REBELOTE, SQUINT, and ULTRAPETALA1 function redundantly in the temporal regulation of floral meristem termination in Arabidopsis thaliana

Abstract

In Arabidopsis thaliana, flowers are determinate, showing a fixed number of whorls. Here, we report on three independent genes, a novel gene REBELOTE (RBL; protein of unknown function), SQUINT (SQN; a cyclophilin), and ULTRAPETALA1 (ULT1; a putative transcription factor) that redundantly influence floral meristem (FM) termination. Their mutations, combined with each other or with crabs claw, the genetic background in which they were isolated, trigger a strong FM indeterminacy with reiterations of extra floral whorls in the center of the flower. The range of phenotypes suggests that, in Arabidopsis, FM termination is initiated from stages 3 to 4 onwards and needs to be maintained through stage 6 and beyond, and that RBL, SQN, and ULT1 are required for this continuous regulation. We show that mutant phenotypes result from a decrease of AGAMOUS (AG) expression in an inner 4th whorl subdomain. However, the defect of AG activity alone does not explain all reported phenotypes, and our genetic data suggest that RBL, SQN, and, to a lesser extent, ULT1 also influence SUPERMAN activity. Finally, from all the molecular and genetic data presented, we argue that these genes contribute to the more stable and uniform development of flowers, termed floral developmental homeostasis.

Figure 1.

Phenotypes of the crc-1 rbl-1, crc-1 sqn-4, and crc-1 ult1-4 Double Mutants. (A) crc-1 flower. (B) From the left to the right, siliques from crc-1, crc-1 rbl-1, crc-1 sqn-4, and crc-1 ult1-4 double mutants. Compared with crc-1, the double mutant siliques are shorter and enclose extra floral organs. (C) to (E) Double mutant (crc-1 rbl-1 in [C] and [E] and crc-1 sqn-4 in [D]) siliques at various developmental stages. Reiterations of stamens (ist), carpels (ica), and sometimes petals (ipet, [E]) occur inside the primary carpels (ca). Such indeterminacy might produce a 5- to 15-mm floral axis bearing groups of stamens and carpels, with visible internodes, according to a whorled phyllotaxy (D). (F) crc-1 ult1-4 flower with more petals. (G) Example of a weak indeterminacy phenotype (observed in crc-1 ult1-4) where extra organs do not grow outside primary carpels. A carpel was removed to show internal structures. (H) Close-up view of the base of a young crc-1 rbl-1 double mutant silique (toluidine staining). The floral axis that bears extra stamens (ist) and carpels (ica) displays elongated internodes (ei) and grows from the bottom of the silique between the two carpels (ca); similar pictures were obtained with crc-1 sqn-4 and crc-1 ult1-4. Bars = 500 μm.

Figure 2.

Structures of the RBL, SQN, and ULT1 Genes and Mapping of the Allelic Mutations. Multiple sequence alignment of RBL, At2g18820, yeast, and animal NOC2/UPF0120 domains. (A) Genomic organization of RBL (At3g55510), SQN (At2g15790), and ULT1 (At4g28190). The positions of the mutations are shown below the sequences. Stars indicate amino acid substitutions and triangle-shaped forms indicate T-DNA insertion. RBL is a single-copy gene and is predicted to consist of eight exons and encode a protein of 594 amino acids. rbl-1 corresponds to the G-to-A transition at nucleotide position 22 relative to the translational initiation site that changes an Ala residue to a Thr residue. rbl-2 (FLAG 299B04) corresponds to a T-DNA insertion in the promoter region, 531 bp upstream the start codon (the right border insertion could not be mapped). rbl-3 (SALK 059.267) contains a T-DNA insertion between positions 2109 and 2122 downstream the ATG, disrupting RBL in the 6th exon. SQN is predicted to consist of eight exons and encode a protein of 361 amino acids. sqn-4 corresponds to a G-to-A transition in the 7th exon to the 7th intron splicing site. sqn-5 (SALK 033.511) bears a T-DNA between positions 673 and 687 (15 bp deletion) downstream of the start codon, disrupting SQN in the 5th intron. The ULT1 complete coding region is 714 bp in length and consists of three exons encoding a protein of 237 amino acids. ult1-3 (SALK 0574.642) contains a T-DNA insertion in the first exon, 159 bp after the START codon. ult1-4 contains a T-DNA inserted 95 bp upstream to the translational START site to 25 bp downstream to the translational STOP site. (B) The RBL NOC2/UPF0120 domain sequence (256 to 582 amino acids) is compared with the NOC2/UPF0120 domain sequences of Arabidopsis At2g18820 and of NOC2 proteins from chicken, cow, dog, fruitfly, human, mouse, rat, and yeast. Such a comparison shows that the NOC2/UPF0120 domains observed in RBL and At2g18220 proteins are significantly conserved. The alignment was done using VectorNTI 10.0.1 (Invitrogen). The black letters on a white background represent different residues. The black letters on a gray background represent conserved residues. The white letters on a black background represent identical residues. Arrowheads above RBL in (A) denote the position of the NOC/UPF0120 domain.

