Chlorophyll fluorescence
Encyclopedia
Chlorophyll fluorescence is light that has been re-emitted after being absorbed by chlorophyll
molecules of plant
leaves
. By measuring the intensity and nature of this fluorescence
, plant ecophysiology
can be investigated.
molecules. Energy in photosystem II
can be converted to chemical energy to drive photosynthesis
(photochemistry). If photochemistry is inefficient, excess energy can damage the leaf. Energy can be emitted (known as energy quenching) in the form of heat (called non-photochemical quenching
) or emitted as chlorophyll fluorescence. These three processes are in competition, so fluorescence yield is high when less energy is emitted as heat or used in photochemistry. Therefore, by measuring the amount of chlorophyll fluorescence, the efficiency of photochemistry and non-photochemical quenching can be assessed.
The fluorescence emitted from a leaf has a longer wavelength than the light absorbed by the leaf. Therefore, fluorescence can be measured by shinning a defined wavelength of light onto a leaf and measuring the level of light emitted at longer wavelengths.
(PSII), followed by a slow decline. First observed by Kautsky et al., 1960, this is called the Kautsky Effect.
The increase in fluorescence is due to PSII reaction centers being in a "closed" state. Reaction centers are "closed" when unable to accept further electrons. This occurs when electron acceptor
s downstream of PSII have not yet passed their electrons to a subsequent electron carrier, so are unable to accept another electron. Closed reaction centres reduce the overall photochemical efficiency, and so increases the level of fluorescence. Transferring a leaf from dark into light increases the proportion of closed PSII reaction centres, so fluorescence levels increase for 1–2 seconds. Subsequently, fluorescence decreases over a few minutes. This is due to; 1. more "photochemical quenching" in which electrons are transported away from PSII due to enzymes involved in carbon fixaton; and 2. more "non-photochemical quenching" in which more energy is converted to heat.
. This is the fluorescence in the absence of photosynthetic light.
To use measurements of chlorophyll fluorescence to analyse photosynthesis, researchers must distinguish between photochemical quenching and non-photochemical quenching
(heat dissipation). This is achieved by stopping photochemistry, which allows researchers to measure fluorescence in the presence of non-photochemical quenching alone. To reduce photochemical quenching to negligible levels, a high intensity, short flash of light is applied to the leaf. This transiently closes all PSII reaction centres, which prevents energy of PSII being passed to downstream electron carriers. Non-photochemical quenching will not be effected if the flash is short. During the flash, the fluorescence reaches the level reached in the absence of any photochemical quenching, known as maximum fluorescence
.
The efficiency of photochemical quenching (which is a proxy of the efficiency of PSII) can be estimated by comparing
to the steady yield of fluorescence in the light 
and the yield of fluorescence in the absence of photosynthetic light 
.
The efficiency of non-photochemical quenching is altered by various internal and external factors. Alterations in heat dissipation mean changes in
. Heat dissipation cannot be totally stopped, so the yield of chlorophyll fluorescence in the absence of non-photochemical quenching cannot be measured. Therefore, researchers use a dark-adapted point (
) with which to compare estimations of non-photochemical quenching.
: Minimal fluorescence (arbitrary units). Fluorescence level when all antenna pigment complexes associated with the photosystem are assumed to be open (dark adapted).
: Maximal fluorescence (arbitrary units). Fluorescence level when a high intensity flash has been applied. All antenna sites are assumed to be closed.
: Terminal fluorescence (arbitrary units). Fluorescence quenching value at the end of the test.
: Half rise time from 
to 
.
is variable fluorescence. Calculated as 
= 
- 
.
is the ratio of variable fluorescence to maximal fluorescence.
Calculated as
. This is a measure of the maximum efficiency of PSII (the efficiency if all PSII centres were open). 
can be used to estimate the potential efficiency of PSII by taking dark-adapted measurements.
measures the efficiency of Photosystem II. Calculated as 
. This parameter measures the proportion of light absorbed by PSII that is used in photochemistry. As such, it can give a measure of the rate of linear electron transport and so indicates overall photosynthesis.
(photochemical quenching). Calculated as 
. This parameter approximates the proportion of PSII reaction centres that are open.
