA and biosynthesis of starch had started in parenchyma cells of the inner adaxial zone of the cotyledons. During later stages, the expression of VfAGPase and synthesis of starch extended over most of the cotyledons but were absent from peripheral cells of the abaxial zone, provascular and procalyptral cells.Abbreviations AGPase ADP-glucose pyrophosphorylase - DAF days after fertilization - Glc1P glucose-1-phosphate - 3-PGA 3-phosphoglycerate - VfAGPC AGPase subunit of Vicia faba mainly expressed in cotyledons - VfAGPP AGPase subunit of Vicia faba mainly expressed in leaves and cotyledons - pVfAGPC, pVfAGPP plasmids containing VfAGPC and VfAGPP, respectively This work was supported by the Bundesministerium für Forschung und Technologie BCT 0389, Molekular- und Zellbiologie von höheren Pflanzen und Pilzen. U.W acknowledges additional support by the Fonds der chemischen Industrie. We thank Elsa Fessel for excellent technical assistance.     

Expression patterns and subcellular localization of a 52 kDa sucrose-binding protein homologue of Vicia faba (VfSBPL) suggest different functions during development

A cDNA coding for a 54 kDa signal sequence containing protein has been isolated from a faba bean cotyledonary library and characterized. The deduced protein is designated Vicia faba SBP-like protein (VfSBPL) since it shares 58% homology to a 62 kDa soybean (Glycine max) protein (GmSBP) which has been described as a sucrose-binding and sucrose-transporting protein (SBP). VfSBPL as well as GmSBP are outgroup members of the large vicilin storage protein family. We were unable to measure any sucrose transport activity in mutant yeast cells expressing VfSBPL. During seed maturation in late (stage VII) cotyledons mRNA was localized by in situ hybridization in the storage parenchyma cells. At the subcellular level, immunolocalization studies proved VfSBPL accumulation in storage protein vacuoles. However, mRNA localization in stage VI cotyledons during the pre-storage/storage transition phase was untypical for a storage protein in that, in addition to storage parenchyma cell labelling, strong labelling was found over seed coat vascular strands and the embryo epidermal transfer cell layer reminiscent of sucrose transporter localization. The VfSBPL gene is composed of 6 exons and 5 introns with introns located at the same sites as in a Vicia faba 50 kDa vicilin storage protein gene. The time pattern of expression as revealed by northern blotting and the GUS accumulation pattern caused by a VfSBPL-promoter/GUS construct in transgenic tobacco seeds was similar to a seed protein gene with increasing expression during seed maturation. Our data suggest different functions of VfSBPL during seed development.     

The identification of gibberellins in immature seeds of Vicia faba,and some chemotaxonomic considerations

GA₁₇, GA₁₉, GA₂₀, GA₂₉, GA₄₄ and 13-hydroxy-GA₁₂, now named GA₅₃, were identified by GC-MS in immature seeds of Vicia faba (broad bean). Also identified were a GA catabolite, two polyhydroxykauranoic acids, and abscisic, phaseic and dihydrophaseic acids. The GAs of Vicia are hydroxylated at C-13, in common with those of other legumes. However the GAs of Vicia are not hydroxylated at C-3, nor do they appear to be readily conjugated. In these respects Vicia resembles Pisum, another member of the tribe Viciae. Vicia differs from Phaseolus and Vigna, of the tribe Phaseoleae, in both these respects.Abbreviations ABA abscisic acid - DPA dihydrophaseic acid - GA_n gibberellin A_n - GC gas chromatography - GC-MS gas chromatography mass spectrometry - KA kauranoic acid - PA phaseic acid - TLC thin layer chromatography     

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     

Polarized microtubule deposition coincides with wall ingrowth formation in transfer cells ofVicia faba L. cotyledons

