Metabolic regulation of ammonia emission in different senescence phenotypes of Nicotiana tabacum

In order to reveal the character of ammonia emission in senescent tobacco (Nicotiana tabacum), the content of NH₄⁺, total nitrogen, and soluble protein, and the activities of nitrogen metabolism-related enzymes were measured in leaves of a quick-leaf-senescence phenotype ZY90 and a slow-leaf-senescence phenotype NC89. Compared with NC89, ZY90 had a higher NH₄⁺ accumulation, a lower glutamine synthetase activity, and a significantly higher stomatal ammonia compensation point, and ammonia emission during 40 to 60 d after leaf emergence. During senescence, the quick-leafsenescence phenotype was characterized by nitrogen re-transfer by ammonia emmission, whereas the slow-leafsenescence phenotype by nitrogen re-assimilation. The ammonia emission was primarily regulated by glutamine synthetase activity, apoplastic pH, and NH₄⁺ content.     

Genotypic variation in photosynthesis in cacao is correlated with stomatal conductance and leaf nitrogen

Variation in photosynthetic parameters was observed between eight contrasting cacao (Theobroma cacao) genotypes. Net photosynthetic rate (P_N) ranged from 3.4 to 5.7 μmol(CO₂) m⁻² s⁻¹ for the genotypes IMC 47 and SCA 6, respectively. Furthermore, genotypic differences were detected in quantum efficiency ranging from 0.020 to 0.043 μmol(CO₂) μmol⁻¹(photon) for UF 676 and AMAZ 15/15, respectively. Differences in PN were correlated with both stomatal conductance (g_s) and leaf nitrogen per unit area. Some variation in water use efficiency was observed between genotypes, both intrinsic (P_N/g_s) and instantaneous (P_N/transpiration rate). Both measures of water use efficiency were a negative function of specific leaf area. Evidence was found for a trade-off mechanism between cacao genotypes in photosynthesis and leaf structure. High photosynthetic rate, expressed on a mass basis was associated with smaller leaves. Furthermore, thinner leaves were compensated for by a higher nitrogen content per unit mass.     

Changes in photosynthesis,fluorescence, and nitrogen metabolism of hawthorn (<Emphasis Type="Italic">Crataegus pinnatifida</Emphasis>) in response to exogenous glutamic acid

Photosynthesis, chlorophyll (Chl) a fluorescence, and nitrogen metabolism of hawthorn (Crataegus pinnatifida Bge.), subjected to exogenous L-glutamic acid (GLA) (200 mg l⁻¹, 400 mg l⁻¹, and 800 mg l⁻¹) that possibly affect secondary metabolic regulation, were measured. The results indicated that photosynthetic and fluorescence characteristics of hawthorn exhibited positive responses to the application of GLA. Different concentrations of GLA caused an increase in Chl content, net photosynthetic rate (P _N) and stomatal conductance (g _s) as well as transpiration rate (E), and improved the carboxylation efficiency (CE), apparent quantum yield (AQY) and maximum carboxylation velocity of Rubisco (V_cmax). Application of GLA could also enhance the maximum ratio of quantum yields of photochemical and concurrent non-photochemical processes in PSII (F_v/F₀), the maximal quantum yield of PSII (F_v/F_m), the probability that an absorbed photon will move an electron into the electron transport chain beyond Q_A (Φ_Eo) as well as the performance index on absorption basis (PI_ABS), but decreased the intercellular CO₂ concentration (C _i) and the minimal fluorescence (F₀). Application of GLA also induced an increase in nitrate reductase (NR; EC 1.6.6.1) and glutamine synthetase (GS; EC 6.3.1.2) activities, and increased the soluble protein content, leaf nitrogen (N) content and N accumulation in leaves as well as the plant biomass. However, the effects were different among different concentrations of GLA, and 800 mg l⁻¹ GLA was better. This finding suggested that application of GLA is recommended to improve the photosynthetic capacity by increasing the light energy conversion and CO₂ transfer as well as the photochemical efficiency of PSII, and enhanced the nitrogen metabolism and growth and development of plants.     

