in vivo 31 P NMR Spectroscopy for the study of P Pools and their Dynamics in Arbuscular Mycorrhizal Fungi

Abstract

The main objective of the studies described in the present P1i.D. thesis was to investigate the phospbate (P) metabolism of arbuscular mycorrhizal (AM) fungi by in viv0 31P nuclear magnetic resonance (NMR) spectroscopy. P is an essential nutrient for all organisms. It is required in relatively large amounts and is often limiting to plant growth. The availability of P is limited by the slow rate of diffusion of inorganic orthopliospliate (Pi) through the soil. As plants remove Pi from the soil solution close to the root an area suii-ounding the root drained for soluble Pi may be formed (the P-depletion zone). AM fungi colonize the roots of most land plants and tbe symbiosis between AM fungi and plants is characterized by bi-directioiial nutrient transport; the AM fungus receiving an indispensable supply of fixed carbon (C) in return for improved inorganic nutrient (mainly P) uptake by the host plant. The extraradical mycelium of an AM fungus forms an extensive hyphal network and allows the plant to access Pi in tlie soil solution beyond the P-depletion zone. Once the association is established, the fungus takes up Pi from the soil through the extraradical mycelium in an active process like plants. However, Pi is accumulated also as polyphosphate (polyp). Polyp is translocated to the intraradical mycelium in vacuoles in a tubular streaming system. At the symbiotic interface inside the root, polyp is hydrolyzed and Pi is subsequently transferred to the plant root cells. Accordingly, polyp is considered to have an important role in tbe Pi trauslocation process. However, the amount, size and other roles of polyp present in .the extraradical and intraradical mycelium is a matter of debate. invasive methods have commonly been used to identify polyp and therefore artifacts of specirnen preparation could possibly have interfered with tbe polyp chain length. More detailed information of P pools and polyp accumulation would benefit from non-invasive and non-destructive measureinents of the dynamic incorporation of Pi into various P pools witliin extraradical mycelium and mycorrhizal roots. In viv0 31P NMR spectroscopy provides an analytical method for identifying and quantifying particular metabolites in liviiig tissue. Moreover, it allows for measuring iiitracellular pH, for probing the subcellular compartmentation of certain ions and for following the flux through metabolic pathways. Thus, in viv031 P NMR spectroscopy is a unique analytical method for the investigation of P pools and their dynamics in AM fungi. The plant chosen for the work was cucumber, in the majority of the work grown in symbiosis with the AM fungus Glomus intruradices in a compartmented growth system. Other species of AM fungi used included Scutellospora caloJpora, G. mosseae and Gigaspora rosea. The cucumber plants were grown in a central mesh-bag, which prevents root penetration but allow free passage of AM fungal hyphae. Tbe extraradical mycelium grew into sand surrounding the mesh-bag and could be collected from the sand, while root matenal could be collected from the mesh-bag. A circulation system was constructed for oxygenating the excised hyphae or roots while in the NMR tube. Both the efficiency of P, uptake and the turn-over of P metabolites by excised hyphae were investigated in order to clarify the metabolic status of excised fungus. Furthermore, an attempt was made to measure phospliatase activity in the extraradical mycelium and mycorrhizal roots using the enzyme-iabeled-fluorescence (ELF) method in order to localize aspects of P metabolism. Alkaline phosphatase activity was observed in all species af AM fungi used, which indicated metabolically active fungi. in this study, polyp of a shori chain length was seen in actively metabolizing extraradical AM fungal hyphae for the first time by the use of in viv0 "P NMR spectroscopy. Furthermore, a time-course "P NMR investigation of the formation af P pools in differently P-treated AM hyphae and mycorrhizal roots was peiformed. It was demonstrated tbat P, taken up by extraradical mycelium accumulated firstly into polyp and suhsequently into vacuolar P, within the extraradical mycelium. Furthermore, a time lag was observed befare auy P metabolites appeared in mycoii-hizal roots. The amount af polyp in extraradical mycelium was considerably higher tlian vacuolar P, and synthesis af polyp was therefore suggested to be important for effective P, uptake in AM fungi. The polyp was located in vacuoles and the measured average chain length was short, supporting a role for polyp iii translocation of P, from soil to host root by AM fungi. Cytoplasmic P, in tlie extraradical mycelium could not be detected by in viv0 "P NMR possibly because af a small cytoplasmic volume ar low concentratiou of cytoplasmic P,. The average polyp chain length was further cliaracterized by the use af extraction procedures and colorimetric measurements. Combiniiig the results obtained from these methods and NMR revealed small ainounts af lang-chain and graiiular polyp in the extraradical mycelium when supplied with high P amounts. Moreover, possible interfungal variation in P pools, polyp content and poiyP average chain length was investigated for the purpose af understanding the diversity in the ability of different species af AM fungi to supply P to the host plant. The results af this preliminary investigation suggested that there are differences between species of AM fungi in P pools sizes within extraradical mycelium and also in effectiveness of translocating the P to the root.