Ferenc Nagy

Brassinosteroids Rescue the Deficiency of CYP90, a Cytochrome P450, Controlling Cell Elongation and De-etiolation in Arabidopsis

The cpd mutation localized by T-DNA tagging on Arabidopsis chromosome 5-14.3 inhibits cell elongation controlled by the ecdysone-like brassinosteroid hormone brassinolide. The cpd mutant displays de-etiolation and derepression of light-induced genes in the dark, as well as dwarfism, male sterility, and activation of stress-regulated genes in the light. The CPD gene encodes a cytochrome P450 (CYP90) sharing homologous domains with steroid hydroxylases. The phenotype of the cpd mutant is restored to wild type both by feeding with C23-hydroxylated brassinolide precursors and by ectopic overexpression of the CPD cDNA. Brassinosteroids also compensate for different cell elongation defects of Arabidopsis det, cop, fus, and axr2 mutants, indicating that these steroids play an essential role in the regulation of plant development.

Perception of UV-B by the Arabidopsis UVR8 Protein

A plant ultraviolet-B photoreceptor uses a tryptophan-based chromophore. To optimize their growth and survival, plants perceive and respond to ultraviolet-B (UV-B) radiation. However, neither the molecular identity of the UV-B photoreceptor nor the photoperception mechanism is known. Here we show that dimers of the UVR8 protein perceive UV-B, probably by a tryptophan-based mechanism. Absorption of UV-B induces instant monomerization of the photoreceptor and interaction with COP1, the central regulator of light signaling. Thereby this signaling cascade controlled by UVR8 mediates UV-B photomorphogenic responses securing plant acclimation and thus promotes survival in sunlight.

Experimental validation of a predicted feedback loop in the multi‐oscillator clock of Arabidopsis thaliana

Our computational model of the circadian clock comprised the feedback loop between LATE ELONGATED HYPOCOTYL (LHY), CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) and TIMING OF CAB EXPRESSION 1 (TOC1), and a predicted, interlocking feedback loop involving TOC1 and a hypothetical component Y. Experiments based on model predictions suggested GIGANTEA (GI) as a candidate for Y. We now extend the model to include a recently demonstrated feedback loop between the TOC1 homologues PSEUDO‐RESPONSE REGULATOR 7 (PRR7), PRR9 and LHY and CCA1. This three‐loop network explains the rhythmic phenotype of toc1 mutant alleles. Model predictions fit closely to new data on the gi;lhy;cca1 mutant, which confirm that GI is a major contributor to Y function. Analysis of the three‐loop network suggests that the plant clock consists of morning and evening oscillators, coupled intracellularly, which may be analogous to coupled, morning and evening clock cells in Drosophila and the mouse.

The ELF4 gene controls circadian rhythms and flowering time in Arabidopsis thaliana

Many plants use day length as an environmental cue to ensure proper timing of the switch from vegetative to reproductive growth. Day-length sensing involves an interaction between the relative length of day and night, and endogenous rhythms that are controlled by the plant circadian clock. Thus, plants with defects in circadian regulation cannot properly regulate the timing of the floral transition. Here we describe the gene EARLY FLOWERING 4 (ELF4), which is involved in photoperiod perception and circadian regulation. ELF4 promotes clock accuracy and is required for sustained rhythms in the absence of daily light/dark cycles. elf4 mutants show attenuated expression of CIRCADIAN CLOCK ASSOCIATED 1 (CCA1), a gene that is thought to function as a central oscillator component. In addition, elf4 plants transiently show output rhythms with highly variable period lengths before becoming arrhythmic. Mutations in elf4 result in early flowering in non-inductive photoperiods, which is probably caused by elevated amounts of CONSTANS (CO), a gene that promotes floral induction.

Light Quality–Dependent Nuclear Import of the Plant Photoreceptors Phytochrome A and B

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.

Streptomycin resistant and sensitive somatic hybrids of Nicotiana tabacum + Nicotiana knightiana: correlation of resistance to N. tabacum plastids.

SummaryProtoplasts of Nicotiana tabacum SRI (streptomycin resistant) and of Nicotiana knightiana (streptomycin sensitive) were fused using polyethylene glycol treatment. From three heterokaryons 500 clones were obtained. From the 43 which were further investigated, 6 resistant, 3 sensitive, and 34 chimeric (consisting of resistant and sensitive sectors) calli were found. From eight clones, a total of 39 plants were regenerated and identified as somatic hybrids. Chloroplast type (N. tabacum = NT or N. knightiana = NK) in the plants was determined on the basis of the species specific EcoRI restriction pattern of the chloroplast DNA. Regenerates contained NT (13 plants) or NK (15 plants) plastids but only the plants with the NT chloroplasts were resistant to streptomycin. This finding and our earlier data on uniparental inheritance points to the chloroplasts as the carriers of the streptomycin resistance factor.

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 ∼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.

Interaction of COP1 and UVR8 regulates UV-B-induced photomorphogenesis and stress acclimation in Arabidopsis

The ultraviolet‐B (UV‐B) portion of the solar radiation functions as an environmental signal for which plants have evolved specific and sensitive UV‐B perception systems. The UV‐B‐specific UV RESPONSE LOCUS 8 (UVR8) and the multifunctional E3 ubiquitin ligase CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1) are key regulators of the UV‐B response. We show here that uvr8‐null mutants are deficient in UV‐B‐induced photomorphogenesis and hypersensitive to UV‐B stress, whereas overexpression of UVR8 results in enhanced UV‐B photomorphogenesis, acclimation and tolerance to UV‐B stress. By using sun simulators, we provide evidence at the physiological level that UV‐B acclimation mediated by the UV‐B‐specific photoregulatory pathway is indeed required for survival in sunlight. At the molecular level, we demonstrate that the wild type but not the mutant UVR8 and COP1 proteins directly interact in a UV‐B‐dependent, rapid manner in planta. These data collectively suggest that UV‐B‐specific interaction of COP1 and UVR8 in the nucleus is a very early step in signalling and responsible for the plants coordinated response to UV‐B ensuring UV‐B acclimation and protection in the natural environment.

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.

Chloroplast transfer in Nicotiana based on metabolic complementation between irradiated and iodoacetate treated protoplasts

Protoplasts of a light sensitive plastome mutant of Nicotiana tabacum (2 n=48) were irradiated and fused with iodoacetate-treated Nicotiana plumbaginifolia (2 n=20) protoplasts. Treated parental protoplasts were unable to divide. Metabolic complementation, however, helped the recovery of interspecific fusion products which survived and formed calli. Altogether 40 clones were investigated. N. plumbaginifolia plants were obtained in 15 clones (38%), somatic hybrids in 23 clones, and both types of regenerates were found in 2 clones. Irradiation therefore significantly increased the frequency of segregant formation with the non-irradiated N. plumbaginifolia nuclei (the frequency was 1.4% in the absence of irradiation). Regenerated plants in most cases (31 out of 34) contained chloroplasts from the irradiated parent. In 6 clones plants were obtained with both types of chloroplast. Thus, irradiated N. tabacum chloroplasts had an improved chance of dominating the heterokaryonderived cells, many of which contained N. plumbaginifolia nucleus. The system described should be generally applicable for the transfer of chloroplasts without the use of selectable genetic markers.