Light-dependent translocation of a phytochrome B-GFP fusion protein to the nucleus in transgenic Arabidopsis.
Phytochrome is a ubiquitous photoreceptor of plants and is encoded by a small multigene family. We have shown recently that a functional nuclear localization signal may reside within the COOH-terminal region of a major member of the family, phytochrome B (phyB) (Sakamoto, K., and A. Nagatani. 1996. Plant J. 10:859-868). In the present study, a fusion protein consisting of full-length phyB and the green fluorescent protein (GFP) was overexpressed in the phyB mutant of Arabidopsis to examine subcellular localization of phyB in intact tissues. The resulting transgenic lines exhibited pleiotropic phenotypes reported previously for phyB overexpressing plants, suggesting that the fusion protein is biologically active. Immunoblot analysis with anti-phyB and anti-GFP monoclonal antibodies confirmed that the fusion protein accumulated to high levels in these lines. Fluorescence microscopy of the seedlings revealed that the phyB-GFP fusion protein was localized to the nucleus in light grown tissues. Interestingly, the fusion protein formed speckles in the nucleus. Analysis of confocal optical sections confirmed that the speckles were distributed within the nucleus. In contrast, phyB-GFP fluorescence was observed throughout the cell in dark-grown seedlings. Therefore, phyB translocates to specific sites within the nucleus upon photoreceptor activation. (+info)
poc1: an Arabidopsis mutant perturbed in phytochrome signaling because of a T DNA insertion in the promoter of PIF3, a gene encoding a phytochrome-interacting bHLH protein.
The phytochrome family of informational photoreceptors has a central role in regulating light-responsive gene expression, but the mechanism of intracellular signal transduction has remained elusive. In a genetic screen for T DNA-tagged Arabidopsis mutants affected in early signaling intermediates, we identified poc1 (photocurrent 1), which exhibits enhanced responsiveness to red light. This phenotype is absent in a phyB (phytochrome B) null mutant background, indicating that the poc1 mutation enhances phyB signal transduction. The T DNA insertion in poc1 was found to be located in the promoter region of PIF3, a gene encoding a basic helix-loop-helix protein. The mutant phenotype seems to result from insertion-induced overexpression of this gene in red-light-grown seedlings, consistent with PIF3 functioning as a positively acting signaling intermediate. These findings, combined with data from a separate yeast two-hybrid screen that identified PIF3 as a phytochrome-interacting factor necessary for normal signaling, provide evidence that phytochrome signal transduction may include a direct pathway to photoresponsive nuclear genes via physical interaction of the photoreceptor molecules with the potential transcriptional regulator PIF3. (+info)
Expansins are conserved in conifers and expressed in hypocotyls in response to exogenous auxin.
Differential display reverse transcription-polymerase chain reaction was used to detect the induction of gene expression during adventitious root formation in loblolly pine (Pinus taeda) after treatment with the exogenous auxin indole-3-butyric acid. A BLAST search of the GenBank database using one of the clones obtained revealed very strong similarity to the alpha-expansin gene family in angiosperms. A near-full-length loblolly pine alpha-expansin sequence was obtained using 5'- and 3'-rapid amplification of cDNA end cloning, and the deduced amino acid sequence was highly conserved relative to those of angiosperm expansins. Northern analysis indicates that alpha-expansin mRNA expression increases 50- to 100-fold in the base of hypocotyl stem cuttings from loblolly pine seedlings in response to indole-3-butyric acid, with peak expression occurring 24 to 48 h after induction. (+info)
Ethylene plays multiple nonprimary roles in modulating the gravitropic response in tomato.
Ethylene is known to interact with auxin in regulating stem growth, and yet evidence for the role of ethylene in tropic responses is contradictory. Our analysis of four mutants of tomato (Lycopersicon esculentum) altered in their response to gravity, auxin, and/or ethylene revealed concentration-dependent modulation of shoot gravitropism by ethylene. Ethylene inhibitors reduce wild-type gravicurvature, and extremely low (0.0005-0.001 microliter L-1) ethylene concentrations can restore the reduced gravitropic response of the auxin-resistant dgt (diageotropica) mutant to wild-type levels. Slightly higher concentrations of ethylene inhibit the gravitropic response of all but the ethylene-insensitive nr (never-ripe) mutant. The gravitropic responses of nr and the constitutive-response mutant epi (epinastic) are slightly and significantly delayed, respectively, but otherwise normal. The reversal of shoot gravicurvature by red light in the lz-2 (lazy-2) mutant is not affected by ethylene. Taken together, these data indicate that, although ethylene does not play a primary role in the gravitropic response of tomato, low levels of ethylene are necessary for a full gravitropic response, and moderate levels of the hormone specifically inhibit gravicurvature in a manner different from ethylene inhibition of overall growth. (+info)
Regulation of differential growth in the apical hook of Arabidopsis.
