FE-SEM visualization of cortical microtubules in plant cells using freeze-fracture techniques

Abstract

Background

Cortical microtubules (CMTs), one of the components of cytoskeleton, control the orientation and localization of newly deposited cellulose microfibrils in cell walls, and thereby determine the shape, size, and structure of plant cells. Imaging of CMTs in plant tissues is generally performed using fluorescently labeled specimens under an optical fluorescence or confocal laser scanning microscope. However, visualizing individual CMTs by optical microscopy is challenging because the observable range is limited to superficial tissue layers accessible to light penetration. In contrast, transmission electron microscopy offers high-resolution visualization of CMTs in plant cells but is restricted to slightly oblique ultrathin sections with an approximate thickness of 70–100 nm. Given that field emission scanning electron microscopy (FE-SEM) provides both high spatial resolution and a broad field of view, the establishment of FE-SEM-based techniques for CMTs visualization is highly desirable.

Results

Herein, we introduce a technique for visualizing CMTs within untreated plant tissues by combining two cryo-fracture techniques, freeze-knife fracture and freeze-tensile fracture, with FE-SEM. We successfully observed the arrangement of CMTs in several plant specimens, including young branches of ginkgo (Ginkgo biloba), calli from the leaves of hybrid poplar (Populus sieboldii × P. grandidentata), and root tips of the adzuki bean (Vigna angularis). CMTs were visualized on the protoplasmic fracture face (PF) using both cryo-FE-SEM and conventional room-temperature FE-SEM. While cryo-FE-SEM was employed to maintain native structures, room-temperature FE-SEM proved to be a viable alternative when specialized cryo-equipment was unavailable.

Conclusions

The combination of freeze-fracture techniques with FE-SEM enables the visualization of CMT arrangement in plant tissues at a high resolution and across a broad area without the need for staining or extraction of cellular components. This technique is applicable to various plant tissues and allows for detailed observation of CMTs within these tissues, providing valuable insights into the role of microtubules in the division and differentiation of plant cells.

Supplementary Information

The online version contains supplementary material available at 10.1186/s13007-026-01510-z.

Introduction

The morphogenesis of plant tissues depends on the orientation and localization of newly deposited cellulose microfibrils (CMFs) on the innermost surface of the primary wall during cell differentiation, which reinforces the cell walls, resulting in resistance to cell expansion in response to turgor pressure [1]. In addition, the orientation of cellulose microfibrils in the secondary walls is closely related to the physical properties of plant cells. The precise control of morphogenesis is crucial for the physical properties of each plant tissue and is essential for the full realization of their functions [2]. Cortical microtubules (CMTs), one of the components of the cytoskeleton, present beneath the plasma membrane regulate the arrangement of CMFs by guiding the movement of cellulose synthase complexes in the plasma membrane in a wide variety of plant cells [3–7]. Additionally, CMTs regulate the deposition of non-cellulosic cell wall components by triggering the release of exocytic vesicles [8, 9]. In interphase plant cells, CMTs assemble into highly organized bundles that regulate the direction of cell elongation [10]. The spatial organization of CMT bundles is also believed to play a significant role in the formation of vascular elements with unique cell wall patterns [11, 12]. Thus, a detailed study of CMTs is important in the broader field of plant research, highlighting the need for techniques that allow researchers to visualize CMTs within plant tissues [13].

CMTs are fine structures, with a diameter of approximately 25 nm [14], and bundles of microtubules are generally aligned at a close distance of approximately 25 nm [15]. Therefore, transmission electron microscopy (TEM), which is capable of high-resolution imaging, has been a useful tool for detailed observations of CMT bundles and of the interactions between their components [13] since the original observations of CMTs in plant cells by Ledbetter and Porter [16]. However, TEM involves many steps for specimen preparation, including chemical or rapid-freeze fixation, dehydration, and resin embedding, which might introduce artifacts. Additionally, its range of observation is extremely limited because it requires slightly oblique sections of cell membrane, approximately 70–100 nm in thickness, to visualize CMTs [5].

