The peculiar NPQ regulation in the pinguiophyte Phaeomonas sp.
challenges the xanthophyll cycle dogma
Nicolas
1
Berne ,
Bastienne
1
Istaz ,
Tereza
1
Fábryová ,
Benjamin
1,2
Bailleul
& Pierre
1
Cardol .
1 Génétique
et Physiologie des Microalgues, Département des Sciences de la vie and PhytoSYSTEMS, Université de Liège, B-4000 Liège, Belgium
2 Present adress: Laboratoire de physiologie membranaire et moléculaire du chloroplaste (UMR 7141), IBPC, UPMC/CNRS, F-75005 Paris, France
A peculiar NPQ regulation in Phaeomonas sp.
NPQ
2.0
1.5
1.0
dark
-2
-1
128 mol quanta. m . s
-2
-1
450 mol quanta. m . s
0.5
0.0
0
2
4
6
time after light onset (min)
8
3
2
1
0
0
1
NPQ
2
3
0.0
-0.5
500
4.
0.8
0.6
8
540
560
580
600
6
4
2
0
-2
-6 -4 -2
0
2
4
6
8 10 12
Linear ECS (542 nm)
0.2
0
100
200
300
400
-1
Irradiance (µmol quanta. m-2. s )
Fig 5. Light dependency of the epoxidation rate, calculated from the NPQ recovery rate
in DTT treated cells.
a) Typical kinetics of NPQ recovery at 0, 128 and 450 μmol quanta. m-2 . s-1.
b) Calculation of the epoxidation rate from the mono-exponential fit of the NPQ
recovery. From 4 independent biological samples (±S.D.). All rates were normalized to
the value at 60 μmol quanta. m-2 . s-1
Fig 6. Linear and quadratic
electrochromic probes allow the
measurement of the dark ΔΨ. Top:
Spectra of the linear (blue) and
quadratic (red) ECS components.
Bottom: Quadratic vs linear ECS
during the decay of a light-induced
pmf, in control and FCCP
conditions (arrow = dark ΔΨ)
Conclusions.
Phaeomonas sp. displays an unusual light dependency of the NPQ, the
latter being maximal in the dark. This is due to:
• the light dependency of the zeaxanthin epoxidase, most likely via the
availability of its substrate NADPH.
• the presence of a proton motive force (ΔΨ and ΔpH) in the dark, which
pre-activates the VDE
Two electrochromic (ECS) probes are
present in Phaeomonas sp. (Fig. 6a),
displaying
linear
and
quadratic
dependencies upon the transthylakoidal
electric field (ΔΨ). Like in diatoms
(Bailleul et al, Nature, in press), the
absolute value of the ΔΨ can be
measured thanks to those two probes. A
ΔΨ is present in the dark, which is
suppressed by the ionophore FCCP (Fig.
6b, red arrow).
In addition, the inhibition of the NPQ
generation in the dark, when nigericin
(H+/K+ antiporter) is added, indicates
that the activity of the VDE is dependent
on the presence of a ΔpH in the dark
(Fig. 7).
Control
FCCP
0.4
0.0
520
(nm)
1.2
1.0
Fig
0.5
Thus, both osmotic and electric
components of the pmf are present in
the dark. The presence of this dark pmf
allows a sufficient acidification of the
lumen to activate the VDE in the
absence of photosynthetic activity.
3
Nigericin
Control
2
NPQ
2.5
4
Quadratic ECS (576nm)
b)
3.0
Epoxidation rate (r.u.)
a)
1.0
5
Fig 4. Zeaxanthin content as a
function of NPQ. Data from
Fig. 1 (open circles), Fig. 2
(close circles) and Fig. 3
(stars).
The light dependency of the zeaxanthin
epoxidation rate mirrors the one of the NPQ (Fig 3
and Fig 5b), strongly suggesting that the regulation
of the ZE plays an important role in the overall XC
regulation in Phaeomonas sp.
quadratic ECS
linear ECS
6
ECS (r.u.)
Zeaxanthin (% total pigment)
When the VDE is inhibited (with DTT) in a sample
quenched beforehand, only the ZE is active and the
recovery of the NPQ is a direct measurement of the
epoxdation activity. The ZE activity is null in the
dark and low under high light, but shows an
optimum under low light conditions (Fig. 5a).
