A Direct ALAD-SSUII Interaction Implies a Potential Link Between Tetrapyrrole and Terpenoid Pathways Toward Chlorophyll Biosynthesis in Plants.

Chlorophylls are the major light-harvesting pigments in photosynthetic organisms. Their biosynthesis requires the coordinated supply of metabolic intermediates from two independent upstream branches: the methylerythritol 4-phosphate (MEP)-derived terpenoid pathway, which supplies the phytyl side chain via geranylgeranyl diphosphate (GGPP), and the tetrapyrrole biosynthesis pathway (TBP), which provides the porphyrin ring. How flux through these two branches is coordinated remains poorly understood. In this study, we report the identification of a direct protein-protein interaction between δ-aminolevulinic acid dehydratase (ALAD), the second enzyme of the TBP, positioned immediately upstream of the first metabolic branch point, and the Type II small subunit of GGPP synthase (SSUII), a key regulator of terpenoid flux toward chlorophyll biosynthesis. ALAD was identified as a candidate SSUII-interacting protein by co-immunoprecipitation coupled with LC-MS analysis of rice leaf tissue, with a sequence coverage of 57.04%. The interactions between OsALAD1 and OsSSUII in rice, and between AtALAD1 and AtSSUII in Arabidopsis thaliana, were validated by yeast two-hybrid assay and bimolecular fluorescence complementation (BiFC) in Arabidopsis protoplasts. BiFC imaging demonstrated that the interaction is localized to the chloroplast. Sequence analysis revealed that plant ALAD proteins are highly conserved, with 92% similarity between OsALAD1 and AtALAD1, and 76.9% similarity between OsALAD1 and the green alga Chlamydomonas reinhardtii CrALAD1, indicating cross-species conservation of the ALAD-SSUII interaction. In vitro enzyme activity assays showed that AtSSUII does not directly alter AtALAD1 catalytic activity, suggesting the interaction operates through post-translational rather than direct catalytic mechanisms. Overexpression of AtALAD1 caused severe chlorosis and seedling lethality, while AtSSUII overexpression produced no distinct phenotype; neither transgene altered the transcript level of the other. Together, our results reveal a conserved cross-pathway protein-protein interaction linking the terpenoid regulatory machinery to the early TBP, suggesting a molecular possibility for the coordinated regulation of chlorophyll biosynthesis.