Howdy Stancho,

First Chlrorphyll fluorescence is not a photophysical or photochemical phenomenon, but a photobiological one.

Chlorophyll fluorescence is light re-emitted by chlorophyll molecules (yes

chlorophyll!) during return from excited to non-excited states. It is used as an indicator of photosynthetic energy conversion in higher plants, algae and bacteria. Excited chlorophyll dissipates the absorbed light energy by driving photosynthesis (photochemical energy conversion), as heat in non-photochemical quenching or by emission as fluorescence radiation. As these processes are complementary processes, the analysis of chlorophyll fluorescence is an important tool in plant research with a wide spectra of applications.

The Kautsky effect

Upon illumination of a dark-adapted leaf, there is a rapid rise in fluorescence from Photosystem II (PSII), followed by a slow decline. First observed by Kautsky et al., 1960, this is called the Kautsky Effect. This variable rise in chlorophyll fluorescence rise is due to photosystem II. Fluorescence from photosystem I is not variable, but constant.

The increase in fluorescence is due to PSII reaction centers being in a "closed" or chemically reduced state. Reaction centers are "closed" when unable to accept further electrons. This occurs when electron acceptors downstream of PSII have not yet passed their electrons to a subsequent electron carrier, so are unable to accept another electron. Closed reaction centres reduce the overall photochemical efficiency, and so increases the level of fluorescence. Transferring a leaf from dark into light increases the proportion of closed PSII reaction centres, so fluorescence levels increase for 1–2 seconds. Subsequently, fluorescence decreases over a few minutes. This is due to; 1. more "photochemical quenching" in which electrons are transported away from PSII due to enzymes involved in carbon fixation; and 2. more "non-photochemical quenching" in which more energy is converted to heat.

PSII yield as a measure of photosynthesis

Chlorophyll fluorescence appears to be a measure of photosynthesis, but this is an over-simplification. Fluorescence can measure the efficiency of PSII photochemistry, which can be used to estimate the rate of linear electron transport by multiplying with light intensity. However, researchers generally mean carbon fixation when they refer to photosynthesis. Electron transport and CO2 fixation correlate well, but may not correlate in the field due to processes such as photorespiration, nitrogen metabolism and the Mehler reaction.

Relating electron transport to carbon fixation

A powerful research technique is to simultaneously measure chlorophyll fluorescence and gas exchange to obtain a full picture of the response of plants to their environment. One technique is to simultaneously measure CO2 fixation and PSII photochemistry at different light intensities, in non-photorespiratory conditions. A plot of CO2 fixation and PSII photochemistry indicates the electron requirement per molecule CO2 fixed. From this estimation, the extent of photorespiration may be estimated. This has been used to explore the significance of photorespiration as a photoprotective mechanism during drought.

Fluorescence analysis can also be applied to understanding the effects of low and high temperatures.

• Sobrado (2008) investigated gas exchange and chlorophyll a fluorescence responses to high intensity light, of pioneer species and forest species. Midday leaf gas exchange was measured using a photosynthesis system, which measured net photosynthetic rate, gs, and intercellular CO2 concentration. His results show that despite pioneer species and forest species occupying different habitats, both showed similar vulnerability to midday photoinhibition in sun-exposed leaves.

Measuring stress and stress tolerance

Chlorophyll fluorescence can measure most types of plant stress. Chlorophyll fluorescence can be used as a proxy of plant stress because environmental stresses, e.g. extremes of temperature, light and water availability, can reduce the ability of a plant to metabolise normally. This can mean an imbalance between the absorption of light energy by chlorophyll and the use of energy in photosynthesis.

• Favaretto et al. (2010) investigated adaptation to a strong light environment in pioneer and late successional species, grown under 100% and 10% light. Numerous parameters, including chlorophyll a fluorescence, were measured. Overall, their results show that pioneer species perform better under high-sun light than late- successional species, suggesting that pioneer plants have more potential tolerance to photo-oxidative damage.
• Neocleous and Vasilakakis (2009) investigated the response of raspberry to Boron and salt stress. Leaf chlorophyll fluorescence was not significantly affected by NaCl concentration when Boron concentration was low. When Boron was increased, leaf chlorophyll fluorescence was reduced under saline conditions. They be concluded that the combined effect of Boron and NaCl on raspberries induces a toxic effect in photochemical parameters.
• Lu and Zhang (1999) studied heat stress in wheat plants and found that temperature stability in Photosystem II of water-stressed leaves correlates positively and well, to the resistance in metabolism during photosynthesis.

Nitrogen Balance Index

A portable multiparametric fluorometer using the ratio between chlorophyll and flavonols can be applied to detect nitrogen deficiency in plants Because of the link between chlorophyll content and nitrogen content in leaves, chlorophyll fluorometers can be used to detect nitrogen deficiency in plants, by several methods.

Based on several years of research and experimentation, polyphenols can be assigned as indicators of the nitrogen status of a plant. For instance, when a plant is under optimal conditions, it favours its primary metabolism and synthesises the proteins (nitrogen molecules) containing chlorophyll, and few flavonols (carbon-based secondary compounds). On the other hand, in case of lack of nitrogen, we will observe an increased production of flavonols by the plant.

The NBI (Nitrogen Balance Index), allows the assessment of nitrogen conditions of a plant by calculating the ratio between Chlorophyll and Flavonols (related to Nitrogen/Carbon allocation) .

