Changes in the cellular localization of cytosolic glutamine synthetase protein in vascular bundles of rice leaves at various stages of development

Cellular localization of cytosolic glutamine synthetase (GS1; EC 6.3.1.2) in vascular bundles of leaf blades of rice (Oryza sativa L.), at the stage at which leaf blades 6 (the lowest position) to 10 were fully expanded, was investigated immunocytologically with an affinity-purified anti-GS1 immunoglobulin G. Strong signals for GS1 protein were detected in companion cells of large vascular bundles when blades 6–8 were tested. Signals for GS1 were also observed in vascular-parenchyma cells of both large and small vascular bundles. The results further support our hypothesis that GS1 is important for the export of leaf nitrogen from senescing leaves. The signals in companion cells were less striking in the younger green leaves and were hardly detected in the non-green portion of the 11th blade. In the non-green blades, strong signals for GS1 protein were detected in sclerenchyma and xylemparenchyma cells. When total GS extracts prepared from the 6th,10th, and the non-green 11th blades were subjected to anion-exchange chromatography, the activity of GS1 was clearly separated from that of chloroplastic GS, indicating that GS1 proteins detected in the vascular tissues were able to synthesize glutamine. The function of GS1 detected in the developing leaves is discussed.Abbreviations Fd-GOGAT ferredoxin-dependent glutamate synthase - GS1 cytosolic glutamine synthetase - GS2 plastidic glutamine synthetase - IgG immunoglobulin G     

The Effect of Mercaptoethanol on the Activity of Enzymes of Nitrogen Metabolism in Leaves from Urtica dioica and Spinacia oleracea

The effect of mercaptoethanol at different concentrations on enzyme activity was investigated in leaves from Urtica dioica and Spinacia oleracea. The interference of mercaptoethanol with enzyme activity is dependent on the type of plant, the configuration of the enzyme and the concentration of mercaptoethanol. A stimulation of GDH (glutamate-dehydrogenase) was obtained in both species, while inhibition of GOT (glutamate-oxaloacetate transaminase) and GPT (glutamate-pyruvate transaminase) was demonstrated in Spinacia. The stimulation of GOT and GPT activity in Urtica was probably due to inhibition of phenol oxidase. Conclusions concerning the effect of mercaptoethanol on NR (nitrate reductase) activity were difficult to draw, since mercaptoethanol itself reduced nitrite and interfered with NR determination in tests in vitro. In Urtica. no activity could be obtained at all with the in vitro method, probably because an inhibitor of NR was liberated during the extraction procedure, Activity of NR could however be obtained in both species when using the in vivo method. Addition of protective agents to the extraction medium has been supposed to influence the protein extractability. In conformity with this increasing amount of fresh matter to the same volume of extraction medium resulted in decreased protein extractability. This led to differences in enzyme activity when expressed on a fresh weight basis but the specific activity remained constant.     

缺氮和复氮对菘蓝幼苗生长及氮代谢的影响

对基质育苗后水培的菘蓝进行缺氮与复氮处理,分析其生长情况及氮代谢产物含量的变化,探讨缺氮和复氮对菘蓝幼苗生长及氮代谢的影响,以提高菘蓝产量和品质以及栽培过程中的氮素利用效率。结果显示:(1)正常供氮条件下,菘蓝幼苗的叶绿素含量、谷氨酰胺合成酶(GS)活性、硝态氮含量、靛玉红含量为最高,而其株高、主根直径、根的鲜重与干重、叶的鲜重与干重、根系活力均最小。(2)缺氮处理增加了菘蓝幼苗的主根直径和根干重,提高其根系活力和硝酸还原酶(NR)活性,促进游离氨基酸在叶中的积累;但降低了GS的活性,也降低了叶中硝态氮、可溶性蛋白、靛玉红及根中游离氨基酸的含量;缺氮对叶中靛蓝的含量无明显影响。(3)复氮处理增加了菘蓝幼苗的株高、主根长、根鲜重、叶鲜重、叶干重,提高了其根系活力,降低了NR和GS的活性;与对照相比,复氮降低了叶中硝态氮含量,提高了叶中可溶性蛋白、靛蓝及根中游离氨基酸的含量,但对叶中游离氨基酸和靛玉红含量影响较小。研究表明,缺氮后再复氮有利于菘蓝幼苗叶的生长,同时有利于增加其叶内靛蓝含量,从而提高其产量和品质。     

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.     

