Apoplast Acidification in Growing Barley (Hordeum vulgare L.) Leaves

Apoplast acidification associated with growth is well documented in roots, coleoptiles, and internodes but not in leaves. In the present study, advantage was taken of the high cuticle permeability in the elongation zone of barley leaves to measure apoplast pH and growth in response to application of test reagents. The role of the plasma membrane H⁺-ATPase (PM-H⁺-ATPase) and K⁺ in this process was of particular interest. pH microelectrodes and an in vitro gel system with bromocresol purple as pH indicator were used to monitor apoplast pH. Growth was measured in parallel or in separate experiments using a linear variable differential transformer. Test reagents that blocked (vanadate) or stimulated (fusicoccin) PM-H⁺-ATPase or that reduced (Cs⁺, tetraethylammonium) K⁺ uptake were applied. Apoplast pH was lower in growing than in nongrowing leaf tissue and increased in the elongation zone with increasing apoplast K⁺. Vanadate increased apoplast pH and reduced growth, whereas fusicoccin caused the opposite effects. It is concluded that barley leaves exhibit acid-growth-type mechanisms in that apoplast pH is lower in elongating leaf tissue. Both growth and apoplast pH depend on the activity of the PM-H⁺-ATPase and K⁺ transport processes. However, not all of the growth displayed by leaves is dependent on a lower apoplast pH in the elongation zone; up to 50 % of growth is retained when apoplast pH in the elongation zone increases to a value observed in mature tissue.     

Lysine Biosynthesis in Barley (Hordeum vulgare L.)

Lysine biosynthesis in seedlings of barley (Hordeum vulgare L. var. Emir) was studied by direct injection of the following precursors into the endosperm of the seedlings: acetate-1-¹⁴C; acetate-2-¹⁴C; pyruvate-1-¹⁴C; pyruvate-2-¹⁴C; pyruvate-3-¹⁴C; alanine-1-¹⁴C; aspartic acid-1-¹⁴C; aspartic acid-2-¹⁴C; aspartic acid-3-¹⁴C; aspartic acid-4-¹⁴C; α-aminoadipic acid-1-¹⁴C; and α, ε-diaminopimelic acid-1-(7)-¹⁴C. The distribution of activity in the individual carbon atoms of lysine in the different biosynthetic experiments was determined by chemical degradation. The incorporation percentages and labeling patterns obtained are in agreement with the occurrence of the diaminopimelic acid pathway. The results do not fit the incorporation percentages and labeling patterns expected if the α-aminoadipic acid pathway was operating. However, the results show that barley seedlings are able to convert a small part of the α-aminoadipic acid administered directly to lysine.     

Proteins in Intercellular Washing Fluids from Leaves of Barley (Hordeum vulgare L.)

Primary leaves of barley were detached, infiltrated with variousbuffers, and centrifuged to yield ‘intercellular washingfluid’ (IWF). Effective pH control of the IWF was obtainedonly with Tris, among all buffers tried. In these liquids, upto 30 proteins were detected by gradient gel electrophoresis.Intracellular protein from injured cells at the cut ends ofleaves was present in IWF but did not contribute significantlyto the total protein recovered in this liquid. The yield ofprotein in the IWF depended on the buffer used for infiltrationand on the concentration of the buffer. Higher concentrationsof buffer yielded more protein. In other experiments leaves were infiltrated with Tris, centrifuged,and then infiltrated a second time with this buffer containingvarious concentrations of the zwitterionic detergent CHAPS,a sulphobetaine derivative of cholate. Gel electrophoresis ofthe IWF obtained after the second centrifugation revealed protein‘bands’ not detected when the detergent had beenomitted from the infiltration buffer. The electrophoretic patternsof protein ‘bands’ in the gels differed dependingon the CHAPS concentration used for infiltration. The effect of CHAPS on plasmalemma integrity was studied byobserving infiltrated tissue with the electron microscope andby treating isolated protoplasts with the detergent. After infiltrationwith CHAPS at 0.6 mM or 2.0 mM no plasmalemma breaks were detectedin leaves, and isolated protoplasts survived exposure to CHAPSat these concentrations for 2 h without bursting. Evidently,CHAPS at these low concentrations did not destroy the integrityof the plasmalemma; the additional protein recovered in theIWF under these conditions probably originated in the cell wall.Infiltration of leaves with 6.0 mM CHAPS resulted in breaksof the plasmalemma, in tissue collapse and leaf tip necrosis.Isolated protoplasts burst within minutes after being exposedto CHAPS at this concentration. Key words: Cell wall permeability, Intercellular space, Detergent, CHAPS, Protoplasts     

