1400 American Journal of Botany 90(9): 1400–1403. 2003. BRIEF COMMUNICATION TRANSGENIC OVERPRODUCTION OF GLUTATHIONE REDUCTASE DOES NOT PROTECT COTTON, GOSSYPIUM HIRSUTUM (MALVACEAE), FROM PHOTOINHIBITION DURING GROWTH UNDER CHILLING CONDITIONS 1 BARRY A. LOGAN, 2 GARY MONTEIRO, 2 DMYTRO KORNYEYEV, 3 PAXTON PAYTON, 3,4 RANDY D. ALLEN, 3 AND A. SCOTT HOLADAY 3 2 Biology Department, Bowdoin College, Brunswick, Maine 04011 USA; and 3 Department of Biological Sciences, Texas Tech University, Lubbock, Texas 79409-3131 USA In some studies, tissues from plants that have been genetically transformed to overproduce antioxidant enzymes sustain less damage when abruptly exposed to short-term chilling in the laboratory. However, few studies have examined the performance of transgenic plants during longer-term growth under chilling conditions. We compared growth of transgenic cotton that overproduces glutathione reductase (GR+; 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 F v /F m (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 - q P (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; photoinhibi- tion. Chilling exacerbates oxidative stress that is experienced by plants during illumination (Baker, 1994; Allen and Ort, 2001). Production of reactive oxygen species (ROS) in excess of a plant’s capacity for detoxification can lead to molecular dam- age 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 chlo- roplastic ROS detoxification pathways. Reduced glutathione is a low-molecular-weight thiol antioxidant (Hausladen and Alscher, 1993) that serves as the reductant for dehydroascor- bate reductase, which reforms ascorbate from dehydroascor- bate (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 reduc- tase (GR), which utilizes NADPH as a reductant. This reaction affords further protection against photoinhibition by forming 1 Manuscript received 18 December 2002; revision accepted 24 April 2003. The authors thank the United States Department of Agriculture, National Research Initiative, Competitive Grants Program for funding this work (grant # 99-35100-7630 to A.S.H., B.A.L., and R.D.A.). 4 Current address: USDA, ARS Plant Stress and Germplasm Development Unit, Lubbock, Texas 79415 USA. NADP + , the preferred electron acceptor for photosynthetic electron transport. Acclimation to chilling temperatures generally leads to in- creased GSH contents and GR activities (Anderson et al., 1992; Logan et al., 1998b). Attempts to enhance chilling tol- erance 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 over- production of chloroplastic GR in cotton decreased the levels of photosystem II (PSII) and photosystem I (PSI) photoinhi- bition by approximately 28% and 20%, respectively, during abruptly imposed, short-term exposure of leaf discs from warm-grown plants to 500 mol 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 exhib- ited 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