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CELL BIOLOGY AND SIGNAL TRANSDUCTION

RED AND FAR-RED INSENSITIVE 2, a RING-Domain Zinc Finger Protein, Mediates Phytochrome-Controlled Seedling Deetiolation Responses¹

Department of Plant Biology, University of Minnesota, St. Paul, Minnesota 55108

	ABSTRACT

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 
Light is arguably the most important resource for plants, and an array of photosensory pigments enables plants to develop optimally in a broad range of ambient-light conditions. The red- and far-red-light-absorbing photosensory pigments or phytochromes (phy) regulate seedling deetiolation responses, photoperiodic flowering, and circadian rhythm. We have identified a long hypocotyl mutant under red and far-red light, rfi2-1 (red and far-red insensitive 2 to 1). rfi2-1 was also impaired in phytochrome-mediated end-of-day far-red light response, cotyledon expansion, far-red light block of greening, and light-induced expression of CHLOROPHYLL A/B BINDING PROTEIN 3 and CHALCONE SYNTHASE. Introduction of rfi2-1 mutation into phyB-9 or phyA-211 did not enhance or suppress the long hypocotyl phenotype of phyB-9 or phyA-211 under red or far-red light, respectively, and RFI2 likely functions downstream of phyB or phyA. RFI2 was identified through the segregation of two T-DNA insertions into different recombinant lines, genetic rescue, and phenotypic characterization of a second mutant allele rfi2-2. RFI2 encodes a protein with a C₃H₂C₃-type zinc finger or RING domain known to mediate protein-protein or protein-DNA interactions, and RFI2 is localized to the nucleus. RFI2 therefore reveals a signaling step that mediates phytochrome control of seedling deetiolation.

Genetic screens have identified mutants that are defective in far-red light-mediated deetiolation responses, and the mutated genes encode phyA-specific signaling components such as FAR1, FHY1, FHY3, FIN2, FIN219, LAF1, LAF6, PAT1, and HFR1 (Whitelam et al., 1993; Hoecker et al., 1998; Soh et al., 1998; Hudson et al., 1999; Bolle et al., 2000; Fairchild et al., 2000; Hsieh et al., 2000; Ballesteros et al., 2001). Genetic screens have also identified several red light-specific mutants including gi, elf3, srr1, aprr1, ztl, pef2, pef3, and red1 (Ahmad and Cashmore, 1996; Wagner et al., 1997; Huq et al., 2000a; Makino et al., 2000; Somers et al., 2000; Liu et al., 2001; Staiger et al., 2003). A few of the mutated genes such as GI, ELF3, SRR1, APRR1, and ZTL have been molecularly characterized (Huq et al., 2000b; Makino et al., 2000; Somers et al., 2000; Liu et al., 2001; Staiger et al., 2003). Five other mutants have defective deetiolation responses under both red and far-red light and include cog1, pef1, psi2, pft1, and prr7 (Ahmad and Cashmore, 1996; Genoud et al., 1998; Cerdán and Chory, 2003; Kaczorowski and Quail, 2003; Park et al., 2003). Both cog1 and psi2 show hypersensitive hypocotyl growth response to red and far-red light, and COG1 and PSI2 therefore negatively regulate phyA and phyB signaling (Genoud et al., 1998; Park et al., 2003). COG1 encodes a Dof family member of transcription factors. In contrast, pef1 and prr7 have hyposensitive hypocotyl growth response to red and far-red light, suggesting that PEF1 and PRR7 regulate phyA and phyB signaling positively (Cerdán and Chory, 2003; Kaczorowski and Quail, 2003). PRR7 encodes a PSEUDO-RESPONSE REGULATOR and the absence of PRR7 also causes a coordinated 3-to-6-h shift in the phasing of the oscillatory expression of CCA1, LHY, and TOC1, the central components of the circadian clock (Kaczorowski and Quail, 2003). Interestingly, pft1 was hyporesponsive to far-red light but hyperresponsive to red light (Cerdán and Chory, 2003). PFT1 may therefore function at a phytochrome-signaling node where antagonistic interactions between phyA and phyB occur.

