Formation of photosystem 11 reaction centers that work as energy sinks in lichen symbiotic Trebouxiophyceae microalgae AJfredo Guéra1 · Francisco Gasulla1,2 · Eva Barreno2
Abstract Lichens are poikilohydric symbiotic organisms that can survive in the absence of water. Photosynthesis must be highly regulated in these organisms, which live under continuous desiccation-rehydration cycles, to avoid photooxidative damage. Analysis of chlorophyll a ftuorescence induction curves in the lichen microalgae of the Trebouxiophyceae Asterochloris erici and in Trebouxia jamesii (TRl) and Trebouxia sp. (TR9) phycobionts, isolated from the lichen Ramalina farinacea, shows differences with higher plants. In the presence of the photosynthetic electron transport inhibitor DCMU, the kinetics of QA reduction is related to variable fluorescence by a sigmoidal function that approaches a horizontal asymptote. An excellent fit to these curves was obtained by applying a model based on the following assumptions: (1) after closure, the reaction centers (RCs) can be converted into " energy sink" centers (sRCs); (2) the probability of energy leaving the sRCs is very low or zero and (3) energy is not transferred from the antenna of PSII units with sRCs to other PSII units. The formation of sRCs units is also induced by repetitive light saturating pulses or at the transition from dark to light and probably requires the
¡:;.:;:¡ Alfredo Guéra alfredo
[email protected]
Departamento de Ciencias de la Vida, Universidad de Alcalá, Edificio de Cien cias, Campus externo, 2887 1 Alcalá de Henares, Madrid, Spain 2
B otánica, ICBIBE, Facultad de Ciencias Biológicas, Universitat de Valencia, C/Dr. Moliner 50, 46100 Burjassot, Valencia, Spain
accumulation of reduced QA, as well as structural changes in the reaction centers of PSII. This type of energy sink would provide a very efficient way to protect symbiotic microalgae against abrupt changes in light intensity.
Keywords Asterochloris erici · Chlorophyll a fluorescence · Energy sinks · Fluorescence transient · PSII reaction center · Trebouxia Abbreviations A Antenna b Photosystem II reaction center Fraction of closed PSII reaction centers at time t Bt Chyp Constant that determines the degree of curvature of fluorescence induction curves el Closed RC DCMU 3-(3 1,4 1-dichlorophenyl)-l, 1-dimethylurea Energy influx E Minimal Chl a fluorescence intensity in darkFo adapted samples Maximal Chl a fluorescence intensity in darkadapted samples Maximum variable Chl a fluorescence (Fv = Fm - F o) An estimate of the maximal quantum yield of PSII photochemistry Fluorescence intensity at time t during exposure of samples to light Effective quantum efficiency of PSII
0.6
0.4
e
Experimental
•
Vert. asymptote
- - - Non-conn ected
0. 2
o
o
0. 2
0.4
0.6
Hor. asymptote
0.8
1.2
Bt Fig. 2 R elative variable chlorophyll a fiuorescence (V,) expressed as a function of the fraction of closed cent.ers (B,). Blue circles exp erimental data; dotted lin e results expected for disconnected RCs; red squares (v ertical asymptot.e), results expect.ed for connect.ed RCs according to m odels for chlorophyll a fiuorescence induction, described by Eq. 2; yellow diarrwnds (horizontal asymptot.e), nonlinear regression fitting of experimental data to Eq. 10. The experimental data are the mean from five different samples. See text for further details
