Gene expression, localization, and protein–protein interaction of Arabidopsis SKP1-like (ASK) 20A and 20B

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Plant Science 174 (2008) 485–495 www.elsevier.com/locate/plantsci

Gene expression, localization, and protein–protein interaction of Arabidopsis SKP1-like (ASK) 20A and 20B Yasunobu Ogura a,1, Norihisa Ihara b,1, Akihiro Komatsu b,1, Yoko Tokioka a, Mami Nishioka b, Tomoyuki Takase a, Tomohiro Kiyosue a,b,* a

Division of Genome Analysis and Genetic Research, Life Science Research Center, Institute of Research Promotion, Kagawa University, 2393 Ikenobe, Miki-cho, Kita-gun, Kagawa 761-0795, Japan b Department of Life Science, Faculty of Agriculture, Kagawa University, 2393 Ikenobe, Miki-cho, Kita-gun, Kagawa 761-0795, Japan Received 23 July 2007; received in revised form 17 January 2008; accepted 20 February 2008 Available online 3 March 2008

Abstract Arabidopsis SKP1-like 20 (ASK20) has two translational products, ASK20A and ASK20B, as a result of alternative splicing. Both ASK20A and ASK20B mRNAs were detected in every organ examined, and only small differences in their accumulation levels were observed in plants exposed to abiotic stress or treated with growth regulators. Transgenic plants possessing ASK20 promoter:GUS (b-glucuronidase) displayed GUS activity in the root, leaf, flower, and seed, in agreement with reverse transcriptase-polymerase chain reaction (RT-PCR) results. Transgenic plants possessing ASK20 promoter:ASK20B-GUS showed a pattern of GUS activity similar to that of ASK20 promoter:GUS plants, whereas transgenic plants possessing ASK20 promoter:ASK20A-GUS displayed little GUS activity in the root vein, cotyledon, sepal, and stamen. GUS activity of the transiently expressed ASK20A-GUS and ASK20B-GUS as well as green fluorescent protein (GFP) signals for ASK20A-GFP and ASK20B-GFP in transgenic plants were localized in the cytosol. In a yeast two-hybrid system, both ASK20A and ASK20B interacted with two F-box proteins (At3g61590 and At5g39250). Neither ASK20A nor ASK20B interacted with CUL1 (cullin1) in the yeast two- or three-hybrid system, and little interaction of ASK20 proteins with CUL1 was detected in a pull-down assay. # 2008 Elsevier Ireland Ltd. All rights reserved. Keywords: Arabidopsis; ASK20A; ASK20B; CUL1; F-box protein; SCF complex

1. Introduction Targeted protein degradation by the ubiquitin–proteasome system is involved in many physiological phenomena. This reaction can be divided into two processes: binding of ubiquitin to a target protein and ATP-dependent degradation of the polyubiquitinated protein by the 26S proteasome [1]. Protein

Abbreviations: ASK, Arabidopsis SKP1-like; 3-AT, 3-amino-1H-1,2,4triazole; CaMV, cauliflower mosaic virus; CTAB, cetyltrimethylammonium bromide; CUL, cullin; DAPI, 40 ,6-diamidino-2-phenylindole; GFP, green-fluorescent protein; GUS, b-glucuronidase; PVDF, polyvinylidene difluoride; RTPCR, reverse transcriptase-polymerase chain reaction; SCF, SKP1–Cullin1–Fbox protein–RBX1; SDS, sodium dodecyl sulfate. * Corresponding author at: Division of Genome Analysis and Genetic Research, Life Science Research Center, Institute of Research Promotion, Kagawa University, 2393 Ikenobe, Miki-cho, Kita-gun, Kagawa 761-0795, Japan. Tel.: +81 87 891 3401; fax: +81 87 891 3405. E-mail address: [email protected] (T. Kiyosue). 1 These authors contributed equally to this work. 0168-9452/$ – see front matter # 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.plantsci.2008.02.010

ubiquitination requires three enzymes: ubiquitin-activating enzyme E1, ubiquitin-conjugating enzyme E2, and ubiquitin– protein ligase E3. E1 activates ubiquitin via ATP-dependent adenylation. The activated ubiquitin is accepted by E2 and transferred to the target protein with the aid of E3. E3 possesses an E2-binding site and a substrate-binding site. Since E3 is involved in the determination of substrate specificity, large and diverse groups of E3s exist in eukaryotes. E3s can be divided into a number of groups on the basis of the structures of their active centers; these groups are Homologous to E6AP Carboxy Terminus (HECT), Really Interesting New Gene (RING), UFD2 homology (U-box), and Plant HomeoDomain (PHD) [2–4]. Many RING-type E3s can function as single subunit enzymes in which the RING domains function as E2-binding sites. However, diverse and complex forms of RING-type E3s, which contain the RING finger protein as a subunit, also function in cells. One of the major E3s is the SCF (SKP1– cullin1–F-box protein–RBX1) complex. In this complex, SKP1 functions as an adaptor between cullin1 (CUL1) and the F-box

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protein [5]. X-ray analysis has shown that CUL1 interacts with SKP1 in its long rod-like domain, and with RBX1, a RING finger protein, in its spherical domain [5]. RBX1 functions in the interaction between E2 and CUL1. F-box protein interacts with SKP1 in its F-box region, which consists of 40–50 amino acid residues, and with each target protein in its C-terminal substrate-binding site, which controls the substrate specificity of the SCF complex [6–8]. In Arabidopsis, 21 SKP1-like, 11 cullin, two RBX1, and more than 700 F-box protein genes have been reported, and their products have been postulated to function as members of SCF complex-type E3 [9–14]. The binding site between human SKP1 and SKP2, an F-box protein, has a four-layered sandwich structure. Three helix domains of SKP1—H5, H6, and H7—bind to the H1, H2, and H3 domains of SKP2, and the H8 domain of SKP1 interacts with the C-terminal LRR motif of SKP2 [15]. The H5, H6, H7, and H8 domains are conserved in proteins encoded by all 21 ASK genes except ASK7, which implies that the ASK gene family may collectively perform a range of functions as a component of the SCF complex [14,16].

Recent studies of ASK genes using reverse transcriptasepolymerase chain reaction (RT-PCR) and promoter:GUS fusion genes have demonstrated that each ASK gene is expressed in certain organs or tissues. ASK1 and ASK18 mRNAs have been detected in the seedling, root, stem, leaf, inflorescence, and fruit; ASK3 mRNA has been detected only in the inflorescence and fruit; ASK16 and ASK17 mRNAs have been detected preferentially in the fruit; and ASK14 mRNA is most abundant in the inflorescence [14,17,18]. However, different research groups have reported different expression patterns of ASK2, ASK11, and ASK13. Such diverse expression patterns suggest that ASK genes may regulate different developmental and physiological processes. Indeed, an ask1 mutant (ask1-1) and ASK1 RNAi lines exhibit abnormal vegetative growth, floral defects, and reduced fertility, whereas T-DNA-insertion mutants of ASK11 and ASK12 and Ds transposon-insertion mutants of ASK14 and ASK18 show no obvious phenotypic changes [18]. ASK1 is predominantly expressed during the leptotene to pachytene stages and represses homologous recombination in male meiosis [19].

