Differential effect of ammonium on the induction of nitrate and nitrite reductase activities in roots of barley (Hordeum vulgare) seedlings

The effect of exogenous NH₄⁺ on the induction of nitrate reductase activity (NRA; EC 1.6.6.1) and nitrite reductase activity (NiRA; EC 1.7.7.1) in roots of 8-day-old intact barley (Hordeum vulgare L.) seedlings was studied. Enzyme activities were induced with 0.1, 1 or 10 mM NO₃⁺ in the presence of 0, 1 or 10 mM NH₄⁺, Exogenous NH₄⁺ partially inhibited the induction of NRA when roots were exposed to 0.1 mM, but not to 1 or 10 mM NO₃⁺, In contrast, the induction of NiRA was inhibited by NH₄⁺ at all NO₃⁺ levels. Maximum inhibition of the enzyme activities occurred at 1.0 mM NH₄⁺ Pre-treatment with NH₄⁺ had no effect on the subsequent induction of NRA in the absence of additional NH₄⁺ whereas the induction of NiRA in NH₄⁺-pretreated roots was inhibited in the absence of NH₄⁺ At 10 mM NO₃⁺ L-methionine sulfoximine stimulated the induction of NRA whether or not exogenous NH₄⁺ was present. In contrast, the induction of NiRA was inhibited by L-methionine sulfoximine irrespective of NH₄⁺ supply. During the postinduction phase, exogenous NH₄⁺ decreased NRA in roots supplied with 0.1 mM but not with 1mM NH₃⁺ whereas, NiRA was unaffected by NH₄⁺ at either substrate concentration. The results indicate that exogenous NH₄⁺ regulates the induction of NRA in roots by limiting the availability of NO₃⁺. Conversely, it has a direct effect, independent of the availability of NO₃⁺, on the induction of NiRA. The lack of an NH₄⁺ effect on NiRA during the postinduction phase is apparently due to a slower turnover rate of that enzyme.     

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.     

Differential localization of two acid proteinases in germinating barley (Hordeum vulgare) seed

A cathepsin D-like aspartic proteinase (EC 3.4.23) is abundant in ungerminated barley ( Hordeum vulgare ) seed while a 30 kDa cysteine endoproteinase (EC 3.4.22) is one of the proteinases synthesized de novo in the germinating seed. In this work, the localization of these two acid proteinases was studied at both the tissue and subcellular levels by immunomicroscopy. The results confirm that they have completely different functions. The aspartic proteinase was present in the ungerminated seed and, during germination, it appeared in all the living tissues of the grain, including the shoot and root. Contrary to previous suggestions, it was not observed in the starchy endosperm. By immunoblotting, the high molecular mass form of the enzyme (32 + 16 kDa) was found in all the living tissues, whereas the low molecular mass form (29 + 11 kDa) was not present in the shoot or root, indicating that the two enzyme forms have different physiological roles. The aspartic proteinase was localized first in the scutellar protein bodies of germinating seed, and later in the vacuoles which are formed by fusion of the protein bodies. In contrast to the aspartic proteinase, the expression of the 30 kDa cysteine proteinase began during the first germination day, and it was secreted into the starchy endosperm; first from the scutellum and later from the aleurone layer. It was not found in either shoots or roots. The 30 kDa cysteine proteinase was detected in the Golgi apparatus and in the putative secretory vesicles of the scutellar epithelium. These results suggest that the aspartic proteinase functions only in the living tissues of the grain, as opposed to the 30 kDa cysteine proteinase which is apparently one of the proteases initiating the hydrolysis of storage proteins in the starchy endosperm.     

Increase in glutamate synthase (NADH) activity in maize seedlings in response to nitrate and ammonium nitrogen

Roots and leaves of Zea mays L. cv. Ganga Safed-2 seedlings grown with nutrient solution containing either 10 m M KNO₃ or NH₄Cl or 5 m M NH₄NO₃ had considerably higher glutamate synthase (NADH, EC 1.4.1.14) activity than the corresponding organs from seedlings grown without any nitrogen. The supply of inorganic nitrogen for a short time, i.e. 3 h, to roots and leaves excised from seedlings grown without nitrogen also increased the enzyme activity in these organs. This increase was more pronounced with nitrate than with ammonium nitrogen. When excised roots and leaves from NH₄NO₃-grown seedlings were incubated in a minus nitrogen medium for 24 h, the enzyme activity declined considerably. This decline was inhibited to some extent by nitrogen, especially by nitrate. Inorganic nitrogen prevented similarly the decline in in vitro enzyme activity during 24 h storage at 25°C, more regularly for the root than for the leaf enzyme. The experiments demonstrate the role of inorganic nitrogen in the regulation of glutamate synthase activity.     

