Enzymes of ammonium metabolism in ectendomycorrhizal and ectomycorrhizal symbionts of pine

Ammonium assimilation enzymes from several strains of ectendo- and ectomycorrhizal fungi were assayed after three weeks culture on a buffered synthetic medium containing ammonium as sole nitrogen source. Activity of NADP-dependent glutamate dehydrogenase (GDH, EC 1.4.1.4) of ectomycorrhizal strains was very low despite excellent mycelial growth. Only ectendomycorrhizal fungus MrgX isolated from roots of Pinus sylvestris showed high GDH activity. Similar results were obtained when the enzyme extracts were subjected to starch gel electrophoresis. Growth of the fungi, except ectendomycorrhizal MrgX, was arrested when inhibitors of glutamine synthetase (GS, EC 6.3.1.2) or glutamate synthase (GOGAT. EC 1.4.7.1) (methionine sulphoximine or albizine, respectively) were included in the culture medium. Glutamine synthetase activity was found in all fungi tested. The results suggest that the GS pathway for ammonium assimilation is potentially operative in ectomycorrhizal fungi and imply only a minor role for GDH in ammonium assimilation by the studied ectomycorrhizal symbionts of pine. Some physiological and ecological implications of these results are discussed.     

Different effects of supplied ammonium on glutamine synthetase activity in mustard (Sinapis alba) and pine (Pinus sylvestris) seedlings

The activity of glutamine synthetase (GS) in mustard ( Sinapis alba L.) and Scots pine ( Pinus sylvestris L.) seedlings was used as an index to evaluate the capacity to cope with excessive ammonium supply. In these 2 species GS activity was differently affected by the application of nitrogen compounds (NH₄⁺ or NO₃⁻). Mustard seedlings older than 5 days showed a considerable increase in GS activity after NH₄⁺ or NO₃⁻ application. This response was independent of the energy flux, but GS activity in general was positively affected by light. Endogenous NH₄⁺ did not accumulate greatly after nitrogen supply. In contrast, seedlings of Scots pine accumulated NH₄⁺ in cotyledons and roots and showed no stimulation of GS activity after the application of ammonium. In addition, root growth was drastically reduced. Thus, the pine seedlings seem to have insufficient capacity to assimilate exogenously supplied ammonium. NO₃⁻, however, did not lead to any harmful effects.     

Porphyrin metabolism in chill-stressed seedlings of Scots pine (Pinus sylvestris)

Scots pine ( Pinus sylvestris L.) is generally resistant to chilling temperatures. Porphyrin metabolism under low temperature stress was studied in etiolated seedlings of Scots pine. Low temperatures affect porphyrin accumulation in at least 3 different temperature sensitive sites: 1) the light activated accumulation of 5-aminolevulinic acid, a porphyrin precursor, 2) the metabolism of 5-aminolevulinic acid to form porphyrins and 3) a preferential accumulation of chlorophyll a over chlorophyll b . The temperature sensitivity of pine is compared to maize ( Zea mays L.), a chilling sensitive plant.     

Appearance of nitrate reductase, nitrite reductase and glutamine synthetase in different organs of the Scots pine (Pinus sylvestris) seedling as affected by light, nitrate and ammonium

Appearance of nitrate reductase (NR, EC 1.6.6.1–3), nitrite reductase (NiR, EC 1.7.7.1) and glutamine synthetase (GS, EC 6.3.1.2) under the control of nitrate, ammonium and light was studied in roots, hypocotyls and needles (cotyledonary whorl) of the Scots pine ( Pinus sylvestris L.) seedling. It was found that appearance of NiR was mainly controlled by nitrate whereas appearance of GS was strongly controlled by light. In principle, the NR activity level showed the same dependency on nitrate and light as that of NiR. In the root, both nitrate and ammonium had a stimulatory effect on GS activity whereas in the whorl the induction was minor. The level of NiR (NR) activity is high in the root and hypocotyl and low in the cotyledonary whorl, whereas the GS activity level per organ increases strongly from the root to the whorl. Thus, in any particular organ the operation of the glutamine synthetase/glutamate synthase (GS/GOGAT) cycle is not closely connected to the operation of the nitrate reduction pathway. The strong control of GS/GOGAT by light and the minor sensitivity to induction by nitrate or ammonium indicate a major role of the GS/GOGAT cycle in reassimilation of endogeniously generated ammonium.     

