Arabidopsis STERILE APETALA, a multifunctional gene regulating inflorescence, flower, and ovule development

Abstract

A recessive mutation in the Arabidopsis STERILE APETALA (SAP) causes severe aberrations in inflorescence and flower and ovule development. In sap flowers, sepals are carpelloid, petals are short and narrow or absent, and anthers are degenerated. Megasporogenesis, the process of meiotic divisions preceding the female gametophyte formation, is arrested in sap ovules during or just after the first meiotic division. More severe aberrations were observed in double mutants between sap and mutant alleles of the floral homeotic gene APETALA2 (AP2) suggesting that both genes are involved in the initiation of female gametophyte development. Together with the organ identity gene AGAMOUS (AG) SAP is required for the maintenance of floral identity acting in a manner similar to APETALA1. In contrast to the outer two floral organs in sap mutant flowers, normal sepals and petals develop in ag/sap double mutants, indicating that SAP negatively regulates AG expression in the perianth whorls. This supposed cadastral function of SAP is supported by in situ hybridization experiments showing ectopic expression of AG in the sap mutant. We have cloned the SAP gene by transposon tagging and revealed that it encodes a novel protein with sequence motifs, that are also present in plant and animal transcription regulators. Consistent with the mutant phenotype, SAP is expressed in inflorescence and floral meristems, floral organ primordia, and ovules. Taken together, we propose that SAP belongs to a new class of transcription regulators essential for a number of processes in Arabidopsis flower development.

Figure 1

Phenotype of wild-type and sap mutant plants and flowers: (A) phenotype of a wild-type Arabidopsis thaliana plant. (B) Phenotype of a unstable sap mutant plant. Plants are sterile and internodes are shorter than in wild-type plants. Revertant branch can be observed (arrow). (C) Wild-type revertant branch (left) and a branch with a sap mutant phenotype (right). Reversion to wild-type phenotype occurred by excision of the transposable element from the sap::I/dSpm allele. (D) Phenotype of a wild-type flower. (E) Phenotype of a sap early-arising flower. Sepals resemble wild-type sepals. Petals are short and narrow. Stamens are short with aberrant anthers. Petals and stamens are reduced in number. (F) Phenotype of a sap late-arising flower. First-whorl organs are sepals with occasionally stigmatic at the tips. Petals are absent. Stamens and pistil are reminiscent with whorl -3 and -4 organs in early-arising sap flowers. (G) Phenotype of a severely affected late-arising flower. First-whorl organs are carpels with stigmatic papillae (p) and ovules at the inner side. Whorl-2 and -3 organs are absent.

Figure 2

Microscopic analysis of wild-type and sap mutant ovules. (A) UV micrograph showing pollen tube growth through a mature pistil of sap. Pollen tubes are visualized by aniline blue. Regular callose plugs inside the pollen tubes (arrow) and strong staining in ovules (arrowhead) can be seen. (B) UV micrograph showing the penetration of pollen tubes into wild-type ovules. The arrowhead points to a successful penetration into the micropyle of the ovule. (C) UV micrograph of two ovules in a pollinated sap mutant plant. The upper ovule is morphologically normal and is penetrated by a pollen tube (arrow). Wild-type ovules appeared rarely in the sap mutant. They are most likely caused by somatic excision of the transposable element from the SAP gene during early stages of ovule development. The lower ovule has a typical sap phenotype with a highly stained plug of callose in the center of the ovule. An arrowhead indicates this callose deposition. (D) Light micrograph of developing wild-type ovules at stage 9 (Smyth et al. 1990). The megasporocyte is indicated by an arrowhead. (E) Light micrograph of developing wild-type ovule at stage 10 (Smyth et al. 1990). The four megaspores are formed in the nucellus after two cycles of meiotic divisions. Simultaneously, the outer and inner integuments appear. (F) Light micrograph of mature wild-type ovules. Outer and inner integuments surround embryo sacs. (G) Light micrograph of developing sap ovules at stage 9 (Smyth et al. 1990). The megasporocyte is indicated by an arrowhead. (H) Light micrograph of developing sap ovule at stage 10 (Smyth et al. 1990). Megasporogenesis is arrested and only two megaspores can be detected. (I) Light micrograph of a mature sap ovule. Embryo sac is absent, but outer and inner integuments are indistinguishable from wild-type integuments. (J) and (K) UV micrographs of aniline blue stained ovules of wild-type plants (J) and sap mutant plants (K). (J) A tetrad and dyad are visible in the wild-type ovules. Callose deposition is indicated by an arrow. (K) Stage 10 (Smyth et al. 1990) of a sap ovule with an aberrant dyad. Callose accumulates inside the megaspores as indicated by an arrow. (L) and (M) Scanning electron micrographs of stage 10 sap ovules (L) and mature sap ovules (M). The sporophytic tissues develop normally and the integuments enclose the nucellus completely. (o) Ovule; (m) megasporocyte; (ms) megaspores; (oi) outer integument; (ii) inner integument; (f) funiculus; (e) embryo sac; (p) placenta; (mp) micropyle. Bar, 10 μm (D,E,G,H,L); 50 μm (F,I) 100 μm (M).

