Below-ground carbon distribution in barley (Hordeum vulgare L.) with and without nitrogen fertilization

The distribution of net assimilated C in barley (Hordeum vulgare L.) grown at two N-levels was determined in a growth chamber. The N-fertilization involved 0 and 3.61 mol N g^-1 dry soil. After growth for seven weeks in an atmosphere with continuously ¹⁴C-labelled CO₂, ¹⁴C was determined in shoots, roots, rhizosphere respiration and soil. At the low N-level, 32% of the net assimilated ¹⁴C was translocated below ground, whereas at the high N-level 27% was translocated below ground. The release of C from roots (root respiration, microbial respiration originating from decomposition of ¹⁴C-labelled root material and ¹⁴C remaining in soil) was greater with no N-supply (19% of net assimilated ¹⁴C) than in the treatment with N-supply (15%). Thus, the effect of N-supply on both translocation of assimilated ¹⁴C below ground and the release of ¹⁴C from growing roots was relatively small.     

Ring chromosomes in barley (Hordeum vulgare L.)

A plant with 2n = 14 + 1 ring chromosomes was obtained in the progeny of a primary trisomie for chromosome 7 of a two-rowed cultivar, Shin Ebisu 16. The morphological characteristics of the trisomic plants with an extra ring chromosome were similar to the primary trisomic for chromosome 7 (Semierect), which suggests that it originated from this chromosome. The ring chromosomes were not completely stable in mitotic cells because of abnormal behavior. Chromosome complements varied in different plants and in different roots within a plant. Root tip cells and spikes with 2n = 14 and 14 + 2 ring chromosomes were observed on plants with 14 + 1 ring chromosomes. Breakage-fusion-bridge cycle was inferred. The ring chromosome was associated with two normal homologues forming a trivalent in 17.6% sporocytes at metaphase I. The transmission of the extra ring chromosome was 23.1% in the progeny of the plant with 14 + 1 ring chromosomes. Trivalent formation may have been much higher at early prophase stages which were difficult to analyze in barley; only 4 of 120 sporocytes analyzed showed an isolated ring at pachytene. The ring chromosome moved to one pole without separation in 24.7% of the sporocytes at AI, and divided in 27.1% sporocytes giving rise to 8-8 separation. Only 10% of the sporocytes showed bridge formation at AI.     

Transgenic barley (Hordeum vulgare L.) by electroporation of protoplasts

Summary Protoplasts isolated from calli derived from cultured microspores of barley (Hordeum vulgare L. cv. Kymppi, an elite cultivar) were transformed with the neomycin phosphotransferase marker gene (nptII) by electroporation. Screening of the regenerated plants for the NPTII activity by gel assay resulted in three positive signals. Southern blot analysis and NPTII assays of second and third generation plants confirmed the genomic integration of the transferred gene and that the new trait was inherited by the progeny.     

Estimation of genetic parameters in barley (Hordeum vulgare L.)

Summary Four exotic and four indigenous strains of barley were used for making diallel crosses. The sets of parents and crosses making full, half and quarter diallel were analysed in a randomized block design for plant height, number of effective tillers, ear length, grain yield per plant, 100 grain weight and number of grains per ear.The three alternatives of diallel were similar with respect to the estimates of degree of dominance, general combining ability and specific combining ability, indicating that all these three methods of diallel were equally efficient. However, as the number of entries are minimum in quarter diallel, it would be economical in terms of cost, time and labour to estimate genetic parameters by this method. Average degree of dominance was found in the range of overdominance. The ranking of parents on the basis of their array mean was similar to the ranking based on gca effects. Similarly, the ranking of crosses on the basis of per se performance was similar to the ranking based on sca effects. This suggests that the selection of best general combiner or best cross combinations may be easier and more effective through array mean for per se performance rather than through high gca and sea effects, respectively. From among 56 crosses, IB-226 X X C-164 was the one which showed superiority for maximum number of characters followed by AB-12/59 X PTS-57. High sea effect for plant height, ear length, grain yield, 100 grain weight and number of grains per ear was the result of cross between parents having high X low general combining ability, indicating additive X dominance type of gene interaction. For number of effective tillers, high sca was produced by low x low general combiners, indicating dominance x dominance gene interaction.Part of a Ph. D. thesis submitted by senior author to Haryana Agricultural University, Hissar.     

