Effects of Pyrocatechol on Some Metabolic Processes of Mature Sugar Beet (Beta vulgaris) Plants

Pyrocatechol (PC), 10^-2M, was applied to the foliage of mature plants of sugar beet (Beta vulgaris L.). Its effect on the activity of nitrate reductase, transaminase, invertase, phosphatases, sucrose synthetase, sucrose phosphate synthetase, and UDPG-pyrophosphorylase were determined 7, 14, and 21 days after treatment. Significant reductions in the activity of nitrate reductase, transaminase, invertase, and phosphatases (including phenyl phosphatase, glucose-1-, glucose-6-, fructose-6-phosphatase, and adenosine triphosphatase) in the treated plants occurred. On the other hand, activities of the enzymes of sucrose biosynthesis, uridine, diphosphate glucose pyrophosphorylase (UDPG-pyrophosphorylase), sucrose synthetase, and sucrose phosphate synthetase were significantly stimulated by the application of pyrocatechol. The results suggest that the growth inhibition following the application of PC to sugar beet plants may stem in part from an amino acid stress resulting from a PC-induced decrement in nitrate reductase and transaminase activity. Its application also creates an enzymatic condition favorable for sucrose biosynthesis and storage.     

Partitioning of Sugar between Growth and Nitrate Reduction in Cotton Roots

The level of endogenous sugars was inversely related to nitrate availability in young cotton (Gossypium hirsutum L.) plants, with high nitrate causing a greater decline in sugar content of roots than of shoots. High nitrate (low sugar) plants also displayed relatively more shoot growth and less root growth than low nitrate (high sugar) plants. These data are consistent with the theory that roots are poor competitors for sugar, and that sugar supply is a major factor limiting root growth in vivo.

The effects of endogenous sugar level on root growth and on nitrate reductase activity in the root were different. When root sugar level was experimentally controlled by varying nitrate concentration in the nutrient solution, root growth was less sensitive than nitrate reductase activity to sugar deficiency. Also, in sterile root tips cultured on media containing a wide range of sucrose concentrations, growth rate was considerably less sensitive to endogenous sugar deficiency than was nitrate assimilation rate. Similarly, in plants which were detopped or girdled, nitrate reductase activity in the roots declined more rapidly than did root sugars, especially glucose and fructose. These results suggest that when sugar is deficient, cotton roots preferentially use it for growth at the expense of nitrate reduction.

     

Activities of enzymes of sugar metabolism in cold-stored tubers of Solanum tuberosum

Storage of tubers of Solanum tuberosum at 10° or 2° for 15 days did not alter significantly the maximum catalytic activities of sucrose phosphate synthetase, sucrose synthetase, glucose-6-phosphate dehydrogenase, aldolase, and glyceraldehydephosphate dehydrogenase. The temperature coefficients of phosphofructokinase, glyceraldehydephosphate dehydrogenase, and pyruvate kinase from the tubers were shown to be higher between 2° and 10° than between 10° and 25°. The rate of sugar accumulation at 2° exceeded the activity of sucrose synthetase but was less than that of sucrose phosphate synthetase. It is suggested that sucrose accumulation at 2° is catalysed by sucrose phosphate synthetase, is not due to changes in the maximum catalytic activities of any of the above enzymes, but may be due, in part, to the susceptibility of key glycolytic enzymes to cold.     

Influence of Carbon Source on Nitrate Removal by Nitrate-Tolerant Klebsiella oxytoca CECT 4460 in Batch and Chemostat Cultures

