The effect of chemical stress on the polypeptide composition of the intercellular fluid of barley leaves

The effect of chemical stress on the polypeptide composition of the intercellular fluid of barley (Hordeum vulgare L.) and tomato (Lycopersicon esculentum Mill.) leaves has been studied. In some dicotyledonous plant species, including tomato, exposure to chemical stress leads to the denovo synthesis of intercellular proteins known as pathogenesis-related proteins which have been implicated to be part of a defence mechanism. In barley, however, no such changes in the polypeptide composition of the intercellular fluid could be detected. On the other hand, similar stress conditions induce in barley a strong accumulation of mRNA encoding leaf-specific thionins. These barley thionins represent a novel class of cell-wall proteins toxic to phytopathogenic fungi and are possibly involved in the defence mechanism. These proteins could not be detected in tomato plants. In contrast to the pathogenesis-related proteins of dicotyledonous plants, the leaf-specific thionins of barley are not present in the intercellular fluid of leaves. These results indicate that barley may have evolved a different mechanism to cope with the presence of stress.Abbreviations PAGE polyacrylamide gel electrophoresis - PR pathogenesis-related - SDS sodium dodecyl sulfate     

Phospholipid changes in wheat and barley leaves under water stress

Total phospholipid content of leaves of wheat and barley increased and phospholipid components changed under water stress. Notable among these were the absence of phosphatidyl serine in barley varieties, decrease in phosphatidyl glycerol content in a less drought-tolerant variety of wheat (S-308) and barley (BG-25), and appearance of phosphatidic acid in both crops. The phospholipid content and its components did not return to normal upon release of the stress by subsequent irrigations. Such observations are indicative of water stress effected alterations in membranes.     

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.     

Plasma membrane vesicles from source and sink leaves : changes in solute transport and polypeptide composition

Plasma membrane vesicles (PMVs) were prepared by phase partitioning from microsomal fractions of either sink or source leaves of sugar beet (Beta vulgaris L.). The purity, the internal volume, the sidedness, and the sealingness of PMVs prepared from sink leaves did not differ from those measured with PMVs from source leaves. Yet, in response to an imposed proton motive force, PMVs from source leaves accumulated about 4-fold more sucrose than PMVs from sink leaves. The developmental stage did not affect the uptake of glucose and valine in PMVs prepared from leaf tissues. It was concluded that the sink/source transition is accompanied either by the incorporation into the plasma membrane of leaf cells of proteins mediating proton-sucrose cotransport, or by their activation. N-ethylmaleimide and a polyclonal ascitic fluid directed against the 42-kD region of the plasma membrane containing a putative sucrose carrier inhibited the uptake of sucrose in PMVs from source leaves, but not in PMVs from sink leaves. Sodium dodecyl sulfate gel electrophoresis and western blot suggested that the 42 polypeptide was more abundant in the PMVs from source leaves than in the PMVs from sink leaves.     

Diurnal carbohydrate metabolism of barley primary leaves

The carbohydrate content of barley (Hordeum vulgare L.) leaves was measured over a 24-hour cycle. Nonstructural carbohydrate accumulation was linear after the 1st hour of light, whereas utilization in the dark was fast initially and slowed as stored reserves were depleted. Sucrose was the most abundant storage form of carbohydrate in the primary leaf. Lesser amounts of starch, fructans, and hexoses were also present. Leaf reserves were almost completely remobilized by the end of the dark period. There was a lag in starch degradation following a light to dark transition. Lower rates of starch accumulation were observed at the beginning and at the end of the day. Fructan synthesis occurred primarily towards the end of the light period as rates of sucrose and starch synthesis decreased. The above results suggested that carbohydrate metabolism in primary barley leaves was controlled by light and by endogenous factors such as foliar sucrose levels. Measurements of specific [¹⁴C]sucrose activity in steady state labeled 7-day-old barley primary leaves suggested the presence of at least two kinetically separate pools. Sucrose levels were higher and apparent turnover rates were lower in barley leaves in comparison to previous studies with other species.     

Phosphatidylethanolamine in wheat and barley leaves under water stress

Phosphatidylethanolamine could not be detected in the leaves of less drought-tolerant varieties of wheat (S-308) and barley (BG-25) when the plants wer     

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.     

Effects of water stress on major photosystem II gene expression and protein metabolism in barley leaves

Although it has long been recognized that water deficit in plants reduces photosystem (PS) II mRNAs and proteins, the detailed mechanisms behind this have not been thoroughly elucidated. In the present study, effects of water stress in barley leaves on degradation of major PSII mRNA and dissociation and migration of PSII proteins were investigated. The results indicated that (1) the steady-state levels of major PSII mRNAs and proteins declined with increasing water stress, as a consequence of increased degradation; under severe water stress, the half-lives of D1 and D2 proteins decreased from 12–14 h to 7–8 h and the half-lives of psbA and psbD mRNA decreased from above 16 to 6–10 h; (2) monomerization of PSII were increased during water stress. Severe water stress accelerated turnover of PSII and inhibited PSII activities.     

