Pollen Tube Elongation Research Articles

ZnO nanoparticles effect on pollen grain germination and pollen tube elongation

Plant germ cells, such as pollen grains, can be affected by exposure to metal nanoparticles (NPs) that have diffused into the environment. The germination and tube elongation of pollen grain (Lilium longiflorum) exposed to low-solubility NPs was observed. The germination rate of pollen grain exposed to 100 mg L−1 ZnO NP dispersion decreased significantly from controls and exposure of the other NPs. On the other hand, when pollens were exposed to ionic solutions in which particles were removed from the 100 mg L−1 ZnO NP dispersion, the germination rates were equivalent to those in the control pollens. On the contrary, decrease of the germination rate compared to controls was small in the exposure to ZnCl2 solution, which contained a larger amount of water-soluble Zn2+ than ZnO NP dispersion. From these results, it was concluded that fine ZnO NP may adhere to pollens, due to the cohesive property of nanoparticles, and Zn2+ dissolved at the interface may be continuously absorbed by pollens. When pollen was exposed to ZnO NP, a spot with a high Ca2+ local concentration was observed at the tip of the pollen tube. On the contrary, simultaneous exposure to antagonistic ZnO NP and CaCl2 resulted in no decrease in the germination rate. From the above, it is considered that upon exposure to ZnO NP, cells absorb Zn2+ depending on the specific solubility of ZnO NP. Despite the low solubility of zinc oxide nanoparticle, pollen cell-attached particles inhibited germination and elongation of pollen tube by continuous Zn2+ dissolution from particles and Zn2+ absorption by the cell.

GORI, encoding the WD40 domain protein, is required for pollen tube germination and elongation in rice.

Successful delivery of sperm cells to the embryo sac in higher plants is mediated by pollen tube growth. The molecular mechanisms underlying pollen germination and tube growth in crop plants remain rather unclear, although these mechanisms are crucial to plant reproduction and seed formation. By screening pollen-specific gene mutants in rice (Oryza sativa), we identified a T-DNA insertional mutant of Germinating modulator of rice pollen (GORI) that showed a one-to-one segregation ratio for wild type (WT) to heterozygous. GORI encodes a seven-WD40-motif protein that is homologous to JINGUBANG/REN4 in Arabidopsis. GORI is specifically expressed in rice pollen, and its protein is localized in the nucleus, cytosol and plasma membrane. Furthermore, a homozygous mutant, gori-2, created through CRISPR-Cas9 clearly exhibited male sterility with disruption of pollen tube germination and elongation. The germinated pollen tube of gori-2 exhibited decreased actin filaments and altered pectin distribution. Transcriptome analysis revealed that 852 pollen-specific genes were downregulated in gori-2 compared with the WT, and Gene Ontology enrichment analysis indicated that these genes are strongly associated with cell wall modification and clathrin coat assembly. Based on the molecular features of GORI, phenotypical observation of the gori mutant and its interaction with endocytic proteins and Rac GTPase, we propose that GORI plays key roles in forming endo-/exocytosis complexes that could mediate pollen tube growth in rice.

Arabidopsis 3-Ketoacyl-CoA Synthase 4 is Essential for Root and Pollen Tube Growth

Very long-chain fatty acids (VLCFAs) are essential precursors of membrane lipids, such as phospholipids and sphingolipids, cuticular waxes, suberins, and Brassica seed oils. The first step of VLCFA synthesis is mediated by 3-ketoacyl-CoA synthase (KCS), which catalyzes the condensation of a C2 unit from malonyl-CoA to acyl-CoA. In the present study, we investigated the role of KCS4 in pollen tube and root growth. KCS4 was predominantly expressed in shoot and root apical meristems, leaf veins, mature and germinated pollen grains, and developing embryos. The fluorescent signals of KCS4 fused with enhanced yellow fluorescent protein (KCS4:eYFP) were detected in the endoplasmic reticulum of tobacco epidermis. KCS4 disruption inhibited pollen tube elongation and root growth, whereas KCS4 promoter-driven KCS4 expression rescued the growth-retarded phenotype to wild type (WT) in kcs4 complementation lines. Root growth assay of WT and kcs4 lines treated with metazachlor and mefluidide, which are specific KCS inhibitors, and fatty acid analysis of their roots and seeds revealed that KCS4 is involved in the elongation of longer than C24 VLCFAs, which are essential for root and pollen tube growth.

