International Journal of Bio-Technology and

Research (IJBTR)
ISSN (P): 2249–6858; ISSN (E): 2249–796X
Vol. 11, Issue 1, Jun 2021, 15-22
© TJPRC Pvt. Ltd.

“CHLOROPHYLL FLUORESCENCE IMAGING: AN INVESTIGATIVE TOOL FOR

PLANT ABIOTIC STRESS”

*AMRUTA. D EESHWARAIAH.B & PRAKASH CHANDRA J

School of Applied Material Sciences, Central University of Gujarat, Gujarat, India
ABSTRACT

Chlorophyll fluorescence is a recent technology characteristically used to monitor the photosynthetic performance of
plants non-invasively. This article reviews a detailed relationship between chlorophyll fluorescence and leaf
photosynthetic performance and their changes in abiotic conditions in the context of applications of fluorescence
measurements to screening programmes which seek to identify improved plant performance owing to its sensitivity and
throughput nature of this technology.

KEYWORDS: Abiotic stress, Photosynthesis, Chlorophyll & Chlorophyll (Chl) fluorescence

Received: Jan 17, 2021; Accepted: Feb 07, 2021; Published: May 08, 2021; PaperId.: IJBTRJUN20213

Original Article
INTRODUCTION

Human actions are lashing rapid changes in the global environment. Among the significant global changes is the
increase in human population, pollution and global warming. These changes primarily affect the atmospheric
variables inflict damage on the photosynthetic apparatus of plants, resulting in a decrease in productivity The key
atmospheric variables that impact crops are solar radiation, air temperature, humidity and precipitation. Hence it is
important to understand the bioenergetics of atmospheric variables and its relationship to plant’s photosynthetic
ability to maintain or develop better crops in future with higher economic yield.

Figure 1: Microclimatic conditions Effecting Crop Yield

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16 *Amruta. D Eeshwaraiah.B & Prakash Chandra J

Major role of weather variables on crop growth and development:

Both the high and low temperature decreases the dry matter production and, at extremes, can causes
Temperature:
production to cease.
Solar- Red and blue light have the greatest effect on plant growth. Blue light induce chlorophyll synthesis in
radiation : plant while red light induce plant development.
Water transport the minerals and nutrients in plants, enlarge cell, increase internode elongation and in
Water : day time increases transpiration by opening of leaf stomata which allows the atmospheric CO 2 enter
into plant which fix the atmospheric carbon into the plant.
RH is low, transpiration increases causing water deficits in the plant. The incidence of insect pests and
Relative
diseases is high under high humidity conditions. Very high or very low RH is not conducive for high
humidity :
grain yield.

Abiotic stress

Stress is an external condition that adversely affects growth, development, and/or productivity of a plant. It triggers a wide
range of plant responses such as:

– Misrepresented gene expression.

– Cellular metabolism.

– Changes in growth rates and crop yields.

Figure 2: Different Abiotic stresses affecting crop yield

Chlorophyll floroscence

Chlorophyll (Chl) is a light harvesting pigment found in the chloroplast and it complex molecule made up of carbon,
nitrogen, hydrogen and a small amount of magnesium, and it is located in thylakoid membranes of the chloroplast. Amount

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“Chlorophyll Fluorescence Imaging: An Investigative Tool for Plant Abiotic Stress” 17

of chloroplast in one leaf is 5× 109 and amount of chlorophyll is 600 million, among them 250-300 chlorophyll absorbed
light energy passes through neighbouring pigments to the “special pair” of chlorophyll pigment in a reaction centre. There
are mainly two types of chlorophyll pigments: Chl-a and Chl-b. The chlorophyll best absorbs light in the blue and red
while poor in the green portion of the electromagnetic spectrum. This energy can be utilized in the process of
photosynthesis.

