Membrane trafficking and polar growth in root hairs and pollen tubes

Root hairs and pollen tubes extend by rapid elongation that occurs exclusively at the tip. Fundamental for such local, tip-focused growth (so-called 'tip growth') is the polarization of the cytoplasm that directs secretory events to the tip, and the presence of internal gradients and transmembrane flux of ions, notably Ca2+, H+, K+, and Cl-. Electrophysiological and imaging studies using fluorescent markers have sought to link ion gradients with growth and membrane trafficking. Current models recognize membrane trafficking as fundamental to tip growth, notably its role in supplying lipid and protein to the new plasma membrane and cell wall that extend the apex of the cell, and a complementary role for endocytosis in retrieving excess membrane and in recycling various protein fractions. The current state of knowledge is reviewed here in order to highlight the major gaps in the present understanding of trafficking as it contributes to polar growth in these cells and recent results, that suggest a role for membrane trafficking in the active regulation of ion channel turnover and activity during polar tip growth, are discussed.     

The Rop GTPase: an emerging signaling switch in plants

G proteins are ubiquitous molecular switches in eukaryotic signal transduction, but their roles in plant signal transduction had not been clearly established until recent studies of the plant-specific Rop subfamily of RHO GTPases. Rop participates in signaling to an array of physiological processes including cell polarity establishment, cell growth, morphogenesis, actin dynamics, H₂O₂ generation, hormone responses, and probably many other cellular processes in plants. Evidence suggests that plants have developed unique molecular mechanisms to control this universal molecular switch through novel GTPase-activating proteins and potentially through a predominant class of plant receptor-like serine/threonine kinases. Furthermore, the mechanism by which Rop regulates specific processes may also be distinct from that for other GTPases. These advances have raised the exciting possibility that the elucidation of Rop GTPase signaling may lead to the establishment of a new paradigm for G protein-dependent signal transduction in plants.     

Oscillatory ROP GTPase activation leads the oscillatory polarized growth of pollen tubes

Oscillation regulates a wide variety of processes ranging from chemotaxis in Dictyostelium through segmentation in vertebrate development to circadian rhythms. Most studies on the molecular mechanisms underlying oscillation have focused on processes requiring a rhythmic change in gene expression, which usually exhibit a periodicity of >10 min. Mechanisms that control oscillation with shorter periods (<10 min), presumably independent of gene expression changes, are poorly understood. Oscillatory pollen tube tip growth provides an excellent model to investigate such mechanisms. It is well established that ROP1, a Rho-like GTPase from plants, plays an essential role in polarized tip growth in pollen tubes. In this article, we demonstrate that tip-localized ROP1 GTPase activity oscillates in the same frequency with growth oscillation, and leads growth both spatially and temporally. Tip growth requires the coordinate action of two ROP1 downstream pathways that promote the accumulation of tip-localized Ca2+ and actin microfilaments (F-actin), respectively. We show that the ROP1 activity oscillates in a similar phase with the apical F-actin but apparently ahead of tip-localized Ca2+. Furthermore, our observations support the hypothesis that the oscillation of tip-localized ROP activity and ROP-dependent tip growth in pollen tubes is modulated by the two temporally coordinated downstream pathways, an early F-actin assembly pathway and a delayed Ca2+ gradient-forming pathway. To our knowledge, our report is the first to demonstrate the oscillation of Rho GTPase signaling, which may be a common mechanism underlying the oscillation of actin-dependent processes such as polar growth, cell movement, and chemotaxis.     

The Rop GTPase switch controls multiple developmental processes in Arabidopsis

G proteins are universal molecular switches in eukaryotic signal transduction. The Arabidopsis genome sequence reveals no RAS small GTPase and only one or a few heterotrimeric G proteins, two predominant classes of signaling G proteins found in animals. In contrast, Arabidopsis possesses a unique family of 11 Rop GTPases that belong to the Rho family of small GTPases. Previous studies indicate that Rop controls actin-dependent pollen tube growth and H(2)O(2)-dependent defense responses. In this study, we tested the hypothesis that the Rop GTPase acts as a versatile molecular switch in signaling to multiple developmental processes in Arabidopsis. Immunolocalization using a general antibody against the Rop family proteins revealed a ubiquitous distribution of Rop proteins in all vegetative and reproductive tissues and cells in Arabidopsis. The cauliflower mosaic virus 35S promoter-directed expression of constitutively active GTP-bound rop2 (CA-rop2) and dominant negative GDP-bound rop2 (DN-rop2) mutant genes impacted many aspects of plant growth and development, including embryo development, seed dormancy, seedling development, lateral root initiation, morphogenesis of lateral organs in the shoot, shoot apical dominance and growth, phyllotaxis, and lateral organ orientation. The rop2 transgenic plants also displayed altered responses to the exogenous application of several hormones, such as abscisic acid-mediated seed dormancy, auxin-dependent lateral shoot initiation, and brassinolide-mediated hypocotyl elongation. CA-rop2 and DN-rop2 expression had opposite effects on most of the affected processes, supporting a direct signaling role for Rop in regulating these processes. Based on these observations and previous results, we propose that Rop2 and other members of the Rop family participate in multiple distinct signaling pathways that control plant growth, development, and responses to the environment.     

