Malate uptake depends on specific transporters whose activities are essential for the initial increase in turgor. reversible hydration of CO2 to form HCO3?. Most reports on plant CAs are concerned with the enzyme from green tissues, but despite its abundance, the physiological role of this protein is still poorly understood (Sltemeyer et al., 1993; Badger and Price, 1994). In leaves two CA isoforms were localized to different subcellular compartments, the chloroplasts and the cytosol (Atkins et al., 1972; Fett and Coleman, 1994; Rumeau et al., 1996). In leaves of C3 plants most of the CA activity resides within the chloroplast stroma. Thus it has been proposed that this enzyme accelerates the dehydration of Rabbit polyclonal to KCTD1 bicarbonate to CO2, providing a constant CO2 supply for Rubisco activity during photosynthesis (Badger and Price, 1994; Majeau and Coleman, 1994). In C4 leaves, however, CA is largely confined to the cytosol of mesophyll cells. The cytosolic enzyme phosphogene was detected in the nodule IC in nitrogen-fixing and ineffective nodules (Coba de la Pe?a et al., 1997), the function of is not likely to be related to nitrogen fixation or malate supply to the bacteroids. To assess the role of in legume nodules, the present study characterized the localization of two forms of CA in determinate and indeterminate nodules, from and isoform in the nodule IC. The results of these experiments have led to proposals for the physiological role of in legume nodules. RESULTS Mature Nodules Contain at Least Two Forms of CA Located in Different Cell Types We have previously identified protein purified from a bacterial culture was used to raise polyclonal antibodies in rabbits. The specificity of these antibodies was tested by western-blot analysis of total soluble protein extracts from roots, nodules, and leaves (Fig. ?(Fig.1A).1A). In nodule extracts, these antibodies strongly recognized a single protein band with a molecular mass of 28.5 kD, very close to that expected for the putative protein extracts. Proteins were separated by SDS-PAGE, electrotransferred onto nitrocellulose, and incubated with antibodies raised against either the recombinant protein (A) or the potato leaf CA (B). Forty micrograms of total soluble proteins from roots (R), mature nodules (N), and leaves (L) were loaded per lane. Arrows indicate the major protein bands recognized by the antibodies. Antibodies raised against a synthetic peptide corresponding to the N-terminal amino acid sequence of the potato leaf chloroplastic CA (kindly provided by G. Peltier) were also tested in western blots. These antibodies cross-reacted also with the cytosolic potato leaf isoform Afzelin (Rumeau et al., 1996). However, this peptide region is not conserved in the band. In leaf extracts, two bands were revealed (the one having a very low molecular mass is probably a degradation product), but not the 39.4-kD isoform that is related to antibodies acknowledged the top and lower bands, respectively, whereas the second option antibody did not recognize any active band in the leaf extracts (Fig. ?(Fig.2B).2B). On the other hand, the recombinant protein was not identified by the leaf antibodies (Fig. ?(Fig.2C).2C). Hence in mature nodules, there are at least two forms of CA: the first is identified by the anti-antibodies (cells expressing recombinant (28.5 kD) were probed with anti-leaf CA and anti-antibodies, remaining and right, respectively. We next tackled where these different isoforms are located in nitrogen-fixing nodules (Fig. ?(Fig.3).3). We carried out an immunolocalization analysis on adult nodules with either the pre-immune serum or the anti-protein antibodies (Fig. ?(Fig.3,3, ACC). A secondary antibody linked to the Cy3 fluo-rophore Afzelin was utilized for exposing the immunological transmission. The antibodies strongly labeled a thin region in the periphery of the nodule equivalent to the IC, whereas no significant signal was recognized in the nitrogen-fixing central region (Fig. ?(Fig.3C).3C). This binding of anti-antibodies was clogged by preincubation with the recombinant protein (data not demonstrated). Moreover, in determinate nodules of proteins are specifically located in the inner cortical cells in indeterminate and determinate nodules. A similar immunolocalization experiment was carried out using the anti-potato leaf CA antibodies (Fig. ?(Fig.3,3, DCF, I). In Afzelin (ACF) and (GCI) nodules with either the pre-immune serum (A, E, and G), the anti-antibodies (B, C, and H), or the anti-leaf CA antibodies (F and I), detection was based on Cy3 fluorescence. A, Pre-immune serum control section indicating the absence of specific labeling in the IC in paraffin-embedded nodules. B, A section related.