Plant Reproduction

In the Brassicaceae, the process of accepting compatible pollen is a key step in successful reproduction and highly regulated following interactions between the pollen and the stigma. Central to this, is the initiation of secretion in the stigma, which is proposed to provide resources to the pollen for hydration and germination and pollen tube growth. Previously, the eight exocyst subunit genes were shown to be required in the Arabidopsis stigma to support these pollen responses. One of the roles of the exocyst is to tether secretory vesicles at the plasma membrane for membrane fusion by the SNARE complex to enable vesicle cargo release. Here, we investigate the role of Arabidopsis SNARE genes in the stigma for pollen responses. Using a combination of different knockout and knockdown SNARE mutant lines, we show that VAMP721, VAMP722, SYP121, SYP122 and SNAP33 are involved in this process. Significant disruptions in pollen hydration were observed following pollination of wildtype pollen on the mutant SNARE stigmas. Overall, these results place the Arabidopsis SNARE complex as a contributor in the stigma for pollen responses and reaffirm the significance of secretion in the stigma to support the pollen-stigma interactions. Key MessageThe VAMP721, VAMP722, SYP121, SYP122 and SNAP33 SNAREs are required in the Arabidopsis stigma for pollen hydration, further supporting a role for vesicle trafficking in the stigmas pollen responses.

The tapetum is a specialized layer of cells within the anther adjacent to the sporogenic tissue. During its short life, it provides nutrients, molecules and materials to the pollen mother cells and microsporocytes being essential during callose degradation and pollen wall formation. However, the acquisition of tapetal cell identity in tomato plants is a process still poorly understood. We report here the identification and characterization of SlTPD1 (Solanum lycopersicum TPD1), a gene specifically required for pollen development in tomato plants. Gene editing was used to generate loss-of-function Sltpd1 mutants that showed absence of tapetal tissue. In these plants, sporogenous cells developed but failed to complete meiosis resulting in complete male sterility. Transcriptomic analysis conducted in wild-type and mutant anthers at an early stage revealed the down regulation of a set of genes related to redox homeostasis. Indeed, Sltpd1 anthers showed a reduction of reactive oxygen species (ROS) accumulation at early stages and altered activity of ROS scavenging enzymes. The obtained results highlight the importance of ROS homeostasis in the interaction between the tapetum and the sporogenous tissue in tomato plants. One sentence summaryThe small protein SlTPD1 is required for tapetum formation in tomato, highlighting the role of this tissue in the regulation of redox homeostasis during male gametogenesis.

In angiosperms, pollen tubes deliver sperm cells to the ovule and communicate with the external environment as they elongate through the pistils. Although pollination alters Ca2+ conditions within the pistil, the effects of extracellular Ca2+ fluctuations on pollen tube growth and guidance remain largely unknown. In this study, we visualized intracellular Ca2+ dynamics using a semi-in vivo assay with the Ca2+-sensitive fluorescent protein GCaMP6s to investigate how pollen tubes respond to changes in extracellular Ca2+ levels. We found that the Ca2+ levels in the apical region of the pollen tubes reflected the extracellular Ca2+ concentrations. The pollen tube growth rate increased depending on the Ca2+ concentration in the growth medium. However, excessive Ca2+ affected the polar growth of pollen tubes. At elevated Ca2+ concentrations of 10 mM, the pollen tube exhibited coiling behavior and failed to maintain directional growth toward the ovule. Moreover, we provided the first evidence that Ca2+ oscillations are not restricted to the apical region but propagate as a wave, reaching 30-50 m from the apex toward the basal regions. As the pollen tube approached the ovule, it coincided with a substantial elevation in Ca2+ levels, which appeared to drive the accelerated nuclear migration toward the tube apex. Our findings demonstrate that the extracellular Ca2+ environment directly regulates intracellular Ca2+ levels in pollen tubes, thereby influencing their growth and guidance.

