Dark induction of nitrate reductase in the halophilic alga Dunaliella salina

The effect of nitrogen starvation on the NO₃-dependent induction of nitrate reductase (NR) and nitrite reductases (NIR) has been investigated in the halophilic alga Dunaliella salina. When D. salina cells previously grown in a medium with NH ₄ ⁺ as the only nitrogen source (NH ₄ ⁺ -cells) were transferred into NO ₃ ^? medium, NR was induced in the light. In contrast, when cells previously grown in N-free medium were transferred into a medium containing NO ₃ ^? , NR was induced in light or in darkness. Nitrate-dependent NR induction, in darkness, in D. salina cells previously grown at a photon flux density of 500 umol · m^?2 s^?1 was observed after 4 h preculture in N-free medium, whilst in cells grown at 100 umol · m^?2 s^?1 NR induction was observed after 7–8 h. An inhibitor of mRNA synthesis (6-methylpurine) did not inhibit NO ₃ ^? -induced NR synthesis when the cells, previously grown in NH ₄ ⁺ medium, were transferred into NO ₃ ^? medium (at time 0 h) after 4-h-N starvation. However, when 6-methylpurine was added simultaneously with the transfer of the cells from NH ₄ ⁺ to NO ₃ ^? medium (at time 0 h), NO ₃ ^? induced NR synthesis was completely inhibited. The activity of NIR decreased in N-starved cells and the addition of NO ₃ ^? to those cells greatly stimulated NIR activity in the light. The ability to induce NR in darkness was observed when glutamine synthetase activity reached its maximal level during N starvation. Although cells grown in NO ₃ ^? medium exhibited high NR activity, only 0.33% of the total NR was found in intact chloroplasts. We suggest that the ability, to induce NR in darkness is dependent on the level of N starvation, and that NR in D. salina is located in the cytosol. Light seems to play an indirect regulatory role on NO ₃ ^? uptake and NR induction due to the expression of NR and NO ₃ ^? -transporter mRNAs.     

NO 3 − transport across the plasma membrane of Arabidopsis thaliana root hairs: Kinetic control by pH and membrane voltage

High-affinity nitrate transport was examined in intact root hair cells of Arabidopsis thaliana using electrophysiological recordings to characterise the response of the plasma membrane to NO ₃ ^? challenge and to quantify transport activity. The NO ₃ ^? -associated membrane current was determined using a three-electrode voltage clamp to bring membrane voltage under experimental control and to compensate for current dissipation along the longitudinal cell axis. Nitrate transport was evident in the roots of seedlings grown in the absence of a nitrogen source, but only 4–6 days postgermination. In 6-day-old seedlings, additions of 5–100 μm NO ₃ ^? to the bathing medium resulted in membrane depolarizations of 8–43 mV, and membrane voltage (V _m) recovered on washing NO ₃ ^? from the bath. Voltage clamp measurements carried out immediately before and following NO ₃ ^? additions showed that the NO ₃ ^? -evoked depolarizations were the consequence of an inward-directed current that appeared across the entire range of accessible voltages (?300 to +50 mV). Both membrane depolarizations and NO ₃ ^? -evoked currents recorded at the free-running voltage displayed quasi-Michaelian kinetics, with apparent values for K_m of 23 ± 6 and 44 ± 11 μm, respectively and, for the current, a maximum of 5.1 ± 0.9 μA cm^?2. The NO ₃ ^? current showed a pronounced voltage sensitivity within the normal physiological range between ?250 and ?100 mV, as could be demonstrated under voltage clamp, and increasing the bathing pH from 6.1 to 7.4–8.0 reduced the current and the associated membrane depolarizations 3- to 8-fold. Analyses showed a well-defined interaction between the kinetic variables of membrane voltage, pH_o and [NO ₃ ^? ]_o. At a constant pH_o of 6.1, depolarization from ?250 to ?150 mV resulted in an approximate 3-fold reduction in the maximum current but a 10% rise in the apparent affinity for NO ₃ ^? . By contrast, the same depolarization effected an approximate 20% fall in the K_m for transport as a function in [H⁺]_o. These, and additional characteristics of the transport current implicate a carrier cycle in which NO ₃ ^? binding is kinetically isolated from the rate-limiting step of membrane charge transit, and they indicate a charge-coupling stoichiometry of 2(H⁺) per NO ₃ ^? anion transported across the membrane. The results concur with previous studies showing a high-affinity NO ₃ ^? transport system in Arabidopsis that is inducible following a period of nitrogen-limiting growth, but they underline the importance of voltage as a kinetic factor controlling NO ₃ ^? transport at the plant plasma membrane.     

