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.