This work was supported by U.S. National Science Foundation grants # MCB-0741914 and MCB-0843627; additional infrastructural support was provided at The City College of New York by National Institutes of Health 2 G12 RR03060-26 from the National Center for Research Resources. We gratefully acknowledge the J.K.C. Rose group in the Cornell University Plant Biology Department for providing M82 (wild type) and CM15 (mutant) tomato cuticles. We thank Dr. Spyros Monastiriotis from the CCNY Chemical Engineering group of Prof. Alexander Couzis for his generous help with the AFM experiments. We thank Ms. Lauren Gohara for graphical design support.

1. Enzymatic Isolation of Tomato Cuticles5
<ol>
 <li>Place several commercial or cultivated tomatoes in a bowl. Peel the skin from the fruits in large sections; discard the inner pericarp tissue. Wash the tomato skins with deionized water and preserve them under water in a beaker.</li>
 <li>Prepare a 50 mM pH 4.0 sodium acetate buffer (at 31 &deg;C) by putting 1.22 g of sodium acetate trihydrate (Mr. 136.08 g/mol) and 2.34 ml glacial acetic acid (17.485 M) in a beaker, adding 200 ml of deionized water, and then adjusting the pH to 4.0 at 31 &deg;C. Prepare a mixture containing 4 ml of pectinase (EC 3.2.1.15; 10 U ml-1, TCI America), 0.2 g of cellulase (EC 232.734.4; 1.3 units/mg solid, Sigma Aldrich), and 13 mg NaN3, and then add 196 ml of sodium acetate buffer to the enzyme mixture to obtain 200 ml of the final enzyme cocktail.5 Completely immerse the peeled tomato skin in the enzyme cocktail and incubate at 31 &deg;C for 24 hours with constant shaking (G24 Environmental Incubator Shaker; New Brunswick Scientific Co.).</li>
 <li>Collect the tomato skins using a kitchen strainer or B&uuml;chner funnel and wash them with deionized water. Thereafter, place them in a vacuum oven at room temperature for an hour. Preserve the dried tomato skins in a labeled and capped bottle for subsequent dewaxing procedures.</li>
</ol>
2. Exhaustive Dewaxing by Soxhlet Extraction6
<ol>
 <li>The equipment used for exhaustive dewaxing consists of a heat source (heating mantle and Variac controller), round bottom flask for a solvent reservoir, Soxhlet extractor, sintered-glass thimble or disposable extraction thimble, anti-bumping chips and a condenser (see Fig. 1). Note that the narrow siphon arm (parts 6 and 7 in Fig. 1) is very delicate and prone to breakage, requiring careful handling.</li>
 <li>Put 0.5-1 g of tomato skin (obtained in step 1.) in a mortar, and grind the sample to a coarse powder with a pestle unless the samples are to be used for AFM measurements (Section 5). Fill a sintered-glass or disposable thimble approximately halfway with the sample and use tweezers to place it carefully at the base of the extraction column.</li>
 <li>Attach the condenser and wrap it with aluminum foil. Heat the methanol solvent (ACS grade) in the presence of a few anti-bumping chips until it boils gently and refluxes on the walls of the flask. Cover the solvent reservoir with both glass wool and aluminum foil. Check the process for an hour, adjusting the Variac voltage so that the reservoir accumulates about one drop per second and siphoning occurs within the Soxhlet apparatus when the thimble is full.</li>
 <li>Continue the extraction process for 12 h, then lower the heating mantle and allow the apparatus to cool down. Remove the extractor and reservoir as a single unit to dispose of the solvent. Use tweezers to raise the thimble to just below the neck of the extraction column, drain excess solvent from it, and place the thimble on a clean surface. Tilt the extraction column to allow siphoning into the flask below. Disconnect the flask and pour the waste into a labeled waste solvent receptacle.</li>
 <li>Repeat steps 2.3 and 2.4 for successive solvents of progressively diminishing polarity, e.g., chloroform and hexane for 12 h in each case.</li>
 <li>Allow the tomato cutin sample to dry inside the thimble, either by blowing a stream of nitrogen gas over it or by placing it in a vacuum oven at room temperature. Finally, measure the mass of the dry sample and store it at room temperature in a screw-top jar sealed with parafilm.</li>
</ol>
3. Selective Isolation of Epicuticular and Intracuticular Waxes3,4
<ol>
 <li>First, wash whole tomatoes (a separate batch of tomatoes from those described in 1.) with distilled water. Dry them with paper towels and Kimwipes, and place them stem downward on a piece of aluminum foil.</li>
 <li>Paint the whole tomatoes with 120% (w/w, mass ratio) gum arabic aqueous solution in a top down fashion and allow about an hour for the gum arabic to dry on the fruit's skin, leaving a thin film. Remove this film using tweezers, being careful not to puncture the tomato skin. Repeat the procedure once more.</li>
 <li>Add the films to vials containing 1:1 (v/v) chloroform-water and mix for three minutes. After vigorous agitation and phase separation, pipette out the chloroform fractions and evaporate them in separate uncovered vials, leaving epicuticular wax. The tomatoes will remain physically intact. Dip them into chloroform for two minutes at room temperature and collect the intracuticular wax after evaporating the solvent. Now, peel the tomatoes and treat them enzymatically (with cellulase and pectinase in sodium acetate buffer) to remove cellulose and pectin, respectively (as described in 1).</li>
