Effect of nitrates and sucrose on the induction of nitrate reductase in etiolated seedling leaves from selected barley genotypes in darkness

The parental genotypes, cv. Aramir and R567 line, as well as the selected DH lines C23, C47/1, C41 and C55, growing in darkness differed significantly in the level of NR activity in crude leaf extracts independently of nitrate concentration in the medium. The highest activity of the enzyme was found in the line C23. When plants grew on the medium with 0.5 mM KNO₃, NR activity in that genotype was almost 10-fold higher than in the parents and lines C41, C55 and also 3.5-fold higher than in the line C47/1. An increase of nitrate concentration in the medium to 10 mM caused a significant increase of NR activity in all the genotypes under study. In the line C23 this enzyme activity was only 20% lower than that found previously in the green leaves of that genotype in light. NR from the leaves of C23 and C41 lines was thermally unstable under in vitro conditions. This enzyme in the leaf extracts from the line C23 was characterized by a considerably lower unstability. The lines DH C23 and C41 growing in the dark on the medium with 0.5 mM KNO₃ did not differ in nitrate accumulation in leaves, whereas a larger nitrate content was found in the leaves of the line C41 when it grew on the medium with 10 mM KNO₃. Independently of nitrate concentration in the medium, leaves of the line C23 were found to have a higher sucrose content than those of the line C41. Excised, etiolated leaves of barley treated with 0.5 and 10 mM KNO₃ in dark under conditions favorable to transpiration had a low NR activity. Leaf treatment with a solution containing 10 mM KNO₃ + 0.2 M sucrose caused, on the average, a 13-fold increase of NR activity in comparison to leaves treated only with 10 mM KNO₃ and about a 6-fold increase of this enzyme in comparison to leaves treated with 0.5 mM KNO₃ + 0.2 M sucrose.     

Optimized nitrate reductase assay predicts the rate of nitrate utilization in the halotolerant microalga Dunaliella viridis

An in situ method for measuring nitrate reductase (NR) activity in Dunaliella viridis was optimized in terms of incubation time, concentration of KNO₃, permeabilisers (1-propanol and toluene), pH, salinity, and reducing power (glucose and NADH). NR activity was measured by following nitrite production and was best assayed with 50 mM KNO₃, 1.2 mM NADH, 5% 1-propanol (v/v), at pH 8.5. The estimated half-saturation constant (K_s) for KNO₃ was 5 mM. Glucose had no effect as external reducing power source, and NADH concentrations >1.2 mM inhibited NR activity. Nitrite production was linear up to 20 min; longer incubation did not lead to higher nitrate reduction. The use of the optimized assay predicted the rate of NO ₃ ⁻ removal from the external medium by D. viridis with high degree of precision. This revised version was published online in September 2006 with corrections to the Cover Date.     

Nitrate reductase activity of radish cotyledons as affected by phytohormones and different nitrogen sources

Radish (Raphanus sativus L.) seedlings pretreated with different hormones viz. kinetin, gibberellic acid and abscisic acid were subjected to different N-forms. The seedlings were treated with different concentrations of KNO₃, NH₄Cl and NH₄NO₃ and the changes in nitrate reductase activity were seen in light and dark conditions in the cotyledons. Nitrate reductase activity was affected differently by hormone application. Nitrate increased and ammonia decreased nitrate reductase activity; in both light and dark-grown seedlings KNO₃ induced more in vitro nitrate reductase activity. NH ₄ ⁺ when combined with NO ₃ ⁻ , however, could level up to some extent, with KNO₃ in light, except in kinetin. A transient response of induction of NR activity was evident with decreased levels after a certain specific ambient N-concentration, despite the presence of high N in the medium. However, the pattern of transition varied with the hormones applied. Further, hormones are found to affect induction of different isoforms of nitrate reductase by NH ₄ ⁺ and NO ₃ ⁻ . NH ₄ ⁺ induced isoform was prominently promoted by kinetin treatment in dark. The data documents a particular kind of interaction between controlling factors (light, N-source and phytohormones) which affect nitrate reductase levels.     

