IiSVP of Isatis indigotica can reduce the size and repress the development of floral organs.

KEY MESSAGE: IiSVP of Isatis indigotica was cloned and its expression pattern was analyzed. Ectopic expression of IiSVP in Arabidopsis could delay the flowering time and reduce the size of the floral organs. SVP (SHORT VEGETATIVE PHASE) can negatively regulate the flowering time of Arabidopsis. In the present work, the cDNA of IiSVP, an orthologous gene of AtSVP in I. indigotica, was cloned. IiSVP was highly expressed in rosette leaves, inflorescences and petals, but weakly expressed in sepals, pistils and young silicles. The results of subcellular localization showed that IiSVP was localized in nucleus. Bioinformatics analysis indicated that this protein was a MADS-box transcription factor. Constitutive expression of IiSVP in Arabidopsis thaliana resulted in decrease of the number of petals and stamens, and curly sepals were formed. In IiSVP transgenic Arabidopsis plants, obvious phenotypic variations in flowers could be observed, especially the size of the floral organs. In comparison with the wild-type plants, the size of petals, stamens and pistil in IiSVP transgenic Arabidopsis plants was decreased significantly. In some transgenic plants, the petals were wrapped by the sepals. Yeast two-hybrid experiments showed that IiSVP could form higher-order complexes with other MADS proteins, including IiSEP1, IiSEP3, IiAP1 and IiSEP4, but could not interact with IiSEP2. In this work, it was proved that the flowering process and the floral development in Arabidopsis could be affected by IiSVP from I. indigotica Fortune.

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

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.

Cloning of a SEPALLATA4-like gene (IiSEP4) in Isatis indigotica Fortune and characterization of its function in Arabidopsis thaliana.

E-class MADS-box genes, SEPALLATA (SEP), participate in various aspects of plant development together with B-, C- and D-class MADS-box genes. IiSEP4, a homologous gene of SEP4, was cloned from Isatis indigotica. IiSEP4 was highly expressed in sepals, and its mRNA was mildly detected in leaves, inflorescences, flowers, stamens and young silicles. Constitutive expression of IiSEP4 in Arabidopsis thaliana caused early flowering, accompanied by the reduction of flowers and floral organs. Moreover, the sepals in some flowers were transformed into carpelloid structures with stigmatic papillae, and obviously accompanied by ovule formation. Yeast two-hybrid assays demonstrated that IiSEP4 interacts with other woad MADS proteins to determine the identity of floral organs. These findings reveal the important roles of IiSEP4 in floral development of I. indigotica. The results of this study can lay a foundation for further study on biological functions of MADS transcriptional factors in I. indigotica.

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

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.

A SEPALLATA1-like gene of Isatis indigotica Fort. regulates flowering time and specifies floral organs.

An orthologous gene of SEPALLATA1, designated as IiSEP1, was isolated from Isatis indigotica. The genomic DNA of IiSEP1 is 3.1 Kb in length. The full-length cDNA of IiSEP1 is 1481 bp and contains a 756 bp ORF encoding a 251-amino-acid protein. Sequence comparison revealed that IiSEP1 belonged to the MADS-box gene family. IiSEP1 contains 7 exons and 6 introns, showing similar exon-intron structure with Arabidopsis SEP1. Phylogenetic analysis suggested that IiSEP1 belonged to AGL2/SEP subfamily and was likely to be an I. indigotica ortholog of Arabidopsis SEP1. Quantitative real-time PCR showed that IiSEP1 was predominantly expressed in the reproductive organs. Ectopic expression of IiSEP1 in Arabidopsis resulted in early flowering, accompanied with the reduction of inflorescence number and the production of terminal flower on the top of the main stems. Moreover, IiSEP1 overexpressing flowers generated numerous variations in phenotype. The sepals were changed into petal-sepal mosaic structures or displayed carpelloid features, and transparent ovules were formed in internal surface of these sepals. In addition, some flowers were constituted by sepals and pistil, but lacked petals and stamens. Taken together, IiSEP1 might play important roles in reproductive growth of I. indigotica and could affect the morphogenesis of flowers and fruits.

Constitutive expression of NtabSPL6-1 in tobacco and Arabidopsis could change the structure of leaves and promote the development of trichomes.

The coding sequence of NtabSPL6-1 was cloned by high-fidelity PCR with specific primers and was used in construction of a binary vector for overexpression. Wild-type Col-0 Arabidopsis plants and Qinyan95 tobacco leaves were transformed using floral dip and leaf disc methods, respectively. Phenotypic observation showed that constitutive expression of NtabSPL6-1 in Arabidopsis could promote the development of trichomes on leaf epidermis and influence the growth pattern of cauline leaves. In tobacco, ectopic expression of NtabSPL6-1 led to dwarfism of the plants and alteration of the leaf structure, accompanied by changes of the glandular trichomes in development. At the same time, the self-regulation capability of NtabSPL6-1 was determined by yeast two-hybrid system. The results indicated that SBP-C terminal domain and C terminal domain of NtabSPL6-1 possessed strong transcriptional activation ability; the intact protein, N terminal domain, and the first peptide fragment in N terminal domain possessed weak transcriptional activation ability; and the second and the third peptide fragments in N terminal domain had no transcriptional activation ability, suggesting the N terminal domain of NtabSPL6-1 could block the activity of the C terminal domain. NtabSPL6-1 may affect the resistance of plants to biotic stress factors indirectly by regulation of the trichome growth.