Arabidopsis seedlings develop a hook-like structure at the apical part of the hypocotyl when grown in darkness. Differential cell growth processes result in the curved hypocotyl hook. Time-dependent analyses of the hypocotyl showed that the apical hook is formed during an early phase of seedling growth and is maintained in a sequential phase by a distinct process. Based on developmental genetic analyses of hook-affected mutants, we show that the hookless mutants (hls1, cop2) are involved in an early aspect of hook development. From time-dependent analyses of ethylene-insensitive mutants, later steps in hook maintenance were found to be ethylene sensitive. Regulation of differential growth was further studied through examination of the spatial pattern of expression of two hormone-regulated genes: an ethylene biosynthetic enzyme and the ethylene receptor ETR1. Accumulation of mRNA for AtACO2, a novel ACC (1-aminocyclopropane-1-carboxylic acid) oxidase gene, occurred within cells predominantly located on the outer-side of the hook and was tightly correlated with ethylene-induced exaggeration in the curvature of the hook. ETR1 expression in the apical hook, however, was reduced by ethylene treatment. Based on the expression pattern of ETR1 and AtACO2 in the hook-affected mutants, a model for hook development and maintenance is proposed. (+info)
Sulfate is both a substrate and an activator of the voltage-dependent anion channel of Arabidopsis hypocotyl cells.
On the basis of the anion content of in vitro-cultured Arabidopsis plantlets, we explored the selectivity of the voltage-dependent anion channel of the plasma membrane of hypocotyl cells. In the whole-cell configuration, substitution of cytosolic Cl(-) by different anions led to the following sequence of relative permeabilities: NO(3)(-) (2.6) >/= SO(4)(2-) (2.0) > Cl(-) (1.0) > HCO(3)(-) (0.8) >> malate(2-) (0.03). Large whole-cell currents were measured for NO(3)(-) and SO(4)(2-), about five to six times higher than the equivalent Cl(-) currents. Since SO(4)(2-) is usually considered to be a weakly permeant or non-permeant ion, the components of the large whole-cell current were explored in more detail. Aside from its permeation through the channel with a unitary conductance, about two-thirds that of Cl(-), SO(4)(2-) had a regulatory effect on channel activity by preventing the run-down of the anion current both in the whole-cell and the outside-out configuration, increasing markedly the whole-cell current. The fact that the voltage-dependent plasma membrane anion channel of hypocotyl cells can mediate large NO(3)(-) and SO(4)(2-) currents and is regulated by nucleotides favors the idea that this anion channel can contribute to the cellular homeostasis of important metabolized anions. (+info)
The relationship between ethylene binding and dominant insensitivity conferred by mutant forms of the ETR1 ethylene receptor.
Ethylene responses in Arabidopsis are mediated by a small family of receptors, including the ETR1 gene product. Specific mutations in the N-terminal ethylene-binding domain of any family member lead to dominant ethylene insensitivity. To investigate the mechanism of ethylene insensitivity, we examined the effects of mutations on the ethylene-binding activity of the ETR1 protein expressed in yeast. The etr1-1 and etr1-4 mutations completely eliminated ethylene binding, while the etr1-3 mutation severely reduced binding. Additional site-directed mutations that disrupted ethylene binding in yeast also conferred dominant ethylene insensitivity when the mutated genes were transferred into wild-type Arabidopsis plants. By contrast, the etr1-2 mutation did not disrupt ethylene binding in yeast. These results indicate that dominant ethylene insensitivity may be conferred by mutations that disrupt ethylene binding or that uncouple ethylene binding from signal output by the receptor. Increased dosage of wild-type alleles in triploid lines led to the partial recovery of ethylene sensitivity, indicating that dominant ethylene insensitivity may involve either interactions between wild-type and mutant receptors or competition between mutant and wild-type receptors for downstream effectors. (+info)
Modification of distinct aspects of photomorphogenesis via targeted expression of mammalian biliverdin reductase in transgenic Arabidopsis plants.
The phenotypic consequences of targeted expression of mammalian biliverdin IXalpha reductase (BVR), an enzyme that metabolically inactivates the linear tetrapyrrole precursors of the phytochrome chromophore, are addressed in this investigation. Through comparative phenotypic analyses of multiple plastid-targeted and cytosolic BVR transgenic Arabidopsis plant lines, we show that the subcellular localization of BVR affects distinct subsets of light-mediated and light-independent processes in plant growth and development. Regardless of its cellular localization, BVR suppresses the phytochrome-modulated responses of hypocotyl growth inhibition, sucrose-stimulated anthocyanin accumulation, and inhibition of floral initiation. By contrast, reduced protochlorophyll levels in dark-grown seedlings and fluence-rate-dependent reduction of chlorophyll occur only in transgenic plants in which BVR is targeted to plastids. Together with companion analyses of the phytochrome chromophore-deficient hy1 mutant, our results suggest a regulatory role for linear tetrapyrroles within the plastid compartment distinct from their assembly with apophytochromes in the cytosol. (+info)