Indirect measurement chlorophyll fluorescence

An alternative to measurement of radiocarbon fixation or oxygen evolution for determining photosynthetic rate is to use measurement of what is referred to as the variable component of chlorophyll fluorescence. Since this has become in recent years a commonly used procedure, a simplified outline of what are believed to be the underlying biochemical processes is presented here. More detailed discussions of this complex topic may be found in Butler (1978), Kolber and Falkowski (1993), Schreiber et al. (1995), Kromkamp and Forster (2003), Suggett et al. (2003) and Falkowski and Raven (2007).

We have already noted that all oxygenic photosynthetic organisms when illuminated, emit some light in the red, with a peak at ^685 nm, as fluorescence at low yield (~1%) from chlorophyll a. Evidence from fluorescence excitation spectra, and action spectra for O2 evolution, together with studies on biochemically fractionated photosynthetic systems, have led to the conclusion that this originates almost entirely from photosystem II. An early indication that fluorescence could be used to measure photosynthesis was the observation by Samuelsson and Oquist (1977) that in cultures of four species of unicellular green algae the fluorescence increase induced by the herbicide, DCMU, which blocks electron flow from photosystem II, correlated closely with the rate of photosynthesis, as measured with H14CO3.

The fluorescence yield of phytoplankton cells, or macrophyte tissue, can be measured using very low levels of activating light, which do not in themselves bring about significant photosynthesis. When the cells or tissue are left for some time in the dark, and then fluorescence is measured in this way, it is found to occur at a low value, commonly indicated with the symbol, F0. If the light intensity is now suddenly increased, so that measureable photosynthesis ensues, or if a short-duration high-intensity (saturating) flash is given, then within a very short interval - ~ 200 milliseconds in leaves and isolated chloroplasts but typically much less in phytoplankton suspensions - the fluorescence emission quickly rises and levels off at a higher value in the region of times F0. This is often referred to as the Kautsky effect, after its 1930s discoverer. The high level of chlorophyll fluorescence is found to be still present if it is measured, once again with a low-intensity activating beam, immediately after switching off the light. In continued darkness, photosynthetic cells quickly (within a millisecond) revert to the much lower F0 fluorescence (when measured with a low-intensity measuring beam).

Any process that lessens the intensity of fluorescence from a system is said to quench that fluorescence. The rapid return of the cells to the F0 state in the dark means that during this dark adaptation something is produced that quenches the fluorescence of chlorophyll a in photosystem II. Duysens and Sweers (1963) proposed that it is the primary electron acceptor of photosystem II, now believed to be QA, a protein-bound form of plastoquinone, which in its oxidized form is the quencher. The reduced version of QA (which, since it has acquired a single electron, we can indicate by QA—), formed by photochemical electron transfer within photosystem II when the light returns, is assumed not to be a quencher, so that fluorescence rises. If, after dark adaptation, the cells are illuminated with continuous light of intensity sufficient to bring about photosynthesis, then the steady-state level of fluorescence (F) exhibited increases with increasing irradiance up to a maximum value (Fm), which is believed to correspond to essentially all of the QA being in the reduced (non-quenching) form. This maximum increase above the dark-adapted level, (Fm-F0), is a measure of the variable component of chlorophyll fluorescence, and is indicated by Fv.

If the progressive increase in fluorescence with increasing ambient light does indeed correspond to the increasingly reduced state of QA, then we may plausibly suppose that at any given light intensity the increase in fluorescence above the dark-adapted value (F-F0), divided by the maximum possible (high light) increase above the dark-adapted value (Fm -F0) corresponds approximately to the proportion of photosystem II reaction centres that contain QA—. Or, if we prefer to think in terms of the extent to which the fluorescence at a given light intensity falls short of the maximum possible fluorescence, we can make the equivalent supposition that (Fm - F) divided by (Fm -F0) is approximately equal to the proportion of photosystem II centres in which QA still exists in the oxidized (quenching) form: this quantity is usually indicated by qP.

Since (Fm - F0) corresponds to the maximum amount by which chlorophyll fluorescence can vary it is, as noted above, indicated by Fv.

A photosystem II in which the reaction centre chlorophyll is already oxidized (i.e. is in the form P(|80), and the protein-bound plastoquinone is already reduced (QA_), cannot make use of any subsequent excitation to achieve photochemical charge separation, and is said to be 'closed', i.e. is not currently available for photosynthetic electron transport. As soon as, in the light, P(|80 once again becomes reduced and QA~ is re-oxidized, another photochemical electron transfer can take place, and the reaction centre is said to be 'open'. Thus, as an alternative to discussing the proportion of photosystem II reaction centres in which QA exists in the oxidized or reduced form, we can refer to the proportion that are open or closed, and this is in fact the common terminology in the literature. qP is thus the proportion of photosystem II centres that are open.