Figure 3.

Expression Patterns of the Three Genes and Subcellular Localizations of RBL and SQN. (A) RT-PCR analysis was performed on RNA extracts from wild-type Landsberg erecta (Ler) tissues: roots (Ro), shoot (Sh), young leaves (yL), old leaves (oL), stem (St), inflorescence (Inflo = iSAM + young buds), flower (Fl), and silique (Si). TCTP (translationally controlled tumor protein; At3g16640) was amplified as a control (Szecsi et al., 2006). (B) to (G) RBL expression pattern monitored by in situ hybridization on longitudinal sections of the iSAM (B), flower buds at stages 3 to 4 (C), stage 6 (D), stage 7 (E), stages 8 to 9 (F), and ovules (G). RBL mRNA is detected throughout the iSAM. The level of RBL expression is stronger in the center of flower buds and weaker in differentiating tissues. (H) Control hybridization with RBL sense probe, showing no signal. (I) to (L) Expression pattern of the GUS reporter gene cloned under the control of the RBL promoter. GUS is ubiquitously detected throughout plant development, in plantlets (I), roots (J), inflorescence (K), and flowers (L), with strong expression in the SAM. (M), (N), and (P) RBL subcellular localization, using the Prom35S:RBL-GFP (M) and the PromRBL:RBL-mRFP (N) constructs in Arabidopsis roots (stable transgenics) and the Prom35S:RBL-GFP (P) construct in tobacco leaf cells (transient expression). RBL displays a nuclear localization. (O) GFP expression pattern in tobacco cells infiltrated by the Prom35S:GFP construct as control. (Q) to (S) Expression pattern of GUS cloned under the control of the SQN promoter (intergenic sequence). As above, GUS is ubiquitously detected in plantlets (Q), inflorescence (R), and flowers (S). (T) and (U) SQN subcellular localization using the Prom35S:SQN-GFP (T) construct in Arabidopsis root cells (stable transgenics) and the Prom35S:SQN-GFP (U) construct in tobacco leaf cells (transient expression). SQN is detected in cytoplasm. Bars = 500 μm in (I) to (N) and (Q) to (S) and 50 μm in (B) to (H) and (O), (P), (T), and (U).

Figure 4.

Analysis of WUS and CLV3 Expression Patterns in crc-1 rbl-1, crc-1 sqn-4, and crc-1 ult1-4 Double Mutant Backgrounds and Phenotypes of the wus-1 rbl-1, wus-1 sqn-4, and wus-1 ult1-4 Double Mutants. (A) to (E) WUS expression (monitored by in situ hybridization). (A) Ler flower, stage 6. (B) crc-1 rbl-1 flower, stage 6. (C) crc-1 sqn-4 flower, stage 8. (D) crc-1 ult1-4 flower, stage 8. (E) crc-1 sqn-4 flower, late stage. The black arrowhead points to the meristematic WUS expression domain. While WUS mRNA is not detected in the center of the FM at stage 6 in Ler (A), it remains detectable in crc-1 rbl-1 (B), crc-1 sqn-4 (C), and crc-1 ult1-4 (D), even at very late stages (E). (F) CLV3-GFP expression pattern in crc-1 sqn-4 flower. The GFP expression in the center of the FM shows that CLV3 also remains expressed long after stage 6 in the center of the FM of the double mutant (white arrowhead). This maintained meristem still produces ectopic primordia (epr) that develop into stamens and carpels (ica). Similar results were obtained with crc-1 rbl-1 and crc-1 ult1-4. (G) wus-1 rbl-1 flower, identical to a wus-1 flower (Laux et al., 1996), with four sepals, four petals, one to two stamens, and no carpel. wus-1 sqn-4 flowers display a similar phenotype. (H) wus-1 ult1-4 flower. Similarly to wus-1 and wus-1 rbl-1, wus-1 ult1-4 flowers display one to two stamens and no carpel. However, they develop more sepals (5.09 ± 0.87, n = 45) and petals (5.84 ± 1.3, n = 45) than wus-1, suggesting that ult1-4 influences sepal and petal numbers independently of wus. Bars = 50 μm in (A) to (F) and 500 μm in (G) and (H).

