Research ArticleResearch Article

ANTHOCYANINLESS2, a Homeobox Gene Affecting Anthocyanin Distribution and Root Development in Arabidopsis

Hiroyoshi Kubo, Anton J. M. Peeters, Mark G. M. Aarts, Andy Pereira, Maarten Koornneef

Published July 1999. DOI: https://doi.org/10.1105/tpc.11.7.1217

Abstract

The ANTHOCYANINLESS2 (ANL2) gene was isolated from Arabidopsis by using the maize Enhancer–Inhibitor transposon tagging system. Sequencing of the ANL2 gene showed that it encodes a homeodomain protein belonging to the HD–GLABRA2 group. As we report here, this homeobox gene is involved in the accumulation of anthocyanin and in root development. Histological observations of the anl2 mutant revealed that the accumulation of anthocyanin was greatly suppressed in subepidermal cells but only slightly reduced in epidermal cells. Furthermore, the primary roots of the anl2 mutant showed an aberrant cellular organization. We discuss a possible role of ANL2 in the accumulation of anthocyanin and cellular organization of the primary root.

INTRODUCTION

Anthocyanins are plant pigments that are responsible for a variety of red, blue, and purple colors. Anthocyanin accumulation occurs in certain plant tissues at specific developmental stages and is subject to control by many environmental factors, such as light, temperature, nutrients, and stress (Blank, 1958; Beggs and Wellmann, 1994). Most enzymes involved in anthocyanin biosynthesis are well characterized, and the structures of the corresponding genes have been determined for many plant species. Several regulatory genes for anthocyanin biosynthesis already have been identified in various plants (Dooner and Robbins, 1991). For example, the maize R/B gene family shows sequence similarity to MYC oncogenes. The R and B loci regulate the tissue specificity of anthocyanin biosynthesis in combination with another gene family, C1/Pl, whose members play a role in light regulation of the pathway and are related to MYB oncogenes. Moreover, the Delila gene of Antirrhinum encodes a transcription factor closely related to the R gene family in maize (Goodrich et al., 1992), and Eluta and Rosea control the pattern of anthocyanin production in Antirrhinum flowers (Martin et al., 1991; Martin and Gerats, 1993). In petunia, the regulatory genes AN1, AN2, and AN11 have been cloned. AN1 and AN2 encode MYC- and MYB-type transcription factors, respectively (Mol et al., 1996). AN11 encodes another type of regulatory protein containing a WD40 repeat motif (de Vetten et al., 1997). Although much research has been done, the mechanisms that control the spatial and temporal regulation of anthocyanin accumulation have not been elucidated.

In Arabidopsis, several mutants lacking anthocyanin have been isolated (Koornneef, 1990). These transparent testa (tt) mutants produce yellow or pale brown seeds because they lack brown pigment in the seed coat. TT3, TT4, and TT5 have been found to encode dihydroflavonol-4-reductase (DFR), chalcone synthase (CHS), and chalcone flavanone isomerase (CHI), respectively (Shirley et al., 1992). TTG and TT8 are regulatory genes that control transcription of DFR but not CHS and CHI (Shirley et al., 1995). The ttg mutant lacks not only anthocyanin but trichomes. The ttg mutant is restored to the wild type when transformed with the maize R gene (Lloyd et al., 1992).

Homeobox genes are another universal group of regulatory genes; however, they have not been associated with anthocyanin biosynthesis. The many homeodomain proteins that have been found in higher plants are divided into five groups: HD–KNOTTED1, HD–BELL1, homeodomain leucine zipper protein (HD–ZIP), plant homeodomain finger protein (PHD–Finger), and HD–GLABRA2 (GL2) (Lu et al., 1996). Recent research has shown that in general, homeobox genes play a role in cell specification and pattern formation in higher plants. KNOTTED1 and SHOOT MERISTEMLESS are considered to be involved in maintaining the indeterminate state of apical meristems (Long et al., 1996; Kerstetter et al., 1997). BELL1 is required for integument development (Reiser et al., 1995). Athb-8, which encodes a protein belonging to the HD–ZIP group, is suggested to regulate vascular development (Baima et al., 1995). GL2 regulates trichome development and suppresses root hair formation (Rerie et al., 1994). ATML1 (for Arabidopsis thaliana meristem L1 layer), which encodes a protein belonging to the HD–GL2 group, is specifically expressed in the L1 layer of the vegetative shoot meristem and the inflorescence meristem. ATML1 is suggested to be involved in setting up morphogenetic boundaries of positional information necessary for controlling cell specification and pattern formation (Lu et al., 1996).

