Christoph Schaeffer

Effects of varied soil nitrogen supply on Norway spruce (Picea abies [L.] Karst.). II. Carbon metabolism in needles and mycorrhizal roots.

The response of carbohydrate metabolism in 3-year-old Norway spruce plants to an increased amount of nitrogen supply to a N-poor forest soil was investigated in a pot experiment. After 7 months of treatment we found a decreased amount of starch in both needles and roots, together with decreased amounts of sucrose in needles of those plants grown under an enhanced inorganic N supply. In addition, the activity and the protein amount of the anaplerotic enzyme phosphoenolpyruvate carboxylase (PEPC) and the activity of NADP-dependent isocitrate dehydrogenase (IDH) were clearly increased. The activity of sucrose phosphate synthase (SPS) and the pool size of fructose 2,6-bisphosphate (F26BP) were not affected by high supply of inorganic N. These data indicate a shift of carbon flow from starch formation towards an enhanced provision of carbon skeletons for N assimilation and shoot growth. In parallel, we found decreased contents of fungus-specific compounds (ergosterol, mannitol, trehalose) in roots, which are indicators of a decreased colonization by ectomycorrhizal fungi, probably as a result of a changed allocation and partitioning of photoassimilates due to an increased N supply.

Mycorrhiza — Carbohydrate and Energy Metabolism

The roots of most trees and shrubs and many herbaceous plants are invaded by mycorrhizal fungi. The most common of these symbiotic associations in trees of temperate forests are ectotrophic. In this type, hyphae extending from a mycelial layer covering the surface of fine roots penetrate between root cells and form characteristic structures (called Hartig net) in the outer cortex. Some trees and many terrestrial flowering plants develop arbuscular mycorrhizas (AM). Estimates are that up to 90% of angiosperms establish this kind of symbiosis (e.g. Law 1985). Here, no superficial mycelial layers nor specific morphological extracellular structures such as the Hartig net are formed. Because of the different kinds of intrusions into the host cells formed by fungal membranes, this type is also called vesicular-arbuscular mycorrhiza (VAM). The development of these symbiotic structures is a complex process which depends on a variety of internal and external factors (e.g. Harley and Smith 1983) of which mineral nutrition and the supply of carbohydrates by the host plant are most important. On the background of the excellent review on mycorrhizal properties published by Harley and Smith in 1983, we focus on literature appearing since then and try to evaluate former conclusions on the basis of recent data. The reader is also referred to other reviews on carbon metabolism in mycorrhizas (Cooper 1984; Lewis 1986; Harris and Paul 1987; Martin et al. 1987; Jakobsen 1991; Schwab et al. 1991; Finlay and Soderstrom 1992; Smith and Read 1996).

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

Abstract 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.

Kinetic and electrophoretic characterization of NADP dependent dehydrogenases from root tissues of Norway spruce [Picea abies (L.) Karst.] employing a rapid one-step extraction procedure

SummaryThe NADPH generating enzymes glucose-6-phosphate dehydrogenase (EC 1.1.1.49), 6-phosphogluconate dehydrogenase (EC 1.1.1.44), and isocitrate-dehydrogenase (NADP dependent; EC 1.1.1.42) have been characterized in spruce [Picea abies (L.) Karst.] roots. Interference from inherent phenolic compounds was minimized by complexation with borate and insoluble polyvinylpyrrolidone in the presence of 2-mercaptoethanol and NADP. A further addition of protective substances had no (bovine serum albumin) or even an inhibitory effect (ascorbate) on the enzyme activities. The enzymes were shown to be strictly NADP specific. The optimal pH values and the apparent Michaelis constants from spruce roots are in good agreement with data from different photosynthetic organisms and gametophytic tissues of conifers. Native electrophoresis and subsequent activity staining showed the same banding patterns for enzymes both from root and needle tissues. In addition, the applicability of a highly sensitive dot-blot assay for the accurate quantification of the extracted protein is shown.

Application of Methods of Quantitative Histochemistry on Mycorrhizal Roots

Knowledge of metabolic interaction in symbiotic root/fungus associations such as ectomycorrhizas is still rather limited because of the small dimensions of the structures formed (less than 0.5 mm diameter, only up to 2 mm in length, dry weight in the μg range). Specific areas of symbiotic interaction along such a mycorrhiza as well as distinguished radial sections such as Hartig net area, cortex, or bundle tissue are obliterated by tissue homogenization which precedes most analytical procedures. It has thus been a challenge to biologists to develop methods that allow for selective sampling of specific cell types, or at least tissue samples, and for biochemical analysis of the resultant small amounts of material. Most important in this context is the conservation of the metabolic state of the intact organ. Ideally, biochemical analysis would be noninvasive, but this is only possible by indirect methods such as NMR spectroscopy and reasonably large sample sizes. Alternatively, histochemical approaches include various implementations of chemical fixation, embedding, or freeze stop. Of these, the latter has the widest utility and will thus form the first step of our description. For details of procedures we do not cover completely, the reader is referred to A Flexible System of Enzymatic Analysis (Lowry and Passonneau 1972; see also new edition 1993) and to a short review with regards to application of these technique on plant tissues (Hampp et al. 1990).