American Journal of Botany 97(4): 535544. 2010.

DEHYDRATION-INDUCED EXPRESSION OF A 31-KDA DEHYDRIN IN POLYPODIUM POLYPODIOIDES (POLYPODIACEAE) MAY ENABLE LARGE, REVERSIBLE DEFORMATION OF CELL WALLS1
Bradley E. Layton2, M. Brent Boyd2, Manuela S. Tripepi3, Beatrice M. Bitonti3, M. Norman R. Dollahon4, and Ronald A. Balsamo4,5
2Department of Mechanical Engineering and Mechanics, 3141 Chestnut St., Drexel University, Philadelphia, Pennsylvania 19104 USA; 3University of Calabria, Dipartimento di Ecologia, Via Pietro Bucci - 87036 Rende Cosenzo, Italy; and 4Villanova University, Department of Biology, 800 Lancaster Ave., Villanova, Pennsylvania 19085 USA

Current and predicted climate changes caused by global warming compel greater understanding of the molecular mechanisms that plants use to survive drought. The desiccation-tolerant fern Polypodium polypodioides exhibits extensive cell wall folding when dried to less than 15% relative water content (RWC) and rapidly (within 24 h) rehydrates when exposed to water and high humidity. A 31-kDa putative dehydrin polypeptide expressed in partially and fully dry tissues detected via western blotting was present only during drying and rapidly dissipated (within 24 h) upon tissue rehydration. Immunostaining indicates the presence of dehydrin near the cell wall of partially and fully dried tissues. Atomic force microscopy of tracheal scalariform perforations indicates that dry vascular tissue does not undergo signicant strain. Additionally, environmental scanning electron microscopy reveals differential hydrophilicity between the abaxial and adaxial leaf surfaces as well as large, reversible deformation. The ability to avoid cell wall damage in some desiccation-tolerant species may be partially attributed to cell wall localization of dehydrins enabling reversible, large cell-wall deformation. Thus, the de novo synthesis of dehydrin proteins and potential localization to the cell walls of these desiccation-tolerant species may play a role in avoiding mechanical failure during drought. Key words: atomic force microscopy; cell wall; cellulose; dehydrin; desiccation tolerance; environmental scanning electron microscopy; material orthotropy; Polypodiaceae; Polypodium polypodioides.

The ability to maintain hydration levels and to survive periods of water scarcity is vital to all life. While many plants are classied as drought resistant because of their ability to endure extended periods without water, a few species that have adapted to survive longer with even less water are categorized as desiccation tolerant (Oliver and Bewley, 1997; Oliver et al., 2000). These are species in which mature plants remain viable to hydration levels as low as 5%. Perhaps most notable for this ability is the resurrection fern, Polypodium polypodioides (L.) Watt (Helseth and Fischer, 2005). Extreme dehydration is known to present challenges not only to proteins, which must be surrounded by water to avoid denaturation (e.g., Tompa et al., 2006), but also to whole cells. When cells are greatly dehydrated, buckling deformation occurs, causing mechanical stress on the cell wall, which may lead to a fatal compromise of the cells integrity (Moore et al., 2007). Presumably, desiccation-tolerant plants have acquired a system of protein expression mechanisms coupled with cell and leaf morphology for recovering from large deformations that in less tolerant species would cause cell rupture (In this context, large deformation has
1 Manuscript received 19 September 2009; revision accepted 13 January 2010.

The authors thank Sally Shrom, Dee Breger, and Ed Basgall for SEM assistance, Craig Hollish for AFM assistance, Andrew McDonald for help with sample preparation, and Kenneth Barbee and the Nanotechnology Institute of Philadelphia for AFM access. This work was supported in part by the USDA/CREES NRI grant 2008-35100-04413. 5 Author for correspondence (e-mail: ronald.balsamo@villanova.edu), phone: 610-519-6621, fax: 610-519-7863 doi:10.3732/ajb.0900285

