The phytochrome interacting proteins ERF55 and ERF58 repress light-induced seed germination in Arabidopsis thaliana.

Seed germination is a critical step in the life cycle of plants controlled by the phytohormones abscisic acid (ABA) and gibberellin (GA), and by phytochromes, an important class of photoreceptors in plants. Here we show that light-dependent germination is enhanced in mutants deficient in the AP2/ERF transcription factors ERF55 and ERF58. Light-activated phytochromes repress ERF55/ERF58 expression and directly bind ERF55/ERF58 to displace them from the promoter of PIF1 and SOM, genes encoding transcriptional regulators that prevent the completion of germination. The same mechanism controls the expression of genes that encode ABA or GA metabolic enzymes to decrease levels of ABA and possibly increase levels of GA. Interestingly, ERF55 and ERF58 are themselves under transcriptional control of ABA and GA, suggesting that they are part of a self-reinforcing signalling loop which controls the completion of germination. Overall, we identified a role of ERF55/ERF58 in phytochrome-mediated regulation of germination completion.

Uncovering a novel function of the CCR4-NOT complex in phytochrome A-mediated light signalling in plants.

Phytochromes are photoreceptors regulating growth and development in plants. Using the model plant Arabidopsis, we identified a novel signalling pathway downstream of the far-red light-sensing phytochrome, phyA, that depends on the highly conserved CCR4-NOT complex. CCR4-NOT is integral to RNA metabolism in yeast and animals, but its function in plants is largely unknown. NOT9B, an Arabidopsis homologue of human CNOT9, is a component of the CCR4-NOT complex, and acts as negative regulator of phyA-specific light signalling when bound to NOT1, the scaffold protein of the complex. Light-activated phyA interacts with and displaces NOT9B from NOT1, suggesting a potential mechanism for light signalling through CCR4-NOT. ARGONAUTE 1 and proteins involved in splicing associate with NOT9B and we show that NOT9B is required for specific phyA-dependent alternative splicing events. Furthermore, association with nuclear localised ARGONAUTE 1 raises the possibility that NOT9B and CCR4-NOT are involved in phyA-modulated gene expression.

Place a seedling on a windowsill, and soon you will notice the fragile stem bending towards the glass to soak in the sun and optimize its growth. Plants can 'sense' light thanks to specialized photoreceptor molecules: for instance, the phytochrome A is responsible for detecting weak and 'far-red' light from the very edge of the visible spectrum. Once the phytochrome has been activated, this message is relayed to the rest of the plant through an intricate process that requires other molecules. The CCR4-NOT protein complex is vital for all plants, animals and fungi, suggesting that it was already present in early life forms. Here, Schwenk et al. examine whether CCR4-NOT could have acquired a new role in plants to help them respond to far-red light. Scanning the genetic information of the plant model Arabidopsis thaliana revealed that the gene encoding the NOT9 subunit of CCR4-NOT had been duplicated in plants during evolution. NOT9B, the protein that the new copy codes for, has a docking site that can attach to both phytochrome A and CCR4-NOT. When NOT9B binds phytochrome A, it is released from the CCR4-NOT complex: this could trigger a cascade of reactions that ultimately changes how A. thaliana responds to far-red light. Plants that had not enough or too much NOT9B were respectively more or less responsive to that type of light, showing that the duplication of the gene coding for this subunit had helped plants respond to certain types of light. The findings by Schwenk et al. illustrate how existing structures can be repurposed during evolution to carry new roles. They also provide a deeper understanding of how plants optimize their growth, a useful piece of information in a world where most people rely on crops as their main source of nutrients.

COLD REGULATED 27 and 28 are targets of CONSTITUTIVELY PHOTOMORPHOGENIC 1 and negatively affect phytochrome B signalling.