Hence Stancho,

I hope this short summary elucidates soma aspects of the photobiology of plant chlorophyll fluorescence. A lot more information is available. For example models to simulate plant fluorescence. An interesting one can be found at:

http://www.ipgp.fr/~jacquemoud/publications/pedros2004.pdf

Success with your studies,

Frank

Please refer the textbook "Plant Physiology" by

Taiz & Zeiger.

Vijaya

Dear Dr. Chitnis, Vijaya R. Chitnis

thank you for recommending that textbook to me. Yes, I have the third edition. Unfortunately, Taiz & Zeiger textbook is a very basic rough introduction to basic plant physiology concepts. It is funny because Taiz and Zeiger make mention very quickly about fluorescence and just once, in a so short and flow way, that you cannot learn absolutely nothing about Chlorophyll Fluorescence from that book. I would not recommend Taiz & Zeiger in any serious discussion about Chlorophyll Fluorescence. Many thanks for your willingness to help me.

Dear colleague,

Please refer the attached files, maybe they can help you.

I am sorry, but I am afraid that nobody has understood what exactly the Dr. Pavlov's question is about.

I think that this question is at the very center of really understanding what fluorescence is and where it come from, and math modeling of chl a fluorescence and computer simulations of electron transport events ocurrying at PSII and PSI should already be a good and sharp starting point. Unfortunately, I am afraid that there is not supporting experimental evidence to answer to Dr Pavlov's question about what molecule complex exactly is emitting fluorescence.

Personally, without any hesitation, I would directly answer that the fluorescence that we measure with any handheld fluorometer is emitted by about 300 Chl a molecules packaged in Light harvesting complexes II and PSII complexes. Today, we know the LHCII is shared with PSI. Looks like plants uses LHC's as a kind of structure that can attach and detach from any Photosystem, I or II, and move in between them. The conditions for attaching and detachment are not elucidated, not yet...

Beside that interesting fact, we know a bit more about the role of carotenoids, and something that is for sure: The special Chl pair at the reaction centres are not the ones producing the fluorescence that we sense with a portable fluorometer. My best guess is that most people interested in plant and fluorescence research will answer "Fluorescence is emitted by Chl a molecules in PSII".

It has been published that Chl a fluorescence from PSI is not variable. Its absorption and emission spectra has been obtained in vitro, with destructive methods at low temperatures. However, have you ever seen Chl a fluorescence spectra at room temperature measured in vivo? Have you ever seen any other instrumental techniques measuring in vivo PSI components activity?

The reaction centre in Photosystem II is assumed to be the trigger of fluorescence produced by Chl a molecules in the LHCII-PSII exciton energy funel and the heat emission by those Chl a molecules as well. Upon fast redox states switching while interacting with the Oxygen Evolving System and Pheophytin and Quinone donor-acceptor molecules, the reaction centre special pair of chl molecules act as a kind of relay or switch, that indeed switch on/off the releasing of trapped excitonic energy by Chl a molecules that built the antenna complex and the PSII core.

Anyway, I will try to find out about in vivo experimental evidence of Chl a fluorescence emitted by PSI and come back to this discussion if I succeed to find something. I am a quite aesceptic about explaning fluorescence using fluorescence. I mean, I have nothing against commercial goals and academicians and private companies profit interests to sell little fancy lab gadgets. The problem that emerges from that practice is the new generation of «press button» «scientists», the new millenial «click and publish» researcher generation.

My Dear Ronald,

The PSI and PSII contributions to the total ChlF emission spectrum can be isolated in order to obtain a correct analysis and interpretation of the fluorescence emission?

Interpretations of this effect are controversial and different, relying on optical effects of light re-absorption and diffusion, or rapid energy distribution changes between PSII and PSI.

My doubt is based on Butler and Kitajima’s model of the fluorescence quenching. This assumption suggest a linear relationship between the two ChlF bands.

my best regards.

Dear Montcharles,

I don't know of any publication about fluorescence lifetime techniques used for measuring the individual contributions of PSII and PSSII to ("total") measured in vivo fluorescence at room temperature. About PSI, I know of researchers speaking about the advantage of mathematical modeling for predicting that PSI could contribute to overall fluorescence emission (see Dusan Lazar paper J Theor Biol. 2013 Oct 21;335:249-64. doi: 10.1016/j.jtbi.2013.06.028 or get it from here Article Simulations show that a small part of variable chlorophyll a...

About Buttler and Kitajima paper, my best guest is that you speak about this one: Energy Distribution in the Photochemical Apparatus of Photosynthesis, that appeared in 1978 in the Annual Review of Plant Physiology, Vol. 29:345-378, or maybe you speak about the Energy transfer between Photosystem II and Photosystem I in chloroplasts, published in 1975 in Biochimica et Biophysica Acta, 396: 72-85. Anyway, in those papers, the authors did mention that:

1. Fo is the energy emission produced by Chl molecules in the antenna and photosystem 2 (PSII), which occurs before any excitation energy can be trapped by the special pair of molecules.

2. Variable fluorescence in PSII is due to excitation energy that goes back from closed reaction centers This happens because of the overlapping resonance energy levels that enables energy exchange between molecular orbitals from the special chl pair in RC's.

In his paper of 1995 (Sixty-Three Years Since Kautsky: Chlorophyll a Fluorescence, Australian Journal of Plant Physiology 22(2) 131 - 160), Govindje stated that even if PSI also contributes to the overall measured Chl fluorescence in a low level, 10%, there is not variable fluorescence coming from PSI because the energy trap in PSI, the P700 reaction centre, is a deeper energy trap, much more efficient than PSII reaction centre, P680. In the case of PSII, the antennae and photosystem II Chl a molecules compete with the P680 RC for the trapped energy. In the case of P700 RC, the energy transfer from antenna to it is highly irreversible, much more than in P680, in any case. Moreover, the main donor in P700 is not a chemical quencher of Chl a, so there is a lower probability for exciton energy competition with it.