With the successful introduction of indirect immunofluorescence staining and confocal laser scanning or fluorescence microscopy for examining much thicker (approximately 40 μm) sections than those suitable for TEM, obtaining 3-D images of microtubules over a relatively large area within several plant tissues such as trees has become possible [6, 17–25]. However, owing to limitations of resolution, detailed observation of higher-order CMT arrays using a high-resolution confocal laser scanning microscopy and structured illumination microscopy would be challenging [26, 27]. Interferometric photoactivation and localization microscopy (iPALM) has successfully been used to measure the diameter of microtubules, approximately 25 nm, by employing fluorescently labeled α-tubulin [28]. However, super-resolution microscopy techniques are sensitive to autofluorescent molecules, which makes them unsuitable for plant cells with abundant pigments and phenolic compounds [13]. Therefore, for the precise visualization of CMTs in a wide variety of plant cells, technical advances are needed [13, 29, 30].

Here, we report a novel method for intact and high-resolution observation of CMT arrays across a wide area in different plant cells by combining a rapid plunge-freezing technique with freeze fracture and observed by field emission scanning electron microscopy (FE-SEM). Microtubules within plant tissues have previously been observed using FE-SEM [31–35]. However, pretreatment with chemical fixation and extraction of cellular components is needed, which potentially leads to the possibility of artifacts during preparation. In contrast, TEM analysis of replica membranes using a freeze-fracture device has yielded significant findings in studies of membrane proteins in plant cells [36, 37]. However, freeze-replica techniques show an overestimation of structures due to the deposition of carbon and platinum films and the loss of positional information during the collection of the replica membrane. In contrast, our method, which uses a freeze-fracture technique, eliminates the need for creating a replica membrane and chemical fixation and dissolution procedures, allowing for direct observation of cellular microstructures. Additionally, FE-SEM enables seamless high-resolution observations across a wide range of magnifications in plant cells [38], providing detailed insights into the higher-order arrangement of CMT bundles in plant tissues. In this study, we conducted a comparative examination of the two primary methods of the freeze-fracture technique, namely knife fracture and tensile fracture (Supplementary Fig. 1) [39], and used multiple experimental materials to visualize CMTs in various plant cells in untreated samples without chemical fixation or extraction of cellular components.

Results

Cryo-FE-SEM images of freeze-knife-fractured young branches of ginkgo (Ginkgo biloba)

Figure 1 presents the terminology used for freeze-fractured faces and surfaces. The fracture face, known as the protoplasmic fracture face (PF) or the exoplasmic fracture face (EF), represents the hydrophobic part of the membrane that is typically exposed by the freeze-fracture process [40]. The protoplasmic surface (PS) or exoplasmic surface (ES) corresponds to the hydrophilic part of the membrane, and is usually revealed through a sublimation process [40].

Fig. 1.

Nomenclature for freeze-fractured faces and surfaces. The fracture face, termed the protoplasmic fracture face (PF) or exoplasmic fracture face (EF), represents the hydrophobic portion of the membrane that is typically exposed during the freeze-fracture process. The protoplasmic surface (PS) or exoplasmic surface (ES) corresponds to the hydrophilic portion of the membrane and is usually revealed through sublimation. E half = exoplasmic half; P half = protoplasmic half

Figure 2 shows cryo-FE-SEM images of young branches of G. biloba. Figure 2A–D show the freeze-knife-fractured faces of regions in the phloem axial parenchyma cells. On the exposed PF, numerous CMTs oriented nearly perpendicular to the direction of cell elongation could be observed (Fig. 2C). These CMTs exhibited a meandering arrangement in the cytoplasm, with some oriented almost parallel to the direction of cell elongation. In this region, although a few CMTs formed bundles, most were arranged individually. When CMTs were bundled, ladder-like structures connecting the microtubules were observed (Fig. 2D). The ladder-like structures connected two microtubules at an interval of approximately 20–40 nm, although this spacing varies depending on the region. Figure 2E, F show the freeze-knife-fractured face of the epidermal parenchyma cells in young branches of G. biloba. CMT bundles oriented at an angle of approximately 45° to the direction of cell elongation were observed at the PF (Fig. 2E, F). Each bundle consisted of 3–4 CMTs, and a few CMTs existed individually. These CMTs were linearly arranged in the cytoplasm, with no meandering microtubules.