Here, we report the isolation of another homeobox gene that encodes a protein belonging to the HD–GL2 group. This gene controls the anthocyanin pigmentation of the leaf subepidermal layer and cellular organization of the primary root.

RESULTS

Genetic Analysis

Heterologous tagging systems that use the maize transposon Enhancer (En)–Inhibitor (I) or Activator–Dissociation system have been used successfully in Arabidopsis (Aarts et al., 1993; Bancroft et al., 1993). In an Arabidopsis line containing the En–I system, we isolated a mutant with strong leaf anthocyanin variegation. This mutant produced normal brown seeds and was therefore different from tt and ttg mutants. We previously isolated an ethyl methanesulfonate–induced anthocyaninless mutant, called anl1, that is not allelic to this new mutant, which we called anl2. The anl2 mutant showed variegation, indicating a high somatic transposon excision frequency. By backcrossing to Landsberg erecta (Ler) plants, we were able to separate the transposon from the transposase source, thus stabilizing the transposon inserted in anl2. This also reduced the number of I elements present in the genome and facilitated further analysis of the mutant.

Pattern of Anthocyanin Accumulation

Under our growth conditions, rosette leaves of Ler plants accumulate much anthocyanin (Figure 1A). This is in contrast to the stable anl2 mutant, in which anthocyanin accumulation is reduced (Figure 1B). The unstable mutants showed variegation of anthocyanin pigmentation (Figure 1C). Variegation was observed only on the adaxial side of rosette leaves, whereas anthocyanin accumulated uniformly on the abaxial side of the leaves (Figures 1D and 1E). Furthermore, anthocyanin content on the adaxial side of rosette leaves of the stable mutant was greatly reduced, although there was also a slight reduction in anthocyanin content on the abaxial side (Figure 2). Cross-sections of rosette leaves were analyzed to determine the precise distribution of anthocyanin accumulation in leaves (Figures 1F and 1G). In the rosette leaves of Ler, anthocyanin was observed to accumulate in subepidermal tissue of the adaxial side but in the epidermis of the abaxial side. In the anl2 mutant, anthocyanin mainly accumulated in the epidermis of the abaxial side. These observations suggest that ANL2 is a regulator of anthocyanin accumulation in subepidermal tissues.

To verify this possibility, we examined the accumulation of anthocyanin in other parts of the anl2 mutant (Table 1). The seedlings of the anl2 mutant accumulated less anthocyanin than did the seedlings of Ler. Anthocyanin was greatly reduced in all organs in which anthocyanin normally accumulates in subepidermal tissue (i.e., the adaxial side of leaves, stems, and flower buds), in contrast to the abaxial side of the rosette leaf. The banyuls (ban) mutant is known to accumulate high levels of anthocyanins in the most internal layer of the seed coat, which is derived from the L1 layer of the meristem (Albert et al., 1997). To examine whether the anthocyanin level in the L1-derived tissue was influenced in the anl2 mutant, we examined anthocyanin accumulation in an anl2 ban double mutant. The anl2 ban double mutant accumulated amounts of anthocyanin similar to those accumulated by the monogenic ban mutant (Table 1). This means that ANL2 is not required for anthocyanin accumulation in the internal layer of the seed coat, which is derived from the L1 layer of the meristem.

Figure 1.
Figure 1.

Pattern of Anthocyanin Accumulation in an anl2 Mutant and a Ler Plant.

(A) to (C) Adaxial side of rosette leaves from Ler, a stable anl2 mutant, and an unstable anl2 mutant showing anthocyanin variegation, respectively.

(D) and (E) Upper and lower surfaces, respectively, of a rosette leaf showing variegation.

(F) and (G) Cross-sections of leaves from a Ler plant and an anl2 mutant, respectively. Bar in (G) = 100 μm for (F) and (G).

Figure 2.
Figure 2.

Time Course of Anthocyanin Accumulation in Rosette Leaves.

