FILAMENTOUS FLOWER, a meristem and organ identity gene of Arabidopsis, encodes a protein with a zinc finger and HMG-related domains

Abstract

Distinctive from that of the animal system, the basic plan of the plant body is the continuous formation of a structural unit, composed of a stem with a meristem at the top and lateral organs continuously forming at the meristem. Therefore, mechanisms controlling the formation, maintenance, and development of a meristem will be a key to understanding the body plan of higher plants. Genetic analyses of filamentous flower (fil) mutants have indicated that FIL is required for the maintenance and growth of inflorescence and floral meristems, and of floral organs of Arabidopsis thaliana. FIL encodes a protein carrying a zinc finger and a HMG box-like domain, which is known to work as a transcription regulator. As expected, the FIL protein was shown to have a nuclear location. In situ hybridization clearly demonstrated that FIL is expressed only at the abaxial side of primordia of leaves and floral organs. Transgenic plants, ectopically expressing FIL, formed filament-like leaves with randomly arranged cells at the leaf margin. Our results indicate that cells at the abaxial side of the lateral organs are responsible for the normal development of the organs as well as for maintaining the activity of meristems.

Figure 1

Inflorescence and flowers of Arabidopsis plants. (A) A wild-type flower; (B) the type-A flower has an aberrant number and arrangement of floral organs; (C) type-B structures are thin filaments interpreted as immature flowers that failed to develop floral organs; (D) a wild-type-looking flower of a transgenic plant carrying a TAC clone, TAC27M5; (E) a flower is converted homeotically to an inflorescence in the fil ap1 double mutant; (F) the fil lfy double mutant forms a cluster of filamentous structures and a cluster of filaments with a sepal-like organ but no type A flowers; (G) schematic drawing of inflorescence of the wild type; (H) schematic drawing of inflorescence of fil mutants. Regions 1–3 are clusters of type-A flowers, type-B structures, and a mixture of both, respectively.

Figure 2

Genomic structure of the FIL region at the lower part of chromosome 2. FIL was isolated by map-based cloning and was mapped between markers SBP3 and ML on clone TAC27M5. Other BAC clones overlapping in the region are shown. Numbers below the molecular markers shown in white boxes indicates the recombination frequency between the marker and the FIL locus (no. of recombinant chromosomes/no. of chromosomes examined).

Figure 3

(A) Nucleotide sequence of the FIL cDNA clone and deduced amino acid sequence of the FIL protein (GenBank accession no. AF074948). Numbering was started from the putative translational initiation site. Positions separated by introns are shown by arrowheads. A zinc finger domain (amino acid residue 22–60) and a region homologous to the HMG-box domain (amino acid number 146–179) are underlined. (*) The position of the fil-1 mutation, which makes a shift of one base in the frame of exon 5. (↑ at nucleotide 89) The mutation in the fil-2 mutant causing a change in residue 30 from Cys to Tyr. (B) The frameshift caused by the fil-1 mutation. The G → A base substitution at the splicing acceptor site of the fourth intron makes a new splicing acceptor site 1 base downward. Novel splicing makes a short polypeptide (shown in A). (C) Amino acid sequence of the HMG domain. Partial sequence of the FIL protein is aligned to partial sequences of the rice homolog osFIL1 (GenBank accession no. AF098752), rice homolog osFIL2 (AF098753), human mitochondrial transcription factor 1 (mtTF1, S38413), and mouse testis-specific HMG (tsHMG, P40630). (*) The consensus sequence of the HMG-box domain (Parisi and Clayton 1991) (D) Amino acid sequence of the amino-terminal conserved region in the FIL protein and in two rice homologs and that of a putative zinc finger protein, LSD1 (GenBank accession no. ATU87833). (*) The position of the putative component of the zinc finger motif. In C and D, residues identical or similar to those of the FIL protein are boxed.

Figure 3

(A) Nucleotide sequence of the FIL cDNA clone and deduced amino acid sequence of the FIL protein (GenBank accession no. AF074948). Numbering was started from the putative translational initiation site. Positions separated by introns are shown by arrowheads. A zinc finger domain (amino acid residue 22–60) and a region homologous to the HMG-box domain (amino acid number 146–179) are underlined. (*) The position of the fil-1 mutation, which makes a shift of one base in the frame of exon 5. (↑ at nucleotide 89) The mutation in the fil-2 mutant causing a change in residue 30 from Cys to Tyr. (B) The frameshift caused by the fil-1 mutation. The G → A base substitution at the splicing acceptor site of the fourth intron makes a new splicing acceptor site 1 base downward. Novel splicing makes a short polypeptide (shown in A). (C) Amino acid sequence of the HMG domain. Partial sequence of the FIL protein is aligned to partial sequences of the rice homolog osFIL1 (GenBank accession no. AF098752), rice homolog osFIL2 (AF098753), human mitochondrial transcription factor 1 (mtTF1, S38413), and mouse testis-specific HMG (tsHMG, P40630). (*) The consensus sequence of the HMG-box domain (Parisi and Clayton 1991) (D) Amino acid sequence of the amino-terminal conserved region in the FIL protein and in two rice homologs and that of a putative zinc finger protein, LSD1 (GenBank accession no. ATU87833). (*) The position of the putative component of the zinc finger motif. In C and D, residues identical or similar to those of the FIL protein are boxed.