Fig. 1. Expression of ASK20A and ASK20B in various organs of Arabidopsis thaliana plants, and in plants exposed to abiotic stress or treated with growth regulators. (A) Schematic representation of genomic structure of ASK20A and ASK20B and oligonucleotide primers used in RT-PCR. Alternative splicing at the sixth intron of ASK20 produces two transcripts, ASK20A and ASK20B. (B) Polyacrylamide gel separation of RT-PCR products. DNA fragments were stained with ethidium bromide and visualized by UV irradiation. Total RNAs of whole plants were used. (C) Total RNAs of root (R), stem (St), rosette leaf (RL), cauline leaf (CL), flower (Fl), dry seed (DS), and fruit (Fr) were used for RT-PCR. (D) Total RNAs of rosette plants exposed to the following conditions: drought (Dry), 250 mM sodium chloride (NaCl), wound (Wound), 4 8C (Cold), or 42 8C (Heat) for 10 h were used for RT-PCR. (E) Total RNAs of rosette plants treated with 0.1 mM 2,4-dichlorophenoxyacetic acid (2,4-D), 0.1 mM gibberellic acid (GA3), 0.1 mM abscisic acid (ABA), 0.05 mM methyl jasmonate (MeJA), 0.1 mM kinetin (Kinetin), or 7 mM ethephon (Ethephon) for 10 h were used for RT-PCR. In each case, UBQ10 was amplified as a control. Relative mRNA levels were calculated using the mRNA level of root (C) or control (D and E). Error bars are S.D. (n = 3).

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Among ASK proteins, ASK20 and ASK21 are distinguishable because, according to a phylogenetic analysis based on amino acid sequences, they belong to another clade different from other typical ASK proteins [16]. The N-terminal regions of ASK20 and ASK21 are especially varied from those of typical ASKs and SKP1. In SKP1, the N-terminal region is the binding site for CUL1 [5]. Furthermore, unlike other ASKs, the ASK20 gene produces two proteins (ASK20A and ASK20B) owing to alternative splicing of its RNA (Fig. 1A, [16]). Both proteins possess extended C-terminal regions that exhibit little similarity in amino acid sequences to those of other proteins listed in the National Center for Biotechnology Information (NCBI) database [16]. In this report, we characterized the mRNA accumulation pattern of each gene, the promoter activity of ASK20, and the organ- and tissue-specific expression of each protein, using GUS (bglucuronidase) as a reporter. Also, we used GUS and green fluorescent protein (GFP) reporters to examine the intracellular localization of both proteins, and we used yeast two- and three-hybrid systems and a pull-down assay to examine the molecular interactions of ASK20A and ASK20B with several F-box proteins and CUL1. 2. Materials and methods 2.1. Plant material, growth conditions, and treatments The Arabidopsis thaliana Columbia accession was used in the experiments. Plants were grown on vermiculite in pots or on 1/3 basal salt Murashige and Skoog (MS) [20] agar (0.8%, w/v) medium containing 1% sucrose in plates at 22 8C under long day (LD) conditions (16 h light and 8 h darkness). White fluorescent tubes (40 W, Hitachi, Japan) were used as the light source (90–100 mmol m2 s1). For selection by kanamycin, surface-sterilized Arabidopsis seeds were sown axenically on 1/ 3 basal salt MS medium containing 1% sucrose, 0.8% agar, 50 mg/l carbenicillin (Wako, Tokyo, Japan), 50 mg/l kanamycin (Nacalai Tesque, Tokyo, Japan), with or without 25 mg/l hygromycin (Wako), incubated at 4 8C in the dark for 3 days to break dormancy, and grown under LD conditions. Two-weekold rosette plants grown on agar plates were used in stress exposure or growth regulator treatment experiments before bolting. For dehydration treatment, plants were harvested from the agar plates and dehydrated on glassine paper (Hakuai, Tokyo, Japan) under dim light. Plants exposed to salt stress or treated with 2,4-dichlorophenoxyacetic acid (2,4-D), gibberellic acid (GA3), abscisic acid (ABA), or kinetin were grown hydroponically under dim light in MS medium containing 250 mM NaCl, or in medium containing 0.1 mM 2,4-D, 0.1 mM GA3, 0.1 mM ABA, or 0.1 mM kinetin. In the case of jasmonic acid or ethylene treatment, plants were grown hydroponically in 1/3 basal salt MS medium containing 0.05 mM methyl jasmonate or 7 mM ethephon. Cold or heat treatment was conducted under dim light by exposing plants grown at 22 8C to a temperature of 4 or 42 8C. In each case, the plants were subjected to the treatment for 1, 2, 5, 10, or 24 h and frozen in liquid nitrogen. For wounding treatment, plants were

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wounded with the tweezers, then allowed to recover for 1, 2, 5, 10, or 24 h before they were frozen in liquid nitrogen. 2.2. RT-PCR Total RNA was isolated from Arabidopsis plants by means of the phenol–sodium dodecyl sulfate (SDS) method [21], or Sepasol–RNA I Super according to the manufacturer’s instructions (Nacalai Tesque, Kyoto, Japan). RNA from seed or fruit was extracted by means of the cetyltrimethylammonium bromide (CTAB) method [22]. One-step RT-PCR was performed using Access quick RT-PCR System (Promega, Madison, WI). For ASK20 cDNA amplification, the forward primer 50 -GACCTTACTGAGGAGGAGAAATTAGAGCCT-30 and reverse primer 50 -TGAAGAAACAATCTTCTGAT-30 were used, and for UBQ10 cDNA amplification the forward primer 50 -CCGGAAAACAATTGGAGGATGGT-30 and reverse primer 50 -GTCATTAGAAAGAAAGAGATAAC-30 were used. PCR conditions were as follows: 40 cycles at 95 8C for 30 s, 52 8C for 1 min, and 72 8C for 1 min for ASK20; and 20 cycles at 95 8C for 30 s, 47 8C for 1 min, and 72 8C for 1 min for UBQ10. PCR products were separated on 5% (w/v) polyacrylamide gel for ASK20 and on 1.5% (w/v) agarose gel for UBQ10, stained with ethidium bromide, and visualized under UV light. Gel images were captured with a Printgraph video capture device (Model AE-6911FX, Atto, Tokyo, Japan) and analyzed with the CS analyzer software (Atto). 2.3. Construction of GUS and GFP fusion genes To construct pASK20:GUS, pASK20:ASK20A-GUS, pASK20:ASK20B-GUS, pASK20:GFP, pASK20:ASK20A-GFP, or pASK20:ASK20B-GFP, genomic DNA that corresponded to the 2-kbp promoter region with the first 15 nucleotides of the ASK20 gene, the entire ASK20A gene without its stop codon or the entire ASK20B gene without its stop codon were amplified from a BAC clone (F4I18) by PCR with KOD-Plus-DNA polymerase (Toyobo, Osaka, Japan). A forward primer (50 -CACCTCTGTCGATATTAGAATAGTCTTTTA-30 ) and a reverse primer (50 -ATCACCTTCTGACATTGATAATAGATTTGA-30 for pASK20, 50 -CAGCCTTGTGATCTGTGAAACAGAATTTTC-30 for pASK20:ASK20A, and 50 TGGAGATTGACCTTCAAATTCAAACCACAA-30 for pASK20:ASK20B) were used. Using GATEWAY technology (Invitrogen, Carlsbad, CA), each PCR product was subcloned into a GATEWAY entry vector, pENTR/D TOPO, sequenced to verify the DNA sequence, and cloned into a destination binary vector, pGWB203 (GUS fusion) or pGWB204 (GFP fusion), which were kindly provided by Dr. T. Nakagawa (Shimane University). The binary vectors were sequenced to verify inframe fusion of PCR fragments to the GUS or GFP gene. 2.4. Analysis of DNA sequences Plasmid DNA templates for sequencing were prepared in automatic plasmid isolation systems (models PI-200 and PI50a, Kurabo, Osaka, Japan). DNA sequences were determined

