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Brief Communication |
2Biology Department, Bowdoin College, Brunswick, Maine 04011 USA; 3Department of Biological Sciences, Texas Tech University, Lubbock, Texas 79409-3131 USA
Received for publication December 18, 2002. Accepted for publication April 24, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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40-fold overproduction) to growth of the wild type in a controlled environment chamber as leaf temperature was lowered from 28° to 14°C over 9 d and for a subsequent 9-d period at 14°C. In wild-type and GR+ cotton, chilling temperatures resulted in decreased dark-adapted Fv/Fm (the ratio of variable to maximal fluorescence; a measure of maximum photosystem II quantum yield) and mid-light period photosystem II quantum yield, coupled with increased 1 – qP (a nonlinear estimate of the reduction state of the primary quinone acceptor of photosystem II). The capacity for photosynthetic oxygen evolution decreased during the first portion of the chilling exposure, but recovered slightly during the second half. At no point during the chilling exposure did the performance of GR+ plants differ significantly from that of wild-type plants in any of the above parameters. The absence of an effect of GR overproduction under longer-term chilling may be explained, in part, by the fact that wild-type cotton acclimated to chilling by upregulating native GR activity.
Key Words: acclimation • antioxidants • chilling • chlorophyll fluorescence • cotton • glutathione reductase • Malvaceae • photoinhibition
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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; Allen and Ort, 2001
). Production of reactive oxygen species (ROS) in excess of a plant's capacity for detoxification can lead to molecular damage and sustained decreases in photosynthetic efficiency that are commonly referred to as photoinhibition (Asada, 1999
; Melis, 1999
; Niyogi, 1999
). Chilling-sensitive plant species, such as cotton, are particularly vulnerable to chilling-induced photoinhibition (Wise, 1995
).
Reduced glutathione (GSH) is a critical constituent of chloroplastic ROS detoxification pathways. Reduced glutathione is a low-molecular-weight thiol antioxidant (Hausladen and Alscher, 1993
) that serves as the reductant for dehydroascorbate reductase, which reforms ascorbate from dehydroascorbate (Hossain and Asada, 1984
). Reduced glutathione can also reduce dehydroascorbate nonenzymatically under the alkaline conditions found in the stroma during illumination (Foyer and Halliwell, 1976
; Winkler et al., 1994
). The glutathione pool is maintained largely in the reduced state by glutathione reductase (GR), which utilizes NADPH as a reductant. This reaction affords further protection against photoinhibition by forming NADP+, the preferred electron acceptor for photosynthetic electron transport.
Acclimation to chilling temperatures generally leads to increased GSH contents and GR activities (Anderson et al., 1992
; Logan et al., 1998b
). Attempts to enhance chilling tolerance via transgenic overproduction of GR have met with some success (Foyer et al., 1995
; Kornyeyev et al., 2001
, 2003
; Payton et al., 2001
). For example, 30- to 40-fold overproduction of chloroplastic GR in cotton decreased the levels of photosystem II (PSII) and photosystem I (PSI) photoinhibition by approximately 28% and 20%, respectively, during abruptly imposed, short-term exposure of leaf discs from warm-grown plants to 500 µmol photons · m–2 · s–1 at 10°C (Kornyeyev et al., 2001
, 2003
). The maintenance of greater rates of photochemistry, along with decreased PSII reduction states, partly explains the enhanced chilling tolerance exhibited by these transgenic plants under these conditions (Melis, 1999
; Kornyeyev et al., 2001
, 2003
).
Much of our understanding of the physiological responses to chilling of transgenic plants that overproduce antioxidant enzymes derives from short-term experiments, such as those described earlier, wherein leaf discs from warm-grown plants are abruptly subjected to conditions that are more extreme than those typically encountered in the field. While these studies have yielded insight into the mechanisms of chilling tolerance and the regulation of oxidative metabolism, there is a need to examine the performance of such transgenic genotypes during growth under longer-term chilling. In the present study, we primarily analyzed chlorophyll fluorescence to compare the performance of cotton overproducing chloroplastic GR to that of wild-type plants during growth in a controlled environment chamber as leaf temperature was lowered from 28° to 14°C over 9 d and for a subsequent 9-d period at 14°C.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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; Payton et al., 2001
). Wild-type cotton (cv. Coker 312) and two independently transformed GR+ lines of cotton were grown from seed in 8-L pots in a greenhouse with a natural photoperiod in December/January. Three weeks after planting, seedlings were transferred to a controlled environment chamber with a photon flux density of 300 µmol photons · m–2 · s–1, a 14-h photoperiod, and a 28°/26°C light/dark temperature regime. After 1 wk in the chamber, the temperature of the chamber was reduced over a 9-d period such that mean leaf temperature fell gradually until it reached 14°C (day and night). The plants were then maintained at this leaf temperature for 9 d (see inset, Fig. 1A). Plant positions within the chamber were random and changed daily. Plants were fertilized with a complete nutrient medium twice weekly. The first fully expanded leaves at the time that the chilling exposure was initiated were used for all measurements.
