1.2.5  Chlorophyll fluorescence

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A dilute solution of leaf chlorophyll in organic solvent appears green when viewed with light transmitted from a white source. Wavelengths corresponding to bands of blue and red have been strongly absorbed (Figure 1.8), whereas mid-range wavelengths corresponding to green light are only weakly absorbed, hence the predominance of those wavelengths in transmitted and reflected light. However, viewed laterally via re-emitted energy, the solution will appear deep red, and that same colour will persist regardless of source light quality. Fluorescence spectra are invariate, and the same spectrum will be obtained (e.g. Figure 1.8 inset) regardless of which wavelengths are used for excitation. This characteristic emission is especially valuable in establishing source pigments responsible for given emission spectra, and for studying changes in their photochemical status during energy transduction.

Fluorescence emission spectra (Figure 1.8 inset) are always displaced towards longer wavelengths compared with corresponding absorption spectra (Stoke’s shift). As quantum physics explains, photons intercepted by the chromophore of a chlorophyll molecule cause an instantaneous rearrangement of certain electrons, lifting that pigment molecule from a ground state to an excited state which has a lifetime of c. 10–9 s. Some of this excitation energy is subsequently converted to vibrational energy which is acquired much more ‘slowly’ by much heavier nuclei. A non-equilibrium state is induced, and molecules so affected begin to vibrate rather like a spring with characteristic periodicity, leading in turn to energy dissipation as heat plus remission of less energetic photons of longer wavelength.

Apart from their role in photon capture and transfer of excitation energy, photosystems function as energy converters because they are able to seize photon energy rather than lose as much as 30% of it through fluorescence as do chlorophylls in solution. Moreover, they can use the trapped energy to lift an electron to a higher energy level from where it can commence a ‘downhill’ flow via a series of electron carriers as summarised in Figure 1.10.

Protein structure confers very strict order on bound chlorophylls. X-ray crystallographic resolution of the bacterial reaction centre has given us a picture of the beautiful asymmetry of pigment and cofactor arrangements in these reaction centres, and electron diffraction has shown us how chlorophylls are arranged with proteins that form the main light-harvesting complexes of PSII. This structural constraint confers precise distance and orientation relationships between the various chlorophylls, as well as between chlorophylls and carotenoids, and between chlorophylls and cofactors enabling the photosystems to become such effective photochemical devices. It also means that only 2–5% of all the energy that is absorbed by a photosystem is lost as fluorescence.

If leaf tissue is held at liquid nitrogen temperature (77 K), photosynthetic electron flow ceases and chlorophyll fluorescence does increase, including some emission from PSI. Induction kinetics of chlorophyll fluorescence at 77 K have been used to probe primary events in energy transduction, and especially the functional state of photosystems. Present discussion is restricted to room temperature fluorescence where even the small amount of fluorescence from PSII is diagnostic of changes in functional state. This is because chlorophyll fluorescence is not emitted simply as a burst of red light following excitation, but in an ordered fashion that varies widely in flux during continuous illumination. These transient events (Figure 1.12) are referred to collectively as fluorescence induction kinetics, fluorescence transients, or simply a Kautsky curve in honour of its discoverer Hans Kautsky (Kautsky and Franck 1943).


Figure 1.12 A representative chart recorder trace of induction kinetics for Chl a fluorescence at room temperature from a mature bean leaf (Phaseolus vulgaris). The leaf was held in darkness for 17 min prior to excitation (zig-zag arrow) at a photon irradiance of 85 µmol quanta m-2 s-1. The overall Kautsky curve is given in (b), and an expanded version of the first 400 ms is shown in (a). See text for explanation of symbols and interpretation of variation in strength for these ‘rich but ambiguous signals’! (Based on Norrish et al. 1983; reproduced with permission of Kluwer Academic Publishers)

At room temperature and under steady-state conditions, in vivo Chl a fluorescence from an immature (greening) bean leaf shows a characteristic emission spectrum with a distinct peak around 680–690 nm and a shoulder near 735 nm. As leaf chlorophyll concentration increases, the intensity of fluorescence at 680–690 nm diminishes compared with emission at 735 nm due to reabsorption of shorter wavelengths by the extra chlorophyll molecules. Fully developed bean leaves thus show a new maximum between 730 and 740 nm. Room temperature fluorometers rely on this secondary peak.

