Comparison of in Vivo and in Vitro Assays of Nitrate Reductase in Wheat (Triticum aestivum L.) Seedlings

The effectiveness of the in vivo and in vitro assays for nitrate reductase (NR) in estimating the amounts of reduced N made available to plants was tested against the daily increases in reduced N (Nesslerization) actually accumulated by the plant. With growth-chamber-grown wheat seedlings, the average ratio values (input of reduced N as estimated by the in vitro assay to actual accumulation of N by the plant) were 3.9 for shoots, 3.7 for the roots, and 4.1 for the entire plant, over a 10-day period. With the in vivo assay, the average ratio values were 0.7 for the shoot, 1.8 for the root, and 0.9 for the entire plant. Although the linear regressions between the accumulated N in the plant and the estimated N input (by both in vitro and in vivo assays) were significant and positive, the in vivo assay provided the closest approximation of the actual amount of N accumulated.     

Nitrate Uptake and the Induction of Nitrate Reductase Activity in Wheat (Triticum aestivum L.) Seedlings

Nitrate uptake and the subsequent induction of in vivo nitratereductase activity in wheat were studied by investigating aeuploid and certain ditelosomic stocks which exhibited in vivoactivity significantly greater than that of the euploid. Thekinetics of nitrate uptake were investigated, but the high activitiesof the ditelosomics were not caused by increased uptake of nitrate,although ditelo-7B^L exhibited unusual uptake dynamics. Analysisof the induction of nitrate reductase activity revealed a biphasicgeneral pattern, with an initial rapid phase being followedby a slower but longer period of induction. The induction rateover the second period, although responsible for only a minorproportion of the total activity induced, was positively correlatedwith the final nitrate reductase level, unlike the rate overthe first induction period. Several stocks exhibited high inductionrates over one or other of the two phases, while ditelo- 1 A^sshowed an abnormal monophasic induction pattern. At the endof the second period of induction, nitrate reductase activitybecame more or less steady, except for activity fluctuationsassociated with the time of application of induction stimuli.     

Biochemical Basis of Variability in Nitrate Reductase Activity in Wheat (Triticum aestivum L.) Genotypes

Significant differences in the activity of nitrate reductasein vivo and in vitro were observed among wheat genotypes, whenexpressed on a dry weight basis. The genotypes with high nitratereductase (HNR) activity exhibited an efficient nitrate inductionsystem. Availability of NADH seemed to be partially responsiblefor the low activities of the enzyme in genotypes categorizedas low nitrate reductase group (‘LNR). Biochemical analysisof the genotypes revealed that ‘HNR’ genotype hadmore active enzyme protein, high rates of enzyme protein synthesisand higher level of steady state nitrate reductase-mRNA relativeto those observed in ‘LNR’ genotype. ¹ Present address: Department of Botany, Faculty of Science,Hamdard University, Hamdard Nagar, New Delhi 110062, India. ² Present address: Institut für Botanik, UniversitätHannover, 3000 Hannover 21, F.R.G.     

Intracellular Localization of Peptide Hydrolases in Wheat (Triticum aestivum L.) Leaves

Protoplasts from 8- to 9-day-old wheat (Triticum aestivum L.) leaves were used to isolate organelles which were examined for their contents of peptide hydrolase enzymes and, in the case of vacuoles, other acid hydrolases. High yields of intact chloroplasts were obtained using both equilibrium density gradient centrifugation and velocity sedimentation centrifugation on sucrose-sorbitol gradients. Aminopeptidase activity was found to be distributed, in approximately equal proportions, between the chloroplasts and cytoplasm. Leucyltyrosine dipeptidase was mainly found in the cytoplasm, although about 27% was associated with the chloroplasts. Vacuoles shown to be free from Cellulysin contamination contained all of the protoplast carboxypeptidase and hemoglobin-degrading activities. The acid hydrolases, phosphodiesterase, acid phosphatase, α-mannosidase, and β-N-acetylglucosamidase were found in the vacuole to varying degrees, but no β-glucosidase was localized in the vacuole.     

