Photoacclimation Strategy in Photosystem II of Prymnesiophyceae

Photoacclimation Strategy in Photosystem II
of Prymnesiophyceae Isochrysis galbana
プリムネシウム藻綱 Isochrysis galbana の光化学系 II における光適応戦略
06D5501 小幡光子
指導教員
山本修一
SYNOPSIS
海洋に生息する藻類は、海水の鉛直混合や昼夜により、弱光から強光までの様々な光強度(Photon flux density; PFD)にさら
される。藻類にとって、弱光は光合成の制限要因となり、強光は光合成の阻害要因となるため、藻類は、光合成とともに
光合成阻害の回避(光保護)を行う。藻類は、さらされる PFD に対して光合成と光保護のバランスが最適となるように適応
すると考えられていることから、藻類の光適応戦略は、供給される光エネルギーと藻類が要求する光エネルギーのバラン
スに関係があることが予想される。光エネルギーの供給と要求の関係から、PFD は光エネルギー供給が要求を下回る「光
制限下」と光エネルギー供給が要求を上回る「光飽和下」に分けられる。また、光合成や光保護の調節は、主に、光吸収
を行う集光アンテナと電子伝達を行う反応中心からなる光化学系 II(Photosystem II; PSII)で行われる。そこで本研究では、
最も簡単な PSII システムを持つプリムネシウム藻綱 Isochrysis galbana をモデル種として、光制限下と光飽和下に対する
光合成と光保護のバランスの適応を調べることにより、藻類の光適応戦略を明らかにすることとした。光合成と光保護の
指標として、１つには、集光アンテナの集光能力と熱放散能力を用い、もう１つには、反応中心の電子伝達とクロロフィ
ル蛍光の放出を用いた。まず、比成長速度と適応させた PFD の関係から、I. galbana では、365μmol photons m-2 s-1 以下が
光制限下、それ以上が光飽和下であることが推定された。集光アンテナの集光能力と熱放散能力のバランスは、365μmol
photons m-2 s-1 付近以下で集光能力が熱放散能力に比べて大きくなったのに対して、それ以上では熱放散能力が集光能力
に比べて大きくなった。一方、反応中心の電子伝達とクロロフィル蛍光の放出のバランスは、365μmol photons m-2 s-1 付近
以下で電子伝達が優占し、それ以上ではクロロフィル蛍光の放出が優占した。本研究は、光制限下では光エネルギーの要
求を満たすために集光能力及び電子伝達に用いられる光エネルギーの割合を増加させて光合成能力を高め、光飽和下では
過剰な光エネルギーを排出するために熱放散とクロロフィル蛍光の放出を増加させて光保護能力を高めるという、PSII
における I. galbana の光適応戦略を明らかにした。また、PSII における光適応戦略が光エネルギーの供給と要求のバラン
スに依存することを明らかにした。さらに、本研究は、アイスアルジ群集の光適応戦略との比較から、本研究が明らかに
した種レベルの光適応戦略を群集レベルに応用できる可能性を示した。
Keywords: electron transport, fluorescence emission, light harvesting, light limited,
light saturated, photoprotection, photosynthesis, thermal dissipation
LL
LS
1.0
1.0
(
(
0
0
KG
) Es/Ed
) μrel
Es/Ed ratio
(relative unit)
Algae live in diverse and highly variable light environments
and are often subjected to changes in the photon flux density
(PFD) imposed by the natural physics of the ocean. The PFD
received by algal cells varies from 0 to 2000 μmol photons m-2
s-1 (Cullen & Lewis 1988). Light is often a limiting factor for
algal growth in the ocean. However, light can be also harmful
at supraoptimal PFD leading to a damage of photosystem II
(PSII) and a reduction in the photosynthetic rate.
Algae have evolved a number of acclimation responses to
accommodate the change in PFD of photosynthetically active
radiation (PAR) to achieve a balance between maximizing rates
of photosynthesis and avoiding the damaging effects of excess
PFD on photosynthesis (La Roche et al. 1991). Algae need to
enhance photosynthetic capability for maximizing rates of
photosynthesis under low PFDs while enhance photoprotective
capability to maintain maximum photosynthetic rate and to
avoid the damage from excess light energy under high PFDs.