(a)  Fluorescence induction kinetics

Strength of emission under steady-state conditions varies according to the fate of photon energy captured by LHCII, and the degree to which energy derived from photosynthetic electron flow is gainfully employed. However, strength of emission fluctuates widely during induction (Figure 1.12) and these rather perplexing dynamics are an outcome of some initial seesawing between photon capture and subsequent electron flow. Taking Figure 1.10 for reference, complexities of a fluorescence transient (Figure 1.12) can be explained as follows. At the instant of excitation (zig-zag arrow), signal strength jumps to a point called F0 which represents energy derived largely from chlorophyll molecules in the distal antennae of the LHCII complex which fail to transfer their excitation energy to another chlorophyll molecule, but lose it immediately as fluorescence. F0 thus varies according to the effectiveness of coupling between antennae chlorophyll and reaction centre chlorophyll, and will increase due to high-temperature stress or photodamage. Manganese-deficient leaves show a dramatic increase in F0 due to loss of functional continuity between photon-harvesting and energy-processing centres of PSII (discussed further in Chapter 16).

Returning to Figure 1.12, the slower rise subsequent to F0 is called I, and is followed by a further rise to Fm. These stages reflect a surge of electrons which fill successive pools of various electron acceptors of PSII. Significantly, Fm is best expressed in leaves that have been held in darkness for at least 10–15 min. During this dark pretreatment, electrons are drawn from QA, leaving this pool in an oxidised state and ready to accept electrons from PSII. An alternative strategy is to irradiate leaves with far-red light to energise PSI preferentially, and so draw electrons from PSII via the Rieske FeS centre. The sharp peak (Fm) is due to a temporary restriction on electron flow downstream from PSII. This constraint results in maximum fluorescence out of PSII at about 500 ms after excitation in Figure 1.12(a). That peak will occur earlier where leaves contain more PSII relative to electron carriers, or in DCMU-treated leaves.

Photochemistry and electron transport activity always quench fluorescence to a major extent unless electron flow out of PSII is blocked. Such blockage can be achieved with the herbicide 3-(3,4-dichlorophenyl)-1,1-dimethyl urea (DCMU) which binds specifically to the D1 protein of PSII and blocks electron flow to QB. DCMU is a very effective herbicide because it inhibits photosynthesis completely. As a consequence, signal rise to Fm is virtually instantaneous, and fluorescence emission stays high.

Variation in strength of a fluorescence signal from F0 to Fm is also called variable fluorescence (Fv) because scale and kinetics of this rise are significantly influenced by all manner of environmental conditions. F0 plus Fv constitute the maximal fluorescence (Fm) a leaf can express within a given measuring system. The Fv/Fm ratio, measured after dark treatment, therefore reflects the proportion of efficiently working PSII units among the total PSII population. Hence it is a measure of the photochemical efficiency of a leaf, and correlates well with other measures of photosynthetic effectiveness (discussed further in Chapter 12).

(b)  Fluorescence relaxation kinetics

Excellent fluorometers for use in laboratory and field such as the Plant Efficiency Analyser (Hansatech, King’s Lynn, UK) make accurate measurements of all the indices of the Kautsky curve and yield rapid information about photochemical capacity and response to environmental stress.