Time courses of anthocyanin accumulation were examined from the adaxial (filled circles and squares) and abaxial (open circles and squares) sides of rosette leaves from Ler (filled and open circles) and anl2 (filled and open squares) plants. Plants grown for 3 weeks at 22°C (7 W m−2) were exposed to 15°C (11 W m−2) for 0 to 6 days to induce anthocyanin accumulation. Anthocyanin was extracted from each layer (adaxial and abaxial sides) of a rosette leaf separately. The amount of anthocyanin was expressed as absorbance (Abs) at 530 nm per leaf area. The average of 10 rosette leaves is shown. Error bars indicate ±se.

Cloning and Genetic Mapping of ANL2

To isolate the ANL2 gene, we isolated genomic DNA from 74 F2 plants (anl2 × Ler), digested it with HindIII, and examined it by using DNA gel blot analysis with an I element–specific probe. A 7-kb HindIII fragment cosegregated with the anl2 allele and was considered to be the candidate for the ANL2 gene into which the I element had been inserted. The flanking regions of the I elements were amplified by inverse polymerase chain reaction (PCR), and the PCR fragment that hybridized to the 7-kb band was cloned to make an ANL2-specific probe. Using this probe, we screened genomic and cDNA libraries. Positive clones were isolated, analyzed, and sequenced. The length of the isolated cDNA clone was 2987 bp, with an open reading frame that predicts a protein of 802 amino acids. Figure 3 shows a physical map of the ANL2 locus. The total length of genomic DNA sequenced is 9.3 kb. ANL2 is a complex gene that contains nine exons interrupted by eight introns. The I element was inserted 13 bp upstream of the seventh exon.

Using restriction fragment length polymorphism (RFLP) and cleaved amplified polymorphic sequence (CAPS) markers on a population of recombinant inbred (RI) lines of Ler/Columbia (Col) (Lister and Dean, 1993) and Ler/Cape Verde Islands (Cvi) (Alonso-Blanco et al., 1998), we were able to map ANL2 to the top of chromosome 4 located between and closely linked to mi122 and g3843. These data were confirmed by cytogenetical analysis showing that ANL2 is located on yeast artificial chromosome CIC10C8 (P. Fransz, S. Armstrong, J.H. de Jong, C. van Drunen, P. Zabel, C. Dean, and G. Jones, personal communication). No other locus affecting anthocyanin accumulation has been identified in this region.

View this table:
Table 1.

Amount of Anthocyanin in Rosette Leaves, Stems, and Buds of Ler and anl2 Plants and in Young Seeds of banyuls (ban) and anl2 ban Double Mutants

Figure 3.
Figure 3.

Physical Map of the ANL2 Locus.

Shown is a physical map of the ANL2 genomic region. Restriction sites are indicated as follows: B, BamHI; E, EcoRI; H, HindIII; S, SalI; X, XhoI. Closed boxes indicate exons. The position of the I element insertion is indicated by an arrowhead.

Predicted Amino Acid Sequence of ANL2

DNA sequencing showed that the ANL2 gene encodes a homeodomain protein that belongs to the HD–GL2 group (Figure 4). The homeodomain of ANL2 shares 23 of 29 amino acids that are conserved in >80% of the homeodomain sequences of various organisms (Gehring et al., 1994). Generally, highly conserved amino acids (L-16, F-20, W-48, F-49, N-51, and R-53) are also conserved in ANL2. In a homology search of GenBank, four other proteins—Arabidopsis ATML1 (GenBank accession number U37589), Phalaenopsis ovule-specific protein O39 (Nadeau et al., 1996; GenBank accession number U34743) , a Helianthus homeodomain protein (GenBank accession number L76588) , and Arabidopsis GL2 (EMBL accession number Z54356)—were found to have a high degree of sequence similarity with ANL2. Among them, ANL2 most resembles ATML1 and O39. Fifty-four percent of the amino acid residues of ATML1 are conserved in ANL2. ANL2 has an extra sequence at the N terminus, and downstream of the homeodomain, there are several regions conserved among the members of the HD–GL2 group. ANL2 contains two introns in the homeobox region (amino acids 18 and 19 and 57 and 58 of the homeodomain). These intron positions are also conserved in GL2.