the same meaning as nite deformation and refers to strains greater than 1%). Recent work in cell wall biochemistry has implicated arabinans (a component of pectin) production as a cell wall component that enables desiccation tolerance through large, reversible, polymeric deformation (Moore et al., 2006). Another class of proteins known as dehydrins (Close, 1996; Allagulova et al., 2003), that are produced in plants and cyanobacteria, (e.g., Close and Lammers, 1993), is also known to play a role in responding to general stresses such as drought, temperature uctuations, and salinity (Rorat, 2006; Kosova et al., 2007) as well as in maintaining cytosolic desiccation tolerance (Bradford and Chandler, 1992; Still et al., 1994; Beardmore and Whittle, 2005; Boudet et al., 2006). And, as proposed in the present work, dehydrins may also play a role in resistance to mechanical failure of the cell wall. We have chosen to investigate the distribution of dehydrins in the fern P. polypodioides because of their conspicuous appearance during volumetric-water-loss-induced mechanical deformation. This species was selected for its relative ease of care and robustness as well as its prevalence in the literature as a desiccation-tolerant species. For a review of dehydrin structure and of the development of dehydrin antibodies, see (Close and Lammers, 1993; Close et al., 1993a, b; Mouillon et al., 2006; Harryson, 2007). Desiccation tolerance in the vegetative tissues of plants is of intense interest for its relevance to both agriculture and to the study of the evolution of terrestrial plants. A number of studies have demonstrated that desiccation-tolerant species synthesize dehydrin (LEA D-11) proteins when challenged with prolonged dehydration stress, resulting in massive loss of cell water to below 20% relative water content (RWC) (Collett et al., 2004; Farrant et al., 2004; Vicre et al., 2004), yet a functional role for these proteins remains elusive. Dehydrins have been localized in the cells of dried tissues to various subcellular structures including 535

American Journal of Botany 97(4): 535544, 2010; http://www.amjbot.org/ 2010 Botanical Society of America

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the cytosol and nucleus (Asghar et al., 1994; Wisniewski et al., 1999), chloroplasts (Godoy et al., 1994), and in developing vascular tissue (Garnczarska et al., 2008). Recently, Rorat (2006) demonstrated that dehydrins also have an afnity to acidic phospholipids. However, little work on the synthesis of dehydrin proteins has been conducted on ferns (Reynolds and Bewley, 1993). Similarly, the mechanisms by which the cell walls of desiccation-tolerant species manage to withstand the strain caused by massive loss of cellular water (and volume) and the changes in mechanical properties of wet vs. dry cell walls remain unresolved (Moore et al., 2006). Previous studies investigating forces generated on the cell wall by the loss of turgor within the cell as well as the loss of water in the cell wall itself have been restricted to gross mechanical experiments on processed cellulose, pieces of or entire laminae (Kneebone, 1960; Vincent, 1982; Greenberg et al., 1989; Balsamo et al., 2003, 2005, 2006). Microscale and nanoscale behavior of cell walls at varying levels of hydration, to our knowledge, has yet to be reported. In this study, we investigate dehydrin expression in the resurrection fern, specically the desiccation-tolerant species P. polypodioides. We present immunolocalization, SDS-PAGE, western blotting, and cryogenic SEM results for determining the presence, prevalence, and spatiotemporal distribution of dehydrins. Environmental scanning electron microscopy (ESEM) was used to observe the real-time dynamic microscopic deformation of whole pinnae, and atomic force microscopy of cell wall in hydrated and dehydrated conditions was used to analyze the nanoscale deformation of cell walls as water content varied. The working hypothesis of the paper is that the synthesis and subsequent transport of dehydrins toward the cell wall enable large, reversible deformations to occur without strain-induced damage during volumetric water loss. This enables drought-tolerant and desiccation-tolerant species to survive dry conditions and subsequent rehydration. While it is still unclear what mechanism may enable preferential distribution of dehydrins at or near the cell wall during dehydration, it may be simply outward radial bulk ow of water. Once dehydrin-enabled water sequestration at or near the cell membranecell wall boundary has occurred, this proteinaceous slurry may act as a lubricant during dehydration-induced cell wall and cell membrane deformation, which in turn dissipates shear stress, thus reducing the likelihood of tissue damage. MATERIALS AND METHODS
Plant materials and growing conditionsTwenty-four rhizomes (each with several fronds present) of the resurrection fern P. polypodioides were purchased from Carolina Biologicals (Burlington, North Carolina, USA). Twelve sets of trays were constructed out of plywood treated with shellac (Zinssers Bulls Eye Shellac Sealer & Finish; Rust-Oleum, Somerset, New Jersey, USA) to prevent water absorption and water leakage with three sets of plants placed into each tray. Plants were maintained and allowed to acclimate in a mixture of 50% peat moss and 50% Metro-mix 200 articial soil (Scotts Miracle-Gro, Marysville, Ohio, USA) for 1 month prior to experiments. Plants were maintained in a greenhouse on the Villanova University campus, Villanova, Pennsylvania, June 2007 through August 2007, with ambient light (10001500 Em2s2, 50% relative humidity, and 24/18C day/night temperatures. Plants were watered using tap water supplemented weekly with half-strength soluble Miracle-Gro Water Soluble All Purpose Plant Food (248-16) Solution (Scotts Miracle-Gro). At the end of 4 weeks (28 d), eight randomly selected trays (groups D and R, Table 1) were selected to receive no water for 2 weeks (14 d), while the other four trays received daily watering and served as controls, group W. At the end of week 6, the relative water content of dried plants (both D and R) was typically between 10 and 15%, compared to group W samples, which remained at 100%. After two additional weeks (weeks 78) in the dry state, the relative water content did not change,