Even more sophisticated is the Pulse Amplitude Modulated (PAM) fluorometer (Walz, Effeltrich, Germany) which employs a number of fluorescence- and/or photosynthesis-activating light beams and probes fluorescence status and quenching properties. With induction kinetics generated by conventional fluorometers (e.g. Figure 1.12) a given source of weak light (commonly a red light-emitting diode producing only 50–100 µmol quanta m–2 s–1) is used for both chlorophyll excitation and as a source of light for photosynthetic reactions. By contrast a PAM fluorometer measures fluorescence excited by a weak modulated light and applies pulses of saturating light for chlorophyll excitation on top of an actinic beam which sustains photosynthesis. A combination of optical filters plus sophisticated electronics ensures that detection of fluorescence emission is locked exclusively onto the modulated signal. In this way, most of the continuous background fluorescence and reflected long-wavelength light is disregarded. The functional condition of PSII in actively photosynthesising leaf tissue is thus amenable to analysis. This instrument also reveals the relative contributions to total fluorescence quenching by photochemical and non-photochemical processes and will help assess any sustained loss of quantum efficiency in PSII. Photosynthetic electron transport rates can be calculated concurrently.

Photochemical quenching (qp) varies according to the oxidation state of electron acceptors on the donor side of PSII. When QA is oxidised (e.g. subsequent to dark pretreatment), quenching is maximised. Equally, qp can be totally eliminated by a saturating pulse of excitation light that reduces QA, so that fluorescence yield will be maximised, as in a PAM fluorometer. Concurrently, a strong beam of actinic light drives photosynthesis (maintaining linear electron flow) and sustaining a pH gradient across thylakoid membranes for ATP synthesis. Those events are a prelude to energy utilisation and contribute to non-photochemical quenching (qn). This qn component can be inferred from a combination of induction plus relaxation kinetics.


Figure 1.13 Induction and relaxation kinetics of in vivo Chl a fluorescence from a well-nourished radish leaf (Raphanus sativus) supplied with a photon irradiance of actinic light at 500 µmol quanta m-2 s-1 and subjected to a saturating pulse of 9000 µmol quanta m-2 s-1 for 0.8 s every 10 s. Output signal was normalised to 1.0 around the value for Fm following 30 min dark pretreatment. Modulated light photon irradiance was <1 µmol quanta m-2 s-1. See text for definition of symbols and interpretation of kinetics . (Original (unpublished) data from John Evans generated on a PAM fluorometer (Heinz Walz GmbH, Germany))

In Figure 1.13, a previously darkened radish leaf (traps now open and QA oxidised) initially receives weak modulated light (<1 µmol quanta m–2 s–1) that is insufficient to close traps but sufficient to establish a base line for constant yield fluorescence (F0). This value will be used in subsequent calculations of fluorescence indices. The leaf is then pulsed with a brief (0.8 s) saturating flash (9000 µmol quanta m–2 s–1) to measure Fm. Pulses follow at 10 s intervals to measure Fm9. Actinic light (500 µmol quanta m–2 s–1) starts with the second pulse and DpH starts to build up in response to photosynthetic electron flow. Photosynthetic energy transduction comes to equilibrium with these conditions after a minute or so, and fluorescence indices qn and qp can then be calculated as follows:

Under these steady-state conditions, saturating pulses of excitation energy are being used to probe the functional state of PSII, and by eliminating qp the quantum efficiency of light-energy conversion by PSII (ΦPSII) can be inferred:

If overall quantum efficiency for O2 evolution is taken as 10 (discussed earlier), then the rate of O2 evolution by this radish leaf will be:

In summary, chlorophyll fluorescence at ambient temperature comes mainly from PSII. This photosystem helps to control overall quantum efficiency of electron flow and its functionality changes according to environmental and internal controls. In response to establishment of a ΔpH across thylakoid membranes, and particularly when irradiance exceeds saturation levels, some PSII units become down-regulated, that is, they change from very efficient photochemical energy converters into very effective energy wasters or dissipators (Chapter 12). Large amounts of the carotenoid pigment zeaxanthin in LHCII ensure harmless dissipation of this energy as heat (other mechanisms may also contribute). PSII also responds to feedback from carbon metabolism and other energy-consuming reactions in chloroplasts, and while variation in pool size of phosphorylated intermediates has been implicated, these mechanisms are not yet understood.