Gérald Zabulon

Characterization of two members of the cryptochrome/photolyase family from Ostreococcus tauri provides insights into the origin and evolution of cryptochromes.

Cryptochromes (Crys) are blue light receptors believed to have evolved from the DNA photolyase protein family, implying that light control and light protection share a common ancient origin. In this paper, we report the identification of five genes of the Cry/photolyase family (CPF) in two green algae of the Ostreococcus genus. Phylogenetic analyses were used to confidently assign three of these sequences to cyclobutane pyrimidine dimer (CPD) photolyases, one of them to a DASH-type Cry, and a third CPF gene has high homology with the recently described diatom CPF1 that displays a bifunctional activity. Both purified OtCPF1 and OtCPF2 proteins show non-covalent binding to flavin adenine dinucleotide (FAD), and additionally to 5,10-methenyl-tetrahydrofolate (MTHF) for OtCPF2. Expression analyses revealed that all five CPF members of Ostreococcus tauri are regulated by light. Furthermore, we show that OtCPF1 and OtCPF2 display photolyase activity and that OtCPF1 is able to interact with the CLOCK:BMAL heterodimer, transcription factors regulating circadian clock function in other organisms. Finally, we provide evidence for the involvement of OtCPF1 in the maintenance of the Ostreococcus circadian clock. This work improves our understanding of the evolutionary transition between photolyases and Crys.

Convergence of two global transcriptional regulators on nitrogen induction of the stress-acclimation gene nblA in the cyanobacterium Synechococcus sp. PCC 7942

Cyanobacteria respond to environmental stress conditions by degrading their phycobilisomes, the light harvesting complexes for photosynthesis. The expression of nblA, a key gene in this process, is controlled by the response regulator NblR in Synechococcus sp. PCC 7942. Here we show that, under nitrogen stress, nblA is also regulated by NtcA, the global regulator for nitrogen control. NtcA activation of nblA was found to be nitrogen‐specific and did not take place under sulphur stress. Transcripts from the two major transcription start points (tsp) for the nblA gene were induced in response to nitrogen and sulphur starvation. The most active one (tspII) required both NblR and NtcA to induce full nblA expression under nitrogen starvation. NblR and NtcA bound in vitro to a DNA fragment from the nblA promoter region, suggesting that, under nitrogen stress, both NblR and NtcA activate the main regulated promoter (PnblA‐2) by direct DNA‐binding. The structure of PnblA‐2 differs from that of the canonical NtcA‐activated promoter and it is therefore proposed to represent a novel type of NtcA‐dependent promoter. We analysed expression patterns from ntcA and selected NtcA targets in NtcA–, NblR– and wild‐type strains, and discuss data suggesting further interrelations between phycobilisome degradation and nitrogen assimilation regulatory pathways.

Histone H2B Monoubiquitination Facilitates the Rapid Modulation of Gene Expression during Arabidopsis Photomorphogenesis

Profiling of DNA and histone modifications has recently allowed the establishment of reference epigenomes from several model organisms. This identified a major chromatin state for active genes that contains monoubiquitinated H2B (H2Bub), a mark linked to transcription elongation. However, assessment of dynamic chromatin changes during the reprogramming of gene expression in response to extrinsic or developmental signals has been more difficult. Here we used the major developmental switch that Arabidopsis thaliana plants undergo upon their initial perception of light, known as photomorphogenesis, as a paradigm to assess spatial and temporal dynamics of monoubiquitinated H2B (H2Bub) and its impact on transcriptional responses. The process involves rapid and extensive transcriptional reprogramming and represents a developmental window well suited to studying cell division–independent chromatin changes. Genome-wide H2Bub distribution was determined together with transcriptome profiles at three time points during early photomorphogenesis. This revealed de novo marking of 177 genes upon the first hour of illumination, illustrating the dynamic nature of H2Bub enrichment in a genomic context. Gene upregulation was associated with H2Bub enrichment, while H2Bub levels generally remained stable during gene downregulation. We further report that H2Bub influences the modulation of gene expression, as both gene up- and downregulation were globally weaker in hub1 mutant plants that lack H2Bub. H2Bub-dependent regulation notably impacted genes with fast and transient light induction, and several circadian clock components whose mRNA levels are tightly regulated by sharp oscillations. Based on these findings, we propose that H2B monoubiquitination is part of a transcription-coupled, chromatin-based mechanism to rapidly modulate gene expression.

Spectro-temporal characterization of the photoactivation mechanism of two new oxidized cryptochrome/photolyase photoreceptors.

