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First published online May 13, 2005; 10.1104/pp.104.057752 Plant Physiology 138:675-685 (2005) © 2005 American Society of Plant Biologists Analysis of a Sugar Response Mutant of Arabidopsis Identified a Novel B3 Domain Protein That Functions as an Active Transcriptional Repressor1Laboratory of Biochemistry, Graduate School of Bioagricultural Science, Nagoya University, Chikusa, Nagoya 464–8601, Japan (H.T., T.S., K.N.); Kazusa DNA Research Institute, Kisarazu, Chiba 292–0818, Japan (D.S.); and College of Bioscience and Biotechnology, Chubu University, Kasugai, Aichi 487–8501, Japan (A.M.)
A recessive mutation hsi2 of Arabidopsis (Arabidopsis thaliana) expressing luciferase (LUC) under control of a short promoter derived from a sweet potato (Ipomoea batatas) sporamin gene (Spomin::LUC) caused enhanced LUC expression under both low- and high-sugar conditions, which was not due to increased level of abscisic acid. The hsi2 mutant contained a nonsense mutation in a gene encoding a protein with B3 DNA-binding domain. HSI2 and two other Arabidopsis proteins appear to constitute a novel subfamily of B3 domain proteins distinct from ABI3, FUS3, and LEC2, which are transcription activators involved in seed development. The C-terminal part of HSI2 subfamily proteins contained a sequence similar to the ERF-associated amphiphilic repression (EAR) motif. Deletion of the C-terminal portion of HSI2 lost in the hsi2 mutant caused reduced nuclear targeting of HSI2. Null allele of HSI2 showed even higher Spomin::LUC expression than the hsi2 mutant, whereas overexpression of HSI2 reduced the LUC expression. Transient coexpression of 35S::HSI2 with Spomin::LUC in protoplasts repressed the expression of LUC activity, and deletion or mutation of the EAR motif significantly reduced the repression activity of HSI2. These results indicate that HSI2 and related proteins are B3 domain-EAR motif active transcription repressors.
In addition to transcriptional activators, transcriptional repressors play important roles in the regulation of transcription. Transcriptional repressors are basically classified into passive repressors and active repressors (Hanna-Rose and Hansen, 1996  ; Thiel et al., 2004). Passive repressors do not have intrinsic repressing activity and inhibit the activation of transcription by inhibiting the function of transcriptional activators through a competition with activators for binding with DNA or a formation of inactive heterodimers with activators. On the other hand, active repressors inhibit transcription in an activator-independent manner by binding with basic transcription factors or corepressors.
The expression of a number of plant genes is regulated by changes in sugar status via multiple signal transduction pathways (for review, see Koch, 1996
The recessive hba1 mutation of Arabidopsis enhances the expression of a gene for the major
Expression of a family of sporamin genes of sweet potato (Ipomoea batatas) is inducible by Suc, Glc, or other metabolizable sugars (Hattori et al., 1990
hsi2 Mutant Exhibits High-Level Expression of the Spomin Promoter
A 210-bp "minimal" promoter derived from a gene for sweet potato sporamin A1 (Spomin; Morikami et al., 2005
In sGsL seedlings grown on medium containing 2% Suc, we observed strong GUS expression in the hypocotyls, which was not observed in seedlings grown without Suc (Fig. 1C). On medium containing 4% Suc, strong GUS staining extended to the cotyledons of the sGsL seedlings. By contrast, the hsi2 seedlings grown without Suc already showed GUS expression in hypocotyls, and strong GUS expression extended to the cotyledons in the seedlings grown with 2% Suc. Unlike Suc or Glc (data not shown), mannitol did not induce expression of LUC and GUS activities in either sGsL or hsi2 plants, except for weak expression in the hypocotyls of hsi2 plants grown on 4% mannitol (Fig. 1C). These results suggest that the hsi2 mutation caused an enhancement of the expression of the Spomin promoter in response to Suc without affecting the spatial pattern of expression or the mode of regulation.
