Fresh âPollen Adhesiveâ Weakens Humidity-Dependent Pollen

Research Article Cite This: ACS Appl. Mater. Interfaces 2019, 11, 24691−24698

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Fresh “Pollen Adhesive” Weakens Humidity-Dependent Pollen Adhesion Shuto Ito* and Stanislav N. Gorb Department of Functional Morphology and Biomechanics, University of Kiel, Am Botanischen Garten 9, D-24118 Kiel, Germany

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S Supporting Information *

ABSTRACT: This study presents a quantitative investigation of pollen adhesion mediated by pollenkitt using pollen grains in their native state. Here, we attempt to clarify whether the exposure time, pollenkitt losses, and the surrounding humidity (relative humidity, RH) levels influence pollen adhesion. Pollen grains of Hypochaeris radicata (Asteraceae) were tested using atomic force microscopy. Regardless of the pollen condition (fresh, aged, and without pollenkitt), higher RH significantly increased pollen adhesion on hydrophilic surfaces, whereas it had little effect on pollen adhesion on hydrophobic surfaces. On hydrophilic surfaces, adhesion of fresh pollen was less dependent on RH than that of aged pollen or without pollenkitt, resulting in reduced adhesion under high RH. On hydrophobic surfaces, adhesion of fresh pollen was significantly lower than that of aged pollen. We utilized capillary models to explain the counterintuitive results obtained and came to the conclusion that the abundant fresh pollenkitt, which is widely accepted as pollen adhesive, can reduce pollen adhesion in some conditions. This study sheds light on the little-known adhesive properties of pollen and on the pollination mechanics. KEYWORDS: pollination, pollen, pollenkitt, adhesion, humidity dependence, wetting, biomechanics, AFM

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pollen grains from other species.17 In contrast, it was recently shown that honeybee’s hairy structures on its eyes and forelegs evolved to enable extremely efficient pollen removal from its body.18 It is known that a layer of viscous sticky substance called pollenkitt covers pollen grains of insect-pollinating species. The pollenkitt is considered a water-in-oil emulsion, composed of lipids, carotenoids, flavonoids, proteins, and carbohydrates.13 The pollenkitt is currently suggested to have a variety of adhesive functions: (1) to hold pollen grains in an anther until dispersal,19 (2) to enable secondary pollen presentation,20 (3) to enable pollen dispersal in clumps,21 and (4) to facilitate pollen adhesion to pollinators and plant stigmas.13,18 In spite of the seemingly wide range of adhesive roles the pollenkitt plays in pollination, only a few quantitative studies have been conducted in this field. It was shown that pollenkitt enhances pollen adhesion up to 3−6 times by forming capillary bridges to a flat surface.15 Sunflower’s pollenkitt absorbs water at high humidity levels, which alters its viscosity and surface tension, resulting in complex dependence of adhesion on humidity.16 The main problems of all previous quantitative studies of pollen adhesion are that they used commercially available pollen grains, which are industrially collected and frozen and

INTRODUCTION Pollination, which is a remarkable biological example of an efficient particle transport system, is a key to successful plant reproduction. In insect-mediated pollination, pollen grains go through multiple attachment/detachment cycles: pollen grains first are released from an anther of a flower and remain attached to an insect until they finally land on a stigma of a flower. Understanding the underlying mechanism of the pollen transport is of importance in botany and agriculture, which are crucial to crop production for human food1−5 and in human respiratory health including pollen allergies and asthma.6,7 In addition, the generalized insight of the particle attachment/ detachment can be applied to a variety of scientific and industrial fields, including environmental pollution,8 drug delivery,9 painting, and semiconductor manufacturing.10,11 Despite a number of detailed studies on morphological and physiological features of pollen,12,13 there are only a few recent quantitative studies that have addressed pollen attachment.14−18 In 2009, atomic force microscopy (AFM) was utilized to quantify van der Waals forces that drive pollen attachment to polymer surfaces.14 The magnitude of van der Waals forces was found to scale with the tip radius of the pollen’s surface ornamentations.15 The ornamentations of pollen were found optimized to facilitate pollen adhesion to other biological surfaces. For example, the morphologies of sunflower’s pollen and stigma are optimized to achieve speciesspecific adhesion, which prevents unwanted deposition of © 2019 American Chemical Society

Received: March 18, 2019 Accepted: June 11, 2019 Published: June 11, 2019 24691

DOI: 10.1021/acsami.9b04817 ACS Appl. Mater. Interfaces 2019, 11, 24691−24698

Research Article

ACS Applied Materials & Interfaces

Figure 1. (a) Typical force−distance curve for fresh Hypochoeris radicata pollen on a hydrophobic glass substrate. Approach curve is shown in blue and retraction curve in orange. The lowest value of the measured force was defined in retraction as the pull-off adhesion force. (b) Scanning electron microscopy (SEM) image of the pollen fixed on an AFM cantilever tip, after the AFM measurements.

