Analyzing the Response of Glutathione Reductase Activity in Taxus cuspidata to Thermal Stress

By Beth A. Brinda

Abstract

Wintertime and summertime Japanese Yew (Taxus cuspidata) needles were assayed spectrophotometrically for glutathione reductase (GR) activity. GR is a part of the antioxidant chain found in the thylakoids of plant chloroplasts, a chain that serves as a photoprotection system for plants that are stressed by cold temperatures. This project investigated whether winter-needle cold tolerance of Taxus cuspidata would be higher than summer-needle tolerance, and whether as assay temperatures decreased, the summer needles would maintain lower GR activity levels than the winter ones.

Published research had indicated that antioxidant enzyme activity was higher in the wintertime than the summertime. It was found that ascorbate peroxidase (APX) activity did not increase statistically during the wintertime, but GR activity increased significantly. Further investigation of winter GR activity showed an average 1.17 µmol s^-1(g protein)^-1 greater activity than summer needles, and the activity was consistently higher at temperatures ranging from 15∫C to 45∫C. The response of winter-needle GR activity to temperature changes was found to be 1.8 times greater than the response of summer-needle GR activity.

Introduction

This project studied glutathione reductase (GR) enzyme activity at varying temperatures in needles of Taxus cuspidata (Japanese Yew) collected in winter and summer. GR is found in the thylakoids of plant chloroplasts and is an antioxidant enzyme (1). Plants that engage in photosynthesis during the winter, such as evergreen trees or bushes, can become overstressed by excess intake of light energy due to their inability to properly fix inorganic carbons in the Calvin cycle when the proteins responsible for photosynthesis are chilled to near inertness. Therefore, plants have developed systems of photoprotection to keep toxins that could otherwise severely damage or kill them from building up in their chloroplasts (1, 2).

One of the photoprotective systems in plants is the antioxidant chain, where antioxidant enzymes remove active oxygen molecules (O₂^-) that form when the ordinary photosynthetic process is inhibited by stresses such as cold temperatures. In regular photosynthesis, NADPH is converted to NADP⁺, and the Calvin cycle makes use of the electrons produced from this conversion. Under cold stress, the Calvin cycle is inhibited, resulting in a backup of electrons in the electron transport chain. Active oxygen is formed when oxygen molecules accept those electrons and form superoxide, a reactive species of oxygen that can cause cellular damage (1).

Plants employ antioxidant enzymes such as GR as a part of their photoprotection system, a system they rely on during times of stress. Figure 1 shows the pathway for the detoxification of active oxygen, which demonstrates the position of the GR enzyme in this system. The pathway involves the following steps: Superoxide is initially converted to hydrogen peroxide (H₂O₂), catalyzed by the enzyme superoxide dismutase (SOD). Oxidized ascorbate or monodehydroascorbate (MDHA) can be reduced immediately via another enzyme, MDHA reductase (MDHAR), using NADPH as a reductant. MDHA can also spontaneously isomerize into dehydroascorbate (DHA) which is reduced by the enzyme DHA reductase (DHAR) using glutathione as the reducing agent. Oxidized glutathione (GSSG) acts as a substrate for GR, which reduces glutathione using electrons from NADPH (1). Overall, the pathway shown in Figure 1 detoxifies active oxygen using successive oxidations and reductions of ascorbate and glutathione, with NADPH serving as the electron donor in the final enzymatic reaction by GR.

Figure 1: Formation of Active Oxygen and Antioxidant Pathways (Dr. Amy Verhoeven)

Early during this project, winter and summer yew needles were analyzed for the activity levels of antioxidant enzymes APX and GR. As seen in Figure 2, comparing winter and summer APX and GR data, preliminary APX activity data showed no difference between the summer yew needles and the winter collection. GR, however, demonstrated a large statistical increase in the activity of wintertime needle enzyme as compared to summer enzyme (p-value: 0.00831). The error bars in Figure 2 represent one standard deviation.

Figure 2: Winter vs. Summer GR Activity (per g protein)

The lack of statistical difference in APX data (p-value: 0.606) contradicted published results of data collected from the needles of evergreens in more temperate wintertime climates than in Minnesota. For example, Logan, et al. found that analysis of Mahonia repens (Oregon Grape Holly) in the mountains of Colorado yielded a statistically notable difference between summer and winter APX activity (4). The difference between the Logan data and the data shown in Figure 2 implied that GR played a more important photoprotective role than APX. This led to an investigation of the impact of temperature alterations on GR activity in both winter and summer yew needles.

The hypotheses for the study reported in this paper were:

· Winter-needle cold tolerance of Taxus cuspidata would be higher than summer-needle tolerance.

· As assay temperatures decreased, the summer needles would maintain lower GR activity levels than the winter ones.

