Estimated
Abstract
Fluorescence-based methodologies are commonly employed to determine a wide spectrum of physiological parameters in intact photosynthetic organisms. These methods rely on the detection of Chlorophyll a fluorescent emission, which exhibits changes in its intensity due to the occurrence of quenching phenomena of either photochemical or non-photochemical nature, as well as in response of the absorption cross-section of the photosystems. At room temperature, it is generally considered that most of the emission stems from Photosystem II, and therefore, most of the physiological parameters rely on the assumption that contribution from Photosystem I is negligible. Moreover, it is often considered that the whole light-harvesting antenna is efficiently coupled to either of the photosystems and does not contribute, independently, to the detected emission. When these caveats are not realised, fluorescence-based indicators might be subjected to biases that tend to underestimate the extent of both photochemical and non-photochemical quenching. The contribution of Photosystem I and partially coupled/antenna components can be assessed through the analysis of the dependency of steady-state emission as a function of both the excitation and the emission wavelengths. On this basis, methods relying on using different combinations of excitation and emission wavelengths will be discussed in order to minimise the bias on the estimation of physiologically relevant parameters.
Keywords
- chlorophyll fluorescence
- maximal photochemical efficiency
- photochemical quenching
- non-photochemical quenching
- Photosystem II
- Photosystem I
- light harvesting complexes
- phycobilisomes
1. Introduction
Methodologies relying on the monitoring of Chlorophyll (Chl)
The most widely employed physiological measurements based on chlorophyll fluorescence detection are the determination of fluorescence quenching processes resulting either from photochemical reactions (photochemical quenching), or regulatory processes affecting the fluorescence emission yield, collectively referred to as non-photochemical quenching (NPQ).
1.1 Photochemical quenching
Photochemical quenching is the process through which the emission yield of protein-bound Chl is decreased in response to energy conversion reactions occurring in photochemically active pigments, the reaction centre (RC), of a photosystem. Historically, the occurrence of fluorescence quenching as a response to the redox state of the terminal electron acceptors,
The maximal fluorescence level can be promoted either by brief, intense, light pulses, or flashes that cause the almost complete reduction of PSII acceptor pool (saturating pulses or flashes), so that
The conclusive demonstration that the differences in the emission intensity under
It can then be straightforwardly demonstrated, and it is therefore commonly accepted, that the maximal photochemical quantum efficiency of PSII,
where
Although the above-presented derivation presents the yield in the form of the so-called “lake model”, in which the quenching induced by photochemistry is shared over a large pool of photosystems, thereby approaching the classic Stern-Volmer description, for the
1.2 Non-photochemical quenching
On top of photochemical quenching processes, fluorescence monitoring finds its natural application in the study of regulative mechanisms of light harvesting efficiency, which manifest themselves principally as a quenching of the Chl fluorescence emission. The establishment of non-photochemical quenching (NPQ) results in the lowering of the
where
NPQ has proven to be a very complex phenomenon, showing several phases of development, upon light exposure, and relaxation, upon return to darkness [15, 16], that despite the generality of its manifestation, depends strongly on the organism or at least the class of organisms in which it is investigated [17, 18]. This large variability is likely due to the generally accepted knowledge that NPQ processes occur principally, when not exclusively, in the external antenna complement of PSII [15, 16, 17, 18]. Whereas the core complex, which serves as an internal light harvesting system as well as the photochemical reaction centre, is well conserved amongst different oxygenic photosynthetic organisms [19], the composition of the external antenna is instead very diversified, having evolved to optimise light harvesting in ecological niches possessing different spectral distributions either in terms of light spectrum or light intensity [20, 21]. Hence, it is not surprising that also adaptive responses to changes in light conditions vary significantly in different organisms.
