The DnaJ-like zinc finger domain-containing proteins are involved in different aspects of plastid function and development. Some of these proteins were recently reported to have dual subcellular localization in the nucleus and plastids. One member of this family, PSA2 (AT2G34860), was found to localize to the thylakoid lumen and regulate the assembly of photosystem I (PSI). However, PSA2 was also annotated as Embryo sac Development Arrest 3 (EDA3) from the observation that its embryo sac development was arrested at the two-nuclear stage. In this study, we characterized the eda3 mutant, and demonstrated that, as compared with the wild-type (WT) plants, the mutant has shorter siliques, fewer siliques per plant, and fewer seeds per silique. Both aborted and undeveloped ovules were observed in siliques of the mutant. By immunoblot analysis, we found that, different from the chloroplast localization in mature leaves, EDA3 localizes in the nucleus in seeds. A nuclear localization signal was identified from the deduced amino acid sequence of EDA3, and also proved to be sufficient for directing its fusion peptide into the nucleus.

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.

Cyanidioschyzon merolae, an extremophilic unicellular red alga thriving in acidic hot springs at temperatures of 40–56 °C and pH 0.5–4.0, faces extreme oxidative stress conditions. This study presents a comprehensive genomic analysis of the carotenoid and vitamin E biosynthetic pathways, which are essential for antioxidant defense in this organism. Through comparative genomics using Arabidopsis thaliana sequences as queries, we identified and characterized genes encoding key enzymes involved in their metabolism. Our analysis reveals that C. merolae exclusively utilizes the methylerythritol-4-phosphate (MEP) pathway for isoprenoid biosynthesis and lacks a complete mevalonate (MVA) pathway. We identified eleven genes involved in terpenoid metabolism and seven genes specifically for carotenoid biosynthesis. Pigment analysis confirmed a streamlined carotenoid profile consisting solely of β-carotene, β-cryptoxanthin, and zeaxanthin, lacking the entire β,ε-branch and part of the β,β-branch. The complete tocopherol biosynthetic pathway produces exclusively α-tocopherol. The absence of the β,ε-carotenoid branch and the exclusive production of α-tocopherol demonstrate metabolic streamlining while maintaining antioxidant efficacy. These findings provide molecular blueprints for biotechnological applications, enabling targeted strategies to enhance antioxidant production through pathway optimization and metabolic engineering, while offering insights into developing stress-tolerant organisms and enhancing nutritional content in crops.