Pollen germination is a process of polarity establishment, through which a single and unique growth axis is established. Although most of the intracellular activities associated with pollen germination are controlled by RHO OF PLANTs (ROPs) and increased ROP activation accompanies pollen germination, a critical role of ROPs in this process has not yet been demonstrated. Here, by genomic editing of all four Arabidopsis (Arabidopsis thaliana) ROPs that are preferentially expressed in pollen, we showed that ROPs are essential for polarity establishment during pollen germination. We further identified and characterized two ROP effectors in pollen germination (REGs) through genome-wide interactor screening, Boundary of ROP domain (BDR) members BDR8 and BDR9, whose functional loss also resulted in no pollen germination. BDR8 and BDR9 were distributed in the cytosol and the vegetative nucleus of mature pollen grains but re-distributed to the plasma membrane (PM) of the germination site and to the apical PM of growing pollen tubes. We demonstrated that the PM re-distribution of BDR8 and BDR9 during pollen germination relies on ROPs but not vice versa. Furthermore, enhanced expression of BDR8 partially restored germination of rop1 pollen but had no effects on that of the quadruple rop pollen, supporting their genetic epistasis. Results presented here demonstrate an ROP signaling route essential for pollen germination, which supports evolutionarily conserved roles of Rho GTPases in polarity establishment.

SUMMARYOvules are female reproductive organs of angiosperms, consisting of sporophytic integuments surrounding female gametophytes, that is, embryo sacs. Synchronization between integument growth and embryo sac development requires intracellular communication. However, signaling routes through which cells of the two generations communicate are unclear. We report that symplastic signals through plasmodesmata (PDs) of integuments are critical for the development of female gametophytes. Genetic interferences of PD biogenesis either by functional loss of CHOLINE TRANSPORTER‐LIKE1 (CTL1) or by integument‐specific expression of a mutated CALLOSE SYNTHASE 3 (cals3m) compromised PD formation in integuments and reduced fertility. Close examination of pINO:cals3m or ctl1 ovules indicated that female gametophytic development was either arrested at various stages after the formation of functional megaspores. In both cases, defective ovules could not attract pollen tubes, leading to the failure of fertilization. Results presented here demonstrate a key role of the symplastic route in sporophytic control of female gametophytic development.

The development of male and female gametophytes is a prerequisite for successful propagation of angiosperms. The small GTPases RAN play fundamental roles in numerous cellular processes. Although RAN GTPases have been characterized in plants, their roles in cellular processes are far from understood. We report here that RAN GTPases in Arabidopsis are critical for gametophytic development. RAN1 loss‐of‐function showed no defects in gametophytic development likely due to redundancy. However, the expression of a dominant negative or constitutively active RAN1 resulted in gametophytic lethality. Genetic interference of RAN GTPases caused the arrest of pollen mitosis I and of mitosis of functional megaspores, implying a key role of properly regulated RAN activity in mitosis during gametophytic development.