Impact of GET3B on chloroplast proteome, PS II assembly and
carbon fixation
The proteomic analysis provided a comprehensive overview of the
downstream effects caused by the absence or reduction of GET3B. This was
crucial in identifying the specific proteins and pathways most affected
by the get3b mutation, shedding light on the multifaceted role of
GET3B in chloroplast biogenesis and function. One of the most striking
findings from the proteomic analysis was the global effect on the
abundance of plastudic proteins in the get3b mutant (Figure 1).
These encompass a wide functional range, suggesting that GET3B has a
broad impact on chloroplast proteostasis.
The entire photosynthetic apparatus of higher plats contains 16 single
pass IMPs (Shi and Schröder, 2004): PS II contains 12 of which 7 have a
TA topology (Wei et al., 2016) PS I comprises two of which none have a
TA topology (Pan et al., 2018) and the ATP synthase encompasses two of
which one shares a TA topology (Hahn et al., 2018). Taking the
postulated function of GET3B into account as the central element of the
stromal TA targeting machinery one would expect the corresponding
substrate to be reduced in a genetic background of a knock-out or
knock-down. Remarkably, the only single pass IMP that was significantly
depleted in get3b was PsbR (Supplemental table S1). Together with
PsbO, PsbP and PsbQ, PsbR forms the OEC and represents its only IMP
(Mishra and Ghanotakis, 1993). To date structural analyses of PS II of
higher plants were unable to resolve the location and topology of PsbR
(Van Bezouwen et al., 2017). However, previous biochemical analyses
corroborated that the majority of the elongated N-terminus is oriented
to the thylakoidal lumen (Ljungberg et al., 1984; Ljungberg et al.,
1986). If this topology is correct, then no bona fide photosynthetic TA
protein was significantly depleted by the absence or reduction of GET3B.
This implies that either GET3B is not involved in the targeting of TA
proteins or another factor can compensate for its absence (see next
section: genetic and physical interactions with STIC component and
SRP54).
The significant reduction of Fv/Fm values during the de novo assembly of
PS II highlight the important role of GET3B in early chloroplast
biogenesis. The partial restoration of emitted fluorescence and
concomitant PS II functionality in the presence of an external carbon
source suggests that GET3B may play a crucial role under autotrophic
conditions (Figure 2A-C). The observed reduction of Fv/Fm values during
de-etiolation could either arise from impediments in OEC
assembly/function or the depletion of key photosynthetic assembly
factors (Supplemental table S1). It is difficult to dissect whether this
reduction actually results from an incomplete assembly of PS II or
solely an impeded OEC of PS II. Further analyses are necessary to
clarify this.
Components of chlorophyll biogenesis pathways such as CHL synthase,
magnesium chelatase and several assembly factors as well as subunits of
RuBisCo were significantly depleted in get3b (Supplemental table
S1). This directly correlates with the reduced chlorophyll content of
young get3b seedlings (Figure 2D) as well as the reduced carbon
assimilation (Figure 3), respectively. These observations further
support the conclusion that GET3B is integral to the proper formation
and function of the entire photosynthetic apparatus. However, at this
stage it is hard to dissect if these are due to the direct function of
GET3B or secondary pleiotropic effects.
The proteomic analysis provided critical insight into the molecular
underpinnings of the get3b mutation. GET3B appears to be vital
for the maintenance of a wide range of chloroplast proteins,
particularly those involved in photosynthesis. The broad impact of GET3B
on chloroplast proteome underscores its central role in chloroplast
biogenesis and function, with potential implications for understanding
how chloroplast proteostasis is maintained under various physiological
conditions.