Arabidopsis phyB Research Articles

Modulation of warm temperature-sensitive growth using a phytochrome B dark reversion variant, phyB[G515E], in Arabidopsis and rice

IntroductionAmbient temperature-induced hypocotyl elongation in Arabidopsis seedlings is sensed by the epidermis-localized phytochrome B (phyB) and transduced into auxin biosynthesis via a basic helix-loop-helix transcription factor, phytochrome-interacting factor 4 (PIF4). Once synthesized, auxin travels down from the cotyledons to the hypocotyl, triggering hypocotyl cell elongation. Thus, the phyB–PIF4 module involved in thermosensing and signal transduction is a potential genetic target for engineering warm temperature-insensitive plants. ObjectivesThis study aims to manipulate warm temperature-induced elongation of plants at the post-translational level using phyB variants with dark reversion, the expression of which is subjected to heat stress. MethodsThe thermosensitive growth response of Arabidopsis was manipulated by expressing the single amino acid substitution variant of phyB (phyB[G515E]), which exhibited a lower dark reversion rate than wild-type phyB. Other variants with slow (phyB[G564E]) or rapid (phyB[S584F]) dark reversion or light insensitivity (phyB[G767R]) were also included in this study for comparison. Warming-induced transient expression of phyB variants was achieved using heat shock-inducible promoters. Arabidopsis PHYB[G515E] and PHYB[G564E] were also constitutively expressed in rice in an attempt to manipulate the heat sensitivity of a monocotyledonous plant species. ResultsAt an elevated temperature, Arabidopsis seedlings transiently expressing PHYB[G515E] under the control of a heat shock-inducible promoter exhibited shorter hypocotyls than those expressing PHYB and other PHYB variant genes. This warm temperature-insensitive growth was related to the lowered PIF4 and auxin responses. In addition, transgenic rice seedlings expressing Arabidopsis PHYB[G515E] and PHYB[G564E] showed warm temperature-insensitive shoot growth. ConclusionTransient expression of phyB variants with altered dark reversion rates could serve as an effective optogenetic technique for manipulating PIF4–auxin-mediated thermomorphogenic responses in plants.

A 5.5-kb LTR-retrotransposon insertion inside phytochrome B gene (CsPHYB) results in long hypocotyl and early flowering in cucumber (Cucumis sativus L.)

The novel spontaneous long hypocotyl and early flowering (lhef) mutation in cucumber is due to a 5551-bp LTR-retrotransposon insertion in CsPHYB gene encoding PHYTOCHROME B, which plays a major role in regulating photomorphogenic hypocotyl growth and flowering. Hypocotyl length and flowering time are important for establishing high-quality seedlings in modern cucumber production, but little is known for the underlying molecular mechanisms of these two traits. In this study, a spontaneous cucumber long hypocotyl and early flowering mutant was identified and characterized. Based on multiple lines of evidence, we show that cucumber phytochrome B (CsPHYB) is the candidate gene for this mutation, and a 5551-bp LTR-retrotransposon insertion in the first exon of CsPHYB was responsible for the mutant phenotypes. Uniqueness of the mutant allele at CsPHYB was verified in 114 natural cucumber lines. Ectopic expression of the CsPHYB in Arabidopsis phyB mutant rescued the long hypocotyl and early flowering phenotype of phyB-9 mutant. The wild-type CsPHYB protein was localized on the membrane and cytoplasm under white light condition, whereas in the nucleus under red light, it is consistent with its roles as a red-light photoreceptor in Arabidopsis. However, the mutant csphyb protein was localized on the membrane and cytoplasm under both white and red-light conditions. Expression dynamics of CsPHYB and several cell elongation-related genes were positively correlated with hypocotyl elongation; the transcription levels of key positive and negative regulators for flowering time were also consistent with the anthesis dates in the mutant and wild-type plants. Yeast two hybrid and bimolecular fluorescence complementation assays identified physical interactions between CsPHYB and phytochrome interacting factor 3/4 (CsPIF3/4). These findings will provide new insights into the roles of the CsPHYB in cucumber hypocotyl growth and flowering time.

