Discussion
The evidence presented here, at both spectral and protein levels,
confirmed that OEC of Z. marina was preferentially inactivated in
response to light exposure: (1)
PSII electron donation capacity
characterized by L 1 and W Kwas significantly decreased already during initial illumination; (2) OEC
activity represented by the contents of OEC peripheral proteins (Bricker
and Frankel, 2008; Popelkova et al.,
2011) greatly decreased, while the
reduction degree of QA at the acceptor-side, represented
by 1 - V J, slightly increased; (3)
PSII photoinhibition occurred upon
light exposure and the photoinhibition rate constant, determined via
first-order kinetic fits, was
positively correlated with PPFD at all tested light intensities. This
was the first in vivo study
of PSII donor-side photoinhibition
with the direct experimental evidence in higher plants under visible
light. The in vivo occurrence of this mechanism in higher
plants
enabled the study of both
integrated photosynthetic characteristics and photoprotective
mechanisms, which
involved
multiple coordinated systems.
The present study investigated the integrated photosynthetic regulatory
mechanisms
involved in light absorption, transfer and conversion, and energy
dissipation via chloroplast
proteome combined with
chlorophyll fluorescence phenotype.
The strong interactions of PsbO with Lhcb3, PGR5, HCF244, Psb29, PsaN,
and PsaH, shown in the PPI network, suggested that
OEC
played a central role in the regulation of
photosynthesis.
The decreased size of the PSI and
PSII
antennas
indicated a decline of the
light-harvesting capacity inZ. marina .
The
interactions of PsbO with PsaH and Lhcb3 were interpreted as a need to
decrease the light-harvesting capacity due to
the partially impaired OEC. It
should be realized that a decrease of PSII antenna size corresponds to
fewer absorbed photons but also to
a faster trapping rate (Croce and Van Amerongen, 2014). Simultaneously,
PSII assembly was activated to retain PSII
functionality.
Because continuously
impaired OEC led to a
relatively oxidized PQ pool
(Makarova et al.,
2007), the
PGR5-dependent
PSI-CEF
in Z. marina was activated to supplement the
PSI linear electron transport.
Activated
PSI-CEF revealed by proteome data in the present study was in agreement
with chlorophyll fluorescence physiological phenotype observed in our
previous study (Yang et al., 2017). The interactions between PsbO and
HCF244, as well as PGR5 and PsaN indicated the potential of the
partially impaired OEC to enhance PSII repair and activate the
PGR5-dependent PSI-CEF.
Continuous
impairment of OEC in this mechanism suggested
electron
deficiency in the electron
transport chain. Unchanged antioxidant levels and unactivated
alternative electron flows, such as
chlororespiration, Mehler reaction,
malic acid synthesis and photorespiration, supported this viewpoint. The
low PSII excitation pressure caused by
electron
deficiency made it difficult to form
a strong trans-thylakoid proton
gradient (ΔpH), and consequently, could not induce strong NPQ.
This was verified by the unchanged
ΔpH sensor PsbS protein, the low
value of NPQmax (Schubert et al.,
2015),
and slightly upregulated ZEP and VDE. NPQ is usually dissected into at
least three kinetic components, in which qE, induced within 20-60 s, was
the dominant NPQ component (Horton et al., 1996; Nilkens et al., 2010).
Further examination of the
dynamics of NPQ induction inZ. marina indicated the
absence of the fast qE component, which accounted for the slow
development of NPQ. Moreover, the decrease of NPQ during the late
illumination indicated that the demand for dissipation
decreased with the increased OEC
impairment. A low NPQ capacity has
also been reported in the seagrass Posidonia sinuosa (Schubert et
al.,
2015).
The low levels of NPQ were conductive to the formation of the reduced
form of NADPH and ATP derived from photosynthetic electron transport,
which in turn contributed to carbon fixation. In C3 plants,
photosynthesis is typically restricted by the Rubisco capacity due to
its exceedingly low catalytic turnover rate and competition with
oxygenation reaction (Farquhar et al., 1980). The significantly enhanced
carboxylase activity of Rubisco and the upregulated NTRC (Nikkanen et
al.,
2016)
suggested an increased CO2 fixation rate in response to
light exposure. Recoveries of
activities of GAPDH and PRK, the
key enzymes of carbon fixation,
were achieved by downregulated CP12
(Marri et al., 2009), suggesting the
activation of carbon fixation
pathway.
