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     

Control of barley root respiration

Evidence from barley [ Hordeum distichum (L.) Lam. cv. Maris Mink], and from many other species, suggests that respiration is controlled by either supply of carbohydrate or demand for ATP. The relationship between root respiration rate (measured as O₂ consumption or CO₂ production) and ethanol-soluble carbohydrate content altered with time following selective pruning, and the change could not be accounted for by buffering of the cytoplasmic carbohydrate concentration by sugars in the vacuole. Exogenous sucrose supplied to the roots prevented any decline of the respiration rate in shoot-pruned plants, and if supplied for 24 h stimulated the respiration rate after any treatment. Root extension responded to sucrose in a similar manner. We suggest that respiration is under fine control by adenylates, but the capacity of the respiratory system is fixed by the supply of sucrose, possibly via coarse control of the respiratory machinery, or of the processes requiring metabolic energy.     

Screening for Barley yellow dwarf virus-Resistant Barley Genotypes by Assessment of Virus Content in Inoculated Seedlings

The content of Barley yellow dwarf virus (BYDV) in roots and leaves of barley seedling plants differing in their level of resistance was assessed by quantitative ELISA 1–42 days after inoculation with the strain of BYDV (PAV). High virus accumulation in roots and low concentration in leaves was characteristic of the period 9–15 days after inoculation. In leaves, the differences in virus content between resistant and susceptible genotypes became significant after 15 days and resistance to virus accumulation was better expressed 30–39 days after inoculation. Roots of resistant materials exhibited evident retardation of virus accumulation and the greatest difference in virus content between resistant and susceptible plants was detected 9 days after inoculation. By these criteria, the selected winter and spring barley cultivars and lines (in total 44 materials) fell in to five groups according to field reactions and the presence or absence of the Yd2 resistance gene. There were highly significant and positive relations between ELISA values and 5‐year field data on symptomatic reactions and grain‐yield reductions due to infection. Using the described method, resistant and moderately resistant genotypes (both Yd2 and non‐Yd2) were significantly differentiated from susceptible genotypes. The possible use of this method in screening for BYDV resistance is discussed.     

Ancestry of American polyploid Hordeum species with the I genome inferred from 5S and 18S-25S rDNA

* BACKGROUND AND AIMS: The genus Hordeum exists at three ploidy levels (2x, 4x and 6x) and presents excellent material for investigating the patterns of polyploid evolution in plants. Here the aim was to clarify the ancestry of American polyploid species with the I genome. * METHODS: Chromosomal locations of 5S and 18S-25S ribosomal RNA genes were determined by fluorescence in situ hybridization (FISH). In both polyploid and diploid species, variation in 18S-25S rDNA repeated sequences was analysed by the RFLP technique. * KEY RESULTS: Six American tetraploid species were divided into two types that differed in the number of rDNA sites and RFLP profiles. Four hexaploid species were similar in number and location of both types of rDNA sites, but the RFLP profiles of 18S-25S rDNA revealed one species, H. arizonicum, with a different ancestry. * CONCLUSIONS: Five American perennial tetraploid species appear to be alloploids having the genomes of an Asian diploid H. roshevitzii and an American diploid species. The North American annual tetraploid H. depressum is probably a segmental alloploid combining the two closely related genomes of American diploid species. A hexaploid species, H. arizonicum, involves a diploid species, H. pusillum, in its ancestry; both species share the annual growth habit and are distributed in North America. Polymorphisms of rDNA sites detected by FISH and RFLP analyses provide useful information to infer the phylogenetic relationships of I-genome Hordeum species because of their highly conserved nature during polyploid evolution.     

Influence of nitrate supply on concentrations and translocation of abscisic acid in barley (Hordeum vulgare)

Spring barley ( Hordeum vulgare L. cv. Golf) was grown at different nitrate supply rates, controlled by using the relative addition rate technique, in order to elucidate the relationship between nitrate-N supply and root and shoot levels of abscisic acid (ABA). The plants were maintained as (1) standard cultures where nitrate was supplied at relative addition rates (RAs) of 0.03, 0.09 and 0.18 day⁻¹, and (2) split-root cultures at RA 0.09 day⁻¹ but with the nitrate distributed between the two root parts in ratios of 100:0, 80:20 and 60:40. Time-dependent changes in root and shoot concentrations of ABA (determined by radioimmunoassay using a monoclonal antibody) were observed in both standard and split-root cultures during 12 days of acclimation to the different nitrate regimes. However, the ABA responses were similar at all nitrate supply rates. Further experiments were performed with split-root cultures where the distribution of nitrate between the two root parts was reversed from 80:20 to 20:80 so that short-term effects to local perturbations of nitrate supply could be studied without altering whole-plant N absorption. Transient increases in ABA concentrations (maximum of 25 to 40% after 3 to 4 h) were observed in both subroot parts, as well as in xylem sap and shoot tissue. By pruning the root system it was demonstrated that the change in ABA had its origin in the subroot part receiving the increased nitrate supply (i.e. switched from 20 to 80% of the total nitrate supply). The data indicate that ABA responses are easily transmitted between different organs, including transmission from one set of seminal roots to another via the shoot. The data do not provide any indication that long-term nitrate supplies or general nitrogen status of barley plants affect, or are otherwise related to, the average tissue ABA concentrations of roots and shoots.     