The nitrate-tolerant organism Klebsiella oxytoca CECT 4460 tolerates nitrate at concentrations up to 1 M and is used to treat wastewater with high nitrate loads in industrial wastewater treatment plants. We studied the influence of the C source (glycerol or sucrose or both) on the growth rate and the efficiency of nitrate removal under laboratory conditions. With sucrose as the sole C source the maximum specific growth rate was 0.3 h⁻¹, whereas with glycerol it was 0.45 h⁻¹. In batch cultures K. oxytoca cells grown on sucrose or glycerol were able to immediately use sucrose as a sole C source, suggesting that sucrose uptake and metabolism were constitutive. In contrast, glycerol uptake occurred preferentially in glycerol-grown cells. Independent of the preculture conditions, when sucrose and glycerol were added simultaneously to batch cultures, the sucrose was used first, and once the supply of sucrose was exhausted, the glycerol was consumed. Utilization of nitrate as an N source occurred without nitrite or ammonium accumulation when glycerol was used, but nitrite accumulated when sucrose was used. In chemostat cultures K. oxytoca CECT 4460 efficiently removed nitrate without accumulation of nitrate or ammonium when sucrose, glycerol, or mixtures of these two C sources were used. The growth yields and the efficiencies of C and N utilization were determined at different growth rates in chemostat cultures. Regardless of the C source, yield carbon (Y_C) ranged between 1.3 and 1.0 g (dry weight) per g of sucrose C or glycerol C consumed. Regardless of the specific growth rate and the C source, yield nitrogen (Y_N) ranged from 17.2 to 12.5 g (dry weight) per g of nitrate N consumed. In contrast to batch cultures, in continuous cultures glycerol and sucrose were utilized simultaneously, although the specific rate of sucrose consumption was higher than the specific rate of glycerol consumption. In continuous cultures double-nutrient-limited growth appeared with respect to the C/N ratio of the feed medium and the dilution rate, so that for a C/N ratio between 10 and 30 and a growth rate of 0.1 h⁻¹ the process led to simultaneous and efficient removal of the C and N sources used. At a growth rate of 0.2 h⁻¹ the zone of double limitation was between 8 and 11. This suggests that the regimen of double limitation is influenced by the C/N ratio and the growth rate. The results of these experiments were validated by pulse assays.     

Sucrose Concentration in Liquid Media Affects Soluble Carbohydrates,Biomass and Storage Quality of Micropropagated Hosta

The effects of sucrose concentration (1, 3, 5, or 7% w/v) in liquid media, in the presence and absence of benzylaminopurine (BAP), on internal carbohydrate status and growth of Hosta tokudama Tratt. Newberry Gold during the multiplication phase (stage II) was investigated. Cultures from all treatment combinations were transferred to media containing 3% (w/v) sucrose during the rooting phase (stage III). At the end of the stage III, these micropropagules were subjected to 5 weeks of storage at 10 °C under low light (photosynthetic photon flux of 5 µmol m^–2s^–1). Endogenous concentrations of soluble sugars (glucose, fructose, and sucrose) in the plantlets increased linearly as the media sucrose concentration increased from 1% to 7% during stage II. Root and shoot biomass increased with increasing media sucrose concentration. BAP increased the biomass and multiplication rate but did not affect internal concentration of soluble sugars. While in storage, endogenous sugar levels and plantlet dry weight remained unchanged for all treatments. Following storage, plants originally cultured in 5% and 7% media sucrose had higher dry weight and less leaf chlorosis than those cultured in 1% and 3% media. Differences in endogenous soluble sugar levels at the end of stage III rooting, and after storage were related to the sucrose concentration of the initial stage II multiplication medium. Increased media sucrose levels during the multiplication cycle has a positive, long-term effect on plant morphology and quality.     

Physiological and Biochemical Characteristics of Sugar Beet Plants Grown at an Increased Carbon Dioxide Concentration and at Various Nitrate Doses

Three-week-old sugar beet (Beta vulgaris L.) seedlings were grown for an additional four weeks under controlled conditions: in river sand watered with a modified Knop mixture containing one half-fold (0.5N), standard (1N), and or threefold (3N) nitrate amount, at the irradiance of 90 W/m² PAR, and at the carbon dioxide concentrations of 0.035% (1C treatment) or 0.07% (2C treatment). The increase in the carbon dioxide concentration and in the nitrogen dose resulted in an increase in the leaf area and the leaf and root dry weight per plant. With the increase in the nitrogen dose, morphological indices characterizing leaf growth increased more noticeably in 1C plants than in 2C plants. And vice versa, the effects of increased CO₂ concentration were reduced with the increase in the nitrogen dose. Roots responded to the changes in the CO₂ and nitrate concentrations otherwise than leaves. At a standard nitrate dose (1N), the contents of proteins and nonstructural carbohydrates (sucrose and starch) in leaves depended little on the CO₂ concentration. At a double CO₂ concentration, the content of chlorophyll somewhat decreased, and the net photosynthesis rate (P _n) calculated per leaf area unit increased. An increase in the nitrogen dose did not affect the leaf carbohydrate content of the 1C and 2C plants except the leaves of the 2C-3N plants, where the carbohydrate content decreased. In 1C and 2C plants, an increase in the nitrogen dose caused an increase in the protein and chlorophyll content. Specific P _n values somewhat decreased in 1C-0.5N plants and had hardly any dependence on the nitrate dose in the 2C plants. The carbohydrate content in roots did not depend on the CO₂ concentration, and the content was the highest at 0.5N. Characteristic nitrogen dose-independent acclimation of photosynthesis to an increased carbon dioxide concentration, which was postulated previously [1], was not observed in our experiments with sugar beet grown at doubled carbon dioxide concentration.     

