Light and gibberellins (GAs) mediate many essential and partially overlapping plant developmental processes. DELLA proteins are GA-signalling repressors that block GA-induced development 1 . GA induces degradation of DELLA proteins via the ubiquitin/ proteasome pathway 2 , but light promotes accumulation of DELLA proteins by reducing GA levels 3 . It was proposed that DELLA proteins restrain plant growth largely through their effect on gene expression 4,5 . However, the precise mechanism of their function in coordinating GA signalling and gene expression remains unknown. Here we characterize a nuclear protein interaction cascade mediating transduction of GA signals to the activity regulation of a light-responsive transcription factor. In the absence of GA, nuclear-localized DELLA proteins accumulate to higher levels, interact with phytochrome-interacting factor 3 (PIF3, a bHLH-type transcription factor) and prevent PIF3 from binding to its target gene promoters and regulating gene expression, and therefore abrogate PIF3-mediated light control of hypocotyl elongation. In the presence of GA, GID1 proteins (GA receptors) elevate their direct interaction with DELLA proteins in the nucleus, trigger DELLA protein's ubiquitination and proteasome-mediated degradation, and thus release PIF3 from the negative effect of DELLA proteins.Light and GA interact during Arabidopsis thaliana seedling development, regulating hypocotyl elongation, cotyledon opening and light-responsive gene expression; their pathways seem to converge at regulation of the abundance of DELLA proteins (GA pathway repressors) 3,6 . Arabidopsis has five DELLA proteins-RGA, GAI, RGL1, RGL2 and RGL3-defined by their unique DELLA domain and a conserved GRAS domain 4 . To analyse them in vivo, we raised antibodies against endogenous RGA and generated transgenic Arabidopsis expressing each of the five DELLA proteins with tandem affinity purification (TAP) tags ( Supplementary Fig. 1). The response of DELLA protein levels to exogenously applied GA 3 (an active form of GA) or PAC (paclobutrazol, a GA biosynthesis inhibitor) was examined. We found that one-hour-long GA treatment eliminates the majority of DELLA proteins, and this GA effect can be largely prevented by 100 mM MG132 (a 26S proteasome-specific inhibitor). PAC, on the other hand, promotes over-accumulation of DELLA proteins (Fig. 1). These results show for the first time in Arabidopsis that all the DELLA proteins are under negative control by GA and the proteasome. Next, we generated lines expressing TAPtagged RGAD17 and GAID17, which lack a 17 amino acid motif within the DELLA domain that is required for GA-induced degradation 7,8 . As expected, TAP-RGAD17 and TAP-GAID17 are completely resistant to GA and accumulate at higher levels than wild-type proteins, which cannot be further increased by PAC (Fig. 1, and *These authors contributed equally to this work. WTAnti-RPN6Anti-RGA Immunoblot analysis of RGA (by anti-RGA antibody) and TAP-DELLA proteins (by anti-MYC antibody) in various light-grown Ara...
Following light-induced nuclear translocation, specific members of the phytochrome (phy) photoreceptor family (phyA to phyE) interact with bHLH transcription factors, such as PIF3, and induce changes in target-gene expression. The biochemical mechanism comprising signal transfer from phy to PIF3 has remained undefined but results in rapid degradation of PIF3. We provide evidence that photoactivation of phy induces rapid in vivo phosphorylation of PIF3 preceding degradation. Both phyA and phyB redundantly induce this PIF3 phosphorylation, as well as nuclear speckle formation and degradation, by direct interaction with PIF3 via separate binding sites. These data suggest that phy-induced phosphorylation of proteins such as PIF3 may represent the primary intermolecular signaling transaction of the activated photoreceptor, tagging the target protein for proteosomal degradation, possibly in nuclear speckles.
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 ;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.
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 lightregulated 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.
The phytochrome (phy) family of plant photoreceptors controls various aspects of photomorphogenesis. Overexpression of rice phyA-green fluorescent protein (GFP) and tobacco phyB-GFP fusion proteins in tobacco results in functional photoreceptors. phyA-GFP and phyB-GFP are localized in the cytosol of dark-adapted plants. In our experiments, red light treatment led to nuclear translocation of phyA-GFP and phyB-GFP, albeit with different kinetics. Red light-induced nuclear import of phyB-GFP, but not that of phyA-GFP, was inhibited by far-red light. Far-red light alone only induced nuclear translocation of phyA-GFP. These observations indicate that nuclear import of phyA-GFP is controlled by a very low fluence response, whereas translocation of phyB-GFP is regulated by a low fluence response of phytochrome. Thus, light-regulated nucleocytoplasmic partitioning of phyA and phyB is a major step in phytochrome signaling.
The phytochrome family of red/far-red photoreceptors is involved in the regulation of a wide range of developmental responses in plants. The Arabidopsis genome contains five phytochromes (phyA-E), among which phyA and phyB play the most important roles. Phytochromes localize to the cytosol in the dark and accumulate in the nucleus under light conditions, inducing specific phytochrome-mediated responses. Light-regulated nuclear accumulation of the phytochrome photoreceptors is therefore considered a key regulatory step of these pathways. In fact, one of the most severe phyA signaling mutants, fhy1 (far red elongated hypocotyl 1), is strongly affected in nuclear accumulation of phyA. The fhy1 fhl (fhy1 like) double mutant, lacking both FHY1 and its only close homolog FHL, is virtually blind to far-red light like phyA null seedlings. Here we show that FHL accounts for residual amounts of phyA in the nucleus in a fhy1 background and that nuclear accumulation of phyA is completely inhibited in an fhy1 FHL RNAi knock-down line. Moreover, we demonstrate that FHL and phyA interact with each other in a light-dependent manner and that they co-localize in light-induced nuclear speckles. We also identify a phyA-binding site at the C-terminus of FHY1 and FHL, and show that the N-terminal 406 amino acids of phyA are sufficient for the interaction with FHY1/FHL.
The phytochrome (phy) family of plant photoreceptors controls various aspects of photomorphogenesis. Overexpression of rice phyA-green fluorescent protein (GFP) and tobacco phyB-GFP fusion proteins in tobacco results in functional photoreceptors. phyA-GFP and phyB-GFP are localized in the cytosol of dark-adapted plants. In our experiments, red light treatment led to nuclear translocation of phyA-GFP and phyB-GFP, albeit with different kinetics. Red light-induced nuclear import of phyB-GFP, but not that of phyA-GFP, was inhibited by far-red light. Far-red light alone only induced nuclear translocation of phyA-GFP. These observations indicate that nuclear import of phyA-GFP is controlled by a very low fluence response, whereas translocation of phyB-GFP is regulated by a low fluence response of phytochrome. Thus, light-regulated nucleocytoplasmic partitioning of phyA and phyB is a major step in phytochrome signaling.
The most hazardous span in the life of green plants is the period after germination when the developing seedling must reach the state of autotrophy before the nutrients stored in the seed are exhausted. The need for an economically optimized utilization of limited resources in this critical period is particularly obvious in species adopting the dispersal strategy of producing a large amount of tiny seeds. The model plant Arabidopsis thaliana belongs to this category. Arabidopsis seedlings promote root development only in the light. This response to light has long been recognized and recently discussed in terms of an organ-autonomous feature of photomorphogenesis directed by the red/blue light absorbing photoreceptors phytochrome and cryptochrome and mediated by hormones such as auxin and/or gibberellin. Here we show that the primary root of young Arabidopsis seedlings responds to an interorgan signal from the cotyledons and that phloem transport of photosynthesis-derived sugar into the root tip is necessary and sufficient for the regulation of root elongation growth by light.
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