Nitrate reductase activity of two leafy vegetables as affected by nickel and different nitrogen forms

The author studied the effect of different nickel concentrations (0, 0.4, 40 and 80 μM Ni) on the nitrate reductase (NR) activity of New Zealand spinach (Tetragonia expansa Murr.) and lettuce (Lactuca sativa L. cv. Justyna) plants supplied with different nitrogen forms (NO₃ ⁻–N, NH₄ ⁺–N, NH₄NO₃). A low concentration of Ni (0.4 μM) did not cause statistically significant changes of the nitrate reductase activity in lettuce plants supplied with nitrate nitrogen (NO₃ ⁻–N) or mixed (NH₄NO₃) nitrogen form, but in New Zealand spinach leaves the enzyme activity decreased and increased, respectively. The introduction of 0.4 μM Ni in the medium containing ammonium ions as a sole source of nitrogen resulted in significantly increased NR activity in lettuce roots, and did not cause statistically significant changes of the enzyme activity in New Zealand spinach plants. At a high nickel level (Ni 40 or 80 μM), a significant decrease in the NR activity was observed in New Zealand spinach plants treated with nitrate or mixed nitrogen form, but it was much more marked in leaves than in roots. An exception was lack of significant changes of the enzyme activity in spinach leaves when plants were treated with 40 μM Ni and supplied with mixed nitrogen form, which resulted in the stronger reduction of the enzyme activity in roots than in leaves. The statistically significant drop in the NR activity was recorded in the aboveground parts of nickel-stressed lettuce plants supplied with NO₃ ⁻–N or NH₄NO₃. At the same time, there were no statistically significant changes recorded in lettuce roots, except for the drop of the enzyme activity in the roots of NO₃ ⁻-fed plants grown in the nutrient solution containing 80 μM Ni. An addition of high nickel doses to the nutrient solution contained ammonium nitrogen (NH₄ ⁺–N) did not affect the NR activity in New Zealand spinach plants and caused a high increase of this enzyme in lettuce organs, especially in roots. It should be stressed that, independently of nickel dose in New Zealand spinach plants supplied with ammonium form, NR activity in roots was dramatically higher than that in leaves. Moreover, in New Zealand spinach plants treated with NH₄ ⁺–N the enzyme activity in roots was even higher than in those supplied with NO₃ ⁻–N.     

Statistical optimization of culture media for production of phycobiliprotein by Synechocystis sp. PCC 6701

Synechocystis sp. PCC 6701 has a brilliantly colored pigment, phycobiliprotein containing phycoerythrin. Culture medium was optimized by sequential designs in order to maximize phycobiliprotein production. The observed fresh weights after 6 days were 0.58 g/L in BG-11, 0.83 g/L in medium for Scenedesmus sp. and 0.03∼0.52 g/L in the other tested media. Medium for Scenedesmus sp. was selected to be optimized by fractional factorial design and central composite design since the medium maintained a more stable pH within a desirable range due to higher contents of phosphate. The fractional factorial design had seven factors with two levels: KNO₃, NaNO₃, NaH₂PO₄, Na₂HPO₄, Ca(NO₃)₂, FeEDTA, and MgSO₄. From the result of fractional factorial design, nitrate and phosphate were identified as significant factors. A central composite design was then applied with four variables at five levels each: nitrate, phosphate, pH, and light intensity. Parameters such as fresh weight and phycobiliprotein contents were used to determine the optimum value of the four variables. The proposed optimum media contains 0.88 g/L of nitrate, 0.32 g/L of phosphate under 25 μE·m⁻²·s⁻¹ of light intensity. The maximum phycobiliprotein contents have been increased over 400%, from 4.9 to 25.9 mg/L after optimization.     

Transport and accumulation of ions in two spring wheat lines differing in salt tolerance

Fifty-two-day old plants of a salt tolerant line, S24 and a salt sensitive, Yecora Rojo were subjected for 15 days to 125 mol·m⁻³ NaCl in Hoagland’s nutrient solution under glass-house conditions. The dry matter of shoots and roots of the salt tolerant line was significantly greater over all time intervals in saline substrate than the salt sensitive line, Yecora Rojo. In the leaves of salt-treated former line concentration of Na⁺ and Cl⁻ was lower as compared to the latter line. The lower Na⁺ and Cl⁻ concentrations in the leaves of S24 were found to be associated with lower transport of these ions to the shoots whereas the reverse was true for Yecora Rojo. The lines did not differ in accumulation of either ion in roots. It is concluded that salt tolerance in these two genotypes of spring wheat is associated with restricted accumulation of toxic Na⁺ and Cl⁻ ions to the shoots or with restricted transport.     