Excited chlorophyll can re-emit a photon and thereby return to its ground state a process known as Chlorophyll
fluorescence. (Taiz and Zeiger, 2006) Light energy that is absorbed by chlorophyll in a leaf can undergo three fates: a)
photosynthesis b) heat dissipation or c) fluorescence. Photosynthesis is inversely proportional to heat dissipation and Chl
fluorescence. These three processes are always antagonistic to each other. As the sum of rate constants is constant, any
increase in the efficiency of one process will eventually result in a decrease in the yield of the other two. When the
reaction center is open (Plastoquinone pool in oxidized state), the photosynthesis is higher and chlorophyll fluorescence is
less but when the reaction center is closed (Plastoquinone pool in reduced state), the photochemistry is less and chlorophyll
fluorescence is high. Fingerprints of excitation spectra of chlorophyll fluorescence can be used to distinguish 'spectral
groups' of plants in vivo and in situ

Figure 3: Chl fluorescence used to detect the responses of plants to Abiotic Stresses

How to Measure Chlorophyll Fluorescence?

A Plant/leaf is kept in dark for 15 to 17 minutes. An Application of measuring light (ML) and saturating flash light (SP)
gives us a minimal fluorescence (Fo) and maximum fluorescence (Fm). Turning the actinic light (AL) on, the fluorescence
reaches at Fp (Peak intensity of fluorescence). The level of fluorescence immediately before the saturating flash light (SP)
is termed Ft. When a leaf of a plant, in the light-adapted state is exposed to a saturating flash light (SP), the maximum
fluorescence (Fm′) obtained and turning off the actinic light (AL) gives minimum fluorescence (Fo') light adapted.

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18 *Amruta. D Eeshwaraiah.B & Prakash Chandra J

Florescence Induction

Photosynthetic metabolism can be distress in different biotic and abiotic conditions. These changes will modify
significantly fluorescence emission characteristics of plants. It is possible to quantitate the changes in fluorescence
induction characteristics resulting from such perturbations by using ratios of fluorescence levels during induction (Habash
et al., 1985).

Photosynthesis is initiated when a dark-adapted leaf is exposed to light. This causes significant changes in
chlorophyll fluorescence. These swift changes in fluorescence are mainly the key to detect the differences in
photosynthetic performance between plants. On immediate exposure to light, fluorescence rises to the minimal level of
fluorescence, which is the level obtained when the PSII reaction centres are mainly in the ‘open’ state (i.e. capable of
photochemistry since QA ,the primary quinone acceptor of PSII, is completely oxidized namely Fo level). The fluorescence
then rises rapidly to the transient inflection level, Fi, before reaching a peak level, Fp. This significant rise to Fi reflects an
rise in the rate of charge stabilization at PSI (QA, is reduced) Changes in the fluorescence level between Fi and Fp are
exclusively due to the increased reduction of the plastoquinone pool, which is largely determined by the relative rates of
PSII photochemistry and oxidation of plastoquinol by electron transfer to PSI. If at this stage, the actinic photosynthetic
photon flux density (PPFD) being used to drive the fluorescence induction is saturating and effects the maximal closure of
PSII reaction centres (maximal reduction of Q A) then the maximal fluorescence level, Fm is obtained. Fv or variable
fluorescence is the difference between Fm and Fo. Chlorophyll fluorescence parameters Fo, Fi, Fm, and Fp, are reliant on
the photochemical and the optical properties of the leaf hence it is necessary to remove the variable of leaf optical
properties when attempting to compare changes in fluorescence characteristics between different leaf samples. Therefore,
determining the yield of chlorophyll fluorescence will provide information about the changes in the efficiency of
photochemistry and heat dissipation. Chlorophyll (Chl) fluorescence is a tool which is widely used to examine
photosynthetic performance in algae and plants. It is a non-invasive analysis that permits to assess photosynthetic
performance in vivo (Baker, 2008)

Chlorophyll fluorescence parameters

Light adapted parameter Dark adapted Parameter Interference

Fo’ Fo Minimum fluorescence
Fm’ Fm Maximum fluorescence
Fv’ = Fm’ - Fo’ Fv = Fm - Fo Variable fluorescence
Fv’/Fm’ = Fm’ - Fo’/Fm’ Fv/Fm = (Fm - Fo)/Fm Maximum quantum use efficiency of PS II
△F/Fm’ = (Fm’ - Fs )/ ∆F/Fm or Y or Φ PS II = Fraction of absorbed photons that are used for
Fm’ (Fm - Fs)/ Fm photochemistry
qN or NPQ = (Fm/ Fm’) --- Non photochemical quenching
–1
qP = (Fm’ - Ft)/(Fm’ - --- Photochemical quenching
Fo’)
ETR = (Fm’ - Ft)/Fm x --- Electron transport rate
PAR
Ft or Fs --- Steady state fluorescence

