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

Correspondence to: Thomas Kretsch, :, kretsch{at}ruf.uni-freiburg.de (E-mail), 49-761-203-2791 (fax)

	ABSTRACT

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION METHODS REFERENCES

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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION METHODS REFERENCES

Phytochromes are a family of photoreceptors that regulate plant development throughout all phases of the plant life cycle. They are involved in the induction of germination, regulation of seedling development, regulation of vegetative growth, and floral induction (Smith 1995 ; Whitelam and Devlin 1997 ). During seedling development in particular, phytochromes can induce drastic morphological changes. In darkness, the seedlings develop according to an etiolated or skotomorphogenic program, with short roots, long hypocotyls, closed hooks, and small, pale cotyledons. When the seedlings are exposed to light, photomorphogenesis begins. At this time, root growth is stimulated, hypocotyl elongation is inhibited, hooks open, the cotyledons expand, and chloroplasts develop.

Phytochromes are dimers of ~125-kD subunits, every monomer of which carries a single, covalently linked phytochromobilin chromophore. In darkness, phytochromes are synthesized in the red light–absorbing Pr form. When red light is absorbed, ~80 to 90% of the total phytochrome can be converted into the far-red-light–absorbing Pfr form; similarly, the Pfr form can be photoconverted back into the Pr form when far-red light is absorbed. Because Pfr acts as a positive effector for phytochrome-mediated responses, the photoreceptor can function as a molecular photoreversible light switch when exposed to treatment with pulses of red/far-red light (Furuya and Song 1994 ; Mancinelli 1994 ).

With respect to light stability, the phytochromes can be subdivided into light-stable and light-labile subclasses. In Arabidopsis, the light-stable type derives from four different genes (PHYTOCHROME B [PHYB], PHYC, PHYD, and PHYE), with the PHYB gene showing the most expression (Mathews and Sharrock 1997 ). Among other reactions, light-stable phytochromes control the responses to strong continuous red light during seedling development (Smith 1995 ; Whitelam and Devlin 1997 ).

The light-labile phytochrome accumulates to a high level in the dark (Clough and Viestra 1997 ) and is encoded by the PHYA gene in Arabidopsis (Mathews and Sharrock 1997 ). Studies using photoreceptor mutants and plants overexpressing phyA indicate that this phytochrome is responsible for the so-called very low fluence responses and for the far-red-light–associated high-irradiance responses (HIRs; Smith 1995 ; Whitelam and Devlin 1997 ). The action spectrum of the far-red-light HIR reveals a maximum at ~720 nm, at which point only 3 to 7% of the total phytochrome molecules remain in the Pfr form. The extent of the HIR depends on the duration and especially the fluence rate of irradiation. The HIR requires continuous irradiation, because even short intervening dark phases result in a breakdown of the response (Mancinelli and Rabino 1975 ; Beggs et al. 1980 ; Holmes and Schafer 1981 ; Heim and Schafer 1982 ; Mancinelli 1994 ).

A powerful approach to identifying components of the light-signaling pathway has been the isolation of photomorphogenic mutants (von Arnim and Deng 1996 ; Fankhauser and Chory 1997 ). Several screening strategies have led to the identification of specific components of the phyA signaling pathway. The fhy1, fhy3, fin2, and far1 mutants show less sensitivity in continuous far-red light, indicating that the respective gene products code for positive-acting components of a phyA-specific signaling cascade (Whitelam et al. 1993 ; Soh et al. 1998 ; Hudson et al. 1999 ). The FAR1 protein is localized in the nucleus and shows no homology with any known protein (Hudson et al. 1999 ). A suppressor of a weak phyA missense allele led to the discovery of the spa1 mutant. The SPA1 protein also is localized in the nucleus and seems to act as a negative regulator of phyA signal transduction (Hoecker et al. 1998 , Hoecker et al. 1999 ).

To search for new, highly specific components of the phyA signal transduction pathway, we decided to use a property of the HIR that was initially described for white mustard seedlings. In mustard, preirradiation with red light, which reduces the amount of phyA, causes a strong reduction in the HIR produced by the subsequent irradiation with far-red light (Beggs et al. 1980 ; Holmes and Schafer 1981 ). By replacing the single preirradiation with repetitive cycles of alternating red/far-red light pulses, the red light–dependent reduction of the HIR could be transferred to Arabidopsis seedlings. We therefore searched for mutants that could overcome the red light–dependent decrease of the HIR. Such mutants were expected either to be impaired in the light-dependent degradation of phyA or to exhibit an increased light sensitivity to compensate for the decrease in phyA. As described in the current work, the second mechanism was demonstrated for the eid1 mutant, which has increased sensitivity to phyA-dependent light regulation.

