Apparent transinhibition of peptide uptake in the scutellum of barley grain

The uptake of glycylsarcosine (Gly-Sar) into scutella separated from germinating grains of barley ( Hordeum vulgare L. cv. Himalaya) is inhibited by other peptides; in most cases the inhibition is not purely competitive but of a mixed type (simultaneous increase in the apparent K_m and decrease in V_max) (Sopanen, T. 1979. FEBS Lett. 108: 447–450). The aim of the present experiments was to elucidate the mechanism of the mixed inhibition by studying how peptides already taken up into the cells affect the uptake of Gly-Sar.
When scutella were preincubated in the presence of various peptides, 11 of the 13 peptides tested inhibited the subsequent uptake of Gly-Sar by 10 to 45%. The inhibition, studied in detail with leucylleucine and prolylproline, was due to a decrease in V_max. The two peptides having no effect were glycylglycine and D-alanyl-L-alanine which are the only peptides known to date acting as purely competitive inhibitors when present together with the substrate Gly-Sar.
Preincubation with leucine, proline and alanine was not inhibitory, although preincubation with the corresponding dipeptides was. This result, together with the demonstration of intact leucylleucine in the scutella after preincubation with leucylleucine, indicates that the inhibition was caused by the intact peptides.
The results support the notion that in the mixed type inhibition the increase in the apparent K_m is due to competition for the carrier at the outside of the membrane, while the decrease in V_max is due to peptides taken up and binding to the carrier at the inside of the membrane.     

Light-mediated release of phytosiderophores in wheat and barley under iron or zinc deficiency

The effect of varied light intensity (50 – 600 mol m-2 s-1) on the rate of phytosiderophore release was studied under zinc (Zn) deficiency using a bread (Triticum aestivum L. cv. Aroona) and a durum wheat cultivar (Triticum durum Desf. cv. Durati) differing in zinc (Zn) efficiency and under iron (Fe) deficiency using a barley cultivar (Hordeum vulgare L. Europe). Plants were grown under controlled environmental conditions in nutrient solution for 15 days (wheat plants) or 11 days (barley plants). Phytosiderophore release was determined by measuring capacity of root exudates to mobilize copper (Cu) from a Cu-loaded resin.With increasing light intensity visual Zn deficiency symptoms such as whitish-brown lesions on leaf blade developed rapidly and severely in wheat, particularly in the durum cultivar Durati. In wheat plants supplied well with Zn, increases in light intensity from 100 to 600 mol m-2 s-1 did not clearly affect the rate of phytosiderophore release. However, under Zn deficiency increases in light intensity markedly enhanced release of phytosiderophores in Zn-deficient Aroona, but not in Zn-inefficient Durati. When Fe-deficient barley cultivar Europe was grown first at 220 mol m-2 s-1 and then exposed to 600 mol m-2 s-1 for 24 and 48 h, the rate of release of phytosiderophores was enhanced about 4-fold and 7-fold, respectively. Transfer of Fe-deficient plants from 600 to 50 mol m-2 s-1 for 48 h reduced the rate of release of phytosiderophores by a factor of 7. The effect of light on phytosiderophore release was similar regardless of whether the rate of phytosiderophore release was expressed per plant or per unit dry weight of roots.The results demonstrate a particular role of light intensity in phytosiderophore release from roots under both Zn and Fe deficiency. It is suggested that in the studies concerning the role of phytosiderophore release in expression of Zn or Fe efficiency among and within cereals, a special attention should be given to the light conditions.     

Organic acids promote the uptake of lanthanum by barley roots

Organic acids play an important role in metal uptake by, and accumulation in, plants. However, the relevant mechanisms remain obscure. Acetic, malic and citric acids increased the uptake of lanthanum (La) by barley (Hordeum vulgare) roots and enhanced La content in shoots under hydroponic conditions. Concentration-dependent net La influx in the absence and presence of organic acids yielded nonsaturating kinetic curves that could be resolved into linear and saturable components. The saturable component followed Michaelis-Menten kinetics. The K(m) values were similar; however, the V(max) values in the presence of acetic, malic and citric acids were 4.3, 2.8, 1.5-times that of the control, respectively. Enhanced uptake of La by organic acids was mediated mainly, but not solely, by Ca(2+) channels. X-ray absorption spectroscopic techniques provided evidence of La-oxygen environment and established that La(III) was coordinated to 11 oxygen atoms that are likely to be involved in the binding of La(III) to barley roots via carboxylate groups and hydration of La(III).     