Efficient transport and delivery of sperm cells (SCs) is vital for angiosperm plant fertility. In Arabidopsis thaliana, SCs are transported through the growing pollen tube by a connection with the vegetative nucleus (VN). During pollen tube growth, the VN leads the way and maintains a fixed distance from the pollen tube tip, while the SCs lag behind the VN. Upon reception at the ovule, the pollen tube bursts and the SCs are released for fertilization. In pollen tubes of Arabidopsis mutants wit12 and wifi, deficient in the outer nuclear membrane component of a plant LINC complex, the SCs precede the VN and the VN falls behind. Subsequently, pollen tubes frequently fail to burst upon reception. In this study, we sought to determine if the pollen tube reception defect observed in wit12 and wifi is due to decreased sensitivity to reactive oxygen species (ROS). Here we show that wit12 and wifi are hyposensitive to exogenous H2O2, and that this hyposensitivity is correlated with decreased proximity of the VN to the pollen tube tip. Additionally, we report the first instance of nuclear Ca2+ spikes in growing pollen tubes, which are disrupted in the wit12 mutant. In the wit12 mutant, nuclear Ca2+ spikes are reduced in response to exogenous ROS, but these spikes are not correlated with pollen tube burst. This study finds that VN proximity to the pollen tube tip is required for both response to exogenous ROS, as well as internal nuclear Ca2+ fluctuations. SummaryMutants deficient in outer nuclear membrane proteins display defects in reactive oxygen species-induced pollen tube burst and nuclear Ca2+ signatures that correlate with the position of the vegetative nucleus.

Major constituents of the plant cell walls are structural proteins that belong to the Hydroxyproline-rich glycoprotein family. Leucine-rich repeat extensis are contain a leucine-rich domain and a C-terminal domain with repetitive Ser-Pro(3-5) motifs plausible to be glycosylated. We have demonstrated that pollen-specific LRX8-11 from Arabidopsis thaliana are necessary to maintain the integrity of the pollen tube cell wall during polarized growth. In classical EXTs and likely in LRXs, proline residues are converted to hydroxyproline by Prolyl-4-hydroxylases, thus defining novel O-glycosylation sites. In this context, we aimed to determine whether hydroxylation and subsequent O-glycosylation of Arabidopsis pollen LRXs are necessary for their proper function and cell wall localization in pollen tubes. We hypothesized that pollen-expressed P4H4 and P4H6 catalyze the hydroxylation of the proline units present in Ser-Pro(3-5) motifs of LRX8-LRX11. Here, we show the p4h4-1 p4h6-1 double mutant exhibits a significant reduction in pollen germination rates and a slight reduction in pollen tube length. Pollen germination is also inhibited by specific P4Hs inhibitors, suggesting that prolyl hydroxylation is required for pollen tube development. Plants expressing pLRX11::LRX11-GFP in the p4h4-1 p4h6-1 background show partial relocalization of LRX11-GFP from the pollen tube tip apoplast to the cytoplasm. Finally, IP-MS- MS analysis revealed a decrease in oxidized prolines in LRX11-GFP in the p4h4-1 p4h6-1 background when compared to lrx11 plants expressing pLRX11::LRX11-GFP. Together, these results suggest that P4H4 and P4H6 are required for pollen germination and are also involved in LRX11 hydroxylation necessary for its localization at the cell wall of pollen tubes. One Sentence SummaryPollen-expressed P4H4 and P4H6 are required for pollen germination and for proper hydroxylation and secretion of LRX11 in pollen tubes.

Live-imaging microscopy technology has been increasingly applied for meiosis study in plants, which largely relies on the set up of a healthy ex vivo culture system for inflorescences ensuring that the captured chromosomes dynamics approaches the natural features of meiosis. Here, we report that Arabidopsis thaliana flowers cultivated in a culture medium (CCM) composed of the half-strength Murashige and Skoog basal salt, MES, Myo-inositol, sucrose and agar produce diploid microspores due to occurrence of meiotic restitution. Cytological studies revealed adjacent nuclei distribution and incomplete cytokinesis at late meiosis II in meiocytes within the CCM flowers. Immunolocalization of -tubulin and the microtubule-associated protein MAP65-3 showed that the orientation of spindles at metaphase II and the organization of radial microtubule arrays at the tetrad stage are interfered, which explains the production of meiotically-restituted microspores. Moreover, the CCM flowers showed a gradually impaired expression of Aborted Microspores (AMS), a key transcription factor regulating tapetum development and meiotic cytokinesis. Interestingly, an increased supply of sucrose in culture medium promoted the expression of AMS and partially rescued haploid microspore formation in the CCM flowers. Taken together, this study suggests a role of sucrose in facilitating meiotic cytokinesis and gametophytic ploidy stability in plants. One-sentence summaryArabidopsis flowers cultivated in culture medium produce unreduced microspores due to interfered meiotic cytokinesis, which is partially rescued by increased sucrose supply.