1.3 Source of possible distortion due to emission from Photosystem I and decoupled light-harvesting antenna
One of the caveats behind Chl-based fluorescence indicators is the assumption that the monitored fluorescence is emitted exclusively or almost exclusively from PSII. This is, in general, a justified assumption since, particularly under closed centres conditions, and at a room temperature or temperatures covering the “physiological interval” of living organisms, the fluorescence yield of PSII could be several folds larger than the one of the other ubiquitous component of the electron transfer chain, Photosystem I (PSI). Measurements on PSI isolated from different species indicate that its average fluorescence lifetime is in the order of 20–40 ps [19, 22, 23], that is, 5–10 times faster than for the PSII-antenna supercomplex at open centres and 200–500 faster at closed centres, in the absence of non-photochemical quenching [6, 7, 8, 9, 10, 11]. Similar results concerning PSI lifetimes are also retrieved for measurements when the photosystem is embedded in the thylakoid membranes [8, 9, 24]. Moreover, PSI does not show a significant change in emission yield upon oxidisation of the terminal donor,
Another possible source of distortion in the determination of these fluorescence levels is the contribution from populations of the external antenna not coupled in terms of energy transfer to the core of either PSII or PSI. Differently from PSI, the core-decoupled pigment-protein complexes of the antenna are very bright in terms of fluorescence, emitting with lifetimes comparable or longer than PSII at closed centres. Typically, the population of decoupled antenna tends to be vanishingly small in most organisms having transmembrane light-harvesting apparatus [27, 29], but it can be sizable in cyanobacteria, accounting for 1–5% of the external antenna [26, 30, 31] and therefore clearly contributing to the cellular emission [26, 27, 28, 32, 33, 34]. Thus, differences between organisms, which harbour diversified external antenna complements, are to be expected.
2. Spectral dependence of the F V/F M ratio
A strategy to verify the distortion originating from the emission due to either PSI or a fraction of decoupled/weakly coupled external antenna components to the determination of the
In the successive paragraphs, experiments performed in model green algae (
Conceptually, these types of experiments are pretty simple, but technically they might be challenging as they require a rather sensitive apparatus in order to collect
2.1 Excitation and emission dependency in model green algae
The emission spectra recorded under
![](http://cdnintech.com/media/chapter/73185/1512345123/media/F1.png)
Figure 1.
Emission spectra recorded under F0' (black lines) and
For ease of comparison, the spectra, at each excitation wavelength, are normalised to the maximum of the
In Figure 2 are shown the
![](http://cdnintech.com/media/chapter/73185/1512345123/media/F2.png)
Figure 2.
Emission wavelength dependency of the
A strategy to extract the
![](http://cdnintech.com/media/chapter/73185/1512345123/media/F3.png)
Figure 3.
Comparison of the normalised
Figure 3 also shows a comparison for the normalised
To better compare the effect of excitation and detection wavelengths in determining the
Excitation | 435 nm | 475 nm | 520 nm | 570 nm | ||||
Interferential (Centre, nm) | ||||||||
650 | 0.302 | 0.698 | 0.325 | 0.675 | 0.310 | 0.690 | 0.319 | 0.681 |
670 | 0.292 | 0.708 | 0.312 | 0.688 | 0.298 | 0.702 | 0.311 | 0.689 |
680 | 0.292 | 0.708 | 0.309 | 0.691 | 0.293 | 0.707 | 0.308 | 0.692 |
690 | 0.304 | 0.696 | 0.315 | 0.685 | 0.305 | 0.695 | 0.319 | 0.681 |
700 | 0.327 | 0.673 | 0.330 | 0.670 | 0.342 | 0.658 | 0.353 | 0.647 |
720 | 0.347 | 0.653 | 0.349 | 0.651 | 0.366 | 0.634 | 0.370 | 0.630 |
Bandpass (cut-on, nm) | ||||||||
655 | 0.314 | 0.686 | 0.325 | 0.675 | 0.323 | 0.677 | 0.334 | 0.666 |
675 | 0.317 | 0.683 | 0.326 | 0.674 | 0.326 | 0.674 | 0.336 | 0.664 |
695 | 0.331 | 0.669 | 0.336 | 0.664 | 0.347 | 0.653 | 0.354 | 0.646 |
720 | 0.327 | 0.673 | 0.335 | 0.665 | 0.341 | 0.659 | 0.348 | 0.652 |
Inteferential (Centre, nm) | ||||||||
650 | 0.343 | 0.657 | 0.365 | 0.635 | 0.348 | 0.652 | 0.371 | 0.629 |
670 | 0.336 | 0.664 | 0.354 | 0.646 | 0.341 | 0.659 | 0.352 | 0.648 |
680 | 0.328 | 0.672 | 0.346 | 0.654 | 0.339 | 0.661 | 0.346 | 0.654 |
690 | 0.330 | 0.670 | 0.353 | 0.647 | 0.350 | 0.650 | 0.355 | 0.645 |
700 | 0.350 | 0.650 | 0.377 | 0.623 | 0.375 | 0.625 | 0.381 | 0.619 |
720 | 0.352 | 0.648 | 0.377 | 0.623 | 0.372 | 0.628 | 0.381 | 0.619 |
Bandpass (Cut-on, nm) | ||||||||
655 | 0.337 | 0.663 | 0.359 | 0.641 | 0.353 | 0.647 | 0.361 | 0.639 |
675 | 0.337 | 0.663 | 0.360 | 0.640 | 0.355 | 0.645 | 0.362 | 0.638 |
695 | 0.345 | 0.655 | 0.369 | 0.631 | 0.365 | 0.635 | 0.374 | 0.626 |
720 | 0.340 | 0.660 | 0.361 | 0.639 | 0.357 | 0.643 | 0.367 | 0.633 |
Table 1.