For cells photosynthesizing at a given light intensity, the overall efficiency with which photosystem II is working at any given time is equal to the steady-state proportion of reaction centres that are open (qP) multiplied by the efficiency of those reaction centres that are open.445 On the basis of a particular model of energy transfer within the photosystem II photosynthetic unit, Butler and Kitajima (1975) showed that the maximum yield of photochemistry (the number of electrons produced by a single charge separation event in photosystem II, per photon absorbed), i.e. the yield when all photosystem II centres are open, is equal to FvjFm Genty et al. (1989) pointed out that since, when some but not all of the photosystem II centres are open, the quantum yield of photosystem II electron transport (fPS2) is equal to the maximum yield (FvjFm) multiplied by the proportion of centres that are open (qP), then we can write

Fm and therefore, from eqn 10.1 and Fv = (Fm - F0), it follows that

Fm where DF = (Fm - F), the amount by which the steady-state fluorescence falls short of the maximum fluorescence. The advantage of this formulation is that it makes it possible to estimate the photosystem II quantum yield without determining the difficult-to-measure quantity, F0. In agreement with the theory, Genty et al., using leaves of a number of terrestrial higher plant species, found that DF/Fm was linearly related to the quantum efficiency of CO2 fixation over a range of light intensities.

In continuous light, particularly at high intensity, the maximum fluorescence emission often settles down to a lower value (F'm) than that measured shortly after a long dark period (Fm). Similarly, the minimum fluorescence, as measured in the dark, is often at a lower level (F00) after the cells have been exposed for some time to high irradiance. It is thus apparent that prolonged high irradiance causes some additional quenching, which, since it is believed not to involve the reaction centre quencher, QA-, is referred to as non-photochemical quenching. There are two main kinds of mechanism by which non-photochemical quenching can occur. One is a reduction in the effective absorption cross-section ofphotosystem II due to a change in its carotenoid composition in the xanthophyll cycle (see later): the reaction centre in this case still functions but has a lower probability of capturing excitation energy. The other is due to reaction centres being rendered non-functional by radiation-induced damage.

Following the approach in the review by Kromkamp and Forster (2003), we can, as a simple initial version of the relationship between photosynthetic rate and fluorescence, write

where ETR is the electron transport rate (mol electrons mg chl a-1 s-1), E is irradiance (mol quanta m-2s-1), a* is the absorption cross-section (m2 mg chl a-1, equivalent to the chlorophyll-specific absorption coefficient of the phytoplankton, §9.4), the 0.5 factor arises from the simplifying assumption that the absorbed light is distributed equally between the two photosystems, and DF/Fm is the quantum efficiency of photosystem II (electrons produced per photon absorbed). To avoid the assumption that photosystem II and photosystem I absorb light at the same rate, we can replace this equation with

where a*PSII is the absorption cross-section of a photosystem II unit (m2 mol photosystem II reaction centres-1) and nPSII is the number of photosystem II units per mg chl a (mol photosystem II mg chl a-1).

If a plausible value for the electron yield of oxygen evolution (Oe, moles of O2 produced per mole of electrons removed from water in photosystem II) can be assumed (ideally Oe = 0.25, 4 electrons per O2 evolved), then the chlorophyll-specific rate of photosynthetic oxygen evolution (mol O2 mg chl a-1 s-1) can be obtained from the fluorescence-derived ETR

For the Kolber and Falkowski (1993) 'pump and probe' fluorescence procedure (see below) the appropriate relationship for P* (O2) is

where sPSII is the functional absorption cross-section of photosystem II (the f factor present in the original Kolber and Falkowski equation has been omitted).753 aPSII can be determined from the flash-intensity saturation curve of variable fluorescence, on the assumption that this corres-


ponds to a cumulative one-hit Poisson function. ,

Experimentally there are two different approaches to the measurement of photosynthesis rates using fluorescence, the single turnover (ST) and the multiple turnover (MT). In the ST method the cells are exposed to an intense but very short (150-400 microseconds) light flash, which fully reduces QA, the primary electron acceptor of photosystem II, and thus brings about a simultaneous single closure event of all photosystem II reaction centres.739,1320,753 In the MT method, a flash of lower intensity but much longer duration (50-1200 milliseconds) is given, which fully reduces not only QA, but also QB and the plastoquinone pool. In the Kolber and Falkowski (1993) 'pump and probe' (ST) method, the fluorometer generates a three-flash sequence: a weak 'probe' flash to measure the initial fluorescence signal, followed after a 500 ms delay by an actinic 'pump' flash of ^200 times greater intensity to bring about the single turnover event, followed after a 70 ms delay by a second 'probe' flash to measure the resultant change in fluorescence. In a further development of this technique, Kolber et al. (1998) use fast repetition rate (FRR) fluorometry to achieve the same effect but with greater flexibility. Instead of a single flash the cells are exposed to a sequence of 80 to 120 'flashlets' of 0.125 to 1.0 ms duration at 0.5 to 2.0 ms intervals.