Experimental groups. Wa = daily watering to saturation. NWa = No water.

Week

Group Control Partially dry Dry control Rehydrated

Designation W PD D R

14 Wa Wa Wa Wa

58 Wa Wa NWa NWa

9 on Wa NWa Wa

and so daily watering to soil saturation (water leaking out the bottom of the trays) was resumed for four of the trays (group R) to collect partially and fully rehydrated (saturated) samples at 1, 4, 24, 48, and 96 h after the beginning of the rehydration process. Plant relative water contentsRelative water content for all samples was obtained by weighing fronds, then placing them into a polyethylene bag with deionized water for 24 h. Surface water was eliminated by blotting the fronds dry with a paper towel. Fronds were then reweighed and placed into a 70C oven containing CaSO4 desiccant) for 48 h and reweighed. Relative water content (Barr and Weatherley, 1962) was then determined as RWC = (We Wd)/ (Wsat Wd) 100%, where We is the experimental mass of the leaf, Wd is the mass of the leaf in the totally desiccated state, and Wsat is the mass of the leaf under full saturation (full turgor). SDS-PAGE and western blottingProteins were extracted from saturated tissues, partially dried laminae, fully dried laminae, partially rehydrated laminae, and fully rehydrated (saturated) laminae over the course of the study according to Wisniewski et al. (1999). Approximately 0.5 g of tissue for each extraction (obtained from at least three plants) was plunged into liquid nitrogen and ground into a ne powder using a mortar and pestle. Next, 1.0 ml of extraction buffer (50 mM ascorbate, 50 mM borate, 1 mM dithiothreitol [DTT], pH 9.0) was added, and tissue was further ground with a mortar and pestle. The liquid was recovered with a glass pipette and placed into 1.0 ml microcentrifuge tubes. This was incubated for 15 min on ice. Samples were centrifuged at 11 000 g for 10 min, the supernatant was collected, and protein concentrations were determined with the Bio-Rad reagent and protocol (Bio-Rad, Hercules, California, USA). The samples were diluted with 2 Laemmli buffer and stored at 80C. For each experiment, 2 g of protein per lane were electrophoretically separated in two identical 12% polyacrylamide gels (Bio-Rad) for 45 min at 150 mV. One gel was subjected to silver staining, and the other was prepared for western blotting. For silver staining, gels were incubated for 90 min in 20% methanol and 0.2% formaldehyde. After being rehydrated in 1% DTT, gels were incubated for 30 min in 0.1% silver nitrate. Gels were then developed in 5% sodium carbonate and 1% formaldehyde. Development was stopped by adding a 5% (w/v) sodium citrate solution. Proteins from unxed gels were transferred to nitrocellulose paper using a Bio-Rad transfer apparatus. The nitrocellulose blot was then blocked with 3% Tris-buffered saline (TBS) solution for 90 min, then washed with 1% gelatin TBS solution. The blot was then incubated for 90 min in a 1 : 1000 dilution of rabbit antidehydrin (PLA-100F, StressGen, San Diego, California, USA) in 1% gelatin TBS. This antibody binds to the lysine-rich C-terminus consensus sequence as rst described by Close et al. (1993b). The blots were then washed three times in 1% gelatin TBS for 7 min each. Blots were then incubated for 2.5 h in a 1 :1 000 goat-anti rabbit AP conjugate (Bio-Rad) in 1% gelatin TBS. The blots were then developed using the BioRad immunoassay developing kit. Developing was stopped with 10% acetic acid. Blots were dried in the dark. In the wet controls (W), data were taken at weekly intervals with no observed change in banding patterns or levels of expression. In the partially dry (PD) samples, data were taken at week 6. For the very dry (D) samples, data were taken at the end of week 8 and over the course of week 9 with no observable changes. For rehydrated samples, data were collected at 4, 24, and 96 h after the reinitiation of watering at the beginning of week 9. ImmunolocalizationFive fully saturated (W) and ve fully dehydrated (D) pinnae from ve plants were xed in half-strength Karnovskys (1965) solution, dehydrated in an ethanol series, and rapidly (24 h) embedded in full strength LR White resin (Electron Microscopy Sciences, Washington, Pennsylvania,

