Crop Science 41:755-758 (2001)
© 2001 Crop Science Society of America
CROP PHYSIOLOGY & METABOLISM
Multipurpose Cryogenic Surface Apparatus
A Liquid Nitrogen-Chilled Sample Tray
N.R. Adam* and
G.W. Wall
U.S. Water Conservation Laboratory, USDA-ARS, 4331 East Broadway, Phoenix, AZ 85040
* Corresponding author (nadam{at}uswcl.ars.ag.gov)
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ABSTRACT
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The use of cryogenic techniques allows collection and storage of large numbers of samples for later processing for biochemical assays. In large field studies it is very difficult to obtain an accurate measure of leaf area as the samples are collected while preserving biological activity. Measurement of leaf area at the time of processing is also difficult because thawing of leaf tissue can cause degradation of leaf biochemical components and processes. The objective of this study was to design a liquid N2–chilled tray that allows cryogenically preserved samples to be sorted and measured without the risk of sample thawing. A stainless steel lid (working surface) was brazed to a stainless steel pan, creating a reservoir that could be filled with liquid N2. The working surface could then be maintained at approximately the temperature of liquid N2. Enzyme activities and polyacrylamide gel separations of ribulose-1,5-bisphosphate carboxylase (Rubisco) and phosphoenol pyruvate carboxylas (PEPCase) of sorghum [Sorghum bicolor (L.) Moench] were used to assess the performance of the tray. Enzyme activity assays and gel separations of Rubisco and PEPCase confirm that biological activity is maintained through use of the multipurpose cryogenic surface (MCS) tray.
Abbreviations: DTT, dithiothreitol • MCS, multipurpose cryogenic surface • PEPCase, phosphoenol pyruvate carboxylase • Rubisco, ribulose-1,5-bisphosphate carboxylase • SDS-PAGE, sodium dodecyl sulfate polyacrylaminde gel eclectrophoresis
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INTRODUCTION
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CRYOGENIC TECHNIQUES are commonly used to preserve leaf tissue for measurements of metabolic processes. To accurately assess treatment effects on biochemical processes, it is necessary to preserve tissues in their physiological state at the time of sampling by preventing the onset of degradation processes, particularly those associated with protease activity. In some case, plant tissue can be frozen and store at -80°C (Crafts-Brandner et al., 1990). Other measurements require that the tissue be harvested and plunged into liquid N2 (-196°C)(Usuda and Shimogawara, 1994). In special cases, such as measuring metabolites that are depleted quickly, it may be necessary to freeze-clamp the leaf while it is still on the plant (Sharkey et al., 1986). Cryotechniques also allow tissue to be collected, frozen, and processed later. This allows larger and more complex experimental designs, increased replication (thereby increasing statistical precision), and the collection of samples from remote field sites.
There are also disadvantages in using cryogenic techniques. One disadvantage is that, with cryofixation, it is often difficult to collect and arrange the smaples in the order that they will be processed in the laboratory, expecially in large field studies. Also time constraints of the laboratory procedures may dictate that only a subset of the samples is processed. These situations require special handling of the samples to avoid damage by partial or complete thawing. Usuda and Shimogawara (1994) showed that thawing of the frozen second leaf of a 24-d-old plant caused a 50% reduction in PEPCase and Rubisco activity within 2 to 3 min. The rates of inactivation were faster for older leaves. Usuda and Shimogawara (1994) also showed, using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), that PEPCase and Rubisco protein content decreased gradually as thawing time increased.
Another disadvantage is the difficulty in accurate measurement of leaf area to express biochemical processes on a comparative basis. The use of cryogenic techniques such as flash-freezing, an increased size of experiment, or sampling from a remote field site may preclude an accurate measure of leaf area. Measurement of leaf area at the time of sampling may be an option in a small study, and Usuda and Shimogawara (1994) showed that a fresh leaf could sit for 10 min without loss of enzyme acitivity. However, the measurement of leaf area can add significantly to the sampling time and in large or remote field studies it often is not possible to collect and measure all the samples. It is especially difficult if enzyme parameters change in situ with time or time of day. To measure sample area in the laboratory prior to processing or grinding can also be deterimental to sample quality because of the time required for measurement. The leaf sample will thaw quickly and sample quality will be compromised (Usuda and Shimogawara, 1994).