Role of guard cell- or mesophyll cell-localized phytochromes in stomatal responses to blue, red, and far-red light.

Guard cell- or mesophyll cell-localized phytochromes do not have a predominant direct light sensory role in red- or blue-light-mediated stomatal opening or far-red-light-mediated stomatal closure of Arabidopsis. The role of phytochromes in blue- and red-light-mediated stomatal opening, and far-red-light- mediated decrease in opening, is still under debate. It is not clear whether reduced stomatal opening in a phytochrome B (phyB) mutant line, is due to phytochrome acting as a direct photosensor or an indirect growth effect. The exact tissue localization of the phytochrome photoreceptor important for stomatal opening is also not known. We studied differences in stomatal opening in an Arabidopsis phyB mutant, and lines showing mesophyll cell-specific or guard cell-specific inactivation of phytochromes. Stomatal conductance (gs) of intact leaves was measured under red, blue, and blue + far-red light. Lines exhibiting guard cell-specific inactivation of phytochrome did not show a change in gs under blue or red light compared to Col-0. phyB consistently exhibited a reduction in gs under both blue and red light. Addition of far-red light did not have a significant impact on the blue- or red-light-mediated stomatal response. Treatment of leaves with DCMU (3-(3,4-dichlorophenyl)-1,1-dimethylurea), a photosynthetic electron transport (PET) inhibitor, eliminated the response to red light in all lines, indicating that stomatal opening under red light is controlled by PET, and not directly by phytochrome. Similar to previous studies, leaves of the phyB mutant line had fewer stomata. Overall, phytochrome does not appear have a predominant direct sensory role in stomatal opening under red or blue light. However, phytochromes likely have an indirect effect on the degree of stomatal opening under light through effects on leaf growth and stomatal development.

Plant Phytochrome B Assembles an Asymmetric Dimer with Unique Signaling Potential

Phytochromes (Phy) sense red and far‐red light and reversibly interconvert between inactive Pr and active Pfr conformers. Phys typically consist of a photo sensory module (PSM) formed by sequential N‐terminal PAS, GAF, and PHY domains, followed by a C‐terminal histidine kinase‐related domain (HKRD). There are four plant isoforms Phy A through D, and they all contain two additional PAS domains (PAS1 and PAS2) inserted between PSM and HKRD. Despite extensive biochemical and structural studies, there has been a lack of a full‐length structure, hindering our understanding of plant Phy signaling. Here we report a 3.3‐Å cryo‐EM structure of Arabidopsis PhyB dimer. Instead of the widely anticipated parallel association of the two monomers resembling the prokaryotic relatives, Plant PhyB adopts a topologically complex dimeric architecture with the two PSMs in a head‐to‐tail antiparallel form while the two HKRDs in a head‐to‐head parallel form. The PAS2 mediates the antiparallel dimerization of PSM that leads to a parallelogram‐shaped platform. And a preceding ‘modulator’ loop assembles tightly with the PSM of its own protomer. We show that these inter‐ and intra‐protomer connections contribute to stabilizing of dimer and accelerate thermal reversion of Pfr back to Pr. Lean of HKRDs towards the platform results in profound asymmetry of PhyB that might enable distinct signaling potentials to the protomers. We propose that this unique structural dynamism creates an extensive photostate‐sensitive surface for interaction with their downstream signaling partners.

Plant phytochrome B is an asymmetric dimer with unique signalling potential.

Many aspects of plant photoperception are mediated by the phytochrome (Phy) family of bilin-containing photoreceptors that reversibly interconvert between inactive Pr and active Pfr conformers1,2. Despite extensive biochemical studies, full understanding of plant Phy signalling has remained unclear due to the absence of relevant 3D models. Here we report a cryo-electron microscopy structure of Arabidopsis PhyB in the Pr state that reveals a topologically complex dimeric organization that is substantially distinct from its prokaryotic relatives. Instead of an anticipated parallel architecture, the C-terminal histidine-kinase-related domains (HKRDs) associate head-to-head, whereas the N-terminal photosensory regions associate head-to-tail to form a parallelogram-shaped platform with near two-fold symmetry. The platform is internally linked by the second of two internal Per/Arnt/Sim domains that binds to the photosensory module of the opposing protomer and a preceding 'modulator' loop that assembles tightly with the photosensory module of its own protomer. Both connections accelerate the thermal reversion of Pfr back to Pr, consistent with an inverse relationship between dimer assembly and Pfr stability. Lopsided contacts between the HKRDs and the platform create profound asymmetry to PhyB that might imbue distinct signalling potentials to the protomers. We propose that this unique structural dynamism creates an extensive photostate-sensitive surface for conformation-dependent interactions between plant Phy photoreceptors and their signalling partners.