Prolonged root hypoxia effects on enzymes involved in nitrogen assimilation pathway in tomato plants

In order to investigate the effects of root hypoxia (1–2% oxygen) on the nitrogen (N) metabolism of tomato plants (Solanum lycopersicum L. cv. Micro-Tom), a range of N compounds and N-assimilating enzymes were performed on roots and leaves of plants submitted to root hypoxia at the second leaf stage for three weeks. Obtained results showed that root hypoxia led to a significant decrease in dry weight (DW) production and nitrate content in roots and leaves. Conversely, shoot to root DW ratio and nitrite content were significantly increased. Contrary to that in leaves, glutamine synthetase activity was significantly enhanced in roots. The activities of nitrate and nitrite reductase were enhanced in roots as well as leaves. The higher increase in the NH₄⁺ content and in the protease activities in roots and leaves of hypoxically treated plants coincide with a greater decrease in soluble protein contents. Taken together, these results suggest that root hypoxia leaded to higher protein degradation. The hypoxia-induced increase in the aminating glutamate dehydrogenase activity may be considered as an alternative N assimilation pathway involved in detoxifying the NH₄⁺, accumulated under hypoxic conditions. With respect to hypoxic stress, the distinct sensitivity of the enzymes involved in N assimilation is discussed.Key words: tomato, hypoxia, nitrogen, glutamine synthetase, protease, glutamate dehydrogenase     

Carbon and nitrogen metabolism in barley plants exposed to UV-B radiation

The effect of UV-B radiation on FW, leaf and stem length, photosynthetic O₂ evolution, levels of carbohydrates and nitrates, and extractable activities of some of the enzymes involved in C and N metabolism was evaluated in barley ( Hordeum vulgare L. cv. Express) seedlings during the 9 days following transfer to an UV-B enriched environment. The results show that under our experimental conditions UV-B radiation scarcely affects the photosynthetic competence of barley leaves, expressed as RuBP carboxylase (EC 4.1.1.39) activity, O₂ evolution rate and chlorophyll content. Nevertheless, this treatment induced significant alterations of the enzyme activity of nitrate reductase (EC 1.6.6.1) and glutamine synthetase (EC 6.3.1.2), although only after a few days of treatment. The effects were not confined to the exposed tissue, but were detectable also at the root level. In fact, nitrate reductase decreased in response to UV-B in both leaf and root tissue, whereas glutamine synthetase was affected only in the root. In contrast, nitrate content was not influenced by the treatment, neither in root nor in leaf tissue, whilst leaf sucrose diminished in exposed plants only on the last day of treatment.     

Biochemical Basis for Partitioning of Photosynthetically Fixed Carbon between Starch and Sucrose in Soybean (Glycine max Merr.) Leaves