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Fig. 1. Gross anatomy of whole Polypodium polypodioides plants. (A) Saturated (100% RWC) and (B) dried for 2 weeks to approximately 12% relative water content [RWC]. Cryogenic SEM of (C) wet and (D) dry P. polypodioides pinna cross sections. Scale bars: 75 m. P: palisade cells; S: spongy cells. (E) Vascular bundle. Arrow indicates scalariform perforations in individual tracheary elements, which are shown in greater detail in Fig. 5. Scale bar: 5 m.
USA). Cross sections of pinnae of 500 nm thickness were cut using a Sorvall MT 6000 ultramicrotome and dried onto glass slides. Slides were blocked in 1% gelatin TBS for 1 h then transferred to 1 : 1000 diluted rabbit antidehydrin in 1% gelatin TBS solution (Asghar et al., 1994). After 2 h, slides were washed three times in 1% TBS solution for 10 min each. Slides were incubated in 15-nm goldconjugated goat-anti rabbit solution (Electron Microscopy Sciences) for 2 h. Slides were washed once in 1% gelatin TBS, twice in distilled water, and allowed to dry. Control slides were stained with 1% aqueous toluidine blue (w/v) for 30 s, and rinsed once with deionized water and air dried. Slides were treated with immersion oil, coverslipped, and viewed on an Olympus BX 60 light microscope using backscattered lighting. Photographs were obtained with an Olympus SC 35 camera. Cryogenic SEMSaturated and fully dehydrated pinnae were cryoxed by rapid plunging into nitrogen slush, cracked under vacuum at 140C, etched at 90C for 10 min, and sputter-coated with carbon/gold (Polaron SC7640, London, UK). Images of cross sections of wet and dry laminae were obtained using a Hitachi S-570 SEM (Tokyo, Japan). Environmental SEM (ESEM)To simulate the natural in vivo drying and resulting gross mechanical cellular deformation, we observed whole pinnae with ESEM during one cycle of drying and rehydration. Samples from the wet controls (W) were harvested at week 9 and prepared for wetmode ESEM imaging on a Phillips XL30 (FEI, Hillsboro, OR) using the protocol of (Layton et al., 2008). Abaxial and adaxial sides were imaged individually at magnications ranging from 125 to 250. Both imaging sessions began with the chamber at room temperature with a 50 L droplet of distilled water placed next to the sample. Chamber hydration was controlled to maintain relative humidity levels between 95 and 10% by changing the chamber pressure from between 720 Pa and 65 Pa, at 20-min intervals while the chamber temperature was maintained at 2C. Images were captured at an image size of 1936 1452 pixels using the software package provided by the manufacturer. Atomic force microscopySamples were imaged according to standard protocols for biological imaging, e.g., (Layton et al., 2004a, b). Thick sections (5 m) were cut from the same preparations used for immuno EM taking care to cut parallel to the vascular bundles. These were mounted on 1% polylysinecoated glass slides (Electron Microscopy Sciences) and etched for 30 min with 8% sodium metaperiodate (Sigma, St. Louis, Missouri, USA) to remove the plastic xative. These were then imaged in air contact mode on a Nanoscope 3100 (Veeco Instruments, Santa Barbara, California, USA) with Veeco DNPS tips with a spring constant of 0.068 N/m. Gains were manually tuned such that integral gain was set to a value just under the ringing threshold, and proportional gain was set to approximately 1.5 of the value of integral gain. The scan rate ranged from 1 m/s to 20 m/s. Throughout imaging, ambient thermal and mechanical vibrations allowed us to maintain a trace-minus-retrace value of less than 1 nm, indicating that we obtained nearly ideal sampletip interactions by minimizing tipsample friction. After scanning was complete, the cross-sectional

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analysis tool in the Digital Instruments (Veeco) v5.12r5 software was used to analyze the image prole axially.

