J. Plant Biochemistry & BiotechnologyVol. 10,143 -146, July 2001
Short Communication
UV-A Inhibition of Alternative Respiration in Pea Leaves and a Unicellular Green Alga Chlamydomonas reinhardtii Doug Mulley, Durba Ghoshal and Arun Goyal* Department of Biology, College of Science & Engineering; Department of Biochemistry and Molecular Biology, School of Medicine, University of Minnesota Duluth, Duluth, MN 55812, USA
Total respiration (vr ) increased after exposure to UV, but a decrease in the capacity of SHAM-sensitive-alternative respiration (Vall) was accompanied by an increase in residual respiration (v res )' The capacity for CN sensitive-cytochrome c respiration (VCYI ) was not inhibited by UV-A. After 4 h of irradiation of high-C0 2-grown cells of Chlamydomonas reinhardtii with UV-A (2 IJW. cm·2) in the presence of white light (300IJE.m·2.s·1), the capacity of Vall was reduced from 10 to 4 IJmol 02' mg·1Chl.h·l, a 60 % reduction. After a similar exposure to UV-A, the capacity of Vall in pea leaves was reduced from 13 to 5 IJmol 02.g·1 fr wt.h·l. Exposure to UV-C was not inhibitory, but UV-B caused up to 25% inhibition of the Vall' Twenty to 48 h after exposure to UV-A radiation, the capacity of alternative respiration had recovered. UV-A inhibition of the alternative respiration was consistent with UV-A absorption by quinones, except that UV-A did not inhibit the cyt c pathway of electron transport that also involves the ubiquinones. Key words: alternative respiration, Chlamydomonas reinhardtii, Pisum sativum, UV-A inhibition.
Destruction of the stratospheric ozone layer due to the release of man-made ozone-destroying chemicals has resulted in increased levels of solar UV radiation reaching at the Earth's surface. Photosynthetic organisms require sunlight for growth and metabolism; thus, they are exposed to UV radiation. The UV radiation includes UVA (320 to 400 nm), UV-B (280 to 320 nm) and UVC (below 280 nm). Normally UV with wavelengths less than 290 nm can not reach the Earth's surface; therefore, UV-C is not a matter of concern. A large number of direct or indirect effects of UV-B on plants have been identified (1,2). The molecular targets of UV irradiation include, but are not limited to, damage to DNA, proteins, membranes, photosynthetic machinery, phytochromes, secondary metabolism, and free-radical scavenging systems (3). UV-A plays an important role in both the inhibition and the repair mechanisms associated with the photosynthetic process and primary aquatic productivity (3, 4). Besides the cyanide-sensitive mitochondrial respiration, plants (5) and algae (6,7) also contain an apparently ubiquitous mitochondrial alternative
* Corresponding author. Email:
[email protected] Abbreviations: SHAM, salicylhydroxamic acid; vr ' measured rate of total respiration; V CN ' oxygen uptake after additon of 1 mM KCN; Valt , capacity of the alternative respiration; Vcy!' capacity of the cytochrome c respiration; v,es' residual respiration.
respiration that is inhibited by SHAM, but not by CN. Although numerous inhibitory effects on plant growth from UV radiation have been reviewed (1), no specific effect of UV on respiration or alternative respiration has been established. In this paper, we report that UV-A inhibits the capacity of alternative respiration, but not the CN-sensitive respiration. Chlamydomonas reinhardtii, strain 137 (UTEX-Algal Culture Collection), cells were grown with 5% CO 2 in air (v/v) , and cultures were harvested in their log phase of growth as described previously (6). Seeds of Pisum sativum L cv Alaska (Atlee Burpee Seed Company, Warminster, PA) were soaked overnight in distilled water, and sown in a 1:1:1 mixture of peat moss, topsoil and vermiculite. Plants were raised in a growth chamber, and watered with Hoagland nutrient solution. Young, fully expanded leaves from two-week-old seedlings were used. Measurements of respiration Endogenous dark respiration by Chlamydomonas reinhardtii cells (2 ml, containing 50 I-Ig Chi) was measured as 02 uptake at 25°C in a Clark-type oxygen electrode (Rank Brothers, Cambridge). The chlorophyll content was 25 I-Ig ml· 1 , determined spectrophotometrically at A652 with a separate aliquot that had been extracted with 80% ethanol. The oxygen uptake was measured for at least 5 min before
144
J Plant Biochem Biotech