Photoacclimation strategy of algae must be associated with
adjustments of the balance between photosynthetic and
photoprotective capability of algae.
Photoacclimation responses depend in part on whether the
PFD is light-limiting or light-saturating for growth (Raven &
Geider 2003). This implies that strategy of algae may be
associated with the balance of light energy supply and demand
of algae. When growth rate depends on growth PFD (light
limited [LL] condition), the light energy supply is insufficient
to algal light energy demand, while growth rate becomes
saturated (light saturated [LS] condition), light energy supply is
enough or excess for the light energy demand (Fig.1). I
hypothesize that algae enhance photosynthetic capability under
LL condition, while algae enhance the photoprotective
capability under LS condition. Photoacclimation strategy of
algae may be explained by adjustments of the balance between
photosynthetic and photoprotective capability to light
conditions.
μrel
(relative unit)
Introduction
0
1500
Growth PFD
(μmol photons m-2 s-1)
Figure 1. Relationship between relative growth rate, μrel or ratio of light
energy supply to demand, Es/Ed and growth PFD. LL, LS and KG
indicate light limited, light saturated conditions and growth saturation
PFD, respectively.
Since both processes of photosynthesis and photoprotection
occur within PSII, photoacclimation strategy of algae is closely
associated with the photoacclimation of PSII. The PS II is one
of the elements of the photosynthetic electron transport system
and is composed of many different proteins and pigment
molecules. A couple of functions of PSII can be categorized for
light-harvesting antenna and reaction center.
To study the balance between photosynthetic and
photoprotective capability in light-harvesting antenna of
Isochrysis galbana, diadinoxanthin (DD) and diatoxanthin
(DT) could be considered as a representative pigments. In fact,
DD can transfer excitation energy to Chl a and play a role in
the light energy acquisition as photosynthetic pigments, while
DT can absorb excitation energy from Chl a and play a role in
the thermal dissipation as photoprotective pigments (Frank et al.
1994). DD and DT have a reversible relation mutually. This
reversible conversion can be considered as a reversible
transition of light-harvesting into thermal dissipation. Thus, DD
and DT are critical to examine the balance between
light-harvesting and thermal dissipative capability of
light-harvesting antenna in PSII of I. galbana.
Information on status and functions of reaction center of
PSII can be derived from the measurement of chlorophyll a
fluorescence induced by light, because fluorescence yield
depends on status and functions of the reaction center (Krause
& Weis 1991). The variable chlorophyll a fluorescence
provides chlorophyll a fluorescence parameters. The
chlorophyll a fluorescence parameters can be categorized by
quantum yields and quenching parameters in the reaction center.
The quantum yields and the quenching parameters in the
reaction center are measured by pulse amplitude modulation
fluorometer (PAM). The quantum yield of PSII (Fv′/Fm′) is
considered as effective efficiency of electron transport in a
reaction center under a light exposure. The maximum quantum
yield of PSII (Fv/Fm) can be measured under dark condition and
is considered as potential efficiency of electron transport in a
reaction center. On the other hand, the quenching parameters
are consisted of operating efficiency (qP) and excitation
pressure (1−qP). These parameters are considered as the
proportions of oxidative and reductive reaction centers,
respectively. Most reaction centers are oxidative at high
operating efficiency, while most reaction centers are reductive
at high excitation pressure.
Allocation of the light energy which is quantifying the fate
of light energy absorbed by the algal cell has become an
important aspect of algal photosynthetic research Recently,
Kato et al. (2003) suggests that the model of the allocation of
light energy absorbed by the algal cell can be estimated from
both quantum yields and quenching parameters. The
determination of the quantum yields provides a measurement of
constitutive energy loss at reaction center (ΦC). The
determination of both quantum yields and quenching
parameters allow estimating electron transport (ΦE) and
fluorescence emission (ΦF). Summation of those three energy
allocations, however, does not equal to the absorbed light
energy. The difference is considered as a non-photochemical
quenching (NPQ). This quenching is derived from a thermal
dissipation of the absorbed light energy through DD-cycle in
light-harvesting antenna. This allocation is estimated as a
regulated thermal dissipation (ΦR). Variability in the allocation
of the absorbed light energy should be associated with
photoacclimation strategy of PSII. In the present study, in order
to study on the photoacclimation in reaction center of PSII, the
allocation of the absorbed light energy is investigated in I.
galbana grown under different PFDs.