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with the BigDye Terminator Cycle Sequencing method on a DNA sequencer (ABI PRISM 3100 Genetic Analyzer; Applied Biosystems, Foster City, CA). The GENETYX (Software Development, Tokyo, Japan) and Sequencher (Gene Codes Corporation, Ann Arbor, MI) software systems were used to analyze the DNA sequences. 2.5. Agrobacterium-mediated transformation Agrobacterium tumefaciens strain GV3101 was transformed with destination binary vectors by tri-parental mating [23]. Arabidopsis plants were transformed by the floral dip method [24]. 2.6. Bombardment experiments Full-length ASK20A or ASK20B cDNA without the stop codon was amplified by PCR with the forward primer 50 AAGATCTGTATGTCAGAAGGTGATTTGGCC-30 and the reverse primer 50 -TAGATCTCAGCCTTGTGATCTGTGAAACAG-30 (for ASK20A) or 50 -TAGATCTTGGAGATTGACCTGTATGCCGTC-30 (for ASK20B). It was then cloned into the pGEM-T vector (Promega, Madison, WI) and sequenced to verify its DNA sequence. The inserts were enzymatically cut out and ligated to the pBI221 vector (Clontech, Palo Alto, CA) to obtain a GUS in-frame fusion construct under the control of the CaMV 35S promoter. One microgram of the resultant plasmid DNA was bombarded into Arabidopsis petiole cells and onion epidermal cells with a PDS-1000/He Biolistic Particle Delivery System (BioRad Laboratories, Inc., Hercules, CA). The bombarded petioles and onion epidermis were maintained at 25 8C for at least 12 h until they were observed microscopically. As a control, pBI221 was also bombarded into Arabidopsis petiole cells and onion epidermal cells. As control for nuclear localization, pUC/GFP-COP1NLS-GUS, which was kindly provided by Dr. Naoki Yamamoto (Ochanomizu University), was used [25]. 2.7. GUS staining, GFP, and DAPI observation For histochemical localization of GUS activity, the transgenic plants were soaked in 90% (v/v) acetone for 1 h at 20 8C, rinsed with 100 mM sodium phosphate buffer (pH 7.0), soaked in GUS staining solution (0.5 mg/ml X-Gluc, 0.5 mM potassium ferricyanide, 0.5 mM potassium ferrocyanide, and 100 mM sodium phosphate buffer, pH 7.0), and incubated at 37 8C in the dark. Optical images of GUS activity were obtained with an Olympus SZX12 stereomicroscope (Olympus, Tokyo, Japan). Optical images of GFP activity and 40 ,6-diamidino-2-phenylindole (DAPI) staining were obtained with an Olympus BX51 microscope equipped with a fluorescent unit and suitable filter units. GFP fluorescence was also visualized with a Leica TCS-E confocal laser scanning microscope equipped with an air-cooled argon ion laser system (Leica Microsystems GmbH, Mannheim, Germany).

2.8. cDNAs cDNAs of ASK1, ASK20A, ASK20B, and AtCUL2 were obtained by RT-PCR using RNAs from rosette plants and the following primer sets: ASK1 forward: 50 -AGGATCCGTATGTCTGCGAAGAAGATTGTG-30 , reverse: 50 -AGGATCCTCATTCAAAAGCCCATTGGTTCT-30 ; ASK20A forward: 5 0 -AAGATCTGTATGTCAGAAGGTGATTTGGCC-3 0 , reverse: 50 -TAGATCTCAGCCTTGTGATCTGTGAAACAG30 ; ASK20B forward: 50 -AAGATCTGTATGTCAGAAGGTGATTTGGCC-30 , reverse: 50 -TAGATCTTGGAGATTGACCTGTATGCCGTC-30 ; and AtCUL2 forward: 50 -CATATGATGGCGAAGAAGGATTCTGTGTTG-30 , reverse: 50 -CATATGCTAAGCCAAATACTTGAAAGTGTT-30 . cDNAs of AtCUL1 were obtained from the RIKEN Bio Resource Center (Tsukuba, Japan) and used as a template for PCR fragment production with the primer set AtCUL1 forward: 50 GGATCCGTATGGAGCGCAAGACTATTGACT-30 , reverse: 50 -GGATCCCTAAGCCAAGTACCTAAACATGTT-30 . Each PCR product was cloned into pGEM-T and sequenced to verify its DNA sequence. 2.9. Yeast two- and three-hybrid assays The yeast two-hybrid assay was performed as described previously [26]. F-box protein constructs in the pGAD424 vector (Invitrogen) were kindly provided by Dr. Hirofumi Kuroda and Dr. Minami Matsui (RIKEN Plant Science Center) [18]. Full-length cDNA clones for ASK1, ASK20A, and ASK20B or those for CUL1 and CUL2 were subcloned into the pGBKT7 vector (Clontech) or the pGADT7 vector (Clontech), respectively, and used for the yeast two-hybrid assay. For the ASK20-N, ASK20A-C, and ASK20B-C constructs, PCR products were amplified, subcloned into pGBKT7, and sequenced. The primer sets used were ASK20-N forward (50 -AAGATCTGTATGTCAGAAGGTGATTTGGCC30 ), ASK20-N reverse (50 -AGATCTCCGTGGATCATCCATTGAGTTCTT-30 ); ASK20A-C forward (50 -AGATCTGTATTCGACTACTGAATAGATTAT-30 ), ASK20A-C reverse (50 -TAGATCTCAGCCTTGTGATCTGTGAAACAG-30 ); and ASK20B-C forward (50 -AGATCTGTATTCGACTACTGAATAGATTAT-30 ), ASK20B-C reverse (50 -TAGATCTTGGAGATTGACCTGTATGCCGTC-30 ). For the yeast three-hybrid assay, ASK1, ASK20A, and ASK20B were amplified by PCR to attach NotI at the 50 end and BglII at the 30 end, then were cloned into pBluescript SK+ (Stratagene, La Jolla, CA) to verify their nucleotide sequences, and subcloned into MCS II of the pBridge vector (Clontech). The primer sets used were ASK1 forward (50 -AGCGGCCGCAGTATGTCTGCGAAGAAGATT-30 ), ASK1 reverse (50 -GAGATCTCATTCAAAAGCCCATTGGTTCTC-30 ), ASK20A and ASK20B forward (50 -AGCGGCCGCAGTATGTCAGAAGGTGATT0 TG-3 ), ASK20A reverse (50 -GAGATCTTACAGCCTTGTGATCTGTGAAAC-30 ), and ASK20B reverse (50 -TAGATCTCATGGAGATTGACCTGTATGCCG-30 ). An EcoRICUL1-SalI fragment from pGBKT7-CUL1 was subcloned into MCS I of pBridge, pBridge-ASK1, pBridge-ASK20A, and

