Seed enhancing treatments: comparative analysis of germination characteristics of 23 key herbaceous species used in European restoration programmes.

The response of seeds from 23 wild plant species to a range of seed enhancing treatments was studied. We tested the hypothesis that sensitivity of the 23 species to these compounds is related to their ecological niche. The three ecological niches considered were open land, open-pioneer and woodland. Hence, the germination of a species is likely adapted to different light conditions and other environmental signals related to the niche. As representatives of environmental signals, the effects of smoke-related compounds (karrikinolide, KAR1 ), nitrate and plant growth regulator (gibberellic acid, GA3 ) on germination were studied. Seeds were exposed to these additives in the imbibition medium; all described as germination cues. We also investigated the effect of light regimes and additives on germination parameters, which included final germination, germination rate and uniformity of germination. Seeds were placed to germinate under three light conditions: constant red light, constant darkness and 12 h white light photoperiod. We observed inhibition by KAR under light in some species, which may have ecological implications. The results showed that no single treatment increased the germination of all the tested species, rather a wide variation of responsiveness of the different species to the three compounds was found. Additionally, no interaction was found between responsiveness to compounds and ecological niche. However, species in the same ecological niche and dormancy class showed a similar responsiveness to light. Species that share a similar environment have similar light requirements for germination, while differences exist among species in their responsiveness to other germination cues.

Genetic and functional characterization of dpp genes encoding a dipeptide transport system in Lactococcus lactis.

The genes encoding a binding-protein-dependent ABC transporter for dipeptides (Dpp) were identified in Lactococcus lactis subsp. cremoris MG1363. Two (dppA and dppP) of the six ORFs (dppAdppPBCDF) encode proteins that are homologous to peptide- and pheromone-binding proteins. The dppP gene contains a chain-terminating nonsense mutation and a frame-shift that may impair its function. The functionality of the dpp genes was proven by the construction of disruption mutants via homologous recombination. The expression of DppA and various other components of the proteolytic system was studied in synthetic and peptide-rich media and by using isogenic peptide-transport mutants that are defective in one or more systems (Opp, DtpT, and/or Dpp). In peptide-rich medium, DppA was maximally expressed in mutants lacking Opp and DtpT. DppA expression also depended on the growth phase and was repressed by tri-leucine and tri-valine. The effect of tri-leucine on DppA expression was abolished when leucine was present in the medium. Importantly, the Dpp system also regulated the expression of other components of the proteolytic system. This regulation was achieved via the internalization of di-valine, which caused a 30-50% inhibition in the expression of the proteinase PrtP and the peptidases PepN and PepC. Similar to the regulation of DppA, the repressing effect was no longer observed when high concentrations of valine were present. The intricate regulation of the components of the proteolytic system by peptides and amino acids is discussed in the light of the new and published data.

Peptides and ATP binding cassette peptide transporters.

In this review our knowledge of ATP binding cassette (ABC) transporters specific for peptides is discussed. Besides serving a role in nutrition of the cell, the systems participate in various signaling processes that allow (micro)organisms to monitor the local environment. In bacteria, these include regulation of gene expression, competence development, sporulation, DNA transfer by conjugation, chemotaxis, and virulence development, and the role of ABC transporters in each of these processes is discussed. Particular attention is paid to the specificity determinants of peptide receptors and transporters in relation to their structure and to the mechanisms of peptide binding.

Combinatorial peptide libraries reveal the ligand-binding mechanism of the oligopeptide receptor OppA of Lactococcus lactis.

The oligopeptide transport system (Opp) of Lactococcus lactis has the unique capacity to mediate the transport of peptides from 4 up to at least 18 residues. The substrate specificity of this binding protein-dependent ATP-binding cassette transporter is determined mainly by the receptor protein OppA. To study the specificity and ligand-binding mechanism of OppA, the following strategy was used: (i) OppA was purified and anchored via the lipid moiety to the surface of liposomes; (ii) the proteoliposomes were used in a rapid filtration-based binding assay with radiolabeled nonameric bradykinin as a reporter peptide; and (iii) combinatorial peptide libraries were used to determine the specificity and selectivity of OppA. The studies show that (i) OppA is able to bind peptides up to at least 35 residues, but there is a clear optimum in affinity for nonameric peptides; (ii) the specificity for nonameric peptides is not equally distributed over the whole peptide, because positions 4, 5, and 6 in the binding site are more selective; and (iii) the differences in affinity for given side chains is relatively small, but overall hydrophobic residues are favored-whereas glycine, proline, and negatively charged residues lower the binding affinity. The data indicate that not only the first six residues (enclosed by the protein) but also the C-terminal three residues interact in a nonopportunistic manner with (the surface of) OppA. This binding mechanism is different from the one generally accepted for receptors of ATP-binding cassette-transporter systems.

