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Leaf Chlorophyll Fluorescence: Background and Fundamentals for Plant Biologists

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Abstract

This review on chlorophyll a fluorescence starts with an overview of the primary photochemistry occurring at PSII and a characterization of the so-called “open” and “closed” states of its reaction centers. This provides the theoretical background for understanding the origin of PSII-emitted fluorescence and how its yield varies with the fraction of open reaction centers. The review proceeds to discuss the changes in fluorescence emission following illumination of a dark-adapted leaf and to define the PSII intrinsic quantum yield of photochemistry, which in turn provides an indication of PSII capacity. In light-adapted leaves, it is discussed how the use of modulated fluorometers and the double lighting technique allow an evaluation of photochemical and non-photochemical quenching, two parameters that give useful information about the plant’s photosynthetic performance under field conditions. Finally, it is described how the PSII operational efficiency can be used to calculate the photosynthetic electron transport rate and the conditions under which this is linearly related to the CO2 assimilation rate. Some requirements for a valid application of the technique as well as some limitations in interpreting its results are discussed.

Resumo

Esta revisão sobre fluorescência da clorofila a começa por sintetizar a fotoquímica primária que ocorre no PSII e caracterizar os chamados estados “aberto” e “fechado” dos seus centros de reacção. Tal fornece os fundamentos teóricos para compreender a origem da fluorescência emitida pelo PSII e perceber como a sua eficiência varia inversamente com a fracção de centros de reacção abertos. A revisão prossegue com uma discussão das variações da fluorescência que se seguem à iluminação de uma folha adaptada ao escuro e define a eficiência quântica intrínseca da fotoquímica do PSII. Em folhas adaptadas à luz, é discutido como a utilização de fluorímetros de luz modulada e a técnica da dupla iluminação permitiram o cálculo da supressão fotoquímica e não-fotoquímica da fluorescência, parâmetros que fornecem informação útil sobre o desempenho fotossintético da planta em condições de campo. Finalmente, é descrito como a eficiência operacional do PSII pode ser utilizada para calcular a taxa de transporte electrónico fotossintético e as condições em que esta está linearmente relacionada com a da assimilação do CO2. São igualmente discutidos alguns requisitos para uma aplicação válida da técnica e algumas limitações à interpretação dos seus resultados.

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Notes

  1. Strictly, this relationship is only valid when a number of assumptions are made, which do not hold under most conditions. In particular, the rate constants for fluorescence and thermal energy dissipation should not change during photochemistry, but it is known that the rate constant for the latter processes increases significantly as the number of closed reaction centers increases. This relationship also implies that all Chl excited states generated in the antenna are equivalent, which is certainly not true given the heterogeneity of PSII organization in the thylakoid membrane.

  2. It should be noted that there is experimental evidence for a light-independent reduction of PQ, possibly with electrons from different origins. Lately, a dark electron transport chain associated with chlororespiration is being characterized that is a probable source of electrons for PQ reduction in the dark, although it is quantitatively a minor process, at least in higher plant chloroplasts. Because of equilibration of PQ with QA it is possible that some PSII reaction centers may remain closed even in the dark. To assure maximal opening of reaction centers, sometimes the sample is initially illuminated with a far-red light that stimulates preferentially PSI activity and reoxidizes any reduced plastoquinone molecules.

  3. There is some simplification in this statement. Indeed, there is always part of excited states that are deactivate non-radiatively, for example, by inter-crossing system. Also, the higher energy of excited states created by absorption of blue photons is partly deactivated as heat loss.

  4. These changes in fluorescence yield following illumination with actinic light of a dark-adapted leaf, termed fluorescence induction, are also known as Kautsky effect, after his discoverer. Indeed, Kautsky and Hirsch (1931) were the first to report such changes and Kautsky postulated that the rise phase of this transient reflected the primary photochemistry of photosynthesis, whereas the declining phase was correlated to the onset of CO2 assimilation. This interpretation was advanced ca. 30 years before Duysens and Sweers (1963) provided the basic understanding of variable fluorescence in PSII and has proven essentially correct, being now considered a cornerstone of photosynthesis research.

  5. Schreiber and Neubauer (1987) used a different notation for the fluorescence rise (O-I1-I2-M) that is currently less used.

  6. The reciprocal of the quantum yield is called quantum requirement and it is often used in efficiency analysis of photosynthetic energy conversion.

  7. The use of saturating light pulses on a background of actinic light is called the “light-doubling” technique and was first introduced by Bradbury and Baker, in 1981; it allows the transient closure of all PSII reaction centers and, thus, momentarily turns off the photochemical quenching, revealing the maximum fluorescence level in the light required to calculate the non-photochemical quenching.