 <li>If desired, perform exhaustive dewaxing on these enzymatically isolated cuticles by Soxhlet extraction (see step 2), using three solvents of varying polarity (methanol, chloroform, and hexane, respectively).</li>
</ol>
4. Molecular Characterization of Tomato Fruit Cutin by Cross-polarization Magic-angle Spinning Solid-state Nuclear Magnetic Resonance (CPMAS ssNMR)6
<ol>
 <li>Place 4-6 mg of fully dewaxed tomato cuticles (cutins) in a 1.6 mm fastMAS zirconia rotor using the vendor-supplied packing tool. (Either ground dewaxed tomato cuticles or very small pieces of partially dewaxed cuticles are suitable.) Ensure that the sample is packed uniformly, but not too tightly, in the rotor. After putting on the top cap, paint half of the cap with a black-ink marker pen to facilitate measurements of the spin rate.</li>
 <li>Adjust the shimming of the NMR spectrometer for minimum spectral line width at half-height and calibrate the proton (1H) and carbon (13C) 90 &deg; pulse widths using a standard compound such as adamantane.</li>
 <li>Using glycine or glutamine as model compounds, obtain maximum signal intensity by optimizing all parameters (Hartmann-Hahn matching power levels, 1H - 13C contact time, heteronuclear 1H decoupling strength) of the cross-polarization magic-angle spinning (CPMAS) experiment. For spectra acquired at a 1H frequency of 600 MHz, recommended conditions include 10 kHz or 15 kHz spinning frequency, a 3-sec delay between acquisitions, and SPINAL heteronuclear proton decoupling7 at a magnetic field strength equivalent to a frequency of 185 kHz.</li>
 <li>Insert the cutin-packed rotor into the probe. Then, place the probe into the magnet. Increase the spinning speed up to 10 kHz gradually to check for good sample packing and rotor stability. Verify the final spinning stability of the rotor to within &plusmn;20 Hz.</li>
 <li>Adjust the tuning and matching capacitors of the probe iteratively to achieve minimum power reflection at both 1H and 13C NMR frequencies. Set the experimental temperature to 25 &deg;C (or room temperature).</li>
 <li>Start the pre-optimized CPMAS experiment corresponding to the Hartmann-Hahn matching condition determined at the 10 kHz spinning frequency.</li>
 <li>Acquire 4096 transients, condition the spectrum with exponential (Lorentzian) line broadening of 50-100 Hz, and do a Fourier transform to generate an NMR spectrum of signal intensity vs. chemical shielding (ppm).</li>
 <li>Reference the 13C chemical shifts externally using adamantane set at 38.4 ppm (-CH2- group)8 as a standard.</li>
 <li>Increase the rotor spinning frequency to 15 kHz and repeat the CPMAS measurement (steps 4.6-4.8) corresponding to the Hartmann-Hahn matching condition determined at this latter spinning frequency.</li>
 <li>Repeat the CPMAS experiments (steps 4.1-4.9) with natural (waxy) and partially dewaxed fruit cuticle samples.</li>
</ol>
5. Probing the Tomato Cuticle Surface with Atomic Force Microscopy (Digital Instruments Nanoscope IIIa; procedures will vary slightly among microscopes)6
<ol>
 <li>Turn on the scanning probe microscope (SPM) (Fig. 2) and make sure that the microscope mode toggle switch is set to the contact Atomic Force Microscopy (AFM) mode.</li>
 <li>Manually raise the SPM head by turning its two user-adjustable front knobs. Detach the AFM tipholder from the SPM head by turning the clamping screw at the back of the head.</li>
 <li>Use tweezers to remove the existing AFM cantilever from the tipholder, then carefully grab a new silicon nitride cantilever (AFM probe) from its package and install it in place of the old cantilever. Use a light microscope to verify that the newly installed AFM cantilever is not broken.</li>
 <li>Attach the tomato cuticle sample (a section of partially dewaxed tomato cuticle ~10 mm x 10 mm) to a stainless steel disk (sample puck) with double-sided tape. Use a light microscope to verify that the cuticle surface remains flat and smooth after placement of the sample on the puck.</li>
 <li>Place the puck with the tomato cuticle sample onto the magnetic region at the top of the SPM scanner.</li>
 <li>Set the front two manual adjustment screws of the scanner high by turning the knobs; set the motorized back adjustment screw to approximately the same level as the other two front screws. Make sure that all three screws are set high enough to avoid breaking the AFM tip when placing the tipholder into the SPM head.</li>
 <li>Reinsert the tipholder into the SPM head and secure it by tightening the clamping screw at the back of the head.</li>
 <li>After turning on the laser, align the laser spot on the AFM cantilever using the central (y) and right (x) laser adjustment knobs on the top of the head. Monitor the reflected laser beam on a piece of paper to position the laser spot exactly at the end of the AFM cantilever.</li>
 <li>Adjust the position of the movable mirror iteratively using the photodetector adjustment knobs to achieve maximum signal (SUM signal), thus ensuring that the reflected laser beam is being received evenly by the four quadrants of the photodetector.</li>