The plant male reproductive system is very sensitive to high temperature stress leading to a reduction in fertility. Damage caused by heat stress is restored by the activation of transcription and the synthesis of chaperones that regulate the heat stress response. Here we report that AtRRB1 is a homolog of the yeast ribosome chaperone protein Rrb1p. AtRRB1 is an essential gene and a T-DNA insertion in the coding region impairs male and female gametogenesis. The heterozygous rrb1-1 mutant shows decreased expression of AtRRB1 and increased transcription of the 60S ribosome proteins RPL3B and RPL4, in line with a chaperone role of AtRRB1 in ribosome biogenesis. Embryo sac development across ovules of a single pistil occurs uncoordinated and about half of the ovules abort. Half of rrb1-1 pollen is substantially smaller and produce shorter pollen tubes than WT pollen. In contrast to the Col-0 pollen, smaller pollen is overly sensitivity to high temperature (24h at 32{degrees}C) treatment, specifically during the early bicellular microspore development stage. Heat stressed rrb1-1 bicellular microspores accumulated excessively rough endoplasmic reticulum stacks, suggesting that loss of AtRRB1 activity causes an arrest in ER associated protein biosynthesis. These findings support a critical requirement for ribosome biogenesis and protein synthesis in bicellular microspores to recover from high temperature stress.

Self-incompatibility is one of the mechanisms preventing inbreeding in populations, based on the failure of pollen grains to germinate or of pollen tubes to grow normally in incompatible crosses. A unique model for studying this process are heterostylous species, in which self-incompatibility alleles are closely linked to flower morphological traits. One such species is L. perenne, a classic distylous species with reciprocal herkogamy and complete self-incompatibility strongly linked. The mechanism of self-pollen recognition is poorly understood in most heterostylous species, including L. perenne. As self-incompatibility in other systems is based on protein-protein interactions and many extracellular proteins are glycosylated, one of the possible participants in this process may be lectins. Therefore, we aimed to investigate the lectin activity in pistils and stamens of L. perenne L-morph and S-morph plants and to establish whether these lectins take part in self-incompatibility. We show that pistil-derived galactose-specific Ca2+-independent lectins from the respective other morph can overcome self-incompatibility in heterostylous L. perenne. This may be due to the presence of some lectins that are specific for arabinose and xylose. Our results suggest that lectins participate in signaling pathways for recognition at the pollen-stigma interface and are involved in the regulation of the self-incompatibility process in L. perenne.

O_LISeeds slowly accumulate damage during storage, which ultimately results in germination failure. The seed coat is the barrier between the embryo and the external environment, and its composition is critical for seed longevity. Flavonols accumulates in the outer integument, but the effect of altering flavonol composition on outer integument development has not been explored. C_LIO_LIGenetic, biochemical, ultrastructural and transcriptomics assays on a battery of loss-of-function mutants in the flavonoid biosynthesis pathway were used to study the effect of altered flavonoid composition in seed development and seed longevity. C_LIO_LIControlled deterioration assays indicate that loss-of-function of the flavonoid 3 hydroxylase TT7 gene dramatically affects seed longevity and seed development. Seed outer integument differentiation is compromised from nine days after pollination in tt7 seeds, with a defective suberine layer and incomplete degradation of seed-coat starch. These distinctive phenotypes are not shared by other mutants showing also altered flavonoid composition. Double-mutant analysis indicate that over-accumulation of kaempferol is the primary cause of the observed phenotypes. Expression analysis suggest that the tt7 flavonoid pattern affects transcriptional and non-transcriptional levels of regulation. C_LIO_LIThe increase of kaempferols in the seed coat influences seed development. This positions TT7 as an essential player modulating seed coat development and seed longevity. C_LI

In Arabidopsis, a dry stigma surface enables a gradual hydration of pollen grains by a controlled release of water. Occasionally the grains may be exposed to extreme precipitations that cause rapid water influx, swelling and eventually lead to pollen membrane (PM) rupture. In metazoans, calcium- and phospholipids-binding proteins, referred to as annexins participate in repair of the plasma membrane damages. It remains unclear, however, how this process is conducted in plants. Here, we examined whether the plant annexin 5 (ANN5), the most abundant member of the annexin family in pollen, is involved in the restoration of PM integrity. We analyzed a cellular dynamics of ANN5 in the pollen grains undergoing in vitro and in vivo hydration. We observed a transient ANN5 association to PM during the in vitro hydration that did not occur in the pollen grains being hydrated on the stigma. To simulate a rainfall, we performed spraying of the pollinated stigma with deionized water that induced ANN5 accumulation at PM. Similarly, calcium or magnesium application affected PM properties and induced ANN5 recruitment to PM. Our data suggest a model, in which ANN5 is involved in the maintenance of membrane integrity in pollen grains exposed to osmotic or ionic imbalances.