On the binding mechanism of the peptide receptor of the oligopeptide transport system of Lactococcus lactis.

Lactococcus lactis degrades exogenous proteins such as beta-casein to peptides of 4-30 amino acids, and uses these as nitrogen sources. The binding protein or receptor (OppA(Ll)) of the oligopeptide transport system (Opp) of L.LACTIS: has the unique capacity to bind peptides from five up to at least 20 residues. To study the binding mechanism of OppA(Ll), nonameric peptides were used in which the cysteine at position 1, 3, 4, 5, 6, 7 or 9 was selectively labeled with either bulky and non-fluorescent or bulky and fluorescent groups. Also, nonameric peptides with a non-natural residue, azatryptophan, at positions 3 or 7 were used. The fluorescence of azatryptophan reports on the polarity of the environment. The studies indicate that the binding protein encloses the first six amino acids of the peptide, whereas the remaining residues stick out and interact with the surface of the binding protein. The peptide binding mechanism of OppA(Ll) is discussed in relation to known three-dimensional structures of members of this class of proteins, and an adaptation of the general binding mechanism is proposed.

Kinetics and structural requirements for the binding protein of the Di-tripeptide transport system of Lactococcus lactis.

The gene (dppA) encoding the binding protein of the di-tripeptide ABC transporter of Lactococcus lactis (DppA) was cloned under the control of the nisin promoter. Amplified expression ( approximately 200-fold increase) of the protein fused to a carboxyl-terminal six-histidine tag allowed the purification of DppA-(His)(6) by nickel-chelate affinity and anion-exchange chromatography. Ligand binding to DppA-(His)(6) elicited an electrophoretic mobility shift, a decrease in the intrinsic fluorescence, and a blue shift of the emission maximum. Each of these parameters detected conformational changes in the protein that reflect ligand binding, and these were used to determine the structural requirements of DppA-(His)(6) for binding peptides. The major features of peptide binding include (i) high affinity for di- and tripeptides, (ii) requirement of a free N-terminal alpha-amino group and an alpha-peptide bound contiguous with the N-terminal amino group, (iii) stereospecificity for L-isomers, and (iv) preference for dipeptides containing methionine or arginine, followed by hydrophobic tripeptides consisting of leucine or valine residues. Maximal binding affinity was detected at pH 6.0, and the K(d) for binding increased 1 order of magnitude for every unit increase in pH. This suggests that the ionization of protein residues (pK > 6.0) in or in close proximity to the binding site is critical in the binding mechanism.

Specificity mutants of the binding protein of the oligopeptide transport system of Lactococcus lactis.

The kinetic properties of wild-type and mutant oligopeptide binding proteins of Lactococcus lactis were determined. To observe the properties of the mutant proteins in vivo, the oppA gene was deleted from the chromosome of L. lactis to produce a strain that was totally defective in oligopeptide transport. Amplified expression of the oppA gene resulted in an 8- to 12-fold increase in OppA protein relative to the wild-type level. The amplified expression was paralleled by increased bradykinin binding activity, but had relatively little effect on the overall transport of bradykinin via Opp. Several site-directed mutants were constructed on the basis of a comparison of the primary sequences of OppA from Salmonella enterica serovar Typhimurium and L. lactis, taking into account the known structure of the serovar Typhimurium protein. Putative peptide binding-site residues were mutated. All the mutant OppA proteins exhibited a decreased binding affinity for the high-affinity peptide bradykinin. Except for OppA(D471R), the mutant OppA proteins displayed highly defective bradykinin uptake, whereas the transport of the low-affinity substrate KYGK was barely affected. Cells expressing OppA(D471R) had a similar K(m) for transport, whereas the V(max) was increased more than twofold as compared to the wild-type protein. The data are discussed in the light of a kinetic model and imply that the rate of transport is determined to a large extent by the donation of the peptide from the OppA protein to the translocator complex.

Kinetics and consequences of binding of nona- and dodecapeptides to the oligopeptide binding protein (OppA) of Lactococcus lactis.

The oligopeptide transport system (Opp) of Lactococcus lactis belongs to the class of binding protein-dependent ABC-transporters. This system has the unique capacity to mediate the uptake of peptides from 4 up to at least 18 residues. Kinetic analysis of peptide binding to the binding protein, OppA, revealed a relationship between the peptide dissociation constants and the length of the ligand. The dissociation constants varied from submicromolar for dodecapeptides to millimolar for pentapeptides. This implies that the residues 6-12 of the peptide contribute to the binding affinity, and, in contrast to the current views on peptide binding by homologous proteins, these residues must interact with OppA. Analysis of pre-steady-state kinetics of binding showed that the observed differences in the -values result primarily from variations in the dissociation rate constants. These results are discussed in relation to the affinity constant for transport of these substrates. Overall, the data suggest that the slow dissociation rate constants for the larger peptides are rate determining in the translocation of peptides across the membrane.

