Chin J Plan Ecolo ›› 2016, Vol. 40 ›› Issue (9): 942-951.doi: 10.17521/cjpe.2016.0001

• Method and Technology • Previous Articles     Next Articles

A new technology of modulated Chl a fluorescence image: In vivo measurement of the PSII maximum photochemical efficiency and its heterogeneity within leaves

Da-Yong FAN*(), Zeng-Juan FU, Zong-Qiang XIE, Rong-Gui LI, Shu-Min ZHANG   

  1. State Key Laboratory of Vegetation and Environmental Change, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China
  • Received:2015-01-03 Accepted:2016-07-19 Online:2016-09-29 Published:2016-09-10
  • Contact: Da-Yong FAN E-mail:fandayong@ibcas.ac.cn

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 (Fv/Fm) images within leaves. Finally, these images were processed and data of Fv/Fm 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 Fv/Fm and its spatial heterogeneity within leaves has been carried out. It was found that Fv/Fm within leaves was not homogonous. Strong light led to a general decrease of Fv/Fm (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 Fv/Fm 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 Fv/Fm 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 Fv/Fm 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

Table 1

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

参数 Index 型号 Type
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?

No
6)
No6)

Yes

Yes
可否得到叶水平面荧光异质性
Can the fluorescence image on the leaf surface be obtained?

No
可以
Yes

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?

No
8)
No8)

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)

Fig. 3

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

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"

Fig. 2

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

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."

Fig. 5

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

Fig. 6

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

Fig. 7

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

Fig. 8

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

Fig. 9

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

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