Through this PhD thesis work, the mechanism for pollen tube growth is studied using combined modeling and experimental approaches.

In chapter 1 of this thesis, we proposed mathematical models for ROP1 activity oscillation at apex of pollen tubes. Our combined theoretical and experimental studies suggested that ROP1 activity oscillation depends on downstream ROP1 signaling events, F-actin assembly and calcium accumulation, which positively and negatively feedback regulate ROP1 activity, respectively. Our findings suggested a key role for calcium in the negative feedback regulation of ROP1 activity.

In chapter 2 of this thesis, we proposed and experimentally validated a model coupling both active ROP1 polarity establishment and pollen tube morphogenesis. We revealed that exocytosis is the key process linking ROP1 GTPase polarity establishment, cell wall pectin distribution and polarized cell growth. Through exocytosis mediated positive feedback loop, RopGAP mediated global inhibition and lateral diffusion of ROP1 GTPase on plasma membrane, active ROP1 GTPases polarity can be established and maintained. Once maintained, active ROP1 controls cell wall mechanics via control of pectin exocytosis and thus determines the shape of pollen tubes. Experimentally, we also validated the model through both genetic (ren1-1 mutant) and chemical (Latrunculin B treatment) perturbation of pollen tube morphogenesis process.

In Chapter 3 of this thesis, we studied the mechanism of endocytosis in pollen tubes. We revealed that two interconvertible phosphoinositides, PI(4,5)P2 and PI4P, function in different steps of clathrin-dependent endocytosis in pollen tubes. Whereas accumulation of PI(4,5)P2 is required for the initial steps of membrane invagination, PI4P is required for the later steps of closing and/or fission of invaginated membrane. Given previous findings that both the accumulation and the hydrolysis of PI(4,5)P2 are required for clathrin-mediated endocytosis in yeast and animal cells, a balance between PI4P and PI(4,5)P2 may provide a common mechanism underlying clathrin-dependent endocytosis in various eukaryotic systems.

Auxin is a multi-functional hormone essential for plant development and pattern formation. A nuclear auxin perception and signaling system controlling auxin-induced gene expression has been established. However this nuclear-based auxin signaling cannot explain all the actions of auxin in plant cells. In this dissertation, we have demonstrated a cytoplasmic auxin signaling mechanism that can regulate cell morphogenesis through ROP GTPase signaling.

In the first charpter, we use leaf pavement cells (PCs) as a model system to investigate how auxin modulate the interdigitation growth between adjacent cells. PCs develop interdigitated lobes and indentations to form a puzzle-piece shape and provide a useful system to investigate the spatial coordination of cell expansion among cells in a two-dimensional plane. PC interdigitation is compromised in leaves deficient in either auxin biosynthesis or its export by the auxin transport facilitator PINFORMED 1 (PIN1) which is localized at the lobe tip. Auxin coordinately activates two Rho GTPases, ROP2 and ROP6, which promotes the formation of complementary lobes and indentations across cells, respectively. Activation of both ROP2 and ROP6 by auxin occurs within 30 seconds and depends on AUXIN-BINDING PROTEIN 1 (ABP1). These findings reveal ROP-based novel auxin signaling mechanisms, which modulate the spatial coordination of cell expansion across a field of cells.

However, the mechanism about how auxin activates ROPs is still unknown since ABP1 localized to the outer surface of plasma membrane and cannot directly bind with ROPs. In the second chapter, we reported a family of receptor like kinases (RLK) called TMK that could bind with ABP1 on plasma membrane and activate ROP GTPase in pavement cells. tmk mutant showed similar phenotype and auxin insensitivity as in abp1 mutant. And both ROP2 signaling and ROP6 signaling are not activated in tmk mutant. ABP1 and TMK1 are confirmed in a same complex on PM by co-immunoprecipitation. In the meanwhile, TMK kinase domain can directly bind with N terminal of SPK1, which is a putative ROPGEF that can activate ROP2. Therefore, we concluded that TMK and ABP1 are forming an auxin receptor complex on PM that mediate auxin activation of ROP signaling pathway.

Tip growth is an extreme form of polar growth and involves highly polarized cellular activities, such as vesicular trafficking and cell surface extension. In the presence of environmental signals, tip growing cells are able to reestablish the axis of polarity and growth in a process called growth guidance. Previous studies showed that in various cell systems, tip growth and growth guidance depend on polarized Rho GTPase signaling and exocytosis. In this study, we combined mathematical modeling and biological experimentation to study the molecular mechanisms underlying the tip growth and guidance of Arabidopsis pollen tubes. Firstly, a mathematical model integrating the intracellular signaling and the cell surface mechanics in pollen tube tip growth is presented. The predictions of the model were confirmed by phenotypic characterization of mutants defective in the ROP1 signaling, exocytosis or cell wall modification. The results supported the hypothesis that in pollen tube tip growth, exocytosis is the central player that couples ROP1 GTPase signaling pathways with cell wall mechanical properties. Secondly, a mathematical model of pollen tube guidance was developed based on the framework of the tip growth model using the Finite Element Method. In this model, the distribution of active ROP1 is biased by the gradient of the extracellular guidance signal, leading to asymmetric growth towards the source of the guidance signal. This model is validated by observing the dynamics of ROP1 activity and cell morphology in pollen tube guidance using the semi-in vitro assay. Finally, a novel method of measuring the exocytosis rate of cell membrane or cell wall proteins is presented. In this method, the protein-of-interest is tagged with Dendra2, a photo-convertible fluorescent protein. Following photoconversion, both exocytosis and other processes that affect fluorescence recovery such as endocytosis and cell surface extension can be tracked simultaneously. We developed an algorithm for data processing and calculation, and applied this method in measuring the exocytosis rate of PRK1 in Arabidopsis pollen tubes. Overall, this study provided a platform for the application of mathematics in the study of plant cell signaling and morphogenesis.