Jasmonate-induced gene expression of barley (Hordeum vulgare) leaves -- the link between jasmonate and abscisic acid

In barley leaves a group of genes is expressed in response to treatment with jasmonates and abscisic acid (ABA) [21]. One of these genes coding for a jasmonate-induced protein of 23 kDa (JIP-23) was analyzed to find out the link between ABA and jasmonates by recording its expression upon modulating independently, the endogenous level of both of them. By use of inhibitors of JA synthesis and ABA degradation, and the ABA-deficient mutant Az34, as well as of cultivar-specific differences, it was shown that endogenous jasmonate increases are necessary and sufficient for expression of this gene. The endogenous rise of ABA did not induce synthesis of JIP-23, whereas exogenous ABA did not act via jasmonates. Different signalling pathways are suggested and discussed.     

Differential effects of abscisic acid and methyl jasmonate on endoproteinases in senescing barley leaves

Endoproteinase activity was analyzed in chloroplasts isolated from barley leaf segments incubated in the dark with various hormonal senescence effectors. As a control, the endoproteinase activity of the supernatant fraction obtained during chloroplast preparation was also analyzed. Measured against azocaseine as substrate, the endoproteinase activity in chloroplasts increased 18 fold during the induction of senescence. This rise in activity was inhibited by kinetin (the activity increased only 10 fold) and very strongly stimulated by abscisic acid (ABA) (117 fold) and methyl jasmonate (Me-JA) (57 fold). Although less so, the endoproteinase activity of the supernatant fraction, mainly vacuolar and with acid pH optimum, was affected in the same way by all three effectors. Among the five endoproteinases (EC) found in chloroplasts, EC2 and EC4 were induced after incubation in water. ABA increased the levels of EC2 and EC4 (5 fold), and induced the development of EC3 and EC5, while Me-JA totally inhibited EC2 and EC4, and induced the development of EC1. At least one of the endoproteinases, EC2, is synthesized in chloroplasts. Among the six endoproteinases found in the supernatant fraction (E), E1, E2, E3 and E5, which are very probably extrachloroplastic endoproteinases, are stimulated by ABA to varying degrees. However, Me-JA stimulates E1 to a greater extent and totally inhibits E3. The differential effects of ABA and Me-JA on chloroplast and supernatant fraction endoproteinases suggest different action mechanisms for both senescence promotors.Abbreviations ABA abscisic acid - DTT dithiothreitol - E supernatant fraction endoproteinase - EC chloroplast endoproteinase - Me-JA methyl jasmonate - PNP p-nitrophenol - SDS-PAGE polyacrylamide gel electrophoresis containing sodium dodecyl sulphate - TCA trichloroacetic acid     

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.     

Ultrastructural demonstration of ferricyanide reductase (Diaphorase) activity in the envelopes of the plastids of etiolated barley (Hordeum vulgare L.) leaves

Electron-dense precipitate was found consistently in the plastid envelope compartment in etiolated barley (Hordeum vulgare L.) leaves, incubated prior to fixation with succinate or malate as substrates and ferricyanide as the electron acceptor. Sulfhydryl reagents p-chloromercuribenzoate and N-ethylmaleimide abolished this reaction, while KCN did not affect it. Prefixation with 0.1% glutaraldehyde followed by incubation in basic media did not change the fine structural localization of precipitate, whereas pretreatment with 1.25% glutaraldehyde resulted in aspecific precipitation. Omission of the subtrate from the medium brought about diminished or negative reaction. Our results indicate that a (possibly not yet assembled) nitrate reductase complex is present in the plastid envelope compartment, the diaphorase part of which is responsible for the observed precipitation.Abbreviations PCMB p-chloromercuribenzoate - NEM N-ethylmaleimide - NR nitrate reductase - SDH succinic dehydrogenase     

Effects of nitrogen deficiency on accumulation of fructan and fructan metabolizing enzyme activities in sink and source leaves of barley (Hordeum vulgare)