Summary The epidermal transfer cells in developingVicia faba L. cotyledons are highly polarized. Extensive wall ingrowths occur on their outer periclinal walls and extend part way down both anticlinal walls. This ingrowth development serves to increase the surface area of the plasma membrane and thus maximize porter-dependent uptake of sugars from the seed apoplasm. In contrast, the inner periclinal walls of these transfer cells do not form wall ingrowths. We have commenced a study of the mechanisms responsible for establishing this polarity by first analysing the microtubule (MT) cytoskeleton in developing transfer cells. Thin sections of fixed cotyledons embedded in methacrylate resin were processed for immunofluorescence microscopy using monoclonal anti--tubulin and counterstained with Calcofluor White to visualize wall ingrowths. In epidermal cells of young cotyledons where wall ingrowths were yet to develop, MT labelling was detected around all cortical regions of the cell. However, in cells where wall ingrowths were clearly established, MT labelling was detected almost exclusively in cortical regions adjacent to the wall ingrowths. Little, if any, MT labelling was detected on the anticlinal or inner periclinal walls of these cells. This distribution of MTs was most prominent in cells with well developed wall ingrowths. In these cells, a subpopulation of MTs were also detected emanating from the subcortex and extending towards the wall ingrowth region. The possible role of MT distribution in establishing transfer cell polarity and wall ingrowth formation is discussed.Abbreviations MT microtubule     

Role of sugars in regulating transfer cell development in cotyledons of developing Vicia faba seeds

Summary. Transfer cell formation in cotyledons of developing faba bean (Vicia faba L.) seeds coincides with an abrupt change in seed apoplasm composition from one dominated by hexoses to one in which sucrose is the principal sugar. On the basis of these observations, we tested the hypothesis that sugars induce and/or sustain transfer cell development. To avoid confounding effects of in planta developmental programs, we exploited the finding that adaxial epidermal cells of cotyledons, which do not become transfer cells in planta, can be induced to form functional transfer cells when cotyledons are cultured on an agar medium. Growth rates of cotyledons cultured on hexose or sucrose media were used to inform choice of sugar concentrations. The same proportion of adaxial epidermal cells of excised cotyledons were induced to form wall ingrowths independent of sugar species and concentration supplied. In all cases, induction of wall ingrowths coincided with a marked increase in the intracellular sucrose-to-hexose ratio. In contrast, further progression of wall ingrowth deposition was correlated positively with intracellular sucrose concentrations that varied depending upon external sugar species and supply. Sucrose symporter induction and subsequent maintenance behaved identically to wall ingrowth formation in response to an external supply of hexoses or sucrose. However, in contrast to wall ingrowth formation, induction of sucrose symporter activity was delayed. We discuss the possibility of intracellular sugars functioning both as signals and substrates that induce and control subsequent development of transfer cells. Correspondence and reprints: School of Environmental and Life Sciences, Biology Building, University of Newcastle, Callaghan, NSW 2308, Australia.     

Comparison of K+-channel activation and deactivation in guard cells from a dicotyledon (Vicia faba L.) and a graminaceous monocotyledon (Zea mays)

We describe and compare inward and outward whole-cell K⁺ currents across the plasma membrane surrounding guard-cell protoplasts from the dicotyledon, Vicia faba, and the graminaceous monocotyledon, Zea mays. Macrosopic whole-cell current is considered in terms of microscopic single-channel activity, which involves discrete steps between conducting (open) and nonconducting (closed) states of the channel protein. Kinetic equations are used to model the number of open and closed states for channels conducting K⁺ influx (K(in)) and K⁺ efflux (K(out)) in the two species, and to calculate the rate at which open-closed transitions occur. The opening and closure of K(in) channels in both Vicia and Zea follow single-exponential timecourses, indicating that K(in)-channel proteins in each species simply fluctuate between one open and one closed state. In both species, opening of K(in) channels is voltage-independent, but closure of K(in) channels is faster at more positive membrane potentials. In response to identical voltage stimuli, K(in) channels in Zea open and close approximately three times as fast as in Vicia. In contrast to K(in), K(out) channels in Zea open and close more slowly than in Vicia. The closure of K(out) channels follows a single-exponential timecourse in each species, indicating one open state. The kinetics of K(out)-channel opening are more complicated and indicate the presence of at least two (Vicia) or three (Zea) closed states. The authors thank Professor N.A. Walker and Dr. D.R. Laver for the use of laboratory equipment, for helpful discussion and for provision of the program, GETHH. Thanks also to Dr. R.J. Ritchie for assistance with statistical analyses and to Ms. Janet Sherwood for maintenance of Vicia and Zea plants. This work was supported by grants from the National Science Foundation (DCB-89-04041) and the McKnight Foundation (S.M.A) and by a Charles Gilbert Heydon Travelling Fellowship (K.F-G).     