Kinetics and specificity of peptide uptake by the oligopeptide transport system of Lactococcus lactis.

To obtain amino acids for growth, Lactococcus lactis uses a proteolytic system to degrade exogenous proteins such as caseins. The extracellular cell wall-attached proteinase PrtP and the oligopeptide transport system Opp mediate the first two steps in the utilization of caseins. beta-Casein is degraded by PrtP to fragments of 5-30 amino acid residues, and only a limited number of peptides are selected from this pool for uptake via Opp. To study the specificity of Opp and the kinetics of peptide uptake in L. lactis in detail, we used the following strategy: (i) the Opp system was overexpressed; (ii) a 4-fold peptidase mutant was used that is unable to degrade KYGK; (iii) iodinated KYGK was used as the reporter peptide; (iv) libraries of peptides, in which one amino acid position is systematically varied, were used as competitive peptides; and (v) peptides were synthesized on the basis of the beta-casein degradation products, their inhibition of KYGK uptake was determined, and the uptake of these peptides was followed by high-performance liquid chromatography (HPLC). These studies indicate that (i) the Opp system can transport a broad range of peptides from 4 up to at least 18 residues with very little preference for particular side chains and (ii) the kinetics of peptide uptake differ for different substrates tested. Whereas class I peptides such as KYGK exhibit normal Michaelis-Menten kinetics, the level of uptake of the majority of peptides (class II) increases sigmoidally with concentration. Different models for explaining the apparent cooperative effects that are observed for peptide uptake are discussed.

Purification of a histidine-tagged plant plasma membrane H+-ATPase expressed in yeast.

In order to facilitate efficient purification of the plant plasma membrane H+-ATPase expressed in yeast, a recombinant H+-ATPase protein with an N-terminal affinity tag of six histidine residues was engineered. When expressed in yeast the recombinant protein accumulated in the endoplasmic reticulum in an active form and showed characteristics comparable with those of the wildtype plasma membrane H+ATPase (Km,ATP, 1.1 mM; pH optimum, 6.6). After solubilization of the membrane proteins from the endoplasmic reticulum with n-dodecyl-beta-d-maltoside, the recombinant protein was purified under nondenaturing conditions by chromatography on Ni2+-nitriloacetic acid-agarose. A fraction was obtained which contained 4.2% of the initial amount of the protein and 26.6% of the ATPase-activity present in the endoplasmic reticulum. The purified protein has a specific activity of 32.6 micromol min-1 mg protein-1 at pH 6.5 and 30 degrees C. This rate is equivalent to a molecular activity of 3400 min-1. The purified plasma membrane H+-ATPase could be reconstituted into liposomes and demonstrated in this configuration the ability to pump protons. The method proves to be a convenient and rapid method for the preparation of purified single isoforms and mutant proteins of the plant plasma membrane H+-ATPase in high and functional quantities. This method might also be useful for achieving purification of other P-type ATPases, normally expressed at very low levels in heterologous systems.

C-terminal deletion analysis of plant plasma membrane H(+)-ATPase: yeast as a model system for solute transport across the plant plasma membrane.

The plasma membrane proton pump (H(+)-ATPase) energizes solute uptake by secondary transporters. Wild-type Arabidopsis plasma membrane H(+)-ATPase (AHA2) and truncated H(+)-ATPase lacking 38, 51, 61, 66, 77, 92, 96, and 104 C-terminal amino acids were produced in yeast. All AHA2 species were correctly targeted to the yeast plasma membrane and, in addition, accumulated in internal membranes. Removal of 38 C-terminal residues from AHA2 produced a high-affinity state of plant H(+)-ATPase with a low Km value (0.1 mM) for ATP. Removal of an additional 12 amino acids from the C terminus resulted in a significant increase in molecular activity of the enzyme. There was a close correlation between molecular activity of the various plant H(+)-ATPase species and their ability to complement mutants of the endogenous yeast plasma membrane H(+)-ATPase (pma1). This correlation demonstrates that, at least in this heterologous host, activation of H(+)-ATPase is a prerequisite for proper energization of the plasma membrane.