	RESULTS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION METHODS REFERENCES

Isolation of Mutants with Enhanced HIRs under Cyclic Red/Far-Red Light Treatments
In mustard seedlings, the far-red HIR can be decreased drastically by two 5-min pulses of red light administered 1 and 2 hr before the onset of irradiation with continuous far-red light (Holmes and Schafer 1981 ). In Arabidopsis, however, even prolonged red light pretreatments lasting as long as 48 hr did not greatly reduce the far-red-light–dependent HIR (data not shown). A clear reduction in the HIR could be obtained only by applying alternating red and far-red light treatments. Continuous cycles of 20 min of red light followed by 20 min of far-red light for 3 days after the induction of germination with 24 hr of irradiation with white light resulted in a clear decrease of HIR compared with that of seedlings that had been treated with continuous cycles of 20 min of darkness followed by 20 min of far-red light for the same period (Fig 1A, Fig 1B, and Fig 2). For Landsberg erecta (Ler) wild-type, phyB-5, and Wassilewskija (WS) wild-type seedlings, irradiation with alternating red/far-red light pulses clearly reduced the expansion and opening of cotyledons, reduced the inhibition of hypocotyl growth, and reduced the stimulation of root growth compared with seedlings that received pulses of far-red light alone. phyA-201 and phyA-201 phyB-5 seedlings exhibited no response to pulses of far-red light alone, because the loss of phyA results in a loss of the HIR induced by this light treatment. The phyA-201 phyB-5 mutant also remained nearly etiolated under the treatment with red/far-red light pulses, whereas the phyA-201 mutant exhibited a partially deetiolated phenotype similar to that of phyB-5 and wild-type plants. We conclude that this partially deetiolated phenotype is caused by the pulses of red light, which induce light responses regulated by the light-stable types of phytochrome.

View larger version (131K): [in this window] [in a new window]   Figure 1. Phenotypes of Ler Wild-Type, phyA-201 phyB-5, phyA-201, phyB-5, phyB-5 eid1-1, phyB-5 eid1-2, the WS Wild-Type, and eid1-3 Seedlings. Seedlings were grown for 3 days after the induction of germination. (A) Seedlings grown under light conditions of the screening program (alternating pulse treatments of 20 min of red followed by 20 min of far-red light). (B) Seedings grown under an alternating pulse treatment consisting of 20 min of darkness followed by 20 min of far-red light. (C) Seedlings grown in darkness. (D) Seedlings grown under strong continuous far-red light (14 µmol m-2 sec-1). (E) Seedlings grown under continuous white light (15 µmol m-2 sec-1). (F) Seedlings grown under weak continuous red light (3 x 10-3 µmol m-2 sec-1). Bar in (A) = 5 mm for (A) to (F).

View larger version (52K): [in this window] [in a new window]   Figure 2. Hypocotyl and Root Lengths of Ler Wild-Type, phyA-201 phyB-5, phyA-201, phyB-5, phyB-5 eid1-1, phyB-5 eid1-2, the WS Wild-Type, and eid1-3 Seedlings. Hypocotyl and root lengths were determined for seedlings grown in darkness, under alternating pulse treatments of 20 min of red followed by 20 min of far-red light used for screening, and under an alternating pulse treatment consisting of 20 min of darkness followed by 20 min of far-red light for 3 days after the induction of germination. Error bars represent standard deviations.

During the first round of screening, ethyl methanesulfonate (EMS)–mutagenized phyB-5 seeds were used. Because of the loss of phyB, the seedlings exhibited only a weak response to the red light treatments, which resulted in a clear phenotype of putative mutants. Additionally, the loss of phyB should allow the finding of specific signaling mutants in the phyA-dependent far-red-light HIR. Considering the strong phenotype of the mutants in the phyB-5 background but the relatively weak phenotype obtained in wild-type seeds (Fig 1A), we decided to start a second round of screening by using T-DNA lines (Feldmann 1991 ).

We searched for mutants that showed enhanced photomorphogenic development, that is, a reduction in hypocotyl growth together with expanded and open cotyledons and a stimulation of root growth (Fig 1A and Fig 2) under the treatment with alternating pulses of red/far-red light. In total, we isolated seven mutant lines from the EMS-mutagenized phyB-5 seeds that exhibited a stable inheritance of the phenotype. One mutant could be isolated from the T-DNA pools. All of these mutant lines remained etiolated in the absence of light (Fig 1C and Fig 2) and thus are clearly different from the constitutively photomorphogenic (cop), deetiolated (det), and fusca (fus) mutants, which are characterized by constitutive photomorphogenesis in darkness (von Arnim and Deng 1996 ; Fankhauser and Chory 1997 ). The mutants also did not show alterations in seedling development under strong, saturating, continuous irradiation with far-red (14 µmol m^-2 sec^-1; Fig 1D) or white light (15 µmol m^-2 sec^-1; Fig 1E) when compared with their respective backgrounds. Hence, the observed phenotype is not the result of a severe, unspecific disturbance of seedling development.

Genetic Analysis
By using complementation analysis, we were able to separate the eid (for empfindlicher im dunkelroten Licht) mutants into six complementation groups (eid1 to eid6). Interestingly, two lines of the EMS-mutagenized phyB-5 seeds (phyB-5 eid1-1 and phyB-5 eid1-2) belong to the same group as the line from the T-DNA pools (eid1-3) and thus should be allelic. In this study, we focused on the eid1 mutant. The monogenic character of the mutation was confirmed by three rounds of backcrosses with the respective background lines, in which the mutation behaved like a recessive Mendelian marker (Table 1). The expression of the phenotype was also independent of the growth substrate used (soil, paper, water agar, and growth medium with agar [ Valvekens et al. 1988 ]; data not shown). For the morphology of adult plants, no differences were observed between the eid1 mutants and their corresponding background plants (data not shown).