Combined biochemical and genetic approaches have also identified a few light-signaling components that function in both phyA and phyB pathways. PIF3, a bHLH transcription factor, was initially isolated by its interaction with phyA and phyB (Ni et al., 1999). A T-DNA knockout allele of PIF3 showed a hypersensitive hypocotyl growth response only to red light. Although PIF3 does not function in phyA-mediated inhibition of hypocotyl elongation, it negatively regulates both phyA- and phyB-mediated cotyledon opening and expansion responses (Kim et al., 2003). Another phyA and phyB interactor, NDPK2, functions positively in phytochrome signaling (Choi et al., 1999). Recombinant NDPK2 preferentially binds to the red light-activated form of phytochrome, and this interaction increases the activity of the recombinant NDPK2 protein. Mutation in NDPK2 showed a partial defect in red and far-red light-mediated cotyledon opening and greening responses (Choi et al., 1999). In contrast, the overexpression lines of PKS1, a phytochrome phosphorylation substrate, were less sensitive only to red light, but retained normal sensitivity to blue and far-red light, indicating that PKS1 acts as an inhibitor of phyB signaling (Fankhauser et al., 1999). phyA and phyB also interact in vitro with PIF1, PIF4, ARR4, ELF3, FyPP, and PAPP5 (Liu et al., 2001; Sweere et al., 2001; Huq and Quail, 2002, 2005; Kim et al., 2002; Ryu et al., 2005).

Although a number of phytochrome-signaling components have been identified, the mechanisms by which phytochromes regulate seedling deetiolation and flowering responses are still largely unknown (Quail, 2002; Simpson and Dean, 2002). We isolated a new long hypocotyl mutant, rfi2-1 (red and far-red insensitive 2 to 1), under both red and far-red light. rfi2-1 also showed other defects in phyA- and phyB-mediated deetiolation responses such as cotyledon expansion and light-induced gene expression. We uncovered two T-DNA insertions on chromosome 2 in rfi2-1, and the recombinant line carrying a T-DNA insertion in front of At2g47700 retained the mutant phenotype. Genetic rescue of rfi2-1 and phenotypic characterization of rfi2-2 further confirmed the identity of RFI2 gene. RFI2 defines a novel step in phytochrome signaling that controls seedling deetiolation responses.

	RESULTS

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 

rfi2-1 Shows Defects in Both phyA and phyB Signaling

To isolate new components in phyB-signaling pathway, we conducted genetic screens for mutants that display a longer hypocotyl than that of wild type under red light from a T-DNA insertion collection (Arabidopsis Biological Resource Center). One individual identified was further characterized for its long hypocotyl phenotype under various monochromatic light conditions. Although the mutant was originally isolated for its reduced sensitivity to red light, it also showed a reduced response to far-red light but not to blue light (Fig. 1, A and B). We named the newly identified mutant rfi2-1. We further examined its hypocotyl growth response under different intensities of red and far-red light. rfi2-1 showed a longer hypocotyl than that of wild type over a wide range of red-light fluence rates, indicating that RFI2 functions in a broad spectrum of red-light intensities (Fig. 2A, left). In contrast, rfi2-1 exhibited a stronger hypocotyl phenotype under relatively weak or intermediate intensities of far-red light, in the range of 1 to 10 pmol m^–2 s^–1. The long hypocotyl phenotype did not persist under relatively strong intensity of far-red light above 20 to 30 pmol m^–2 s^–1 (Fig. 2A, right).

View larger version (69K): [in this window] [in a new window]   Figure 1. rfi2-1 shows reduced response to red and far-red light inhibition of hypocotyl elongation. A, The longer hypocotyl phenotype of rfi2-1 under red and far-red light, but not under blue light. Seedlings were grown at 23°C under continuous red (10 µmol m–2 s–1), far-red (0.02 µmol m–2 s–1), or blue (15 µmol m–2 s–1) light for 4 d. At least 30 seedlings were included for each measurement, and data are presented as means ± SE. B, Four-day-old Ws and rfi2-1 seedlings grown in darkness or under different light conditions as specified in A. Bar = 2.5 mm.