Modulation of H+-ATPase Activity by Fusicoccin in Plasma Membrane Vesicles from Oat (Avena sativa L.) Roots (A Comparison of Modulation by Fusicoccin, Trypsin, and Lysophosphatidylcholine).

The fungal phytotoxin fusicoccin affects various transport processes in the plasma membrane of plant cells. The plasma membrane (PM) H+-ATPase (EC 3.6.1.35) seems to be the primary target of fusicoccin action. The kinetics of the stimulation of the PM H+-ATPase by fusicoccin was studied in PM vesicles isolated from oat (Avena sativa cv Adamo) roots by aqueous two-phase partitioning. Considerable stimulation of activity was observed only when roots were treated with fusicoccin prior to the PM isolation. Fusicoccin treatment shifted the pH optimum of the ATPase toward more alkaline values and increased Vmax. No effects on Km were observed. Treatment with trypsin resulted in stimulation of ATPase activity in control vesicles but not in the fusicoccin-treated vesicles. The characteristics of stimulation by trypsin in control vesicles were comparable with those of stimulation by fusicoccin. This result and the change of the polypeptide pattern on western blots suggest the involvement of the C-terminal inhibitory domain in the fusicoccin signal transduction chain. On the other hand, stimulation by lyso-PC demonstrated other characteristics than stimulation by fusicoccin. Lyso-PC was able to stimulate ATPase activity at both acidic and alkaline pH values. Kinetic analysis of the pH dependency curves revealed different mechanisms for activation by fusicoccin and by lyso-PC. Whereas fusicoccin shifted the pH dependency of formation of phosphorylated intermediate to more alkaline values, lyso-PC seemed to increase dephosphorylation independently of pH.

Compartmental analysis of amino-acid release from attached and detached pea seed coats.

Using the empty-seed-coat technique, we have studied the release of amino acids from pea (Pisum sativum L. cv. Marzia) seed coats, either attached to or detached from the mother plant, at various stages of development. During an experimental period of 8 h, about 70% of the amino-acid content of the seed coat was released into the solution with which the seed-coat cup was filled. Major components of the released amino-acid mixture were glutamine (25%), alanine (20%) and threonine (15%). At the end of the experimental period (t≥6 h) the amino-acid efflux from attached seed coats was approx. 0.15 µmol · h(-1)·(seed coat)(-1) greater than that from detached seed coats. This difference may be attributed to the import of amino acids into attached seed coats. It is equivalent to approx. 0.3 µmol N · h(-1) · (seed coat)(-1), whereas the nitrogen demand of the embryo amounted to 0.50 ± 0.04 µmol N · h(-1) · embryo(-1). The time course of the release from detached seed coats could be described by the sum of two exponentials with t1/2 = 0.4-0.7 h and t(1/2) = 3.3-6.9 h, respectively, which probably represent the emptying of the cytoplasmic and vacuolar compartments. During development the vacuolar amino-acid pool decreased considerably (from 6.2 to 1.4 µmol · (seed coat)(-1)), whereas the cytoplasmic pool was much more constant (2.1-1.2 µmol · (seed coat)(-1)). For attached seed coats it will be shown that the time course of the amino-acid release could be fully accounted for by the sum of two exponentials and a linear term, where the parameters of the exponentials were, within error, the same as for detached seed coats and the linear term represented the import of amino acids. The results will be discussed with reference to prevailing models of phloem unloading in the seed coat, and in relation to the flux of amino acids from seed coat to the developing embryo.

Osmosensitivity of Sucrose Uptake by Immature Pea Cotyledons Disappears during Development.

Sucrose uptake was studied in isolated, immature pea cotyledons (Pisum sativum L. cv Marzia) in relation to their developmental stage. During the developmental period examined the water content of the cotyledons decreased from approximately 80% "stage 1" to approximately 55% "stage 2". When assayed in an isotonic medium (400 osmoles per cubic meter) the influx capacity per gram fresh weight for sucrose was almost constant during this developmental period. The influx could be analyzed into a saturable component (K(m) approximately 9 moles per cubic meter; V(max) approximately 150 nanomoles per minute per gram fresh weight) and an unsaturable component (k(i) approximately 0.5 nanomoles per minute per gram fresh weight [per mole per cubic meter]). Incubation in a hypotonic medium reduced the sucrose influx in stage 1 cotyledons, up to 80% reduction at 0 milliosmole (medium without mannitol), but had no effect on sucrose uptake by stage 2 cotyledons. Reduced uptake in a hypotonic medium (100 osmoles per cubic meter) could be attributed to a lowering of the V(max) from 150 to 36 nanomoles per minute per gram fresh weight. During incubation of stage 1 cotyledons and stage 2-cotyledons in a hypotonic medium (200 osmoles per cubic meter) their volume increased by 16% and 5.6%, respectively, while the calculated turgor pressure increased from 0.2 to 0.6 megapascal for cotyledons of both developmental stages. Reduced sucrose influx in hypotonic medium, therefore, seems to be related to cell swelling (membrane stretching) rather than to increased turgor pressure.

