Abstract
<title>Abstract</title> <p> The green alga <italic>Haematococcus pluvialis</italic> is of great economic importance in astaxanthin hyperaccumulation, yet the dynamic remodeling of lipid droplet proteomes during encystment remains poorly understood, hindering bioprocess optimization. Here, we constructed the first integrated spatiotemporal proteomic atlas of <italic>H. pluvialis</italic> lipid droplets (HPLDs) across five critical encystment stages, quantifying 3,396 high-confidence proteins. We reveal that HPLDs undergo profound stage-specific proteomic remodeling, redefine them as dynamic metabolic and communication hubs. Bioinformatic analysis revealed distinct functional shifts in HPLDs, including lipid metabolism, β-carotene biosynthesis, vesicular trafficking, antioxidant defense, and inter-organellar communication. Time-resolved clustering and pathway enrichment uncover four distinct expression modules, delineating a precise functional succession: from precursor mobilization and transient biosynthetic bursts (e.g., phospholipid:diacylglycerol acyltransferase surges 13.3-fold at HPLDs-3), through sustained ABC transporter-mediated trafficking, to dramatic structural reinforcement (Caleosin rises 11.5-fold at HPLDs-5) and the assembly of a compartmentalized antioxidant shield (Catalase surges 20-fold). Notably, terminal astaxanthin synthesis enzymes (BKT/CHYb) are absent from HPLDs, indicating spatial uncoupling of synthesis from storage and leading us to propose a conserved two‑step sequestration and stabilization model for ketocarotenoids. These findings establish HPLDs as protein-governed platforms that integrate chloroplast, endoplasmic reticulum, and mitochondrial functions to drive carbon repartitioning, redox homeostasis, and astaxanthin ester deposition. Beyond providing a mechanistic blueprint for engineering astaxanthin hyperaccumulation, this spatiotemporal proteome resource illuminates fundamental principles of LD functionalization in photosynthetic organisms—principles that are broadly shared with land plants and offer new targets for improving stress tolerance and lipid-based metabolite storage in green lineages. </p>