In a paper from 1991, Holzwarth shows that the fluorescence lifetime of PSI Chl a molecules is much shorter than the fluorescence lifetime of PSII Chl a molecules. See Excited state kinetics in chlorophyll systems and its relationship to the functional organization of the photosystems. In 'Chlorophylls'. (Ed. H. Scheer.) pp. 1125-1 151. (CRC Press: Boca Raton.). I can send a copy to you in case that you have not access to that book. Anyway, the faster the lifetime the lower its fluorescence quantum yield. It is worth to note that PSII radical special pair reaction centre has been associated to residual energy sinking by delayed luminescence. I guess, this is a question quite related to the molecular structure of PSI and its components, protein structure, donors, acceptors, reaction centres, etc.

Unfortunately, right now I do not have time to check for new publications that proof variable fluorescence coming from PSI. As soos as possible, I will check and come back to this discussion.

I would like to thank Dr Pavlov for starting this interesting discussion. I hope this exchange will help you to understand some basic points about Chl a fluorescence emission. For me is also a good opportunity to get inside again into these intriguing mysteries about PSI fluorescence.

Cheers,

Ronald

By the way, I agree with Dr Pavlov about the poor attetion that Taiz and Zeiger textbook pays to Chllorophyll fluorescence, I am quite surprised to see it with my own eyes. It is a pity that these authors still keep ignoring the significance of fluorescence and the advances, contributions, and progress made thanks to the fluorescence techniques that has fostered so much toward a better understanding of plant physiology.

I will try to briefly answer your questions.

Variable fluorescence is associated with the chl a of the light-harvesting complexes (LHC) of PS2. The PS2 consists of a core in which a reaction center (RC) is incorporated. The RC consists of D1 and D2 proteins and cyt b-559. The core also includes chl- containing proteins of the internal antenna of PS2 and components of water oxidizing complex. The core is the minimum structural unit capable of oxidizing water and reducing plastoquinone. Thus, the proper photochemical processes take place in the PS2 core. Another structural part of PS2 is the external antenna consisting of minor LHCs and the main mobile external complex Lhcb1-3. The function of al LHCs is absorption of the scattered light energy. Once absorbed, this energy can be 1) directed to the RC (to the special pair located on D1 and D2 proteins) and used in photochemical processes; 2) emitted in the form of fluorescence; 3) scattering in the form of heat. If due to some reasons the photochemical processes are blocked, the energy output in variants 2 and 3 increases. “The narrow neck” of the photochemical pathway is the relatively slow redox transformations of the plastoquinone pool, associated with the absorption of protons. The variable character of the fluorescence of the chlorophylls of PS2 is related to this site. If plastoquinone pool is fully reduced the further electron transfer becomes impossible (closed RC) - fluorescence increases and vice versa. The such problem is not exist in the PS21.

Dear Colleagues, thank you very much for your kind answers!

I would like to resume the information from the contributed answers, in order to be sure that I have well understood your main points:

1. Initial fluorescence (Fo) is deexcitation energy that is emitted by chl a molecules in PSII and PSI complexes and light harvesting antennae before any trapping of exciton energy occurs in the reaction center of PSI and/or PSII.

2. Fluorescence is mainly produced by chlorophyll a molecules in the photosystem II (PSII) and the light harvesting antenna complex attached to PSII. The chlorophyll a molecules in Photosystem I (PSI) is about 10% of the overall measured fluorescence.

3. Delayed fluorescence comes from residual deexcitation energy at reaction centres in both PSII and PSI.

4. Fluorescence from PSI is also produced by chlorophyll a molecules. It is not variable because the efficiency of the PSI core is so high compared to the antenna chlorophylls that there is not competition between antenna chlorophylls and the special pair in the reaction center core, because of the nature of the donor and acceptor side around PSI core reaction centre, that do not compete against Chl a antenna molecules for exciton energy. Therefore, not variable fluorescence can be generated.

5. Maximal fluorescence (Fm) is the point where all of the plastoquinone molecules have been saturated and cannot accept more electrons coming from the core reaction center. Therefore, no more energy is being trapped at the reaction centers and it follows that no competition for exciton trapped energy between the antennae chlorophyll a molecules and the reaction center can occurs. The only possible mechanisms for deexcitation of exciton energy trapped in the antenna chlorophyll a molecules is heat dissipation, or heat sinking, and residual fluorescence emission that decrease until it reach a stationary steady fluorescence level.

Please, could you be so kind to validate (check, correct, confirm, or complete) these 5 statements?

Your help is greatly appreciated! Thank you so much in advance.

Stancho V Pavlov I would like to add one more point

FV/FM ratio represents the relative state of PSII and gives information on the functional damage of photosynthetic apparatus in plants.

Howdy Stancho,

First Chlrorphyll fluorescence is not a photophysical or photochemical phenomenon, but a photobiological one.

Chlorophyll fluorescence is light re-emitted by chlorophyll molecules (yes

chlorophyll!) during return from excited to non-excited states. It is used as an indicator of photosynthetic energy conversion in higher plants, algae and bacteria. Excited chlorophyll dissipates the absorbed light energy by driving photosynthesis (photochemical energy conversion), as heat in non-photochemical quenching or by emission as fluorescence radiation. As these processes are complementary processes, the analysis of chlorophyll fluorescence is an important tool in plant research with a wide spectra of applications.