Values of
2.2 Excitation and emission dependency in model cyanobacteria
The emission spectra recorded under
![](http://cdnintech.com/media/chapter/73185/1512345123/media/F4.png)
Figure 4.
Emission spectra recorded under
This is further highlighted in Figure 5, where the
![](http://cdnintech.com/media/chapter/73185/1512345123/media/F5.png)
Figure 5.
Emission wavelength dependency of the
Furthermore, the
However, the bandshape of the
![](http://cdnintech.com/media/chapter/73185/1512345123/media/F6.png)
Figure 6.
Comparison of the normalised
In Figure 6 are also shown the normalised
To further highlight the importance of measurement conditions on the determination of the
Excitation | 435 nm | 475 nm | 520 nm | 570 nm | ||||
Interferential (Centre, nm) | ||||||||
650 | 0.633 | 0.367 | 0.802 | 0.198 | 0.917 | 0.083 | 0.927 | 0.073 |
670 | 0.454 | 0.546 | 0.614 | 0.386 | 0.729 | 0.271 | 0.724 | 0.276 |
680 | 0.419 | 0.581 | 0.529 | 0.471 | 0.585 | 0.415 | 0.568 | 0.432 |
690 | 0.467 | 0.533 | 0.552 | 0.448 | 0.584 | 0.416 | 0.571 | 0.429 |
700 | 0.559 | 0.441 | 0.626 | 0.374 | 0.684 | 0.316 | 0.675 | 0.325 |
720 | 0.613 | 0.387 | 0.694 | 0.306 | 0.742 | 0.258 | 0.729 | 0.271 |
Bandpass (cut-on, nm) | ||||||||
655 | 0.504 | 0.496 | 0.612 | 0.388 | 0.688 | 0.312 | 0.683 | 0.317 |
675 | 0.508 | 0.492 | 0.598 | 0.402 | 0.649 | 0.351 | 0.636 | 0.364 |
695 | 0.556 | 0.444 | 0.635 | 0.365 | 0.691 | 0.309 | 0.680 | 0.320 |
720 | 0.534 | 0.466 | 0.608 | 0.392 | 0.675 | 0.325 | 0.663 | 0.337 |
Interferential (Centre, nm) | ||||||||
650 | 0.750 | 0.250 | 0.866 | 0.134 | 0.955 | 0.045 | 0.972 | 0.028 |
670 | 0.487 | 0.513 | 0.678 | 0.322 | 0.775 | 0.225 | 0.803 | 0.197 |
680 | 0.413 | 0.587 | 0.598 | 0.402 | 0.629 | 0.371 | 0.663 | 0.337 |
690 | 0.446 | 0.554 | 0.631 | 0.369 | 0.630 | 0.370 | 0.666 | 0.334 |
700 | 0.530 | 0.470 | 0.710 | 0.290 | 0.729 | 0.271 | 0.766 | 0.234 |
720 | 0.547 | 0.453 | 0.722 | 0.278 | 0.774 | 0.226 | 0.806 | 0.194 |
Bandpass (cut-on, nm) | ||||||||
655 | 0.491 | 0.509 | 0.677 | 0.323 | 0.731 | 0.269 | 0.761 | 0.239 |
675 | 0.479 | 0.521 | 0.663 | 0.337 | 0.691 | 0.309 | 0.720 | 0.280 |
695 | 0.516 | 0.484 | 0.697 | 0.303 | 0.728 | 0.272 | 0.756 | 0.244 |
720 | 0.498 | 0.502 | 0.677 | 0.323 | 0.708 | 0.292 | 0.730 | 0.270 |
Table 2.
