Carbon and nitrogen metabolism in a barley (Hordeum vulgare L.) mutant with impaired chloroplast dicarboxylate transport

A mutant line, RPr79/2, of barley (Hordeum vulgare L. cv. Maris Mink) has been isolated that has an apparent defect in photorespiratory nitrogen metabolism. The metabolism of ¹⁴C-labelled glutamine, glutamate and 2-oxoglutarate indicates that the mutant has a greatly reduced ability to synthesise glutamate, especially in air, although in-vitro enzyme analysis indicates the presence of wild-type activities of glutamine synthetase (EC 6.3.1.2) glutamate synthase (EC 1.4.7.1 and EC 1.4.1.14) and glutamate dehydrogenase (EC 1.4.1.2). Several characteristics of RPr79/2 are very similar to those described for glutamate-synthase-deficient barley and Arabidopsis thaliana mutants, including the pattern of labelling following fixation of ¹⁴CO₂, and the rapid rise in glutamine content and fall in glutamate in leaves on transfer to air. The CO₂-fixation rate in RPr79/2 declines much more slowly on transfer from 1% O₂ to air than do the rates in glutamate-synthase-deficient plants, and RPr79/2 plants do not die in air unless the temperature and irradiance are high. Analysis of (glutamine+NH₃+2-oxoglutarate)-dependent O₂ evolution by isolated chloroplasts shows that chloroplasts from RPr79/2 require a fivefold greater concentration of 2-oxoglutarate than does the wild-type for maximum activity. The levels of 2-oxoglutarate in illuminated leaves of RPr79/2 in air are sevenfold higher than in Maris Mink. It is suggested that RPr79/2 is defective in chloroplast dicarboxylate transport.     

Cloning and sequence analysis of a cDNA for barley ferredoxin-dependent glutamate synthase and molecular analysis of photorespiratory mutants deficient in the enzyme

The NH₂-terminal sequences of ferredoxin-dependent glutamate synthase (Fd-GOGAT; EC 1.4.7.1) purified from barley (Hordeum vulgare L.) and Chlamydomonas reinhardtii (Dangeard), and of a barley peptide, were determined and the barley sequences were used to design oligonucleotide primers for the polymerase chain reaction. A specific 1.3-kilobase (kb) cDNA fragment specifying the NH₂-terminal one-third of the mature barley polypeptide, was amplified, cloned and sequenced. The NH₂-terminus of plant Fd-GOGAT is highly conserved and homologous to the NH₂-terminus of the heavy subunit of Escherichia coli NADPH-GOGAT. Based on sequence homologies, we tentatively identified the NH₂-terminal region of Fd-GOGAT as the glutamine-amidotransferase domain, which is related to the corresponding domain of the purF-type amidotransferases. The Fd-GOGAT cDNA clone, and polyclonal antibodies raised against the barley enzyme, were used to analyse four Fd-GOGAT-deficient photorespiratory mutants. Three mutants (RPr 82/1, RPr 82/9 and RPr 84/82) had no detectable Fd-GOGAT protein in leaves, while the fourth (RPr 84/42) had a small amount of cross-reacting material. Hybridization to Northern blots of total leaf RNA revealed that both RPr 82/9 and RPr 84/82 were indistinguishable from the parental line (Maris Mink), having normal amounts of a 5.7-kb mRNA species. On the other hand, RPr 82/2 and RPr 84/42 each contained two distinct hybridizing RNA species, one of which was larger than 5.7 kb, the other smaller. Using a set of wheat-barley telosomic addition lines we have assigned the Fd-GOGAT structural locus to the short arm of chromosome 2.Abbreviations bp kbase pairs - cDNA copy DNA - Fd ferredoxin - GOGAT glutamate synthase - GAT glutamine amidotransferase - kb kilobase - PCR polymerase chain reaction C.A. was the holder of a Fleming award from the British Council and the Spanish Ministry of Education and Science. A.J.M. was for part of the work the recipient of a European Molecular Biology Organization postdoctoral fellowship. The research was also partly supported by contract no. BAP/O354/E of the Biotechnology Action Programme of the E.C., by an Acciones Integradas award (no. 40/125) from the British Council and the Spanish Ministry of Education and Science, by the Junta de Andalucia (to Group 3263) and by project PB91-0613 from DGICYT (Spain). We thank Daryl Pappin (Department of Biochemistry, University of Leeds) for amino-acid sequencing, and Martin Cornelius (Rothamsted Experimental Station, Harpenden, Herts., UK) for synthesis of oligonuleotides.     

