Hordeum Vulgare Var Research Articles

Vanadate inhibits fructose-2,6-bisphosphatase and leads to an inhibition of sucrose synthesis in barley leaves

Vanadate (0.1–1 mM) was supplied to leaves of barley (Hordeum vulgare var. Roland) via the transpiration stream. It led to a selective inhibition of the rate of photosynthesis at high light without altering the initial slope of the light response curve, produced markedly biphasic photosynthesis induction kinetics, and selectively decreased sucrose synthesis compared to starch synthesis. There was a 3-fold increase of the steady state level of the signal metabolite fructose-2,6-bisphosphate in near saturating light. Fructose-2,6-bisphosphate is a potent inhibitor of cytosolic fruc-tose-l,6-bisphosphatase and, in agreement, the fructose-1,6-bisphosphatc level doubled. The increase of fructose-2,6-bisphosphate could not be accounted for by the known regulation of fructose-6-phosphate,2-kinase and fructose 2,6-bisphosphatase by 3-phosphoglycerate and fiuctose-6-phosphate, because these metabolites remained constant or even changed in the opposite direction to that required to generate an increase of fructose-2,6-bisphosphate. Instead, vanadate strongly inhibited the hydrolysis of fructose-2,6-bisphosphate in extracts, producing a half maximal inhibition at 2 \nM and 50 \iM in assays designed to preferentially measure the high-and low-affinity forms of fructose-2,6-bisphosphatase, respectively. Vanadale had no effect on fructosc-6-phosphate,2-kinase activity at these concentrations. Vanadate also led to a deactivation of sucrose phosphate synthase. The results are discussed in relation to the role of fructose-2,6-bisphosphate in regulating sucrose synthesis, and its interaction with the ‘coarse’ control of sucrose phosphate synthase.

Vanadate inhibits fructose‐2,6‐bisphosphatase and leads to an inhibition of sucrose synthesis in barley leaves

Vanadate (0.1–1 mM) was supplied to leaves of barley (Hordeum vulgare var. Roland) via the transpiration stream. It led to a selective inhibition of the rate of photosynthesis at high light without altering the initial slope of the light response curve, produced markedly biphasic photosynthesis induction kinetics, and selectively decreased sucrose synthesis compared to starch synthesis. There was a 3‐fold increase of the steady state level of the signal metabolite fructose‐2,6‐bisphosphate in near saturating light. Fructose‐2,6‐bisphosphate is a potent inhibitor of cytosolic fruc‐tose‐l,6‐bisphosphatase and, in agreement, the fructose‐1,6‐bisphosphatc level doubled. The increase of fructose‐2,6‐bisphosphate could not be accounted for by the known regulation of fructose‐6‐phosphate,2‐kinase and fructose 2,6‐bisphosphatase by 3‐phosphoglycerate and fiuctose‐6‐phosphate, because these metabolites remained constant or even changed in the opposite direction to that required to generate an increase of fructose‐2,6‐bisphosphate. Instead, vanadate strongly inhibited the hydrolysis of fructose‐2,6‐bisphosphate in extracts, producing a half maximal inhibition at 2 \nM and 50 \iM in assays designed to preferentially measure the high‐and low‐affinity forms of fructose‐2,6‐bisphosphatase, respectively. Vanadale had no effect on fructosc‐6‐phosphate,2‐kinase activity at these concentrations. Vanadate also led to a deactivation of sucrose phosphate synthase. The results are discussed in relation to the role of fructose‐2,6‐bisphosphate in regulating sucrose synthesis, and its interaction with the ‘coarse’ control of sucrose phosphate synthase.

