SEPALLATA--like genes of Isatis indigotica can affect the architecture of the inflorescences and the development of the floral organs

Abstract

Background: The architecture of inflorescence and the development of floral organs can influence the yield of seeds and have a significant impact on plant propagation. E-class floral homeotic MADS-box genes exhibit important roles in regulation of floral transition and differentiation of floral organs. Woad (Isatis indigotica) possesses unique inflorescence, floral organs and fruit. However, very little research has been carried out to determine the function of MADS-box genes in this medicinal cruciferous plant species.

Results: SEPALLATA orthologs in I. indigotica were cloned by degenerate PCR. The sequence possessing the highest identity with SEP2 and SEP4 of Arabidopsis were named as IiSEP2 and IiSEP4, respectively. Constitutive expression of IiSEP2 in Columbia (Col-0) ecotype of Arabidopsis led to early flowering, and the number of the flowers and the lateral branches was reduced, indicating an alteration in architecture of the inflorescences. Moreover, the number of the floral organs was declined, the sepals were turned into carpelloid tissues bearing stigmatic papillae and ovules, and secondary flower could be produced in apetalous terminal flowers. In 35S::IiSEP4-GFP transgenic Arabidopsis plants in Landsberg erecta (Ler) genetic background, the number of the floral organs was decreased, sepals were converted into curly carpelloid structures, accompanied by generation of ovules. Simultaneously, the size of petals, stamens and siliques was diminished. In 35S::IiSEP4-GFP transgenic plants of apetalous ap1 cal double mutant in Ler genetic background, the cauliflower phenotype was attenuated significantly, and the petal formation could be rescued. Occasionally, chimeric organs composed of petaloid and sepaloid tissues, or petaloid and stamineous tissues, were produced in IiSEP4 transgenic plants of apl cal double mutant. It suggested that overexpression of IiSEP4 could restore the capacity in petal differentiation. Silencing of IiSEP4 by Virus-Induced Gene Silencing (VIGS) can delay the flowering time, and reduce the number and size of the floral organs in woad flowers.

Conclusion: All the results showed that SEPALLATA-like genes could influence the architecture of the inflorescence and the determinacy of the floral meristems, and was also related to development of the floral organs.

Keywords: Arabidopsis thaliana; Floral homeotic conversion; Floral meristem determinacy; Floral organ differentiation; Floral transition; Inflorescence architecture; Isatis indigotica Fortune; Overexpression; Petal rescue; SEPALLATA-like genes.

Figure 1. Expression pattern of IiSEP2 in I. indigotica analyzed by qRT-PCR.

Error bars represent the standard deviation. Root was used as normalization sample. Multiple testing correction was conducted with SPSS software, different letters indicate significant difference at 5% level.

Figure 2. Effects of IiSEP2 overexpression on aerial architecture of Arabidopsis plants.

(A) Comparison of rosette leaf number produced by 35S::IiSEP2 transgenic and wild-type Col-0 plants before flowering. (B) Comparison of the angle between the cauline leaves and the stems. (C) Comparison of the flower bud number produced by 35S::IiSEP2 transgenic and wild-type Col-0 plants. Values correspond to mean ± standard error (n = 15). Multiple testing correction was conducted with SPSS software; different letters indicate significant difference at 5% level.

Figure 3. Morphological comparison of the cauline leaves between wild-type Col-0 and IiSEP2 transgenic plants.

(A) The rosette leaves and cauline leaves of wild-type Col-0 plants, the angles between the oval-shaped cauline leaves and the stems are larger. (B) The upper part of a wild-type Col-0 plant in the flowering stage. (C) IiSEP2 transgenic Arabidopsis plants. (D) IiSEP2-OE-6#. (E) IiSEP2-OE-7#. (F) IiSEP2-OE-8#. (G) IiSEP2-OE-9#. IiSEP2 transgenic Arabidopsis plants in (F) and (G) show the inward-curling cauline leaves, and the angles between the cauline leaves and the stems are reduced. In (A–C), scale bars represent 1 cm. In (D–G), scale bars represent 1 mm.