Excess irradiance causes early symptoms of senescence during leaf expansion in photoautotrophically <Emphasis Type="Italic">in vitro</Emphasis> grown tobacco plants

Photosynthetic parameters, growth, and pigment contents were determined during expansion of the fourth leaf of in vitro photoautotrophically cultured Nicotiana tabacum L. plants at three irradiances [photosynthetically active radiation (400–700 nm): low, LI 60 μmol m⁻² s⁻¹; middle, MI 180 μmol m⁻² s⁻¹; and high, HI 270 μmol m⁻² s⁻¹]. During leaf expansion, several symptoms usually accompanying leaf senescence appeared very early in HI and then in MI plants. Symptoms of senescence in developing leaves were: decreasing chlorophyll (Chl) a+b content and Chl a/b ratio, decreasing both maximum (F_V/F_M) and actual (Φ_PS2) photochemical efficiency of photosystem 2, and increasing non-photochemical quenching. Nevertheless, net photosynthetic oxygen evolution rate (P _N) did not decrease consistently with decrease in Chl content, but exhibited a typical ontogenetic course with gradual increase. P _N reached its maximum before full leaf expansion and then tended to decline. Thus excess irradiance during in vitro cultivation did not cause early start of leaf senescence, but impaired photosynthetic performance and Chl content in leaves and changed their typical ontogenetic course.     

Chlorophyll fluorescence imaging of photosynthetic activity in sun and shade leaves of trees

The differences in pigment levels, photosynthetic activity and the chlorophyll fluorescence decrease ratio R _Fd (as indicator of photosynthetic rates) of green sun and shade leaves of three broadleaf trees (Platanus acerifolia Willd., Populus alba L., Tilia cordata Mill.) were compared. Sun leaves were characterized by higher levels of total chlorophylls a + b and total carotenoids x + c as well as higher values for the weight ratio chlorophyll (Chl) a/b (sun leaves 3.23–3.45; shade leaves: 2.74–2.81), and lower values for the ratio chlorophylls to carotenoids (a + b)/(x + c) (with 4.44–4.70 in sun leaves and 5.04–5.72 in shade leaves). Sun leaves exhibited higher photosynthetic rates P _N on a leaf area basis (mean of 9.1–10.1 μmol CO₂ m⁻² s⁻¹) and Chl basis, which correlated well with the higher values of stomatal conductance G _s (range 105–180 mmol m⁻² s⁻¹), as compared to shade leaves (G _s range 25–77 mmol m⁻² s⁻¹; P _N: 3.2–3.7 μmol CO₂ m⁻² s⁻¹). The higher photosynthetic rates could also be detected via imaging the Chl fluorescence decrease ratio R _Fd, which possessed higher values in sun leaves (2.8–3.0) as compared to shade leaves (1.4–1.8). In addition, via R _Fd images it was shown that the photosynthetic activity of the leaves of all trees exhibits a large heterogeneity across the leaf area, and in general to a higher extent in sun leaves than in shade leaves.     

Ontogenetic patterns of leaf CO2 exchange, morphology and chemistry in Betula pendula trees

In order to explore ontogenetic variation in leaf-level physiological traits of Betula pendula trees, we measured changes in mass- (A _mass) and area-based (A _area) net photosynthesis under light-saturated conditions, mass- (RS_mass) and area-based (RS_area) leaf respiration, relative growth rate, leaf mass per area (LMA), total nonstructural carbohydrates (TNC), and macro- and micronutrient concentrations. Expanding leaves maintained high rates of A _area, but due to high growth respiration rates, net CO₂ fixation occurred only at irradiances >200 μmol photons m^–2 s^–1. We found that full structural leaf development is not a necessary prerequisite for maintaining positive CO₂ balance in young birch leaves. Maximum rates of A _area were realized in late June and early July, whereas the highest values of A _mass occurred in May and steadily declined thereafter. The maintenance respiration rate averaged ≈8 nmol CO₂ g^–1 s^–1, whereas growth respiration varied between 0 and 65 nmol CO₂ g^–1 s^–1. After reaching its lowest point in mid-June, leaf respiration increased gradually until the end of the growing season. Mass and area-based dark respiration were significantly positively correlated with LMA at stages of leaf maturity, and senescence. Concentrations of P and K decreased during leaf development and stabilized or increased during maturity, and concentrations of immobile elements such as Ca, Mn and B increased throughout the growing season. Identification of interrelations between leaf development, CO₂ exchange, TNC and leaf nutrients allowed us to define factors related to ontogenetic variation in leaf-level physiological traits and can be helpful in establishing periods appropriate for sampling birch leaves for diagnostic purposes such as assessment of plant and site productivity or effects of biotic or abiotic factors. Received: 29 December 1998 / Accepted: 26 July 1999     