Figure 3

SAP genomic structure and sequences of SAP cDNA and mutant alleles. (A) Genomic structure of the SAP gene. Solid bars represent exons and the single intron of approximately 3 kb in length is indicated by an interrupted line. The locations of the start (ATG) and stop codon (GTA) are shown. The thin line represents genomic DNA outside the SAP gene. The position of the I/dSpm insertion is indicated by the large open triangle. Primer 4 was used for 5′ RACE. (E) EcoRI; (H) HinfI restriction sites. (B) Nucleotide sequence of SAP cDNA and the deduced amino acid sequence. The position of the intron is shown (▾); the position of the I/dSpm insertion is indicated (). The serine–glysine-rich domain is underlined. (*) The stop codon of the ORF. (C) Footprint alleles generated after excision of the I/dSpm element from the sap:I/dSpm allele. The phenotype is either as wild-type or sap mutant plants. The sequence of the duplications is underlined. In allele SAP-5.510 a duplication of four nucleotides results in the generation of a stopcodon indicated by an asterisks. In allele SAP-4.42 a duplication of two nucleotides (TA) is accompanied by a GC deletion, restoring the ORF.

Figure 3

SAP genomic structure and sequences of SAP cDNA and mutant alleles. (A) Genomic structure of the SAP gene. Solid bars represent exons and the single intron of approximately 3 kb in length is indicated by an interrupted line. The locations of the start (ATG) and stop codon (GTA) are shown. The thin line represents genomic DNA outside the SAP gene. The position of the I/dSpm insertion is indicated by the large open triangle. Primer 4 was used for 5′ RACE. (E) EcoRI; (H) HinfI restriction sites. (B) Nucleotide sequence of SAP cDNA and the deduced amino acid sequence. The position of the intron is shown (▾); the position of the I/dSpm insertion is indicated (). The serine–glysine-rich domain is underlined. (*) The stop codon of the ORF. (C) Footprint alleles generated after excision of the I/dSpm element from the sap:I/dSpm allele. The phenotype is either as wild-type or sap mutant plants. The sequence of the duplications is underlined. In allele SAP-5.510 a duplication of four nucleotides results in the generation of a stopcodon indicated by an asterisks. In allele SAP-4.42 a duplication of two nucleotides (TA) is accompanied by a GC deletion, restoring the ORF.

Figure 4

Expression of SAP mRNA in wild-type Landsberg erecta inflorescence and flowers. Numbers refer to the stage of flower development according to Smyth et al. (1990). Expression of SAP was determined by in situ hybridization with ³⁵S-labeled antisense RNA corresponding to the SAP cDNA. Hybridization signals were viewed using dark-field microscopy. (i) Inflorescence meristem; (b) bract; (p) petal; (a) anther; (s) stigma; (ow) ovary wall; (oi) outer integuments; (ii) inner integument; (f) funiculus; (n) nucellus. (A,B) Longitudinal sections through inflorescence. Strong expression is detected in the inflorescence meristem and at the flanks of the apex, corresponding to the initiation of a floral primordium and young floral buds (stages 1 and 2). At flower stages 3–4 expression is detectable in the central region between the sepal primordia. At stage 6, hybridization signals are visible in the primordia of the petals, the stamens and the central gynoecium but not in the sepals. (C) Cross section through an inflorescence apex. The developing floral buds surround the inflorescence meristem in a spiral pattern. Expression is maintained throughout stages 2–5 and decreases during stage 6, whereas expression is abolished after stage 7. (D) Longitudinal section of a stage-12 flower bud. Strong SAP expression is present in ovules (arrow), but not in other floral organs. (E,F) Cross sections through a pistil of stage 11 imaged under bright field (E) or dark-field (F). Strong hybridization signals are present in the developing outer and inner integuments, and weaker signals are detectable in the nucellus. Bars, 100 μm (A–D); 50 μm (E,F).

