Comparative Induction of Nitrate and Nitrite Uptake and Reduction Systems by Ambient Nitrate and Nitrite in Intact Roots of Barley (Hordeum vulgare L.) Seedlings

The induction by ambient NO3- and NO2- of the NO3- and NO2- uptake and reduction systems in roots of 8-d-old intact barley (Hordeum vulgare L.) seedlings was studied. Seedlings were induced with concentrations of NaNO3 or NaNO2 ranging from 0.25 to 1000 [mu]M. Uptake was determined by measuring the depletion of either NO3- or NO2- from uptake solutions. Enzyme activities were assayed in vitro using cell-free extracts. Uptake and reduction systems for both NO3- and NO2- were induced by either ion. The Km values for NO3- and NO2- uptake induced by NO2- were similar to those for uptake induced by NO3-. Induction of both the uptake and reduction systems was detected well before any NO3- or NO2- was found in the roots. At lower substrate concentrations of both NO3- and NO2- (5-10 [mu]M), the durations of the lag periods preceding induction were similar. Induction of uptake, as a function of concentration, proceeded linearly and similarly for both ions up to about 10 [mu]M. Then, while induction by NO3- continued to increase more slowly, induction by NO2- sharply decreased between 10 and 1000 [mu]M, apparently due to NO2- toxicity. In contrast, induction of NO3- reductase (NR) and NO2- reductase (NiR) by NO2- did not decrease above 10 [mu]M but rather continued to increase up to a substrate concentration of 1000 [mu]M. NO3- was a more effective inducer of NR than was NO2-; however, both ions equally induced NiR. Cycloheximide inhibited the induction of both uptake systems as well as NR and NiR activities whether induced by NO3- or NO2-. The results indicate that in situ NO3- and NO2- induce both uptake and reduction systems, and the accumulation of the substrates per se is not obligatory.     

Stimulation of Nitrate and Nitrite Efflux by Ammonium in Barley (Hordeum vulgare L.) Seedlings

The inhibitory effect of NH4+ on net NO3- uptake has been attributed to an enhancement of efflux and, recently, to an inhibition of influx. To study this controversy, we devised treatments to distinguish the effects of NH4+ on these two processes. Roots of intact barley (Hordeum vulgare L.) seedlings, uninduced or induced with NO3- or NO2-, were used. Net uptake and efflux, respectively, were determined by following the depletion and accumulation in the external solutions. In roots of both uninduced and NO2- -induced seedlings, NO3- efflux was negligible; hence, the initial uptake rates were equivalent to influx. Under these conditions, NH4+ had little effect on NO3- uptake (influx) rates by either the low- or high-Km uptake systems. In contrast, in plants preloaded with NO3-, NH4+ and its analog CH3NH3+ decreased net uptake, presumably by enhancing NO3- efflux. The stimulatory effect of NH4+ on NO3- efflux was a function of external NH4+ and internal NO3- concentration. These results were corroborated by the absence of any effect of NH4+ on NO2- uptake unless the roots were preloaded with NO2-. In this case NH4+ increased efflux and decreased net uptake. Hence, the main effect of NH4+ on net NO3- and NO2- uptake appears to be due to enhancement of efflux and not to inhibition of influx.     

The Uptake and Transport of Chromium by Barley Seedlings (Hordeum vulgare L.)

Uptake of ⁵¹CrO^2–₄ by intact barley seedlings was linearover 24 h and was stimulated by Ca2⁺ but inhibited by SO^2–₄and other Group VI anions. Uptake increased with increasingchromate concentration, but unless the concentration was high(100 µM) less than 1 per cent of the isotope absorbedwas transported to the shoots. The results of solvent extraction,subcellular fractionation and efflux studies indicated thatmost of the isotope accumulated by the roots was present ina soluble non-particulate form in the vacuoles. The possiblereasons for the restriction in chromate transport are discussedin relation to the metabolism of the element.     

