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cgMT1 is a metallothionein (MT)-like gene that was isolated from a cDNA library of young nitrogen-fixing nodules resulting from the symbiotic interaction between Frankia spp. and the actinorhizal tree Casuarina glauca. cgMT1 is highly transcribed in the lateral roots and nitrogen-fixing cells of actinorhizal nodules; it encodes a class I type 1 MT. To obtain insight into the function of cgMT1, we studied factors regulating the expression of the MT promoter region (PcgMT1) using a β-glucuronidase (gus) fusion approach in transgenic plants of Arabidopsis thaliana. We found that copper, zinc, and cadmium ions had no significant effect on the regulation of PcgMT1-gus expression whereas wounding and H2O2 treatments led to an increase in reporter gene activity in transgenic leaves. Strong PcgMT1-gus expression also was observed when transgenic plants were inoculated with a virulent strain of the bacterial pathogen Xanthomonas campestris pv. campestris. Transgenic Arabidopsis plants expressing cgMT1 under the control of the constitutive 35S promoter were characterized by reduced accumulation of H2O2 when leaves were wounded and by increased susceptibility to the bacterial pathogen X. campestris. These results suggest that cgMT1 could play a role during the oxidative response linked to biotic and abiotic stresses.

cg12 is an early actinorhizal nodulin gene from Casuarina glauca encoding a subtilisin-like serine protease. Using transgenic Casuarinaceae plants carrying cg12-gus and cg12-gfp fusions, we have studied the expression pattern conferred by the cg12 promoter region after inoculation with Frankia. cg12 was found to be expressed in root hairs and in root and nodule cortical cells containing Frankia infection threads. cg12 expression was also monitored after inoculation with ineffective Frankia strains, during my-corrhizae formation, and after diverse hormonal treatments. None of these treatments was able to induce its expression, therefore suggesting that cg12 expression is linked to plant cell infection by Frankia strains. Possible roles of cg12 in actinorhizal symbiosis are discussed.

Recent phylogenetic studies have implied that all plants able to enter root nodule symbioses with nitrogen-fixing bacteria go back to a common ancestor (D. E. Soltis, P. S. Soltis, D. R. Morgan, S. M. Swensen, B. C. Mullin, J. M. Dowd, and P. G. Martin, Proc. Natl. Acad. Sci. USA, 92:2647–2651, 1995). However, nodules formed by plants from different groups are distinct in nodule organogenesis and structure. In most groups, nodule organogenesis involves the induction of cortical cell divisions. In legumes these divisions lead to the formation of a nodule primordium, while in non-legumes they lead to the formation of a so-called prenodule consisting of infected and un-infected cells. Nodule primordium formation does not involve prenodule cells, and the function of prenodules is not known. Here, we examine the differentiation of actinorhizal prenodule cells in comparison to nodule cells with regard to both symbionts. Our findings indicate that prenodules represent primitive symbiotic organs whose cell types display the same characteristics as their nodule counterparts. The results are discussed in the context of the evolution of root nodule symbioses.

In search of plant genes expressed during early interactions between Casuarina glauca and Frankia, we have isolated and characterized a C. glauca gene that has strong homology to subtilisin-like protease gene families of several plants including the actinorhizal nodulin gene ag12 of another actinorhizal plant, Alnus glutinosa. Based on the expression pattern of cg12 in the course of nodule development, it represents an early actinorhizal nodulin gene. Our results suggest that subtilisin-like proteases may be a common element in the process of infection of plant cells by Frankia in both Betulaceae (Alnus glutinosa) and Casuarinaceae (Casuarina glauca) symbioses.

The purpose of this study was to compare the control of expression of legume and nonlegume hemoglobin genes. We used the Casuarina glauca and Allocasuarina verticillata transformation system to examine the properties of the soybean (lbc3), Parasponia andersonii, and Trema tomentosa hemoglobin gene promoters in actinorhizal plants. Expression of the hemoglobin promoters gus genes was examined by fluorometric and histochemical assays. The fluorometric assays in various organs showed that the soybean and P. andersonii promoters were most active in nodules whereas the T. tomentosa promoter gave a very high activity in roots. The histochemical study showed that GUS activity directed by the soybean and the P. andersonii gus chimeric genes appeared mainly confined to the infected cells of the C. glauca and A. verticillata nodules. The T. tomentosa hemoglobin promoter was primarily expressed in the root's cortex and vascular tissue. The results indicate that the soybean, P. andersonii, and T. tomentosa hemoglobin promoters retain their cell-specific expression in transgenic members of the Casuarinaceae, suggesting a close relationship between legume, Ulmaceae member, and actinorhizal hemoglobin genes. The conservation of the mechanism for nodule-specific expression of soybean, P. andersonii, and C. glauca and A. verticillata hemoglobin genes is discussed in view of recent molecular phylogenetic data that suggest a single origin for the predisposition to form root nodule symbioses.