Whilst
gives an estimation of the efficiency, 
and 
tell us which processes which have altered the efficiency. Closure of reaction centers as a result of a high intensity light will alter the value of 
. Changes in the efficiency of non-photochemical quenching will alter the ratio 
.
when they refer to photosynthesis. Electron transport and CO2 fixation can correlate well, but may not correlate in the field due to processes such as photorespiration, nitrogen metabolism and the Mehler reaction.
to obtain a full picture of the response of plants to their environment. One technique is to simultaneously measure CO2 fixation and PSII photochemistry at different light intensities, in non-photorespiratory conditions. A plot of CO2 fixation and PSII photochemistry indicates the electron requirement per molecule CO2 fixed. From this estimation, the extent of photorespiration
may be estimated. This has been used to explore the significance of photorespiration as a photoprotective mechanism during drought.
Fluorescence analysis can also be applied to understanding the effects of low and high temperatures.
. Chlorophyll fluorescence can be used as a proxy of plant stress because environmental stresses, e.g. extremes of temperature, light and water availability, can reduce the ability of a plant to metabolise normally. This can mean an imbalance between the absorption of light energy by chlorophyll and the use of energy in photosynthesis.
s has allowed chlorophyll fluorescence to become a common method of measuring plant stress in plant ecophysiology studies. Chlorophyll fluorescence has been revolutionized by the use of modulated chlorophyll fluorometers in which the light source is modulated (rapidly switched on and off) and the detector is tuned to detect only fluorescence excited by the measuring light. This means the relative yield of fluorescence can be measured in the presence of background light. Crucially, this means chlorophyll fluorescence can be measured in the field in full sunlight. Most modern fluorometers are modulated.
Some modulated fluorometers can determine both ambient light and dark adaptation parameters (Fo, Fm, Fv/Fm, Y, Ft, Foq, Fms and OJIP transients) and can calculate photochemical and non-photochemical quenching coefficients (qP, qN and NPQ). In contrast, some fluorometers are slimmed down, and designed to be portable and operated in one hand.
A recent advancement in chlorophyll fluorescence is the development of imaging fluorometers which can visualize spatial heterogeneities in photosynthetic activity of a sample. The Walz Imaging-PAM M-Series is such an imaging system having several versions which allow study of chlorophyll fluorescence in sample sizes ranging from whole plants to single cells.
Because of the link between chlorophyll content and nitrogen
content in leaves, chlorophyll fluorometers can be used to detect nitrogen deficiency. Main article; Chlorophyll fluorometers for nutrient tissue tests
Chlorophyll
Chlorophyll is a green pigment found in almost all plants, algae, and cyanobacteria. Its name is derived from the Greek words χλωρος, chloros and φύλλον, phyllon . Chlorophyll is an extremely important biomolecule, critical in photosynthesis, which allows plants to obtain energy from light...
molecules of plant
Plant
Plants are living organisms belonging to the kingdom Plantae. Precise definitions of the kingdom vary, but as the term is used here, plants include familiar organisms such as trees, flowers, herbs, bushes, grasses, vines, ferns, mosses, and green algae. The group is also called green plants or...
leaves
Leaves
-History:Vocalist Arnar Gudjonsson was formerly the guitarist with Mower, and he was joined by Hallur Hallsson , Arnar Ólafsson , Bjarni Grímsson , and Andri Ásgrímsson . Late in 2001 they played with Emiliana Torrini and drew early praise from the New York Times...
. By measuring the intensity and nature of this fluorescence
Fluorescence
Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation of a different wavelength. It is a form of luminescence. In most cases, emitted light has a longer wavelength, and therefore lower energy, than the absorbed radiation...
, plant ecophysiology
Ecophysiology
Ecophysiology or environmental physiology is a biological discipline which studies the adaptation of organism's physiology to environmental conditions...
can be investigated.
Assessing plant physiology with Chlorophyll fluorescence
Light energy that has been absorbed by a leaf will excite electrons in chlorophyllChlorophyll
Chlorophyll is a green pigment found in almost all plants, algae, and cyanobacteria. Its name is derived from the Greek words χλωρος, chloros and φύλλον, phyllon . Chlorophyll is an extremely important biomolecule, critical in photosynthesis, which allows plants to obtain energy from light...
molecules. Energy in photosystem II
Photosystem II
Photosystem II is the first protein complex in the Light-dependent reactions. It is located in the thylakoid membrane of plants, algae, and cyanobacteria. The enzyme uses photons of light to energize electrons that are then transferred through a variety of coenzymes and cofactors to reduce...
can be converted to chemical energy to drive photosynthesis
Photosynthesis
Photosynthesis is a chemical process that converts carbon dioxide into organic compounds, especially sugars, using the energy from sunlight. Photosynthesis occurs in plants, algae, and many species of bacteria, but not in archaea. Photosynthetic organisms are called photoautotrophs, since they can...