Given that the Spomin promoter is induced by both sugars and ABA and that sugar enhances the endogenous level of ABA (Arenas-Huertero et al., 2000
When the hsi2 plants (Columbia [Col]-0 ecotype) were crossed with the wild-type Wassilewskija (Ws) ecotype, all of the F1 plants showed LUC activities similar to the sGsL line. In the F2 generations, 502 out of 2,074 kanamycin-resistant plants displayed high LUC activities in the leaves (
For further mapping, hsi2 was crossed with the wild-type Landsberg erecta (Ler), and DNAs were isolated from 363 kanamycin-resistant F2 individuals showing high LUC activity in water-treated leaves. Mapping with CAPS and SSLP markers indicated that hsi2 was located near the C4H marker and between the TB20-R85 and TB20-R32 markers (Fig. 2B). A genomic DNA carried on a transformation-competent bacterial artificial chromosome (TAC) clone, K24I13, covering the C4H and TB20-R32 loci was introduced into the hsi2 mutant. However, transformed plants showed high LUC activities similar to the hsi2 plants (data not shown). These results indicated that the hsi2 mutation is located within a 25-kb region between the TB20-R85 and C4H markers.
According to the public database, the 25-kb region contained four putative protein-coding genes (Fig. 2B). Sequencing of genomic DNAs covering these putative genes from both the sGsL and hsi2 plants identified one base substitution in the At2g30470 gene. This G-to-A substitution was present in the 10th exon, and it could result in nonsense mutation from the Trp codon (TGG) in sGsL to TGA in hsi2 (Fig. 2C). When a genomic fragment of sGsL covering the At2g30470 gene was introduced into the hsi2 plant, transformed plants showed low LUC activities in leaves treated with water or Suc (Fig. 2D). Based on these results, we concluded that the At2g30470 gene is the HSI2 gene. Determination of the nucleotide sequence of a full-length cDNA clone for HSI2 (RAFL07-13-K13; Seki et al., 2002
The coding sequence of the HSI2 gene was composed of 2,370 bp, and it could encode a polypeptide of 790 amino acids (Fig. 3A). The HSI2 protein contained a plant-specific B3 DNA-binding domain. The Arabidopsis genome contains 42 genes that could code for proteins with B3 DNA-binding domain. Based on the amino acid sequence of the encoded proteins, these genes could be classified into three families that are represented by ABI3 (abscisic acid-insensitive 3; Giraudat et al., 1992
Other than the B3 domain, the HSI2 subfamily proteins do not share sequence similarities with ABA3, FUS3, and LEC2. However, in addition to the B3 domain, the three HSI2 subfamily proteins shared conserved sequences from C1 to C4 (Fig. 3A), which may have some functional significance. The C-terminal part of the C4 region contains a sequence similar to a bipartite nuclear localization signal (NLS; Liu et al., 1999 ). In addition to these, the C-terminal region of the three HSI2 subfamily proteins contains sequences similar to the EAR motif, which is involved in active repression of class II ERF transcriptional repressors and TFIIIA-type zinc-finger proteins ([L/F]DLN[L/F]xP; Ohta et al., 2001). The Trp-553 that was mutated in the hsi2 mutant is located between the C3 and C4 regions.
We examined the expression of mRNAs for HSI2 and two other related genes in various organs of Arabidopsis by semiquantitative reverse transcription (RT)-PCR. Actin 2 (ACT2) mRNA served as a control. The HSI2 mRNA was detected in roots, flowers, stems, and leaves, and the highest levels appeared to be in the flowers (Fig. 4A). The mRNA for HSI2-L1 (At4g32010) was also detected in all of these organs at similar levels. The level of mRNA for HSI2-L2 (At4g21550), on the other hand, was very low in organs other than the flowers.