Sodium fluxes in corn roots: comparison to Cl and K fluxes, and to Na-fluxes in barley

Abstract. Carbonylcyanide, m -chlorophenyl hydra-zone (CCCP) decreased the ATP content of barley and corn roots by 80% within 5 min. The protonophore inhibited K and Cl absorption by largely unvacuolated root tips, and vacuolated root segments of barley and corn. The protonophore also inhibited Na absorption by root segments and Na extrusion by root tips of barley; it did not affect these Na fluxes in corn root tips and segments, and Na Influx in barley root tips. It was concluded that corn roots lack a metabolic mechanism for Na extrusion from the cytoplasm to the external solution or vacuole, which is functional in barley roots.     

Enhancement of ferric-mugineic acid uptake by iron deficient barley roots in the presence of excess free mugineic acid in the medium

To investigate the mechanism of mugineic acid-Fe^III uptake by barley roots, plasma membrane fractions were isolated from Fe-deficient barley roots using an aqueous two-phase partition method. Utilizing the plasma membrane vesicles, we developed an assay system for studying mugineic acid-⁵⁵Fe^III binding to the plasma membrane. However, no efficient active transport of mugineic acid-⁵⁵Fe^III into the plasma membrane vesicle was detected, because of large amount of non-specific adsorption of ⁵⁵Fe^III onto the vesicle. And the adsorption could be decreased by adding excess amount of free mugineic acid to the assay system. From the results it is speculated that an excess of free mugineic acids is necessary in the medium for effective uptake of mugineic acid-Fe^III by Fe-deficient barley roots. Support for this speculation came from a multi-compartment transport box experiment with excised roots of Fe-deficient barley.Abbreviations CCCP carbonylcyanide-m-chlorophenylhydrazone - MA mugineic acid     

A specific transporter for iron(III)-phytosiderophore in barley roots

Iron acquisition of graminaceous plants is characterized by the synthesis and secretion of the iron-chelating phytosiderophore, mugineic acid (MA), and by a specific uptake system for iron(III)-phytosiderophore complexes. We identified a gene specifically encoding an iron-phytosiderophore transporter (HvYS1) in barley, which is the most tolerant species to iron deficiency among graminaceous plants. HvYS1 was predicted to encode a polypeptide of 678 amino acids and to have 72.7% identity with ZmYS1, a first protein identified as an iron(III)-phytosiderophore transporter in maize. Real-time RT-PCR analysis showed that the HvYS1 gene was mainly expressed in the roots, and its expression was enhanced under iron deficiency. In situ hybridization analysis of iron-deficient barley roots revealed that the mRNA of HvYS1 was localized in epidermal root cells. Furthermore, immunohistological staining with anti-HvYS1 polyclonal antibody showed the same localization as the mRNA. HvYS1 functionally complemented yeast strains defective in iron uptake on media containing iron(III)-MA, but not iron-nicotianamine (NA). Expression of HvYS1 in Xenopus oocytes showed strict specificity for both metals and ligands: HvYS1 transports only iron(III) chelated with phytosiderophore. The localization and substrate specificity of HvYS1 is different from those of ZmYS1, indicating that HvYS1 is a specific transporter for iron(III)-phytosiderophore involved in primary iron acquisition from soil in barley roots.     

Movement of fluorescein into isolated caryopses of wheat and barley

Abstract. The movement of fluorescein, a symplastic fluorescent tracer, into isolated caryopses of wheat and barley is described. The dye followed the pathway to the endosperm which has been proposed previously from anatomical studies, namely a movement from the phloem, through cells of the pigment strand and nucellar projection, followed by a radial spread of the dye from the endosperm cavity into the starchy endosperm. By contrast, the fluorochromes calcofluor white M2R and ANS remained confined to the apoplast and failed to cross the 'xylem discontinuity' at the base of the caryopses.     

Plasmodesmatal frequency in relation to short-distance transport and phloem loading in leaves of barley (Hordeum vulgare). Phloem is not loaded directly from the symplast

We investigated the phloem loading pathway in barley, by determining plasmodesmatal frequencies at the electron microscope level for both intermediate and small blade bundles of mature barley leaves. Lucifer yellow was injected intercellularly into bundle sheath, vascular parenchyma, and thin-walled sieve tubes. Passage of this symplastically transported dye was monitored with an epifluorescence microscope under blue light. Low plasmodesmatal frequencies endarch to the bundle sheath cells are relatively low for most interfaces terminating at the thin- and thick-walled sieve tubes within this C₃ species. Lack of connections between vascular parenchyma and sieve tubes, and low frequencies (0.5% plasmodesmata per μm cell wall interface) of connections between vascular parenchyma and companion cells, as well as the very low frequency of pore-plasmodesmatal connections between companion cells and sieve tubes in small bundles (0.2% plasmodesmata per μm cell wall interface), suggest that the companion cell-sieve tube complex is symplastically isolated from other vascular parenchyma cells in small bundles. The degree of cellular connectivity and the potential isolation of the companion cell-sieve tube complex was determined electrophysiologically, using an electrometer coupled to microcapillary electrodes. The less negative cell potential (average –52 mV) from mesophyll to the vascular parenchyma cells contrasted sharply with the more negative potential (–122.5 mV) recorded for the companion cell-thin-walled sieve tube complex. Although intercellular injection of lucifer yellow clearly demonstrated rapid (0.75 μm s^-1) longitudinal and radial transport in the bundle sheath-vascular parenchyma complex, as well as from the bundle sheath through transverse veins to adjacent longitudinal veins, we were neither able to detect nor present unequivocal evidence in support of the symplastic connectivity of the sieve tubes to the vascular parenchyma. Injection of the companion cell-sieve tube complex, did not demonstrate backward connectivity to the bundle sheath. We conclude that the low plasmodesmatal frequencies, coupled with a two-domain electropotential zonation configuration, and the negative transport experiments using lucifer yellow, precludes symplastic phloem loading in barley leaves.     