inhibitors were added to final concentrations of 1 mM cyanide (CN) or 2.5 mM SHAM. Oxygen content in the air-saturated medium was assumed to be 250 ~M. A stock solution of 250 mM SHAM was prepared in 100% ethanol, and there was no effect on respiration by the amount of ethanol carried into the assays. Dark oxygen uptake by leaf disks was measured with oxygen electrode at 25°C in 10 mM potassium phosphate buffer (pH 7.5, 3 ml). Fifteen to twenty leaf disks (100 mg fresh weight) were punched out of leaves immediately before the assay, equilibrated for 5 min in the buffer with continuous stirri'ng, after which time the rate of respiration, vT was determined. After about 5 min either 1 mM KCN (final concentration) or 2.5 mM SHAM was added to the suspension and 02 uptake measurements were recorded for the next 5 min, after which the other inhibitor, SHAM or KCN was added. V has been designated as the "capacity" for respiration and v for the measured rate of respiration. The method, calculations, and definitions of the capacities and velocity of the cytochrome c and alternative pathways are described elsewhere (6). vT was the measured rate of total respiration without inhibitors. The capacity of the cyt c pathway, VCyt ' was calculated as the difference in the rate of 02 uptake in the presence of SHAM caused by the addition of KCN. In this case vT was measured first without inhibitor, then the SHAM inhibited rate was determined, and finally KCN was added. The difference in the rate of 02 uptake before and after addition of KCN was Vcyr Similarly the capacity of the alternative pathway, Valt ' was calculated from the difference in the rate of 02 uptake inhibited by SHAM in the presence of KCN. In this case KCN was added first and then SHAM was added to provide Vall' VCN was the rate of 02 uptake upon addition of KCN. UV treatment and recovery - For UV-A (2 ~W . cm- 2 ) or UV-C (2 ~W . cm- 2) irradiation, a dilute Chlamydomonas reinhardtii cell suspension was stirred in a beaker and exposed to the radiation from a short or long wavelength UV lamp (UVGL-58, Ultra-Violet
Products, Inc., San Gabriel, CA, USA) UV was measured by a Metrologic laser power meter. For UV-8 radiation, algae in a 3 ml quartz cuvette were stirred by bubbling air and exposed to 300 nm of light in a Gilford Spectrophotometer with the slit wide open. UV-8 light intensity was about 2 ~W . cm- 2. During the UV treatments, the algae were also illuminated with an overhead white light of 300 ~E.m-2.s-1 after filtration of light through a copper sulfate solution to remove infra red heat. For treating pea seedlings, each tray with 50 to 75 seedlings was exposed to UV-A by a hand-held UV lamp «UVGL-58, Ultra-Violet Products, Inc., San Gabriel, CA, USA) by placing the lamp 3 feet above the top of the plants for 8 h. The UV light had an intensity of 2 ~W. cm- 2. White light of 300 ~E. m-2. S-1 was also provided to the plants from an incandenscent 300 W reflector lamp filtered through a solution of CuS0 4 . For control plants, only white light was provided. The rate of respiration and capacities were measured before and after the UV treatment. Plants were also held for recovery from the UV damage, and respiration was measured at 0, 20 and 48h after the UV exposure. During the recovery, a 10 h light and 14 h dark cycle was used, and plants were watered every 24 h. Effect of UV on the capacity of the alternative respiration by Chlamydomonas reinhardtii - The rate of dark respiration, measured as oxygen uptake (vT), increased by about 25% over the control from UVtreatment (Table 1). However, based on the increased residual respiration (vres ~ oxygen uptake that is not sensitive to CN or SHAM),· the increased oxygen uptake in UV-treated cells may not be due to true respiratory activity. It appears that during 4 h of UV-8/UV-C treatment, the capacity of cyt c pathway (V cy1) increased by about 60%, but the capacity of the alternative pathway (Vall) was essentially unchanged. The reason for the increase in dark respiration and the capacity of cyt c respiration is not known. The Chlamydomonas cells treated with UVA showed increased oxygen uptake, similar to the other two treatments, but the capacity of the alternative
Table 1. Effect of UV radiation on respiration by Chlamydomonas reinhardtii Time of exposure (h)
-UV
+UV-A V cyt
Valt
+ UV-8 Valt
Respiration rate (~mol
°
2
+ UV-C Valt
,
V cyt
Valt
10
mg· 1 Chi. h· 1)
0
14
14
10
14
14
10
14
14
10
14
14
2
18
14
10
24
14
4
24
24
10
24
24
8
4
18
13
9
22
11
4
25
20
10
24
24
8
Short Communication
pathway (Vall) was decreased by 60% with no change in the capacity of cyt c pathway (V cyl) (Table 1). Inhibition of alternative respiration in pea leaves by UV-A - Leaf disks in the dark took up 02 at a rate of 42 tJmol 02. g- 1fr wt.h- 1 (vT) , and the capacity of cyt c pathway (V cyt) , was about 60%, and the capacity of the alternative pathway (Vall) was about 30%, of the total respiration (vT ) (Table 2). About 10% of the total respiration was residual that was not inhibited by both KCN and SHAM. After exposure to UV-A and white light for 6 to 8 h, there was little change in the capacity of cyt c pathway (V Cyl) , but the capacity of the alternative pathway was reduced by 60% and the residual respiration (v res ) had increased proportionally (Table 2). When the UV-A irradiated plants were allowed to recover, the Vall returned to almost to normal after two days. The recovery of the capacity of the alternative pathway (Vall) was accompanied by a gradual decline in the residual respiration, vres to normal levels. The SHAM-sensitive, cyanide-resistant alternative pathway of mitochondrial respiration represents an oxidation of a reduced ubiquinone pool by a quinol oxidase (5). Its function is unknown, other than to waste energy and produce heat. Quinones, plastoquinone and ubiquinones, have an absorption spectra for UV-A (8) that may be consistent with UV-A inhibition of the alternative quinol oxidase. However, electron transport by both the cyt c and the alternative oxidase is thought to involve similar ubiquinones. Because the cyt c pathway was not inhibited by UV-A, it would seem that a quinol