The well
photosynthetic performance during
photoinhibition was also supported by the normal photosynthetic rate
characterized by O2 evolution rate
in Z. marina .
1O2 is usually formed by a
photosensitization reaction in which an oxygen molecule reacts with3P680 (Pospíšil,
2016).
The sensitizer molecule3P680, produced
by3[P680+Phe−]
which is generated either via spin conversion of primary radical pair
P680+Phe− or via recombination of
P680+QA−, is
triggered by the over-reduction of QA (Vass et al.,
1992; Hakala et al., 2005; Ohnishi et
al., 2005; Vass, 2011).
The reduction degree of
QA was low in Z. marina , which was evidenced by a
slight decrease of 1 - V J. Therefore, the
conditions for 1O2 production were
not appropriate. In fact, light
exposure neither caused an upregulation of PsbH and GPX, nor an
increase of1O2levels. The strong interaction between PsbO and PsbH indicated that
there was no need for PsbH to stabilize the combination of excess
β-carotene and PSII (Hall et al.,
2016) to eliminate1O2. Moreover, the inhibition of
alternative electron donation in our study resulted in a significant
damage of PSII RC described
by
the net losses of D1 and CP43
proteins, indicating that the photodamage of
PSII was attributed to the
long-lived P680+rather than1O2.
Thus, the long-lived P680+ resulted from the
interrupted electron supply was usually considered as the damage source
in the PSII donor-side photoinhibition, which has been suggested by Vass
(2012) and Tyystjärvi (2008).
Noticeably, a distinct low descending amplitude ofF v/F mwas observed during light exposure, which could be explained by most
PSII RC remaining open to contribute to the depletion of the pool of
high-potential P680+ via facilitating the direct
charge recombination of the
P680+QA−. Because
the competition between3[P680+Phe−]
formation and the direct recombination of the
P680+QA− was
controlled by PSII excitation pressure (Vass and Cser,
2009),
the low reduction degree of QAin Z. marina prevented the
formation of3[P680+Phe−].
The existence of alternative PSII electron donors has been reported in
some species (Thompson and Brudvig, 1988; Tóth et al., 2009,
2011).
Based on in vivo data, the increased AsA level and the upregulated GLDH
content involved in AsA synthesis following light exposure provided a
clue of AsA as alternative electron donor. Similarly, in contrast to 62
μM DMBQ enhancing PSII-CEF, 125 μM DMBQ uncoupling PSII-CEF induced the
lower PSII electron donation capacity and PSII photochemical activity,
providing a clue of PSII-CEF as
another alternative donation pathway. Furthermore, a factorial design
experiment with different combinations of AsA and PSII-CEF inhibitions
demonstrated that the suppression of alternative electron donation
damaged the PSII component, including a decrease inF v/F m, an increase inW K, as well as net losses of PSII RC proteins and
OEC peripheral proteins. With the duration of light exposure, the
inhibition effect became more
significant, of which, the most severe damage of PSII was caused by
the dual inhibition of AsA and
PSII-CEF. Based on these results, we suggested that both AsA and
PSII-CEF were important photoprotective mechanisms, which provided
electrons to remit the oxidative stress from long-lived
P680+ during light exposure.
In conclusion, the water-splitting dysfunction caused by OEC
photoinactivation interrupted the
electron supply from water to the oxidized primary donor
P680+, resulting in the damage to the PSII RC ofZ. marina . At least
PSII-CEF and the alternative donor
AsA exerted photoprotective roles in the depletion of
P680+ by donating electrons to
PSII (Fig. 7). In contrast to
acceptor-side photoinhibition caused by electron excess, continuous
impairment of OEC resulted in electron deficiency in the electron
transport chain during the PSII photoinhibition. For the efficient use
of the limited electrons, NPQ was inefficient and the alternative
electron flows associated with energy dissipation, such as
chlororespiration, Mehler reaction, malic acid synthesis and
photorespiration, were not significantly activated to
decrease unnecessary consumption
(Fig. 7). The extremely sensitivity of OEC to visible light was
presumably a result of insufficient polyphenol levels
caused by a lack of blue light
photoreceptors in a habitat that is rich in blue light (Peng and
Moriguchi, 2013; Jiang et al., 2016).