Leaf traits variation during leaf expansion in <Emphasis Type="Italic">Quercus ilex</Emphasis> L.

The morphological, anatomical and physiological variations of leaf traits were analysed during Quercus ilex L. leaf expansion. The leaf water content (LWC), leaf area relative growth rate (RGR_l) and leaf dry mass relative growth rate (RGR_m) were the highest (76±2 %, 0.413 cm² cm⁻² d⁻¹, 0.709 mg mg⁻¹ d⁻¹, respectively) at the beginning of the leaf expansion process (7 days after bud break). Leaf expansion lasted 84±2 days when air temperature ranged from 13.3±0.8 to 27.6±0.9 °C. The net photosynthetic rate (P _N), stomatal conductance (g _s), and chlorophyll content per fresh mass (Chl) increased during leaf expansion, having the highest values [12.62±1.64 μmol (CO₂) m⁻² s⁻¹, 0.090 mol (H₂O) m⁻² s⁻¹, and 1.03±0.08 mg g⁻¹, respectively] 56 days after bud break. Chl was directly correlated with leaf dry mass (DM) and P _N. The thickness of palisade parenchyma contributed to the total leaf thickness (263.1±1.5 μm) by 47 %, spongy layer thickness 38 %, adaxial epidermis and cuticle thickness 9 %, and abaxial epidermis and cuticle thickness 6 %. Variation in leaf size during leaf expansion might be attributed to a combination of cells density and length, and it is confirmed by the significant (p<0.001) correlations among these traits. Q. ilex leaves reached 90 % of their definitive structure before the most severe drought period (beginning of June — end of August). The high leaf mass area (LMA, 15.1±0.6 mg cm⁻²) at full leaf expansion was indicative of compact leaves (2028±100 cells mm⁻²). Air temperature increasing might shorten the favourable period for leaf expansion, thus changing the final amount of biomass per unit leaf area of Q. ilex.     

Seasonal variation in ammonia compensation point and nitrogen pools in beech leaves (Fagus sylvatica)

Ammonia (NH₃) fluxes between beech leaves (Fagus sylvatica) and the atmosphere were investigated in a 90-year-old forest canopy and related to leaf nitrogen (N) pools and glutamine synthetase (GS) activities. The stomatal ammonia compensation point, ?? _NH3, was measured by both a twig cuvette and bioassay techniques involving measurements of pH and ammonium (NH ₄ ⁺ ) concentration in the leaf apoplastic solution. The ?? _NH3 determined on the basis of the gas exchange measurements followed a seasonal variation with early-season peaks during leaf expansion (9.6 nmol NH₃ mol^?1 air) and late-season peaks during leaf senescence (7.3 nmol NH₃ mol^?1 air). In the mid-season, the ?? _NH3 of mature green leaves was much lower (around 3 nmol NH₃ mol^?1 air) and dropped below the NH₃ concentration in the ambient atmosphere. For comparison, ?? _NH3 obtained by the apoplastic bioassay were 7.0, 3.7 and 6.4 nmol NH₃ mol^?1 air in early-, mid-, and late -season, thus agreeing reasonably well with ?? _NH3 values derived from the gas exchange measurements. Potential NH₃ emission fluxes during early and late season were 1.31 and 0.51 nmol m^?2 leaf surface area s^?1, respectively, while leaves were a sink for NH₃ during mid-season. During leaf establishment and senescence, both apoplastic and bulk tissue NH ₄ ⁺ concentrations were relatively high coinciding with low activities of glutamine synthetase, which is a key enzyme in leaf N metabolism. In conclusion, the exchange of NH₃ between beech leaves and the atmosphere followed a seasonal variation with NH₃ emission peaks being related to N mobilization during early leaf establishment and remobilization during late leaf senescence.     

