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1 Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya 464-8602, Japan
2 Plant Biology Research Center, Chubu University, Kasugai 487-8501, Japan
3 College of Bioscience and Biotechnology, Chubu University, Kasugai 487-8501, Japan
4 Department of Biological Sciences, Graduate School of Science, the University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
5 Molecular Membrane Biology Laboratory, RIKEN, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan
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Abstract |
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 Top Abstract IntroductionResultsDiscussionExperimental proceduresReferences |
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Introduction |
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TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
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The SAM is divided into three regions with distinct functions (Steeves & Sussex 1989; Meyerowitz 1997; Carles & Fletcher 2003). The central zone is located at the apex of the meristem. The peripheral zone surrounds the central zone, and the rib meristem underlies the central zone. The division of cells in the central zone maintains of the meristem itself and it is believed, also, to provide new cells for the peripheral zone and rib meristem. Continued division of the cells in the rib meristem and the peripheral zone leads to growth of the stem below the SAM and formation of leaves on the flanks of the SAM. The functional domains of the SAM, namely, the central zone, the peripheral zone, and the rib meristem, are established during embryonic development and their relative proportions, in terms of volume, are maintained throughout post-embryonic life. Genetic approaches have identified several genes in Arabidopsis that participate in the establishment and maintenance of the SAM. These genes encode, for example, a small extracellular protein, transmembrane receptor proteins and homeodomain-containing transcription factors (Carles & Fletcher 2003). Their expression is confined to distinct domains within the SAM (Long et al. 1996; Clark et al. 1997; Mayer et al. 1998; Fletcher et al. 1999). Communication between these domains through extracellular signaling controls the mass of the central zone relative to that of the peripheral zone and the overall volume of the SAM (Clark et al. 1993, 1995; Reddy & Meyerowitz 2005). Several mutants that develop disorganized tumorous tissue in the shoot apex have been isolated. In tumorous shoot development (tsd) mutants (Frank et al. 2002), disorganized tumor-like shoot tissue is derived from the SAM or the base of leaf. Cell division in the SAM of tsd mutants is abnormal and no normal lateral leaf organs are formed. A mutant allele of the KORRIGAN1 gene, encoding a membrane-bound endo-1,4-β-D-glucanase that is involved in cellulose biosynthesis, leads to the random production of leaf-like organs from the SAM and, also, callus formation (Nicol et al. 1998; Zuo et al. 2000; Sato et al. 2001). These observations suggest the existence of some unknown molecular mechanism(s) that participates in the organization of the SAM and might be related to the nature or organization of the extracellular matrix.
The development of plant organs depends on cell-fate determination along axes and on the organized proliferation and expansion of cells. The expansion of plant cells includes the organized secretion of Golgi vesicles; the organized deposition of cell wall materials, mainly cellulose microfibrils; and modification of cell walls. In plant cells in interphase, microtubules are often organized in a parallel cortical array. The cortical array of microtubules appears to serve as a scaffold for the deposition of cellulose microfibrils (Baskin 2001; Paredez et al. 2006). Primary cell walls consist mainly of cellulose, cellulose-binding hemicelluloses, and pectins. The latter two classes of cell wall components are synthesized within Golgi cisternae (Driouich et al. 1993), whereas cellulose is generated at the plasma membrane in the form of paracrystalline microfibrils (Kimura et al. 1999; Somerville et al. 2004). The relationship between organ formation and the synthesis and organization of cell wall materials has not been extensively investigated.
Leaves are flat organs with left-right symmetry and generally develop along three axes: the proximal-distal, medial-lateral and adaxial–abaxial axes (Steeves & Sussex 1989; Waites et al. 1998; Hudson 2000; Byrne et al. 2001). It has been proposed that the flat shape of leaves is dependent on the finely tuned integration of cell proliferation and cell expansion (Tsukaya 2005). Various mutants have been identified with alterations in leaf morphology that are related to the development of leaf shape along each of the three axes, to adaxial–abaxial identity, and to the overall shape of leaves (Waites et al. 1998; Hudson 2000; Byrne et al. 2001; Semiarti et al. 2001; Iwakawa et al. 2002; Carraro et al. 2006; Tsukaya 2006). However, our understanding of the molecular mechanisms that control leaf development is still very limited.
