Dark inactivation of ferredoxin-NADP reductase and cyclic electron flow under far-red light in sunflower leaves

Abstract

The oxidation kinetics under far-red light (FRL) of photosystem I (PSI) high potential donors P700, plastocyanin (PC), and cytochrome f (Cyt f) were investigated in sunflower leaves with the help of a new high-sensitivity photometer at 810 nm. The slopes of the 810 nm signal were measured immediately before and after FRL was turned on or off. The same derivatives (slopes) were calculated from a mathematical model based on redox equilibrium between P700, PC and Cyt f and the parameters of the model were varied to fit the model to the measurements. Typical best-fit pool sizes were 1.0–1.5 μmol m⁻² of P700, 3 PC/P700 and 1 Cyt f/P700, apparent equilibrium constants were 15 between P700 and PC and 3 between PC and Cyt f. Cyclic electron flow (CET) was calculated from the slope of the signal after FRL was turned off. CET activated as soon as electrons accumulated on the PSI acceptor side. The quantum yield of CET was close to unity. Consequently, all PSI in the leaf were able to perform in cycle, questioning the model of compartmentation of photosynthetic functions between the stroma and grana thylakoids. The induction of CET was very fast, showing that it was directly redox-controlled. After longer dark exposures CET dominated, because linear e⁻ transport was temporarily hindered by the dark inactivation of ferredoxin-NADP reductase.

Appendix

Redox equilibrium-based modeling of the 810 nm signal

For convenience, we defined the following denotations:

$$ \varepsilon _{{{\text{PC}}}} \; = \;\frac{{{\text{extP}}700}} {{{\text{extPC}}}}\;\;\;\;{\text{(ratio of extinction coefficients for the two compounds),}} $$

$$ g_{{{\text{PC}}}} \; = \;\frac{{{\text{P}}700_{{{\text{ox}}}} /{\text{P}}700_{{{\text{red}}}} }} {{{\text{PC}}_{{{\text{ox}}}} /{\text{PC}}_{{{\text{red}}}} }}\;\;\;\;( = 1/K_{{\text{P}}} {\text{ in Fig}}{\text{. }}2), $$

$$ g_{{\text{f}}} \; = \;\frac{{{\text{P}}700_{{{\text{ox}}}} /{\text{P}}700_{{{\text{red}}}} }} {{{\text{Cyt f}}_{{{\text{ox}}}} /{\text{Cyt f}}_{{{\text{red}}}} }}\;\;\;\;( = 1/(K_{{\text{P}}} \; \cdot \;K_{{\text{C}}} ){\text{ in Fig}}{\text{. }}2), $$

where g denote the reciprocal redox equilibrium constants between the specified compound and P700. The system of the redox-equilibrated compounds is resolved with the help of an assistant variable x, reflecting the oxidation state of P700:

$$ x\; = \;\frac{{P700_{{{\text{ox}}}} }} {{P700_{{{\text{red}}}} }}. $$

Since the optical signal is generated by oxidized fractions, the model is based on the oxidized fractions of the redox-equilibrated compounds. Using these denotations, the sum of all redox-equilibrated oxidized fractions is

$$ e_{x} \; = \;P700_{{\text{T}}} {\left( {\frac{x} {{1\; + \;x}}\; + \;{\text{PC}}\frac{x} {{g_{{{\text{PC}}}} \; + \;x}}\; + \;{\text{Cyt f}}\frac{x} {{g_{{\text{f}}} \; + \;x}}} \right)},\;{\text{$\upmu$mol}}\;{\text{missing}}\;{\text{e}}^{-} \,{\text{m}}^{{ - 2}} . $$

(1)

Here P700_T denotes the only involved total pool in μmol m⁻², other donor side electron carriers PC and Cyt f are expressed relative to P700_T. The 810 nm optical signal T is generated by oxidized P700 and PC as follows:

$$ T\; = \;{\text{extP}}700\; \cdot \;{\text{P}}700_{{\text{T}}} {\left( {\frac{x} {{1\; + \;x}}\; + \;\frac{{{\text{PC}}}} {{\varepsilon _{{{\text{PC}}}} }}\; \cdot \;\frac{x} {{g_{{{\text{PC}}}} \; + \;x}}} \right)}, $$

(2)

where extP700 is the absolute extinction coefficient of P700, the fractional transmittance change per μmol m⁻² of oxidized P700. The maximum signal is expressed from (2) by letting x → ∞, as follows

$$ T_{{\max }} \; = \;{\text{extP}}700\; \cdot \;{\text{P}}700_{{\text{T}}} {\left( {1\; + \;\frac{{{\text{PC}}}} {{\varepsilon _{{{\text{PC}}}} }}} \right)}. $$

(2a)

Under FRL the rate of oxidation of all equilibrated PSI donors is

$$ \frac{{de_{x} }} {{dt}}\; = \;\eta \; \cdot \;{\text{FRL}}\; \cdot \;{\text{P}}700\; = \;\frac{{{\text{FRL}}\; \cdot \;\eta }} {{1\; + \;x}},\;\;\upmu {\text{mol}}\;{\text{e}}^{ - } \;{\text{m}}^{{ - 2}} \,{\text{s}}^{{ - 1}} , $$

(3)

where η is the quantum efficiency of PSI electron transport under FRL (assumed to be unity), FRL is the flux density absorbed by PSI and P700 is now denoting the reduced fraction, $P700\; = \;1/(1\; + \;x)$.

