Okayama S, Butler W L
Department of Biology, Revelle College, University of California, San Diego, La Jolla, California 92037.
Plant Physiol. 1972 May;49(5):769-74. doi: 10.1104/pp.49.5.769.
Hill activity (oxygen evolution with ferricyanide as the electron acceptor), light-induced absorbance changes at liquid nitrogen temperature associated with the primary activity of photosystem II, and fluorescence yield changes at both low temperature and room temperature were measured with lyophilized spinach chloroplasts before and after extraction with hexane and reconstitution with beta-carotene and plastoquinone A. Extraction eliminated the Hill activity, and both beta-carotene and plastoquinone A were required for maximal restoration of activity to the reconstituted chloroplasts.Extraction also eliminated the light-induced absorbance changes at -196 C due to the photoreduction of C-550 and photooxidation of cytochrome b(559), and reconstitution with beta-carotene and plastoquinone A restored the low temperature photoreactions. However, only beta-carotene was essential for the restoration of the photoreactions. Cytochrome b(559) was modified, as a result of the extraction, to a lower redox potential, autooxidizable form and remained as such after reconstitution with beta-carotene. The beta-carotene-restored chloroplasts showed the photoreduction of C-550 but not the photooxidation of cytochrome b(559) because the cytochrome was already oxidized. When beta-carotene-reconstituted chloroplasts were suspended in buffer containing ascorbate prior to freezing, the cytochrome b(559) was reduced and could be photooxidized by irradiation at low temperature. After reconstitution with beta-carotene plus plastoquinone A the cytochrome b(559) was partially restored to its original high potential form and was in the reduced state so that both the photoreduction of C-550 and the photooxidation of cytochrome b(559) occurred on irradiation of the beta-carotene plus plastoquinone A-reconstituted chloroplasts. Reconstitution with plastoquinone A alone had essentially no effect on restoring the photoreactions.The fluorescence yield of dark-adapted lyophilized chloroplasts at -196 C showed an irreversible increase of about 2.5-fold during irradiation. After extraction the fluorescence yield of the chloroplasts was high (at the maximal light-induced level of the lyophilized control chloroplasts) and showed very little change in the light. Reconstitution with beta-carotene alone restored some fluorescence quenching which was relieved by irradiation at low temperature. Reconstitution with plastoquinone A alone restored a high degree of quenching, but this quenching was not relieved by light at low temperature. Fluorescence emission spectra at -196 C showed that the fluorescence of variable yield in the lyophilized and beta-carotene-reconstituted chloroplasts involved only the 680 and 695 nm emission bands but not the larger 730 nm emission band, whereas the irreversible quenching in plastoquinone A-reconstituted chloroplasts involved all wavelengths of emission. Extraction of the chloroplasts also eliminated the sharp 695 nm emission band at low temperature, and reconstitution with beta-carotene partially restored it.The fluorescence yield changes at room temperature differed from the low temperature measurements in that the strong fluorescence quenching restored to the plastoquinone A-reconstituted chloroplasts was relieved by light and reappeared in the dark. Thus plastoquinone A appeared to be much more effective than beta-carotene in restoring the fluorescence of variable yield in room temperature measurements. However, it is argued from the results at low temperature that the quenching in plastoquinone A-reconstituted chloroplasts, which is probably due to the oxidized form of the quinone, is nonspecific and a different quenching mechanism from that which obtains in normal chloroplasts.The results suggest that extraction with hexane removes plastoquinone A, which interrupts electron transport, and beta-carotene, which disrupts the primary photochemical activity of photosystem II. Reconstitution of the extracted chloroplasts with beta-carotene alone restores C-550 and the primary photochemical activity of photosystem II, and when the photosystem II reaction centers are restored the additional requirement of plastoquinone A for the Hill reaction can be demonstrated.
用冻干的菠菜叶绿体在己烷提取前后以及用β - 胡萝卜素和质体醌A重构后，测量了希尔活性（以铁氰化物作为电子受体的放氧活性）、与光系统II初级活性相关的液氮温度下光诱导的吸光度变化以及低温和室温下的荧光产量变化。提取消除了希尔活性，并且β - 胡萝卜素和质体醌A都是使重构叶绿体的活性最大程度恢复所必需的。提取还消除了由于C - 550的光还原和细胞色素b(559)的光氧化导致的-196℃下光诱导的吸光度变化，用β - 胡萝卜素和质体醌A重构恢复了低温光反应。然而，对于光反应的恢复，只有β - 胡萝卜素是必需的。细胞色素b(559)由于提取而被修饰为较低氧化还原电位的、可自动氧化的形式，在用β - 胡萝卜素重构后仍保持如此。β - 胡萝卜素恢复的叶绿体显示出C - 550的光还原，但没有细胞色素b(559)的光氧化，因为细胞色素已经被氧化。当β - 胡萝卜素重构的叶绿体在冷冻前悬浮于含有抗坏血酸的缓冲液中时，细胞色素b(559)被还原并且在低温下照射时可被光氧化。在用β - 胡萝卜素加质体醌A重构后，细胞色素b(559)部分恢复到其原来的高电位形式并且处于还原状态，因此在照射β - 胡萝卜素加质体醌A重构的叶绿体时，C - 550的光还原和细胞色素b(559)的光氧化都发生。仅用质体醌A重构对恢复光反应基本上没有影响。-196℃下暗适应的冻干叶绿体的荧光产量在照射期间显示出约2.5倍的不可逆增加。提取后叶绿体的荧光产量很高（处于冻干对照叶绿体的最大光诱导水平）并且在光照下变化很小。仅用β - 胡萝卜素重构恢复了一些荧光猝灭，这种猝灭在低温照射时被解除。仅用质体醌A重构恢复了高度的猝灭，但这种猝灭在低温下不被光解除。-196℃下荧光发射光谱表明，冻干的和β - 胡萝卜素重构的叶绿体中可变产量的荧光仅涉及680和695nm发射带，而不是较大的730nm发射带，而质体醌A重构的叶绿体中的不可逆猝灭涉及所有发射波长。叶绿体的提取也消除了低温下尖锐的695nm发射带，用β - 胡萝卜素重构部分恢复了它。室温下的荧光产量变化与低温测量不同，因为恢复到质体醌A重构叶绿体的强荧光猝灭被光解除并且在黑暗中重新出现。因此，在室温测量中，质体醌A在恢复可变产量荧光方面似乎比β - 胡萝卜素有效得多。然而，从低温结果来看，质体醌A重构的叶绿体中的猝灭可能是由于醌的氧化形式，是非特异性的，并且与正常叶绿体中的猝灭机制不同。结果表明，用己烷提取去除了中断电子传递的质体醌A和破坏光系统II初级光化学活性的β - 胡萝卜素。仅用β - 胡萝卜素对提取的叶绿体进行重构恢复了C - 550和光系统II的初级光化学活性，并且当光系统II反应中心恢复时，可以证明质体醌A对希尔反应的额外需求。