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pBridge-ASK20B. Positive control vectors (pBridge-g1-s1 and pGAD-b1) were kindly provided by Dr. Kazuhisa Nakayama (Kyoto University) [27]. 2.10. Pull-down assay To construct vectors for insect cell expression, cDNAs of FLAG-tagged ASK1, FLAG-ASK20A, and CUL1-HA were amplified by PCR. The primer sets used were FLAGASK1 forward (50 -AAATGGACTACAAGGATGACGACGACAAGTCTGCGAAGAAGATTGTGTTGAAGAG-30 ), ASK1 reverse (50 -AATCATTCAAAAGCCCATTGGTTCTCTC-30 ); FLAG-ASK20A forward (50 -AGAATTCATGGACTACAAGGATGACGACGACAAGATGTCAGAAGGTGATTTGGC-30 ), ASK20A reverse (50 -ACTCGAGTTACAGCCTTGTGATCTGTGAA-30 ); and CUL1 forward (50 -AGGATCCGTATGGAGCGCAAGACTATTGAC-30 ), CUL1-HA reverse (50 -AACTAAGCGTAATCTGGTACGTCGTAAGCCAAGTACCTAAACATGTTAG-30 ). PCR-amplified fragments were subcloned into pBluescriptSK+ (Stratagene) to verify their nucleotide sequences, and then inserted into a multi-cloning site of pFastBac1 (Invitrogen). To construct GST-At3g61590 and GST-At5g39250 expression vectors, cDNAs encoding At3g61590 and At5g39250 were amplified by PCR, subcloned into pBluescriptSK+ (Stratagene) to verify their nucleotide sequences, and then inserted into pFastBac1-GST, which was provided by Dr. Yoshinao Katsu (National Institute for Basic Biology). The primer sets used were At3g61590 forward (50 AGTCGACGAATGGAAGCAGAAACGTCTTGG-30 ), At3g61590 reverse (50 - AGCGGCCGCTAAGGAGCAATCTCGAGTCTT-30 ); and At5g39250 forward (50 - AGAATTCATGTTTAGTGAAGAAGTATTGAA-30 ), At5g39250 reverse (50 TGCGGCCGCTCACTTCCAATCAAGCATATC-30 ). These plasmids were introduced into Escherichia coli DH10Bac (Invitrogen) to generate recombinant bacmid DNAs. Recombinant bacmid DNAs were transfected into Sf21 insect cells derived from Spodoptera frugiperda. Recombinant baculoviruses were harvested and amplified. For protein expression, Sf21 cells were co-infected with recombinant baculoviruses encoding FLAG-ASK, CUL1-HA, and GST-F-box proteins. After 2 days of culture at 28 8C, cells were harvested, resuspended in 1 TBS (50 mM Tris–HCl [pH 7.4], 150 mM NaCl), and disrupted by sonication of four times of 5-s-long pulses. Soluble fractions were collected by centrifugation and incubated with ANTI-FLAG M2 affinity gel (Sigma, Saint Louis, MO). After four washes with 1 TBS, bound proteins were eluted with 0.1 M glycine HCl (pH 3.5). The soluble fraction and eluates were separated by SDS-PAGE, transferred onto polyvinylidene difluoride (PVDF) membranes, and crossreacted with anti-FLAG M2 monoclonal antibody (Sigma), the anti-GST monoclonal antibody GTX70196 (GeneTex, Inc., San Antonio, TX), and anti-CUL1 polyclonal antibody. The anti-CUL1 polyclonal antibody was generated with a synthetic polypeptide cocktail (Operon Biotechnologies, K.K., Tokyo, Japan). Signals were developed by ECL Advance (GE Healthcare UK Ltd., Little Chalfont, UK), captured by a Light capture (Model AE-6962; Atto), and analyzed with the CS analyzer software (Atto).

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2.11. T-DNA insertion mutant analysis Seeds were sown from a seed pool, SALK_042202, which contained a T-DNA insertion line in the Columbia accession for ASK20, from the Arabidopsis Biological Resource Center (Columbus, OH), and genomic DNA was isolated from the leaves of 4-week-old plants. The genomic DNA was used for PCR with the primer set 50 -GATCCAGCCATGAGGGAACTGCTTGATAGG-30 (primer 1 in Supplementary Fig. S1D), which corresponded to ASK20, and 50 -TGGTTCACGTAGTGGGCCATCG-30 (primer 2 in Supplementary Fig. S1D), which corresponded to the T-DNA left border, or 50 -AAGCAATTGATCTTGATCAAGGAACTTAAA-30 (primer 3 in Supplementary Fig. S1D), which corresponded to ASK20, to identify homo T-DNA insertion plants. 2.12. Sense plants, anti-sense plants, and RNAi plants pBE2113 expression vector, which contained CaMV 35S promoter and omega translational enhancer [28], was used for the overexpression of ASK20A and ASK20B cDNA in either the sense or antisense orientation. The RNAi vector pHellstage8 [29] was kindly provided by Dr. Peter M. Waterhouse (CSIRO Plant Industry, Canberra, Australia). To generate the ASK20 RNAi construct, a 400-bp ASK20 coding region from the initiation ATG codon was amplified from ASK20 cDNA with the primers 50 -CACCATGTCAGAAGGTGATTTGGCCGTCAT-30 and 50 TTCCTTCAATGATTCTTGCAAGTGCGCGAC-30 . The PCR product was cloned into pENTR/D-TOPO, sequenced to verify the DNA sequence, cloned into pHellstage8 with the Gateway cloning technology (Invitrogen), and sequenced again to verify the DNA sequence. Transgenic plants were generated by Agrobacterium-mediated transformation. 3. Results 3.1. Expression of ASK20A and ASK20B genes The mRNA expression levels of ASK20A and ASK20B were examined by RT-PCR using a primer set that corresponded to a junction of the fourth and fifth exons and to the internal sequence of the seventh exon (Fig. 1A). The PCR product of ASK20A was 35 bp longer than that of ASK20B, and they were separated on a polyacrylamide gel (Fig. 1B). Both mRNAs were detected in every organ tested; in all of these organs, the ASK20B mRNA expression levels were higher than those of ASK20A (Fig. 1B and C). Exposure to stress or treatment with growth regulators did not severely affect mRNA expression levels (Fig. 1D and E). 3.2. Promoter activity of ASK20 Transgenic Arabidopsis plants containing the ASK20 promoter:GUS construct were generated to monitor the promoter activity of ASK20 (Fig. 2A). GUS signals were detected in the shoot apex, root vein, root tip, cotyledon, rosette leaf, sepal, stamen, and seed (Fig. 2B–H).