Figure 2. (a, b) Comparison of pull-off adhesion forces (fa) of H. radicata pollen on hydrophilic cleaned glass (a) and hydrophobic silanized glass (b) at different relative humidity (RH) levels (N = 9, n = 1323). (c, d) A series of pull-off adhesion force (fa) measurements of fresh (green plots) and aged pollen (orange plots) on hydrophilic cleaned glass surfaces (c) and on hydrophobic silanized glass surfaces (d) under controlled fluctuations of RH.

reported,22 but neither adhesive properties nor other physical properties during the exposure are known yet. As pollenkitt seems to have different physicochemical properties depending on the hydration level and the exposure time, one would assume that the adhesion properties are also altered by them. In this study, we report the first quantitative analysis of pollen adhesion in the native state by using AFM with an emphasis on the influence of the exposure time and the surrounding humidity on pollen adhesion.

dried for much longer periods of time than the time scale of pollination in nature.14−17 Considering that the pollenkitt contains volatile compounds, carbohydrates, and proteins, which may irreversibly change its proportion and physical properties after freezing and dehydration, it is reasonable to investigate the adhesion properties of pollen grains in their native state. In plants relying on insect-mediated pollination, pollen grains are first presented in highly humid environments such as boundary layers of anthers or other parts of plants, and therefore they would experience dehydration after the attachment to pollinators until they finally become rehydrated on stigmas. During the pollen exposure and transport, noteworthy changes of pollenkitt in color have been previously

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RESULTS In the AFM force measurements, each approach−retraction cycle to the surface generated a corresponding force−distance curve, such as shown in Figure 1a. Using the force−distance 24692


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ACS Applied Materials & Interfaces curves, the adhesion force was defined: pull-off adhesion force fa as the lowest measured force in retraction. In other words, fa is the amount of force required to break contact generated in approach between the pollen grain and the substrate. In Figure 2a,b, fa of fresh and aged pollen grains are compared as bar plots that contain all of the measurements (N = 9, n = 1323). The thick vertical bars are medians, and the error bars are 75th and 25th quantiles. We also have plotted typical results of fa transitions from consecutive force measurements of fresh and aged pollen grains under fluctuating relative humidity (RH) (Figure 2c,d). We focused on the exposure time on an hourly time scale because the opening and presentation of a flower of H. radicata lasts for 3−7 h,23 and the pollen transport by pollinators is likely to be completed within a day. On cleaned glass surfaces (Figure 2a), significantly higher adhesion forces were detected under high RH than under low RH in both fresh (Welch’s t-test, t = −12.0, df = 321.5, p < 2.2 × 10−16) and aged (Welch’s t-test, t = −21.1, df = 232.0, p < 2.2 × 10−16) grains. High RH caused stronger increase (108%) in the aged grains than in the fresh ones (36%). This trend was quite prominent if fa was plotted versus elapsed time t, such as in Figure 2c. fa of a pollen grain in the fresh state (green line) exhibited less RH dependence than that in the aged state (orange line). In the very fresh state (green line: 0 < t < 50) of grains, fa under high RH was occasionally even lower than that under low RH. However, the RH dependence of fa (higher RH caused larger fa) gradually became noticeable as the pollen with increased exposure time. After 19 h from the collection (orange line), the RH dependence became more obvious. Similar RH dependence to aged pollen was found in washed pollen without pollenkitt (Figure 3a, blue bar plots). Under high RH, fa was 73% significantly higher than that under low RH (Welch’s t-test, t = −18.4, df = 593.3, p < 2.2 × 10−16). However, in the case of silanized glass surfaces (Figures 2b and 3 red plots), there was no practically significant difference of fa depending on RH levels regarding all of the pollen conditions: fresh, aged, and washed. However, the comparison between the fresh and the aged state enabled us to see an interesting phenomenon: the adhesion of aged pollen grains became significantly higher than that of fresh ones (Welch’s ttest, t = −12.2, df = 560.7, p < 0.001). While fa of fresh grains remained constant over time, that of aged grains gradually increased without being affected by RH (Figure 2d). SEM observations were performed to define the pollen geometry and to visualize pollenkitt and water on pollen surfaces (Figure 4). First, the fresh pollen (Figure 4a) and the month-old pollen (Figure 4b) were compared on hydrophilic glass surfaces in cryo-SEM. One can observe the thick layer of pollenkitt covering grains both in the fresh and in the aged state. Apparent shrinkage or volume loss of pollenkitt was not observed from the comparison. After pollenkitt removal (Figure 4c,d), pollen geometry was analyzed: a single pollen grain was found to have two convex faces with conical spikes on it and 15 concave faces, each of which was surrounded by conical spikes located at the edge. There were numerous pores and cavities on the surface of pollen. In the conventional SEM mode, the conical spikes were found shrunk to cause a number of nanosized wrinkles on the surface (Figure S1), whereas in the cryo-SEM mode, the surface was smooth and neither shrinkage nor nanowrinkles were found. Occasionally, a small amount of remained pollenkitt was found to wet the pollen surface (see arrow in Figure 4c). To further study wettability of