Effects of La3+ on Surface Charges, Dielectrophoresis, and Electrofusion of Barley Protoplasts

When dielectrophoresis and electrofusion of barley (Hordeum vulgare var Moor) leaf protoplasts were assayed in the presence of 0.1 to 1 millimolar lanthanum ion (La(3+)) in the basal medium (0.7 molar mannitol, 1 millimolar piperazine-N, N-bis[2-ethanesulfonic acid]-Na [pH 6.7], 0.1 millimolar CaCl(2)), dielectrophoresis and induction of electrofusion were strongly inhibited. The latter remained inhibited and the former recovered by about 60% after washing the La(3+) -treated protoplasts without EDTA. These inhibitions were almost completely abolished by washing the La(3+) -treated protoplasts with 1 millimolar EDTA. Inductively coupled plasma atomic emission spectroscopic analysis revealed that protoplasts retained a considerable amount of La(3+) after washing without EDTA and released most of the bound La(3+) by washing with 1 millimolar EDTA. This tightly bound La(3+) seemed responsible for the inhibition of electrofusion and dielectrophoresis that was observed in the La(3+) -treated protoplasts after washing. zeta-potentials of protoplasts were -39.0+/-3.2 millivolts, -16.7 +/- 2.6 millivolts, and virtually zero in media containing 0, 0.1, and 0.3 millimolar La(3+) (I = 7.2 millimolar), respectively, and had a positive value (+ 14.2 +/- 2.2 millivolts) in the presence of 1 millimolar La(3+). These effects of La(3+) on zeta-potentials were easily abolished by washing without EDTA. This indicates that charged species located at the surface of plasma membrane of protoplasts cannot account for the sites at which La(3+) exerts its inhibition of dielectrophoresis and electrofusion. In contrast, the promotion of spherical fusion and the reduction of broken fusion products observed in the presence of La(3+) were almost completely abolished by washing without EDTA. Our results also indicate that the initial induction and development of electrofusion can be studied independently.

Reconstitution of the Light Harvesting Chlorophyll a/b Pigment-Protein Complex into Developing Chloroplast Membranes Using a Dialyzable Detergent

Conditions were developed to isolate the light-harvesting chlorophyll-protein complex serving photosystem II (LHC-II) using a dialyzable detergent, octylpolyoxyethylene. This LHC-II was successfully reconstituted into partially developed chloroplast thylakoids of Hordeum vulgare var Morex (barley) seedlings which were deficient in LHC-II. Functional association of LHC-II with the photosystem II (PSII) core complex was measured by two independent functional assays of PSII sensitization by LHC-II. A 3-fold excess of reconstituted LHC-II was required to equal the activity of LHC developing in vivo. We suggest that a linker component may be absent in the partially developed membranes which is required for specific association of the PSII core complex and LHC-II.

Relationship between Stress-Induced ABA and Proline Accumulations and ABA-Induced Proline Accumulation in Excised Barley Leaves

When excised second leaves from 2-week-old barley (Hordeum vulgare var Larker) plants were incubated in a wilted condition, abscisic acid (ABA) levels increased to 0.6 nanomole per gram fresh weight at 4 hours then declined to about 0.3 nanomole per gram fresh weight and remained at that level until rehydrated. Proline levels began to increase at about 4 hours and continued to increase as long as the ABA levels were 0.3 nanomole per gram fresh weight or greater. Upon rehydration, proline levels declined when the ABA levels fell below 0.3 nanomole per gram fresh weight.Proline accumulation was induced in turgid barley leaves by ABA addition. When the amount of ABA added to leaves was varied, it was observed that a level of 0.3 nanomole ABA per gram fresh weight for a period of about 2 hours was required before proline accumulation was induced. However, the rate of proline accumulation was slower in ABA-treated leaves than in wilted leaves at comparable ABA levels. Thus, the threshold level of ABA for proline accumulation appeared to be similar for wilted leaves where ABA increased endogenously and for turgid leaves where ABA was added exogenously. However, the rate of proline accumulation was more dependent on ABA levels in turgid leaves to which ABA was added exogenously than in wilted leaves.Salt-induced proline accumulation was not preceded by increases in ABA levels comparable to those observed in wilted leaves. Levels of less than 0.2 nanomole ABA per gram fresh weight were measured 1 hour after exposure to salt and they declined rapidly to the control level by 3 hours. Proline accumulation commenced at about 9 hours. Thus, ABA accumulation did not appear to be involved in salt-induced proline accumulation.