Triple hybridization with cultivated barley (Hordeum vulgare L.)

Summary A crossing programme for trispecific hybridization including cultivated barley (Hordeum vulgare L.) as the third parent was carried out. The primary hybrids comprised 11 interspecific combinations, each of which had either H. jubatum or H. lechleri as one of the parents. The second parent represented species closely or distantly related to H. jubatum and H. lechleri. In trispecific crosses with diploid barley, the seed set was 5.7%. Crosses with tetraploid barley were highly unsuccessful (0.2% seed set). Three lines of diploid barley were used in the crosses, i.e. Gull, Golden Promise and Vada. Generally, cv Gull had high crossability in crosses with related species in the primary hybrid. It is suggested that Gull has a genetic factor for crossability not present in cv Vada and cv Golden Promise. One accession of H. brachyantherum used in the primary hybrid had a very high crossability (seed set 54.7%) in combination with cv Vada but no viable offspring was produced. In all, two trispecific hybrids were raised, viz. (H. lechleri x H. brevisubulatum) x Gull (2n=7–30) and (H. jubatum x H. lechleri) x Gull (2n=20–22). The first combination invariably had a full complement of seven barley chromosomes plus an additional chromosome no. 7, but a varying number of chromosomes (19–22) of the wild-species hybrid. The second combination had a full set of barley chromosomes. The meiotic pairing was low in both combinations.     

Estimation of genetic parameters in barley (Hordeum vulgare L.)

Summary Four different sets of partial diallels were analysed for their relative efficiencies for estimating the genetic parameters in barley: (1) partial diallel with 12 parents, each involved in only 5 crosses; (2) partial diallel with 12 parents, each involved in only 3 crosses; (3) partial diallel with 8 parents, each involved in only 5 crosses; and (4) partial diallel with 8 parents, each involved in only 3 crosses. In partial diallel experiments, the estimates of gca effects were higher than in those of full diallel. Ranking pattern of the parents on the basis of gca effects in partial diallels deviated considerably from the ranking in full diallel. With decreasing s per parent, the deviation in ranking was also more. This clearly suggests the unsuitability of partial diallel analysis for screening high general combiners. Selection of best cross combinations is also not possible because only a sample of crosses (s out of n) is analysed under partial diallel so that there is every possibility of the best cross being excluded from the sample. In general, overdominance was exhibited, indicating that there is ample scope for heterosis breeding in barley.     

Impact of biocontrol strain Pseudomonas fluorescens CHA0 on rhizosphere bacteria isolated from barley (Hordeum vulgare L.) with special reference to Cytophaga-like bacteria