Figure 3. (a) Comparison of adhesion pull-off force (fa) of washed H. radicata pollen grains on hydrophilic cleaned glass and on hydrophobic silanized glass at different RH levels (N = 2, n = 1003). (b) Series of pull-off force (fa) measurements of washed pollen grains on hydrophilic cleaned glass (blue plot) and on hydrophobic silanized glass (red plot) under controlled fluctuations of RH.

the pollen exine, we attempted to visualize water on the pollen grains (Figure 4e,f). The film of water was found to wet through the pores and cavities of pollen and form bridges between the conical spikes (Figure 4f). To relate the measured adhesion force to the gravitational force, the mass of a single pollen grain was determined. The calculated mean mass of an H. radicata pollen grain was 15.0 ng (Figure S2b). The grain counting from the image processing seems to be accurate enough so that the experimentally obtained data for mass and corresponding grain number were perfectly fitted by linear regression (R2 > 0.99).

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DISCUSSION RH Dependence of Pollen Adhesion. The measured force between a pollen grain and a target surface is given as F = Fvdw + Fe + Fw (1) where Fvdw is van der Waals forces, Fe is electrostatic force, and Fw is the force arising from capillary attraction. The capillary attraction can be explained by two components Fw = Fl + Fp

(2)

where Fp is the force deriving from the pressure difference across the liquid−vapor interface (Laplace pressure) and Fl is the capillary force that acts on the three-phase contact line (TCL) of pollen surfaces. Electrostatic effect is quite distinguishable because of its long-range attraction in a 24693


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Figure 5. (a) Components of capillary attraction (Fl, Fp) were calculated versus filling angles based on two different contact angles of θ1. (b) Total capillary attraction Fw was calculated versus contact angle θ1 based on three filling angles. The illustration defines parameters for the calculation: θ2 = 23°, R = 151 nm, and a = 0.165 nm.

and the target surface, and θ1 is the contact angle of the liquid on sporopollenin (major constituent of pollen exine). Based on the cryo-SEM images, we considered that the conical spikes have smooth surfaces and therefore have roughness factor r = 1. We also confirmed that water is able to wet in pores and cavities on pollen surfaces and, therefore, we considered that the contact angle of water on sporopollenin is θ1 < 90°. The force induced by Laplace pressure is

Figure 4. (a, b) Cryo-SEM images of a fresh pollen grain (a) and a 1 month-old pollen grain (b) in contact with a glass substrate. (c, d) SEM images of a cleaned pollen in different magnifications: (c) equatorial view of the whole pollen, (d) sub-microstructure of one of the faces of the pollen having spikes on its perimeter. (e, f) Cryo-SEM images showing bulk water on a cleaned pollen grain.

Fp = πR12ΔP

ij 1 1 yzz ΔP = γ jjj − z jR R1 zz{ (5) k 2 where R1 is the lateral curvature radius and R2 is the vertical curvature radius of the capillary bridge, and ΔP is the Laplace pressure. R1 and R2 can be calculated based on the circular assumption of the capillary bridge geometry R1 = R sin φ (6)

force−distance curve (Figure S4). In this study, the electrostatic force was eliminated using an ionizing gun, when the long-range attraction was observed. The experimental results exhibited the clear RH dependence of pollen adhesion on cleaned glass surfaces except for the pollen grains in the very fresh state. This dependence was increasingly enhanced over the exposure time or increased number of indentations. Interestingly, adhesion of the washed pollen without pollenkitt also responded to the fluctuating RH in a quite similar way as the aged pollen. Given that Fe is negligible and that Fvdw should not be enhanced by high RH, then Fw should arise from the condensed water and/or pollenkitt on pollen surfaces and should be the key for the RH dependence. Since the pollen without pollenkitt had similar adhesive characteristics as the pollen in the aged state, it seems reasonable to focus on the potential contribution of the condensed water on the RH dependence. If we consider the contact formation at the tip of the conical spike, the force induced by capillary attraction Fw can be calculated based on the assumption of the meniscus geometry as shown in the inset illustration of Figure 5. The capillary force applied along the TCL on a conical spike was calculated as follows Fl = 2πRrγ sin φ sin(φ + θ1)