Characterization of the epidermis from barley primary leaves

A method is described for isolating epidermal protoplasts from the primary leaves of barley (Hordeum vulgare L.). Epidermal protoplasts are lighter than mesophyll protoplasts because of their smaller ratio of cytoplasm to vacuole, and can be separated from the latter by density-gradient centrifugation after complete digestion of the leaves. We have started a basic characterization of the epidermal protoplast fraction in comparison with mesophyll protoplasts. Epidermal protoplasts had a mean diameter of 63.5 m, whereas that of mesophyll protoplasts was 35.7 m. Their respiratory oxygen consumption was not influenced by light. They contained acid hydrolases and cytoplasmic enzymes in relative activities different from those of mesophyll protoplasts. Their polypeptide pattern as judged from two-dimensional separations was, in principle, similar to that of mesophyll cells after elimination of the plastids from the latter by the preparation of vacuoplasts. However, in addition, a considerable number of epidermis-specific polypeptides were observed. Isolated epidermal protoplasts were viable and efficiently incorporated [³⁵S]methionine into newly synthesized proteins. The results show that epidermal protoplasts are suitable for the investigation of the physiological and molecular properties of epidermal cells in leaves.Abbreviation SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis We are grateful to Professor U. Heber (Lehrstuhl Botanik 1, Würzburg) for his continuous support. This work was supported by the DFG and the University of Würzburg within the Sonderforschungsbereich 176.     

Targeted modification of storage protein content resulting in improved amino acid composition of barley grain

C-hordein in barley and ω-gliadins in wheat are members of the prolamins protein families. Prolamins are the major component of cereal storage proteins and composed of non-essential amino acids (AA) such as proline and glutamine therefore have low nutritional value. Using double stranded RNAi silencing technology directed towards C-hordein we obtained transgenic barley lines with up to 94.7 % reduction in the levels of C-hordein protein relative to the parental line. The composition of the prolamin fraction of the barley parental line cv. Golden Promise was resolved using SDS-PAGE electrophoresis, the protein band were excised and the proteins identified by quadrupole-time-of-flight mass spectrometry. Subsequent SDS-PAGE separation and analysis of the prolamin fraction of the transgenic lines revealed a reduction in the amounts of C-hordeins and increases in the content of other hordein family members. Analysis of the AA composition of the transgenic lines showed that the level of essential amino acids increased with a concomitant reduction in proline and glutamine. Both the barley C-hordein and wheat ω-gliadin genes proved successful for RNAi-gene mediated suppression of barley C-hordein level. All transgenic lines that exhibited a reduction for C-hordein showed off-target effects: the lines exhibited increased level of B/γ-hordein while D-hordein level was reduced. Furthermore, the multicopy insertions correlated negatively with silencing.     

Alterations in glycolipids of wheat and barley leaves under water stress

Glycolipids of leaves from water-stressed and stress-recovered wheat and barley plants were studied. A decrease in the content of total glycolipid, mon     

Environmentally-induced changes in protein composition in developing grains of wheat are related to changes in total protein content

Nitrogen (N) nutrition, post-anthesis temperature and drought-induced changes in the kinetics of accumulation of dry mass, total grain N and protein fractions (albumins-globulins, amphiphils, gliadins, and glutenins) contents were examined for winter wheat (Triticum aestivum L.). Crops were grown in controlled environment tunnels in 1994 and 1998. In 1994, five post-anthesis temperatures averaging from 15-25 degrees C were applied during grain-filling. In 1998 two post-anthesis temperatures averaging 13 degrees C and 20 degrees C were applied and factorized with two post-anthesis water regimes. In 1994 crops also were grown in the field, where different application rates and timing of N nutrition were tested. When expressed in thermal time, the kinetics of accumulation of the protein fractions were not significantly affected by post-anthesis temperature or drought; whereas N nutrition significantly increased the rate and duration of accumulation of storage proteins. Albumin-globulin proteins accumulated during the early stage of grain development. The rate of accumulation of that fraction decreased significantly at c. 250 degrees Cd after anthesis, when the storage proteins (gliadins and glutenins) started to accumulate significantly. Single allometric relationships for the different environmental conditions exist between the quantity of each protein fraction and the total quantity of N per grain. From these results it was concluded that (1) the process of N partitioning is neither significantly affected by post-anthesis temperature or drought nor by the rate and timing of N nutrition and (2) at maturity, variations in protein fraction composition are mainly because of differences in the total quantity of N accumulated during grain-filling.     