As part of our efforts to characterize the molecular mechanisms that control the organization of the SAM and leaf development, we isolated a novel mutant of Arabidopsis, designated embryo yellow (eye), which failed to develop beyond the embryonic or, sometimes, the seedling stage. The eye mutant did not exhibit any apparent abnormalities until the late stage of embryogenesis, when the mutant embryos were yellow and which contained cells with defects in their expansion. Some mutant seeds failed to germinate and, even when seeds germinated, the plant organs did not develop properly: cotyledons barely expanded; leaf primordia were formed but were arrested at very early developmental stages; and the organization of the SAM was disrupted. The EYE gene encodes a protein that is homologous to Cog7, a subunit of the conserved oligomeric Golgi (COG) complex. In eye mutant cells, a Golgi protein was mislocalized and Golgi morphology was distorted. Moreover, our analysis of cell surfaces also suggested that the extracellular matrix of eye mutant cells might be abnormal. On the basis of our results, we propose that the plant COG complex, which includes the EYE protein, might control the proper formation of the extracellular matrix, which, in turn, is required for appropriate intercellular interactions and the organization of the SAM that is essential for organ development in plants.
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Results |
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We isolated a mutant line of Arabidopsis, designated #039, which produced morphologically abnormal seeds at a frequency of approximately 25%, from T-DNA-transformed lines. Abnormal seeds were not observed within the same siliques as normal seeds until 11 days after flowering (DAF; Fig. 1A). Thirteen DAF, abnormal yellow seeds were distinguishable from normal green seeds within the single siliques (Fig. 1A). Of 919 immature seeds from 10 plants, 721 (78.5%) were green and 198 (21.5%) were yellow. The value of 21.5% was significantly different from the ideal value (25%; 
2 = 5.85; P = 0.02). A few mutant seeds might have been indistinguishable from wild-type seeds. Abnormal seeds germinated at a frequency of 11.5% and grew into dwarf seedlings on plates (Fig. 2B). We examined whether any normal seeds grew into seedlings with the abnormal phenotype of eye mutant seedlings. Eleven (2%) of 556 seedlings grown from normal seeds were dwarfed. These results suggested that the defects in the development of seeds and seedlings were caused by a single recessive mutation. We designated this mutation eye.
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Phenotype of eye plants after germination
After germination of eye mutant seeds, seedlings displayed a pleiotropic phenotype. Figure 2B shows an eye plant seven days after vernalization (DAV). The eye mutant was dwarfed and had a radially swollen hypocotyl. The lengths of eye hypocotyls were 35% of those of the wild-type (0.73 ± 0.15 mm (n = 20) vs. 2.06 ± 0.36 mm (n = 15), respectively). Figure 2C shows root growth from 1 to 5 days after germination (DAG). The eye mutant roots did not grow well: the growth rate of eye roots from 2 to 5 DAG was 17% of that of the wild-type (0.99 ± 1.5 mm/day (n = 22) vs. 5.7 ± 1.1 mm/day (n = 10), respectively). From 5 to 8 DAG, some of growth rates of eye roots were 0.1 mm/day or lower (data not shown), indicating that growth of these eye roots had ceased almost entirely (the average of growth rate of wild-type root from 5 to 10 DAG was 7.3 ± 1.0 mm/day (n = 10)).
We measured the lengths of epidermal cells in fixed and cleared plants under the light microscope with Normarski optics. The lengths of epidermal cells of eye mutant hypocotyls and roots were 37% and 35% of wild-type values, respectively (Fig. 2D). These results suggested that the reduction in cell length might account for the decrease in hypocotyl length, but that only the reduction in cell length of roots might not account for the decrease in the growth rate of roots. Therefore, in addition to a reduction in cell length, slower proliferation of root cells was probably responsible for the decrease in the growth rate of roots.
Figures 2E and 3I show epidermal cells in the hypocotyl and cotyledons, respectively. Many epidermal cells in these organs of eye mutant plants bulged (Fig. 3A). Rounded epidermal cells were often seen in eye leaves also (Fig. 3K).
