调制式荧光影像新技术: 叶片内部最大光化学量子效率及其异质性的活体测定

doi:10.17521/cjpe.2016.0001

摘要/Abstract

摘要：

研究叶片内部光合能力及其异质性, 是光合作用生化模型、光抑制机理、光保护机理等光合生理生态热点问题的重要前提, 但目前缺少可以进行活体测定的装置。该文作者对Vogelmann和Evans (2002)的装置进行了改进, 获得了叶片内部光系统II (PSII)最大光化学量子效率(F_v/F_m)及其异质性影像, 并采用基于Matlab软件自编的图像处理程序对其进行了分析, 研究了不同时间光抑制处理导致的叶内F_v/F_m及其异质性的变化。研究发现: 叶片内部不同层叶肉组织F_v/F_m存在异质性。强光照射导致叶片内部不同层叶肉组织F_v/F_m值下降, 但靠近上表皮的栅栏组织具有较强的抗光抑制能力。光抑制导致叶片内部F_v/F_m异质性的变化, 短期光抑制导致F_v/F_m异质性变大, 这可能与部分叶绿体避光运动相关, 长期光抑制导致F_v/F_m异质性变小, 说明叶绿体避光运动这种光保护机制已经失效。相比于其他类型叶绿素荧光仪, 此装置可以完整获得活体叶片内部F_v/F_m及其异质性影像数据, 这对于系统研究叶片内部光合能力及其异质性机制, 以及为进一步研究一些光合生理生态热点问题提供了有力工具, 具有进一步研发和应用的前景。

关键词: 菠菜, 叶绿素荧光影像, 光抑制

Abstract:

The spatial photosynthetic heterogeneity within leaves is an important prerequisite for the studies on the photosynthetic model, the mechanism(s) of photoinhibition and light protection, etc. However, currently the in vivo measurement of the spatial photosynthetic heterogeneity within leaves is difficult. The present study improved the device assembled by Vogelmann & Evans (2002), thereby acquired the photosystem II (PSII) maximum photochemical efficiency (F_v/F_m) images within leaves. Finally, these images were processed and data of F_v/F_m and its spatial variations could be obtained, with the aid of MATLAB software. Based on the innovative technique, an investigation of the effects of strong light on the F_v/F_m and its spatial heterogeneity within leaves has been carried out. It was found that F_v/F_m within leaves was not homogonous. Strong light led to a general decrease of F_v/F_m (PSII photoinhibition) across leaf section, and the palisade tissue close to the epidermis layer had high tolerance to photoinhibition. Compared with control, short-term photoinhibition caused a larger spatial variation of F_v/F_m within leaves, which may be related to the chloroplast-avoidance response induced by high-fluence. On the contrary, long-term light inhibition led to a smaller spatial variation of F_v/F_m within leaves, indicating such mechanism is no longer effective. Compared to other types of chlorophyll fluoremeter, the device in the present study can in vivo obtain the panoramic picture of F_v/F_m within leaves, providing a powerful tool for the studies on the mechanism(s) attributed to the spatial heterogeneity of photosynthetic capacity of leaf, which is critical for the understanding on several hot spots in the research field of photosynthesis.

Key words: Spinacia oleracea, chlorophyll fluorescence image, photoinhibition

樊大勇, 付增娟, 谢宗强, 李荣贵, 张淑敏. 调制式荧光影像新技术: 叶片内部最大光化学量子效率及其异质性的活体测定. 植物生态学报, 2016, 40(9): 942-951. DOI: 10.17521/cjpe.2016.0001

Da-Yong FAN, Zeng-Juan FU, Zong-Qiang XIE, Rong-Gui LI, Shu-Min ZHANG. A new technology of modulated Chl a fluorescence image: In vivo measurement of the PSII maximum photochemical efficiency and its heterogeneity within leaves. Chinese Journal of Plant Ecology, 2016, 40(9): 942-951. DOI: 10.17521/cjpe.2016.0001

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链接本文: https://www.plant-ecology.com/CN/10.17521/cjpe.2016.0001

https://www.plant-ecology.com/CN/Y2016/V40/I9/942

图/表 10

表1 目前常见进行叶绿素荧光最大光化学量子效率(Fv/Fm)测定的荧光仪性能对比

Table 1 The comparison of current fluorometers which can measure maximum photochemical efficiency (Fv/Fm)