Electrophoretic seed albumin patterns and species relationships inVicia sect.Faba (Fabaceae)

Electrophoretic analysis of seed albumins (PAGE) covered 173 accessions representing nine species ofVicia sect.Faba. The number of albumin bands recorded in particular species varied from three inV. eristaloides to 23 inV. faba; in total, 38 bands were distinguished in the investigated material. The examined species, exceptV. eristalioides, showed intraspecific variation with respect to the number and relative staining intensity of albumin bands; individual variation was especially marked inV. faba and inV. narbonensis. Hierarchical clustering of the investigated taxa was based onBhattacharyya distances calculated from the electrophoretic data. The taxa grouped in three main clusters.Vicia faba and the rather remotely relatedV. kalakhensis formed one cluster. The second cluster was composed ofV. narbonensis distantly related toV. hyaeniscyamus. The third cluster comprised three subgroups: 1.V. johannis, V. galilaea andV. serratifolia, 2.V. eristalioides, and 3.V. bithynica. The obtained results are discussed with reference to taxonomic relationships inVicia sect.Faba.     

Retrotransposon populations of <Emphasis Type="Italic">Vicia</Emphasis> species with varying genome size

The (non-LTR) LINE and Ty3-gypsy-type LTR retrotransposon populations of three Vicia species that differ in genome size (Vicia faba, Vicia melanops and Vicia sativa) have been characterised. In each species the LINE retrotransposons comprise a complex, very heterogeneous set of sequences, while the Ty3-gypsy elements are much more homogeneous. Copy numbers of all three retrotransposon groups (Ty1-copia, Ty3-gypsy and LINE) in these species have been estimated by random genomic sequencing and Southern hybridisation analysis. The Ty3-gypsy elements are extremely numerous in all species, accounting for 18–35% of their genomes. The Ty1-copia group elements are somewhat less abundant and LINE elements are present in still lower amounts. Collectively, 20–45% of the genomes of these three Vicia species are comprised of retrotransposons. These data show that the three retrotransposon groups have proliferated to different extents in members of the Vicia genus and high proliferation has been associated with homogenisation of the retrotransposon population.Electronic Supplementary Material Supplementary material is available for this article at .     

Purification of an endopeptidase involved with storage-protein degradation in Phaseolus vulgaris L. cotyledons

Phaseolin, the major seed storage protein of Phaseolus vulgaris L., is degraded in the cotyledons in the first 7–10 d following seed germination. We assayed cotyledon extracts for protease activity by using [³H]phaseolin as a substrate and then fractionated the digestion mixtures by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in order to identify the cleavage products. The cotyledons of 4-d-old seedlings contain an endopeptidase which cleaves the polypeptides of [³H]phaseolin (apparent molecular weights=51 000, 48 000, 46 000 and 43 000) into three discrete clusters of proteolytic fragments (M _rs=27 000, 25 000 and 23 000). Endopeptidase activity is not detected in the cotyledons until the protein content of these organs starts to decline, shortly after the first day of seedling growth. Endopeptidase activity increases to a maximum level in the cotyledons of 5-d-old seedlings and then declines to a minimum value by day 10. The enzyme was purified 335-fold by ammonium-sulfate precipitation, organomercurial-agarose chromatography, gel filtration and ion-exchange chromatography. The endopeptidase constitutes 0.3% of the protein content in the cotyledons of 4-d-old seedlings. It is a cysteine protease with a single polypeptide chain (M _r=30 000). Optimum hydrolysis of [³H]phaseolin occurs at pH 5. The enzyme is irreversibly inactivated at pH values above 7 and at temperatures above 45° C. The endopeptidase attacks only a limited number of peptide bonds in [³H]phaseolin, without causing any appreciable change in the native molecular weight of the storage protein. The endopeptidase is also able to hydrolyze the bean-seed lectin, phytohemagglutinin. Thus, this enzyme may play a general role in degrading cotyledon proteins of P. vulgaris following seed germination.Abbreviations Da dalton - DTT dithiothreitol - M _r apparent molecular weight - PAGE polyacrylamide gel electrophoresis - PHA phytohemagglutinin - SDS sodium dodecyl sulfate     

