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Leghaemoglobin within bacteroid-enclosing membrane envelopes from soybean root nodules
1Division of Plant Industry, CSIRO, P.O. Box 1600, 2601, Canberra City, A.C.T., Australia.
Planta
|December 5, 2013
Summary
Researchers isolated intact membrane envelopes from soybean root nodules containing bacteroids. This study confirms leghaemoglobin is located within these envelopes, not from the cytosol, with concentrations up to 523 μM.
Area of Science:
- Plant Molecular Biology
- Biochemistry
- Cell Biology
Background:
- Soybean (Glycine max (L.) Merr.) root nodules house nitrogen-fixing bacteria (bacteroids).
- Leghaemoglobin, a vital oxygen-binding protein, is abundant in these nodules, but its precise subcellular localization has been debated.
- Understanding leghaemoglobin's location is crucial for elucidating nitrogen fixation efficiency and nodule physiology.
Purpose of the Study:
- To develop and validate a method for isolating intact membrane envelopes containing bacteroids from soybean root nodules.
- To determine the subcellular localization of leghaemoglobin within these isolated nodule preparations.
- To quantify the concentration of leghaemoglobin within the identified subcellular compartment.
Main Methods:
- Isolation of intact membrane envelopes from soybean root nodules using low-speed centrifugation (approx. 150 g) at an optimal osmotic strength of 600 mOsm/kg H2O.
- Recovery of envelope contents via osmotic shock and/or high-speed centrifugation (>10,000 g).
- Spectrophotometric identification of leghaemoglobin, analysis of fluorescent materials, and immunochemical assays using (131)I-labelled leghaemoglobin and bovine serum albumin.
Main Results:
- Intact membrane envelopes, significantly less dense than bacteroids, were successfully isolated.
- Leghaemoglobin was detected within the envelopes and did not dissociate upon rupture, unlike cytosol-derived proteins.
- Estimated leghaemoglobin concentration within the envelopes ranged from 178-523 μM, representing approximately one-third of the total nodule leghaemoglobin.
Conclusions:
- The study provides strong evidence that leghaemoglobin is primarily located within the membrane envelope space surrounding bacteroids in soybean root nodules.
- This localization suggests a specific functional role for leghaemoglobin within the symbiosome, distinct from the general cytosol.
- Isoelectric focusing revealed all known soybean leghaemoglobin components plus a novel minor component within the isolated envelopes.
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