145
specific for the alternative oxidase pathway may be UVA sensitive. In the chloroplast thylakoid PS " system, the 01 protein that transf\3rs electrons into the plastiquinone pool is also UV-A sensitive (9). Because the 0 I protein rapidly turns over, recovery of the PS " system from UV-A irradiation occurs. Likewise after UV-A inhibition of alternative respiration in pea leaves, recovery occurred in 1 to 2 days. The results indicate that the duration and dose of UV-A used in this study caused an elastic damage to the capacity or component of the alternative pathway in C. reinhardtii or peas. A longer and more intense exposure to UV-A may result in a plastic injury to the capacity or component of the alternative pathway in plants and algae. The recovery of the capacity of the alternative pathway appears to be related to repair mechanism(s) of the plant responses to environmental stress. A decrease in the capacity of the alternative respiration by UV-A does not represent the actual engagement of the pathway. The simultaneous increase in residual oxygen uptake (not inhibited by KCN and SHAM) is likely due to an increased lipoxygenase activity. The oxidation of membrane lipids as precursors for the synthesis of molecules that have intracellular or longrange signaling activities is well known (10). Therefore, it is possible that the transient damage by UV-A could have induced lipoxygenase activity, the product(s) of which are involved in the synthesis of signaling molecules (10), such as jasmonate. In the past few years, the
Table 2. Elfect of UV-A radiation on the capacity of the cytochrome pathway (V cyt) and alternative pathway (V alt) in leaf discs of pea cv Alaska
(I-Imol
°
2 •
vT g-1 fr wI. h·1 )
V cyt
Valt (% of vT )
vres
Zero time Control (No UV-A)
42 ± 2.5
60
30
9
After 6 or 8 h exposure to UV-A
'31 ± 0.5
56
13
37
Control (No UV-A)
42 ± 0
60
28
10
UV treated plants
:f)
± 0
70
21
28
44 ± 0
64
29
11
40 ± 0
62
28
12
20 h after UV treatment and recovery in light
48 h after UV treatment and recovery in light Control (No UV-A) UV treated plants
Note - Values are expressed as a percentage of total dark respiration (vT). Results are mean ± SE (n == 6). vT is expressed as I-Imol 02 . mg-I fr wt . h-I. Theoretically, if both the cyt c pathway and the alternative pathway were operating to their full capacities, vT == Vr:f. + Va It + Vrss · Normally either one or both pathways do not operate at full capacity for the measured total velocity, vT ; therefore, v T was always less than the sum of V cyt + V aIt + V res . Thus V cyt'aIt V and vres do not always add up to 100 .
146
J Plant Biochem Biotech
role of jasmonate as a key component of a wound signaling has been established. It is therefore, likely that an unknown signaling molecule may have been produced by irradiation with UV-A for which lipoxygenase activity may have been a pre-requisite, thus causing increased oxygen uptake. This is a speculation on the possible mechanism for increased oxygen uptake; however, it is likely that the UV-A radiation causes very complex effects. The UV-A inhibition of the Val! may provide another research tool for studying alternative respiration.
Acknowledgements We thank Dr Neil Nelson for critically reading the manuscript. This work was supported in part by the Undergraduate Research Opportunity Program of the University of Minnesota to OM. Received 8 May, 2001; revised 29 June, 2001.
References Worrest RC & Caldwell MM, Stratospheric ozone reduction, solar ultraviolet radiation and plant life, Springer Verlag, Berlin (1986). 2
3 4 5 6 7 8 9 10
Jansen AKM, Gaba V & Greenberg BM, TIPS, 3 (1998) 131. Nielsen T & Ekelund NGA, FEMS Microbiol Ecol, 18 (1995) 281. Helbling EW, Villafane V, Ferrario M Hoi-Hanson 0, Mar Ecol Prog Ser, 80 (1992) 89. Douce R, Mitochondria in higher plants, Academic Press, New York (1985). Goyal A & Tolbert NE, Plant Physiol, 89 (1989) 958. Sasa T, Plant Cell Physiol, 2 (1961) 253. Brodie AF, Methods Enzymol, 6 (1963) 295. Greenberg BM, Gaba V, Canaani 0, Malkin S, Mattoo AK & Edelmann M, Proc Nat! Acad Sci, USA, 86 (1989) 6617. Buchanan BB, Gruissem W & Jones RL, Biochemistry and molecular biology of plants, American Soc Plant Physiol, Rockville (2000) p 505.