Uptake of amino acids and peptides by developing barley embryos

Developing embryos of barley ( Hordeum vulgare L. cv. Bomi) detached 21–27 days after anthesis took up 1 mM [¹⁴C]-glutamine at pH 5 and 30°C at a rate of about 20 nmol embryo^−l h⁻¹ (5 μol g⁻¹h⁻¹). The uptake was inhibited by about 50% by di-nitrophenol and by about 80% by 300 m M unlabelled glutamine or alanine. The bulk of the uptake appeared, therefore, to be due to carrier-mediated active transport. The pH optimum of the uptake was 4.5. Leucine, proline, lysine, arginine and as-paragine were taken up at approximately similar rates as glutamine, and they also inhibited the uptake of glutamine. This, suggests that the uptake of glutamine was at least partly due to an unspecific carrier(s) also shared by other amino acids. The embryos also took up the dipepti.de glycykarcosine; the rate was about 6 nmol embryo⁻¹h⁻¹ (1.5 μol g⁻¹h⁻¹) (2 mM glycylsarcosine, pH 4.5, 30°C). The uptake was inhibited by about 70% by dinitrophenol or by 300 m M glycylglycine. This indicates that the bulk of the uptake was due to carrier-mediated active transport. The pH optimum of the uptake was about 4.5.
The rates of glutamine and glycylsarcosine uptake increased during the early and middle stages of embryo development (until day 28 after anthesis), but decreased towards the end of the maturation of the grain. These changes, as well as the relatively high activities, suggest that carrier-mediated active uptake of amino acids, and possibly also that of peptides, plays a role in the nutrition of the developing embryo.     

Regeneration from intact and sectioned immature embryos of barley (Hordeum vulgare L.): the scutellum exhibits an apico-basal gradient of embryogenic capacity

Immature zygotic embryos from spring barley cv. Dissa were used to induce somatic embryogenenesis. Up to 158 germinated somatic embryos could be recovered per plated zygotic embryo. Critical factors for obtaining a high yield of regenerants were the size of the explant, the level of 2,4-D used for callus induction and the careful division of callus at each subculture. Use of microsections of immature embryos as explants revealed a pronounced gradient of callus formation and embryogenic response across the scutellum. Sections from the scutellar tissue at the coleoptilar end of the embryo gave the most callus and were highly embryogenic. The regeneration response of sectioned explants was comparable to that recovered from intact embryos of similar size.     

Location of sugars in barley seeds during germination by NMR microscopy

The distribution and fluctuation of sugars in germinating barley seeds were examined by ¹³C nuclear magnetic resonance (NMR) spectroscopy, ¹H-NMR imaging and ¹H-NMR localized spectroscopy in relation to morphology. Maltose, sucrose, fructose and oils were detected in intact imbibed seeds by ¹³C-NMR spectra. During the first 6 d of germination, the maltose content increased and the oil content gradually decreased, whilst the levels of sucrose and fructose remained constant. Sugars were located by ¹H-NMR images and ¹H-NMR localized spectra in the vascular bundle of the seeds as well as in the solubilized endosperm. They were also detected in the shoots. The sugars detected in an 80% ethanol shoot extract were sucrose and glucose, which were located in the vascular bundles but not in the mesophyll cells of the coleoptile. They were also located in the basal part of the shoot, but not above 7 mm from the scutellum. The data suggest that the sugars are primarily transported through the vascular bundles and, at the same time, rapidly incorporated into mesophyll cells in the leaves.     