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. Analyses of the effect of chilling on foliar GR activities employed the same assay.
Chlorophyll fluorescence
Chlorophyll fluorescence emission was measured from attached leaves in their growth environment with a pulse amplitude-modulated fluorometer (FMS2, Hansatech, King's Lynn, Norfolk, UK). Measurements were taken immediately before and in the middle of the light period. The experimental protocol described by Schreiber et al. (1986)
and nomenclature of van Kooten and Snel (1990)
were used. The ratio of variable to maximal fluorescence emission (Fv/Fm) was calculated as (Fm – Fo)/Fm, where Fo and Fm are the pre-light period minimal and maximal levels of fluorescence, respectively. The fraction of light energy absorbed by PSII antennae that was utilized for photochemistry (PSII efficiency) was estimated as (Fm' – F)/Fm', where Fm' is maximal fluorescence during illumination and F is steady state fluorescence during illumination (Genty et al., 1989
). The value 1 – qP was estimated as (F – Fo')/(Fm' – Fo'), where Fo' is minimal fluorescence of leaves in the light-acclimated state. Minimal fluorescence was measured after a brief application of low-intensity far-red light. Saturating light pulses of 1-s duration were provided by a white light source embedded in the fluorometer.
Oxygen evolution
The capacity for oxygen evolution was measured at the beginning, middle (day 7), and end (day 18) of the chilling exposure. Measurements were performed on leaf discs (1.1 cm2) exposed to 1700 µmol · m–2 · s–1 at 25°C in an atmosphere of humidified 5% CO2, 21% O2, and the balance N2 in the chamber of a gas-phase oxygen electrode (Model LD-2, equipped with an LS-2 light source, Hansatech). Steady-state rates of oxygen evolution were determined, followed by measurement of respiration upon return to darkness.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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40-fold higher foliar GR activities than wild-type plants. No significant differences were observed between the physiological performances of the two independently transformed lines of GR+ cotton (data not shown); therefore, data from each were combined. We reported previously that transgenic overproduction of GR has no effect on the size of the foliar glutathione pool in cotton (Kornyeyev et al., 2003
).
In both wild-type and GR+ cotton, the onset of chilling temperatures led to decreased pre-light period Fv/Fm and mid-light period PSII efficiency along with increased mid-light period values for 1 – qP (Fig. 1; the results of one chilling exposure are reported; the entire experiment was repeated and yielded similar results). All three of these effects of decreasing leaf temperature were statistically significant for both wild-type and GR+ cotton (analyses of covariance, P < 0.0001). These responses to the chilling exposure are hallmarks of chilling stress on chilling-sensitive plant species such as cotton (Melis, 1999
; Allen and Ort, 2001
). Decreased pre-light period Fv/Fm is a classical manifestation of chilling-induced photoinhibition. Inhibition of Calvin cycle activity and the resultant decrease in the demand for photochemically generated reductant is thought to underlie chilling-induced increases in 1 – qP, which is a nonlinear estimate of the reduction state of the primary quinone acceptor of PSII. An increase in the reduction state renders PSII more vulnerable to photoinactivation by increasing the probability of charge recombination in the reaction center that can lead to triplet chlorophyll and, ultimately, singlet oxygen formation (Melis, 1999
).
For all three chlorophyll fluorescence parameters depicted in Fig. 1, there was no significant difference between the response of wild-type and GR+ cotton. The absence of differences in the performance of GR+ vs. wild-type cotton contrasts with previous studies of short-term (e.g., 3-h) exposure of leaf discs from warm-grown cotton to either 10° or 15°C at 500 µmol photons · m–2 · s–1 wherein GR+ cotton maintained elevated rates of photochemistry and sustained 28% less PSII photoinhibition (Kornyeyev et al., 2001
, 2003
).
In both wild-type and GR+ cotton, the capacity for photosynthetic oxygen evolution decreased during the first half of the chilling exposure (analysis of variance, P < 0.0001) and exhibited a small but statistically significant increase over the second half of the chilling exposure (P = 0.04; Fig. 2). However, the response of photosynthetic oxygen evolution did not differ between GR+ and wild-type cotton (P = 0.24).
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; Logan et al., 1999
). In chilling-tolerant evergreen species such as Pinus strobus (Anderson et al., 1992
) and Mahonia repens (Logan et al., 1998b
), acclimation to winter involves profound increases in the capacity to detoxify ROS. Acclimation to environmental stress can occur over a period of days and has been shown to exhibit faster kinetics in crop species (Logan et al., 1998a
). In the present study, the GR activities of wild-type cotton doubled over the course of the chilling exposure (Fig. 3). The chilling exposure had no statistically significant effect on the GR activities of GR+ plants (data not shown). While a large difference in GR activity between wild-type and GR+ genotypes remained even after acclimation to chilling, the twofold increase in wild-type GR activity may have been sufficient to meet the demand for enzymatic capacity to reduce oxidized glutathione under the growth conditions.