Nucleotide Sequences of the I Element Insertion Site in the Revertants

After crossing the stable mutant with a plant that has only the En element in the genome, we observed variegation in the F2 plants. This shows that the anl2 mutation is caused by I element insertion. We selected several independent stable revertants from progenies of variegated plants and amplified the insertion site of the I element by PCR. The sequences of these insertion sites were determined to confirm that we had isolated the ANL2 gene.

In the anl2 mutant, the trinucleotide target site sequence (AAG) was duplicated upon insertion of the I element. Three types of excision footprints were found in the revertants. Compared with the wild type, two revertants had an addition of 5 bp (CTAAG); another two had an addition of 3 bp (AAG). One revertant had the same sequence as the wild type. All the excision events restored the wild-type phenotype because the I element was inserted in intron 6 of the ANL2 gene.

Expression of ANL2

Reverse transcription–PCR (RT-PCR) was performed using primers specific for the sixth and eighth exon sequences (Figures 5A and 5B). A 0.7-kb fragment was amplified in Ler but not in the stable anl2 mutant. No signal was detected by DNA gel blot analysis in the anl2 mutant (data not shown), which means that the anl2 mutant did not contain any complete ANL2 mRNA. ANL2 was expressed in wild-type leaves, stems, buds, and roots. Another primer set designed for the sequence upstream of the I element insertion site (specific for the first and fourth exon sequences) gave the same results for both the wild type and the anl2 mutant (data not shown). This shows that the insertion of the I element in the sixth intron does not affect transcription of the ANL2 gene.

Effect of ANL2 on Cellular Organization of the Primary Root

Prompted by the fact that the ANL2 gene is a homeobox gene, we examined morphological aspects of the anl2 mutant. Cell number and location are invariant in the primary root of Arabidopsis (Dolan et al., 1993). In the primary root of Ler, an endodermis made up of eight cells is surrounded by a cortex of eight cells and then by an epidermal layer of ~20 cells. The eight hair cell files that stained deeply with toluidine blue overlaid the radial walls of the underlying cortical cells (Figure 6A). The anl2 mutant had an endodermis made up of eight cells and a cortex of eight cells. However, there were several extra cells between the cortical and epidermal layers (Figure 6B). We call these extra cells intervening cells. The staining pattern of toluidine blue was irregular, and the intervening cells and cortical cells stained similarly. No wild-type plants and no revertant plants had such intervening cells (Table 2). This indicates that the mutation in the ANL2 gene induced an aberrant arrangement of the cellular organization in the primary root.

The files of hair cells are separated by one or two files of non-hair cells in wild-type plants. In the anl2 mutant, files of hair cells adjoining each other were sometimes observed (data not shown).

Figure 4.
Figure 4.

Primary Structure of the ANL2 Protein.

The deduced amino acid sequence of ANL2 (GenBank accession number AF077335) is compared with that of ATML1 (Lu et al., 1996; GenBank accession number U37589) and GL2 (Cristina et al., 1996; EMBL accession number Z54356) from Arabidopsis and O39 (Nadeau et al., 1996; GenBank accession number U34743) from Phalaenopsis. Dots indicate amino acids identical to the ANL2 sequence. Dashes indicate gaps in the sequence to allow for maximal alignment. The homeobox is marked by a box. The arrowheads indicate the position of introns. The stars indicate acidic amino acids. The bar indicates a proline- and glutamine-rich region.

The number of cells in cortical and endodermal layers is invariant in both Ler and the anl2 mutant, although the number of cells in the epidermal layer is variable. However, there was no correlation between the number of epidermal cells and the number of intervening cells (Figure 7). This suggests that the intervening cells are formed not by positional shifts of epidermal cells but by abnormal cell divisions.

DISCUSSION

ANL2 Encodes a Homeodomain Protein

In the En–I two-element system modified by Aarts et al. (1995), a T-DNA construct carrying an immobilized En transposase source activates the transposition of I elements, which cause mutations when inserted into a gene. The original anl2 mutant contained the En transposase source and showed variegation of anthocyanin pigmentation in rosette leaves. This phenotype is evidence that the anl2 mutation is caused by I element insertion. DNA gel blot analysis showed that there is one I element that cosegregates with the anl2 mutation. In all revertants, this element was excised from an anl2::I allele, restoring the ANL2 transcript. This proves that we have isolated ANL2.