(4)

R2 =

a + R(1 − cos φ) cos(θ1 + φ) + cos θ2

(7)

where a is the separation distance and θ2 is the contact angle of the liquid on the target substrate. Here, we attempted to reason the RH dependence that was observed in the experiments with this analytical approach. The parameters used for the calculation were as follows: a = 0.165 nm, θ2 = 23° (see Table 1, water contact angle on cleaned glass surfaces), and R = 151 nm, which was measured from SEM images. We consider the contact radius of a conical spike constant over approach−retraction cycles since pollen exine is sufficiently hard so that the applied contact pressure would not be able to deform it.24 If the preload force leads to the deformation of spikes, higher preload would enhance the pollen adhesion due to the expanded contact radius. A supporting experiment, to test the effect of preload on pollen adhesion, has proved that there is no such effect (Figure S3). In Figure 5a, the capillary force Fl and the force induced by Laplace pressure Fp on cleaned glass surfaces were calculated versus the filling angle φ based on two extreme contact angles

(3)

where R is the contact radius of a conical spike, r is the roughness factor, γ is the surface tension of water, φ is the filling angle that defines the amount of liquid between the tip 24694


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ACS Applied Materials & Interfaces Table 1. Contact Angles on Three Different Substrates with Three Different Probe Liquidsa contact angle (deg)

surface energy (mN/m)

substrate

water

diiodomethane

ethylene glycol

polar

dispolar

total

cleaned glass silanized glass pollenkitt

23 ± 2 113 ± 2 63 ± 2*

37 ± 3 80 ± 2 29 ± 2*

21 ± 2 103 ± 2 60 ± 2*

46 ± 2 0 4±1

13 ± 1 16 ± 1 39 ± 1

60 ± 2 16 ± 1 42 ± 1

a

The data with stars were referred to the previous study.15 The surface energies of the substrates were calculated based on the contact angle measurements.

however, water may not be able to hold itself and be squeezed out from the interface as the surfaces come into contact due to the poor wettability of the silanized surfaces. Pollenkitt as Pollen Adhesive. As previously mentioned, pollenkitt has been previously considered to be adhesive materials facilitating pollen adhesion. However, this study has revealed the more complex adhesive properties of pollenkitt depending on its freshness and the surrounding humidity. The abundant pollenkitt in the fresh state (1) weakens adhesion on hydrophilic glass surfaces under high RH and (2) exhibits lower adhesion on hydrophobic silanized surfaces than that in the aged state (Figure 2a,b). We were able to explain the first phenomenon with the water capillary model in the previous section. That is to say, the hydrophobicity of the pollenkitt weakens the water capillary attraction to the cleaned glass surfaces. On the other hand, we could not utilize the water capillary model for the silanized surfaces. However, in the case of pollenkitt, its lower surface energy possibly enables the formation of a bridge and causes capillary attraction. We calculated that the pollenkitt contact angle on the silanized surface is 81° based on our contact angle measurements and the data from the literature,15 according to the OWRK theory. Taking this into consideration, we plotted the relationship between the pollenkitt capillary attraction Fw and the filling angle φ (Figure 6). In this calculation, we regard that the

on the pollen surfaces (θ1 = 0, 90°). Assuming highly wettable pollen surfaces (blue plots), increasing filling angle φ enhances Fl (blue dashed-curve) due to the expanding length of TCL, whereas it decreases Fp (blue solid-curve) except the range of the small filling angle (φ < 10°). On the poorly wettable pollen surfaces (red plots), unlike the highly wettable surfaces, increasing φ leads to the decrease of Fl (red dashed-curve) in the range of large filling angles (φ > 45°). Regardless of the wettability, the existence of water at the interface tends to cause positive capillary attraction Fw. The only condition that causes capillary repulsion (negative Fw) is a large filling angle on the poorly wettable surfaces. Under high RH, the ambient water is likely to be condensed or absorbed on the pollen surfaces, which causes the capillary bridge at the contact interface. In Figure 5b, the relationship between the total capillary attraction Fw and the water contact angle on the pollen surfaces θ1 is plotted. Even a single capillary bridge with a relatively small amount of water (φ < 10°, R1 < 27 nm) can produce the comparable force to the difference of the measured adhesion forces between high and low RH. The water contact angle on pollen surfaces θ1 plays a key role in determining the magnitude of capillary attraction. As mentioned above, very fresh pollen showed less RH dependence. However, the more the contact-and-breakage cycles were performed and the more time the pollen grains were exposed, the more prominent the RH-dependent pollen adhesion became (similar to the one of the washed pollen without pollenkitt) on the cleaned glass surfaces. When pollen surfaces are covered by the thick fresh pollenkitt, whose major constituent is a hydrophobic lipid, the water capillary attraction presumably reduced with high θ1 on the pollenkitt. Without pollenkitt, water is likely to wet more on pollen surfaces (low θ1), resulting in high capillary attraction. Each of the contactand-breakage cycle in our experiment can cause a pollenkitt loss, and the exposure of pollen can alter the wettability of pollenkitt, both of which can change θ1. It could be an explanation for the transition from the low RH dependence of the fresh pollen to the high one of the aged and washed pollen grains. In contrast to cleaned glass surfaces, none of the pollen conditions showed RH-dependent adhesion on the silanized glass surfaces. The silanization renders the glass surfaces hydrophobic due to the low surface energy (Table 1). Even on such surfaces, if the water capillary is formed at the interface, there should be an influence on adhesion to some extent. However, the experiments revealed that the fluctuating RH had no effect on the pollen adhesion on silanized glass surfaces (Figures 2b,d and 3a,b). Therefore, the water capillarity seems unlikely to occur on the silanized surfaces. To achieve the water capillary bridge at the interface, (1) surfaces require the water that is condensed on the topmost surfaces, which may not occur on low-energy surfaces, and (2) water should form a bridge to connect the pollen surface and the silanized surface;