Effect of circularly polarized light on germination, hypocotyl elongation and biomass production of arabidopsis and lettuce: Involvement of phytochrome B.

Circular dichroism (CD), defined as the differential absorption of left- and right-handed circularly polarized light (CPL), is a useful spectroscopic technique for structural studies of biological systems composed of chiral molecules. The present study evaluated the effects of CPL on germination, hypocotyl elongation and biomass production of Arabidopsis and lettuce. Higher germination rates were observed when Arabidopsis and lettuce seedlings were irradiated with red right-handed CPL (R-CPL) than with red left-handed CPL (L-CPL). Hypocotyl elongation was effectively inhibited when Arabidopsis and lettuce seedlings were irradiated with red R-CPL than with red L-CPL. This difference was not observed when a phytochrome B (phyB) deficient mutant of Arabidopsis was irradiated, suggesting that inhibition of elongation by red R-CPL was mediated by phyB. White R-CPL induced greater biomass production by adult Arabidopsis plants, as determined by their fresh shoot weight, than white L-CPL. To determine the molecular basis of these CPL effects, CD spectra and the effect of CPL on the photoreaction of a sensory module of Arabidopsis phyB were measured. The red light-absorbing form of phyB showed a negative CD in the red light-absorbing region, consistent with the results of germination, inhibition of hypocotyl elongation and biomass production. L-CPL and R-CPL, however, did not differ in their ability to induce the interconversion of the red light-absorbing and far-red light-absorbing forms of phyB. These findings suggest that these CPL effects involve phyB, along with other photoreceptors and the photosynthetic process.

Photosensing and Thermosensing by Phytochrome B Require Both Proximal and Distal Allosteric Features within the Dimeric Photoreceptor

Phytochromes (Phys) encompass a diverse collection of bilin-containing photoreceptors that help plants and microorganisms perceive light through photointerconversion between red light (Pr) and far-red light (Pfr)-absorbing states. In addition, Pfr reverts thermally back to Pr via a highly enthalpic process that enables temperature sensation in plants and possibly other organisms. Through domain analysis of the Arabidopsis PhyB isoform assembled recombinantly, coupled with measurements of solution size, photoconversion, and thermal reversion, we identified both proximal and distal features that influence all three metrics. Included are the downstream C-terminal histidine kinase-related domain known to promote dimerization and a conserved patch just upstream of an N-terminal Period/Arnt/Sim (PAS) domain, which upon removal dramatically accelerates thermal reversion. We also discovered that the nature of the bilin strongly influences Pfr stability. Whereas incorporation of the native bilin phytochromobilin into PhyB confers robust Pfr → Pr thermal reversion, that assembled with the cyanobacterial version phycocyanobilin, often used for optogenetics, has a dramatically stabilized Pfr state. Taken together, we conclude that Pfr acquisition and stability are impacted by a collection of opposing allosteric features that inhibit or promote photoconversion and reversion of Pfr back to Pr, thus allowing Phys to dynamically measure light, temperature, and possibly time.

Unearthing the transition rates between photoreceptor conformers.