Heavy metals differentially affect date palm pollen germination and tube elongation

Heavy metals differentially affect date palm pollen germination and tube elongation

The Peptide PbrPSK2 From Phytosulfokine Family Induces Reactive Oxygen Species (ROS) Production to Regulate Pear Pollen Tube Growth.

Phytosulfokines (PSKs) are plant peptide growth factors that participate in multiple biological processes, including cell elongation and immune signaling. However, little is known about PSKs in Rosaceae species. Here, we identified 10 PSK genes in pear (Pyrus bretschneideri), 11 in apple (Malus × domestica), four in peach (Prunus persica), six in strawberry (Fragaria vesca), and five in Chinese plum (Prunus mume). In addition, we undertook comparative analysis of the PSK gene family in pear and the four other species. Evolutionary analysis indicated that whole genome duplication events (WGD) may have contributed to the expansion of the PSK gene family in Rosaceae. Transcriptomes, reverse transcription-PCR and quantitative real-time-PCR analyses were undertaken to demonstrate that PbrPSK2 is highly expressed in pear pollen. In addition, by adding purified E. coli-expressed PbrPSK2 to pollen and using an antisense oligonucleotide approach, we showed that PbrPSK2 can promote pear pollen tube elongation in a dose-dependent manner. Furthermore, PbrPSK2 was found to mediate the production of reactive oxygen species to regulate pear pollen tube growth.

Establishing Pollination Requirements in Japanese Plum by Phenological Monitoring, Hand Pollinations, Fluorescence Microscopy and Molecular Genotyping.

The Japanese plum cultivars commonly grown are interspecific hybrids derived from crosses between the original Prunus salicina with other Prunus species. Most hybrids exhibit gametophytic self-incompatibility, which is controlled by a single and highly polymorphic S-locus that contains multiple alleles. Most cultivated hybrids are self-incompatible and need pollen from a compatible donor to fertilize their flowers. Establishing pollination requirements in Japanese plum is becoming increasingly important due to the high number of new cultivars with unknown pollination requirements. In this work, a methodology for the determination of pollination requirements in Japanese plum-type hybrids is described. Self-(in)compatibility is determined by hand-pollinations in both the field and in the laboratory, followed by monitoring pollen tube elongation with fluorescence microscopy, and also monitoring fruit maturation in the field. Selection of pollinizer cultivars is assessed by combining the identification of S-genotypes by PCR analysis with the monitoring of flowering time in the field. Knowing the pollination requirements of cultivars facilitates the selection of cultivars for the design of new orchards and allows the early detection of productivity problems related with pollination deficiency in established orchards.

Naturally occurring triploid birch hybrids from woodlands in Iceland are partially fertile

