This experiment uses biophysical techniques to assess the cross-linking capacity of the tomato, cute and protective polymer, and to monitor selective removal of epi and intra cuticular waxes from fruit cuticles. The isolation of the cuticle from the whole fruit is achieved by enzymatic treatment of the tomato skins and adhering cell walls. In a second step, ate extraction is used to dewax the fruit cuticle exhaustively so as to obtain the cutin molecular characterization of the isolated cutin.
Bio polyester is conducted by solid state nuclear magnetic resonance. Solid state NMR spectral analysis reveals that in cutin from wild type and engineered tomato fruit cuticles, there are differences in the relative proportions of oxygenated allophatic moieties that are capable of cross-linking. Alternatively, the whole fruit is painted and peeled with gum Arabic to remove epic cuticular waxes and then dipped in chloroform to remove intra cuticular waxes from the fruit cuticle.
Then the partially dew waxed cuticles isolated by the same enzymatic treatment as the tomato skins are examined using solid state NMR and atomic force microscopy. Ultimately, the sequential removal of epi and intra cuticular waxes is apparent through observation of a progressively diminishing wax peak in the NMR spectra and alterations in the surface profile of the fruit cuticle observed by concurrent A FM imaging Above ground organs of plants are protected by a bio bio polyester wax assembly called the cuticle. We will present protocols to evaluate the selective removal of internal and external waxes from tomato fruit cuticles using solid state nuclear magnetic resonance and atomic force microscopy.
These methods can also be used to evaluate the cross-linking capacity of engineered cuticular materials. Demonstrating the procedures will be three stark group members, Dr.Ishish Chatterjee, PhD candidate Shantani Sarkar, and former high school student Julia Tik. An additional demonstrator is Dr.Olivia Nisau, a postdoctoral associate in the lab of our chemical engineering colleague, Dr.Alexander S.To begin enzymatic isolation of the tomato skin, place three commercial or cultivated fruits in a bowl, peel the skin in large sections and discard the inner peri carp tissue.
Then wash the tomato skins with deionized water and preserve them underwater in a beaker, completely immersed the peeled tomato skin in an enzyme cocktail prepared as described in the written protocol. Incubate at 31 degrees Celsius for 24 hours with continuous shaking. Collect the tomato skins using a buer funnel or kitchen strainer and wash them with deionized water.
After placing the skins in a vacuum oven at room temperature for one hour, the dried skins can be stored in a labeled and capped bottle. For subsequent de waxing procedures, fill a centered glass or disposable thimble approximately halfway with the tomato cuticle sample used tweezers to place it carefully at the base of the extraction column to be used in the soli extraction. Continue to set up the so lit apparatus using methanol solvent as described in the written protocol.
Monitor the process for an hour, adjusting the variac voltage so that the reservoir accumulates about one drop per second and siphoning occurs within the socks that apparatus. When the thimble is full, subsequent to a 12 hour extraction, allow the apparatus to cool, then lower the heating mantle and remove the extractor and reservoir as a single unit to dispose of the solvent following extraction. Tilt the column to allow siphoning into the flask below.
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. Disconnect the flask and pour the waste into a labeled solvent receptacle.
Repeat the extraction procedure for successive solvents of progressively diminishing polarity, such as chloroform and taxane for 12 hours each. Once the tomato cutten sample is dry, measure the mass and store it room temperature and a screw top jar sealed with param. To prepare the sample for NMR place four to six milligrams of fully dew waxed tomato cuticles called cutin in a 1.6 millimeter fast MAS zirconia rotor Using the vendor supplied packing tool.
After installing the top cap, paint half of the cap with a black ink marker pen to allow measurement of the spin rate. Initial setup of the solid state NMR experiment is a technically demanding process and based achieved under the superficial of experienced staff. Begin by adjusting the magnetic field homogeneity and optimizing experimental parameters using a model compound as described in the written protocol, Insert the cute and packed rotor into the probe and then place the probe into the magnet.
Gradually increase the spinning speed up to 10 kilohertz to verify good sample packing and ability of the rotor to spin stably with variation of less than 20 hertz. Adjust the tuning and matching capacitors of the probe iteratively to achieve minimum power reflection at both proton and carbon 13 NMR frequencies. Set the experimental temperature to 25 degrees Celsius.
Start the pre optimized CPMA experiment corresponding to the Hartman Han proton carbon matching condition determined at the 10 kilohertz spinning frequency After acquiring 4, 096 transients. Condition the spectrum with exponential line broadening of 50 to 100 hertz and do a Fourier transform to generate an NMR spectrum of signal intensity versus chemical shielding in parts per million. Reference the carbon 13 chemical shifts as described in the text.
