Carbohydrate metabolism in one- and two-year-old spruce needles, and stem carbohydrates from three months before until three months after bud break

Starch and sucrose metabolism of one- and two-year-old needles of Norway spruce (Picea abies [L.] Karst., about 30 years old) was investigated from three months before until three months after bud break at a natural site. We distinguish different metabolic states according to the extractable activities of enzymes (α-amylase [EC 3.2.1.1], ADP-glucose pyrophosphorylase [AGP, EC 2.7.7.27], D-enzyme [EC 2.4.1.25], starch phosphorylase [STP. EC 2.4.1.1]), sucrose phosphate synthase [SPS, EC 2.4.1.14], sucrose syntbase [SS, EC 2.4.1.13]. acid invertase [AI, EC 3.2.1.261) and pool sizes of related metabolites (starch, glucose, fructose, sucrose, raffinose, stachyose, fructose 6-phosphate [F6P], glucose 6-phosphate [G6P], fructose 2,6-bisphosphate [F26BP], and inorganic phosphate [P₁]). The period ending with bud break was characterized by high rates of net photosynthesis, a pronounced decrease in the amount of soluble sugars, and a steep rise in starch (from the detection limit to approximately 600 nmol glycosyl units [mg dry weight]_-1). In parallel, the extractable activity of AGP increased, while D-enzyme was on a relative high level when compared with the period after bud break. With respect to sucrose metabolism, F26BP, an inhibitor of sucrose synthesis, decreased from 1 to 0.4 pmol (mg dry weight)_-1. This was complemented by SPS activity, which was due to both increased protein levels shown by immunoblotting and activation under metabolite control (high levels of G6P and a low P_i/G6P ratio). This indicates a high capacity of synthesis of starch and sucrose in the period before bud break. These observations are in accordance with estimates of photosynthetic carbon gain, which indicate that in early spring large amounts of carbon from current photosynthesis are exported out of the needles. In addition, the content of nonstructural carbohydrates (expressed as hexoses) increased in the bark of the stem. This could also be a consequence of an enhanced carbon export from the needles. After the onset of bud break, starch concentration decreased in all tissues under investigation. In contrast, the level of total nonstructural carbohydrates in the outermost sapwood nearly doubled from bud break until the end of sampling. In the needles, net photosynthesis was reduced by about 75% and a decrease in SPS activity and protein level were found together with lower G6P concentration, and an increased P_i/G6P ratio. These results suggest that during that period sucrose synthesis was reduced in the older needles. In addition, under conditions of reduced photosynthesis, carbon demand of current year needles was in part ensured by the mobilization of starch in the older needles. Taken together our data show that before bud break carbon metabolism of mature leaves is related with the sink demands of storage organs. After bud break the accumulated assimilate pools in needles and stem, mainly the bark, are mobilized and support carbon supply to new tissues.     

Mycorrhiza formation on Norway spruce (Picea abies) roots affects the pathway of anaplerotic CO2 fixation

Activities of carboxylation enzymes were analyzed in the mycelium of the mycorrhizal fungus Amanita muscaria (L. ex Fr.) Hooker, in non-mycorrhizal short roots of Norway spruce ( Picea abies [L.] Karst.) and in myconhizas of these two partners. While pyruvale carboxylase (PC, EC 6.4.1.1) and phosphoenolpyruvate carboxykinase activities (PEPCK.EC 4.1.1.49) could be detected in the mycelium of A. muscaria , phosphoenolpyruvate carboxyknase (PEPC, EC 4.1.1.31) was only active in root tissue. In A. muscaria , PC activity was generally low (around 10 nmol mg^−tprotein min⁻) but PEPCK activity was above 250 nmol mg⁻¹ protein min⁻¹. Mycorrhizal development on short roots decreased PEPC activity by more than 75%, although dilution by the fungal biomass in mycorrhizas was only 35%. This reduction in activity was paralleled by a decreased content of PEPC protein. By means of micro-analytical methods it was shown that PEPC activity was lowest in the central zones of the mycorrhizas, Whereas PEPC activity was highest in the corresponding central sections in non-mycorrhizal short roots. ¹⁴CO₂ labelling, on the other hand, revealed that in vivo CO₂ fixation was higher in mycorrhizas compared to non-mycorrhizal short roots. It is concluded that fungal carboxylases (probably PEPCK) are important for anaplerotic CO₂ fixation during nitrogen assimilation in mycorrhizas of Norway spruce.     