The Kautsky effect

Upon illumination of a dark-adapted leaf, there is a rapid rise in fluorescence from Photosystem II (PSII), followed by a slow decline. First observed by Kautsky et al., 1960, this is called the Kautsky Effect. This variable rise in chlorophyll fluorescence rise is due to photosystem II. Fluorescence from photosystem I is not variable, but constant.

The increase in fluorescence is due to PSII reaction centers being in a "closed" or chemically reduced state. Reaction centers are "closed" when unable to accept further electrons. This occurs when electron acceptors downstream of PSII have not yet passed their electrons to a subsequent electron carrier, so are unable to accept another electron. Closed reaction centres reduce the overall photochemical efficiency, and so increases the level of fluorescence. Transferring a leaf from dark into light increases the proportion of closed PSII reaction centres, so fluorescence levels increase for 1–2 seconds. Subsequently, fluorescence decreases over a few minutes. This is due to; 1. more "photochemical quenching" in which electrons are transported away from PSII due to enzymes involved in carbon fixation; and 2. more "non-photochemical quenching" in which more energy is converted to heat.

PSII yield as a measure of photosynthesis

Chlorophyll fluorescence appears to be a measure of photosynthesis, but this is an over-simplification. Fluorescence can measure the efficiency of PSII photochemistry, which can be used to estimate the rate of linear electron transport by multiplying with light intensity. However, researchers generally mean carbon fixation when they refer to photosynthesis. Electron transport and CO2 fixation correlate well, but may not correlate in the field due to processes such as photorespiration, nitrogen metabolism and the Mehler reaction.

Relating electron transport to carbon fixation

A powerful research technique is to simultaneously measure chlorophyll fluorescence and gas exchange to obtain a full picture of the response of plants to their environment. One technique is to simultaneously measure CO2 fixation and PSII photochemistry at different light intensities, in non-photorespiratory conditions. A plot of CO2 fixation and PSII photochemistry indicates the electron requirement per molecule CO2 fixed. From this estimation, the extent of photorespiration may be estimated. This has been used to explore the significance of photorespiration as a photoprotective mechanism during drought.

Fluorescence analysis can also be applied to understanding the effects of low and high temperatures.

• Sobrado (2008) investigated gas exchange and chlorophyll a fluorescence responses to high intensity light, of pioneer species and forest species. Midday leaf gas exchange was measured using a photosynthesis system, which measured net photosynthetic rate, gs, and intercellular CO2 concentration. His results show that despite pioneer species and forest species occupying different habitats, both showed similar vulnerability to midday photoinhibition in sun-exposed leaves.

Measuring stress and stress tolerance

Chlorophyll fluorescence can measure most types of plant stress. Chlorophyll fluorescence can be used as a proxy of plant stress because environmental stresses, e.g. extremes of temperature, light and water availability, can reduce the ability of a plant to metabolise normally. This can mean an imbalance between the absorption of light energy by chlorophyll and the use of energy in photosynthesis.

• Favaretto et al. (2010) investigated adaptation to a strong light environment in pioneer and late successional species, grown under 100% and 10% light. Numerous parameters, including chlorophyll a fluorescence, were measured. Overall, their results show that pioneer species perform better under high-sun light than late- successional species, suggesting that pioneer plants have more potential tolerance to photo-oxidative damage.
• Neocleous and Vasilakakis (2009) investigated the response of raspberry to Boron and salt stress. Leaf chlorophyll fluorescence was not significantly affected by NaCl concentration when Boron concentration was low. When Boron was increased, leaf chlorophyll fluorescence was reduced under saline conditions. They be concluded that the combined effect of Boron and NaCl on raspberries induces a toxic effect in photochemical parameters.
• Lu and Zhang (1999) studied heat stress in wheat plants and found that temperature stability in Photosystem II of water-stressed leaves correlates positively and well, to the resistance in metabolism during photosynthesis.

Nitrogen Balance Index

A portable multiparametric fluorometer using the ratio between chlorophyll and flavonols can be applied to detect nitrogen deficiency in plants Because of the link between chlorophyll content and nitrogen content in leaves, chlorophyll fluorometers can be used to detect nitrogen deficiency in plants, by several methods.

Based on several years of research and experimentation, polyphenols can be assigned as indicators of the nitrogen status of a plant. For instance, when a plant is under optimal conditions, it favours its primary metabolism and synthesises the proteins (nitrogen molecules) containing chlorophyll, and few flavonols (carbon-based secondary compounds). On the other hand, in case of lack of nitrogen, we will observe an increased production of flavonols by the plant.

The NBI (Nitrogen Balance Index), allows the assessment of nitrogen conditions of a plant by calculating the ratio between Chlorophyll and Flavonols (related to Nitrogen/Carbon allocation) .

Hence Stancho,

I hope this short summary elucidates soma aspects of the photobiology of plant chlorophyll fluorescence. A lot more information is available. For example models to simulate plant fluorescence. An interesting one can be found at:

http://www.ipgp.fr/~jacquemoud/publications/pedros2004.pdf

Success with your studies,

Frank

Thanks Frank Veroustraete , for this very detailed, complex answer. That was really good for a beginner in this field as I am. Have a nice day, Petra

You are welcome Petra,

Cheers,

Frank

Respected Dr. Frank Veroustraete

I am beginner in the plant physiology research and just wanted to ask, why the PSII reaction centers are “closed” and unable to accept further electrons in a dark adapted leaf? In other words, why the PSII is unable to pass its electrons to subsequent electron carriers as explained by Kautsky effect?