 <li>Lower the AFM tip by retracting the manual adjustment front screws and the motorized back adjustment screw of the SPM scanner, visually monitoring the approach of the AFM tip toward the sample surface with an inverted microscope. Make sure that all three screws are at the same level to avoid artifacts from imaging a tilted sample. Bring the tip toward the sample, but not so close that the tip touches or breaks through the sample surface.</li>
 <li>At this point, the laser light reflected off the AFM cantilever will bounce off the movable mirror to the photodetector. For the contact AFM mode with a silicon nitride AFM tip, set the OUTPUT signal (Vertical Deflection) voltage to -2 V for a 0 V Setpoint and the DIFF Signal (vertical/horizontal difference) to 0 V by adjusting the position of the mirror.</li>
 <li>Using the Nanoscope software, click on the microscope icon and select the appropriate profile (Contact mode AFM).</li>
 <li>Using the "Scan Controls Settings" panel, set the scan rate and scan size e.g., 10 micron scan size and 2 Hz scan rate.</li>
 <li>Allow the AFM tip to engage the sample surface by clicking the engage-tip icon. By controlling the motorized back screw of the SPM base, the program will now lower the tip until it engages the sample surface. The scanning process will automatically start once the tip has engaged successfully.</li>
 <li>Monitor the scanning process using both image and scope modes of the software, adjusting parameters such as setpoint, integral gain, proportional gain, scan size, scan rate, lines and samples/line iteratively to achieve the highest resolution images. Start scanning with a large z-axis range (data scale); then carefully reduce the data scale value to observe the best contrast of surface features on the image. Minimize the occurrence of white areas in the scanned image, which indicate that the height of the surface features exceeds the available z-axis range.</li>
 <li>Capture the image to save the data file; then process the data using flattening functions to remove image artifacts due to vertical (Z) scanner drift, image bows, and vertical offset between scan lines;9 finally calculate the average roughness. After saving the data, retract the AFM tip by reversing the action of the computer-controlled screw motor that was used to engage it. Image processing may be performed in offline image analysis mode and/or using a different computer.</li>
 <li>Use the front bottom gray metal knobs of the tip holder to change the x and y positions of the scanning area; then repeat the measurement at five sample locations to check reproducibility, replacing the AFM cantilever if the image quality deteriorates.</li>
</ol>
6. Representative Results
Chemical shift analysis of the CPMAS 13C NMR spectra (Fig. 3) identified the major functional groups present in the exhaustively dewaxed tomato cuticle (cutin). The key carbon moieties in the cutin biopolyester were found to be long-chain aliphatics (0-45 ppm), oxygenated aliphatics (45-110 ppm), multiply-bonded and aromatics (110-160 ppm), and carbonyls (160-220 ppm). The oxygenated alkyl moieties (CHO + CH2O) play a crucial role in establishing covalent connections between the monomeric units of the cutin biopolymer, thereby forming the molecular architecture of the cutin matrix. Differences in relative peak areas observed in the spectral region between 45 and 100 ppm suggest that the mutant cutin has a relatively large proportion of cross-link forming CHO structural moieties compared to the wild-type cutin; careful quantitative measurements using direct polarization (DPMAS) NMR5 methods support this inference (data not shown).
CPMAS 13C NMR spectra also showed a progressively diminishing wax peak at 31 ppm (Fig. 4), indicating the sequential removal of epi- and intracuticular waxes from the cutin-wax composite while retaining the principal chemical architecture of the cutin biopolymer. Parallel AFM image analysis (Fig. 5) revealed surface irregularities due to the stepwise extraction of epi- and intracuticular waxes from the fruit cuticle, signifying alterations in the organization of the cuticular assembly.
<img alt="Figure 1" src="/files/ftp_upload/3529/3529fig1.jpg" /> 
Figure 1. Soxhlet extractor (image source: Wikipedia).
<img alt="Figure 2" src="/files/ftp_upload/3529/3529fig2.jpg" /> 
Figure 2. A) Scanning probe microscope. B) SPM head (adapted from the AFM training manual provided by Digital Instruments).
<img alt="Figure 3" src="/files/ftp_upload/3529/3529fig3.jpg" /> 
Figure 3. 150 MHz CPMAS 13C NMR spectra of exhaustively dewaxed red ripe tomato fruit cuticles (cutins) from wild-type (M82) and mutant (CM15) show resonances with chemical shifts corresponding to the functional groups of a cross-linked hydroxyfatty acid-based polyester and also some multiply bonded moieties. All spectra were acquired with a 10 kHz spinning frequency.
<img alt="Figure 4" src="/files/ftp_upload/3529/3529fig4.jpg" /> 
Figure 4. 150 MHz 13CCPMAS NMR spectra of commercial red ripe tomato fruit cuticles exhibit compositional changes upon sequential removal of epicuticular and intracuticular waxes by gum Arabic mechanical extraction and a two-minute chloroform dip, respectively. All spectra were acquired with a 15 kHz spinning frequency.
<img alt="Figure 5" src="/files/ftp_upload/3529/3529fig5.jpg" /> 
Figure 5. AFM images and roughness estimates for the partially dewaxed commercial tomato cuticles described in Figure 4 after the stepwise removal of epicuticular (left) and intracuticular (right) waxes.