AIMS: To assess the impact of the biocontrol strain Pseudomonas fluorescens CHA0 on a collection of barley rhizosphere bacteria using an agar plate inhibition assay and a plant microcosm, focusing on a CHA0-sensitive member of the Cytophaga-like bacteria (CLB). METHODS AND RESULTS: The effect of strain CHA0 on a collection of barley rhizosphere bacteria, in particular CLB and fluorescent pseudomonads sampled during a growth season, was assessed by a growth inhibition assay. On average, 85% of the bacteria were sensitive in the May sample, while the effect was reduced to around 68% in the July and August samples. In the May sample, around 95% of the CLB and around 45% of the fluorescent pseudomonads were sensitive to strain CHA0. The proportion of CHA0-sensitive CLB and fluorescent pseudomonad isolates decreased during the plant growth season, i.e. in the July and August samples. A particularly sensitive CLB isolate, CLB23, was selected, exposed to strain CHA0 (wild type) and its genetically modified derivatives in the rhizosphere of barley grown in gnotobiotic soil microcosms. Two dry-stress periods were imposed during the experiment. Derivatives of strain CHA0 included antibiotic or exopolysaccharide (EPS) overproducing strains and a dry-stress-sensitive mutant. Despite their inhibitory activity against CLB23 in vitro, neither wild-type strain CHA0, nor any of its derivatives, had a major effect on culturable and total cell numbers of CLB23 during the 23-day microcosm experiment. Populations of all inoculants declined during the two dry-stress periods, with soil water contents below 5% and plants reaching the wilting point, but they recovered after re-wetting the soil. Survival of the dry-stress-sensitive mutant of CHA0 was most affected by the dry periods; however, this did not result in an increased population density of CLB23. CONCLUSIONS: CLB comprise a large fraction of barley rhizosphere bacteria that are sensitive to the biocontrol pseudomonad CHA0 in vitro. However, in plant microcosm experiments with varying soil humidity conditions, CHA0 or its derivatives had no major impact on the survival of the highly sensitive CLB strain, CLB23, during two dry-stress periods and a re-wetting period; all co-existed well in the rhizosphere of barley plants. SIGNIFICANCE AND IMPACT OF THE STUDY: Results indicate a lack of interaction between the biocontrol pseudomonad CHA0 and a sensitive CLB when the complexity increases from agar plate assays to plant microcosm experiments. This suggests the occurrence of low levels of antibiotic production and/or that the two bacterial genera occupy different niches in the rhizosphere.     

Advanced backcross QTL analysis in barley (Hordeum vulgare L.)

This paper reports on the first advanced backcross-QTL (quantitative trait locus) project which utilizes spring barley as a model. A BC(2)F(2) population was derived from the initial cross Apex ( Hordeum vulgare ssp. vulgare, hereafter abbreviated with Hv) x ISR101-23 ( H. v. ssp. spontaneum, hereafter abbreviated with Hsp). Altogether 136 BC(2)F(2) individuals were genotyped with 45 SSR (simple sequence repeat) markers. Subsequently, field data for 136 BC(2)F(2) families were collected for 13 quantitative traits measured in a maximum of six environments. QTLs were detected by means of a two-factorial ANOVA with a significance level of P < 0.01 for a marker main effect and a marker x environment (M x E) interaction, respectively. Among 585 marker x trait combinations tested, 86 putative QTLs were identified. At 64 putative QTLs, the marker main effect and at 27 putative QTLs, the M x E interaction were significant. In five cases, both effects were significant. Among the putative QTLs, 29 (34%) favorable effects were identified from the exotic parent. At these marker loci the homozygous Hsp genotype was associated with an improvement of the trait compared to the homozygous Hv genotype. In one case, the Hsp allele was associated with a yield increase of 7.7% averaged across the six environments tested. A yield QTL in the same chromosomal region was already reported in earlier barley QTL studies.     

Aquaporin-facilitated water uptake in barley (Hordeum vulgare L.) roots

It is not known to what degree aquaporin-facilitated water uptake differs between root developmental regions and types of root. The aim of this study was to measure aquaporin-dependent water flow in the main types of root and root developmental regions of 14- to 17-d-old barley plants and to identify candidate aquaporins which mediate this flow. Water flow at root level was related to flow at cell and plant level. Plants were grown hydroponically. Hydraulic conductivity of cells and roots was determined with a pressure probe and through exudation, respectively, and whole-plant water flow (transpiration) determined gravimetrically in response to the commonly used aquaporin inhibitor HgCl(2). Expression of aquaporins was analysed by real-time PCR and in situ hybridization. Hydraulic conductivity of cortical cells in seminal roots was largest in lateral roots; it was smallest in the fully mature zone and intermediate in the not fully mature 'transition' zone along the main root axis. Adventitious roots displayed an even higher (3- to 4-fold) cortical cell hydraulic conductivity in the transition zone. This coincided with 3- to 4-fold higher expression of three aquaporins (HvPIP2;2, HvPIP2;5, HvTIP1:1). These were expressed (also) in cortical tissue. The largest inhibition of water flow (83-95%) in response to HgCl(2) was observed in cortical cells. Water flow through roots and plants was reduced less (40-74%). It is concluded that aquaporins contribute substantially to root water uptake in 14- to 17-d-old barley plants. Most water uptake occurs through lateral roots. HvPIP2;5, HvPIP2;2, and HvTIP1;1 are prime candidates to mediate water flow in cortical tissue.     