Arabidopsis lines with loss-of-function mutations in the gene encoding Embryo sac Pectin MethylEsterase Inhibitor (Atepmei) were found to have short silique and high seed sterility. Examination of tissue-cleared mature ovules (FG7-stage) revealed irregularly positioned nuclei within the embryo sacs. Instead of horse-shoe-shaped ovules, defective globular ovules without proper micropylar and chalazal ends were found. Embryo sac cell-type-specific GFP marker expression studies confirmed gamete and accessory cell identity alterations. Egg cell-specific marker (DD45) expression analysis confirmed the presence of multiple egg cells in the mutant embryo sacs, possibly due to defect in embryo sac cellularization. These supernumerary egg cells were functional as evident from the production of twin embryos when supernumerary sperm cells were provided. The results of Ruthenium red and tannic acid-ferric chloride staining of Atepmei mutant developing ovules, conferred its interaction with the specific PME in proper cell wall formation and maintenance around embryo sac nuclei which also coincide with its fate as a specific gamete. This is the first report implicating role of cell wall in gamete cell fate determination by altering cell-cell communication. Our analysis of the twin-embryo phenotype of epmei mutants also sheds light on the boundary conditions for double fertilization in plant reproduction.

In Brassicaceae self-incompatibility (SI), self-pollen rejection is initiated by the S-haplotype specific interactions between the pollen SCR/SP11 ligand and the stigma S Receptor kinase (SRK). In Brassica SI, a member of the Plant U-Box (PUB) E3 ubiquitin ligases, ARC1, is then activated by SRK in this stigma and cellular events downstream of this cause SI pollen rejection by inhibiting pollen hydration and pollen tube growth. During the transition to selfing, Arabidopsis thaliana lost the SI components, SCR, SRK, and ARC1. However, this trait can be reintroduced into A. thaliana by adding back functional copies of these genes from closely related SI species. Both SCR and SRK are required for this, though the degree of SI pollen rejection varies between accessions, and ARC1 is not always needed to produce a strong SI response. For A. thaliana C24, only transforming with A. lyrata SCR and SRK confers a strong SI trait, and so here we investigated if ARC1-related PUBs were involved in the SI pathway. Two close ARC1 paralogs, PUB17 and PUB16, were selected, and CRISPR/Cas9 technology was used to generate pub17 and pub16 mutations in the C24 accession. These mutants were then crossed into a transgenic A. thaliana SI-C24 line and their potential impact on SI pollen rejection was investigated. Overall, we did not observe any significant differences to implicate PUB17 and PUB16 functioning in the transgenic A. thaliana SI-C24 stigma to reject SI pollen.

In flowering plants, the growth of pollen tubes carrying sperm cells to the female gametophyte is necessary for double fertilization and seed development. The rate of growth of the pollen tube tip determines the fertility of many artificially cultivated plants and depends on the vesicle-mediated transport of cell membrane and cell wall components. However, the investigation of vesicle transport is hampered by the lack of transgenic methods for economically important plants. Here, we developed a method to transiently inhibit vesicle activity using brefeldin A (BFA) and antisense oligodeoxynucleotides (AS-ODNs) targeting key genes in the wishbone flower (Torenia fournieri), which has been used in studies of sexual reproduction in plants. BFA disrupted vesicle gradient homeostasis, thereby altering cell wall deposition and pollen tube morphology. The AS-ODN targeting Torenia fournieri ANXUR (TfANX), which is implicated in maintaining pollen tube growth and is used as a marker to assess inhibition by AS-ODNs, penetrated cell membranes and inhibited the expression of target genes. The treatment of pollen tubes with AS-ODN against TfRABA4D, which is specifically expressed in pollen and involved in regulating vesicle targeting, resulted in pollen tubes with a bulging phenotype and disrupted pectin deposition, effects similar to those of BFA. In summary, vesicle-mediated mechanisms regulate the patterning of the pollen tube cell wall in T. fournieri and our findings could facilitate the genetic manipulation of horticultural crops.