In Vivo Nitrate Reduction in Roots and Shoots of Barley (Hordeum vulgare L.) Seedlings in Light and Darkness

In vivo NO₃⁻ reduction in roots and shoots of intact barley (Hordeum vulgare L. var Numar) seedlings was estimated in light and darkness. Seedlings were placed in darkness for 24 hours to make them carbohydrate-deficient. During darkness, the leaves lost 75% of their soluble carbohydrates, whereas the roots lost only 15%. Detached leaves from these plants reduced only 7% of the NO₃⁻ absorbed in darkness. By contrast, detached roots from the seedlings reduced the same proportion of absorbed NO₃⁻, as did roots from normal light-grown plants. The rate of NO₃⁻ reduction in the roots accounted for that found in the intact dark-treated carbohydrate-deficient seedlings. The rates of NO₃⁻ reduction in roots of intact plants were the same for approximately 12 hours, both in light and darkness, after which the NO₃⁻ reduction rate in roots of plants placed in darkness slowly declined. In the dark, approximately 40% of the NO₃⁻ reduction occurred in the roots, whereas in light only 20% of the total NO₃⁻ reduction occurred in roots. A lesser proportion was reduced in roots because the leaves reduced more nitrate in light than in darkness.     

Nitrate Reduction in Roots and Shoots of Barley (Hordeum vulgare L.) and Corn (Zea mays L.) Seedlings: I. N Study

Nitrate reduction in roots and shoots of 7-day-old barley seedlings, and 9-day-old corn seedlings was investigated. The N-depleted seedlings were transferred for 24 h or 48 h of continuous light to a mixed nitrogen medium containing both nitrate and ammonium. Total nitrate reduction was determined by ¹⁵N incorporation from ¹⁵NO₃⁻, translocation of reduced ¹⁵N from the roots to the shoots was estimated with reduced ¹⁵N from ¹⁵NH₄⁺ assimilation as tracer, and the translocation from the shoots to the roots was measured on plants grown with a split root system. A model was proposed to calculate the nitrate reduction by roots from these data. For both species, the induction phase was characterized by a high contribution of the roots which accounted for 65% of the whole plant nitrate reduction in barley, and for 70% in corn. However, during the second period of the experiment, once this induction process was finished, roots only accounted for 20% of the whole plant nitrate reduction in barley seedlings, and for 27% in corn. This reversal in nitrate reduction localization was due to both increased shoot reduction and decreased root reduction. The pattern of N exchanges between the organs showed that the cycling of reduced N through the plant was important for both species. In particular, the downward transport of reduced N increased while nitrate assimilation in roots decreased. As a result, when induction was achieved, the N feeding of the roots appeared to be highly dependent on translocation from the leaves.     

Proton Flux and Elongation in Primary Roots of Barley (Hordeum vulgare L.)

The elongation zone of the primary root of barley (Hordeum vulgare L.) has been reported to be markedly basic in pH, in apparent contradiction of the acid-growth theory. We determined simultaneously the location of the elongation zone and the basic zone in these roots and found them indeed to be the same. However, sections of barley root elongation zones were found to respond to acidic, basic, and neutral solutions as predicted by the acid-growth theory.     

Growth and Development of Young Roots of Barley (Hordeum vulgare L.) Genotypes

Barley (Hordeum vulgare L.) genotypes from countries with aMediterranean climate grown in temperature-controlled glasshousein nutrient solution to determine whether the co-ordinationof root branching and growth found by other workers appliedto a wider of up to 14 genotypes. There was substantial variationin the number of seminal axes produced by the genotypes, rangingfrom about seven for Hoshimasari and Swanneck to about fourfor Gerbel 'B'. The number of nodal axes was linearly relatedto the number of leaves and typically between one and two mainstemleaves were required before nodal axes appeared. There weresmall genotypic differences in the number of axes produced perleaf with values ranging from 1·5 to 2·3. Theproduction and growth of lateral roots were coordinated so thatthe mean length of laterals generally increased with time. Landraces(Arabic abiad and Arabic aswad) produced more lateral rootswith a faster rate extension compared with other genotypes.The length and number of primary and secondary lateral rootswere related linearly, but no genotypic differences in thisrelation were evident. Length of primary lateral roots increasedmore rapidly than that of secondary lateral roots throughoutthe three to five leaf stage. The ratio of root weight to totalplant weight decreased with time but there were only small differenceswithin this range of genotypes.Copyright 1995, 1999 AcademicPress Barley, seminal axes, nodal axes, primary lateral roots, relative extension rates, relative multiplication rates     

Properties and Regulation of Aspartate Kinase from Barley Seedlings (Hordeum vulgare L.)

