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
- 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 Ámol 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'
- Why do chlorophyll pigments fluoresce?
- In an intact leaf, which pigments are primarily
responsible for fluorescence
xanthophyll, carotenoids? Is it the entire "light
harvesting complex" (LHC) or primarily P680 and
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)?
- Why is the maximum fluorescence (Fm) higher in a
dark-adapted leaf than a leaf exposed to bright light?
- 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?
- If a leaf were moved to a cold refrigerator, causing
metabolism to slow down significantly, would fluorescence
remain high or decrease? Why?
- If Fo is 100 units and Fm 420 units, what is Fv/Fm? Olle
Bj÷rkman 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?
- Why should a leaf in bright sunlight have a lower Fv/Fm
than one in deep shade or in darkness?
- If Fs (steady-state fluorescence in light) is 120 units
and Fm' is 250 units, what is the fluorescence yield (Y)
in the light?
- If Fm is (420 units), what is the non-photosynthetic
quenching (NPQ) of the leaf in question number 8 above?
- Under the conditions of question 8 and 9 above, what is
the rate at which electrons in Ámol 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 Ámol photons m-2 s-1
- 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,
- 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).
- In bright light, would leaves primarily be in a
zeaxanthin form of xanthophyll or a violaxanthin form?
- Which type of leaf would probably have an overall higher
xanthophyll content, a shade leaf or sun leaf? Why?
- 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?