The control of photosynthetic starch/sucrose formation in leaves of soybean (Glycine max L. Merr.) cultivars was studied in relation to stage of plant development, photosynthetic photoperiod, and nitrogen source. At each sampling, leaf tissue was analyzed for starch content, activities of sucrose-metabolizing enzymes, and labeling of starch and sucrose (by ¹⁴CO₂ assimilation) in isolated cells. In three of the four varieties tested, nodulated plants had lower leaf starch levels and higher activities of sucrose phosphate synthetase (SPS), and isolated mesophyll cells incorporated more carbon (percentage of total ¹⁴CO₂ fixed) into sucrose and less into starch as compared to nonnodulated (nitrate-dependent) plants. The variation among cultivars and nitrogen treatments observed in the activity of SPS in leaf extracts was positively correlated with labeling of sucrose in isolated cells (r = 0.81) and negatively correlated with whole leaf starch content (r = −0.66). The results suggested that increased demand for assimilates by nodulated roots may be accommodated by greater partitioning of carbon into sucrose in the mesophyll cells. We have also confirmed the earlier report (Chatterton, Silvius 1979 Plant Physiol 64: 749-753) that photoperiod affects partitioning of fixed carbon into starch. Within two days of transfer of nodulated soybean Ransom plants from a 14-hour to a 7-hour photoperiod, leaf starch accumulation rates doubled, and this effect was associated with increased labeling of starch and decreased labeling of sucrose in isolated cells. Concurrently, activities of SPS, sucrose synthase, and uridine diphosphatase in leaves were decreased.     

Sucrose metabolism during the growth of Camellia japonica pollen

The free sugar in the mature pollen grains of Camellia japonica is almost all sucrose and the sucrose content decreases rapidly during pollen growth. The activity of soluble invertase increases during culturing and a high constant activity was found at the later stages of pollen tube growth. By contrast, the level of sucrose synthetase activity remains constant during pollen growth and that of wall-bound invertase activity is very low. Cycloheximide has little effect on the activity of these enzymes. Exogenous sucrose or glucose was simultaneously incorporated into the pollen grains when they absorbed water and swelled. The free sugar levels in growing pollen depend on the nature of the exogenous sugar. The sugar metabolism in the pollen at the stage of germination differs from that during tube growth, the latter being particularly influenced by exogenous sugar.     

Regulation of sulfate assimilation by nitrogen and sulfur nutrition in poplar trees

Plants cover their need for sulfur by taking up inorganic sulfate, reducing it to sulfide, and incorporating it into the amino acid cysteine. In herbaceous plants the pathway of assimilatory sulfate reduction is highly regulated by the availability of the nutrients sulfate and nitrate. To investigate the regulation of sulfate assimilation in deciduous trees we used the poplar hybrid Populus tremula × P. alba as a model. The enzymes of the pathway are present in several isoforms, except for sulfite reductase and -glutamylcysteine synthetase; the genomic organization of the pathway is thus similar to herbaceous plants. The mRNA level of APS reductase, the key enzyme of the pathway, was induced by 3 days of sulfur deficiency and reduced by nitrogen deficiency in the roots, whereas in the leaves it was affected only by the withdrawal of nitrogen. When both nutrients were absent, the mRNA levels did not differ from those in control plants. Four weeks of sulfur deficiency did not affect growth of the poplar plants, but the content of glutathione, the most abundant low molecular thiol, was reduced compared to control plants. Sulfur limitation resulted in an increase in mRNA levels of ATP sulfurylase, APS reductase, and sulfite reductase, probably as an adaptation mechanism to increase the efficiency of the sulfate assimilation pathway. Altogether, although distinct differences were found, e.g. no effect of sulfate deficiency on APR in poplar leaves, the regulation of sulfate assimilation by nutrient availability observed in poplar was similar to the regulation described for herbaceous plants.     

Influence of phloem transport, N-fertilization and ion accumulation on sucrose storage in the taproots of fodder beet and sugar beet

To investigate the factors governing the accumulation of sucroseand amino acids in the taproots of sugar beet, their contentswere measured in the leaves, phloem sap and the taproots ofsugar beet, fodder beet and a hybrid between both, grown oneither 3.0 or 0.5 mM nitrate. In the taproots the contents ofmalate, citrate and inorganic ions were also determined. Forthe high sucrose accumulation in sugar beet as compared to theother varieties three factors were found. (a) In sugar beet,less amino acids and more sucrose are taken up into the phloemthan in fodder beet. (b) In sugar beet, the sucrose and aminoacid syntheses are less sensitive to the nitrate concentrationsthat are required for optimal plant growth than in other varieties.In fodder beet, upon raising the nitrate concentration from0.5 mM to 3 mM, the synthesis and storage of sucrose is decreasedand that of amino acids increased. The corresponding valuesin sugar beet (0.5 mM) are similar to those in fodder beet andare not much affected by an increase of nitrate. (c) The sucroseaccumulation is limited by the accumulation of inorganic ionsin the taproots. The sucrose content in the taproots is negativelycorrelated to the total ion content. Whereas sucrose representstwo-third of all solutes in the taproots of sugar beet, it amountsto only one-third of the solutes in fodder beet taproots. Key words: Amino acids, Beta vulgans L, phloem sap, potassium, sucrose storage, sugar beet, taproots, transport     

Effect of sulfur on nitrate reductase and ATP sulfurylase Activities in groundnut (Arachis hypogea L.)

