Thomas Kretsch

eid1: A New Arabidopsis Mutant Hypersensitive in Phytochrome A–Dependent High-Irradiance Responses

To identify specific mutants for components of phytochrome A (phyA) signaling in Arabidopsis, we established a light program consisting of multiple treatments with alternating red and far-red light. In wild-type seedlings, irradiation with multiple red light pulses can reduce the amount of phyA, which in turn decreases the high-irradiance responses (HIRs) mediated by the subsequent treatments with far-red light. Our mutants were able to avoid this red light–dependent reduction of the HIR. Here, we describe eid1, a new recessive mutant with increased sensitivity to far-red light. The eid1 mutation maps to the top of chromosome 4. The mutants showed no change in phenotype in darkness or under continuous white light, but they exhibited an increased sensitivity to red light and an increased persistence of HIR during prolonged dark phases after multiple short pulses of far-red light. The eid1 seedlings accumulated normal amounts of phytochrome and showed no alterations in the degradation or de novo synthesis of phyA. The expression of the Eid1 phenotype requires the presence of phyA. Our data provide evidence that EID1 is a negatively acting component in the phyA-dependent HIR-signaling pathway.

EDL3 is an F-box protein involved in the regulation of abscisic acid signalling in Arabidopsis thaliana

The EID1-like protein 3 (EDL3) shows high similarity to EID1 (Empfindlicher im dunkelroten Licht 1), an F-box protein that functions as a negative regulator in the signalling cascade downstream of the phytochrome A photoreceptor in Arabidopsis thaliana. Analyses revealed a strong and rapid induction of EDL3 gene expression under osmotic stress, high salinity, and upon abscisic acid (ABA) application. Therefore, it was speculated that EDL3 is involved in the regulation of responses controlled by this plant hormone, which not only regulates many aspects of plant development but also integrates responses towards temperature, drought, osmotic, and salt stresses. Physiological data obtained with over-expresser lines and a conditional knock-down mutant demonstrated that EDL3 functions as a positive regulator in ABA-dependent signalling cascades that control seed germination, root growth, greening of etiolated seedlings, and transition to flowering. Results further demonstrate that EDL3 regulates anthocyanin accumulation under drought stress. The observed effects on physiological responses fit to tissue-specific expression patterns obtained with EDL3-promoter:GUS lines. Bimolecular Fluorescence Complementation assays and yeast two-hybrid analyses showed that EDL3 carries a functional F-box domain. Thus, the protein is presumed to act as a component of a ubiquitin ligase complex that specifically directs negatively acting factors in ABA signalling to degradation via the proteasome.

Promoter elements of the mustard CHS1 gene are sufficient for light regulation in transgenic plants

The expression of chalcone synthase (CHS) genes, which encode the first enzyme of the flavonoid pathway, is under developmental control as well as affected by external stimuli such as light. Varying fragments of the 1 kb upstream region of the CHS1 gene from white mustard (Sinapis alba L.) were fused to the GUS-coding region, and the light-regulated expression of these constructs was analysed in transgenic Arabidopsis and tobacco plants. Studies performed with Arabidopsis seedlings indicate the presence of two elements within the CHS1 promoter mediating light responses via different photoreceptors. One element, located about 150 bp upstream of the transcription start site, is homologous to Unit 1 of the parsley CHS gene, the second, far more upstream element carries sequences similar to Unit 2 of the same gene. Detailed studies on Unit 1-driven expression indicate that this element transfers the expression characteristics of the original gene to both Arabidopsis and tobacco. Although the expression characteristics of Unit 1 are indistinguishable from those of the full-length promoter within the same species, we observed differences in mustard CHS promoter regulation between Arabidopsis and tobacco plants transgenic for the identical construct. The difference in photoreceptor usage by the same promoter element in different transgenic species (Unit 1 from mustard in Arabidopsis vs. tobacco) was also observed for different but homologous promoter elements in the same transgenic species (Unit 1 from mustard and parsley in tobacco). We therefore conclude that the same promoter and even the same promoter element (Unit 1) can mediate different spatial patterns of expression and modes of light regulation in different transgenic species.