Fig. 2.

Cryo-FE-SEM images of freeze-knife-fractured faces of young branches of ginkgo (Ginkgo biloba). Cryo-FE-SEM images of freeze-knife-fractured faces of young branches of gingko (G. biloba). A, B Low-magnification images. Boxed areas indicate the location of the phloem axial parenchyma cells, as shown in B–D. C Numerous cortical microtubules (CMTs) were observed on the protoplasmic fracture face (PF) of phloem axial parenchyma cells. The CMTs were generally oriented nearly perpendicular to the direction of cell elongation, with most exhibiting a meandering pattern across the cytoplasm. However, some CMTs were aligned nearly parallel to the direction of elongation. D High-magnification image of the PF. In regions where CMTs were bundled, ladder-like structures connecting adjacent microtubules were observed (boxed area). The ladder-like structures bridged two microtubules with an inter-microtubule spacing of approximately 20–40 nm. E, F In the PF of the epidermal parenchyma cells, the CMT bundles were arranged at an angle of approximately 45 ° relative to the direction of elongation. Each CMT bundle consisted of 3–4 microtubules. Black arrows = direction of cell elongation; White arrows = CMTs; CW = cell wall. Scale bars = 50 μm (A), 5 μm (B), 1 μm (C, E), 500 nm (D, F)

Cryo-FE-SEM images of freeze-knife-fractured calli of hybrid poplar (Populus sieboldii × P. grandidentata)

Figure 3 shows cryo-FE-SEM images of the calli of hybrid poplar (P. sieboldii × P. grandidentata). In the exposed PF region, the cell surface exhibited a smooth dome-like shape (Fig. 3A). In the PF, CMTs were oriented nearly perpendicular to the longitudinal axis of the cells, which corresponded to the direction of cell elongation (Fig. 3B). Most CMTs were bundled, although some existed as individual units. In regions where CMTs were densely bundled, each CMT bundle consisted of 4–6 microtubules (Fig. 3C). Beneath the CMTs, organelles presumed to be mitochondria were observed in the cytoplasm (Fig. 3B).

Fig. 3.

Cryo-FE-SEM images of freeze-knife-fractured calli of hybrid poplar (Populus sieboldii × P. grandidentata). Cryo-FE-SEM image of a freeze-knife-fractured face of the calli of hybrid poplar (P. sieboldii × P. grandidentata). A The cell surface exhibited a smooth, dome-like shape on the protoplasmic fracture face (PF). B On the PF, cortical microtubules (CMTs) were aligned nearly perpendicular to the longitudinal axis of the cell. C In regions where CMTs were densely bundled, each CMT bundle consisted of 4–6 microtubules. Black arrow = longitudinal axis of the cell; White arrows = CMTs; M = mitochondria. Scale bars = 3 μm (A), 1 μm (B), 500 nm (C)

Cryo-FE-SEM images of freeze-tensile-fractured face of root tips of adzuki beans (Vigna angularis)

Figure 4 shows cryo-FE-SEM images of a freeze-tensile-fractured face of the root tips of adzuki bean (V. angularis). The PF and exoplasmic fracture face (EF) were reproducibly observed at the sites where separation occurred between the lipid bilayers (Fig. 4A and B). Island-like depressions were present on the EF because of the partial absence of the cell membrane (Fig. 4B and D). Although difficult to identify in the cryo-FE-SEM image, detached ES was easily observed partially adhering to the PF after freeze substitution (FS) and critical point drying (CPD), as described later. On the PF, CMTs were oriented approximately perpendicular to the direction of cell elongation (Figs. 4C and 5). The diameters of the CMTs in V. angularis were greater than those in the young branches of G. biloba and calli from the hybrid poplar (P. sieboldii × P. grandid). In areas where the cell membrane was absent in the EF, CMFs were exposed (Fig. 4D). The orientations of the CMFs and CMTs were nearly identical, aligning approximately perpendicular to the direction of tissue elongation, confirming that CMTs control the orientation of CMFs and, therefore, the cell shape.