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Seedlings of the eye mutant began to form many leaves with the shoot apex from 10 DAV (Fig. 3A–C). Leaves formed by eye plants were not arranged in the spiral pattern of wild-type plants (Fig. 3B,D); they did not expand and they were glossy (Fig. 3C). Because of the protruding and rounded-shape of epidermal cells and the glossy surfaces of eye leaves (Fig. 3B,C), we postulated that the deposition of wax and/or cuticle on the surfaces of epidermal cells might be abnormal. To examine this hypothesis, we used the toluidine blue (TB) test, in which plants with defective hydrophobic layers are stained but wild-type plants are not (Tanaka et al. 2004). As shown in Fig. 2I, the surfaces of eye seedlings were clearly stained with TB, indicating that the formation and/or the permeability of the hydrophobic layers had been affected by the eye mutation. Our eye seedlings became senesce at 20 DAV and did not generate inflorescence stems (data not shown).
We examined the shoot apex of the eye mutant in detail. Figure 3E, F show longitudinal sections of the shoot apices of plants seven DAV. The SAM of wild-type plants is dome-shaped. Leaf primordia are produced on the flanks of the meristem dome (Fig. 3E). By contrast, the SAM of the eye plant was flat (Fig. 3F). Figure 3G–J show scanning electron micrographs of the shoot apex. Although many eye plants produced primordia of the first and second leaves at a right angle to the axis of cotyledons like wild-type plants (Fig. 3G,H), subsequent leaf primordia were produced at irregular positions that did not flank the SAM (Fig. 3K,J).
Cloning of the EYE gene
No recombination between the site of insertion of the T-DNA and the eye mutation was detected during 104 independent meioses, indicating that the insertion of T-DNA was tightly linked to the eye mutation. Therefore, we postulated that the gene responsible for the mutation had been disrupted by insertion of the T-DNA. To identify the mutated gene, we isolated segments of the plant genome that included plant DNA–T-DNA junctions from EYE heterozygotic plants, as described in "Experimental procedures". Analysis of nucleotide sequences revealed that nucleotide sequences adjacent to the left and the right border of the T-DNA in these segments were, respectively, identical to part of the sequence of the hypothetical gene AT5G51430 and its upstream region. We used junction segments to isolate genomic DNA clones for use as probes. We identified a transcript by RNA blot analysis of wild-type plants using a genomic fragment that contained part of the putative coding region of the AT5G51430 gene (data not shown). Then we used this fragment to isolate cDNA clones. The longest cDNA (2695 bp) contained an open reading frame of 2511 bp (DDBJ/GenBank/EMBL accession no.: AB429304 [GenBank] ). As shown in Fig. 4A, the AT5G51430 gene consisted of ten exons and covered 3.8 kbp of the Arabidopsis genome. The T-DNA was inserted in the 5'UTR of this gene (Fig. 4A) and the level of accumulation of transcripts of this gene in entire eye mutant seedlings 15 DAV was lower than that in the wild-type (Fig. 4B).
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We examined the sites of accumulation of EYE transcripts in wild-type Arabidopsis plants. Transcripts of the EYE gene appeared to be ubiquitous (Fig. 4C).
Putative product of the EYE gene
The amino acid sequence of the EYE protein was deduced from the longest open reading frame in the cDNA (Fig. 5A). The cDNA encoded a putative protein of 836 amino acid residues. The nearest in-frame termination codon preceding the predicted initiation codon was located 39 nucleotides upstream of the ATG codon.
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Restoration of a normal phenotype upon introduction of truncated EYE cDNAs into the eye mutant
To examine the significance of the conserved region near the amino terminus, we created constructs that expressed either the entire EYE protein (35S:EYE), as a positive control, or truncated EYE proteins that consisted of amino acid residues 1–274 (35S:EYE
1), 1–123 (35S:EYE2) and 1–53 (35S:EYE3) (Fig. 4E), and we introduced these constructs into heterozygous eye plants. Table 1 shows the results of the complementation experiment with these cDNAs. Transformed plants were designated E, E1, E2 and E3, respectively. We examined the genotype of T1 plants and the frequency of eye seeds, as described above. The E transgenic plants (#6, #11 and #13 in Table 1) that were heterozygous for eye yielded aborted seeds at frequencies that were not significantly different from 6.25% (Table 1, lanes 1–3). In addition, E transgenic plants that were homozygous for the eye locus developed normally (Fig. 4F). Transgenic plants designated E1 (#7, #27 and #31) and E2 (#5, #9 and #15) were heterozygous for eye and yielded aborted seeds at frequencies that were not significantly different from 6.25% (Table 1, lanes 4–9). Two E2 transgenic plants (#20 and #41) were homozygous for the eye locus and yielded aborted seeds at frequencies that were not significantly different from 25% (Table 1, lanes 10 and 11). E1 and E2 transgenic plants that were homozygous for the eye locus developed normally and were morphologically indistinguishable from wild-type plants and from E transgenic plants (Fig. 4F). By contrast, E3 transgenic plants (#2, #3 and #7) that were heterozygous for eye yielded aborted seeds at frequencies that were not significantly different from 25% (Table 1, lanes 12–14). These results indicate that 35S:EYE1 and 35S:EYE2, in which corresponded to truncated EYE cDNAs, reversed the abnormal phenotype of the eye mutant, while 35S:EYE3 did not.
