Photoconversion and nuclear trafficking cycles determine phytochrome A's response profile to far-red light.

Phytochrome A (phyA) is the only photoreceptor in plants, initiating responses in far-red light and, as such, essential for survival in canopy shade. Although the absorption and the ratio of active versus total phyA are maximal in red light, far-red light is the most efficient trigger of phyA-dependent responses. Using a joint experimental-theoretical approach, we unravel the mechanism underlying this shift of the phyA action peak from red to far-red light and show that it relies on specific molecular interactions rather than on intrinsic changes to phyA's spectral properties. According to our model, the dissociation rate of the phyA-FHY1/FHL nuclear import complex is a principle determinant of the phyA action peak. The findings suggest how higher plants acquired the ability to sense far-red light from an ancestral photoreceptor tuned to respond to red light.

A DELAY OF GERMINATION 1 (DOG1)-like protein regulates spore germination in the moss Physcomitrium patens.

DELAY OF GERMINATION 1 is a key regulator of dormancy in flowering plants before seed germination. Bryophytes develop haploid spores with an analogous function to seeds. Here, we investigate whether DOG1 function during germination is conserved between bryophytes and flowering plants and analyse the underlying mechanism of DOG1 action in the moss Physcomitrium patens. Phylogenetic and in silico expression analyses were performed to identify and characterise DOG1 domain-containing genes in P. patens. Germination assays were performed to characterise a Ppdog1-like1 mutant, and replacement with AtDOG1 was carried out. Yeast two-hybrid assays were used to test the interaction of the PpDOG1-like protein with DELLA proteins from P. patens and A. thaliana. P. patens possesses nine DOG1 domain-containing genes. The DOG1-like protein PpDOG1-L1 (Pp3c3_9650) interacts with PpDELLAa and PpDELLAb and the A. thaliana DELLA protein AtRGA in yeast. Protein truncations revealed the DOG1 domain as necessary and sufficient for interaction with PpDELLA proteins. Spores of Ppdog1-l1 mutant germinate faster than wild type, but replacement with AtDOG1 reverses this effect. Our data demonstrate a role for the PpDOG1-LIKE1 protein in moss spore germination, possibly alongside PpDELLAs. This suggests a conserved DOG1 domain function in germination, albeit with differential adaptation of regulatory networks in seed and spore germination.

COLD REGULATED GENE 27 and 28 antagonize the transcriptional activity of the RVE8/LNK1/LNK2 circadian complex.

Many molecular and physiological processes in plants occur at a specific time of day. These daily rhythms are coordinated in part by the circadian clock, a timekeeper that uses daylength and temperature to maintain rhythms of ∼24 h in various clock-regulated phenotypes. The circadian MYB-like transcription factor REVEILLE 8 (RVE8) interacts with its transcriptional coactivators NIGHT LIGHT-INDUCIBLE AND CLOCK-REGULATED 1 (LNK1) and LNK2 to promote the expression of evening-phased clock genes and cold tolerance factors. While genetic approaches have commonly been used to discover connections within the clock and between clock elements and other pathways, here, we used affinity purification coupled with mass spectrometry (APMS) to identify time-of-day-specific protein interactors of the RVE8-LNK1/LNK2 complex in Arabidopsis (Arabidopsis thaliana). Among the interactors of RVE8/LNK1/LNK2 were COLD-REGULATED GENE 27 (COR27) and COR28, which coprecipitated in an evening-specific manner. In addition to COR27 and COR28, we found an enrichment of temperature-related interactors that led us to establish a previously uncharacterized role for LNK1 and LNK2 in temperature entrainment of the clock. We established that RVE8, LNK1, and either COR27 or COR28 form a tripartite complex in yeast (Saccharomyces cerevisiae) and that the effect of this interaction in planta serves to antagonize transcriptional activation of RVE8 target genes, potentially through mediating RVE8 protein degradation in the evening. Together, these results illustrate how a proteomic approach can be used to identify time-of-day-specific protein interactions. Discovery of the RVE8-LNK-COR protein complex indicates a previously unknown regulatory mechanism for circadian and temperature signaling pathways.