Control of photosynthesis in barley leaves with reduced activities of glutamine synthetase or glutamate synthase

Wild-type and mutant plants of barley (Hordeum vulgare L. cv. Maris Mink) lacking activities of chloroplastic glutamine synthetase (GS) and of ferredox-in-dependent glutamate synthase (Fd-GOGAT) were crossed to generate heterozygous plants. Crosses of the F² generation containing GS activities between 47 and 97 of the wild-type and Fd-GOGAT activities down to 63 of the wild-type have been selected to study the control of both enzymes on photorespiratory carbon and nitrogen metabolism. There were no major pleiotropic effects. Decreased GS had a small impact on leaf protein and the total activity of ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco). The activation state of Rubisco was unaffected in air, but a decrease in GS influenced the activation state of Rubisco in low CO₂. In illuminated leaves, the amino-acid content decreased with decreasing GS, while the content of ammonium rose, showing that even small reductions in GS limit ammonium re-assimilation and may bring about a loss of nitrogen from the plants, and hence a reduction in protein and Rubisco. Leaf amino-acid contents were restored, and ammonium and nitrate contents decreased, by leaving plants in the dark for 24 h. The ratios of serine to glycine decreased with a decrease in GS when plants were kept at moderate photon flux densities in air, suggesting a possible feedback on glycine decarboxylation. This effect was absent in high light and low CO₂. Under these conditions ammonium contents exhibited an optimum and amino-acid contents a minimum at a GS activity of 65 of the wild-type, suggesting an inhibition of ammonium release in mutants with less than 65 GS. The leaf contents of glutamate, glutamine, aspartate, asparagine, and alanine largely followed changes in the total amino-acid contents determined under different environmental conditions. Decreased Fd-GOGAT resulted in a decrease in leaf protein, chlorophyll, Rubisco and nitrate contents. Chlorophyll a/b ratios and specific leaf fresh weight were lower than in the wild-type. Leaf ammonium contents were similar to the wild-type and total leaf amino-acid contents were only affected in low CO₂ at high photon flux densities, but mutants with decreased Fd-GOGAT accumulated glutamine and contained less glutamate.Abbreviations Chl chlorophyll - FBPase fructose-1,6-bisphosphatase - Fd-GOGAT ferredoxin-dependent glutamine: 2-oxoglutarate aminotransferase - GS glutamine synthetase - PEP phosphoenolpyruvate - PFD photon flux density - Rubisco ribulose-1,5-bisphosphate carboxylase-oxygenase This research was jointly supported by the Agricultural and Food Research Council and the Science and Engineering Research Council, U.K. in the programme on Biochemistry of Metabolic Regulation in Plants (PG50/555).     

The development of NAD(P)H-dependent and ferredoxin-dependent glutamate synthase in greening barley and pea leaves

The activity of NAD(P)H-dependent glutamate synthase (E.C. 1.4.1.14) has been demonstrated in extracts from etiolated shoots of pea (Pisum sativum L.) and barley (Hordeum vulgare L.). This activity does not significantly alter upon greening of the etiolated shoots, and is at a similar level in light-grown material. Ferredoxin-dependent glutamate synthase (E.C. 1.4.7.1) has low activity in etiolated shoots but increases rapidly on greening. In light grown leaves ferredoxin-dependent activity is 30–40-fold higher than NAD(P)H-dependent activity. It is not considered that the NAD(P)H-dependent glutamate synthase plays an important role in ammonia assimilation in the photosynthetic tissue of higher plants.     

