Glucose-6-phosphate dehydrogenase in barley roots: kinetic properties and localisation of the isoforms

Two different isoforms of glucose-6-phosphate dehydrogenase (Glc6PDH; EC 1.1.1.49) have been partially purified from barley (Hordeum vulgare L., cv. Alfeo) roots. The procedure included an ammonium sulfate step, Q-Sepharose and Reactive Blue agarose chromatography, and led to 60-fold and 150-fold purification for the two enzymes, respectively. The Glc6PDH 1 isoform accounts for 17% of total activity of the enzyme in roots, and is very sensitive to the effects of NADP⁺/NADPH ratio and dithiothreitol; the Glc6PDH 2 isoform is less affected by reducing power and represents 83% of the total activity. The isoforms showed distinct pH optima, isoelectric points, K _m for glucose-6-phosphate and a different electrophoretic mobility. The kinetic properties for the two enzymes were affected by ATP and metabolites. Both enzymes are inhibited to different extents by ATP when magnesium is omitted from the assay mixture, whereas the addition of ATP-Mg²⁺ had no effect on Glc6PDH activities. The Glc6PDH isoforms are usually present in the plastids and cytosol of plant cells. To verify the intracellular locations of the enzymes purified from barley roots, Glc6PDH was purified from isolated barley root plastids; this isoform showed kinetic parameters coincident with those found for Glc6PDH 1, suggesting a plastid location; the enzyme purified from the soluble fraction had kinetic parameters resembling those of Glc6PDH 2, confirming that this isoform is present in the cytosol of barley roots. Received: 21 June 2000 / Accepted: 28 July 2000     

The stress- and abscisic acid-induced barley gene HVA22: developmental regulation and homologues in diverse organisms

Abscisic acid (ABA) induces the expression of a battery of genes in mediating plant responses to environmental stresses. Here we report one of the early ABA-inducible genes in barley (Hordeum vulgare L.), HVA22, which shares little homology with other ABA-responsive genes such as LEA (late embryogenesis-abundant) and RAB (responsive to ABA) genes. In grains, the expression of HVA22 gene appears to be correlated with the dormancy status. The level of HVA22 mRNA increases during grain development, and declines to an undetectable level within 12 h after imbibition of non-dormant grains. In contrast, the HVA22 mRNA level remains high in dormant grains even after five days of imbibition. Treatment of dormant grains with gibberellin (GA) effectively breaks dormancy with a concomitant decline of the level of HVA22 mRNA. The expression of HVA22 appears to be tissue-specific with the level of its mRNA readily detectable in aleurone layers and embryos, yet undetectable in the starchy endosperm. The expression of HVA22 in vegetative tissues can be induced by ABA and environmental stresses, such as cold and drought. Apparent homologues of this barley gene are found in phylogenetically divergent eukaryotic organisms, including cereals, Arabidopsis, Caenorhabitis elegans, man, mouse and yeast, but not in any prokaryotes. Interestingly, similar to barley HVA22, the yeast homologue is also stress-inducible. These observations suggest that the HVA22 and its homologues encode a highly conserved stress-inducible protein which may play an important role in protecting cells from damage under stress conditions in many eukaryotic organisms.     

Analysis of simple sequence repeats (SSRs) in wild barley from the Fertile Crescent: associations with ecology,geography and flowering time

Wild barley, Hordeum spontaneum C. Koch, is the progenitor of cultivated barley, Hordeum vulgare. The centre of diversity is in the Fertile Crescent of the Near East, where wild barley grows in a wide range of conditions (temperature, water availability, day length, etc.). The genetic diversity of 39 wild barley genotypes collected from Israel, Turkey and Iran was studied with 33 SSRs of known map location. Analysis of molecular variance (AMOVA) was performed to partition the genetic variation present within from the variation between the three countries of origin. Using classification tree analysis, two (or three) specific SSRs were identified which could correctly classify most of the wild barley genotypes according to country of origin. Associations of SSR variation with flowering time and adaptation to site-of-origin ecology and geography were investigated by two contrasting statistical approaches, linear regression based on SSR length variation and linear regression based on SSR allele class differences. A number of SSRs were significantly associated with flowering time under four different growing regimes (short days, long days, unvernalised and vernalised). Most of the associations observed could be accounted for by close linkage of the SSR loci to earliness per se genes. No associations were found with photoperiodic and vernalisation response genes known to control flowering in cultivated barley suggesting that different genetic factors may be active in wild barley. Novel genomic regions controlling flowering time in wild barley were detected on chromosomes 1HS, 2HL, 3HS and 4HS. Associations of SSRs with site-of-origin ecological and geographic data were found primarily in genomic regions determining plant development. This study shows that the analyses of SSR variation by allele class and repeat length are complementary, and that some SSRs are not necessarily selectively neutral.     

Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Specific Proteolysis in Barley Chloroplasts During Dark Induced Senescence

Intact chloroplasts were isolated from dark-senescing primary barley (Hordeum vulgare L.) leaves in order to study selective ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCO) degradation by the stromal and membrane fractions. RuBPCO specific degradation was estimated and characterised applying sensitive avidin-biotin ELISA method with non-modified or oxidatively modified biotinylated RuBPCO (BR) as substrates. Distinct proteolytic activities were detected. They differed in ATP and divalent metal ion dependence, protease inhibitory profile, and dynamics in the time-course of dark-induced senescence. The results supported involvement of ATP- and metal ion-dependent serine type proteolytic activity against non-modified BR early in induced senescence and appearance of ATP-independent activity at later stage. Active oxygen-modified BR was degraded by ATP-independent serine-type protease probably containing essential SH-groups and requiring divalent metal ions.     

Effects of Soil Drought and Atmospheric Humidity on Yield,Gas Exchange,and Stable Carbon Isotope Composition of Barley

The combined effects of water status, vapour pressure deficit (VPD), and elevated temperature from heading to maturity were studied in barley. Plants growing at high VPD, either under well-watered or water deficit conditions, had higher grain yield and grain filling rate than plants growing at low VPD. By contrast, water stress decreased grain yield and individual grain dry matter at any VPD. Water regime and to a lesser extent VPD affected ¹³C of plant parts sampled at mid-grain filling and maturity. The differences between treatments were maximal in mature grains, where high VPD increased ¹³C for both water regimes. However, the total amount of water used by the plant during grain filling did not change as response to a higher VPD whereas transpiration efficiency (TE) decreased. The net photosynthetic rate (P _N) of the flag leaves decreased significantly under water stress at both VPD regimes. However, P _N of the ears was higher at high VPD than at low VPD, and did not decrease as response to water stress. The higher correlation of grain yield with P _N of the ear compared with that of the flag leaf support the role of ear as the main photosynthetic organ during grain filling under water deficit and high VPD. The deleterious effects of combined moderately high temperature and drought on yield were attenuated at high VPD.     

Efficient fine mapping of the naked caryopsis gene (<Emphasis Type="Italic">nud</Emphasis>) by HEGS (High Efficiency Genome Scanning)/AFLP in barley

The hulled or naked caryopsis character of barley (Hordeum vulgare L.) is an important trait for edibility and to follow its domestication process. A single recessive gene, nud, controls the naked caryopsis character, and is located on the long arm of chromosome 7H. To develop a fine map around the nud locus efficiently, the HEGS (High Efficiency Genome Scanning) electrophoresis system was combined with amplified fragment length polymorphism (AFLP). From bulked segregant analysis of 1,894 primer combinations, 12 AFLP fragments were selected as linked markers. For mapping, an F₂ population of 151 individuals derived from a cross between Kobinkatagi (naked type) and Triumph (hulled type) was used. Seven AFLP markers were localized near the nud region. A fine map was developed with one-order higher resolution than before, along with the seven anchor markers. Among the seven linked AFLP markers (KT1–7), KT1, KT2 and KT6 were co-dominant, and the former two were detected for their single-nucleotide polymorphisms (SNPs) in the same length of fragments after electrophoresis with the non-denaturing gels of HEGS. The nud locus has co-segregated with KT3 and KT7, and was flanked by KT2 and KT4, at the 0.3-cM proximal and the 1.2-cM distal side, respectively. Four of these AFLP markers were converted into sequence-characterized amplified region (SCAR) markers, one of which was a dominant marker co-segregating with the nud gene.Communicated by G. Wenzel     

A cytoplasmically inherited mutant controlling early chloroplast development in barley seedlings