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2) that encoded a mutated EYE cDNA that included nucleotides from positions 1 to 369 with a single-nucleotide deletion at position 122, generating a frameshift mutation (counting position 1 as the first nucleotide of cDNA sequences that encoded the EYE protein; Fig. 4E). We introduced this construct into heterozygous eye plants but it did not reverse the abnormal phenotype of the eye mutant (Table 1). We concluded that the amino acid sequences encoded by truncated EYE cDNAs, in particular, residues 54–123, which included the conserved putative coiled-coil, were important for complementation of the eye mutation. Intracellular distribution of two Golgi marker proteins, ERD2-GFP and GFP-MEMB12, in the eye mutant
We examined whether two fluorescently labeled Golgi marker proteins, ERD2-GFP (Saint-Jore et al. 2002; Takeuchi et al. 2002) and GFP-MEMB12 (Uemura et al. 2004), were properly localized in the Golgi apparatus in the eye mutant. In the cells of cotyledons of wild-type embryos, we detected the fluorescence of ERD2-GFP as disks and bars, which reflected typical Golgi morphology (Fig. 6A). By contrast, some cells of eye embryos had no fluorescent disks and bars (Fig. 6B and Supplementary Fig. S1). In addition, fluorescence was detected as a polygonal network (Fig. 6B and Supplementary Fig. S1). The fluorescence pattern resembled the reticulate network that has been described in previous studies to represent the endoplasmic reticulum network (Haseloff et al. 1997; Ridge et al. 1999; Runions et al. 2006). Thus, the eye mutation might cause mislocalization of ERD2-GFP proteins to the endoplasmic reticulum.
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Discussion |
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The eye mutant displayed pleiotropic defects in morphology. A defect in cell shape and the random production of leaf primordia in the shoot apex were particularly prominent abnormalities (Figs 2 and 3). These features of the eye mutant resemble those of mutants with a decrease in cellulose synthesis. For example, a mutation in the ROOT SWELLING1 gene, which encodes the catalytic subunit of cellulose synthase, causes a defect in cell shape (Arioli et al. 1998). A mutant allele of the KORRIGAN1 (KOR1) gene for an endo-1,4-β-D-glucanase leads to the random production of leaf-like organs from the SAM and to callus formation (Nicol et al. 1998; Zuo et al. 2000). In plants with the tumorous shoot development (tsd) mutation, reduced cell adhesion results development of callus-like tissue with leaf-like organs from shoot apices (Frank et al. 2002). These findings suggest that integrity of the cell wall might be crucial for proper cell shape and the production of leaf primordia with the regular phyllotaxy. Therefore, it seems plausible that features of the eye mutant might be caused by defects in the cell wall. In addition, in the TB test, the surfaces of eye mutant leaves were stained with the hydrophilic dye (Fig. 2I). It has been reported that waxy cuticular components must move through the epidermal cell wall to reach the cuticle (Kunst & Samuels 2003). Thus, the eye phenotype might be due to defects in extracellular matrix structures including cuticular layers and cell walls.
EYE gene encodes a protein with homology to Cog7
The COG complex regulates Golgi retrograde trafficking (Suvorova et al. 2002; Zolov & Lupashin 2005). This secretory pathway is essential for the maintenance of Golgi structure and function and is thought to be required for the retention and/or retrieval of a subset of Golgi-localized proteins to the appropriate intra-Golgi cisternae (Kingsley et al. 1986; Ungar et al. 2002; Oka et al. 2004; Oka & Krieger 2005; Zolov & Lupashin 2005).