Lysine Catabolism in Barley (Hordeum vulgare L.)

Lysine catabolism in seedlings of barley (Hordeum vulgare L. var. Emir) was studied by direct injection of the following tracers into the endosperm of the seedlings: aspartic acid-3-(14)C, 2-aminoadipic acid-1-(14)C, saccharopine-(14)C, 2,6-diaminopimelic acid-1-(7)-(14)C, and lysine-1-(14)C. Labeled saccharopine was formed only after the administration of either labeled 2,6-diaminopimelic acid or labeled lysine to the seedlings. The metabolic fate of the other tracers administered also supported a catabolic lysine pathway via saccharopine, and apparently proceeding by a reversal of some of the biosynthetic steps of the 2-aminoadipic acid pathway known from lysine biosynthesis in most fungi. Pipecolic acid seems not to be on the main pathway of l-lysine catabolism in barley seedlings.     

Phosphoinositides in Barley (Hordeum vulgare L.) Aleurone Tissue

[3H]Inositol labeling of barley (Hordeum vulgare L. cv Himalaya) aleurone layers and analysis of phospholipids by deacylation revealed the presence of phosphatidylinositol (PtdIns), PtdIns3P, and PtdIns4P but not PtdInsP2 species. In contrast to an earlier report (P.P.N. Murthy, G. Pliska-Matyshak, L.M. Keranen, P. Lam, H.H. Mueller, N. Bhuvarahamurthy [1992] Plant Physiol 98: 1498-1501) systematic chemical degradation of PtdIns revealed no evidence of a second isomer of PtdIns. Evidence of the widespread occurrence of 3-phosphorylated PtdIns within the plant kingdom is presented.     

The Effect of Water Stress upon Polyamine Levels in Barley (Hordeum vulgare L.) Leaves

The effect of water stress on leaf polyamine content of fourHordeum vulgare varieties, Alger/Ceres, Palmella Blue, Rihaneand Roho, with different drought characters was studied After6 d without water Alger/Ceres, Palmella Blue and Rihane hadaccumulated putrescine, although only in concentrations up totwice those found in the controls, but Roho had a decreasedputrescine content. However, one common response was identified;the accumulation and subsequent loss of putrescine was dependentupon the maintenance and loss of leaf turgor respectively. Consequentlyvarietal differences in putrescine accumulation were relatedto water consumption rates and the extent of osmotic adjustment.Spermine behaved in a similar manner to putrescine but spermidinelevels always decreased. Polyamine levels were never high enoughto be an important component of solute accumulation. Prolinelevels were 150-fold higher and glycine-betaine levels 50-foldhigher than polyamines in stressed plants. Proline and glycine-betaineaccumulation occurred once a threshold turgor was reached, whichin Roho appeared to be a reduction by 0•2–0•25MPa or 30–40%. The importance of polyamine accumulationduring water stress is discussed. Key words: Hordeum vulgare, polyamine, water stress     

Characterization of the Major Protein Component from Aleurone Cells of Barley (Hordeum vulgare L.)

