The Sequence of Appearance of Leghaemoglobin and Nitrogenase Components I and II in Root Nodules of Pisum sativum

Abstract

~~~ ~ ~~The sequence of appearance of nitrogenase components I and TI and leghaemoglobin in root nodules of pea plants inoculated with Rhizobium Zeguminosarum (strain PRE) was studied using radioimmunoassays. Leghaemoglobin was detected before nitrogenase activity. The MoFe component (I) of nitrogenase was detected at the same time as leghaemoglobin, while the Fe component (11) of nitrogenase and nitrogenase activity appeared 1 and 2 d later, respectively. The same order of appearance of nitrogenase activity and leghaemoglobin was found in root nodules of cowpea plants inoculated with Rhizobium sp. 32H 1. I N T R O D U C T I O NIn an earlier paper (Bisseling et al., 1979) we demonstrated that leghaemoglobin in pea root nodules induced by Rhizobium Zeguminosarzm was synthesized before nitrogenase activity could be measured. We also found that in the early phase of the development of nitrogen-fixing activity, the synthesis of the MoFe component (CI) of nitrogenase exceeded the synthesis of the Fe component (CII) (Bisseling et al., 1979). In this paper we report further experiments on the order of synthesis of leghaemoglobin and CI and CII of nitrogenase, which are three essential components for nitrogen fixation in pea-rhizobium symbiosis. A radioimmunoassay has been developed to quantify leghaemoglobin and C1 and CIJ. This method is a few orders of magnitude more sensitive than the Mancini immunodiffusion assay (Mancini et al., 1965), which we used previously for the determination of leghaemoglobin; it has allowed us to extend the measurements towards the beginning of nodule development and to confirm that leghaemoglobin is present before nitrogenase activity is measurable. Moreover, we have now found that CI is synthesized before the presence of CII can be demonstrated in bacteroid extracts.Fast-and slow-growing rhizobia differ in many respects (e.g. Paau, 1978). To investigate whether slow-growing rhizobium bacteroids differ from the fast-growing rhizobia (R.legurninusarum) in the order of appearance of nitrogenase activity and leghaemoglobin synthesis, we have studied the appearance of leghaemoglobin and nitrogenase activity in root nodules of cowpea plants inoculated with Rhizobium sp. 32Hl. The results were similar to those obtained with the pea root nodules. METHODS Growth of plants.Pea plants (Pisum sativunz var. Rondo) were cultured and nodulated with Rhizobium ieguminosarum (strain PRE), as described earlier (Bisseling et al., 1978), at 19 to 20 "C with an 8 h dark and 16 h light period. Rhizobium leguminosarum was cultured in a yeast/mannitol medium (Bisseling et al., 1978). Vigna unguiculata T . B I S S E L I N G A N D O T H E R SAssay of nitrogenase. Nitrogenase activity of intact nodules was measured by the acetylene reduction method on pieces of the main root carrying the root nodules. Ethylene concentrations were measured in a Pye 104 gas chromatograph with a Porapak R column (Bisseling et al., 1978).Preparation of bacteroid and plant proteins. The isolation of bacteroids and the preparation of s...

cDNA cloning and developmental expression of pea nodulin genes

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Nodule-Specific Polypeptides from Effective Alfalfa Root Nodules and from Ineffective Nodules Lacking Nitrogenase

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Identification and characterization of a novel Bradyrhizobium japonicum gene involved in host-specific nitrogen fixation

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