Effect of phytohormones and tungsten on light-induced and nitrate-induced nitrate reductase activity of mustard cotyledons

Mustard (Brassica juncea Coss cv. T-59 ‘Varuna’) seedlings pretreated with gibberellic acid (GA) and kinetin (KiN) were grown in light. In vivo nitrate reductase (NR) activity was estimated and effect of tungsten on light-induced and NO ₃ ^su− -induced NR activity was investigated. Different concentrations of GA did not show any effect on induction of light-induced NR; addition of nitrate promoted in vivo NR activity but no concentration effect of GA was evident. Light-induced NR was promoted by KiN and like in GA treatment, addition of nitrate increased NR activity. Addition of Na-tungstate inhibited NO ₃ ⁻ induced NR while light-induced NR was not much affected in both GA and KiN treated seedlings. The two forms of NRs were further characterized by studying the decay kinetics using Na-tungstate. In light-induced NR, tungstate did not affect NR activity up to 11 h, while at later periods, a slight decay was observed. On the other hand, NO ₃ ⁻ -induced NR activity increased up to 4 h and subsequently a rapid fall was observed. It was therefore apparent that light-induced NR had a very low turnover rate as compared to NO ₃ ⁻ -induced NR. These results further support the earlier conclusion that in mustard seedlings two distinct types of NR enzyme exist and that nitrate requirement for NR induction is not absolute.     

Effects of attachment substrates on the growth and differentiation of LLC-PK1 cells

Summary The growth and differentiation of an established renal epithelial cell line, LLC-PK₁, on membrane bound mussel adhesive protein (MAP), collagen, and extracellular matrix (ECM) in serum-containing medium was studied. Cell attachment and growth on uncoated- vs. protein-coated cellulose nitrate and acetate membranes did not differ significantly, and confluence was achieved on all membranes. However, cells remained in a single monolayer only when plated on collagen or ECM. LLC-PK₁ monolayers grown on ECM-coated membranes displayed the highest transepitheliald-glucose transport (333 ± 22 ng·cm⁻²·min⁻¹) whereas cells plated on collagen-coated membranes displayed the lowest (94 ± 23 ng·cm⁻²·min⁻¹). Glucose flux values increased with age of the culture, reaching a plateau at 28 d postseeding. These results indicate that the underlying substratum and cell age can affect differentiation of renal epithelial cells in vitro.     

Diurnal and seasonal changes in the intensity of photosynthesis in stems of lilac (Syringa vulgaris L.)

It has been demonstrated that during the whole year the stems are photosyntheticaly active and capable of assimilating atmospheric CO₂. The intensity of photosynthesis varies. During the vegetation period the registered net photosynthesis lasted up to 13 hours per day, and in the leafless period for 2–3 hours a day. Photosynthesis was registered also at temperatures below zero (−3 °C) as a reduced CO₂ evolution in light in comparison with darkness. The maximal net photosynthesis values during the vegetation period amounted to 6 up 8 μmol (CO₂)·m⁻²·s⁻¹, and in the leafless period 0.5 – 1 μmol (CO₂)·m⁻²·s⁻¹, and they were close to being up to twice as big as the values obtained of darkness respiration. An increase of the photosynthetic activity of stems preceded the spring development of the leaves.     