Seedlings of barley ( Hordeum vulgare L. cv. Agneta) were grown hydroponically under continuous light, constant temperature and relative humidity. During the first two weeks, the relative growth rate (RGR) was kept at 25% by limiting only the supply of nitrogen. The cultures were then transferred to nitrogen-free media and the amounts of fructan, starch, sucrose, glucose and fructose in sink and source leaves were measured at 0, 12, 24, 48, 72, 120 and 156 h. The activities of two key enzymes in fructan metabolism, sucrose:sucrose fructosyltransferase (SST), fructan exohydrolase (FEH), as well as acid invertase were also measured in the two types of leaves.
The fructan and starch levels in both sink and source leaves increased during nitrogen deficiency. The highest increase in starch was 200% of the control while for fmctans a 700% increase was recorded. The activity of SST increased parallel to fructan accumulation in sink leaves. However the FEH activity was constant and not affected by nitrogen deficiency. The invertase activity both in sink and source leaves was reduced by nitrogen deficiency. More fructans as well as sucrose and fructose accumulated in source leaves compared to sink leaves both before and after nitrogen starvation. The results show that fructan is the major carbohydrate reserve accumulating under nitrogen deficiency both in sink and source leaves in barley plants. The induction of fructan accumulation in sink leaves caused by nitrogen deficiency is intimately connected with the regulation of SST     

Tissue specificity and induction of class I, II and III chitinases in barley (Hordeum vulgare)

Barley ( Hordeum vulgare L.) chitinases (EC 3.2.1.14) were found to be distributed and induced in highly tissue specific patterns. Out of 6 chitinases investigated 3 were present in leaves and only a class II chitinase (molecular mass 24 846 ± 5 Da, pI≥9.8) was markedly induced in leaves heavily infected with powdery mildew ( Erysiphe graminis f. sp. hordei ). The class II chitinase and a novel class III chitinase (molecular mass 30 kDa, pI≥9.8) were found in intercellular washing fluid of leaves, suggesting extracellular deposition. Neither of these two proteins were induced after infiltration of sodium salicylate (2 m M , pH 6.5) or nickel chloride (2 m M ). The class III chitinase showed exochitinase activity in addition to endochitinase activity. No grain specific chitinases were found in leaves after any of the stresses applied. In contrast, 3 grain specific chitinases and one of the leaf chitinases were found in in vitro cultures.     

Light-independent accumulation of chlorophyll a and b and protochlorophyllide in green barley (Hordeum vulgare)

Barley ( Hordeum vulgare L. cvs Clipper, Procter, Astrix) seedlings were transferred from daylight to darkness and changes in chlorophyll a , chlorophyll b , protochlorophyllide and chlorophyllide (μ leaf⁻¹) in either the first or second leaf determined spectrophotometrically after separating the esterified from unesterified pigments by partitioning between ammoniacal acetone and light petroleum ether. Chlorophyll a and b as well as protochlorophyllide accumulated in the dark. The ratio of chlorophyll to protochlorophyllide formed in the absence of light was 18:1. 5-aminolevulinic acid (10 m M ) promoted the synthesis of chlorophyll a and b and protochlorophyllide. Pigment synthesis and response to 5-aminolevulinic acid addition was related to tissue age. Mature tissue in the apical third of the leaf accumulated most chlorophyll, but per μg chlorophyll present at the time of transfer to darkness, was less efficient than immature tissue towards the base of the leaf. Immature tissue was also most responsive to added 5-aminolevulinic acid. Chlorophyll synthesis in the dark was accompanied by chloroplast development. Chloroplasts in immature leaf tissue increased in size and extent of thylakoid development when transferred from daylight to darkness. The results indicate that chlorophyll synthesis and chloroplast membrane development in light-grown barley continue into the dark phase of the diurnal cycle. A light-independent protochlorophyllide reductase in light-grown barley seedlings is postulated.     

Variation within flax (Linum usitatissimum) and barley (Hordeum vulgare) in response to allelopathic chemicals

Summary A possible method of manipulating allelopathy would be to develop crop varieties showing an increased tolerance to allelopathic chemicals. We therefore examined four flax (Linum usitatissimum) varieties and two wild Linum species in the presence of p-coumaric acid and four barley (Hordeum vulgare) varieties in the presence of p-coumaric acid, scopoletin and wild oat (Avena fatua) extract. Analysis of variance indicates significant interaction between variety and treatment for shoot and root growth for seedling flax, shoot growth for older flax, and root growth for seedling barley. These differences in tolerance between varieties could be exploited to develop-varieties with greater tolerances to the allelochemicals produced by weeds or in crop residues and therefore potentially more tolerant of the presence of weeds.     