Small-conductance chloride channels in human peripheral T lymphocytes

During whole-cell patch-clamp recording from normal (nontransformed) human T lymphocytes a chloride current spontaneously activated in >98% of cells (n > 200) in the absence of applied osmotic or pressure gradients. However, some volume sensitivity was observed, as negative pressure pulses reduced the current. With iso-osmotic bath and pipette solutions the peak amplitude built up (time constant ≈23 sec at room temperature), a variable-duration plateau phase followed, then the current ran down spontaneously (time constant ≈280 sec). The anion permeability sequence, calculated from reversal potentials was I^?, Br^? > NO ₃ ^? , Cl^? > CH₃SO ₃ ^? , HCO ₃ ^? > CH₃COO^? > F^? > aspartate, gluconate, SO ₄ ^2? and there was no measurable monovalent cation permeability. The Cl^? current was independent of time during long voltage steps and there was no evidence of voltage-dependent gating; however, the current showed intrinsic outward rectification in symmetrical Cl^? solutions. The conductance of the channels underlying the whole-cell current was calculated from fluctuation analysis, using power-spectral density and variance-vs.-mean analysis. Both methods yielded a single channel conductance of about 0.6 pS at ?70 mV (close to the normal resting potential of T lymphocytes). The power spectral density function was best fit by the sum of two Lorentzian functions, with corner frequencies of 30 and 295 Hz, corresponding to mean open times of 0.54 and 5.13 msec. The pharmacological profile included rapid block by external application of flufenamic acid (50 μm), 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB, 100 μm, [6,7-dichloro-2-cyclopentyl-2,3-dihydro-2-methyl-1-oxo-1H-inden-5-y1) oxy] acetic acid (IAA-94, 250 μm) or 100 μm 1,9-dideoxyforskolin. The stilbene derivatives DIDS (4,4′-diisothiocyano-2,2′ di-sulphonic acid stilbene, 500 μm) and SITS (4-acetamido-4′-isothiocyano-2, 2′-disulphonic acid stilbene, 500 μm) prevented buildup of Cl^? current after a 30-min preincubation at 500 μm. When tested in a mitogenic assay, DIDS, flufenamic acid, NPPB and IAA-94 all inhibited T-cell proliferation, suggesting a physiological function in addition to the observed volume sensitivity.     

Ammonium nutrition increases water absorption in rice seedlings (Oryza sativa L.) under water stress

Water stress is a primary limitation on plant growth. In previous studies, it has been found that ammonium enhances the tolerance of rice plants to water stress, but how water is related to nitrogen form and water stress remains unknown. To study the effects of nitrogen form (NH ₄ ⁺ , NO ₃ ^? , and a mixture of NH ₄ ⁺ and NO ₃ ^? ) on the growth and water absorption of rice (Oryza sativa L.) seedlings, a hydroponic experiment with water stress, simulated by the addition of polyethylene glycol (PEG, 10% w/v, MW 6000), was conducted in a greenhouse. The results showed that, compared with non-water stress, under water stress, the fresh weight of rice seedlings increased by 14% with NH ₄ ⁺ nutrition, whereas it had decreased by about 20% with either NO ₃ ^? or mixed nitrogen nutrition. No significant difference was found in the transpiration rate of excised shoots or in xylem exudation of excised roots in NH ₄ ⁺ supply between the two water situations, whereas xylem flow decreased by 57% and 24% under water stress in NO ₃ ^? and mixed nutrition, and root hydraulic conductivity decreased by 29% and 54% in plants in NH ₄ ⁺ and NO ₃ ^? nutrition conditions, respectively. Although water absorption ability decreased in both NH ₄ ⁺ and NO ₃ ^? nutrition, aquaporin activity was higher in NH ₄ ⁺ than in NO ₃ ^? nutrition, regardless of water stress. We conclude that NH ₄ ⁺ nutrition can improve water handling in rice seedlings and subsequently enhance their resistance to drought.     

