Functional identification of the different regions in B-class floral homeotic MADS-box proteins IiAP3 and IiPI from Isatis indigotica.

APETALA3 (AP3) and PISTILLATA (PI) are B-class MADS-box floral homeotic genes of Arabidopsis and are involved in specifying the identity of petals and stamens. In the present work, IiAP3 and IiPI, the respective orthologous genes of AP3 and PI, were cloned from Isatis indigotica. By expressing in ap3-6 and pi-1 homozygous mutant and in wild-type Arabidopsis under the control of AP3 promoter or CaMV 35S promoter, we demonstrated that IiAP3 and IiPI were functionally equivalent to AP3 and PI of Arabidopsis. Referring to previous reports and the research results in the present work, expression patterns of AP3 and PI homologs are not the same in different angiosperms possessing diverse floral structures. It suggests that the alterations in expression may contribute to the changing morphology of flowers. To further determine the relationship between IiAP3 and IiPI, the coding sequences of the different structural regions in these two proteins were swapped with each other, and the data collected from transgenic Arabidopsis plants of the chimeric constructs suggested that MADS domain was irreplaceable for the function of IiAP3, K domain of IiAP3 was involved in specifying the identity of stamens, K domain of IiPI was mainly related to the formation of petals, and C-terminal region of IiPI was involved in characterization of stamens. In addition, a complete KC region of these two proteins was more effective in phenotypic complementation of the mutants.

IiAGL6 participates in the regulation of stamen development and pollen formation in Isatis indigotica.

The AGL6 (AGMOUSE LIKE 6) gene is a member of the SEP subfamily and functions as an E-class floral homeotic gene in the development of floral organs. In this study, we cloned IiAGL6, the orthologous gene of AGL6 in Isatis indigotica. The constitutive expression of IiAGL6 in Arabidopsis thaliana resulted in a late-flowering phenotype and the development of curly leaves during the vegetative growth period. Abnormal changes in floral organ development were observed during the reproductive stage. In woad plants, suppression of IiAGL6 using TRV-VIGS (tobacco rattle virus-mediated virus-induced gene silencing) decreased the number of stamens and led to the formation of aberrant anthers. Similar changes in stamen development were also observed in miRNA-AGL6 transgenic Arabidopsis plants. Yeast two-hybrid and BiFC tests showed that IiAGL6 can interact with other MADS-box proteins in woad; thus, playing a key role in defining the identities of floral organs, particularly during stamen formation. These findings might provide novel insights and help investigate the biological roles of MADS transcription factors in I. indigotica.

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.

Tobacco NtabSPL6-2 can enhance local and systemic resistances of Arabidopsis thaliana to bacterial and fungal pathogens.

NtabSPL6-2 of Nicotiana tabacum was introduced into Arabidopsis by Agrobacterium-mediated floral-dip method. Compared to wild-type Col-0 plants, the arrangement of cauline leaves in NtabSPL6-2 transgenic plants was converted into opposite from simple and alternate, and the margin of rosette leaves was serrated. NtabSPL6-2 transgenic plants possessed a significantly greater fresh weight. Subcellular localization by fusion with GFP confirmed that the encoded product of NtabSPL6-2 existed in the nucleus. The leaves of NtabSPL6-2 transgenic plants exhibited an enhanced capacity to restrain the bacterial reproduction after infection by Pseudomonas syringae, accompanied by higher expression of the pathogenesis-related gene PR1 in the infiltrated leaves, indicating NtabSPL6-2 could improve the defense response of Arabidopsis to P. syringae at the local sites. Similarly, it was confirmed that NtabSPL6-2 could enhance the systemic acquired resistance of Arabidopsis in response to P. syringae. In addition, the area of necrotic plaque appearing on the transgenic leaves inoculated with Botrytis cinerea was smaller and accompanied by an upregulation of PR1 and PR5, indicating NtabSPL6-2 transgenic leaves were less susceptible to the fungal pathogen. Moreover, there was less accumulation of reactive oxygen species (H2O2 and O2-) and malondialdehyde in the local infected sites of transgenic plants, whereas the wild-type Col-0 plants were more oxidatively injured after infestation by B. cinerea.