INNER NO OUTER regulates abaxial- adaxial patterning in Arabidopsis ovules

Abstract

The Arabidopsis INNER NO OUTER (INO) gene is essential for formation and asymmetric growth of the ovule outer integument. INO encodes a member of the newly described YABBY family of putative transcription factors that contain apparent Cys(2)-Cys(2) zinc-finger domains and regions of similarity to the high mobility group (HMG) transcription factors. In wild-type plants, INO is expressed specifically on one side of the central region of each ovule primordium in the cells that give rise to the outer integument. Alterations in the INO expression pattern in mutant backgrounds implicate INO as a positive regulator of its own expression, and ANT, HLL, BEL1, and SUP as direct or indirect negative regulators that help to establish the spatial pattern of INO expression. We hypothesize that INO is necessary for polarity determination in the central part of the ovule. Maintenance of polarity in other parts of ino ovules indicates the existence of additional regulators and provides further evidence that the ovule is a compound structure.

Figure 1

Scanning electron micrographs of wild-type and ino ovules. In all panels, the base of the ovary (and therefore, the abaxial side of the ovules) is at left. Bars, 10 μm. (A) Wild-type ovules at stage 2-III (stages according to Schneitz et al. 1995). The nucellus, funiculus, and inner and outer integument primordia are visible. The outer integument initiates on the abaxial side of the wild-type ovule. (B) Wild-type ovules at stage 3-I. The outer integument encloses most of the inner integument and nucellus. (C) Wild-type ovules at anthesis (stage 4-I). The outer integument completely covers the inner integument and nucellus. (D) ino-1 ovules at stage 2-III. The outer integument on the abaxial side of the ovule is absent. A protuberance (arrowheads) forms on the adaxial side of each ino-1 ovule. (E) ino-1 ovules at stage 3-I. (F) ino-1 ovules at stage 4-I. The inner integument completely covers the nucellus and the adaxial protuberance (arrowheads) is enlarged. (G) ino-4 ovules at stage 2-III. Initiation of the outer integuments on the abaxial sides is apparent (arrows). (H) ino-4 ovules at stage 3-I. The outer integuments are reduced relative to wild type. The adaxial protuberance is visible on some ovules (arrowhead). (I,J) ino-4 ovules at stage 4-I. The outer integuments are reduced relative to wild type. The adaxial protuberance (arrowheads) protrudes between the inner and outer integuments. (f) funiculus; (ii) inner integument (primordium); (n) nucellus; (oi) outer integument (primordium).

Figure 2

INO DNA and protein sequences. (A) INO cDNA and predicted protein sequence (Co-3 ecotype). Coding region of cDNA is shown in uppercase and 5′- and 3′-untranslated regions are in lowercase. Nucleotide differences in the coding region of the Ler ecotype are shown above the cDNA sequence. Single and double underlines indicate the putative zinc finger and YABBY domains, respectively. Conserved cysteine residues in the putative zinc finger region are in boldface type. (▿) Intron locations, and introns containing the ino-1, ino-2, and ino-4 mutations are indicated. (Asterisks) Stop codons. (B) Mutations lead to altered splicing. Wild-type genomic sequences of introns 5 and 4, with some flanking exon sequence, are illustrated at top of each panel. Wild-type exons and the amino acids they encode are shown in boldface and donor (GT) and acceptor (AG) intron splice sites are overlined. Vertical gray bars indicate G-to-A transitions identified in the mutant genomic sequences. Mutant cDNA sequences are illustrated below each genomic sequence. Mutant donor and acceptor sites are indicated by black underlines. Alterations in the protein sequence resulting from aberrant splicing in the mutants are indicated by gray underlines. (C) Alignment of conserved putative zinc finger (top) and YABBY (bottom) domains. Black boxes indicate identical and gray boxes indicate conserved residues between INO and other proteins. Broken lines indicate gaps in the sequences. The overline indicates predicted α-helical domains in INO (by the method of Chou et al. 1978); the underline indicates α helices in SRY (Werner et al. 1995).

Figure 2

INO DNA and protein sequences. (A) INO cDNA and predicted protein sequence (Co-3 ecotype). Coding region of cDNA is shown in uppercase and 5′- and 3′-untranslated regions are in lowercase. Nucleotide differences in the coding region of the Ler ecotype are shown above the cDNA sequence. Single and double underlines indicate the putative zinc finger and YABBY domains, respectively. Conserved cysteine residues in the putative zinc finger region are in boldface type. (▿) Intron locations, and introns containing the ino-1, ino-2, and ino-4 mutations are indicated. (Asterisks) Stop codons. (B) Mutations lead to altered splicing. Wild-type genomic sequences of introns 5 and 4, with some flanking exon sequence, are illustrated at top of each panel. Wild-type exons and the amino acids they encode are shown in boldface and donor (GT) and acceptor (AG) intron splice sites are overlined. Vertical gray bars indicate G-to-A transitions identified in the mutant genomic sequences. Mutant cDNA sequences are illustrated below each genomic sequence. Mutant donor and acceptor sites are indicated by black underlines. Alterations in the protein sequence resulting from aberrant splicing in the mutants are indicated by gray underlines. (C) Alignment of conserved putative zinc finger (top) and YABBY (bottom) domains. Black boxes indicate identical and gray boxes indicate conserved residues between INO and other proteins. Broken lines indicate gaps in the sequences. The overline indicates predicted α-helical domains in INO (by the method of Chou et al. 1978); the underline indicates α helices in SRY (Werner et al. 1995).

Figure 3

Histochemical detection of in situ hybridization with an INO antisense RNA probe in wild-type ovules. In all panels, the base of the carpel and the abaxial side of the ovules, is at left. Bars, 10 μm. (A,B) Stage 2-I ovule primordia (before emergence of integuments). (A) Longitudinal sections show INO mRNA at the site of origination of the outer integument on the abaxial sides of three ovule primordia (two pointing upward and one pointing downward). (B) Cross sections show that INO mRNA is confined to a semicircular collar of cells corresponding to the site of initiation of the outer integument. (C) Longitudinal section of ovules at stage 2-II. INO mRNA is in the abaxial layers of only the outer integument primordium. (f) Funiculus; (ii) inner integument primordium; (n) nucellus; (oi) outer integument primordium.

Figure 4

Distribution of INO mRNA in mutant ovules. Scanning electron micrographs (A,C,E,G,I) and in situ hybridizations with an INO antisense RNA probe (B,D,F,H,J) of mutant ovules. In all panels, the base of the carpel and the adaxial side of the ovules is at left. Bars, 10 μm. (A,B) Stage 4-I hll-1 ovules. INO mRNA is detected in the integumentary ridge and nucellus but not in the funiculus. Note that the ridge is not evident in all ovules. (C,D) Stage 4-I ant-4 ovules. INO mRNA is detected throughout the integumentary ridge. (E,F) Stage 4-1 bel1-1 ovules. INO mRNA is detected uniformly throughout the integument-like structure of bel1 ovules. (G,H) Stage 4-I sup-5 ovules. INO mRNA is detected on both sides of the base of the outer integument and in the funiculus. (I) Stage 4-I and (J) stage 2-III ats ovules. ats ovules have only a single integument (Leon-Kloosterziel et al. 1994). During early development of this structure, ino mRNA was detected only in the basal half of the abaxial side of the single integument primordium. (ir) Integumentary ridge; (n) nucellus; (f) funiculus; (il) integument-like structure; (i) single integument; (oi) outer integument.