SummaryWe have focused our interest on two cis-regulatory elements, named site II motif and telo box, identi®ed within the promoter of plant proliferating cellular nuclear antigen (PCNA) and putatively involved in meristematic expression of the gene. A conserved topological association between site II motifs and telo boxes is observed in the promoter of numerous genes expressed in cycling cells, including several cell cyclerelated genes and 153 Arabidopsis genes encoding ribosomal proteins. Meristematic expression of a GUS reporter gene was observed in plants under the control of Arabidopsis site II motif within a minimal promoter. This expression is strongly enhanced by addition of a telo box within this chimaeric promoter. We showed by gel retardation experiments that the site II motif is a target for several DNA-binding activities present in Arabidopsis crude cell extract and can bind a transcription factor, At-TCP20, from the Teosinte branched 1, Cycloidea, PCF (TCP)-domain protein family. In yeast two-hybrid experiments, At-TCP20 appears to be a potential partner of AtPura, which was previously shown to bind telo boxes. An important consequence of this analysis is to reveal new and conserved regulatory processes concerning the regulation of plant ribosomal gene expression in cycling cells. The implication of these observations in plant-speci®c developmental pathways is discussed.

Cadmium is suspected to exert its toxic action on cells through oxidative damage. However, the transition metal is unable to directly generate reactive oxygen species (ROS) via redox reactions with molecular oxygen in a biological environment. Here, we show that bright yellow-2 (BY-2) tobacco cells exposed to millimolar concentrations of CdCl 2 developed cell death within 2-3 h. The death process was preceded by two successive waves of ROS differing in their nature and subcellular localization. Firstly, these consisted in the transient NADPH oxidase-dependent accumulation of H 2 O 2 followed by the accumulation of O 2 -. in mitochondria. A third wave of ROS consisting in fatty acid hydroperoxide accumulation was concomitant with cell death. Accumulation of H 2 O 2 was preceded by an increase in cytosolic free calcium concentration originating from internal pools that was essential to activate the NADPH oxidase. The cell line gp3, impaired in NADPH oxidase activity, and that was unable to accumulate H 2 O 2 in response to Cd 2 + , was nevertheless poisoned by the metal. Therefore, this first wave of ROS was not sufficient to trigger all the cadmiumdependent deleterious effects. However, we show that the accumulation of O 2 -. of mitochondrial origin and membrane peroxidation are key players in Cd 2 + -induced cell death.

Cell division often occurs at specific times of the day in animal and photosynthetic organisms. Studies in unicellular photosynthetic algae, such as Chlamydomonas or Euglena, have shown that the photoperiodic control of cell division is mediated through the circadian clock. However, the underlying mechanisms remain unknown. We have studied the molecular basis of light-dependent control of cell division in the unicellular green alga Ostreococcus. We found that cell division obeys a circadian oscillator in Ostreococcus. We provide evidence suggesting that the clock may, at least in part, regulate directly cell division independently of the metabolism. Combined microarray and quantitative real-time reverse transcription-polymerase chain reaction analysis of the main core cell cycle gene expression revealed an extensive transcriptional regulation of cell division by the photoperiod in Ostreococcus. Finally, transcription of the main core cell cycle genes, including cyclins and cyclin-dependent kinases, was shown to be under circadian control in Ostreococcus, suggesting that these genes are potential targets of the circadian clock in the control of cell division.

). † These authors contributed equally to this work. SummaryVarious physiological imbalances lead to reactive oxygen species (ROS) overproduction and/or increases in lipoxygenase (LOX) activities, both events ending in lipid peroxidation of polyunsaturated fatty acids (PUFAs). Besides the quantification of such a process, the development of tools is necessary in order to allow the identification of the primary cause of its development and localization. A biochemical method assessing 9 LOX, 13 LOX and ROS-mediated peroxidation of membrane-bound and free PUFAs has been improved. The assay is based on the analysis of hydroxy fatty acids derived from PUFA hydroperoxides by both the straight and chiral phase high-performance liquid chromatography. Besides the upstream products of peroxidation of the 18:2 and 18:3 PUFAs, products coming from the 16:3 were characterized and their steady-state level quantified. Moreover, the observation that the relative amounts of the ROS-mediated peroxidation isomers of 18:3 were constant in leaves allowed us to circumvent the chiral analyses for the discrimination and quantification of 9 LOX, 13 LOX and ROS-mediated processes in routine experiments. The methodology has been successfully applied to decipher lipid peroxidation in Arabidopsis leaves submitted to biotic and abiotic stresses. We provide evidence of the relative timing of enzymatic and non-enzymatic lipid peroxidation processes. The 13 LOX pathway is activated early whatever the nature of the stress, leading to the peroxidation of chloroplast lipids. Under cadmium stress, the 9 LOX pathway added to the 13 LOX one. ROS-mediated peroxidation was mainly driven by light and always appeared as a late process.

Fourier transform infrared (FTIR) difference spectroscopy allows the study of molecular changes occurring at active sites in proteins with high sensitivity. Reactions are triggered by light, potential, or temperature steps and more recently by the diffusion of buffers containing effectors above membrane proteins deposited as films on ATR crystals. We have adapted a microdialysis system to an ATR, to study metal sites in soluble proteins. In this study, we identified a Cd(2+)- or Zn(2+)-binding site in cytochrome c with dissociation constants of 17 and 42 microM, respectively, which affects the oxidation rate of ferrocytochrome c by hydrogen peroxide. Using the microdialysis ATR-FTIR setup, we determined that a histidine and the carboxylate group of a glutamate are involved in Zn(2+) binding. The implication of His 33 and Glu 104 in the binding site was deduced from the comparison of FTIR data recorded with horse heart and the variant tuna cytochrome c lacking these two amino acids. A two-dimensional NMR analysis of the Zn(2+)-binding site in horse heart cytochrome c confirmed that His 33 and residues close to the C terminus are sensitive to Zn(2+) binding. This study demonstrates that the microdialysis ATR-FTIR setup is promising for the analysis of metal sites in proteins. From H(2)O/(2)H(2)O exchange experiments, we concluded that the impact of Zn(2+) and Cd(2+) binding on the oxidation kinetics of ferrocytochrome c by H(2)O(2) is associated to the perturbation of a hydrogen-bonding network involving His 33 that is sensitive to the redox state of cytochrome c.

No abstract

No abstract