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Fig. 2. Histochemical localization of GUS activity in transgenic Arabidopsis plants possessing pASK20:GUS. (A) Schematic representation of the pASK20:GUS construct. GUS activity in shoot apex (B), root (C), cotyledon (D), and rosette leaf (E) of transgenic Arabidopsis seedling, and in flower (F), stamen (G), and seed (H) of adult transgenic Arabidopsis plant possessing the pASK20:GUS construct.

3.3. GUS activity in transgenic plants possessing the pASK20:ASK20A-GUS or pASK20:ASK20B-GUS constructs To monitor organ- and tissue-specific expression of ASK20A and ASK20B, transgenic plants containing a GUS in-frame fusion of ASK20A ( pASK20:ASK20A-GUS) or ASK20B (pASK20:ASK20B-GUS) were generated (Fig. 3A). GUS activity was detected in a large area of the shoot apex in pASK20:ASK20B-GUS plants, whereas it was restricted to a small area in pASK20:ASK20A-GUS plants (Fig. 3B and F). GUS activity was detected in the root vein and tip of pASK20:ASK20B-GUS plants, whereas it was restricted to the tip, and only a little activity was detected in the vein, of pASK20:ASK20A-GUS plants (Fig. 3C and G). GUS activity was detected in the cotyledon of pASK20:ASK20B-GUS plants but not of pASK20:ASK20A-GUS plants (Fig. 3D and H). GUS activity was detected in the entire rosette leaf of pASK20:ASK20B-GUS plants, whereas it was restricted to the petiole and basal part of the rosette leaf in pASK20:ASK20AGUS plants (Fig. 3E and I). GUS activity was detected in the sepal, stamen, and seed of pASK20:ASK20B-GUS plants, whereas little activity was detected in these organs or the seeds of pASK20:ASK20A-GUS plants (Fig. 3J–O). 3.4. Intracellular localization of GUS- and GFP-fusion of ASK20A and ASK20B ASK20A-GUS and ASK20B-GUS fusion proteins were transiently expressed in Arabidopsis petiole cells and onion

epidermal cells. In both cases, GUS activity was observed in the cytosol (Fig. 4A). Cytosolic localization of ASK20A-GUS and ASK20B-GUS activity was also observed in cells that expressed pASK20:ASK20A-GUS and pASK20:ASK20B-GUS (Fig. 3B–O). Similarly, GFP signals were observed in the cytosol, but not in the nuclei, of cells that expressed pASK20:ASK20A-GFP or pASK20:ASK20B-GFP (Fig. 4B). These results indicated that neither ASK20A nor ASK20B exhibits any nuclear localization signals. 3.5. Interaction of ASK20A and ASK20B with F-box proteins Although ASK20A and ASK20B have extended C-terminal regions, they possess four helical regions that are conserved among SKP1 and ASK proteins (Fig. 5A, [16]), suggesting that ASK20A and ASK20B can interact with F-box proteins. To examine this point, a yeast two-hybrid assay was performed with several F-box protein cDNAs [18]. Both ASK20A and ASK20B interacted with At3g61590 and At5g39250 proteins, but not with At5g43190, At4g18380, At5g56370, At4g39550, At2g02360, At5g38590, At3g18980, At5g48990, At3g16740, or At4g38870 protein (Fig. 5B). ASK1, which interacts with a broad range of F-box proteins [16], interacted with the At2g02360, At3g61590, and At5g39250 proteins in our system. A deletion experiment with ASK20 proteins revealed that the F-box proteins interacted not with the extended C-terminal region of ASK20A or ASK20B, but with the N-terminal region containing the conserved four-helix domains (Fig. 5A and C).

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Fig. 3. Histochemical localization of GUS activity in transgenic Arabidopsis plants possessing pASK20:ASK20A-GUS or pASK20:ASK20B-GUS. (A) Schematic representation of pASK20:ASK20A-GUS and pASK20:ASK20B-GUS constructs. GUS activity in shoot apex (B and F), root (C and G), cotyledon (D and H), and rosette leaf (E and I) of transgenic Arabidopsis seedling, and in flower (J and M), stamen (K and N), and seed (L and O) of adult transgenic Arabidopsis plant possessing pASK20:ASK20A-GUS (B–E, J–L) or pASK20:ASK20B-GUS (F–I, M–O).

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Fig. 4. Subcellular localization of ASK20A-GUS, ASK20B-GUS, ASK20A-GFP, and ASK20B-GFP. (A) Plasmids containing p35S:ASK20A-GUS or p35S:ASK20B-GUS were bombarded into Arabidopsis petiole cells (left panels) and onion epidermal cells (right panel). DAPI staining of the cell nucleus is also shown (arrowed). The empty vector pBI221 was used as control for general cytoplasmic GUS staining. GFP-COP1NLS-GUS was used as a control for nuclear localization. (B) Roots of transgenic Arabidopsis seedlings possessing pASK20:GFP, pASK20:ASK20A-GFP, or pASK20:ASK20B-GFP were observed by fluorescence and optical microscopy. Bars = 10 mm.