Therefore an apparatus is needed that would allow samples to be sorted and measured without allowing the samples to thaw, thereby compromising sample integrity. The objective of this study was to design a liquid N2-chilled tray (MCS tray) that allows cryogenically preserved samples to be sorted and measured without the risk of sample thawing.
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MATERIALS AND METHODS
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Tray Construction
The MCS tray was designed to allow measurment and sorting of frozen leaf samples without the risk of thawing (Fig. 1). A 225.6-mm-wide, 290.6-mm-long, and 9.5-mm-thick plate of stainless steel was brazed just inside the top of a 25-mm-deep stainless steel pan, forming a covered reservoir with a raised edge of 4.8 mm. The upper surface of the plate functions as the working surface. A 158.8-mm-tall, 35.1-mm-diameter pipe was attached at one end of the upper surface so that the reservoir could be filled with liquid N2. A 107-mm-tall, 6.4-mm-diameter vent tube was attached at the other end of the upper surface, and the tray was tilted at an angle of 
5° so the bottom of the vent tube was 19 mm higher than the bottom of the fill tube. This allowed air bubbles to escape through the vent tube. The tray was placed on a layer of 25-mm styrofoam inside a 508 by 406 by 127 mm plastic pan. One or more layers of 25-mm styrofoam were placed between the steel pan and the plastic pan on all four sides and the interstices were filled with expanding foam insulation.
Test Sample Collection
An experiment was conducted to ensure that activity of enzymes was maintained throughout the use of the tray. Sorghum was planted in 6.6-L pots in the glasshouse. Approximately 55 to 60 d after emergence, six pairs of leaf discs were collected from the same sorghum leaf, placed in perforated 2-mL plastic vials, and frozen and stored in liquid N2. One pair of leaf discs was retained in the liquid N2 and one pair removed from the vial and allowed to sit on the cold tray for 10 min before being returned to the liquid N2 for later assay. Other pairs of discs were removed from the vials and allowed to thaw at 28°C for 1, 2, 5, and 10 min before being returned to the liquid N2. The experiment was replicatd by collecting discs from another sorghum leaf and subjecting them to the same treatments. Assays for Rubisco inital and full activity and PEPCase activity were conducted the following day for all treatments. In addition, to ensure that the process of freezing did not affect the measurement of leaf area, sorghum leaf discs of a known diameter and area were frozen and measured on the MCS tray.
Assay Procedure
Leaf samples were removed from liquid N2 and quickly ground in a chilled ground glass homogenizer using a 100 mM Tricine (pH 8.0) buffer with the following components: 10 mM MgCl2, 1 mM ethylene diamine tetraacetic acid (EDTA), 14 mM dithiothreitol (DTT), soluble polyvinylpyrrolidone (PVP, 20 mg mL-1), glycerol (0.4 mL mL-1), 1 mM phenyl methyl sulfonyl fluoride (PMSF), and 1 mM NaFl. Aliquots from the same leaf homgenate were used to measure both PEPCase and Rubisco activities. An additional aliquot was boiled in SDS and used for SDS-PAGE to determine whether protein content was affected by thawing.
Rubisco was assayed according to Salvucci and Anderson (1987) and PEPCase was assayed using a 14C adaptation of a standard assay procedure (Jiao et al., 1991). The Rubisco assay buffer included Tricine (100 mM, pH 8.0), 10 mM MgCl2, 2 mM DDT, and 10 mM NaH14CO3. RuBP was added at 0.4 mM. Phosphoenol pyruvate carboxylase was assayed using 50 mM HEPES-KOH (pH 8.0), 5 mM MgCl2, 10 mM NaH14CO3, and 10 unites mL-1 MDH. Phosphoenolpyruvate was added at 5 mM. Reactions were initiated within 2 min of homogenization. To full activate Rubisco, the homogenate was allowed to incubate with the bicarbonate solution for 10 min.