The photoactivation dynamics of two new flavoproteins (OtCPF1 and OtCPF2) of the cryptochrome photolyase family (CPF), belonging to the green alga Ostreococcus tauri , was studied by broadband UV-vis femtosecond absorption spectroscopy. Upon excitation of the protein chromophoric cofactor, flavin adenine dinucleotide in its oxidized form (FAD(ox)), we observed in both cases the ultrafast photoreduction of FAD(ox): in 390 fs for OtCPF1 and 590 fs for OtCPF2. Although such ultrafast electron transfer has already been reported for other flavoproteins and CPF members, the present result is the first demonstration with full spectral characterization of the mechanism. Analysis of the photoproduct spectra allowed identifying tryptophan as the primary electron donor. This residue is found to be oxidized to its protonated radical cation form (WH(*+)), while FAD(ox) is reduced to FAD(*-). Subsequent kinetics were observed in the picosecond and subnanosecond regime, mostly described by a biexponential partial decay of the photoproduct transient signal (9 and 81 ps for OtCPF1, and 13 and 340 ps for OtCPF2), with reduced spectral changes, while a long-lived photoproduct remains in the nanosecond time scale. We interpret these observations within the model proposed by the groups of Brettel and Vos, which describes the photoreduction of FADH(*) within E. coli CPD photolyase (EcCPD) as a sequential electron transfer along a chain of three tryptophan residues, although in that case the rate limiting step was the primary photoreduction in 30 ps. In the present study, excitation of FAD(ox) permitted to reveal the following steps and spectroscopically assign them to the hole-hopping process along the tryptophan chain, accompanied by partial charge recombination at each step. In addition, structural analysis performed by homology modeling allowed us to propose a tentative structure of the relative orientations of FAD and the conserved tryptophan triad. The results of preliminary transient anisotropy measurements performed on OtCPF2 finally showed good compatibility with the oxidation of the distal tryptophan residue (WH(351)) in 340 ps, hence, with the overall Brettel-Vos mechanism.

The conserved factor DE‐ETIOLATED 1 cooperates with CUL4–DDB1DDB2 to maintain genome integrity upon UV stress

Plants and many other eukaryotes can make use of two major pathways to cope with mutagenic effects of light, photoreactivation and nucleotide excision repair (NER). While photoreactivation allows direct repair by photolyase enzymes using light energy, NER requires a stepwise mechanism with several protein complexes acting at the levels of lesion detection, DNA incision and resynthesis. Here we investigated the involvement in NER of DE‐ETIOLATED 1 (DET1), an evolutionarily conserved factor that associates with components of the ubiquitylation machinery in plants and mammals and acts as a negative repressor of light‐driven photomorphogenic development in Arabidopsis. Evidence is provided that plant DET1 acts with CULLIN4‐based ubiquitin E3 ligase, and that appropriate dosage of DET1 protein is necessary for efficient removal of UV photoproducts through the NER pathway. Moreover, DET1 is required for CULLIN4‐dependent targeted degradation of the UV‐lesion recognition factor DDB2. Finally, DET1 protein is degraded concomitantly with DDB2 upon UV irradiation in a CUL4‐dependent mechanism. Altogether, these data suggest that DET1 and DDB2 cooperate during the excision repair process.

Light signaling controls nuclear architecture reorganization during seedling establishment.

Significance Nuclear organization and genome expression are subject to massive reprogramming during most developmental transitions. The mechanisms triggering them in response to environmental stimuli are only poorly understood. Here we describe that dynamic changes in higher-order nuclear organization occurring in cotyledons (embryonic leaves) upon germination are impacted by light availability in the plant Arabidopsis thaliana. Upon light perception, master regulators of the light signaling pathway trigger rapid and massive nuclear expansion, condensation of large chromatin domains, and increased transcriptional activity of genes. These findings establish a foundation for deciphering the relationships between topological genome organization and transcriptional regulation associated with the establishment of photosynthesis. The spatial organization of chromatin can be subject to extensive remodeling in plant somatic cells in response to developmental and environmental signals. However, the mechanisms controlling these dynamic changes and their functional impact on nuclear activity are poorly understood. Here, we determined that light perception triggers a switch between two different nuclear architectural schemes during Arabidopsis postembryonic development. Whereas progressive nucleus expansion and heterochromatin rearrangements in cotyledon cells are achieved similarly under light and dark conditions during germination, the later steps that lead to mature nuclear phenotypes are intimately associated with the photomorphogenic transition in an organ-specific manner. The light signaling integrators DE-ETIOLATED 1 and CONSTITUTIVE PHOTOMORPHOGENIC 1 maintain heterochromatin in a decondensed state in etiolated cotyledons. In contrast, under light conditions cryptochrome-mediated photoperception releases nuclear expansion and heterochromatin compaction within conspicuous chromocenters. For all tested loci, chromatin condensation during photomorphogenesis does not detectably rely on DNA methylation-based processes. Notwithstanding, the efficiency of transcriptional gene silencing may be impacted during the transition, as based on the reactivation of transposable element-driven reporter genes. Finally, we report that global engagement of RNA polymerase II in transcription is highly increased under light conditions, suggesting that cotyledon photomorphogenesis involves a transition from globally quiescent to more active transcriptional states. Given these findings, we propose that light-triggered changes in nuclear architecture underlie interplays between heterochromatin reorganization and transcriptional reprogramming associated with the establishment of photosynthesis.