Estimated
Values of
2.3 Contribution of different chromophore-pigment super-complexes to photosynthetic membrane emission
The spectral dependencies of
where
which satisfies the lack of excitation wavelength dependency of
Recent reports from our laboratory have demonstrated that
where the absorption cross-section (
In Figure 7 are shown examples of the decomposition of the emission spectra of the green alga
![](http://cdnintech.com/media/chapter/73185/1512345123/media/F7.png)
Figure 7.
Decomposition of the
![](http://cdnintech.com/media/chapter/73185/1512345123/media/F8.png)
Figure 8.
Decomposition of the
In accordance with qualitative data inspection of the spectra, in green algae, the emission spectra are largely dominated by PSII, and, even though the contribution of PSI is not negligible, the one of the uncoupled antenna, fundamentally, is so. The values of
A relatively simple way to compensate for the contribution of the emission from pigment-protein complexes and supercomplexes that do not show variable fluorescence is that of applying a correction to the measured fluorescence levels. This however would in principle require independent evidences that the variable fluorescence spectrum is effectively independent of the excitation wavelength (as shown in Figures 3 and 6) and knowledge of the relative emission contribution of
A list of
Excitation | 435 nm | 475 nm | 520 nm | 570 nm | 435 nm | 475 nm | 520 nm | 570 nm |
Interferential (Centre, nm) | ||||||||
650 | 0.02 | 0.03 | 0.05 | 0.03 | 0.02 | 0.03 | 0.02 | 0.03 |
670 | 0.02 | 0.02 | 0.03 | 0.02 | 0.02 | 0.03 | 0.02 | 0.03 |
680 | 0.02 | 0.01 | 0.02 | 0.02 | 0.01 | 0.02 | 0.02 | 0.02 |
690 | 0.02 | 0.02 | 0.03 | 0.02 | 0.02 | 0.03 | 0.03 | 0.04 |
700 | 0.06 | 0.04 | 0.07 | 0.06 | 0.04 | 0.07 | 0.06 | 0.08 |
720 | 0.09 | 0.07 | 0.11 | 0.09 | 0.04 | 0.07 | 0.06 | 0.08 |
Bandpass (cut-on, nm) | ||||||||
655 | 0.04 | 0.03 | 0.06 | 0.04 | 0.03 | 0.04 | 0.03 | 0.05 |
675 | 0.05 | 0.04 | 0.06 | 0.05 | 0.03 | 0.04 | 0.04 | 0.05 |
695 | 0.07 | 0.05 | 0.09 | 0.07 | 0.04 | 0.06 | 0.05 | 0.06 |
720 | 0.06 | 0.05 | 0.08 | 0.06 | 0.03 | 0.04 | 0.04 | 0.05 |
Excitation | 435 nm | 475 nm | 520 nm | 570 nm | 435 nm | 475 nm | 520 nm | 570 nm |
Interferential (Centre, nm) | ||||||||
650 | 0.10 | 0.89 | 0.83 | 0.83 | 0.62 | 0.79 | 0.93 | 0.95 |
670 | 0.06 | 0.41 | 0.47 | 0.47 | 0.23 | 0.45 | 0.65 | 0.68 |
680 | 0.05 | 0.23 | 0.25 | 0.25 | 0.13 | 0.29 | 0.42 | 0.45 |
690 | 0.08 | 0.27 | 0.25 | 0.25 | 0.15 | 0.33 | 0.42 | 0.44 |
700 | 0.15 | 0.51 | 0.43 | 0.43 | 0.29 | 0.52 | 0.58 | 0.60 |
720 | 0.20 | 0.64 | 0.52 | 0.52 | 0.37 | 0.60 | 0.64 | 0.66 |
Bandpass (Cut-on, nm) | ||||||||
655 | 0.11 | 0.46 | 0.42 | 0.42 | 0.24 | 0.46 | 0.57 | 0.59 |
675 | 0.12 | 0.43 | 0.37 | 0.37 | 0.22 | 0.43 | 0.50 | 0.51 |
695 | 0.16 | 0.58 | 0.44 | 0.44 | 0.28 | 0.51 | 0.55 | 0.56 |
720 | 0.15 | 0.57 | 0.41 | 0.41 | 0.24 | 0.46 | 0.48 | 0.49 |
Table 3.