Some effects of nitrate abundance and starvation on metabolism and accumulation of nitrogen in barley (Hordeum vulgare L. cv Sonja)

Nitrate and nitrite reductases were both induced by adding three concentrations of nitrate to the nutrient supply of nitrate-starved barley seedlings. Enzyme induction was not proportional to the amount of nitrate introduced. Glutamine synthetase also increased above a high endogenous activity but the increase did not differ significantly between any of the three nitrate treatments. Nitrate accumulated rapidly in leaves of plants given 4.0 mM or 0.5 mM nitrate but not with 0.1 mM nitrate. In all treatments, amino acids in leaves increased for 2 d, chiefly attributable to glutamine, then declined. Transferring plants from the three nitrate treatments to nitrate-free nutrient produced an immediate decline in nitrate reductase but nitrite reductase continued to increase for 2 d, before declining. Glutamine-synthetase activity was not affected by withdrawal of nitrate, nor did nitrate withdrawal retard plant growth during the 9-d period of the experiment. The disparity between accumulated nitrate and nitrate-reducing capacity and the rapid decrease in leaf nitrate when nutrient nitrate supply was removed, indicated the presence of a nitrate-storage pool that could be called upon to maintain amino-acid production in times of nitrogen starvation.Abbreviations GS glutamine synthetase - NR nitrate reductase - NiR nitrite reductase     

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     

Production and diurnal utilization of assimilates in leaves of spinach (Spinacia oleracea L.) and barley (Hordeum vulgare L.)

Rates of CO₂ fixation during the light period and the rates of CO₂ release during the night period were measured using mature leaves from 39- to 49-d-old spinach (Spinacia oleracea L., US Hybrid 424; grown in 9 h light, 15 h darkness, daily) and mature leaves from 21-d-old barley (Hordeum vulgare L., cv. Apex; grown in 14 h light, 10 h darkness, daily). At certain times during the light and dark periods leaves were harvested for assay of their contents of soluble carbohydrates, starch, malate and the various amino acids. Evaluation of the results of these measurements shows that in spinach and barley leaves 46% and 26%, respectively, of the carbon assimilated during the light period is deposited in the leaves for export during the night period. Taking into account the carbon consumption in the source leaves by dark respiration, it is evaluated that rates of assimilate export during the light period from spinach and barley leaves [38 and 42 atom C · (mg Chl)^–1 · h^–1] are reduced in the dark period to 16 atom C · (mg Chl)^–1 · h^–1 in both species. The calculated C/N ratios of the photoassimilates exported during the dark period were 0.029 and 0.015 for spinach and barley leaves, respectively.This work was supported by the Deutsche Forschungsgemeinschaft. We thank Dr. Dieter Heineke for stimulating discussions and Mrs. Petra Hoferichter and Mrs. Marita Feldkämper for their technical assistance.     

Synthesis of wax esters by a cell-free system from barley (Hordeum vulgare L.)

Experimental evidence for a membranebound microsomal ester synthetase from Bonus barley primary leaves is reported. The results are consistent with at least two mechanisms for the synthesis of barley wax esters: an acyl-CoA-fattyalcohol-transacylase-type reaction and an apparent direct esterification of alcohols with fatty acids. Biosynthesis of wax esters was not specific with regard to the chain length of the tested alcohols. The microsomal preparation readily catalyzed the esterification of C₁₆-, C₁₈-, C₂₂- or C₂₄-labelled alcohols with fatty acids of endogenous origin. Exogenous long-chain alcohols were exclusively incorporated into the alkyl moieties of the esters. Addition of ATP, CoA and-or free fatty acids was not effective in stimulating or depressing the esterifying activity of the microsomal fraction. Partial solubilization of the ester synthetase was obtained using phosphate-buffered saline.Abbreviations P pellet - PBS phosphate-buffered saline - S supernatant - SDS sodium dodecyl sulphate     

Initiation of morphogenic cell-suspension and protoplast cultures of barley (Hordeum vulgare L.)