Phytochrome higher order mutants reveal a complex set of light responses in the moss Physcomitrium patens.

Phytochromes are photoreceptors enabling plants to respond to various light conditions. Independent gene duplications resulted in small phytochrome families in mosses, ferns and seed plants. This phytochrome diversity is hypothesised to be critical for sensing and adapting to different light conditions, but experimental evidence for this idea is lacking for mosses and ferns. The moss model species Physcomitrium patens contains seven phytochromes grouped into three clades, PHY1/3, PHY2/4 and PHY5. Here, we used CRISPR/Cas9-generated single and higher order mutants to investigate their role in light regulation of protonema and gametophore growth, protonema branching and induction of gametophores. We found both specific and partially overlapping roles for the three phytochrome clades in regulating these responses in different light conditions. PHY1/3 clade phytochromes act as primary far-red light receptors, while PHY5 clade phytochromes are the primary red light receptors. PHY2/4 clade phytochromes have functions in both red and far-red light. We also observed that PHY1/3 and PHY2/4 clade phytochromes promote gametophore growth in simulated canopy shade and also play a role in blue light. Similar to seed plants, gene duplications in the phytochrome lineage in mosses were followed by functional diversification into red and far-red light-sensing phytochromes.

Phytochrome B and PCH1 protein dynamics store night temperature information.

Plants experience temperature fluctuations during the course of the daily cycle, and although stem growth responds rapidly to these changes we largely ignore whether there is a short-term memory of previous conditions. Here we show that nighttime temperatures affect the growth of the hypocotyl of Arabidopsis thaliana seedlings not only during the night but also during the subsequent photoperiod. Active phytochrome B (phyB) represses nighttime growth and warm temperatures reduce active phyB via thermal reversion. The function of PHOTOPERIODIC CONTROL OF HYPOCOTYL1 (PCH1) is to stabilise active phyB in nuclear bodies but, surprisingly, warmth reduces PCH1 gene expression and PCH1 stability. When phyB was active at the beginning of the night, warm night temperatures enhanced the levels of nuclear phyB and reduced hypocotyl growth rate during the following day. However, when end-of-day far-red light minimised phyB activity, warm night temperatures reduced the levels of nuclear phyB and enhanced the hypocotyl growth rate during the following day. This complex growth pattern was absent in the phyB mutant. We propose that temperature-induced changes in the levels of PCH1 and in the size of the physiologically relevant nuclear pool of phyB amplify the impact of phyB-mediated temperature sensing.

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.

Differential phosphorylation of the N-terminal extension regulates phytochrome B signaling.

Phytochrome B (phyB) is an excellent light quality and quantity sensor that can detect subtle changes in the light environment. The relative amounts of the biologically active photoreceptor (phyB Pfr) are determined by the light conditions and light independent thermal relaxation of Pfr into the inactive phyB Pr, termed thermal reversion. Little is known about the regulation of thermal reversion and how it affects plants' light sensitivity. In this study we identified several serine/threonine residues on the N-terminal extension (NTE) of Arabidopsis thaliana phyB that are differentially phosphorylated in response to light and temperature, and examined transgenic plants expressing nonphosphorylatable and phosphomimic phyB mutants. The NTE of phyB is essential for thermal stability of the Pfr form, and phosphorylation of S86 particularly enhances the thermal reversion rate of the phyB Pfr-Pr heterodimer in vivo. We demonstrate that S86 phosphorylation is especially critical for phyB signaling compared with phosphorylation of the more N-terminal residues. Interestingly, S86 phosphorylation is reduced in light, paralleled by a progressive Pfr stabilization under prolonged irradiation. By investigating other phytochromes (phyD and phyE) we provide evidence that acceleration of thermal reversion by phosphorylation represents a general mechanism for attenuating phytochrome signaling.