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RESULTS
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Tray Performance
The reservoir was filled with liquid N2 and the level was maintained within 20 to 50 mm from the top of the fill pipe. The temperature of the working surface was monitored with a thermocouple. The working surface reached -184 to -190°C in 20 min (Fig. 2a). Although 4 to 6 L of liquid N2 were necessary to obtain the working temperature, less was needed to maintain that temperature. Once the working temperature was reached, it was possible to visually estimate the level of liquid N2 within the reservoir due to the appearance of a "frostline". Below the frostline, the temperature of the working surface was uniform and stable near -190°C. Replenishing the liquid N2 at the same rate that it vaporized minimized frost accumulation on the working area. Maintaining the level of liquid N2 ensured a working surface area of 275 by 220 mm. The presence of the raised edge allowed the accumulation of overflow to form a small pool of liquid N2 on the lower end of the working sufrace that could be sued to prechill storage vials (Fig. 2b).
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Fig. 2. (a) View of multipurpose cryogenic surface working surface and thermometer; temperature readout is -188.3°C. (b) Measurement of leaf area with digital calipers.
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After the desired working surface temperature was reached, leaf samples, could be processed. Leaf discs were placed on the tray, and the diameters of the discs were measured using a digital caliper accurate to 0.01 mm (Fig. 2b). Despite frost accumulation on the calipers that caused the digital readout to function more slowly, the measurements themselves remained accurate and reproducible. As a control of the accuracy of the readings, a penny of known diameter (18.93 mm) was placed on the working surface and measured periodically. The measured diameter never varied by >0.05% (0.01 mm). In addition, the process of freezing did not alter leaf dimensions; measurements of the fresh leaf discs were identical to the measurements after they were frozen. This is beneficial for large or remote field studies in which it is not feasible to measure leaf area or process samples at the time of collection.
Assay Results
Rubisco activity was not reduced by 10 min on the MCS tray. Inital activity of Rubisco was reduced by 33% following 1 min of thawing (Fig. 3a). Activity continued to decrease gradually until the activity was reduced by 70% after 10 min of thawing. In contrast, fully activated Rubisco showed less that a 10% decline in activity following 10 min of thawing (Fig. 3b). The quantity of Rubisco protein observed by SDS-PAGE was unchanged by thawing (Fig. 4).
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Fig. 3. (a) Initial and (b) full activity of sorghum leaf ribulose-1,5-bisphosphate carboxylase and (c) phosphoenol pyruvate carboxylase activity after designated period of thawing or time on the multipurpose cryogenic surface apparatus. Error bars present for all data points, but may be hidden by symbols.
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Fig. 4. Sodium dodecyl sulfacte polyacrylamide gel electrophoresis of sorghum leaf proteins. Lane 0, molecular markers; Lane 1, upon removal from liquid N2; Lane 2, after 1 min of thawing; Lane 3, after 2 min thawing; Lane 4, after 5 min thawing; Lane 5, after 10 min thawing; and Lane 6, after 10 min exposure on multipurpose cryogenic surface apparatus. Abbreviations: R LSU, ribulose-1,5-bisphosphate carboxylase (Rubisco) large subunit; LHCII, light-harvesting complex II; R SSU, Rubisco small subunit.
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Activities of PEPCase were not significantly reduced by 10 min on the cold tray (Fig. 3c). Acitivity of PEPCase was reduced from 280 µmol m-2 s-1 to 25 µmol m-2 s-1 following only 1 min of thawing. Results of SDS-PAGE (Fig. 4) indicated that the quantiy of PEPCase protein was also severly decreased in the thawed tissue.