Transfer of a dominant gene for powdery mildew resistance and DNA from Hordeum bulbosum into cultivated barley (H. vulgare)

Summary In an attempt to transfer traits of agronomic importance from H. bulbosum into H. vulgare we carried out crosses between four diploid barley cultivars and a tetraploid H. bulbosum. Eleven viable triploid F₁ plants were produced by means of embryo rescue techniques. Meiotic pairing between H. vulgare and H. bulbosum chromosomes was evidenced by the formation of trivalents at a mean frequency of 1.3 with a maximum of five per cell. The resulting triploid hybrids were backcrossed to diploid barley, and nine DC₁ plants were obtained. Three of the BC₁ plants exhibited H. bulbosum DNA or disease resistance. A species specific 611-bp DNA probe, pSc119.2, located in telomeres of the H. bulbosum genome, clearly detected five H. bulbosum DNA fragments of about 2.1, 2.4, 3.4, 4.0 and 4.8 kb in size present in one of the BC₁ plants (BC₁-5) in BamHI-digésted genomic Southern blots. Plant BC₁-5 also contained a heterozygous chromosomal interchange involving chromosomes 3 and 4 as identified by N-banding. One of the two translocated chromosomes had the H. bulbosum sequence in the telomeric region as detected using in situ hybridization with pSc119.2. Two other BC₁ plants (BC₁-1 and BC₁-2) were resistant to the powdery mildew isolates to which the barley cultivars were susceptible. Seventy-nine BC₂ plants from plant BC₁-2 segregated 32 mildew resistant to 47 susceptible, which fits a ratio of 11, indicating that the transferred resistance was conditioned by a single dominant gene. Reciprocal crosses showed a tendency towards gametoselection that was relative to the resistance. Mildew resistant plant BC₁-2 also had a 1-kb H. bulbosum DNA fragment identified with a ten-base random primer using polymerase chain reaction (PCR). Forty-three BC₁ plants, randomly sampled from the 79 BC₁ plants, also segregated 2320 for the presence versus absence of this 1-kb H. bulbosum DNA fragment, thereby fitting a 11 ratio and indicating that the PCR product originated from a single locus. The 1-kb DNA fragment and disease resistance were independently inherited as detected by PCR analysis of bulked DNA from 17 resistant and 17 susceptible plants as well as by trait segregation in the 43 individual plants. The progenies produced could serve as an important resistant source in plant breeding. This is the first conclusive report of the stable transfer of disease resistance and DNA from H. bulbosum to H. vulgare.     

Chlorophyll b reduction during senescence of barley seedlings

During senescence of flowering plants, only breakdown products derived from chlorophyll a were detected although  b disappears, too (Matile et al., 1996, Plant Physiol 112: 1403–1409). We investigated the possibility of chlorophyll b reduction during dark-induced senescence of barley (Hordeum vulgare L.) leaves. Plastids isolated from senescing leaves were lysed and incubated with NADPH. We found 7¹-hydroxy-chlorophyll a, 7¹-hydroxy-chlorophyllide a, and, after incubation with Zn-pheophorbide b, also Zn-7¹-hydroxy-pheophorbide a, indicating activity of chlorophyll(ide) b reductase. The highest activity was found at day 2 of senescence when chlorophyll breakdown reached its highest rate. Chlorophyllase reached its highest activity under the same conditions only at days 4–6 of senescence. Based on the chlorophyll b reductase activity of plastids at day 2.5 of senescence (=100%), the bulk of activity (83%) was found in the thylakoids and only traces (5%) in the envelope fraction. Chlorophyll b reduction is considered to be an early and obligatory step of chlorophyll b breakdown. Received: 22 February 1999 / Accepted: 24 March 1999     

甘露糖对大麦品种不同外植体生长的影响

以大麦栽培品种的茎尖、成熟胚为外植体,研究了不同甘露糖浓度对这些外植体愈伤组织诱导和生长的影响。结果表明:甘露糖浓度为20 g/L时,茎尖的叶片伸长和生根受到明显抑制;甘露糖浓度为10 g/L或15 g/L时,成熟胚的愈伤组织诱导率降低50%,甘露糖浓度为20 g/L或25 g/L时,成熟胚愈伤诱导和生长完全受到抑制,因此在以大麦茎尖和成熟胚为外植体的磷酸甘露糖异构酶(PM I)/甘露糖筛选中,可分别以20 g/L、25 g/L甘露糖作筛选压。另外,在培养早期阶段筛选比较适宜。