Cell-suspension cultures were initiated from embryogenic calli of various barley cultivars. Seven fast-growing suspension lines were obtained from four different cultivars (cvs. Dissa, Emir, Golden Promise and Igri). Two of these cell suspensions showed morphogenic capacity. From a cell suspension of cv. Dissa, albino plantlets were regenerated when aggregates were cultured on solid medium. Aggregates of cv. Igri usually stopped differentiation at the globular stage, but occasionally formed scutellum-like structures. Five suspension lines were used for protoplast isolation and culture. Dividing protoplasts were obtained from all lines, but with cv. Igri a few divisions only and no further development were observed. Protoplasts from the various lines differed in the time of first division (2–14 d), division frequency (0.09–70.9%) and efficiency of colony formation (0.09–7.3%). Protoplasts isolated from the morphogenic cell suspension of cv. Dissa developed compact calli which sporadically regenerated albino plantlets.Abbreviations CC, MS, N6, SH, Kao8p culture media; see Material and methods - cv chltivar - dicamba 3,6-dichloro-o-anisic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - picloram 4-amino-3,5,6-trichloropicolinic acid     

Control of photosynthesis in barley mutants with reduced activities of glutamine synthetase and glutamate synthase

Heterozygous mutants of barley (Hordeum vulgare L. cv. Maris Mink) with decreased activities of chloroplastic glutamine synthetase (GS) between 97 and 47% of the wild type and ferredoxin dependent glutamate synthase (Fd-GOGAT) down to 64% of the wild type have been used to study aspects of glyoxylate metabolism and the effect of glyoxylate on the activation state of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) in vivo. In the leaf, the extractable activities of serine:glyoxylate aminotransferase decreased with a decrease in GS whereas activities of glutamate and alanine:glyoxylate aminotransferase increased, pointing to a re direction of amino donors from serine to glutamate and alanine. Under ambient conditions, the leaf contents of glutamate and alanine declined continuously with a decrease in GS, in parallel with the decrease in total amino acids. Glycine, serine and asparagine contents decreased with a decrease in GS to approximately 70% of the wild type, but increased again with a further decrease in GS. At high irradiances and at low CO₂ concentrations, glyoxylate contents exhibited a pronounced minimum between 60% and 80% GS. With a further decrease in GS, glyoxylate contents recovered and approached values similar to the wild type. The activation state of Rubisco showed a negative correlation with glyoxylate contents, indicating that a decrease in GS feeds back on the first step of carbon assimilation and photorespiration. The activation state of stromal fructose-1,6-bisphosphatase was unaffected by a decrease in GS or Fd-GOGAT, whereas the activation state of NADP dependent malate dehydrogenase changed in a complex manner. The CO₂photocompensation point, ^*, was appreciably increased in mutants with 47% GS. Mitochondrial respiration in the light (R_d) was reduced with a decrease in GS. Relative rates of CO₂ release into CO₂-free air between the wild type and the 47%-GS mutant correlated with determinations of ^*. These data are consistent with the view that when GS is decreased there is an increased oxidative decarboxylation of glyoxylate resulting from a decreased availability of amino donors for the transamination of glyoxylate to glycine, and that when GS activities are lower than 70% of the wild type an additional mechanism operates to reduce the photorespiratory loss of ammonia.Abbreviations AGAT nine:glyoxylate aminotransferase - FBPase fructose-1,6-bisphosphatase - Fd-GOGAT ferredoxin dependent glutamate synthase - GGAT glutamate:glyoxylate aminotransferase - GS glutamine synthetase - MDH malate dehydrogenase - PFD photon flux density - Rubisco ribulose-1,5-bisphosphate carboxylase-oxygenase - SGAT serine:glyoxylate aminotransferase This research was supported by the Biotechnology and Biological Sciences Research Council initiative on the Biochemistry of Metabolic Regulation in Plants (PG 50/555).     