3.6. Interaction of ASK20A and ASK20B with CUL1 and CUL2 To function as part of the SCF complex, ASK20 proteins must interact not only with F-box proteins but also with CUL1. To examine this point, a yeast two-hybrid assay was performed with CUL1 and CUL2 cDNAs. ASK1 interacted with CUL1, but not with CUL2. Neither ASK20A nor ASK20B interacted with CUL1 or CUL2 (Fig. 6A). Marrocco et al. [30] demonstrated that the interaction of CUL1 with ASK proteins was undetectable in either yeast or plant cells, whereas it was detectable when EID1, an F-box protein, was added to the assay. This suggested that the heterodimer of F-box protein and ASK protein could form a stable trimeric complex with CUL1. Therefore, a three-hybrid assay was performed to check whether F-box proteins, which had been shown to interact with ASK20 proteins, could function as stabilizers of the CUL1– ASK20–F-box protein complex. Neither ASK20A nor ASK20B functioned as a bridge between CUL1 and the Fbox proteins At3g61590 and At5g39250, whereas ASK1 functioned as such (Fig. 6B). To obtain additional information on the CUL1–ASK–F-box protein complex, a pull-down assay was performed from a crude extract of insect cells expressing FLAG-tagged ASK proteins, HA-tagged CUL1, and GST-tagged F-box proteins. When FLAG-ASK1 or ASK20A protein was immunoprecipitated with anti-FLAG affinity gel, GST-At3g61590 and GSTAt5g39250 were co-immunoprecipitated (Fig. 6C and D). CUL1-HA was also co-immunoprecipitated with FLAG-ASK1. However, only a very weak co-immunoprecipitated CUL1-HA was obtained with FLAG-ASK20A (Fig. 6C and D). 3.7. Analysis of T-DNA insertion mutant, sense- and antisense plants, and RNAi plants of ASK20 Among the 21 ASK genes, only the ask1 mutant has a distinct phenotype: small vegetative and floral organs, abnormal development of floral organs, and reduced fertility to repress homologous recombination in male meiosis [19,31,32]. To

address the functions of the ASK20 gene, a T-DNA insertion mutant, sense- and anti-sense plants, and RNAi plants were analyzed (Supplementary Fig. S1). The life cycle of these plants, ranging from the seed to senescence stages, was examined. However, no obvious differences in phenotype were observed when compared with the wild-type (data not shown). 4. Discussion RT-PCR demonstrated that the expression level of ASK20B mRNA was higher than that of ASK20A. ASK20A and ASK20B expression was detected in all organs examined, in contrast to that of ASK3, 5, 7, 8, and 11–17 [14]. Furthermore, ASK20A and ASK20B expression was not largely affected by exposure to abiotic stress or treatment with growth regulators. These results point to the basic role played by ASK20A and ASK20B in plant cells. Promoter activity of ASK20 was detected in many tissues and organs, in agreement with the RT-PCR results. The promoter activities of several ASK genes, namely, ASK1–7, 9, and 11–19, have been reported, but none of those activities showed a pattern identical to that of ASK20. For example, ASK1 promoter activity was observed in almost all tissues except the root tip and vascular tissue [14,17,18], where strong ASK20 promoter activity was detected. Promoter activities of both ASK18 and ASK20 were observed in the shoot apex and root tip, whereas that of ASK18 was not detected in the cotyledon or seed. Promoter activities of ASK3, 6, 14, 17, and 19 were observed in the leaf hydathode, whereas that of ASK20 was observed in the whole leaf [14,17,18]. Although accumulation of ASK20A and ASK20B mRNAs was observed in every organ tested, the patterns of GUS activity in pASK20:ASK20A-GUS and pASK20:ASK20B-GUS plants suggested differences in the tissue- or cell-specific expression of ASK20A and ASK20B proteins (Fig. 3). Since GUS activity in pASK20:GUS plants was very similar, or almost identical, to that in pASK20:ASK20B-GUS plants, ASK20 promoter activity probably determines the type of cell in which ASK20B is to be expressed. In contrast, GUS activity differed between

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Fig. 5. Yeast two-hybrid interactions between ASK and F-box proteins. (A) Schematic representation of ASK20A, ASK20B, and their deletion constructs. Four a-helix domains conserved among SKP1 and ASK proteins are shown by gray boxes (H5–8). (B) A single colony of yeast AH109 containing a GAL4 DNA-binding domain fusion of ASK1, ASK20A, or ASK20B, and a GAL4 activation domain fusion of F-box proteins grown on SD (synthetic defined)– Leu/– Trp (SD – LW) (upper) or SD – Ade/– His/– Leu/– Trp (SD – AHLW) (lower) agar medium. Empty two-hybrid vectors were used as negative controls. The yeasts were grown at 30 8C. (C) A single colony of yeast AH109 containing a GAL4 DNA-binding domain fusion of one of the ASK20 protein deletions, and a GAL4 activation domain fusion of two F-box proteins, grown on SD – Leu/– Trp (SD – LW) or AD –Ade/– His/– Leu/– Trp (SD – AHLW) agar medium. Empty two-hybrid vectors were used as negative controls. The yeasts were grown at 30 8C.

pASK20:GUS and pASK20:ASK20A-GUS plants: activity was not observed in the cotyledon, frontal area of the rosette leaf, root vein, sepal, or stamen of pASK20:ASK20A-GUS plants. This indicated that ASK20A expression is determined not only by ASK20 promoter activity but also by another factor or factors. One candidate factor is unique splicing machinery that generates ASK20A mRNA. ASK20 protein may be produced in cells where promoter activity and splicing activity overlap. Other possible factors are protein degradation and transportation.

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The presence of four conserved helix domains in both ASK20A and ASK20B suggested the possibility of their interaction with F-box proteins. Indeed, we showed that both ASK20A and ASK20B interacted with two F-box proteins (At3g61590 and At5g39250). At3g61590 was recently identified as HAWAIIAN SKIRT (HWS), which regulates organ fusion and growth [33], whereas the function of At5g39250 is unknown, although examination of the microarray database Genevestigator (https://www.genevestigator.ethz.ch/) reveals that its mRNA is detected in many organs and tissues. Using yeast two- or three-hybrid systems, we also showed that neither ASK20A nor ASK20B interacted with CUL1 in the presence or absence of F-box proteins that interacted with both proteins. Furthermore, the interaction of ASK20A with CUL1 was very weak in a pull-down assay. By contrast, ASK1–4, 10, 11, 13–16, 18, and 19 interact with CUL1 regardless of the presence or absence of F-box proteins [30]. The N-terminal regions of these ASK proteins and SKP1 are postulated to be CUL1-binding sites [5], and the N-terminal regions of the ASK20A and ASK20B proteins showed low levels of similarity to those of other ASK proteins, with the exception of ASK21 (Supplementary Fig. S2). In the case of human SKP1, Asn 49, Asn 108, and Tyr 109 were demonstrated to be important for the interaction with CUL1 [5]. These amino acids are well conserved in ASK1–6, 9, 11–16, 18, and 19, but not in ASK20A, ASK20B, or ASK21, the corresponding amino acids of which are changed to Gln 62, Asp 112, and Ser 113 (Supplementary Fig. S2). Furthermore, ASK20A, ASK20B, and ASK21 have an additional amino acid insertion immediately after Gln 62. These findings support our result that ASK20A and ASK20B do not strongly interact with CUL1. ASK20A and ASK20B might prevent certain F-box proteins from forming SCF complexes by competing with other ASK proteins that are able to act as components of the SCF complex. Another possibility is that ASK20A and ASK20B function in a manner different from the SCF complex. In yeast, several F-box proteins, such as RCY1 and MFB1, are known to form a non-SCF complex and to function with SKP1 but not CUL1. The complex of SKP1 and RCY1 is involved in the recycling of SNC1, a v-SNARE protein, which is involved in the fusion of Golgi-derived secretory vesicles to the plasma membrane [34]. On the other hand, the complex of SKP1 and MFB1 negatively regulates the association of MFB1 with mitochondria in the bud [35]. To gain an understanding of the function of ASK20, we performed a phenotypic analysis of a series of ASK20overexpressing plants, T-DNA insertion plants, and knockdown plants, but no obvious differences in phenotype were observed. T-DNA was inserted into the ninth exon (Supplementary Fig. S1). Therefore, full-length ASK20A protein may be expressed in plant cells. It is also possible that the short Nterminal truncated version of ASK20B protein retains its own function. T-DNA insertion plants of ASK11 and ASK12 and transposon-insertion plants of ASK14 and ASK18 showed no obvious differences in phenotype compared with wild-type plants [18]. ASK11 RNAi plants also have no distinct phenotypes [14]. Since the amino acid sequence of ASK20 resembles that of ASK21, analysis of ask20 ask21 double