  8. To achieve full closure of PSII reaction centers in the light, a pulse of very high light intensity, of the order of several thousand μmol photons m-2 s-1 as to be used. In the field, when the plant is under high PFD and excitation energy is being effectively dissipated by non-photochemical quenching, maximum fluorescence level is often not attained and this leads to errors in the estimation of several fluorescence parameters.

  9. Note that in the light it is not possible to fully turn off the non-photochemical quenching in order to obtain a fluorescence level that could be used as a reference (zero level) for calculation of the extent of that process; in dark-adapted leaves, however, there is no qN, and thus measurements of non-photochemical quenching are made using as reference the Fm level measured in the dark-adapted state.

  10. It becomes clear, then, that the quantity 1 − qP = (Fs′ − F0′) / (Fm′ − F0′) doesn’t exactly indicate the proportion of closed RCII. This quantity is sometimes referred to as “excitation pressure”.

  11. Formation of triplet states and state 2 transitions, that occur preferentially under high and low irradiance, respectively, also account for some fluorescence quenching, but generally for a very minor fraction of this.

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Acknowledgements

The author would like to thank his colleague Victor Conceição Martins for drawing the figures included in this review.

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Authors and Affiliations

  1. Plant Biology Unit, Department of Life Sciences, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516, Caparica, Portugal

    Fernando S. Henriques

  2. Grupo de Disciplinas de Biologia Vegetal, Departamento de Ciências da Vida, Faculdade de Ciências e Tecnologia da UNL, Quinta da Torre, 2829-516, Caparica, Portugal

    Fernando S. Henriques

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Appendix

Appendix

BOX: PSII and singlet oxygen production

When the PSII reaction center is in its state P680.Pheo.QA − (closed), charge separation between P680 and Pheo is strongly restrained by the repulsive electrostatic effect of QA −, but it still can occur. Subsequent stabilization of the separated charges, however, is prevented because QA is already reduced. The high energy electron in Pheo.− can then return to P680 .+, where it may undergo spin reversion to generate the lower energy triplet state of the excited primary electron donor of PSII, 3P680*. This long-lived triplet state is unable to initiate any productive photochemistry, but it can react rather readily with ground state molecular oxygen, 3O2, exciting it to the higher energy singlet state, 1O2*. Remember that the ground state of oxygen is a triplet state with two unpaired electrons of parallel spins, which makes its effectiveness as an oxidant very low; in the new excited singlet state, the spin of one of the unpaired electrons is reversed and the oxidizing ability of oxygen towards organic molecules is greatly enhanced. Singlet oxygen is thus a highly reactive form of oxygen that can cause extensive modifications to lipids, proteins and nucleic acids, and the chloroplasts have developed a number of defence mechanisms against its potential damaging effects. Among the molecules that protect the thylakoid membranes against singlet oxygen are carotenoids and tocopherols and it is proposed that a β-carotene molecule located at the PSII reaction center serves to quench singlet oxygen that may there be produced.

3P680* + 3O2 → 1P680 + 1O2*

1O2* + 1β-Car → 3O2 + 3β-Car*

3β-Car* → 1β-Car + heat

In the PSII triplet chlorophyll molecules can also be formed directly in the antenna by intersystem crossing of singlet excited chlorophylls. This occurs particularly under conditions where there is an accumulation of excitation energy which lengthens the lifetime of singlet excited chlorophylls and increases the likelihood of spin inversion. Carotenoid molecules appropriately located throughout the antenna complex can directly quench these chlorophyll triplets and subsequently loose their excess energy as heat, thus preventing the formation of the singlet oxygen and, therefore of oxidative stress.

3Chl* + 1Car → 1Chl + 3Car*

3Car* → 1Car + heat

Because of its short lifetime in vivo and its high reactivity, it is likely that singlet oxygen damages principally thylakoid components close to the site of its production. The subunit D1 of PSII reaction center is a preferential target of 1O2*, and this protein was shown to display an extremely high turnover rate, particularly under excess light conditions in which chlorophyll triplets are mainly formed. This selective destruction of D1, followed by its repair, has thus been regarded as a safety valve that serves to prevent further damage to PSII.

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Henriques, F.S. Leaf Chlorophyll Fluorescence: Background and Fundamentals for Plant Biologists. Bot. Rev. 75, 249–270 (2009). https://doi.org/10.1007/s12229-009-9035-y

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