BackgroundObtaining accurate estimates of biological or enzymatic reaction rates is critical in understanding the design principles of a network and how biological processes can be experimentally manipulated on demand. In many cases experimental limitations mean that some enzymatic rates cannot be measured directly, requiring mathematical algorithms to estimate them. Here, we describe a methodology that calculates rates at which light-regulated proteins switch between conformational states. We focus our analysis on the phytochrome family of photoreceptors found in cyanobacteria, plants and many optogenetic tools. Phytochrome proteins change between active (P A) and inactive (P I) states at rates that are proportional to photoconversion cross-sections and influenced by light quality, light intensity, thermal reactions and dimerisation. This work presents a method that can accurately calculate these photoconversion cross-sections in the presence of multiple non-light regulated reactions.ResultsOur approach to calculating the photoconversion cross-sections comprises three steps: i) calculate the thermal reversion reaction rate(s); ii) develop search spaces from which all possible sets of photoconversion cross-sections exist, and; iii) estimate extinction coefficients that describe our absorption spectra. We confirm that the presented approach yields accurate results through the use of simulated test cases. Our test cases were further expanded to more realistic scenarios where noise, multiple thermal reactions and dimerisation are considered. Finally, we present the photoconversion cross-sections of an Arabidopsis phyB N-terminal fragment commonly used in optogenetic tools.ConclusionsThe calculation of photoconversion cross-sections has implications for both photoreceptor and synthetic biologists. Our method allows, for the first time, direct comparisons of photoconversion cross-sections and response speeds of photoreceptors in different cellular environments and synthetic tools. Due to the generality of our procedure, as shown by the application to multiple test cases, the photoconversion cross-sections and quantum yields of any photoreceptor might now, in principle, be obtained.Electronic supplementary materialThe online version of this article (doi:10.1186/s12918-016-0368-y) contains supplementary material, which is available to authorized users.

NtPHYB1K326, a homologous gene of Arabidopsis PHYB, positively regulates the content of phenolic compounds in tobacco

Polyphenols are important secondary metabolites and bioactive compounds in plants. Light is a vital abiotic factor that greatly impacts the content of polyphenols in plants. In spite of their importance the mechanism of polyphenol regulation still remains unknown in tobacco. A phytochrome B homolog, NtPHYB1K326, was isolated from Nicotiana tabacum cv. K326 to investigate the role of light receptors in the regulation of polyphenol metabolism in tobacco leaves. Furthermore, role of NtPHYB1K326 in polyphenol metabolism was analyzed by over-expression and RNAi-silencing approaches. Consistent and complemented results indicated involvement of NtPHYB1K326 in the regulation of polyphenol metabolism in tobacco leaves. Moreover, high levels of NtPHYB1K326 transcripts favor the accumulation of chlorogenic acid and its isomers, the key polyphenol component in tobacco leaves. Transcriptome analysis was also carried out for exploring the regulation mechanism of NtPHYB1K326 in the polyphenol metabolism. Compared with WT, 1665 and 1421 differentially-expressed genes were found in NtPHYB1K326-GFP and NtPHYB1K326-RNAi transgenic lines, respectively. Among these, about 30 genes were related to phenylpropanoid pathway, which is predominantly involved in synthesis of polyphenols. Further evidences from quantitative RT-PCR confirmed that NtPHYB1K326 may control phenylpropanoid pathway through regulating the transcription of PAL4 (phenylalanine ammonialyase 4), 4CL1 (4-coumarate:coenzyme A ligase 1) and COMT (caffeic acid 3-O-methyltransferase) genes.

Rice Phytochrome B (OsPhyB) Negatively Regulates Dark- and Starvation-Induced Leaf Senescence.

Light regulates leaf senescence and light deprivation causes large-scale transcriptional reprogramming to dismantle cellular components and remobilize nutrients to sink organs, such as seeds and storage tissue. We recently reported that in Arabidopsis (Arabidopsis thaliana), Phytochrome-Interacting Factor4 (PIF4) and PIF5 promote dark-induced senescence and natural senescence by directly activating the expression of typical senescence-associated genes (SAGs), including ORESARA1 (ORE1) and ETHYLENE INSENSITIVE3 (EIN3). In contrast, phytochrome B (PhyB) inhibits leaf senescence by repressing PIF4 and PIF5 at the post-translational level. Although we found how red light signaling represses leaf senescence in Arabidopsis, it remains unknown whether PhyB and/or PhyA are involved in leaf senescence in rice (Oryza sativa). Here we show that rice phyB knockout mutants (osphyB-1, -2, and -3) exhibited an early senescence phenotype during dark-induced senescence, but an osphyA knockout mutant (osphyA-3) senesced normally. The RT-qPCR analysis revealed that several senescence-associated genes, including OsORE1 and OsEIN3, were significantly up-regulated in osphyB-2 mutants, indicating that OsPhyB also inhibits leaf senescence, like Arabidopsis PhyB. We also found that leaf segments of osphyB-2 senesced faster even under light conditions. Supplementation with nitrogen compounds, such as KNO3 and NH4NO3, rescued the early senescence phenotype of osphyB-2, indicating that starvation is one of the major signaling factors in the OsPhyB-dependent leaf senescence pathway.

