The SCL30a SR protein regulates ABA-dependent seed traits and germination under stress.

SR proteins are conserved RNA-binding proteins best known as splicing regulators that have also been implicated in other steps of gene expression. Despite mounting evidence for a role in plant development and stress responses, the molecular pathways underlying SR protein regulation of these processes remain poorly understood. Here we show that the plant-specific SCL30a SR protein negatively regulates ABA signaling to control seed traits and stress responses during germination in Arabidopsis. Transcriptome-wide analyses revealed that loss of SCL30a function barely affects splicing, but largely induces ABA-responsive gene expression and genes repressed during germination. Accordingly, scl30a mutant seeds display delayed germination and hypersensitivity to ABA and high salinity, while transgenic plants overexpressing SCL30a exhibit reduced ABA and salt stress sensitivity. An ABA biosynthesis inhibitor rescues the enhanced mutant seed stress sensitivity, and epistatic analyses confirm that this hypersensitivity requires a functional ABA pathway. Finally, seed ABA levels are unchanged by altered SCL30a expression, indicating that the gene promotes seed germination under stress by reducing sensitivity to the phytohormone. Our results reveal a new player in ABA-mediated control of early development and stress response.

Physiological Analysis of Phototropic Responses to Blue and Red Light in Arabidopsis.

Plants utilize light as sole energy source. To maximize light capture, they are able to detect the light direction and orient themselves toward the light source. This phototropic response is mediated by the plant blue-light photoreceptors phototropin1 and phototropin2 (phot1 and phot2). Although fully differentiated plants also exhibit this response, it can be best observed in etiolated seedlings. Differences in light between the illuminated and shaded site of a seedling stem lead to changes in the auxin distribution, resulting in cell elongation on the shaded site. Since phototropism connects light perception, signaling, and auxin transport, it is of great interest to analyze this response with a fast and simple method. Moreover, pre-exposure to red light enhances the phototropic response via phytochrome A (phyA) and phyB action. Here we describe a method to analyze the phototropic response of Arabidopsis seedlings to blue light and the enhanced response with a red-light pretreatment. With numerous mutants available, its fast germination, and its small size, Arabidopsis is well suited for this analysis. Different genotypes can be simultaneously probed in less than a week.

Analysis of Phytochrome-Dependent Seed Germination in Arabidopsis.

Light-dependent seed germination guarantees seedling proximity to the soil surface, enabling quick photosynthetic energy supply. While seedling hypocotyl length is mainly used in phytochrome physiological assays to determine the functional impact of photoreceptor point mutations, different intracellular localizations, or the function of signal transduction components, phytochrome-controlled seed germination offers a different, very sensitive tool to test the phytochrome photoreceptor network. Photon fluences as low as 1 nmol m-2 are sufficient to elicit the phytochrome A (phyA)-dependent very low fluence response (VLFR), whereas higher fluences (> 10 µmol m-2) are needed to elicit the phyB-controlled and phyB-photoreversible low fluence response (LFR). Taking advantage of the different sensitivities of both phytochromes to different light qualities and quantities, a screening protocol is presented to score germination under different light conditions.

Arabidopsis phytochrome A nuclear translocation is mediated by a far-red elongated hypocotyl 1-importin complex.

Phytochrome A (phyA) is a red and far-red (FR) sensing photoreceptor regulating plant growth and development. Its biologically active FR-absorbing form Pfr translocates into the nucleus and subsequently regulates gene expression. Two transport facilitators, FR elongated hypocotyl 1 (FHY1) and FHY1-like (FHL), are crucial for its cytoplasmic-nuclear translocation. FHY1 interacts preferentially with activated phyA (Pfr) in assays with recombinant phyA and FHY1 and in vivo. Nuclear translocation of the phyA-FHY1 complex depends on a nuclear localization signal (NLS) of FHY1, which is recognized by IMPαs independently of phyA. The complex is guided along the actin cytoskeleton. Additionally, FHY1 has the ability to exit the nucleus via the exportin route, thus is able to repeatedly transport phyA molecules to the nucleus, balancing the nucleo-cytoplasmic distribution. The direction of FHY1s transport appears to depend on its phosphorylation state in different compartments. Phosphorylated serins close to the NLS prevent FHY1 binding to IMPα. The work presented here elucidates key steps of the mechanism by which photoactivated phyA translocates to the nucleus.

Physiological Analysis of Phototropic Responses in Arabidopsis.

Plants utilize light as sole energy source. To maximize light capture they are able to detect the light direction and orient themselves towards the light source. This phototropic response is mediated by the plant blue light photoreceptors phototropin1 and 2 (phot1 and phot2). Although fully differentiated plants also exhibit this response it can be best observed in etiolated seedlings. Differences in light between the illuminated and shaded site of a seedling stem lead to changes in the auxin-distribution, resulting in cell elongation on the shaded site. Since phototropism connects light perception, signaling, and auxin transport, it is of great interest to analyze this response with a fast and simple method.Here we describe a method to analyze the phototropic response of Arabidopsis seedlings. With numerous mutants available, its fast germination and its small size Arabidopsis is well suited for this analysis. Different genotypes can be simultaneously probed in less than a week.

