Light-regulated nuclear import and degradation of Arabidopsis phytochrome-A N-terminal fragments.

The photoreceptor phytochrome-A (phyA) regulates germination and seedling establishment by mediating very low fluence (VLFR) and far-red high irradiance (FR-HIR) responses in Arabidopsis thaliana. In darkness, phyA homodimers exist in the biologically inactive Pr form and are localized in the cytoplasm. Light induces formation of the biologically active Pfr form and subsequent rapid nuclear import. PhyA Pfr, in contrast to the Pr form, is labile and has a half-life of ∼30 min. We produced transgenic plants in a phyA-201 null background that express the PHYA-yellow fluorescent protein (YFP) or the PHYA686-YFP-dimerization domain (DD) and PHYA686-YFP-DD-nuclear localization signal (NLS) or PHYA686-YFP-DD-nuclear exclusion signal (NES) fusion proteins. The PHYA686-YFP fusion proteins contained the N-terminal domain of phyA (686 amino acid residues), a short DD and the YFP. Here we report that (i) PHYA686-YFP-DD fusion protein is imported into the nucleus in a light-dependent fashion; (ii) neither of the PHYA686 fusion proteins is functional in FR-HIR and nuclear VLFR; and (iii) the phyA-dependent, blue light-induced inhibition of hypocotyl growth is mediated by the PHYA686-YFP-DD-NES but not by the PHYA686-YFP-DD-NLS and PHYA686-YFP-DD fusion proteins. We demonstrate that (i) light induces degradation of all PHYA N-terminal-containing fusion proteins and (ii) these N-terminal domain-containing fusion proteins including the constitutively nuclear PHYA686-YFP-DD-NLS and predominantly cytoplasmic PHYA686-YFP-DD-NES degrade at comparable rates but markedly more slowly than PHYA-YFP, whereas (iii) light-induced degradation of the native phyA is faster compared with PHYA-YFP.

Functional cross-talk between two-component and phytochrome B signal transduction in Arabidopsis.

The A-type response regulator ARR4 is an element in the two-component signalling network of Arabidopsis. ARR4 interacts with the N-terminus of the red/far-red light photoreceptor phytochrome B (phyB) and functions as a modulator of photomorphogenesis. In concert with other A-type response regulators, ARR4 also participates in the modulation of the cytokinin response pathway. Here evidence is presented that ARR4 directly modulates the activity state of phyB in planta, not only under inductive but also under extended irradiation with red light. Mutation of the phosphorylatable aspartate to asparagine within the receiver domain creates a version of ARR4 that negatively affects photomorphogenesis. Additional evidence suggests that ARR4 activity is regulated by a phosphorelay mechanism that depends on the AHK family of cytokinin receptors. Accordingly, the ability of ARR4 to function on phyB is modified by exogenous application of cytokinin. These results implicate a cross-talk between cytokinin and light signalling mediated by ARR4. This cross-talk enables the plant to adjust light reponsiveness to endogenous requirements in growth and development.

Functional characterization of phytochrome interacting factor 3 for the Arabidopsis thaliana circadian clockwork.

Light, in a quality- and quantity-dependent fashion, induces nuclear import of the plant photoreceptors phytochromes and promotes interaction of these receptors with transcription factors including PHYTOCHROME INTERACTING FACTOR 3 (PIF3). PIF3 was shown to form in vitro a ternary complex with the G-box element of the promoters of LATE ELONGATED HYPOCOTYL (LHY) and CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) and the Pfr conformer of phytochromes. CCA1 and LHY together with TIMING OF CAB EXPRESSION 1 (TOC1) constitute a transcriptional feed-back loop that is essential for a functional circadian clock in Arabidopsis. These findings led to the hypothesis that the PIF3-containing ternary complex regulates transcription of light-responsive genes and is involved in phototransduction to the central circadian clockwork. Here we report that (i) overexpression or lack of biologically functional PIF3 does not affect period length of rhythmic gene expression or red-light-induced resetting of the circadian clock and (ii) the transcription of PIF3 displays a low-amplitude circadian rhythm. We demonstrated previously that irradiation of etiolated seedlings induces rapid, phytochrome-controlled degradation of PIF3. Here we show that nuclear-localized PIF3 accumulates to relatively high levels by the end of the light phase in seedlings grown under diurnal conditions. Taken together, we show that (i) PIF3 does not play a significant role in controlling light input to and function of the circadian clockwork and (ii) a yet unknown mechanism limits phytochrome-induced degradation of PIF3 at the end of the day under diurnal conditions.

