Our comparative studies on the promoter (pr) activity of Enod40 in the model legume Lotus japonicus in stably transformed GusA reporter lines and in hairy roots of L. japonicus demonstrate a... Show moreOur comparative studies on the promoter (pr) activity of Enod40 in the model legume Lotus japonicus in stably transformed GusA reporter lines and in hairy roots of L. japonicus demonstrate a stringent regulation of the Enod40 promoter in the root cortex and root hairs in response to Nod factors. Interestingly, the L. japonicus Enod40-2 promoter fragment also shows symbiotic activity in the reverse orientation. Deletion analyses of the Glycine max (Gm) Enod40 promoter revealed the presence of a minimal region -185 bp upstream of the transcription start. Stable transgenic L. japonicus reporter lines were used in bioassays to test the effect of different compounds on early symbiotic signaling. The responses of prGmEnod40 reporter lines were compared with the responses of L. japonicus (Lj) reporter lines based on the LjNin promoter. Both reporter lines show very early activity postinoculation in root hairs of the responsive zone of the root and later in the dividing cells of nodule primordia. The LjNin promoter was found to be more responsive than the GmEnod40 promoter to Nod factors and related compounds. The use of prGmEnod40 reporter lines to analyze the effect of nodulin genes on the GmEnod40 promoter activity indicates that LJNIN has a positive effect on the regulation of the Enod40 promoter, whereas the latter is not influenced by ectopic overexpression of its own gene product. In addition to pointing to a difference in the regulation of the two nodulin genes Enod40 and Nin during early time points of symbiosis, the bioassays revealed a difference in the response to the synthetic cytokinin 6-ben-zylaminopurine (BAP) between alfalfa and clover and L. japonicus. In alfalfa and clover, Enod40 expression was induced upon BAP treatment, whereas this seems not to be the case in L japonicus; these results correlate with effects at the cellular level because BAP can induce pseudonodules in alfalfa and clover but not in L. japonicus. In conclusion, we demonstrate the applicability of the described L. japonicus reporter lines in analyses of the specificity of compounds related to nodulation as well as for the dissection of the interplay between different nodulin genes. Show less
We have isolated and characterized a Lotus japonicus gene (Ljsbp) encoding a putative polypeptide with striking homology to the mammalian 56-kDa selenium-binding protein (SBP). cDNA clones... Show moreWe have isolated and characterized a Lotus japonicus gene (Ljsbp) encoding a putative polypeptide with striking homology to the mammalian 56-kDa selenium-binding protein (SBP). cDNA clones homologous to LjSBP were also isolated from soybean, Medicago sativa, and Arabidopsis thaliana. Comparative expression studies in L japonicus and A. thaliana showed that sbp transcripts are present in various tissues and at different levels. Especially in L. japonicus nodules and seedpods and A. thaliana siliques, sbp expression appears to be developmentally up-regulated. sbp Gene transcripts were localized by in situ hybridization in the infected cells and vascular bundles of young nodules, while in mature nodules, low levels of expression were only detected in the parenchymatous cells. Expression of sbp transcripts in young seedpods and siliques was clearly visible in vascular tissues and embryos, while in embryos, low levels of expression were detected in the root epidermis and the vascular bundles. Polyclonal antibodies raised against a truncated LjSBP recombinant protein recognized a polypeptide of about 60 kDa in nodule extracts. Immunohistochemical experiments showed that accumulation of LjSBP occurred in root hairs, in the root epidermis above the nodule primordium, in the phloem of the vasculature, and abundantly in the infected cells of young nodules. Irrespective of the presence of rhizobia, expression of SBP was also observed in root tips, where it was confined in the root epidermis and protophloem cells. We hypothesize that LjSBP may have more than one physiological role and can be implicated in controlling the oxidation/reduction status of target proteins, in vesicular Golgi transport, or both. Show less