Growth and physiological changes in saplings of Minquartia guianensis and Swietenia macrophylla during acclimation to full sunlight

Low light availability under a forest canopy often limits plant growth; however, sudden increase in light intensity may induce photoinhibition of photosynthesis. The aim of this study was to evaluate the ecophysiological changes that occur in potted plants of Minquartia guianensis and Swietenia macrophylla during the acclimation process to full sunlight. We used six full-sun independent acclimation periods (30, 60, 90, 120, 150, and 180 days) and a control kept in the shade. Shading was obtained by placing plants under the canopy of a small forest. The F_v/F_m ratio, net photosynthetic rate (P _N), the maximum carboxylation velocity of Rubisco (V _cmax), maximum electron transport rate (J _max), specific leaf area (SLA), and growth were assessed at the end of each of the six acclimation periods. Plant exposure to full sunlight caused a sudden decrease in the F_v/F_m ratio (photoinhibition) particularly in Minquartia. Photooxidation (necrotic patches) of the leaf tissue was observed in upper leaves of Minquartia. The higher P _N values were observed in Swietenia under full sun, about 12 μmol(CO₂) m⁻² s⁻¹. V _cmax25 values were higher after 90 days of acclimation, about 14 μmol(CO₂) m⁻² s⁻¹ for Minquartia, and 35 μmol(CO₂) m⁻² s⁻¹ for Swietenia. At the end of a 180-d acclimation period J _max25 was 35 μmol(electron) m⁻² s⁻¹ for Minquartia and 60 μmol(electron) m⁻² s⁻¹ for Swietenia. SLA was higher in Swietenia than in Minquartia. In Minquartia, monthly rate of leaf production per plant (MRLP) was positive (0.22 leaf month⁻¹) after four months in the open. Whereas, in Swietenia MRLP was positive (0.56 leaf month⁻¹) after an acclimation period of two months. After six months in the open, height growth rates were 3.5 and 28 mm month⁻¹ for Minquartia and Swietenia, respectively. The greater acclimation capacity of Swietenia was associated to an enhanced photosynthetic plasticity under full sun. In Minquartia, transition to full-sun conditions and lack of physiological adjustment resulted in severe photoinhibition and loss of leaves.     

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

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

Changes in stomatal frequency, stomatal conductance and cuticle thickness during leaf expansion in the broad-leaved evergreen species, Eucalyptus regnans

Aspects of leaf anatomical and physiological development were investigated in the broad-leaved evergreen species, Eucalyptus regnans F.Muell. Newly emergent leaves were tagged in the field and measured for stomatal conductance while a subset was collected every 14 days for the measurement of stomata and cuticle over a 113-day period. Cuticle thickness increased during leaf expansion, the increase following a sigmoid curve. Stomatal frequency (no. mm⁻²) decreased from 56 to 113 days after leaf emergence. The frequency of both immature and intermediate developmental stages of stomata also decreased over this time, but the total number of stomata per leaf remained relatively constant. Stomatal conductance (g _s) of young expanding leaves increased during expansion, and was significantly linearly correlated with stomatal frequency (excluding immature stomata), and with cuticle thickness. The progressive increase in g _s in young developing leaves was contrary to the observed changes in structural characteristics (increased cuticle thickness and decreased stomatal frequency). This increase in g _s with development may be related to the progressive increase in number of mature stomata with larger apertures and, therefore, a higher total pore area in fully expanded leaves.     

Effect of fertilizer application on photosynthesis and oil yield of <Emphasis Type="Italic">Jatropha curcas</Emphasis> L.