Woodlands in Iceland are characterized by shrub-like birch, a result of introgressive hybridization between diploid dwarf birch, Betula nana, and tetraploid downy birch, B. pubescens. Introgression occurs via triploid hybrids. We evaluated the fertility of triploid birch by examining pollen viability and seed germination. Pollen samples were collected from woodlands throughout Iceland from 99 plants, including 22 triploid hybrids. The germination of pollen from the triploid hybrids was about one third that of the diploid or tetraploid species (mean values: 9% vs. 26% and 29%). Most triploid plants had a pollen germination of less than 2%. However, some showed exceptionally high pollen fertility (six plants: 11–79%). Microscopic examination revealed normal pollen tube growth and elongation in hybrids with low germination, while other hybrids showed abnormal tube growth. Fluorescence microscopy indicated pollen viability in all ploidy groups, with green autofluorescence from the cytoplasmic contents and red fluorescence from the exine wall. The Aniline test of pollen viability showed that grains with three pores (normal, triporate grains) stained positively, but non-triporate grains were unstained and empty. Germination tests were performed on seeds collected from 246 plants, including 21 triploid hybrids. The germination percentage of seeds from triploid hybrid plants was about 20 times lower than that of the diploid or the tetraploid species (population means: 0–4% vs. 3–41% and 6–54%). The present study shows that triploid birch hybrids are not sterile. Both the paternal and maternal fertility of the hybrids should be sufficient to facilitate gene flow via backcrossing with the parental species.

Mechanics of Pollen Tube Elongation: A Perspective.

Pollen tube (PT) serves as a vehicle that delivers male gametes (sperm cells) to a female gametophyte during double fertilization, which eventually leads to the seed formation. It is one of the fastest elongating structures in plants. Normally, PTs traverse through the extracellular matrix at the transmitting tract after penetrating the stigma. While the endeavor may appear simple, the molecular processes and mechanics of the PT elongation is yet to be fully resolved. Although it is the most studied “tip–growing” structure in plants, several features of the structure (e.g., Membrane dynamics, growth behavior, mechanosensing etc.) are only partially understood. In many aspects, PTs are still considered as a tissue rather than a “unique cell.” In this review, we have attempted to discuss mainly on the mechanics behind PT–elongation and briefly on the molecular players involved in the process. Four aspects of PTs are particularly discussed: the PT as a cell, its membrane dynamics, mechanics of its elongation, and the potential mechanosensors involved in its elongation based on relevant findings in both plant and non-plant models.

AtMYB109 negatively regulates stomatal closure under osmotic stress in Arabidopsis thaliana

Osmotic stress, caused by drought, salinity, or PEG (polyethylene glycol), is one of the most important abiotic factors that hinder plant growth and development. In Arabidopsis, more than 100 R2R3-MYB transcription factors (TFs) have been identified, and many of them are involved in the transcriptional regulation of a variety of biological processes related to growth and development, as well as responses to biotic and abiotic stresses. However, the MYB TF involving in both plant development and stress response has rarely been reported. We report here that Arabidopsis AtMYB109, a R2R3-MYB TF, functions as a negative regulator of stomatal closure under osmotic stress as well as of pollen tube elongation. Under PEG-induced osmotic stress, whole leaves of AtMYB109-OXs were intensely wilted, while leaves of the wild-type (WT) and myb109 were weakly affected. Moreover, we confirmed that the wilting in AtMYB109-OXs was more severe than in WT and myb109 under drought conditions, and that after re-watering, WT and myb109 plants promptly recovered, while AtMYB109-OXs failed to survive. In addition, stomatal closure was delayed in the AtMYB109-OXs compared to the WT and myb109. However, proline content and the expression of stress-induced and proline synthesis genes were higher in the overexpression lines than in WT and myb109. Then, we observed that the expression of ICS1, a key gene in SA biosynthesis, was greatly suppressed in AtMYB109-OXs. In addition, we found that AtMYB109 expression gradually increased until the flowers were fully opened and thereafter dramatically decreased during silique development. The pollen tube growth was significantly suppressed in AtMYB109-OXs compared to the WT and myb109. Using EMSA and ChIP-qPCR, we confirmed that AtMYB109 bound to the promoter of RABA4D, a gene encoding a pollen development regulator. Taken together, we suggest the delayed stomatal closing and vulnerable phenotypes in the AtMYB109-OXs under osmotic stress are possibly directly or indirectly associated with a SA-mediated mechanism, and that AtMYB109 suppresses RABA4D that modulates pollen tube growth.