For mapping analysis, the phyB-5 eid1-1 and phyB-5 eid1-2 lines were crossed with the phyB-9 mutant from the Columbia (Col) background, and the eid1-3 line was crossed with wild-type plants from the Ler and Col ecotype. The segregation analysis in the F₁ and F₂ generations again demonstrated that the eid1 mutation is a recessive Mendelian marker (Table 1). For linkage analysis, polymerase chain reaction (PCR)–based markers were used. The eid1 mutation was located on the top of chromosome 4 at ~0.5 centimorgans from the GA1 cleavable amplified polymorphic sequence (CAPS) marker (one recombination event in 200 analyzed chromatids). The close proximity of the mutant gene to GA1 was confirmed by linkage analyses using PHYD, Det1, and nga8 as markers (data not shown). Mapping analysis revealed that the eid1 locus clearly can be separated from det1, PHYD, PHYE, hy4, shy4-2, and far1, six loci on chromosome 4 that also are involved in photomorphogenesis.

Expression of the Eid1 Phenotype Is Coupled to the Presence of phyA
To verify the dependency of the Eid1 phenotype on the presence of phyA by genetic means, we crossed eid1-3 derived from the WS background with phyA-201 and phyA-211 plants, two different phyA null mutants in the Ler and Col background. We used phyA mutants from two different ecotypes to minimize possible influences from differences in the light sensitivity of different background lines. The results of the crossings were analyzed by specific light treatments to test for the presence of two copies of the recessive eid1-3 and phyA alleles.

As a test for the presence of the Eid1 phenotype, seedlings were induced to germinate and then were grown under weak, continuous red light (3 x 10^-3 µmol m^-2 sec^-1) for 3 days. Under these conditions, only plants with two copies of the recessive eid1-3 allele showed a reduction in hypocotyl elongation and open, expanded cotyledons, whereas all other lines used for crossing remained completely etiolated (Fig 1E). We applied weak red light instead of the alternating red/far-red light pulses, because seedlings showing the Eid1 phenotype could be separated easily from their etiolated counterparts.

Under strong far-red light (14 µmol m^-2 sec^-1 for 3 days after the induction of germination), ordinarily only seedlings with at least one intact PHYA allele are able to mediate the HIR with anthocyanin accumulation, a reduced elongation of the hypocotyl, and open and expanded cotyledons (Whitelam et al. 1993 ). Therefore, only plants with two copies of the recessive phyA null alleles remain completely etiolated under these light conditions (Fig 1D).

In the F₁ generation, no mutant phenotype was observed under the weak red light and under the strong far-red light (data not shown). This was as expected, because both the phyA null alleles and the eid1 alleles are recessive. After selfing, the F₂ generation was analyzed again under the two different light conditions. Because the PHYA and the EID1 genes are located on different chromosomes (chromosomes 1 and 4, respectively), both genes and their respective mutant alleles should segregate independently in the F₂. Therefore, a segregation ratio of 1:3 for deetiolated to etiolated seedlings should be observable after treatment with weak red light, if the expression of the Eid1 phenotype occurs independent of the presence of an intact PHYA gene. On the other hand, if the expression of the Eid1 phenotype is strictly phyA dependent, the ratio of deetiolated F₂ seedlings in the weak red light should be reduced to 3:13 because phyA-201 eid1-3 and phyA-211 eid1-3 double mutants would no longer show a light response. Under strong far-red light, the inverse result would be expected. If eid1-3 can function independently of phyA, then the ratio of deetiolated to etiolated seedlings should be reduced to 3:13. However, if the eid1 mutation remains silent in the absence of phyA, the F₂ generation should segregate at a ratio of 1:3 for etiolated to deetiolated seedlings under continuous irradiation with strong far-red light. Under both light conditions, the data obtained for the segregation behavior in the F₂ were consistent with a strict phyA dependency of the expression of the Eid1 phenotype (Table 2).

For additional proof, we isolated 25 F₃ plants homozygous for phyA-211 by selecting seedlings from the F₂ generation that remained etiolated under strong far-red light. After verifying the homozygosity for phyA-211 by cultivation in the strong far-red light, we further analyzed the F₃ seed pools under the weak red light. None of the 25 F₃ plants lacking phyA showed an Eid1 phenotype. Assuming a phyA-independent function of eid1-3 and a probability of P = 0.25 (1:3) for the homozygosity of the eid1-3 allele in the F₃ progenies, such a result (k = 0 out of n = 25 plants) would occur by chance with a probability of P (0.25, 0, 25) = 7.5 x 10^-4 = 0.075%; for binomial distributions, P (P, k, n) = (ⁿ_k) x P^k x (1 - P)^n-k, in which P = probability of an event, k = number of events, and n = number of trials.

The Eid1 phenotype is clearly detectable in a phyB null background because both the eid1-1 and the eid1-2 alleles were isolated from the phyB-5 mutant. This phenotype also can occur in the absence of phyD, because the eid1-3 allele is derived from a WS wild-type line lacking this phytochrome (Aukerman et al. 1997 ; data not shown). Therefore, in contrast to the strict phyA dependency, the Eid1 phenotype can occur independent of the presence of phyB and phyD.

Analysis of phyA Degradation
One reason for the increased light sensitivity might be an enhanced abundance of phyA in the eid1 mutants, which could be caused by an overexpression of the photoreceptor or by reduced degradation when irradiated. Both the immunoblot data (see Fig 4A) and the spectroscopic measurements in vivo (Table 3) argue against overexpression of phytochromes, because their amounts in the eid1 alleles were similar.