Figure 6. Total capillary attraction Fw and its components Fl and Fp arising from a pollenkitt capillary bridge on silanized glass were calculated versus filling angle.

pollenkitt can fully wet the sporopollenin (θ1 = 0°). The result demonstrates that Fw declines with an increasing filling angle except for the range of small filling angles (φ < 10°). Unlike the case of the water capillarity, the volume of pollenkitt on pollen surfaces is quite abundant,15 and therefore, the range of small filling angles (φ < 10°) is not realistic. If one focuses on the range of mid- or high filling angles, the result indicates that the larger the volume of pollenkitt is at the interface, the lower is the contribution of the capillary attraction. In capillarymediated adhesion, two surfaces are separated when the 24695


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possibly performed for the wide range of plant species and may enrich various branches of science fields including agricultural sciences and physics/engineering of particle adhesion.

breakage of the capillary bridge occurs. After the breakage, a small amount of liquid can remain attached to the surface, and therefore, a series of contact-and-breakage cycles lead to potential losses of the pollenkitt from the pollen surfaces. The quantitative discussion about the pollenkitt losses is difficult due to some missing information, such as pollenkitt viscosity and contact angle hysteresis.25 However, the theory could explain why adhesion of aged pollen grains is higher than that of fresh ones. Interpreting the counterintuitive results that are different from the traditional understanding of pollenkitt as pollen glue is quite a challenge. Let us first discuss the magnitude of adhesion that we observed in the experiments. The minimum adhesion force was measured between fresh pollen and silanized surfaces, which is 38 nN. Even this amount of force has the safety factor of 258.5 (force/mass of a grain). Therefore, the amount of adhesion can be interpreted to be sufficient enough to assure the pollen attachment against gravity. On the contrary, the too strong adhesion can potentially make it difficult for pollen grains to fulfill their biological tasks, namely, dispersal and pollination. The safety factor of washed pollen on cleaned glass surfaces under high RH reached 1300. Without pollenkitt, pollen would be firmly fixated on high-energy surfaces under high humidity, potentially leading to the failure of dispersal. Speaking of the pollination process, when pollen grains presented on anthers or other auxiliary floral structures, they require detaching from the wet surfaces under high local humidity to attach to insect body parts, most of which are waxy hydrophobic surfaces. If water capillarity were dominant in adhesion during this phase, the pollen transport from the wet plant surfaces under high local humidity to the greasy insect body could be difficult. During the pollen transport by pollinators, the adhesive properties of pollen may become more dependent on humidity due to exposure and pollenkitt losses, resulting in higher adhesion on stigma. And therefore, the aging effects, which may occur much quicker than laboratory conditions (due to high temperature, solar radiation, etc.), may enhance the pollen capture on stigma from pollinators. Although our experimental results seem to be reasonable, even when considering the natural process of pollination, additional quantitative studies using biological surfaces (e.g., anther, insect, and stigma) are required to further comprehend the details of adhesive interactions between pollen grains and surfaces involved in the pollination mechanics.