View larger version (40K): [in this window] [in a new window]   Figure 2. rfi2-1 shows a long hypocotyl phenotype over a range of red and far-red light intensities and has a reduced EOD far-red light response. A, Hypocotyl elongation fluence rate responses of Ws and rfi2-1 seedlings to continuous-red (left) or far-red (right) light. B, Seedlings were grown under a 9-h red-light/15-h-dark cycle for 4 d and were treated with or without a 10-min saturating far-red light pulse at the end of each light period. Data are presented as means ± SE.

phyB is the major receptor for red light-mediated deetiolation responses and controls many light responses in a red and far-red light-reversible manner. A saturating far-red light pulse provided at the end of the day can enhance hypocotyl elongation, and this end-of-day (EOD) far-red light response is mediated by phyB (Robson et al., 1993; Aukerman et al., 1997). The EOD far-red light response was almost absent in phyB-9, and was also reduced in rfi2-1 (Fig. 2B). The ratio of ecotype Wassilewskija (Ws) R – EODFR (EOD far-red response) to Ws R + EODFR hypocotyl length was 0.79 (SD = 0.132, n = 30), and the ratio of rfi2-1 R – EODFR to rfi2-1 R + EODFR hypocotyl length was 0.91 (SD = 0.146, n = 30). Student's t tests show that the ratios are significantly different from each other (P < 0.05). The reduced EOD far-red light response in rfi2-1 suggests a function of RFI2 in phyB-mediated red-light signaling. In addition, phyB also regulates light-induced cotyledon expansion, and phyB-9 develops small cotyledons under red or white light (Gyula et al., 2003). Consistent with a positive role of RFI2 in phyB signaling, rfi2-1 developed smaller cotyledons compared to wild type when grown under continuous white light (Fig. 3A).

View larger version (45K): [in this window] [in a new window]   Figure 3. rfi2-1 has reduced cotyledon expansion and a reduced response to far-red light preconditioned blocking of greening. A, Seedlings were grown under continuous white light for 8 d, and cotyledon area of 30 seedlings was measured. B, Seedlings were grown either in darkness or under far-red light for 5 d, and then transferred to white light for additional 4 d. Each treatment included at least 40 seedlings, and chlorophyll content was measured spectrophotometrically.

RFI2 Is Required for Light-Induced Expression of CAB3 and CHS

The photophysiological experiments described above demonstrate that the rfi2-1 mutation impairs several red and far-red light-mediated responses. We further examined the light-induced expression of CHLOROPHYLL A/B BINDING PROTEIN 3 (CAB3) and CHALCONE SYNTHASE (CHS) in Ws and rfi2-1. The CAB3 gene is often used as a molecular marker to assess phytochrome-mediated responses since its expression can be quickly turned on by red or far-red light signals. Another molecular marker is CHS gene, which is involved in anthocyanin production and its expression is also tightly regulated by red and far-red light (Mancinelli, 1985, 1991). Compared to wild type, the induction of CAB3 expression by red light in rfi2-1 was reduced 5-fold, but the induction of CAB3 expression by far-red light was reduced even more dramatically (Fig. 4, A and B). The red and far-red light-induced expression of CHS in rfi2-1 was also greatly reduced. The reduced expression of CAB3 and CHS under red and far-red light in rfi2-1 is consistent with its hyposensitive hypocotyl growth response to red and far-red light.