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Fig. 6. Interactions among CUL, ASK, and F-box proteins. (A) Yeast two-hybrid interactions between ASK and CUL proteins. A single colony of yeast AH109 containing a GAL4 DNA-binding domain fusion of CUL1 or CUL2, and a GAL4 activation domain fusion of ASK1, ASK20A, or ASK20B grown on SD – Leu/– Trp (– LW) or AD – Ade/– His/– Leu/– Trp (– AHLW) agar medium. Empty two-hybrid vectors were used as negative controls. The yeasts were grown at 30 8C. (B) Yeast three-hybrid interactions among ASK, CUL, and F-box proteins. A single colony of yeast AH109 containing a GAL4 DNA-binding domain fusion of CUL1, HAtagged ASKs (ASK1, ASK20A, or ASK20B), and a GAL4 activation domain fusion of F-box proteins (At3g61590 or At5g39250) grown on SD – Met/– Leu/– Trp (– MLW) or AD – Met/– Ade/– His/– Leu/– Trp (– MAHLW) agar medium. Empty two-hybrid vectors were used as negative controls. As positive control, b- and gadaptin and s subunits of adaptor protein (AP) complex were used [27]. The yeasts were grown at 20 8C. (C and D) Pull-down assay of ASK, CUL, and F-box proteins. CUL1-HA and GST-tagged F-box proteins were co-expressed in insect cells with FLAG-ASK1 or FLAG-ASK20A. FLAG-tagged ASK proteins were immunoprecipitated from the soluble fraction of cells using anti-FLAG-antibody-conjugated agarose and eluted from agarose by 0.1 M glycine–HCl (pH 3.5). Soluble fractions (Input) and eluates (IP) were analyzed by immunoblotting using anti-FLAG monoclonal antibodies, anti-CUL1 polyclonal antibodies, and anti-GST monoclonal antibodies.

mutants and knockdown plants seems to be necessary if we are to understand the functions of these extraordinary ASK proteins. Acknowledgments We thank Dr. Hirofumi Kuroda and Dr. Minami Matsui (Plant Science Center, RIKEN) for providing the plasmids for the F-box protein cDNAs; Dr. Tsuyoshi Nakagawa (Shimane University) for providing binary vectors; Dr. Kazuhisa Nakayama (Kyoto University) for providing positive control vectors for the three-hybrid assay; Dr. Peter Waterhouse (CSIRO Plant Industry) for providing pHellstage8; and Dr. Naoki Yamamoto (Ochanomizu University) for providing pUC/ GFP-COP1NLS-GUS. We thank Dr. Keiji Tanaka (The Tokyo Metropolitan Institute of Medical Science); Dr. Norihisa Matsuda (RIKEN); Dr. Tomoki Chiba (University of Tsukuba); and Dr. Yoshinao Katsu (National Institute for Basic Biology)

for their helpful technical advice on protein expression in insect cells. We thank ABRC (Arabidopsis Biological Resource Center), the Salk Institute Genomic Analysis Laboratory, and RIKEN Bio Resource Center for providing the BAC clone, the sequenced-indexed Arabidopsis T-DNA insertion mutant, and cDNAs for ASK and CUL. We also thank Mss. Kayo Sato, Emi Takata, and Noriko Uemura for their technical assistance. This work was partly supported by the Ministry of Education, Culture, Sports, Science, and Technology of Japan (Grants-in-Aid for Scientific Research [KAKENHI; no. 17084003 to T.K.]), and by a grant from the Program for Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN) to T.K. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.plantsci. 2008.02.010.

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References [1] A.R. Willems, M. Schwab, M. Tyers, A Hitchhiker’s guide to the cullin ubiquitin ligases: SCF and its kin, Biochim. Biophys. Acta 1695 (2004) 133–170. [2] A.N. Hegde, Ubiquitin–proteasome-mediated local protein degradation and synaptic plasticity, Prog. Neurobiol. 73 (2004) 311–357. [3] J.A. Johnston, K. Madura, Rings, chains and ladders: ubiquitin goes to work in the neuron, Prog. Neurobiol 73 (2004) 227–257. [4] C.M. Pickart, M.J. Eddins, Ubiquitin: structures, functions, mechanisms, Biochim. Biophys. Acta 1695 (2004) 55–72. [5] N. Zheng, B.A. Schulma, L. Song, J.J. Miller, P.D. Jeffrey, P. Wang, C. Chu, D.M. Koepp, S.J. Elledge, M. Pagano, R.C. Conaway, J.W. Conaway, J.W. Harper, N.P. Pavletich, Structure of the Cul1-Rbx1Skp1-F boxSkp2 SCF ubiquitin ligase complex, Nature 416 (2002) 703–709. [6] K.L. Crai, M. Tyers, The F-box: a new motif for ubiquitin-dependent proteolysis in cell cycle regulation and signal transduction, Prog. Biophys. Mol. Biol. 72 (1999) 299–328. [7] W. Xiao, J.-C. Jang, F-box proteins in Arabidopsis, Trend. Plant Sci. 5 (2000) 454–457. [8] T. Cardozo, M. Pagano, The SCF ubiquitin ligase: insights into a molecular machine, Nat. Rev. Mol. Cell. Biol. 5 (2004) 739–751. [9] J.M. Gagne, B.P. Downe, S.H. Shiu, A.M. Durski, R.D. Vierstra, The Fbox subunit of the SCF E3 complex is encoded by a diverse superfamily of genes in Arabidopsis, Proc. Natl. Acad. Sci. USA 99 (2002) 11519–11524. [10] W.M. Gray, H. Hellmann, S. Dharmasiri, M. Estelle, Role of the Arabidopsis RING-H2 protein RBX1 in RUB modification and SCF function, Plant Cell 14 (2002) 2137–2144. [11] H. Kuroda, N. Takahashi, H. Shimada, M. Seki, K. Shinozaki, M. Matsui, Classification and expression analysis of Arabidopsis F-box-containing protein genes, Plant Cell Physiol. 43 (2002) 1073–1085. [12] E. Lechner, D. Xie, S. Grava, E. Pigaglio, S. Planchais, J.A. Murray, Y. Parmentier, J. Mutterer, B. Dubreucq, W.H. Shen, P. Genschik, The AtRbx1 protein is part of plant SCF complexes, and its down-regulation causes severe growth and developmental defects, J. Biol. Chem. 277 (2002) 50069–50080. [13] W.H. Shen, Y. Parmentier, H. Hellmann, E. Lechner, A. Dong, J. Masson, F. Granier, L. Lepiniec, M. Estelle, P. Genschik, Null mutation of AtCUL1 causes arrest in early embryogenesis in Arabidopsis, Mol. Biol. Cell 13 (2002) 1916–1928. [14] D. Zhao, W. Ni, B. Feng, T. Han, M.G. Petrasek, H. Ma, Members of the Arabidopsis-SKP1-like gene family exhibit a variety of expression patterns and may play diverse roles in Arabidopsis, Plant Physiol. 133 (2003) 203–217. [15] B.A. Schulman, A.C. Carrano, P.D. Jeffrey, Z. Bowen, E.R. Kinnucan, M.S. Finnin, S.J. Elledge, J.W. Harper, M. Pagano, N.P. Pavletich, Insights into SCF ubiquitin ligases from the structure of the Skp1–Skp2 complex, Nature 408 (2000) 381–386. [16] E.P. Risseeuw, T.E. Daskalchuk, T.W. Banks, E. Liu, J. Cotelesage, H. Hellmann, M. Estelle, D.E. Somers, W.L. Crosby, Protein interaction analysis of SCF ubiquitin E3 ligase subunits from Arabidopsis, Plant J. 34 (2003) 753–767. [17] K. Marrocco, A. Lecureuil, P. Nicolas, P. Guerche, The Arabidopsis SKP1like genes present a spectrum of expression profiles, Plant Mol. Biol. 52 (2003) 715–727. [18] N. Takahashi, H. Kuroda, T. Kuromori, T. Hirayama, M. Seki, K. Shinozaki, H. Shimada, M. Matsui, Expression and interaction analysis of Arabidopsis Skp1-related genes, Plant Cell Physiol. 45 (2004) 83–91.