Pectin Synthesis and Pollen Tube Growth in Arabidopsis Involves Three GAUT1 Golgi-Anchoring Proteins: GAUT5, GAUT6, and GAUT7.

The major cell wall pectic glycan homogalacturonan (HG) is crucial for plant growth, development, and reproduction. HG synthesis occurs in the Golgi and is catalyzed by members of the galacturonosyltransferase (GAUT) family with GAUT1 being the archetypal and best studied family member. In Arabidopsis suspension culture cells and tobacco leaves, the Golgi localization of Arabidopsis GAUT1 has been shown to require protein-protein interactions with its homolog GAUT7. Here we show that in pollen tubes GAUT5 and GAUT6, homologs of GAUT7, also target GAUT1 to the Golgi apparatus. Pollen tube germination and elongation in double homozygous knock-out mutants (gaut5 gaut6, gaut5 gaut7, and gaut6 gaut7) are moderately impaired, whereas gaut5 −/− gaut6 −/− gaut7 +/− triple mutant is severely impaired and male infertile. Amounts and distributions of methylesterified HG in the pollen tube tip were severely distorted in the double and heterozygous triple mutants. A chimeric protein comprising GAUT1 and a non-cleavable membrane anchor domain was able to partially restore pollen tube germination and elongation and to reverse male sterility in the triple mutant. These results indicate that GAUT5, GAUT6, and GAUT7 are required for synthesis of native HG in growing pollen tubes and have critical roles in pollen tube growth and male fertility in Arabidopsis.

Cytological Observations and In Vitro Germination of Nipa (Nypa fruticans Wurmb., Arecaceae) Pollen

The staminate rachillae from laterals of nipa (Nypa fruticans Wurmb.) at different stages of inflorescence development were sampled for cytological observations using squash technique, with 2% acetocarmine as the stain. Results showed that the dividing microsporocytes exhibited a tetrad in tetragonal shape enclosed in thick callose walls at the end of meiosis II. The microspore products consisted not only of uninucleate but also of binucleate and trinucleate pollen during anthesis. Meiotic stages were interpreted as highly normal, which suggests high pollen viability. In vitro germination observed every 4-h intervals for 24 h showed that sucrose level at 15% attained significantly highest germination at 53%. The germinated pollen developed a knob protrusion instead of an elongated pollen tube. There was a significant difference in pollen knob length at 15% sucrose measuring 9.6 µm, the longest observed, compared to the control. With significant interaction between sucrose and boron levels, 10% and 15% sucrose levels achieved higher in vitro germination when combined with 0.01% and 0.005% boron, respectively. Indeed, sucrose and boric acid consisting of the liquid medium can initiate nipa pollen germination, confirming earlier results with lower concentrations.

The nuclear envelope protein KAKU4 determines the migration order of the vegetative nucleus and sperm cells in pollen tubes.

A putative component protein of the nuclear lamina, KAKU4, modulates nuclear morphology in Arabidopsis thaliana seedlings, but its physiological significance is unknown. KAKU4 was highly expressed in mature pollen grains, each of which has a vegetative cell and two sperm cells. KAKU4 protein was highly abundant on the envelopes of vegetative nuclei and less abundant on the envelopes of sperm cell nuclei in pollen grains and elongating pollen tubes. Vegetative nuclei are irregularly shaped in wild-type pollen. However, KAKU4 deficiency caused them to become more spherical. After a pollen grain germinates, the vegetative nuclei and sperm cells enter and move along the pollen tube. In the wild type, the vegetative nucleus preceded the sperm cell nuclei in >90% of the pollen tubes, whereas, in kaku4 mutants, the vegetative nucleus preceded the sperm cell nuclei in only about half of the pollen tubes. kaku4 pollen was less competitive for fertilization than wild-type pollen after pollination. These results led us to hypothesize that the nuclear shape in vegetative cells of pollen grains affects the orderly migration of the vegetative nucleus and sperm cells in pollen tubes.