View larger version (25K): [in this window] [in a new window]   Figure 3. The eid1 Mutants Are Not Altered in the Light-Dependent Degradation Rate of phyA. (A) Immunoblot analysis of phyA degradation. Three-day-old dark-grown seedlings were irradiated by continuous red light (39 µmol m-2 sec-1). The seedlings were harvested at indicated time points after the onset of irradiation. (B) phyA degradation in continuous far-red light (14 µmol m-2 sec-1). Degradation kinetics of 3-day-old etiolated seedlings were determined spectroscopically during irradiation with continuous far-red light. Error bars represent standard deviations. (C) Measurement of phyA degradation after a red light pulse (39 µmol m-2 sec-1) lasting 5 min. Degradation kinetics of 3-day-old etiolated seedlings were determined spectroscopically. Error bars represent standard deviations.

View larger version (32K): [in this window] [in a new window]   Figure 4. Hypocotyl-Length Inhibition and Anthocyanin Accumulation in Different Mutants and Wild Types. (A) Fluence rate response curves for the inhibition of hypocotyl growth in continuous far-red light. Hypocotyl length was analyzed 3 days after the induction of germination. The relative lengths of the hypocotyls were determined in relation to the lengths of etiolated seedlings for each line. The hypocotyl length of the dark controls was 11.0 ± 1.6 mm for Col, 9.0 ± 1.2 mm for phyA-211, and 10.7 ± 0.9 mm for phyA-211 eid1-3. The hypocotyl lengths of etiolated seedlings of WS and the other mutants correspond to those shown in Fig 2. Standard deviations were between 5 and 19%. (B) Anthocyanin content of seedlings grown in darkness or in continuous weak far-red light (1.4 µmol m-2 sec-1) for 3 days after the induction of germination. The amount of anthocyanin was determined spectroscopically in phyB-5, phyB-5 eid1-1, phyB-5 eid1-2, the WS wild type, and eid1-3. Error bars represent standard deviations.

To analyze phyA degradation, we transferred 3-day-old dark-grown seedlings to continuous red light (14.3 µmol m^-2 sec^-1) and followed the degradation kinetics by immunoblot analyses (Fig 3A). For all three eid1 alleles and their respective wild-type plants, very similar degradation kinetics were observed. This result was confirmed by in vivo spectroscopy (data not shown).

Furthermore, we measured the amount of phyA in 3-day-old etiolated seedlings during 8 hr of continuous irradiation with far-red light. The degradation kinetics of eid1-3 and WS were similar to one another (Fig 3B). The same result was obtained for phyB-5 eid1-1, phyB-5 eid1-2, and phyB-5 seedlings (data not shown). Because the degradation rate depends on the amount of the Pfr form of phyA (Pfr-A) present, the apparent rate of phyA degradation in far-red light remained low compared with that in continuous red light irradiations.

To test for possible differences in phytochrome de novo synthesis, we monitored the phytochrome amounts in eid1-3 and WS seedlings kept in darkness after a saturating 5-min red light pulse. Again, no difference between the strong eid1-3 allele and its respective wild type occurred (Fig 3C). No dark reversion was detectable with eid1-3 and WS seedlings (data not shown), which is similar to the results obtained for the Ler ecotype (Hennig et al. 1999 ).

The eid1 Mutation Leads to Increased Sensitivity to Continuous Far-Red Light
The idea of this approach was to isolate mutants with defects in the phyA signaling pathway. A typical photoresponse driven by phyA is the HIR under continuous far-red light. To test whether this response is affected in the eid1 mutants, we measured fluence rate response curves for hypocotyl elongation (Fig 4A). For all three eid1 alleles, sensitivity to continuous far-red light was increased with regard to their respective background lines, in which phyB-5 eid1-1 and eid1-3 seedlings displayed a strong sensitivity. The lower sensitivity of phyB-5 eid1-2 seedlings in the fluence rate response curves is in good agreement with the results obtained for hypocotyl elongation and root growth under the red/far-red light pulses used for screening and under the alternating dark/far-red light treatment (Fig 2). For phyB-5, WS, and Col seedlings, approximately the same fluence rate response curves were detected (Fig 4A). phyA-211 and phyA-211 eid1-3 plants were unable to show an HIR for hypocotyl elongation (Fig 4A), which is similar to the behavior of other phyA null mutants (Smith 1995 ; Whitelam and Devlin 1997 ).

A second HIR in Arabidopsis seedlings is the accumulation of anthocyanins. Therefore eid1, phyB-5, and WS seedlings were irradiated with weak continuous far-red light (1.4 µmol m^-2 sec^-1) for 3 days after the induction of germination, and the anthocyanin content was determined spectroscopically. Similar to the results obtained for hypocotyl elongation, phyB-5 eid1-1 and eid1-3 clearly exhibited a stronger response than did their background lines, whereas in phyB-5 eid1-2, only a weak effect was detectable (Fig 4B). The absolute anthocyanin contents of eid1-1 and eid1-3 cannot be compared directly, because the ability for anthocyanin formation also varies widely in the respective background lines. For phyA-201, phyA-211, phyA-201 phyB-5, and phyA-211 eid1-3 seedlings, no anthocyanin accumulation was detectable, even under strong far-red light (Kunkel et al. 1996 ; data not shown). A greater far-red light sensitivity of the eid1 mutants also was seen for the expansion and opening of cotyledons, root growth, and inhibition of the negative gravitropic response (data not shown).