Extrachloroplastic PP7L Functions in Chloroplast Development and Abiotic Stress Tolerance.

Chloroplast biogenesis is indispensable for proper plant development and environmental acclimation. In a screen for mutants affected in photosynthesis, we identified the protein phosphatase7-like (pp7l) mutant, which displayed delayed chloroplast development in cotyledons and young leaves. PP7L, PP7, and PP7-long constitute a subfamily of phosphoprotein phosphatases. PP7 is thought to transduce a blue-light signal perceived by crys and phy a that induces expression of SIGMA FACTOR5 (SIG5). We observed that, like PP7, PP7L was predominantly localized to the nucleus in Arabidopsis (Arabidopsis thaliana), and the pp7l phenotype was similar to that of the sig6 mutant. However, SIG6 expression was unaltered in pp7l mutants. Instead, loss of PP7L compromised translation and ribosomal RNA (rRNA) maturation in chloroplasts, pointing to a distinct mechanism influencing chloroplast development. Promoters of genes deregulated in pp7l-1 were enriched in PHYTOCHROME-INTERACTING FACTOR (PIF)-binding motifs and the transcriptome of pp7l-1 resembled those of pif and CONSTITUTIVE PHOTOMORPHOGENESIS1 (COP1) signalosome complex (csn) mutants. However, pif and csn mutants, as well as cop1, cryptochromes (cry)1 cry2, and phytochromes (phy)A phyB mutants, do not share the pp7l photosynthesis phenotype. PhyB protein levels were elevated in pp7l mutants, but phyB overexpression plants did not resemble pp7l These results indicate that PP7L operates through a different pathway and that the control of greening and photosystem biogenesis can be separated. The lack of PP7L increased susceptibility to salt and high-light stress, whereas PP7L overexpression conferred resistance to high-light stress. Strikingly, PP7L was specifically recruited to Brassicales for the regulation of chloroplast development. This study adds another player involved in chloroplast biogenesis.

Characterization of Phytochrome Interacting Factors from the Moss Physcomitrella patens Illustrates Conservation of Phytochrome Signaling Modules in Land Plants.

Across the plant kingdom, phytochrome (PHY) photoreceptors play an important role during adaptive and developmental responses to light. In Arabidopsis thaliana, light-activated PHYs accumulate in the nucleus, where they regulate downstream signaling components, such as phytochrome interacting factors (PIFs). PIFs are transcription factors that act as repressors of photomorphogenesis; their inhibition by PHYs leads to substantial changes in gene expression. The nuclear function of PHYs, however, has so far been investigated in only a few non-seed plants. Here, we identified putative target genes of PHY signaling in the moss Physcomitrella patens and found light-regulated genes that are putative orthologs of PIF-controlled genes in Arabidopsis. Phylogenetic analyses revealed that an ancestral PIF-like gene was already present in streptophyte algae, i.e., before the water-to-land transition of plants. The PIF homologs in the genome of P. patens resemble Arabidopsis PIFs in their protein domain structure, molecular properties, and physiological effects, albeit with notable differences in the motif-dependent PHY interaction. Our results suggest that P. patens PIFs are involved in PHY signaling. The PHY-PIF signaling node that relays light signals to target genes has been largely conserved during land plant evolution, with evidence of lineage-specific diversification.

PHYTOCHROME INTERACTING FACTORs in the moss Physcomitrella patens regulate light-controlled gene expression.