An approach to explain the abnormal behaviour of A. erici was obtained from the model shown in Fig. l B. Here, we propose that in A. erici, after DCMU treatment, closed
respectively and Fm is the maximal fluorescence emission when all centers are sink centers. Thus,
RCs are converted into energy sink centers, where most of the energy is dissipated as heat and fluorescence. We assume that the probability of energy leaving the PSII units containing sink RCs, including their antenna, is very low or zero; i.e. these PSII units act, as a whole, as true energy traps. In accordance with the theory of energy fluxes (Strasser 1978, 1981, 1986; Strasser et al. 2004), the equations defining this model are as follows:
_ E°,,P _ op (1 - P22)(l - Bs) Fop,t - AtotP2f EAO l ' ' -p22 (l - B) s P2f (1 - P22)(l - Bs) = Fo------1 - p22(l - Bs)
+ E'J:p22(l E~ = l + E'J:P22(l E~P
=
l
E"Amax
'
= Fs m
Bs)
+ Psf) Bs
=
Bs)
1 -p22 (l - B) (p2f s
+ P2sPst)
(9)
Bs l -p22(l -Bs)
(3)
= E~PP2b
Eb
Fs,t = EA,tot (plf
Ft = Fop,t
+ Fs,t =
Fo[(l - pn)(l - Bs)] + F:,.Bs 1 - P22(l - Bs) '
Es= E~2s where "s" is a "modified" RC acting as an energy sink. Equation 3 can be easily solved to give the expressions describing the total energy influxes in the antenna and RCs: op
s
l-p22(l-Bs)
-
lp2b
-
(4)
1- pn(l - Bs) fp2s
Es = - - - - - 1 - p22(l - Bs) For extreme cases in which all centers are open (E°1,0 ) or are sinks (E'A,M): op -_ _ J__ EAO '
1 - P22
E~,M
(5)
l
=
Thus, A,O
1 - Pn . J!'..P _ Jf'.P ( 1 - Pn ) (l ) 1 - Pn(l - B,)' A,tot - A,0 1 - Pn( l - B,) - B,
E'. _ E'. A -
E,=
A,max
s EA,rnn
1
. E'.
l _ Pn (l _ B,) '
_ E'.
A,tot -
E',
P2s
l _ Pn(l _ B,) ;
E s,tot
=
A,max
P2sBs
l _ Pn(l _ B,)
(6) where E°1,10 P E~,tot> and Es,tot are the total energy influxes in all open antenna, "sink" antenna, and sink RCs, respectively. As
(7)
and, assuming that all centers are converted to sRCs F:U
=
E~,max (p1f
+ PlsPsf) '
1 - p22(l - Bs)
= Bc1 + Bs
(10)
(11)
and
dt = kBc1,
1 _ Pn( l _ B,)
Fo = E°1,0P2t
B
8 = -----
dBs
B,
A,max
Vt
This function yields a hyperbola with a horizontal asymptote and it can be applied to the experimental data (Fig. 2, yellow diamonds), albeit very roughly. Note that in this model PzG = p 22 . A better approximation is obtained if we consider that the conversion from closed to sRCs needs sorne kind of structural, conformational or biochemical change, which starts after the light is switched on and takes a time ( T) until it is completed (Fig. l C). In this case, after a certain period there should be a variable population that would include opRCs, clRCs and sRCs. Both closed and sink centers are centers with QA reduced and then B
J!'..P _ Jf'.P A -
F -F
0 -1 - =
F:O - Fo
J EA = EA = - - - - - -
Eb
which can be converted by substitution and simplification to:
(12)
where k is the rate constant for the conversion of Bc1 to B 8 • Therefore, after a time (t) the fraction of Bc1 = Be-k1 (Eq. 12) and
(13) and then, B = 1 + Chyp (1-B) e- kt ( see Eq. 1)
Vc1
(14)
and
(8)
where P zt and Pst are the probabilities of fluorescence emission from the antenna and from sink centers,
V8
=
B l-p22 (l-B)
and consequently:
- kt
(1-e)
(15)
B
V=
e-kt
1 +Chyp(l-B)
+
B l -p22(l-B)
(1 - e-kt)
'
~
(16)
.~
"'e
2l
where Chyp
=
(~: - 1)P22,
.:
A. erici 2000
1500
QJ
(17)
for this final model (Fig. lC).
