Check and Balance Between RIC3 and RIC4 Pathways Roles in Actin Dynamics

As discussed above, active ROP 1 stimulates two important downstream pathways, the assembly of tip-localized F-actin and the tip-focused [Ca2+ ]c gradient, via two distinct mediators, RIC3 and RIC4 (Gu et al. 2005) (see Fig. 3). The assembly of tip-localized F-actin could provide forces for targeted delivery and accumulation of secretory vesicles to the future growth site; too much and less dynamic F-actin could be the physical barrier to vesicle fusion (Giner et al. 2005). Cytosolic Ca2+ is also required for vesicle fusion, but high levels of cytosolic Ca2+ disrupt F-actin (see the chapter by Yokota and Shimmen, this volume). Efficient tip growth thus requires a critical balance between Ca2+ and dynamic F-actin. Not surprisingly, RIC3-dependent Ca2+ and RIC4-dependent F-actin pathways counteract each other (Gu et al. 2005). RIC4-overexpressing pollen tubes showed enhanced F-actin assembly but a less pronounced tip-focused Ca2+ gradient. In contrast, RIC3-overexpressing tubes developed higher tip [Ca2+]c but completely lost the tip fine F-actin structure. RIC4 coexpression suppressed RIC3-induced depolarized growth in an F-actin dependent manner, and LatB treatment blocked the suppression of RIC3 signaling by RIC4 coexpression. RIC3 coexpression suppressed RIC4-induced depolarized growth in a Ca2+-dependent manner, and blocking of Ca2+ influx by La3+ inhibited the counteracting effect of RIC3 on RIC4 signaling (Gu et al. 2005). Clearly, control mechanisms are needed to check the activation of these two pathways and make them balanced for optimum growth. It was found that when RIC3 and RIC4 were expressed at 1: 2 molecular ratio in tobacco pollen tubes, the apical morphology was comparable to the control tubes and a normal tip F-actin structure was restored (Gu et al. 2005). Interestingly, ric3 and ric4 double loss-of-function Arabidopsis pollen tubes grew with normal apical morphology (although shorter than wild-type tubes), whereas single loss-of-function mutants displayed retarded growth (Gu et al. 2005). In the RNAi ric3 and ric4 double mutant, cells could still express diminute amounts of RIC3 and RIC4, thus justifying normal morphology but shorter growth.

How do the RIC3- and RIC4-dependent pathways counteract each other? An increase of cytosolic [Ca2+ ] activates several actin binding proteins (ABPs) (see chapter by Yokota and Shimmen, this volume, for detailed dis-

Fig. 3 A model for a ROP signaling network regulating pollen tube growth. Active ROP1 stimulates F-actin assembly and [Ca2+]c increase via RIC4- and RIC3-dependent pathways. F-actin assembly may control the targeted accumulation of secretory vesicles, and late [Ca2+]c increase induces actin depolymerization, promoting vesicle fusion. By regulating targeted vesicle delivery and fusion, tip-localized active ROP1 couples the temporal and spatial aspects of tip growth. F-actin assembly feed-forwardly promotes the accumulation of active ROP1 to the tip, forming a positive feedback loop of signal amplification. A [Ca2+]c increase terminates the active ROP1 amplification probably by counteracting the F-actin pathway. It remains to be answered whether tip high Ca2+ directly activates the downregulation of ROP1 activity via, or independently of, F-actin

Fig. 3 A model for a ROP signaling network regulating pollen tube growth. Active ROP1 stimulates F-actin assembly and [Ca2+]c increase via RIC4- and RIC3-dependent pathways. F-actin assembly may control the targeted accumulation of secretory vesicles, and late [Ca2+]c increase induces actin depolymerization, promoting vesicle fusion. By regulating targeted vesicle delivery and fusion, tip-localized active ROP1 couples the temporal and spatial aspects of tip growth. F-actin assembly feed-forwardly promotes the accumulation of active ROP1 to the tip, forming a positive feedback loop of signal amplification. A [Ca2+]c increase terminates the active ROP1 amplification probably by counteracting the F-actin pathway. It remains to be answered whether tip high Ca2+ directly activates the downregulation of ROP1 activity via, or independently of, F-actin cussion). For example, in tobacco pollen tubes, profilin and RIC3 completely restored the RIC4 overexpression phenotype (Gu et al. 2005).

Ca2+ channels at the pollen tube plasma membrane are also putative targets for F-actin to suppress a [Ca2+]c increase. RIC3-induced Ca2+ increase is dependent on the influx of extracellular Ca2+ (Gu et al. 2005), and recent studies of Ca2+ channel activities in pollen protoplasts provide some hints to explain why increased F-actin assembly by RIC4 suppressed RIC3-dependent Ca2+ accumulation in the tip. Wang et al. (2004) have shown that the voltage-operated inward Ca2+ channel activity found in the plasma membrane of pollen protoplasts was stimulated by actin depolymerization. Increased tip-localized F-actin assembly by RIC4 may affect the activities of these Ca2+ influx channels in the tip plasma membrane, thereby inhibiting the increase of [Ca2+ ]c.

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