The NblAI protein from the filamentous cyanobacterium Tolypothrix PCC 7601: regulation of its expression and interactions with phycobilisome components

Cyanobacteria respond to changes in light or nutrient availability by modifications in their photosynthetic light harvesting antenna. In unicellular cyanobacteria a small polypeptide (NblA) is required for phycobilisome degradation following environmental stresses. In the filamentous strain Tolypothrix sp. PCC 7601 the nblAI gene, encoding a NblA homologue, is located upstream of the operon coding for phycoerythrin (cpeBA). The nblAI transcripts all originate from a single transcription start point; their intracellular levels vary according to nitrogen regimes but not with light spectral quality. Using recombinant His‐tagged NblAI protein, we found that in vitro NblAI has affinity for both phycocyanin and phycoerythrin subunits from Tolypothrix sp. PCC 7601, but not for allophycocyanin from this cyanobacterium or for phycobiliproteins from other cyanobacterial species. We also observed that although nblAI is mainly expressed under nitrogen starvation, NblAI polypeptides are always present in the cell; a significant portion of them co‐purify with phycobilisome preparations but only if cells were grown under red light. Our data indicate that NblAI attaches to the phycobilisomes even under non‐inducing conditions and suggest a preferential affinity of NblAI for phycocyanin.

Plants Release Precursors of Histone Deacetylase Inhibitors to Suppress Growth of Competitors

Chemical compounds in plant root exudates influence the growth of neighboring plants by interfering with their chromatin configuration and gene expression. To secure their access to water, light, and nutrients, many plant species have developed allelopathic strategies to suppress competitors. To this end, they release into the rhizosphere phytotoxic substances that inhibit the germination and growth of neighbors. Despite the importance of allelopathy in shaping natural plant communities and for agricultural production, the underlying molecular mechanisms are largely unknown. Here, we report that allelochemicals derived from the common class of cyclic hydroxamic acid root exudates directly affect the chromatin-modifying machinery in Arabidopsis thaliana. These allelochemicals inhibit histone deacetylases both in vitro and in vivo and exert their activity through locus-specific alterations of histone acetylation and associated gene expression. Our multilevel analysis collectively shows how plant-plant interactions interfere with a fundamental cellular process, histone acetylation, by targeting an evolutionarily highly conserved class of enzymes.

Spectroscopic characterization of a (6-4) photolyase from the green alga Ostreococcus tauri

The cofactor content of OtCPF1, a (6-4) photolyase isolated from the green marine alga Ostreococcus tauri, was characterized by steady-state absorption and fluorescence spectroscopy. The heterologously expressed, GST-fused, purified protein (MW: 89kDa) is non-covalently bound to flavin adenine dinucleotide (FAD), with a flavin to apoprotein molecular ratio of 64%. No light-harvesting chromophore was found in this protein. In freshly purified OtCPF1, FAD is present in three different redox states: the fully oxidized form (FAD(ox), 82%), the neutral semiquinone (FADH*, 14%) and the fully reduced anion (FADH-, 4%). Keeping the sample in the dark, at 5 degrees C, yields oxidation of FADH* and FADH-, partial release of FAD to the solution and slow degradation of the protein. Upon steady-state blue-light irradiation of OtCPF1 at 450nm, photoreduction processes leading to an accumulation of stable FADH* and FADH- species are observed. We demonstrate that this accumulation is due to the presence of an external electron donor agent in the purification buffer. Composition changes observed under steady-state photoexcitation are interpreted in terms of photoinduced reductions of FAD(ox) and FADH* states and competitive back reactions. Specific irradiation by red light at 620 nm shows both photoreduction of FADH* to FADH- and irreversible oxidation of FADH* to FAD(ox). The photoinduced oxidation reaction is believed to be indirectly caused by the external donor agent present in the buffer. Photoexcitation is also shown to stabilize the binding of FAD to the protein. We suggest this effect to be due to slight changes in the protein conformation, possibly strengthening the hydrogen-bonding network surrounding FAD.

NblA Gene Expression in Synechocystis PCC 6803 Strains Lacking DspA (Hik33) and a NblR-like Protein

Cyanobacteria respond to nutrient-limiting conditions by degrading their phycobilisomes (PBS), the light-harvesting complexes for photosynthesis. In Synechococcus sp. PCC 7942, the expression of nblA, an essential gene in this process, is controlled by the response regulator NblR and the sensor NblS. Here we study the effect of inactivation of dspA (an nblS homologue) and an nblR-like gene on phycobilisome degradation in Synechocystis sp. PCC 6803, under nitrogen starvation. In each mutant, the expression of nblA was found to be unaffected and sequential PBS degradation occurred after nitrogen deprivation (although it was slightly delayed). Our results demonstrate that dspA and nblR-like do not exert a major control of PBS degradation in Synechocystis sp. PCC 6803.