SYMMETRIC PETALS 1 Encodes an ALOG Domain Protein that Controls Floral Organ Internal Asymmetry in Pea (Pisum sativum L.)

Abstract

In contrast to typical radially symmetrical flowers, zygomorphic flowers, such as those produced by pea (Pisum sativum L.), have bilateral symmetry, manifesting dorsoventral (DV) and organ internal (IN) asymmetry. However, the molecular mechanism controlling IN asymmetry remains largely unclear. Here, we used a comparative mapping approach to clone SYMMETRIC PETALS 1 (SYP1), which encodes a key regulator of floral organ internal asymmetry. Phylogenetic analysis showed that SYP1 is an ortholog of Arabidopsis thaliana LIGHT-DEPENDENT SHORT HYPOCOTYL 3 (LSH3), an ALOG (Arabidopsis LSH1 and Oryza G1) family transcription factor. Genetic analysis and physical interaction assays showed that COCHLEATA (COCH, Arabidopsis BLADE-ON-PETIOLE ortholog), a known regulator of compound leaf and nodule identity in pea, is involved in organ internal asymmetry and interacts with SYP1. COCH and SYP1 had similar expression patterns and COCH and SYP1 target to the nucleus. Furthermore, our results suggested that COCH represses the 26S proteasome-mediated degradation of SYP1 and regulates its abundance. Our study suggested that the COCH-SYP1 module plays a pivotal role in floral organ internal asymmetry development in legumes.

Keywords: ALOG family; COCHLEATA; IN asymmetry; LSH3; Pisum sativum; SYMMETRIC PETALS 1.

Figure 1

Phenotypes of the cochleata (coch) and symmetric petals (syp1) mutants in pea. (A) Petals of the wild type (JI116) and the coch mutant (JI2757) possess dorsal-ventral (DV) differentiation. (B) Petals of the wild type (Terese) and the syp1 mutant (JI2757) possess DV differentiation. (C) Petals of the F₁ plants (coch × Terese, coch × syp1) possess DV differentiation. (D) Petals of the coch syp1 double mutant possess DV differentiation. The red lines indicate the internal (IN) asymmetry and the dotted lines indicate the abolishment of IN asymmetry. The arrows indicate the cutting at the ventral petals so as to flatten the petals. DP, the dorsal petal; LP, the lateral petal; VP, the ventral petal. (A–D) Scale bar = 1 cm.

Figure 2

Molecular characterization of SYP1. (A) Comparative mapping and syntenic analysis of syp1 in pea and Medicago truncatula. The dotted lines indicate the homologous markers in the syntenic region. (B) The gene structure of Psat6g053880. The black boxes represent the exons and the black line represents the intron. (C) The gene expression levels in the different organs of the syp1 mutant and the wild-type plants. (D) PCR amplification of the genomic fragment of Psat6g053880 in Terese, the syp1 mutant, JI992, and JI2822. (E) The lateral petals and ventral petals of the wild-type plants and VIGS-SYP1 silenced plants with strong and weak phenotypes. The red lines indicate the IN asymmetry and the dotted lines indicate the abolishment of IN asymmetry. VA, vegetative apices; RA, reproductive apices RA; FBS, 2-mm floral buds; FBL, 5-mm floral buds; DP, the dorsal petal; LP, the lateral petal; VP, the ventral petal.

Figure 3

Phylogenetic analysis and sequence alignments of ALOG proteins. (A) The neighbor-joining tree of members of the ALOG gene family in pea and Arabidopsis. The bootstrapping value is located in each node as percentages along the branches. Red line indicates the branch including AtLSH3, SYP1 and SYL1. (B) The alignment of SYP1, SYL1, Medtr1g069825, Medtr7g097030, Lj5g3v1083950, Lj1g3v4515410, and AtLSH3 proteins using full-length amino acid sequences. The ALOG domain is indicated by a black line.

Figure 4

Phylogenetic analysis of LSH3 orthologs in legumes. (A) The neighbor-joining tree of AtLSH3 orthologs in legume plants. The bootstrapping value is located in each node as percentages along the branches. Red lines indicate two proteins in pea, SYP1 and SYL1. (B) The lateral petals and ventral petals of the wild type and VIGS-SYP1 and VIGS-SYL1 silenced plants. The red lines indicate the IN asymmetry and the dotted lines indicate the abolishment of IN asymmetry. The arrow indicates where the ventral petal was cut to flatten the petal. DP, dorsal petal; LP, lateral petal; VP, ventral petal.

Figure 5

Spatiotemporal expression pattern and subcellular localization of SYP1. (A,C) SYP1 gene expression was detected in developing flowers. (B,D) COCH gene expression was detected in developing flowers. (E,F) The SYP1 and COCH sense probes were used as the negative controls. No hybridization signal was detected in developing flowers. St, Stamen; Sp, Sepal; Pe, Petal; Ca, Carpel. (A–F) Scale bars = 100 μm. (G–J) Subcellular localization of SYP1 fusion proteins in mung bean protoplasts by YFP or mCherry fluorescence. The fluorescent fusion proteins were expressed in mung bean protoplasts and visualized by confocal microscopy. (G–J) Subcellular localization analysis of SYP1-YFP together with the nuclear marker OsARF4-mCherry. The cells were analyzed for yellow fluorescence emission, mCherry fluorescence emission, and under bright-field illumination 12 h after transformation. Scale bar = 5 μm.

Figure 6

COCH interacted with SYP1. (A) Yeast two-hybrid assays for COCH and SYP1. The bait protein was expressed as a binding domain fusion and the indicated prey proteins were expressed as activating domain fusions in yeast AH109 cells. Transformed yeast was grown on selective media lacking Leu and Trp (-2) or lacking Ade, His, Leu, and Trp (-4) plus X-α-Gal to test protein interaction. 10⁰, 10⁻¹, 10⁻² and 10⁻³ represent dilution series. (B) COCH was co-immunoprecipitated with SYP1. (C) Degradation assay of His-SYP1 performed in a pea cell-free system with or without MG132. (D) Degradation assay of His-SYP1 performed in a pea cell-free system with COCH or GST. (E) Degradation assay of His-SYP1 performed in a pea cell-free system with or without MG132 and different amounts of COCH.