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FOOTNOTES |
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4 Current address: USDA, ARS Plant Stress and Germplasm Development Unit, Lubbock, Texas 79415 USA
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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Anderson J. V. B. I. Chevone J. L. Hess 1992 Seasonal variation in the antioxidant system of eastern white pine needles. Plant Physiology 98: 501-508
Asada K. 1999 The water-water cycle in chloroplasts: scavenging of active oxygens and dissipation of excess photons. Annual Review of Plant Physiology and Plant Molecular Biology 50: 601-639[CrossRef][ISI]
Baker N. R. 1994 Chilling stress and photosynthesis. In C. Foyer and P. Mullineaux [eds.], Causes of photooxidative stress and amelioration of defense systems in plants, 127–154. CRC Press, Boca Raton, Florida, USA
Foyer C. H. B. Halliwell 1976 The presence of glutathione and glutathione reductase in chloroplasts: a proposed role in ascorbic acid metabolism. Planta 133: 21-25[CrossRef][ISI]
Foyer C. H. N. Souriau S. Perret M. Lelandais K.-J. Kunert C. Pruvost L. Jouanin 1995 Overexpression of glutathione reductase but not glutathione synthetase leads to increases in antioxidant capacity and resistance to photoinhibition in poplar trees. Plant Physiology 109: 1047-1057[Abstract]
Genty B. J. M. Briantais N. R. Baker 1989 The relationship between the quantum yield of photosynthetic electron transport and quenching of chlorophyll fluorescence. Biochimica et Biophysica Acta 990: 87-90[ISI]
Hausladen A. R. G. Alscher 1993 Glutathione. In R. Alscher and J. Hess [eds.], Antioxidants in higher plants, 31–58. CRC Press, Boca Raton, Florida, USA
Hossain H. A. K. Asada 1984 Purification of dehydroascorbate reductase from spinach and its characterisation as a thiol enzyme. Plant and Cell Physiology 25: 85-95
Kornyeyev D. B. A. Logan P. Payton R. D. Allen A. S. Holaday 2001 Enhanced photochemical light utilization and decreased chilling-induced photoinhibition of photosystem II in cotton overexpressing genes encoding chloroplast-targeted antioxidant enzymes. Physiologia Plantarum 113: 323-331[CrossRef][Medline]
Kornyeyev D. B. A. Logan P. Payton R. D. Allen A. S. Holaday 2003 Elevated chloroplastic glutathione reductase activities decrease chilling-induced photoinhibition by increasing rates of photochemistry, but not thermal energy dissipation, in transgenic cotton. Functional Plant Biology 30: 101-110[CrossRef][ISI]
Logan B. A. B. Demmig-Adams W. W. Adams III 1999 Acclimation of photosynthesis to the environment. In G. Singhal, G. Renger, S. Sopory, K. Irrgang, and Govindjee [eds.], Concepts in photobiology: photosynthesis and photomorphogenesis, 477–512. Narosa Publishing House, New Dehli, India
Logan B. A. B. Demmig-Adams W. W. Adams III S. C. Grace 1998a Antioxidants and xanthophyll cycle-dependent energy dissipation in Cucurbita pepo L. and Vinca major L. acclimated to four growth PPFDs in the field. Journal of Experimental Botany 49: 1869-1879
Logan B. A. S. C. Grace W. W. Adams III B. Demmig-Adams 1998b Seasonal differences in xanthophyll cycle characteristics and antioxidants in Mahonia repens growing in different light environments. Oecologia 116: 9-17[CrossRef][ISI]
Melis A. 1999 Photosystem-II damage and repair cycle in chloroplasts: what modulates the rate of photodamage in vivo?. Trends in Plant Science 4: 130-135[CrossRef][ISI][Medline]
Niyogi K. K. 1999 Photoprotection revisited: genetic and molecular approaches. Annual Review of Plant Physiology and Plant Molecular Biology 50: 333-359[CrossRef][ISI]
Payton P. R. Webb D. Kornyeyev R. Allen A. S. Holaday 2001 Protecting cotton photosynthesis during moderate chilling at high light intensity by increasing chloroplastic anti-oxidant enzyme activity. Journal of Experimental Botany 52: 2345-2354
Schreiber U. U. Schliwa W. Bilger 1986 Continuous recording of photochemical chlorophyll fluorescence quenching with a new type of modulation fluorometer. Photosynthesis Research 10: 51-62
van Kooten O. J. F. H. Snel 1990 The use of chlorophyll fluorescence nomenclature in plant stress physiology. Photosynthesis Research 25: 147-150[CrossRef]
Winkler B. S. S. M. Orselli T. S. Rex 1994 The redox couple between glutathione and ascorbic acid: a chemical and physiological perspective. Free Radical Biology and Medicine 17: 333-349[CrossRef][ISI][Medline]
Wise R. R. 1995 Chilling-enhanced photooxidation: the production, action, and study of reactive oxygen species produced during chilling in the light. Photosynthesis Research 45: 79-97[CrossRef]
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