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No conflicts of interest declared.

The protocols described herein allow for detailed molecular and microscale characterization of a complex intractable plant material without the need for destructive chemical breakdown. To investigate the blending of the cutin biopolyester with various lipids (waxes) that control the structural organization of the cuticular assembly,10 we conducted and monitored procedures for selective removal of epicuticular and intracuticular waxes from the heterogeneous cuticular blend. Solid-state 13C NMR was used to gauge extraction of wax molecular components, and atomic force microscopy served to examine concomitant changes in surface roughness.6,11 To compare the cross-linking capabilities of cutins from cultivated wild-type and single-gene mutant tomato fruits, solid-state 13C NMR was also used to estimate the relative numbers of CHO and CH2O chemical moieties.
A number of design features of this protocol are notable. As the wax materials encompass a wide range of lipids, treating the fruit cuticle with a series of solvents having divergent polarities is essential to achieve exhaustive dewaxing. In addition, dewaxing time can vary from 8 hours to 24 hours depending on the nature of the cuticle samples. To extract epicuticular waxes consistently from the intact fruit cuticle, it is imperative to apply the adhesive coating uniformly to the surface.
Solid-state CPMAS 13C NMR12 is a rapid qualitative method for identifying various structural components of highly heterogeneous and insoluble plant biopolymers while preserving their native physical characteristics;13 traditional solution-state NMR can also be used to characterize the extracted wax mixtures. If quantitative estimation of functional groups is desired for the intact plant polymers,5 high-fidelity direct-polarization magic-angle spinning (DPMAS) 13C NMR5,14 should be used as a complementary method. Accurate quantitation of the functional groups requires careful optimization of recycle times, excitation pulse lengths, and the strength of heteronuclear decoupling.15 The heteronuclear decoupling can be set for a 1H field strength ranging from 50 kHz to 185 kHz by using the TPPM16 or SPINAL7 methodologies. In addition to these parameters, the sensitivity of CPMAS measurements depends on the spin-lock time and Hartmann-Hahn matching condition.15 In place of traditional CPMAS, a ramped-amplitude CP (RAMP-CP) technique can be implemented to maximize the cross-polarization efficiency by varying the 1H amplitude linearly (~20-50 %) or tangentially while keeping the amplitude of 13C field strength constant during the spin-lock period (or vice versa).17,18 Carrying out the CPMAS measurements at a minimum of two different rotor-spinning frequencies is imperative to distinguish spinning sidebands from the main spectral peaks.
Concurrent AFM measurements conducted in contact mode enable direct imaging of the cuticle surface condition with high scanning speeds and high resolution,19 for instance during sequential removal of waxy constituents. Operating AFM in tapping (non-contact) mode can be used as an alternative for surface characterization of delicate "soft" plant materials, avoiding possible damage due to lateral (shear) forces and scraping of the sample surface.5,20 In either case, sequential acquisition of multiple images of the same spot on the surface serves to identify any surface damage due to "probe-surface interactions" in AFM measurements.6,21 For optimal reproducibility, AFM probes with spring constants suitable for soft cuticular surfaces should be used, and constancy of temperature and humidity should be maintained.6,15,20 Whereas solid-state NMR offers a molecular profile of ensemble average (bulk) properties in tomato fruit cuticles, atomic force imaging provides a complementary noninvasive probe 22,23 for tracking the surface topography of these exquisitely complex macromolecular assemblies.1,2

