Abstract: Despite its importance in determining seed composition, and hence quality, regulation of the development of legume seeds is incompletely understood. Because of the cardinal role played by the nucleus in gene expression and regulation, we have characterized the nuclear proteome of Medicago truncatula at the 12 days after pollination (dap) stage that marks the switch towards seed filling. Nano-liquid chromatography-tandem mass spectrometry analysis of nuclear protein bands excised from one-dimensional SDS-PAGE identified 179 polypeptides (143 different proteins), providing an insight into the complexity and distinctive feature of the seed nuclear proteome and highlighting new plant nuclear proteins with possible roles in the biogenesis of ribosomal subunits (PESCADILLO-like) or nucleocytoplasmic trafficking (dynamin-like GTPase). The results revealed that nuclei of 12-dap seeds store a pool of ribosomal proteins in preparation for intense protein synthesis activity, occurring subsequently during seed filling. Diverse proteins of the molecular machinery leading to the synthesis of ribosomal subunits were identified along with proteins involved in transcriptional regulation, RNA processing or transport. Some had already been shown to play a role during the early stages of seed formation whereas for others the findings are novel (e.g. the DIP2 and ES43 transcriptional regulators or the RNA silencing-related ARGONAUTE proteins). This study also revealed the presence of chromatin-modifying enzymes and RNA interference proteins that have roles in RNA-directed DNA methylation and may be involved in modifying genome architecture and accessibility during seed filling and maturation.
Abstract: Plants possess a broad range of strategies to cope with cadmium (Cd) stress, including the arbuscular mycorrhizal (AM) symbiosis. In cell responses towards Cd, the contribution of changes in ploidy levels is still unclear. We used flow cytometry to investigate if nuclear ploidy changes are involved in response mechanisms toward Cd and to analyze the effect of the symbiotic status on populations of nuclei. The impact of Cd was investigated in roots of two pea (Pisum sativum L.) genotypes differing in their Cd-sensitivity (Cd-sensitive VIR4788 and Cd-tolerant VIR7128). In pea seedlings grown under hydropony, 25 and 250 microM Cd concentrations lead to an increase in 4 C together with a decrease in 2 C nuclei. The same genotypes, grown in soil/sand substrate, were inoculated or not with the AM fungus Glomus mosseae BEG12 and treated or not with Cd at transplanting (Cd1) or 2 weeks after (Cd2). The Cd2 increased the proportion of 6 and 8 C nuclei in the mycorrhizal VIR4788 and in the non-mycorrhizal VIR7128 genotypes. Thus, changes in ploidy levels reflect pea responses towards Cd, which are modulated by the symbiotic interaction. The Cd-induced increase in ploidy may account for changes in DNA transcription and/or translation.
Abstract: The effects of cadmium (Cd) administration on primary root growth, mitotic activity of apical meristems, mitotic aberrations and percentage of nucleus ploidy classes of differentiated roots were examined in Pisum sativum L. cv. Frisson. Cadmium caused a reduction of root length related to concentration, with an almost complete block of growth in plants treated with 250 μM Cd, from 24 h of treatment. Root lengthening is generally related to apical meristem activity, however, in the examined pea plants, mitotic activity was suppressed by 2.5 and 25 μM Cd treatment, while the highest Cd concentration, 250 μM, caused the occurrence of mitotic figures consisting almost exclusively of prophases. The lack of relation between root lengthening and mitotic activity was explained by the meristematic activity in the first period of treatment and by a different cell elongation. Lower (0.25, 0.5 and 1 μM), non-blocking Cd concentrations induced a number of mitotic aberrations, mainly consisting of sticky metaphases and anaphase bridges, whose frequency increased with Cd concentration. Besides, Cd induced variations of the percentages of nucleus populations in the differentiated roots, increasing the percentage of 4C nuclei and decreasing that of 2C. The mechanisms involved in the nuclear response to Cd, and the possible relations between Cd alteration of meristem cell activity and nuclear ploidy of differentiated cells are discussed.
Abstract: Arbuscular mycorrhiza (AM) can increase plant tolerance to heavy metals. A targeted proteomic approach was used to determine the putative identity of some of the proteins induced/modulated by cadmium (Cd) and to analyse the impact of the mycorrhizal process. • The effect of Cd (100 mg Cd kg<sup>-1</sup> substrate) applied either at planting or 15 d later on two pea (Pisum sativum) genotypes, differing in sensitivity to Cd inoculated or not with the AM fungus Glomus mosseae, was studied at three levels: plant biomass production, development of G. mosseae and root differential protein display with one- and two-dimensional gel electrophoresis (1-DE and 2-DE) analyses. • Cd-induced growth inhibition was significantly alleviated by mycorrhiza in the Cd-sensitive genotype. The AM symbiosis modulated the expression of several proteins, identified by liquid chromatography-tandem mass spectrometry, newly induced and upregulated or downregulated by Cd. • The protective effect of AM symbiosis towards Cd stress was observed in the Cd-sensitive genotype. Our results demonstrate the usefulness of proteomics to better understand the possible role of AM symbiosis in detoxification/response mechanisms towards Cd in pea plants.
Abstract: Nuclear changes in roots of tomato (Lycopersicon esculentum), a plant with a small genome, during the establishment of arbuscular mycorrhizal (AM) colonization were studied using light and electron microscopy, as well as flow and static cytometry. Nuclei of mycorrhizal root cortex cells were larger and had more decondensed chromatin than those of controls. Significant ploidy distribution differences were observed between nuclei of AM colonized and control roots, and a strong correlation between nuclear polyploidization and AM colonization was found. Polyploidization and decondensation are usually associated with high metabolic activity. The metabolic activity of mycorrhizal root cells, evaluated in this work as respiratory activity by using a cytochemical assay for succinate dehydrogenase combined with image analysis, increased in comparison to controls. The meaning of polyploidization is discussed in relation to the structural and metabolic modifications induced by mycorrhization.