Field experiments were conducted to determine the effect of sulfur (S) and Nitrogen (N) on nitrate reductase (NR) and ATP-sulfurylase activities in groundnut cultivars (Arachis hypogea L. cv. Ambar and Kaushal). Two combinations of S (in kg ha^-1): OS (-S) and 20S (+S) were used with 20 kg ha^-1 N. The application of S enhanced the NR and ATP-sulfurylase activities in both the cultivars at all the growth stages. The application of S also increased soluble protein and chlorophyll content in the all growth stages of both the cultivars. NR and ATP-sulfurylase activities in the leaves were measured at various growth stages as the two enzymes catalyze the rate limiting steps of the assimilatory pathways of nitrate and sulfate, respectively.     

Induction of leaf senescence by low nitrogen nutrition in sunflower (Helianthus annuus) plants

Different parameters which vary during the leaf development in sunflower plants grown with nitrate (2 or 20 mM) for a 42‐day period have been determined. The plants grown with 20 mM nitrate (N+) showed greater leaf area and specific leaf mass than the plants grown with 2 mM nitrate (N?). The total chlorophyll content decreased with leaf senescence, like the photosynthetic rate. This decline of photosynthetic activity was greater in plants grown with low nitrogen level (N?), showing more pronounced senescence symptoms than with high nitrogen (N+). In both treatments, soluble sugars increased with aging, while starch content decreased. A significant increase of hexose to sucrose ratio was observed at the beginning of senescence, and this raise was higher in N? plants than in N+ plants. These results show that sugar senescence regulation is dependent on nitrogen, supporting the hypothesis that leaf senescence is regulated by the C/N balance. In N+ and N? plants, ammonium and free amino acid concentrations were high in young leaves and decreased progressively in the senescent leaves. In both treatments, asparagine, and in a lower extent glutamine, increased after senescence start. The drop in the (Glu+Asp)/(Gln+Asn) ratio associated with the leaf development level suggests a greater nitrogen mobilization. Besides, the decline in this ratio occurred earlier and more rapidly in N? plants than in N+ plants, suggesting that the N? remobilization rate correlates with leaf senescence severity. In both N+ and N? plants, an important oxidative stress was generated in vivo during sunflower leaf senescence, as revealed by lipid peroxidation and hydrogen peroxide accumulation. In senescent leaves, the increase in hydrogen peroxide levels occurred in parallel with a decline in the activity of antioxidant enzymes. In N+ plants, the activities of catalase and ascorbate peroxidase (APX) increased to reach their highest values at 28 days, and later decreased during senescence, whereas in N? plants these activities started to decrease earlier, APX after 16 days and catalase after 22 days, suggesting that senescence is accelerated in N‐leaves. It is probable that systemic signals, such as a deficit in amino acids or other metabolites associated with the nitrogen metabolism produced in plants grown with low nitrogen, lead to an early senescence and a higher oxidation state of the cells of these plant leaves.     

Fate and effects on yield components of extra applications of nitrogen on spring wheat (Triticum aestivum L.) grown in solution culture