RESULTS Gross anatomy of wet and dry P. polypodioides We observed consistent leaf/pinnae curling about all three axes: axial, transverse, and normal in the dry samples and consistently expanded leaves/pinnae in the saturated plants at the organism scale (Fig. 1A, B). The adaxial side underwent negative (compressive) strain to a greater extent than the abaxial side, resulting in the pinnae becoming highly concave. Cryogenic SEM indicates large deformation Transverse pinna cross sections of saturated P. polypodioides revealed typical columnar-shaped palisade parenchyma and spherical spongy parenchyma (Fig. 1C). Fully dehydrated laminae of P. polypodiodes exhibited extensive wall deformation and loss of volume in both the palisade and spongy parenchyma cells (Fig. 1D). The palisade cells, which are tubular structures on the adaxial side (sunward side) of the pinna, buckled slightly in the through-plane direction and shrank in the in-plane direction causing pinna folding through a mechanism whereby the adaxial side of the pinna loses water mass and thus volume, at a rate, and to an extent greater than that of the abaxial side. The pinna thus curled upward about both the major (axial) and minor (transverse) axes of the pinna. In addition, the pinnae curl about their normal axes, belying the orthotropic material structure of the pinna. Furthermore, even in the fully dry state, D, tracheal members of the vascular tissue running through the center of the pinna appeared to remain open (Fig. 1E). Plant relative water content The mean dry mass of the laminae was 31% 3.77 (N = 30) of the mass of the fully hydrated laminae. This value was used as a baseline for the following sample measurements: partially dehydrated, PD = 58%; fully dehydrated, D = 12%; partially rehydrated, 4R = 64%; resaturated, 96R = 100%. SDS-PAGE and western blotting resolve timing of dehydrin expression A comparison of tissues that ranged from fully hydrated (W), partially dry (PD), and fully dry (D) and through rehydration, (4 h [4R], 24 h [24R], and 96 h [96R]) of soluble protein extracts of P. polypodioides revealed a general trend of polypeptides of 3134 kD (Fig. 2A). However, western blot analysis using a polyclonal antibody raised against the C-terminus consensus sequence (Close et al., 1993a) revealed a single polypeptide in the PD, D, and 4R samples of approximately 31 kDa (Fig. 2B). This putative dehydrin was not detected thereafter, implying that it is preferentially expressed only when the plant is undergoing volumetric water loss. Immunolocalization of dehydrins near cell wall Unlabeled transverse cross sections of fully hydrated (W) laminae exhibited low-aspect-ratio cells with single, large, central vacuoles (Fig. 3A). Unlabeled cross sections of partially dried, PD (58% RWC) laminae revealed cells with normal architecture, some degree of wall infolding, and multiple, small vacuoles distributed throughout the cell (Fig. 3B). Only the partially (PD) and fully dehydrated (D) samples showed any label of the antidehydrin. Furthermore, the labeling was primarily restricted to the periphery (Fig. 3D, E), with no detectable labeling in the
Fig. 2. Protein extracts of Polypodium polypodioides at various hydration levels. (A) SDS-PAGE. Right arrows indicate the 31-kDa dehydrin. Lower bands were only expressed in dry material but were not detected by western blotting. (B) Western blot. MWM, molecular weight marker; W, wet; PD, partially dry; D, dry; 4R, 4-h postrecovery; 24R, 24-h postrecovery; 96R, 96-h postrecovery.

fully hydrated (W) samples, which were used as a negative control (Fig. 3C). ESEM observations reveal cell buckling Under ESEM, whole pinnae behaved in a manner similar to those observed in vivo. As the humidity in the chamber was lowered from fully saturated to 5% humidity, pinnae underwent considerable mechanical strain (ca. 70%), primarily on the adaxial side (Fig. 4 AE). This strain was dramatically illustrated by the antler-like appearance of buckled cells (Fig. 4C). The abaxial side underwent considerably less strain (ca. 20%). Also notable was the greater degree of hydrophilicity of the abaxial surface (Fig. 4FJ). With the exception of imaging artifacts (e.g., bubbling around imaged regions) caused by the electron beam, pinnae regained their original morphology upon rehydration. Atomic force microscopy reveals cell wall layers We chose to focus our attention on the sclariform perforations of the tracheary elements (Luna et al., 2008) to resolve both the laminar structure of cell wall and as a method for evaluating nanoscale deformation of individual cells. As can be seen from Fig. 5, the cell wall of P. polypodioides cellulose structure has a distinct orthotropic architecture. To our knowledge, this is the rst in situ AFM image of the scalariform lamina in vascular tissue from any organism. Other examples of directly observed cellulose structures in the cell wall may be found in the burgeoning biofuels literature (e.g., Ding and Himmel, 2006; Himmel et al., 2007) or in processed samples (e.g., Davies and Harris, 2003). Cross-sectional proles of the hydrated and dehydrated specimen were taken in the axial direction using the Digital Instruments cross-sectional analysis tool, and peak-to-peak measurements were made of the scalariform perforations. Because sample preparation and subsequent imaging proved to be tedious, we were not able to obtain sufcient data to perform a statistical analysis (Table 2). However, there was not an observable strain in the two scalariform perforations measured in the wet state and the two measured in the dry state, indicating that the tissue remains relatively undeformed even in the fully dehydrated (D) state, consistent with our cryogenic SEM imaging results. In the fully hydrated state, individual laminae within scalariform perforations were observable and had thicknesses on the order of 5065 nm.