Comparison of the SDS-PAGE patterns of salt-soluble proteinsfrom aleurone protoplasts, enriched aleurone layers preparedby pearling, and isolated embryos of mature barley showed threegroups of bands that reacted with antiserum raised against the7S globulins of oat embryos. These had M_rs of about 50 000,40 000 and 25 000. The enriched aleurones also contained a thirdgroup of immunoreactive bands (M_r about 70 000), which did notco-purify when the proteins were fractionated by ammonium sulphateprecipitation, ion exchange chromatography and immunoaffinitychromatography. The purified protein gave a single sharp peakon RP-HPLC, and contained a fourth minor group of subunits ofM_r about 20 000, in addition to those of M_rs about 50 000, 40000 and 25 000. The holoprotein and the ‘major groups’ of subunitsall had similar major N-terminal amino acid sequences whichwere related to the N-terminal amino acid sequences of pea andbean vicilins, and to sequences in the putative N-terminal regionsof the mature 7S ¹-globulins of cottonseed, confirming the homologywith these groups of proteins. Key words: Barley, seeds, 7S globulin, vicilin, ¹-globulin, amino acid sequence, protein homology     

Inositol Trisphosphate Metabolism in Subcellular Fractions of Barley (Hordeum vulgare L.) Mesophyll Cells

Phosphatases in cytosolic fractions, vacuoles, and vacuolar membranes from barley (Hordeum vulgare L.) leaves were found to dephosphorylate inositol 1,4,5-trisphosphate (IP3). 1,4-inositol bisphosphate (1,4-IP2) is the main product of IP3 dephosphorylation by the cytosolic fraction. The activity was strictly Mg2+ dependent. In contrast, IP3 dephosphorylation activity of both the soluble vacuolar and the tonoplast fractions was inhibited up to 50% by Mg2+. When vacuolar membranes were incubated with IP3, 1,4-IP2 was produced only under neutral and slightly alkaline conditions. Under acidic conditions, however, dephosphorylation yielded putative 4,5-inositol bisphosphate. Li+ (20 mM) and Ca2+ (100 [mu]M) strongly inhibited activity in the soluble vacuolar fraction but had only a slight effect on the activities of the cytosolic and tonoplast fractions.     

大麦转基因座位结构的研究

利用质粒营救法获得基因枪法转化的4种转绿色荧光蛋白基因(green fluorescent protein,GFP)大麦的转基因座位序列,序列分析显示4种材料的转基因座位中均有完整栽体的串联重复现象,表明转基因整合是同源重组的结果.同时转基因座位中也存在不完整载体片段、基因组片段的混杂排列,说明转基因整合时也发生异常重组.微粒轰击的转基因整合是由异常重组和同源重组共同完成的.     

Heat Shock Response in Hypocotyl of Barley (Hordeum vulgare L.)

In vivo protein synthesis in barley (Hordeum vulgare L.) hypocotyl was maximum at 35°C and 40°C.HPLC analysis of soluble proteins showed 10 different types of proteins, out of which the peak corresponding to retention time 13.3 min was present at 25°C but was absent at 35°C and 40°C. Instead, another peak with retention time 13.7 min was noticed at 35°C and 40°C. Amino acid analysis showed that heat shock resulted in an increase in lysine and histidine and decrease in arginine. Heat shock also resulted in increase in peroxidase, protease and ribonuclease activity at 35°C and 37°C in comparison to 25°C. The incorporation of (3H)-uridine was significantly decreased at 37°C in comparison to 25°C.     

Flow Cytometric Analysis and Chromosome Sorting of Barley (Hordeum vulgare L.)

