Reflections on “Chlorophyll fluorescence – a practical guide”

Philip White talks to Giles Johnson and finds out that what some people call a waste of time, other people call a citation classic.

Contemporary Citation Classic: Maxwell K and Johnson GN (2000) Chlorophyll fluorescence – a practical guide. Journal of Experimental Botany 51, 659-668. https://doi.org/10.1093/jxb/51.345.659

Philip White writes: Recently, I compiled a list of Contemporary Citation Classics in Plant Sciences and promised to investigate the historical context of these papers, the authors’ motivations in writing them, and why the authors believed their paper had become so well cited. I posed these questions to Giles Johnson (University of Manchester, UK) about the seminal paper he published with Kate Maxwell in 2000. This paper was ranked #4 in my list of Contemporary Citation Classics and has been cited more than six thousand times according to Google Scholar. Here are his reflections on the paper.


It started, for me at least, at the boozy end of a conference (at the University of Leicester, I think). What do academics do when they get together in a bar? They moan.

My co-author Kate Maxwell and I were both early career scientists. Kate was a Royal Society research fellow at the University of Newcastle. I was a relatively newly appointed lecturer at the University of Manchester. We were both learning the hard way the challenges of transitioning from post-doc to PI. As a post-doc, you are busy, but you have focus. You have a project, you carry it out. As a young PI, you still have (at least one) project and you still carry it out. But you are also suddenly expected to manage other projects, write grant applications, teach, attend meetings and, above all else, train students. Training young scientists is possibly the most fun I have on a weekly basis, but it is time-consuming and to do it to the best of your ability can take more time than you routinely have.

A fluorescent leaf

So, Kate and I were moaning. In this case, we were moaning about the time it takes to teach students the basics of the technique of chlorophyll fluorescence. Fluorescence had become an amazingly important and widely used technique in plant physiology. Starting with the pioneering work of Kautsky and Hirsch (1931), who showed that fluorescence yield varies depending on conditions, fluorescence analysis had developed to a stage where it was becoming accessible to anyone. Important breakthroughs such as the development of the “light doubling” technique (Bradbury and Baker, 1981) and the introduction of modulated measuring systems (Quick and Horton, 1984) made it possible to deconvolute the signals detected from leaves and to infer information about their photosynthetic performance. The introduction of new ways of analysing data, especially the intuitive parameter we called at the time the “Genty factor” FPSII (Genty et al., 1989), provided parameters that were easy to understand and explain conceptually. Technological developments, especially driven by Ulrich Schreiber and his collaborators at Heinz Walz, had led to the availability of easy to use and reliable instruments. The introduction, in 1991, of the Walz PAM-2000 had brought chlorophyll fluorescence to the field.

To fully understand chlorophyll fluorescence, it is not essential to understand all the background theory behind it, but it helps. When I started my PhD in Peter Horton’s lab in Sheffield, I had a wonderful post-doc Debbie Rees (now a Reader of Plant Physiology at the University of Greenwich) who was willing to dedicate hours talking me through it. I also spent further days of my own time going back to the basics, deriving for myself the equations of Butler’s bipartite model (Butler, 1984). I also made use of the excellent, if rather technical, classic review by Krause and Weis (Krause and Weis, 1991). Along the way, I got totally absorbed and sometimes excited; for example, when I derived a new way of analysing fluorescence data to estimate non-photochemical quenching, an approach which was published only a few weeks later by Bilger and Björkman (1990). Understanding the theory is important in being able to understand the strengths and limitations of the technique but not everyone has the time or support needed to examine this is such depth.