Comprehensive analysis of polygalacturonase gene family highlights candidate genes related to pollen development and male fertility in wheat (Triticum aestivum L.).

Four polygalacturonase gene family members were highlighted that contribute to elucidate the roles of polygalacturonase during the fertility conversion process in male-sterile wheat. Polygalacturonase (PG) belongs to a large family of hydrolases with important functions in cell separation during plant growth and development via the degradation of pectin. Specific expressed PGs in anthers may be significant for male sterility research and hybrid wheat breeding, but they have not been characterized in wheat (Triticum aestivum L.). In this study, we systematically studied the PG gene family using the latest published wheat reference genomic information. In total, 113 wheat PG genes were identified, which could be classified into six categories A-F according to their structure characteristics and phylogenetic comparisons with Arabidopsis and rice. Polyploidy and segmental duplications in wheat were proved to be mainly responsible for the expansion of the wheat PG gene family. RNA-seq showed that TaPGs have specific temporal and spatial expression characteristics, in which 12 TaPGs with spike-specific expression patterns were detected by qRT-PCR in different fertility anthers of KTM3315A, a thermo-sensitive cytoplasmic male-sterile wheat. Four of them specific upregulated (TaPG09, TaPG95, and TaPG93) or downregulated (TaPG87) at trinucleate stage of fertile anthers, and further aligning with the homologous in Arabidopsis revealed that they may undertake functions such as anther dehiscence, separation of pollen, pollen development, and pollen tube elongation, thereby inducing male fertility conversion in KTM3315A. These findings facilitate function investigations of the wheat PG gene family and provide new insights into the fertility conversion mechanism in male-sterile wheat.

Effect of methyl jasmonate on in-vitro pollen germination and tube elongation of Pinus nigra.

The purpose of the main research was to investigate the effects of methyl jasmonate (MeJA) (0.05, 0.25, 0.5, and 2.5mM) on the pollen germination and tube elongation of Pinus nigra. Total pollen germination rate increased after MeJA treatments while the most enhancement was observed at 0.05-mM MeJA. No germination was observed at 2.5-mM MeJA. Although the unipolar and bipolar germination were observed in all groups, no significant changes were observed in unipolar and bipolar pollen germination rates after MeJA treatments. Tube length increased only at 0.05-mM MeJA. Although branched tubes were observed in all groups, branched tube rate increased only at 0.05-mM MeJA. Although two branched, three branched, and consecutive branched tubes were observed in all groups, the most common branching type was two branched type in all groups. Although anisotropy of actin filaments in the shank and apex of unbranched tubes decreased after MeJA treatments, the most decrease was observed at 0.05-mM MeJA. Also, anisotropy of actin filaments in the shank and in pre-branching region of branched tubes decreased only at 0.25-mM MeJA. Anisotropy of both two apexes of a branched tube changed only at 0.25- and 0.5-mM MeJA. Callose accumulation in the apex of unbranched and branched tubes increased in parallel with the increase in MeJA concentration. However, more callose is accumulated in one apex than the other apex of a branched tube. In conclusion, MeJA affected the actin organization, changed the callose distribution, and altered the pollen tube growth of Pinus nigra.

OsAPT1 a Pollen Preferentially Expressed Gene Is Essential for Pollen Tube Germination and Elongation in Rice