Influence of Osmotic Stress on Nitrate Reductase Activity in Wheat (Triticum aestivum L.) and the Role of Abscisic Acid

Wheat seedlings (Triticum aestivum L. cv. Timmo) were treatedwith up to 20% (w/v) polyethylene glycol (PEG, mol. wt. 3350)in the nutrient medium for 6 d. Shoot growth and nitrate transportand metabolism were substantially affected by PEG treatment.At 20% PEG (corresponding to a water potential of approximately–1.6 MPa), which caused plants to wilt within 1–2h, activity of nitrate reductase (NR) declined with a half-lifeof approximately 5 h in both roots and shoots. The decline wasconsiderably slower at lower PEG concentrations. Significantincreases in levels of abscisic acid (ABA) only occurred inshoots. Application of ABA to intact plants or excised shootsdid not induce or accelerate decline in shoot NR activity. Therapid decline in NR activity during wilting appears unrelatedto both nitrate flux and ABA. At lower PEG concentrations andin the long-term, however, NR activity corroborates rates ofboth transport and growth-related utilization of nitrate. Therole of ABA in this context appears to be indirect through itsaction on stomatal function which reduces water flux and gasexchange. Key words: Stress, nitrate reductase, abscisic acid (ABA)     

Nitrate Reductase Activity in Maize (Zea mays L.) Leaves: I. Regulation by Nitrate Flux

The roles that leaf nitrate content and nitrate flux play in regulating the levels of nitrate reductase activity (NRA) were investigated in 8- to 14-day old maize (Zea mays L.) plants containing high nitrate levels while other environmental and endogenous factors were constant. The nitrate flux of intact plants was measured from the product of the transpiration rate and the concentration of nitrate in the xylem. NRA decreased when the seedlings were deprived of nitrate. The nitrate flux and the leaf nitrate content also decreased. When nitrate was resupplied to the roots, all three parameters increased.     

Elicitor-Induced Cinnamyl Alcohol Dehydrogenase Activity in Lignifying Wheat (Triticum aestivum L.) Leaves

The substrate-specific induction of wheat (Triticum aestivum L. cv Fenman) leaf cinnamyl alcohol dehydrogenase (CAD, EC 1.1.1.195) was examined in relation to its role in regulating the composition of defensive lignin induced at wound margins. Treatment of wounds with a partially acetylated chitosan hydrolysate or spores of the nonpathogen Botrytis cinerea elicited lignification at wound margins and invoked significant increases in phenylalanine ammonia-lyase (EC 4.3.1.5), peroxidase (EC 1.11.1.7), and CAD activities. The substrate-specific induction of CAD with time was determined in elicitor-treated leaves and in excised lignifying wounds. In whole leaf extracts no significant increases in p-cou-maryl and coniferyl alcohol dehydrogenase activities were detectable, but a significant 5-fold increase in sinapyl alcohol dehydrogenase activity was evident 32 h after elicitor treatment. Similarly, fungal challenge resulted in elevated levels of only sinapyl alcohol dehydrogenase in whole-leaf extracts. In excised lignifying tissues p-coumaryl alcohol dehydrogenase levels were similar to those observed in healthy tissue. A small yet significant increase in coniferyl alcohol dehydrogenase was apparent, but the most dramatic increase occurred in sinapyl alcohol dehydrogenase activity, which increased to values approximately 10 times higher than the untreated controls. Our results show for the first time that CAD induction in lignifying tissues of wheat is predominantly attributable to highly localized increases in sinapyl alcohol dehydrogenase activity.     

Light and Dark Controls of Nitrate Reduction in Wheat (Triticum aestivum L.) Protoplasts

Protoplasts were isolated from the leaves of nitrate-cultured wheat (Triticum aestivum L. var. Frederick) seedlings. When incubated in the dark, protoplasts accumulated nitrite under anaerobic, but not under aerobic, conditions. The assimilation of [¹⁵N]nitrite by protoplasts was strictly light-dependent, and no loss of nitrite from the assay medium was observed under dark aerobic conditions. Therefore, the absence of nitrite accumulation under dark aerobic conditions was the result of an O₂ inhibition of nitrate reduction and not a stimulation of nitrite reduction. In the presence of antimycin A, protoplasts accumulated nitrite under dark aerobic conditions. The oxygen inhibition of nitrate reduction was apparently due to a competition between nitrate reduction and dark respiration for cytoplasmic-reducing equivalents.     

