Gene Expression of Medicago sativa Inoculated with Sinorhizobium meliloti as Modulated by the Xenobiotics Cadmium and Fluoranthene

Abstract

Alfalfa plants (Medicago sativa cv. Europe) inoculated with Sinorhizobium meliloti 2011 (formerly Rhizobium meliloti, de Lajudie et al., 1994) were cultivated for 14 days under standardized growth conditions in mineral medium with addition of the heavy metal cadmium or the polycyclic aromatic hydrocarbon fluoranthene. These xenobiotics significantly reduced the numbers of root nodules before any visible damage to the plant could be detected. EC10. EC50, and EC90 (effective concentrations reducing nodulation, shoot and root fresh weight by 10, 50, or 90% compared to the control without pollutant) were calculated. EC50 for cadmium ranged from 5.8 jam (nodulation) to more than 20 μᴍ (root fresh weight). Testing fluoranthene resulted in an EC50 of 2.5 μg cm-2 for nodulation, and EC50 values of more than 35 μg cm-2 for shoot and root biomass production, indicating that the effect parameter nodulation is 10-fold more sensitive than shoot and root fresh weight. With m RNA differential display techniques the effects of both xenobiotics on gene expression in alfalfa root systems were studied. 37 differentially displayed transcripts were detected. Two of them, called DDMs1 and DDMs2, were confirmed by northern hybridization to be down-regulated in the presence of the xenobiotics. The expression of transcript DDMs1 was enhanced in alfalfa control plants inoculated with rhizobia, the transcript level was increased 2.5-3-fold compared to non-inoculated plants. This positive effect of nodulation was suppressed, partly by 35 μg cm-2 fluoranthene and totally by 20 μᴍ cadmium. The decrease in DDMsl transcription was highly affected by the cadmium concentration with an EC50 of 5.9 μᴍ . Compared to nodulation, almost identical EC10, EC50. and EC90 values were found for DDMsl expression. Sequence analysis of DDMsl revealed a significant overall homology (50% identity) to a hypothetical protein from Arabidopsis thaliana with high similarity to a copper transporting ATPase. High levels of transcript DDMs2 were observed in control plants with a 50% decrease in the xenobiotic-treated plants. DDM s2 gave a strong homology (82% identity) to the cytoplasmatic 60S ribosomal protein L18 from Arabidopsis thaliana.

Flavodoxin overexpression reduces cadmium-induced damage in alfalfa root nodules

“…Cd toxicity results in increased generation of ROS, and both ROS and antioxidants play a critical role in nodule formation (Pauly et al 2006). A reduction in the number of nodules in alfalfa plants subjected to Cd treatment has been previously observed (Neumann and Werner 2000). We did not detect any major differences in the number of nodules or in the kinetics of nodule formation between wt and Fld-expressing rhizobia.…”

“…The presence of Cd inhibits nitrogenase activity and photosynthesis, affecting the number of nodules and shoot, root, leaf and nodule biomass (Neumann and Werner 2000;Balestrasse et al 2003Balestrasse et al , 2005a and inducing nodule senescence (Balestrasse et al 2004). Cd is also toxic to the microsymbiont (Ibekwe and Angle 1996).…”

Carbon Cycles, Nitrogen Fixation and the Legume-Rhizobia Symbiosis as Soil Contaminant Biotest System

“…Using mRNA differential display techniques in the symbiotic test system affected by cadmium and fl uoranthene, 37 differentially displayed transcripts were detected. Two of them called DDMs1 and DDMs2 were confi rmed by northern hybridization, regulated by the presence of the two xenobiotics (Neumann and Werner, 2000). Transcription of DDMs1 was highly affected by the cadmium concentrations with an EC50 of 5.9 M. This is almost identical to the EC50 found for nodulation.…”

Differential display analysis of gene expression in plants

“…Several defense genes which are expressed in response to attacks by pathogens [103][104][105][106][107][108][109][110][111][112][113] and herbivores [114,115] were obtained. In plant-microbe interactions such as symbiosis, induced genes were isolated and characterized [116][117][118][119][120][121][122]. Information about the functions of these genes will provide agriculturally valuable leads.…”