Genetic analysis of the accumulation of COR14 proteins in wild (Hordeum spontaneum) and cultivated (Hordeum vulgare) barley

The cold-regulated (COR14) protein of 14 kDa is a polypeptide accumulated under low-temperature conditions in the chloroplasts of barley leaves. In H. vulgare the COR14 antibody cross-reacts with two proteins, with a slightly different relative molecular weight around the marker of 14.4 kDa, referred to as COR14a and COR14b (high and low relative molecular weight, respectively). In a collection of H. spontaneum genotypes a clear polymorphism was found for the corresponding COR proteins. While some accessions showed the same COR pattern as cultivated barley, in 38 out of 61 accessions examined the COR14 antibody cross-reacted with an additional coldregulated protein with a relative molecular weight of about 24 kDa (COR24). The accumulation of COR24 was often associated with the absence of COR14b; the relationship between the COR14b/COR24 polymorphism and the adaptation of H. spontaneum to different environments is discussed. By studying COR14 accumulation in cultivated barley we have found that the threshold induction-temperature of COR14a is associated with the loci controlling winter hardiness. This association was demonstrated by using either a set of 30 cultivars of different origin, or two sets of frost-tolerant and frost-sensitive F1 doubled-haploid lines derived from the cross Dicktoo (winter type) x Morex (spring type). These results suggest that the threshold induction-temperature of COR14a can be a potential biochemical marker for the identification of superior frostresistant barley genotypes.     

Genotype-specific differences in chilling tolerance of maize in relation to chilling-induced changes in water status and abscisic acid accumulation

Four inbred maize lines differing in chilling tolerance were used to study changes in water status and abscisic acid (ABA) levels before, during and after a chilling period. Seedlings were raised in fertilized soil at 24/22°C (day/night), 70% relative humidity. and a 12-h photoperiod with 200 μmol m⁻² s⁻¹ from fluorescent tubes. At an age of 2 weeks the plants were conditioned at 14/12°C for 4 days and then chilled for 5 days at 5/3°C. The other conditions (relative humidity, quantum flux, photoperiod) were unchanged. After the chilling period the plants were transferred to the original conditions for recovery. The third leaves were used to study changes in leaf necrosis, ion efflux, transpiration, water status and ABA accumulation. Pronounced differences in chilling tolerance between the 4 lines as estimated by necrotic leaf areas, ion efflux and whole plant survival were observed. Conditioning significantly increased tolerance against chilling at 5/3°C in all genotypes. The genotypes with low chilling tolerance had lower water and osmotic potentials than the more tolerant genotypes during a chilling period at 5/3°C. These differences were related to higher transpiration rates and lower diffusive resistance values of the more susceptible lines. During chilling stress at 5/3°C ABA levels were quadrupled. Only a small rise was measurable during conditioning at 14/12°C. However, conditioning enhanced the rise of ABA during subsequent chilling. ABA accumulation in the two lines with a higher chilling tolerance was triggered at a higher leaf water potential and reached higher levels than in the less tolerant lines. We conclude that chilling tolerance in maize is related to the ability for fast and pronounced formation of ABA as a protective agent against chilling injury.     

Water stress‐induced abscisic acid accumulation in relation to reducing agents and sulfhydryl modifiers in maize plant

Signalling process of water stress‐induced abscisic acid (ABA) accumulation was investigated in maize (Zea mays L.) leaf and root tissues. Potent free‐radical scavengers and reducing agents, N‐acetyl cysteine (NAC) and cystein (Cys), significantly inhibited or nearly completely blocked dehydration‐induced ABA accumulation. Dithiothreitol (DTT), a reducing agent but not a free‐radical scavenger, also significantly inhibited such accumulation whereas solely free‐radical scavengers, dimethyl sulphoxide (DMSO) and melatonin (Mela), had no effects. Moreover, water stress‐induced ABA accumulation was not affected either by free radicals, such as superoxide anion and hydrogen peroxide, or by oxidants such as KIO_4. These observations suggest that the blocking of water stress‐induced ABA accumulation resulted from the reducing effect, rather than from anything associated with free radicals. The disulphide bond might be crucial to the reactivity of some signal element(s) in the signalling process of water stress‐induced ABA accumulation. To test the hypothesis, we used a sulfhydryl modifier, iodoacetamide (IOA), and found that it nearly totally blocked the water stress‐induced ABA accumulation. Furthermore, an impermeable sulfhydryl modifier, p‐chloromercuriphenylsulphonic acid (PCMBS), could also inhibit the water stress‐induced ABA accumulation in the leaf tissues. These results indicate that water stress‐perception protein(s) or receptor(s) may be located on the plasmalemma and a sulfhydryl group in the extracellular domain is critical to the reactivity of the speculated water stress receptors. Cys, DTT and IOA did not lead to a decrease of the baseline ABA level, i.e. in non‐stressed roots. Result indicates that their blocking of water stress‐induced ABA accumulation occurred upstream of the ABA biosynthesis pathway, i.e. in the signalling process that initiates such accumulation.     

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