Figure 3

(A) Nucleotide sequence of the FIL cDNA clone and deduced amino acid sequence of the FIL protein (GenBank accession no. AF074948). Numbering was started from the putative translational initiation site. Positions separated by introns are shown by arrowheads. A zinc finger domain (amino acid residue 22–60) and a region homologous to the HMG-box domain (amino acid number 146–179) are underlined. (*) The position of the fil-1 mutation, which makes a shift of one base in the frame of exon 5. (↑ at nucleotide 89) The mutation in the fil-2 mutant causing a change in residue 30 from Cys to Tyr. (B) The frameshift caused by the fil-1 mutation. The G → A base substitution at the splicing acceptor site of the fourth intron makes a new splicing acceptor site 1 base downward. Novel splicing makes a short polypeptide (shown in A). (C) Amino acid sequence of the HMG domain. Partial sequence of the FIL protein is aligned to partial sequences of the rice homolog osFIL1 (GenBank accession no. AF098752), rice homolog osFIL2 (AF098753), human mitochondrial transcription factor 1 (mtTF1, S38413), and mouse testis-specific HMG (tsHMG, P40630). (*) The consensus sequence of the HMG-box domain (Parisi and Clayton 1991) (D) Amino acid sequence of the amino-terminal conserved region in the FIL protein and in two rice homologs and that of a putative zinc finger protein, LSD1 (GenBank accession no. ATU87833). (*) The position of the putative component of the zinc finger motif. In C and D, residues identical or similar to those of the FIL protein are boxed.

Figure 3

(A) Nucleotide sequence of the FIL cDNA clone and deduced amino acid sequence of the FIL protein (GenBank accession no. AF074948). Numbering was started from the putative translational initiation site. Positions separated by introns are shown by arrowheads. A zinc finger domain (amino acid residue 22–60) and a region homologous to the HMG-box domain (amino acid number 146–179) are underlined. (*) The position of the fil-1 mutation, which makes a shift of one base in the frame of exon 5. (↑ at nucleotide 89) The mutation in the fil-2 mutant causing a change in residue 30 from Cys to Tyr. (B) The frameshift caused by the fil-1 mutation. The G → A base substitution at the splicing acceptor site of the fourth intron makes a new splicing acceptor site 1 base downward. Novel splicing makes a short polypeptide (shown in A). (C) Amino acid sequence of the HMG domain. Partial sequence of the FIL protein is aligned to partial sequences of the rice homolog osFIL1 (GenBank accession no. AF098752), rice homolog osFIL2 (AF098753), human mitochondrial transcription factor 1 (mtTF1, S38413), and mouse testis-specific HMG (tsHMG, P40630). (*) The consensus sequence of the HMG-box domain (Parisi and Clayton 1991) (D) Amino acid sequence of the amino-terminal conserved region in the FIL protein and in two rice homologs and that of a putative zinc finger protein, LSD1 (GenBank accession no. ATU87833). (*) The position of the putative component of the zinc finger motif. In C and D, residues identical or similar to those of the FIL protein are boxed.

Figure 4

Subcellular localization of the FIL protein by biolistic bombardment of onion skin cells with GUS as a vital marker. (A,B) Transiently transformed with 35S promoter::GUS. (C,D) Transiently transformed with 35S promoter::FIL–GUS translational fusion construct. The same cells were stained with 2 mg/ml X-Gluc (A,C) or with 1 μg/ml DAPI (B,D). Scale bar, 100 μm.

Figure 5

In situ localization of FIL mRNA in wild-type Arabidopsis plants. Photographs were taken with double exposures of the dark field with a red filter and the bright field without a color filter. (A) Vegetative meristem. FIL is expressed on the abaxial side of leaves, in young leaf primordia, and in a small region in the peripheral zone of the meristem under a young leaf primordium (arrow). (B) Inflorescence meristem with very young floral meristems. FIL is expressed in a region in the peripheral zone where the floral meristem will be formed (arrow 1) and in a floral meristem of stage 1 (arrow 2). (C) A stage-3 floral meristem. FIL is expressed in sepal primordia and in their base regions in the meristem. (D) A stage-5 floral bud. FIL is expressed on the abaxial side of sepals (arrow 1) and in stamen primordia (arrow 2). (E) A stage-7 floral bud. FIL is strongly expressed on the abaxial side of sepals, young stamens (arrow 1), and young carpels (arrow 2). (F) A stage-10 floral bud. FIL expression continues on the abaxial side of floral organs, including petals (arrows 1). Note that FIL is expressed on the abaxial side of the filaments of stamens (arrow 2).

Figure 6

Morphology of the 35S::FIL transgenic plants. (A) A young wild-type leaf. Immature trichomes are formed on the adaxial surface. Thick and long cells (leaf margin cells) are observed at the margin (arrow). (B) A wild-type leaf. Trichomes are developing. Three files of leaf margin cells (arrow 1) are proliferating from the top to the bottom of the leaves. Arrow 2 indicates the proliferating front of the leaf margin cells. (C) A vegetative shoot apex of a transgenic plant forming filamentous leaves. A cotyledon at the bottom side was removed. (D) Enlarged picture of a filamentous leaf of the transgenic plant. No trichomes are observed. Formation of the leaf margin cells occurs only at the bottom of the leaf (arrow). (E) Enlarged picture of a young wrinkled leaf of a transgenic plant forming the leaf margin cells at random positions (arrowheads). (F) A developed wrinkled leaf of the transgenic plant. Epidermal cells are shown on the adaxial (G) and the abaxial (H) side of a wild-type leaf. (I) Epidermal cells on the adaxial side of the wrinkled leaf of a transgenic plant (F). Arrowheads indicate small patches of cells similar to the cells of the abaxial side. (J) Epidermal cells on the abaxial side of a wrinkled leaf (F). (K) A transverse section of a 35S::FIL filamentous leaf. Scale bars, 50 μm (A,B,D,E); 100 μm (C,G–J).