Influence of different mineral nitrogen sources (NO 3 − -N vs. NH 4 + -N) on arbuscular mycorrhiza development and N transfer in a Glomus intraradices–cowpea symbiosis

Labeled nitrogen (¹⁵?N) was applied to a soil-based substrate in order to study the uptake of N by Glomus intraradices extraradical mycelium (ERM) from different mineral N (NO ₃ ^? vs. NH ₄ ⁺ ) sources and the subsequent transfer to cowpea plants. Fungal compartments (FCs) were placed within the plant growth substrate to simulate soil patches containing root-inaccessible, but mycorrhiza-accessible, N. The fungus was able to take up both N-forms, NO ₃ ^? and NH ₄ ⁺ . However, the amount of N transferred from the FC to the plant was higher when NO ₃ ^? was applied to the FC. In contrast, analysis of ERM harvested from the FC showed a higher ¹⁵?N enrichment when the FC was supplied with ¹⁵NH ₄ ⁺ compared with ¹⁵NO ₃ ^? . The ¹⁵?N shoot/root ratio of plants supplied with ¹⁵NO ₃ ^? was much higher than that of plants supplied with ¹⁵NH ₄ ⁺ , indicative of a faster transfer of ¹⁵NO ₃ ^? from the root to the shoot and a higher accumulation of ¹⁵NH ₄ ⁺ in the root and/or intraradical mycelium. It is concluded that hyphae of the arbuscular mycorrhizal fungus may absorb NH ₄ ⁺ preferentially over NO ₃ ^? but that export of N from the hyphae to the root and shoot may be greater following NO ₃ ^? uptake. The need for NH ₄ ⁺ to be assimilated into organically bound N prior to transport into the plant is discussed.     

Characterization of ammonium and nitrate uptake and assimilation in roots of tea plants

It has been pointed out that tea (Camellia sinensis (L.) O. Kuntze) prefers ammonium (NH ₄ ⁺ ) over nitrate (NO ₃ ^? ) as an inorganic nitrogen (N) source. ¹⁵N studies were conducted using hydroponically grown tea plants to clarify the characteristics of uptake and assimilation of NH ₄ ⁺ and NO ₃ ^? by tea roots. The total ¹⁵N was detected, and kinetic parameters were calculated after feeding ¹⁵NH ₄ ⁺ or ¹⁵NO ₃ ^? to tea plants. The process of N assimilation was studied by monitoring the dynamic ¹⁵N abundance in the free amino acids of tea plant roots by GC-MS. Tea plants supplied with ¹⁵NH ₄ ⁺ absorbed significantly more ¹⁵N than those supplied with ¹⁵NO ₃ ^? . The kinetics of ¹⁵NH ₄ ⁺ and ¹⁵NO ₃ ^? influx into tea plants followed a classic biphasic pattern, demonstrating the action of a high affinity transport system (HATS) and a low affinity transport system (LATS). The V _max value for NH ₄ ⁺ uptake was 54.5 nmol/(g dry wt min), which was higher than that observed for NO ₃ ^? (39.3 nmol/(g dry wt min)). K_M estimates were approximately 0.06 mM for NH ₄ ⁺ and 0.16 mM for NO ₃ ^? , indicating a higher rate of NH ₄ ⁺ absorption by tea plant roots. Tea plants fed with ¹⁵NH ₄ ⁺ accumulated larger amounts of assimilated N, especially glutamine (Gln), compared with those fed with ¹⁵NO ₃ ^? . Gln, Glu, theanine (Thea), Ser, and Asp were the main free amino acids that were labeled with ¹⁵N under both conditions. The rate of N assimilation into Thea in the roots of NO ₃ ^? -supplied tea plants was quicker than in NH ₄ ⁺ -supplied tea plants. NO ₃ ^? uptake by roots, rather than reduction or transport within the plant, seems to be the main factor limiting the growth of tea plants supplied with NO ₃ ^? as the sole N source. The NH ₄ ⁺ absorbed by tea plants directly, as well as that produced by NO ₃ ^? reduction, was assimilated through the glutamine synthetase-glutamine oxoglutarate aminotransferase pathway in tea plant roots. The ¹⁵N labeling experiments showed that there was no direct relationship between the Thea synthesis and the preference of tea plants for NH ₄ ⁺ .     