参数 Index	型号 Type
参数 Index	PAM101-102-103¹⁾	IMAGING-PAM	PEA	本仪器 This set-up
信号采集方式 Signal acquisition mode	调制-锁相放大 Lock-in amplifier	CCD连续采集 CCD continuous collection	光电连续采集 Photoelectric continuous collection	CCD连续采集 CCD continuous collection
荧光数据形式 Fluorescence data format	点式 Point	图像 Image	点式 Point	图像 Image
测量光光强 The measuring light intensity	20-30²⁾	10-1 000	3 000	1 000³⁾
测量光脉冲宽度 The measuring light pulse width	2 μs⁴⁾	1.8 ms	2 s	160 μs
测量光周期 The measuring cycle duration	600 μs⁵⁾	125-1 000 ms	NA	3.3 ms
测量光脉冲宽度在OJIP的位置 The position of the measuring light pulse in OJIP curve	O段 At O stage	接近J段 Close to J stage	NA	接近O段 Close to O stage
测量光占空比 The measuring light duty cycle	1/300	1.8/(125-1000)	NA	1/30
饱和光光强 Saturating light intensity (μmol·m^-2·s^-1)	>3 000	800-1 200	3 000	1 000
测量光光强是否与饱和光光强一致 Is the measuring light intensity consistent with the saturating light intensity?	否 No	否⁶⁾ No⁶⁾	是 Yes	是 Yes
可否得到叶水平面荧光异质性 Can the fluorescence image on the leaf surface be obtained?	否 No	可以 Yes	否 No	可以⁷⁾ Yes⁷⁾
可否得到叶横截面荧光异质性 Can the fluorescence across leaf section be obtained?	否 No	否⁸⁾ No⁸⁾	否 No	可以⁹⁾ Yes⁹⁾
测量光和饱和光的均匀性 The uniformity of measuring and saturating light source	均匀 Uniform	一定范围内均匀 Uniform within a specific range	均匀 Uniform	均匀¹⁰⁾ Uniform¹⁰⁾

图3 本仪器设置的调制光对叶绿素荧光最大光化学量子效率(Fv/Fm)测定值的影响(平均值±标准偏差)。

Fig. 3 Effect of the current set-up of modulated measuring light on maximum photochemical efficiency (Fv/Fm) values (mean ± SD).

图1 仪器装置图。1 叶夹; 2 垫片; 3 叶片; 4 显微镜物镜; 5 入射调制测量光(用于测定Fo)及饱和强光(用于测定Fm); 6 蓝色激光LED; 7 斩波器(在测定Fo时打开, 产生调制测量光, 在测定Fm时关闭, 使得饱和强光通过); 8 恒流源; 9 显微镜二向分光镜; 10 叶绿素荧光; 11 短波截止滤光片(RG9); 12 显微镜自带CCD; 13 计算机。Fo, 暗适应状态下的最小荧光产量; Fm, 暗适应状态下的最大荧光产量。

Fig. 1 Block diagram of the experimental set-up of measurement of chlorophyll fluorescence yield within leaves. 1, leaf clip; 2, leaf pad; 3, leaf disk; 4, microscope objective; 5, incident modulated measuring light (for the determination of Fo) and saturate light (for the determination of Fm); 6, blue laser LED; 7, chopper (when turned on, modulated measuring light is created, for the determination of Fo; when turned off, continuous strong light is created, for the determination of Fm); 8, constant current source; 9, beam splitter; 10, chl a fluorescence; 11, short-wave cut-off filter (RG9); 12, charge coupled device (CCD); 13, computer. Fo, minimum fluorescence yield in dark-adapted state; Fm, maximum fluorescence yield in dark- adapted state

图2 1 W大功率蓝色激光二极管的光谱。用AvaSpec- ULS2048×64光纤光谱仪(Avantes, Apeldoorn, the Netherlands)测定。

Fig. 2 Spectrum of 1 W high power blue laser diode. Measured by a fiber optic spectrometer AvaSpec-ULS2048×64 (Avantes, Apeldoorn, the Netherlands).

图4 对照叶片和光抑制处理后沿叶横截面的最大光化学量子效率(Fv/Fm)异质性假彩色影像。A, 徒手切片。B, 0小时。C, 1小时。D, 3小时。

Fig. 4 The false color images of maximum photochemical efficiency (Fv/Fm) across leaf section after photoinhibition. A, free-hand section showing the thickness of leaf. B, 0 h. C, 1 h. D, 3 h.

图5 对照叶片沿叶横截面的最大光化学量子效率(Fv/Fm)异质性。

Fig. 5 Maximum photochemical efficiency (Fv/Fm) heterogeneity across leaf section of control leaf.

图6 光抑制1小时后叶片沿叶横截面的最大光化学量子效率(Fv/Fm)异质性。

Fig. 6 Maximum photochemical efficiency (Fv/Fm) heterogeneity across leaf section of leaf with 1 hour photoinhibition.

图7 光抑制3 h后沿叶横截面的最大光化学量子效率(Fv/Fm)异质性。

Fig. 7 Maximum photochemical efficiency (Fv/Fm) heterogeneity across leaf section of leaf with 3 h photoinhibition.

图8 对照和光抑制处理后沿叶横截面的最大光化学量子效率(Fv/Fm)差异。

Fig. 8 Maximum photochemical efficiency (Fv/Fm) curves across leaf section of the control and photoinhibited leaves.

图9 对照和光抑制处理后沿叶横截面的最大光化学量子效率(Fv/Fm)变异性(S.D.)。

Fig. 9 Maximum photochemical efficiency (Fv/Fm) variability (S.D.) along the cross section of control and photoinhibited leaves.

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