Before we can express the speed of signal change versus time, we express the change versus the P700 oxidation parameter x by differentiating (1) and (2) with respect to x:

$$ \frac{{de_{x} }} {{dx}}\; = \;{\text{P}}700_{{\text{T}}} {\left[ {\frac{1} {{{\left( {1\;+\;x} \right)}^{2} }}\;+\;{\text{PC}}\frac{{g_{{{\text{PC}}}} }} {{{\left( {g_{{{\text{PC}}}} \;+\;x} \right)}^{2} }}\;+\;{\text{Cyt}}\;{\text{f}}\frac{{g_{{\text{f}}}}} {{{\left( {g_{{\text{f}}}\;+\;x} \right)}^{2} }}} \right]} $$

(4)

$$ \frac{{dT}} {{dx}}\; = \;{\text{extP}}700\; \cdot \;{\text{P}}700_{{\text{T}}} {\left[ {\frac{1} {{{\left( {1\; + \;x} \right)}^{2} }}\; + \;\frac{{{\text{PC}}}} {{\varepsilon _{{{\text{PC}}}} }}\; \cdot \;\frac{{g_{{{\text{PC}}}} }} {{{\left( {g_{{{\text{PC}}}} \; \cdot \;{\text{PC}}\; + \;x} \right)}^{2} }}} \right]} $$

(5)

The change of the signal per change of electrons in the equilibrated compounds is expressed as

$$ \frac{{dT}} {{de_{x} }}\; = \;\frac{{dT/dx}} {{de_{x} /dx}} $$

(6)

where the numerator is from Eq. 5 and denominator from Eq. 4. Considering that

$$ \frac{{dT}} {{dt}}\; = \;\frac{{dT}} {{de_{x} }}\; \cdot \;\frac{{de_{x} }} {{dt}}, $$

(7)

we obtain, considering Eq. 3

$$ \frac{{dT}} {{dt}}\; = \;{\text{extP}}700\; \cdot \;{\text{FRL}}\; \cdot \;\eta \frac{{\frac{1} {{{\left( {1\; + \;x} \right)}^{2} }}\; + \;\frac{{{\text{PC}}}} {{\varepsilon _{{{\text{PC}}}} }}\; \cdot \;\frac{{g_{{{\text{PC}}}} }} {{{\left( {g_{{{\text{PC}}}} \; + \;x} \right)}^{2} }}}} {{{\left( {1\; + \;x} \right)}{\left( {\frac{1} {{{\left( {1\; + \;x} \right)}^{2} }}\; + \;{\text{PC}}\frac{{g_{{{\text{PC}}}} }} {{{\left( {g_{{{\text{PC}}}} \; + \;x} \right)}^{2} }}\; + \;{\text{Cyt f}}\frac{{g_{{\text{f}}} }} {{{\left( {g_{{\text{f}}} \; + \;x} \right)}^{2} }}} \right)}}}. $$

(8)

In Eq. 8 extP700 is presented as a fraction of transmittance change per μmol m⁻² of P700, FRL is the absorbed flux density, μmol quanta m⁻² s⁻¹ and η is dimensionless net efficiency of PSI electron transport. For the oxidative titration, x was varied and the corresponding abscissa value of Fig. 3 was calculated as the signal normalized to the maximum signal:

$$ \frac{T} {{T_{{\max }} }}\; = \;\frac{{\frac{x} {{1\; + \;x}} + \frac{{{\text{PC}}}} {{\varepsilon _{{{\text{PC}}}} }}\; \cdot \;\frac{x} {{g_{{{\text{PC}}}} \; + \;x}}}} {{1\; + \;\frac{{{\text{PC}}}} {{\varepsilon _{{{\text{PC}}}} }}}} $$

(9)

The corresponding ordinate was calculated from Eq. 8. The whole modeled curve was obtained as a series of pairs obtained from Eqs. 8 and 9 by varying x from 0 to ∞. Model parameters P700, PC and Cyt f, as well as equilibrium constants g _PC and g _f were varied until the calculated curve best fitted to the data points. Later, when the corresponding number of electrons was calculated from a certain optical signal, the value of x corresponding to the particular measured T/T _max value was expressed from Eq. 9 and the corresponding number of electrons in PSI donors was calculated from Eq. 1, separately in Cyt f, PC and P700, as well as their sum.

The solution of Eq. 9 with respect to x results in a quadratic equation

$$ x\; = \;\frac{{ - B\; - \;{\sqrt {B^{2} \; - \;4AC} }}} {{2A}}, $$

(10)

where

$$ A\;=\;a\;- \;1\; - \;\frac{{{\text{PC}}}} {{\varepsilon_{{{\text{PC}}}} }};\;\;B\;=\;a\; + \;a\;\cdot \;g_{{{\text{PC}}}}\;-\;g_{{{\text{PC}}}}\;-\;\frac{{{\text{PC}}}} {{\varepsilon _{{{\text{PC}}}} }};\;\;C\;=\;a\;\cdot\;g_{{{\text{PC}}}} , $$

where $a = T/T_{{\max }} \; \cdot \;(1\; + \;(PC/\varepsilon _{{{\text{PC}}}})).$