(photochemistry). If photochemistry is inefficient, excess energy can damage the leaf. Energy can be emitted (known as energy quenching) in the form of heat (called non-photochemical quenching
Non-photochemical quenching
Non-photochemical quenching is a mechanism employed by plants and algae to protect themselves from the adverse effects of high light intensity. It involves the quenching of singlet excited state chlorophylls via enhanced internal conversion to the ground state , thus harmlessly dissipating...
) or emitted as chlorophyll fluorescence. These three processes are in competition, so fluorescence yield is high when less energy is emitted as heat or used in photochemistry. Therefore, by measuring the amount of chlorophyll fluorescence, the efficiency of photochemistry and non-photochemical quenching can be assessed.
The fluorescence emitted from a leaf has a longer wavelength than the light absorbed by the leaf. Therefore, fluorescence can be measured by shinning a defined wavelength of light onto a leaf and measuring the level of light emitted at longer wavelengths.
The Kautsky effect
Upon illumination of a dark-adapted leaf, there is a rapid rise in fluorescence from Photosystem IIPhotosystem II
Photosystem II is the first protein complex in the Light-dependent reactions. It is located in the thylakoid membrane of plants, algae, and cyanobacteria. The enzyme uses photons of light to energize electrons that are then transferred through a variety of coenzymes and cofactors to reduce...
(PSII), followed by a slow decline. First observed by Kautsky et al., 1960, this is called the Kautsky Effect.
The increase in fluorescence is due to PSII reaction centers being in a "closed" state. Reaction centers are "closed" when unable to accept further electrons. This occurs when electron acceptor
Electron acceptor
An electron acceptor is a chemical entity that accepts electrons transferred to it from another compound. It is an oxidizing agent that, by virtue of its accepting electrons, is itself reduced in the process....
s downstream of PSII have not yet passed their electrons to a subsequent electron carrier, so are unable to accept another electron. Closed reaction centres reduce the overall photochemical efficiency, and so increases the level of fluorescence. Transferring a leaf from dark into light increases the proportion of closed PSII reaction centres, so fluorescence levels increase for 1–2 seconds. Subsequently, fluorescence decreases over a few minutes. This is due to; 1. more "photochemical quenching" in which electrons are transported away from PSII due to enzymes involved in carbon fixaton; and 2. more "non-photochemical quenching" in which more energy is converted to heat.
Measuring fluorescence
Usually the initial measurement is the minimal level of fluorescence,. This is the fluorescence in the absence of photosynthetic light.To use measurements of chlorophyll fluorescence to analyse photosynthesis, researchers must distinguish between photochemical quenching and non-photochemical quenching
Non-photochemical quenching
Non-photochemical quenching is a mechanism employed by plants and algae to protect themselves from the adverse effects of high light intensity. It involves the quenching of singlet excited state chlorophylls via enhanced internal conversion to the ground state , thus harmlessly dissipating...
(heat dissipation). This is achieved by stopping photochemistry, which allows researchers to measure fluorescence in the presence of non-photochemical quenching alone. To reduce photochemical quenching to negligible levels, a high intensity, short flash of light is applied to the leaf. This transiently closes all PSII reaction centres, which prevents energy of PSII being passed to downstream electron carriers. Non-photochemical quenching will not be effected if the flash is short. During the flash, the fluorescence reaches the level reached in the absence of any photochemical quenching, known as maximum fluorescence
.The efficiency of photochemical quenching (which is a proxy of the efficiency of PSII) can be estimated by comparing
to the steady yield of fluorescence in the light and the yield of fluorescence in the absence of photosynthetic light .The efficiency of non-photochemical quenching is altered by various internal and external factors. Alterations in heat dissipation mean changes in
. Heat dissipation cannot be totally stopped, so the yield of chlorophyll fluorescence in the absence of non-photochemical quenching cannot be measured. Therefore, researchers use a dark-adapted point () with which to compare estimations of non-photochemical quenching.Common fluorescence parameters
: Minimal fluorescence (arbitrary units). Fluorescence level when all antenna pigment complexes associated with the photosystem are assumed to be open (dark adapted).: Maximal fluorescence (arbitrary units). Fluorescence level when a high intensity flash has been applied. All antenna sites are assumed to be closed.: Terminal fluorescence (arbitrary units). Fluorescence quenching value at the end of the test.: Half rise time from to .Calculated parameters