Influence of fruit load and environmental factors on nitrate reductase activity and on concentration of nitrate and carbohydrates in leaves of eggplant (Solanum melongena)

Both the in vivo (+ nitrate) nitrate reductase (NR) activity (leaf disks incubated in the presence of KNO₃) and the in vivo (? nitrate) NR activity (leaf disks incubated without KNO₃) in leaves of eggplant (Solanum melongena L. cv. Bonica) were affected by rapidly growing fruits. Plants with a fruit load showed more pronounced diurnal variation in (+ nitrate) NR activity and higher (? nitrate) NR activity than plants without fruit. The higher (? nitrate) NR activity was accompanied by higher nitrate and lower sucrose and starch contents of leaves. The more pronounced diurnal changes in (+ nitrate) NR activity were paralleled by more pronounced diurnal variation in carbohydrate content of leaves. Fruit removal led to a decrease in both (? nitrate) NR activity and nitrate concentration in leaves, while the carbohydrate content increased. Plants supplied with ammonium instead of nitrate showed only slightly lower (+ nitrate) but no (? nitrate) NR activity. As for plants treated with nitrate, diurnal changes in (+ nitrate) NR activity were most pronounced in leaves of plants with fruit and this again was paralleled by a more pronounced diurnal variation in the carbohydrate concentration in the leaves. Increasing the oxygen level of the atmosphere to 50% led to a dramatic decrease in the (+ nitrate) NR activity and to an increase in both (? nitrate) NR activity and nitrate concentration, which was accompanied by decreasing carbohydrate contents of the leaves. Low light intensities and extended dark periods caused similar changes in NR activity and nitrate and carbohydrate concentrations in leaves. Increasing the nitrate concentration in the nutrient solution led to a rise in (+ nitrate) and (? nitrate) NR activity, but only the (? nitrate) NR activity paralleled the nitrate concentration in the leaves. This increase in the nitrate concentration was accompanied by a decrease in the carbohydrate content of the leaves. It is concluded that the level of and the diurnal changes in both (+ nitrate) and (? nitrate) NR activity and the concentration of nitrate in the leaves are dependent upon their carbohydrate status.     

Photoinhibition of photosynthesis in intact kiwifruit (Actinidia deliciosa) leaves: effect of growth temperature on photoinhibition and recovery

Intact leaves of kiwifruit (Actinidia deliciosa (A. Chev.) C.F. Liang et A.R. Ferguson) from plants grown in a range of controlled temperatures from 15/10 to 30/25°C were exposed to a photon flux density (PFD) of 1500 μmol·m⁻²·s⁻¹ at leaf temperatures between 10 and 25°C. Photoinhibition and recovery were followed at the same temperatures and at a PFD of 20 μmol·m⁻²·s⁻¹, by measuring chlorophyll fluorescence at 77 K and 692 nm, by measuring the photon yield of photosynthetic O₂ evolution and light-saturated net photosynthetic CO₂ uptake. The growth of plants at low temperatures resulted in chronic photoinhibition as evident from reduced fluorescence and photon yields. However, low-temperature-grown plants apparently had a higher capacity to dissipate excess excitation energy than leaves from plants grown at high temperatures. Induced photoinhibition, from exposure to a PFD above that during growth, was less severe in low-temperature-grown plants, particularly at high exposure temperatures. Net changes in the instantaneous fluorescence,F ₀, indicated that little or no photoinhibition occurred when low-temperature-grown plants were exposed to high-light at high temperatures. In contrast, high-temperature-grown plants were highly susceptible to photoinhibitory damage at all exposure temperatures. These data indicate acclimation in photosynthesis and changes in the capacity to dissipate excess excitation energy occurred in kiwifruit leaves with changes in growth temperature. Both processes contributed to changes in susceptibility to photoinhibition at the different growth temperatures. However, growth temperature also affected the capacity for recovery, with leaves from plants grown at low temperatures having moderate rates of recovery at low temperatures compared with leaves from plants grown at high temperatures which had negligible recovery. This also contributed to the reduced susceptibility to photoinhibition in low-temperature-grown plants. However, extreme photoinhibition resulted in severe reductions in the efficiency and capacity for photosynthesis.     