Effect of atmospheric ammonia on the nitrogen metabolism of Scots pine (Pinus sylvestris) needles

Four-year-old seedlings of Scots pine ( Pinus sylvestris L.) were exposed to filtered air (FA), and to FA supplemented with NH₃ (60 and 240 μg m⁻³) in controlled-environment chambers for 14 weeks. Exposure to the higher NH₃ concentration resulted in an increased activity of glutamine synthetase (GS, EC 6.3.1.2), and an increase in the concentrations of soluble proteins, total nitrogen, free amino acids and leaf pigments in the needles. The GS activity (μmol g⁻¹ fresh weight h⁻¹) in the needle extract increased to levels 69% higher than in FA and the soluble protein concentration to levels 22% higher. Total nitrogen concentration in the needles was 42% higher than in FA, while the free amino acid concentration was 300% higher, which was caused by an increase in arginine, glutamate, aspartate and glutamine. Chlorophyll a , chlorophyll b and carotenoid concentrations were 29, 38 and 11% higher, respectively. Neither the glutamate dehydrogenase (GDH, EC 1.4.1.2) activity nor the concentrations of free NH₄⁺ and glucose in the needles were affected by exposure to NH₃. After NH₃ fumigation at 240 μg m⁻³ the starch concentration decreased by 39% relative to the FA. The results indicate that the metabolism of Scots pine acclimates to concentrations of NH₃ which are 3 to 10 times higher than the average concentration in areas with intensive stock farming. The possible mechanisms underlying acclimation to NH₃ are discussed.     

Persistent effects of low concentrations of SO2 and NO2 on photosynthesis in Scots pine (Pinus sylvestris) needles

In an open-field experiment, 50-year-old trees of Scots pine (Pinus sylvestris L.) were fumigated with low concentrations of SO₂ and NO₂ (10–15 nl I^?1) during the growing season in four consecutive years (1988 to 1991). Results from the autumn and early winter of 1991 and 1992 are presented. The maximum photochemical efficiency of photosystem II (PSII), as indicated by the ratio of variable to maximum fluorescence (F_v/F_M) was assessed in current and one-year-old needles from the top and the bottom of the canopy. Furthermore, simultaneous measurements of photosynthetic O₂ evolution and chlorophyll fluorescence were made in current-year needles at 20°C. In general, the F_v/F_M ratio as well as the gross rate of O₂ evolution in needles of fumigated trees was not significantly different from that in needles of control trees during the fumigation period. However, both current and one-year-old needles sampled in November and December 1991 from the top of the canopy of fumigated trees had significantly lower F_v/F_M values than corresponding needles of control trees. Similar differences in F_v/F_M correlated with the treatments were observed in needles from the bottom of the canopy, indicating that the depression of F_v/F_M in needles of fumigated trees was not due to an increased susceptibility to photoinhibition. In 1992, when no fumigation occurred, differences in F_v/F_M between the treatments were not significant during autumn and early winter. The gross rate of O₂ evolution at high irradiances was significantly lower in current-year needles of fumigated trees sampled in November and December 1991 than in those of control trees. Furthermore, a nearly identical linear relationship between the quantum yield of PSII electron transport determined from chlorophyll fluorescence and the quantum yield of O₂ evolution (gross rate of O₂ evolution/PPFD) was found during autumn and early winter. This appeared to be largely a result of changes in the thermal energy dissipation within PSII. The observed differences in photosynthetic characteristics correlated with the different treatments after the fumigation period is suggested to be mainly caused by increased sensitivity of the needles of fumigated trees to low and subfreezing temperatures. However, current-year needles of fumigated trees tended to have a lower N content than those of control trees, which may partly explain the differences in gross photosynthesis between fumigated and control trees.     

The effects of long-term elevation of air temperature and CO on the frost hardiness of Scots pine

The frost hardiness of 20 to 25-year-old Scots pine (Pinus sylvestris L.) saplings was followed for 2 years in an experiment that attempted to simulate the predicted climatic conditions of the future, i.e. increased atmospheric CO₂ concentration and/or elevated air temperature. Frost hardiness was determined by an electrolyte leakage method and visual damage scoring on needles. Elevated temperatures caused needles to harden later and deharden earlier than the controls. In the first year, elevated CO₂ enhanced hardening at elevated temperatures, but this effect disappeared the next year. Dehardening was hastened by elevating CO₂ in both springs. The frost hardiness was high (相似文献   

Inhibitors of endogenous proteinases in the seeds of Scots pine, Pinus sylvestris