Aspartate kinase (EC 2.7.2.4) has been purified 8-fold and characterized from germinating barley (Hordeum vulgare) seedlings. The enzyme is inhibited 50% by 0.7 mm l-lysine and almost completely at 5 mm. l-Methionine does not affect the enzyme on its own, but at low concentrations (0.1-1 mm) increases the inhibition in the presence of lysine, indicating that the two amino acids act as cooperative feedback regulators.     

Lysine Biosynthesis in Barley (Hordeum vulgare L.)

Lysine biosynthesis in seedlings of barley (Hordeum vulgare L. var. Emir) was studied by direct injection of the following precursors into the endosperm of the seedlings: acetate-1-¹⁴C; acetate-2-¹⁴C; pyruvate-1-¹⁴C; pyruvate-2-¹⁴C; pyruvate-3-¹⁴C; alanine-1-¹⁴C; aspartic acid-1-¹⁴C; aspartic acid-2-¹⁴C; aspartic acid-3-¹⁴C; aspartic acid-4-¹⁴C; α-aminoadipic acid-1-¹⁴C; and α, ε-diaminopimelic acid-1-(7)-¹⁴C. The distribution of activity in the individual carbon atoms of lysine in the different biosynthetic experiments was determined by chemical degradation. The incorporation percentages and labeling patterns obtained are in agreement with the occurrence of the diaminopimelic acid pathway. The results do not fit the incorporation percentages and labeling patterns expected if the α-aminoadipic acid pathway was operating. However, the results show that barley seedlings are able to convert a small part of the α-aminoadipic acid administered directly to lysine.     

Lysine Catabolism in Barley (Hordeum vulgare L.)

Lysine catabolism in seedlings of barley (Hordeum vulgare L. var. Emir) was studied by direct injection of the following tracers into the endosperm of the seedlings: aspartic acid-3-(14)C, 2-aminoadipic acid-1-(14)C, saccharopine-(14)C, 2,6-diaminopimelic acid-1-(7)-(14)C, and lysine-1-(14)C. Labeled saccharopine was formed only after the administration of either labeled 2,6-diaminopimelic acid or labeled lysine to the seedlings. The metabolic fate of the other tracers administered also supported a catabolic lysine pathway via saccharopine, and apparently proceeding by a reversal of some of the biosynthetic steps of the 2-aminoadipic acid pathway known from lysine biosynthesis in most fungi. Pipecolic acid seems not to be on the main pathway of l-lysine catabolism in barley seedlings.     

In vivo Nitrate Reductase Activity in Barley (Hordeum vulgare L.) during Ear Development

Experiments were conducted during the 1974–75 and 1975–76winter season with the barley (Hordeum vulgare L.) cultivarJyoti. From amongst the various plant parts, the flag leaf bladehad higher in vivo nitrate reductase (NR) activity than thelower two leaf blades, glumes, and grains. However, the potentialof a plant part to reduce NO₃^– is a function of its freshweight and the NR per unit fresh weight. On this basis, thesecond and third leaf blades could reduce more NO₃^– thanthe flag leaf blade. N fertilizer application resulted in enhancementof the activity of the leaf blades alone. N fertilizer appliedduring the reproductive phase was taken up and assimilated bythe various plant parts. The studies suggest that, even whenthe fertilizer is applied at optimum levels for obtaining maximumyields, the upper leaf blades have sub-optimal NR activity andthat there is a likelihood of either a preferential flow ofNO₃^– to the leaf blades or transnational barriers to NO₃^–movement to the ear.     

Phosphoinositides in Barley (Hordeum vulgare L.) Aleurone Tissue

[3H]Inositol labeling of barley (Hordeum vulgare L. cv Himalaya) aleurone layers and analysis of phospholipids by deacylation revealed the presence of phosphatidylinositol (PtdIns), PtdIns3P, and PtdIns4P but not PtdInsP2 species. In contrast to an earlier report (P.P.N. Murthy, G. Pliska-Matyshak, L.M. Keranen, P. Lam, H.H. Mueller, N. Bhuvarahamurthy [1992] Plant Physiol 98: 1498-1501) systematic chemical degradation of PtdIns revealed no evidence of a second isomer of PtdIns. Evidence of the widespread occurrence of 3-phosphorylated PtdIns within the plant kingdom is presented.     

