Phosphorylation in two acts: Marchantia phototropin undergoes sequential cis- and trans-autophosphorylation

Abstract

Plants optimise photosynthetic performance and protect themselves from light-induced damage by sensing the intensity, periodicity, composition and direction of light. Consequently, plants have evolved several sets of photoreceptors to monitor their light environment. Phototropins are UV-A and blue light receptors that regulate responses such as phototropism, leaf flattening, chloroplast positioning and stomatal opening (Hart & Gardner, 2021). Among these, light-induced chloroplast movement is one of the most conserved processes and can be observed not only in seed plants, but also in green algae, mosses and liverworts. Under low light conditions, chloroplasts accumulate at cell walls and orient perpendicular to the incoming light, maximising photosynthetic efficiency. Strong light intensities instead elicit an avoidance response where chloroplasts reorient parallel to the incoming light. This is generally thought of as a photoprotective mechanism, avoiding light-induced damage to the photosynthetic apparatus, but also allowing light to penetrate into deeper tissues (Łabuz et al., 2022).

Phototropins contain two light-sensing LOV (Light, Oxygen, or Voltage-sensing) domains and a C-terminal serine/threonine kinase domain. In darkness, the LOV2 domain represses the kinase activity. Upon light absorption, this repression is relieved, triggering autophosphorylation of the kinase domain at multiple serine and threonine residues – an essential step in phototropin signalling (Hart & Gardner, 2021). Phototropins form dimers, suggesting that autophosphorylation can occur both in cis (by the molecule's own kinase domain) and in trans (by its dimer partner). Arabidopsis phototropin 1 (Atphot1) was shown to undergo trans-autophosphorylation (Kaiserli et al., 2009; Petersen et al., 2017), but such mechanistic insight remains scarce for phototropins of other species.

Yutaka Kodama got interested in phototropins because of their major role in chloroplast movement. He worked on chloroplast biology during his postdoc in the lab of Masamitsu Wada at the National Institute for Biology and Kyushu University, after which he gained further expertise in bioimaging with Chang-Deng Hu at Purdue University. He started his own lab at Utsunomiya University in 2011, where his research focuses on organelle biology and developing innovative imaging technology. Studying chloroplast dynamics in response to external stimuli led him to explore phototropin function. Minoru Noguchi, currently a PhD candidate in the group, is particularly interested in phototropin signalling pathways and has been studying phototropin function in the liverwort Marchantia polymorpha (Marchantia).

Marchantia harbours a single copy phototropin gene (MpPHOT) that mediates chloroplast accumulation and avoidance responses under weak and strong light, respectively (Komatsu et al., 2014), as well as avoidance responses at low temperatures (Fujii et al., 2017). In the highlighted publication, Noguchi and colleagues investigated the effects of these light and temperature conditions on Mpphot autophosphorylation. They observed that the Mpphot protein in extracts from blue-light-grown plants appeared larger (i.e. exhibited slower mobility) on an immunoblot than protein from dark-grown plants, suggesting increased autophosphorylation. Interestingly, the level of phosphorylation differed with light intensity and temperature: while weak blue light caused a moderate mobility shift at 22°C, both strong blue light at 22°C and weak blue light at low temperatures of 5°C resulted in a stronger mobility shift, suggesting a further increase in phosphorylation (hyperphosphorylation) (Figure 1a).

To identify Mpphot's phosphorylation sites, Noguchi et al. generated Marchantia lines expressing Citrine-tagged Mpphot (Mpphot-Cit). Mass spectrometry analysis of purified Mpphot-Cit identified a total of 23 serine/threonine phosphosites, 21 of which were also detected in dark-treated samples. Weak blue light resulted in an overall increase in the number of phosphopeptides at all 23 sites, with further increases observed under low temperature, suggesting that activation of Mpphot occurs via a global rise in serine/threonine phosphorylation across the entire protein, rather than via site-specific modifications.

Noguchi et al. then generated a kinase-inactive Mpphot variant (Mpphot-KI-Cit) by mutating a conserved aspartic acid residue in the kinase domain to prevent cis-autophosphorylation. When introduced into wild-type Marchantia plants, Mpphot-KI-Cit could only undergo trans-autophosphorylation by endogenous Mpphot and did not display a mobility shift under weak blue light, suggesting that primary autophosphorylation under these conditions requires cis-autophosphorylation. However, a mobility shift was detected for Mpphot-KI-Cit under strong blue light or at low temperature, suggesting that these conditions promote trans-autophosphorylation. A time course analysis during a shift from darkness to strong blue light showed that cis-autophosphorylation preceded trans-autophosphorylation: endogenous Mpphot, but not Mpphot-KI-Cit, underwent primary autophosphorylation within a minute, while both proteins displayed a similar increase in phosphorylation from 3 min onward. Trans-autophosphorylation did not require blue-light perception: a mutant version lacking the chromophore binding sites in both LOV domains (Mpphot-LKI-Cit) showed comparable phosphorylation levels to that of Mpphot-KI-Cit.

Interestingly, Mpphot-KI-Cit exerted a dominant negative effect on endogenous Mpphot. While Mpphot autophosphorylation levels were not affected under weak blue light, Mpphot-KI-Cit suppressed Mpphot trans-autophosphorylation under strong blue light or at low temperature. This suppression also impaired Mpphot function: Mpphot-KI-Cit lines showed a wild-type-like chloroplast accumulation response under weak blue light, but the avoidance responses were attenuated under strong blue light or at low temperature, suggesting that trans-autophosphorylation of endogenous Mpphot is critical for cold and high light acclimation in Marchantia.

Noguchi et al. show for the first time that a phototropin undergoes both cis- and trans-autophosphorylation in a coordinated manner, providing novel insight into phototropin's mode of action in Marchantia. The combination of cis- and trans-autophosphorylation allows for rapid signal amplification, which may increase the speed and efficiency of cellular responses to changing light environments. However, the exact consequences of distinct autophosphorylation events for Mpphot's function within the cell, as well as their effect on downstream signalling components, are still unknown. While some phosphorylation sites in the kinase domains are conserved, many other sites differ between Arabidopsis and Marchantia phototropins, including specific residues linked to interactions between Arabidopsis phototorpins and 14–3-3 proteins (Sullivan et al., 2021; Tseng et al., 2012). These observations suggest that, while the activation mechanism of phototropin kinases is likely conserved, species-specific signalling cascades exist that remain to be explored.