Nitrogen Metabolism in Senescent Flag Leaves of Wheat (Triticum aestivum L.) in the Light

Nitrogen metabolism was examined in senescent flag leaves of 90- to 93-day-old wheat (Triticum aestivum L. cv Yecora 70) plants. CO₂ assimilation and the levels of protein, chlorophyll, and nitrogen in the leaves decreased with age. Glutamine synthetase activity decreased to one-eighth of the level in young flag leaves. Detached leaves were incubated (with the cut base) in ¹⁵N-labeled NH₃, glutamate, or glycine in the light (1.8 millieinstein per square meter per second) at 25°C in an open gas exchange system under normal atmospheric conditions for up to 135 minutes. The ¹⁵N-enrichment of various amino acids derived from these ¹⁵N-substrates were examined. The amido-N of glutamine was the first ¹⁵N-labeled product in leaves incubated with ¹⁵NH₄Cl whereas serine, closely followed by the amido- and amino-N of glutamine, were the most highly ¹⁵N-labeled products during incubation with [¹⁵N]glycine. In contrast, aspartate and alanine were the first ¹⁵N-labeled products when [¹⁵N] glutamate was used. These results indicate that NH₃ was assimilated via glutamine synthetase and glutamate synthase activities and the photorespiratory nitrogen cycle remained functional in these senescent wheat flag leaves. In contrast, an involvement of glutamate dehydrogenase in the assimilation of ammonia could not be detected in these tissues.     

Free Polyamines in Senescing Wheat(Triticum aestivum L.)

The free polyamine content of flag leaves, peduncles, rachis,glumes, and grains of wheat (Triticum aestivum L., cv. Castell)plants, ripening under field conditions, has been investigatedduring three consecutive growing seasons. Putrescine was quantitativelythe most important of all polyamines detected in these organs.Concentrations were highest in the grains, glumes and flag leaves.No correlation was found between polyamine content and the onsetof senescence of flag leaves and other organs. Excised primaryleaves, however, showed a decrease in polyamine content in thedark and also in light/dark cycles, but in the latter case onlyafter an initial increase. Sink removal of otherwise intactwheat plants caused an accumulation of putrescine in flag leavesat the later stages of senescence, whereas removal of all otherleaves was without any significant effect. Putrescine was alsorecovered in phloem-exudate samples collected throughout theperiod of grain development. In both grains and glumes, peakconcentrations of polyamines were found early during seed development. Key words: Triticum aestivum, polyamines, ripening, senescence     

In Vivo Determination of Parameters of Nitrate Utilization in Wheat (Triticum aestivum L.) Seedlings Grown with Low Concentration of Nitrate in the Nutrient Solution

Six genotypes of winter wheat (Triticum aestivum L.) differing in grain protein concentration were grown on a nutrient solution containing low concentrations of NO₃⁻ (2 millimolar). Total NO₃⁻ uptake varied between genotypes but was not related to grain protein content. An in vivo nitrate reductase assay was used to determine the affinity of the enzyme for NO₃⁻, and large phenotypic variations were observed. In vivo estimations of the concentration and size of the metabolic pool were variable. However, the three genotypes with the higher ratios of metabolic pool size to leaf total NO₃⁻ concentration were the high protein varieties. It is proposed that a high affinity of nitrate reductase for nitrate might be a biochemical marker for the capacity of the plant to continue assimilating NO₃⁻ for a longer period during the last stage of growth.     