Fig. 4.

Cryo-FE-SEM image of freeze-tensile-fractured surface of roots tips of adzuki beans (Vigna angularis). Cryo-FE-SEM images of a freeze-tensile-fractured face of the root tips of adzuki bean (V. angularis). A, B Both the protoplasmic fracture face (PF) and exoplasmic fracture face (EF) were reproducibly observed at the interfacial fracture of the lipid bilayer. Island-shaped depressions (white arrowheads) caused by the absence of the cell membrane were formed on the EF. C High-magnification image of the PF showing numerous cortical microtubules (CMTs) oriented approximately perpendicular to the direction of cell elongation. Occasionally, CMT bundles consisting of two microtubules were observed. D Enlarged image of EF shows cellulose microfibrils (CMFs) in areas where the cell membrane was absent (white arrowheads). The orientations of CMFs and CMTs were nearly identical. Black arrow = direction of cell elongation; White arrows = CMTs; CW = cell wall. Scale bars = 2 μm (A), 1 μm (B), 500 nm (C, D)

Fig. 5.

Tracing of cortical microtubules on the protoplasmic fracture face of root tips of adzuki bean (Vigna angularis). A Cryo-FE-SEM image of the protoplasmic fracture face (PF) from the root tips of adzuki bean (V. angularis). B Tracing of cortical microtubules (CMTs) on the PF, created based on the SEM image shown in A. The identification of CMTs was performed via visual confirmation. Black arrow = direction of cell elongation; Scale bar = 1 μm

FE-SEM images captured at room temperature of freeze-tensile-fractured face of root tops of adzuki bean (Vigna angularis) after freeze substitution and critical point drying

Figure 6 shows FE-SEM images of a freeze-tensile-fractured face of root tips of V. angularis at room temperature (25 °C). The samples were subjected to treatments of FS and CPD. The PF was exposed over a wide area in the region fractured between the lipid bilayers (Fig. 6C and D). The EFs in the cryo-FE-SEM images (Fig. 4A and B) were not observed in the samples after treatment with FS and CPD. The CMTs that were oriented approximately perpendicular to the direction of cell elongation were observed in the PF (Figs. 6E and 7). The surfaces of CMTs appeared to be covered with the protoplasmic half (P half) of the lipid bilayer and a tissue-derived extract. The diameters of these CMTs were greater than those of the ginkgo and hybrid poplar calli (P. sieboldii × P. grandidentata). Although some CMTs were bundled together, they appeared to exist individually. ESs, with diameters of several hundred nanometer, were also observed in some regions, exhibiting an island-like shape on the PF.

Fig. 6.

FE-SEM images captured at room temperature of freeze-tensile-fractured face of root tips of adzuki bean (Vigna angularis). FE-SEM images of a freeze-tensile fractured surface of the root tips of adzuki bean (V. angularis) captured at room temperature after freeze substitution and critical point drying treatment. A, B Low-magnification images. The boxed areas indicate the positions of the cells observed at high magnification in C–F. C, D A widespread protoplasmic fracture face (PF) with numerous cortical microtubules (CMTs). The exoplasmic fracture face (EF), which complementarily pairs with the PF observed via cryo-FE-SEM, was absent. E, F Enlarged views of the PF. The CMTs were oriented approximately perpendicular to the direction of cell elongation and their surfaces appeared to be covered by the protoplasmic half (P half) of the lipid bilayer. Island-like shapes of the ESs were also observed in some regions. Black arrow = direction of cell elongation; White arrows = CMTs; CW = cell wall. Scale bars = 100 μm (A), 10 μm (B), 3 μm (C), 1 μm (D), 500 nm (E), 200 nm (F)

Fig. 7.

