Chlorophyll Fluorescence in vivo: A Theory (Part I)
Most part of the photosynthetic pigments in phytoplankton cell reside in peripheral pigment-protein complexes of the light-harvesting antenna (I, see Fig. 1). Absorption of light quantum induces the transition of the pigment molecule into excited state. From peripheral antenna complexes, excitation is efficiently transferred to core antenna complexes near photosynthetic reaction centers (II, Fig. 1), where it can be used in the primary photochemical reaction of photosynthesis. But a small fraction of excitons is reemitted as fluorescence or thermally dissipated while they migrate in the core antenna complex to the reaction center (Fig. 1).
Fig.1. The constant and variable fluorescence origin mechanism.
The fate of exciton is determined by the relative values of the rate constants of three concurrent deactivation processes in the core complex:
P* P (1)
P and P* - the ground and excited states of chlorophyll ‘a’ molecule;
kf, kd and kph - the rate constants of the radiative (fluorescence), nonradiative (thermal dissipation), and photochemical (phothosynthetical) deactivation of excitons.
The quantum yields of the primary photosynthetic reaction and fluorescence are equal, respectively, to
fZ = kph /(kf + kd + kph) and fFo= kf /(kf + kd + kph) (2)
The rate constant values are dependent on the molecular organization of the photosynthetic reaction centers and, probably, do not change with taxonomic composition of phytoplankton. Under optimal conditions and with active reaction centers, kph is the greatest from these three constants. As a result, the quantum yield of the excitation energy use (fZ) is near to unit, and only a small part of the excitons (about 0.03%) is lost in the form of fluorescence during exiton migration to the reaction centers.
The relation between constant fluorescence (Fo)
and phytoplankton concentration
Measuring the fluorescence yirld with open reaction centers (Fo) is a fairly simple and convenient method for estimating phytoplankton concentration. The fluorescence sensors in PrimProd fluorometer are commonly calibrated in chlorophyll ‘a’ concentration units, because the fluorescence intensity fairly good correlates with this phytoplankton concentration parameter (Yentsch and Menzel, 1963), especially for individual algae species (Fig. 2).
Fig.2. Fo vs. chlorophyll ‘a’ concentration in Chlorella sp. suspension.
Data were obtained using PrimProd fluorometer.
But such correlation may be low for natural phytoplankton communities, because the pigment composition of the pripheral light-harvesting antenna complexes is variable in different algae taxons. Besides, the ratio of pigments in light-harvesting complexes varies in response to ambient light intensity, nutrition supply etc. As a result, the proportion of chlorophyll in bulk photosynthetic pigments changes in a wide range depending on taxonomic composition and physiological condition of the phytoplankton studied.
Despite the fact that only chlorophyll ‘a’ is the fluorescence emitter, all the light-harvesting pigments, including the pigments of the peripheral complexes, supply excitons for the fluorescence.
Fluorescence intensity from a water sample is given by the equation:
Fo = G*NRC*fFo*Ifl*ifl (l)*S(l)dl (3)
G = const - a factor determined by the geometry and sensitivity of the fluorescence sensor;
NRC - the concentration of the photosynthetic reaction centers in a unit volume;
fFo - the fluorescence quantum yield with reaction centers being in the open state;
Ifl=Ifl(l)dl - the total measuring (probe) light intensity integrated over the spectral range, where Ifl(l) - spectral distribution of the light intensity;
ifl(l) = Ifl(l)/Ifl - normalized spectral distribution of the probe light;
S(l)- the absorption spectrom of all the pigments supplying excitons to reaction centers (i. e. the dependence of the absorption cross section of the light-harvesting antenna on the exciting light wavelength).
Considaring that G*Ifl is constant for available fluorometer one can write:
Fo = Q * fFo * NRC * S (4)
where S = ifl(l)*S(l)dl - the absorption cross section of the light-harvesting antenna of single reaction center for given spectral distribution of the exciting light ifl(l);
Q is constant value.
If the light intensity is uniformly distributed in a spectral range (ifl(l) = const) and assuming fFo as constant, then S is an integral of the absorption spectrum of the light-harvesting antenna and Fo is a linear function of the product NRC*S, i. e. the total absorptivity of all the reaction centers present in the water sample, thus Fo could be characteristic of light absorbtion capacity of phytoplankton (so called aPSP [details in Part II]).
The value Fo reflects light absorption by the given phytoplankton community and can be a more adequate index of phytoplankton concentration than chlorophyll concentration.
In the present model of the fluorometer, the probe flash is given from a xenon lamp through a blue-green absorption filter SZS-22. This combination provides nearly uniform spectral distribution of the exciting light in the range from 380 to 540 nm. Fig. 2 shows, that in chrysophytum (N. salina) and diatomea (Th. weissflogii) algae, in which chlorophyll comprises small part of the light-harvesting antenna, Fo intensity per unit of chlorophyll ‘a’ is about three times higher than in green alga, which has chlorophyll ‘a’ as a major light-harvesting pigment. These relation correlates with the pigment index, which reflects carotenoid contribution in the light absorption.
Fig. 3. The dependences of Fo intensity on chlorophyll ‘a’ concentration for four marine species relating to different algae taxons: diatomea - Thalasiosera weissflogii (1); chrysophyte - Nephrochloris salina (2) and green algae - Ankistrodesmus sp. (3) and Platimonas viridis (4). The data were obtained using a PrimProd fluorometer.
The variable chlorophyll fluorescence and
the photosynthetical activity
The light energy conversion in the reaction center takes some time to be completed. During this time (turnover time), the reaction center is in so called closed state and can not process a next exciton. In this state, the rate constant of the photochemical exiton quenching is equal to zero and the quantum yield of the chlorophyll fluorescence reaches its maximum level (Fm):
fZ = 0 fFm = kf /( kf + kd ) (5)
The difference between fluorescence intensities in closed and open reaction centers (Fv=Fm-Fo) is known as the variable chlorophyll fluorescence; it corresponds to that part of the absorbed light energy, which would be used in photosynthesis if the reaction centers were in the open state. It follows from (2) and (5), that the ratio of the variable to maximum fluorescence yield is equal to the quantum efficiency of the primary charge separation in photosynthetic reaction centers:
(fFm - fFo)/fFm = kph /( kf + kd +kph)=qZ (6)
Therefore, measuring the fluorescence intensities Fo and Fm enables to estimate the efficiency of the photochemical conversion of absorbed light energy in reaction centers of PS II:
fZ =Fv/Fm (7)
Relation Fv/Fm can be used as characteristic of photosynthetical activity of phytoplankton.