Introduction of the Arabidopsis PHYB gene increases resistance of photosynthetic apparatus in transgenic Solanum tuberosum plants to UV-B radiation

The influence of phytochrome B overproduction in 50- to 60-day-old transgenic potato plants (Solanum tuberosum L., lines Dara 5 and Dara 12 with moderate and intense expression of PHYB, respectively) on the resistance of photosynthetic apparatus to UV-B irradiation was investigated. In plants unexposed to UV-B, there was no significant difference in photosynthetic rates (P n) and fluorescence parameters (F v/F m, qN, qP) between the nontransformed (NT) line and Dara-5 and Dara-12 lines, whereas the content of photosynthetic pigments per 1 cm2 leaf area was higher in the transgenic plants. Irradiation with UV-B resulted in the decrease in photosynthetic rate in NT plants by 35–45%, whereas in Dara-12 line this rate was lowered by only 20–25%. Exposure to UV-B reduced the amplitudes of both fast and slow components of delayed fluorescence (DF), which indicated the diminished efficiency of photosystem II (PSII). The decrease in the maximum amplitude of the slow DF component was markedly lower in Dara-12 (19%) than in NT line (33%). The maximal photochemical quantum yield of PSII (F v/F m ratio) in plants exposed to UV-B was also suppressed stronger in NT line than in Dara-12. The line Dara-5 had intermediate position among other lines in terms of UV-B resistance of photosynthesis and PSII activity, but it was closer to NT than to Dara-12. Thus, the potato plants actively expressing the gene of Arabidopsis phytochrome B apoprotein (PHYB) demonstrated a higher resistance of photosynthetic apparatus to UV-B radiation compared to nontransformed plants. The elevated UV-B tolerance is most likely related to the increased leaf content of chlorophyll, carotenoids, and flavonoids.

The homeodomain‐leucine zipper ATHB23, a phytochrome B‐interacting protein, is important for phytochrome B‐mediated red light signaling

Phytochromes are red (R)/far-red (FR) photoreceptors that are central to the regulation of plant growth and development. Although it is well known that photoactivated phytochromes are translocated into the nucleus where they interact with a variety of nuclear proteins and ultimately regulate genome-wide transcription, the mechanisms by which these photoreceptors function are not completely understood. In an effort to enhance our understanding of phytochrome-mediated light signaling networks, we attempted to identify novel proteins interacting with phytochrome B (phyB). Using affinity purification in Arabidopsis phyB overexpressor, coupled with mass spectrometry analysis, 16 proteins that interact with phyB in vivo were identified. Interactions between phyB and six putative phyB-interacting proteins were confirmed by bimolecular fluorescence complementation (BiFC) analysis. Involvement of these proteins in phyB-mediated signaling pathways was also revealed by physiological analysis of the mutants defective in each phyB-interacting protein. We further characterized the athb23 mutant impaired in the homeobox protein 23 (ATHB23) gene. The athb23 mutant displayed altered hypocotyl growth under R light, as well as defects in phyB-dependent seed germination and phyB-mediated cotyledon expansion. Taken together, these results suggest that the ATHB23 transcription factor is a novel component of the phyB-mediated R light signaling pathway.