ROP GTPase regulation of pollen tube growth through the dynamics of tip-localized F-actin

Pollen tubes expand by tip growth and extend directionally toward the ovule to deliver sperms during pollination. They provide an excellent model system for the study of cell polarity control and tip growth, because they grow into uniformly shaped cylindrical cells in culture. Mechanisms underlying tip growth are poorly understood in pollen tubes. It has been demonstrated that ROP1, a pollen-specific member of the plant-specific Rop subfamily of Rho GTPases, is a central regulator of pollen tube tip growth. Recent studies in pollen from Arabidopsis and other species have revealed a ROP-mediated signalling network that is localized to the apical PM region of pollen tubes. The results provide evidence that the localization of this signalling network establishes the site for tip growth and the localized activation of this signalling network regulates the dynamics of tip F-actin. These results have shown that the ROP1-mediated dynamics of tip F-actin is a key cellular mechanism behind tip growth in pollen tubes. Current understanding of the molecular basis for the regulation of the tip actin dynamics will be discussed.     

SEC1A and SEC6 synergistically regulate pollen tube polar growth

Pollen tube polar growth is a key physiological activity for angiosperms to complete double fertilization, which is highly dependent on the transport of polar substances mediated by secretory vesicles. The exocyst and Sec1/Munc18 (SM) proteins are involved in the regulation of the tethering and fusion of vesicles and plasma membranes, but the molecular mechanism by which they regulate pollen tube polar growth is still unclear. In this study, we found that loss of function of SEC1A, a member of the SM protein family in Arabidopsis thaliana, resulted in reducing pollen tube growth and a significant increase in pollen tube width. SEC1A was diffusely distributed in the pollen tube cytoplasm, and was more concentrated at the tip of the pollen tube. Through co-immunoprecipitation-mass spectrometry screening, protein interaction analysis and in vivo microscopy, we found that SEC1A interacted with the exocyst subunit SEC6, and they mutually affected the distribution and secretion rate at the tip of the pollen tube. Meanwhile, the functional loss of SEC1A and SEC6 significantly affected the distribution of the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex member SYP125 at the tip of the pollen tube, and led to the disorder of pollen tube cell wall components. Genetic analysis revealed that the pollen tube-related phenotype of the sec1a sec6 double mutant was significantly enhanced compared with their respective single mutants. Therefore, we speculated that SEC1A and SEC6 cooperatively regulate the fusion of secretory vesicles and plasma membranes in pollen tubes, thereby affecting the length and the width of pollen tubes.     

Dynamin GTPase,a force-generating molecular switch

Dynamin is a GTPase that regulates late events in clathrin-coated vesicle formation. Our current working model suggests that dynamin is targeted to coated pits in its unoccupied or GDP-bound form, where it is initially distributed uniformly throughout the clathrin lattice. GTP/GDP exchange triggers its release from these sites and its assembly into short helices that encircle the necks of invaginated coated pits like a collar. GTP hydrolysis, which is required for vesicle detachment, presumably induces a concerted conformation change, tightening the collar. Unlike most of its GTPase cousins that serve as molecular switches, dynamin has a low affinity for GTP, a very high intrinsic rate of GTP hydrolysis and functions as a homo-oligomer. A concerted conformational change resulting from coordinated GTP hydrolysis by the dynamin oligomer might be sufficient to generate force. In this case, dynamin would be the first GTPase identified that acts as a structural protein with mechano-chemical function.     

Total polar lipid biosynthesis during growth of Crotalaria juncea pollen tubes

[1-¹⁴C]-Acetate incorporation into total and polar lipids was studied in the growing pollen tubes of Crotalaria juncea. Ungerminated pollen had phosphatidyl inositol, phosphatidyl serine, phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl glycerol, monogalactosyl diglyceride, digalactosyl diglyceride, sulpholipid and steryl glycosides. In the growing pollen tubes considerable [1-¹⁴C]-acetate incorporation was observed into the individual polar lipids. The exogenous carbon source significantly influenced lipid biosynthesis. Boric acid (20mg/l.) promoted both pollen tube growth and acetate incorporation into phospholipids. In comparison to 5′-adenosine monophosphate, cyclic-3′,5′-adenosine monophosphate (cAMP) promoted tube growth and also enhanced phospho-and glycolipid biosynthesis. The regulation of membrane component biosynthesis by cAMP is suggested.     

