Regulation of pollen tube polarity

Abstract

Targeted delivery of immotile sperm through growing pollen tubes is a crucial step in achieving sexual reproduction in angiosperms. Unlike diffuse-growing cells, the growth of a pollen tube is restricted to the very apical region where targeted exocytosis and regulated endocytosis occur. The plant-specific Rho GTPases, Rops, are central organizers for pollen tube polarity. Through effector binding, Rops regulate the tip-focused Ca²⁺ gradient and the actin cytoskeleton during pollen tube growth. Therefore, understanding the spatiotemporal regulation of Rop activity would reveal how establishment and maintenance of tube polarity as well as re-orientation of the growth axis are accomplished. Recent findings indicated that two feedback loops may be fundamental in maintaining a fine-tuned and dynamic Rop activity. The concerted activities of RopGAP and RopGDI prevent lateral diffusion of activated Rop, restricting Rop activity to the apical plasma membrane. Conversely, pollen receptor kinases (PRKs) and RopGEFs positively feedback regulate Rop activity through protein binding and membrane recruitment. Feedback loops would also be essential for pollen tube re-orientation. Shallow extracellular cues amplified by concerted activities of feedback loops would lead to asymmetric activation of Rop and result in tube re-orientation.

Plant-Specific Rho GTPases, Rops/Racs, Are Central Organizers During Pollen Tube Growth

Polarized growth of pollen tubes is a critical step for sexual reproduction of angiosperms. Pollen tubes are initiated upon landing on the stigmatic surface. Growing tubes penetrate deep inside the pistil to deliver immotile sperm to the embryo sac, a process that requires the maintenance of polarity over a certain distance and the perception of guidance cues.1 How such a targeted delivery can be accomplished is a fascinating area not only because of its agricultural significance, but because its understanding will shed more light on the evolutionary origin of signaling cascades that control cell polarization and environmental sensing.

As in other polarized cells, such as neurite growth cones and neutrophils, plant-specific Rho GTPases (designated Rops/Racs) are central organizers of intracellular activities. Rho GTPases act as molecular switches, alternating between a GTP-bound active and a GDP-bound inactive state.2 In addition, membrane association of Rho GTPases is essential for their functionality. Rop associates with the apical plasma membrane in pollen tubes,3 where targeted exocytosis and regulated endocytosis occur to drive rapid growth.4 The GTP-bound Rop regulates both a tip-focused Ca²⁺ gradient and apical actin cytoskeleton organization through two antagonistic effector pathways.5 Constitutively active Rop resulted in isotropic growth,6,7 i.e., radial expansion rather than tip growth, whereas reduced Rop activity inhibited tube growth.6–8

Evolutionarily Conserved Negative Feedback Loop

Considering the critical roles that Rops play, the key to pollen tube growth would be a fine-tuned and dynamically regulated Rop activity. Recent advances indicated the existence of both negative and positive feedback loops in spatiotemporally regulating Rop activity. The negative feedback loop includes the GTPase activating proteins (GAPs) and the guanine nucleotide dissociation inhibitors (GDIs). RopGAPs accelerate the intrinsic GTPase activity of Rop, thereby preventing the lateral diffusion of activated Rop during tube growth.9,10 The localization and activity of RopGAPs are in turn regulated by Rop through a CRIB domain (Cdc42/Rac interactive binding motif) within RopGAPs, which facilitates Rop-binding and promotes GAP activity.9,10 RopGDIs sequester GDP-bound Rop in the cytosol where GTP is abundant.2 Because RopGDIs preferentially bind to prenylated, GDP-bound Rop at the plasma membrane,11 they are critical to actively remove Rop at the junction between the apical and subapical plasma membrane during pollen tube growth, and by doing so, restrict the membrane distribution of Rop to the apical region. Both the catalytic domains of RopGAPs and RopGDIs are evolutionarily conserved and a similar negative feedback loop also exists in metazoans.12