Figure 4. Influence of IiSEP2 on development of inflorescences.

(A & B) Indefinite inflorescences of wild-type Col-0 plants. The inflorescence meristem could produce new flower buds continuously. (C) Inflorescence of IiSEP2 overexpressing Arabidopsis plant, only a few flower buds could be observed. (D) Wilted inflorescence with undersized flower buds at early development stage in IiSEP2 transgenic Arabidopsis plant. (E & F) The inflorescences converted into defective terminal flowers in IiSEP2 transgenic Arabidopsis plant, arrow indicate stamens. (G) Comparison of the number of stamens per flower. Scale bars represent 1 mm. Multiple testing correction was conducted with SPSS software, different letters indicate significant difference at 5% level.

Figure 5. Abnormal phenotypes of various floral organs induced by IiSEP2 overexpression.

(A–C) The flowers of wild-type Col-0 Arabidopsis plants. The sepals and petals have been removed from Arabidopsis flower in (C). (D) IiSEP2 transgenic line 3, red arrow indicate terminal flower containing a secondary flower, blue arrow indicate lateral flower. (E) IiSEP2 transgenic line 7, the sepals were converted into carpelloid tissues. (F) IiSEP2 transgenic line 4, red arrow indicate terminal flower containing a secondary flower. (G) IiSEP2 transgenic line 5, five petals could be observed. (H) IiSEP2 transgenic line 6, apetalous flower was generated. (I) Terminal flowers and secondary flower in crossing progeny of IiSEP2 transgenic line 8 and wild-type Col-0. (J) IiSEP2 transgenic line 8, abnormal flowers were produced. (K) Flower lacking petal in crossing progeny of IiSEP2 transgenic line 8 and wild-type Col-0 plant. (L) Abnormal floral organs in crossing progeny of IiSEP2 transgenic line 8 and wild-type Col-0 plant. Scale bars represent 1 mm.

Figure 6. Phenotypic variations of the floral organs in IiSEP4 transgenic Arabidopsis (in Ler genetic background).

(A) The flower of the wild-type Ler plant. (B–E) The abnormal phenotype of the flowers in 35S::IiSEP4-GFP transgenic plants. (F) The silique in 35S::IiSEP4-GFP transgenic plant (left) and wild-type Ler plant (right). Bar = 1 mm.

Figure 7. Phenotypic variations of IiSEP4 transgenic plants of ap1 cal double mutant in Ler genetic background.

(A & B) The inflorescence of ap1 cal double mutant. (C–H) The inflorescence of IiSEP4 transgenic plants of ap1 cal double mutant. (C) shows an incomplete cauliflower phenotype and petals reappeared. (D) is an enlarged photo of (C), a number of sepals, stamens and pistils can be found in one flower; In comparision with ap1 cal double mutant, multiple petals can be produced. (E) is the photo of the flower in (D) taken in different direction, arrow shows a chimeric organ composed of petaloid and stamineous tissues, representing fusion of petal and stamen. In (F and G), arrows show the chimeric organ composed of petaloid and sepaloid tissues, representing fusion of petal and sepal. (G and H) are the photos of the flower in (F) taken in different directions. Bar = 1 mm.

Figure 8. Phenotypic observation of the floral organs in woad plants treated with TRV-IiSEP4.

(A) Wild-type woad flower. (B–L) Phenotypic variations of the flowers in woad plants infiltrated with TRV-IiSEP4. (B) a flower constituted by one sepal, one petal, one stamen and one pistil. (C and D) anatomy view of the flower in (B). (E) a flower with three sepals, three petals, three stamens and one pistil. (F–H) comparison of the size of the floral organs in the flower of (E) (up) with the floral organs in wild-type flower (down). (I) a flower contains four sepals, five petals, five stamens and one pistil. (J–L) comparison of the size of the floral organs in the flower of (I) (up) with the floral organs in wild-type flower (down). Bar = 1 mm.