Nt-RhoGDI2 regulates Rac/Rop signaling and polar cell growth in tobacco pollen tubes

Rac/Rop-type Rho-family small GTPases accumulate at the plasma membrane in the tip of pollen tubes and control the polar growth of these cells. Nt-RhoGDI2, a homolog of guanine nucleotide dissociation inhibitors (GDIs) regulating Rho signaling in animals and yeast, is co-expressed with the Rac/Rop GTPase Nt-Rac5 specifically in tobacco (Nicotiana tabacum) pollen tubes. The two proteins interact with each other in yeast two-hybrid assays, preferentially when Nt-Rac5 is prenylated. Transient over-expression of Nt-Rac5 and Nt-RhoGDI2 depolarized or inhibited tobacco pollen tube growth, respectively. Interestingly, pollen tubes over-expressing both proteins grew normally, demonstrating that the two proteins functionally interact in vivo. Nt-RhoGDI2 was localized to the pollen tube cytoplasm and effectively transferred co-over-expressed YFP-Nt-Rac5 fusion proteins from the plasma membrane to this compartment. A single amino acid exchange (R69A), which abolished binding to Nt-RhoGDI2, caused Nt-Rac5 to be mis-localized to the flanks of pollen tubes and strongly compromised its ability to depolarize pollen tube growth upon over-expression. Based on these observations, we propose that Nt-RhoGDI2-mediated recycling of Nt-Rac5 from the flanks of the tip to the apex has an essential function in the maintenance of polarized Rac/Rop signaling and cell expansion in pollen tubes. Similar mechanisms may generally play a role in the polarized accumulation of Rho GTPases in specific membrane domains, an important process whose regulation has not been well characterized in any cell type to date.     

A Rho family GTPase controls actin dynamics and tip growth via two counteracting downstream pathways in pollen tubes

Tip growth in neuronal cells, plant cells, and fungal hyphae is known to require tip-localized Rho GTPase, calcium, and filamentous actin (F-actin), but how they interact with each other is unclear. The pollen tube is an exciting model to study spatiotemporal regulation of tip growth and F-actin dynamics. An Arabidopsis thaliana Rho family GTPase, ROP1, controls pollen tube growth by regulating apical F-actin dynamics. This paper shows that ROP1 activates two counteracting pathways involving the direct targets of tip-localized ROP1: RIC3 and RIC4. RIC4 promotes F-actin assembly, whereas RIC3 activates Ca(2+) signaling that leads to F-actin disassembly. Overproduction or depletion of either RIC4 or RIC3 causes tip growth defects that are rescued by overproduction or depletion of RIC3 or RIC4, respectively. Thus, ROP1 controls actin dynamics and tip growth through a check and balance between the two pathways. The dual and antagonistic roles of this GTPase may provide a unifying mechanism by which Rho modulates various processes dependent on actin dynamics in eukaryotic cells.     

Small GTPase 'Rop': molecular switch for plant defense responses

The conserved Rho family of GTPases (Rho, Rac, and Cdc42) in fungi and mammals has emerged as a key regulator of diverse cellular activities, such as cytoskeletal rearrangements, programmed cell death, stress-induced signaling, and cell growth and differentiation. In plants, a unique class of Rho-like proteins, most closely related to mammalian Rac, has only been found and termed 'Rop' (Rho-related GTPase from plant [Li et al. (1998) Plant Physiol. 118, 407-417; Yang (2002) Plant Cell 14, S375-S388]). ROPs have been implicated in regulating various plant cellular responses including defense against pathogens. It has been shown that ROPs, like mammalian Rac, trigger hydrogen peroxide production and hence the 'oxidative burst', a crucial component associated with the cell death, most likely via activation of nicotinamide adenine dinucleotide phosphate oxidase in both monocotyledonous and dicotyledonous species. Recent studies have established that ROPs also function as a molecular switch for defense signaling pathway(s) linked with disease resistance. As discerning the defense pathway remains one of the priority research areas in the field of plant biology, this review is therefore particularly focused on recent progresses that have been made towards understanding the plant defense responses mediated by ROPs.     

Rop GTPase: a master switch of cell polarity development in plants

Cell polarity is fundamentally important to plant growth and development, yet the mechanism governing its development is understood poorly. Several studies have revealed a role for Rop GTPases in pollen polar tip growth. Rop is also localized to the future site of root hair development and the tip of root hairs, and expression of constitutively active Rop mutants impacts on the morphogenesis of tip-growing root hairs as well as on non-tip-growing cells. These findings highlight the importance of Rop as a common switch in cell polarity control in plants.     