The Mutation in eid1 Leads to Increased Sensitivity to Red Light
As shown in Fig 1F, the eid1 plants also exhibited an enhanced red light sensitivity. To analyze the responsiveness toward continuous red light in more detail, we determined fluence rate response curves for hypocotyl elongation (Fig 5A). The eid1-3 mutant exhibited a clearly enhanced sensitivity to red light at low fluence rates (between 1.4 x 10^-4 and 1.4 x 10^-2 µmol m^-2 sec^-1) compared with that of WS wild type. Astonishingly, in eid1-3 seedlings the red light response decreased at increasing fluence rates. At fluence rates >1.4 x 10^-2 µmol m^-2 sec^-1, the red light effect in mutant and WS seedlings became nearly identical. The phyB-5 mutant remained red light insensitive over the whole range of fluence rates tested. In contrast to its background, phyB-5 eid1-1 exhibited a minimum curve with the greatest light responses between 1.4 x 10^-1 and 1.4 x 10^-2 µmol m^-2 sec^-1. The phyB-5 eid1-1 seedlings were not able to respond to red light at very low and very high fluence rates. phyA-211 and phyA-211 eid1-3 seedlings exhibited nearly the same fluence rate response curves, which were also very similar to the fluence rate response curves obtained for WS and Col seedlings (Fig 5A). These results demonstrate that the observed increase in red light sensitivity in eid1 mutants at very low fluence rates is phyA dependent and can be separated from the phyB-dependent red light response at high fluence rates.

View larger version (47K): [in this window] [in a new window]   Figure 5. Sensitivity to Red Light Is Increased in eid1 Seedlings. (A) Fluence rate response curves for hypocotyl growth in continuous red light. Seedlings were irradiated at various photon fluence rates for 3 days after the induction of germination. The relative lengths of the hypocotyls were determined in relation to the lengths of etiolated seedlings for each line. Standard deviations were between 5 and 15%. For further details, refer to Fig 4. (B) Immunoblot analysis of the phyA content after irradiation in continuous red light at different photon fluences. Seedlings were harvested after irradiation for 3 days at the same photon fluences as in (A).

In a parallel experiment, we determined by immunoblot analysis the amount of phyA accumulated at the different fluence rates of red light (Fig 5B). With increasing fluence rates, the steady state level of phyA decreased whereby the eid1 mutants and their respective background lines exhibited the same dependency on fluence rate. The comparison of the data in Fig 5A and Fig 5B revealed a good correlation between the red light responses for hypocotyl elongation and the phyA level in phyB-5 eid1-1 and in eid1-3. As long as the amount of phyA remained high (Fig 5B), the sensitivity of hypocotyl elongation in eid1 mutants was greater than in their background lines (Fig 5A). When the amount of phyA fell below the detection limit, the eid1-3 mutant and wild-type seedlings exhibited the same degree of red light responsiveness, and the red light sensitivity in phyB-5 eid1-1 mutants decreased drastically.

The Lesion in eid1 Results in an Enhanced Persistence of the HIR Response
A characteristic property of the HIR is its dependence on continuous irradiation. Normally, even short, intervening dark phases lead to a loss of the response (Mancinelli and Rabino 1975 ). To study this effect in Arabidopsis seedlings, we administered repetitive far-red light pulses of 6 µmol m^-2 sec^-1 for 2.5 min over 3 days after the induction of germination, and the intermitting dark phases were varied. With the WS wild type, even a dark period as short as 5 min leads to a clear reduction in the inhibition of hypocotyl elongation, and only a weak response (if any) was detectable if the intervening dark period was extended to 17.5 min (Fig 6). For the strong eid1-3 allele, even extended dark periods of 17.5 or 27.5 min did not lead to a clear reduction of the HIR. Indeed, a 2.5-min far-red light pulse given only once an hour remained very effective. If the light intensity of the far-red light pulse was decreased to 0.6 µmol m^-2 sec^-1, the difference between the eid1-3 mutant and its respective background became even more pronounced (Fig 6). The above observations also could be verified for phyB-5 eid1-1 and phyB-5 eid1-2, in which phyB-5 eid1-2 again exhibited a weaker phenotype (data not shown).

View larger version (36K): [in this window] [in a new window]   Figure 6. Mutation in eid1 Increases the Persistence of the HIR. The WS and eid1-3 seedlings were irradiated with multiple far-red light pulses for 2.5 min, varying the duration of the dark phases between the light pulses. The seedlings were analyzed after running the pulse program for 3 days after the induction of germination. Photon fluence rates of the far-red light pulses were 6 µmol m-2 sec-1 or 0.6 µmol m-2 sec-1. Error bars represent standard deviations.

	DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION METHODS REFERENCES

To find new, light-specific mutations of the phyA signal transduction pathway, we used a physiologic approach, addressing a phyA-specific response mode. Using intermittent pulses of red light, we lowered the phyA content, which resulted in a strong reduction of the far-red-light–induced HIR in Arabidopsis wild-type seedlings. We suggest that the eid1 mutants described here can compensate for the decrease in phyA by an enhanced light sensitivity. Our analyses provide evidence for a new locus, encoding a product that is specifically involved in a phyA-related signal transduction chain responsible for expression of the HIR.