NPQ
Due to the presence of a pmf in the dark,
the VDE is already active in the absence of
photosynthesis
ZE shows a strong light dependency,
mirroring NPQ
In Phaeomonas sp., like in diatoms (Lavaud et al,
Plant Physiol, 2002), the NPQ is proportional to the
de-epoxidized xanthophyll (here zeaxanthin, Fig. 4).
This provides a fast and non-invasive measurement
of the ZE activity.
% of total pigments
Zea (% of total pigments)
Zea (% of total pigments)
NPQ
NPQ
When photosynthetic organisms experience fluctuating light condition, they
3
4
3
Control
can develop different strategies to finely balance light harvesting,
DTT
3
photochemistry, and protection from excess photons. One of those
2
2
strategies is the high energy state quenching (qE), a component of the NPQ
2
(Non Photochemical Quenching), consisting in the dissipation of excess light
1
1
DCMU
1
energy in the form of heat within photosystem II.
DTT
Control
This mechanism is tightly related
0
0
0
to the xanthophyll cycle (XC),
10
6
Violaxanthin
which consists in the reversible
4
5
8
Zeaxanthin
de-epoxidation
of
some
4
3
6
carotenoids. In viridiplantae, the
(ascorbate,
3
2
4
ΔpH)
regulation of the XC is made (NADPH,
2
DCMU
2
1
DTT
through the activation of the O2)
1
Control
from Niyogi et al, PNAS, 1997
0
0
violaxanthin de-epoxidase (VDE)
0
0
100
200
300
400
500
0
2
4
6
8 10 12 14
0
5
10
15
20
-1
by the light-driven ΔpH.
Irradiance
(µmol
quanta.
m-2.
s
)
time after light onset (min)
Time in the dark (min)
Surprisingly, the pinguiophyte Phaeomonas sp, displays a huge NPQ in the
Fig 1. Generation of a xanthophyll Fig 2. Relaxation of the
Fig 3. Light dependency of
dark (Fig. 1), which is dependent on the XC: it is suppressed by the VDE cycle dependent NPQ after a lightNPQ and XC pigments in
NPQ and XC
inhibitor dithiothreitol (DTT).
to-dark transition
Phaeomonas sp.
after a dark-to-moderate
This NPQ recovers under low light (Fig. 2), following the activity of the
light transition
zeaxanthin epoxidase (ZE). ZE activity and NPQ recovery depend on the
photosynthetic electron transfer from PSII to NADP+ : they are suppressed in
Because of the peculiar dark behavior, the conventional nomenclature for the
the presence of DCMU or DBMIB.
calculation of NPQ is not adapted. Instead we calculated NPQ as (Fmll Fm’)/Fm’, where Fm’ is the maximal fluorescence in a given light condition,
NPQ and violaxanthin de-epoxidation also occur under high light (Fig. 3).
and Fmll the one under a “reference” low light (30 μmol quanta. m-2. s-1).
The peculiar light dependencies of NPQ and XC in this species , shown in Fig.
3, offers a unique opportunity to study the xanthophyll cycle, and to dissect
its different regulators.
1
0
0
5
10
15
20
Time in the dark (min)
25
Fig 7.
Nigericin
inhibition of
the NPQ
generated
after a lightto-dark
transition.
Pending Questions and Future Work
Is the ZE, and in turn NPQ, regulated by
the amount of NADPH in the stroma?
Why this “dark NPQ” ? Does it provide
photoprotection to photosystem II ?
Are those features specific to Phaeomonas
sp. ?
Probe the NADPH/NADP+
reduction state in the light in
vivo, and correlate with the
activity of the ZE
Probe the PSII photo-inhibition
rate at the onset of light
(control vs DTT).
Testing other stramenopiles,
like diatoms.
Acknowledgement: This work has been supported by the Belgian Fonds de la Recherche Scientifique F.R.S.-F.N.R.S. (F.R.F.C. 2.4597.11, CDR J.0032.15, and Incentive Grant for Scientific Research F.4520). The strain Phaeomonas sp.
(RCC 503) was provided by the Roscoff Culture Collection (Roscoff, France).