Figure 5

Phenotypes of sap/ap2 double mutants. (A) Phenotypes of wild-type (1), ap2-1 (2), and sap (3) mutant flowers. (B) Phenotype of sap/ap2-1 double mutant flower. Sepals are leaf-like as in ap2-1 single mutants and petals are completely absent. The number of stamens is reduced and anthers are malformed or carpelloid. Pistil is smaller than in ap2-1 and sap single mutants. (C) Longitudinal section through mature wild-type ovules. Outer and inner integuments surround the embryo sacs. (D) Longitudinal section through a mature sap/ap2-1 ovule. These ovules exhibit protruding nucellus (n) and the outer integument (oi) does not enclose the ovule completely. Bar, 50 μm. (E) UV micrograph of cleared wild-type ovule at stage 9 (Smyth et al. 1990) stained with aniline blue to visualize callose deposition. A tetrad is visible and the arrows indicate callose deposition. (F) UV micrograph of a cleared sap/ap2-1 ovule at stage 9 (Smyth et al. 1990) stained with aniline blue to visualize callose deposition. Callose accumulates (arrow) in the nucellus before megaspore formation. (G) Phenotype of an early-arising sap flower. (H) Phenotype of an ap2-5 mutant flower. First whorl organs show carpelloid features with occasionally stigmatic tissue (arrow). Petals are reduced in size. Stamens are reduced in number but develop normally. The pistil is not affected. (I) Phenotype of an ap2-6 mutant flower. Whorl-one organs are transformed into carpels, petals are absent and the number of stamens is reduced. (J) Phenotype of an early-arising ap2-5/sap double mutant flower. Four carpels are formed in whorl one, which are partially fused to a gynoecium. Petals and stamens are completely absent. The pistil contains ovules with similar aberrations as is observed in sap/ap2-1 double mutants. (K) Inflorescence of an ap2-5/sap double mutant. (L) Phenotype of a late-arising ap2-5/sap double mutant flower. Four completely fused carpels are formed in whorl one, which covers the central gynoecium. (M) Light micrograph of a cross section through a wild-type flower. The floral whorls are indicated. The central gynoecium is composed of two fused carpels. (N) Light micrograph of a cross section through a sap/ap2-5 flower. The four carpels in whorl 1 are fused to a tube. Ovules in the outer and inner gynoecium are indicated with arrows. Bars, M and N, 100 μm.

Figure 6

Phenotypes of sap/ag double mutants and expression pattern of AG in wild-type and sap inflorescences. (A) Schematic representation of position and identity of the floral organs in wild-type (wt), sap, ag-1, and sap/ag-1 mutant flowers. Organs are marked as follows: sepals (green); petals (blue); degenerated petals in sap (thin blue lines); stamens (yellow), carpels with ovules (black); new floral buds (red). (B) Phenotype an ag-1 mutant flower. (C) Phenotype of an early-arising sap/ag-1 double mutant flower. One sepal was removed to show the inner floral organs. sap/ag-1 flowers are indeterminate with a repeated pattern of sepals-petals-petals from the outermost whorl to the center as in ag-1 single mutants. Compared to wild-type petals, petals of sap/ag-1 are shorter and narrower as in sap. (D–E) Developing sap/ag-1 flowers resembling inflorescences. Secondary flowers, indicated by an arrowhead, develop in axils of the second (p2) and third (p3) whorl petals and all internal floral organs. Internodes are formed between the floral whorls (arrow). (F) Old sap/ag-1 flower producing an inflorescence-like branch. (G–K) Expression of AG was determined by in situ hybridization with ³⁵S-labeled antisense RNA. Hybridization signals were viewed using dark-field microscopy. (i) Inflorescence meristem; (b) bud; (fm) floral meristem. (G) Distribution of AG mRNA in a wild-type inflorescence. Signal appears in the center of buds at early stage 3 and in older buds only in stamens and carpels. (H–I) Longitudinal section through a sap inflorescence imaged under bright field (H) or dark field (I). Strong hybridizing signal is visible in the inflorescence meristem and in the entire bud (b). These buds are composed of only carpelloid sepals and a gynoecium in the fourth whorl. (J–K) Longitudinal section through a sap floral meristem at early stage 3 of flower development imaged under bright field (J) or dark field (K). Strong hybridizing signal is visible in a ring around the floral apex. The first-whorl primordia just emerge at the flanks of the meristem.