Steady State Proline Levels in Salt-Shocked Barley Leaves

Excised barley (Hordeum vulgare var Larker) leaves were treated with salt solutions or wilted. After the treatment period, the leaves were allowed to recover in a 50 millimolar sucrose and 1 millimolar glutamate solution, and proline, Na(+), and K(+) were measured at intervals. Na(+) and K(+) concentrations stayed at a constant high level after the salt treatments, and proline increased to a steady state concentration in response. The relationship between the maximum rate of proline accumulation and the Na(+) concentration reached in each experiment was linear. The final steady state proline concentration reached was also directly proportional to the Na(+) concentration. For a given Na(+) concentration in the leaves, the steady state proline level was greater when 410 millimolar NaCl was added to the leaves than when 205 millimolar NaCl was added. These results are consistent with proline acting as a compatible cytoplasmic solute, balancing an accumulation of salts outside of the cytoplasm.In contrast to the proline levels in salt-shocked leaves, the concentrations in wilted leaves decreased to near control levels within 24 hours of relief of stress.

Source of Endoproteolytic Activity Associated with Purified Ribulose Bisphosphate Carboxylase

The association of endoproteolytic activity with purified ribulose bisphosphate carboxylase (RuBPCase) from barley (Hordeum vulgare var Numar) leaves was investigated. RuBPCase purified by chromatography on agarose gel and diethylaminoethyl-cellulose was free of associated endoproteolytic activity. The addition of leupeptin and casein to the extraction buffer completely eliminated proteolysis during initial extraction of RuBPCase. Endoproteolytic activity previously found associated with RuBPCase was identified as being due to contamination of endoproteinases from broken vacuoles.

Effects of NaCl on Proline Synthesis and Utilization in Excised Barley Leaves

Proline accumulation in NaCl-treated excised barley (Hordeum vulgare var Larker) leaves was studied. Leaves were treated by placing the cut end in NaCl solutions and allowing the salt to enter the leaf via the transpiration stream. Leaves treated this way maintained turgor while the sodium content increased and the osmotic potential decreased. Proline began accumulating after 12 hours and continued accumulating over the subsequent 12-hour period at an average rate of 0.6 micromoles per hour per gram fresh weight.During the time proline was accumulating, [(14)C]glutamate was added to measure the effects of salt on proline synthesis from glutamate and [(14)C] proline was added in separate experiments to determine the effect of salt on proline utilization. Salt treatment dramatically increased proline synthesis from glutamate. Proline utilization by oxidation and for protein synthesis was decreased by 50 and 60%, respectively, by the salt treatment.These effects are similar to the effects of drought and abscisic acid in barley leaves. The results indicate that common mechanisms cause proline to accumulate under these different stresses.

Effect of Methionine Sulfoximine on the Accumulation of Ammonia in C3 and C4 Leaves

Additions of methionine sulfoximine (MSX), an inhibitor of glutamine synthetase (GS), result in an increase in NH(3) in seedling leaves of C(3) (wheat [Triticum aestivum cv. Kolibri] and barley [Hordeum vulgare var Perth]) and C(4) (corn [Zea mays W6A x W182E] and sorghum [Sorghum Vulgare var MK300]) plants. NH(3) accumulation is higher in C(3) (about 17.8 micromoles per gram fresh weight per hour) than in C(4) (about 4.7 micromoles) leaves. Under ideal conditions, when photosynthesis is not yet inhibited by the accumulation of NH(3), the rate of NH(3) accumulation is about 16% of the apparent rate of photosynthesis. A maximum accumulation of NH(3) was elicited by 2.5 millimolar MSX and was essentially independent of the addition of NO(3) (-) during either the growth or experimental period. When O(2) levels in the air were reduced to 2%, MSX resulted in some accumulation of NH(3) (6.0 micromoles per gram fresh weight per hour). At these levels of NH(3), there was no significant inhibition of rates of CO(2) fixation. There was also a minor, but significant, accumulation of NH(3) in corn roots treated with MSX. Inhibitors of photorespiration (isonicotinic hydrazide, 70 millimolar; 2-pyridylhydroxymethanesulfonic acid, 20 millimolar) or transaminase reactions (aminooxyacetate, 1 millimolar) inhibited the accumulation of NH(3) in both C(3) and C(4) leaves. These results support the hypothesis that GS is important in the assimilation of NH(3) in leaves and that the glycine-serine conversion is a major source of that NH(3).