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Fig. 3. Wet and dry pinnae cross sections of Polypodium polypodioides. (A, B) Bright-eld images of lamina stained with toluidine blue: (A) fully hydrated, W tissue (100% relative water content [RWC]); (B) partially dried, PD tissue (58% RWC). (CE) Backscatter images of immunogold-labeled sections of lamina stained for dehydrins: (C) fully hydrated, W tissue (100% RWC); (D) partially dried, PD tissue (58% RWC); (E) detail of (D). Scale bars: 80 m. V = vascular tissue; M = mesophyll cells; arrows indicate dehydrin labeling near the cell wall.

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Fig. 4. Wet-mode ESEM imaging of full pinnae. (AE) Adaxial side of the pinna at (A) 100%, (B) 50%, (C) 5%, (D) 50%, (E) 90% humidity. (FJ) Abaxial side of pinna at (F) 100%, (G) 50%, (H) 5%, (I) 50%, (J) 90% humidity. Arrows indicate a single feature (stoma). Asterisks indicate water. Scale bar = 20 m.

DISCUSSION Overview In an effort to elucidate the molecular mechanisms that plants use to survive drought, we have presented the results of a multifaceted approach. Primarily, we have suggested a role that dehydrins may play in drought and desiccation survivability. (1) Dehydrins were expressed only during drying and in the fully dry state, (2) these dehydrins were localized at the cell periphery, and (3) leaves/pinnae demonstrated the ability to undergo large-scale, reversible deformation. This study is the rst in which the localization of dehydrins near the cell walls of dehydrated specimens of a desiccation-tolerant species of fern has been observed simultaneously with large deformation of the cell wall. While previous studies have focused on the presence of arabinans in cell wall as a molecule responsible for dehydration or desiccation tolerance (e.g., Moore et al., 2006), we present evidence that the presence of dehydrins may permit large deformations to occur, thus allowing cell walls to deform without rupturing during drastic uctuations in plant water content. With this information, it may become

possible to look for a similar dehydrin synthesis, localization, and large-scale deformation in plants that also possess the ability to resist drought or desiccation. Furthermore, it may be possible to confer upon species that are not able to resist drought, this ability by introducing the dehydrin gene of P polypodioides for agricultural purposes. Our work also represents the rst AFM images of the discrete layers of cell wall organization in plant vascular tissue. Atomic force microscopy and high-resolution imaging of prepared cellulose structures The theoretical maximum resolution as predicted by the equal partition theorem for ideal imaging conditions of atomic force microscopy is less than 0.1 nm. Typical imaging conditions of biological AFM imaging, however, limit the practical resolution to approximately 0.5 nm in the z-direction (vertical) and 12 nm in the xy plane (horizontal direction). A common practice for visualizing individual membranes and proteins is to prepare a monolayer prior to imaging to reduce artifacts caused by thermal drift and frictional effects. Our strategy was to resolve molecular-scale features in

Fig. 5. Air contact mode image of scalariform perforations in tracheal members. (A) Fully hydrated sample. (B) Dry sample. Arrows depict individual cell wall lamina. Scale bars = 1 m.