The present study focused on the adjustment of the balance
between photosynthetic and photoprotective capability to meet
the balance between light energy supply and demand in the
both light-harvesting antenna and reaction center of PSII in
Prymnesiophyceae Isochrysis galbana. I. galbana which
belong to algal group has one main light-harvesting system and
energy regulation mechanism. The objectives in this study are
(1) to examine the relationship between growth rate and growth
PFD to delimit the two light conditions in relative to light
energy supply and demand, (2) to determine the
photoacclimation of DD and DT in the light-harvesting antenna
in relative to the light conditions, and (3) to determine similarly
the photoacclimation of variable chlorophyll a fluorescence in
the reaction center. Finally, a model of the photoacclimation
strategy in PSII of I. galbana and requirements for future
research are presented.
Materials and Methods
Study 1. The relationship between light energy supply and
demand of I. galbana
I. galbana (NEPECC633) was obtained from the North
East Pacific Culture Collection at the University of British
Columbia. I. galbana was grown in 250 ml batch cultures in
enriched f/2 seawater medium at 25 ºC and PFD of 30, 60, 125,
250, 500 and 996 μmol photons m-2 s-1 provided by cool-white
fluorescent lamps with a 12h light and 12h dark cycle. Cells of
I. galbana were counted on an inverted microscope (Olympus).
The growth rate and growth PFD curve was estimated by fitting
the following equations;
μrel = 1 − exp (-E/KE)
(1)
where μrel is relative growth rate, E is the growth PFD, KE is
light saturation parameter. Growth saturation PFD (KG) was
defined as the growth PFD value when μrel was assumed to be
equal to 0.99.
Study 2. Photoacclimation of light-harvesting antenna of I.
galbana
I. galbana was preconditioned in 3 or 5 L continuous
culture which was maintained in enriched f/2 seawater medium
at 25 ºC and 35PSU salinity. Growth PFD of 45, 250, 425 and
1370 μmol photons m-2 s-1 provided by cool-white fluorescent
lamps with a 12h light and 12h dark cycle. The steady state of
growth rates was established at 0.3 day-1 by controlling the
dilution rate with a peristaltic pump. The PFD was determined
by a scalar quantum sensor. Subsamples were collected every 3
hours for the analysis of Chl a, DD and DT concentrations by
HPLC. Light-harvesting capability (LHC) was estimated from a
slope of least square regression analysis between Chl a specific
DD (DDChl a) and Chl a specific DD+DT ([DD+DT]Chl a). On
Results and Discussions
Study 1. The relationship between light energy supply and
demand of I. galbana
The intrinsic growth rate and PFD curve of I. galbana was
obtained by normalizing growth rate to the maximum growth
rate (μrel) (Fig.2). The growth saturation PFD, KG of I. galbana
was 365 μmol photons m-2 s-1. This suggests that a range of
light limited (LL) and light saturated (LS) conditions for I.
galbana correspond to lower and higher than 365 μmol photons
m-2 s-1, respectively.
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0
LS
1.0
365
Growth PFD
(μmol photons m-2 s-1)
Figure 2. Relationship between the relative growth rate, μrel and growth
PFD in I. galbana. LL, LS and KG indicate light limited, light saturated
conditions and growth saturation PFD, respectively. Data sources are as
follows: Falkowski et al. (1985) (square), Tzovenis et al. (1997)
(triangle), Jokiel and York (1984) (diamond) and the present study
(circle).