Expression of the Eid1 Phenotype Is Light Dependent
In darkness, eid1 seedlings did not exhibit any deetiolation phenotype. The expression of the Eid1 phenotype was always light dependent. Hence, the eid1 mutants are clearly distinct from the class of cop/det/fus mutants (von Arnim and Deng 1996 ; Fankhauser and Chory 1997 ) and some suppressor mutants (Kim et al. 1996 ; Reed et al. 1998 ) that confer constitutive photomorphogenesis. According to their increased light sensitivity, the eid1 mutants also can be clearly separated from mutations that result in a light insensitivity (Koornneef et al. 1980 ; Whitelam et al. 1993 ; Wagner et al. 1997 ). The phenotype of the eid1 mutant resembles that of the nonallelic spa1 (Hoecker et al. 1998 ) and psi2 mutants (Genoud et al. 1998 ), which results in similarly enhanced sensitivity to red and far-red light.

Effect of the eid1 Mutation Is phyA Dependent
The strict phyA dependency of the eid1 mutation was confirmed by genetic and physiologic means. The F₂ segregation behavior of crosses between the eid1-3 and phyA null mutants deviated markedly from the ratios that would have been expected for a phyA-independent effect of the eid1 mutation. Furthermore, an Eid1 phenotype was never observed in phyA null mutants of the F₃ generation. In contrast, expression of the Eid1 phenotype seems to be independent of the lack of phyB in phyB-5 eid1-1 and phyB-5 eid1-2 or the lack of phyD in eid1-3. Therefore, EID1 most probably is not involved in the signaling cascade of these light-stable phytochromes.

The red and far-red-light fluence rate response curves of the phyA-211 eid1-3 double mutant also were consistent with a strict phyA dependency of the Eid1 phenotype. With both light qualities, the hypersensitivity of the eid1 mutation was completely abolished because of the lack of phyA.

Finally, the comparison of the fluence rate response curves in red light and the amount of immunologically detectable phyA in the seedlings of the wild type and the mutant eid1-3 further confirms that the physiologic effect of the eid1 mutation requires the presence of phyA. The increased red light sensitivity of the eid1 mutant was observed only as long as the steady state level of phyA remained high. At higher fluence rates of red light, de novo synthesis can no longer compensate for the increased degradation rate of Pfr-A, resulting in a strong decrease of phyA. Under these conditions, the red light responses in the eid1 mutant became identical to that of the wild-type seedlings.

The eid1 Mutation Seems to Alter phyA Signal Transduction
Overexpression of phyA in transgenic Arabidopsis plants causes hypersensitivity to continuous red and far-red light (Boylan and Quail 1991 ; Stockhaus et al. 1992 ). Therefore, the enhanced light sensitivity in eid1 could be caused by increased steady state amounts of phyA because of either increased synthesis or decreased degradation (Mancinelli 1994 ). In eid1, however, neither of these parameters seemed to be altered. As shown by immunoblot analysis and in vivo spectroscopy, the amount of phyA protein present always remained normal under all light conditions tested. Moreover, phyA degradation rates in continuous red and far-red light were identical between eid1 and wild-type seedlings. The same is true for the fluence rate dependency of phyA degradation under continuous red light. Finally, the kinetics after one saturating pulse of red light argues against changes in reaccumulation rates. These findings, together with the demonstration of phyA dependence, provide evidence that EID1 functions as a component of a phyA-specific signaling cascade, similar to the functioning of FHY1, FHY3, FIN2, FAR1, and SPA1 (Whitelam et al. 1993 ; Hoecker et al. 1998 , Hoecker et al. 1999 ; Soh et al. 1998 ; Hudson et al. 1999 ).

Role of EID1 in phyA Signaling
The pleiotropic effects in the eid1 mutants and the specificity for phyA signaling indicate a role for EID1 during an early step of the phyA signal transduction chain. The function of EID1 seems to be restricted to HIR in that none of the very low fluence responses tested (Lhcb mRNA accumulation and seed germination) exhibited an increased responsiveness (data not shown). The recessive nature of the eid1 mutations and the increased light sensitivity of the mutant seedlings suggest that EID1 functions as a negative regulator in the signaling cascade.

A negative regulator might be necessary to adapt phyA signaling after strong or prolonged stimulation of the sensory system. However, such a desensitizing function is not very likely for EID1, which seems to exert its function predominantly at weak light intensities. Thus, it might be necessary to suppress photoresponses that should not be induced by weak light stimuli.

An attenuation of light signals also can improve the temporal resolution of the sensory system. Without an inactivation mechanism, even a single light pulse would persist in the signaling cascade for an extended time and hence would interfere with subsequent light stimuli. The observed increased persistence of far-red light pulses in eid1 mutants is in good agreement with this hypothesis.

EID1 and Its Relation to the HIR Signaling Cascade: A Model
A good temporal resolution might be a prerequisite for the phyA signaling system that leads to HIR. The most important characteristic of this signaling system is its ability to measure light fluences under far-red light conditions in which only ~5% of the phyA molecules remains in the active Pfr form. The slow degradation of phyA and consequently the relatively high amounts of Pfr-A remaining are regarded as one reason for the strong effects observed under far-red light conditions (Beggs et al. 1980 ; Holmes and Schafer 1981 ). Nevertheless, explaining the fluence rate dependency of the HIR only in terms of high Pfr-A contents is difficult, because this value does not change substantially with increasing light intensities, whereas the amplitude of the HIR does change (Schafer and Mohr 1974 ; Schafer 1975 ). In addition, the high amounts of persisting Pfr-A under these light conditions cannot adequately explain why the HIR is fully manifested only under continuous far-red light and why it can be only partially induced by pulse treatments that adjust approximately the same amount of Pfr-A (Mancinelli and Rabino 1975 ; Heim and Schafer 1982 ; Mancinelli 1994 ). Hence, the sensory system involved is expected to include components that allow measuring time in addition to measuring the amount of photons.