Phytochromes are red/far-red light receptors in plants involved in the regulation of growth and development. Phytochromes can sense the light environment and contribute to measuring day length; thereby, they allow plants to respond and adapt to changes in the ambient environment. Two well-characterized signalling pathways act downstream of phytochromes and link light perception to the regulation of gene expression. The CONSTITUTIVELY PHOTOMORPHOGENIC 1/SUPPRESSOR OF PHYA-105 (COP1/SPA) E3 ubiquitin ligase complex and the PHYTOCHROME INTERACTING FACTORs (PIFs) are key components of these pathways and repress light responses in the dark. In light-grown seedlings, phytochromes inhibit COP1/SPA and PIF activity and thereby promote light signalling. In a yeast-two-hybrid screen for proteins binding to light-activated phytochromes, we identified COLD-REGULATED GENE 27 (COR27). COR27 and its homologue COR28 bind to phyA and phyB, the two primary phytochromes in seed plants. COR27 and COR28 have been described previously with regard to a function in the regulation of freezing tolerance, flowering and the circadian clock. Here, we show that COR27 and COR28 repress early seedling development in blue, far-red and in particular red light. COR27 and COR28 contain a conserved Val-Pro (VP)-peptide motif, which mediates binding to the COP1/SPA complex. COR27 and COR28 are targeted for degradation by COP1/SPA and mutant versions with a VP to AA amino acid substitution in the VP-peptide motif are stabilized. Overall, our data suggest that COR27 and COR28 accumulate in light but act as negative regulators of light signalling during early seedling development, thereby preventing an exaggerated response to light.

Investigation of Light-Regulated Protein-Protein Interactions Using Yeast Two-Hybrid Assays.

Yeast two-hybrid allows for investigation of interactions between two proteins in vivo. Yeast media can be supplemented with phycocyanobilin, allowing the formation of photoactive phytochromes, and the investigation of their light-regulated interactions. Three reporter assays are described; the histidine growth selection assay for the HIS3 reporter, and the filter lift and quantitative ONPG assays for the LacZ reporter. Design considerations for yeast two- and three-hybrid experiments are also covered.

PCH1 and PCHL promote photomorphogenesis in plants by controlling phytochrome B dark reversion.

Phytochrome B (phyB) is the primary red light photoreceptor in plants, and regulates both growth and development. The relative levels of phyB in the active state are determined by the light conditions, such as direct sunlight or shade, but are also affected by light-independent dark reversion. Dark reversion is a temperature-dependent thermal relaxation process, by which phyB reverts from the active to the inactive state. Here, we show that the homologous phyB-binding proteins PCH1 and PCHL suppress phyB dark reversion, resulting in plants with dramatically enhanced light sensitivity. Moreover, far-red and blue light upregulate the expression of PCH1 and PCHL in a phyB independent manner, thereby increasing the response to red light perceived by phyB. PCH1 and PCHL therefore provide a node for the molecular integration of different light qualities by regulation of phyB dark reversion, allowing plants to adapt growth and development to the ambient environment.

Molecular mechanisms and ecological function of far-red light signalling.

Land plants possess the ability to sense and respond to far-red light (700-760 nm), which serves as an important environmental cue. Due to the nature of far-red light, it is not absorbed by chlorophyll and thus is enriched in canopy shade and will also penetrate deeper into soil than other visible wavelengths. Far-red light responses include regulation of seed germination, suppression of hypocotyl growth, induction of flowering and accumulation of anthocyanins, which depend on one member of the phytochrome photoreceptor family, phytochrome A (phyA). Here, we review the current understanding of the underlying molecular mechanisms of how plants sense far-red light through phyA and the physiological responses to this light quality. Light-activated phytochromes act on two primary pathways within the nucleus; suppression of the E3 ubiquitin ligase complex CUL4/DDB1COP1/SPA and inactivation of the PHYTOCHROME INTERACTING FACTOR (PIF) family of bHLH transcription factors. These pathways integrate with other signal transduction pathways, including phytohormones, for tissue and developmental stage specific responses. Unlike other phytochromes that mediate red-light responses, phyA is transported from the cytoplasm to the nucleus in far-red light by the shuttle proteins FAR-RED ELONGATED HYPOCOTYL 1 (FHY1) and FHY1-LIKE (FHL). However, additional mechanisms must exist that shift the action of phyA to far-red light; current hypotheses are discussed.

