A carregar...
Publicação
In silico, in vitro, and in vivo characterization of thiamin-binding proteins from plant seeds
| Nome: | Descrição: | Tamanho: | Formato: | |
|---|---|---|---|---|
| 3.15 MB | Adobe PDF |
Orientador(es)
Resumo(s)
Thiamin, an essential micronutrient, is a cofactor for enzymes involved in the central carbon metabolism and amino acid pathways. Despite efforts to enhance thiamin content in rice by incorporating thiamin biosynthetic genes, increasing thiamin content in the endosperm remains challenging, possibly due to a lack of thiamin stability and/or a local sink. The introduction of storage proteins has been successful in several biofortification strategies, and similar efforts targeting thiamin have been performed, leading to a 3–4-fold increase in white rice. However, only one thiamin-binding protein (TBP) sequence has been described in plants, more specifically from sesame seeds. Therefore, we aimed to identify and characterize TBPs, as well as to evaluate the effect of their expression on thiamin concentration, using a comprehensive approach integrating in silico, in vitro, and in vivo methods. We identified the sequences of putative TBPs from Oryza sativa (Os, rice), Fagopyrum esculentum (Fe, buckwheat), and Zea mays (Zm, maize) and pinpointed the thiamin-binding pockets through molecular docking. FeTBP and OsTBP contained one pocket with binding affinities similar to the Escherichia coli TBP, a well-characterized TBP, supporting their function as TBPs. In vivo expression studies of TBPs in tobacco leaves and rice callus resulted in increased thiamin levels, with FeTBP and OsTBP showing the most pronounced effects. Additionally, thermal shift assays confirmed the thiamin-binding capabilities of FeTBP and OsTBP, as observed by the significant increases in melting temperatures upon thiamin binding, indicating protein stabilization. These findings offer new insights into the diversity and function of plant TBPs and highlight the potential of FeTBP and OsTBP to modulate thiamin levels in crop plants.
Descrição
Funding Information: This work was supported by the Collen-Francqui Research Professorship (STI.DIV.2022.0014.01) awarded to DVDS by the Francqui Foundation, the Research Foundation Flanders (FWO; G064119N to DVDS and senior postdoctoral fellowship 1288923N to RSM), Ghent University (Bijzonder Onderzoeksfonds (BOF) GOA 01G00409 and BOF-BAS) and BiodivERsA under the BiodivClim ERA-Net (BIOFAIR project, co-funded by the Research Foundation Flanders), as well as by FCT (Funda\u00E7\u00E3o para a Ci\u00EAncia e a Tecnologia) (2022.02916.PTDC, https://doi.org/10.54499/2022.02916.PTDC), the Plants for Life PhD fellowship of MF (PD/BD/148694/2019), the contract of TL (CEECIND/03641/2017), the Research Unit \"GREEN-IT \u2013 Bioresources for Sustainability\" (10.54499/ UIDB/04551/2020, https://doi.org/10.54499/UIDB/04551/2020 and 10.54499/UIDP/04551/2020, https://doi.org/10.54499/UIDP/04551/2020). Publisher Copyright: © 2025 The Author(s).
Palavras-chave
Biochemistry Molecular Biology Cell Biology