Differences between Effects of Undissociated and Anionic 2,4-Dinitrophenol on Permeability of Barley Roots

Effects of 2,4-dinitrophenol (DNP) and several other substituted phenols on permeability of barley roots (Hordeum vulgare var. Trebi) to ions were assayed as a function of pH and phenol concentration. Solutions containing 0.1 micromolar undissociated DNP increase the permeability of barley root cells to small ions such as K(+), Na(+), Ca(2+), and Cl(-) with no inhibition of respiration. Undissociated forms of the other phenols increase permeability also, but they are less effective than DNP. Only the undissociated DNP is effective. Anionic DNP does not increase permeability or inhibit ion uptake, although it is the major species accumulated by the roots, both at pH 5 and pH 7. At pH 7, in contrast to pH 5, 10 micromolar DNP has no effect on ion permeability of barley roots yet it uncouples oxidative phosphorylation of barley root mitochondria. This indicates that the all too common use of DNP as a test for active transport or involvement of ATP synthesis can be misleading.

Effects of 2,4-Dinitrophenol on Membrane Lipids of Roots

Previous work has shown that the undissociated form of 2,4-dinitrophenol (DNP) increases the permeability of barley (Hordeum vulgare var. trebi) roots to ions. The present studies were undertaken to determine whether the effects of undissociated DNP were directly on membrane lipids. Relative amounts of the principal fatty acids from the lipids of barley root membranes were assayed as a function of DNP concentration, pH, and time of treatment under conditions similar to the previous studies of DNP effects on permeability. Undissociated DNP increases the proportions of palmitic and oleic acids and decreases linoleic and linolenic acids with no changes in the amounts of total fatty acids. The effects are immediate, as are the effects on permeability. Only the undissociated DNP is effective. Anionic DNP has no effect, although it is the major species taken up by the roots both at pH 5 and pH 7. DNP has no effect on respiration at either pH, indicating that undissociated DNP effects are on the membranes and not a general metabolic effect. The close parallelism between the effects of DNP on the composition of membrane lipids and on permeability suggests that the increase in permeability produced by undissociated DNP is due to a direct effect on the root membranes.

Identification of the Leaf Vacuole as a Major Nitrate Storage Pool

Highly purified vacuoles were isolated from protoplasts derived from green barley (Hordeum vulgare var. Numar) leaves, in order to determine their role as a NO(3) (-) storage sink. alpha-Mannosidase and acid phosphatase activities were used as markers to identify vacuoles, alpha-mannosidase being the more suitable. Nitrate and alpha-mannosidase, which were released from vacuoles destroyed during lysis of protoplasts, moved at unequal rates in the density gradient used for vacuole isolation. Purified vacuoles retained less NO(3) (-) than alpha-mannosidase during a single washing. Empirically determined corrections were used to account for NO(3) (-) movement in estimating the percentage of total cellular nitrate found in the vacuole. Vacuoles from plants grown in the presence of NO(3) (-) contained 58% of the total cellular NO(3) (-) and therefore represent a major NO(3) (-) storage pool.

Cadmium Accumulations and Bioavailability in Soils From Long‐Term Phosphorus Fertilization