Phytochromes: More Than Meets the Eye.

Phytochromes play a key role in the regulation of plant growth and development. Phytochrome-related proteins also occur in some bacteria, fungi, and algae. We highlight recent findings on the evolution of phytochromes and discuss novel hypotheses on the function of phytochromes in diatoms, a group of mainly pelagic algae.

SPA proteins: SPAnning the gap between visible light and gene expression.

MAIN CONCLUSION: In this review we focus on the role of SPA proteins in light signalling and discuss different aspects, including molecular mechanisms, specificity, and evolution. The ability of plants to perceive and respond to their environment is key to their survival under ever-changing conditions. The abiotic factor light is of particular importance for plants. Light provides plants energy for carbon fixation through photosynthesis, but also is a source of information for the adaptation of growth and development to the environment. Cryptochromes and phytochromes are major photoreceptors involved in control of developmental decisions in response to light cues, including seed germination, seedling de-etiolation, and induction of flowering. The SPA protein family acts in complex with the E3 ubiquitin ligase COP1 to target positive regulators of light responses for degradation by the 26S proteasome to suppress photomorphogenic development in darkness. Light-activated cryptochromes and phytochromes both repress the function of COP1, allowing accumulation of positive photomorphogenic factors in light. In this review, we highlight the role of the SPA proteins in this process and discuss recent advances in understanding how SPAs link light-activation of photoreceptors and downstream signaling.

High-level expression and phosphorylation of phytochrome B modulates flowering time in Arabidopsis.

Optimal timing of flowering in higher plants is crucial for successful reproduction and is coordinated by external and internal factors, including light and the circadian clock. In Arabidopsis, light-dependent stabilization of the rhythmically expressed CONSTANS (CO) is required for the activation of FLOWERING LOCUS T (FT), resulting in the initiation of flowering. Phytochrome A and cryptochrome photoreceptors stabilize CO in the evening by attenuating the activity of the CONSTITUTIVE PHOTOMORPHOGENIC 1-SUPPRESSOR OF PHYA-105 1 (COP1-SPA1) ubiquitin ligase complex, which promotes turnover of CO. In contrast, phytochrome B (phyB) facilitates degradation of CO in the morning and delays flowering. Accordingly, flowering is accelerated in phyB mutants. Paradoxically, plants overexpressing phyB also show early flowering, which may arise from an early phase of rhythmic CO expression. Here we demonstrate that overexpression of phyB induces FT transcription at dusk and in the night without affecting the phase or level of CO transcription. This response depends on the light-activated Pfr form of phyB that inhibits the function of the COP1-SPA1 complex by direct interactions. Our data suggest that attenuation of COP1 activity results in the accumulation of CO protein and subsequent induction of FT. We show that phosphorylation of Ser-86 inhibits this function of phyB by accelerating dark reversion and thus depletion of Pfr forms in the night. Our results explain the early flowering phenotype of phyB overexpression and reveal additional features of the molecular machinery by which photoreceptors mediate photoperiodism.

Light-activated phytochrome A and B interact with members of the SPA family to promote photomorphogenesis in Arabidopsis by reorganizing the COP1/SPA complex.

