SUMMARYThe number of root nodules developing on legume roots after rhizobial infection is controlled by the plant shoot through autoregulation and mutational inactivation of this mechanism leads to hypernodulation. We have characterised the Pisum sativum (pea) Sym28 locus involved in autoregulation and shown that it encodes a protein similar to the Arabidopsis CLAVATA2 (CLV2) protein. Inactivation of the PsClv2 gene in four independent sym28 mutant alleles, carrying premature stop codons, results in hypernodulation of the root and changes to the shoot architecture. In the reproductive phase sym28 shoots develops additional flowers, the stem fasciates, and the normal phyllotaxis is perturbed. Mutational substitution of an amino acid in one leucine rich repeat of the corresponding Lotus japonicus LjCLV2 protein results in increased nodulation. Similarly, down-regulation of the Lotus Clv2 gene by RNAi mediated reduction of the transcript level also resulted in increased nodulation. Gene expression analysis of LjClv2 and Lotus hypernodulation aberrant root formation Har1 (previously shown to regulate nodule numbers) indicated they have overlapping organ expression patterns. However, we were unable to demonstrate a direct protein-protein interaction between LjCLV2 and LjHAR1 proteins in contrast to the situation between equivalent proteins in Arabidopsis. LjHAR1 was localised to the plasma membrane using a YFP fusion whereas LjCLV2-YFP localised to the endoplasmic reticulum when transiently expressed in Nicotiana benthamiana leaves. This finding is the most likely explanation for the lack of interaction between these two proteins.

A novel hypernodulation mutant line was isolated from Lotus japonicus Miyakojima MG-20 by irradiation with a helium ion beam. This mutant, named klavier (klv), had roots that were densely covered with small nodules. The nodulation zone of klv was significantly wider than that of the wild type. Grafting experiments showed that klv is impaired in the long-distance shoot-to-root autoregulatory mechanism. Thus the shoot genotype was found to be responsible for the negative regulation of nodule development by KLV. Nodulation of klv showed a higher tolerance to nitrogen (KNO 3 ) than the wild type, which is a common feature of hypernodulating mutants. In addition to an increased number of nodules, the klv mutant showed convex leaf veins on the adaxial leaf surface, markedly delayed flowering and dwarf phenotypes. Microscopic examination of the leaf veins revealed that they were discontinuous. Other phenotypes such as fasciated stems, increased number of flowers and bifurcated pistils were also frequently observed in the klv mutant. Among these phenotypes, hypernodulation, aberrant leaf vein formation and significantly delayed flowering were all linked in a monogenic and recessive manner, indicating that these phenotypes are caused by either a single mutation, or tightly linked mutations. KLV was mapped within 0.29 cM on the long arm of chromosome 1.

In central Europe, soybean cultivation is gaining increasing importance to reduce protein imports from overseas and make cropping systems more sustainable. In the field, despite the inoculation of soybean with commercial rhizobia, its nodulation is low. In many parts of Europe, limited information is currently available on the genetic diversity of rhizobia and, thus, biological resources for selecting high nitrogen-fixing rhizobia are inadequate. These resources are urgently needed to improve soybean production in central Europe. The objective of the present study was to identify strains that have the potential to increase nitrogen fixation by and the yield of soybean in German soils. We isolated and characterized 77 soybean rhizobia from 18 different sampling sites. Based on a multilocus sequence analysis (MLSA), 71% of isolates were identified as Bradyrhizobium and 29% as Rhizobium. A comparative analysis of the nodD and nifH genes showed no significant differences, which indicated that the soybean rhizobia symbiotic genes in the present study belong to only one type. One isolate, GMF14 which was tolerant of a low temperature (4°C), exhibited higher nitrogen fixation in root nodules and a greater plant biomass than USDA 110 under cold conditions. These results strongly suggest that some indigenous rhizobia enhance biological nitrogen fixation and soybean yield due to their adaption to local conditions.

The reduction or absence of TCR ζ-chain (ζ) expression in systemic lupus erythematosus (SLE) patients is thought to be related to the pathogenesis of SLE. Recently, we reported the predominant expression of ζ mRNA containing an alternatively spliced 3′-untranslated region (3′UTR; ζmRNA/as-3′UTR) and a reduction in the expression of ζ mRNA containing the wild-type 3′UTR (ζmRNA/w-3′UTR) in T cells from SLE patients. Here we show that AS3′UTR mutants (MA5.8 cells deficient in ζ protein that have been transfected with ζmRNA/as-3′UTR) exhibit a reduction in the expression of TCR/CD3 complex and ζ protein on their cell surface as well as a reduction in the production of IL-2 after stimulation with anti-CD3 Ab compared with that in wild-type 3′UTR mutants (MA5.8 cells transfected with ζmRNA/w-3′UTR). Furthermore, the real-time PCR analyses demonstrated that the half-life of ζmRNA/as-3′UTR in AS3′UTR mutants (3 h) was much shorter than that of ζmRNA/w-3′UTR in wild-type 3′UTR mutants (15 h). Thus, the lower stability of ζmRNA/as-3′UTR, which is predominant in SLE T cells, may be responsible for the reduced expression of the TCR/CD3 complex, including ζ protein, in SLE T cells.