View larger version (45K): [in this window] [in a new window]   Figure 4. rfi2-1 mutation affects light-induced expression of CAB3 and CHS. A, Northern-hybridization analysis of CAB3 and CHS expression in 5-d-old dark-grown Ws or rfi2-1 seedlings that received no light treatment (D) or were irradiated for 4 h with red (R), far-red (FR), or blue light (B) at intensities as specified in Figure 1A. Seedlings were harvested under green safety light and 10 µg total RNA was loaded in each lane. B, Normalization of CAB3 and CHS mRNA levels to EF1- signals. The hybridization signals were detected with PhosphorImager screen and quantified using Imagequant program. Data are presented as means ± SE from three independent experiments.

rfi2-1 was originally isolated from a T-DNA transformed Arabidopsis population in Ws background (Valvekens et al., 1988). The original rfi2-1 mutant was back crossed to Ws wild type for further genetic analysis. All F1 seedlings (about 51) displayed a normal hypocotyl phenotype as wild type under red light, whereas F2 population showed a segregation of three short hypocotyl to one long hypocotyl under red light (306:114), suggesting that rfi2-1 is caused by a single recessive mutation. F2 seeds were also scored directly on kanamycin plates, and the ratio of resistant seedlings to sensitive seedlings is nearly 3 to 1 (227:81), suggesting that the line contains a single T-DNA or tightly linked T-DNAs. The long hypocotyl seedlings, scored on regular medium under red light, were then transferred to kanamycin plates, and all of them survived the kanamycin selection. Data thus indicate that the T-DNA insertion is tightly linked to the long hypocotyl phenotype.

We discovered two T-DNA insertions on chromosome 2 using thermal asymmetric interlaced-PCR technique, in which three nested T-DNA border primers were used in successive PCR reactions together with an 18-bp degenerate primer (Liu et al., 1995). The identification of two T-DNA insertions on chromosome 2 is still consistent with our genetic analysis since the two T-DNA insertions are genetically linked on a single chromosome (Fig. 5A). The two insertions were 1.36 Mega bp apart and were further verified using PCR with gene-specific primers and T-DNA border primers (data not shown). Each T-DNA insertion appeared to carry an intact kanamycin gene. The first T-DNA was inserted about 89 bp upstream of the ATG start codon of At2g43290, which encodes a putative calcium-binding protein. The T-DNA insertion also caused a 49-bp deletion of the plant genomic sequence flanking the T-DNA left border. The second T-DNA was inserted about 148 bp upstream of the ATG start codon of At2g47700, which encodes a putative C₃H₂C₃-type RING-domain zinc finger protein (Fig. 5A).

View larger version (44K): [in this window] [in a new window]   Figure 5. Both rfi2-1 and rfi2-2 are knock-down mutants. A, The T-DNA insertion in rfi2-1 was identified in the promoter regions of At2g47700 (148 bp upstream of the ATG codon) on chromosome II. A second rfi2 allele, rfi2-2, was identified from SALK T-DNA insertion lines and contains a T-DNA insertion 167 bp downstream of At2g47700 stop codon. Lb, T-DNA left border. B, Northern-hybridization analysis on the expression of At2g47700 in Ws, rfi2-1, Col, and rfi2-2. 18S rRNA was probed as loading control. C, Hypocotyl growth responses of Ws, rfi2-1, rfi2-1 recombinant lines (T47700 and T43290), rfi2-1 rescue lines (C6-2 and C10-7), Col, and rfi2-2 to red light and far-red light at intensities specified in Figure 1A. Recombinant lines that carry a single T-DNA insertion in front of At2g43290 or At2g47700 are abbreviated as T43290 and T47700, respectively. Genetic complementation lines 6-2 and 10-7 are abbreviated as C6-2 and C10-7, respectively.