Natural H Currents Traverse Growing Roots and Root Hairs of Barley (Hordeum vulgare L.)

With the aid of an extracellular vibrating electrode, natural electric fields were detected and measured in the medium near growing roots and root hairs of barley seedlings. An exploration of these fields indicates that both the root as a whole, as well as individual root hairs, drive large steady currents through themselves. Current consistently enters both the main elongation zone of the root as well as the growing tips of elongating root hairs; it leaves the surface of the root beneath the root hairs. These currents enter with a density of about 2 microamperes per square centimeter, leave with a density of about 0.5 to 1 microampere per square centimeter, and total about 30 nanoamperes.     

Apoplast Acidification in Growing Barley (Hordeum vulgare L.) Leaves

Apoplast acidification associated with growth is well documented in roots, coleoptiles, and internodes but not in leaves. In the present study, advantage was taken of the high cuticle permeability in the elongation zone of barley leaves to measure apoplast pH and growth in response to application of test reagents. The role of the plasma membrane H⁺-ATPase (PM-H⁺-ATPase) and K⁺ in this process was of particular interest. pH microelectrodes and an in vitro gel system with bromocresol purple as pH indicator were used to monitor apoplast pH. Growth was measured in parallel or in separate experiments using a linear variable differential transformer. Test reagents that blocked (vanadate) or stimulated (fusicoccin) PM-H⁺-ATPase or that reduced (Cs⁺, tetraethylammonium) K⁺ uptake were applied. Apoplast pH was lower in growing than in nongrowing leaf tissue and increased in the elongation zone with increasing apoplast K⁺. Vanadate increased apoplast pH and reduced growth, whereas fusicoccin caused the opposite effects. It is concluded that barley leaves exhibit acid-growth-type mechanisms in that apoplast pH is lower in elongating leaf tissue. Both growth and apoplast pH depend on the activity of the PM-H⁺-ATPase and K⁺ transport processes. However, not all of the growth displayed by leaves is dependent on a lower apoplast pH in the elongation zone; up to 50 % of growth is retained when apoplast pH in the elongation zone increases to a value observed in mature tissue.     

Heat Shock Response in Hypocotyl of Barley (Hordeum vulgare L.)

In vivo protein synthesis in barley (Hordeum vulgare L.) hypocotyl was maximum at 35°C and 40°C.HPLC analysis of soluble proteins showed 10 different types of proteins, out of which the peak corresponding to retention time 13.3 min was present at 25°C but was absent at 35°C and 40°C. Instead, another peak with retention time 13.7 min was noticed at 35°C and 40°C. Amino acid analysis showed that heat shock resulted in an increase in lysine and histidine and decrease in arginine. Heat shock also resulted in increase in peroxidase, protease and ribonuclease activity at 35°C and 37°C in comparison to 25°C. The incorporation of (3H)-uridine was significantly decreased at 37°C in comparison to 25°C.     

大麦转基因座位结构的研究

利用质粒营救法获得基因枪法转化的4种转绿色荧光蛋白基因(green fluorescent protein,GFP)大麦的转基因座位序列,序列分析显示4种材料的转基因座位中均有完整栽体的串联重复现象,表明转基因整合是同源重组的结果.同时转基因座位中也存在不完整载体片段、基因组片段的混杂排列,说明转基因整合时也发生异常重组.微粒轰击的转基因整合是由异常重组和同源重组共同完成的.     