Initial characterization of shade avoidance response suggests functional diversity between Populus phytochrome B genes

Shade avoidance signaling involves perception of incident red/far-red (R/FR) light by phytochromes (PHYs) and modulation of downstream transcriptional networks. Although these responses are well studied in Arabidopsis, little is known about the role of PHYs and the transcriptional responses to shade in the woody perennial Populus. Tissue expression and subcellular localization of Populus PHYs was studied by quantitative real-time PCR (qRT-PCR) and protoplast transient assay. Transgenic lines with altered PHYB1 and/or PHYB2 expression were used in phenotypic assays and transcript profiling with qRT-PCR. RNA-Seq was used to identify transcriptional responses to enriched FR light. All three PHYs were differentially expressed among tissue types and PHYBs were targeted to the nucleus under white light. Populus PHYB1 rescued Arabidopsis phyB mutant phenotypes. Phenotypes of Populus transgenic lines and the expression of candidate shade response genes suggested that PHYB1 and PHYB2have distinct yet overlapping functions. RNA-Seq analysis indicated that genes associated with cell wall modification and brassinosteroid signaling were induced under enriched FR light in Populus. This study is an initial attempt at deciphering the role of Populus PHYs by evaluating transcriptional reprogramming to enriched FR and demonstrates functional diversity and overlap of the Populus PHYB1 and PHYB2 in regulating shade responses.

Light-dependent, Dark-promoted Interaction between Arabidopsis Cryptochrome 1 and Phytochrome B Proteins

Plant photoreceptors transduce environmental light cues to downstream signaling pathways, regulating a wide array of processes during growth and development. Two major plant photoreceptors with critical roles in photomorphogenesis are phytochrome B (phyB), a red/far-red absorbing photoreceptor, and cryptochrome 1 (CRY1), a UV-A/blue photoreceptor. Despite substantial genetic evidence for cross-talk between phyB and CRY1 pathways, a direct interaction between these proteins has not been observed. Here, we report that Arabidopsis phyB interacts directly with CRY1 in a light-dependent interaction. Surprisingly, the interaction is light-dissociated; CRY1 interacts specifically with the dark/far-red (Pr) state of phyB, but not with the red light-activated (Pfr) or the chromophore unconjugated form of the enzyme. The interaction is also regulated by light activation of CRY1; phyB Pr interacts only with the unstimulated form of CRY1 but not with the photostimulated protein. Further studies reveal that a small domain extending from the photolyase homology region (PHR) of CRY1 regulates the specificity of the interaction with different conformational states of phyB. We hypothesize that in plants, the phyB/CRY1 interaction may mediate cross-talk between the red/far-red- and blue/UV-sensing pathways, enabling fine-tuning of light responses to different spectral inputs.

A Non-Covalently Attached Chromophore can Mediate Phytochrome B Signaling in Arabidopsis

Phytochrome B (phyB) is the major informational photoreceptor in light-grown plants. The phyB polypeptide is folded into two domains, the N-terminal domain and the C-terminal domain. The N-terminal domain covalently binds to the chromophore via a particular cysteine residue, which allows the holoprotein to absorb light and undergo a photoreversible conformational change. The N-terminal domain of phyB interacts with transcription factors, such as PIF3 (PHYTOCHROME-INTERACTING FACTOR 3), to transduce the light signal to downstream components. Since substitution of the chromophore attachment site, Cys357, with alanine (C357A) abolishes the biological activity of Arabidopsis phyB, the covalent attachment with the chromophore is widely assumed to be necessary for phyB signal transduction. In this study, we show that Arabidopsis phyB is capable of transducing signals with a non-covalently retained chromophore. Substituting the Tyr276 residue of phyB with histidine (Y276H) is known to confer constitutive phyB signaling. PhyB containing both Y276H and C357A substitutions exhibited light-independent biological activity in transgenic Arabidopsis plants in a chromophore-dependent manner. Spectrophotometric analysis showed that the N-terminal domain of phyB containing just the C357A substitution could retain the chromophore non-covalently. The N-terminal domain containing both the Y276H and C357A substitutions interacted with PIF3 in a light-independent but chromophore-dependent fashion in yeast two-hybrid assays. From these results, we conclude that the constitutive phyB signaling conferred by Y276H requires the chromophore, but that the chromophore does not need to be covalently bonded to phyB.