Sexual reproduction is a critical process in life cycle of flowering plants. To achieve successful reproduction, some important steps are required including gametogenesis, pollination and fertilization. Gametogenesis refers to the development of haploid gametes (egg and sperm). Pollination requires pollen-pistil recognition to promote pollen tube growth toward the ovule. During fertilization, the reproductive gametes fuse to each other and embryo is produced. In this study, different approaches were used to clarify the biological function of three leucin rich repeat receptor-like kinases (LRR-RLKs); RLK B, RLK C and RLK D, during sexual reproduction in Arabidopsis. RLK B, RLK C and RLK D belong to LRR VI, LRR III and LRR VIII-type of the LRR-RLK subfamily, respectively. According to the phenotypic characterization of different combination of mutants, we show that RLK C function redundantly with RLK B and RLK D to regulate male and female fertilities. In rlk c, the synergids were not specified in some embryo sacs resulted in an increase of unfertilized ovules. Mutations in RLK B and RLK D could enhance phenotypes of rlk c. The rlk c rlk b plant exhibited severe defect in pollen tube guidance and seed production. It is most likely that RLK C and RLK B function redundantly to elicit a signal transduction pathway involved in synergid specification or female gametophyte development. By contrast, rlk c rlk d-2 caused more severe defect in pollen fertility. In both cases, the major contribution for gamete fertility is from RLK C signaling due to no apparent phenotype in the rlk b and rlk d single mutants. In addition, the rlk b rlk d-2 mutant did not show phenotype, indicating their unique functions in reproduction. According to dominant-negative (DN) study, the LAT52p::DN-RLK C receptor lacking the kinase domain enhanced a defect in male fertility in the rlk c mutant. This confirms that the functional redundancy in RLK C is essential to regulate male fertility. Taken together, we provide evidence that RLK C, RLK B and RLK D function to achieve fully fertility of the reproductive gametes.

Polarized growth in pollen tube requires complex signaling events including ROP1 GTPase pathway and calcium signaling. The role of tip calcium in negative feedback regulation of ROP1 remains a question and potentially involves ROP1 negative regulator, REN1GAP, whose activity seems to be regulated by calcium. Calcium-dependent protein kinases (CDPKs/CPKs) are calcium sensors with known function in regulating pollen tube tip growth. As such, we hypothesized that CPK substrate(s) in pollen tube may be component of tip growth regulator such as REN1. Here we report that REN1 is phosphorylated by pollen-expressed CPK16 with a relative EC50 value of ~4.6 µM. MS/MS analysis revealed calcium-dependent phosphorylation sites within REN1 which include Ser70 and Ser267. Functional analyses suggested that REN1 phosphorylation at Ser267 is required for its activity while at Ser70,71 affected its localization and subsequently activity. Mutation analysis of CPK16 loss-of-function, cpk16-3, revealed enhanced pollen tube growth and germination when grown in low calcium media. Treatment of cpk16-3 tubes with either brefeldin A or latrunculin B induced tip swelling phenotype similar to the same effects produced by chemical treatments of partially complemented ren1-1 Lat52::GFP-REN1, Overall, these results suggest a link between calcium and a major signaling pathway, ROP1, via calcium-dependent protein kinase and its substrate REN1.

To dissect the causal and phasal relationships between oscillations of growth, active ROP1, F-actin dynamics, and tip-focused calcium, a chemical genetics approach was utilized with the goal to identify small molecules that would specifically activate ROP1. To this end, 20000 chemicals were screened in a cell-based yeast two hybrid assay to target inhibitors of active ROP and GAP. One compound, #7, inhibited ROP-GAP interaction as confirmed by in vitro assays as well as slightly enhanced pollen tube tip width. Treatment of ROP1 OX severely enhanced tip swelling suggesting that it primarily targets ROP1. Docking analyses of compound 7 to the protein interaction interface of RhoA and p190RhoGAP revealed possible binding sites within the GTP-binding pockets of Rho-GAP interface in potentially disrupting protein-protein interaction. This study leads to a potential activator of ROP1 which may be useful for future ROP1 studies.