Automatic Chloroplast Movement Analysis.

In response to low or high intensities of light, the chloroplasts in the mesophyll cells of the leaf are able to increase or decrease their exposure to light by accumulating at the upper and lower sides or along the side walls of the cell respectively. This movement, regulated by the phototropin blue light photoreceptors phot1 and phot2, results in a decreased or increased transmission of light through the leaf. This way the plant is able to optimize harvesting of the incoming light or avoid damage caused by excess light. Here we describe a method that indirectly measures the movement of chloroplasts by taking advantage of the resulting change in leaf transmittance. By using a microplate reader, quantitative measurements of chloroplast accumulation or avoidance can be monitored over time, for multiple samples with relatively little hands-on time.

Tyrosine 263 in cyanobacterial phytochrome Cph1 optimizes photochemistry at the prelumi-R→lumi-R step.

We report a low-temperature fluorescence spectroscopy study of the PAS-GAF-PHY sensory module of Cph1 phytochrome, its Y263F mutant (both with known 3D structures) as well as Y263H and Y263S to connect their photochemical parameters with intramolecular interactions. None of the holoproteins showed photochemical activity at low temperature, and the activation barriers for the Pr→lumi-R photoreaction (2.5-3.1 kJ mol(-1)) and fluorescence quantum yields (0.29-0.42) were similar. The effect of the mutations on Pr→Pfr photoconversion efficiency (ΦPr→Pfr) was observed primarily at the prelumi-R S0 bifurcation point corresponding to the conical intersection of the energy surfaces at which the molecule relaxes to form lumi-R or Pr, lowering ΦPr→Pfr from 0.13 in the wild type to 0.05-0.07 in the mutants. We suggest that the Ea activation barrier in the Pr* S1 excited state might correspond to the D-ring (C19) carbonyl - H290 hydrogen bond or possibly to the hindrance caused by the C13(1) /C17(1) methyl groups of the C and D rings. The critical role of the tyrosine hydroxyl group can be at the prelumi-R bifurcation point to optimize the yield of the photoprocess and energy storage in the form of lumi-R for subsequent rearrangement processes culminating in Pfr formation.

Mutants of phospholipase A (pPLA-I) have a red light and auxin phenotype.

pPLA-I is the evolutionarily oldest patatin-related phospholipase A (pPLA) in plants, which have previously been implicated to function in auxin and defence signalling. Molecular and physiological analysis of two allelic null mutants for pPLA-I [ppla-I-1 in Wassilewskija (Ws) and ppla-I-3 in Columbia (Col) ] revealed pPLA-I functions in auxin and light signalling. The enzyme is localized in the cytosol and to membranes. After auxin application expression of early auxin-induced genes is significantly slower compared with wild type and both alleles show a slower gravitropic response of hypocotyls, indicating compromised auxin signalling. Additionally, phytochrome-modulated responses like abrogation of gravitropism, enhancement of phototropism and growth in far red-enriched light are decreased in both alleles. While early flowering, root coils and delayed phototropism are only observed in the Ws mutant devoid of phyD, the light-related phenotypes observed in both alleles point to an involvement of pPLA-I in phytochrome signalling.

Protein phosphatase activity and acidic/alkaline balance as factors regulating the state of phytochrome A and its two native pools in the plant cell.

Phytochrome A (phyA), the most versatile plant phytochrome, exists in the two isoforms, phyA' and phyA'', differing by the character of its posttranslational modification, possibly, by phosphorylation at the N-terminal extension [Sineshchekov, V. (2010) J. Botany 2010, Article ID 358372]. This heterogeneity may explain the diverse modes of phyA action. We investigated possible roles of protein phosphatases activity and pH in regulation of the phyA pools' content in etiolated seedlings of maize and their extracts using fluorescence spectroscopy and photochemistry of the pigment. The phyA'/phyA'' ratio varied depending on the state of development of seedlings and the plant tissue/organ used. This ratio qualitatively correlated with the pH in maize root tips. In extracts, it reached a maximum at pH ≈ 7.5 characteristic for the cell cytoplasm. Inhibition of phosphatases of the PP1 and PP2A types with okadaic and cantharidic acids brought about phyA' decline and/or concomitant increase of phyA'' in coleoptiles and mesocotyls, but had no effect in roots, revealing a tissue/organ specificity. Thus, pH and phosphorylation status regulate the phyA'/phyA'' equilibrium and content in the etiolated (maize) cells and this regulation is connected with alteration of the processes of phyA' destruction and/or its transformation into the more stable phyA''.