Tracing of cortical microtubules on the protoplasmic fracture face of root tips of adzuki bean (Vigna angularis) after freeze substitution and critical point drying treatment. A FE-SEM image of the protoplasmic fracture face (PF) of the root tips of adzuki bean (V. angularis) captured at room temperature. B Tracing of cortical microtubules (CMTs) on the PF, created based on the SEM image shown in A. The identification of CMTs was performed via visual confirmation. Black arrow = direction of cell elongation; Scale bar = 1 μm

Discussion

In this study, we demonstrated that the arrangement of CMTs could be visualized at high resolution by rapidly freezing the samples and performing high-resolution FE-SEM observations of freeze-fractured faces.

Cryo-FE-SEM observations

During the freeze-fracture process, cracks are formed between the lipid bilayers of the cell membrane, exposing the EF and PF [41]. In the freeze-fracture replica method, which involves vacuum deposition of Pt and C, membrane proteins are observed in the PF³⁹. In contrast, because our method did not involve vacuum deposition, we observed CMTs located beneath the cell membrane (Figs. 2, 3, 4, 5, 6 and 7). The escape depth of secondary electrons is within 10 nm of the outermost surface of the samples [42]; therefore, secondary electron images obtained using SEM primarily provide information about the outermost surface of the samples. However, because the P half of the lipid bilayer is estimated to be several nanometers thick, CMTs located beneath the PF could be observed using FE-SEM. The CMTs observed using FE-SEM in this study, as well as in previous studies, had slightly larger diameters than those observed using TEM (Figs. 2, 3, 4, 5, 6 and 7) [31, 35]. These differences were particularly noticeable in CMTs of young branches of G. biloba. In cryo-FE-SEM observations, any frost or ice that adheres during transportation is sublimated in the high vacuum of the FE-SEM device before observation. As ice formed from water between the cytoplasm and cell membrane sublimates, the P half of the lipid bilayer is presumed to cover the CMTs on the PF. This attachment of the P half likely contributed to the slight increase in the thickness of CMTs in this study.

In addition, we observed cross-linked structures that connected microtubules at consistent distances in the bundled regions of CMTs of young branches of G. biloba (Fig. 2D). The microtubule-associated protein MAP65 connects CMTs, maintaining a parallel spacing of 25–30 nm, which appears as filamentous crosslinks in TEM observations [12]. The crosslinked structures connecting CMTs observed in this study may be CMT-associated proteins (MAPs) that play an important role in maintaining parallelism of CMTs. Because FE-SEM observations using our method reflect the state modified by MAPs, the differences in the diameters of CMTs among the different plant samples might reflect variations in their modification states.

Samples after freeze substitution and critical point drying

The CMTs were also observed using room-temperature FE-SEM after the samples underwent FS and CPD (Fig. 6). Compared with the cryo-FE-SEM images, PF after FS and CPD appeared to have more surface deposits (Fig. 6E, F). This was likely due to components leaking from the plant tissues during the FS process and adhering to the surface of the samples.

The EF fragments that were complementary paired with the PS in cryo-FE-SEM (Fig. 4A and B) were not observed in room-temperature FE-SEM following FS and CPD. As the EF fragments were weakly attached to the sample, most of them might have been lost during FS or CPD. If cryo-FE-SEM is not available, FE-SEM at room temperature after FS and CPD might be effective in observing CMTs. However, it should be noted that this pretreatment may increase the accumulation of deposits on the sample surface. In adzuki bean root tips, although a slight increase in surface deposits was observed in samples after freeze substitution and critical point drying, tracing of cortical microtubules on the protoplasmic fracture face was successfully achieved in both room-temperature SEM and cryo-SEM images (Figs. 5 and 7). However, in lipid-rich plant tissues, when a large amount of lipids is extracted during immersion in osmium-acetone in the freeze-substitution process, room-temperature FE-SEM observation of microtubules is expected to become difficult. In such cases, because the observation surface becomes covered with lipids, FE-SEM after freeze substitution and critical point drying is not considered applicable.