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MATERIALS AND METHODS

Pollen-Tip Cantilever Preparation. H. radicata (Asteraceae) was adopted as a model species to study adhesive properties of pollen in an insect-pollinated plant. The yellow flowering plant is one of the most abundant flowering herbs in grassy areas in Kiel (Germany) and the surrounding areas during summertime. H. radicata is known to be self-incompatible, which means that successful transport of pollen grains to different individual plants of the same species is imperative for its healthy reproduction.26 In summertime, flowering stems of H. radicata were collected in the morning from grassy areas of Kiel. To test fresh pollen grains, the collected stems were put into water to allow the youngest ray flowers to push the styles through the anthers and present the fresh pollen grains on surfaces of the styles. The newly opened ray flowers were picked up, and then individual fresh grains were quickly collected using individual hairs of human eyelashes to be finally glued upon the tip of AFM tipless-cantilevers (Nanosensors, Neuchatel, Switzerland) using a slight amount of epoxy glue (Figure 1b). The epoxy glue was cured for 15 min before the onset of AFM adhesion measurements for the pollen grains in the fresh state. After the cycles of adhesion measurements, the pollen-tip cantilevers were stored under controlled laboratory conditions (temperature: 20−25 °C, RH: 40−60%). On the following day, adhesion of the grains was tested again for the comparison between pollen in fresh and aged states. To study the effect of pollenkitt on pollen adhesion, the pollen grains without pollenkitt were prepared. The collected grains were soaked in a mixture of chloroform and methanol (3:1), and the solvent that contains pollenkitt was filtered out 10 times. Then, adhesion of the washed grains was examined with AFM in the same way as described above for the fresh and aged grains. Substrate Preparation. In this study, we utilized two types of glass substrates with distinct wettabilities: hydrophilic substrates (water contact angle = 23°) and hydrophobic substrates (water contact angle = 113°). Hydrophilic glass substrates were prepared by thoroughly washing borosilicate glass coverslips. The coverslips were rinsed and sonicated in an ultrasound bath (Bandelin electronic, Berlin, Germany) for 5 min with ethanol (70%) and distilled water. This washing procedure was repeated several times and the remaining liquid on the substrate was blown by compressed air. Hydrophobic glass substrates were prepared using the silanization procedure. First, cleaned glass coverslips were subjected to air plasma treatment (Diener electronic, Ebhausen, Germany). Then, the treated coverslips were vacuum-pumped (BÜ CHI Labortechnik, Flawil, Switzerland) in a desiccator together with a glass vial, containing 200 μL of dichlorodimethylsilane (Merck Schuchardt, Hohenbrunn, Germany). The vacuum pump was disconnected from the desiccator once the silane started to evaporate faster, and the desiccator was left closed for 5 h to achieve sufficient deposition of the silane on the coverslips. The surface-modified coverslips were rinsed thoroughly with isopropanol and distilled water and were blown dry by compressed air. Humidity-Controlled AFM Force Measurements. We used atomic force microscopy (AFM) with the pollen-tip cantilevers to track adhesive properties of pollen over time. A pollen-tip cantilever was mounted on a NanoWizard scanning probe microscope (JPK Instruments, Berlin, Germany) to perform consecutive force measurements on target substrates. In case that force curves showed longrange attraction between a cantilever and a substrate, which is a characteristic of electrical attraction (Figure S3), the system was discharged using an ionizing gun (Simco, TX). The force measurements were performed in an enclosed chamber connected to N2 supply capable of setting different levels of relative humidity (RH) within the chamber. Here, we defined high RH and low RH as above 75% and below 25%, respectively. After the measurements, the spring constants of the tested cantilevers were determined by the thermal noise method incorporated in the AFM program. To measure the

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CONCLUSIONS To the best of our knowledge, this study provided the first quantitative insight into nonphysiological adhesive properties of pollen in their native state. In contrast to the traditional understanding of pollenkitt, as an adhesive that sticks pollen grains to other surfaces, the quantitative adhesion measurements exhibited that pollenkitt of H. radicata in the fresh state reduced humidity dependence of pollen adhesion, resulting in the decreased adhesion on high surface energy surfaces under high humidity conditions. In addition, we found that the pollen adhesion on low surface energy surfaces increased as the pollenkitt was exposed and the cycles of attachment/ detachment were repeated. These counterintuitive phenomena were supported here with capillary models of condensed water and pollenkitt. Our findings on the unique adhesive properties of fresh pollen grains may foster further quantitative studies using pollen in their native state. Further studies will be 24696


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All authors discussed the results and gave the final approval for publication.

