Light-Harvesting Protein LHCB4 Is Important for Stable Dimeric Organization of Photosystem II

Light-harvesting protein LHCB4 is a monomeric antenna of photosystem II (PSII) that connects the PSII core (C) with strongly (S) and moderately (M) bound LHCII trimers, forming the stable dimeric supercomplex C₂S₂M₂. LHCB4 is known to stabilize LHCII attachment to the PSII core dimer (C₂). Here, we show that LHCB4 is essential for the intrinsic stability of the PSII core dimer itself, a fundamental structural unit required for efficient photochemistry. In the lhcb4 mutant, clear-native PAGE revealed the absence of C₂S₂M₍₂₎ and only small amounts of C₂S₂ and C₂S supercomplexes. Single-particle electron microscopy identified two types of C₂S₍₂₎ supercomplexes: a canonical configuration with an empty LHCB4 site and a more abundant "packed" structure, arising from a relative shift of the C(S) monomers. However, most PSII particles were present as monomeric CS complexes with attached LHCB5 antenna. In contrast, PSII analyzed directly in thylakoid membranes retained the canonical C₂ organization and even formed nonparallel associations of C₂S₂ supercomplexes, indicating that the "packed" C₂S₂ and CS forms arise during isolation due to reduced PSII dimer stability. The PSII core dimer also readily disintegrates in the mutant lacking all monomeric antenna proteins (NoM), implying that LHCB4 is critical for the stability of the C₂ dimer itself. Consistently, lhcb4 plants exhibited reduced thermal stability of PSII macro-organization and photochemistry in vivo. Our findings demonstrate that LHCB4 is not merely a peripheral antenna connector, but also an important structural determinant of PSII core dimer stability, underscoring its essential role in efficient photochemistry and photoprotection.