So, back to the bar. Both Kate and I knew the frustration of not having a simple introduction to give to our students. We were also both spending a lot of our time refereeing papers in which there were glaring errors in the application of fluorescence. Cheap and easy to use machines were being bought by labs with no background in the technique and with little support in how to use them. I think Kate had already had the idea that a simple introduction was needed to the technique and was looking for a writing partner. The meeting was being supported by a special issue of Journal of Experimental Botany and was looking for contributions. The beer was flowing, so I agreed to come in and to provide the more theoretical parts of the paper, while Kate contributed the parts on applications. Our experience and interests were complimentary, with me being more lab-based and she a field scientist. We knew there was a genuine need for this paper (a point that was sadly missed by one of the referees of the paper, who thought it a waste of time) and we thought it would be popular. We drank a toast to our “citation classic”.

The title was suggested later by Howard Griffiths. The paper took some time to come to fruition and missed publication in the JxB special issue. As a result, it was published later and we were actually paid for writing it! The paper is now somewhat out of date. Several new parameters have since been added to the pantheon of analysis including for example qL introduced by David Kramer, another citation classic on the list (Kramer et al., 2004), and a number of excellent new reviews have appeared (e.g. Murchie and Lawson, 2013). Nevertheless, our paper remains widely used and cited, probably, I think, because it gives exactly what it promises: a practical guide.

Further reading

Bilger, W., & Björkman, O. (1990). Role of the xanthophyll cycle in photoprotection elucidated by measurements of light-induced absorbance changes, fluorescence and photosynthesis in leaves of Hedera canariensis. Photosynthesis Research, 25(3), 173–185. https://doi.org/10.1007/BF00033159

Bradbury, M., & Baker, N. R. (1981). Analysis of the slow phases of the in vivo chlorophyll fluorescence induction curve. Changes in the redox state of Photosystem II electron acceptors and fluorescence emission from Photosystems I and II. Biochimica et Biophysica Acta (BBA) – Bioenergetics, 635(3), 542–551. https://doi.org/10.1016/0005-2728(81)90113-4

Butler, W. L. (1984). Exciton Transfer Out of Open Photosystem II Reaction Centers. Photochemistry and Photobiology, 40(4), 513–518. https://doi.org/10.1111/j.1751-1097.1984.tb04626.x

Genty, B., Briantais, J.-M., & Baker, N. R. (1989). The relationship between the quantum yield of photosynthetic electron transport and quenching of chlorophyll fluorescence. Biochimica et Biophysica Acta (BBA) – General Subjects, 990(1), 87–92. https://doi.org/10.1016/S0304-4165(89)80016-9

Kautsky, H., & Hirsch, A. (1931). Neue Versuche zurKohlensäureassimilation. Die Naturwissenschaften, 19(48), 964–964. https://doi.org/10.1007/BF01516164

Kramer, D. M., Johnson, G., Kiirats, O., & Edwards, G. E. (2004). New Fluorescence Parameters for the Determination of QARedox State and Excitation Energy Fluxes. Photosynthesis Research, 79(2), 209–218. https://doi.org/10.1023/B:PRES.0000015391.99477.0d

Krause, G. H., & Weis, E. (1991). Chlorophyll Fluorescence and Photosynthesis: The Basics. Annual Review of Plant Physiology and Plant Molecular Biology, 42(1), 313–349. https://doi.org/10.1146/annurev.pp.42.060191.001525

Maxwell, K., & Johnson, G. N. (2000). Chlorophyll fluorescence—a practical guide. Journal of Experimental Botany, 51(345), 659–668. https://doi.org/10.1093/jxb/51.345.659

Murchie, E. H., & Lawson, T. (2013). Chlorophyll fluorescence analysis: a guide to good practice and understanding some new applications. Journal of Experimental Botany, 64(13), 3983–3998. https://doi.org/10.1093/jxb/ert208

Quick, W.P., Horton, P. (1984), Studies on the induction of chlorophyll fluorescence in barley protoplasts. I. Factors affecting the observation of oscillations in the yield of chlorophyll fluorescence and the rate of oxygen evolution. Proceedings of the Royal Society of London. Series B. Biological Sciences, 220(1220), 361–370. https://doi.org/10.1098/rspb.1984.0006