fresh pollen adhesion, we have tested nine cantilevers with freshly collected pollen grains (five cantilevers on cleaned glass surfaces, and four on silanized glass surface). For the comparison between pollen adhesion in fresh and aged states, eight cantilevers out of the cantilevers that were used to test fresh pollen were used again to measure adhesion in an aged state (four cantilevers on the cleaned glass surfaces and four cantilevers on the silanized glass surfaces). To disclose the adhesive function of pollenkitt, we have tested three cantilevers with washed pollen grains on cleaned glass surfaces, and two out of the three cantilevers were tested on silanized glass surfaces as well. Pollen Mass Determination. If one knows the single pollen grain mass, the magnitude of the measured pollen adhesion in AFM can be related to the gravitational force acting on the pollen. To determine the single grain mass, clumps of fresh pollen that were composed of 1000−2000 grains were weighed with an ultra-microbalance (Sartorius Lab Instruments, Göttingen, Germany). The grains were sandwiched between a glass slide and a glass coverslip to spread out as one layer (Figure S2a), to be photographed in a binocular microscope (Leica Microsystem, Wetzlar, Germany). From the images, the grains were counted by a software custom written using Matlab (Mathworks, Natick). The results of the image processing were confirmed by the experimenter and manually corrected, if required. The single pollen grain mass was determined using least-squares linear regression from the data obtained from weighting and counting. Cryo-Scanning Electron Microscopy (Cryo-SEM). Cryo-SEM was used to visualize the presence of liquid on pollen surfaces. In other cases, unless specified otherwise, conventional warm SEM was used. Pollen grains were gently shed on cleaned coverslips or carbon tapes and then mounted on SEM stubs. The stubs were directly transferred to a sputtering chamber, where samples were frozen at −140 °C. Then, they were sputter-coated with 10 nm Au−Pd in the frozen state and observed in a Hitachi S 4800 scanning electron microscope (Hitachi Ltd, Tokyo, Japan) at the cryostage at −120°C. When we attempted to visualize water on pollen exine, the washed pollen grains were soaked with distilled water on the carbon tapes, and excess water was removed by a filter paper. The stubs were first frozen in the liquid N2 before being transferred to a sputter chamber. Contact Angle Measurements. Contact angles of three probe liquids (distilled water, diiodomethane, and ethylene glycol) on hydrophilic and hydrophobic glass surfaces were measured using a contact angle measurement device OCA20 (Dataphysics Instruments, Filderstadt, Germany). The contact angles on pollenkitt were referred to the previous work.15 The surface energies of solid substrates were calculated based on the measurements according to the OWRK theory.27

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Funding

“DAAD Research grantdoctoral programs in Germany” to S.I. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors are grateful to Dr Alexander Kovalev and Esther Appel for technical support. They are also thankful to Dr. Hamed Rajabi, Dr. Alexander Kovalev, Halvor Tramsen, Dr. Emre Kizilkan, Dr. Yoko Matsumura, and Prof. Agnieszka Kreitschitz for valuable discussion.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.9b04817. SEM images of conical pollen spikes, mass determination of pollen grains, electrostatic interaction in force−distance curves, and comparison of pull-off forces with different preloads (PDF)

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REFERENCES

(1) Ashman, T. L.; Knight, T. M.; Steets, J. A.; Amarasekare, P.; Burd, M.; Campbell, D. R.; Dudash, M. R.; Johnston, M. O.; Mazer, S. J.; Mitchell, R. J.; Morgan, M. T.; et al. Pollen Limitation of Plant Reproduction: Ecological and Evolutionary Causes and Consequences. Ecology 2004, 85, 2408−2421. (2) Aguilar, R.; Ashworth, L.; Galetto, L.; Aizen, M. A. Plant Reproductive Susceptibility to Habitat Fragmentation: Review and Synthesis through a Meta-Analysis. Ecol. Lett. 2006, 9, 968−980. (3) Potts, S. G.; Biesmeijer, J. C.; Kremen, C.; Neumann, P.; Schweiger, O.; Kunin, W. E. Global Pollinator Declines: Trends, Impacts and Drivers. Trends Ecol. Evol. 2010, 25, 345−353. (4) Klein, A. M.; Vaissiere, B. E.; Cane, J. H.; Steffan-Dewenter, I.; Cunningham, S. A.; Kremen, C.; Tscharntke, T. Importance of Pollinators in Changing Landscapes for World Crops. Proc. R. Soc. B 2006, 274, 303−313. (5) Ricketts, T. H.; Regetz, J.; Steffan-Dewenter, I.; Cunningham, S. A.; Kremen, C.; Bogdanski, A.; Gemmill-Herren, B.; Greenleaf, S. S.; Klein, A. M.; Mayfield, M. M.; Morandin, L. A.; et al. Landscape Effects on Crop Pollination Services: Are There General Patterns? Ecol. Lett. 2008, 11, 499−515. (6) Gilmour, M. I.; Jaakkola, M. S.; London, S. J.; Nel, A. E.; Rogers, C. A. How Exposure to Environmental Tobacco Smoke, Outdoor Air Pollutants, and Increased Pollen Burdens Influences the Incidence of Asthma. Environ. Health Perspect. 2006, 114, 627−633. (7) D’Amato, G.; Cecchi, L.; Bonini, S.; Nunes, C.; Annesi-Maesano, I.; Behrendt, H.; Liccardi, G.; Popov, T.; Van Cauwenberge, P. Allergenic Pollen and Pollen Allergy in Europe. Allergy 2007, 62, 976−990. (8) Nel, A. Air Pollution-related Illness: Effects of Particles. Science 2005, 308, 804−806. (9) Champion, J. A.; Katare, Y. K.; Mitragotri, S. Particle Shape: a New Design Parameter for Micro-and Nanoscale Drug Delivery Carriers. J. Controlled Release 2007, 121, 3−9. (10) Itano, M.; Kezuka, T.; Ishii, M.; Unemoto, T.; Kubo, M.; Ohmi, T. Minimization of particle contamination during wet processing of Si wafers. J. Electrochem. Soc. 1995, 142, 971−978. (11) Kraus, T.; Malaquin, L.; Schmid, H.; Riess, W.; Spencer, N. D.; Wolf, H. Nanoparticle Printing with Single-particle Resolution. Nat. Nanotechnol. 2007, 2, 570−576. (12) Grant-Downton, R. Pollen Terminology: An Illustrated Handbook; Springer, 2009. (13) Pacini, E.; Hesse, M. Pollenkittits Composition, Forms and Functions. Flora 2005, 200, 399−415. (14) Thio, B. J. R.; Lee, J. H.; Meredith, J. C. Characterization of Ragweed Pollen Adhesion to Polyamides and Polystyrene Using Atomic Force Microscopy. Environ. Sci. Technol. 2009, 43, 4308− 4313. (15) Lin, H.; Gomez, I.; Meredith, J. C. Pollenkitt Wetting Mechanism Enables Species-specific Tunable Pollen Adhesion. Langmuir 2013, 29, 3012−3023.

