In order to study the molecular response of plants to heavy metal stress, an experimental system using the model cereal plant barley (Hordeum vulgare L.) was performed. The reaction of barley seedlings to heavy metal stress was analysed by the two photosynthesis related stress parameters: chlorophyll content and PSII efficiency. By comparing cDNA populations derived from chromium-stressed primary leaves of barley (Hordeum vulgare L.) with controls using restriction fragment differential display-PCR, 48 differentially expressed cDNA fragments could be identified. On the basis of sequence analyses and expression studies two of them, representing novel heavy metal induced genes from barley with putative regulatory functions; C2 domain protein and LysM receptor like kinase 1 were investigated in more detail in this study. The deduced amino acid sequence of one of these cDNAs [named 'C2 domain 1' (HvC2d1)] exhibits a motif that is similar to the known C2 domain and a nuclear localization signal (NLS). Expression of this member of a novel class of plant C2 domain-like proteins was studied using quantitative real-time PCR. For the first time, the results obtained in this study show that, HvC2d1 is induced after exposure to different heavy metals treatments. Its mRNA accumulates also during the phase of leaf senescence. In further experiments, using overexpressed and purified HvC2d1 protein, the binding of calcium to the C2 domain protein could be confirmed biochemically. Using chimeric HvC2d1-GFP, protein localization at the plasma membrane, cytoplasm and the nucleus could be shown in onion epidermal cells. Interestingly, after addition of calcium ionophore A23187 the green fluorescence was only visible in the nucleus. These data suggest a calcium dependent translocation of HvC2d1 to the nucleus. A second gene encodes for a LysM receptor-like protein kinase, was also identified in this study. The expression of HvLysMR1 is transiently induced during exposure to high concentration and is also induced during exposure to low concentrations of different heavy metals (Cr, Cu and Cd). During senescence, HvLysMR1 transcript accumulates. In vitro phosphorylation of HvLysMR1 could be proven with radioactive 32P-ATP. Using overexpressed and purified HvLysMR1-kinase domain. The phosphorylation of HvLysMR1 could also be confirmed by nano-liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) at amino acids localised at the juxtamembrane region. For the first time, the data obtained in this study suggest a possible role of HvC2d1, HvLysMR1 in heavy metal stress- and development-dependent signalling indicating overlapping regulatory pathways during heavy metal stress response and leaf senescence. Further experiments are needed to elucidate the functions of each protein in the signalling processes. |