Materials and Methods

Preparation of Sample Extracts
Freshly collected yew needles from the same part of one yew bush were packaged, labeled, and immersed in liquid nitrogen for storage. To prepare extract samples, one package was removed from liquid nitrogen, and five green needles were chosen, weighed, and then ground to powder while maintaining an ice-cold temperature. An extraction buffer was made with 50 mM K₂HPO₄, 4% PVP-10, 50 mM KH₂PO₄, 0.1 mM EDTA, and 0.3% Triton X-100 to a total volume of 100 mL as described in Logan, et al. (4), and stored at 4∫C. Immediately prior to using the extraction buffer in the extraction, 1.3 mM ascorbic acid solution was added in a ratio of 1:13 ascorbic acid solution to extraction buffer. A total volume of 2 mL extraction buffer was added to the powdered needles. These were centrifuged at 11,000 rpm for ten minutes, and the supernatant was removed and placed into new eppendorf tubes. The sample extracts were stored on ice during experimentation and in a -85∫C freezer for longer lengths of time.

Preparation of Assay Buffer
Using 100 mM Tris, 1 mM EDTA, and HCl to a pH of 8.0, 500 mL of assay buffer was prepared and stored at room temperature.

Preparation of Substrates
NADPH was prepared to a concentration of 5 mM using deionized water, and 50 mM GSSG was prepared using assay buffer. Both were stored on ice and prepared fresh daily.

Spectrophotometric Determination of Activity
The spectrophotometer was set to 340 nm and programmed to chart absorbance readings every second for 3.5 minutes. Each experimental cuvette contained 50 µL sample extract, 20 µL 50 mM GSSG solution, 1.91 mL assay buffer, and 20 µL 5 mM NADPH added just before beginning the run. A background cuvette was prepared for each sample extract, containing 50 µL sample extract, 1.93 mL assay buffer, and 20 µL 5 mM NADPH added just before the run; this was used to determine the GR activity without the presence of GSSG substrate. The spectrophotometer generated a graph of absorbance versus time and calculated the rate of change of absorbance. This rate of change was used to find the activity per gram protein by using the published extinction coefficient value for glutathione of 0.0062 in the following equation:

0.0062 = absorbance / (concentration x length of cuvette)

Determination of Protein Content
Protein content of each five-needle sample was analyzed using Bio-Rad protein dye. The spectrophotometer was set to 595 nm, and a blank was prepared containing 200 µL Bio-Rad protein dye and 800 µL deionized water. In three eppendorf tubes,10 µL of the extract samples for protein analysis were combined with 200 µL Bio-Rad protein dye and 790 µL deionized water for each sample extract. The samples were allowed to incubate at room temperature for five minutes before testing, and the spectrophotometer was reset with the blank between each absorbance reading.

Electrophoretic Determination of Activity
For gel electrophoresis, 7.5% resolving gel was prepared with 4.9 mL deionized water, 2.5 mL 30% acrylamide solution, 2.5 mL 1.5 M Tris buffer with a pH of 8.8, 100 µL 10% ammonium persulfate solution, and 6 µL tetramethylethylenediamine (TEMED). Also prepared was a 5% stacking gel, which contained 3.45 mL deionized water, 830 µL of 30% acrylamide solution, 630 µL 1.0 M Tris buffer with a pH of 6.8, 50 µL 10% ammonium persulfate solution, and 5 µL TEMED. Gel-loading buffer was made of 50 mM Tris, 0.1% bromophenol blue, and 10% glycerol; it was mixed with the yew sample extracts in a 1:1 ratio. After electrophoresis, the gels were stained for GR activity using a solution of 10 mg MTT, 10 mg 2,6-dichlorophenol-indophenol (DPIP), 3.4 mM GSSG, 400 µM NADPH, and 50 mM Tris, as described in Anderson, et al. (6).

Results

Figure 3 shows the average GR activity calculated spectrophotometrically for summer and winter Taxus cuspidata needles at varying temperatures. The lowest temperature, 15∫C, displays the smallest difference of 0.338 µmol s^-1(g protein)^-1, and the largest difference is at the highest temperature; the average difference in activity is 2.09 µmol s^-1(g protein)^-1 for the 45∫C activity measurement. Each of the error bars represents one standard deviation.

Figure 3: GR Activity of Summer vs. Winter Yew Needles at Different Temperatures (per g protein)

Figure 4 shows a temperature-response curve for change in temperature and change in activity for both the summer and winter yew needles. These data values are linear, confirmed by high correlations for both linear fit models: the r2 values are 0.99 for both. The slope of the winter activity line is 0.10 µmol s^-1(g protein)^-1 ∫C^-1, while the slope of the summer activity line is 0.045 µmol s^-1(g protein)^-1 ∫C^-1. Each of the error bars represents one standard deviation.

Figure 4: Temperature Response Curve for GR Activity (per g protein)

Table 1 shows that GR activity for the summer and winter Taxus cuspidata needles. The p-values less than 0.05 demonstrate a very strong statistical significance in all cases.

Temperature (∫C)	p-value
15	0.0000758
25	0.00000243
35	0.000101
45	0.000103

Figure 4: Activity comparison p-values

Discussion

Previous research indicated that antioxidant enzyme activity in evergreen needles was higher in the wintertime than the summertime, and this was strongly supported by the data collected here (5). Not reported in previous research was that wintertime GR activity increase was significantly greater than seasonal variation in APX activity.