Fluorescence transients in chloroplasts of Chara corallina cells during transmission of photoinduced signal with the streaming cytoplasm

Intracellular transport assisted by rotatory cytoplasmic movement in characean green algae exerts regulatory influence on plasmalemmal ion channels and photosynthetic activity of chloroplasts. In internodal cells of Chara corallina Klein ex Willd., the photoinduced signal transmitted with the flow of streaming cytoplasm for a distance of 1–3 mm from the site of its emergence was found to release or enhance non-photochemical quenching of chlorophyll fluorescence, depending on the intensity of background illumination in the analyzed area. Under dim background irradiance (10–30 μmol quanta/(m²s)), the distant signal transferred from brightly illuminated 0.4-mm-wide area elicited a transient increase in maximal (F′_m) and actual (F) fluorescence. However, at higher background irradiances, the arrival of the same signal resulted in strong quenching of F′_m and in transitory changes of F. The transformation of “low light response” to F′_m changes of opposite polarity occurred at some threshold irradiance. Hence, even slight variations in irradiance at the chloroplast layer, attributed to structural features of characean internodes, might promote formation of uneven photosynthetic profile under the influence of signals transmitted along the cell with the cytoplasmic flow. Analysis of chloroplast fluorescence in situ as a function of pH in experiments with intracellular perfusion indicated that the initial response to a distant light stimulus is caused by the transient increase in cytoplasmic pH in the area of fluorescence measurements.     

Mechanisms of cell volume regulation in the proximal segment of the Malpighian tubule of Rhodnius neglectus

The cell volume regulation of the lower segment cells of the Malpighian tubule of Rhodnius neglectus in anisosmotic media was evaluated by using videooptic techniques. When the medium osmolality was increased with addition of 100 mm mannitol the cells shrank to a minimum of 16.84±2.62% and subsequently swelled towards their initial volume undergoing a typical regulatory volume increase (RVI). Replacement of either K⁺ or Cl^? or HCO ₃ ^? by Na⁺, gluconate and phosphate, respectively, abolished the RVI response. Furthermore, the substitution of Na⁺ by tetramethylammonium (TMA⁺) in isosmotic conditions led to cellular swelling and death. Addition of either amiloride 10^?4 m, anthracene-9-COOH 5×10^?4 m, furosemide 5×10^?4 m or ethacrynic acid 5×10^?5 m, also abolished RVI. On the other hand, addition of either Ba²⁺ 10^?3 m, SITS 5× 10^?4 m, ouabain 10^?3 m or vanadate 10^?3 m, did not change the RVI response. When the tubules were incubated in hyperosmotic media with EGTA 2 mm or verapamil 10^?6 m, the RVI response was abolished. In contrast, a decrease of NaCl concentration from 129 to 79 mm induced a cell swelling to a maximum of 33.11+1.73%, but the cells maintained swollen, only partially regulating their volume. These results show that the proximal cells of Malpighian tubule of R. neglectus are able to regulate their volume in hyperosmotic but only partially regulating in hyposmotic solutions. The mechanisms in RVI involve Na⁺, K⁺, Cl^?, Ca²⁺ and HCO ₃ ^? transport pathways and a ouabain-insensitive ATPase stimulated by Na⁺. This work was supported by grants from the Fundação de Amparo à Pesquisa do Estado de São Paulo—FAPESP; Conselho Nacional de Desenvolvimento Científico e Tecnológico—CNPq e Financiadora de Projetos e Pesquisas-FINEP.     

Nitrate-use traits of understory plants as potential regulators of vegetation distribution on a slope in a Japanese cedar plantation

Background and aims

Plant physiological traits and their relation to soil N availability was investigated as regulators of the distribution of understory shrub species along a slope in a Japanese cedar (Cryptomeria japonica) plantation in central Japan.

Methods

At the study site, previous studies demonstrated that both net and gross soil nitrification rates are high on the lower slope and there are dramatic declines in different sections of the slope gradient. We examined the distributions of understory plant species and their nitrate (NO ₃ ^? -N) use traits, and compared the results with the soil traits.

Results

Our results show that boundaries between different dominant understory species correspond to boundaries between different soil types. Leucosceptrum stellipilum occurs on soil with high net and gross nitrification rates. Hydrangea hirta is dominant on soil with high net and low gross nitrification rates. Pieris japonica occurs on soil with very low net and gross nitrification rates. Dominant understory species have species-specific physiological traits in their use of NO ₃ ^? -N. Pieris japonica lacks the capacity to use NO ₃ ^? -N as a N source, but other species do use NO ₃ ^? -N. Lindera triloba, whose distribution is unrelated to soil NO ₃ ^? -N availability, changes the extent to which it uses NO ₃ ^? -N in response to soil NO ₃ ^? -N availability.

