Independent roles of Arp2/3 complex and RIC4 protein in the control of epidermal cell shape

Our results show that RIC4 and the Arp2/3 complex function largely independently in the control of pavement cell shape. Genetic analyses indicate that they act in parallel rather than in one signaling cascade. We further demonstrate that RIC4 functions as a negative regulator of lobe formation, as its loss increases cell shape complexity whereas its overexpression increases cell circularity. RIC4-induced cell shape changes occur even in the absence of a functional Arp2/3 complex, excluding Arp2/3 as a downstream effector of RIC4 or ROP signaling. Finally, 22 no correlation between cortical actin dynamics and cell shape phenotypes was detected, which suggests that global actin dynamics alone cannot explain pavement cell morphogenesis. The aim of this study was to determine whether a Cdc42/Rac interactive binding (CRIB) domain-containing protein 4 (RIC4) that functions as an effector of ROP GTPases, and the Arp2/3 complex, an actin nucleator, functionally cooperate in controlling the shape of Arabidopsis cotyledon epidermal cells. The combination of knock-out mutants demonstrated that loss of RIC4 and loss of the Arp2/3 complex results in completely opposite epidermal cell shape phenotypes. The double knock-out (KO) mutation phenotype is similar to the Arp2/3 mutation, and the effect of RIC4 loss is completely eliminated. Analysis of overexpression revealed that excess RIC4 significantly suppresses the formation of pavement cell lobes. However, RIC4 does not require an active Arp2/3 complex for this effect. Our data further show that overexpression of RIC4 has a specific actin stabilization effect in cotyledon epidermal cells. Interestingly, while RIC4 overexpression induced actin stabilization and reduced cell-shape complexity, the loss of Arp2/3 with a similar cell-shape phenotype did not show reduced actin dynamics. In conclusion, RIC4 and the Arp2/3 complex do not share the same signaling pathway in the control of cotyledon epidermal cell shape.