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.

det1-1-induced UV-C hyposensitivity through UVR3 and PHR1 photolyase gene over-expression.

Obligate photoautotrophs such as plants must capture energy from sunlight and are therefore exposed to the damaging collateral effects of ultraviolet (UV) irradiation, especially on DNA. Here we investigated the interconnection between light signaling and DNA repair, two concomitant pathways during photomorphogenesis, the developmental transition associated with the first light exposure. It is shown that combination of an enhanced sunscreen effect and photoreactivation confers a greater level of tolerance to damaging UV-C doses in the constitutive photomorphogenic de-etiolated1-1 (det1--1) Arabidopsis mutant. In darkness, expression of the PHR1 and UVR3 photolyase genes, responsible for photoreactivation, is maintained at a basal level through the positive action of HY5 and HYH photomorphogenesis-promoting transcription factors and the repressive effects of DET1 and COP1. Upon light exposure, HY5 and HYH activate PHR1 gene expression while the constitutively expressed nuclear-localized DET1 protein exerts a strong inhibitory effect. Altogether, the data presented indicate a dual role for DET1 in controlling expression of light-responsive and DNA repair genes, and describe more precisely the contribution of photomorphogenic regulators in the control of light-dependent DNA repair.

A functional connection between the clock component TOC1 and abscisic acid signaling pathways.

The hormone abscisic acid (ABA) regulates the stress signals crucial for plant tolerance to adverse environmental conditions. The circadian clock also uses environmental cues for appropriate timing of plant physiology and metabolism. Despite previous studies showing the connections between ABA and clock signalling pathways, the molecular nodes underlying these connections remained unknown. In a recent study, we have shown that the essential clock component TOC1 (Timing of CAB expression 1) regulates the diurnal expression of the ABA-related gene ABAR/CHLH/GUN5 by direct binding to its promoter. Treatment with ABA specifically induces TOC1 at midday and this induction controls both the phase of TOC1 binding and the expression of ABAR. TOC1 induction by ABA is abolished in ABAR RNAi plants revealing a new feedback loop that reciprocally links ABAR and TOC1 expression. This regulation is essential for ABA function as TOC1 and ABAR overexpressing and mutant plants display altered ABA-mediated tolerance to drought conditions. Notably, TOC1 is also implicated in ABA-mediated inhibition of seed germination but in an opposite direction to that observed for dehydration responses. These opposing functions open interesting questions about the spatial and temporal networks connecting ABA and clock signaling pathways.

Signalling through kinase-defective domains: the prevalence of atypical receptor-like kinases in plants.

The structure of plant receptor-like kinases (RLKs) is similar to that of animal receptor tyrosine kinases (RTKs), and consists of an extracellular domain, a transmembrane span, and a cytoplasmic domain containing the conserved kinase domain. The mechanism by which animal RTKs, and probably plant RLKs, signal includes the dimerization of the receptor, their intermolecular phosphorylation, and the phosphorylation of downstream signalling proteins. However, atypical RTKs with a kinase-dead domain that signal through phosphorylation-independent mechanisms have also been described in animals. In the last few years, some atypical RLKs have also been reported in plants. Here these examples and their possible signalling mechanisms are reviewed. Plant genomes contain a much larger number of genes coding for receptor kinases than other organisms. The prevalence of atypical RLKs in plants is analysed here. A sequence analysis of the Arabidopsis kinome revealed that 13% of the kinase genes do not retain some of the residues that are considered as invariant within kinase catalytic domains, and are thus putatively kinase-defective. This percentage rises to close to 20% when analysing RLKs, suggesting that phosphorylation-independent mechanisms mediated by atypical RLKs are particularly important for signal transduction in plants.

Regulation of the kinase activity of the MIK GCK-like MAP4K by alternative splicing.

Alternative splicing of introns is essential to ensure the complexity of mammalian genome functions. In particular, the generation of a high number of different isoforms by alternative splicing is an important characteristic of genes coding for signalling proteins such as mitogen activated protein kinases (MAPKs). This is thought to allow these proteins to transduce multiple stimuli in a highly regulated manner. Plant genes are also subjected to alternative splicing. Nevertheless, clear examples of the functional consequences of this phenomenon are still scarce in plants. MIK is a maize gene coding for a GCK-like MAP4K that can be activated by interaction with maize atypical receptor kinase (MARK), an atypical receptor kinase. Here we show that MIK is subjected to alternative splicing. Expression of MIK leads to, at least, 4 different mature mRNAs that accumulate with particular expression profiles during maize development. Our results show that the polypeptides encoded by the different MIK mRNAs display different kinase activity and are differentially activated by interaction with the MARK receptor. Two MIK isoforms display constitutive kinase activity, one isoform is inactive but can be activated by MARK, and the fourth MIK isoform is inactive and cannot be activated by MARK. Our results constitute a clear example of the biochemical consequences of alternative splicing in plants. The selective conservation during evolution of the intron-exon structure of the region coding for the regulator domain of MIK, as well as the maintenance in maize, rice and Arabidopsis of the alternative splicing of some of these introns, are strong indications of its functional importance.

The direct activation of MIK, a germinal center kinase (GCK)-like kinase, by MARK, a maize atypical receptor kinase, suggests a new mechanism for signaling through kinase-dead receptors.

Signaling by receptor protein kinases (RPKs) involves their dimerization and transphosphorylation. However, atypical RPKs with kinase-defective domains have been described recently. Some of them are essential for proper signaling in animal systems, although the precise mechanism involved is unknown in most cases. Here we describe the cloning and characterization of an atypical plant receptor kinase from maize, MARK, which does not phosphorylate in vitro. A yeast two-hybrid approach has allowed us to identify a new germinal center kinase (GCK)-related protein, MIK, that interacts with MARK. Interestingly, the interaction of the intracellular domain of MARK with the regulator domain of MIK strongly induces MIK kinase activity. As some GCK-related proteins connect cell-surface receptors to the intracellular MAPK cascades, the activation of MIK by direct interaction with MARK could illustrate a new mechanism for signaling through atypical RPKs.