Conclusions

Our results indicate that differences in the physiological capabilities and adaptabilities of plant species in using NO ₃ ^? -N as a N source regulate their distribution ranges. The identity of the major form of available soil N is therefore an environmental factor that influences plant distributions.     

The interrelationship between HCO 3 − , NO 3 − , and Cl− as effective anions in the photosynthetic electron transport

Thylakoids were isolated from the leaves of three different plants (Pisum sativium L., Lactuca sativa L., and Raphanus sativus L.). The addition of HCO ₃ ^? to a suspension of salt-and HCO ₃ ^? -epleted thylakoids (suspended in salt-free medium) raised the rate of O₂ evolution up to fourfold. This stimulation could be partially replaced by the addition of chloride or nitrate ions. However, the addition of HCO ₃ ^? in the presence of Cl^? or NO ₃ ^? resulted in a higher stimulation of O₂ evolution (sixfold in the presence of nitrate and sevenfold in the presence of chloride). On the other hand, the addition of HCO ₃ ^? to the thylakoids depleted from salt only raised the rate of O₂ evolution by 10–15%, whereas 40–70% was obtained by the addition of nitrate or chloride ions. The fluorescence induction studies indicated a significant decrease in the yield of the variable fluorescence of the salt- and HCO ₃ ^? -depleted thylakoids. A partial increase in the fluorescence yield was obtained by the addition of HCO ₃ ^? alone. A typical fluorescence induction curves were obtained by the addition of HCO ₃ ^? in the presence of Cl^? or NO ₃ ^? ions. The data obtained suggest a similar role for chloride and nitrate ions in O₂ evolution in the Hill-reaction, which is restricted at the donor side of photosystem II, whereas bicarbonate plays its role at both sides (acceptor and donor sides). The presented data are those obtained for the thylakoids of P. sativium, which were more or less similar to those obtained for L. sativa and R. sativus.     

Effects of NO 3 − -N on the growth and salinity tolerance of Tamarix laxa Willd

The influence of NO ₃ ^? -N on growth and osmotic adjustment was studied in Tamarix laxa Willd., a halophyte with salt glands on its twigs. Seedlings of T. laxa Willd. were exposed to 1 mM (control) or 300 mM NaCl, with 0.05, 1 or 10 mM NO ₃ ^? -N for 24 days. The relative growth rate of seedlings at 300 mM NaCl was lower than that of control plants at all NO ₃ ^? -N levels, but the concentrations of organic N and total N in the twigs did not differ between the two NaCl treatments. Increasing NO ₃ ^? supply under 300 mM NaCl improved the growth of T. laxa, indicating that NO ₃ ^? played positive roles in improving salt resistance of the plant. The twigs of T. laxa Willd. accumulated mainly inorganic ions, especially Na⁺ and Cl^?, to lower osmotic potential (Ψs): the contributions of Na⁺ and Cl^? to Ψs were estimated at 31% and 27% respectively, at the highest levels of supply of both NaCl and NO ₃ ^? -N. The estimated contribution of NO ₃ ^? -N to Ψs was as high as 20% in the twigs in these conditions, indicating that NO ₃ ^? was also involved in osmotic adjustment in the twigs. Furthermore, increases in tissue NO ₃ ^? were accompanied by decreases in tissue Cl^? and proline under 300 mM NaCl. The estimated contribution of proline to Ψs declined as with NO ₃ ^? -N supply increased from 1 to 10 mM, while the contributions of nitrate to Ψs were enhanced under 300 mM NaCl. This suggested that higher accumulation of nitrate in the vacuole alleviated the effects of salinity stress on the plant by balancing the osmotic potential. In conclusion, NO ₃ ^? -N played both nutritional and osmotic roles in T. laxa Willd. in saline conditions.     

Interaction between NO 3 − and abscisic acid in the regulation of lateral root elongation in Zea mays L.

