Localization Of Carbonic Anhydrase In Legume Nodules

C.A. Atkins \ P.M.C. Smith A.J. Mann P.P. Thumfort2

'Dept Botany, Univ. Western Australia, Crawley WA 6009 Australia

2Dept Biology, MIT, Cambridge, MA 02319, USA

The plant tissue fraction of nodules from a range of legume species shows significant rates of carbonic anhydrase (CA) activity compared to the supporting root. The partially purified native CA protein from bean (Phaseolus vulgaris L.) nodules differed from the leaf isozyme in size, and in sensitivity to inhibitors. This evidence led to the suggestion that the smaller isozyme in legumes was a nodule-specific form, associated in some functional way with nitrogen fixation (Atkins 1974). Separate extracts of the central infected zone (CZ) and the cortex of nodules from Lupinus angustifolius L., Vigna unguiculata L. [Walp], Pisum sativum L., Vicia faba L. and Medicago sativa L. as well as those of Phaseolus vulgaris L. both contained significant CA activities (Atkins et al. 2001). Immunoassay using antisera to a putative nodule CA (Mscal) cloned from M. sativa (Coba de la Pena et al. 1997) also indicated expression in both tissue types. Quantitative confocal microscopy using laser scanning imaging and a fluorescent CA-specific probe (5-dimethylaminonaphthalene-1-sulfonamide [DNSA]) localized expression to the infected cells in the CZ tissue and to a narrow band of 2-3 files of cells in the cortical tissue that corresponded to the inner cortex. In the infected cells the enzyme activity was evenly distributed in the cytosol but in the inner cortical cells was restricted to the periphery.

It is reasonable to suppose that in the infected cells CA will be coordinately expressed with PEPC in the cytosol, to ensure an adequate rate of CO2 hydration in a neutral or slightly acidic environment for C4 acid synthesis (cf. mesophyll cells of the C4-leaf; Burnell, Hatch 1988). Despite this apparently obvious role for CA, Raven and Newman (1994) have calculated that the uncatalyzed rate of CO2 hydration was adequate for organic acid synthesis in soybean nodules and other possibilities, such as the short term (transient) buffering of intracellular pH change or ion transport, should not be ignored.

With the realization that nitrogenase activity is regulated by the flux of O2, and that this may be varied as a result of reversible changes to diffusive resistance in the cortex (Hunt, Layzell 1993), there has been renewed interest in events in this tissue. While there is general acceptance that the mechanism probably involves a variable "aqueous barrier" to the diffusion of gases, and especially O2, the underlying processes that cause reversible changes to the water relations of these cells are yet to be described. However, a number of observations have led to the idea of a mechanism(s) closely related to that in stomatal guard cells relying on synthesis of malate through PEPC. An alternative role, unrelated to malate synthesis, is plausible. Convection, as well as diffusion, may contribute to the aeration of plant tissues faced with a significant aqueous barrier (Beckett et al. 1988).

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