Pathways of starch and sucrose biosynthesis in developing tubers of potato (Solanum tuberosum L.) and seeds of faba bean (Vicia faba L.)

Tissue slices from developing potato tubers (Solanum tuberosum L.) and developing cotyledons of faba bean (Vicia faba L.) were incubated with specifically labelled [¹³C]glucose and [¹³C]ribose. Enriched[¹³C]glucose released from starch granules was analysed by nuclear magnetic resonance (NMR). Spectral analyses were also performed on sucrose purified by high-performance liquid chromatography. In both tissues a low degree of randomisation (< 11 % in potato and < 14% in Vicia) was observed between carbon positions 1 and 6 in glucose released from starch when material was incubated with [¹³C]glucose labelled in positions 6 and 1, respectively. Similarly, with [2-¹³C]glucose a low degree of randomisation was observed in position 5. These findings indicate that extensive transport of three-carbon compounds across the amyloplast membrane does not occur in storage organs of either species. This is in agreement with previously published data which indicates that sixcarbon compounds are transported into the plastids during active starch synthesis. When [1-¹³C]ribose was used as a substrate, ¹³C-NMR spectra of starch indicated the operation of a classical pentose-phosphate pathway. However, with [2-¹³C]glucose there was no preferential enrichment in either carbon positions 1 or 3 relative to 4 or 6 of sucrose and starch (glucose). This provides evidence that entry of glucose in this pathway may be restricted in vivo. In both faba bean and potato the distribution of isotope between glucosyl and fructosyl moieties of sucrose approximated 50%. The degree of randomisation within glucosyl and fructosyl moieties ranged between 11 and 19.5%, indicating extensive recycling of triose phosphates.Abbreviation NMR nuclear magnetic resonance We are grateful to Dr. George Ratcliffe for his critical reading of the text and Dr. Bernard Goodman for helpful suggestions on the NMR measurements. The research was funded by a European Economic Community research grant, which the authors duly acknowledge.     

Transplantation of isolated nuclei into plant protoplasts

The uptake of isolated nuclei from Vicia hajastana Grossh. cells into protoplasts of an auxotrophic cell line of Datura innoxia P. Mill. was induced under the influence of polyethylene glycol and Ca²⁺ at pH 6.8. The frequency of nuclear uptake varied from 0.8 to 2.3% and that of the recovery of prototrophic clones from 10^-5 to 6·10^-4. The prototrophic nuclear fusion products following nuclear uptake could be rescued by initial culture of the protoplasts in non-selective conditions and by the subsequent use of feeder cell layers to support the growth of surviving colonies on a selective medium. The presence of Vicia genomic DNA in some prototrophic clones was confirmed by dot-blot hybridization using Datura and Vicia DNA probes. In certain transformed clones, the recovery of prototrophy was accompanied by the restoration of morphogenetic potential. Welldeveloped shoots typical of wild-type Datura could be regenerated employing an appropriate regeneration medium.Abbreviations MS Murashige and Skoog (1962) - PEG polyethylene glycol