3-PHOSPHOINOSITIDE-DEPENDENT PROTEIN KINASE1 (PDK1), a conserved master regulator of AGC kinases, is encoded by two redundant genes in Arabidopsis thaliana, PDK1 and PDK2. pdk1 pdk2 mutants exhibit a broad range of defects, including apolar or arrested pollen tube growth, a phenotype also observed in agc1.5 agc1.7 mutants. Pollen-specific expression of constitutively active AGC1.5 in pdk1 pdk2 restores polar pollen tube growth, indicating that PDK1 functions upstream of redundant AGC1.5/AGC1.7 signaling in this process. In contrast, the PDK1 splice variant PDK1S0, lacking the phospholipid-binding PH domain, cannot restore polar pollen tube growth. Our results indicate a key role for the phospholipid PI(4,5)P2 in recruiting PDK1 through its PH domain to establish polar pollen tube growth, as PI(4,5)P2 marks the pollen germination initiation site together with PDK1, it forms a dome at the plasma-membrane of the pollen tube tip beneath which PDK1 remains largely cytosolic and exhibits reciprocal feedback regulation with the PDK1-AGC1.5/1.7 kinases. Defects in endocytosis and actin organization further support that phospholipid-dependent PDK1-AGC signaling maintains pollen tube growth polarity.

Leaf meristem is a cell proliferative zone present in the lateral organ primordia. In this study, we investigated how the proliferative zone affects the final morphology of the lateral organs. We examined how cell proliferative zones differ in the primordia of planar floral organs and polar auxin transport inhibitor (PATI)-treated leaves from normal foliage leaf primordia of Arabidopsis thaliana with a focus on the spatial accumulation pattern of ANGUSTIFOLIA3 (AN3), a key element for leaf meristem positioning. We found that organ shape changes by PATI treatment were correlated to the angle of the cell division plane relative to the leaf primordia axis in the leaf meristem (cell division angle), but not with leaf-meristem positioning, size of the leaf meristem, or the localization pattern of AN3 protein. In contrast, different shapes between sepals and petals compared with foliage leaves were associated with both altered meristem position associated with altered AN3 expression patterns and different distributions of cell division angles. These results suggest that lateral organ shapes are regulated via two aspects: position of meristem and cell division angles Summary statementDifferent lateral organs with different morphology possess different properties of meristems; cell division angles, position of cell proliferative area and AN3 localization patterns.

The pollen tube plays a pivotal role in double fertilization by delivering sperm cells (SCs) to the ovule. In Arabidopsis thaliana, a pair of SCs tightly connects with the vegetative nucleus (VN) to form the male germ unit (MGU), which is located in the apical region during pollen-tube growth, keeping the VN ahead of the SCs. MGU transport relies on independent motility of VN and SC pairs. However, the complexity of this dual motive force has hindered our understanding of MGU behavior, including its positioning and nuclear order. We used Arabidopsis mutants or transgenic plants that produced semi-motile MGUs with aberrant VNs or SCs to analyze the positioning of VN or SCs after stochastically disconnecting the MGU. In pollen tubes with an immotile SC pair, the VN was [~]70 m away from the tip, whereas in pollen tubes with an immotile VN, the SC pair was [~]100 m away from the tip, implying that the VN and SCs have independent home positions. The position of MGU moved forward owing to the loss of the microtubule-destabilizing kinesin KINESIN-13A. Conversely, microtubule depolymerization by oryzalin treatment or introducing mutations in TUBULIN BETA 4 (TUB4) deregulated the position of the MGU and shifted its position backward. In addition, tub4 plants exhibited reduced fertility. These data indicate a significant role of microtubules in stable MGU positioning to ensure reproductive success.

Secondary metabolites produced by the phenylpropanoid pathway, which is regulated by transcription factors of the MYB family, play crucial roles in this early phase of fruit development. The MYB46 transcription factor is a key regulator of secondary cell wall structure and lignin and flavonoid biosynthesis in many plants, but little is known about its activity in flowers and berries in F. vesca. For functional analysis of FvMYB46, we designed a CRISPR-Cas9 construct with an endogenous F. vesca specific U6-promoter for efficient and specific expression of two gRNAs targeting the first exon of FvMYB46. This generated mutants with an in frame 81-bp deletion of the first conserved MYB-domain or an out of frame 82-bp deletion potentially knocking out the gene function. In both types of mutant plants, pollen germination and the frequency of flowers developing to mature berries was significantly reduced compared to wild type. Transcriptomic analysis of flowers demonstrated that FvMYB46 is positively regulating the expression of genes involved in pollen germination, homeostasis of reactive oxygen species (ROS) and the phenylpropanoid pathway, including secondary cell wall biosynthesis and flavonoid biosynthesis, while has a negative impact on carbohydrate metabolism. In FvMYB46-mutant flowers, the flavonols and flavan-3-olscontent, especially epicatechin, quercetin-glucoside and kaempferol-3-coumaroylhexoside were reduced, and we observed a local reduction of lignin content in anthers. Together these results suggest that MYB46 control fertility and efficient fruit set by regulating cell wall structure, flavonoid biosynthesis, carbohydrate metabolism and ROS-signaling in flowers and early fruit development in F. vesca.

