Exfiltration from the chloroplast: A shuttle protein shows the way to the nucleus

Plastids originated from an endosymbiosis event that occurred between an ancient cyanobacterium and a eukaryotic cell. The evolution of the endosymbiont towards an organelle was accompanied by the appearance of an intimate organelle-to-nucleus communication system inducing gene expression changes in the nucleus, called retrograde signaling. Retrograde signaling is induced by a sharp increase in light intensity and therefore may have contributed to the colonization of earth by the algal ancestor of land plants (Calderon and Strand, 2021).

An important contributor to retrograde signaling is the singlet oxygen (¹O₂) reactive oxygen species (ROS) produced through free chlorophyll or its precursors. Genetic screens have identified a number of genes required for ¹O₂-dependent gene expression changes. They encode either chloroplast-localized proteins such as EXECUTER1 (EX1; Wang et al., 2016) or nuclei-localized proteins such as Topoisomerase VI subunits (Simkova et al., 2012). However, the nature of the mobile molecule(s) between the chloroplast and nucleus, downstream of EX1, remained elusive. To identify new components of ¹O₂ signaling, Yuhong Li and colleagues (Li et al., 2022) screened for second-site mutations that suppressed the phenotype of PHYTOCHROME-INTERACTING FACTORS1 (PIF1) and PIF3 double mutants. This double mutant develops photobleached cotyledons under high light and accumulates higher levels of ¹O₂ than the wild-type.

Out of 100 independent suppressors of cotyledon photobleaching, 24 lines contained point mutations in either GENOME UNCOUPLED 4 (GUN4) or GUN5, two genes encoding factors required for the synthesis of chlorophyll precursors that can mediate ¹O₂ production upon increasing light intensity. Surprisingly, the authors showed that GUN4/5 interacted with EX1 in plastids by co-immunopurification (co-IP) and split luciferase experiments. Interestingly, GUN-EX interactions decreased with increasing light or upon treatment with rose bengal (RB), which induces ¹O₂ production (see Figure). By using fluorescence microscopy, the authors unexpectedly observed that EX1-GFP decreased in plastids, whereas it increased in the nucleus upon light irradiation or RB treatment. The shift in localization still occurred after blocking translation with cycloheximide, suggesting that it was caused by the movement of EX1 from the plastid to the nucleus. The above results suggest that upon the triggering of ¹O₂ production, the GUN-EX complex dissociates to release EX1 that relocalizes to the nucleus by a mechanism that remains unclear (see Figure). Importantly, the authors identified a motif of four lysines that acted as a nuclear localization signal and that was required in EX1 to complement the ex1 mutant phenotype, suggesting that EX1 relocalization to the nucleus is essential for its function.

Figure.

As a mobile signal in retrograde signaling. Upon an increase in light intensity, the production of ¹O₂ induces the dissociation of EX-GUN interactions to allow EX1 to relocalize to the nucleus. In the nucleus, EX1 interacts with WRKYs to regulate the expression of ¹O₂-responsive genes. Image credit: L.V. Méteignier, protein, chloroplast, and nucleus images from bioicons.com.

To elucidate the molecular role of EX1 in the nucleus, the authors examined the set of ¹O₂-responsive genes and found that a surprisingly high proportion contained a W-box, a cis DNA motif responsible for the binding of WRKY transcription factors, that is enriched in ¹O₂-responsive genes. They tested whether EX1 interacted with ROS-responsive WRKYs with yeast two-hybrid experiments and observed that EX1 physically associated with all the WRKYs tested. Furthermore, the authors then showed via transient co-IP experiments in planta that EX1 interacted more with WRKY18 and 40 in the light than in the dark, likewise after RB treatment. Importantly, overexpression of WRKY18 or 40 complemented the ex1 mutant phenotype, confirming that WRKY18/40 act downstream of EX1 to transduce the ¹O₂ signal. The authors further documented the interaction of WRKY18/40 with three ¹O₂-responsive gene promoters by yeast one-hybrid and chromatin IP (see Figure).

Taken together, these results suggest that upon ¹O₂ sensing, EX1 relocalizes from the chloroplast to the nucleus where it interacts with WRKY transcription factors to regulate ¹O₂-responsive genes upon sensing increasing light. Importantly, a previous study has shown that the post-translational modification and degradation of EX1 upon ¹O₂ sensing is important for ¹O₂ signaling (Dogra et al., 2019). Therefore, a number of questions are raised by the work of Li and colleagues, including: How is EX1 relocalized to the nucleus? How are the multiple interactions of EX1 with WRKYs orchestrated in planta? Future studies are required to clarify the hierarchy of events involving EX1 degradation, relocalization, and interactions during ¹O₂ signaling.