What we know is that NADPH is oxidized during carbon fixation reactions to NADP+ and reduced back to NADPH during the light reactions. Isn’t that correct that a dark adapted leaf will have all its NADPH converted to NADP+? If yes, then why upon exposure to light, a dark adapted leaf undergoes Kautsky Effect and exhibit fluorescence instead of passing electrons to downstream electron acceptors?

Please explain and thank you so much for the previous detailed explanation.

Hi Fahad,

In a dark adapted leaf the chlorophyll concentration is very low, and sometimes with seedlings even zero. Hence there are no or not enough receptors for electrons when the dark adapted is transferred to a light phase. Hence with very low number of PSII electron receptors they are immediately closed, due to a lack of PSII receptors. In that case fluorescence and also heat dissipation (non-photochemicla quenching) wil increase strongly,to dump the excess of incoming EM energy as heat or fluorescence. This has evidently also its impact on the Kautsky effect.

In light conditioned leaves fluorescence will be much lower due to a much larger availability of PSII electron recptors. Heat disspation will still be large since only a small part of incoming radiation is dessipated as fluorescence ( only a few percent at maximum) the large majority is dissipated as heat and is important for the evapotranspiration at leaf level.

Cheers,

Frank

Respected Prof. Dr. Frank Veroustraete , Many thanks for the fine explanation.

Best Regards

Fahad

With my deepest respect, I disagree with Prof. Frank Veroustraete upon his answer to Fahad Shafiq about closed reaction centers. Any debate and discussion can teach us something new. I will try to explain why I disagree and to be as short as possible:

First : Prof. Veroustraete's concept of dark-adapted leaves is not wrong if we speak literally, but is wrong whithin the specific research framework/field that we are considering here. So, technically speaking, the term use is out of our concrete context. I am afraid that Prof. Veroustraete did not manage the already classical term used in Chlorophyll Fluorescence Research about "dak-adapting" leaves just before a Chl fluorescence induction sampling/measurement, a use or abuse that can also be questioned. Anyway, I think, for helping Fahad, it is necessary to make clear that fact in order to help him to understand that Prof. Veroustraete´s answer, even if well-intentioned and willingful to help, is a missleading one.

Second: When applying the Chl Fluorescence technique for measuring Kautsky curves with direct method (in the pump-&-probe PAM method dark-adaptation is not necessary), chlorophyll concentration does not change along a 30 minutes or 60 minutes dark-adaption period. Technically speaking, we may induce a decrease in Chl concentration, for example, if the leaf is detached and exposed to drying, which could lead to Chl degradation or else, by applying a treatment that may cause chlorophyll degradation (heating, chemical poisoning, induced nutrient deficiency, etc). Otherwise, if the measured leaf is attached, there will be not important (statistically speaking) changes in Chlorophyll content. In the context of his answer, I deduce that Prof. Veroustraete was speaking about etiolated plants and chlorosis cases, which has nothing to do with Fahad's question.

Third, exposing etiolated plants (chlorotic plants) to a continuous actinic light for measuring their fluorescence response is something very tricky. In that case, we need to have a control group of plants cultivated under normal growth conditions, in order to compare their fluorescence response to the respective response in etiolated plants of the same species. Here, you will observe different behavior because of the strategy adopted by the plant to cope with low light conditions. The Light Harvesting complexes are organised otherwise, the Chl a/b ratio is different, being Chl b higher in etiolated than in normal plants. Not necessarily we will observe higher fluorescence nor higher heat sinking, when compared to "normal" plants. It will be exactly the other way around.

Forth: Prof. Veroustraete said "Hence with very low number of PSII electron receptors they are immediately closed, due to a lack of PSII receptors", sounds to me "complètement à coté de la plaque". Absolutely nothing to do with the topic. We speak about electron transfer reactions, and about aceptors and donors. We speak about a thylakoidal transmembrane potential... that may influence also the process... we speak about quinones and a quinone pool... and pheophytin and a Oxygen Evolving System... from what is known about Chl a/b ratio, we may predict a higher light use efficiency because of a specific plant strategy mechanism that will favour a decrease of the Chl a/b ratio, which increases the efficiency of exciton trapping and consequently optimize light trapping mechanisms, less excitons goes back to the antenna and consequently, less energy dissipation as fluorescence and as heat. we could expect lower Fo and Fm levels. But then, we must think about carotenoids and their photoprotective role.

There are more points to disagree with, but I stop here. Hope this answer will contribute with something to this highly interesting discussion.

Dear Prof. Frank Veroustraete , thank you so much for your kind and detailed explanation about Ch fluorescence. It is deeply appreciated. I would like to add some brief remarks on top of it.