Translocation of N to and from barley roots: its dependence on local nitrate supply in split-root culture

The relationship between availability of external nitrate and N translocation between root and shoot was studied in N-limited barley ( Hordeum vulgare L. cv. Golf). Nitrate-N was added at a relative rate (i.e. N added per unit time and unit N in plant biomass) of (1.09 da-¹, and distributed between the subroots at ratios of 50:50 or 80:20. The plants were grown for 13 days under these conditions of nitrate nutrition, and for another three days with the nitrate distribution reversed from 80:20 to 20:80. The nitrale-N doses thus experienced by individual subroots ranged from 2 to 11 mg N g-¹ root dry weight day-¹ . ¹⁵N-Nitrate labellings were performed after 2 to 3 and 12 to 13 days of nitrate nutrition. and 2 to 3 days after reversal of nitrate additions. For all treatments, between 60 and 82% of the absorbed label initially left the root, and between 25 and 55% of the label recovered in roots had been supplied (cycled) via the shoot. Labelling of xylem N at the end of the 24-h labelling period ranged from to 36 to 46% indicating that a substantial fraction of the N in the xylem had been absorbed by the plant prior to labelling. It is concluded that cycling of N to roots, and cycling of N in the plant as a whole, is substantial also during N-limited growth. N allocation to roots increased with external nitrate dose. An increased utilization of non-translocated N as well as an increased translocation of N from the shoot contributed to this effect. Thus, the results indicate that increased external availability of N also increased the sink strength of the root for cycling N.     

Contact with ballotini (glass spheres) stimulates exudation of iron reducing and iron chelating substances from barley roots

Contact between roots and Fe-containing solid substrate is known to facilitate acquisition of iron by plants, but the actual mechanism of this contact effect is not yet clear. This study was undertaken to evaluate the effect of root contact with ballotini (glass spheres) on exudation of substances capable of reducing or chelating insoluble Fe(III) compounds by the roots of barley (Hordeum vulgare L. cv. Europa) seedlings. Seedlings with roots encountering mechanical impedance (i.e., in contact with ballotini) produced more lateral roots than the seedlings with unimpeded (i.e., freely suspended) roots in the nutrient solution. Nutrient solution bathing roots in contact with ballotini showed higher concentrations of Fe(III)-chelating (83% on day 7) and Fe(III)-reducing (107% on day 12) substances than solutions bathing unimpeded roots. The pH of all solutions rose continuously during the course of the experiment but was always lower (by a nonsignificant degree) in the solutions with roots in contact with ballotini than in those with unimpeded roots. The data indicate that under natural soil conditions the amount of Fe-chelating and Fe-reducing root exudates may be higher than is usually measured from roots of terrestrial plants artificially suspended in nutrient solution.     

The effect of ammonium ions on membrane potential and anion flux in roots of barley and tomato

The effects of ammonium (0–5 mol m^?3) on root hair membrane potential and on the influx of nitrate and phosphate were investigated in roots of intact barley and tomato plants. In both species, addition of ammonium to the medium bathing the roots caused an almost immediate depolarization of the membrane potential; the depolarization was greater at higher concentrations of ammonium. Influx of ¹³NC₃^? and ³²Pi was inhibited over the same time scale and concentration range. In tomato roots, there was little further depolarization of the membrane potential or inhibition of anion influx at ammonium concentrations above 0.4 mol m^?3. In barley roots, the inhibition of nitrate influx and the depolarization of the membrane potential did not saturate below 5 mol m^?3 ammonium.     

Compartmental analysis of Rb+ efflux from roots of intact high-salt barley plants

The validity of compartmental analysis of Rb⁺ efflux from roots of intact high-salt barley plants ( Hordeum vulgare L. cv. Salve) was examined. ⁸⁶Rb⁺ was used as a tracer. Rb⁺ (1 m M ) was included together with 3 m M K⁺ in the growth medium, and steady-state conditions were assumed to prevail during the experiment. Three phases of efflux were revealed with half-times of 23 min, 109 min and 12 h, respectively; and the time span of the experiment had to be at least 20 h to make determination of the slow phase possible. We cannot state what compartments in the root the 3 different slopes represent. A comparison of slopes was made between the plots of In efflux vs time and In content vs time. In spite of correction for tracer transport from the roots to the shoot, the slopes for the slow phase did not agree unless up to 85% of the root content of Rb⁺ is assumed not to participate in efflux.     

Membrane- and cell wall-associated heat shock proteins in two genotypes of barley seedlings

ABSTRACT

The heat-shock (HS) response of two genotypes (cv. Onice, winter type, and cv. Georgie, spring type) of barley (Hordeum vulgare L.) was compared. Protein synthesis was markedly reduced by HS in roots and coleoptiles of both genotypes. The reduction in cv. Onice was higher than in cv. Georgie. The pattern of cytosolic, membrane and cell wall HSPs was analysed by SDS-PAGE in coleoptiles and roots of the two genotypes. Differences and similarities in coleoptiles and roots of the same genotype and between the two genotypes were observed. In roots of the genotype Onice, LMW and HMW HSPs isolated from the cytosol, membranes and cell walls were resolved into a diverse array of polypeptides by two-dimensional gel electrophoresis. Present results confirm the de novo synthesis of cytosolic and membrane HSPs, and demonstrate, for the first time, their presence in cell walls.