Extracts of resting pine seeds inhibited the proteinase activities present in extracts of endosperms of germinating seeds (hydrolysis of haemoglobin at pH 3.7 and hydrolysis of casein at pH 5.4 and 7.0). Heating the extracts of resting seeds at 60°C destroyed their own proteinase activity but their proteinase inhibitor activity decreased by only 25 to 30%. Some properties of the inhibitor(s) were studied using extracts treated at 60°C. The inhibitor activities were non-dialysable. the inhibition increased linearly with increasing inhibitor concentration up to 80% of total proteinase activity, and the maximal inhibition was 80% at pH 3.7. 90% at pH 5.4. and 97% at pH 7.0. The extracts of resting seeds did not inhibit the pepsin-like acid pine proteinase that accounts for a minor part of the proteolytic activity of endosperm extracts at pH 3.7. Neither did they have any effect on the acid pine carboxypeptidase or trypsin and chymotrypsin. Fresh extracts of endosperms of germinating seeds contained relatively high proteinase activity (assayed directly) and moderate inhibitor activity (assayed after treatment at 60°C). When fresh extracts were dialysed at 50°C for 48 h their proteinase activities increased considerably while the corresponding inhibitor activities disappeared. It is concluded that the decrease of inhibitors during dialysis is due to enzymatic inactivation and that the corresponding increase of proteinase activities is at least partly due to the destruction of the inhibitors.     

Reduced realised fecundity in the pine looper Bupalus piniarius caused by host plant defoliation

1. Whether the potential fecundity of herbivorous insects is realised or not may depend on female behaviour, which in turn may be influenced by host plant acceptability. Female Bupalus piniarius were observed to discriminate against needles growing out the year following defoliation (current‐year needles) of its host plant Pinus sylvestris. 2. It was hypothesised that the discriminatory behaviour was due to current‐year needles being less secure as a substrate. Field and laboratory experiments were designed to test this hypothesis and to estimate the discrepancy between potential and realised fecundity when females were offered defoliated branches. 3. In a laboratory oviposition experiment, B. piniarius females were exposed to branches bearing either current‐year needles only or both mature and current‐year needles. Daily oviposition rate, egg batch size, longevity, and mature eggs and fat retained at death were recorded for each female. In field experiments, the rate at which eggs dropped from expanding needles and the capacity of neonate larvae hatching from the dropped eggs to colonise a tree were assessed. 4. Significantly fewer eggs were laid when females were exposed to defoliated branches. 5. Twenty‐six and 16% of the eggs laid on current‐year needles dropped from the needles in 1998 and 1999 respectively, whereas no eggs dropped from mature needles in 1998 and only one egg (< 1%) dropped in 1999. 6. A very small proportion of larvae hatching on the forest floor (simulated egg drop) was able to recolonise host trees. 7. These results emphasise the importance of oviposition behaviour on realised fecundity when analysing insect population dynamics. In the case of B. piniarius, egg placement, although a minor detail during the normal course of events, became of key importance when defoliation deprived females of their preferred egg attachment site.     

Assimilation of nitrate and ammonium by the Scots pine (Pinus sylvestris) seedling under conditions of high nitrogen supply

Seedlings of Scots pine ( Pinus sylvestris L.) were grown on perlite for 21 days under controlled conditions. Apart from the water control, KNO₃ (15 m M ), (NH₄)₂SO₄ (7.5 m M ), and NH₄NO₃ (15 m M ) were offered to study the effects of a high nitrogen supply on nitrogen assimilation. In some experiments 1.3 m M potassium was added to the basic ammonium solutions. In labelling studies nitrate and ammonium were 2.3 atom%¹⁵N-enriched. It was found that over the 21-day period approximately three times more ammonium-N was taken up than nitrate-N. However, nitrate and ammonium, applied simultaneously, were taken up to the same extent as if they were applied separately (additivity). The presence of K⁺ in the medium did not affect N-uptake. Among the soluble N-containing compounds nitrate, ammonium and 8 amino acids were quantified. It was found that assimilation of nitrate can cope with the uptake of NO⁻₃ under all circumstances. Neither free nitrate nor ammonium or amino acids accumulated to an extent exceeding the values of water-grown seedlings. On the other hand, in case of high ammonium supply considerably more nitrogen was taken up than could be incorporated into nonsoluble N-containing substance ('protein'). The remaining nitrogen was found to accumulate in intermediary storage pools (free NH₄⁺, glutamine, asparagine, arginine). Part of this accumulated N could be incorporated into protein when potassium was offered in the nutrient solution. It is concluded that potassium is a requirement for a high rate of protein synthesis not only in crop plants but also in conifers.