A pgrl1ab suppressor screen uncovers mutations in the metal-binding site of PGRL2 and the copper-transporting ATPase...

PROTON GRADIENT REGULATION5 (PGR5) promotes cyclic electron flow (CEF) and is essential for plant survival under fluctuating light conditions. PGR5 interacts with PGR5-LIKE1 (PGRL1), which stabilizes PGR5 and modulates its activity. In the absence of PGRL1, PGR5 is degraded through the action of PGR5-LIKE2 (PGRL2). However, when both PGRL1 and PGRL2 are missing, PGR5 accumulates but exhibits altered functionality. A suppressor screen in Arabidopsis (Arabidopsis thaliana) pgrl1ab plants identified 24 mutations affecting 14 proteins, including five previously implicated in pgr5 suppression. Six of the mutations disrupted photosystem II function, one affected cytosolic enzymes, six impaired cytochrome b6f assembly, and one influenced the regulation of tetrapyrrole biosynthesis. PGRL2 and the copper-transporting ATPase PAA1 were each targeted by five mutations. PGRL2 mutations restored growth and CEF in pgrl1ab plants by stabilizing PGR5 levels, with one truncating mutation rendering PGRL2 non-functional. AlphaFold3 predictions suggested a ternary PGR5-PGRL1-PGRL2 complex where the conserved cysteine pairs of PGRL2 coordinate Fe3⁺/Zn2⁺ ions near PGR5. Other PGRL2 mutations were clustered around these metal-binding sites, where they potentially destabilize PGRL2-PGR5 interactions by disrupting ion coordination. Mutations in PAA1 suppressed pgrl1ab lethality by perturbing linear electron flow more effectively than other perturbations of plastocyanin (PC) levels (caused by mutations in PAA2 or the PETE PC isoforms), primarily by restoring photosystem I donor-side limitation.