Genetic Complementation and Isolation of a Second Mutant Allele rfi2-2

To further confirm that At2g47700 is the candidate gene for RFI2, we cloned a genomic DNA fragment, including a 1.0-kb 5'-untranslated region sequence, At2g47700 coding sequence, and a 541-bp 3'-untranslated region, into binary vector pCAMBIA 3300. The construct was transformed into rfi2-1, and multiple independent-transgenic lines were generated and tested for their hypocotyl elongation responses under red or far-red light. We presented data for two representative lines, C6-2 and C10-7, with a wild-type hypocotyl growth response, confirming that the loss of At2g47700 function is responsible for the rfi2-1 mutant phenotype (Fig. 5C). In contrast, delivering a genomic DNA fragment spanning At2g43290 gene into rfi2-1 failed to rescue the mutant phenotype (data not shown). We also searched public databases for additional T-DNA insertion alleles in either the At2g43290 or At2g47700 gene. A T-DNA inserted 15 bp upstream of the At2g43290 ATG start codon (SAIL-77-G08) did not exhibit a visible hypocotyl phenotype under either red or far-red light condition (data not shown). In contrast, a T-DNA inserted 167 bp downstream of the At2g47700 stop codon (SALK-113269 line or rfi2-2) caused a long hypocotyl phenotype under both red and far-red light conditions, identical to that of rfi2-1 (Fig. 5C). The T-DNA insertion also reduced the expression of At2g47700 below a detection level (Fig. 5B, right).

RFI2 Functions Downstream of phyA or phyB

The level of phyA and phyB proteins were unaltered in rfi2-1 compared to wild type, suggesting that RFI2 affects processes downstream of phyA and phyB (data not shown). We constructed phyA-211/rfi2-1 and phyB-9/rfi2-1 double mutants. The phyA-211/rfi2-1 double mutant showed a strong hypocotyl phenotype similar to single phyA-211 mutant under far-red light, whereas the phyB-9/rfi2-1 double mutant showed a strong hypocotyl phenotype similar to single phyB-9 mutant under red light (Fig. 6). Mutation in RFI2 apparently is unable to enhance or suppress phyA-211 or phyB-9 phenotype. RFI2 likely functions downstream of phyA or phyB since the hypocotyl growth phenotype of the double mutants are not additive.

View larger version (49K): [in this window] [in a new window]   Figure 6. Hypocotyl growth responses of Ws, rfi2-1, Col, phyA-211, Ws/Col, and multiple rfi2-1/phyA-211 lines to far-red light (0.02 µmol m–2 s–1; A) or of Ws, rfi2-1, Col, phyB-9, Ws/Col, and multiple rfi2-1/phyB-9 lines to red light (10 µmol m–2 s–1; B).

Using BLAST and PSI-BLAST, we found that RFI2 encodes a C₃H₂C₃-type zinc finger or RING-domain protein (Freemont et al., 1991). A typical RING domain contains three Cys residues followed by two His residues and another set of three Cys residues (Fig. 7A). RFI2 homologs were also found in other plants including rice (Oryza sativa) and Avena fatual. One close relative of RFI2, AfVIP2 (gi6996144) from A. fatual, specifically interacts with AfVP1 to regulate embryo dormancy in mature imbibed seeds (Jones et al., 2000). We then selected protein sequences that contain a conserved RING domain and an overall sequence identity above 30% for phylogenetic analysis. In the phylogenetic analysis, RFI2 occupies a distinct branch but is very closely related to At3g05545 and At4g13490 from Arabidopsis and gi38175560 from rice (Fig. 7B). Organisms other than plants also contain sequences similar to the RING domain found in RFI2 N terminus, and thus RING domain is highly conserved between animals and plants.

View larger version (68K): [in this window] [in a new window]   Figure 7. RFI2 encodes a RING-finger protein and is localized to the nucleus. A, Alignment of RFI2 amino acid sequence (At2g47700) with protein sequences from Arabidopsis (At3g05545 and At4g13490), rice (gi34895186, gi34897618, and gi38175560), and Avena fatual (gi6996144). Asterisks represent identical amino acid residues and dots represent similar amino acid residues. The conserved Cys and His residues are indicated as C and H, respectively. B, Phylogenetic tree generated among RFI2 and RFI2 homologs with ClustalX software program. C, Subcellular localization of the RFI2:GFP fusion protein in transiently transfected onion epidermal cells. Green fluorescence (left) and 4',6-diamino-phenylindole staining (right) images were shown for cells exposed to white light (30 µmol m–2 s–1). Bar = 100 µm.