A candidate for a central component of the necessary clockwork mechanism might be a positive effector that can oscillate between active and inactive states with a certain frequency. According to our model (Fig 7), the amount of the activated effector formed at a given time should be determined by the rate of its formation, which should depend on the actual absolute amount of Pfr-A. The activated state of the positive effector then should be reverted to the ground state by inactivating components of the signaling cascade, which should exert their function constitutively and at a constant rate. Hence, the measurement of photon fluence rates would be obtained in the case of a Pfr-dependent modulation of the formation rate of the positive effector. In accordance with the proposed model, a mutation in a negative component of the oscillating system would result in what was observed—an increased sensitivity to continuous red and far-red light—because smaller amounts of Pfr-A still could induce the HIR. But most important, the loss of such a negative factor could explain the observed increased persistence to far-red light pulses during subsequent dark phases.

View larger version (15K): [in this window] [in a new window]   Figure 7. Schematic Diagram of the Model Proposed for phyA Signaling Leading to HIR. The positive regulators FHY1, FHY3, FIN2, and FAR1 (Whitelam et al. 1993 ; Soh et al. 1998 ; Hudson et al. 1999 ) are potential candidates for oscillating positive effectors (symbolized by a dashed arrow) or for components that are involved in the interaction between Pfr-A and this positive effector. Negative regulators such as SPA1 (Hoecker et al. 1998 , Hoecker et al. 1999 ) and EID1 might be responsible for the inactivation mechanism.

	METHODS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION METHODS REFERENCES

Plant Material and Mutagenesis
For genetic crossing analysis and mapping, the following ecotypes and photomorphogenic mutants of Arabidopsis thaliana were used: Columbia (Col), Landsberg erecta (Ler), Wassilewskija (WS; obtained from Lehle Seed, Tuscon, AZ), phyB-5 (ecotype Ler; Reed et al.., 1993 ), phyA-201 (ecotype Ler), phyA-211 (ecotype Col), and the phyA-201 phyB-5 (ecotype Ler) double mutant (Nagatani et al. 1993 ). Seeds of the phyB-5 mutant line (1 g) were mutagenized by imbibition in 0.3% ethyl methanesulfonate (EMS) for 13 hr, followed by extensive washing with distilled water as described by Redei and Koncz 1992 . Approximately 50,000 seeds were sown directly into soil in 100 separate pots. Pots containing the M₁ families were harvested independently to obtain the M₂ generation. For a second round of screening, 40 pools of 100 independent T-DNA lines (Feldmann 1991 ) were analyzed (obtained from Nottingham Seed Stock Center, Nottingham, UK). Except for the T-DNA lines, all seeds used were derived from plants grown in a phytochamber under 16 hr of white light (300 µmol m^-2 sec^-1, produced by 10 Osram HQIL 400-W lamps plus four Osram L40/W60 fluorescent bulbs; Osram, Berlin, Germany) and 8 hr of darkness at 21°C.

Seedling Growth, Screening for Mutants, and Light Sources
For all analyses, seeds were sown on four layers of water-soaked filter paper (2043 BMGL; Schleicher and Schüll, Dassel, Germany), which were placed into clear plastic boxes. The standard sowing procedure was followed by a 48-hr cold treatment at 4°C in the dark and 24 hr of white light induction (15 µmol m^-2 sec^-1) of germination before irradiation with different light qualities.

Approximately 5000 seedlings were screened per M₂ family for a hypersensitive phenotype (short hypocotyl and expanded cotyledons). In screening for mutants, the seedlings were treated with alternating pulses of far-red light (5.4 µmol m^-2 sec^-1) and red light (5 µmol m^-2 sec^-1) for 20 min for 3 days after the induction of germination (4-day-old seedlings). Putative mutants were planted into soil and transferred into a phytochamber (see above). M₃ seeds were harvested from individual M₂ plants, and a subset was screened again in the screening program. Putative mutants were then backcrossed three times to their respective background lines (phyB-5 for EMS mutants and WS for T-DNA lines).

For the screening program, modified Leitz Prado 500-W universal projectors (Leitz, Wetzlar, Germany) were used as light sources for pulse irradiation with Osram xenophot longlife lamps. Red light was obtained by passing the light beam through a KG65 (Balzers, Liechtenstein) filter with a maximal transmission at 650 nm (band path, 15 nm). To get far-red light, we used a 715-nm DAL filter (band path, 15 nm; Schott, Mainz, Germany). For all further experiments, standard white light, red light, and far-red light fields were used for the irradiations, as described in Heim and Schafer 1982 . Light intensities were attenuated by neutral glasses (Balzers). The fluence rate was measured by using a J16 photometer and a J6512 radiant energy probe (Tektronix, Beaverton, OR).