The predicted amino acid sequence indicates that ANL2 codes for a homeodomain protein of the HD–GL2 group. The helix-turn-helix structure and most of the invariant amino acids of the homeodomain (L-16, F-20, W-48, F-49, N-51, and R-53) are conserved in ANL2. There are several motifs that have been implicated in activation of transcription: a proline–glutamine stretch and an acidic region. Therefore, ANL2 probably has DNA binding activity and most likely plays a role as a transcription factor. Within the HD–GL2 group, ANL2 is most similar to the ATML1 protein, which shares several conserved regions downstream of the homeodomain in these two proteins. These regions are conserved for dicots (ANL2 and ATML1) and monocots (O39), suggesting that these regions are important for common processes required for the function of HD–GL2. Despite the similarity in amino acid sequence, ANL2 showed a different expression pattern from ATML1. ANL2 was expressed in all organs examined, whereas ATML1 was expressed only in floral buds and siliques (Lu et al., 1996).

Figure 5.
Figure 5.

Detection of ANL2 mRNA by RT-PCR in Ler and anl2 Seedlings and Plants.

(A) ANL2 expression in Ler and anl2 seedlings. Total RNA was extracted from 4-day-old Ler or anl2 seedlings. The PCR product was detected by ethidium bromide staining after 30 amplification cycles.

(B) ANL2 expression in leaves, stems, buds, and roots of Ler plants. Total RNA was extracted from various organs of Ler plants grown for 3 weeks under continuous light. The PCR product was detected by DNA gel blot analysis after 18 amplification cycles.

Size of the PCR product is shown at right.

Effect on Anthocyanin Accumulation

The mutant allele of ANL2 had no effect on the pigmentation of seed coat, although it affected the pigmentation of the seedling and mature plant tissues. Thus, the phenotype of the anl2 mutant was opposite to tt1, tt2, and tt8 mutants lacking seed pigment but containing a normal amount of anthocyanin in plant tissues (Koornneef, 1990). This suggests that anthocyanin accumulation in the seed coat is under a different control from that in seedling and mature plant tissues and that ANL2 is specific for seedling and mature tissues. This suggestion is supported by the experiment using the anl2 ban double mutant (Table 1).

In maize, the pattern of anthocyanin pigmentation is determined by the individual members of the two gene families R/B and C1/Pl. Pigmentation of kernel requires R and C1, whereas pigmentation of mature tissues of the plant requires B and Pl (Mol et al., 1996). It is suggested that R and C1 homologs are involved in anthocyanin biosynthesis of Arabidopsis (Lloyd et al., 1992). These regulatory genes may control the tissue specificity of anthocyanin biosynthesis in Arabidopsis.

Figure 6.
Figure 6.

Transverse Sections of the Primary Root of Ler and anl2 Plants.

(A) Ler primary root.

(B) anl2 primary root.

Asterisks and arrowheads indicate cortical and intervening cells, respectively. Bar in (B) = 25 μm for (A) and (B).

View this table:
Table 2.

Numbers of Endodermal, Cortical, Intervening, and Epidermal Cells of the Primary Roots of Ler, anl2, and ANL2 Revertant Plants

Anthocyanin accumulated in subepidermal tissue on the adaxial side of rosette leaves and in epidermal tissue on the abaxial side of the leaves. There are several observations that the pattern of anthocyanin accumulation in the epidermis is different from that in the subepidermal layer. For example, Parkin (1903) examined tissue localization of anthocyanin in foliage leaves of many species and showed that inducible anthocyanins accumulate in mesophyll but permanent anthocyanins accumulate mainly in epidermis. There is a possibility that ANL2 is involved in an induction process of anthocyanin synthesis in subepidermal cells. In Arabidopsis, however, both epidermal and subepidermal anthocyanins are similarly induced by light. The effects of other stimuli on the tissue specificity of anthocyanin accumulation remain unknown.

