The Integrated Single-Nucleus Transcriptome and High-resolution Spatial Multi-Omics Atlas of Cotton Ovule (Gossypium barbadense)

Cotton (Gossypium barbadense), prized for its superior fiber quality, undergoes critical ovule development from -1 to +1 days post-anthesis, yet the spatiotemporal molecular networks driving fiber initiation and elongation remain poorly understood. Here, we constructed a high-resolution multi-omics atlas of G. barbadense ovules by integrating single-nucleus RNA sequencing, spatial transcriptomics, matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI), and laser capture microdissection (LCM)-based proteomics. Pseudotime trajectory analysis uncovered a biphasic progression from preparatory metabolism to dynamic expansion, including an early auxin-repressive mechanism. This approach delineated 11 spatial domains, revealing epidermal fiber subtypes and novel metabolic marker (steryl lipid ST 30:0;O accumulating progressively during elongation) and transcripts markers (DUF538 and GH17). Notably, we identified a novel bilayer metabolic architecture in the inner integument: the inner layer serves as a TCA cycle energy hub providing ATP and carbon flux, while the outer layer functions as an upstream flavonoid biosynthesis center for defense and signaling. These findings elucidate the molecular orchestration of superior fiber traits in G. barbadense, offering actionable targets for genetic engineering and breeding to enhance fiber length, fineness, and strength. This integrated framework advances multi-omics applications in plant developmental biology.