The level of HSI2 mRNA in leaves showed a slight increase following a 24-h treatment with 6% Suc, whereas treatment with 50 µM ABA did not affect the level of HSI2 mRNA (Fig. 4B). The levels of mRNAs for HSI2-L1 and HSI2-L2 were not affected by treatment of plants with Suc or ABA (data not shown). The hsi2 plants contained the mutant HSI2 mRNA at levels similar to the HSI2 mRNA in the sGsL plants. To examine the cellular localization of HSI2, genes for translational fusions of synthetic green fluorescent protein (sGFP) at either the C (HSI2-GFP) or N terminus (GFP-HSI2) of HSI2 were constructed downstream of the cauliflower mosaic virus (CaMV) 35S promoter. These fusion gene constructs were introduced into onion (Allium cepa) epidermal cells by particle bombardment, and GFP expression was monitored by fluorescence microscopy. In contrast to cells expressing the control GFP, in which both cytoplasm and nucleus showed fluorescence, strong fluorescent signals were detected only in the nucleus of cells expressing HSI2-GFP and GFP-HSI2 (Fig. 5).
Because a nonsense hsi2 mutation was located between the B3 DNA-binding domain and the C4 region containing the putative NLS (Fig. 3A) and because a mutant transcript of HSI2 was detected in the hsi2 plants (Fig. 4B), it seemed likely that the hsi2 plants produce a truncated HSI2 protein with a B3 domain that is not efficiently targeted to the nucleus. To examine this possibility, sGFP was fused to the C or N terminus of a truncated hsi2 mutant form of HSI2 (HSI2[  553-790]). Unlike GFP fusions with the full length of HSI2, fluorescence from these fusion proteins was observed both in nucleus and cytoplasm (Fig. 5). The residual nuclear localization of GFP-HSI2[553-790] could be due to remaining weak NLS or interaction of HSI2[553-790] with other nuclear protein.
A T-DNA insertion line, K_24I13, from the Salk Institute (La Jolla, CA) contained a T-DNA inserted in the seventh exon of the HSI2 gene (Fig. 2C). This T-DNA insertion line was crossed with the sGsL line, and F2 plants harboring the sGsL dual reporters and homozygous for disruption of the HSI2 gene were selected. Unlike the hsi2 mutant, the HSI2 mRNA was not detected in the
To determine whether overexpression of HSI2 causes negative effects on the expression of Spomin, full-length HSI2 cDNA was placed downstream of the CaMV 35S promoter and used to transform the sGsL plants. The T2 generation of the transformed plants expressed higher levels of HSI2 mRNA than the sGsL plant (Fig. 6C). In the T2 generation, 14 out of 20 independent transformants (e.g. OX4, OX5, OX7, and OX10) had LUC activities in the leaves that were less than 20% of the levels in the sGsL plants (Fig. 6D). Some of the transformants (e.g. OX1 and OX2) had LUC activities that were similar to those in the sGsL plants, despite an increased level of HSI2 mRNA.
To further characterize HSI2 as a negative regulator of Spomin, we conducted transient coexpression of the Spomin::LUC reporter and the 35S::HSI2 effector in protoplasts derived from suspension-cultured Arabidopsis cells. The 35S::GUS plasmid DNA was also added to each assay, and LUC activity was normalized according to the GUS activity. The expression of LUC activity from the Spomin::LUC reporter was strongly repressed when the HSI2 effector was coexpressed, and the activity was reduced to less than 20% of the level with the empty vector (Fig. 7B). In these assays, the level of GUS activity did not vary significantly, and the expression of the 35S::LUC reporter was not affected by coexpression of 35S::HSI2 (Fig. 7B). These results suggest that repression of LUC reporter expression by coexpression of HSI2 is promoter dependent.