The MT method makes use of what is referred to as pulse amplitude modulation (PAM) fluorometry, developed by Schreiber et al. (1986, 1993, 1995). Fluorescence is monitored continuously with weak ms light pulses at a frequency selectable at 1.6 to 100 KHz. Actinic (MT) pulses of longer duration, as above, are given at specified intervals, e.g. 16 Hz. The modulated nature of the light used to activate fluorescence makes it possible to detect the resulting modulated fluorescence signal against a large background of continuous signals from the actinic illumination (stray light and fluorescence). Instruments for studying photosynthesis using FRR or PAM fluorometry are now commercially available, for submersible or laboratory operation. They do not, however, give identical results since they do not measure precisely the same quantity. The fluorescence measured with an MT flash is, for example, somewhat larger than that achieved with an ST flash. For estimating photosynthetic rate in benthic organisms, Gorbunov et al. (2000) have developed an underwater FRR fluorometer, which can be hand held by a scuba diver or permanently moored, and which is pointed at whatever part of the benthos is under study.

Fluorescence-based measurements of photosynthesis are quick and convenient to use, but in aquatic systems do not always show such reliably linear relationships between the fluorescence-derived and the directly measured 14C, O2, CO2 values as those obtained by Genty et al. (1989) with terrestrial higher plant leaves. In a natural bloom of freshwater cyanobacteria, Masojidek et al. (2001), using PAM fluorome-try, found that the relationship between fluorescence-derived ETR and photosynthetic O2 evolution was linear at irradiance values up to 800 mmole quantam~2s_1, but non-linear at higher irradiances. Similarly, using cultures of the green alga, Dunaliella tertiolecta, Fujiki et al. (2007) concluded that FRR fluorometry can be used as a good indicator of photosynthetic rates from low to middle light levels, but becomes increasingly questionable as the maximum photosynthetic rate is approached. In species of green, brown and red macroalgae, Beer and Axelsson (2004) found a clear positive correlation between O2 evolution and fluorescence-based ETR at low irradiances, but at high irradiance there was a decrease in ETR while O2 evolution remained relatively constant. Smyth et al. (2004) compared FRR fluorometry and 14C incorporation measurements of phytoplankton photosynthesis in the Celtic Sea. The light-saturated photosynthetic rates estimated by fluorometry were consistent with those measured using radiocarbon, but there were systematic differences when the estimates of the initial slope (a) and the saturation onset parameter (Ek, see below for definitions) of the photosynthesis versus irradiance curve were compared. On the other hand, Kolber and Falkowski (1993) found a linear correlation (r = 0.86) between the fluorescence and the 14C estimates of integral primary production in the western North Atlantic. Suggett et al. (2003) express the view that while the PAM and FRR approaches can make a major contribution to understanding variability of photosynthesis in aquatic systems, whether these techniques can improve estimates of primary productivity remains an open question. Until we arrive at a better understanding of the underlying fundamental processes that are controlling fluorescence emission at the molecular level, the data must be interpreted with caution.

In all the work described above, the fluorescence emission studied has been activated by artificial light supplied by the experimenter. Some studies have also been carried out to determine to what extent information on photosynthetic rates can be obtained by in situ measurements of the natural, solar-stimulated fluorescence of phytoplankton at ^683 nm. On the basis of measurements at 76 stations, from oligotrophic oceanic to productive inshore waters, Chamberlin et al. (1990) found that photosynthetic rate (14C, in situ) was highly correlated with natural fluorescence emission. At higher irradiance values the quantum yield of photosynthesis decreased more rapidly than the quantum yield of fluorescence. In addition, Chamberlin and Marra (1992), using data from the North Atlantic and the Weddell-Scotia Seas (Antarctica) found that at lower temperatures the ratio of quantum yield of photosynthesis to that of fluorescence decreased, there being an approximately linear relationship between the ratio of quantum yields and temperature. On the basis of their measurements they arrived at a relationship expressing photosyn-thetic rate as a function of the temperature, scalar irradiance of PAR, and fluorescence emission, together with certain empirically determined constants. A log-log plot of measured versus predicted photosynthesis exhibited approximate linearity for stations covering a wide range of productivity. For Mexican coastal waters (California Current and Gulf of Mexico), Garcia-Mendoza and Maske (1996) found natural fluorescence data to be useful as a proxy of primary production (r2 = 0.85) for volumetric rates of photosynthesis up to ^300 nmol C m~3 s_1.

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