Study 2. Photoacclimation of light-harvesting antenna of I.
galbana
Light-harvesting capability (LHC) and thermal dissipative
capability (TDC) of I. galbana showed sigmoid response to
growth PFD (Fig.3). This sigmoid relationship was crossed at
349 μmol photons m-2 s-1. The maximum LHC and minimum
TDC observed under LL condition suggest that I. galbana
might have to enhance the light-harvesting capability to gain
much light energy. In contrast, the minimum LHC and the
0.2
500
1000
1500
Figure 3. Light dependence of light-harvesting capability, LHC
(circles) and thermal dissipative capability, TDC (squares) in I.
galbana.
Study 3. Photacclimation of reaction center of I. galbana
Decrease of the operating efficiency (qP) and increase of
the excitation pressure (1−qP) with actinic PFD were observed
regardless of growth PFD (Fig.4). In contrast, the maximum
quantum yield of PSII (Fv/Fm) and quantum yield (Fv'/Fm') were
relatively constant regardless of growth PFD. These results
suggest that the photoacclimation of reaction center can be
characterized for the redox state of reaction centers rather than
the efficiency of electron transport of a reaction center.
The qP and the 1−qP crossed under all growth PFD (Fig.4).
Average of the PFD at the cross point was 294±57 μmol
photons m-2 s-1. These results suggests that the state of reaction
centers of PSII in Isochrysis galbana change from oxidative to
reductive state upon a shift LL and LS condition.
1.0
1500
0.4
Growth PFD
(μmol photons m-2 s-1)
0.8
0
0.6
0
KG
n=18
r2 =0.94
p<0.0001
0.8
0
(A)
(B)
(C)
(D)
0.6
qP and 1−qP (relative unit)
μrel (relative unit)
LL
maximum TDC obtained under LS condition suggest that I.
galbana do not require much light energy and might have to
enhance the performance of dissipation of excitation energy
rather than absorption of photons.
LHC and TDC
(relative unit)
the other hand, thermal dissipative capability (TDC) was
estimated from a slope of least square regression analysis
between Chl a specific DT (DTChl a) and (DD+DT)Chl a.
Study 3. Photacclimation of reaction center of I. galbana
I. galbana was preconditioned in continuous culture and
was acclimated under the same experimental conditions
described in study 2. Subsamples for the measurements of
quantum yields of PSII and quenching parameters were
collected every 3 hours. The quantum yields of PSII under dark
and light conditions (Fv/Fm and Fv'/Fm'), and the quenching
parameters (qP and 1−qP) were measured by PAM (Obata et al.
2009). The Fv'/Fm', qP and 1−qP were determined at step-wise
increasing actinic light up to 2000 μmol photons m-2 s-1 with 30
sec illumination periods at each. Allocation of light energy
absorbed by algal cell to constitutive energy loss (ΦC), electron
transport (ΦE), fluorescence emission (ΦF), and regulated
thermal dissipation by DT (ΦR) were estimated by qP·Fv'/Fm',
1−Fv/Fm, (1−qP)·Fv'/Fm', and Fv/Fm−Fv'/Fm', respectively
(Demming-Adams et al. 1996).
0.4
0.2
0.0
1.0
0.8
0.6
0.4
0.2
0.0
0
500
1000
1500
2000 0
500
1000
1500
2000
Actinic PFD
(μmol photons m-2 s-1)
Figure 4. Response of operating efficiency, qP (circles) and excitation
pressure, 1-qP (squares) to actinic PFD in I. galbana acclimated at 45
(A), 250 (B), 425 (C) and 1370 (D) μmol photons m-2 s-1.
Allocation of light energy absorbed by the algal cell varied
with growth PFD in I. galbana (Fig.5). The ΦC ranged from 0.3
to 0.4. In contrast, the ΦE decreased from 0.6 to 0.1 and the ΦF
increased from 0.1 to 0.5 with growth PFD. The ΦE was high
under LL condition while the ΦF was high under LS condition.
This result suggests that I. galbana enhance the electron
transport under LL condition and the fluorescence emission
under LS condition. At the highest growth PFD, the ΦF reached
to 0.5 and ΦR was appeared. These results suggested that
energy loss depend on ΦF in I. galbana. Fluorescence emission
through the adjustment of the redox state of reaction centers
may be a critical photoprotective mechanism for I. galbana.