Compared to the 80% reduction of the level of expression of Spomin::LUC by HSI2, only about 25% reduction of the expression of Spomin::LUC was observed when C-terminally truncated hsi2 effector was coexpressed instead of HSI2 (Fig. 7, A and B). Coexpression of the EAR or mEAR effector, in which the EAR motif-like sequence (IDLNSDP) in the C-terminal region of HSI2 was either deleted or mutated to IAANADP (Fig. 7A), respectively, also only weakly repressed the LUC activity (Fig. 7B). These results indicate that the EAR motif is important for the transrepression activity of HSI2. When the activation domain of VP16 was fused to the N terminus of EAR and mEAR effectors, these VP16-EAR and VP16-mEAR caused more than 2-fold transactivation of Spomin::LUC (Fig. 7B). These results suggest that loss of transrepression activity in the EAR and mEAR effectors is not due to loss of DNA-binding activity or rapid degradation of these modified effectors. In contrast to EAR and mEAR effectors, HSI2 showed strong transrepression activity even when the activation domain of VP16 was fused to its N terminus (Fig. 7B), indicating that the EAR motif of HSI2 is an active repression domain that can negate the function of VP16 activation domain. Although deletion of the N-terminal C1 region did not affect the repressor activity of HSI2, deletion of both the C1 and C2 regions resulted in significant reduction of the repressor activity. These N-terminal regions could be required for correct DNA-binding activity or repression activity of HSI2.
The hsi2 mutant exhibiting highly enhanced expression of Spomin::LUC was due to a single recessive mutation. In the hsi2 mutant, the levels of expression of Spomin::LUC were always higher than the sGsL line not only under noninductive condition but also after treatment of plants with Suc or ABA. In addition, the spatial pattern of expression of Spomin::GUS in the hsi2 mutant was not changed compared to the sGsL line, and the hsi2 mutant did not exhibit increased levels of ABA or defective starch synthesis. These results suggest that HSI2 has negative effects on the basal transcription level from the Spomin promoter.
The hsi2 mutation was a nonsense mutation in a gene encoding a protein with B3 DNA-binding domain. The HSI2-GFP fusion protein was localized in the nucleus, while truncation of HSI2 at the hsi2 mutation (GFP-HSI2[ In addition to HSI2, the Arabidopsis genome contained two other expressed genes, HSI2-L1 (At4g320110) and HSI2-L2 (At4g215510), which code for proteins with high similarities to HSI2. Despite the similarity of the B3 domain, the HSI2 subfamily proteins do not show structural similarities other than the B3 domain with ABI3, FUS3, and LEC2. Three HSI2 subfamily proteins share conserved C1 to C4 regions in addition to the B3 domain. They also contain sequences similar to the EAR motif in the C-terminal region. The rice (Oryza sativa) genome contains one gene that could code for a protein with overall similarities to HSI2, and sequences conserved among three HSI2 subfamily proteins of Arabidopsis are also conserved in this rice protein (data not shown).
The EAR motif was first identified in class II ERFs, such as NtERF3, AtERF3, and AtERF4 (Fujimoto et al., 2000
The presence of EAR motif-like sequences in the C-terminal region of HSI2 and its conservation among the HSI2-related proteins suggested that HSI2 functions as an active transcriptional repressor. Coexpression of HSI2 in Arabidopsis protoplasts reduced the transient expression of the Spomin::LUC reporter to less than 20% of the level in the absence of effector. On the other hand, coexpression of a truncated hsi2 mutant form of HSI2 (HSI2[
