A 31P nuclear magnetic resonance study of phosphate levels in roots of ectomycorrhizal and nonmycorrhizal plants of Castanea sativa Mill.

TL;DR: The level of orthophosphate in mycorrhizal roots was significantly lower than in nonmycorrhIZal ones, which indicates that this system relies upon the fungal polyphosphates as a major source of phosphate.

Abstract: 31P-Nuclear Magnetic Resonance (NMR) was used to assess phosphate distribution in ectomycorrhizal and nonmycorrhizal roots of Castanea sativa Mill. as well as in the mycorrhizal fungus Pisolithus tinctorius in order to gain insight into phosphate trafficking in these systems. The fungus P. tinctorius accumulated high levels of polyphosphates during the rapid phase of growth. Mycorrhizal and nonmycorrhizal roots accumulate orthophosphate. Only mycorrhizal roots presented polyphosphates. The content in polyphosphates increased along the 3 months of mycorrhiza formation. In mycorrhizal roots of plants cultured under axenic conditions, the orthophosphate pool decreased along the culture time. In nonmycorrhizal roots the decrease in the orthophosphate content was less pronounced. The level of orthophosphate in mycorrhizal roots was significantly lower than in nonmycorrhizal ones, which indicates that this system relies upon the fungal polyphosphates as a major source of phosphate.

Summary

Introduction

• The increase in phosphate concentration in mycorrhizal plants was also correlated with increased photosynthetic rates, as the phosphate level of the chloroplast influences photosynthetic rates (Osmond 1981; Coombs 1985; Foyer and Spencer 1986; Jakobsen 1991) .
• It was suggested that phosphate is translocated along hyphae as polyphosphate granules inside vacuoles (Cox et al. 1980 ).

Materials and methods

• Castanea sativa micropropagated plants were obtained as previously described (Martins et al. 1996) .
• The fungus Pisolithus tinctorius (Pers.) Coker and Couch isolate 289/Marx kindly supplied by Dr. Ingrid Kottke (Tübingen University) was maintained on a MMN solid medium (Marx 1969) .

Mycorrhizal synthesis

• Fungus was inoculated 3 weeks before plant transfer.
• In vitro mycorrhization of micropropagated plants was induced 4-5 weeks after plant rooting, by transfer of rooted plants to the petri dishes inoculated 3 weeks before with mycelium.
• A portion of the petri dishes with roots was covered with aluminum foil to prevent oxidation of root phenols by light.
• Control plants were transferred to petri dishes without fungus and maintained under the same conditions.

Sample preparation for NMR

• Samples of excised root systems of mycorrhizal or control plants as well as samples of pure cultures of the fungus were prepared for NMR analysis.
• Similar masses of material and similar packing were used in the different experiments.
• Two glass capillaries were fitted into the NMR tube, one positioned a few millimeters from the bottom (inlet of medium) and the other just above the cotton wool filter that was used to restrict the volume of the biological material (outlet of medium).
• The acquisition time of each spectrum was approximately 40 min.
• In all the experiments, the probe head temperature was kept at 25°C.

Phosphate quantification

• Extraction of total P from roots leaves and stems of mycorrhizal and nonmycorrhizal plants was performed according to Bowman (1988) .
• Phosphate quantification was made 30, 40, 60 and 90 days after mycorrhizal induction in vitro by the molybdenum blue method according to John (1970) .

Results and discussion

• 31 P-NMR spectra of the fungus samples, either in pure culture or extramatrical hyphae collected from petri dishes containing 1-month-old mycorrhizas, showed the presence of resonances due to the vacuolar orthophosphate pool and the polyphosphates pool (Fig. 1 ) as al-ready reported for P. tinctorius (Ashford et al. 1994 ) and for other fungi (Martin et al.
• When present in a P-limiting medium, the stored polyphosphates are hydrolyzed, releasing Pi and maintaining Pi concentration within the fungal cell (Martin et al. 1985; MacFall et al. 1992) .
• Chestnut mycorrhizas are able to accumulate phosphate, mostly in the form of polyphosphates, in contrast to nonmycorrhizal roots, which accumulate only orthophosphate (Fig. 2 ).
• The differences in phosphate content between mycorrhizal and nonmycorrhizal plants decreased with time.

Figures

Fig. 2 31P-NMR spectra of control (A) and mycorrhizal (B) chestnut roots at different mycorrhization times: (a) 3 weeks after mycorrhizal induction; (b) 1 month after mycorrhizal induction; (c) 3 months after mycorrhizal induction. Pi, intracellular orthophosphate; polyP, polyphosphates&/fig.c:

Fig. 1 31P-NMR spectra of the fungus Pisolithus tinctoriusgrown in axenic conditions with different ages: (a) 7 days, (b) 15 days and (c) 1.5 months. Pi, intracellular orthophosphate; tP, terminal phosphate; UDP-Hexose, uridine diphosphate hexose; PP, penultimate phosphate; polyP, polyphosphates&/fig.c:

Frequently asked questions

Q1. What are the contributions in this paper?

Martins et al. this paper found that mycorrhizal and nonmycizal roots accumulate orthophosphate, which indicates that this system relies upon the fungal polyphosphates as a major source of phosphate.