Meiosis‐specific protein co‐expression in <i>Arabidopsis thaliana</i> : a protein delivery tool in meiotic cells

Meiosis is a key process in sexual reproduction, orchestrated by numerous proteins that differ in their spatiotemporal expression level and dynamics. Employing meiotic gene promoters typically leads to varying levels of transgene expression within meiotic and non-meiotic cells in plants. Hence, their application for protein expression during meiosis is limited. Here, we present a tool for meiosis-specific protein co-expression in Arabidopsis thaliana. By utilizing ribosome-skipping peptides, we leverage the distinct expression patterns of the two meiotic genes ASY1 and MTOPVIB to co-express various proteins through a single polyprotein precursor containing the linker peptides P2a or IntF2a with 'self-cleaving' abilities. During translation of ASY1 or MTOPVIB in prophase I, those linkers of viral origin facilitate the separation of the polyprotein precursor components through a ribosomal 'skip' and their release as individual proteins in a one-to-one ratio. Compared with P2a, IntF2a achieves more efficient cleavage in male meiocytes. We used our system for meiosis-specific co-expression of up to two additional proteins. For example, by applying light sheet fluorescence microscopy, we studied the spatiotemporal dynamics of H2B, CENH3, and alpha-tubulin isoform TUA6 in combination with three different fluorescent proteins during male meiosis. Overall, we achieved coordinated co-synthesis of up to three proteins from a single polyprotein precursor, allowing synchronized co-expression of proteins at the level of either ASY1 or MTOPVIB during meiosis. In summary, the approach provides a versatile and efficient tool for studying or potentially modifying meiotic processes through precise and coordinated protein expression during meiosis or other biotechnological applications requiring coordinated protein co-synthesis in plants.