Carbon and nitrogen metabolism in barley (Hordeum vulgare L.) mutants lacking ferredoxin-dependent glutamate synthase

Five mutant lines of barley (Hordeum vulgare L.), which are only able to grow at elevated levels of CO₂, contain less than 5% of the wild-type activity of ferredoxin-dependent glutamate synthase (EC 1.4.7.1). Two of these lines (RPr 82/1 and RPr 82/9) have been studied in detail. Leaves and roots of both lines contain normal activities of NADH-dependent glutamate synthase (EC 1.4.1.14) and the other enzymes of ammonia assimilation. Under conditions that minimise photorespiration, both mutants fix CO₂ at normal rates; on transfer to air, the rates drop rapidly to 15% of the wild-type. Incorporation of ¹⁴CO₂ into sugar phosphates and glycollate is increased under such conditions, whilst incorporation of radioactivity into serine, glycine, glycerate and sucrose is decreased; continuous exposure to air leads to an accumulation of ¹⁴C in malate. The concentrations of malate, glutamine, asparagine and ammonia are all high in air, whilst aspartate, alanine, glutamate, glycine and serine are low, by comparison with the wild-type parent line (cv. Maris Mink), under the same conditions. The metabolism of [¹⁴C]glutamate and [¹⁴C]glutamine by leaves of the mutants indicates a very much reduced ability to convert glutamine to glutamate. Genetic analysis has shown that the mutation in RPr 82/9 segregates as a single recessive nuclear gene.Abbreviations GDH glutamate dehydrogenase (EC 1.4.1.2) - GS glutamine synthetase (EC 6.3.1.2) - RuBP ribulose 1,5-bisphosphate     

Root contribution to nitrate reduction in barley seedlings (Hordeum vulgare L.)

Summary The leaf and root nitrate reductase activities were measured in 7 day-old barley seedlings by anoxic nitrite accumulation in darkness, during 48h after the transfer from a N-starved medium to a 1.5 mM K¹⁵NO₃ medium. Thisin situ nitrate reduction was compared with the¹⁵N incorporation in the reduced N fraction of the whole seedlings.The nitrate reduction integrated fromin situ measurements was lower than the reduced¹⁵N accumulation. The rootin situ nitrate reductase activity seemed to account for only the third of the real root nitrate reduction, which may have been responsible for the overall underestimation. This discrepancy was partly explained by the ability of the root to reduce nitrite in an anoxic environment.These results suggest that, after correction of thein situ estimation of the nitrate reduction. the roots contribute to about 50% of the total assimilation.     

Chromium uptake and transport in barley seedlings (Hordeum vulgare L.)

Summary Potassium chromate is more toxic to the growth of barley in solution culture than chromic chloride, though apparent uptake of the latter is much faster. Inhibitor studies indicate that CrO₄ ^2- uptake is active whereas Cr³⁺ uptake is passive, demonstrating that the two forms do not share a common uptake mechanism. Studies on the form of Cr inside root cells show that in plants fed CrO₄ ^2- the Cr remains largely unchanged whereas in plants fed Cr³⁺ a little CrO₄ ^2- (0.5 per cent) is produced. This conversion is dependent on the presence of living material and is probably enzymatic. Chromate uptake follows Michaelis-Menten kinetics at low concentration and is competitively inhibited by sulphate. Transport of chromium up the root is very slow, accounting for the low levels of Cr in the shoots. Chromate is transported better than Cr³⁺ though still to a very limited extent. These experiments provide a physiological basis for previous observations.     