The use of Jatropha curcas oil as a source of biofuel has been well-explored. However, the physiological and growth studies of J. curcas have received considerably lesser attention. In this study, leaf gas exchange measurements and leaf nitrogen content were determined for four varieties of J. curcas, grown in the field or in pots. Based on stable carbon isotope analysis (δ¹³C) and gas-exchange studies, J. curcas is a C₃ sun plant and the range of leaf photosynthetic rates (or CO₂ assimilation rates, P _Nmax) were typically between 7 and 25 μmol(CO₂) m⁻² s⁻¹ and light saturation generally occurred beyond 800 μmol(quanta) m⁻² s⁻¹. Higher rates of leaf photosynthesis were generally obtained with the mature leaves. In addition, increased foliar P _Nmax were recorded in potted J. curcas variety Indiana with increasing nitrogen (N) nutrition levels. These plants also showed greater growth, increased leaf N content, higher maximum CO₂ assimilation capacity (P _NhighCO2) and chlorophyll (Chl) content, indicating the potential of optimizing the growth of Jatropha by varying fertilizer nutrient levels. A rapid assessment for leaf N using a nondestructive and portable Chl meter had been established for J. curcas. This approach will allow repeated sampling of the same plant over time and thus enable the monitoring of the appropriate levels of soil fertility to achieve good Jatropha plantation productivity. High N nutrition improved the overall plant oil yield by increasing the total number of fruits/seeds produced per plant, while not affecting the intrinsic seed oil content.     

Entropy budgets of deciduous plant leaves and a theorem of oscillating entropy production

From an energy budget of a deciduous plant leaf in moderate conditions, entropy fluxes into or out of the leaf due to solar radiation, infrared radiation, evaporation of water and heat conduction are calculated. Net entropy flow into the leaf is negative. On the assumption that the entropy in the leaf is in a steady state, the entropy production in the typical deciduous leaf in moderate conditions [the solar energy absorbed by both sides of the leaf isE _solar=0.0602 (J cm⁻² s⁻¹)] becomesS _prod=1.8×10⁻⁴ (J cm⁻² s⁻¹ K⁻¹). The positiveness of the entropy production shows that the Second Law of Thermodynamics certainly holds in the plant leaf. Entropy productions in other conditions are also calculated. The entropy production in the leafS _prod becomes a linear function of the solar energy absorbed by the leafE _solar:S _prod≈-(29.5E _solar)×10⁻⁴. A theorem is presented: the entropy production in plant leaves oscillates during the period of one day, paralleling the daily solar energy absorbed by leaves.     

Photosynthetically versatile thin shade leaves: A paradox of irradiance-response curves

Thick sun leaves have a larger construction cost per unit leaf area than thin shade leaves. To re-evaluate the adaptive roles of sun and shade leaves, we compared the photosynthetic benefits relative to the construction cost of the leaves. We drew photosynthetically active radiation (PAR)-response curves using the leaf-mass-based photosynthetic rate to reflect the cost. The dark respiration rates of the sun and shade leaves of mulberry (Morus bombycis Koidzumi) seedlings did not differ significantly. At irradiances below 250 μmol m⁻² s⁻¹, the shade leaves tended to have a significantly larger net photosynthetic rate (P _N) than the sun leaves. At irradiances above 250 μmol m⁻² s⁻¹, the P _N did not differ significantly. The curves indicate that plants with thin shade leaves have a larger daily CO₂ assimilation rate per construction cost than those with thick sun leaves, even in an open habitat. These results are consistently explained by a simple model of PAR extinction in a leaf. We must target factors other than the effective assimilation when we consider the adaptive roles of thick sun leaves.     

Expression of a ferredoxin-dependent glutamate synthase gene in mesophyll and vascular cells and functions of the enzyme in ammonium assimilation in Nicotiana tabacum (L.)