The mechanism by which the EAR motif inhibits transcription is not known. Generally, the repression domain of active repressors inhibits the activation of transcription by interacting with basic transcription factors, activator/coactivator, or corepressor (Hanna-Rose and Hansen, 1996
The B3 domains of ABI3, FUS3, and LEC2 exhibit binding to sequences containing the RY motif [CATGCA] (Suzuki et al., 1997
Plant Materials and Treatment with Sugar or ABA
The Arabidopsis (Arabidopsis thaliana L.) Heynh. (ecotype Col-0) harboring one copy of the Spomin::GUS-Spomin::LUC transgene on the upper arm of chromosome 5 (referred to as the sGsL line or the wild type) and the screening of mutants displaying abnormal patterns of LUC reporter expression will be described elsewhere (A. Morikami, T. Saijo, M. Yamada, H. Tsukagoshi, T. Hattori, and K. Nakamura, unpublished data). Unless otherwise indicated, seeds were sterilized in sterile water, kept at 4°C for 4 d, and sown on gellan gum plates containing Murashige and Skoog medium, pH 5.8, 100 mg/L myoinositol, 10 mg/L thiamine-HCl, 1 mg/L nicotinic acid, 1 mg/L pyridoxine HCl, and 1.5% Suc. Plates were incubated in a growth chamber at 22°C under continuous fluorescent light at an intensity of 65 µmol m–2 s–1. Mature leaves of the 3-week-old plants were excised with a sharp razor blade, and the cut edges of petioles were immersed in a sterile solution of sugar or ABA and incubated at 22°C under continuous light (Mita et al., 1995
For luminescence imaging of LUC activity, leaves treated with Suc or ABA for 48 h were sprayed with 0.8 mM D-luciferin (Molecular Probes, Eugene, OR) in 0.01% Triton X-100 and kept in the dark for 5 min before imaging. Luminescence images were taken with a 5-min exposure time by a CCD system and processed with Argus-50 (Hamamatsu Photonics, Shizuoka, Japan). For determination of LUC activity, leaves were homogenized in an extraction buffer composed of 100 mM potassium phosphate, pH 7.5, and 1 mM dithiothreitol and centrifuged at 15,000g for 10 min at 4°C. The resulting supernatants were used as protein extracts. The protein concentrations were determined using a protein assay (Bio-Rad, Hercules, CA) with bovine serum albumin as a standard. LUC activity was determined using a commercial assay kit (Picagene Luminescence kit; Toyo-Ink, Tokyo).
Whole plants or leaves were immersed in 1 mM 5-bromo-4-chloro-3-indolyl-
To quantify the ABA content in leaves, 100 mg of leaves were frozen in liquid N2 and freeze-dried before extraction with 1 mL of sterile distilled water by shaking constantly overnight at 4°C. The amount of ABA was quantified with a Phytodetek ABA kit (AGDIA, Elkhart, IN) following the protocol provided by the manufacturer.
For genetic mapping, the hsi2 mutant was crossed with the ecotype Ws and Ler, and the F1 plants were allowed to self-pollinate. Among F2 plants descended from crosses with Ws and Ler, 502 and 363 homozygous hsi2 mutant plants, respectively, were selected based on high LUC activity in leaves treated with water. The genomic DNA isolated from these homozygous hsi2 mutants were subjected to genetic mapping using SSLP and CAPS markers. For fine mapping, new SSLP and CAPS markers were designed based on DNA sequences. The new SSLP and CAPS markers used for fine mapping were as follows (for SSLP markers, the relevant restriction enzyme and the size of fragments for Col-0/Ler in base pairs are indicated in parentheses): TD9-4in-f, 5'-ACAATTAGAGGTACGTGGGAAT-3'; TD9-4in-r, 5'-TCATATTGATTAATGGGTTCCA-3' (310/280); TB20-85-f, 5'-GGACCGTAGTGCTACTTGTGACG-3'; TB20-85-r, 5'-GACCTATTACTATTAGTACTACGAATG-3' (Tru9I, 220/120 + 100); TB20-R32-f, 5'-GGAAAAGAGATGGAACGTGGGTG-3'; and TB20-R32-r, 5'-GAATCGAATTGAATCTAAGTTTTGTGG-3' (HhaI, 120 + 110/230).