Phytochromes are red and far-red light receptors in plants that control growth and development in response to changes in the environment. Light-activated phytochromes enter the nucleus and act on a set of downstream signalling components to regulate gene expression. PHYTOCHROME INTERACTING FACTORs (PIFs) belong to the basic helix-loop-helix family of transcription factors and directly bind to light-activated phytochromes. Potential homologues of PIFs have been identified in ferns, bryophytes and streptophyte algae, and it has been shown that the potential PIF homologues from Physcomitrella patens, PIF1 to PIF4, have PIF function when expressed in Arabidopsis. However, their function in Physcomitrella is still unknown. Seed plant PIFs bind to G-box-containing promoters and, therefore, we searched the Physcomitrella genome for genes that contain G-boxes in their promoter. Here, we show that Physcomitrella PIFs activate these promoters in a G-box-dependent manner, suggesting that they could be direct PIF targets. Furthermore, we generated Physcomitrella pif1, pif2, pif3 and pif4 knock out mutant lines and quantified the expression of potential PIF direct target genes. The expression of these genes was generally reduced in pif mutants compared to the wildtype, but for several genes, the relative induction upon a short light treatment was higher in pif mutants than the wildtype. In contrast, expression of these genes was strongly repressed in continuous light, and pif mutants showed partial downregulation of these genes in the dark. Thus, the overall function of PIFs in light-regulated gene expression might be an ancient property of PIFs.

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.

The protein Slr1143 is an active diguanylate cyclase in Synechocystis sp. PCC 6803 and interacts with the photoreceptor Cph2.

Cyclic-di-GMP is an ubiquitous second messenger in bacteria. Several c-di-GMP receptor proteins have been identified to date, and downstream signalling pathways are often mediated through protein-protein interactions. The photoreceptor Cph2 from the cyanobacterium Synechocystis sp. PCC 6803 comprises three domains related to c-di-GMP metabolism: two GGDEF and one EAL domain. It has been shown that the C-terminal GGDEF domain acts as blue-light triggered c-di-GMP producer thereby inhibiting motility of the cells in blue light. The specific function of the other two c-di-GMP related domains remained unclear. In this study, we test knockout mutants of potential interaction partners of Cph2 for altered phototactic behaviour. Whereas wild-type cells are non-motile under high-intensity red light of 640 nm, the mutant Δslr1143 displays positive phototaxis. This phenotype can be complemented by overexpression of full-length Slr1143, which also results in an increased cellular c-di-GMP concentration. However, the non-motile phenotype of wild-type cells under high-intensity red light appears not to be due to an elevated cellular c-di-GMP content. Using co-precipitation and yeast two-hybrid assays, we demonstrate that the GGDEF domain of Slr1143 interacts with the EAL and the GGDEF domains of Cph2. However, under the test conditions, the interaction of the two proteins is not light-dependent. We conclude that Slr1143 is a new Cph2-interacting regulatory factor which modulates motility under red light and accordingly we propose Cip1 (Cph2-interacting protein 1) as a new designation for this gene product.

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.

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.

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.

Characterization of photomorphogenic responses and signaling cascades controlled by phytochrome-A expressed in different tissues.

The photoreceptor phytochrome A acts as a light-dependent molecular switch and regulates responses initiated by very low fluences of light (VLFR) and high fluences (HIR) of far-red light. PhyA is expressed ubiquitously, but how phyA signaling is orchestrated to regulate photomorphogenesis is poorly understood. To address this issue, we generated transgenic Arabidopsis thaliana phyA-201 mutant lines expressing the biologically active phyA-YFP photoreceptor in different tissues, and analyzed the expression of several reporter genes, including ProHY5:HY5-GFP and Pro35S:CFP-PIF1, and various FR-HIR-dependent physiological responses. We show that phyA action in one tissue is critical and sufficient to regulate flowering time and root growth; control of cotyledon and hypocotyl growth requires simultaneous phyA activity in different tissues; and changes detected in the expression of reporters are not restricted to phyA-containing cells. We conclude that FR-HIR-controlled morphogenesis in Arabidopsis is mediated partly by tissue-specific and partly by intercellular signaling initiated by phyA. Intercellular signaling is critical for many FR-HIR induced responses, yet it appears that phyA modulates the abundance and activity of key regulatory transcription factors in a tissue-autonomous fashion.

