Heme protein in tuberculate ectomycorrhizae on lodgepole pine.

PAUL, LESLIE¹, BILL CHAPMAN², CHRIS CHANWAY¹ & BRIAN HOLL⁴.

¹Department of Soil Science, University of British Columbia, Vancouver, B.C., CANADA. ²Research Department, B.C. Ministry of Forests, Williams Lake, B.C., CANADA. ³Department of Plant Science, University of British Columbia, Vancouver, B.C., CANADA.

Heme protein has been found within the tissue of tuberculate ectomycorrhizae (TEM) on lodgepole pine (Pinus contorta var. latifolia Engelm. ex S. Wats.). Acetylene reduction assays demonstrated that nitrogen fixation occurs in lodgepole pine TEM, and N₂-fixing bacteria have been isolated from within the tubercles. A pinkish cast to many of the TEM was reminiscent of nitrogen fixing nodules and caused us to look for haemoglobin (in spite of the fact that some of the fungi involved in forming these mycorrhizae are known to stain pink) Initially, the Drabkin's Reagent test was used to determine the presence of heme protein in homogenized TEM tissue. Samples weighing 0.5 g resulted in a range of total heme protein concentrations from 5.6 - 9.7 mg/dl (n=15). Cellulose acetate electrophoresis of extracts of TEM showed positive detection of protein (Ponceau S staining) and haem (o-dianisidine) components using human haemoglobin (1mg/ml) as a comparative standard.
This study is the first to report the occurrence of a heme protein associated with TEM or a tripartite structure (fungus/ plant/ bacteria) of this type. Since this study found nitrogen fixing bacteria within tubercles and since these bacteria fix nitrogen optimally under microaerophillic conditions, the presence of heme protein within the tubercle suggests that TEM are formed to regulate the amount of oxygen being supplied to symbiotic N₂-fixing bacteria and are a structure analogous to legume or Frankia nodules.