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalog no.</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Sodium acetate trihydrate</td>
			<td>Sigma-Aldrich</td>
			<td>S8625-500G</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Pectinase</td>
			<td>TCI America</td>
			<td>P0026</td>
			<td>EC 3.2.1.15; 10 U ml-1, store in refrigerator</td>
		</tr>
		<tr>
			<td>Cellulase</td>
			<td>Sigma-Aldrich</td>
			<td>C1184-100KU</td>
			<td>EC232.734.4; 1.3 units/mg, store in refrigerator</td>
		</tr>
		<tr>
			<td>Glacial Acetic acid</td>
			<td>Sigma-Aldrich</td>
			<td>A9967</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sodium azide</td>
			<td>Sigma-Aldrich</td>
			<td>S2002-100G</td>
			<td>Extremely hazardous</td>
		</tr>
		<tr>
			<td>Incubator/shaker</td>
			<td>New Brunswick Scientific Co.</td>
			<td>Model No.G24</td>
			<td>MFG No.M1036-000G</td>
		</tr>
		<tr>
			<td>Vacuum Oven</td>
			<td>Precision Scientific</td>
			<td>31566</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Variac Controller</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sintered glass thimble (85 mm/25mm)</td>
			<td>VWR</td>
			<td>89056</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Disposable extraction thimble ( 80 mm/ 25 mm)</td>
			<td>VWR</td>
			<td>28320</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>VWR</td>
			<td>EMD-MX0485-7</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Glass wool</td>
			<td>VWR</td>
			<td>RK20789</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Aluminum foil</td>
			<td>Fisher</td>
			<td>01-213-100</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Tweezers</td>
			<td>VWR</td>
			<td>82027-452</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>VWR</td>
			<td>EM-CX1050-1</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Hexane</td>
			<td>Fisher Scientific</td>
			<td>H302-4</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Nitrogen gas</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>VWR</td>
			<td>52858</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Paper towels</td>
			<td>VWR</td>
			<td>89002-984</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Kim wipes</td>
			<td>VWR</td>
			<td>21905-026</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Gum arabic</td>
			<td>Sigma</td>
			<td>G9752</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>1.6 mm fastMAS zirconia rotor</td>
			<td>Varian (Agilent)</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>NMR spectrometer</td>
			<td>Varian 600 NMRS</td>
			<td>standard bore magnet</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Sigma-Aldrich</td>
			<td>50046</td>
			<td>Model compound for CPMAS</td>
		</tr>
		<tr>
			<td>Glutamine</td>
			<td>Sigma-Aldrich</td>
			<td>49419</td>
			<td>Model compound for CPMAS</td>
		</tr>
		<tr>
			<td>Adamantane</td>
			<td>Sigma-Aldrich</td>
			<td>100277</td>
			<td>To calibrate 90&deg; pulse in NMR</td>
		</tr>
		<tr>
			<td>Multimode Scanning Probe Microscope (Nanoscope IIIA)</td>
			<td>Digital Instruments (Bruker AXS)</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Nanoscope software</td>
			<td>Digital Instruments (Bruker AXS)</td>
			<td>&nbsp;</td>
			<td>Version 5.30r3sr3 (2005)</td>
		</tr>
		<tr>
			<td>AFM probe (Nonconductive silicon nitride tip)</td>
			<td>Veeco (Bruker AXS)</td>
			<td>Model NP-20</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Light microscope</td>
			<td>Digital Instruments</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Magnetic puck</td>
			<td>Digital Instruments</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Double sided tape</td>
			<td>VWR</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Fruit Peeler</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>B&uuml;chner funnel</td>
			<td>VWR</td>
			<td>89038</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

isolation and biophysical study of fruit cuticles

The cuticle, a hydrophobic protective layer on the aerial parts of terrestrial plants, functions as a versatile defensive barrier to various biotic and abiotic stresses and also regulates water flow from the external environment.1 A biopolyester (cutin) and long-chain fatty acids (waxes) form the principal structural framework of the cuticle; the functional integrity of the cuticular layer depends on the outer 'epicuticular' layer as well as the blend consisting of the cutin biopolymer and 'intracuticular' waxes.2 Herein, we describe a comprehensive protocol to extract waxes exhaustively from commercial tomato (Solanum lycopersicum) fruit cuticles or to remove epicuticular and intracuticular waxes sequentially and selectively from the cuticle composite. The method of Jetter and Sch&auml;ffer (2001) was adapted for the stepwise extraction of epicuticular and intracuticular waxes from the fruit cuticle.3,4 To monitor the process of sequential wax removal, solid-state cross-polarization magic-angle-spinning (CPMAS) 13C NMR spectroscopy was used in parallel with atomic force microscopy (AFM), providing molecular-level structural profiles of the bulk materials complemented by information on the microscale topography and roughness of the cuticular surfaces. To evaluate the cross-linking capabilities of dewaxed cuticles from cultivated wild-type and single-gene mutant tomato fruits, MAS 13C NMR was used to compare the relative proportions of oxygenated aliphatic (CHO and CH2O) chemical moieties.
Exhaustive dewaxing by stepwise Soxhlet extraction with a panel of solvents of varying polarity provides an effective means to isolate wax moieties based on the hydrophobic characteristics of their aliphatic and aromatic constituents, while preserving the chemical structure of the cutin biopolyester. The mechanical extraction of epicuticular waxes and selective removal of intracuticular waxes, when monitored by complementary physical methodologies, provides an unprecedented means to investigate the cuticle assembly: this approach reveals the supramolecular organization and structural integration of various types of waxes, the architecture of the cutin-wax matrix, and the chemical composition of each constituent. In addition, solid-state 13C NMR reveals differences in the relative numbers of CHO and CH2O chemical moieties for wild-type and mutant red ripe tomato fruits. The NMR techniques offer exceptional tools to fingerprint the molecular structure of cuticular materials that are insoluble, amorphous, and chemically heterogeneous. As a noninvasive surface-selective imaging technique, AFM furnishes an effective and direct means to probe the structural organization of the cuticular assembly on the nm-&mu;m length scale.