Increase the rotor spinning frequency to 15 kilohertz and repeat the C-P-M-A-S measurement corresponding to the Hartman Han Matching condition determined at the ladder spinning frequency chemical shift analysis of the C-P-M-A-S carbon. 13 NMR Spectra identified the major functional groups present in the exhaustively, DW waxed tomato cuticle, long chain PHAs oxygenated PHAs, multiply, bonded and aromatics and carbon yields. The oxygenated almo 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 parts per million. Suggest that the mutant cutin has a relatively large proportion of cross-link forming CHO structural moieties as compared to the wild type apin. To begin selective isolation of epi cuticular and intra cuticular waxes.
Wash a second batch of whole tomatoes with distilled water. Dry them with paper towels and Kim wipes and place them stem downward on a piece of aluminum foil. Paint the whole tomatoes from top to bottom with a 120%gum Arabic aqueous solution.
Allow the gum Arabic to dry on the fruit skin for one hour, leaving a thin film. Remove this film using tweezers, but avoid puncturing the tomato skin. Further treatment of the film as described in the text yields the epic cuticular wax.
The tomatoes will remain physically intact. You dip them into chloroform for two minutes. At room temperature, the intra cuticular wax can be collected after evaporating the solvent, peel the tomatoes and treat them enzymatically with cellulase and pectinate and sodium acetate puffer to remove cellulose and pectin respectively.
A sali extraction can then be performed as described earlier to exhaustively dewax the tomatoes. The C-P-M-A-S experiments can be repeated with natural and partially dew waxed fruit cuticle samples as described in the text Setup of the atomic force microscope is a complicated process and is best achieved under the initial supervision of experienced technical stuff. A FM begins by turning on the scanning probe microscope, abbreviated SPM, and ensuring that the microscope mode toggle switch is set to the contact atomic force microscopy mode.
Next, manually raise the SPM head by turning its two user adjustable front knobs. Detach the a FM tip holder from the SPM head by turning the clamping screw at the back of the head. Use tweezers to remove the existing a FM cantilever from its holder.
Then carefully grab a new cantilever with silicon nitride tip from its package and install it in place of the old cantilever. Use a light microscope to verify that the newly installed a FM cantilever is not broken. Attach the tomato cuticle sample to a sample puck with double-sided tape.
Use a light microscope to verify that the cuticle sample remains flat and smooth. After placement of the sample on the puck, place the tomato cuticle sample that is attached to the puck onto the magnetic region at the top of the SPM scanner. By turning the knobs set the front two manual screws of the scanner high enough to avoid breaking the A FM tip.
When the tip holder is placed into the SPM head, set the motorized back screw to approximately the same level as the other two front screws. Reinsert the tip holder into the SPM head and secure it by tightening the back clamping screw of the head. After turning on the laser, align the laser spot on the a FM cantilever using the central and right 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 a FM cantilever. Adjust the position of the laser spot to achieve maximum sum signal. Regulate the position of the reflected laser spot on the photo detector by adjusting the position of the movable mirror horizontally and vertically to ensure that the incident light is being received evenly by the four quadrants.
The detector lower the a FM tip by retracting the user controlled front screws and the motorized back screw of the SPM scanner. Visually monitoring the approach of the A FM 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 it touches or breaks through the sample surface. The laser light reflected off the A FM cantilever will bounce off a movable mirror to a photo sensitive detector for contact a FM mode with a silicon nitride a FM probe. Set the output signal voltage to negative two volts for zero volt setpoint and the differential signal to zero volts by adjusting the position of the mirror.
Using the nano scope software. Click on the microscope icon and select the appropriate profile. Using the scan control settings panel, set the scan rate and scan size.
Allow the A FM 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.
Monitor the scanning process using both image and scope modes of the software, adjust parameters iteratively to achieve the highest resolution images as described in the written protocol. For example, start scanning with a large Z axis data scale value. Then carefully reduce the Z data scale value to obtain best contrast of surface features on the image.
Capture the image to save the data file. Process the data using flattening functions to remove image artifacts due to vertical scanner drift image bows and vertical offset between scan lines. Finally, calculate the average roughness carbon.
13 C-P-M-A-S-N-M-R Spectra showed a progressively diminishing wax peak at 31 parts per million, indicating the sequential removal of epi and intra cuticular waxes from the cute and wax composite while retaining the principle chemical architecture of the cutin biopolymer parallel. A FM image analysis revealed surface irregularities due to the stepwise extraction of epic cuticular wax and intra cuticular wax from the fruit cuticle signifying alterations in the organization of the Cuticular assembly. Once you roasted this technique, the sequential removal of epi and intra cuticular waxes from the Tomato fruit cuticles can be done in two hours.
When conducting soled extraction for the exhaustive daxing of the fruit cuticles, it is important to use a panel of solvents of varying polarity after using the contact node. A FM protocol illustrated here. Additional A FM experiments can be used to measure surface elasticity of the cuticle.
After watching this video, you should have a good understanding of how to systematically isolate components from the cutin WAX composite and to conduct molecular ization and rapid surface examination of the cuticle materials using solid state MR and a FM respectively. The techniques illustrated in this video make it possible for researchers in plant biology to determine the molecular composition and super molecular architecture of natural and engineered plant tissues.