Genome-Wide Analysis of Light-Dependent Transcript Accumulation Patterns during Early Stages of Arabidopsis Seedling Deetiolation

Light is among the most important exogenous factors that regulate plant development. To sense light quality, intensity, direction, and duration, plants have evolved multiple photoreceptors that enable the detection of photons from the ultraviolet B (UV-B) to the far-red spectrum. To study the effect of different light qualities on early gene expression, dark-grown Arabidopsis (Arabidopsis thaliana) seedlings were either irradiated with continuous far-red, red, or blue light or received pulses of red, UV-A, or UV-A/B light. The expression profiles of seedlings harvested at 45 min and 4 h were determined on a full genome level and compared with the profiles of dark controls. Data were used to identify light-regulated genes and to group these genes according to their light responses. While most of the genes were regulated by more than one light quality, a considerable number of UV-B-specific gene expression responses were obtained. An extraordinarily high similarity in gene expression patterns was obtained for samples that perceived continuous irradiation with either far-red or blue light for 4 h. Mutant analyses hint that this coincidence is caused by a convergence of the signaling cascades that regulate gene expression downstream of cryptochrome blue light photoreceptors and phytochrome A. Whereas many early light-regulated genes exhibited uniform responses to all applied light treatments, highly divergent expression patterns developed at 4 h. These data clearly indicate that light signaling during early deetiolation undergoes a switch from a rapid, but unspecific, response mode to regulatory systems that measure the spectral composition and duration of incident light.

The Negatively Acting Factors EID1 and SPA1 Have Distinct Functions in Phytochrome A-Specific Light Signaling

EID1 (empfindlicher im dunkelroten Licht) and SPA1 (suppressor of phytochrome A[phyA]-105) function as negatively acting components in phyA-specific light signaling. Mutants in the respective genes led to very similar phenotypes under weak-light conditions. To examine whether both genes are functionally redundant, detailed physiological and genetic analyses were performed witheid1 and spa1 mutants isolated from the same wild-type background. Measurements of hypocotyl elongation, anthocyanin accumulation, and Lhcb1-transcript accumulation under different light treatments demonstrated that SPA1 has a strong influence on the regulation of very low fluence responses and a weaker influence on high-irradiance responses. In contrast, EID1 severely altered high-irradiance responses and caused almost no change on very low fluence responses. Analyses on eid1 phyA-105double mutants demonstrated that EID1 could not suppress the phenotype of the weak phyA allele under continuous far-red light. Measurements on eid1 spa1 double mutants exhibited a strong interference of both genes in the regulation of hypocotyl elongation. These results indicate that EID1 and SPA1 are involved in different but interacting phyA-dependent signal transduction chains.

The Mediator Complex Subunit PFT1 Interferes with COP1 and HY5 in the Regulation of Arabidopsis Light Signaling

Arabidopsis (Arabidopsis thaliana) mutants hypersensitive to far-red light were isolated under a light program of alternating red and far-red light pulses and were named eid (for empfindlicher im dunkelroten Licht). The dominant eid3 mutant carries a missense mutation in a conserved domain of PHYTOCHROME AND FLOWERING TIME1 (PFT1), an important component of the plant mediator coactivator complex, which links promoter-bound transcriptional regulators to RNA polymerase II complexes. Epistatic analyses were performed to obtain information about the coaction between the mutated PFT1eid3 and positively and negatively acting components of light signaling cascades. The data presented here provide clear evidence that the mutation mainly enhances light sensitivity downstream of phytochrome A (phyA) and modulates phyB function. Our results demonstrate that the Mediator component cooperates with CONSTITUTIVE PHOTORMORPHOGENIC1 in the regulation of light responses and that the hypersensitive phenotype strictly depends on the presence of the ELONGATED HYPOCOTYL5 transcription factor, an important positive regulator of light-dependent gene expression. Expression profile analyses revealed that PFT1eid3 alters the transcript accumulation of light-regulated genes even in darkness. Our data further indicate that PFT1 regulates the floral transition downstream of phyA. The PFT1 missense mutation seems to create a constitutively active transcription factor by mimicking an early step in light signaling.

Characterization of a Novel Non-Constitutive Photomorphogenic cop1 Allele

A specific light program consisting of multiple treatments with alternating red and far-red light pulses was used to isolate mutants in phytochrome A-dependent signal transduction in Arabidopsis seedlings. Because of their phenotype, the mutants were called eid (empfindlicher im dunkelroten Licht, which means hypersensitive in far-red light). One of the isolated mutants, eid6, is a novel recessive allele of the COP1 gene (constitutive photomorphogenic 1) that carries an amino acid transition in a conserved histidine residue of the RING finger domain. Mutant seedlings exhibited an extreme hypersensitivity towards all tested light qualities, but in contrast to known cop1 alleles, no constitutive photomorphogenic phenotype was detectable in darkness. Thus, the novel cop1eid6 allele seems to encode for a protein whose remaining activity is sufficient for the suppression of photomorphogenesis in dark-grown plants. In adult cop1eid6 plants, the development of the Cop1 phenotype is dominated by phytochrome B. Comparison of the phenotype of the novel cop1eid6 and the weak cop1-4 allele under continuous far-red light indicates that the RING finger and coiled-coil domains of COP1 are sufficient for some specific regulatory function in phytochrome A-dependent high irradiance responses.

