get3b mutation affects de novo assembly of photosystem II
The data from the comparative proteomics indicated that the light reaction as well as assembly factors of the photosynthetic machinery, like YCF3 and CRR6, were affected in get3b (Figure 1E-G, supplemental table S1). This prompted us to examine a possible involvement of GET3B function in photosystem assembly. In order to address this, we employed PAM measurements during de-etiolation ofA. thaliana seedlings. These measurements exploit the fluorescence solely emitted by PS II, since PS I is a fluorescence quencher (Brettel, 1997). We utilized Fv/Fm values as a read out for PS II functionality. By employing this during de-etiolation we were able to assess the extent of de novo formation of PS II. For this, seedlings were germinated and cultivated in vitro on Murashige Skoog (MS) medium (Murashige and Skoog, 1962) for 4 days in the dark and in the morning of the fifth day illuminated normally. The course of de-etiolation and concomitant de novo PS II assembly was encompassed by PAM measurements. This occurred 1, 7 and 24 hours after the initial illumination.
When cultivated without a carbon source, get3b was significantly hampered in the assembly of PSII in comparison to wild-type after 7 and 24 hours (Figure 2A; top panel). In the presence of a carbon source, this difference was only apparent after 24 hours and not to such a high degree of significance, indicating that PS II functionality was still hindered in the absence or reduction of GET3B when grown heterotrophically (Figure 2A; bottom panel).
As a control we grew the same plates for five days without etiolation and analyzed these fluorometrically. When cultivated autotrophically,get3b was still significantly obstructed in PS II assembly (Figure 2B; top panel). Conversely, when grown in the presence of a carbon source, no difference was detectable with PAM measurements (Figure 2B; bottom panel).
Being able to observe significant differences after five days of growth under normal irradiation, prompted us to analyze 4-day-old seedlings under regular illumination in the absence of sucrose. The corresponding PAM measurements mirrored the previous assessment and confirmed an impaired PSII assembly in get3b (Figure 2C). Consistently,get3b also exhibited a significant reduction in chlorophyll content (Figure 2D).
Taken together these data indicated that, in get3b mutants, not only the initial stages (up to 24 hours) of de novo assembly of PS II were affected but also the later stages (up to 5 days). Additionally, the reduced chlorophyll content of get3b illustrated the obstruction of the photosynthetic machinery during early plant development.