Axenic mycorrhization of wild type and transgenic hybrid aspen expressing T-DNA indoleacetic acid-biosynthetic genes

 We describe and document the in vitro synthesis of ectomycorrhiza between roots of wild type and transgenic aspen (Populus tremula × P. tremuloides), expressing Agrobacterium tumefaciens T-DNA indoleacetic acid (IAA)-biosynthetic genes, and Amanita muscaria. Plantlets were raised from tissue culture. The root system of approximately 4-week-old plantlets was transferred to Petri dishes and incubated together with fungal mycelia under sterile conditions. Ectomycorrhiza showing both a well developed hyphal mantle and Hartig net were established within 3 to 4 weeks. Formation and morphology of ectomycorrhiza were not affected by the transformation of aspen, expressing the IAA biosynthetic genes in roots. As both hybrid aspen and fungal cells can be genetically engineered, this system offers a new approach to the study of mycorrhizal symbioses. Received: 19 January 1996 / Accepted: 23 January 1996     

The effect of acid irrigation on enzyme activities of the single partners of ectomycorrhizas from a limed stand of Norway spruce (Picea abies [L.] Karst.)

The aim of our investigations was to elucidate the effects of acid precipitation on some enzymes of the primary metabolism of ectomycorrhizas. Mycorrhizas of the type of Piceirhiza nigra Gronbach and of Russula ochroleuca (Pers.) Fr. and Tuber puberulum Berk. and Br. were collected from a stand of Norway spruce (Picea abies [L.] Karst.) during the growing seasons of 1991 and 1992. The experimental plots had been limed (Ca: 22 kmol ha_-1, Mg: 20 kmol ha_-1) in 1984 and exposed to acid irrigation (pH 2.7–2.8, H₂SO₄: 2 kmol ha_-1 a_-1) from 1984 to 1990. Crude extracts of mycorrhizas were assayed for the activities of glucose-6-phosphate dehydrogenase (G6P-DH, EC 1.1.1.49), 6-phosphogluconate dehydrogenase (EC 1.1.1.44), NADP-dependent isocitrate dehydrogenase (EC 1.1.1.42) and NAD-dependent glutamate dehydrogenase (EC 1.4.1.2). The influence of the experimental treatments on these enzyme activities of the primary metabolism was generally low. For P. nigra, the activity of G6P-DH was decreased on the irrigated plot (photometric determinations). This seems to be a selective effect on the fungal partner, since quantitative enzyme electrophoresis revealed a decrease in the percentage of the fungal enzyme activity in relation to the total enzyme activity, whereas the content of the fungal compound ergosterol was not affected. A decrease in the fungal G6P-DH activity could also be detected in mycorrhizas of Tuber puberulum. There was also a seasonal variation in the proportion of fungal activity of G6P-DH in relation to the total G6P-DH activity. In the photometric assay (total activity) the effect was not discernible. This is indicative of a degree of regulation between the two partners, which could only be detected by quantitative enzyme electrophoresis. In addition, it could be deduced from the electrophoretograms, that in the case of G6P-DH and 6PG-DH the fungal enzyme activity was dominating in all mycorrhizas studied whereas in the case of ICA-DH the fungal band varied from being conspicuous to absent in different species of mycorrhizas. The banding pattern of G6P-DH was reproducibly different for all investigated species of mycorrhizas.     

Effects of varied soil nitrogen supply on Norway spruce (Picea abies [L.] Karst.)

During a seven-month period the effect of different nitrogen (N) availability in soil on growth and nutrient uptake was studied in three-year-old Norway spruce (Picea abies [L.] Karst.) trees. The plants were grown in pots on N-poor forest soil supplied with various amounts and forms (inorganic and organic) of N. Increasing supply of inorganic N (as NH₄NO₃) increased the formation of new shoots and shoot dry weight. The root/shoot dry weight ratio of new growth was drastically decreased from 1.6 in plants without N supply to 0.5 in plants supplied with high levels of NH₄NO₃. This decrease in root/shoot dry weight ratio was associated with distinct changes in root morphology in favour of shorter and thicker roots. The addition of keratin as organic N source did neither affect growth nor root morphology of the trees. The amount of N taken up by plants was closely related to the supply of inorganic N, and trees supplied with highest levels of NH₄NO₃ also had the highest N contents in the dry matter of needles and roots. In contrast, N contents in needles of trees grown without additional N, or with keratin supply, were in the deficiency range. Supply of NH₄NO₃ decreased the contents of phosphate (P) and potassium (K) and therefore markedly increased N/P and N/K ratios in the needles. On the other hand, the contents of calcium (Ca), magnesium (Mg), and manganese (Mn) in the needles were increased in the plants supplied with inorganic N, suggesting high soil availability and promotion of uptake of these divalent cations by high nitrate uptake. The observed effects on root/shoot dry weight ratio, root morphology, and mineral nutrient composition of the needles indicated that high inorganic N supply may increase above-ground productivity but at the same time decrease the tolerance of trees against soil-borne (e.g. deficiency of other mineral nutrients) stress factors. Deceased 21 September 1996 Deceased 21 September 1996