# Chlorophyll fluorescence

A chlorophyll extract in alcohol shown under white light (above) and UV light inducing fluorescence (below).

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.

## Assessing plant physiology

Integrated fluorometer being used to measure photosynthesis and chlorophyll fluorescence simultaneously

Light energy that has been absorbed by a leaf will excite electrons in chlorophyll 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 shining a defined wavelength of light onto a leaf and measuring the level of light emitted at longer wavelengths.

By measuring chlorophyll fluorescence, plant ecophysiology can be investigated. Chlorophyll fluorometers are used by plant researchers to assess plant stress. Gitelson (1999) states, "The ratio between chlorophyll fluorescence at 735 nm and the wavelength range 700nm to 710 nm, F735/F700 was found to be linearly proportional to the chlorophyll content (with determination coefficient, r2, more than 0.95) and thus this ratio can be used as a precise indicator of chlorophyll content in plant leaves."[1]

## The Kautsky effect

Main article: Kautsky effect

Upon illumination of a dark-adapted leaf, there is a rapid rise in fluorescence from Photosystem II (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 acceptors 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 fixation; and 2. more "non-photochemical quenching" in which more energy is converted to heat.

## Measuring fluorescence

Fluorometer being used to measure Fv/Fm in the field

Usually the initial measurement is the minimal level of fluorescence, $\,F_0$. This is the fluorescence in the absence of photosynthetic light.[2]

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 affected 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 $\,F_m$.[2]

The efficiency of photochemical quenching (which is a proxy of the efficiency of PSII) can be estimated by comparing $\,F_m$ to the steady yield of fluorescence in the light $\,F_t$ and the yield of fluorescence in the absence of photosynthetic light $\,F_0$.[3] The efficiency of non-photochemical quenching is altered by various internal and external factors. Alterations in heat dissipation mean changes in $\,F_m$. 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 ($F_m^0$) with which to compare estimations of non-photochemical quenching.[2][3]

### Common fluorescence parameters

$\,F_0$: Minimal fluorescence (arbitrary units). Fluorescence level when all antenna pigment complexes associated with the photosystem are assumed to be open (dark adapted).[3]

$\,F_m$: Maximal fluorescence (arbitrary units). Fluorescence level when a high intensity flash has been applied. All antenna sites are assumed to be closed.[3]

$\,F_{tr}$: Terminal fluorescence (arbitrary units). Fluorescence quenching value at the end of the test.

$\,T_{1/2}$: Half rise time from $\,F_0$ to $\,F_m$.

### Calculated parameters

$\,F_v$ is variable fluorescence. Calculated as $\,F_v$ = $\,F_m$ - $\,F_0$.[4]

$\tfrac{F_v}{F_m}$ is the ratio of variable fluorescence to maximal fluorescence. Calculated as $\frac{F_m-F_0}{F_m}$. This is a measure of the maximum efficiency of PSII (the efficiency if all PSII centres were open). $\tfrac{F_v}{F_m}$ can be used to estimate the potential efficiency of PSII by taking dark-adapted measurements.[3]

$\,\Phi_{PSII}$ measures the efficiency of Photosystem II. Calculated as $\frac{F_m-F_{tr}}{F_m}$. 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.

$\,qP$ (photochemical quenching). Calculated as $\,\frac{F_m-F_{tr}}{F_m-F_0}$. This parameter approximates the proportion of PSII reaction centres that are open.

Whilst $\,\Phi_{PSII}$ gives an estimation of the efficiency, $\,qP$ and $\tfrac{F_v}{F_m}$ 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 $\,qP$. Changes in the efficiency of non-photochemical quenching will alter the ratio $\tfrac{F_v}{F_m}$.[3]

## Applications of the Theory

### PSII yield as a measure of photosynthesis

Modulated fluorometer measuring photosynthetic yield Y(II) in the field

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 fixation 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)[5] examined banana peel degreening after treatment with 1-methylcyclopropene (1-MCP). Many parameters were measured, including chlorophyll fluorescence since fluorescense is a measure of chloroplast 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 $\,F_m$ were responsible for the patterns of de-greening. After 7 and 8 days, $\,F_m$ 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 exchange 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.

• Sobrado (2008)[6] 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 system, which measured net photosynthetic rate, gs, and intercellular CO2 concentration ($C_i$). In the same leaves used for gas exchange measurements, chlorophyll a fluorescence parameters (initial, $\,F_0$; maximum, $\,F_m$; and variable, $\,F_v$) 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 stress. 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.[7]

• Favaretto et al. (2010)[8] 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 $\tfrac{F_v}{F_m}$ 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)[4] investigated the response of raspberry to boron and salt stress. An chlorophyll fluorometer was used to measure $\,F_0$, $\,F_m$ and $\,F_v$. 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

Example of a portable multiparametric fluorometer that uses the ratio between chlorophyll and flavonols to detect the nitrogen deficiency of plants

The development of portable fluorometers 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.[2] 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.

