OsTIP3;1 regulates rice seed germination via aleurone layer lipid metabolism: a transcriptome analysis

Tonoplast intrinsic proteins (TIPs) are essential for water transport and cell physiology, however, their specific roles in seed development and germination, particularly in lipid metabolism, remain poorly understood. This study elucidates the role of rice OsTIP3;1 using knockout (KN, tip3;1-ii ) and overexpression (OE, OE- TIP3;1-III ) lines. OsTIP3;1 knockout led to significant defects in glume morphology and aleurone grain structure, whereas overexpression did not exhibit any adverse effects. During germination, KN seeds showed delayed radicle and plumule elongation, reduced lateral root development, lower germination energy (GE) and germination index (GI), although the final germination rate (GR) remained unchanged. In contrast, OE seeds displayed only transient inhibition of early radicle and plumule growth. Laser confocal microscopy (LCM) revealed asynchronous vacuolar fusion in aleurone cells, which was more pronounced in KN and OE lines than in the wild-type (WT). Transcriptome analysis identified distinct molecular pathways among genotypes. KN lines exhibited delayed fatty acid degradation, downregulated early-stage lipid metabolic genes, and enriched late-stage endoplasmic reticulum protein processing pathways, while OE lines showed enhanced vesicle transport processes. Protein-protein interaction network analysis identified lipid metabolism genes LOC4335609 and LOC4340986 as central hubs across all genotypes. Notably, RNA-seq and qRT-PCR analyses showed two genes were downregulated in KN lines at 1 d and 3 d of germination compared to the WT. These findings suggest that OsTIP3;1 regulates rice seed germination potentially via modulating vacuolar fusion in the aleurone layer and coordinating lipid metabolic pathways to support efficient germination and seedling establishment.