Distribution and Partitioning of Newly Taken-up Boron in Sunflower

Distribution and partitioning of newly acquired boron (B) in a mature sunflower (Helianthus annuus L., cv. 3101) plant was investigated. In leaf blades of sunflower plants grown under 0.93, 2.8, and 9.3 mmol B m⁻³, the level of cell-wall-bound B was rather uniform, irrespective of leaf position and B concentration. Boron concentration gradients among leaf positions were produced mainly by different levels of water-soluble B. To determine the distribution of newly taken-up B in plant parts, ¹⁰B-labeled boric acid at a concentration of 2.8 mmol B m⁻³ was applied. The majority of newly acquired B was delivered to the younger leaves, however, approximately one-fourth of the B in the top and second leaves was the older B which was taken up before the 6 d treatment period. In the root tissues, two-fifth of the water-soluble B was new B taken up in the last 6 d, however, within 6 h of the application new B contributed to approximately 80% of the xylem sap B, suggesting that newly taken-up B is preferentially transported to the shoots. When B was withdrawn from the culture solution, the B concentration per leaf area of the lower leaves decreased slightly over 9 d. However, there was an abrupt decrease in the younger leaves, even when taking into account the rapid expansion of the leaf blade, suggesting that B moves more rapidly from the younger leaves than from the older leaves     

Role of light and CO2 fixation in the control of nitrate-reductase activity in barley leaves

Nitrate reductase (NR, NADH:nitrate oxidoreductase, EC 1.6.6.1) from barley (Hordeum vulgare L. cv. Hassan) leaves was inactivated during a light-dark transition, losing approx. 50% of activity after 30 min of darkness. The dark inactivation was reversed by illumination of the seedlings, the kinetics of reactivation being similar to those of inactivation. High extractable NR activity and significant differences between illuminated and darkened leaves were observed in media containing EDTA and inorganic phosphate (P_i). Addition of Ca²⁺ ions during extraction and assay decreased NR activity from illuminated and darkened leaves, enhancing the light-dark difference. While no clear correlation could be found between irradiance and NR activity, a hyperbolic correlation appeared between extractable NR activity and in-vivo rates of CO₂ fixation, indicating that NR activation follows saturation kinetics with respect to CO₂ fixation. Furthermore, hexoses and hexose-phosphates fed to the leaves via the transpiration stream protected against the dark-inactivation of NR. The results indicate that carbon-assimilation products are regulatory factors of NR activity in barley leaves, mediating both the light-dark modulation of NR and its dependence upon CO₂ fixation.     

Nitrate Assimilation in Rice Grown under Lowland Conditions

Pattern and extent to which the main shoot of rice (Oryza sativa L.) cv. Pusa 33 assimilates NO₃^- when grown under lowland conditions was determined in a field study. The in vivo NR (nitrate reductase) activity is low as compared to the value in other cereals grown under aerobic soil conditions. The leaf blades had higher NR activity (g fr. wt.)^-1 than the sheaths and stem. Calculation of total NO₃^- (mol) reduced in the main shoot, obtained by integrating the in vivo NR assay values per plant part and per day over the duration for which the various plant parts on the main shoot remained metabolically active, showed that out of the total reduced N at harvest, 16.6% was assimilated via the enzyme nitrate reductase. In the leaf sheaths and stem the NO₃^- was reduced to slightly over 50% of the total NO₃^- that was reduced in the main shoot. The rest of the amount was reduced in the leaf blades.     

Interaction between barley yellow dwarf virus and rust in wheat, barley and oats, and the effects on grain yield and quality

In three separate experiments, the upper leaf surface of the fifth formed leaf of wheat cv. Highbury, the fourth and fifth leaves of barley cv. Julia and the third and fourth leaves of oat cv. Mostyn were inoculated in a spore settling tower with wheat brown rust (Puccinia recondita f. sp. tritici), barley brown rust (P. hordei) or oat crown rust (P. coronata f. sp. avenae), respectively. Fewer pustules developed on distal portions of leaves of plants infected with barley yellow dwarf virus (BYDV) than on similar portions of leaves from virus-free plants. There were no significant differences in the number of pustules on proximal leaf portions. In barley and oats, the number of pustules on distal leaf portions was negatively correlated with the amount of yellowing of the leaf areas scored. In wheat, symptoms of BYDV were mild and leaves were little affected by yellowing. The latent period of rust on wheat and oats was not affected by BYDV. In barley, BYDV reduced the latent period of rust on leaf 5, but not on leaf 4, and reduced it on proximal, but not distal, leaf portions. In other experiments, BYDV reduced the yield of wheat and oats by 44% and 66%, respectively, while BYDV-infected barley was almost sterile. The appropriate rust reduced the yield of wheat, barley and oats by 33%, 13% and 86%, respectively. When infected with both BYDV and rust, yield of wheat and oats was reduced by 63% and 91%, respectively. Neither BYDV nor rust affected the percentage crude protein content of wheat grain, nor did rust affect that of barley. In oats, BYDV and rust each significantly increased crude protein of grain, but rust infection of BYDV-infected plants tended to reduce it.     