A phytochrome-phototropin light signaling complex at the plasma membrane.

Phytochromes are red/far-red photochromic photoreceptors central to regulating plant development. Although they are known to enter the nucleus upon light activation and, once there, regulate transcription, this is not the complete picture. Various phytochrome effects are manifested much too rapidly to derive from changes in gene expression, whereas others seem to occur without phytochrome entering the nucleus. Phytochromes also guide directional responses to light, excluding a genetic signaling route and implying instead plasma membrane association and a direct cytoplasmic signal. However, to date, no such association has been demonstrated. Here we report that a phytochrome subpopulation indeed associates physically with another photoreceptor, phototropin, at the plasma membrane. Yeast two-hybrid methods using functional photoreceptor molecules showed that the phytochrome steering growth direction in Physcomitrella protonemata binds several phototropins specifically in the photoactivated Pfr state. Split-YFP studies in planta showed that the interaction occurs exclusively at the plasma membrane. Coimmunoprecipitation experiments provided independent confirmation of in vivo phy-phot binding. Consistent with this interaction being associated with a cellular signal, we found that phytochrome-mediated tropic responses are impaired in Physcomitrella phot(-) mutants. Split-YFP revealed a similar interaction between Arabidopsis phytochrome A and phototropin 1 at the plasma membrane. These associations additionally provide a functional explanation for the evolution of neochrome photoreceptors. Our results imply that the elusive phytochrome cytoplasmic signal arises through binding and coaction with phototropin at the plasma membrane.

Cytoplasmic phytochrome action.

Phytochrome photoperception is a common mechanism for the detection of red and far-red light in bacteria, cyanobacteria, fungi and plants. However, the responses following phytochrome activation appear to be quite diverse between species. Lower plants, such as mosses, show phytochrome-mediated directional responses, namely phototropism and polarotropism. These cannot be explained by nuclear gene regulation and are thought to be triggered by phytochromes in the cytoplasm or at the plasma membrane. In higher plants, similar directional responses are mediated via phototropin, a blue light receptor, with phytochromes mainly controlling morphogenetic responses through gene regulation. However, cytoplasmic phytochrome responses exist in higher plants too, which appear to be intertwined with directional blue light perception. By summarizing the respective findings, a possible conservation of cytoplasmic phytochrome function in higher and lower plants is addressed here.

Arabidopsis fhl/fhy1 double mutant reveals a distinct cytoplasmic action of phytochrome A.

Phytochrome A (phyA) plays an important role during germination and early seedling development. Because phyA is the primary photoreceptor for the high-irradiance response and the very-low-fluence response, it can trigger development not only in red and far-red (FR) light but also in a wider range of light qualities. Although phyA action is generally associated with translocation to the nucleus and regulation of transcription, there is evidence for additional cytoplasmic functions. Because nuclear accumulation of phyA has been shown to depend on far-red-elongated hypocotyl 1 (FHY1) and FHL (FHY1-like), investigation of phyA function in a double fhl/fhy1 mutant might be valuable in revealing the mechanism of phyA translocation and possible cytoplasmic functions. In fhl/fhy1, the FR-triggered nuclear translocation of phyA could no longer be detected but could be restored by transgenic expression of CFP:FHY1. Whereas the fhl/fhy1 mutant showed a phyA phenotype in respect to hypocotyl elongation and cotyledon opening under high-irradiance response conditions as well as a typical phyA germination phenotype under very-low-fluence response conditions, fhl/fhy1 showed no phenotype with respect to the phyA-dependent abrogation of negative gravitropism in blue light and in red-enhanced phototropism, demonstrating clear cytoplasmic functions of phyA. Disturbance of phyA nuclear import in fhl/fhy1 led to formation of FR-induced phyA:GFP cytoplasmic foci resembling the sequestered areas of phytochrome. FHY1 and FHL play crucial roles in phyA nuclear translocation and signaling. Thus the double-mutant fhl/fhy1 allows nuclear and cytoplasmic phyA functions to be separated, leading to the novel identification of cytoplasmic phyA responses.

Real time spectral analysis during phytochrome chromophore and chromoprotein purification.