Watch this Scientific Journal Video about Isolation and Biophysical Study of Fruit Cuticles at JoVE.com

Isolation and Biophysical Study of Fruit Cuticles

Aerial plant organs are protected by the cuticle, a supramolecular biopolyester-wax assembly. We present protocols to monitor selective removal of epi- and intracuticular waxes from tomato fruit cuticles on molecular and micro scales by solid-state NMR and atomic force microscopy, respectively, and to assess the cross-linking capacity of engineered cuticular biopolyesters.

The cuticle, a hydrophobic protective layer on the aerial parts of terrestrial plants, functions as a versatile defensive barrier to various biotic and ...

isolation-and-biophysical-study-of-fruit-cuticles

Research

JoVE Journal

Biology

16.6K Views.  City College of New York, City University of New York Graduate Center and Institute for Macromolecular Assemblies. Aerial plant organs are protected by the cuticle, a supramolecular biopolyester-wax assembly. We present protocols to monitor selective removal of epi- and intracuticular waxes from tomato fruit cuticles on molecular and micro scales by solid-state NMR and atomic force microscopy, respectively, and to assess the cross-linking capacity of engineered cuticular biopolyesters.

Video: Isolation and Biophysical Study of Fruit Cuticles

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

Studying Aggression in Drosophila (fruit flies)

Aggression is an innate behavior that evolved in the framework of defending or obtaining resources. This complex social behavior is influenced by genetic, hormonal and environmental factors. In many organisms, aggression is critical to survival but controlling and suppressing aggression in distinct contexts also has become increasingly important. In recent years, invertebrates have become increasingly useful as model systems for investigating the genetic and systems biological basis of complex social behavior. This is in part due to the diverse repertoire of behaviors exhibited by these organisms. In the accompanying video, we outline a method for analyzing aggression in Drosophila whose design encompasses important eco-ethological constraints. Details include steps for: making a fighting chamber; isolating and painting flies; adding flies to the fight chamber; and video taping fights. This approach is currently being used to identify candidate genes important in aggression and in elaborating the neuronal circuitry that underlies the output of aggression and other social behaviors.

Studying Aggression in Drosophila fruit flies

Aggression is an innate behavior that evolved in the framework of defending or obtaining resources.  This complex social behavior is influenced by ...

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite morphogenesis at the functional and molecular levels. To aid in these analyses, we have developed a strategy for the isolation of a subclass of PNS neurons called dendritic arborization (da) neurons that have been widely used for studying dendrite morphogenesis1,2. These neurons are very difficult to isolate as a pure population, due in part to their extremely low occurrence and their difficult-to-reach location below the tough chitinous larval cuticle. Our newly developed method overcomes these challenges, and is based on a fast and specific cell enrichment using antibody-coated magnetic beads. For our magnetic bead sorting studies, we have used age-matched third instar larvae expressing a mouse CD8 tagged GFP fusion protein (UAS-mCD8-GFP)3 under the control of either the class IV dendritic arborization (da) neuron-specific pickpocket (ppk)-GAL4 driver4 or the control of the pan-da neuron-specific GAL421-7 driver5. Although this protocol has been optimized for isolating PNS cells which are attached to the inner wall of the larval cuticle, by varying a few parameters, the same protocol could be used to isolate many different cell types attached to the cuticle at larval or pupal stages of development (e.g. epithelia, muscle, oenocytes etc.), or other cell types from larval organs depending upon the GAL4-specific driver expression pattern. The RNA isolated by this method is of high quality and can be readily used for downstream genomic analyses such as microarray gene expression profiling studies. This approach offers a powerful new tool to perform studies on isolated Drosophila dendritic arborization (da) neurons thereby providing novel insights into the molecular mechanisms underlying dendrite morphogenesis.

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

In this video-article we present a method for the isolation and purification of Drosophila peripheral neurons using a fast magnetic bead assisted cell sorting strategy. RNA obtained from the isolated cells can be readily used for downstream applications including microarray analyses.

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite ...

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of muscle specification, formation and function in model systems has provided valuable insight into muscle physiology. Therefore, identifying and characterizing molecular mechanisms that underlie muscle damage is critical. The structure of adult Drosophila multi-fiber muscles resemble vertebrate striated muscles 1 and the genetic tractability of Drosophila has made it a great system to analyze dystrophic muscle morphology and characterize the processes affecting muscular function in ageing adult flies 2. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. These preparations allow for the tissue to be stained with classical histological stains and labeled with protein detecting dyes, and specifically cryosections are ideal for immunohistochemical detection of proteins in intact muscles. This allows for analysis of muscle tissue structure, identification of morphological defects, and detection of the expression pattern for muscle/neuron-specific proteins in Drosophila adult muscles. These techniques can also be slightly modified for sectioning of other body parts.

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

Identification of mechanisms underlying muscle damage is crucial. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. This allows analysis of muscle morphology and localization of protein and other muscle cell components.

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of ...