The eye mutation caused mislocalization of one of two Golgi marker proteins, ERD2-GFP (Fig. 6B). This result suggests that EYE might influence the localization of certain Golgi proteins in two ways. First, EYE might directly or indirectly protect some Golgi-localized proteins from inappropriate trafficking. Second, EYE might be required for exit from the ER of some Golgi proteins that are either being newly synthesized or constitutively recycled. In addition, the second Golgi marker protein that we examined, GFP-MEMB12, seemed to be localized in the Golgi apparatus in the eye mutant. However, the profile of the fluorescent signals derived from it was abnormal, and smaller fluorescent dots were also observed in the eye mutant (Fig. 6D). These results suggest that EYE might be required for maintenance of normal Golgi morphology and that the eye mutation might induce some diminution in the size of the Golgi vesicles.
These phenotypic features of the eye mutant are somewhat similar to those of mammalian cells that are deficient in a subunit of the COG complex. In mutant mammalian somatic cells that that lack the Cog1 or Cog2 subunit, some Golgi membrane proteins are abnormally localized in the endoplasmic reticulum and/or degraded (Oka et al. 2004). In HeLa cells, depletion of the Cog3 subunit by RNA interference induces fragmentation of Golgi membranes and accumulation of transport vesicles (Zolov & Lupashin 2005). In addition, mammalian homologs of Cog7 are required for normal Golgi trafficking (Wu et al. 2004; Shestakova et al. 2006). These observations suggest that the Arabidopsis COG complex, which includes EYE, might play a role in maintaining Golgi structure and function at the molecular level. However, the GFP-fused EYE proteins, which were capable of restoring the phenotype of the eye mutant, did not show the obvious localization at the Golgi apparatus (T. Ishikawa & Y.M., unpublished data). In plants, processes that maintain Golgi structure remain poorly described in molecular terms. However, it was recently reported that mutations in the GNOM-LIKE1 gene, which encodes the ARF-GEF protein in Arabidopsis, caused an increase in the diameter of Golgi stacks (Richter et al. 2007; Teh & Moore 2007). Therefore, the machinery that regulates vesicle trafficking might play critical roles for maintaining Golgi morphology in Arabidopsis.
Conserved amino-terminal region of EYE is functional
Introduction of 35S:EYE2 for the expression of truncated EYE cDNAs normalized the phenotype of eye mutant plants and resultant plants were morphologically indistinguishable from the wild-type, while 35S:EYE3 and 35S:mEYE2 were ineffective (Fig. 4E). These results indicate that a region of 70 amino acids (residues 54-123), which includes a conserved putative short coiled coil, is important for the functioning of the EYE/Cog7 protein. This region might be involved in interactions with other subunits of the COG complex. In the mammalian COG complex, Cog7 is required for formation of the Cog5–Cog7 and Cog5–Cog8 subcomplexes in vitro (Ungar et al. 2005). The conserved amino acids at the amino terminus (residues 1–123) of EYE might be sufficient for the formation of a COG subcomplex that contains EYE/Cog7 and a functional COG complex when this region is overproduced. Since the T-DNA in the eye mutant is integrated into the region upstream of the EYE ORF (Fig. 4A) and the full length of EYE transcripts was detectable at a reduced level (Fig. 4B) (T. Ishikawa & Y.M., unpublished data), small amounts of functional EYE protein might be synthesized in the mutant. If EYE proteins form a multimer, a multimer containing one or a few entire EYE proteins plus the truncated EYE2 proteins might be functional.
Role of EYE in development
Mutant mammalian somatic cells lacking Cog1 or Cog2 exhibit pleiotropic defects in the processing of Golgi-associated glycoproteins and glycolipids (Kingsley et al. 1986). Depletion of Cog3 or Cog7 by RNA interference leads to defects in protein glycosylation (Shestakova et al. 2006). It has been proposed that these defects might be due to the mislocalization and/or instability of some of the proteins responsible for the synthesis of glycoconjugate (Kingsley et al. 1986; Oka et al. 2004; Shestakova et al. 2006). In addition, it was reported recently that the COG complex of Caenorhabditis elegans plays a role in morphogenesis of the gonads by regulating Golgi enzymes, thereby affecting the glycosylation (Kubota et al. 2006). Therefore, the eye mutant might be expected to exhibit defects in the processing of Golgi-associated glycoproteins and glycolipids, which might lead to a pleiotropic abnormal phenotype. In addition, some Arabidopsis mutants that have defects in the processing of N-linked glycans are defective in cellulose synthesis (Boisson et al. 2001; Burn et al. 2002; Gillmor et al. 2002). The eye mutant might also affect cellulose synthesis as a consequence of defects in synthesis of N-linked glycoconjugates in the Golgi.