Effect of culture conditions on growth of green alga — <Emphasis Type="Italic">Haematococcus pluvialis</Emphasis> and astaxanthin production

Influence of culture conditions such as light, temperature and C/N ratio was studied on growth of Haematococcus pluvialis and astaxanthin production. Light had significant effect on astaxanthin production and it varied with its intensity and direction of illumination and effective culture ratio (ECR, volume of culture medium/volume of flask). A 6-fold increase in astaxanthin production (37 mg/L) was achieved with 5.1468·10⁷ erg·m⁻²·s⁻¹ light intensity (high light, HL) at effective culture ratio of 0.13 compared to that at 0.52 ECR, while the difference in the astaxanthin production was less than 2 — fold between the effective culture ratios at 1.6175·10⁷ erg·m⁻²·s⁻¹ light intensity (low light, LL). Multidirectional (three-directional) light illumination considerably enhanced the astaxanthin production (4-fold) compared to unidirectional illumination. Cell count was high at low temperature (25 °C) while astaxanthin content was high at 35 °C in both autotrophic and heterotrophic media. In a heterotrophic medium at low C/N ratio H. pluvialis growth was higher with prolonged vegetative phase, while high C/N ratio favoured early encystment and higher astaxanthin formation.     

Mutation of the regulatory phosphorylation site of tobacco nitrate reductase results in high nitrite excretion and NO emission from leaf and root tissue

In wild-type Nicotiana plumbaginifolia Viv. and other higher plants, nitrate reductase (NR) is regulated at the post-translational level and is rapidly inactivated in response to, for example, a light-to-dark transition. This inactivation is caused by phosphorylation of a conserved regulatory serine residue, Ser 521 in tobacco, and interaction with divalent cations or polyamines, and 14-3-3 proteins. The physiological importance of the post-translational NR modulation is presently under investigation using a transgenic N. plumbaginifolia line. This line expresses a mutated tobacco NR where Ser 521 has been changed into aspartic acid (Asp) by site-directed mutagenesis, resulting in a permanently active NR enzyme [C. Lillo et al. (2003) Plant J 35:566–573]. When cut leaves or roots of this line (S₅₂₁) were placed in darkness in a buffer containing 50 mM KNO₃, nitrite was excreted from the tissue at rates of 0.08–0.2 mol (g FW)^–1 h^–1 for at least 5 h. For the control transgenic plant (C1), which had the regulatory serine of NR intact, nitrite excretion was low and halted completely after 1–3 h. Without nitrate in the buffer in which the tissue was immersed, nitrite excretion was also low for S₅₂₁, although 20–40 mol (g FW)^–1 nitrate was present inside the tissue. Apparently, stored nitrate was not readily available for reduction in darkness. Leaf tissue and root segments of S₅₂₁ also emitted much more nitric oxide (NO) than the control. Importantly, NO emission from leaf tissue of S₅₂₁ was higher in the dark than in the light, opposite to what was usually observed when post-translational NR modulation was operating.Abbreviations NR Nitrate reductase - NO Nitric oxide - Ser Serine - WT Wild type     

Critical evaluation of thein situ nitrate reductase assay

Anin situ method, derived from anin vivo method, was used to determine nitrate reductase activity (NRA) in:i) excised barley and corn shoots and excised soybean leaves during a N-depletion experiment and; ii) roots and shoots of N-depleted barley and corn seedlings during induction of nitrate, reductase (NR). Nitrate reduction, calculated from thesein situ RNA measurements, was compared with estimates of each organ's nitrate reduction in light aerobic conditions from NO ₃ ⁻ consumption and a¹⁵N model (Gojonet al., 1986b). Thein situ RNA of roots strongly underestimated their¹⁵NO ₃ ⁻ reduction. In contrast, in barley and corn shoots and in the first trifoliolate leaves from 26-day-old, soybean, thein situ NRA assay gave a fair approximation of the true NO ₃ ⁻ reduction rate (relative differences ranging from −14 to +32%). In young soybean leaves (from 20-day-old plants), however, thein situ NRA strongly underestimated the actual NO ₃ ⁻ reduction. The physiological significance of thein situ NRA assay in shoots and roots, and its value for field studies are discussed from these results.     