For complementation of the hsi2 mutation, the DNA from TAC clone K06D02 was digested with PvuII. A 10-kb genomic fragment covering the entire At2g30470 locus (HSI2 gene), including an approximately 2,000-bp sequence upstream from the putative initiation ATG codon, was cloned into the SmaI site of the binary vector pBIB-Hyg (Becker, 1990
To construct genes for GFP fusion proteins, we used Gateway cloning technology (Invitrogen, Carlsbad, CA) with pGWB2, pGWB5, or pGWB6 vectors, which was developed by Dr. T. Nakagawa (Research Institute for Molecular Genetics, Shimane University, Shimane, Japan). The resulting plasmids, pGWB2-HSI2, pGWB5-HSI2, pGWB6-HSI2, pGWB5-HSI2[
Transient expression assays of GFP localization in onion (Allium cepa) epidermal cells were carried out by particle bombardment (IDERA GIE III; Tanaka, Hokkaido, Japan) as described (Takeuchi et al., 1992
RNA was isolated from plant tissues using Trizol reagent (Invitrogen) and dissolved in nuclease-free water. For quantitative real-time PCR, the first-strand cDNA was synthesized from 5 µg of total RNA with oligo(dT)20 primers using SUPERSCRIPT III (Invitrogen) and diluted with 10 volumes of RNase-free water. The real-time PCR reaction mixture was performed in 25 µL containing 5 µL of diluted cDNA solution, 12.5 µL of Cybergreen dye set (Bio-Rad), and 0.5 µL of each primer (final concentration of 200 nM). PCR was initiated with denaturation at 95°C for 5 min, followed by 40 cycles of denaturation at 95°C for 15 s, annealing at 60°C for 15 s, and extension at 72°C for 30 s. The comparative threshold cycle method was used to determine the relative mRNA levels. ACT2 was used as an internal reference, and expression levels were expressed as relative to the control treatment. The following primer sets were used: HSI2, 5'-CTTCCATATCAGCTTGAAACTCTC-3' and 5'-TGGCTCAAGACGCCAGTGATGTTT-3'; and ACT2, 5'-CTGTTGACTACGAGCAGGAGATGGA-3' and 5'-GACTTCTGGGCATCTGAATCTCTCA-3'. The forward and reverse primer pairs used for RT-PCR analysis of mRNA expression were as follows: HSI2-L1, 5'-ATGAGGCTTCTCCAAGCTGCAGCGT-3' and 5'-GAACCGTGTTCTGTGCTGACCATAT-3'; and HSI2-L2, 5'-AGTGCTCATAGCCAAAATAACAAGG-3' and 5'-CTACATGGAGCTTGTGGTGGTGGTG-3'.
Arabidopsis protoplasts were isolated from T87 suspension-cultured cells (Axelos et al., 1992 Sequence data from this article have been deposited with the DDBJ/EMBL/GenBank data libraries under accession numbers AB206553 and AB206554.
We thank Dr. T. Hattori and Dr. S. Ishiguro for valuable discussions and suggestions; Dr. M. Seki and Dr. K. Shinozaki of RIKEN for the RAFL07-13-K13 cDNA clone; Dr. T. Nakagawa of the Research Institute of Molecular Genetics, Shimane University, for the sGFP binary vectors; and the Salk Institute Genomic Analysis Laboratory for providing the sequence-indexed Arabidopsis T-DNA insertion mutant. Received December 7, 2004; returned for revision January 18, 2005; accepted January 21, 2005.
1 This work was supported in part by the Research for the Future program of the Japan Society for the Promotion of Science (grant no. 00L01603) and a Grant-in-Aid for Scientific Research on Priority Areas (Molecular Mechanisms of Storage Activity in Plants) from the Ministry of Education, Culture, Sports, Science, and Technology, Japan (grant no. 12138203 to K.N. and A.M.). 
2 Present address: Faculty of Agriculture, Meijo University, Tenpaku, Nagoya 468–8502, Japan. Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.104.057752. * Corresponding author; e-mail i032011d{at}mbox.nagoya-u.ac.jp; fax 81–52–789–4094.
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ABSTRACT
RESULTS
-amylase under conditions of low sugar (Mita et al., 1997a
2 for a ratio of 3:1 = 0.639; P > 0.05), suggesting that hsi2 is a single recessive mutation. Using simple sequence length polymorphism (SSLP) markers, hsi2 was located near the nga361 marker (63 cM) on the lower arm of chromosome 2, and further analysis with new cleaved-amplified polymorphic sequence (CAPS) markers located hsi2 within 1 cM of the 3C3F marker (61.8 cM; 