Genetic Analysis and Mapping
Complementation and segregation analyses were performed as described by Koornneef and Stam 1991 . We mapped the eid1 mutation by using polymerase chain reaction (PCR)–based simple sequence length polymorphism and cleavable amplified polymorphic sequence (CAPS) markers that were polymorphic between the ecotypic backgrounds Ler and Col or Ler, Col, and WS, respectively (Konieczny and Ausubel 1993 ; Bell and Ecker 1994 ). We crossed phyB-5 eid1-1 and phyB-5 eid1-2 mutant alleles (Ler background) with phyB-9 plants (Col background; Reed et al. 1993 ), and we crossed eid1-3 mutant plants (WS background) with Ler and Col wild-type plants. DNA was assayed from individual F₂ progeny for WS-, Ler-, or Col-specific polymorphisms. The homozygosity of the F₂ plants was verified by analyzing the F₃ progenies. In total, we analyzed 40 F₂ plants from crosses between eid1-3 and Ler or Col, 30 F₂ plants from crosses between phyB-5 eid1-1 and phyB-9, and 30 F₂ plants from crosses between phyB-5 eid1-2 and phyB-9. The PCR primers for CAPS and simple sequence length polymorphism markers were purchased from Research Genetics (Huntsville, AL). Map distances were calculated on the basis of Kosambi, as described by Koornneef and Stam 1991 , by using summarized data from all 100 F₂ plants analyzed.

Measurement of Hypocotyl Length and Anthocyanin Accumulation
For the measurements, seeds were sown in 85 x 85 x 50-mm plastic boxes on four layers of 2043 BMGL filter paper that had been presoaked in distilled water and supplemented with 4 mL of distilled water. Germination induction was performed as described above. Seedlings were analyzed 3 days after induction of germination (at 4 days old).

Hypocotyl length was measured manually with a ruler. All data represent the mean of at least 60 seedlings analyzed in at least two independent experiments. For anthocyanin extraction, 50 seedlings were transferred to ice-cold extraction buffer (17% propanol and 1% concentrated HCl [v/v]). The seedlings were boiled in a water bath for 1 min and then cooled on ice immediately. The samples were shaken overnight at 8°C in darkness. All further manipulations and the correction of the spectroscopic measurements were done as described by Schmidt and Mohr 1981 . All data represent the mean of at least five independent experiments.

In Vivo Spectroscopy
Destruction of phyA was measured in a dual wavelength ratio spectrophotometer at 4°C as described by Gross et al. 1984 . Changes in absorbance (A) values were normalized to the amount of fresh weight, which was determined immediately after the measurements. The data are the means of at least three independent measurements.

Protein Extraction and Immunoblotting
Seedlings were extracted with SDS sample buffer (65 mM Tris-HCl, pH 7.8, 4 M urea, 10 mM DTE, and 0.5% [w/v] bromphenol blue) by sonification for 1 min with a Sonopuls GM 70 MS 72/D Sonifier (Bandelin Electronic, Berlin, Germany) at maximal intensity. Afterward, the probes were heated to 95°C for 5 min. The crude extracts were clarified by centrifugation for 15 min at 20,000g and 25°C. Protein extracts (25 µg) were separated by SDS-PAGE (Schagger and von Jagow 1987 ). Protein concentration was determined by using amidoblack (Popov et al. 1975 ). Protein gel blotting and immunodetection were performed as described by Harter et al. 1993 . The phyA-specific monoclonal antibody was obtained from Brian Thomas (Warwick, UK).

Construction of phyA-211 eid1-3 Double Mutants
F₁ plants obtained by crossing phyA-211 with eid1-3 were allowed to self-pollinate to produce the F₂. To test for the presence of two phyA-211 alleles, we grew seeds from the F₂ under strong far-red light (14 µmol m^-2 sec^-1). Etiolated F₂ seedlings were selected and grown to maturity to obtain independent F₃ lines by self-pollination. These F₃ lines again were tested under strong far-red light. Because the Eid1 phenotype is strictly dependent on the presence of phyA, the homozygosity of eid1-3 could not be tested directly in the F₃ lines that were homozygous for phyA-211. Therefore, randomly chosen plants from the different F₃ lines were used as pollen donators for crosses with eid1-3. Progenies of these crossings were checked in weak red light (3 x 10^-3 µmol m^-2 sec^-1) to determine their segregation ratios for the Eid1 phenotype. F₃ plants with progeny homozygous for the recessive Eid1 phenotype were regarded as being homozygous for eid1-3, and their seeds were used for further analysis. In total, we were able to isolate seven phyA-211 eid1-3 lines that exhibited a similar response under all light conditions tested so far. The data shown in the fluence rate response curves were obtained by measuring hypocotyl length from seven seedlings from each phyA-211 eid1-3 line for each light intensity.

	FOOTNOTES

¹ Current address: Staatliches Weinbauinstitut, Merzhauserstrasse 119, D-79100 Freiburg, Germany.

	ACKNOWLEDGMENTS

We thank Martina Krenz and Beate Kiefer for their excellent technical assistance throughout the project; Peter Nick for help with the manuscript; Masaki Furuya for the gift of the phyB-5, phyA-201, and phyA-201 phyB-5 mutants; and Brian Thomas for the gift of the phyA antibody. This research was supported by a grant from the DFG Arabidopsis Schwerpunktprogramm (SPP 294 "Arabidopsis").

Received October 15, 1999; accepted February 5, 2000.

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