Correction factors compensating for non-variable fluorescence contributions at
3. Impact of the excitation/emission dependency on the NPQ estimations
Non-photochemical quenching of Chl fluorescence is one of the most intensively investigated regulative processes in oxygenic photosynthesis. As stated in the introduction, NPQ being an excited state quenching process, it is natural that fluorescence detection has been by far the most popular instrument to investigate it, particularly
In land plants, and most green algae, the larger qE component of the NPQ process is dependent on the establishment of large pH gradients across the thylakoid membrane [15, 16, 17, 39]. However, whereas in land-plant, the presence of the PsbS subunit is crucial for a rapid and large NPQ establishment [15, 16, 17, 18, 19, 39, 40], in green algae, this is less fundamental, and the expression of specific light harvesting complexes (known as lhcs) has a more central role [39, 40]. Carotenoids, particularly those of the so-called xanthophyll cycle, also influence either the extent or the kinetics of formation/relaxation of NPQ [15, 16, 17, 19, 39, 40]. In both cases, the main effect is that of promoting the formation of quenching centres within the external antenna matrix of PSII, despite the site of quenching being still a matter of contention. As excited state equilibration within PSII is reached rapidly with respect to the excited state decay at
The mechanisms of NPQ in cyanobacteria are understood in less detail. In these organisms, it has been shown that non-photochemical centres are also located in the external PBS antenna (reviewed in [17, 18, 40]) and that NPQ can occur even in mutants lacking the reaction centres [41, 42]. The induction of NPQ has a pronounced actinic light dependency, whose action spectrum closely matches the absorption of the orange carotenoid protein (OCP), which is a key factor in the sensing and possibly the induction of NPQ in this organism. In this scenario, it should then be considered that non-photochemical quenching could occur in the whole PSII-PBS supercomplex as well as in the uncoupled PBS fraction (as well as in both).
For the purposes of this chapter, a detailed discussion of the mechanisms of NPQ is not required, being the focus on the distortion of the measured Chl-based parameters, which for this process is through the “
It is possible to estimate the extent of the bias originating from the emission of PSI and of the energetically uncoupled antenna fraction in a parameter, which shall reflect changes in the fluorescence yield of PSII, by simulating the cellular emission, starting from the decompositions obtained under measured
Using the same formalism adopted so far, when NPQ occurs in PSII only, the non-photochemically quenched emission at
And when quenching in the uncoupled antenna fraction is necessary,
3.1 Excitation and emission dependency of NPQ estimation in model green algae
The simulated fluorescence emission spectra in
![](http://cdnintech.com/media/chapter/73185/1512345123/media/F9.png)
Figure 9.
Simulations of
The deviations between
An alternative strategy to quantify non-photochemical quenching is via the non-photochemical quenching yield, (
3.2 Excitation and emission dependency of NPQ estimation in model cyanobacteria
Using the same approach described above, the
![](http://cdnintech.com/media/chapter/73185/1512345123/media/F10.png)
Figure 10.
Simulations of
![](http://cdnintech.com/media/chapter/73185/1512345123/media/F11.png)
Figure 11.
Simulations of
It is immediately apparent that, for the simulations of the
The scenario is markedly different when considering quenching in both PSII-PBS and the uncoupled PBS. As shown in Figure 11, quenching is more obvious across a larger portion of the emission bandwidth, and this leads to a closer correspondence between
The non-photochemical quenching yield,
It is in principle possible to correct the measurement parameters, analogously to what is described in Eq. (8), provided the same caveats and even further recommendation of parsimony of use, so that the “corrected” NPQ parameter results:
and the photochemical quenching yield:
4. Conclusions
In this chapter, we discussed the influence of measurement settings on the determination of the two parameters,
Simple expressions for empirical correction of the measured
Acknowledgments
This research was supported by: Fondazione Cariplo through the project “Cyanobacterial Platform Optimised for Bioproductions” (CYAO, ref. 2016-0667); sPATIALS3 project, financed by the European Regional Development Fund under the ROP of the Lombardy Region ERDF 2014-2020 - Axis I “Strengthen technological research, development and innovation” - Action 1.b.1.3 “Support for co-operative R&D activities to develop new sustainable technologies, products and services” - Call Hub; CNR project FOE-2019 DBA.AD003.139, BIO-ECO.
Conflict of interest
The authors declare no conflict of interest.
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