Figure 5.

AG Expression Patterns in Wild-Type and crc-1 sqn-4 and crc-1 ult1-4 Double Mutant Backgrounds. AG expression was monitored by in situ hybridization. (A) to (D) AG expression pattern in Ler flowers at stages 3 (A), 4 (B), and early and late 6 ([C] and [D], respectively). AG expression is uniform throughout the FM center until stage 4 ([A] and [B]) and then throughout the 3rd and 4th whorls ([C] and [D]). (E) to (H) AG expression pattern in crc-1 sqn-4 flowers at stages 3 (E), 5 (F), 6 (G), and 7 (H). (I) to (L) AG expression pattern in crc-1 ult1-4 at stages 3 (I), 4 (J), 6 (K), and 7 (L). In both double mutants, AG expression is lower in the very center of the FM from stage 3. This region with a lower AG expression later defines a 4th whorl subdomain in the intercarpellary space (arrows in [H] and [L]). Bars = 30 μm.

Figure 6.

Phenotypes of the rbl-1, sqn-4, and ult1-4 Single, Double, and Triple Mutants. (A) Ler flower. (B) From the left to the right, siliques from Ler, rbl-1, sqn-4, and ult1-4 single mutants. Compared with the wild type, siliques from the single mutants display extra carpels that can develop in the upper half of the gynoecium. (C) ult1-4 flower, with extra petals. (D) From the left to the right, siliques from Ler, rbl-1 sqn-4, rbl-1 ult1-4, and sqn-4 ult1-4 double mutants. Compared with Ler, siliques from the double mutants are much shorter, develop extra carpels, and are borne by a gynophore. (E) Dissection of rbl-1 sqn-4 silique. Stamens and carpels are reiterated inside the primary carpels (similar results were obtained with rbl-1 ult1-4 and sqn-4 ult1-4). (F) to (L) Flowers of the rbl-1 sqn-4 ult1-4 triple mutant. (F) to (H) Weak phenotype. The flowers develop extra stamens and fused carpels that enclose extra floral organs. Chimerical petaloid stamens regularly develop in place of stamens in flowers of the rbl-1 sqn-4 ult1-4 triple mutant ([G] and [H]). (I) and (J) Intermediate phenotypes. The flowers reiterate whorls of stamens that eventually become carpelloid: they bear ovules (arrowhead) along the connective and stigmatic papillae at their top (J). (K) and (L) Strong phenotype. After the two first whorls, the flowers become totally indeterminate, with an indeterminate meristem (arrows) that produces only stamens following a spiraled phyllotaxy. (J) and (L) are scanning electron microscopy close-up views of (I) and (K), respectively. Bars = 500 μm, except in (J) and (L), were bars = 80 μm.

Figure 7.

Phenotypes of ag-4 and ag-6 Single Mutants and of ag rbl-1, ag sqn-4, and ag ult1-4 Double Mutants. (A) ag-4 (weak allele) flower. (B) ag-4 rbl-1 flower. Successive whorls of petals (with few remaining stamens) alternate with whorls of sepals. This flower resembles ag-6 flowers. (C) ag-4 sqn-4 flower. This flower develops more organs than ag-4 but does not show homeotic transformations. (D) ag-4 ult1-4 flower. This flower resembles ag-4 flowers but also displays some partial stamen-to-petal transformations. (E) ag-6 (strong allele) flower. (F) ag-6 rbl-1 flower (we observed a similar phenotype with ag-6 sqn-4). Successive whorls of petals, without obvious alternating sepals, make ag-6 rbl-1 flower different from ag-6 flowers but similar to ag-6 sup-1 flower. (G) Second ring sepals from ag-6 rbl-1 (right) and ag-6 flowers (left). The ag-6 rbl-1 ones are petaloid compared with the ag-6 ones. (H) ag-6 sup-1 flower. Such a flower produces only petals, after a single whorl of sepals. (I) From the left to the right, siliques from Ler, crc-1 rbl-1, and ag-4/+ rbl-1 double mutants (similar phenotype was observed with ag-4/+ sqn-4 and ag-4/+ ult1-4). ag-4/+ rbl-1 siliques are shorter than Ler siliques and are borne by gynophores. (J) Dissection of an ag-4/+ rbl-1 silique. Extra floral organs develop inside primary carpels. For (I) and (J), crosses with ag-4 and ag-6 give similar results. Bars = 500 μm.

