Changes in the protein composition of cytoplasmic ribsomes during the greening of etiolated barley leaves

We examined changes in the protein composition of cytoplasmic ribosomes in etiolated barley leaves following illumination. Cytoplasmic ribosomes were isolated from greening barley leaves by sucrose density gradient centrifugation, and were analyzed by radical-free highly reducing polyacrylamide gel electrophoresis (RFHR-PAGE). Eighty-nine proteins were resolved from the ribosomal fraction; among them, 8 proteins changed their copy numbers depending on the stage of greening. We designated these as phase dependent ribosomal proteins (PD1–PD8). Two of the proteins (PD1 and 5) present in the ribosomes of etiolated leaves showed a decrease in level during greening. In contrast, the levels of 6 ribosomal proteins (PD2, 3, 4, 6, 7 and 8) increased as greening proceeded. N-terminal amino acid sequence of PD8 showed high homology to rat ribosomal protein L34. The ribosomal proteins that appeared after illumination were not found in any fraction of the etiolated leaves, suggesting that they were synthesized after the onset of illumination. Copy numbers of other ribosomal proteins did not change during greening.     

Diurnal variations of nitrate reductase activity and stability in barley leaves

Diurnal variations of nitrate reductase (NR) activity and stability have been studied in leaves of barley seedlings ( Hordeum vulgare L. cv. Herta) grown in an 8 h light/16 h darkness regime. Stability (decay) of NR was tested both in the extracts and in the plants. In the morning, when the plants were transferred to light, NR activity increased rapidly during the first hour and then remained constant. After the photoperiod, activity decreased rapidly during the first hour of darkness and then remained fairly constant during the rest of the dark period. The high NR activity during the photoperiod was associated with low NR stability both in the extracts and in the plants. On the other hand the low NR activity during the dark period was associated with high stability in the extracts and in the plants.     

Regulation of nitrate and nitrite reduction in barley leaves

Reduction of nitrate and accumulation of nitrite were studied in barley (Hordeum vulgare L. cv. Gars Clipper ex Napier) leaf sections in the dark and in the light, under aerobic (air and mixtures of O₂ and N₂) or anaerobic (N₂) conditions. Oxygen prevented nitrite accumulation but had no effect on accumulated or infiltrated nitrite. Most of the nitrite accumulated under dark-anaerobic conditions was in the "cytoplasmic" (the cell section between the plasma lemma and the tonoplast) fraction of the tissue. Reduction of nitrate was stimulated by 2, 4-dinitrophenol in tissue under dark-air and by 3-(3', 4'-dichlorophenyl)-l, l-dimethyl urea (DCMU) and carbonyl cyanide m -chlorophenylhydrazone (CCCP) in tissue under all environmental conditions studied. Nitrite accumulated in the light in DCMU-treated tissue under N₂ or under aerobic conditions in the presence of CCCP. On its own, CCCP did not promote accumulation of nitrite in leaf sections under light-air. A model for the reduction of nitrate and nitrite is proposed.     

A minimal model of light-induced circadian rhythms of nitrate reductase activity in leaves of barley

Observed circadian rhythms of nitrate reductase (NR) (EC 1.6.6.1) activity in leaves of barley ( Hordeum vulgare L. cv. Herta) under continuous light conditions are described by a simple kinetic model. The oscillatory mechanism has been decomposed into the negative and positive feedback loops which are necessary according to present theories of chemical oscillating systems. Our results indicate that the decrease of NR activity in darkness can be considered as a reversible unimolecular conversion of the active form of NR into an inactive form, forming a negative stabilizing feedback loop. The light-induced increase of NR activity is related to a positive destabilizing feedback loop. In our treatment this process is represented as an autocatalytical reaction.     

Ultrastructural changes in aging leaves of a light-grown achlorophyllous mutant of barley

The pattern and sequence of cellular degradation during the course of leaf senescence remains obscure and the nature of the trigger that induces cell senescence is unknown. In order to probe the pre-mortem phase of senescence temporal changes in cell ultrastucture were studied in aging leaves of light-grown achlorophyllous Hordeum vulgare L. cv. Dyan mutant seedlings. Electron microscope examination of the ultrastructure of mesophyll cell plastids revealed the absence of ribosomes and a highly disorganized prolamellar body. Both the number and size of plastoglobuli increased with aging and this change coincided with depletion of starch grains and dilation of lamellar membranes. Aging of mesophyll cells occurred coincident with a decline in ribosome content of the cytoplasm and loss of matrix granularity. Loss of ribosomes associated with the outer nuclear envelope membrane and a reduction in chromatin were also apparent. Only after 10 days was there evidence of loss of internal membrane integrity and swelling of mitochondrial cristae. Compartmentation was thus maintained during the aging process with membrane dissolution occurring late in senescence. These results suggest that an inability to produce chlorophyll and carotenoids and form thylakoid stacks due to the absence of plastid ribosomes, contributes to the rapid onset of senescence in light-grown achlorophyllous seedlings. Furthermore, disruption of chloroplast ribosome synthesis/assembly may constitute part of the plastid signal involved in triggering cell senescence.     