Yes, Chl fluorescence may be a phenomenon studied by photobiologists , but lucky the rest of us, common mortals, it is not a patent-made by a Universal Society of Photobiology for the strictly use of photobiologists only, isn't? Chl fluorescence is just a natural phenomenon as natural as sex or a black hole. There is nothing mystic or mythic in it. If we want to argue, then, as far as I know, there is not a Nobel prize in Photobiology. And never will be. There is a Chemistry Nobel Prize. There is a Physics Nobel Prize. There is even a Physiology Nobel medal. Period. When speaking about photochemistry and photophysics, I already speak about two interdisciplinary fields. I should add Spectroscopy and Mathematics. Any natural phenomenon can be studied and approached using any kind of tools, like mathematics, chemistry, computer sciences, physics, biology, and many more and then, if we want to go into a higher detail level, then we speak about spectroscopy, computer sciences, quantum mechanics, math modeling, Boltzmann statistics, just to mention a few interdisciplinary fields. Chlorophyll is a term first introduced in 1817 by two French pharmacists (Joseph Bienaimé Caventou et Pierre-Joseph Pelletier) who first isolated the green pigment from plants and described it, proposing this name from greek "chloros" (color) and "fillon" (leaf). As for variable fluorescence, it has was first described by a chemist named Hans Kautsky and his lab assistant, Albert Hirsch. By the way, nobody in photobiology research knows that his first name was Albert. Did you know it? In 1931, Kaustky was a researcher at Heilderberg. Being son of a Jewish mother and surviving in Hitler´s Germany was kind of difficult as arial arrogance was triumphally and overwhelmingly marching to its zenith and so, intellectual and moral blindness was reaching its critical mass even far beyond Germany´s frontiers. The greatest photosynthesis specialists of Kautsky´s time were terribly disturbed on why an unknown jewish chemist who was not even a spectroscopist, dared to put his nose into a scientific field that was not its own… and moreover, he was just a simple chemist of an unknown dark chemistry lab in a little German town! Yes, sometimes some people feel like somebody else is trying to invade their sacred territory… Lucky us, today, scientific progress is made possible thanks to interdisciplinarity, global collaboration and open-mindedness. Back to fluorescence, she has already nearly 500 years history maybe more. We may recall the interesting luminescence phenomenon first mentioned by the Spanish medical doctor Nicolas Monardes in his Encyclopedic book "Historia Medicinal" (Sevilla, 1565) while being in Mexico and describing the glooming properties of an extract of "Lignum nephriticum", Kidney wood, a Mexican tree species used by the Azteca surgeons in 16th century to treat kidney ailments; In 1834, it was described again by the Scottish preacher Sir David Brewster, and again observed in 1846 by Sir John Herschel, a telescope builder, who called this phenomenon "epipolic dispersion". It was a British mathematician, Sir G.G. Stokes, who in 1852 gave to fluorescence its current name. Yes, it is the same GG Stokes who also discovered that the emission bands are shifted to wavelengths longer than the absorption bands, a phenomenon that we call today "Stokes shift". Can you see? Mathematicians, we too have made some quite remarkable contributions to this research field… Photosynthesis has intrigued many people from so many different fields through last 500 years… If we follow the sixteen century works of Spanish missionaries and scholars, like Fr. Bernardino de Sahagun and Francisco Hernandez, who in 1527 compiled a huge amount of information about the Aztec culture, then we discover that fluorescence was first observed by Pre-hispanic Native American doctors that had already noticed the blue fluorescence emission from the infusion of the "Coatli" tree, a Eysenhardtia tree species of the Leguminosae family, who could possibly be the source of the Lignum nephriticum described by Monardes. Today, we know that Eysenhardtia sp. contains huge amounts of Coatline B that gives rise to a fluorescent product when in alkaline water solution at room temperature. Many people, from many fields, and not only from scientific fields, has participated in the construction of this beautiful and exotic intellectual building. Boyle, newton, Priestley, just to name a few. Long before top talented "intellectuals" started to pronounce "photobiology" in their academic roosters, Chl fluorescence was already a well-known natural phenomenon. Photobiology is a relatively new interdisciplinary research field, in which biology plays a central role, yes, but it is thanks to the contributions of many other research fields, like maths, physiology, physics, chemistry, biochemistry, biophysics, informatics, spectroscopy, etc., and thanks to so many other interdisciplinary fields, that photobiology became a scientific discipline as such. The first American Society for Photobiology was formed in 1972 and the first European Society of Photobiologist was born only after 1984. Any advances in Science are made also thanks to solid financial support, technology progress and education. Photochemistry and Photophysics, are two fundamental research fields (also built as result of an enormous interdisciplinary collaboration), upon which photobiology has been built and successfully established itself as an independent research field on it own. I propose to nail it down by agreeing that Chl fluorescence is today a research field on its own too, built itself upon a tremendous interdisciplinary collaboration that is currently being integrated into a holistic scientific discipline of its own too: the Biology Systems Theory.

Dear Iqbal Mir , I am a bit skeptical about your statement on Fv/Fm and its relation to a linked Qa oxidation model. Do not worry. That interpretation belong to Prof. Lou Duysens and Sweers, back in 1963... if there is somebody to blame, that will be him... and, of course, Prof. Butler, who first proposed to use Fv/Fm as an indicator of PSII redox state fluorescence yield.

Now, in the following lines, I'll try to explain why I put in doubt that so widely extended misbelief:

First, we will define the meaning of Fo and Fm

First thing: let's make clear what the experiment is. Following a reasonable short period for a photosynthetic material to remain in darkness the so called "dark-adaption period", we illuminate that photosynthetic sample, typically a plant leaf, with a continuous 650 nm excitation/actinic light source of about 600 W/m2. This irradiation energy will be absorbed by the photosynthetic machinery; A fraction of the absorbed light will be trapped by the reaction centers (RC) in PSI and PSII and converted into chemical energy; another fraction will be dissipated as heat and as fluorescence (measured at 730 nm). What is not absorbed at all is transmitance and/or reflectance.

Some post above, @Stancho V Pavlov made an excellent short resume of the main points discussed around his two questions related to

1) the exciton energy dissipation when the RC is not available to trap excitonic energy by the chlorophylls special pair, and

2) Variable fluorescence produced only by PSII and not by PSI.