Flow cytometric analysis was systematically performed to optimize the concentration and duration of hydroxyurea (DNA synthesis inhibitor) and trifluralin (metaphase blocking reagent) treatments for synchronizing the cell cycle and accumulating metaphase chromosomes in barley root tips. A high metaphase index (76.5% in the root tip meristematic area) was routinely achieved. Seedlings of about 1.0-cm length were treated with 1.25 mM hydroxyurea for 14 h to synchronize the root tip meristem cells at the S/G2 phase. After rinsing with hydroxyurea, the seedlings were incubated in a hydroxyurea-free solution for 2 h and were treated with 1 microM trifluralin for 4 h to accumulate mitotic cells in the metaphase. The consistent high metaphase index depended on the uniform germination of seeds prior to treatment. High-quality and high-quantity isolated metaphase chromosomes were suitable for flow cytometric analysis and sorting. Flow karyotypes of barley chromosomes were established via univariate and bivariate analysis. A variation of flow karyotypes was detected among barley lines. Two single chromosome types were identified and sorted. Bivariate analysis showed no variation among barley individual chromosomes in AT and GC content.     

Control of Some Enzymes of Nitrogen Metabolism during Senescence of Detached Barley (Hordeum vulgare L.) Leaves

The effects of chloramphenicol, cycloheximide and kinetin onthe changes in activity of glutamate dehydrogenase (GDH), glutamatepyruvate transaminase (GPT), glutamate oxaloacetate transaminase(GOT) and nitrite reductase were studied during the senescenceof detached barley leaves in the light and dark. The four enzymesseemed to be synthesized at least during the first hours ofsenescence. The rate of synthesis of GDH was clearly higherthan that of its degradation, thus continuously increasing duringsenescence. Chloramphenicol and kinetin delayed the enzyme degradationprocesses of senescence in the dark. However, chloramphenicolaccelerated senescence in the light. Kinetin had no significanteffect on the enzyme activities in the light. Cycloheximidetreatments produced lower enzyme levels than their respectivecontrols in both the light and dark, but the enzyme levels werehigher in cycloheximide treated leaves in the light than inthe controls in water in the dark. The results are discussedwith reference to the requirement for protein synthesis in thedifferent processes of senescence. (Received August 17, 1981; Accepted February 22, 1982)     

Growth Regulators and the Effect of Barley Yellow Dwarf Virus on Barley (Hordeum vulgare L.)

Estimation of cell number in the third leaf of barley (Hordeumvulgare L. C I 666) infected with barley yellow dwarf virus(BYDV) showed a marked decrease in the mitotic activity of theinfected plants Assay of endogenous gibberellins revealed adecrease in the level of a substance corresponding to gibberellicacid (GA₃) in BYDV-infected plants No significant differencein the level of endogenous auxins was observed Application ofgibberellic acid to infected plants reversed the dwarfing effectbut the response was not significantly different from the responseof healthy plants and was found to be due to increased cellelongation. It is suggested that the dwarfing of BYDV-infectedplants is a result of reduced mitotic activity This may be relatedto the reduced level of endogenous gibberellins.     

Effects of Mannitol Pretreatment on Androgenesis of Barley (Hordeum vulgare L.)

The effects of mannitol pretreatment on androgenesis of barley were systematically studied in comparison with that of cold pretreatment and control. The results showed that mannitol pretreatment could significantly increase the frequency of pollen survival reaching 19.0% on the eighth day, while in cold pretreatment and control they were 8.4% and 6.6 %, respectively. Mannitol pretreatment could also improve the quality of pollen and inhibit starch production from microspore, which were quite advantageous to microspore division and development. The developing period was shortened 2--3 days as compared with cold pretreatment and control. The major developmental pathways of androgenesis after mannitol pretreatment were the equal division (B pathway). In addition, the majority of microspore nuclei were diploids. On the contrary, the major microspores pretreated with low temperature had fewer chromosomes than with mannitol pretreatment, the microspore nuclei were haploids.     