不同浓度硝态氮供应下小麦生长、硝态氮累积及根系钙信号特征

以小麦品种‘石麦15’和‘衡观35’为材料进行营养液水培试验,研究不同浓度硝态氮供应对小麦苗期根系形态、钙离子流特征及钙调蛋白(CaM)含量的影响。结果表明,与适宜浓度硝态氮处理(2.5mmol/L)相比,无外源硝态氮供应时小麦地上部鲜重、硝态氮含量均降低,侧根数量显著减少;高浓度硝态氮处理(50mmol/L)下两个小麦品种地上部硝态氮含量升高,根系总长度降低,‘石麦15’侧根数量减少。无硝态氮和高浓度硝态氮处理下,根系中钙调蛋白含量降低,且‘衡观35’的降低幅度大于‘石麦15’。无外源硝态氮供应时小麦根尖表现出较为明显的钙离子外流特征;与适宜浓度硝态氮处理相比,高硝态氮处理下小麦根尖Ca2+的内流速度显著下降。说明硝态氮供应不足和高浓度硝态氮供应会影响小麦根系生长,根系Ca2+外流或Ca2+内流速度下降,CaM含量减少,Ca2+/CaM可能介导硝态氮调控小麦根系生长发育。     

The Morphology and Development of Cross Veins in the Leaves of Bread Wheat (Triticum aestivum L.)

In the leaves of bread wheat Triticum aestivum L. the longitudinalvascular bundles are linked by small transverse bundles Pairsof similar small vascular bundles also link the upper ends ofminor longitudinal bundles to their neighbours in a Y-shapedarrangement The cross-vein procambial strands arise from unexpanded cellsof one layer of the mesophyll tissue. Lines of these cells connectone longitudinal procambial strand to the next The procambialcells subsequently undergo two tangential divisions to producecells which differentiate to form the conducting and parenchymatouselements of the mature cross veins. Anomalous cross veins are sometimes found. possible modes oforigin of these anomalous cross veins are considered.     

QTL Mapping of Diffuse Reflectance Indices of Leaves in Hexaploid Bread Wheat (Triticum aestivum L.)

Russian Journal of Plant Physiology - Quantitative trait loci (QTL) mapping of diffuse reflectance indices of laminas in bread wheat (Triticum aestivum L.) has been first performed under controlled...     

Colchicine-induced Paracrystals in Root Cells of Wheat (Triticum aestivum L.)

Tubulin conformations other than microtubules in the meristematiccells of wheat roots grown in the presence of 2 mM colchicinesolution were investigated by immunofluorescence and electronmicroscopy. In the affected cells microtubules disappeared andwere replaced by tubulin fluorescent strands that occurred inthe cortical cytoplasm. With increasing time of exposure tocolchicine the tubulin strands became better organized and occurredalso in the subcortical cytoplasm and finally they were restrictedto the area around the nucleus. In prophase and preprophasecells thick strands occupied the cortical cytoplasmic zone wherein normal cells a preprophase microtubule band (PMB) was expectedto be assembled. In the colchicine-treated cells electron microscopy revealedan accumulation of paracrystalline aggregates, which initiallyoccurred along the cell wall and later deeper in the cytoplasm,in the perinuclear regions and the cytoplasmic invaginationsof the nucleus. In transverse planes the paracrystalline strandsappear to consist of hexagonal subunits in a 'honeycomb' arrangement,while in longitudinal and oblique sections they exhibit variableimages. Since their distribution coincides with that of thetubulin strands visualized by immunofluorescence, they are consideredto be the same structure. Therefore, the paracrystals consistof, or at least contain, tubulin. They are most likely to bepolymers of tubulin-colchicine complexes.Copyright 1995, 1999Academic Press Wheat roots, colchicine, immunofluorescence, electron microscopy, tubulin paracrystals, Triticum aestivum L     

Floret Initiation and Development in Spring Wheat (Triticum aestivum L.)

The number and developmental stages of florets were determinedin each spikelet of the spike in the main shoots of spring wheat.Samples were taken frequently from plants grown in a phytotronand in a nitrogen application field-test. Ten stages of development,from floret initiation until anthesis, were recognized and described. Inter-spikelet variation in the total number of initiated floretswas rather small. However, the number of florets at advancedstages of development, as well as the number of grains, washighest in the central spikelets in which florets initiatedfirst. Floret initiation did not proceed beyond spike emergence,whereafter the distal florets and the spikelet apex degenerated.Grain-set was restricted to florets which had developed at leastto the stage of visible anther lobes at spike emergence. Thenumber of these florets was increased significantly by nitrogenapplication. Wheat, Triticum aestivum L., spikelet, floret, grain set, nitrogen     

A Study of Floret Development in Wheat (Triticum aestivum L.)