Auxin-indnced changes in barley coleoptile cell wall composition

Auxin induces extension growth of barley coleoptile segments,causing cell extension and cell wall loosening represented bya change in mechanical properties of the cell wall. This responsedecreased after the segments were starved for more than 12 hrin buffer solution. Auxin decreased the noncellulosic glucosecontent of the cell wall of the segments starved for 0 and 6hr, but very little that of segments starved for 12 and 18 hr.The contents of arabinose, xylose and galactose, among noncellulosicpolysaccharides, and -cellulose of the cell wall increased duringthe starvation, but auxin did not affect them. The auxin-induceddecrease in glucose content was inhibited by nojirimycin, apotent inhibitor of ß-glucanase, which inhibited auxin-inducedextension and changes in mechanical properties of the cell wall,suggesting that cell wall loosening, and thus cell extension,resulted from partial degradation of ß-glucan of thecell wall. (Received April 20, 1978; )     

Hornet venom induces abundant expression of specific polypeptides in young barley leaves

Venom of Vespa crabro induced in barley an abundant expression of at least five polypeptides, having the same apparent molecular masses as the jasmonate-induced polypeptides. Treatment of venom by trypsin, as well as addition of indomethacin prevented the appearance of the polypeptides. Separation of the venom by gel-filtration chromatography showed that the fraction containing phospholipase activity was able to induce expression of the 23 kDa polypeptide as revealed by 2-D electrophoresis, but the induction was much stronger when this fraction was applied together with the fraction of the low molecular weight peptides. The treatment by venom did not promote senescence of the detached leaves as the jasmonate did and kept the photosynthesis, transpiration, protein content, and the intensity of labelled amino acid incorporation into proteins near the control values.     

High irradiance and water stress induce alterations in pigment composition and chloroplast activities of primary wheat leaves

Remarkable changes were observed in chlorophyll (Chl) (a+b), carotenoids (Car), and protein content of leaves and fluorescence emission, polarisation, excitation energy transfer, lipid peroxidation and DCPIP photoreduction activity in isolated chloroplasts of wheat leaves grown under moderate irradiance (MI, 15 W m⁻², control) and subsequently exposed to high irradiance stress (HIS, 250 W m⁻²), water stress (WS, 5 % aqueous polyethylene glycol-4000 solution) and HIS+WS simultaneously, during mature and senescence phase. In the stress exposed samples the Chl, Car and protein contents and kinetics of Hill activity significantly declined. Decrease in excitation energy transfer and increase in membrane polarisation and accumulation of malondialdehyde (MDA) in chloroplasts were also observed. The effect was more pronounced when the seedlings were treated with HIS+WS simultaneously. These observations suggest additive and a possible synergetic action of HIS and WS causing faster loss of pigments and protein content, intense changes in membrane properties including photochemical function, compared to samples exposed to either of the stresses individually.     

Lipid composition of plasma membranes from barley leaves and roots, spinach leaves and cauliflower inflorescences

Highly purified plasma membranes (PM) were obtained from barley (Hordeum vulgare L. cv. Kristina) leaves and roots, spinach (Spinacia oleracea L. cv. Viking II) leaves, and cauliflower (Brassica oleracea) inflorescences by partitioning in an aqueous polymer two-phase system. The sterol and polar lipid composition of the PM, including the fatty acid composition of the glycerolipids, was determined. Dominating lipids were free sterols, glucocerebroside, phosphatidylcholine (PC) and phosphatidylethanolamine (PE), although large variations in content were observed between the PM of the different species and organs. Thus, the spinach leaf PM contained only 7% (mol %) free sterol compared to over 30% free sterol in the other PM analysed, with the barley root PM as the other extreme (57% free sterol). On the other hand, sterol derivatives were more abundant in the spinach leaf PM, containing 13% acylated sterol glycosides. Cerebroside constituted 16% of the lipids in the barley leaf PM but only 3% in cauliflower. The phospholipids PC and PE ranged from 25 and 24%, respectively, in the spinach leaf PM to 8 and 7%, respectively, in the barley root PM. As a result of the large variations in sterol and phospholipid content, the ratio of free sterol to phospholipid varied from 2.2 in the barley root PM to only 0.1 in the spinach leaf PM. Sitosterol, campesterol and stigmasterol were the completely dominating sterols in the barley and cauliflower PM, whereas the unique sterol composition of spinach was dominated by spinasterol. Palmitic (16:0), linoleic (18:2) and linolenic (18:3) acid were the major glycerolipid fatty acids. The fatty acid composition of the barley root PM was the most saturated (44% 16:0, 13% 18:3), whereas that of the cauliflower PM was the most unsaturated (21% 16:0,42% 18:3). Thus, very large variations were observed in both total lipid and fatty acid composition of the PM investigated, which represent both mono— and dicotyledons, as well as both photosynthetic and non-photosynthetic tissue. The consequences of this large diversity in composition of the lipid bilayer for the function of integral PM proteins are discussed.     

