Expression studies of superoxide dismutases in nodules and leaves of transgenic alfalfa reveal abundance of iron-containing isozymes, posttranslational regulation, and compensation of isozyme activities.

The composition of antioxidant enzymes, especially superoxide dismutase (SOD), was studied in one nontransgenic and three transgenic lines of nodulated alfalfa plants. Transgenic lines overproduced MnSOD in the mitochondria of nodules and leaves (line 1-10), MnSOD in the chloroplasts (line 4-6), and FeSOD in the chloroplasts (line 10-7). In nodules of line 10-7, the absence of transgene-encoded FeSOD activity was due to a lack of mRNA, whereas in nodules of line 4-6 the absence of transgene-encoded MnSOD activity was due to enzyme inactivation or degradation. Transgenic alfalfa showed a novel compensatory effect in the activities of MnSOD (mitochondrial) and FeSOD (plastidic) in the leaves, which was not caused by changes in the mRNA levels. These findings imply that SOD activity in plant tissues and organelles is regulated, at least partially, at the posttranslational level. All four lines had low CuZnSOD activities and an abundant FeSOD isozyme, especially in nodules, indicating that FeSOD performs important antioxidant functions other than the scavenging of superoxide radicals generated in photosynthesis. This was confirmed by the detection of FeSOD cDNAs and proteins in nodules of other legumes such as cowpea, pea, and soybean. The cDNA encoding alfalfa nodule FeSOD was characterized and the deduced protein found to contain a plastid transit peptide. A comparison of sequences and other properties reveals that there are two types of FeSODs in nodules.

Mesorhizobium loti increases root-specific expression of a calcium-binding protein homologue identified by promoter tagging in Lotus japonicus.

A promoter tagging program in the legume Lotus japonicus was initiated to identify plant genes involved in the nitrogen-fixing symbiosis between legumes and rhizobia. Seven transformed plant lines expressing the promoterless reporter gene uidA (beta-glucuronidase; GUS) specifically in roots and/or nodules were identified. Four of these expressed GUS in the roots only after inoculation with nodule-forming Mesorhizobium loti. In one line (T90), GUS activity was found in the root epidermis, including root hairs. During seedling growth, GUS expression gradually became focused in developing nodules and disappeared from root tissue. No GUS activity was detected when a non-nodulating mutant of M. loti was used to inoculate the plants. The T-DNA insertion in this plant line was located 1.3 kb upstream of a putative coding sequence with strong homology to calcium-binding proteins. Four motifs were identified, which were very similar to the "EF hands" in calmodulin-related proteins, each binding one Ca2+. We have named the gene LjCbp1 (calcium-binding protein). Northern (RNA) analyses showed that this gene is expressed specifically in roots of L. japonicus. Expression was reduced in roots inoculated with non-nodulating M. loti mutants and in progeny homozygous for the T-DNA insertion, suggesting a link between the T-DNA insertion and this gene.