In the eye mutant, embryo development and callus formation appeared to be normal (T. Ishikawa & Y.M., unpublished data) and organ primordia were produced, suggesting that cell proliferation was not significantly affected by the eye mutation. Similarly, a defect in Cog3 subunit does not have a severe negative effect on the rate of proliferation of mammalian cells (Zolov & Lupashin 2005). By contrast, the expansion of cells and the organization of SAM were strikingly affected by the eye mutation. Therefore, we propose that Golgi-associated reactions, maintained by EYE, play important roles in cell expansion and the organization of the meristem.
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Experimental procedures |
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Arabidopsis thaliana ecotype Landsberg erecta was used as the wild-type strain. The #039 line was isolated from T-DNA-transformed lines generated in our laboratory. For analysis of the phenotype of seeds and embryos, plants were grown on soil at 22 °C under continuous white light. For analysis of the phenotype of plants after germination, sterilized seeds were sown on plates of 0.2% gellan gum-solidified Murashige and Skoog medium (Wako Pure Chemical Industries, Ltd., Osaka, Japan). Plates with seeds were stored for two days at 4 °C for vernalization and then transferred to a growth chamber (22 °C; continuous light). The age of plants is given in terms of numbers of DAV.
Phenotypic analysis
Embryos and seedlings were cleared as described by Ishikawa et al. (2003). Leaf surfaces were stained by the TB method as described by Tanaka et al. (2004). Sections were prepared and examined as follows. Tissues were fixed in 4% paraformaldehyde, dehydrated in a graded ethanol series and mounted in Technovit® 7100 resin (Heraeus Kulzer, Wehrheim, Germany) as described in the instructions from the manufacturer. Sections of 3–5 µm in thickness were cut with a microtome (HM360; Carl Zeiss, Tokyo, Japan) and stained with 0.1% TB. For examination of shoot apices of eye plants, samples were frozen in liquid nitrogen and observed with a scanning electron microscope (XL30; Philips Electron Optics, Eindhoven, The Netherlands).
Cloning of genomic and cDNA sequences that contained the EYE-coding region
To amplify fragments of genomic DNA that flanked the T-DNA, we synthesized primers with sequences specific to the T-DNA (LBrev1, 5'-CGCGCAATATTTACACATAGACACACACAT CATCTC-3'; LBrev3, 5'-CTCGAAATCAGCCAATTTTA GACAAGTATCAAACGG-3'; Tnosrev2, 5'-CAGCCTCTC GATTGCTCATCGTCATTACACAGTAC-3'; and Tnosrev4, 5'-CAAGTGTTCGGACGTGGGTTTTCGATGG-3') and the cassette (CPC1, 5'-GTACATATTGTCGTTAGAACGCG TAATACGACTCA-3'; and CPC2, 5'-CGTTAGAACGCG TAATACGACTCACTATAGGGAGA-3'). To amplify a DNA fragment that included plant DNA and the left border of the T-DNA junction, we used primers LBrev1 and CPC1 for the first PCR and primers LBrev3 and CPC2 for the second PCR. To amplify a DNA fragment that included plant DNA and the right border of the T-DNA junction, we used primers Tnosrev4 and CPC1 for the first PCR and primers Tnosrev2 and CPC2 for the second PCR. Each amplified fragment was subcloned into pBluescript SK– (STRATAGENE, La Jolla, CA). A Landsberg erecta genomic library (CD4-8) was obtained from the Arabidopsis Biological Resource Center (ABRC, Ohio State University, Columbus, OH). We used a previously constructed cDNA library (Nishihama et al. 1997) and we gave the designation pEYE-7A to the cDNA clone with the longest insert that contained a coding strand of the EYE gene.