Water stress enhances expression of an alpha-amylase gene in barley leaves

The amylases of the second leaves of barley seedlings (Hordeum vulgare L. cv Betzes) were resolved into eight isozymes by isoelectric focusing, seven of which were β-amylase and the other, α-amylase. The α-amylase had the same isoelectric point as one of the gibberellin-induced α-amylase isozymes in the aleurone layer. This and other enzyme characteristics indicated that the leaf isozyme corresponded to the type A aleurone α-amylase (low pI group). Crossing experiments indicated that leaf and type A aleurone isozymes resulted from expression of the same genes.

In unwatered seedlings, leaf α-amylase increased as leaf water potential decreased and ABA increased. Water stress had no effect on β-amylase. α-Amylase occurred uniformly along the length of the leaf but β-amylase was concentrated in the basal half of the leaf. Cell fractionation studies indicated that none of the leaf α-amylase occurred inside chloroplasts.

Leaf radiolabeling experiments followed by extraction of α-amylase by affinity chromatography and immunoprecipitation showed that increase of α-amylase activity involved synthesis of the enzyme. However, water stress caused no major change in total protein synthesis. Hybridization of a radiolabeled α-amylase-related cDNA clone to size fractionated RNA showed that water-stressed leaves contained much more α-amylase mRNA than unstressed plants. The results of these and other studies indicate that regulation of gene expression may be a component in water-stress induced metabolic changes.

     

Aging induced changes in photosynthetic electron transport of detached barley leaves

Primary leaf segments of 11-day-old seedlings of barley (Hordtumvulgare L. cv IB 65) were floated on distilled water in darknessat 25°C to induce senescence. This stress induced agingbrings significant loss in the total content of pigments, proteinsand nucleic acids (DNA, RNA) of the leaves and of chloroplastsisolated from the senescing leaves. Of the three macromolecularcomponents, RNA content of theisolated chloroplasts was foundmost susceptible to stress-induced aging. Loss of DCPIP Hill activity of the isolated chloroplasts couldbe correlated, in a general way, with the loss of pigments,proteins and nucleic acids of the leaves and chloroplasts isolatedfrom them. However, during the stress period, the ability ofdifferent exogenous electron donors like MnCl₂ and diphenylcarbazide(DPC) to feed electrons to Photo System II (PS II) was foundto be different. MnCl₂ supported photoreduction of DCPIP onlyup to the fourth day, whereas DPC sustained its ability to donateelectrons up to the seventh day of incubation of the leavesin darkness. These results suggest a sequential alteration ofthe sites in the electron-transport chain between H₂O and PSII reaction centers of chloroplasts during dark-induced senescence.Kinetin not only prevented the loss of pigments and proteinsduring senescence, but also preserved the integrity of the electron-transportchain. (Received November 15, 1975; )     

Lipid transfer protein genes specifically expressed in barley leaves and coleoptiles

Genes/cDNAs encoding so-called lipid-transfer proteins (LTPs) have been isolated from a variety of tissues from different plants, but the in-vivo function of the LTP proteins is not yet known. In barley (Hordeum vulgare L.), the LTP1 gene (encoding a probable amylase/ protease inhibitor, Mundy and Rogers 1986, Planta 169, 51–63) is active in aleurone tissue, and in this paper two LTP-encoding cDNAs isolated from green leaves are described. The encoded proteins start with signal sequences, they are 75% homologous to each other, 60–63% homologous to rice aleurone LTP and maize seed/ coleoptile LTP, but only 48% homologous to barley aleurone LTP. Northern hybridization experiments established that the two seedling-specific genes are both highly expressed in leaves and coleoptiles whereas the LTP1 gene is inactive in seedlings. No LTP gene expression was detected in roots using either seedling or aleurone cDNA clones as probes. Tissue-print hybridization indicates that the LTP genes are first expressed in young epidermal cells in leaves and coleoptiles, and subsequently expressed in the vascular strands. Genomic Southern analysis indicates that the barley LTP gene family has four to six members.Abbreviation LTP lipid transfer protein I thank Dr. J. Mundy, Carlsberg Research Laboratory, Copenhagen, Denmark for the PAPI cDNA clone and R. Barkardottir, Department of Molceular Biology, University of Aarhus, Denmark for providing RNA for some of the Northern analyses. I also thank I. Bjørndal and L. Kjeldbjerg for excellent technical assistance. This work was supported by the The Danish Biotechnology Programme.