Optimization of culture medium for the continuous cultivation of the microalga Haematococcus pluvialis

The freshwater microalga Haematococcus pluvialis is one of the best microbial sources of the carotenoid astaxanthin, but this microalga shows low growth rates and low final cell densities when cultured with traditional media. A single-variable optimization strategy was applied to 18 components of the culture media in order to maximize the productivity of vegetative cells of H. pluvialis in semicontinuous culture. The steady-state cell density obtained with the optimized culture medium at a daily volume exchange of 20% was 3.77 · 10⁵ cells ml⁻¹, three times higher than the cell density obtained with Bold basal medium and with the initial formulation. The formulation of the optimal Haematococcus medium (OHM) is (in g l⁻¹) KNO₃ 0.41, Na₂HPO₄ 0.03, MgSO₄ · 7H₂O 0.246, CaCl₂ · 2H₂O 0.11, (in mg l⁻¹) Fe(III)citrate · H₂O 2.62, CoCl₂ · 6H₂O 0.011, CuSO₄ · 5H₂O 0.012, Cr₂O₃ 0.075, MnCl₂ · 4H₂O 0.98, Na₂MoO₄ · 2H₂O 0.12, SeO₂ 0.005 and (in μg l⁻¹]) biotin 25, thiamine 17.5 and B₁₂ 15. Vanadium, iodine, boron and zinc were demonstrated to be non-essential for the growth of H. pluvialis. Higher steady-state cell densities were obtained by a three-fold increase of all nutrient concentrations but a high nitrate concentration remained in the culture medium under such conditions. The high cell productivities obtained with the new optimized medium can serve as a basis for the development of a two-stage technology for the production of astaxanthin from H. pluvialis. Received: 10 September 1999 / Received revision: 2 December 1999 / Accepted: 3 December 1999     

RETRACTED ARTICLE: Effect of root-zone salinity and form of N on photosynthate partitioning in wheat (Triticum aestivum L.)

Carbon-14 pulse labeling technique was used to study the effect of rooting medium salinity and form and availability of N on growth and rhizodeposition of wheat (Triticum aestivum L.). Thirty days old plants grown in continuously aerated Arnon and Hoagland nutrient solution were subjected to ¹⁴C pulse labeling for 24 h and transferred to aqueous rooting medium containing 0, 150, and 300 mM NaCl in all combinations with different forms (calcium nitrate, ammonium sulphate, and ammonium nitrate) and amounts (0.5, 1.0, 1.5, and 2.0 times the standard N concentration (150 ppm) of Arnon and Hoagland plant growth medium). Plant samples immediately after pulse labeling, following 7 days of growth under different rooting medium conditions, and the freeze-dried rooting medium were analyzed for total C and ¹⁴C. Length and fresh/dry weight of root and shoot portions and calculated values of unaccounted ¹⁴C were determined. Presence of NaCl in the rooting medium led to a decrease in root and shoot portions. However, NO₃ ⁻-fed plants showed better growth than NH₄ ⁺-fed plants at all the three salinity levels. Salinity in rooting medium led to higher rhizodeposition and lower loss of ¹⁴C. Relatively higher proportion of ¹⁴C was released as rhizodeposits and retained in root/shoot portions of plants fed with NH₄ ⁺ or NH₄ ⁺+NO₃ ⁻, than those with NO₃ ⁻, while less was respired. The specific activity of the rhizodeposits (kBq ¹⁴C g⁻¹ C) was also higher under saline conditions. The rhizodeposits in NH₄ ⁺-fed plants were more highly labeled as compared to NO₃ ⁻-plants.     

Response of Cleopatra mandarin seedlings to a polyamine-biosynthesis inhibitor under salt stress

Six-month-old, uniform-sized seedlings of Cleopatra mandarin growing in hydroponics in Hoagland nutrient solution under glasshouse conditions were subjected to salinity treatment (NaCl 75 mM). Addition of NaCl to the nutrient medium reduced plant growth (shoot height and leaf number), leaf chlorophyll content, chlorophyll fluorescence yields (Fv/Fm), net photosynthesis, stomatal conductance, transpiration rate, intracellular CO₂ concentration, N, K⁺ and Ca²⁺ + Mg²⁺ contents of the leaves; and increased total putrescine (Put), proline and Na⁺ and Cl⁻ contents of the leaves. Addition of d-arginine (1 mM) to the saline medium further reduced the values of all the parameters except Fv/Fm, photosynthesis and related parameters and Ca²⁺ + Mg²⁺ contents of the leaves, and increased total spermine (Spm) content of the leaves. However, total Put contents were decreased; spermidine (Spd) contents and Na⁺ and Cl⁻ titres of the leaves were unaffected. Weekly spray of d-arginine (5 mM) was less harmful than its addition to the nutrient medium (1 mM). Addition of 0.5 mM Spd to the medium alleviated the adverse effects of d-arginine by improving plant growth, leaf chlorophyll content, total Put, Spd, Spm, N, P, K⁺ and Ca²⁺ + Mg²⁺ contents of the leaves. Weekly spray of Spd (5 mM) was less effective than its addition in the nutrient medium (0.5 mM).     