GLU1 encodes the major ferredoxin-dependent glutamate synthase (Fd-GOGAT, EC 1.4.7.1) in Arabidopsis thaliana (ecotype Columbia). With the aim of providing clues on the role of Fd-GOGAT, we analyzed the expression of Fd-GOGAT in tobacco (Nicotiana tabacum L. cv. Xanthi). The 5′ flanking element of GLU1 directed the expression of the uidA reporter gene in the palisade and spongy parenchyma of mesophyll, in the phloem cells of vascular tissue and in the roots of tobacco. White light, red light or sucrose induced GUS expression in the dark-grown seedlings in a pattern similar to the GLU1 mRNA accumulation in Arabidopsis. The levels of GLU2 mRNA encoding the second Fd-GOGAT and NADH-glutamate synthase (NADH-GOGAT, EC 1.4.1.14) were not affected by light. Both in the light and in darkness, ¹⁵NH₄⁺ was incorporated into [5−¹⁵N]glutamine and [2−¹⁵N]glutamate by glutamine synthetase (GS, EC 6.3.1.2) and Fd-GOGAT in leaf disks of transgenic tobacco expressing antisense Fd-GOGAT mRNA and in wild-type tobacco. In the light, low level of Fd-glutamate synthase limited the [2−¹⁵N]glutamate synthesis in transgenic leaf disks. The efficient dark labeling of [2−¹⁵N]glutamate in the antisense transgenic tobacco leaves indicates that the remaining Fd-GOGAT (15–20% of the wild-type activity) was not the main limiting factor in the dark ammonium assimilation. The antisense tobacco under high CO₂ contained glutamine, glutamate, asparagine and aspartate as the bulk of the nitrogen carriers in leaves (62.5%), roots (69.9%) and phloem exudates (53.2%). The levels of glutamate, asparagine and aspartate in the transgenic phloem exudates were similar to the wild-type levels while the glutamine level increased. The proportion of these amino acids remained unchanged in the roots of the transgenic plants. Expression of GLU1 in mesophyll cells implies that Fd-GOGAT assimilates photorespiratory and primary ammonium. GLU1 expression in vascular cells indicates that Fd-GOGAT provides amino acids for nitrogen translocation. The nucleotide sequence data of the GLU1 gene reported in the present study is available from GenBank with the following accession number: AY189525     

Through-flow of water in leaves of a submerged plant is influenced by the apical opening

Submerged aquatic higher plants maintain acropetal water transport to the young leaves in active growth to satisfy their demand for nutrients and hormones derived from the roots. We here present the first measurements of hydraulic properties for a submerged plant, the monocotyledon Sparganium emersum Rehman. The hydraulic conductance per unit length, K_h, was measured in leaf segments without the leaf tip and shown to be greater in old, fully developed leaves (1.5 · 10⁻¹⁰ · m⁴ · MPa⁻¹ · s⁻¹) than in young leaves (1.0 · 10⁻¹⁰ · m⁴ · MPa⁻¹ · s⁻¹). In leaves with intact leaf tips, however, K_h was significantly greater in the youngest leaves, which suggests that the leaf tip with the hydathode influences resistance and thus flow. Microscopy confirmed that the hydathodal area, which is an apical opening, undergoes structural changes with leaf age; a matrix of microorganisms develops in the older leaves and probably restricts water flow by clogging the hydathodes. The leaf specific conductivity expressing transport capacity relative to the leaf area supplied, of S. emersum (0.1 · 10⁻⁸ to 9 · 10⁻⁸ · m² MPa⁻¹· s⁻¹) was within the same range as for various species of terrestrial ferns, vines and trees. This finding does not support the traditional concept of functionally reduced vascular transport in Received: 15 July 1996 / Accepted: 30 November 1996     

Dark respiration of leaves and traps of terrestrial carnivorous plants: are there greater energetic costs in traps?