Freeze-knife fracture and freeze-tensile fracture method

The freeze-knife fracture method enables micrometer-scale precision in controlling fracture depth through the use of the microtome integrated into the freeze-fracture device (Supplementary Fig. 2). A primary advantage of this method is its ability to reliably fracture the well-preserved, vitreous ice layer near the frozen surface. If the CMTs of interest are situated near the sample surface, this method offers high versatility and is applicable to a broad range of plant tissues.

The freeze-tensile fracture method produced PF with a larger area than did the freeze-knife fracture method. This approach allowed the observation of both sides of the PF and EF, which are complementary (Fig. 4A and B). In addition, by excising a specific area of interest, sandwiching it between copper plates, and then peeling it off, this method enables the observation of CMTs within the interior of plant tissues, demonstrating an additional advantage. This method is effective for plant samples in which the CMTs of interest are located in the interior and thin sectioning of the tissue is feasible.

In cross-sections prepared using the freeze-tensile fracture method, island-like fragments of the ES were observed on the PF (Fig. 6E and F). However, no such island-like ES fragments were observed on freeze-knife-fractured faces (Figs. 2 and 3). Therefore, the island-shaped ES fragments, which resisted the freeze-tensile process, might correspond to local changes in membrane composition such as lipid rafts where some membrane proteins may accumulate.

The cross-sections prepared via the freeze-knife fracture and freeze-tensile fracture methods were highly cracked between lipid bilayers and exhibited various fracture patterns. Consequently, it is difficult to selectively observe the CMTs in specific regions using these methods. Moreover, because the depth of vitreous ice formation in plunge rapid freezing is limited to approximately 20 μm [43], it is advisable to perform fracturing close to the frozen surface. However, for samples to which precise and intact plant tissue sectioning techniques (e.g., laser dissection or vibratome sectioning) are applicable, combining these approaches with freeze-tensile fracture is expected to increase the likelihood of observing CMTs in targeted regions of interest.

Conclusions

Our study demonstrates that the combination of cryofracture techniques with FE-SEM observation is effective for the high-resolution imaging of fine CMTs in plant tissues. This method is applicable to various plant tissues and is particularly useful for visualizing CMTs within plant tissues that are challenging to observe with an optical microscope. In contrast, it is important to note that when using our method, the CMTs are covered by the P half of the thin cell membranes at the nanoscale, and the freeze-fractured cross-sections can display various fracture patterns. Therefore, careful consideration is needed when interpreting FE-SEM images of CMTs.

Methods

The materials used in this study were young branches of G. biloba growing on the campus of Tokyo University of Agriculture and Technology in Fuchu, Tokyo, calli derived from leaves of hybrid poplar (P. sieboldii × P. grandidentata) growing on the same campus [44], and root tips of V. angularis (adzuki bean; commercial Tamba Dainagon) approximately one week after sowing.

Plunge freezing

An aluminum metal cup was cooled to liquid nitrogen temperature and filled with ethane gas (99.5% purity; GL Sciences, Tokyo, Japan) to produce liquefied ethane. The cryoembedding medium was prepared using bovine albumin (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) dissolved in four-volumes of distilled water. The samples were secured by applying a thin layer of bovine albumin solution to the sample stage during rapid freezing. Young branches of G. biloba and calli derived from the petioles of hybrid poplar (P. sieboldii × P. grandidentata) used in the freeze-knife fracture experiments were cut into pieces several millimeters in length and positioned on a copper hat-type sample stage for the freeze-fracture device. Rapid freezing was performed by immersing the sample in liquid ethane while being fixed on the sample stage. The root tips of V. angularis used in the freeze-tensile fracture experiments were sliced to a thickness of several hundred micrometers with a razor and then securely placed between two copper disks (0.1 mm thick, approximately 3 mm in diameter, Nisshin EM, Tokyo, Japan). The sample sandwiched between the copper disks was rapidly frozen by pinching with tweezers and immersing in liquid ethane [45]. The frozen samples were then attached to the sample stage of the freeze-fracture device and stored in liquid nitrogen.