The epidermis is the first barrier that protects organisms from surrounding stresses. Similar to the hyphae of filamentous pathogens that penetrate and invade the outer tissues of the host, the pollen germinates and grows a tube within epidermal cells of the stigma. Early responses of the epidermal layer are therefore decisive for the outcome of these two-cell interaction processes. Here, we aim at characterizing and comparing how the papillae of the stigma respond to intrusion attempts, either by hypha of the hemibiotrophic oomycete root pathogen, Phytophthora parasitica or by the pollen tube. We found that P. parasitica spores attach to the papillae and hyphae subsequently invade the entire pistil. Using transmission electron microscopy, we examined in detail the invasive growth characteristics of P. parasitica and found that the hypha passed through the stigmatic cell wall to grow in contact with the plasma membrane, contrary to the pollen tube that advanced engulfed within the two cell wall layers of the papilla. Further quantitative image analysis revealed that the pathogen and the pollen tube trigger reorganization of the endomembrane system (trans Golgi network, late endosome) and the actin cytoskeleton. Some of these remodeling processes are common to both invaders, while others appear to be more specific showing that the stigmatic cells trigger an appropriate response to the invading structure and somehow can recognize the invader that attempts to penetrate.

Pollen, a neighbor-less cell that contains the male gametes, undergoes multiple mechanical challenges during plant sexual reproduction, including desiccation and rehydration. It was previously showed that the pollen-specific mechanosensitive ion channel MscS-Like (MSL)8 is essential for pollen survival during hydration and proposed that it functions as a tension-gated osmoregulator. Here we test this hypothesis with a combination of mathematical modeling and laboratory experiments. Time-lapse imaging revealed that wild-type pollen grains swell and then stabilize in volume rapidly during hydration. msl8 mutant pollen grains, however, continue to expand and eventually burst. We found that a mathematical model wherein MSL8 acts as a simple tension-gated osmoregulator does not replicate this behavior. A better fit was obtained from variations of the model wherein MSL8 inactivation is independent of its membrane tension gating threshold or MSL8 strengthens the cell wall without osmotic regulation. Experimental and computational testing of several perturbations, including hydration in an osmolyte-rich solution, hyper-desiccation of the grains, and MSL8-YFP overexpression, indicated that the Cell Wall Strengthening Model best simulated experimental responses. Finally, expression of a non-conducting MSL8 variant did not complement the msl8 overexpansion phenotype. These data indicate that, contrary to our hypothesis and to known MS ion channel function in single-cell systems, MSL8 does not act as a simple membrane tension-gated osmoregulator. Instead, they support a model wherein ion flux through MSL8 is required to alter pollen cell wall properties. These results demonstrate the utility of pollen as a cellular-scale model system and illustrate how mathematical models can correct intuitive hypotheses.

Pollen tubes are dynamic tip-growing cells that deliver sperm nuclei to female gametes in flowering plants, allowing for sexual reproduction and seed formation. Actin and microtubule cytoskeletons both play important roles in directional pollen tube growth and guidance. While actin dynamics are well-studied in pollen tubes, the role of microtubules and the interactions between these two cytoskeletal filaments are less well understood. To address this knowledge gap, we imaged growing Arabidopsis thaliana pollen tubes co-expressing fluorescently-labeled tubulin and actin markers and observed partial co-localization of actin and microtubule filaments. We found that treatment with microtubule disrupting drugs did not affect the actin cytoskeleton. In contrast, when actin filaments were depolymerized, microtubules in the medial region of pollen tubes were disrupted, while microtubules at the cell cortex remained intact. Thus, the microtubule cytoskeleton in A. thaliana pollen tubes relies on the actin cytoskeleton in a spatially dependent manner. Furthermore, we utilized native expression of the microtubule plus-end binding protein EB1b to track microtubule orientation in growing pollen tubes. We found the microtubule array to be largely parallel, with plus ends growing away from the tube apex. Together, these findings offer new insights into the dynamics and organization of microtubules in growing pollen tubes and the interactions between actin filaments and microtubules.