Phytochromes function as red/far-red photoreceptors in plants and are essential for light-regulated growth and development. Photomorphogenesis, the developmental program in light, is the default program in seed plants. In dark-grown seedlings, photomorphogenic growth is suppressed by the action of the CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1)/SUPPRESSOR OF phyA-105 (SPA) complex, which targets positive regulators of photomorphogenic growth for degradation by the proteasome. Phytochromes inhibit the COP1/SPA complex, leading to the accumulation of transcription factors promoting photomorphogenesis; yet, the mechanism by which they inactivate COP1/SPA is still unknown. Here, we show that light-activated phytochrome A (phyA) and phytochrome B (phyB) interact with SPA1 and other SPA proteins. Fluorescence resonance energy transfer-fluorescence lifetime imaging microscopy analyses show that SPAs and phytochromes colocalize and interact in nuclear bodies. Furthermore, light-activated phyA and phyB disrupt the interaction between COP1 and SPAs, resulting in reorganization of the COP1/SPA complex in planta. The light-induced stabilization of HFR1, a photomorphogenic factor targeted for degradation by COP1/SPA, correlates temporally with the accumulation of phyA in the nucleus and localization of phyA to nuclear bodies. Overall, these data provide a molecular mechanism for the inactivation of the COP1/SPA complex by phyA- and phyB-mediated light perception.

Inter- and intra-molecular interactions of Arabidopsis thaliana DELLA protein RGL1.

The phytohormone gibberellin and the DELLA proteins act together to control key aspects of plant development. Gibberellin induces degradation of DELLA proteins by recruitment of an F-box protein using a molecular switch: a gibberellin-bound nuclear receptor interacts with the N-terminal domain of DELLA proteins, and this event primes the DELLA C-terminal domain for interaction with the F-box protein. However, the mechanism of signalling between the N- and C-terminal domains of DELLA proteins is unresolved. In the present study, we used in vivo and in vitro approaches to characterize di- and tri-partite interactions of the DELLA protein RGL1 (REPRESSOR OF GA1-3-LIKE 1) of Arabidopsis thaliana with the gibberellin receptor GID1A (GIBBERELLIC ACID-INSENSITIVE DWARF-1A) and the F-box protein SLY1 (SLEEPY1). Deuterium-exchange MS unequivocally showed that the entire N-terminal domain of RGL1 is disordered prior to interaction with the GID1A; furthermore, association/dissociation kinetics, determined by surface plasmon resonance, predicts a two-state conformational change of the RGL1 N-terminal domain upon interaction with GID1A. Additionally, competition assays with monoclonal antibodies revealed that contacts mediated by the short helix Asp-Glu-Leu-Leu of the hallmark DELLA motif are not essential for the GID1A-RGL1 N-terminal domain interaction. Finally, yeast two- and three-hybrid experiments determined that unabated communication between N- and C-terminal domains of RGL1 is required for recruitment of the F-box protein SLY1.

N-terminal domains of DELLA proteins are intrinsically unstructured in the absence of interaction with GID1/gibberellic acid receptors.

The plant growth-repressing DELLA proteins (DELLAs) are known to represent a convergence point in integration of multiple developmental and environmental signals in planta, one of which is hormone gibberellic acid (GA). Binding of the liganded GA receptor (GID1/GA) to the N-terminal domain of DELLAs is required for GA-induced degradation of DELLAs via the ubiquitin-proteasome pathway, thus derepressing plant growth. However, the conformational changes of DELLAs upon binding to GID1/GA, which are the key to understanding the precise mechanism of GID1/GA-mediated degradation of DELLAs, remain unclear. Using biophysical, biochemical, and bioinformatics approaches, we demonstrated for the first time that the unbound N-terminal domains of DELLAs are intrinsically unstructured proteins under physiological conditions. Within the intrinsically disordered N-terminal domain of DELLAs, we have identified several molecular recognition features, sequences known to undergo disorder-to-order transitions upon binding to interacting proteins in intrinsically unstructured proteins. In accordance with the molecular recognition feature analyses, we have observed the binding-induced folding of N-terminal domains of DELLAs upon interaction with AtGID1/GA. Our results also indicate that DELLA proteins can be divided into two subgroups in terms of their molecular compactness and their interactions with monoclonal antibodies.