To further understand its cellular function, we analyzed RFI2 sequence with the PSORT program (http://psort.ims.u-tokyo.ac.jp/) but failed to identify any signature sequences that can predict RFI2's localization site. However, a search with RFI2 sequence through the Subloc program (www.bioinfo.tsinghua.edu.cn/SubLoc) predicted a nuclear localization of RFI2 with a reliability index 3 and an expected accuracy 84%. We then performed an in vivo transient localization assay with onion (Allium cepa) epidermal cells, and found that an RFI2:green fluorescent protein (GFP) fusion protein, under the control of the cauliflower mosaic virus 35S promoter, was localized to the nucleus either in darkness or under white light (Fig. 7C).

	DISCUSSION

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 

RFI2 Functions Positively in Both phyA- and phyB-Mediated Deetiolation Responses

Mutation in RFI2 shows hyposensitivity in several phyA- and phyB-mediated red or far-red light responses, including the inhibition of hypocotyl elongation, cotyledon expansion, EOD far-red light response, far-red light-preconditioned block of greening, and light-induced expression of CAB and CHS genes. In addition, rfi2-1 has much elongated petioles under white light, which is a characteristic of phyB-deficient mutants (Koornneef et al., 1995; data not shown). rfi2-1 also flowers early (data not shown). Since both rfi2-1 and rfi2-2 are recessive and loss-of-function mutants, RFI2 thus encodes a positive regulator of phyA and phyB signaling that controls seedling deetiolation responses.

Other red and far-red light mutants include cog1, pef1, psi2, pft1, and prr7 (Ahmad and Cashmore, 1996; Genoud et al., 1998; Cerdán and Chory, 2003; Kaczorowski and Quail, 2003; Park et al., 2003). Among these mutants, pef1 and prr7 show a hyposensitive hypocotyl growth response to both red and far-red light similar to rfi2 (Cerdán and Chory, 2003; Kaczorowski and Quail, 2003). PRR7 encodes a PSEUDO-RESPONSE REGULATOR and the absence of PRR7 also causes a coordinated 3-to-6-h shift in the phasing of the oscillatory expression of CCA1, LHY, and TOC1 (Kaczorowski and Quail, 2003). Interestingly, pef1 also has an early flowering phenotype under both long-day and short-day conditions similar to rfi2 (Ahmad and Cashmore, 1996). pef1 mutation was mapped to the upper arm on chromosome 3, whereas RFI2 gene is on chromosome 2, excluding the possibility that rfi2 is allelic to pef1 (Ahmad and Cashmore, 1996).

RFI2 Encodes a Nuclear RING-Domain Protein

RING domain or RING-finger domain is a specialized type of zinc finger of 40 to 60 residues that binds two atoms of zinc. RING domain is defined by a cross-brace motif C-X2-C-X(9–39)-C-X(1–3)-H-X(2–3)-(N/C/H)-X2-C-X(4–48)-C-X2-C. RING domain has two variants, the C₃HC₄ type and the C₃H₂C₃ type (RING-H2 finger) as defined by their different Cys and His patterns (Freemont et al., 1991). RFI2 belongs to the C₃H₂C₃ type (Fig. 7A). In other organisms, RING-domain-containing proteins function in a wide range of physiological and cellular processes, including development, oncogenesis, apoptosis, viral replication, transcription, RNA processing, organelle transport, and peroxisomal biogenesis (Borden, 2000). The RING-domain proteins are found throughout the nucleus and cytoplasm, and the unifying theme appears to be the ability of RING domains mediating protein-protein interactions, particularly those involved in the formation of large macromolecular complexes. Other RING-domain proteins are involved in DNA binding and DNA-dependent regulation of transcription. For example, RING1 can bind to HPC3 (human polycomb 3) in vivo and is involved in repression of transcriptional activity (Bardos et al., 2000). TRAF2A (TNFR [tumor necrosis factor receptor]-associated factor 2A), another RING-finger protein, inhibits TNFR2-mediated NF-KappaB activation (Brink and Lodish, 1998). In A. fatual, AfVIP2 (gi6996144), a close relative of RFI2 in plants, specifically interacts with AfVP1, a putative transcription factor, to control embryo dormancy in mature imbibed seeds (Jones et al., 2000).