Pollen tube germination and elongation are important processes for double fertilization in flowering plants. However, little is known about the regulatory mechanism of pollen germination and tube growth in rice. Here, we reported that OsAPT1, a pollen-high expression gene and encoding a putative APT1 protein in Oryza sativa, regulating pollen tube germination and elongation in rice. Real-time quantitative PCR (qRT-PCR) and in situ hybridization analysis showed high expression level of OsAPT1 at late stage of microsporogenesis including late uninucleate stage, binucleate pollen stage, and mature pollen grains. OsAPT1 RNA interference (OsAPT1-RNAi) lines showed that the pollen tube germination and elongation rate were decreased. Compared with wild type (WT), the integrity and assembly of actin microfilaments were abnormal in hydrated pollen grains of OsAPT1-RNAi plants, and the fluorescence signals of actin microfilaments were altered in the pollen tube of OsAPT1-RNAi plants. GO analysis of the co-expressing genes of OsAPT1 showed that the GO terms such as pollination, cell growth, cellular cytoskeleton organization, and motor activity were enriched. In addition, the qRT-PCR results showed variations in the expression of some genes related to pollen tube growth. Overall, our results indicated that OsAPT1 is required for pollen tube germination and elongation in rice.

Interaction Between AtCML9 and AtMLO10 Regulates Pollen Tube Development and Seed Setting.

In higher-plant reproduction, the compatibility of pollen tube germination in the pistil is essential for successful double fertilization. It has been reported that Mildew Locus O (MLO) family gene NTA (MLO7), expressing in synergid cells, can correctly guide pollen tubes. However, the molecular mechanism underlying the interacting partners to MLOs in the fertilization is still unknown. In our study, we identified the direct protein interaction between CML9 and MLO10 within a non-canonical CaMBD. In GUS reporter assays, CML9 expresses in a high level in pollens, whereas MLO10 can be specifically detected in stigma which reaches up to a peaking level before fertilization. Therefore, the spatio-temporal expression patterns of MLO10 and CML9 are required for the time-window of pollination. When we observed the pollen germination in vitro, two cml9 mutant alleles dramatically reduced germination rate by 15% compared to wild-type. Consistently, the elongation rate of pollen tubes in planta was obviously slow while manually pollinating cml9-1 pollens to mlo10-1 stigmas. Additionally, cml9-1 mlo10-1 double mutant alleles had relatively lower rate of seed setting. Taken together, protein interaction between MLO10 and CML9 is supposed to affect pollen tube elongation and further affect seed development.

Tomato SlIDA has a critical role in tomato fertilization by modifying reactive oxygen species homeostasis.

Anther development and pollen tube elongation are key steps for pollination and fertilization. The timing and spatial distribution of reactive oxygen species (ROS) and programmed cell death are central to these processes, but the regulatory mechanism of ROS production is not well understood. Inflorescence deficient in abscission (IDA) is implicated in many plant development and responses to environmental stimuli. However, their role in reproductive development is still unknown. We generated tomato knockout lines (CR-slida) of an IDA homolog (SlIDA), which is expressed in the tapetum, septum and pollen tube, and observed a severe defect in male gametes. Further analysis indicated that there was a programmed cell death defect in the tapetum and septum and a failure of anther dehiscence in the CR-slida lines, likely related to insufficient ROS signal. Liquid chromatography-tandem mass spectrometry identified mature SlIDA as a 14-mer EPIP peptide, which was shown to be secreted, and a complementation experiment showed that application of a synthetic 14-mer EPIP peptide rescued the CR-slida defect and enhanced the ROS signal. Moreover, the application of the ROS scavengers diphenyleneiodonium or Mn-TMPP suppressed peptide function. Collectively, our results revealed that SlIDA plays an essential role in pollen development and pollen tube elongation by modulating ROS homeostasis.

Physiological Importance of Pectin Modifying Genes During Rice Pollen Development.