Lateral root (LR) elongation rate of 7–8-day maize seedlings depends on the availability of NO ₃ ^? , NO ₂ ^? , and abscisic acid (ABA) in an environment. Four-hour exposure to 0.01–1.5 mM NO ₂ ^? increases the relative LR elongation rate; in the case of NO ₂ ^? , the stimulation occurs only at an NO ₂ ^? concentration equal to 0.01 mM. Exogenous ABA (10^?6 M) inhibits the LR elongation process. In the case of a combined influence of NO ₃ ^? and ABA or NO ₂ ^? and ABA, the character of the response elongation reaction is different. The NO role in the regulation of LR elongation is discussed.     

Ionic balance,proton efflux,nitrate reductase activity and growth ofHippophaë rhamnoides L. ssp.rhamnoides as influenced by combined-N nutrition or N2 fixation

Growth of 2-month-old nonnodulatedHippophaë rhamnoides seedlings supplied with combined N was compared with that of nodulated seedlings grown on zero N. Plant growth was significantly better with combined N than with N₂ fixation and, although not statistically significant for individual harvests, tended to be highest in the presence of NH ₄ ⁺ , a mixture of NH ₄ ⁺ and NO ₃ ^? producing the highest yields. Growth was severely reduced when solely dependent on N₂ fixation and, unlike the combined-N plants, shoot to root ratios had only slightly increased after an initial decrease. An apparently insufficient nodule mass (nodule weight ratio <5 per cent) during the greater part of the experimental period is suggested as the main cause of the growth reduction in N₂-fixing plants. Thein vivo nitrate reductase activity (NRA) of NO ₃ ^? dependent plants was almost entirely located in the roots. However, when grown with a combination of NO ₃ ^? and NH ₄ ⁺ , root NRA was decreased by approximately 85 per cent.H. rhamnoides demonstrated in the mixed supply a strong preference for uptake of N as NH ₄ ⁺ , NO ₃ ^? contributing only for approximately 20 per cent to the total N assimilation. Specific rates of N acquisition and ion uptake were generally highest in NO ₃ ^? +NH ₄ ⁺ plants. The generation of organic anions per unit total plant dry weight was approximately 40 per cent less in the NH ₄ ⁺ plants than in the NO ₃ ^? plants. Measured extrusions of H⁺ or OH^? (HCO ₃ ^? ) were generally in good agreement with calculated values on the basis of plant composition, and the acidity generated with N₂ fixation amounted to 0.45–0.55 meq H⁺. (mmol N_org)^?1. Without acidity control and in the presence of NH ₄ ⁺ , specific rates of ion uptake and carboxylate generation were strongly depressed and growth was reduced by 30–35 per cent. Growth of nonnodulatedH. rhamnoides plants ceased at the lower pH limit of 3.1–3.2 and deterioration set in; in the case of N₂-fixing plants the nutrient solution pH stabilized at a value of 3.8–3.9 without any apparent adverse effects upon plant performance. The chemical composition of experimental and field-growing plants is being compared and some comments are made on the nitrogen supply characteristics of their natural sites.     

Partitioning of previously-accumulated nitrate to translocation,reduction, and efflux in corn roots

The effect of nitrate uptake, or its absence, on the utilization of nitrate previously accumulated by dark-grown, decpitated maize (Zea mays L., cv. DeKalb XL-45) seedlings was examined. Five-d-old plants that had been pretreated with 50 mM ¹⁴NO ₃ ^? for 20 h were exposed for 8 h to nutrient solutions containing either no nitrate or 50 mM ¹⁵NO ₃ ^? , 98.7 atom % ¹⁵N. The ambient solution, xylem exudate, and plant tissue were analyzed to determine the quantities of previously-accumulated (endogenous) ¹⁴NO ₃ ^? that were translocated to the xylem, lost to the solution, or reduced within the tissue during the 8-h period. Energy was continuously available to the roots from the attached endosperm. In the absence of incoming nitrate, appreciable reduction and translocation of the endogenous ¹⁴NO ₃ ^? occurred, but efflux of ¹⁴NO ₃ ^? to the external solution was minimal. In contrast, during ¹⁵NO ₃ ^? uptake, there was considerable efflux of ¹⁴NO ₃ ^? as well as translocation of ¹⁴NO ₃ ^? to the xylem, but little ¹⁴NO ₃ ^? was reduced. Thus there appeared to be an inverse relationship between ¹⁴NO ₃ ^? efflux and reduction. The data are tentatively interpreted on the basis of a model which envisages (a) two storage locations within roots, one of which primarily supplies nitrate for translocation and the other of which primarily supplies nitrate for outward passage through plasmalemma, and (b) the majority of nitrate reduction as occurring during or immediately following influx across the plasmalemma, with endogenous ¹⁴NO ₃ ^? initially moving outward being recycled inward and thereby being reduced.     