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Shuto Ito: 0000-0003-2939-1693 Author Contributions

S.I. and S.N.G. conceptualized and designed the study. S.I. conducted the research and collected the data. S.I. and S.N.G. performed cryo-SEM. S.I. analyzed the data and wrote the manuscript. S.N.G. provided corrections for the manuscript. 24697


Research Article

ACS Applied Materials & Interfaces (16) Lin, H.; Lizarraga, L.; Bottomley, L. A.; Meredith, J. C. Effect of Water Absorption on Pollen Adhesion. J. Colloid Interface Sci. 2015, 442, 133−139. (17) Lin, H.; Qu, Z.; Meredith, J. C. Pressure Sensitive Microparticle Adhesion through Biomimicry of the Pollen−stigma Interaction. Soft Matter 2016, 12, 2965−2975. (18) Amador, G. J.; Matherne, M.; Mathews, M.; Gorb, S. N.; Hu, D. L. Honey bee hairs and pollenkitt are essential for pollen capture and removal. Bioinspiration Biomimetics 2017, 12, No. 026015. (19) Proctor, M.; Yeo, P.; Lack, A. The Natural History of Pollination; Harper Collins Publishers, 1996. (20) Erbar, C.; Leins, P. Portioned Pollen Release and the Syndromes of Secondary Pollen Presentation in the CampanulalesAsterales-complex1. Flora 1995, 190, 323−338. (21) Lisci, M.; Cardinali, G.; Pacini, E. Pollen Dispersal and Role of Pollenkitt in Mercurialis annua L.(Euphorbiaceae). Flora 1996, 191, 385−391. (22) Lunau, K. Notes on the Colour of Pollen. Plant Syst. Evol. 1995, 198, 235−252. (23) Turkington, R.; Aarssen, L. W. Hypochoeris radicata L. (Achyrophorus Radicatus (L.) Scop.). J. Ecol. 1983, 71, 999−1022. (24) Qu, Z.; Meredith, J. C. The Atypically High Modulus of Pollen Exine. J. R. Soc., Interface 2018, 15, No. 20180533. (25) Vagharchakian, L.; Restagno, F.; Léger, L. Capillary Bridge Formation and Breakage: A Test to Characterize Antiadhesive Surfaces. J. Phys. Chem. B. 2008, 113, 3769−3775. (26) Pico, F. X.; Ouborg, N. J.; Van Groenendael, J. Influence of Selfing and Maternal Effects on Life-cycle Traits and Dispersal Ability in the Herb Hypochaeris radicata (Asteraceae). Bot. J. Linn. Soc. 2004, 146, 163−170. (27) Kaelble, D. H. Dispersion-polar Surface Tension Properties of Organic Solids. J. Adhes. 1970, 2, 66−81.

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Fresh âPollen Adhesiveâ Weakens Humidity-Dependent Pollen

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