Growth and Development of Young Roots of Barley (Hordeum vulgare L.) Genotypes

Barley (Hordeum vulgare L.) genotypes from countries with aMediterranean climate grown in temperature-controlled glasshousein nutrient solution to determine whether the co-ordinationof root branching and growth found by other workers appliedto a wider of up to 14 genotypes. There was substantial variationin the number of seminal axes produced by the genotypes, rangingfrom about seven for Hoshimasari and Swanneck to about fourfor Gerbel 'B'. The number of nodal axes was linearly relatedto the number of leaves and typically between one and two mainstemleaves were required before nodal axes appeared. There weresmall genotypic differences in the number of axes produced perleaf with values ranging from 1·5 to 2·3. Theproduction and growth of lateral roots were coordinated so thatthe mean length of laterals generally increased with time. Landraces(Arabic abiad and Arabic aswad) produced more lateral rootswith a faster rate extension compared with other genotypes.The length and number of primary and secondary lateral rootswere related linearly, but no genotypic differences in thisrelation were evident. Length of primary lateral roots increasedmore rapidly than that of secondary lateral roots throughoutthe three to five leaf stage. The ratio of root weight to totalplant weight decreased with time but there were only small differenceswithin this range of genotypes.Copyright 1995, 1999 AcademicPress Barley, seminal axes, nodal axes, primary lateral roots, relative extension rates, relative multiplication rates     

Proton Flux and Elongation in Primary Roots of Barley (Hordeum vulgare L.)

The elongation zone of the primary root of barley (Hordeum vulgare L.) has been reported to be markedly basic in pH, in apparent contradiction of the acid-growth theory. We determined simultaneously the location of the elongation zone and the basic zone in these roots and found them indeed to be the same. However, sections of barley root elongation zones were found to respond to acidic, basic, and neutral solutions as predicted by the acid-growth theory.     

The Uptake and Transport of Chromium by Barley Seedlings (Hordeum vulgare L.)

Uptake of ⁵¹CrO^2–₄ by intact barley seedlings was linearover 24 h and was stimulated by Ca2⁺ but inhibited by SO^2–₄and other Group VI anions. Uptake increased with increasingchromate concentration, but unless the concentration was high(100 µM) less than 1 per cent of the isotope absorbedwas transported to the shoots. The results of solvent extraction,subcellular fractionation and efflux studies indicated thatmost of the isotope accumulated by the roots was present ina soluble non-particulate form in the vacuoles. The possiblereasons for the restriction in chromate transport are discussedin relation to the metabolism of the element.     

Properties and Regulation of Aspartate Kinase from Barley Seedlings (Hordeum vulgare L.)

Aspartate kinase (EC 2.7.2.4) has been purified 8-fold and characterized from germinating barley (Hordeum vulgare) seedlings. The enzyme is inhibited 50% by 0.7 mm l-lysine and almost completely at 5 mm. l-Methionine does not affect the enzyme on its own, but at low concentrations (0.1-1 mm) increases the inhibition in the presence of lysine, indicating that the two amino acids act as cooperative feedback regulators.     

Localization of ATPase Activity on the Plasmalemma of Scutellar Epithelial Cells of Germinating Barley (Hordeum vulgare L.)

Chaffey, N. J. and Harris, N. 1985. Localization of ATPase activityon the plasmalemma of scutellar epithelial cells of germinatingbarley (Hordeum vulgare L.).—J. exp. Bot 36: 1612–1619. ATPase activity has been localized at an ultrastructural levelin the absorptive region of the scutella of germinating barley(Hordeum vulgare L.). The enzyme is localized on the plasmalemmaof the epithelial cells. Using the Gomori reaction the depositionof reaction product on the plasmalemma, which is dependent uponthe presence of supplied ATP, was precluded or reduced by theinhibitors orthovanadate, mercuric chloride and DCCD, whilstß-glycerophosphate would not act as an alternativesubstrate. The mitochondria demonstrated phosphatase activitywith both ATP and ß-glycerophosphate as substrate.The results are discussed in relation to the active uptake ofmetabolites by the scutellum during germination and the structuralmodification of the plasmalemma of the epithelial cells to formplasmatubules. Key words: ATPase, Hordeum vulgare L., localization (ultrastructural)