Characterization of ribose-5-phosphate isomerase converting d-psicose to d-allose from Thermotoga lettingae TMO

The gene coding for ribose-5-phosphate isomerase (Rpi) from Thermotoga lettingae TMO was cloned and expressed in E. coli. The recombinant enzyme was purified by Ni-affinity chromatography. It converted d-psicose to d-allose maximally at 75 °C and pH 8.0 with a 32 % conversion yield. The k _m, turnover number (k _cat), and catalytic efficiency (k _cat k _m ^?1 ) for substrate d-psicose were 64 mM, 6.98 min^?1 and 0.11 mM^?1 min^?1 respectively.     

Denitrification in soil amended with thermophile-fermented compost suppresses nitrate accumulation in plants

NO ₃ ^? is a major nitrogen source for plant nutrition, and plant cells store NO ₃ ^? in their vacuoles. Here, we report that a unique compost made from marine animal resources by thermophiles represses NO ₃ ^? accumulation in plants. A decrease in the leaf NO ₃ ^? content occurred in parallel with a decrease in the soil NO ₃ ^? level, and the degree of the soil NO ₃ ^? decrease was proportional to the compost concentration in the soil. The compost-induced reduction of the soil NO ₃ ^? level was blocked by incubation with chloramphenicol, indicating that the soil NO ₃ ^? was reduced by chloramphenicol-sensitive microbes. The compost-induced denitrification activity was assessed by the acetylene block method. To eliminate denitrification by the soil bacterial habitants, soil was sterilized with γ irradiation and then compost was amended. After the 24-h incubation, the N₂O level in the compost soil with presence of acetylene was approximately fourfold higher than that in the compost soil with absence of acetylene. These results indicate that the low NO ₃ ^? levels that are often found in the leaves of organic vegetables can be explained by compost-mediated denitrification in the soil.     

Dominance of biologically produced nitrate in upland waters of Great Britain indicated by stable isotopes

Atmospheric deposition of nitrogen (N) compounds is the major source of anthropogenic N to most upland ecosystems, where leaching of nitrate (NO ₃ ^? ) into surface waters contributes to eutrophication and acidification as well as indicating an excess of N in the terrestrial catchment ecosystems. Natural abundance stable isotopes ratios, ¹⁵N/¹⁴N and ¹⁸O/¹⁶O (the “dual isotope” technique) have previously been used in biogeochemical studies of alpine and forested ecosystems to demonstrate that most of the NO ₃ ^? in upland surface waters has been microbially produced. Here we present an application of the technique to four moorland catchments in the British uplands including a comparison of lakes and their stream inflows at two sites. The NO ₃ ^? concentrations of bulk deposition and surface waters at three sites are very similar. While noting the constraints imposed by uncertainty in the precise δ¹⁸O value for microbial NO ₃ ^? , however, we estimate that 79–98% of the annual mean NO ₃ ^? has been microbially produced. Direct leaching of atmospheric NO ₃ ^? is a minor component of catchment NO ₃ ^? export, although greater than in many similar studies in forested watersheds. A greater proportion of atmospheric NO ₃ ^? is seen in the two lake sites relative to their inflow streams, demonstrating the importance of direct NO ₃ ^? deposition to lake surfaces in catchments where terrestrial ecosystems intercept a large proportion of deposited N. The dominance of microbial sources of NO ₃ ^? in upland waters suggests that reduced and oxidised N deposition may have similar implications in terms of contributing to NO ₃ ^? leaching.     

Chlorophyll fluorescence induction,chlorophyll content,and chromaticity characteristics of leaves as indicators of photosynthetic apparatus senescence in arboreous plants

