Accumulation of Plastoquinone A during Low Temperature Growth of Winter Rye

Abstract

Chloroplasts isolated from rye (Secale cereale L. cv Puma) grown at 5°C (RH) accumulated 260% more plastoquinone A (PQA) per plastid than chloroplasts isolated from rye grown at 20°C (RNH). The number of plastoglobuli increased by 270% in RH chloroplasts compared with RNH plastids. When RH plastids were lysed and washed, the number of plastoglobuli associated with thylakoid membranes decreased significantly , yet the PQA levels remained high. Room temperature fluorescence induction indicated that (a) there is no change in the size of the PQA pool immediately available for photochemistry in RNH and RH thyla-koids and (b) there is a pool of oxidized PQA present in RNH and RH thylakoids which is not available for photochemistry. The accumulated PQA in RH thylakoids may reflect an increased nonphotochemical function such as regulation of thylakoid protein phosphorylation or protection against photoinhibition. In studies of chloroplasts isolated from overwintering Pinus sylvestris, Oquist reported that PSII activity was inhibited 60% by freezing temperatures (18). Using fluorescence induction and photosynthetic partial reactions, Oquist and Hellgren (20) correlated the inhibition of electron transport with a decrease in the plastoquinone pool size. It was also shown that the amount of extractable plastoquinone decreased in chloroplasts isolated from overwintering P. sylvestris, although the ratio of extractable plastoquinone to Chl (w/w) remained relatively constant (16). Winter rye requires exposure to low environmental temperatures in order to acquire and maintain freezing resistance. Rye leaves developed at 5°C can withstand freezing to-22°C (13). When thylakoids are isolated from these leaves, they exhibit the same rate of PSII activity at 26°C as the thylakoids isolated from rye grown at 20°C. However, when PSII activity is measured at 160C or 8°C, thylakoids isolated from cold-grown plants exhibit higher rates than RNH2 thylakoids (12). Studies of thylakoids isolated from both 5°C and 200C leaves have revealed few com-positional differences in polypeptides (7, 9) or lipids (8), although a 1.67-fold accumulation of Chl per plastid has been reported in RH plants (11). In view of the proposed role of plastoquinone as a site of freezing injury in pine thylakoids, we decided to investigate the role ofplastoquinone in cold acclimation ofwinter rye. MATERIALS AND METHODS Plant Materials. Secale cereale L. cv Puma was potted in vermiculite, watered with Hoagland solution, and provided with 'Recipient of a Killam Postdoctoral Fellowship from the University of British Columbia, Vancouver, Canada. 2Abbreviations: RNH, nonhardened rye; RH, cold-hardened rye; PQA, plastoquinone A. 200 ME m-2 s-' PAR during a 16-h photoperiod. Unhardened rye plants were grown at 20 to 25°C/15C (day/night) for 4 weeks, while rye plants to be cold hardened were transferred after 1 week to a growth chamber set at 7 to 5C for an additional 10 weeks. Chloroplast Isolation. Rye leaves were homogenized in grinding buffer containing 0.4 M sorbitol (0.7 M sorbitol for RH leaves), 2 mm EDTA, 2 mm ascorbic acid, and 25 mm Hepes/NaOH (pH 7.6). After filtering through four layers of Miracloth, the homog-enate was centrifuged at 6,000g for 15 s. To obtain intact chloroplasts, half the pellets were washed and resuspended in grinding buffer. When checked by phase contrast microscopy, both RNH and RH chloroplast preparations contained 90% intact chloroplasts. To obtain thylakoids, the remaining pellets were washed four times by resuspending the pellets in 10 mM