Spring wheat (Triticum aestivum L.) was grown with daily additions of nitrate-N. The relative addition rate of nitrate-N was decreased stepwise, and after 125 days of growth, 58 mg N plant^-1 had been introduced. The fate and effect of an extra addition of nitrate (20 mg N plant^-1) at six different times during the ontogeny (37, 54, 66, 79, 94 and 108 days from sowing) on grain yield and grain protein concentration was investigated. The plants absorbed all or most of the extra nitrate at all stages of development evaluated. Dry matter production of both aerial vegetative parts and grains, but not roots, generally increased as a result of the extra nitrate addition. The increase in grain dry matter was mainly an effect of an increased number of grains per plant. Extra nitrate applications had large effects on grain nitrogen content at all stages, but the effect on main shoot and tiller ears varied depending on the time of application. Early applications, i.e. before anthesis, mainly led to increased yield with unchanged protein concentration whereas late applications also led to increased grain protein concentration. The largest effect on grain nitrogen concentration (25–30% increase) was obtained when the extra nitrate was applied late after sowing, i.e. less than four weeks before final harvest. As the extra dose of nitrate was labelled with ¹⁵N, it was possible to follow the movement of the extra nitrogen addition within the plant. Samples were taken at one and five days after ¹⁵N-addition and at final harvest. There were differences in the movement of ¹⁵N depending on when it was introduced. Generally, net movement of the ¹⁵N-labelled N into the grain increased with age at application until 94 days after sowing when a maximum of 90% of the added ¹⁵N ended up in the grain.Abbreviations RN Relative increase in nitrogen content - RA Relative nitrogen addition rate - RG Relative growth rate - N nitrogen     

Role of sugars in nitrate utilization by roots of dwarf bean

Nitrate uptake and in vivo, nitrate reductase activity (NRA) in roots of Phaseolus vulgaris, L. cv. Witte Krombek were measured in nitrogen-depleted plants of varying sugar status, Variation in sugar status was achieved at the start of nitrate nutrition by excision, ringing, darkness or administration of sugars to the root medium. The shape of the apparent induction pattern of nitrate uptake was not influenced by the sugar status of the absorbing tissue. When measured after 6 h of nitrate nutrition (0.1 mol m^?3), steady state nitrate uptake and root NRA were in the order intact>dark>ringed>excised. Exogenous sucrose restored NRA in excised roots to the level of intact plants. The nitrate uptake rate of excised roots, however, was not fully restored by sucrose (0.03–300 mol m^?3). When plants were decapitated after an 18 h NO₃^? pretreatment, the net uptake rate declined gradually to become negative after three hours. This decline was slowed down by exogenous fructose, whilst glucose rapidly (sometimes within 5 min) stimulated NG^?₃ uptake. Presumably due to a difference in NO₃^? due to a difference in NO₃^? uptake, the NRA of excised roots was also higher in the presence of glucose than in the presence of fructose after 6 h of nitrate nutrition. The sugar-stimulation of, oxygen consumption as well as the release of ¹⁴CO₂ from freshly absorbed (U-¹⁴C) sugar was the same for glucose and fructose. Therefore, we propose a glucose-specific effect on NO₃^? uptake that is due to the presence of glucose rather than to its utilization in root respiration. A differential glucose-fructose effect on nitrate reductase activity independent of the effect on NO₃^? uptake was not indicated. A constant level of NRA occurred in roots of NO₃^? induced plants. Removal of nutrient nitrate from these plants caused an exponential NRA decay with an approximate half-life of 12 h in intact plants and 5.5 h in excised roots. The latter value was also found in roots that were excised in the presence of nitrate, indicating that the sugar status primarily determines the apparent rate of nitrate reductase decay in excised roots.     

外源α-萘乙酸对花期干旱大豆碳代谢的影响

以耐旱性大豆品种晋豆21和干旱敏感性大豆品种徐豆22为试验材料,通过盆栽试验,研究α-萘乙酸(NAA)对花期干旱大豆碳代谢的影响.结果表明: 干旱胁迫下,与徐豆22相比,晋豆21净光合速率(P_n)下降幅度较小,光呼吸速率(P_r)和叶片可溶性糖含量增加幅度较小,而叶片蔗糖磷酸合成酶(SPS)、蔗糖合成酶(SS)(合成方向)活性、根系蔗糖含量增加幅度较大.NAA处理提高了干旱胁迫下P_n,并降低了P_r,进而明显缓解了干旱胁迫对大豆植株的生长抑制;降低了叶片淀粉分解酶、酸性转化酶(AI)和SS(分解方向)活性,从而抑制了干旱胁迫诱导的可溶性糖积累;NAA处理也能增加干旱胁迫下叶片SPS、SS(合成方向)活性、根系蔗糖含量、根冠比,表明NAA处理促进了叶片中蔗糖向根系的转运.总之,在干旱胁迫下,外源NAA能够通过调控碳代谢增强大豆植株对干旱胁迫的耐受性.     