Fruit Volatile Analysis Using an Electronic Nose

Numerous and diverse physiological changes occur during fruit ripening, including the development of a specific volatile blend that characterizes fruit aroma. Maturity at harvest is one of the key factors influencing the flavor quality of fruits and vegetables1. The validation of robust methods that rapidly assess fruit maturity and aroma quality would allow improved management of advanced breeding programs, production practices and postharvest handling. 
Over the last three decades, much research has been conducted to develop so-called electronic noses, which are devices able to rapidly detect odors and flavors2-4. Currently there are several commercially available electronic noses able to perform volatile analysis, based on different technologies. The electronic nose used in our work (zNose, EST, Newbury Park, CA, USA), consists of ultra-fast gas chromatography coupled with a surface acoustic wave sensor (UFGC-SAW). This technology has already been tested for its ability to monitor quality of various commodities, including detection of deterioration in apple5; ripeness and rot evaluation in mango6; aroma profiling of thymus species7; C6 volatile compounds in grape berries8; characterization of vegetable oil9 and detection of adulterants in virgin coconut oil10. 
This system can perform the three major steps of aroma analysis: headspace sampling, separation of volatile compounds, and detection. In about one minute, the output, a chromatogram, is produced and, after a purging cycle, the instrument is ready for further analysis. The results obtained with the zNose can be compared to those of other gas-chromatographic systems by calculation of Kovats Indices (KI). Once the instrument has been tuned with an alkane standard solution, the retention times are automatically converted into KIs. However, slight changes in temperature and flow rate are expected to occur over time, causing retention times to drift. Also, depending on the polarity of the column stationary phase, the reproducibility of KI calculations can vary by several index units11. A series of programs and graphical interfaces were therefore developed to compare calculated KIs among samples in a semi-automated fashion. These programs reduce the time required for chromatogram analysis of large data sets and minimize the potential for misinterpretation of the data when chromatograms are not perfectly aligned.
We present a method for rapid volatile compound analysis in fruit. Sample preparation, data acquisition and handling procedures are also discussed.

A rapid method for volatile compound analysis in fruit is described. The volatile compounds present in the headspace of a homogenate of the sample are rapidly separated and detected with ultra-fast gas chromatography (GC) coupled with a surface acoustic wave (SAW) sensor. A procedure for data handling and analysis is also discussed.

Numerous and diverse physiological changes occur during fruit ripening, including the development of a specific volatile blend that characterizes fruit ...

Selective Capture of 5-hydroxymethylcytosine from Genomic DNA

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene expression, maintenance of genome integrity, parental imprinting, X-chromosome inactivation, regulation of development, aging, and cancer1. Recently, the presence of an oxidized 5-mC, 5-hydroxymethylcytosine (5-hmC), was discovered in mammalian cells, in particular in embryonic stem (ES) cells and neuronal cells2-4. 5-hmC is generated by oxidation of 5-mC catalyzed by TET family iron (II)/&alpha;-ketoglutarate-dependent dioxygenases2, 3. 5-hmC is proposed to be involved in the maintenance of embryonic stem (mES) cell, normal hematopoiesis and malignancies, and zygote development2, 5-10. To better understand the function of 5-hmC, a reliable and straightforward sequencing system is essential. Traditional bisulfite sequencing cannot distinguish 5-hmC from 5-mC11. To unravel the biology of 5-hmC, we have developed a highly efficient and selective chemical approach to label and capture 5-hmC, taking advantage of a bacteriophage enzyme that adds a glucose moiety to 5-hmC specifically12.
Here we describe a straightforward two-step procedure for selective chemical labeling of 5-hmC. In the first labeling step, 5-hmC in genomic DNA is labeled with a 6-azide-glucose catalyzed by &beta;-GT, a glucosyltransferase from T4 bacteriophage, in a way that transfers the 6-azide-glucose to 5-hmC from the modified cofactor, UDP-6-N3-Glc (6-N3UDPG). In the second step, biotinylation, a disulfide biotin linker is attached to the azide group by click chemistry. Both steps are highly specific and efficient, leading to complete labeling regardless of the abundance of 5-hmC in genomic regions and giving extremely low background. Following biotinylation of 5-hmC, the 5-hmC-containing DNA fragments are then selectively captured using streptavidin beads in a density-independent manner. The resulting 5-hmC-enriched DNA fragments could be used for downstream analyses, including next-generation sequencing.
Our selective labeling and capture protocol confers high sensitivity, applicable to any source of genomic DNA with variable/diverse 5-hmC abundances. Although the main purpose of this protocol is its downstream application (i.e., next-generation sequencing to map out the 5-hmC distribution in genome), it is compatible with single-molecule, real-time SMRT (DNA) sequencing, which is capable of delivering single-base resolution sequencing of 5-hmC.

Described is a two-step labeling process using &beta;-glucosyltransferase (&beta;-GT) to transfer an azide-glucose to 5-hmC, followed by click chemistry to transfer a biotin linker for easy and density-independent enrichment. This efficient and specific labeling method enables enrichment of 5-hmC with extremely low background and high-throughput epigenomic mapping via next-generation sequencing.

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene ...