Plants of wheat (Triticum aestlvum L.) cv. Aotea were grownat high or low nitrogen levels and in a natural photoperiodor continuous light. Starting 17–21 days from the double-ridgestage, eight plants from each treatment were sampled every 3days until anthesis, and the two basal, the sixth, and the terminalspikelets were sectioned longitudinally. A developmental scorewas assigned to each floret and rates of development calculated.Continuous light hastened development but reduced the numberof spikelets per ear, while high nitrogen delayed developmentbut increased spikelet numbers. The number of florets initiatedin each spikelet varied within narrow limits, but grain settingdepended strongly on spikelet position and on treatment. Althoughflorets were initiated in acropetal succession, the rate ofdevelopment tended to increase up to floret 4 but then declinedmarkedly. As a result grain setting was confined to basal floretpositions, although the two basal spikelets developed so slowlythat they contributed relatively little to grain yield. Distalflorets degenerated almost simultaneously at or before ear emergence,but those in intermediate positions continued to develop untilafter fertilization in the lower florets. It is argued thatthe spikelet is an integrated system in which correlative mechanismsplay a part throughout the development of the florets.     

部分耐盐小麦品种(系)SSR位点遗传多样性研究

选择有多态性的32对SSR引物对80个小麦耐盐品种(系)进行遗传差异研究,共检测出155个等位变异,平均每个位点上有4.75个等位变异;供试80份耐盐小麦品种(系)来源广泛,遗传基础丰富,表现出较高的遗传多样性,遗传相似系数范围在0.26～0.81;聚类分析结果显示,冬性小麦品种(系)聚为一大类;春性小麦品种(系)也聚为一大类;一些系谱相同或相近的品种(系)遗传相似系数较大;A、B、D基因组中SSR位点平均等位变异差异不大,以B基因组较高.     

外源水杨酸对光抑制条件下小麦叶片光合作用的影响

以浓度为50、100、200 mg·kg-1的水杨酸(SA)预先处理灌浆期的小麦叶片,可有效防护强光所致的氧化损伤,维持较高的超氧化物歧化酶(SOD)和抗坏血酸过氧化物酶(APX)活性,减轻丙二醛(MDA)积累.叶片在光抑制条件下,可维持较高的通过PSⅡ电子传递速率(Fm/Fo)、PSⅡ原初光化学效率(Fv/Fm)、PSⅡ量子效率(ΦPSⅡ)、光化学猝灭系数(qP)、净光合速率(Pn)和较低的非光化学猝灭系数(qNP).其中,以较低浓度SA(50 mg·kg-1)的效果较好.     

Reassessment of the in vivo Assay for Nitrate Reductase in Leaves

The in vivo assay procedure for nitrate reductase and its dependence on the concentration of nitrate and other ions were examined. It was found that high ion concentrations led to an increased release of nitrite to the reaction media which could be interpreted as a stimulated nitrate reductase activity. This phenomenon is not an osmotic effect, since equivalent concentrations of mannitol did not lead to identical results. The effect of ions on the enhanced nitrite production was attributed to changes in cell membrane permeability rather than to a supply of substrate. This conclusion is based on several findings: (a) in in vitro assays, the rate of nitrite production was not affected by ion concentrations: (b) the stimulation of nitrite production was obtained by various ions and not only by nitrate; (c) pretreatment of alfalfa leaves with nitrate did not increase the NO₂^? release rate to the external solution; and (d) nitrate and nitrite export from leaf discs to the solution was stimulated even in discs which were enzymatically inactive. Calcium ions in the presence of KNO₃ inhibited the enhanced nitrite production, probably due to alteration of membrane stability. The effect of ions on the rate of nitrite production was reversible and the high rate of nitrite production was reduced to the control rate when discs were transferred to a solution of low concentration.