Residues Clustered in the Light-Sensing Knot of Phytochrome B are Necessary for Conformer-Specific Binding to Signaling Partner PIF3

The bHLH transcription factor, PHYTOCHROME INTERACTING FACTOR 3 (PIF3), interacts specifically with the photoactivated, Pfr, form of Arabidopsis phytochrome B (phyB). This interaction induces PIF3 phosphorylation and degradation in vivo and modulates phyB-mediated seedling deetiolation in response to red light. To identify missense mutations in the phyB N-terminal domain that disrupt this interaction, we developed a yeast reverse-hybrid screen. Fifteen individual mutations identified in this screen, or in previous genetic screens for Arabidopsis mutants showing reduced sensitivity to red light, were shown to also disrupt light-induced binding of phyB to PIF3 in in vitro co-immunoprecipitation assays. These phyB missense mutants fall into two general classes: Class I (eleven mutants) containing those defective in light signal perception, due to aberrant chromophore attachment or photoconversion, and Class II (four mutants) containing those normal in signal perception, but defective in the capacity to transduce this signal to PIF3. By generating a homology model for the three-dimensional structure of the Arabidopsis phyB chromophore-binding region, based on the crystal structure of Deinococcus radiodurans phytochrome, we predict that three of the four Class II mutated phyB residues are solvent exposed in a cleft between the presumptive PAS and GAF domains. This deduction suggests that these residues could be directly required for the physical interaction of phyB with PIF3. Because these three residues are also necessary for phyB-imposed inhibition of hypocotyl elongation in response to red light, they are functionally necessary for signal transfer from photoactivated phyB, not only to PIF3 and other related bHLH transcription factors tested here, but also to other downstream signaling components involved in regulating seedling deetiolation.

Regulation of Phytochrome B Nuclear Localization through Light-Dependent Unmasking of Nuclear-Localization Signals

Phytochromes are red and far-red photoreceptors that regulate plant growth and development in response to environmental light cues. Phytochromes exist in two photo-interconvertible conformational states: an inactive Pr form and an active Pfr form. The alteration of phytochromes' subcellular location functions as a major regulatory mechanism of their biological activities. Whereas phytochromes in the Pr form localize in the cytoplasm, phytochromes in the Pfr form accumulate in the nucleus, where they interact with transcription factors to regulate gene expression. The molecular details of the regulation of phytochrome translocation by light are poorly understood. Using Arabidopsis phyB as a model, we demonstrate that the C-terminal PAS-related domain (PRD) is both necessary and sufficient for phyB nuclear import and that the entire C terminus is required for nuclear-body (NB) localization. We also show that phyB's N-terminal bilin lyase domain (BLD) and PHY domain interact directly with the PRD in a light-dependent manner. In vivo localization studies indicate that BLD-PHY is sufficient to regulate phyB's nuclear accumulation. For phyB nuclear localization, our results suggest a molecular mechanism in which the nuclear-localization signal in the PRD is masked by interactions with phyB's chromophore-attachment domains and unmasked by light-dependent conformational changes.

Signaling activities among the Arabidopsis phyB/D/E-type phytochromes: a major role for the central region of the apoprotein.

The Arabidopsis phyB, phyD, and phyE phytochromes regulate plant developmental and growth responses to continuous red light (R) and to the ratio of R to far-red (FR) light. The activities of these three photoreceptors in the control of seedling growth have been compared using a transgenic assay based upon induction of R-hypersensitivity of hypocotyl elongation by overexpression of the apoproteins from the 35S promoter. 35S-phyB, 35S-phyD, and 35S-phyE lines expressing similar levels of the respective phytochromes were isolated. Under pulses of R, phyB is very active in inducing a dwarf hypocotyl phenotype, whereas phyD and phyE are inactive. Under high-fluence continuous R, phyD shows a gain in activity whereas phyE does not. These results demonstrate significant differences in the inherent regulatory activities of these receptor apoproteins. To localize the sequence determinants of these functional differences, chimeric proteins were constructed by shuffling amino-terminal, central, and carboxy-terminal regions of phyB and phyD. Overexpression analysis of the phyB/D chimeras shows that it is the central region of these proteins that is most critical in determining their respective activities.

Differential activities of the Arabidopsis phyB/D/E phytochromes in complementing phyB mutant phenotypes.