u
eQJ
1000
u
• DCMU
"'~
g
• Control
500
;:¡: o-r-~~~.-~~--,.-~~~~~~~.-~~-
Results Analysis of chlorophyll a fiuorescence induction curves in the presence of DCMU Chlorophyll a fluorescence transient usually shows three characteristic steps between minimal (O) and maximal (P) fluorescence emission intensity; these are K, J and I, which were clearly distinguished when plotted on a logarithmic time scale (Strasser and Govindjee 1991, 1992; Strasser et al. 2004; Lázar 1999). The K, J , I, and P steps corresponded, in the two Trebouxiophyceae genera studied here, to time intervals of about 0.3, 3, 60-80, and 300 ms, respectively (Fig. 3). The herbicide DCMU is an inhibitor of electron transport between the quinones QA and QB of PSII (Velthuys 198 1; Stirbet and Govindjee 2011); it works by displacing QB from its binding site at the D 1 protein of the PSII reaction center complex (Velthuys 198 1). The fluorescence kinetics in the presence of DCMU represents the photosynthetic events leading to the complete closure of RCs (Lázar 1999; Strasser et al. 2004). Figure 3 shows the results obtained during the recording of fluorescence induction with a PEA fluorometer for samples m aintained for 30 min in the dark and in the presence of 100 µM DCMU by careful addition of 500 µl ofDCMU solution on to the algae disc in darkness. In contrast to control samples, Chl a fluorescence rise in DCMU-treated algae had a sigmoidal shape and did not show the K, J, I inflection points. DCMU-treated algae showed higher values of F 0 than control samples. Fm values did not change, but the time needed to get Fm was shorter in DCMU-treated algae than in control ones (the rise time of DCMU-treated algae to Fm is similar to the rise time of the J level in uninhibited samples). Most of these results wer e similar to those extensively described for many species in previous works (Tóth et al. 2005 ; Stirbet and Govindjee 2012 and references therein). Tóth et al. (2005) reported for Pisum sativum leaves treated with DCMU that when the DCMU treatment was carried out in the presence of low light, the value of F 0 significantly increased (at 1 µmol photons m - 2 s- 1 light intensity the F 0 was ~48 . 5 % higher, and at
0.0001
0.00001
0.001
0.0 1
0.1
0.01
0.1
time (s) ~
3500
~
3000
~ ·¡¡; e
2500
.....: QJ
QJ
u
e QJ
2000 1500
u
1000
o
500
"'~ :::s
;:¡:
o 0.0000 1
0.0001
0.001
time (s)
p
=! >
3500
~
3000
.~
"'e
2500
1::
2000
QJ
~ e ~
1500
QJ
1000
...
500
o"'
..:!
O .¡--~~--,~~~-,~~~.-~~---,~~~~
0.00001
0.0001
0.001
0.01
0.1
Time (s)
Fig. 3 Chlorophyll a fluorescence induction curves obtained in control (red squares) and DCMU-treated (blue diamonds) cultures of Asterochloris erici, and the R. farinacea phycobionts TRl and TR9
0.3 µmol photons m - 2 s- 1 , it was ~ 39 % higher than the F 0 of the non-DCMU-treated leaf discs). Therefore, it can be assumed that the small increase of F 0 in DCMU-treated algae cells was a consequence of exposure to dim light (< 2 µmol photons m - 2 s- 1 ) during the addition ofDCMU or during the transport of the leaf discs from the petri dishes to the fluorometer. To estímate the kinetics of QA reduction, the curve of fluorescence induction was normalized as vt (Strasser et al. 2004), where V1 = Jr ~FFº . Then, following Strasser et al. (2004), m
o
t
f Bt
=
1.2
(1 - V 1 )dt
º lfm
f
o
A. erici :==::
S1i 0.8
(1 - V1 )dt