Energy allocation
(dimension less)
1.0
Light
LL
0.8
ΦR
0.6
ΦF
0.4
ΦE
0.2
ΦC
0.0
under LS condition (Fig.6). In I. galbana, main photoprotective
mechanism may be the fluorescence emission through the
adjustment of the redox state of reaction centers rather than
thermal dissipation by DT. A better knowledge of the
photoacclimation strategy is required to take account of the
balance between light energy supply and demand.
45
250
425
Fluorescence
emission
1370
Figure 5. Allocation of absorbed light energy for I. galbana. Bars of
white, right striped line, left striped line and black indicate constitutive
energy loss, ΦC, electron transport, ΦE, fluorescence emission, ΦF, and
regulated thermal dissipation by DT, ΦR, respectively. Sum of these
four fractions become unity.
Conclusions
Photoacclimation strategy of algae can be explained by
adjustments of the balance between photosynthetic and
photoprotective capability of PSII to meet the balance of light
energy supply and demand of algae. The growth PFD was
delimited at growth saturation PFD (KG) for two light
conditions; light limited (LL) and light saturated (LS)
conditions. Under the LL condition, the algal light energy
demand is overwhelmed the light energy supply while the light
energy supply is enough or excess for the light energy demand
under the LS condition. In both the light-harvesting antenna
and reaction center, photosynthetic and photoprotective
capability of Isochrysis galbana depend on the light conditions.
The acclimation of the light-harvesting antenna and the reaction
centers leads enhancement of allocation of absorbed light
energy to electron transport under LL condition and to
fluorescence emission under LS condition. Thermal dissipation
by DT is also occurred under LS condition. The similarity in all
estimates suggests that the photoacclimation strategy in PSII of
I. galbana shift from photosynthesis to photoprotection around
KG (Table 1).
Table 1. The growth saturation PFD, KG, the PFD of the cross point of
LHC and TDC and that of qP and 1-qP of I. galbana.
KG
Cross point of LHC and TDC
Cross point of qP and 1-qP
Chl a
Chl a
Electron transport
P680
Growth PFD
(μmol photons m-2 s-1)
PFD
DT
DD
μmol photons m-2 s-1
Source
365
Study 1
349
Study 2
294±57
Study 3
Under LL condition, I. galbana enhance photosynthetic
capability of PSII by increase of light-harvesting capability and
electron transport for maximizing photosynthetic rate while
enhance photoprotective capability of PSII by increase of
thermal dissipative capability in addition to fluorescence
emission for avoiding the damage from excess light energy
Light
LS
Fluorescence
emission
DD
Chl a
DT
Thermal
dissipation
Chl a
Electron transport
P680
Figure 6. Simplified schema of PSII related to photoacclimation
strategy in PSII of I. galbana under light limited (LL) and light
saturated (LS) condition. Dotted and shaded areas indicate
light-harvesting antenna while stripe area indicates reaction center.
Expansive application
The findings in the present study can support the
importance of the scaling of growth PFD for understanding of
photoacclimation strategy. The growth PFD normalized to KG
can serve effectively as the scaling of photoacclimiation for
comparative studies. By scaling of growth PFD in this manner,
photoacclimation strategy can be incorporated into the
photoadaptation strategy of algal community. I tested the
applicability of the photoacclimation strategy at the levels of
species to photoadaptation strategy at the level of community
by investigation of photoadaptation in PSII of ice algal
community which inhabits under LL condition defined in study
1. I conducted in situ incubation experiment at Saroma-ko
Lagoon, Hokkaido, Japan (Obata & Taguchi 2009). Ice algal
community in Saroma-Ko Lagoon, Hokkaido enhanced the
light-harvesting capability and electron transport in PSII and
allocation of absorbed light energy to electron transport. The
validity in applicability of the photoacclimation strategy to the
photoadaptation strategy was demonstrated by the experimental
observations of the ice algal community as well as I. galbana
under LL condition. This can be supported by the
photophysiological studies of ice algal community in
Saroma-Ko Lagoon, Hokkaido (Obata & Taguchi 2009).
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