Effect of Light Quality on Nitrogen Metabolism of Radish Plants

The long-term action of blue or red light on nitrogen metabolism was studied in radish (Raphanus sativus L.) plants. The potential activity of nitrate reductase (NR) in vivo and its maximum activity in vitro, the content of soluble protein and free amino acids were determined in the course of the growth of a third leaf of radish plants. The effect of light quality on NR activity was found to depend significantly on the stage of leaf development. Blue light (BL) stimulated NR activity in leaves, when their areas were about 11–13% of the fully developed leaves. The efficiency of red light (RL) was significantly lower, because the maximum NR activity was observed in the leaves developed to the stage, when their areas were 38–40% of the final one. The comparative analysis of the pool of free amino acids in expanding leaves of BL- or RL-grown plants revealed significant changes in the contents of individual amino acids. Despite a higher accumulation of two amino acids in the leaves of BL-grown plants, namely, Asp (27% as compared to 13–16% in the RL-grown leaves) and Gly (5% against 2.5% in RL-grown leaves), the BL-grown leaves also demonstrated a significant decrease in Ala (10% as compared to 23% in the RL-grown leaves) and some decrease in the amounts of Ser and Gly. The content of soluble protein in a juvenile BL-grown leaf was observed to decrease gradually during leaf development. However, the protein content in the BL-grown leaf was always higher than in the RL-grown leaf of the same age. We concluded that the photoregulatory action of BL on NR activity determined the different rates of nitrogen assimilation in BL- and RL-grown plants.__________Translated from Fiziologiya Rastenii, Vol. 52, No. 3, 2005, pp. 349–356.Original Russian Text Copyright © 2005 by Maevskaya, Bukhov.     

Oviposition site selection on oats: the effect of plant architecture,plant and leaf age,tissue toughness,and hardness on cereal leaf beetle,Oulema melanopus

In sequentially planted oat stands, the cereal leaf beetle (CLB), Oulema melanopus (L.) (Coleoptera: Chrysomelidae: Lemini), is found in greater numbers, and lays more eggs, on later planted (younger) oats (Avena sativa L.) (Poaceae). Plant characteristics that could explain this ovipositional preference were examined in a series of experiments. Cage and open field whole plant preference tests confirmed the attraction of ovipositing females to younger oats. A cage effect illustrated the role of plant architecture (plant height) in CLB host selection. Two multiple‐choice and one no‐choice excised leaf experiments determined that characteristics of individual leaves associated with leaf insertion level (leaf number from base to apex) and age influence ovipositional site selection. Leaves of higher insertion level have higher nitrogen content, but fewer eggs are laid on those leaves. Two experiments examining the interaction between total leaf nitrogen and leaf insertion level showed that only leaf insertion level affected oviposition choice. Published literature suggests variation in secondary plant compounds cannot explain O. melanopus ovipositional preference among leaves. Grass leaves of higher insertion level have more extensively developed cells associated with tissue toughness and hardness. The data and supporting literature suggest tissue toughness and hardness are deterring oviposition on oat leaves of higher insertion level. However, newly eclosed larvae are able to feed on leaves usually avoided as oviposition sites. The explanation for this result may be a lack of correlation between host suitability and ovipositional preference.     

Factors of between- and within-plant distribution of Metopolophium dirhodum (Hom., Aphididae) on small grain cereals

Abstract: The relationship between abundance of rose‐grain aphid Metopolophium dirhodum (Walker) and leaf chlorophyll content of spring wheat, spring oats and winter barley was investigated. Within production stands of each crop 18–25 plots were established, located at places with different plant quality where aphids were counted on particular leaves of 50–100 tillers, and the leaf chlorophyll content and area were determined. In all stands aphid numbers × tiller^?1 increased exponentially with chlorophyll content (r² = 0.783–0.933). This parallel variation may be explained by increased nitrogen content and assimilate production of vigorous chlorophyll‐rich plants. The aphid numbers on leaves of particular order (within‐plant distribution) were also correlated with leaf chlorophyll content (r² = 0.373–0.827). However, in oats and barley the analysis of variance of residuals of log aphids × leaf^?1 versus leaf chlorophyll regression revealed a significant effect of leaf position (the order of the leaf from the top of the plant). The magnitude of residuals was positively related to leaf size and may be related to the intensity of phloem transport from the source leaves to sink organs.     