Metabolism of maltose and sucrose by microspores isolated from barley (Hordeum vulgare L.)

The aim of this work was to discover why barley (Hordeum vulgare L.) microspores die when cultured on media containing 40 mM sucrose but undergo embryogenesis on 40 mM maltose. Freshly isolated microspores were cultured for 6–24 h on media containing either [U-¹⁴C]maltose or [U-¹⁴C]sucrose at 40 mM, and the detailed distribution of ¹⁴C was determined. The amounts of glycolytic intermediates, ATP, ADP and AMP, in microspores were also measured. Cultures on sucrose differed from those on maltose in that the initial rate of metabolism was faster but declined rapidly, less ¹⁴C was recovered in polymers and more in alanine, there was extensive leakage of assimilated carbon, significant accumulation of ethanol and a lower adenylate energy charge. It is argued that microspores cultured on 40 mM sucrose die because they metabolize the sugar rapidly, become hypoxic and, as a result, accumulate large quantities of ethanol within the cells. Metabolism of maltose is slower and there is sufficient oxygen available to allow cells to survive in culture. Consequently some of the cultured cells undergo embryogenesis.P.S. thanks the Science and Engineering Research Council and Shell Research Ltd., Sittingbourne, for a Cooperative Award in Science and Engineering studentship.     

Photorespiratory N donors,aminotransferase specificity and photosynthesis in a mutant of barley deficient in serine: glyoxylate aminotransferase activity

A mutant of Hordeum vulgare L. (LaPr 85/84) deficient in serine: glyoxylate aminotransferase (EC 2.6.1.45) activity has been isolated. The plant also lacks serine: pyruvate aminotransferase and asparagine: glyoxylate aminotransferase activities. Genetic analysis of the mutation strongly indicates that these three activities are all carried on the same enzyme protein. The mutant is incapable of normal rates of photosynthesis in air but can be maintained at 0.7% CO₂. The rate of photosynthesis cannot be restored by supplying hydroxypyruvate, glycerate, glutamate or ammonium sulphate through the xylem stream. This photorespiratory mutant demonstrates convincingly that photorespiration still occurs under conditions in which photosynthesis becomes insensitive to oxygen levels. Two major peaks and one minor peak of serine: glyoxylate aminotransferase activity can be separated in extracts of leaves of wild-type barley by diethylaminoethyl-sephacel chromatography. All three peaks are missing from the mutant, LaPr 85/84. The mutant showed the expected rate (50%) of ammonia release during photorespiration but produced CO₂ at twice the wild-type rate when it was fed [¹⁴C]glyoxylate. The large accumulation of serine detected in the mutant under photorespiratory conditions shows the importance of the enzyme activity in vivo. The effect of the mutation on transient changes in chlorophyll a fluorescence initiated by changing the atmospheric CO₂ concentration are presented and the role of the enzyme activity under nonphotorespiratory conditions is discussed.Abbreviations DEAE diethylaminoethyl - PFR photon fluence rate - SGAT serine:glyoxylate aminotransferase     

Sulphate deprivation depresses the transport of nitrogen to the xylem and the hydraulic conductivity of barley (Hordeum vulgare L.) roots