The effect of temperature on pollen germination, pollen tube growth, and stigmatic receptivity in peach

Temperature is a major climatic factor that limits geographical distribution of plant species, and the reproductive phase has proven to be one of the most temperature-vulnerable stages. Here, we have used peach to evaluate the effect of temperature on some processes of the progamic phase, from pollination to the arrival of pollen tubes in the ovary. Within the range of temperatures studied, 20 degrees C in the laboratory and, on average, 5.7 degrees C in the field, the results show an accelerating effect of increasing temperature on pollen germination and pollen tube growth kinetics, as well as an increase in the number of pollen tubes that reach the style base. For the last two parameters, although the range of temperature registered in the field was much lower, the results obtained in the laboratory paralleled those obtained in the field. Increasing temperatures drastically reduced stigmatic receptivity. Reduction was sequential, with stigmas first losing the capacity to sustain pollen tube penetration to the transmitting tissue, then their capacity to offer support for pollen germination and, finally, their capacity to support pollen grain adhesion. Within a species-specific range of temperature, this apparent opposite effect of temperature on the male and female side could provide plants with the plasticity to withstand changing environmental effects, ensuring a good level of fertilization.     

The function of actin-binding proteins in pollen tube growth

Pollen tube growth is a key step in sexual reproduction of higher plants. The pollen tube is a typical example of tip-growing cells and shows a polarized cytoplasm. To develop and maintain polarized growth, pollen tubes need a carefully regulated actin cytoskeleton. It is well known that actin-binding proteins are responsible for the direct control of dynamic actin filaments and serve as a link between signal transduction pathways and dynamic actin changes in determining cellular architecture. Several of these classes have been identified in pollen tubes and their detailed characterisation is progressing rapidly. Here, we aim to survey what is known about the major actin-binding proteins that affect actin assembly and dynamics, and their higher-order organisation in pollen tube growth.     

Effect of toluidine blue on pollen germination and pollen tube growth

Toluidine blue is known to induce gynogenic haploids in significant numbersin Populus]. Because the efficacy of a chemical in inducing gynogenesis depends largely on its effeot on pollen germination, on pollen tube growth, and on male gamete formation, the effect of toluidine blue (0, 1, 10 and 100 mgl-1) on these processes was studied in treated pistils ofSolatium nigrum (4 X), as well as on cultured pollen grains ofS. nigrum andTrigonella foenumgraecum. Irrespective of the time of application, toluidine blue (1 and 10 mg I-1) had no effect on pollen germination or pollen tube growth in pistils ofS. nigrum; at 100 mg I-1 it invariably inhibited both the processes. Almost similar responses were elicited by cultured pollen grains. InT. foenum-graecum toluidine blue had no effeot on pollen germination and suppressed tube growth. Gamete formation was inhibited, to various degrees, at all the concentrations tested; at 100 ing I-1 hardly any pollen tube showed gamete formation. Based on our results, and those on other systems, the potentiality of toluidine blue as an inducer of gynogenesis has been analysed.     

Phospholipids can switch the GTPase substrate preference of a GTPase-activating protein

The major cellular inhibitors of the small GTPases of the Ras superfamily are the GTPase-activating proteins (GAPs), which stimulate the intrinsic GTP hydrolyzing activity of GTPases, thereby inactivating them. The catalytic activity of several GAPs is reportedly inhibited or stimulated by various phospholipids and fatty acids in vitro, indicating a likely physiological role for lipids in regulating small GTPases. We find that the p190 RhoGAP, a potent GAP for the Rho and Rac GTPases, is similarly sensitive to phospholipids. Interestingly, however, several of the tested phospholipids were found to effectively inhibit the RhoGAP activity of p190 but stimulate its RacGAP activity. Thus, phospholipids have the ability to "switch" the GTPase substrate preference of a GAP, thereby providing a novel regulatory mechanism for the small GTPases.     

The Ran GTPase mediates chromatin signaling to control cortical polarity during polar body extrusion in mouse oocytes

The molecular basis for asymmetric meiotic divisions in mammalian oocytes that give rise to mature eggs and polar bodies remains poorly understood. Previous studies demonstrated that the asymmetrically positioned meiotic chromosomes provide the cue for cortical polarity in mouse oocytes. Here we show that the chromatin-induced cortical response can be fully reconstituted by injecting DNA-coated beads into metaphase II-arrested eggs. The injected DNA beads induce a cortical actin cap, surrounded by a myosin II ring, in a manner that depends on the number of beads and their distance from the cortex. The Ran GTPase plays a critical role in this process, because dominant-negative and constitutively active Ran mutants disrupt DNA-induced cortical polarization. The Ran-mediated signaling to the cortex is independent of the spindle but requires cortical myosin II assembly. We hypothesize that a Ran(GTP) gradient serves as a molecular ruler to interpret the asymmetric position of the meiotic chromatin.