The Arabidopsis phyB, phyD, and phyE phytochromes regulate plant developmental and growth responses to continuous red light and to the ratio of red to far-red light. They are also more highly related in sequence to each other and more recently derived evolutionarily than phyA and phyC. In order to directly compare the signaling activities of these three photoreceptor apoproteins, an assay was developed based upon complementation of the phyB-1 null mutant phenotype with transgenes consisting of the PHYB promoter (PB) driving expression of the PHYB, PHYD, or PHYE coding sequences. Expression analysis indicates that the PB-phyB, PB-phyD, and PB-phyE transgenes are transcriptionally and translationally active. However, whereas the PB-phyB transgene complements the phyB-1 red light hypocotyl growth phenotype completely, the PB-phyD and PB-phyE transgenes are only weakly active in restoring seedling growth regulation. Red light fluence curves indicate that this difference is not likely to be due to differences in dark reversion rates. The PB-phyD and PB-phyE transgenes also both partially restore the rosette leaf morphology phenotype of the phyB-1 mutant. However, the PB-phyD gene complements the early flowering phenotype of phyB-1 very strongly whereas PB-phyE does not. These results demonstrate that Arabidopsis phyB-related apoproteins differ significantly in their capacities to signal in various seedling and adult plant phytochrome responses.

The flowering transition and florigen

What is the flowering transition? It marks the end of vegetative growth and the beginning of reproductive development in flowering plants. The physiological changes that cause it are referred to as floral induction.What is SAM? The shoot apical meristem — a group of undifferentiated progenitor cells that occupies the tip of the stem. It gives rise to both vegetative organs (leaves) and reproductive structures (inflorescence and flower).How is the flowering transition regulated? By environmental cues such as the photoperiod, temperature, and water and nutrient availability. Photoperiod is a measure of the season, and most plants time their reproductive development to the appropriate season. Most annuals flower in late summer and fall when daylight becomes short and are called short-day plants. Biennials or perennials have sufficient vegetative growth in spring and flower in spring or early summer to take advantage of the resourceful summer for reproductive development. These are called long-day plants. Some plants flower at a certain developmental stage regardless of the photoperiod, and are called autonomous-flowering or day-neutral plants.What is florigen? Florigen represents a signal that triggers flowering, and was thought to be a specific hormone. Early grafting experiments showed that photoperiod is sensed by leaves, yet affects flowering and the SAM. Therefore, a signal must be produced in the leaves and transported to the SAM. It remains a controversial proposition. More recently, others have proposed that substances are transmitted from leaves to the SAM and together trigger flowering. In fact, both plant hormones and chemical assimilates such as sugar can regulate flowering time.Is that all that's known? Recent molecular genetic studies in Arabidopsis have made considerable progress in understanding the control of floral induction. The perception of photoperiod depends on plant photoreceptors, phytochromes and cryptochromes, which detect red/far-red and blue/UV-A light, respectively. In particular the Arabidopsis phytochromes PHYA and PHYB play a role in regulating flowering time. And the Arabidopsis cryptochrome CRY2 is important for sensing photoperiod and promotes flowering.How do photoreceptors affect floral initiation? Photoreceptors regulate the plant circadian clock which regulates flowering. The photoreceptors regulate the expression of flowering time genes such as CONSTANTS (CO). CO promotes floral induction only under long-day conditions. Expression of CO is regulated by photoperiod and is decreased in cry2 mutants. CO likely acts between the circadian clock and the control of flowering.Are CO and florigen connected?CO is in leaves, yet can activate the expression of two genes, LEAFY and APETALA1, expressed in reproductive meristems, that directly control the initiation of flower development. Therefore CO could regulate the synthesis of a chemical that might fit the definition of florigen. Alternatively, CO could control the levels of several substances, which together regulate the flowering transition. Similar arguments can be made for a role of the maize gene indeterminate in regulating the synthesis of transportable substances because of its function in regulating floral initiation and its expression in leaves.Now the future… Recent studies provide no evidence to distinguish different models for substances that are transmitted from leaves to the SAM. Further studies are required to provide new insights into the area of flower transition, including understanding the properties of the proposed florigen.Where can I find out more?