I'm using the Walz Diving PAM to measure Fv/Fm (after 30 min darkness) and F'/Fm' (light-adapted) in the same algae samples. Fv/Fm values are consistently lower than F'/Fm'. Any ideas would be greatly appreciated!
Dear Tom,
that dpends if you have green plants/algae, brown or red algae.
During darkness the chloroplast respiration might be coupled to the photophosporylation, well known for several organism. It is known as chlororespiration. During a daily course you can observe that Fv/Fm increase with sunrise, but reaching darkness it decreasees again, because electrons from the respiratory pathways become couple to the electrone transport chain of photosynthesis, reducing plastochinon and increase again fluorescence quenching mechanisms and thus a decrease of Fv/Fm. I got the quite often when measures yield in the field during a daily course with brown macroalgae.
Probably it is a side effect of antennae assembly and connectivity, you may see the literature of late 90s in https://books.google.it/books?id=_MN3HPuWln4C&pg=PA13&lpg=PA13&dq=algae+chlororespiration+fvfm&source=bl&ots=6mSwWtYRE4&sig=6r2iWLodk-RwPwrfeBUgeK8B4J4&hl=it&sa=X&ved=0ahUKEwiO162gk8fKAhXEWBQKHQS_AQoQ6AEIOjAD#v=onepage&q=algae%20chlororespiration%20fvfm&f=false
In dark adapted leaves or algal cells, enzymes in ARC (antennae and reaction center) and PET pathway downstream to ARC becomes active in presence of light. In dark, the enzymes becomes active very late or do not express well. So, Fv/Fm is lower in dark adapted leaves. Besides, in dark adapted leaves, photosynthesizing enzymes generally become less active in dark and it is instinct of the leaf mesophyll cells or algal cells. But in light adapted leaves, higher photochemical yielding enzymes becomes more active in presence of light, so Fv/Fm is higher. It is instinct of the light adapted leaves. That is, light adapted leaves develop enzymes of this type, so the enzymes becomes more active in presence of light. I have not handled the electron transport studying equipment. But, still I think on the matter. I have handled a flurometer for a few minutes.
Are you sure that your fluoromoeter settings are properly establised, i.e. that you are using proper light intensities? I use Hansatech's PAM and I had the same problem at the beginning, working with Desmodesmus (green alga) - I had to try different intensities of saturating pulses and actinic light to obtain reliable results. It is very important to have proper ratio of saturating-light/actinic-light. I know that this is purely technical issue, but I know from my experience that it is very important.
Yep, temperature conditions are the same. I suspect that it's something to do with PSII not quite relaxing completely or being influenced by the chlororespiration pathways mentioned above. In these same samples, a second saturating pulse (in the dark adapted state) usually results in a higher PSII yield, which is kind of counter-intuitive. Thanks for the input folks!
It is important to consider temperature. Iám using a LICOR equipment with constant temperature for a chamber
State transitions are more probable than chlororespiration, criteria and samples can be found in Gabor et al. manuscript on cyanobacteria http://pcp.oxfordjournals.org/content/early/2012/02/01/pcp.pcs009.full.pdf
State transitions would not lower Fv/Fm below deltaF/Fm'. All quenching mechanisms have to relax (i.e.qE and qT should be zero) for measurements of Fv/Fm, only qI will be still there. Sorry, but it demonstrate people did not understand what they measure with PAM-fluorometry, if these things are published. If the yield in darkness (maximum yield) is lower than the effective yield in light, it is a failure in the measurement or this can be affected only by chlororespiration. Even changes in temperature would not cause such strong effects, or was this very high?
Thanks for your comments, Hanelt. To be explicit, the algae being measured are residing within the tissues of a symbiotic marine invertebrate. This makes it difficult to repeatedly measure exactly the same algal cells between light- and dark phases. Light-adapted yields are being measured at midday in the light : dark cycle, with Fv/Fm measured 30-min after the end of the light phase. Water temperatures and PAM excitation/detection settings are the same for both measurements. When the yields are measured in quick succession (dark-adapted: Fv/Fm and then actinic light exposure: F'/Fm) then the results are more intuitive (F'/Fm < Fv/Fm).
Just to add some details to the first comment of Dieter Hanelt: Also in diatoms the highest Fv/Fm is often only obtained after low light acclimation but not after dark acclimation. It has been shown that in darkness a significant amount of diatoxanthin can be produced, which may induce some NPQ, thus lowering Fv/Fm (Jakob et al. 1999, Plant Biology). This has been interpreted in terms of chlororespiration involving the PQ-pool, as described in Dieters' comment. However, recent results of Bailleul et al. (2015, Nature) explain this by a backwards reaction of the plastidic ATPase, using mitochondrial produced ATP, which then generates a proton gradient over the thylakoid membrane.
in primitive algae and bacteria state transition may occur in the dark for a very different reaction of antennae phosporilation, compared with terrestrial thylakoid plant reaction; and I can not figure what it has to do with q components. Please take in mind Fig 3 of the Gabor Bernat paper of have a word with him at Bochum University http://pcp.oxfordjournals.org/content/early/2012/02/01/pcp.pcs009.full.pdf
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