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AFM results indicating that the scalariform perforation spacing in tracheary elements was relatively undeformed in the dry state (D) relative to the fully hydrated state (W). L = length of cross section sampled, i = number of scalariform perforations sampled, Ls = average axial length of scalariform perforation.
L (m) 9.160 5.234 10.719 5.359 i 5 3 5 3 Ls (m) 1.832 1.745 2.144 1.786

stresses develop in the cell wall, the water-bound dehydrins could serve as a lubricant, thus reducing the probability of cell wall fracture and minimizing cell membrane disruption. Atomic force microscopy and high-resolution imaging of natural cellulose systems Only two other studies used AFM to examine the nanobrous structure of plants. Fukumoto et al. (2005) studied embryonic conifer cell bers from Larix leptolepis with AFM and resolved individual callose brils and subbrils with diameters of 700 nm and 170 nm, respectively. In the other study, Mine and Okuda (2007) imaged prepared cellulose from the alga Vaucheria terrestris with AFM and resolved brils with diameters of ca. 20 nm, but no axial banding or orthotropic architecture. While this is insufcient evidence to demonstrate that the orthotropic structure we resolved in P. polypodioides is unique, it does offer evidence that it may be an atypical feature, perhaps limited to desiccationtolerant ferns. Reports of AFM on cellulose or cell wall as it appears in vivo or in situ are also scarce. We found three papers of relevance to the current work. Kirby et al. (1996) performed contact mode AFM on a variety of freshly prepared homogenized and SDStreated plant cell walls from the parenchymal cells of a variety of fruits, imaging them as they dried. Their primary ndings, consistent with ours, were that the walls exhibited a laminar structure with microbrils having diameters of approximately 25 nm. Pesacreta et al. (1997) imaged native-state and peroxide-treated cotton microbrils, revealing bril structures ranging from a few nanometers to a few hundred nanometers. Thimm et al. (2000), viewing celery (Apium graveolens) parenchyma cell walls with AFM under dehydration conditions, reported a microbril diameter of 625 nm and observed that the cellulose microbrils appear smaller in dehydrated conditions. Mats of faintly ordered wavy cellulose microbrils were clearly seen at the 3 m scan size, but with no observable periodicity along the bril axis. Protein synthesis and cell wall mechanics under growth and deformation conditions We propose that the desiccation tolerance in P. polypodioides is related to the interaction strength between the laminas of the cellulose cell wall. While other protein mechanisms such as expansin production have been proposed to explain regulation of cell wall expansion (Cho and Cosgrove, 2000), these mechanisms have not been related to desiccation tolerance. Another recent publication (DeBolt et al., 2007) cites a newly discovered compound, morlin, which may be partially responsible for cellulose synthase movement. In addition, morlin may be partially responsible for the organization of cellulose and, thus, the relative ability of cells to resist turgor pressure. However, the role of morlin in desiccation tolerance has not been addressed. Specically, the dehydrins found in the current study may be responsible for providing a means of attracting and localizing water, which then serves as a lubricant between cell wall laminae undergoing large deformation, and the rate at which these proteins are expressed may affect the survivability of various desiccationtolerant species. Molecular mechanics We also hypothesize that in a growing plant, freshly synthesized cellulose polymer, as it is extruded from the rosette structures, supplies sufcient mechanical pressure to either rupture or displace existing laminae and that this synthesis may be anisotropically distributed along the length of

Specimen W D

their native anatomical conguration. Our images were obtained from relatively rough fragments of material, which nevertheless had vertical dimensions less than the total z-range of the microscope, 5 m. Multiscale relevance For the present work, it is of great importance to relate the nanoscale deformation of cellulose as plants uctuate between hydrated and dehydrated states. For example, knowing whether the individual laminae of the cell wall are capable of sliding past each other during dehydrationinduced deformation, will enable deeper understanding of the trifaceted problem of systematically resolving the relationships between nanoscale biomechanics, cell wall biochemistry, and organism survivability. Cellular to molecular scale Of particular importance for understanding how individual cells deform under dehydration conditions is the ability to directly observe changes in morphology at the nanoscale and to build models for the biomechanics of individual cellulose bers. Recently, Kondo et al. (2004) imaged a processed preparation consisting of bleached cotton linters with a degree of polymerization of 1300 at stretch ratios of 1.0, 1.5, 1.7, and 2.0 with AFM and resolved a Poisson effect in both the in-plane and through-plane directions. Prior work by the same group reveled a molecular periodicity for cellulose of 0.46 nm (Kondo et al., 2001). Togawa and Kondo (1999) employed x-ray diffraction analysis of drawn, thin lms of cellulose to quantify crystallinity. They attributed the reluctance of cellulose to crystallize in their drawing process to the hydrogen bonding between polymer chains, citing earlier work (Postema et al., 1990) that estimated a single hydrogen bond energy to be approximately 5% of a single covalent bond. Based on our observations, this balance of forces between the largely hydrogen-bond-dominated interlaminar interactions and the largely covalent-bond-dominated intralaminar interactions likely determines the deformation modes of orthotropically aligned cellulose observed in the current study. In particular, a mechanism such as dehydrin-enabled water localization near the cell wall during drying that enables interlaminar sliding while the cell wall is undergoing large deformation could help explain the survivability of the resurrection fern and other related plants. During dehydration at the cellular scale, when dehydrin synthesis is induced via increased levels of abscisic acid (ABA) (Close et al., 1989; Robertson, 2003), there must also exist a radial transport ow eld from the interior of the cell to the exterior. It is reasonable to postulate that water-bound dehydrins are transported along with this ow. If they pass through the membrane, then they could potentially serve a lubricating role. Specically, as deformation-induced, interlaminar shear