Plant-specific Positive Feedback Loop

In contrast to the evolutionarily conserved negative feedback loop, plants evolved a distinct positive feedback loop for controlling Rop activity. Polarized metazoan cells generally use transmembrane G protein-coupled receptors (GPCRs) to sense extracellular stimuli and Dbl-type RhoGEFs to relay signals for spatiotemporal Rho activation.12 No conventional GPCRs have been identified in plants. Instead, receptor-like kinases are ideal candidates for transmembrane signaling relays in plants, due to their large numbers and diversified extracellular domains.13 Plants also do not have homologs of Dbl-type RhoGEFs. Rather, a plant-specific novel class of RopGEFs has recently been characterized.14–16 RopGEFs contain a central catalytic domain (PRONE) flanked by regulatory domains.14,15,17 We recently showed that pollen-specific RopGEFs interact with pollen receptor kinases (PRKs).16,17 Such an interaction releases the C-terminal inhibition of RopGEF activity.17 The interaction between PRKs and RopGEFs recruits RopGEFs to the plasma membrane, thus serving as a positive feedback loop to maintain continued activation of Rop at the apical plasma membrane. There is as yet no evidence showing direct regulation of PRKs by Rop. However, given that PRKs are transmembrane receptors, it is plausible that the spatiotemporal membrane distribution and concentration of PRKs are influenced by vesicle trafficking, which in turn is regulated through Rop-mediated Ca²⁺ gradient maintenance and actin polymerization.5

It is not surprising that components in the negative feedback loop are conserved during evolution while those in the positive feedback loop are not. The negative feedback loop is only involved in self-organizing cycles for cell polarization, while the positive feedback loop is also responsible for receiving and responding to ever-changing extracellular cues. It has always been an intriguing thought that pollen tubes can sense and respond to a myriad of signaling cues. How such a complexity can be achieved may also lie in the positive feedback loop. Multiple PRKs,18 RopGEFs17 and Rops19 are expressed in pollen. Supposing there are interaction specificities conferred by extracellular stimuli, many different combinations of these three components can be envisioned.

Pollen Tube Growth: It is All About Coordinated Activities of Feedback Loops

We propose that coordinated activities of feedback loops are fundamental to the establishment, maintenance and re-localization of spatially restricted Rop activity during pollen tube growth. In this model (Fig. 1), random membrane association of Rop before pollen germination results in small concentration differences along the pollen plasma membrane. Feedback loops amplify such slight differences to establish a stable growth axis in which Rop resides at the apical plasma membrane. During rapid growth, when there is no need for re-orientation, the growth axis is constant, as is the case during in vitro pollen tube growth.20 Such a self-organizing property of pollen tubes requires dynamically restricted apical Rop localization, made possible by feedback loops. In addition, a positive feedback loop composed of PRKs and RopGEFs has the ability to perceive shallow gradients of extracellular stimuli. To transduce the perceived signals, the positive feedback loop asymmetrically activates Rop, which then is stabilized by the negative feedback loop. A spatiotemporal switch of Rop activity at plasma membrane finally redirects intracellular activities, leading to pollen tube re-orientation.

Abbreviations

RopGEF	=	guanine nucleotide exchange factor for Rop
RopGAP	=	GTPase activating protein for Rop
RopGDI	=	guanine nucleotide dissociation inhibitor for Rop
PRK	=	pollen receptor kinase
CRIB	=	Cdc42/Rac interactive binding motif
GPCRs	=	G protein-coupled receptors
RLKs	=	receptor like kinases

Figures and Tables

Figure 1 Feedback loops of Rop control polarized growth of pollen tubes. As described in the text, RopGAPs inhibit Rop activity by accelerating intrinsic GTPase activity while RopGDIs extract prenylated, GDP-bound Rop from the membrane for its recycling to the apical plasma membrane. Both are regulated by Rop through direct interactions, forming a negative feedback loop. PRKs interact with RopGEFs to facilitate their membrane association. The apical plasma membrane-localized Rop and membrane PRKs bind to different domains of RopGEFs, forming a positive feedback loop that regulates Rop activity. PRKs may be regulated by Rop-mediated vesicle trafficking, shown with a dashed arrow. Extracellular cues directly alter the activity of the positive feedback loop, resulting in tube re-orientation.

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