植物学报 ›› 2021, Vol. 56 ›› Issue (5): 594-604.DOI: 10.11983/CBB21052
张春艳1,2,*(
), 庞肖杰2
首页收稿日期:2021-03-23
接受日期:2021-06-18
出版日期:2021-09-01
发布日期:2021-08-31
通讯作者:
张春艳
作者简介:*E-mail: zhangchunyan@ibcas.ac.cn基金资助:
Chunyan Zhang1,2,*(), Xiaojie Pang2
Received:2021-03-23
Accepted:2021-06-18
Online:2021-09-01
Published:2021-08-31
Contact:
Chunyan Zhang
摘要： 光谱技术已广泛应用于光合研究领域, 如光吸收信号P515和P700氧化还原动力学以及叶绿素荧光等, 可快速、准确地检测植物的光合活性。P515信号广泛存在于高等植物和藻类中, 是类囊体膜上的色素分子吸收光能后, 其吸收光谱发生位移造成。利用光诱导的P515快速和慢速动力学, 可检测PSI和PSII反应中心的比值、ATP合酶的质子传导性、围绕PSI的环式电子传递速率、质子动力势及其组分, 还可通过同步检测叶绿素荧光和P515信号研究光保护机制。该文总结了P515的主要测量原理、方法及其应用, 旨在为深入研究光合作用机理提供技术支持。
张春艳, 庞肖杰. P515的测量原理、方法和应用. 植物学报, 2021, 56(5): 594-604.
Chunyan Zhang, Xiaojie Pang. The Measurement Principles, Methods and Applications of P515. Chinese Bulletin of Botany, 2021, 56(5): 594-604.
图1 饱和闪光诱导的P515快速动力学 (A) 饱和闪光诱导的P515快速动力学示意图, 包括快速上升阶段(橙)、缓慢上升阶段(蓝)和缓慢下降阶段(绿), 箭头表示1次单周转饱和闪光(ST); (B) 饱和闪光诱导莱茵衣藻在520-550 nm的快速吸收变化(单周转饱和闪光波长635 nm, 光强200000 μmol·m-2·s-1, 持续时间5 μs), 莱茵衣藻野生型(WT) (浓度约25 μg·mL-1)加入(红)和不加入(黑) 10 µmol·L-1二氯苯基二甲脲(DCMU)和1 mmol·L-1羟胺(HA), 测量前莱茵衣藻暗适应20分钟; (C) 预照光对P515快速动力学的影响, 莱茵衣藻野生型在暗适应20分钟(黑)以及在光下持续照光2分钟(光强40 μmol·m-2·s-1), 黑暗1分钟(红)后的P515快速动力学。
Figure 1 The P515 fast kinetics induced by a single turnover saturating flash (A) The diagram of P515 fast kinetics induced by a single turnover saturating flash, rapid ascent stage (orange), slow ascent stage (blue) and slow descent stage (green), the arrow indicated a single turnover saturating flash (ST); (B) The rapid absorption changes of Chlamydomonas reinhardtii cells (WT) at 520-550 nm induced by a ST (the wavelength of ST was 635 nm, the light intensity was 200000 μmol·m-2·s-1 and the duration was 5 μs), WT (concentration 25 μg·mL-1) was incubated with (red) and without (black) 10 μmol·L-1 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and 1 mmol·L-1 hydroxylamine (HA) for 20 min in darkness; (C) The affection of preillumination to the P515 fast kinetics, WT algae was incubated in darkness for 20 min (black) and preilluminated for 2 min at 40 μmol·m-2·s-1 followed by 1 min dark (red).
图2 测量拟南芥叶绿体ATP合酶的质子传导性 拟南芥离体叶片在光强为500 μmol·m-2·s-1的活化光(635 nm)下照光10分钟, 照光期间提供稳定的CO2 (380 μmol·mol-1)、湿度(60%大气湿度)和温度(23°C)。检测黑暗250 ms的P515衰减动力学, 重复100次后取平均值。ATP合酶的质子传导率为P515衰减动力学曲线半衰期(t1/2)的倒数, 即94.3·s-1。箭头表示活化光关闭。
Figure 2 Detection the H+ conductivity of chloroplast ATP synthase in Arabidopsis thaliana The detached leaf of Arabidopsis thaliana was exposed to light (635 nm, 500 μmol·m-2·s-1) for 10 min, and provided stable CO2 (380 μmol·mol-1), humidity (60% atmospheric humidity) and temperature (23°C) during illumination. Detected the P515 decay kinetics curve in darkness that lasted 250 ms, the curve corresponded to 100 independent biological replicates. The H+ conductivity of ATP synthase was 94.3·s-1, which is the reciprocal of the half-time (t1/2) of P515 decay kinetics curve. The arrow indicated light off.