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a growing wall structure (e.g., Geitmann and Parre, 2004). Because cellulose laminae must provide mechanical stability to the underlying cellular structures, the outer structures must expand as cells grow. See Niklas et al. (2006) for review. However, because it is likely that the molecular lattices are only deformable over small strains prior to failure and because plants do not express cellulase for remodeling, then the outer lamina must either rupture and reform, or slide and displace as the underlying cells grow. The laminae observed in P. polypodioides in conjunction with their associated proteins such as the transiently expressed dehydrins may provide a mechanism for laminae to slide past each other rather than rupture during the large deformation induced by dehydration-induced cell wall buckling. Recently, it has been hypothesized that individual lamina are frequently crosslinked with calcium or hemicellulose (Koehler and Telewski, 2006). These crosslinks purportedly serve the purpose of maintaining resistance to vertical bending. If these crosslinks were not present, then a plant structure would perhaps be too compliant, and unable to support its own mass as it grew to compete for light. The confounding requirements of limited interlaminar crosslinking to avoid buckling failure, and sufcient interlaminar crosslinking to maintain vertical structural integrity, suggest a global optimization function for cellulose deposition and cellulose-associated proteins or hemicellulose. The role of lignin content and distribution in individual leaves also cannot be understated. In fact, the distribution of lignin in Pinus nigra and P. resinosa (Pinaceae) was recently revealed to be primarily responsible for the fracture mechanics in these two closely related species (Meicenheimer et al., 2008). These data support previous studies Balsamo et al. (2003) that demonstrated the importance of leaf architecture to mechanical stability during drought. Testable models of the cellulose deposition strategies and the subsequent interactions of cell walls with stress-related proteins such as dehydrins are critical if these mechanisms of drought survivability, especially with regard to cell wall deformation and recovery, are to be revealed. Future work Future work will include testing the hypothesis that plant species that thrive under conditions favoring rapid growth have a greater degree of interlaminar crosslinking, thus giving them a bending stiffness sufcient to resist environmental forces such as wind and rain. The downside of this greater degree of enhanced interlaminar crosslinking required for sufcient bending stiffness, however, may diminish the ability to resist radial buckling loads under drought conditions. Thus, other drought- and desiccation-tolerant plant species such as the genus Eragrostis (Balsamo et al., 2006), that may possess the diminished interlaminar crosslinking compared with those found in rapidly growing plants, may possess the ability to undergo large deformation without failure may also have the ability to survive under drought conditions. It is reasonable to assume that, because the cellulose is extruded in single polymer chains from the rosettes, the lateral association happens after extrusion, with younger layers coming in under older layers. What remains unclear is how existing outer layers expand to allow new inner layers to form. It could be that there is an exchange between layers in cases where an outer lamina becomes damaged, allowing for available binding sites. It is also possible that uncrystallized polymer chains that are being extruded have enough mobility to patch holes in the lattice structure where damage has occurred.

Conclusions We have found a potential correlation between the nanoscale architecture of the cell wall of P. polypodioides, a desiccation-tolerant species of fern, the temporal prole of its dehydrin expression, and the spatial distribution of dehydrins near the cell periphery and possibly within the cell wall. The cell wall of P. polypodioides may be uniquely adapted to survive large-scale deformation associated with dehydration and desiccation. Additional work using functional AFM to resolve the spatial distribution of individual dehydrin molecules potentially present in the cell wall (Lee et al., 2007) may prove valuable for quantifying the nanoscale localization of individual dehydrins or other drought-associated proteins. LITERATURE CITED
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