Cytoplasmic line 2 (CL2) has been previously reported as a cytoplasmically inherited chlorophyll-deficient mutant selected from a chloroplast-mutator genotype of barley. It was characterized by a localized effect on the upper part of the first-leaf blade. At emergence the CL2 seedlings-phenotype varied from a grainy light green to an albino color. They gradually greened during the following days, starting from the base of the blade and extending to cover most of its surface when it was fully grown. The present results, from both light microscopy and transmission electron microscopy (TEM), confirmed the previously described positional and time-dependent expression of the CL2 syndrome along the first-leaf blade. During the first days after emergence, light microscopy showed a normally developed chloroplast at the middle part of the CL2 first-leaf blade, meanwhile at the tip only small plastids were observed. TEM showed that the shapes and the internal structure of the small plastids were abnormal, presenting features of proplastids, amyloplasts and/or senescent gerontoplasts. Besides, they lack plastid ribosomes, contrasting with what was observed inside chloroplasts from normal tips, which presented abundant ribosomes. Phenotypic observations and spectrophotometric analysis of seedlings produced by mother plants that had been grown under different temperatures indicated that higher temperatures during seed formation were negatively associated with pigment content in CL2 seedlings. In contrast, higher temperatures during the growth of CL2 seedlings have been associated with increased pigment content. Aqueous solution with kanamycin and streptomycin, which are antibiotics known to interfere with plastid gene translation, were used for imbibition of wild-type and CL2 seeds. Antibiotic treatments differentially reduced the chlorophyll content in the upper part of the first-leaf blade in CL2, but not in wild-type seedlings. These results suggest that in the wild-type, plastid-gene proteins which are necessary for chloroplast development and chlorophyll synthesis in the upper part of the first-leaf blade are usually synthesized during embryogenesis. However, under certain circumstances, in CL2 seedlings, they would be synthesized after germination. In addition, a shortening of the sheath has been observed in association with pigment decrease suggesting the existence of plastid factors affecting the expression of some nuclear genes. We consider the CL2 mutant a unique experimental material useful to study biological phenomena and external factors regulating plastid, and nuclear gene expression during embryogenesis and early seedling development.Communicated by R. Hagemann     

Barley Mla and Rar mutants compromised in the hypersensitive cell death response against Blumeria graminis f.sp. hordei are modified in their ability to accumulate reactive oxygen intermediates at sites of fungal invasion

 The pathogenesis-related accumulation of superoxide radical anions (O^·− ₂) and hydrogen peroxide (H₂O₂) was comparatively analyzed in a barley line (Hordeum vulgare L. cv Sultan-5) carrying the powdery mildew (Blumeria graminis f.sp. hordei, Speer, Bgh) resistance gene Mla12, and in susceptible mutants defective in Mla12 or in genes “required for Mla12-specified disease resistance” (Rar1 and Rar2). In-situ localization of reactive oxygen intermediates was performed both by microscopic detection of azide-insensitive nitroblue tetrazolium (NBT) reduction or diaminobenzidine (DAB) polymerization, and by an NBT-DAB double-staining procedure. The Mla12-mediated hypersensitive cell death occurred either in attacked epidermal cells or adjacent mesophyll cells of wild-type plants. Whole-cell H₂O₂ accumulation was detected in dying cells, while O^·− ₂ emerged in adjacent cells. Importantly, all susceptible mutants lacked these reactions. An oxalate oxidase, which is known to generate H₂O₂ and has been implicated in barley resistance against the powdery mildew fungus, was not differentially expressed between the wild type and all mutants. The results demonstrate that the Rar1 and Rar2 gene products, which are control elements of R-gene-mediated programmed cell death, also control accumulation of reactive oxygen intermediates but not the pathogenesis-related expression of oxalate oxidase. Received: 7 January 2000 / Accepted: 2 June 2000     

Accumulation of pathogenesis-related proteins in barley leaf intercellular spaces during leaf senescence

Accumulation of the pathogenesis-related (PR) proteins localised in intercellular spaces of barley primary leaves, chlorophyll content, structure of chloroplasts, and photosynthesis were examined during natural and in vitro induced leaf senescence (cultivation of whole plants in the dark or detached leaves under nutrient deficiency). Some of PR proteins accumulated during natural senescence, but their accumulation pattern was different from those of pathogen-induced as well as during in vitro-induced senescence, which indicate different molecular bases of these processes. Photosynthetic rate and chlorophyll content indicate that natural senescence of barley primary leaves began from 15th day after sowing. In 35-d-old first leaves, the chloroplasts showed typical characteristics of senescence as significant decrease of size, greater grana, and prominent plastoglobuli. The chloroplasts contained more grana under in vitro induced senescence and they had reduced length in the dark. Correspondingly, accumulation of PR proteins was detectable on about the 15th day but the content of some PR proteins increased in later stages of senescence. This revised version was published online in July 2006 with corrections to the Cover Date.