Background

Being the basis of life on Earth, photosynthesis has become a major research focus from the mid-19^th century (Hill, 1937 and 1939; Benson and Calvin, 1950; Benson et al., 1950; Porter, 1950; Huzisige and Ke, 1993). Among several biochemical characterization methods, separation in polyacrylamide gel-based matrix of photosynthetic multi-subunit complexes, such as Photosystem I and II (PSI and PSII), was optimized from previous methods (Schägger and von Jagow, 1991; Kügler et al., 1997; Järvi et al., 2011; Haniewicz et al., 2015; Farci et al., 2017; Rantala et al., 2017). Furthermore, the solubilization approach to extract those complexes evolved over the last decades, moving from “harsh” toward “gentle” procedures by using detergents with lower critical micelle concentration (CMC) and at lower concentrations. The same is true for all procedures aimed at extracting membrane protein complexes for structural and functional analyses. Yet, sample preparation and biochemical characterization remain challenging.

In almost all biochemical studies, the isolation of protein complexes is of fundamental importance for the success of any project. Protocols need to be robust and reproducible to minimize variations between extractions. A crucial factor for successful isolation and characterization is the employment of the best-suited detergent depending on the material used, considering the CMC, incubation time, and the temperature during solubilization but also the mixing procedure. It is an even more delicate task to obtain protocols that keep the isolated membrane protein complexes intact, in a close-to-native state. A protein complex in solution does not necessarily have a native and stable structure; additionally, a detergent useful for extraction can be unsuitable for purification procedures and subsequent studies. Thylakoid membranes are solubilized with different detergents, such as n-Dodecyl-β-D-Maltopyranoside (β-DDM), n-Dodecyl-α-D-Maltopyranoside (α-DDM), and digitonin, at an efficiency depending on the thylakoid region (grana or stoma lamellae) and species (Barera et al., 2012; Haniewicz et al., 2013 and 2015; Albanese et al., 2016; Rantala et al., 2017). Isolated photosynthetic complexes have been fully characterized in Arabidopsis thaliana, Pisum sativum L., and Spinacia oleracea L. (Albanese et al., 2016; Rantala et al., 2017; Wood et al., 2018), and partially characterized in other species, such as Nicotiana tabacum L., Physcomitrella patens, Picea abies L. (Norway spruce), Selaginella martensii, Oryza sativa L., and Zea mays L. (D’Amici et al., 2008; Romanowska et al., 2008; Haniewicz et al., 2013 and 2015; Ferroni et al., 2014; Shen et al., 2017; Iwai et al., 2018; Grebe et al., 2019; Kouřil et al., 2020). Different detergent concentrations, mixtures of two detergents, and multi-step strategies (e.g., differential solubilization) are also used to optimize solubilization (Wientjes et al., 2013; Haniewicz et al., 2015). These approaches gained detailed knowledge on oligomeric states, subunit composition, and dynamics in PSII. To achieve reproducibility, most of these studies were performed on chamber-grown plants, but they do not necessarily yield answers to how photosynthetic regulation takes place under natural conditions. Conversely, studies on tree species (e.g., P. abies) growing in a challenging condition (like a boreal winter) provided a more complex picture of photosynthetic acclimation to environmental stress (Bag et al., 2020; Grebe et al., 2020; Chang et al., 2021). For these species, several differences in composition and abundance in photosynthetic complexes were reported: the presence of triple phosphorylation of the novel LHCB1 variant, needed to achieve winter-adapted chloroplast structures (Bag et al., 2020; Grebe et al., 2020); higher-order mega complexes (Kouřil et al., 2020), which are still a matter of debate in higher plants; and the absence of the PSI-NDH complex, mega-complexes, and the so-called M-LHCII complex (Bassi and Dainese, 1992; Nystedt et al., 2013; Kouřil et al., 2016). The latter is most likely an LHCII assembly containing both Lhcb3 and Lhcb6 that are missing in the Pinaceae family of conifers (Kouřil et al., 2016; Grebe et al., 2019) but present in A. thaliana.

Consequently, optimized biochemical procedures for the isolation and characterization of membrane complexes are needed to pursue such challenging studies, compare different species, and gain meaningful insights on structural/functional differences. In this manuscript, we use digitonin to apply and improve a mild-solubilization procedure for isolating thylakoid complexes from Norway spruce (P. abies) and Scots pine (P. sylvestris). The complexes are characterized by Blue-Native gel electrophoresis (BN-PAGE) and denaturing second dimension gel electrophoresis (2D SDS-PAGE). This newly optimized protocol will enable further characterization of PSI and PSII complexes by improving the extraction procedure in a close-to-native state, which, in these species, cannot be achieved by classic α-/β-DDM solubilization. Finally, the availability of this method opens paths for obtaining an in-depth understanding of the winter adaptation processes in evergreen pine trees.