Properties of nitrate reductase from seedling leaves of selected barley genotypes

Crude extracts from leaves of 6-day barley seedlings of parental genotypes (cv. Aramir and primitive line R567) and selected doubled haploid (DH) lines were not found to have significant differences in the NADH:NR activity, while considerable differences between these genotypes were shown by the NAD(P)H:NR activity. The cv. Aramir and DH lines did not differ by nitrate accumulation in the leaves. However, the primitive line R567, as compared to the remaining genotypes, was characterized by an appreciably lower ability to accumulate nitrates. In partially purified leaf extracts, significant differences in total NADH:NR activity and in distal activity dependent on methyl viologen (MV:NR) were found between the parental genotypes and selected DH lines. The studied genotypes differed also in dehydrogenase NR activity, i.e. cytochrome c reductase activity in crude extracts. In the studied genotypes, the NADH:NR activity in partially purified leaf extracts did not substantially differ by Km values for nitrates. Calculated Vmax values for NADH:NR in these genotypes were similar to total NR activity in partially purified extracts. Significant differences between the parental genotypes and selected DH lines were found in the thermal NADH:NR stability in crude and partially purified leaf extracts. From the performed studies it follows that different NR stability was one of the reasons of revealed differences in total activity and in partial NR activities in the leaf extracts between the studied genotypes of spring barley. Besides, it is suggested that varied NR gene expression in the leaves of these barley genotypes could also influence NR activity.     

Use of protein in extraction and stabilization of nitrate reductase

The in vitro instability of nitrate reductase (EC 1.6.6.1) activity from leaves of several species of higher plants was investigated. Decay of activity was exponential with time, suggesting that an enzyme-catalyzed reaction was involved. The rate of decay of nitrate reductase activity increased as leaf age increased in all species studied. Activity was relatively stable in certain genotypes of Zea mays L., but extremely unstable in others. In all genotypes of Avena sativa L. and Nicotiana tabacum L. studied, nitrate reductase was unstable. Addition of 3% (w/v) bovine serum albumin or casein to extraction media prevented or retarded the decay of nitrate reductase activity for several hours. In addition, the presence of bovine serum albumin or casein in the enzyme homogenate markedly increased nitrate reductase activity (up to 15-fold), especially in older leaf tissue.     

The effect of leaf insertion on nitrate reductase and glutamine synthetase activity and NO3-N content inPisum sativum L.

Nitrate reductase (NR) activity estimated byin vivo andin vitro methods in 17 days old pea seedlings is the highest in young fully expanded leaves (4th leaf from the stem base). NO₃-N content is parallel to the NR activity being also highest in the 4th leaf. On the other hand the activity of glutamine synthetase (GS) is the highest in the youngest leaves which had not yet reached their maximum size (5th leaf). Equilibrium between NO₃-N content and activity of enzymes involved in assimilation of nitrogen containing compounds in individual leaves is discussed in relation to their insertion.     

Fructose 2,6-bisphosphate levels in mature leaves of tobacco (Nicotiana tabacum) and potato (Solanum tuberosum)

Here we show that fructose 2,6-bisphosphate cannot be reliably measured in mature leaves of tobacco (Nicotiana tabacum L.), potato (Solanum tuberosum L.), or stinging nettle (Urtica dioica L.) using conventional extraction techniques, since the recoveries of fructose 2,6-bisphosphate added during extraction are poor. However, fructose 2,6-bisphosphate could be extracted by boiling leaves in ethanol and aqueous buffer. Evidence for the reliability of this technique is provided by high recovery measurements of fructose 2,6-bisphosphate added to the leaves before extraction. This extraction method was used to measure changes in the level of fructose 2,6-bisphosphate throughout the photoperiod in tobacco and potato leaves. These changes are compared with the rate of accumulation of sucrose and starch in the leaf samples. Variations in the levels of fructose 2,6-bisphosphate, and the relationship between this metabolite and sucrose and starch accumulation in these leaves during the photoperiod are similar to the pattern observed in leaves of other plant species.Abbreviations BSA bovine serum albumin - Fru-2,6-P₂ fructose 2,6-bisphosphate This research was supported by the Agricultural and Food Research Council (Grant no. PG43/531), and the Royal Society.     