Complementation of the eye mutation
For complementation of the eye mutation, we introduced, separately, a 6.0-kbp Bst1107I-SplI genomic fragment that included the entire putative EYE gene and a 1.2-kbp Bst1107I-XhoI genomic fragment, in which part of the EYE gene had been deleted, into the pBIBAR binary vector (Ishikawa et al. 2003) to generate EYE6.0 and EYE1.2, respectively. To generate p35S:EYE1, p35S:EYE2 and p35S:EYE3, individual PCR-amplified fragments that contained EYE cDNA that corresponded to amino acid residues 1–274, 1–123 and 1–53, respectively, and the termination codon were cloned into pTH-2 (Chiu et al. 1996) from which the DNA fragment that contained the green fluorescent protein-coding (GFP-coding) region had been removed. To generate p35S:EYE, the DNA fragment excised by XhoI and NotI from pEYE-7A was ligated into the XhoI and NotI sites of p35S:EYE1. To generate p35S:mEYE2, the PCR-amplified fragment that contained a single nucleotide deletion and the termination codon was inserted at the XhoI and NotI sites of p35S:EYE1. Individual DNA fragments containing the 35S promoter of cauliflower mosaic virus, the EYE cDNA and the nos terminator were excised from each plasmid and cloned into the binary vector pBIBAR. The GV3101 strain of Agrobacterium tumefaciens (Koncz & Schell 1986) was transfected with each construct. The transformed Agrobacterium cells were used to introduce DNA into eye heterozygous plants by Agrobacterium-mediated transformation by the vacuum-infiltration method (Galbiati et al. 2000). Transgenic plants were selected on soil that contained 0.01% Basta (AgroEvo, Frankfurt, Germany). The genotype of T1 plants was examined by DNA blot analysis with the genomic DNA fragment that flanked the T-DNA as probe or by PCR with primers 039LB11 and 039RB11, with primers specific for the plant DNA adjacent to the left and the right border of the T-DNA, respectively, and the LBrev4 primer that was specific for the left border of the T-DNA. The sequences were 039LB11, 5'-GAGAGTAGAA GAAGCTGCTAGAGGCTAGAGAGTC-3'; 039RB11, 5'-CCAGATCCACAAGATGTTTCTCGAGTGAATCCTGTG-3'; and LBrev4, 5'-CGTCCGCAATGTGTTATTAAGTTGTCTA AGCGTC-3'.
RT-PCR
Total RNA was isolated from wild-type and eye plants with a QIAGEN RNeasy® Mini kit (QIAGEN GmbH, Hilden, Germany). Poly(A)+ RNA was purified from total RNA with Dynabeads® Oligo(dT)25 (DYNAL BIOTECK, Oslo, Norway) and first-strand cDNA was synthesized with Ready-To-Go You-Prime first-strand beads (GE Healthcare UK Ltd., Little Chalfont, UK). Equal aliquots from each first-strand reaction were used as template to amplify EYE cDNA. RT-PCR was performed with primers specific for the EYE gene, namely, 039RB12, 5'-GGATCTAGGTCCATTCTCAGATGAGAAGTTCGATG-3'; and LRPE63, 5'-ACTAGCCCCCAGTACTCCCATGG-3'. As an internal control for RT-PCR, we amplified cDNA using primers for the gene for 
-tubulin (Semiarti et al. 2001). PCR was performed with 21, 24, 27 or 30 cycles of incubations at 94 °C for 1 min, 60 °C for 30 s, and 72 °C for 1.5 min. Products of PCR were separated on a 0.7% agarose gel, stained with ethidium bromide, and visualized under UV-transillumination.
Intracellular distribution of Golgi marker proteins
We amplified MEMB12 cDNA that included the entire coding region of the EYE gene from pooled cDNA derived from the Columbia ecotype of Arabidopsis by PCR with the gene-specific primers: AtMEMB12F1BglII, 5'-TTAGATCTATGGCGTCT GGGACAGTGG-3'; and AtMEMB12R1Not, 5'-TTGCG GCCGCTAGCGTGTCCATCTTATG-3'. We subcloned the cDNA into the pENTR 3C vector (Invitrogen Corp., Carlsbad, CA), and then integrated it into the GATEWAY binary vector using LR clonase (Invitrogen). The resulting plasmid was introduced into Agrobacterium strain GV3101, which was used to transform Arabidopsis ecotype Landsberg erecta by the floral dip method (Galbiati et al. 2000). Transgenic lines were obtained by selection on Murashige and Skoog's medium supplemented with 15 mg/L hygromycin and 300 mg/L carbenicillin. The fluorescence due to GFP was visualized with a confocal laser scanning microscope (LSM 510; Carl Zeiss, Oberkochen, Germany) and an argon laser (488 nm).
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Acknowledgements |
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Footnotes |
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* Correspondence: Email: yas{at}bio.nagoya-u.ac.jp
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References |
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Accepted: 26 February 2008
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