In Vivo Characterization of Chimeric Phytochromes in Yeast

Phytochromes are plant photoreceptors that play a major role in photomorphogenesis. Two members of the phytochrome family have been characterized in some detail. Phytochrome A, which controls very low fluence and high irradiance responses, is rapidly degraded in the light, forms sequestered areas of phytochrome (SAPs), and does not exhibit dark reversion in monocotyledonous seedlings. Phytochrome B mediates red/far-red reversible responses, is stable in the light, and does not form SAPs. We report on the behavior in yeast of the phytochrome apoproteins of rice PHYA, tobacco PHYB, and chimeric PHYAB and PHYBA and on the behavior of the respective holoprotein adducts after assembly with phycocyanobilin chromophore (PHY*). SAP-like formation in yeast was not observed for PHYB, but was detectable for PHYA, PHYAB, and PHYBA. Rice PHYA* did not undergo dark reversion in yeast. Surprisingly, all other tested phytochrome constructs did exhibit dark reversion, including chimeric phytochromes with a short N-terminal part of tobacco PHYB or parsley PHYA fused to rice PHYA. Furthermore, the proportion of phytochrome undergoing dark reversion and the rate of reversion were increased for both the N terminus-swapped constructs and PHYBA*. These results are discussed with respect to structure/function analysis of phytochromes A and B.

The Histidine Kinase-Related Domain of Arabidopsis Phytochrome A Controls the Spectral Sensitivity and the Subcellular Distribution of the Photoreceptor

Phytochrome A (phyA) is the primary photoreceptor for sensing extremely low amounts of light and for mediating various far-red light-induced responses in higher plants. Translocation from the cytosol to the nucleus is an essential step in phyA signal transduction. EID1 (for EMPFINDLICHER IM DUNKELROTEN LICHT1) is an F-box protein that functions as a negative regulator in far-red light signaling downstream of the phyA in Arabidopsis (Arabidopsis thaliana). To identify factors involved in EID1-dependent light signal transduction, pools of ethylmethylsulfonate-treated eid1-3 seeds were screened for seedlings that suppress the hypersensitive phenotype of the mutant. The phenotype of the suppressor mutant presented here is caused by a missense mutation in the PHYA gene that leads to an amino acid transition in its histidine kinase-related domain. The novel phyA-402 allele alters the spectral sensitivity and the persistence of far-red light-induced high-irradiance responses. The strong eid1-3 suppressor phenotype of phyA-402 contrasts with the moderate phenotype observed when phyA-402 is introgressed into the wild-type background, which indicates that the mutation mainly alters functions in an EID1-dependent signaling cascade. The mutation specifically inhibits nuclear accumulation of the photoreceptor molecule upon red light irradiation, even though it still interacts with FHY1 (for far-red long hypocotyl 1) and FHL (for FHY1-like protein), two factors that are essential for nuclear accumulation of phyA. Degradation of the mutated phyA is unaltered even under light conditions that inhibit its nuclear accumulation, indicating that phyA degradation may occur mostly in the cytoplasm.

Phenotypic Characterization of Photomorphogenic Responses During Plant Development

Light is one of the most important exogenous factors regulating plant development throughout the entire life cycle. Light is involved in the breaking of seed dormancy, the regulation of photomorphogenic seedling development, the adaptation of plant morphology toward spectral composition of incident light, and the transition to flowering. Plants have evolved with several photoreceptor families that sense UV-A, blue, red, and far-red light. Here, basal methods to measure light-regulated changes in plant morphology and pigment accumulation will be described. The methods include the determination of apical hook angle and cotyledon opening, the measurement of stem elongation, the determination of leaf surface area, the measurements that characterize light-controlled transition to flowering, and the determination of anthocyanin and chlorophyll accumulation. Furthermore, different light programs are listed that can be used to test for the functional involvement of separate light response modes controlling photomorphogenic plant development.