Assimilatory nitrate uptake in Pseudomonas fluorescens studied using nitrogen-13

The mechanism of nitrate uptake for assimilation in procaryotes is not known. We used the radioactive isotope, ¹³N as NO₃ ^-, to study this process in a prevalent soil bacterium, Pseudomonas fluorescens. Cultures grown on ammonium sulfate or ammonium nitrate failed to take up labeled nitrate, indicating ammonium repressed synthesis of the assimilatory enzymes. Cultures grown on nitrite or under ammonium limitation had measurable nitrate reductase activity, indicating that the assimilatory enzymes need not be induced by nitrate. In cultures with an active nitrate reductase, the form of ¹³N internally was ammonium and amino acids; the amino acid labeling pattern indicated that ¹³NO₃ ^- was assimilated via glutamine synthetase and glutamate synthase. Cultures grown on tungstate to inactivate the reductase concentrated NO₃ ^- at least sixfold. Chlorate had no effect on nitrate transport or assimilation, nor on reduction in cell-free extracts. Ammonium inhibited nitrate uptake in cells with and without active nitrate reductases, but had no effect on cell-free nitrate reduction, indicating the site of inhibition was nitrate transport into the cytoplasm. Nitrate assimilation in cells grown on nitrate and nitrate uptake into cells grown with tungstate on nitrite both followed Michaelis-Menten kinetics with similar K _mvalues, 7 M. Both azide and cyanide inhibited nitrate assimilation. Our findings suggest that Pseudomonas fluorescens can take up nitrate via active transport and that nitrate assimilation is both inhibited and repressed by ammonium.     

Changes in the activities of nitrogen assimilation enzymes ofLolium perenne L. during regrowth after cutting

The activities of key enzymes involved in N assimilation were investigated after defoliation of 6-week-old ryegrass plants grown in water culture conditions. In a first experiment, nitrate reductase, glutamine synthetase and glutamate dehydrogenase activities were measured in roots, stubble and leaves on the day of cutting and at 7-day intervals over the following 5-week period of regrowth. Ammonia assimilation enzymes showed little change whereas the nitrate reductase activity sharply decreased 2 weeks after clipping. In a second experiment, the nitrate reductase activity was measured at 2- or 3-day intervals 1 week before and 3 weeks after clipping.In vivo andin vitro assays both showed an increasing activity in leaves up to 8 days after cutting while root activity decreased. The opposite changes then occurred and both organs recovered their initial nitrate reductase activity levels after 12–14 days of regrowth. These fluctuations in nitrate reductase activity were considered to be related to the capacity for C assimilation and the nitrate availability.     

Physiological and metabolic enzymes activity changes in transgenic rice plants with increased phosphoenolpyruvate carboxylase activity during the flowering stage

To compare the differences in physiology and metabolism between phosphoenolpyruvate carboxylase (PEPC) transgenic rice and its control, untransformed wild rice, dry matter accumulation, soluble sugar, starch and protein contents and enzyme activities were determined in different plant parts during flowering. Results revealed that PEPC transgenic rice had higher dry weights for leaf, stem and sheath as well as panicle than the untransformed wild rice did, with the largest increase in the panicle. Soluble sugar and protein content in the grains of PEPC transgenic rice were significantly enhanced while starch content changed less. PEPC transgenic rice exhibited high levels of PEPC activity, manifesting in high net photosynthetic rates during flowering. Moreover, transgenic rice with high PEPC expression levels also had elevated levels of the enzymes such as sucrose-p-synthase and sucrose synthase, which may confer a higher capacity to assimilate CO₂ into sucrose. Little increase in grain starch content was observed in transgenic plants due to the stable activities of starch synthase and Q enzyme. However, the PEPC transgenic rice plant induced the activities of nitrate reductase, glutamate oxaloacetate transaminase, glutamate pyruvate transaminase, glutamine synthetase, and asparagine synthase to high levels, as compared with the untransformed rice plant. PEPC activity was correlated with protein content in grains and the enzymes of nitrogen metabolism, suggesting that high PEPC activity in transgenic rice might be able to redirect carbon and nitrogen flow by regulating some enzymes related to carbon or nitrogen metabolisms. These results may help to understand how the C₃ plants possessing a C₄-like photosynthesis pathway worked by expression of PEPC.