Arabidopsis <scp>RabGDIs</scp> are essential for the asymmetric division of zygotes and embryonic development

Summary In angiosperms, the asymmetric division of the zygote establishes the plant's primary body axis. While vesicular trafficking is known to be critical for this process, its key regulators are not fully understood. This study investigates the role of Arabidopsis guanine nucleotide dissociation inhibitors (RabGDIs) in zygotic division and early embryogenesis. We generated gdi1 and gdi2 single and double mutants using Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR‐associated protein 9 (CRISPR/Cas9). The effects of RabGDI loss‐of‐function on embryogenesis, cell fate determination, vacuolar dynamics, and Rab5 localization were analyzed using confocal microscopy and genetic complementation. A dominant‐negative Rab5 (ARA7 DN ) was expressed to validate its role. RabGDI1 and RabGDI2 are constitutively expressed and interact specifically with canonical Rab GTPases. The gdi1 gdi2 double mutant is embryo‐lethal, showing defective zygotic asymmetry, disrupted apical–basal patterning, and mislocalization of auxin transporters. Vacuolar biogenesis and polarity are severely compromised in mutant zygotes, correlating with mis‐targeted Rab5. Expressing ARA7 DN in the zygote phenocopied the gdi1 gdi2 division defects. We conclude that Arabidopsis RabGDI1 and RabGDI2 are essential regulators of early embryogenesis. They function by controlling the targeting of canonical Rab GTPases, particularly Rab5, thereby ensuring proper vacuolar dynamics necessary for the asymmetric division of the zygote and subsequent embryonic patterning.