Extraction and Affinity Purification of NADH:Nitrate Reductase from Barley (Hordeum distichum L.) Roots

Conditions suited for the extraction and purification of NADH:nitratereductase (NR) from barley (Hordeum distichum L.) roots wereexamined. The addition of 10 mM EDTA to the extraction mediumproduced an 8-fold increase in the NR activity in the crudeextract, whereas the presence of cysteine in the medium causedan appreciable decrease in this activity. EDTA and FAD stimulatedNR activity in the crude extract; cysteine inhibited it. Theeffect of EDTA seemed to be due to the inhibition of the contaminatingNADH-oxidizing system. The NADH:NR was purified 300-fold by ammonium sulfate fractionationand blue dextran-Sepharose affinity chromatography. The specificactivity was 1,420 nmol nitrite formed min^–1 mg protein^–1at 30?C; the highest specific activity among the NR preparationsobtained thus far from root tissues of higher plants. EDTA,as well as cysteine behaved as an inhibitor for the purifiedNR. (Received January 27, 1982; Accepted June 21, 1982)     

Variability in Proline-Accumulating Ability of Barley (Hordeum vulgare L.) Cultivars Induced by Vapor Pressure Deficit

This work was undertaken in an effort to reconcile the conflicting proline-accumulating responses of the barley (Hordeum vulgare L.) cultivars, Excelsior and Proctor, reported by Singh et al. (1972) and Hanson et al. (1976). It deals with the effects of different vapor pressure deficits (VPD) during growth and subsequent drought stress on several barley cultivars. A higher VPD (1.2 kilopascals) during Clipper seedling growth resulted in higher solute-accumulating ability, seemingly independently of leaf water potential, than a lower VPD (0.12 kilopascals). The higher VPD during stress also resulted in higher solute contents, and this response may be more closely related to leaf water potential. When the responses of Excelsior and Proctor were examined in detail, it was found that the relative proline-accumulating ability of the two cultivars was dependent upon the VPD under which they were grown. At low VPD, Proctor accumulated significantly more proline than did Excelsior; whereas at higher VPD, Excelsior accumulated more proline than did Proctor. The crossover occurred at a VPD of about 0.72 kilopascals. This reversal of cultivar response was enhanced by multiplying seed under the two VPD extremes. Glycinebetaine accumulation did not demonstrate the crossover effect, although the concentration of this compound in all cultivars also depended on the VPD prevailing during growth and/or stress. Solute levels, in general, were more closely related to the decrease in relative water content than to a decrease in leaf water potential. It is concluded that the conflicting proline-accumulating responses of Excelsior and Proctor could be explained by these findings.     

Flow Cytometric Analysis and Chromosome Sorting of Barley (Hordeum vulgare L.)

Flow cytometric analysis was systematically performed to optimize the concentration and duration of hydroxyurea (DNA synthesis inhibitor) and trifluralin (metaphase blocking reagent) treatments for synchronizing the cell cycle and accumulating metaphase chromosomes in barley root tips. A high metaphase index (76.5% in the root tip meristematic area) was routinely achieved. Seedlings of about 1.0-cm length were treated with 1.25 mM hydroxyurea for 14 h to synchronize the root tip meristem cells at the S/G2 phase. After rinsing with hydroxyurea, the seedlings were incubated in a hydroxyurea-free solution for 2 h and were treated with 1 microM trifluralin for 4 h to accumulate mitotic cells in the metaphase. The consistent high metaphase index depended on the uniform germination of seeds prior to treatment. High-quality and high-quantity isolated metaphase chromosomes were suitable for flow cytometric analysis and sorting. Flow karyotypes of barley chromosomes were established via univariate and bivariate analysis. A variation of flow karyotypes was detected among barley lines. Two single chromosome types were identified and sorted. Bivariate analysis showed no variation among barley individual chromosomes in AT and GC content.     

Effects of Mannitol Pretreatment on Androgenesis of Barley (Hordeum vulgare L.)

The effects of mannitol pretreatment on androgenesis of barley were systematically studied in comparison with that of cold pretreatment and control. The results showed that mannitol pretreatment could significantly increase the frequency of pollen survival reaching 19.0% on the eighth day, while in cold pretreatment and control they were 8.4% and 6.6 %, respectively. Mannitol pretreatment could also improve the quality of pollen and inhibit starch production from microspore, which were quite advantageous to microspore division and development. The developing period was shortened 2--3 days as compared with cold pretreatment and control. The major developmental pathways of androgenesis after mannitol pretreatment were the equal division (B pathway). In addition, the majority of microspore nuclei were diploids. On the contrary, the major microspores pretreated with low temperature had fewer chromosomes than with mannitol pretreatment, the microspore nuclei were haploids.