In this study, O₂-based dark respiration rate (R_D) in leaf and trap cuttings was compared in 9 terrestrial carnivorous plant species of 5 genera to decide whether traps represent a greater energetic (maintanence) cost than leaves or photosynthetic parts of traps. R_D values of cut strips of traps or leaves of terrestrial carnivorous plants submerged in water ranged between 2.2 and 8.4 nmol g⁻¹ s⁻¹ (per unit dry weight) in pitcher traps of the genera Sarracenia, Nepenthes, and Cephalotus, while between 7.2 and 25 nmol g⁻¹ _DW s⁻¹ in fly-paper or snapping traps or leaves of Dionaea and Drosera. No clear relationship between R_D values of traps (or pitcher walls) and leaves (or pitcher wings or petioles) was found. However, RD values of separated Drosera prolifera tentacles exceeded those of leaf lamina 7.3 times.     

Activity profiles of enzymes involved in glutamine and glutamate metabolism in the apple during autumnal senescence

Seasonal changes in glutamine synthetase (EC 6.3.1.2), glutamate synthase (EC 2.6.1.53), and glutamate dehydrogenase (EC 1.4.1.3) were measured in both senescing leaf and bark tissues of ‘Golden Delicious’ apple trees (Malus domestica Borkh.). From the measured enzyme activities we attempted to estimate the in vivo catalytic potentials of the enzymes with special reference to nitrogen mobilization and conservation of senescing apple trees. The cumulative glutamine synthetase activity of leaf tissue was about three times higher than that of bark. The estimated catalytic potential of leaf glutamine synthetase was 800-fold higher than the actual protein nitrogen loss of senescing leaves. The cumulative glutamate synthase activity of bark was about six times higher than that of leaf. The estimated catalytic potential of bark glutamate synthase was 160-times higher than the actual protein nitrogen gain in that tissue. The cumulative glutamate dehydrogenase activities in leaf and bark tissue were approximately the same. However, the catalytic potential of leaf glutamate dehydrogenase was twice that of leaf glutamate synthase. It is thus concluded that the physiological role of glutamine synthetase in senescing leaf tissue is to furnish the amide(s) prior to mobilization of nitrogen to storage tissue. The higher activity of glutamate synthase in bark tissue could provide a mechanism to transform the imported amide nitrogen to amino nitrogen of glutamate for storage protein synthesis. The possible regulatory factors upon the activity of these enzymes in the tissues of senescing apple trees are discussed.     

Overexpression of a soybean gene encoding cytosolic glutamine synthetase in shoots of transgenic Lotus corniculatus L. plants triggers changes in ammonium assimilation and plant development

A soybean cytosolic glutamine synthetase gene (GS15) was fused with the constitutive 35S cauliflower mosaic virus (CaMV) promoter in order to direct overexpression in Lotus corniculatus L. plants. Following transformation with Agrobacterium rhizogenes, eight independent Lotus transformants were obtained which synthesized additional cytosolic glutamine synthetase (GS) in the shoots. To eliminate any interference caused by the T-DNA from the Ri plasmid, three primary transformants were crossed with untransformed plants and progeny devoid of T_L- and T_R-DNA sequences were chosen for further analyses. These plants had a 50–80% increase in total leaf GS activity. Plants were grown under different nitrogen regimes (4 or 12 mM NH₄ ⁺) and aspects of carbon and nitrogen metabolism were examined. In roots, an increase in free amino acids and ammonium was accompanied by a decrease in soluble carbohydrates in the transgenic plants cultivated with 12 mM NH₄ ⁺ in comparison to the wild type grown under the same conditions. Labelling experiments using ¹⁵NH₄ ⁺ were carried out in order to monitor the influx of ammonium and its subsequent incorporation into amino acids. This experiment showed that both ammonium uptake in the roots and the subsequent translocation of amino acids to the shoots was lower in plants overexpressing GS. It was concluded that the build up of ammonium and the increase in amino acid concentration in the roots was the result of shoot protein degradation. Moreover, following three weeks of hydroponic culture early floral development was observed in the transformed plants. As all these properties are characteristic of senescent plants, these findings suggest that expression of cytosolic GS in the shoots may accelerate plant development, leading to early senescence and premature flowering when plants are grown on an ammonium-rich medium. Received: 17 July 1996 / Accepted: 16 October 1996