RING domain was also found in proteins with E3 ubiquitin-protein ligase activity and various RING fingers exhibit binding activity toward E2 ubiquitin-conjugating enzymes within an ubiquitin ligase complex (Saijo et al., 2003). A plant protein that contains a less homologous RING domain to that of RFI2 is COP1 in Arabidopsis. COP1 also contains a coiled-coil and a WD40 domain, acts as an ubiquitin-protein ligase, and functions as a constitutive repressor of photomorphogenesis within the nucleus (Deng et al., 1992; Saijo et al., 2003). Despite the diverse cellular function of RING-domain proteins, RING domain appears to be involved in mediating either protein-protein interactions or protein-DNA interactions. Considering the overall sequence homology and domain structure of RFI2 to AfVIP2, RFI2 likely interacts with other nuclear regulatory proteins such as Arabidopsis AfVP1-like proteins. The nuclear function might be required for its involvement in regulating both seedling deetiolation and photoperiodic flowering responses in Arabidopsis.

	MATERIALS AND METHODS

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 

Plant Materials and Photobiology

T-DNA insertion lines (ecotype Ws) transformed with pD991-AP3, a derivative of T-DNA vector pD991 (Valvekens et al., 1988), were acquired from the Arabidopsis Biological Resource Center. Seeds were surface sterilized, plated on agar growth medium without Suc, stratified in darkness at 4°C for 4 d, treated with 1 h white light, and transferred to appropriate light-emitting diodes Snap-Lite sources (Quantum Devices) for 4 d. Light intensity and peak wavelength were measured with a SPEC-UV/PAR spectroradiometer (Apogee Instruments). Hypocotyl, cotyledon, and leaf images were taken using an Olympus digital Camedia C-700, and their length and area were measured using ImageJ. Petiole length and leaf area were measured for the first fully expanded true leaf. In the EOD far-red light experiments, seedlings were grown under a 9-h red-light/15-h-dark cycle for 4 d. At the end of each day, a pulse of 10-min saturating far-red light pulse was delivered. To assess far-red light block of the greening response, chlorophyll was extracted from seedlings with absolute ethanol and the absorbance of the extract was measured spectrophotometrically at 665 and 649 nm (Barnes et al., 1996).

Genetic Analysis and Molecular Cloning of RFI2

For genetic analysis, rfi2-1 was crossed to Ws wild type, and F2 seedlings were examined for their hypocotyl growth responses under red light to determine the segregation ratio of long versus short hypocotyl individuals. The long hypocotyl seedlings were then transferred to soil, and the F3 seedlings were genotyped on kanamycin plates. RFI2 was cloned by thermal asymmetric interlaced-PCR on rfi2-1 genomic DNA prepared with DNeasy plant mini kit (Qiagen; Liu et al., 1995). The T-DNA left-border primers used are JL1 (5'-CGACGGATCGTAATTTGTCGTTTTATCAA-3'), JL202 (5'-CATTTTATAATAACGCTGCGGACATCTAC-3'), and JL270 (5'-TTTCTCCATATTGACCATCATACTCATTG-3'). The PCR fragments were directly sequenced to anchor the T-DNA insertions.

Northern-Blot Analysis

To examine light-induced gene expression in rfi2-1, seedlings were treated with monochromatic red, far-red, or blue light for 4 h after an initial growth for 5 d in darkness. Total RNA was prepared with the SV Total RNA isolation system (Promega). Ten micrograms of total RNA was loaded onto each lane on formaldehyde MOPS gels and was transferred to Hybond-N (Amersham Biosciences) using 10xSSC. Probes were generated with Rediprime II Random Primer labeling system (Amersham Biosciences). Northern hybridization was performed at 65°C in Church buffer and washed at 65°C for 40 min with 0.1xSSC and 0.2% SDS (Ni et al., 1998). The blots were exposed to both x-ray films and PhosphorImager screens, and the band intensity was quantified using Imagequant program (Molecular Dynamics).