is variable fluorescence. Calculated as = - . is the ratio of variable fluorescence to maximal fluorescence.Calculated as
. This is a measure of the maximum efficiency of PSII (the efficiency if all PSII centres were open). can be used to estimate the potential efficiency of PSII by taking dark-adapted measurements. measures the efficiency of Photosystem II. Calculated as . This parameter measures the proportion of light absorbed by PSII that is used in photochemistry. As such, it can give a measure of the rate of linear electron transport and so indicates overall photosynthesis. (photochemical quenching). Calculated as . This parameter approximates the proportion of PSII reaction centres that are open.Whilst
gives an estimation of the efficiency, and tell us which processes which have altered the efficiency. Closure of reaction centers as a result of a high intensity light will alter the value of . Changes in the efficiency of non-photochemical quenching will alter the ratio .PSII yield as a measure of photosynthesis
Chlorophyll fluorescence appears to measure of photosynthesis, but this is an over-simplification. Fluorescence can measure the efficiency of PSII photochemistry, which can be used to estimate the rate of linear electron transport by multiplying by the light intensity. However, researchers generally mean carbon fixationCarbon fixation
In biology, carbon fixation is the reduction of carbon dioxide to organic compounds by living organisms. The obvious example is photosynthesis. Carbon fixation requires both a source of energy such as sunlight, and an electron donor such as water. All life depends on fixed carbon. Organisms that...
when they refer to photosynthesis. Electron transport and CO2 fixation can correlate well, but may not correlate in the field due to processes such as photorespiration, nitrogen metabolism and the Mehler reaction.
- De Martino et al. (2007) examined banana peel degreening after treatment with 1-methylcyclopropene1-Methylcyclopropene1-Methylcyclopropene is a cyclopropene derivative used as a synthetic plant growth regulator. It is structurally related to the natural plant hormone ethylene and it is used commercially to slow down the ripening of fruit and to help maintain the freshness of cut flowers.-Chemical...
(1-MCP). Many parameters were measured, including chlorophyll fluorescence since fluorescense is a measure of chloroplastChloroplastChloroplasts are organelles found in plant cells and other eukaryotic organisms that conduct photosynthesis. Chloroplasts capture light energy to conserve free energy in the form of ATP and reduce NADP to NADPH through a complex set of processes called photosynthesis.Chloroplasts are green...
photochemical efficiency. 1-MCP treatment was shown to delay the decrease in chlorophyll fluorescence. Greenness and photochemical efficiency decreased simultaneously after ethylene treatment, suggesting that changes in chlorophyll fluorescence
were responsible for the patterns of de-greening. After 7 and 8 days, 
values of 1-MCP peel were ca. 3-fold the values of the other treatments, showing that 1-MCP treated fruit maintain their photochemical efficiency when the other treatment regimes do not.
Relating electron transport to carbon fixation
A powerful research technique is to simultaneously measure chlorophyll fluorescence and gas exchangeGas exchange
Gas exchange is a process in biology where gases contained in an organism and atmosphere transfer or exchange. In human gas-exchange, gases contained in the blood of human bodies exchange with gases contained in the atmosphere. Human gas-exchange occurs in the lungs...
to obtain a full picture of the response of plants to their environment. One technique is to simultaneously measure CO2 fixation and PSII photochemistry at different light intensities, in non-photorespiratory conditions. A plot of CO2 fixation and PSII photochemistry indicates the electron requirement per molecule CO2 fixed. From this estimation, the extent of photorespiration
Photorespiration
Photorespiration, or "'photo-respiration'", is a process in plant metabolism by which RuBP has oxygen added to it by the enzyme , instead of carbon dioxide during normal photosynthesis. This is the beginning step of the Calvin-Benson cycle...
may be estimated. This has been used to explore the significance of photorespiration as a photoprotective mechanism during drought.
Fluorescence analysis can also be applied to understanding the effects of low and high temperatures.
- Sobrado (2008) investigated gas exchange and chlorophyll a fluorescence responses to high intensity light, of pioneer species and forest species. Midday leaf gas exchange was measured using a photosynthesis systemPhotosynthesis systemPhotosynthesis systems are electronic scientific instruments designed for non-destructive measurement of photosynthetic rates in the field. Photosynthesis systems are commonly used in agronomic and environmental research, as well as studies of the global carbon cycle.- How photosynthesis systems...