In that resume, Prof. Pavlov very nicely explained what Fo is. He wrote "Initial or ground fluorescence (Fo) is deexcitation energy that is emitted by chl a molecules in PSII and PSI light harvesting antennae before any trapping of exciton energy occurs in the reaction center of PSI and/or PSII" .

So, at this point, we come to a very clear conclusion: Fo does not reflect any activity in the RC's, however, it may be a measure or indicator of the inactivity of the RCs. The moment when we measure Fo, we call it "time zero" (To), the reaction centers are in a "open" state. We know that only a fraction of the total reaction centers in the photosynthetic units (PSU) will be in a open state. There is a probability that some of them will remain in a "sinking" state along the entire period of illumination with 650 nm light. The question is, what fraction of RCs will remain in a sinking state? We cannot deduce it from the Fo value. Moreover, Fo does not reflect any activity in RC's.

Variable fluorescence, emitted by PSII reach a max value, at time Tfm, and we call that fluorescence level as Fm, Fp, P... or maximal fluorescence. After Tfm, the fluorescence will decrease, until it reach a lower steady level. At Fm, we say that all of the RC's are closed. There is not more measurable activity coming from RC's at time Tfm. The primary and secondary redox reactions between acceptor quinones and the plastoquinone pool will reach a quasiequilibrium state, an optimally coordinated state. Fm does not reflect any activity in the RC's. Fm is reached only after multiple PSII turnovers.

So, if Fo and Fm don't reflect any activity in the RC's, why do we use them ? What exactly does Fo and Fm reflect? Can you see my point?

Respected Prof. Ronald Maldonado Rodriguez

Thank you so much for elucidating different pros and cons on my question. A lot of aspects are explained in your detailed answer. First of all, it became clear that the Prof Frank Veroustraete indicated etiolated leaf. Photomorphogenesis results in establishment of thylakoid membranes and functional architecture of photosystems from proplastids containing pro-lamellar bodies. So, it is quite obvious, the Kautsky effect would be evident for an etiolated leaf.

Secondly, you are also right about the Chl a/b ratio that fluctuate dramatically in response to light quality. Even under stress conditions, plants tend to incorporate chlorophyll b molecules in the outer core of antenna complexes to cope with limited photo-chemical efficiency. It is also right that, caroteonids and other pigments interfere with fluorescence directly through VDE cycle or indirectly via non-photochemical quenching etc. So many factors to consider, for sure.

Now please, re-assume a dark-adapting leaf?

You stated that " chlorophyll concentration does not change along a 30 minutes or 60 minutes dark-adaption period". For a short term acclimation period, this is pretty acceptable that changes may not be significant as you stated. But what about the long term acclimation period? Isn't that correct that the plants dynamically acclimate to high or low light via up or down regulation of GLK transcripts, resulting in regulation of thylakoid stacks density and subsequent LHCs regulation (Waters and Langdale, 2009).

Consequently, low light may increase thylakoid stacks and LHCs to increase light harvesting efficiency. With increased thylakoids density under low light conditions, Kautsky effect remained unexplained for a dark adapted leaf?

Please respond Sir

Reference: Waters and Langdale (2009). The making of a chloroplast. EMBO J. 28(19): 2861–2873.

Hello Fahad, Fahad Shafiq

please, don't call me professor, I am not a professor. I would love to be, but somebody kicked me out from academics... so, just calling me Ronald will be nice.

About your questions, keep in mind that Photosynthesis occurs in several stages or phases, we mainly differentiate two: the light phase and the dark phase. Please, do not mix these two different things, ok? They occurs in different time scales, and the speed of the light reaction processes are so much more faster than darkreactions, that we cannot simply start to mix them as if they were happening in about the same time scale. Please, keep in mind, these processes, light and dark phases, occurs in two very different time scales. that is the first thing to keep in mind. Is you mix them, is like trying to compare what's going with the firing force of the spark plug that fires up inside the combustion chamber of the piston in the engine of your car, the first time that you switch it on and comparing it to what the emission gases that the engines produces after keeping that car switched on for several years... I mean, these two things are part of the car, same machine, but the mechanics, chemistry, the processes involved are two worlds apart.

Now, let-s back to the term "dark-adapted" leaves for measuring Chl fluorescence induction. I hope you have clear in your mind that this has nothing to do with acclimation, ok? This "dark-adaptaton" is just a "wise" technique to help us to be sure that the RCs and its associated redox actors around it (donors/acceptors/whatever else) have reached the lowest possible activity state. Because, in Chl a fluorescence induction experiments, when you irradiate during 1 second, for example, with continuous 650 nm light, about 600 W/m2, that acts as continuous measuring and actinic light simultaneously, the goal is to check the capacity of those RCs to reach their max potential. You see? It's a kind of pump-and-probe method, if you want. You elongate a spring to a max distance, the max possible, without deforming it, and then you release it, and count the time for it to reach again its initial static state. OK, withing chloroplast does not happens the same, it is just an analogy that I used for illustrating the idea of pump-and-probe.

Anyway, when dark-adapting a photosynthetic sample, our aim is to reproduce the necessary conditions for the photosynthetic machinery to reach a ground, a basal lowest possible state, a common floor, the zero point if zou want to call it like that, where all of the processes that will participate in fluorescence emission (during that 1 second illumination pulse) can start from a commong ground. Can you see? The goal is to bring Qp to a value of 1 and Qn to a value of zero. Here, we are not speaking about modying the chemistry of the plant or modifying the enzymatic activity or NADPH or the conformation of the chloroplast or the shape of the leaf or its alkaloids... or any other sophisticated stuff like that. dark adapting a leaf is much simpler than that.