where S 1 represents the normalized complementary area at time t during the induction (the area between a line parallel to the t-axis joining F m to the ordinates axis, and the induction curve represented as V¡) with respect to the total normalized complementary area (i.e. from t = O to t = max), and B1 is the fraction of closed RCs (identical to the fraction of reduced QA) that has a value of zero when all the RCs are open, and 1 when all the RCs are closed. The model based on the energy flux theory (Strasser 1978, 1981; Strasser et al. 2004; Tsimilli-Michael and Strasser 2013) predicts that the B1 versus V¡ curves approach a hyperbolic curve with a vertical asymptote (Fig. l A). However, our experimental data for A. erici and for the TRl (Trebouxia jamesii) and TR9 (Trebouxia sp.) phycobionts isolated from R. farinacea showed sigmoidal curves approaching a horizontal asymptote (Fig. 4A-C, blue circles). Therefore, an alternative model was needed to explain the behaviour of these microalgae. We consider that sorne RCs could be converted from the closed state (closed RCs, clRCs) to a kind of energy sink (sink RCs, abbreviated as sRCs), where most of the energy input is converted into heat and fluorescenc e (Fig. lB , C). This model is described by the following equation (see "Theory" section): V =
B
1 +Chyp(l-B)
e- kt
+
B
l -p22(l-B)
>
0.6 0.4 0.2
0.6
0.2
0.8
1.2
Bt 1.2
TRl 0.8
>
•
0.6 0.4 0.2
0.2
0.6
0.8
1.2
Bt 1.2
TR9 0.8
>
•
0.6
~·· •• •
0.4
(1 - e-kt) '
0.2
(16) Figure 4A-C shows that this model is consistent with the experimental data for A. erici and Trebouxia TRl and TR9 (blue circles). Multiparametric non-linear regression was performed using the programs Datafit 9® (Oakdale Engineering, Oakdale, PA, USA) and Sigma plot 10®. The computed R 2 values for the three studied phycobionts were higher than 0.999 (Table 1) and the model-predicted values (Fig. 4, red triangles) fitted well with the experimental values (Fig. 4, blue circles). The parameters calculated are shown in Table 1. The value of p22, the probability of interconnection between RCs, was high in the three species, with maximal values for TR9 ( ~ 0.83) and minimal for TRl ( ~ 0.65). On the other hand, TRl had the highest value for the rate constant k for the conversion of closed into sink centers ( ~ 9500 s- 1 ) and TR9 the lowest ( ~ 3100 s- 1 ). These results show that a very active exchange of energy between PSII units is present in trebouxoid phycobionts. Equation 16, derived from this
0.2
0.4
0.6
0.8
1. 2
Bt Fig. 4 Fitting of 1he model described by Eq. 16 to experimental data. Blue circles experimental data obtained for DCMU-treated cells; red triangles non-linear regression
Table 1 B, (proportion of closed reaction centers) vs. V, (variable fluorescence) curve parameters Species
Pw
0.7 ± O.O
8912 ± 96
0.9996
TRl (Trebou.xia jamesii)
0.6476 ± 0.0029
9483 ± 157
0.9998
TR9 (Trebou.xia sp .)
0.8 266 ± 0.0461
3093 ± 8
0.9998
Asterochloris erici
p 2 G global probability for excitation en er gy transfer ("excitan" transfer) from one PSII unit to ano1her, K rate constant for the transformation of closed centers to en ergy sink centers, R 2 r egression coefticient Results are 1he m ean ± SE of 3-5 independent experiments
model, can be easily transformed to represent Ft as a function of time and it also fits very well to the original experimental data (Fig. 5A, C). Green triangles and purple circles, in Fig. 5A-C, represent the computed time-course for closed and sink units, respectively.
1.2
~-------------------~
,,,,---
A. erici
0.8
>
••
0.6 0.4 0.2
o
•
Experimen tal
- - - Predicted
I
.o.
closed cent res
•
sin k cent res
l _ (F~/F;,_)
(Fv/Fm)'
- •t.6.