Assays of enzyme activities demonstrate that Rubisco and PEPCase are preserved even after 10 min on the MCS tray. For typical use, the samples are not on the tray for this long. Thawing for only 1 to 2 min was shown to severly reduce enzyme activity. It was surprising that only 1 min of thawing resulted in the loss of 90% of the PEPCase activity. This is a much greater and much faster reduction than that shown by Usuda and Shimogawara (1994), who showed a 50% reduction in PEPCase activity within 3.2 min after the start of thawing for 24-d-old plants. However, the plants in this study were older (60 d old), and according to Usuda and Shimogawara (1994), rates of inactivation are faster for older leaves. Also, leaves in this experiment were thawed and then refrozen, whereas Usuda and Shimogawara (1994) assayed the leaves immediately after thawing. Possibly, refreezing caused as much damage as the initial thawing.
On the other hand, Usuda and Shimogawara (1994) showed a 50% reduction in Rubisco activity within 2.4 min of thawing, while our results indicate that the activity of fully activated Rubisco was reduced only 9% after 5 min of thawing and 13% after 10 min of thawing. The initial activity of Rubisco was more strongly affected. Initial activity was reduced 30% after 1 min of thawing and 41% by 2 min of thawing. These results are in agreement with those of Usuda and Shimogawara (1994).
The SDS-PAGE (Fig. 4) showed that Rubisco and PEPCase were affected differently by thawing. The content of Rubisco was not affected by thawing treatment, whereas the content of PEPCase was sharply reduced by only 1 min of thawing. Rubisco content and activity (Fig. 3a, 3b, and 4) showed that it was the activation state of Rubisco that was most affected by thawing. Loss of PEPCase protein after 1 min of thawing (Fig. 4) showed that the decline in PEPCase activity was due to degradation of the protein. This work agrees with the work of Usuda and Shimogawara (1994), which showed that Rubisco content was decreased only slightly after 15 min of thawing. However, the reductions in PEPCase protein content after only 1 min in this study were seen by Usuda and Shimogawara (1994) only after 30 min of thawing, possibly because the samples in this study were refrozen after the thawing period. These results indicate that samples collected for SDS-PAGE must be handled as carefully as samples collected for enzyme activity assays.
In conclusion, this work demonstrates the utility of the MCS tray in preventing the leaves from thawing when dividing leaf tissue and measuring the leaf area. This preserves the activity of Rubisco and PEPCase at physiological levels and also prevents degradation of the proteins. Although these experiments were performed on relatively small leaf pieces stored in small vials, larger leaves could be processed as well. In other experiments, liquid N2-stored foil-wrapped leaves were unwrapped while maintaining contact with the tray surface, divided, and measured without thawing. In addition, the shallow pool of liquid N2 near the raised edge would allow thicker samples or vials to be sorted or measured without the risk of thawing. The MCS tray allows more flexibility in experimental design when working with cryogenically preserved samples.
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ACKNOWLEDGMENTS
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The authors thank Andrew Webber of Arizona State University, Tempe, AZ, for the use of his laboratory and critical reading of the manuscript; John Replogle of the U.S. Water Conservation Laboratory, Phoenix, AZ, for helpful advice; Bud Lewis for technical support; and Jotham Austin III and Jose Olivieri for their assistance in preparation of the figures. This research was supported by Interagency Agreement No. IBN-9652614 between the National Science Foundation and the USDA-ARS U.S. Water Conservation Laboratory as part of the NSF/DOE/NASA/USDA Joint Program on Terrestrial Ecology and Global Change (TECO II; G.W. Wall, principal investigator). Operation support was also provided by the USDA-ARS U.S. Water Conservation Laboratory, Phoenix, AZ.
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NOTES
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Operational Support contributed by the NSF/DOE/NASA/USDA Interagency Program on Terrestrial Ecology and Global Change (TECO II, NSF-95-27, proposal no. IBN-9652614).
Received for publication June 22, 1999.
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REFERENCES
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- Salvucci, M.E., and J.C. Anderson. 1987. Factors affecting the activation state and the level of total activity of ribulose bisphosphate carboxylase in tobacco protoplasts. Plant Physiol. 85:66–71.[Abstract/Free Full Text]
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