Constitutive photomorphogenesis 1 and multiple photoreceptors control degradation of phytochrome interacting factor 3, a transcription factor required for light signaling in Arabidopsis.

Light, in a quality- and quantity-dependent fashion, induces nuclear import of the plant photoreceptors phytochrome, promotes interaction of phytochrome A (phyA) and phyB with transcription factors including phytochrome interacting factor 3 (PIF3), and is thought to trigger a transcriptional cascade to regulate the expression of approximately 2500 genes in Arabidopsis thaliana. Here, we show that controlled degradation of the transcription factor PIF3 is a major regulatory step in light signaling. We demonstrate that accumulation of PIF3 in the nucleus in dark requires constitutive photomorphogenesis 1 (COP1), a negative regulator of photomorphogenesis, and show that red (R) and far-red light (FR) induce rapid degradation of the PIF3 protein. This process is controlled by the concerted action of the R/FR absorbing phyA, phyB, and phyD photoreceptors, and it is not affected by COP1. Rapid light-induced degradation of PIF3 indicates that interaction of PIF3 with these phytochrome species is transient. In addition, we provide evidence that the poc1 mutant, a postulated PIF3 overexpressor that displays hypersensitivity to R but not to FR, lacks detectable amounts of the PIF3 protein. Thus, we propose that PIF3 acts transiently, and its major function is to mediate phytochrome-induced signaling during the developmental switch from skotomorphogenesis to photomorphogenesis and/or dark to light transitions.

Nucleocytoplasmic partitioning of the plant photoreceptors phytochrome A, B, C, D, and E is regulated differentially by light and exhibits a diurnal rhythm.

The phytochrome family of plant photoreceptors has a central role in the adaptation of plant development to changes in ambient light conditions. The individual phytochrome species regulate different or partly overlapping physiological responses. We generated transgenic Arabidopsis plants expressing phytochrome A to E:green fluorescent protein (GFP) fusion proteins to assess the biological role of intracellular compartmentation of these photoreceptors in light-regulated signaling. We show that all phytochrome:GFP fusion proteins were imported into the nuclei. Translocation of these photoreceptors into the nuclei was regulated differentially by light. Light-induced accumulation of phytochrome species in the nuclei resulted in the formation of speckles. The appearance of these nuclear structures exhibited distinctly different kinetics, wavelengths, and fluence dependence and was regulated by a diurnal rhythm. Furthermore, we demonstrate that the import of mutant phytochrome B:GFP and phytochrome A:GFP fusion proteins, shown to be defective in signaling in vivo, is regulated by light but is not accompanied by the formation of speckles. These results suggest that (1) the differential regulation of the translocation of phytochrome A to E into nuclei plays a role in the specification of functions, and (2) the appearance of speckles is a functional feature of phytochrome-regulated signaling.

Plant RanGAPs are localized at the nuclear envelope in interphase and associated with microtubules in mitotic cells.

In animals and yeast, the small GTP-binding protein Ran has multiple functions - it is involved in mediating (i) the directional passage of proteins and RNA through the nuclear pores in interphase cells; and (ii) the formation of spindle asters, the polymerization of microtubules, and the re-assembly of the nuclear envelope in mitotic cells. Nucleotide binding of Ran is modulated by a series of accessory proteins. For instance, the hydrolysis of RanGTP requires stimulation by the RanGTPase protein RanGAP. Here we report the complementation of the yeast RanGAP mutant rna1 with Medicago sativa and Arabidopsis thaliana cDNAs encoding RanGAP-like proteins. Confocal laser microscopy of Arabidopsis plants overexpressing chimeric constructs of GFP with AtRanGAP1 and 2 demonstrated that the fusion protein is localized to patchy areas at the nuclear envelope of interphase cells. In contrast, the cellular distribution of RanGAPs in synchronized tobacco cells undergoing mitosis is characteristically different. Double-immunofluorescence shows that RanGAPs are co-localized with spindle microtubules during anaphase, with the microtubular phragmoplast and the surface of the daughter nuclei during telophase. Co-assembly of RanGAPs with tubulin correlates with these in vivo observations. The detected localization pattern is consistent with the postulated function of plant RanGAPs in the regulation of nuclear transport during interphase, and suggests a role for these proteins in the organization of the microtubular mitotic structures.