Effect on Cellular Organization

In addition to the reduction in anthocyanin content in leaves, we found that the cellular organization of the primary root was distorted in the anl2 mutant. This is not surprising, considering that gl2, another homeobox gene, also has an effect on both the leaf and root. Several root developmental mutants have been isolated in Arabidopsis (Scheres et al., 1996; Schiefelbein et al., 1997). Some of these mutants have abnormal roots because of excessive cell expansion of a particular tissue (Benfey et al., 1993). Others are root patterning mutants that lack a particular cell layer (Scheres et al., 1995). The anl2 mutant clearly shows a different phenotype compared with these mutants.

Files of hair cells that adjoined each other were sometimes observed in the anl2 mutant. This may be caused by the abnormal organization of the root cells. Precise observations regarding the relative positions of the hair cell files and the intervening cells are being made. The intervening cells stained similarly to the cortical cells, but the character of the intervening cells has not been clear. Further studies using epidermis- and cortex-specific reporter genes are required to characterize the intervening cells.

No histological differences were found upon examination of rosette leaf and stem tissues of the anl2 mutant and the wild type. Because the number and size of cells in these tissues are quite different, depending on the developmental stage, it is possible that we were not able to detect small changes in cellular organization in these tissues. Therefore, we examined cotyledons and hypocotyl tissues but found no differences between Ler and anl2 plants. Mutation of ANL2 apparently does not induce morphological changes in the cotyledons and the hypocotyl, although the accumulation of anthocyanin was affected in these organs.

Function of ANL2

A homeodomain protein is generally thought to regulate the transcription of specific target genes (Gehring et al., 1994). Therefore, ANL2 is unlikely to be a structural gene of anthocyanin biosynthesis. In addition, we found no difference in anthocyanin composition between adaxial and abaxial sides of rosette leaves in wild-type plants by using thin-layer chromatography (data not shown). Thus ANL2 is probably a regulatory gene that is involved in regulating the tissue-specific accumulation of anthocyanins and in organizing the root.

ANL2 affected anthocyanin accumulation in subepidermal tissues as well as the cellular organization of the primary root. Some regulatory genes are known to affect several independent phenotypes. The gl2 mutant is defective in trichome development, root hair formation, and mucilage production in the seed coat (Rerie et al., 1994; Masucci et al., 1996). The ttg mutant shows not only a morphological defect similar to gl2 but also is unable to synthesize anthocyanins (Koornneef, 1981). One possibility is that ANL2 regulates two independent pathways: one is anthocyanin accumulation in subepidermal tissues, and the other is development of cellular organization in the primary root. Another possibility, which is an attractive one, is that ANL2 determines the identity of subepidermal cells and acts on a developmental process that subsequently interferes with the accumulation of anthocyanin.

Figure 7.
Figure 7.

Absence of Correlation between the Number of Epidermal Cells and the Number of Intervening Cells.

The numbers of epidermal cells and intervening cells of the primary root of anl2 plants were counted under a microscope. Each plot shows the numbers of epidermal cells and intervening cells of one primary root.

METHODS

Plant Material and Growth Conditions

Plant material (Arabidopsis thaliana) and normal growth conditions were as described previously (Koornneef et al., 1991). For the experiments in which anthocyanin content was measured, plants were grown on agar plates (5 mM KNO3, 2.5 mM KH2PO4, 2 mM MgSO4, 2 mM Ca[NO3]2, 0.05 mM Fe-EDTA, micronutrients, 1% sucrose, and 0.8% agar) for 4 days or in soil for 3 weeks at 22°C (7 W m−2) and then at 15°C under continuous white light (11 W m−2) to induce anthocyanin production.

Measurement of Anthocyanin Content

Anthocyanin was extracted from leaves with 1 mL of 0.5 M HCl-methanol at 4°C for 24 hr. To compensate for the absorbance of chlorophyll, we bleached the anthocyanins with H2O2, with the difference of A530 nm before bleaching and after bleaching representing anthocyanin content (Swain and Hillis, 1959). To measure the anthocyanin content of adaxial and abaxial sides separately, we peeled off the epidermis of the abaxial side of a rosette leaf with adhesive tape.

Morphological Observations

For observation of leaf tissue, we embedded fresh tissues in 5% agarose and sliced them with a microslicer (model DTK-1000; Dosaka EM Co., Kyoto, Japan). For observation of the primary root, we fixed seedlings for 4 hr in 50% ethanol, 5% acetic acid, and 3.7% formaldehyde at room temperature, dehydrated them in a graded ethanol series, permeated them with xylene, and then infiltrated them with paraffin. Eight-micrometer sections were made with a microtome and stained with 0.05% toluidine blue. The number of cells was counted under a microscope.