Super-resolution Imaging of the Bacterial Division Machinery

Bacterial cell division requires the coordinated assembly of more than ten essential proteins at midcell1,2. Central to this process is the formation of a ring-like suprastructure (Z-ring) by the FtsZ protein at the division plan3,4. The Z-ring consists of multiple single-stranded FtsZ protofilaments, and understanding the arrangement of the protofilaments inside the Z-ring will provide insight into the mechanism of Z-ring assembly and its function as a force generator5,6. This information has remained elusive due to current limitations in conventional fluorescence microscopy and electron microscopy. Conventional fluorescence microscopy is unable to provide a high-resolution image of the Z-ring due to the diffraction limit of light (~200 nm). Electron cryotomographic imaging has detected scattered FtsZ protofilaments in small C. crescentus cells7, but is difficult to apply to larger cells such as E. coli or B. subtilis. Here we describe the application of a super-resolution fluorescence microscopy method, Photoactivated Localization Microscopy (PALM), to quantitatively characterize the structural organization of the E. coli Z-ring8.
	PALM imaging offers both high spatial resolution (~35 nm) and specific labeling to enable unambiguous identification of target proteins. We labeled FtsZ with the photoactivatable fluorescent protein mEos2, which switches from green fluorescence (excitation = 488 nm) to red fluorescence (excitation = 561 nm) upon activation at 405 nm9. During a PALM experiment, single FtsZ-mEos2 molecules are stochastically activated and the corresponding centroid positions of the single molecules are determined with &lt;20 nm precision. A super-resolution image of the Z-ring is then reconstructed by superimposing the centroid positions of all detected FtsZ-mEos2 molecules.
	Using this method, we found that the Z-ring has a fixed width of ~100 nm and is composed of a loose bundle of FtsZ protofilaments that overlap with each other in three dimensions. These data provide a springboard for further investigations of the cell cycle dependent changes of the Z-ring10 and can be applied to other proteins of interest.

We describe a super-resolution imaging method to probe the structural organization of the bacterial FtsZ-ring, an essential apparatus for cell division. This method is based on quantitative analyses of photoactivated localization microscopy (PALM) images and can be applied to other bacterial cytoskeletal proteins.

Bacterial cell division requires the coordinated  assembly of more than ten essential proteins at midcell1,2. Central  to this process is the formation of ...

Efficient Production and Purification of Recombinant Murine Kindlin-3 from Insect Cells for Biophysical Studies

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and the control of gene transcription in the cell nucleus. The kindlins are ~75 kDa multidomain proteins and bind to an NPxY motif and upstream T/S cluster of the integrin &#946;-subunit cytoplasmic tail. The hematopoietically-important kindlin isoform, kindlin-3, is critical for platelet aggregation during thrombus formation, leukocyte rolling in response to infection and inflammation and osteoclast podocyte formation in bone resorption. Kindlin-3&#39;s role in these processes has resulted in extensive cellular and physiological studies. However, there is a need for an efficient method of acquiring high quality milligram quantities of the protein for further studies. We have developed a protocol, here described, for the efficient expression and purification of recombinant murine kindlin-3 by use of a baculovirus-driven expression system in Sf9 cells yielding sufficient amounts of high purity full-length protein to allow its biophysical characterization. The same approach could be taken in the study of the other mammalian kindlin isoforms.

Kindlins are fundamental to cell adhesion through integrins but studies of them have been hampered by the difficulty encountered in expressing them recombinantly in bacterial hosts. We describe here methods for their efficient production in baculovirus-infected insect cells.

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and ...

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial scientific task. Many disease causing genes in humans have a fly homologue, making Drosophila a good model to study signaling pathways involved in the development of different disorders. Additionally, the tractability of Drosophila simplifies genetic screens to aid in identifying novel therapeutic targets that may regulate metabolism. In order to perform such a screen a simple and fast method to identify changes in the metabolic state of flies is necessary. In general, carbon dioxide production is a good indicator of substrate oxidation and energy expenditure providing information about metabolic state. In this protocol we introduce a simple method to measure CO2 output from flies. This technique can potentially aid in the identification of genetic perturbations affecting metabolic rate.

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are among one of the most common diseases in humans. The genetically tractable model organism D. melanogaster can be used to identify novel genes that regulate metabolism. This paper describes a relatively simple method which allows studying the metabolic rate in flies by measuring their CO2 production.

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial ...

Biophysical Characterization of Flagellar Motor Functions

The role of flagellar motors in bacterial motility and chemotaxis is well-understood. Recent discoveries suggest that flagellar motors are able to remodel in response to a variety of environmental stimuli and are among the triggers for surface colonization and infections. The precise mechanisms by which motors remodel and promote cellular adaptation likely depend on key motor attributes. The photomultiplier-based bead-tracking technique presented here enables accurate biophysical characterization of motor functions, including adaptations in motor speeds and switch-dynamics. This approach offers the advantage of real-time tracking and the ability to probe motor behavior over extended durations. The protocols discussed can be readily extended to study flagellar motors in a variety of bacterial species.

Recent findings suggest that bacterial flagellar motors sense a variety of environmental signals and remodel in response. The bead-assays discussed here are expected to help explain the role of remodeling in cellular adaptation to environmental stressors.

The role of flagellar motors in bacterial motility and chemotaxis is well-understood. Recent discoveries suggest that flagellar motors are able to remodel ...