Figure 8.

Phenotypes of sup-1 rbl-1, sup-1 sqn-4, sup-1 ult1-4, and sup-1 ag-4 Double Mutants. Analysis of WUS expression patterns in sup-1. (A) sup-1 flower. (B) and (C) WUS expression (monitored by in situ hybridization). While WUS mRNA is not detected in Ler FM at stage 6 (B), it remains detectable in sup-1 (C) at a similar or slightly older stage, when two consecutive rows of stamen (str1 and str2) are produced in the FM center. (D) sup-1 rbl-1 double mutant flower. The flower does not develop carpels but only stamens. (E) Scanning electron microscopy magnification of the center of a sup-1 rbl-1 double mutant flower. Stamens are produced by an everlasting meristem following a spiraled phyllotaxy. Numbers correspond to the successive primordia. (F) and (G) Young and old flower, respectively, of a sup-1 sqn-4 double mutant. Flowers produce a very high number of stamens, but end with unfused chimerical stamen-carpel organs. (H) sup-1 ult1-4 double mutant flower. The number of stamens produced is lower than in sup-1 rbl-1 and sup-1 sqn-4 and flower ends with unfused chimerical stamen-carpel organs. (I) sup-1 ag-4 double mutant flower. The flower does not develop carpels but only stamens following a spiraled phyllotaxy, thus mimicking rbl-1 sup-1. Bars = 500 μm in (A), (D), and (F) to (H), 200 μm in (I), and 50 μm in (B), (C), and (E).

Figure 9.

Phenotypes of Triple Mutants Combining crc-1 rbl-1, crc-1 sqn-4, or crc-1 ult1-4 and ap3-3 or pi-1 Mutations. (A) ap3-3 flower. (B) pi-1 flower. (C) and (D) Flowers from crc-1 rbl-1 pi-1 and crc-1 ult1-4 ap3-3 triple mutants, respectively. The triple mutant flowers are still strongly indeterminate and reiterate a high number of carpels. Bars = 500 μm.

Figure 10.

Model for RBL, SQN, and ULT1 Action in the Spatial and Temporal Control of FM Determinacy. (A) Sketch summarizing the phenotypes of flowers in different mutant backgrounds. Genotypes are indicated under the schemes. Class I groups together mutants that exhibit reiterations of stamens and carpels inside the primary carpels. Such FM indeterminacy is mainly due to a recurrent reset of the FM identity and results from events at stage 6 and beyond. Class II groups together mutants that exhibit an increase of primary stamen number. These mutants display a strong expansion of whorl 3, which, in the most extreme cases, gives rise to a spiral of stamens. Such a phenotype suggests that Class II type of indeterminacy results from earlier events, starting at stages 3 to 4. (B) and (C) Stage 4 and 6 situations, respectively. AG expression domain is colored in green, with the inner subdomain, where AG is underexpressed in crc-1 sqn-4 and crc-1 ult1-4 backgrounds (Figure 5), in light green. (B) At stage 4, we propose that AG and SUP both promote FM termination by repressing WUS expression (probably indirectly for SUP, by preventing B class gene expression in the center of the FM). This process corresponds to the early events of FM termination. We also propose that mutations in RBL, SQN, and ULT1 decrease both AG and SUP activities in the FM center and thus result in Class II phenotypes. (C) At stage 6, AG promotes FM termination by further repressing WUS expression. CRC also contributes (directly or indirectly) to FM termination, very likely, in an AG-independent manner. These events correspond to the late events of FM termination. We propose that AG activity therefore needs to be maintained in the inner 4th whorl subdomain for a persistent repression of the FM indeterminacy potential and that RBL, SQN, and ULT1 appear to be required in this process.