Genomic DNA Isolation and DNA Gel Blot Analysis

Genomic DNA was isolated from three expanded leaves by a down-scaled version of a protocol for tomato DNA isolation (Bernatzky and Tanksley, 1986). Total DNA (0.5 to 1 μg) was digested with HindIII, electrophoresed, and transferred to a nylon membrane (Hybond N+; Amersham) by vacuum blotting (Pharmacia). A 600-bp probe of the left junction of the Inhibitor (I) element was used to detect restriction fragments containing I elements. 32P-labeled probes were made by random prime labeling (Feinberg and Vogelstein, 1984) using α-32P-dATP (110 TBq/mmol; Amersham). After overnight hybridization in 5 × SSC (1 × SSC is 0.15 M NaCl and 0.015 M sodium citrate) and 0.1% SDS at 65°C, we washed the membrane twice with 2 × SSC and 0.1% SDS for 10 min at 65°C before autoradiography.

Reverse Transcription–Polymerase Chain Reaction

The Titan one-tube reverse transcription–polymerase chain reaction (RT-PCR) system (Boehringer Mannheim) was used for RT-PCR. Total RNA was extracted by a standard phenol-SDS extraction method. The same amount of total RNA was used for template, and reactions were performed by using a scaled-down version of the manufacturer's instructions. Two pairs of primers (anl6′ and anl8R′; anl2138′ and anl3320R′) were used to detect the ANL2 transcript. The sequences of the primers were anl6′ (5′-TTGTCTCCACTGGTTCCGGT-3′), anl8R′ (5′-ATTGGAGCGGAGCAGAGAGA-3′), anl2138′ (5′-ATGAACTTCGGTAGTCTCTT-3′), and anl3320R′ (5′-TGATCAATCCCATTAATCAC-3′). PCR conditions were denaturing at 94°C for 30 sec, annealing at 58°C (anl6′ and anl8R′ primer set) or 50°C (anl2138′ and anl3320R′ primer set) for 30 sec, and extension at 68°C for 90 sec. After 18 cycles (samples for leaves, stems, buds, and roots) or 30 cycles (samples isolated from seedlings of Landsberg erecta [Ler] and anl2) of PCR, the products were electrophoresed, transferred to a nylon membrane, and detected by DNA gel blot analysis. 32P-labeled probe was made by using the ANL2 cDNA as a template.

Screening of cDNA and Genomic Libraries and Sequencing

DNA flanking the I element was amplified by inverse PCR, as described previously (Aarts et al., 1995), using HinfI-digested genomic DNA and three sets of nested primers.

The cDNA library that we used, λPRL2, from Columbia (Col) mRNA (Newman et al., 1994), was a gift from the Arabidopsis Biological Resource Center (Columbus, OH) and was constructed in λZipLox (Gibco BRL). Approximately 105 plaque-forming units were screened, which resulted in one positive clone of 3 kb. The genomic library was constructed with Sau3AI partially digested genomic DNA from Ler in λFIX II (Stratagene, La Jolla, CA; C. Alonso-Blanco and A.J.M. Peeters, unpublished data), according to the manufacturer's protocol. Two positive clones (21 and 33) were isolated, and hybridization and restriction analysis showed that these were partially overlapping. Parts of the cDNA were subcloned into pSPORT1 (Gibco BRL) to enable efficient double-strand sequencing, after Taq cycle PCR with SP6 and T7 primers, on an ABI 373 sequencer (Applied Biosystems, Foster City, CA).

Genomic Sequencing

A mapping kit (Stratagene) was used for mapping genomic clones. A 4.6-kb HindIII fragment and a 2.6-kb SalI fragment of phage 21 and a 2.3-kb EcoRI-NotI fragment and a 3.3-kb SalI fragment of phage 33 were subcloned in pBluescript SK+ (Stratagene). A series of deletion fragments to obtain various sizes of deletion clones was made using an ExoIII exonuclease deletion kit (Takara Shuzo, Kyoto, Japan). A DNA sequencer (model 373; Applied Biosystems) was used for sequencing. All cloning procedures were performed with standard methods (Sambrook et al., 1989).