Parameters of chlorophyll fluorescence induction (CFI) are widely used for assessment of the physiological state of higher plant leaves in biochemical, physiological, and ecological studies and in agricultural applications. In this work we have analyzed data on variability of some CFI parameters — Φ _PSII ^max = F _v/F _m (relative value of variable fluorescence), q _NPQ (non-photochemical quenching coefficient), R _Fd (“vitality index”) — in autumnal leaves of ten arboreous plant species of the temperate climatic zone. The correlation between the chlorophyll content in the leaves and fluorescence parameters characterizing photosynthetic activity is shown for two representative species, the small-leaved linden Tilia cordata and the rowan tree Sorbus aucuparia. During the period of mass yellowing of the leaves, the Φ _PSII ^max value can be used as an adequate characteristic of their photochemical activity, while in summer the q _NPQ or R _Fd values are more informative. We have established a correlation between the Φ _PSII ^max value, which characterizes the maximal photochemical activity of the photosystem II, and “chromaticity coordinates” of a leaf characterizing its color features. The chromaticity coordinates determined from the optical reflection spectra of the leaves serve as a quantitative measure of their hues, and this creates certain prerequisites for a visual expert assessment of the physiological state of the leaves.     

Culture medium optimization for improved puerarin production by cell suspension cultures of Pueraria tuberosa (Roxb. ex Willd.) DC.

The effects of carbon, nitrogen, phosphate, and copper on cell growth and production of the isoflavone puerarin by suspension cultures of Pueraria tuberosa (Roxb. ex. Willd.) DC were investigated. Among the various sugars evaluated (glucose, galactose, fructose, maltose, and sucrose), use of sucrose in the medium led to the maximum accumulation of puerarin. A sucrose-feeding strategy in which additional sucrose was added to the flasks 15?d into the culture cycle stimulated both cell biomass and puerarin production. The maximum production of puerarin was obtained when a concentration balance of 20:60?mM NH ₄ ⁺ /NO ₃ ^? was used as the nitrogen source. Alteration in the concentration balance of nitrogen components (NH ₄ ⁺ /NO ₃ ^? 60:20?mM) or the use of either NH ₄ ⁺ or NO ₃ ^? alone decreased biomass production and puerarin accumulation compared with the control culture (NH ₄ ⁺ /NO ₃ ^? 20:20?mM). High amounts of phosphate (2.5 and 5?mM) in the medium inhibited puerarin production whereas 0.625?mM phosphate promoted puerarin production (68.3???g/g DW on day?25). An increase in Cu²⁺ concentration from 0.025 to 0.05?mg/l in the P. tuberosa cell culture medium resulted in a 2.2-fold increase in puerarin production (up to 141???g/g DW on day?25) but reduced cell culture biomass.     

Alteration of nitrogen metabolism in rice variety 'Nipponbare' induced by alkali stress

Aims

Alkali stress (AS) is an important agricultural contaminant and has complex effects on plant metabolism, specifically root physiology. The aim of this study was to test the role of nitrogen metabolism regulation in alkali tolerance of rice variety 'Nipponbare'.

Methods

In this study, the rice seedlings were subjected to salinity stress (SS) or AS. Growth, the contents of inorganic ions, NH ₄ ⁺ -nitrogen (free amino acids), and NO ₃ ^? -nitrogen in the stressed seedlings were then measured. The expression of some critical genes involved in nitrogen metabolism were also assayed to test their roles in the regulation of nitrogen metabolism during adaptation of rice variety 'Nipponbare' to AS.

Results

AS showed a stronger inhibiting effect on rice variety 'Nipponbare' growth than SS. AS may have more complex effects on nitrogen metabolism than SS.

Conclusions

Effects of AS on the nitrogen metabolism of rice variety 'Nipponbare' mainly comprised two mechanisms. Firstly, in roots, AS caused the reduction of NO ₃ ^? content, which caused two harmful consequences, the large downregulation of OsNR1 expression and the subsequent reduction of NH ₄ ⁺ production in roots. On the other hand, under AS (pH, 9.11), almost all the NH ₄ ⁺ was changed to NH₃, which caused a severe deficiency of NH ₄ ⁺ surrounding the roots. Both events might cause a severe deficiency of NH ₄ ⁺ in roots. Under AS, the increased expression of several OsAMT family members in roots might be an adaptative response to the reduction of NH ₄ ⁺ content in roots or the NH ₄ ⁺ deficiency in rhizosphere. Also, the down-regulation of OsNADH-GOGAT and OsGS1;2 in roots might be due to NH ₄ ⁺ deficiency in roots. Secondly, in shoots, AS caused a larger acuumulatiuon of Na⁺, which possibly affected photorespiration and led to a continuous decrease of NH ₄ ⁺ production in shoots, and inhibited the expression of OsFd-GOGAT and OsGS2 in chloroplasts.