RESUMEN
Theobromine is an important quality component in tea plants (Camellia sinensis), which is produced from 7-methylxanthine by theobromine synthase (CsTbS), the key rate-limiting enzyme in theobromine biosynthetic pathway. Our transcriptomics and widely targeted metabolomics analyses suggested that CsMYB114 acted as a potential hub gene involved in the regulation of theobromine biosynthesis. The inhibition of CsMYB114 expression using antisense oligonucleotides (ASO) led to a 70.21% reduction of theobromine level in leaves of the tea plant, which verified the involvement of CsMYB114 in theobromine biosynthesis. Furthermore, we found that CsMYB114 was located in the nucleus of the cells and showed the characteristic of a transcription factor. The dual luciferase analysis, a yeast one-hybrid assay, and an electrophoretic mobility shift assay (EMSA) showed that CsMYB114 activated the transcription of CsTbS, through binding to CsTbS promoter. In addition, a microRNA, miR828a, was identified that directly cleaved the mRNA of CsMYB114. Therefore, we conclude that CsMYB114, as a transcription factor of CsTbS, promotes the production of theobromine, which is inhibited by miR828a through cleaving the mRNA of CsMYB114.
Asunto(s)
Camellia sinensis , Camellia sinensis/genética , Camellia sinensis/metabolismo , Teobromina/metabolismo , Cafeína/metabolismo , Hojas de la Planta/metabolismo , Té/metabolismo , Factores de Transcripción/genética , ARN Mensajero/metabolismo , Regulación de la Expresión Génica de las Plantas , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismoRESUMEN
Stamen development is an important developmental process controlled by multiple internal and external factors. Developmental abnormalities of stamens can disrupt the structure and function of anthers, and then result in male sterility. As well known, APETELA 3 (AP3) has a clear function in regulating stamen development, which may impact in male sterility. However, the mechanisms of stamen development and male sterility controlled by AP3 are still not very clear, particular in Pak-choi (Brassica rapa ssp. chinensis). In this work, BcAP3 encoded a protein containing a MADS-box domain, which was a homolog of AtAP3, was identified in Pak-choi. Sequence alignments and phylogenetic analysis indicated that BcAP3 was highly similar to AtAP3. BcAP3 was shown to be localized to the nucleus and exhibited the potential of transcription factor. The transcript of BcAP3 was only expressed in flowers of Pak-choi, indicating that it may act in flower development. Overexpression of BcAP3 in Arabidopsis resulted in developmental abnormalities of anther wall and low vigor pollen, which were associated with the phenotype of male sterility. Expression levels of NST1 and NST2, involved in secondary wall thickening in anther walls, were significantly higher in the BcAP3-transgenic plants than in control plants, suggesting that BcAP3 may affect anther wall development by regulating NST1 and NST2. Taken together, our study demonstrated that BcAP3 could play an essential role in stamen development and male sterility.
Asunto(s)
Brassica rapa/crecimiento & desarrollo , Brassica rapa/genética , Flores/crecimiento & desarrollo , Flores/genética , Genes de Plantas , Proteínas de Dominio MADS/genética , Infertilidad Vegetal/genética , Proteínas de Plantas/genética , Secuencia de Aminoácidos , Regulación de la Expresión Génica de las Plantas , Proteínas de Dominio MADS/metabolismo , Fenotipo , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente , Polen , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Cytoplasmic male sterility (CMS) is a common trait in higher plants, and several transcription factors regulate pollen development. Previously, we obtained a basic helix-loop-helix transcription factor, BcbHLHpol, via suppression subtractive hybridization in non-heading Chinese cabbage. However, the regulatory function of BcbHLHpol during anther and pollen development remains unclear. In this study, BcbHLHpol was cloned, and its tissue-specific expression profile was analyzed. The results of real-time polymerase chain reaction showed that BcbHLHpol was highly expressed in maintainer buds and that the transcripts of BcbHLHpol significantly decreased in the buds of pol CMS. A virus-induced gene silencing vector that targets BcbHLHpol was constructed and transformed into Brassica campestris plants to further explore the function of BcbHLHpol. Male sterility and short stature were observed in BcbHLHpol-silenced plants. The degradation of tapetal cells was inhibited in BcbHLHpol-silenced plants, and nutrients were insufficiently supplied to the microspore. These phenomena resulted in pollen abortion. This result indicates that BcbHLHpol functions as a positive regulator in pollen development. Yeast two-hybrid and bimolecular fluorescence complementation assays revealed that BcbHLHpol interacted with BcSKP1 in the nucleus. This finding suggests that BcbHLHpol and BcSKP1 are positive coordinating regulators of pollen development. Quantitative real-time PCR indicated that BcbHLHpol and BcSKP1 can be induced at low temperatures. Thus, we propose that BcbHLHpol is necessary for meiosis. This study provides insights into the regulatory functions of the BcbHLHpol network during anther development.