Questions Concerning Fluorescence

For light to be active in leaf photosynthesis it must be absorbed. Light energy that is absorbed can be dissipated in one of three ways -- A) lost as heat, B) re-irradiated as red light (fluorescence) or C) used in photosynthesis (photochemistry). These three processes are competitive. That is, an increase in the efficiency of one process will decrease the yield of the other two. Even though chlorophyll fluorescence is only about 1 to 2% of the total light energy absorbed; we can use the fluorescence signal of a leaf to estimate rates of photosynthesis (photochemistry) and heat dissipation. We will see that:

• The maximum quantum yield of PSII in a leaf (dark adapted) is Fv/Fm which is proportional to the quantum yield of photosynthesis (QY = CO2 absorbed/photons absorbed = O2 evolved/photons absorbed)
• The steady-state quantum yield (Y) of PSII of a leaf (in bright light) is Fv'/Fm'
• We can calculate the rate at which electrons are being transported by the light reaction of photosynthesis in a leaf because, according to Einstein, for each electron excited, one photon must be absorbed. The electron transport rate (ETR) will be in units of µmol electrons m^-2 s^-1. We will simply multiply the amount of light hitting the leaf (PAR) by the absorptance of the leaf (~0.84), times 0.5 (because the same electrons must be excited twice, once in PSII and once again in PSI) times the fluorescence yield of electron shuttling (Fv'/Fm').

ETR = Fv'/Fm' x PAR x 0.5 x 0.84

• Quenching of fluorescence can occur by photosynthetic (qP) and non-photosynthetic (qNP) mechanisms. We are interested in non-photosynthetic quenching because it represents photoprotection (heat dissipation, xanthophyll cycling, photorespiration) or photodamage (chlorophyll bleaching, protein denaturing, etc.). A straight forward way to estimate non-photochemical quenching (NPQ) from fluorescence transients is :

NPQ = Fm - Fm'/Fm'

Questions

1. Why do chlorophyll pigments fluoresce?
2. In an intact leaf, which pigments are primarily responsible for fluorescence

   a) Chlorophyll, xanthophyll, carotenoids? Is it the entire "light harvesting complex" (LHC) or primarily P680 and P700?

   b) Why are P680 and P700 the primary chlorophyll molecules that fluorescent in intact leaves? Why is this not the case in a leaf extract, alcohol solution?

   c) Why is most of the fluorescence measured with a fluorometer coming from PSII and not PSI (hint, recall that variable fluorescence is measured over a very short time span, usually in ms)?

3. Why is the maximum fluorescence (Fm) higher in a dark-adapted leaf than a leaf exposed to bright light?
4. If you were to place a chemical that blocks photosynthesis (electron shuttling in the light reaction) on a leaf, such as the herbicide DCMU (dichloro-methyl-urea), would fluorescence remain high or dramatically decrease? Why?
5. If a leaf were moved to a cold refrigerator, causing metabolism to slow down significantly, would fluorescence remain high or decrease? Why?
6. If Fo is 100 units and Fm 420 units, what is Fv/Fm? Olle Björkman has shown that the quantum yield of PSII (Fv/Fm) of a healthy leaf (dark-adapted) is usually around 0.83. That is, 83% of the photons absorbed by the leaf are being used to shuttle electrons via PSII. In bright light, this number decreases, why?
7. Why should a leaf in bright sunlight have a lower Fv/Fm than one in deep shade or in darkness?
8. If Fs (steady-state fluorescence in light) is 120 units and Fm' is 250 units, what is the fluorescence yield (Y) in the light?
9. If Fm is (420 units), what is the non-photosynthetic quenching (NPQ) of the leaf in question number 8 above?
10. Under the conditions of question 8 and 9 above, what is the rate at which electrons in µmol electrons m^-2 s^-1 are being shuttled in the light reaction of photosynthesis? Assume leaf absorptivity is 0.84 (84% of the PAR is being absorbed) and the light level in PAR is 2,000 µmol photons m^-2 s^-1 (noonday sun)?
11. Leaves must dissipate excess light energy (they can not run and hide in the shade or put up a beach umbrella). Excess light energy occurs at midday or when stomata close in response to drought or when leaves are cold (slows down Calvin cycle). The "xanthophyll cycle" is one means of photoprotection against excessive light energy. Xanthopyll cycling is thought to dissipate excess light energy through enhanced heat loss and a reduction in the transmembrane-proton gradient (thylakoid proton gradient), although the mechanisms are not fully understood. Dissipation of excess energy can exceed 60% of the total absorbed. Xanthophyll pigments are interconvertable from Violoxanthin (in darkness) to Antheroxanthin and finally to Zeaxanthin (in bright light). The conversion of Violaxanthin to Zeaxanthin is by removal of oxygen (de-epoxidation) catalyzed by the enzyme "De-epoxidase." But the De-epoxidase enzyme is unusual because its pH optimum is very low, around 5.2.
12. Why would xanthophyll cycling be initiated by a low pH? (hint: consider that the underlying mechanism by which light energy is converted to chemical energy in thylakoid membranes of chloroplasts is via a transmembrane-proton gradient, via the chemiosmotic theory of Peter Mitchell).
13. In bright light, would leaves primarily be in a zeaxanthin form of xanthophyll or a violaxanthin form? Why?
14. Which type of leaf would probably have an overall higher xanthophyll content, a shade leaf or sun leaf? Why?
15. When would alpine conifers (e.g. pine trees at high elevation) have the highest xanthophyll content in their leaves, spring or winter? How about their zeaxanthin content at midday, spring or winter?