Protein synthesis by chloroplasts during the senescence of barley leaves

Chloroplasts were isolated from senescent leaf segments of barley ( Hordeum vulgare L. var. Mozoncillo) and assayed for protein synthesis. Protein synthesis activity of the chloroplasts greatly increased after 10–20 h of incubation of leaf segments in the dark in spite of an intense degradation of chloroplast rRNA. The rise in the activity of protein synthesis was more pronounced when kinetin was present in the incubation medium. However, as deduced from SDS-polyacrylamide gel electrophoresis of the products, different proteins were synthesized under the two conditions of incubation of the leaf segments. The activity of protein synthesis of the chloroplasts decreased during the first hours of incubation of the leaf segments in the light.
Cutting and incubation in the dark of the leaf segments enhanced the synthesis of a few proteins also formed by chloroplasts in attached senescing leaves. Hormone and senescence treatments changed the type and the rate of the protein synthesized by chloroplasts, which suggests that hormones may control senescence through a modulation of the protein synthesized by the chloroplasts.     

Circadian rhythmicity of nitrate reductase activity in barley leaves

Nitrate reductase (EC 1.6.6.1) activity showed circadian rhythmicity in the first leaf of 8–11 days old barley ( Hordeum vulgare L. cv. Herta) plants. Circadian rhythms were found using both the in vitro and in vivo method for testing the enzyme activity. When the light intensity was reduced from 65 to 20 W m⁻², the amplitude was smaller and the oscillations were damped sooner. In continuous darkness nitrate reductase activity decreased in a two step process. Three different light qualities were tested which all gave the same results.     

Effects of low temperature acclimation upon photosynthetic induction in barley primary leaves

Oxygen evolution and chlorophyll fluorescence were measured in cold-hardened and unhardened leaves of barley ( Hordeum vulgare L. cv. Asa) during the induction period of photosynthesis. The lag phase of light-saturated photosynthesis was increased and steady-state rates of photosynthesis were higher in cold-hardened than in unhardened barley leaves. Fluorescence was quenched more rapidly during the first minutes of induction in hardened than unhardened leaves, largely because of greater energy-dependent quenching (q_E). Also, slow fluorescence transients through the M peak were delayed and less pronounced in cold-hardened than in unhardened leaves. Based upon the combined fluorescence and oxygen evolution data it was concluded that cold-hardening delayed light activation of the energy consuming carbon reduction cycle, thereby delaying the use of ATP and NADPH formed in the light reaction. Measurements of oxygen evolution and fluorescence kinetics during photosynthetic induction under oxygenic and anoxygenic conditions suggest that oxygen photoreduction is important for additional ATP generation during both the onset of photosynthetic carbon assimilation and during steady-state photosynthesis.     

XET-related genes and growth kinematics in barley leaves

DV, displacement velocity
GA₃, gibberellic acid
REGR, relative elemental growth rate
XET, xyloglucan-endotransglycosylase

Recently Schünmann et al. (1997 ; Plant, Cell and Environment 20, 1439–1450) investigated the correlation of spatial patterns of xyloglucan-endotransglycosylase (XET) activity, XET-related mRNAs, and growth in elongating barley ( Hordeum vulgare L.) leaves. Here, methodological difficulties in the kinematic growth analysis are discussed, and it is concluded that the role that XET-related gene activity plays in the control of spatial growth patterns remains undetermined.     

Transport-related amino acid metabolism in germinating barley grains

When eight [¹⁴C]-labelled amino acids were separately injected into the endosperm of germinating (4 days at 20°C) barley (Hordeum vulgare L. cv. Himalaya) grains, the label was rapidly taken up by the scutellum and further transported to the shoot and roots. Some of the amino acids (leucine, lysine and asparagine) were transported in an intact form through the scutellum to the seedling, whilst glutamic acid and aspartic acid were largely converted to glutamine in the scutellum. Proline was mainly transported unchanged, but a small part of the label appeared in glutamine. Arginine was mostly broken down in the scutellum, possibly providing ammonia for the synthesis of glutamine. During further transport in the seedling there was a partial transfer of label from glutamine to asparagine, particularly in the shoot. None of the amino acids used supplied carbon for the synthesis of sucrose, glucose or fructose. Glutamine synthetase activity was particularly high in the scutellum during the period of rapid amino acid transport.     