Complementation of rfi2-1 and Double Mutant Analysis

rfi2-1 was genetically complemented by introducing a 3-Kb DNA fragment spanning the RFI2 gene coding sequence. The DNA fragment was amplified from Ws genomic DNA using primer pair 5'-CGGAATTCACAGGATATACAAGGAGGAGC-3' and 5'-GCTCTAGAGCTCTTGTTGTGGGAAGCCATGG-3' with end-incorporated EcoRI and XbaI restriction sites. After restriction digestion, the genomic fragment was cloned into EcoRI and XbaI sites of pCMBIA3300 binary vector. The construct was introduced into Agrobacterium tumefaciens GV3101 and was then transformed into rfi2-1 plants with vacuum infiltration (Bechtold et al., 1993). The rfi2-1/phyA-211 or rfi2-1/phyB-9 double mutants were generated in the same way as previously described (Kang et al., 2005). To correct the different ecotype backgrounds in the double mutants, F2 or F3 plants, which were wild type for both loci, were used as controls.

RFI2 Sequence Alignment, Phylogenetic Analysis, and Subcellular Localization

Sequence alignment of RFI2 with other proteins was performed using ClustalW (www.hgsc.bcm.tmc.edu/searchlauncher) and Boxshade (www.ch.embnet.org/software/Box_form.html) software programs. Phylogenetic tree was generated by ClustalX (ftp://ftp-igbmc.u-strasbg.fr/pub/clustalX/) and viewed by Treeview (taxonomy.zoology.gla.ac.uk/rod/treeview.html). The subcellular localization of RFI2 was predicted with Subloc online software program (www.bioinfo.tsinghua.edu.cn/SubLoc).

To study the subcellular localization, RFI2 coding sequence was PCR amplified using a primer pair 5'-GCTCTAGGCCGGAGCTAAAGATT-3' and 5'-TCCCCCCGGGGAAGTGTCTATGCCACAAGCT-3' with end-incorporated XbaI and XmaI restriction sites. The PCR fragment was then cloned into the XbaI and XmaI sites of a modified PBI121 binary vector, in which the -glucuronidase coding sequence was replaced with a GFP coding sequence. The construct was delivered into onion (Allium cepa) epidermal cells using Bio-Rad PDS-1000 helium biolistic gun (Donpont). The transformed tissues were then incubated on agar plate under white light or in darkness for 24 h at 25°C. GFP fluorescence and 4',6-diamino-phenylindole nuclear images were acquired using a Nikon Eclipse E800 microscope with a Cool Cam color CCD camera (Cool Camera) and Image Pro Plus version 3.0 software (Media Cybermetics).

Sequence data from this article can be found in the GenBank/EMBL data libraries under accession number gi42470189.

	ACKNOWLEDGMENTS

 
We thank Drs. Neil Olszewski and David Marks for comments on the manuscript, and Dr. David Marks for help and use of Nikon Eclipse E800 microscope. We also thank the Ohio State Stock Center for Arabidopsis T-DNA insertion collections and mutant seeds.

Received October 20, 2005; returned for revision November 19, 2005; accepted November 29, 2005.

	FOOTNOTES

 
¹ This work was supported by the University of Minnesota start-up and Grants-in-Aid funds, and by the National Research Initiative of the U.S. Department of Agriculture Cooperative State Research, Education and Extension Service (grant no. 2004–35304–14939).

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Min Ni (nixxx008{at}tc.umn.edu).

Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.105.073163.

^* Corresponding author; e-mail nixxx008{at}tc.umn.edu; fax 612–625–1738.

	LITERATURE CITED

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