Effects of medium osmolarity on the release of amino acids from isolated cotyledons of developing pea seeds : Evidence for vacuolar amino-acid release at increased turgor.

The release of endogenous amino acids from isolated, immature pea (Pisum sativum L. cv. Marzia) cotyledons was investigated in relation to their developmental stage and the osmolarity of the bathing medium. The water potential of the cotyledons was about-1.1 MPa from which it could be inferred that the osmolarity of their apoplastic fluids will be approximately 450 mosmol·l(-1). The time course of amino-acid release conformed to an exponential function. Rate constants of the release were in the range 0.3 to 0.9 · h (-1). No indication was found for increased permeability of the plasmamembrane for amino acids at low medium osmolarity. Rate constants were even 1.5-fold lower in 0 mM mannitol than in medium with 400 mM mannitol. This effect could be ascribed to reduced protein synthesis in hypotonic media. In the presence of 400 mM mannitol the release was nearly proportional to the total amino-acid pool of the cotyledons and ranged from 12% to 8% for the various developmental stages. Amino-acid release was stimulated by incubation in a hypotonic medium (< 400 mM mannitol), up to fourfold in a medium without mannitol where as much as 45% of the cotyledonary amino-acid content could be released. The extra aminoacid release induced by the hypotonic condition declined during development and eventually vanished completely. Release of amino acids into a medium with 400 mM mannitol was more selective than into a medium without mannitol. For instance, arginine was one of the main constituents of the cotyledonary amino-acid pool (19%) as well as of the released amino-acid mixture when the medium contained no mannitol (10%), whereas it was virtually absent when the medium contained 400 mM mannitol. As an overall interpretation of these results, it is proposed that the hypotonic condition greatly enhances the permeability of the tonoplast (not that of the plasmalemma) for amino acids so that the otherwise well-sequestered amino acids in the vacuole become available for release into the bathing medium.

Changing kinetics of L-valine uptake by immature pea cotyledons during development : An unsaturable pathway is supplemented by a saturable system.

The influx of L-[(14)C]valine was measured over a wide concentration range (1 µM to 100 mM) in immature, isolated pea (Pisum sativum L. cv. Marzia) cotyledons at two developmental stages, both in the absence and in the presence of 0.4 M mannitol. At an early developmental stage (water content of the cotyledons ≈80%) the valine influx was strictly proportional to the external amino-acid concentration over the whole concentration range (so-called linear component). This system renders the plasmalemma of the cotyledonary cells approximately 1000-fold more permeable to valine than the presumed basic permeability of the membrane for valine. At a later stage of development (water content of the cotyledons ∼55%) this transport pathway was supplemented by a saturable system (K m = 5mM; V max = 9.5 µmol · gFW(-1)). The saturable system emerged when the water content of the cotyledons was about 65%, and its activity increased steadily up to the latest developmental stage examined (water content of the cotyledons =50%). As a result, uptake rates of L-valine at low concentrations, expressed on a fresh-weight basis, increased about 15-fold in this phase of development. Low osmolarity of the bathing medium (0 mM mannitol) had no effect on the linear component, and reduced the uptake by the saturable component only slightly by increasing its K m from 3.8 mM to 5.6 mM. The possible role of the saturable system in the acquisition of amino acids by the developing embryo is discussed.

Depolarization of Cell Membrane Potential during Trans-Plasma Membrane Electron Transfer to Extracellular Electron Acceptors in Iron-Deficient Roots of Phaseolus vulgaris L.

Transfer of electrons from the cytosol of bean (Phaseolus vulgaris L.) root cells to extracellular acceptors such as ferricyanide and Fe(III)EDTA causes a rapid depolarization of the membrane potential. This effect is most pronounced (30-40 millivolts) with root cells of Fe-deficient plants, which have an increased capacity to reduce extracellular ferric salts. Ferrocyanide has no effect. In the state of ferricyanide reduction, H(+) (1H(+)/2 electrons) and K(+) ions are excreted. The reduction of extracellular ferric salts by roots of Fe-deficient bean plants is driven by cellular NADPH (Sijmons, van den Briel, Bienfait 1984 Plant Physiol 75: 219-221). From this and from the membrane potential depolarization, we conclude that trans-plasma membrane electron transfer from NADPH is the primary process in the reduction of extracellular ferric salts.