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.     

Variability in mesophyll conductance between barley genotypes,and effects on transpiration efficiency and carbon isotope discrimination

Leaf internal, or mesophyll, conductance to CO₂ (g_m ) is a significant and variable limitation of photosynthesis that also affects leaf transpiration efficiency (TE). Genotypic variation in g_m and the effect of g_m on TE were assessed in six barley genotypes (four Hordeum vulgare and two H. bulbosum). Significant variation in g_m was found between genotypes, and was correlated with photosynthetic rate. The genotype with the highest g_m also had the highest TE and the lowest carbon isotope discrimination as recorded in leaf tissue (Δ_p). These results suggest g_m has unexplored potential to provide TE improvement within crop breeding programmes.     

Preparation and polypeptide composition of chlorophyll-free plasma membranes from leaves of light-grown spinach and barley

Chlorophyf l-free preparations of plasma membranes from leaves of barley (Hordeum vulgare L. cv. Kristina) and spinach (Spinada oleracea L. cv. Viking II) were obtained by partition in an aqueous dextran-polyethylene glycol two-phase system. CJlu-can synthetase II (EC 2.4,1.34), a marker for the plasma membrane, was highly enriched in both preparations. Silicotungstic acid, a specific stain for the plasma membrane, indicated a purity close to 100% for the barley preparation. Both plasma membrane preparations contained a light-reducible b-cytochrome, as shown by low temperature spectroscopy. The plasma membranes had a tow protein content compared to the bulk of intracellular membranes. The polypeptide composition of the barley and spinach plasma membranes showed striking similarities, with.the most prominent polypeptides in the 49-58 kdalton region, and some further prominent bands in the 30 kcialton region. Some high molecular weight polypeptides in the 73-110 kdalton region were also typical for the plasma membranes compared to the microsomal fractions.     

Polyamines in discrete regions of barley leaves infected with the powdery mildew fungus, Erysiphe graminis

The concentrations of putrescine, spermidine and spermine and the activities of arginine decarboxylase (ADC; EC 4.1.1.19) and ornithine decarboxylase (ODC: EC 4.1.1.17) were determined in discrete regions of barley leaves ( Hordeum vulgare L. cv. Golden Promise) infected with the powdery mildew fungus ( Erysiphe graminis f.sp. hordei Marchal). Polyamine concentrations and the activities of both enzymes were always greatest within the region surrounding the fungal pustule, with the lowest values always being found in the region furthest away from the pustule. Although the concentrations of the three amines and ADC and ODC activities within the fungal pustule were always less than values from the zone surrounding the pustule, these differences were never significant. Polyamine concentrations and ODC activity were not significantly reduced, and ADC activity remained unchanged in mildewed leaves with all surface fungal growth removed. It would appear therefore that not only does most of the increase in amines and ODC activity reside in the leaf itself, but that very little of this increase is due to fungal growth and sporulation. Furthermore, it seems possible that the increase in polyamines in mildewed barley could be involved in 'green-island' formation, where regions around mildew pustules remain green and physiologically active while the rest of the leaf senesces.     

Diurnal variations of nitrite reductase, glutamine synthetase, glutamate synthase, alanine aminotransferase and aspartate aminotransferase in barley leaves

Diurnal variations of in vitro activity of 5 enzymes of nitrogen metabolism were studied. Barley ( Hordeum vulgare L. cv. Herta) seedlings were grown in 8 h short days, in daylight or under fluorescent lamps. During, the photoperiod nitrite reductase (EC 1.7.7.1) increased by an average of 18% in daylight and 10% under fluorescent lamps. Glutamine synthetase (EC 6.3.1.2) activity increased by 14 and 10%, respectively. The increase in enzyme activity reflected the overall increase in soluble proteins which was 8% in daylight and 3% under fluorescent lamps. Alanine aminotransferase (EC 2.6.1.2) increased by 82% in daylight and 37% under fluorescent lamps. Desalting of the extracts did not alter the enzyme activity and thus supported the assumption that changes in extractable enzyme activity are due to changes in the amount of (active) enzyme protein. Glutamate synthase (EC 1.4.7.1) activity did not show regular diurnal variations, and aspartate aminotransferase (EC 2.6.1.1) activity was almost constant.