Primary productivity and nutrients in the sediment retention basin of Lake Monroe

Lake Monroe is the largest body of water in Indiana with a daily mean productivity of 220 mg · C · m⁻² · day⁻¹ in an observed range from 26 to 714 m · C · M⁻² day⁻¹. It is a medium soft reservoir; the acid combining capacity varies from 0.28 to 0.71 meq · l⁻¹ with a mean slightly above 0.5 meq · l⁻¹. The results of diurnal changes in major nutrients, the C, N, and P ratios, and bioassay experiments indicated that phosphorus is the major limiting nutrient on algal photosynthesis in this lake. Surface photo-inhibition may be used to indicate the sufficiency of light for the species of algae in the water. The low productivity in December, January, February, and early March can be attributed to light limitation due to low water transparency. Contribution no. 312 of the Great Lakes Research Division, University of Michigan. Contribution no. 312 of the Great Lakes Research Division, University of Michigan.     

Modulation of carbon and nitrogen metabolism, and of nitrate reductase, in untransformed and transformed Nicotiana plumbaginifolia during CO2 enrichment of plants grown in pots and in hydroponic culture

Transformed plants of Nicotiana plumbaginifolia Viv. constitutively expressing nitrate reductase (35S-NR) or β-glucuronidase (35S-GUS) and untransformed controls were grown for two weeks in a CO₂-enriched atmosphere. Whereas CO₂ enrichment (1000 μl · l⁻¹) resulted in an increase in the carbon (C) to nitrogen (N) ratio of both the tobacco lines grown in pots with vermiculite, the C/N ratio was only slightly modified when plants were grown in hydroponic culture in high CO₂ compared to those grown in air. Constitutive nitrate reductase (NR) expression per se did not change the C/N ratio of the shoots or roots. Biomass accumulation was similar in both types of plant when hydroponic or pot-grown material, grown in air or high CO₂, were compared. Shoot dry matter accumulation was primarily related to the presence of stored carbohydrate (starch and sucrose) in the leaves. In the pot-grown tobacco, growth at elevated CO₂ levels caused a concomitant decrease in the N content of the leaves involving losses in NO⁻ ₃ and amino acid levels. In contrast, the N content and composition were similar in all plants grown in hydroponic culture. The 35S-NR plants grown in air had higher foliar maximum extractable NR activities and increased glutamine levels (on a chlorophyll or protein basis) than the untransformed controls. These increases were maintained following CO₂ enrichment when the plants were grown in hydroponic culture, suggesting that an increased flux through nitrogen assimilation was possible in the 35S-NR plants. Under CO₂ enrichment the NR activation state in the leaves was similar in all plants. When the 35S-NR plants were grown in pots, however, foliar NR activity and glutamine content fell in the 35S-NR transformants to levels similar to those of the untransformed controls. The differences in NR activity between untransformed and 35S-NR leaves were much less pronounced in the hydroponic than in the pot-grown material but the difference in total extractable NR activity was more marked following CO₂ enrichment. Foliar NR message levels were decreased by CO₂ enrichment in all growth conditions but this was much more pronounced in pot-grown material than in that grown hydroponically. Since β-glucuronidase (GUS) activity and message levels in 35S-GUS plants grown under the same conditions of CO₂ enrichment (to test the effects of CO₂ enrichment on the activity of the 35S promoter) were found to be constant, we conclude that NR message turnover was specifically accelerated in the 35S-NR plants as well as in the untransformed controls as a result of CO₂ enrichment. The molecular and metabolic signals involved in increased NR message and protein turnover are not known but possible effectors include NO₃ ⁻, glutamine and asparagine. We conclude that plants grown in hydroponic culture have greater access to N than those grown in pots. Regardless of the culture method, CO₂ enrichment has a direct effect on NR mRNA stability. Received: 17 October 1996 / Accepted: 11 February 1997