Diagnostic tools of chlorophyll fluoroscence

Pulse –Amplitude-Modulated Fluorometers

Impact Factor (JCC): 5.9084 NAAS Rating: 3.80

“Chlorophyll Fluorescence Imaging: An Investigative Tool for Plant Abiotic Stress” 19

Fo is determined for a dark-adapted leaf, using a very low PPFD (generally below 1 µmol m-2 s -1), this guarantees all of
the PSII reaction centres in the open state (i.e. capable of photochemistry). The dark-adapted leaf is then exposed to a short
actinic light pulse of very high PPFD (generally less than 1 s at several thousand µmol m-2s-1), this results in maximum
level of fluorescence (Fm) as the majority of the PSII reaction centres is now closed (i.e. incapable of photochemistry). The
ratio of Fv=Fm provides an approximate value of the maximum quantum efficiency of PSII photochemistry (Butler, 1978).
Fv=Fm has been extensively used to detect stress-induced perturbations in the photosynthetic apparatus, since the
development of slowly relaxing quenching processes and photodamage to PSII reaction centres, can result in decrease in
Fv=Fm which reduces the maximum quantum efficiency of PSII photochemistry.

The application of fluorescence measurements is to study the changes leaf photosynthetic performance increased
dramatically with the development of the light addition technique which could resolve fluorescence quenching into
photochemical and non-photochemical components (Bradbury and Baker, 1981, 1984).

Chlorophyll fluorescence imaging system.

This utilizes the technology of imaging of fluorescence signals using charge coupled device (CCD) cameras One of the
major advantage of this technology is that images of fluorescence parameters can be obtained from whole leaves or large
numbers of plants.

Chlorophyll fluorescence Parameters for different physiological stresses.

Physiological
Parameter Interference
stress
Decrease in the rate of utilization of ATP and NADPH in photosynthetic
Drought F`q=F`m
metabolism will consequently, decreases in F`q=F`m
High Rise in Fo and
It indicates the critical temperature for PSII inactivation
temperatures decrease in Fv=Fm
Leaf metabolism is highly inhibited and the potential for slowly
Freezing Fv=Fm
recovering downregulation and photodamage to PSII is high.
As the rate of utilization of photoreductants and ATP decreases, itresults
Chilling F`q=F`v
in a decrease in the PSII efficiency factor.
Soil toxicity Decrease in Fv=Fm Down regulation and photo damage to PSII

Advantages

(1) Non-destructive (2) Non-invasive and (3) Rapid sensitive in real time.

LIMITATIONS

Fv/Fm – Dark adapted test

Fv/Fm is not sensitive to nitrogen and sulfur stress until very low levels are reached. (Baker 2004) It is not sensitive to
nickel and Zinc stress. (Joshi 2004)

Yield or △F/Fm’ - Light adapted test

Yield is not sensitive to sulfur stress until starvation levels are reached. (Baker 2004)

Applications

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20 *Amruta. D Eeshwaraiah.B & Prakash Chandra J

 Precursor for the onset of harmful algal blooms.

 Oceanographic, estuarine, limnological and riverine studies.

 Indicator of contaminants in the water body.

 Diagnosis of environmental stresses.

 Screening of tolerant plants to environmental stresses.

 Thermal and chlorophyll fluorescence imaging provide specific signatures for diagnosis of distinct diseases and
abiotic stresses.

CONCLUSIONS

It is conclusive that chlorophyll fluorescence can provide very swift assessment of plant performance in a wide range of
situations. A key factor for the successful application of chlorophyll fluorescence measurements in crop improvement
program would be proper selection of appropriate fluorescence parameters to identify changes in plant performance. This
technique should enable the agricultural and horticultural industries to improve the quality and rate of many of their plant
screening programmes.

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