An evolutionarily conserved signaling mechanism mediates far-red light responses in land plants.

Phytochromes are plant photoreceptors important for development and adaptation to the environment. Phytochrome A (PHYA) is essential for the far-red (FR) high-irradiance responses (HIRs), which are of particular ecological relevance as they enable plants to establish under shade conditions. PHYA and HIRs have been considered unique to seed plants because the divergence of seed plants and cryptogams (e.g., ferns and mosses) preceded the evolution of PHYA. Seed plant phytochromes translocate into the nucleus and regulate gene expression. By contrast, there has been little evidence of a nuclear localization and function of cryptogam phytochromes. Here, we identified responses to FR light in cryptogams, which are highly reminiscent of PHYA signaling in seed plants. In the moss Physcomitrella patens and the fern Adiantum capillus-veneris, phytochromes accumulate in the nucleus in response to light. Although P. patens phytochromes evolved independently of PHYA, we have found that one clade of P. patens phytochromes exhibits the molecular properties of PHYA. We suggest that HIR-like responses had evolved in the last common ancestor of modern seed plants and cryptogams and that HIR signaling is more ancient than PHYA. Thus, other phytochromes in seed plants may have lost the capacity to mediate HIRs during evolution, rather than that PHYA acquired it.

Nuclear phytochrome A signaling promotes phototropism in Arabidopsis.

Phototropin photoreceptors (phot1 and phot2 in Arabidopsis thaliana) enable responses to directional light cues (e.g., positive phototropism in the hypocotyl). In Arabidopsis, phot1 is essential for phototropism in response to low light, a response that is also modulated by phytochrome A (phyA), representing a classical example of photoreceptor coaction. The molecular mechanisms underlying promotion of phototropism by phyA remain unclear. Most phyA responses require nuclear accumulation of the photoreceptor, but interestingly, it has been proposed that cytosolic phyA promotes phototropism. By comparing the kinetics of phototropism in seedlings with different subcellular localizations of phyA, we show that nuclear phyA accelerates the phototropic response, whereas in the fhy1 fhl mutant, in which phyA remains in the cytosol, phototropic bending is slower than in the wild type. Consistent with this data, we find that transcription factors needed for full phyA responses are needed for normal phototropism. Moreover, we show that phyA is the primary photoreceptor promoting the expression of phototropism regulators in low light (e.g., PHYTOCHROME KINASE SUBSTRATE1 [PKS1] and ROOT PHOTO TROPISM2 [RPT2]). Although phyA remains cytosolic in fhy1 fhl, induction of PKS1 and RPT2 expression still occurs in fhy1 fhl, indicating that a low level of nuclear phyA signaling is still present in fhy1 fhl.

Interaction with plant transcription factors can mediate nuclear import of phytochrome B.

Phytochromes (phy) are red/far-red-absorbing photoreceptors that regulate the adaption of plant growth and development to changes in ambient light conditions. The nuclear transport of the phytochromes upon light activation is regarded as a key step in phytochrome signaling. Although nuclear import of phyA is regulated by the transport facilitators far red elongated hypocotyl 1 (FHY1) and fhy1-like, an intrinsic nuclear localization signal was proposed to be involved in the nuclear accumulation of phyB. We recently showed that nuclear import of phytochromes can be analyzed in a cell-free system consisting of isolated nuclei of the unicellular green algae Acetabularia acetabulum. We now show that this system is also versatile to elucidate the mechanism of the nuclear transport of phyB. We tested the nuclear transport characteristics of full-length phyB as well as N- and C-terminal phyB fragments in vitro and showed that the nuclear import of phyB can be facilitated by phytochrome-interacting factor 3 (PIF3). In vivo measurements of phyB nuclear accumulation in the absence of PIF1, -3, -4, and -5 indicate that these PIFs are the major transport facilitators during the first hours of deetiolation. Under prolonged irradiations additional factors might be responsible for phyB nuclear transport in the plant.