Influence of nitrogen source and concentration on nitrogen isotopic discrimination in two barley genotypes (Hordeum vulgare L.)

The occurrence of nitrogen isotope discrimination with absorption and assimilation of nitrate (NO₃^–) and ammonium (NH₄⁺) was investigated using two genotypes of barley, Hordeum vulgare L. cv. Steptoe and Az12 : Az70, the latter of which lacks the characterized nitrate reductase isozymes. Plants were grown under two situations: a closed system with limited nitrogen or an open system with unlimited nitrogen, to elucidate the conditions and processes that influence discrimination. There was no discrimination observed for Az12 : Az70 when supplied with limited nitrogen. Discrimination was observed for Steptoe seedlings at high external NO₃^– concentrations, but not with low NO₃^– when assimilation is probably rapid and complete. The same pattern was observed for Steptoe when NH₄⁺ was supplied; indicating that for both nitrogen forms discrimination is dependent upon the presence of the assimilatory enzyme and the external concentration. The implications of this study are that both internal (assimilatory enzyme distribution) and external (source concentration) factors may have a larger impact on tissue δ ¹⁵N than the form of nitrogen utilized. This suggests that tissue δ ¹⁵N may not always be a reliable indicator of a plant's integrated nitrogen nutrition.     

Mapping of QTL associated with nitrogen storage and remobilization in barley (Hordeum vulgare L.) leaves

Nitrogen uptake and metabolism are central for vegetative and reproductive plant growth. This is reflected by the fact that nitrogen can be remobilized and reused within a plant, and this process is crucial for yield in most annual crops. A population of 146 recombinant inbred barley lines (F(8) and F(9) plants, grown in 2000 and 2001), derived from a cross between two varieties differing markedly in grain protein concentration, was used to compare the location of QTL associated with nitrogen uptake, storage and remobilization in flag leaves relative to QTL controlling developmental parameters and grain protein accumulation. Overlaps of support intervals for such QTL were found on several chromosomes, with chromosomes 3 and 6 being especially important. For QTL on these chromosomes, alleles associated with inefficient N remobilization were associated with depressed yield and higher levels of total or soluble organic nitrogen during grain filling and vice versa; therefore, genes directly involved in N recycling or genes regulating N recycling may be located on these chromosomes. Interestingly, the most prominent QTL for grain protein concentration (on chromosome 6) did not co-localize with QTL for nitrogen remobilization. However, QTL peaks for nitrate and soluble organic nitrogen were detected at this locus for plants grown in 2001 (but not in 2000). For these, alleles associated with low grain protein concentration were associated with higher soluble nitrogen levels in leaves during grain filling; therefore, gene(s) found at this locus might influence the nitrogen sink strength of developing barley grains.     

Carbon and nitrogen in the root-zone of barley (Hordeum vulgare L.) supplied with nitrogen fertilizer at two rates