Although cell wall dynamics, particularly modification of homogalacturonan (HGA, a major component of pectin) during pollen tube growth, have been extensively studied in dicot plants, little is known about how modification of the pollen tube cell wall regulates growth in monocot plants. In this study, we assessed the role of HGA modification during elongation of the rice pollen tube by adding a pectin methylesterase (PME) enzyme or a PME-inhibiting catechin extract (Polyphenon 60) to in vitro germination medium. Both treatments led to a severe decrease in the pollen germination rate and elongation. Furthermore, using monoclonal antibodies toward methyl-esterified and de-esterified HGA epitopes, it was found that exogenous treatment of PME and Polyphenon 60 resulted in the disruption of the distribution patterns of low- and high-methylesterified pectins upon pollen germination and during pollen tube elongation. Eleven PMEs and 13 PME inhibitors (PMEIs) were identified by publicly available transcriptome datasets and their specific expression was validated by qRT-PCR. Enzyme activity assays and subcellular localization using a heterologous expression system in tobacco leaves demonstrated that some of the pollen-specific PMEs and PMEIs possessed distinct enzymatic activities and targeted either the cell wall or other compartments. Taken together, our findings are the first line of evidence showing the essentiality of HGA methyl-esterification status during the germination and elongation of pollen tubes in rice, which is primarily governed by the fine-tuning of PME and PMEI activities.

Secondary Outcomes of the Ole e 1 Proteins Involved in Pollen Tube Development: Impact on Allergies.

Ole e 1 protein is involved in olive fertilization mechanisms controlling pollen tube development. Similarly to the process by which pollen grains hydrated and form a pollen tube upon arrival at the female gametophyte, when pollen grains fall on the nasal mucosa the expression of Ole e 1 protein induce allergic reaction in sensitive individuals. The research was conducted in Ourense (North-western Spain), during the 2009–2018 period. Ole e 1 protein was collected using a Cyclone Sampler and processed with the ELISA methodology. Airborne Olea pollen were monitored using a Hirst type volumetric sampler. Allergy risk episodes identified by pollen concentrations were detected in five of the 10 studied years, all with moderate risk. Actual risk episodes of allergy increased when the combination of pollen and Ole e 1 concentrations were considered. Moderate risk episodes were detected during 9 years and high-risk episodes during 3 years. In addition, some years of low annual pollen concentrations recorded high total amounts of Ole e 1. During the years with lower pollen production, the tree increases the synthesis of Ole e 1 to ensure proper pollen tube elongation in order to complete a successful fertilization. This fact could justify higher sensitization rates in years in which a lower pollen production is expected. The present method contributes to the determination of the real exposure to Ole e 1 allergen evaluating the role of this protein as an aeroallergen for sensitized population. The allergen content in the atmosphere should be considered to enhance the prevention of pollinosis clinical symptomatology and the reduction of medicine consumption.

Quantitative Structural Organization of Bulk Apical Membrane Traffic in Pollen Tubes.

Pollen tube tip growth depends on balancing secretion of cell wall material with endocytic recycling of excess material incorporated into the plasma membrane (PM). The classical model of tip growth, which predicts bulk secretion, occurs apically, and is compensated by subapical endocytosis, has been challenged in recent years. Many signaling proteins and lipids with important functions in the regulation of membrane traffic underlying tip growth associate with distinct regions of the pollen tube PM, and understanding the mechanisms responsible for the targeting of these regulatory factors to specific PM domains requires quantitative information concerning the sites of bulk secretion and endocytosis. Here, we quantitatively characterized the spatial organization of membrane traffic during tip growth by analyzing steady-state distributions and dynamics of FM4-64-labeled lipids and YFP-tagged transmembrane (TM) proteins in tobacco (Nicotiana tabacum) pollen tubes growing normally or treated with Brefeldin A to block secretion. We established that (1) secretion delivers TM proteins and recycled membrane lipids to the same apical PM domain, and (2) FM4-64-labeled lipids, but not the analyzed TM proteins, undergo endocytic recycling within a clearly defined subapical region. We mathematically modeled the steady-state PM distributions of all analyzed markers to better understand differences between them and to support the experimental data. Finally, we mapped subapical F-actin fringe and trans-Golgi network positioning relative to sites of bulk secretion and endocytosis to further characterize functions of these structures in apical membrane traffic. Our results support and further define the classical model of apical membrane traffic at the tip of elongating pollen tubes.