With dark-adaptation, we simply look specially to be sure that all of the Qa activity reaches, if not a full total complete max relaxation state, at least to get closer to it as much as possible. Using this technique, dark-daption, we push the photosynthetic sample to go into a state of full relaxation of all of the non-photochemical quenching reactions. Also, we push it into a state where all of the enzymatic reactions that were activated during the Calvin-Benson-Bassham cycle reach a minimum level of activity, or no activity at all. Also, by applying the dark-adaptation trick, we look to push the leaf to manage to adjust its complex mechanism of stomatal aperture.

How we do proceed for it to happen? Usually, we put the plant in a dark room or use a leafclip for 15 to 20 minutes, that should be enough for dark-adapting a sample. But you have to test it by yourself and decide how long is ok for your plant. Each plant species has its own optimal dark-adaption period. You must test it by your self. The technique is simple. You just put for example your plant with enough leaves that look about the same, nice homogenous green all of them, in a dark room or put a closed leafclip in the leaves, on leaves of about the same age, about same color, same appearance, and then, measure the first leave after a 10 minute period dark adaption. the second one after a 15 minutes dark adapting, the third one after 20 minutes, and so on... after that, just compare them and determine with which of them you get the lowest possible Fo and that will be your optimal dark-adaption period time. Clear enough? I hope so.

Worthwhile to mention this: The Qa oxydation state can be decreased to its lower level by using a far-red light pulse, previous to the 1 second pulse with 600 W/m2 650 nm light. This is the technique used since long time ago in the Walz fluorometers and since 2003 with the introduction of the Senior-PEA by Hansatech UK. this is valid only and only for the Qa relaxation stuffs. For the rest of enzymatic reactions and stomatal aperture regulation and the rest of the baseball team, I am so sorry for the bad news but you have to do "dark-adaption" if you want to get good results.

Now, back to your questions>

1) But what about the long term acclimation period?

2) Isn't that correct that the plants dynamically acclimate to high or low light via up or down regulation of GLK transcripts, resulting in regulation of thylakoid stacks density and subsequent LHCs regulation (Waters and Langdale, 2009)?

3) As low light may increase thylakoid stacks and LHCs to increase light harvesting efficiency, with increased thylakoids density under low light conditions, Kautsky effect remained unexplained for a dark adapted leaf?

Right, the question on longer dark adaption periods is about GLK transcrioption expression. Sorry, that is a topic absolutely related to, yes, but the answer cannot come from fluorescence. And we are speaking again about two different time scales. In the case of Golden-2-like transcriptors or GNC trranscription factors, we speak about gene regulation during chloroplast biogenesis... different time scales, my friend. Without the experimental observations here, is like trying to give an answer without having the question, Two different things, related, yes, because in that case you are coping with complex enzymatic reactions, biogenesis, conformational changes at chloroplast development and protein activity, signal transduction and huge conformatical changes that will finally it simply has nothing to do with the 20 minutes dark-adaption technique. For that, I would recommend, if you want to measure all that, don't bother with fluorescence. Get out your Western blot machine and your molecular biology kits, get the correct mutants in shape, run your microplate assays for enzymatic activity again, kick out the dust from your old Beckham spectrometer, and for sure, electronic microscopy and GC-MS and HPLC cannot miss the party. All that could be run in parallel with fluorescence induction, if you want. After the full experiment, then we can talk about it.

Thanks a lot Ronald Maldonado Rodriguez for a wonderful answer once again. My best wishes for your appointment as Professor somewhere, you deserve it for sure.

The concept of spatial and temporal separation of all these phenomenon explained almost everything. In addition, you explained everything comprehensively which is quite good actually. Towards the last part of your reply about the experimentation, unfortunately I can't do it now. I am PhD candidate in Botany and currently waiting for my thesis defense. But, it can be performed surely in future.

once again, my best regards for being so nice and supportive.

Fahad

You are welcome Fahad Shafiq!!!!

Dear Colleagues,

I agree with Prof. Stancho V Pavlov about Chl fluorescence being a complex photophysical and photochemical phenomenon. Of course, it is approached by photobiology but the relevance and remarkable contributions of other disciplines to its understanding should not be neglected, not at all. What Prof Frank Veroustraete says is right, Ch fluorescence is studied by Photobiology, but all of its related phenomena cannot be understood without a good background of photochemistry and photophysics. I am sorry but the tools to study what's going on in electron and exciton transfer in primary reactions of photosynthesis don't come at all from Photobiology. I will mention spectroscopy therefore biophysics and maths, chemistry, optics, electronics, thermodynamics, and so many other disciplines. We are prone to create complex terminology to call simple things in a very sophisticated way, which may create interdisciplinarity barriers that may block scientific progress. It seems that we live in a World that love to build "Walls". I am not sure that is a good idea. Thanks Dr. Pavlov for defending the importance of interdisciplinarity in scientific research.

Thanks Dr Ronald Maldonado Rodriguez!

Thanks Ronald Maldonado Rodriguez for clarification, will look more into it.

No, no, please, it is me who thank you Prof. Stancho V Pavlov for giving us the opportunity to exchange so many interesting ideas about fluorescence!

ChI is known as antena complex which is responsible for absorbing for photo synthetic phosphorylation reaction , which mainly occur inside chlorophyll .