The slopes of the lines fit to the data in Figure 4 demonstrate the stronger response of winter enzyme in Taxus cuspidata needles to temperature alterations. Especially remarkable was that winter-needle GR not only had an average increase in activity of 1.17 µmol s^-1(g protein)^-1 than summer-needle GR under the same temperature stresses, but it also showed a response 1.8 times greater to those stresses. Therefore, wintertime increases in activity can likely be attributed either to an increase in the activity of that enzyme already present in the plant tissue or an increase in the quantity of GR enzyme itself that is produced.

The data in Figure 3 indicate that at all four temperatures, higher GR activity was demonstrated in the wintertime Taxus cuspidata needles than in the summertime needles. GR activity has been shown to be statistically higher in the wintertime for several other species of plants, but GR activity in Taxus cuspidata had not been studied previously. In addition, Minnesota, where this plant enzyme was tested, has notably colder wintertime temperatures (average winter low ñ16.2∫C) and a larger range between high and low temperatures (average summer high 28.9∫C) than Virginia (high 31.3∫C, low ñ3.2∫C) and Colorado (high 33.4∫C, low ñ9.8∫C) where previous experimentation with antioxidant enzymes was done (7, 8).

Further study could be done to determine if the increase in GR activity was due to additional synthesis GR enzyme in the plant or to an upregulation in activity of the present quantity of enzyme. Three separate attempts to pinpoint GR activity via gel assay, including one run in a 4∫C refrigerator, were unsuccessful in this project, producing completely blank gels even after incubation in staining solution. Were the gel assay to function, this test would likely demonstrate whether the wintertime increase in activity is related to an increase in enzyme concentration.

Acknowledgements

I would like to extend sincere thanks to Dr. Amy Verhoeven, Assistant Professor at the University of St. Thomas, for sharing her knowledge and her laboratory with me throughout the course of my research and for demonstrating how far an honest creative interest in research can take a person. In addition, I am very appreciative of Ms. Lois Fruen for her constant assistance and willingness to share her intelligence, and all of my fellow research classmates, namely Mike Hektner, Jonathan Schwalbe, Stephen Morris, and Adam Timm. Much gratitude also goes to my father, Paul Brinda, for teaching me how to write clearly, and to my brilliant and humorous biology instructor, Dr. Jacob Miller. Finally, graduate students John Whiteman, Mai Thao, and Annie Swanberg deserve credit for teaching me not only the physical means by which laboratory work is done, but also a real love of science.

Works Cited

1.	Alscher, R.G., Donahue, J.L, Cramer, C.L. (1997) Reactive oxygen species and antioxidants: Relationships in green cells. Physiologia Plantarium 100: 224-233.
2.	Demmig-Adams, B., Adams III, W.W. (1992) Photoprotection and other responses of plants to high light stress. Plant Physiol. 43: 599-626.
3.	Demmig-Adams, B., Adams III, W.W. (1996) The role of xanthophyll cycle carotenoids in the protection of photosynthesis. Trends in Plant Science 1: 21-26.
4.	Logan, B.A., Grace, S.C., Adams III, W.W., Demmig-Adams, B. (1998) Seasonal differences in xanthophyll cycle characteristics and antioxidants in Mahonia repens growing in different light environments. Oecologia 116: 9-17.
5.	Anderson, J.V., Chevone, B.I., Hess, J.L. (1992) Seasonal variation in the antioxidant system of eastern white pine needles. Plant Physiol. 98: 501-508.
6.	Anderson, J.V., Hess, J.L., Chevone, B.I. (1990) Purification, characterization, and immunological properties for two isoforms of glutathione reductase from eastern white pine needles. Plant Physiol. 94: 1402-1409.
7.	The Minnesota Climatology Working Group (2001) The Minnesota Climatology Working Group Home Page. October, 2001. http://www.climate.umn.edu (November 2, 2001).
8.	NState (2001) High and Low Temperatures of the 50 States. 2001. http://www.netstate.com/states/tables/st_temp.htm (November 9, 2001).

Bibliography

ï	Esterbauer, H., Grill, D. (1978) Seasonal variation of glutathione and glutathione reductase in needles of Picea abies. Plant Physiol. 61: 119-121.
ï	Hausladen, A., Alscher, R.G. (1994) Cold-hardiness-specific glutathione reductase isozymes in red spruce. Plant Physiol. 105: 215-223.
ï	Hausladen, A., Alscher, R. G. (1994) Purification and characterization of glutathione reductase isozymes specific for the state of cold hardiness of red spruce. Plant Physiol. 105: 205-213.
ï	Raven, P.H., Johnson, G.B. Biology. 5th ed. Boston: WCB McGraw-Hill (1999).
ï	Xiang, C., Werner, B.L., Christensen, E.M., Oliver, D.J. (2001) The biological functions of glutathione revisted in Arabidopsis transgenic plants with altered glutathione levels. Plant Physiol. 126: 564-574.