During the first 4 d after the removal of SO ₄ ^2- from cultures of young barley plants, the net uptake of ¹⁵N-nitrate and the transport of labelled N to the shoot both decline. This occurred during a period in which there was no measurable change in plant growth rate and where the incorporation of [³H]leucine into membrane and soluble proteins was unaffected. Reduced N translocation was associated with six- to eightfold increases in the level of asparagine and two- to fourfold increases in glutamine in root tissue; during the first 4 d of SO ₄ ^2- deprivation there were no corresponding increases in amides in leaf tissue. The provision of 1 mol · m^–3 methionine halted, and to some extent reversed the decline in NO ₃ ^- uptake and N translocation which occurred during continued SO ₄ ^2- deprivation. This treatment had relatively little effect in lowering amide levels in roots. Experiments with excised root systems indicated that SO ₄ ^2- deprivation progressively lowered the hydraulic conductivity, Lp, of roots; after 4 d the Lp of SO ₄ ^2- -deprived excised roots was only 20% of that of +S controls. In the expanding leaves of intact plants, SO ₄ ^2- deprivation for 5 d was found to lower stomatal conductance, transpiration and photosynthesis, in the order given, to 33%, 37% and 18% of control values. The accumulation of amides in roots is probably explained by a failure to export either the products of root nitrate assimilation or phloem-delivered amino-N. This may be correlated with the lowered hydraulic conductivity. Enhanced glutamine and-or asparagine levels probably repressed net uptake of NO ₃ ^- and ¹³NO ₃ ^- influx reported earlier (Clarkson et al. 1989, J. Exp. Bot. 40, 953–963). Attention is drawn to the similar hydraulic signals occurring in the early stages of several different types of mineral-nutrient stresses.Abbreviations Asn asparagine - Gln glutamine - Lp hydraulic conductivity J.L.K. is extremely grateful to the British Council for supporting his working visit to Long Ashton. We thank John Radin for helpful discussion and encouragement.     

Biosynthesis of indole-3-acetic acid in protoplasts,chloroplasts and a cytoplasmic fraction from barley (Hordeum vulgare L.)

Protoplast preparations from barley (Hordeum vulgare L.) enzymatically converted [5-³H]tryptophan to [³H]indole-3-acetic acid (IAA). Both a chloroplast and a crude cytoplasmic fraction, isolated from protoplasts that had previously been fed [5-³H]tryptophan, contained [³H]IAA. Chloroplast and cytoplasmic preparations, isolated from protoplasts and thereafter incubated with [5-³H]tryptophan, also synthesized [³H]IAA, although, in both instances the pool size was less than 50% of that detected in the in-vivo feeds. There were no significant differences in the amounts of [³H]IAA that accumulated in protoplast and chloroplast preparations incubated in light and darkness.Abbreviations HPLC high-performance liquid chromatography - IAA indole-3-acetic acid - RC radiocounting     

The effects of DNA hypomethylating drugs on androgenesis in barley (Hordeum vulgare L.)

Summary The effects of DNA hypomethylating drugs (azacytidine and ethionine) on induction of microspore-derived calluses and embryos were studied in barley (Hordeum vulgare L.) ev. Igri. The results were as follows: (1) Yield of calluses and embryos pretreated with the different concentrations of azacytidine for 3 d was several-fold higher than that of the control. The highest yield of calluses and embryos in all treatments appeared at a concentration of 3 mg l⁻¹, which reached 11.03 per anther. It was 110-fold higher than the control. (2) There was a significant difference in yield of calluses and embryos between the different days of pretreatment. The highest yield was obtained at a 3-d pretreatment. If the period of pretreatment was shorter or longer than 3 d, yield of calluses and embryos was reduced sharply, and was similar to that of the control. (3) The data obtained with ethionine pretreatment were very similar to those obtained with azacytidine. (4) Tests on the different methods of pretreatment showed that yield of calluses and embryos pretreated with distilled H₂O, mannitol, azacytidine, and ethionine was much higher than other pretreatments and the control, and reached 6.53–11.39 per anther. The yield of calluses and embryos pretreated with DNA hypomethylating drugs was higher than with mannitol. However, pretreatment with hypomethylation drugs supplemented with induction medium was not effective.     

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.     

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.