Below-ground carbon (C) production and nitrogen (N) flows in the root-zone of barley supplied with high or low amounts of N-fertilizer were investigated. Interest was focused on the effect of the level of N-fertilizer on the production of root-derived C and on gross immobilization (i) and gross mineralization (m) rates. The plants were grown for 46 days in a sandy loam soil. Principles of pool dilution and changes in ¹⁵N pool abundances were used in conjunction with mathematical modelling to calculate the flows of N. N was applied at a high or a low rate, as (¹⁵NH₄)₂SO₄ solution (17.11 atom% ¹⁵N excess), before sowing. Nitrification was inhibited by using nitrapyrin (N-Serve). Pots were sampled four or five times during the experimental period, i.e. 0, 22, 30, 38 and 46 days after germination. On the three last sampling occasions, samples were also collected from pots in a growth chamber with ¹⁴C-labelled atmosphere.The release of ¹⁴C, measured as the proportion of the total ¹⁴C translocated below ground, was higher in the high-N treatment, but the differences between treatments were small. Our results were not conclusive in demonstrating that high-N levels stimulate the decomposition and microbial utilization of root-released materials. However, the internal circulation of soil-N, calculated N fluxes (m), which were in accordance with C mineralization rates and amounts of unlabelled N found in the plants (PU), suggested that the decomposition of native soil organic matter was hampered in the high-N treatment. Apparently, towards the end of the experimental period, microorganisms in the low-N treatment used C from soil organic matter to a greater extent than C they used from root released material, presumably because lower amounts of mineral N were available to microorganisms in the low-N treatment. Immobilization of N appeared to be soil driven (organisms decomposing soil organic matter account for the N demand) at low-N and root-driven (organisms decomposing roots and root-derived C account for the N demand) at high-N.Abbreviations AU Ammonium N-unlabelled - AL Ammonium N-labelled - AT Ammonium N-labelled and unlabelled (total) - NU Nitrate N-unlabelled - OU Organic N-unlabelled - OL Organic N-labelled - OT Organic N-total - PU Plant N-unlabelled (shoots and roots) - PL Plant N-labelled (shoots and roots) - PT Plant N-total (shoots and roots) - SL Sink or source of N-labelled - S Source or sink of N, mainly to and from the outer part of the cylinder - SU Sink or source of N-unlabelled - m Mineralization rate - i Immobilization rate - ua Uptake of ammonium - un Uptake of nitrate - la Loss of ammonium.     

Salinity tolerance in different cultivars of barley (Hordeum vulgare L.)

Seven barley(Hordeum vulgäre L.) cultivars tested varied greatly in their responses to root medium salinity (electrical conductivity of 3, 5, 10, 15 and 20 dS nr^-1)-lant growth was relatively more adversely affected than seed germination. Dry/fresh mass ratio increased at higher salinity levels in all barley cultivars indicating reduced water uptake. Higher K/Na ratio in plant shoots compared to that in the root medium solution indicated selective uptake of K that seems to be among processes involved in tolerance of cultivars to salinity stress.     

Regulation of threonine biosynthesis in barley seedlings (Hordeum vulgare L.)

Homoserine kinase was purified 700-fold by fractional ammonium sulfate precipitation, heat treatment, CM-Sephadex C-50 and DEAE-Sephadex A-50 ion exchange chromatography, and Sephadex G-100 gel filtration. The reaction products O-phosphohomoserine and ADP were the only compounds which caused considerable inhibition of homoserine kinase activity. Product inhibition studies showed non-competitive inhibition between ATP and O-phosphohomoserine and between homoserine and O-phosphohomoserine, and competitive inhibition between ATP and ADP. ADP showed non-competitive inhibition versus homoserine at suboptimal concentrations of ATP. At saturating concentrations of ATP no effect of ADP was observed. The homoserine kinase activity was negligible in the absence of K⁺ and the K_m value for K⁺ was observed to be 4.3 mmol l^–1. A non-competitive pattern was observed with respect to the substrates homoserine and ATP. Threonine synthase in the first green leaf of 6-day-old barley seedlings was partially purified 15-fold by ammonium sulfate fractionation and Sephadex G-100 gel chromatography. Threonine synthase was shown to require pyridoxal 5-phosphate as coenzyme for optimum activity and the enzyme was strongly activated by S-adenosyl-L-methionine. The optimum pH for threonine synthase activity was 7 to 8.Abbreviations PLP Pyridoxal 5-phosphate - SAM S-adenosyl-L-methionine - HSP O-phosphohomoserine