Genetic Mapping of ANL2

Mapping was conducted in three separate ways. By using phage 33 as a restriction fragment length polymorphism (RFLP) probe, we identified an EcoRI polymorphism between Ler and Col. DNA from 96 Ler/Col recombinant inbred (RI) lines (Lister and Dean, 1993) was isolated, and 400 ng of digested DNA was separated on a 0.7% agarose gel and blotted onto a nylon membrane (Hybond N+; Amersham). By using 32P-labeled phage 33 as an RFLP probe, we were able to score the genotype of each line, and we calculated the map position by using Joinmap (Stam, 1993). DNA was isolated from Ler/Cape Verde Islands (Cvi) RI lines constructed in our laboratory (Alonso-Blanco et al., 1998). The sequences of the primers used to amplify the DNA by PCR were anl3p2′ (5′-TATGTAGCGCGAGTAAGCCA-3′) and anl3p3′ (5′-CCATCTTTGACCACTTCCC-3′). Fifty nanograms of genomic DNA was used as a template. The conditions were 35 cycles of denaturing at 94°C for 30 sec, annealing at 56.5°C for 30 sec, and extension at 72°C for 90 sec, using 1 unit of Taq DNA polymerase (Pharmacia) in a total volume of 50 μL and the GeneAmp PCR System 9600 (Perkin Elmer). The DNA amplified from template DNA of ecotypes Ler and Cvi was digested with several restriction enzymes to identify polymorphic restriction sites. A polymorphism was found for TaqI. The primers were then used to produce a cleaved amplified polymorphic sequence (CAPS) marker on DNA of all the Ler/Cvi RI lines, which after digestion with TaqI and separation on an agarose gel could be scored. Computer analysis using Joinmap calculated the map position.

Cytogenetical methods using fluorescent in situ hybridization on extended DNA fibers (Fransz et al., 1996) confirmed the outcome of the methods described above (P. Fransz, S. Armstrong, J.H. de Jong, C. van Drunen, P. Zabel, C. Dean, and G. Jones, personal communication).

Analysis of Revertants

Revertants were selected from among the progeny of variegated plants obtained by crossing a stable mutant line and an Enhancer (En) transposase-containing line. Two primers were designed to amplify a part of the genomic sequence ranging from exons 4 to 7, including the I element insertion site. The sequences of the primers used to amplify the DNA by PCR were anl3′ (5′-GTAGCGAGCCATCTTTGAG-3′) and anl4′ (5′-AACCGGTTTAGCTACTGAA-3′). Fifty nanograms of genomic DNA was used as a template. The conditions were 35 cycles of denaturing at 94°C for 30 sec, annealing at 54°C for 30 sec, and extension at 72°C for 60 sec, using 1 unit of Taq DNA polymerase (Pharmacia) in a total volume of 50 μL in a GeneAmp PCR System 9600 (Perkin Elmer). This method produced a single band of 1581 to 1586 bp in all the revertant lines. Each PCR product was directly sequenced using primer anl3′ as sequencing primer.

ACKNOWLEDGMENTS

We thank Dr. Isabera Debeaujon for the gift of ban mutant seeds. This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture of Japan (Nos. 07740614 and 09640766) and by the European Union BRIDGE/BIOTECH project (No. BIOT-CT90-0207). Tony van Kampen, Dorien Haarsma, Suzanne Hesemans, Mitsuru Futatsugi, and Hirotaka Hayashi are acknowledged for technical assistance. The cDNA library as well as the expressed sequence tag clones were a gift from the Arabidopsis Biological Resource Center, Columbus, OH.

Footnotes

  • ↵1 These authors contributed equally to this work.

  • Received October 20, 1998.
  • Accepted April 23, 1999.
  • Published July 1, 1999.

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ANTHOCYANINLESS2, a Homeobox Gene Affecting Anthocyanin Distribution and Root Development in Arabidopsis
Hiroyoshi Kubo, Anton J. M. Peeters, Mark G. M. Aarts, Andy Pereira, Maarten Koornneef
The Plant Cell Jul 1999, 11 (7) 1217-1226; DOI: 10.1105/tpc.11.7.1217
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