Ectopic expression of IiSHP2 from Isatis indigotica Fortune, a PLE-lineage MADS-box gene, influences leaf, floral organ and silique morphology in Arabidopsis thaliana

Abstract

In order to ascertain the regulatory mechanism of fruit development in Isatis indigotica Fortune, the complementary DNA (cDNA) sequence of the SHATTERPROOF 2 (SHP2) orthologous gene was identified by Rapid Amplification of cDNA Ends technology and the corresponding gene was named IiSHP2. The expression pattern of IiSHP2 was determined by quantitative reverse transcription-polymerase chain reaction and wild-type Col-0 Arabidopsis plants were transformed with the IiSHP2 gene using Agrobacterium tumefaciens and the floral-dip method. Expression analyses indicated that IiSHP2 was highly expressed in flowers, silicles and seeds. Compared to wild-type plants, IiSHP2 transgenic lines bolted earlier. Detailed phenotypic observations showed that the size of the rosette and cauline leaves in transgenic lines was reduced and the cauline leaves of the transgenic lines were incurved and displayed a funnel-like shape. During the reproductive growth stage, IiSHP2 transgenic plants produced shortened sepals and the flower buds were not encapsulated completely. Moreover, the petals of the transgenic lines were converted into stamineous tissues, accompanied by exposed stamens, short malformed siliques and wrinkled valves, indicating a severe decline in fertility. These experimental conclusions are valuable as a reference for the breeding of medicinal plants.

Keywords: Floral differentiation; IiSHP2; Isatis indigotica Fortune; MADS-box gene; Silique development.

Fig. 1

Alignment of the amino acid sequences of SHP2 homologous proteins. Green indicates identical amino acids, pink and orange indicate different amino acids, black line shows MADS domain, purple line shows K domain. The AG motif I and AG motif II in the C-terminal region are framed with black boxes. SHP2, Arabidopsis thaliana (NP_850377); EsAGL5, Eutrema salsugineum (XP_024016862.1); RsAGL5, Raphanus sativus (XP_018483390.1); LcSHP2, Lepidium campestre (CBY05405.1) (color figure online)

Fig. 2

Phylogenetic tree of MADS proteins from different plant species. The tree was constructed with neighbor-joining algorithm in MEGA 7. IiSHP2 is framed in red box. SHP1, NP_001190130.1; SHP2, NP_850377.1; CsAGL5, XP_010506124.1; EsAGL5, XP_024016862.1; LcSHP1, CBY05404.1; LcSHP2, CBY05405.1; RsAGL5, XP_018483390.1; BnSHP1, NP_001302702.1; PrtSHP, AEI01160.1; TrSHP, ABB59995.1; PLENA, AAB25101.1; FAR, BAI68392.1; AG, NP_567569.3; TrAG, ABB59994.1; BnAG, XP_013719485.1; EsAG, XP_024005545.1; CbAG, ACD76828.1; CrAG, XP_023633847.1; STK, NP_001078364.1; PeAGL11, XP_011031538.1; RsAGL11, XP_018473770.1; BnAGL11, XP_013732865.1; EsAGL11, XP_006397127.1; FUL, NP_568929.1; TrFUL, ABB59991.1; AP1, NP_177074.1; TrAP1, ABB59990.1

Fig. 3

Expression pattern of IiSHP2 in woad in different tissues (a), different floral organs (b), the silicles at different development stages and different parts of the silicles in stage 3 (c). Error bars represent the standard deviation

Fig. 4

Expression analysis of IiSHP2 in transgenic Arabidopsis by qRT-PCR. Error bars represent the standard deviation. Asterisks indicate a significant difference (P < 0.05) between transgenic lines and wild-type Col-0 plants

Fig. 5

Comparison of phenotypic characteristics and the number of rosette leaves between wild-type Col-0 and IiSHP2 transgenic plants. a The 35S::IiSHP2 plants (shown at right) flowered after producing 5–6 rosette leaves, while the wild-type plant (shown at left) did not flower until the eighth leaf was produced. b The rosette leaves of a wild-type Col-0. c The rosette leaves of an 35S::IiSHP2 plant. Bar = 1 cm

Fig. 6

Statistical analysis of rosette leaf number between 35S::IiSHP2 transgenic plants and wild-type Col-0 plants (n = 10). Error bars represent the standard deviation. Asterisks indicate a significant difference (P < 0.05) between transgenic lines and wild-type Col-0 plants

Fig. 7

Influence of IiSHP2 overexpression on morphological development of flowers. a, b Flower buds and flowers with four normal whorls of floral organs in wild-type Col-0 plants. c–g Flowers with four whorls of floral organs produced by IiSHP2 transgenic Arabidopsis plants. h–m Abnormal inflorescences and floral organs produced by IiSHP2 transgenic Arabidopsis plants. Bar = 1 mm

Fig. 8

Influence of IiSHP2 overexpression on silique and seed development of Arabidopsis. a Siliques of a wild-type Col-0 plant (lower) and an IiSHP2 transgenic plant (upper). b–g Siliques of IiSHP2 transgenic plants, with non-shedding perianth, wrinkled valves or curled shape. Bar = 1 mm

Fig. 9

Statistical analysis of the silique length of wild-type Col-0 and 35S::IiSHP2 transgenic Arabidopsis. Error bars represent the standard deviation. Asterisks indicate a significant difference (P < 0.05) between transgenic lines and wild-type Col-0 plants

Fig. 10

Influence of IiSHP2 overexpression on cauline leaf development of Arabidopsis. a Fully-developed cauline leaf of wild-type Arabidopsis plant. b–d, f Fully-developed cauline leaves in IiSHP2 transgenic Arabidopsis plants. e, g The cauline leaves at an earlier stage of production of lateral branches. h Cauline leaves from wild-type (left) and transgenic (right) plants. In a–g, bar = 1 mm. In H, bar = 1 cm