图3 测量莱茵衣藻围绕PSI的环式电子传递速率 莱茵衣藻野生型(WT)和PGR5突变体(Mutant)在光强为80 μmol·m-2·s-1的活化光(635 nm)下照光5分钟, 关光后检测围绕PSI的环式电子传递速率, 黑暗持续时间为250 ms。测量前, 藻细胞(浓度约25 μg·mL-1)加入10 µmol·L-1二氯苯基二甲脲(DCMU)、1 mmol·L-1羟胺(HA)和20%的聚蔗糖, 暗适应20分钟。箭头表示活化光关闭。
Figure 3 Detection the cyclic electron flow rate around photosystem I in Chlamydomonas reinhardtii The C. reinhardtii cells (WT) and PGR5 mutant (Mutant) were exposed to light (635 nm, 80 μmol·m-2·s-1) for 5 min. The cyclic electron flow rate was detected after the light was turned off and the darkness duration was 250 ms. Before measurement, algal cells (about 25 μg·mL-1) were incubated with 10 μmol·L-1 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), 1 mmol·L-1 hydroxylamine (HA) and 20% ficoll for 20 min in darkness. The arrow indicated light off.
图4 拟南芥质子动力势(pmf)及其组分(ΔΨ和ΔpH)的测定 拟南芥离体叶片在光强为100 μmol·m-2·s-1 (WT 100, 黑)和750 μmol·m-2·s-1 (WT 750, 红)的活化光(635 nm)下照光3分钟, 照光期间提供稳定的CO2 (380 μmol·mol-1)、湿度(60%大气湿度)和温度(23°C)。黑暗时间60秒, 曲线为3次重复的平均值。测量前, 拟南芥叶片暗适应至少3小时。向上箭头表示活化光打开, 向下箭头表示活化光关闭。
Figure 4 Detection the proton motive force (pmf) and its two components (Δψ and ΔpH) in Arabidopsis thaliana The detached leaf of Arabidopsis thaliana was exposed to light (635 nm, 100 μmol·m-2·s-1, labeled as WT 100, black and 750 μmol·m-2·s-1, labeled as WT 750, red) for 3 min, and provided stable CO2 (380 μmol·mol-1), humidity (60% atmospheric humidity) and temperature (23°C) during illumination. The dark time was 60 s and the curve corresponded to 3 independent biological replicates. The Arabidopsis leaf was darkly adapted for at least 3 hours before measurement. The up arrow indicated light on, and the down arrow indicated light off.
图5 同步测量莱茵衣藻P515信号和叶绿素荧光 将莱茵衣藻野生型(浓度约30 μg·mL-1)暗适应20分钟, 放入荧光仪的测量室, 分别打开P515/535模块和叶绿素荧光模块的测量光, 测定初始荧光产量(Fo)。打开饱和闪光(635 nm, 6000 μmol·m-2·s-1, 持续时间250 ms), 测定黑暗状态下的最大荧光产量(Fm)。之后打开活化光(635 nm, 1500 μmol·m-2·s-1), 持续照光140秒, 期间每隔60秒施加1次饱和闪光, 测定光下最大荧光产量(Fm′)。活化光关闭后测定质子动力势(pmf)及其组分(ΔΨ和ΔpH), 与此同时每隔60秒施加1次饱和闪光, 检测黑暗状态下的荧光产量是否恢复到最大荧光产量(Fm)。Fo: 初始荧光产量; Fm: 最大荧光产量; Fm′: 光下最大荧光产量; Fs: 稳态荧光产量; Actinic light: 活化光; Saturating pulse: 饱和闪光; pmf: 质子动力势; ΔΨ: 跨膜电势; ΔpH: 跨膜质子梯度
Figure 5 Simultaneous measurement of the P515 signal and fluorescence in Chlamydomonas reinhardtii The C. reinhardtii cells (WT, about 30 μg·mL-1) were placed in the chamber of the chlorophyll fluorometer after dark adaptation for 20 minutes. The measuring lights of the P515/535 module and the chlorophyll fluorescence module were turned on respectively to determine the original fluorescence yield (Fo). Then a saturating pulse (635 nm, 6000 μmol·m-2·s-1, duration 250 ms) was turned on to detect the maximal fluorescence yield (Fm) in darkness. Then an actinic light (635 nm, 1500 μmol·m-2·s-1) was turned on for 140 seconds, during which a saturating flash was applied every 60 seconds to detect the maximum fluorescence yield (Fm') under light. The proton motive force (pmf) and its components (ΔΨ and ΔpH) were measured after the actinic light was turned off, meanwhile a saturating pulse was applied every 60 seconds to check whether the fluorescence yield was restored to the maximum fluorescence yield (Fm) in darkness. Fo: Original fluorescence yield; Fm: Maximal fluorescence yield; Fm′: Maximal fluorescence yield under light; Fs: Steady state fluorescence yield; pmf: Proton motive force; ΔΨ: Transmembrane potential; ΔpH: Transmembrane proton gradient
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| [1] | 王强 温晓刚 张其德. 光合作用光抑制的研究进展[J]. 植物学报, 2003, 20(05): 539-548. |
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