RT Journal Article
T1 Characterization of Phytoene Desaturase Knockout Carotenoid-Deficient Microalgal Mutants Generated by Cas9-Ribonucleoprotein Complexes
A1 Molina Márquez, Ana María
A1 Kelterborn, Simon
A1 Hegemann, Peter
A1 Pérez Rodríguez, Miguel
A1 Vigara Fernández, Javier
A1 León Bañares, Rosa María
AB Phytoene desaturase (PDS; EC 1.3.5.5) is a key enzyme of the carotenoid biosynthetic pathway, catalyzing the desaturation of phytoene, precursor of all carotenoids. In this study, several PDS-knockout (PDS-KO) transformants of the chlorophyte microalga Chlamydomonas reinhardtii were generated using a reverse genetics strategy. Two single guide RNAs (sgRNA) were designed to target the first exon of the PDS gene, and pre-assembled Cas9 ribonucleoprotein (RNPs) complexes were delivered into microalgal nuclei by electroporation. Multiple white PDS-KO transformants were successfully obtained by this approach, and three independent transformant lines were subsequently characterized. By integrating ultrastructural, pigment and transcriptomic analyses of dark-grown cells of several PDS-KO carotenoid-deficient mutants in comparison with the parental strain, it was demonstrated that carotenoids are indispensable components of multiple cellular architectures. Chromatographic analysis confirmed that the only carotenoid accumulated in these transformants was phytoene, which lacks the critical structural and photoprotective functions of its colored derivatives. Transmission Electron Microscopy (TEM)  observations revealed profound ultrastructure alterations, including poorly developed chloroplasts and effects on other cellular structures that were either absent or severely disorganized. Consistently, clustering differentially expressed genes into functional groups revealed downregulation of pathways associated with photosynthesis, chlorophyll and carotenoid biosynthesis, ribosome biogenesis, and vesicle and membrane trafficking in the PDS-KO lines. Conversely, upregulation of regulatory and retrotransposon-inducing genes was observed. These findings underscore the central metabolic role of colored carotenoids in plant cells, highlighting their essential contribution to cellular homeostasis and photosynthetic competence.
PB Wiley
SN 0031-9317
SN 1399-3054 (electrónico)
YR 2026
FD 2026
LK https://hdl.handle.net/10272/28192
UL https://hdl.handle.net/10272/28192
LA eng
NO Molina‐Márquez, A., Kelterborn, S., Hegemann, P., Pérez‐Rodríguez, M., Vigara, J., & León, R. (2026). Characterization of Phytoene Desaturase Knockout Carotenoid‐Deficient Microalgal Mutants Generated by Cas9‐Ribonucleoprotein Complexes. Physiologia Plantarum, 178(2). https://doi.org/10.1111/ppl.70811
NO This work was supported by the Regional Development Fund through the Agencia Estatal de Investigación-MCIN/ AEI/10.13039/5011000110 33-( research grants 2019-110438RB- C22 and PID2022-140995OB- C21) and the German Research Foundation (DFG) (Project: 426566805, PH). P.H is a Hertie Professor and funded by the Hertie Foundation. Funding for open access charge: Universidad de Huelva/CBUA is acknowledged.
DS Repositorio Institucional de la Universidad de Huelva
RD 31 may 2026