495

[19] Y. Wang, M. Yang, The ARABIDOPSIS SKP1-LIKE1 (ASK1) protein acts predominately from leptotene to pachytene and represses homologous recombination in male meiosis, Planta 227 (2006) 613–617. [20] T. Murashige, F. Skoog, A revised medium for rapid growth and bioassays with tobacco tissue cultures, Physiol. Plant. 15 (1962) 473–497. [21] T. Kiyosue, K. Yamaguchi-Shinozaki, K. Shinozaki, K. Higashi, S. Satoh, H. Kamada, H. Harada, Isolation and characterization of a cDNA that encodes ECP31, an embryogenic-cell protein from carrot, Plant Mol. Biol. 19 (1992) 239–249. [22] M.G. Murray, W.F. Thompson, Rapid isolation of high molecular weight plant DNA, Nucleic Acids Res. 8 (1980) 4321–4325. [23] D.H. Figurski, D.R. Helinski, Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans, Proc. Natl. Acad. Sci. USA 76 (1979) 1648–1652. [24] S.J. Clough, A.F. Bent, Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana, Plant J. 16 (1998) 735–743. [25] N. Matsumoto, T. Hirano, T. Iwasaki, N. Yamamoto, Functional analysis and intercellular localization of rice Cryptochrome, Plant Physiol. 133 (2003) 1494–1503. [26] M. Yasuhara, S. Mitsui, H. Hirano, R. Takanabe, Y. Tokioka, N. Ihara, A. Komatsu, M. Seki, K. Shinozaki, T. Kiyosue, Identification of ASK and clock-associated proteins as molecular partners of LKP2 (LOV kelch protein 2) in Arabidopsis, J. Exp. Bot. 55 (2004) 2015–2027. [27] H. Takatsu, M. Futatsumori, K. Yoshino, Y. Yoshida, H.W. Shin, K. Nakayama, Similar subunit interactions contribute to assembly of clathrin adaptor complexes and COPI complex, Biochem. Biophys. Res. Commun. 284 (2001) 1084–1089. [28] I. Mitsuhara, M. Ugaki, H. Hirochika, M. Ohshima, T. Murakami, Y. Gotoh, Y. Katayose, S. Nakamura, R. Honkura, S. Nishimiya, K. Ueno, A. Mochizuki, H. Tanimoto, H. Tsugawa, Y. Otsuki, Y. Ohashi, Efficient promoter cassettes for enhanced expression of foreign genes in dicotyledonous and monocotyledonous plants, Plant Cell Physiol. 37 (1996) 49–59. [29] S.V. Wesley, C.A. Helliwell, N.A. Smith, M.B. Wang, D.T. Rouse, Q. Liu, P.S. Gooding, S.P. Singh, D. Abbott, P.A. Stoutjesdijk, S.P. Robinson, A.P. Gleave, A.G. Green, P.M. Waterhouse, Construct design for efficient, effective and high-throughput gene silencing in plants, Plant J. 27 (2001) 581–590. [30] K. Marrocco, Y. Zhou, E. Bury, M. Dieterle, M. Funk, P. Genschik, M. Krenz, T. Stolpe, T. Kretsch, Functional analysis of EID1, an F-box protein involved in phytochrome A-dependent light signal transduction, Plant J. 45 (2006) 423–438. [31] M. Yang, Y. Hu, M. Lodhi, W.R. McCombie, H. Ma, The Arabidopsis SKP1-LIKE1 gene is essential for male meiosis and may control homologue separation, Proc. Natl. Acad. Sci. USA 96 (1999) 11416–11421. [32] D. Zhao, M. Yang, J. Solava, H. Ma, The ASK1 gene regulates development and interacts with the UFO gene to control floral organ identity in Arabidopsis, Dev. Genet. 25 (1999) 209–223. [33] Z.H. Gonzalez-Carranza, U. Rompa, J.L. Peters, A.M. Bhatt, C. Wagstaff, A.D. Stead, J.A. Roberts, HAWAIIAN SKIRT: an F-box gene that regulates organ fusion and growth in Arabidopsis, Plant Physiol. 144 (2007) 1370–1382. [34] J.M. Galan, A. Wiederkehr, J.H. Seol, R. Haguenauer-Tsapis, R.J. Deshaies, H. Riezman, M. Peter, Skp1p and the F-box protein Rcy1p form a non-SCF complex involved in recycling of the SNARE Snc1p in yeast, Mol. Cell. Biol. 21 (2001) 3105–3117. [35] N. Kondo-Okamoto, K. Ohkuni, K. Kitagawa, J.M. McCaffery, J.M. Shaw, K. Okamoto, The novel F-box protein Mfb1p regulates mitochondrial connectivity and exhibits asymmetric localization in yeast, Mol. Biol. Cell 17 (2006) 3756–3767.