Characterization and Expression Analysis of PtAGL24, a SHORT VEGETATIVE PHASE/AGAMOUS-LIKE 24 (SVP/AGL24)-Type MADS-Box Gene from Trifoliate Orange (Poncirus trifoliata L. Raf.)

Abstract

The transition from vegetative to reproductive growth in perennial woody plants does not occur until after several years of repeated seasonal changes and alternative growth. To better understand the molecular basis of flowering regulation in citrus, a MADS-box gene was isolated from trifoliate orange (precocious trifoliate orange, Poncirus trifoliata L. Raf.). Sequence alignment and phylogenetic analysis showed that the MADS-box gene is more closely related to the homologs of the AGAMOUS-LIKE 24 (AGL24) lineage than to any of the other MADS-box lineages known from Arabidopsis; it is named PtAGL24. Expression analysis indicated that PtAGL24 was widely expressed in the most organs of trifoliate orange, with the higher expression in mature flowers discovered by real-time PCR. Ectopic expression of PtAGL24 in wild-type Arabidopsis promoted early flowering and caused morphological changes in class I transgenic Arabidopsis. Yeast two-hybrid assay revealed that PtAGL24 interacted with Arabidopsis AtAGL24 and other partners of AtAGL24, suggesting that the abnormal morphology of PtAGL24 overexpression in transgenic Arabidopsis was likely due to the inappropriate interactions between exogenous and endogenous proteins. Also, PtAGL24 interacted with SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (PtSOC1) and APETALA1 (PtAP1) of citrus. These results suggest that PtAGL24 may play an important role in the process of floral transition but may have diverse functions in citrus development.

Keywords: MADS-box; PtAGL24; floral development; flowering; trifoliate orange.

FIGURE 1

Sequence alignment and structure analysis of PtAGL24. (A) Comparison of PtAGL24 protein with related STMADS subfamily proteins from Populus trichocarpa (PtrMADS9: XM_002301057), Petunia hybrida (PhMADS20: GU129907.1), Arabidopsis (AGL24: NM_118587.5; SVP: NM_127820.3), Poncirus trifoliata (PtSVP, FJ373210.1), and Vitis vinifera (VvSVP, XM_002285651.2). Identical amino acids are shaded in black. The heavy black line indicates conserved MADS-box and the dashed line represents K-box. (B) Schematic representation of gene structure of PtAGL24 and its putative homolog in Populus trichocarpa (PtrMADS9), Arabidopsis (AGL24), Brassica napus (BnAGL24), and Solanum tuberosum (SVP/AGL24).

FIGURE 2

Phylogenetic relationship of PtAGL24 and other STMADS proteins from various plants. Bootstrap values in 1000 replicates are shown in percentages at the nodes. St, Solanum tuberosum; At, Arabidopsis thaliana; Bn, Brassica napus; Cc, Carya cathayensis; Pt, Poncirus trifoliata; Rc, Rafflesia cantleyi; Ca, Coffea arabica; Ib, Ipomoea batatas; Ph, Petunia hybrida; Pp, Physalis pubescens; Ws, Withania somnifera; Br, Brassica rapa; Eo, Eucalyptus occidentalis; Pk, Paulownia kawakamii; Am, Antirrhinum majus; Mt, Medicago truncatula; Ac, Actinidia chinensis; Md, Malus domestica; and Ps, Paeonia suffruticosa.

FIGURE 3

Subcellular localization of PtAGL24 protein. (A) Schematic representation of 35S::PtAGL24-GFP fusion construct and 35S::GFP construct; (B) subcellular localization of PtAGL24 protein in onion epidermal cells; the fluorescence signals were examined by a confocal microscopy. Nuclei of the onion cells were stained with DAPI; overlay: merged DAPI and bright-field images (scale bars: 50μm).

FIGURE 4

The expression pattern of the PtAGL24 gene in precocious trifoliate orange. The fruit is whole fruit at 30 days after flowering. (A,B) Spatial expression of PtAGL24 in various tissues and different whorls of mature flower. (C) The expression profile of PtAGL24 at different developmental stages of flower (scale bar: 1 cm). (D) Gene expression pattern of PtSOC1 in different tissues. The expression results were normalized to β-actin. Data represent the mean ± SD of four replicate reactions for the relative expression. (E) Gene expression pattern of PtAP1 in different tissues.

FIGURE 5

Phenotype analysis of PtAGL24 transgenic Arabidopsis. (a) Accelerated flowering of class I 35S::PtAGL24 plants (right) compared with wild-type control (left). (b) The leaf morphologies of 35S::PtAGL24 plants before inflorescences emerged. An inverted triangle indicates the juvenile to adult transition point on the basis of the abaxial trichomes appearance. (c) Wild-type Arabidopsis inflorescence. (d,e) Comparison of flowers (d) and siliques (e) from wild-type (left), 35S::PtAGL24 severe phenotype with conversion of sepals into leaf-like structures (middle) and mild phenotype similar to wild-type (right). Arrows indicate leaf-like sepals. (f) A solitary flower of 35S::PtAGL24 after fertilization. (g) Mature flowers with leaf-like sepals after anthesis in transgenic plants. (h,i) Scanning electron microscopy (SEM) pictures of inflorescence (h) and mature flower (i) of class I 35S::PtAGL24 lines. (j) SEM pictures of 35S::PtAGL24 sepal (right) with enriched trichomes (arrow) compared to wild-type sepal (left). (k–n) SEM analysis of the cell surface morphology in wild-type sepal (k) and carpel (l) and class I 35S::PtAGL24 sepal (m) and carpel (n), respectively. Scale bars: 1 mm (a–g) and 50 μm (h–n).

FIGURE 6

Expression analysis of PtAGL24 and endogenous flowering regulators in wild-type and 35S::PtAGL24 transgenic Arabidopsis. (A) PtAGL24 transcript levels in class I and class II transgenic lines by real-time PCR. (B) Expression patterns of endogenous flowering regulators in wild-type and class I. AP1, LFY, AGL24, SEP3, and TFL1 from Arabidopsis were used in the analysis. The data were normalized against the expression of β-actin. Error bars indicate standard deviation. (C) A schematic representation of the involvement of PtAGL24 in flowering regulation.