, which measured net photosynthetic rate, gs, and intercellular CO2 concentration (
). In the same leaves used for gas exchange measurements, chlorophyll a fluorescence parameters (initial, 
; maximum, 
; and variable, 
) were measured using a fluorometer. The results showed that despite pioneer species and forest species occupying different habitats, both showed similar vulnerability to midday photoinhibition in sun-exposed leaves.
Measuring stress and stress tolerance
Chlorophyll fluorescence can measure most types of plant stressPlant stress measurement
Plant stress measurement is the quantification of environmental effects on photosynthesis, and plant health.When plants are subjected to less than ideal growing conditions, plants are considered to be under stress. Plant stress can affect plant growth, plant survival and crop yields...
. Chlorophyll fluorescence can be used as a proxy of plant stress because environmental stresses, e.g. extremes of temperature, light and water availability, can reduce the ability of a plant to metabolise normally. This can mean an imbalance between the absorption of light energy by chlorophyll and the use of energy in photosynthesis.
- Favaretto et al. (2010) investigated adaptation to a strong light environment in pioneer and late successional species, grown under 100% and 10% light. Numerous parameters, including chlorophyll a fluorescence, were measured. A greater decline in
under full sun light in the late-successional species than in the pioneer species was observed. Overall, their results show that pioneer species perform better under high-sun light than late- successional species, suggesting that pioneer plants have more potential tolerance to photo-oxidative damage.
- Neocleous and Vasilakakis (2009) investigated the response of raspberryRaspberryThe raspberry or hindberry is the edible fruit of a multitude of plant species in the genus Rubus, most of which are in the subgenus Idaeobatus; the name also applies to these plants themselves...
to boronBoronBoron is the chemical element with atomic number 5 and the chemical symbol B. Boron is a metalloid. Because boron is not produced by stellar nucleosynthesis, it is a low-abundance element in both the solar system and the Earth's crust. However, boron is concentrated on Earth by the...
and saltSaltIn chemistry, salts are ionic compounds that result from the neutralization reaction of an acid and a base. They are composed of cations and anions so that the product is electrically neutral...
stress. An chlorophyll fluorometer was used to measure
, 
and 
. The leaf chlorophyll fluorescence was not significantly affected by NaCl concentration when B concentration was low. When B was increased, leaf chlorophyll fluorescence was reduced under saline conditions. It could be concluded that the combined effect of B and NaCl on raspberries induces a toxic effect in photochemical parameters.
Chlorophyll fluorometers
The development of portable fluorometerFluorometer
A fluorometer or fluorimeter is a device used to measure parameters of fluorescence: its intensity and wavelength distribution of emission spectrum after excitation by a certain spectrum of light. These parameters are used to identify the presence and the amount of specific molecules in a medium...
s has allowed chlorophyll fluorescence to become a common method of measuring plant stress in plant ecophysiology studies. Chlorophyll fluorescence has been revolutionized by the use of modulated chlorophyll fluorometers in which the light source is modulated (rapidly switched on and off) and the detector is tuned to detect only fluorescence excited by the measuring light. This means the relative yield of fluorescence can be measured in the presence of background light. Crucially, this means chlorophyll fluorescence can be measured in the field in full sunlight. Most modern fluorometers are modulated.
Some modulated fluorometers can determine both ambient light and dark adaptation parameters (Fo, Fm, Fv/Fm, Y, Ft, Foq, Fms and OJIP transients) and can calculate photochemical and non-photochemical quenching coefficients (qP, qN and NPQ). In contrast, some fluorometers are slimmed down, and designed to be portable and operated in one hand.
A recent advancement in chlorophyll fluorescence is the development of imaging fluorometers which can visualize spatial heterogeneities in photosynthetic activity of a sample. The Walz Imaging-PAM M-Series is such an imaging system having several versions which allow study of chlorophyll fluorescence in sample sizes ranging from whole plants to single cells.
Because of the link between chlorophyll content and nitrogen
Nitrogen
Nitrogen is a chemical element that has the symbol N, atomic number of 7 and atomic mass 14.00674 u. Elemental nitrogen is a colorless, odorless, tasteless, and mostly inert diatomic gas at standard conditions, constituting 78.08% by volume of Earth's atmosphere...
content in leaves, chlorophyll fluorometers can be used to detect nitrogen deficiency. Main article; Chlorophyll fluorometers for nutrient tissue tests
External links
- http://www.kalaji.pl/
- http://www.hansatech-instruments.com/
- http://www.optisci.com/cf.htm
- http://jxb.oxfordjournals.org/content/51/345/659.full.pdf+html