Characterization and Localization of a Novel Protein (HFN 40) in Maize Genotypes Without Husk Leaf Blades

Some maize (Zea mays L.) genotypes produced husk leaves without leaf blades. However, the physiological implication of this leaf deformity is unclear. Difference in protein pattern was observed between maize with and without husk leaf blades. A clear band around 38[sim ]40 kDa in seeds of maize genotypes without husk leaf blades appeared, while it was not detected in ones with husk leaf blades. These protein might be involved in leaf blade intiation.     

Cytokinin activity inCitrus jambhiri Lush. seedlings colonized by vesicular-arbuscular mycorrhizal fungi

Summary The influence of vesicular-arbuscular mycorrhizal symbiosis on cytokinin activity in Citrus jambhiri Lush, seedlings was investigated. C. jambhiri inoculated with cultures of Glomus caledonium (Nicol. and Gerd.), G. epigaeum (Dan. and Trappe), G. etunicatum (Becker and Gerd.), G. fasciculatum Thaxt. (Gerd, and Trappe) or G. mosseae (Nicol and Gerd.) was grown from seed for 105 days in a glasshouse. Cytokinin activity in roots and leaves of seedlings was analyzed using high-performance liquid chromatography, mass spectrometry and a bioassay. Seedling leaf tissue had greater cytokinin activity than root tissue. Zeatin, zeatin riboside, and their dihydro- and glucoside derivatives were isolated from leaves of 105-day-old seedlings inoculated with G. fasciculatum and G. mosseae. Cytokinin activity in roots and leaves was associated with differences in seedling total dry weight and vesicular-arbuscular mycorrhizal colonization. The ribose moiety and the saturated side chain apparently influence cytokinin transport and physiological activity in Citrus seedlings.     

Sulfur deprivation and nitrogen metabolism in maize seedlings

The objective of this experiment was to elucidate the manner in which N metabolism is influenced by S nutrition. Maize (Zea mays L.) seedlings supplied with Hoagland solution minus SO₄²⁻ exhibited S deficiency symptoms 12 days after emergence. Prior to development of these symptoms, a decline in leaf blade nitrate reductase (NR, EC 1.6.6.1) activity was observed in S-deprived seedlings compared to normal seedlings. Twelve days after emergence, in vitro NR activity was diminished 50% compared to normal seedlings. Glutamine synthetase (EC 6.3.1.2) and NAD-glutamate dehydrogenase (EC 1.4.1.2) activities were less severely affected (19 and 13%, respectively, at day 12). NADP-glutamate dehydrogenase (EC 1.4.1.4) activity and leaf blade fresh weight were not altered by S deprivation. Concentrations of soluble protein and chlorophyll (a and b) in leaf blades were reduced 18 and 25%, respectively, at day 12. A significantly higher concentration of NO₃⁻-N was observed for leaf blade and stem (culms, leaf sheaths, and unfurled leaves) fractions (46 and 31%, respectively) in S-deprived plants. In contrast to the other parameters measured, NR activity in S-deprived seedlings could be readily restored to the normal level by addition of SO₄²⁻. The apparent preferential effect of S deprivation on NR activity could be causally related to the observed changes in NO₃⁻-N and soluble protein concentration.     

Purification and Kinetics of Higher Plant NADH:Nitrate Reductase

Squash cotyledon (Cucurbita pepo L.) NADH:nitrate reductase (NR) was purified 150-fold with 50% recovery by a single step procedure based on the affinity of the NR for blue-Sepharose. Blue-Sepharose, which is prepared by direct coupling of Cibacron blue to Sepharose, appears to bind squash NR at the NADH site. The NR can be purified in 2 to 3 hours to a specific activity of 2 μmol of NADH oxidized/minute • milligram of protein. Corn (Zea mays L.) leaf NR was also purified to a specific activity of 6.9 μmol of NADH oxidized/minute • milligram of protein using a blue-Sepharose affinity step. The blue-Sepharose method offers the advantages of a rapid purification of plant NR to a high specific activity with reasonable recovery of total activity.

The kinetic mechanism of higher plant NR was investigated using these highly purified squash and corn NR preparations. Based on initial velocity and product inhibition studies utilizing both enzymes, a two-site ping-pong mechanism is proposed for NR. This kinetic mechanism incorporates the concept of the reduced NR transferring electrons from the NADH site to a physically separated nitrate site.