• Nitrogen Balance Index

Because of the link between chlorophyll content and nitrogen content in leaves, chlorophyll fluorometers can be used to detect nitrogen deficiency in plants, by several methods.

Based on several years of research and experimentation, polyphenols can be the indicators of nitrogen status of a plant. For instance, when a plant is under optimal conditions, it favours its primary metabolism and synthesises the proteins (nitrogen molecules) containing chlorophyll, and few flavonols (carbon-based secondary compounds). On the other hand, in case of lack of nitrogen, we will observe an increased production of flavonols by the plant.[9]

The NBI (Nitrogen Balance Index) by Force-A, allows the assessment of nitrogen conditions of a culture by calculating the ratio between Chlorophyll and Flavonols (related to Nitrogen/Carbon allocation) .

## Alternative approaches

### LIF sensors

Techniques based on the Kautsky effect do not exhaust the variety of detection and evaluation methods based on the chlorophyll fluorescence. In particular, recent advances in the area of laser-induced fluorescence (LIF) also provide an opportunity of developing sufficiently compact and efficient sensors for photophysiological status and biomass assessments. Instead of measuring the evolution of the total fluorescence flux, such sensors record the spectral density of this flux exited by strong monochromatic laser light pulses of nanoseconds duration. Requiring no 15- 20 min dark adaptation period (as is the case for the Kautsky effect methods[10]) and being capable to excite the sample from considerable distance, the LIF sensors can provide fast and remote evaluation.

• Application of the LIF technique to the assessment of drought stress in cork oak (Quercus suber) and maritime pine (Pinus pinaster) on the basis of chlorophyll emission ratio I685/I740 is described in Ref.[11] Recently the LIF sensing technique was harnessed to address the role of pPLAIIα protein in the protection of the photosynthetic metabolism during drought stress using genetically modified Arabidopsis plants.[12]
• In 2011, Vieira et al. applied a compact low-cost LIF sensor[13] (built around a frequency-doubled solid-state Q-switched Nd:YAG laser and a specially modified commercial miniature fiber optic spectrometer Ocean Optics USB4000) to study intertidal microphytobenthos communities. Chlorophyll emission enabled the researchers to adequately assess the surface biomass and track migratory rhythms of epipelic benthic microalgae in muddy sediments.[14]

## References

1. ^ Gitelson, Anatoly A; Buschmann, Claus; Lichtenthaler, Hartmut K (1999). "The Chlorophyll Fluorescence Ratio F735/F700 as an Accurate Measure of the Chlorophyll Content in Plants". Remote Sensing of Environment 69 (3): 296. doi:10.1016/S0034-4257(99)00023-1.
2. ^ a b c d "Chlorophyll fluorescence—a practical guide". Jxb.oxfordjournals.org. 2000-04-01. Retrieved 2011-03-28.
3. "Stress Testing". Optisci.com. Retrieved 2011-03-28.
4. ^ a b "Effects of Boron and Salinity on Red Raspberry in Vitro - International Journal of Fruit Science". Informaworld.com. 2008-12-03. Retrieved 2011-03-28.
5. ^ De Martino et al. (2007). "Abstract:Preliminary investigation into the uneven ripening of banana peel after 1-MCP treatment". SpringerLink. doi:10.1007/978-1-4020-6014-4_53.
6. ^
7. ^ "Plant Stress Biology". Personalpages.manchester.ac.uk. Retrieved 2011-03-28.
8. ^
9. ^ A. Cartelat, Z.G. Cerovic, Y. Goulas, S. Meyer, C. Lelarge, J.-L. Prioul, A. Barbottin, M.-H. Jeuffroy, P. Gate, G. Agati, I. Moya (2005). Optically assessed contents of leaf polyphenolics and chlorophyll as indicators of nitrogen deficiency in wheat (Triticum aestivum L.). Field Crops Research Volume 91, Issue 1, pages 35-49.
10. ^ Handy PEA: Continuous Excitation Plant Efficiency Analyser. Norfolk: Hansatech Instruments. 2012. p. 2.
11. ^ Lavrov et al. (2012). "Water stress assessment of cork oak leaves and maritime pine needles based on LIF spectra". Optics and Spectroscopy (Springer) 112 (2): 271–279. doi:10.1134/S0030400X12020166.
12. ^ Silvestre et al. Contribution of pPLAIIα to drought tolerance using genetically modified arabidopsis plants: II. Effects on photosynthetic metabolism. Int. Meeting Prog. Plant Symposium of the SEB: Oxidative stress and cell death in plants: mechanisms and implications, Florence, Italy, 26–28 June 2013, p. 5
13. ^ Utkin et al. (2013). "Compact low-cost detector for in vivo assessment of microphytobenthos using laser induced fluorescence". Optics and Spectroscopy (Springer) 114 (3): 471–477. doi:10.1134/S0030400X13030259.
14. ^ Vieira et al. (2011). "Effects of intertidal microphytobenthos migration on biomass determination via laser-induced fluorescence". Marine Ecology Progress Series 432: 45–52. doi:10.3354/meps09157.