AbstractSoils from citrus groves that had been fertilized with the equivalent of approximately 175 kg P/ha per year [as treble superphosphate (TSP)] over a 36‐year period were sampled and analyzed for total P, Cd, and Zn as well as water‐soluble P and Cd. A P fertilization rate of 175 kg/ha (about 400 kg P2O5/ha) is much higher than normal field rates, which are typically about 30 kg/ha. Concentrations of total Cd in surface soil were highly correlated (r = 0.89) with concentrations of total P. The concentrations of Cd in surface soil receiving broadcast P for 36 years averaged 1.0 µg/g, and were considerably greater than those of the controls, which showed a mean concentration of 0.07 µg/g. Phosphorus in soil saturation paste extracts ranged from 0.10 µg/ml (controls) to 8.87 µg/ml in P fertilized soils. Water‐soluble saturation extract Cd ranged from 0.008 µg/ml in controls to 0.017 µg/ml in fertilized soils, and was not well correlated with watersoluble P.Cadmium levels in barley (Hordeum vulgare var. U.C. 566) grain and leaves grown in the field on soil subject to long‐term heavy P fertilization were not elevated above levels in barley grown on the control soil. Swiss chard (Beta vulgaris var. cicla) was grown in the greenhouse on the above surface soils collected from the field. Although Cd levels averaging 1.6 µg/g in plant tissue were significantly elevated over those on the control soil (0.26 µg/gm), no yield depression was observed.Long‐term heavy application of TSP caused measurable increases in total Cd of surface soil, but the accumulated Cd burden per year was much lower than levels reported from application of sewage sludge. Cadmium accumulating in soils as a result of P fertilization seemed to be less available to Swiss chard than Cd accumulations resulting from applications of sewage sludge reported in the literature.

Characterization of Protoheme Levels in Etiolated and Greening Plant Tissues

The protoheme content of etiolated, greening, and fully greened bean (Phaseolus vulgaris L. var. Light Red Kidney) leaves has been studied. The protoheme level in etiolated and fully greened leaf tissue stays relatively constant from age 7 to 14 days. In agreement with the studies reported for barley (Castelfranco and Jones 1975 Plant Physiol 55: 485-490), the protoheme content of greening bean and barley (Hordeum vulgare var. Larker) leaves does not change appreciably during the first 9 hours of illumination, but the level rises significantly by the 24th hour of illumination (cf. Hendry and Stobart 1977 Phytochemistry 16: 1545-1548). This increase also occurs in seedlings returned to the dark for 24 to 48 hours following a 10-minute pulse of light. These results demonstrate a limited correlation with previous studies on the development of b-type cytochromes during greening of these tissues (Gregory and Bradbeer 1973; Planta 109: 317-326).

Production of Cell Wall Hydrolyzing Enzymes by Barley Aleurone Layers in Response to Gibberellic Acid

The cell walls of barley (Hordeum vulgare var. Himalaya) aleurone layers undergo extensive degradation during the tissue's response to gibberellic acid. Previous work had shown that these cell walls consist almost entirely of arabinoxylan. In this study we show that gibberellic acid stimulates endo-beta-1,4-xylanase activity in isolated aleurone layers. In addition, gibberellic acid enhances the activity of two glycosidases: beta-xylopyranosidase and alpha-arabinofuranosidase. No gibberellic acid-stimulated cellulase activity was detected. Germination studies showed a similar pattern of enzyme development in intact seeds.

Alkylguanidines as Inhibitors of K+ Transport in Isolated Barley Roots

It has been shown that plants can accumulate K(+) through an energy-dependent process. The effect of alkylguanidines, in particular octylguanidine on the uptake of (86)Rb(+) by excised barley roots (Hordeum vulgare var. Apizaco LV-72), has been studied. (86)Rb(+) was used as tracer of K(+). The uptake of (86)Rb(+) which is linear with time and shows saturation kinetics is inhibited by octylguanidine. Half-maximal inhibition of (86)Rb(+) uptake is attained at 50 muM octylguanidine. Octylguanidine induces a decrease in the V(max) of the process and increases the Km of the system for Rb(+). When the effects of various alkylguanidines were studied, the following order of effectiveness was encountered; octylguanidine = hexilguanidine > butylguanidine > ethylguanidine > guanidine. This suggests that guanidines inhibit Rb(+) uptake by interacting through its positively charged guanidinium group with a Rb(+) carrier while the alkyl chain interacts with the hydrophobic milieu of the membrane.

