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Simple Summary: Capacitation confers competency to spermatozoa to fertilize the oocyte, yet its regulatory mechanisms are not fully understood. Thus, we aimed to investigate the comparative proteomic profiling in ram spermatozoa under non-capacitating (NC) and capacitating (CAP) conditions in vitro using a liquid chromatography–tandem mass spectrometry (LCMS/MS) combined with tandem mass tag (TMT) labeling strategy. Functional enrichment analysis indicated that the differentially abundant proteins Prune Exopolyphosphatase 1, Galactose-1-Phosphate Uridylyltransferase, and ATP Citrate Lyase were strictly related to energy production and conversion, and Phosphoglycolate phosphatase, Glucosamine-6-Phosphate Deaminase 1 and 2 were related to metabolism, RNA processing, and vesicular transport pathways. Furthermore, the networks of protein–protein interaction indicated a strong interaction among these differential proteins in annotated pathways such as ubiquitin and transport metabolism. Together, our results provided the database for studying the differentially expressed proteins during ram sperm capacitation. Ram sperm undergo a sequence of physiological and biochemical changes collectively termed as capacitation to perform oocyte fertilization. However, the protein changes induced by capacitation remain in need of further exploration. Thus, the present study investigated the comparative proteomic profiling in ram spermatozoa under non-capacitating (NC) and capacitating (CAP) conditions in vitro using a liquid chromatography–tandem mass spectrometry combined with tandem mass tag labeling strategy. As a results, 2050 proteins were identified and quantified; 348 of them were differentially abundant, with 280 of the proteins upregulated and 68 of the proteins downregulated between the CAP and NC spermatozoa, respectively. Functional enrichment analysis indicated that the differentially abundant proteins Prune Exopolyphosphatase 1, Galactose-1-Phosphate Uridylyltransferase, and ATP Citrate Lyase were strictly related to energy production and conversion, and Phosphoglycolate phosphatase, Glucosamine-6-Phosphate Deaminase 1 and 2 were related to metabolism, RNA processing, and vesicular transport pathways. Furthermore, the networks of protein–protein interaction indicated a strong interaction among these differential proteins in annotated pathways such as ubiquitin and transport metabolism. Our findings indicate that capacitation progress might be regulated through different pathways, providing insights into mechanisms involved in ram sperm capacitation and fertility. [ABSTRACT FROM AUTHOR]

Seed germination is a complex process that is regulated by various exogenous and endogenous factors, in which abscisic acid (ABA) plays a crucial role. The triphosphate tunnel metalloenzyme (TTM) superfamily exists in all living organisms, but research on its biological role is limited. Here, we reveal that TTM2 functions in ABA-mediated seed germination. Our study indicates that TTM2 expression is enhanced but repressed by ABA during seed germination. Promoted TTM2 expression in 35S::TTM2-FLAG rescues ABA-mediated inhibition of seed germination and early seedling development and ttm2 mutants exhibit lower seed germination rate and reduced cotyledon greening compared with the wild type, revealing that the repression of TTM2 expression is required for ABA-mediated inhibition of seed germination and early seedling development. Further, ABA inhibits TTM2 expression by ABA insensitive 4 (ABI4) binding of TTM2 promoter and the ABA-insensitive phenotype of abi4-1 with higher TTM2 expression can be rescued by mutation of TTM2 in abi4-1 ttm2-1 mutant, indicating that TTM2 acts downstream of ABI4. In addition, TTM1, a homolog of TTM2, is not involved in ABA-mediated regulation of seed germination. In summary, our findings reveal that TTM2 acts as a downstream factor of ABI4 in ABA-mediated seed germination and early seedling growth. [ABSTRACT FROM AUTHOR]