Freeze-fracture and cryo-FE-SEM observations

The frozen samples were placed into a freeze-fracture device (EM-19501JFDV, JEOL, Tokyo, Japan) for freeze-knife fracture and freeze-tensile fracture at a sample stage temperature of − 110 °C and a knife temperature of − 180 °C (Supplementary Fig. 2). Young branches of G. biloba and calli of hybrid poplar (P. sieboldii × P. grandidentata) were freeze-fractured a few micrometers from the outermost surface of the specimen using a knife attached to a freeze-fracture device. The freeze-tensile fracture process for the root tips of V. angularis was performed by removing the copper plate from the sample using a knife attached to the freeze-fracture device. The samples remaining on the copper disk above the sample stage after the tensile fracture were used for SEM observations. After freeze-fracturing, the samples were stored in liquid nitrogen and inserted into the FE-SEM (JSM-7100 F, JEOL) equipped with a cryo-unit (MP-Z08181T, JEOL) (Supplementary Fig. 3). Cryo-FE-SEM observations were carried out at an accelerating voltage of 1 kV and a sample stage temperature of − 90 °C. Images were captured using a secondary electron detector. Before observation, any frost that accumulated during the transportation of the sample was sublimated within the cryo-FE-SEM device. No conductive coating was applied for the cryo-FE-SEM observations.

Freeze substitution and critical point drying

FS was performed by immersing the freeze-tensile fractured root tips of V. angularis in 2% OsO₄ in acetone and storing them at − 85 °C for 2 days, at − 20 °C for 4 h, and at 4 °C for 2 h. The samples were then warmed to 25 °C and washed several times with 100% acetone. The samples were placed in a critical point dryer (SYSGLCP-8; Sanyu-Gijutsu, Tokyo, Japan). CPD was carried out at a flow rate of 1.0 L/min and a temperature of 40 °C. After CPD, the samples were coated with a 1.5 nm-thick layer of osmium using an osmium plasma coater (HPC-20; Vacuum Device, Ibaraki, Japan). Observations were made using an FE-SEM device (JSM-7900F, JEOL) at an accelerating voltage of 1 kV, and the images were acquired using a secondary electron detector.

Supplementary Information

Below is the link to the electronic supplementary material.

Abbreviations

CPD

Critical point drying

CMFs

Cellulose microfibrils

CMT

Cortical microtubule

EF

Endoplasmic fracture face

E half

Exoplasmic half

ES

Exoplasmic surface

FE-SEM

Field emission scanning electron microscopy

FS

Freeze-substitution

PF

Protoplasmic fracture face

P half

Protoplasmic half

PS

Protoplasmic surface

TEM

Transmission electron microscopy

Author contributions

Tomohiro Hatano (TH), Satoshi Nakaba (SN), Yoshiki Horikawa (YH), Satoshi Kimura (SK), Hikaru Nagata (HN), Iwao Doi (ID), and Ryo Funada (RF) contributed to the study conception and design. Tomohiro Hatano (TH) performed the experiments . Tomohiro Hatano (TH) and Ryo Funada (RF) wrote the manuscript. Hikaru Nagata (HN) and Iwao Doi (DI) cultivated hybrid poplar calli. All authors read and approved the final manuscript.

Funding

This work was supported, in part, by Grants-in-Aids for Scientific Research from the Japan Society for the Promotion of Science (JSPS) (nos. 18H02251, 21H02253 and 24 K01822).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Statement of compliance

Plant experiments and the collection of plant materials were performed in accordance with relevant institutional, national, and international guidelines and legislation.

Competing interests

The authors declare no competing interests.