Interactions of Micronutrients in Barley Grown on Zinc‐polluted Soils

AbstractBarley (Hordeum vulgare var. Varde) grown in zinc‐polluted soils with variable levels of Zn, Mn, and Fe showed no marked differences in the yield attributable to these variables in both soils investigated. However, in direct contrast to results reported by other investigators, concentrations of 65Zn and total zinc were found to increase with increased levels of added Mn and/or Fe with few exceptions. Similarly, concentrations of 54Mn and total Mn also increased with increased levels of Zn and/or Fe. The effects of Zn and Mn on the concentrations of 59Fe and total Fe were inconsistent. The concentration of Mo was found to decrease with increased levels of Zn, Mn, and Fe and the decrease was more pronounced in one soil than in the other. Although concentration of Cu also showed a slight tendency to decrease with Zn, Mn, and Fe application, the results were inconsistent in both the soils. In general, labelled nutrient elements were more mobile in barley plants than corresponding soil‐derived nutrients as evident in the relative percentage distributions between roots and tops.The percentage distribution of Zn, Mn, Fe, Mo, and Cu between roots and tops was apparently not affected by the presence of their interacting ions. The effects of Zn on Mn distribution and that of Mn on Mo distribution, were, however, noticeable.It is concluded that though Zn, Fe, and Mn ions in barley appear to have limited influence on translocation within the plant, these ions exert strong synergistic effects at the site of transport. There appeared to be pronounced antagonism between Mo‐Zn, Mo‐Fe, and Mo‐Mn in the metabolism of barley.

Effect of Deuterium Water on the Mitotic Cycle, the Deoxyribonucleic Acid Stability, and the Frequency of Radiation-Induced Chromosome Aberrations in Barley

Hordeum vulgare var. Himalaya seeds were irradiated with gamma rays and hydrated in protium or deuterium water. The frequency of radiation-induced chromosome aberrations was lower in the deuterated material. The reduced frequency of chromosome aberrations was coupled with a delay of the early stages, an inhibition of the late stages of mitotic division, and an increase in the melting temperature (Tm) of the deoxyribonucleic acid. The decrease in the radiation-induced damage, in the presence of deuterium water, is attributed to the immobilization of the broken ends of the chromosomes due to the delay of the mitosis and the increased stability of the deoxyribonucleic acid molecule, both of them favoring the reconstitution of the broken ends.

The Structure of Plant Cell Walls

The walls of barley (Hordeum vulgare var. Himalaya) aleurone cells are composed of two major polysaccharides, arabinoxylan (85%) and cellulose (8%). The cell wall preparations contain 6% protein, but this protein does not contain detectable amounts of hydroxyproline. The arabinoxylan has a linear 1,4-xylan backbone; 33% of the xylosyl residues are substituted at the 2 and/or 3 position with single arabinofuranosyl residues. The results of in vitro cellulose binding experiments support the hypothesis that noncovalent bonds between the arabinoxylan chains and cellulose fibers play a part in maintaining wall structure. It is suggested that bonding between the arabinoxylan chains themselves is also utilized in forming the walls.

Use of Rubidium as a Chemical Tracer for Potassium in Long‐term Experiments in Cotton and Barley1

AbstractConsiderable controversy surrounds the technique of using Rb to chemically or isotopically label K. A series of experiments were conducted to determine the usefulness of this technique when other, potentially interactive, ions are present in the media and the plants are exposed to these solutions for extended periods.Cotton (Gossypium hirsutum var. ‘Acala, SJ‐1’) and barley (Hordeum vulgare var ‘Arivat’) were grown in the greenhouse for 45 days in culture solutions containing various ratios of Rb/K. Na and NH4 were added to determine the extent of the interaction of these cations with Rb and K.The distribution of Rb and K in different plant parts was determined chemically at the end of the growth period. Quantitative tagging of Rb for K depends upon factors of ionic concentration of both ions, the ratio of Rb/K in the substrate, plant species, and the presence of other monovalent cations, such as NH4 or Na ions.Roots of both cotton and barley plants retain Rb preferentially over K. Ammonium ions compete more with K than with Rb uptake. Cotton blades reflect the Rb/K ratios of the culture solution, with a maximum deviation of 20% over the fiftyfold range of K concentration examined.