0.00001
0.0001
0.001
0.01
0.1
Time{s) 1.2
,,,,-·•
TRl 0.8
.... >
•
0.6 0.4 0. 2
o
/
-•"'A
0.00001
0.0001
Figure 6 shows results obtained for TR9 when several saturating pulses (3000 µmol photons m- 2 s- 1 , 1 s) were applied, separated by dark time intervals of 1 s. In the presence of DCMU, there was a large increase in F 0 between the first and second saturating pulses. The following pulses had only a small effect on the increase in F 0 in DCMU-treated samples. The increase in F 0 without a decrease in Fm in DCMU-treated samples indicated the presence of closed centers (centers with reduced QA) at the start of the second and successi ve saturating pulses. A quantification of closed centers at the start of the second and successive pulses is shown as an inset in Fig. 6. The proportion of closed RCs was calculated as:
where F~ and F:r, are respectively the variable Chl a fluorescence and the relative maximal fluorescence values for the second and successive pulses. Following the first pulse, around 39 % of the RCs were closed in DCMU-treated cells after 1 s of darkness; meanwhile there were only about 15 % clRCs in the untreated cells. The proportion of clRCs increased up to 46 % after another four successive saturation pulses in DCMU-treated cells, but remained constant in non-DCMU-treated cells. These results were
4000
TR9 3500
•
0.00 1
0.01
·-'?'
0.1 3000
time {s) 1.2
2500
TR9
• p ulse DCMU 3
/
0.8
::i
~ u..
,.
> 0.6
• p ulse DCMU 4
2000
1 500
0.4 1000
..'.."
.: ··~:~ a·~ ~
... • • •
3
0. 2
o 0.00001
0.000 1
0.001
0.01
0.1
• p ulse DCMU 5
" pulse 3 • p ulse 4 • pulse S
3.5
.e
:10
~
zs zo
u
1S
"'
500
a pulse 1 " p ulse 2
• " ·DCMU"
¡;;; 10
~
time {s)
puhcl pulscZ pulsc:I pulic4 pulses O+----~---~---~---~---~
Fig. 5 Time course of relative variable fluor escence (V,= F, - F 0 ! Fm - F 0 ) in DCMU-treated cells. Blue c ircles experim ental data obtain ed for DCMU-treated cells incubated for 30 min in the dark; red discontinous line non-linear regression results ; green triangles calculated time course for closed RCs; black circles calculated timecourse for sink centers. Results presented h er e are the m ean from five different samples. There were no significant differ ences found wh en the experiments were repeated (five times, n = 2- 5 per experiment) with different cultures (R 2 was always > 0. 99)
0. 00001
0.0001
0.001
0. 01
0.1
Time{s)
Fig. 6 Effect of su ccessive high light pulses (3000 µmol ph otons m 2 s 1) on the chlorophyll a flu or escen ce transient of TR9 control cells and those treated with DCMU. l nset fraction of closed reaction centers at the start of the second to the fifth saturation pulse in DCMU-treated (blue bars) and untreated (red bars) cells. See text for further details
expected because DCMU blocks electron transport beyond
from fluorescence curves of DCMU-treated cells. The llPtest is a frequently used tool for the rapid screening of many samples providing information about the structure, conformation and function of their photosynthetic apparatus (Strasser et al. 2000). The main disadvantage of this test is that the derived parameters (see below) are not direct measurements, but inferred quantities based on many assumptions (see Supplementary material). The use of spider-plots (also called "radar-plots") was recommended for the presentation of JIP-test results by Strasser et al. (2000) as a multi-parametric description of structure and function of each photosynthetic sample, presented by a polygonal line. According to Strasser et al. (2000), the spider-plot provides a direct visualization of the behaviour of a sample and thus facilitates the comparison of plant material. Strasser et al. (2004) defined the specific trapping flux (TR/RC) as the rate per RC, by which excitons are trapped by RCs resulting in the reduction of QA, i.e. in the increase in fraction B of closed RCs; hence TRt/RC (in arbitrary units), which is equal to dB/dt. TRo/RC expressing the specific trapping flux at time O, can be calculated, according to Strasser's definition, by the extrapolation of dB/dt versus Vt curves to Vt = O (Supplementary material). We note than in the Strasser's original llP-test, TRo/RC is calculated in a different way, taking asan assumption that the shape of the 0 - J part of a normal transient (t < 2 m s) measured in vi vo is quite proportional to the 0-J rise in the presence of DCMU (Strasser and Strasser 1995; Strasser and Stirbet 1998). Once the value of TRo/RC is obtained, it is easy to calculate other parameters of the llP-test, as the absorption flux per active RC (ABS/RC = TRo/RC (l/