The plant photoreceptor phytochrome senses light quality and quantity in the red region of the spectrum, directing adaptation and development. The functional holo-protein is a dimeric chromoprotein which is formed by an autoassembly reaction between the translation product and the open chain tetrapyrroles phytochromobilin (PPhiB) or phycocyanobilin (PCB). We are interested in structure/function relationships within the phytochrome molecule, in particular chromophore/protein interaction during the assembly and photoactivation, using IR and NMR spectroscopy. For this we use an automated F/HPLC system running in a darkroom to purify large amounts of protein and chromophore separately. To obtain highly pure PCB chromophore we developed improved extraction and purification methods in which the final step is RPC-18 HPLC. As there are many spectrally only slightly different tetrapyrroles in the extract, the triple-wavelength monitoring offered by the F/HPLC detector was inadequate for distinguishing between PCB and impurities. Furthermore, lambda(max) for the phytochrome Pfr signalling state lies between 705 and 730 nm, beyond the range of the detector. Also, as both holo-protein and chromophore are photoactive, we wished to minimize light exposure of the eluate. We therefore implemented a miniature CCD-based flow UV-vis spectrophotometer using a xenon flash and quartz fiber optics enabling us monitor the entire 250-800 nm spectrum of the eluate to an accuracy of +/-3 x 10(-3)A in real time. The instrumentation described can be added to any chromatographic system, thereby allowing the purification of any molecule to be monitored easily and efficiently.

FHL is required for full phytochrome A signaling and shares overlapping functions with FHY1.

Phytochrome A (phyA) plays a primary role in initiating seedling de-etiolation and is the only plant photoreceptor known to be activated by far-red light (FR). The signaling intermediate FHY1 appears to either participate directly in relaying the phyA signal or to positively regulate a critical signaling event(s) downstream of phyA activation. Here we identify a homolog of FHY1 named FHL (FHY1-like) as a novel signaling factor essential for complete responsiveness to phyA. FHL possesses functional nuclear localization and nuclear export signals. Lines in which FHL function was abolished by insertional mutagenesis or attenuated by RNAi-mediated suppression displayed a weaker hyposensitivity to continuous FR than fhy1 null mutants and most reported phyA signaling mutants. However, hypocotyl elongation assays indicated that suppression of FHL expression in fhy1-3 caused an insensitivity of hypocotyl elongation to FR and blue light (B) indistinguishable from that seen in phyA. Real-time PCR indicates that in FR, FHY1 transcripts are approximately 15-fold more abundant than FHL transcripts. Although both FHY1 and FHL are capable of homo- and hetero-interaction via their C-termini, the ability of FHL overexpression to restore wild-type (WT) morphological and molecular phenotypes to fhy1-3 seedlings suggests that the extreme insensitivity to FR associated with suppression of FHL expression in fhy1-3 cannot be accounted for by a critical role for FHY1-FHL heterodimers in phyA signal transmission. Rather, we suggest that the relative abundances of FHY1 and FHL in WT plants account for the differences in the severity of fhy1 and fhl mutations. As for FHY1, FHL transcript accumulation is dependent on FHY3 and is decreased after exposure to FR, R or B light. These findings reiterate the prevalence of partial degeneracy in plant signaling networks that regulate responses crucial to survival.

The nuclear localization signal and the C-terminal region of FHY1 are required for transmission of phytochrome A signals.

Plants use the family of phytochrome photoreceptors to sense their light environment in the red/far-red region of the spectrum. Phytochrome A (phyA) is the primary photoreceptor that regulates germination and early seedling development. This phytochrome mediates seedling de-etiolation for the developmental transition from heterotrophic to photoauxotrophic growth. High intensity far-red light provides a way to specifically assess the role of phyA in this process and was used to isolate phyA-signaling intermediates. fhy1 and pat3 (renamed fhy1-3) are independently isolated alleles of a gene encoding a phyA signal transduction component. FHY1 is a small 24 kDa protein that shows no homology to known functional motifs, besides a small conserved septin-related domain at the C-terminus, a putative nuclear localization signal (NLS) and a putative nuclear exclusion signal (NES). Here we demonstrate that the septin-related domain is important for FHY1 to transmit phyA signals. Moreover, the putative NLS and NES of FHY1 are indeed involved in its nuclear localization and exclusion. Nuclear localization of FHY1 is needed for it to execute responses downstream of phyA. Together with the results from global expression analysis, our findings point to an important role of FHY1 in phyA signaling through its nuclear translocation and induction of gene expression.

Targeted knockout in Physcomitrella reveals direct actions of phytochrome in the cytoplasm.

The plant photoreceptor phytochrome plays an important role in the nucleus as a regulator of transcription. Numerous studies imply, however, that phytochromes in both higher and lower plants mediate physiological reactions within the cytoplasm. In particular, the tip cells of moss protonemal filaments use phytochrome to sense light direction, requiring a signaling system that transmits the directional information directly to the microfilaments that direct tip growth. In this work we describe four canonical phytochrome genes in the model moss species Physcomitrella patens, each of which was successfully targeted via homologous recombination and the distinct physiological functions of each gene product thereby identified. One homolog in particular mediates positive phototropism, polarotropism, and chloroplast movement in polarized light. This photoreceptor thus interacts with a cytoplasmic signal/response system. This is our first step in elucidating the cytoplasmic signaling function of phytochrome at the molecular level.