Effect of warm and cold stratification on ¹H-NMR profiles, endogenous gibberellins and abscisic acid in Styrax japonicus seeds

Germination of Styrax japonicus seeds is promoted by warm stratification (WS) at 18-20°C followed by cold stratification (CS) at 4-5°. The objective of this work was to analyze the state and mobility of water molecules measured by ¹H-NMR and endogenous gibberellins (GAs) and abscisic acid (ABA) by ultra performance liquid chromatography/mass spectrometry/mass spectrometry (UPLC-MS/MS) as influenced by WS and CS treatments had not previously been investigated. Styrax seeds that received 35 days of WS (35D WS) followed by 63 days of CS (63D CS) (35D WS + 63D CS) germinated. Seeds that received only 35D WS failed to germinate. Endogenous GA₁, GA₈, GA₁₉, GA₂₀, and GA₅₃ were identified as well as GA₁₇, GA₂₃, GA₂₈, GA₂₉, and GA₉₇ by gas chromatography/MS (GC/MS) and UPLC-MS/MS in seeds that were treated with warm and cold stratification (WS + CS). This suggests that the early C-13 hydroxylation pathway [-GA₅₃-(GA₄₄)-GA₁₉-GA₂₀-GA₁-GA₈] of GAs is a major biosynthetic pathway in the seeds. The concentration of GA₅₃ and GA₁₉ increased following WS and that of GA₅₃ increased after WS + CS. The concentration of GA₁₉ increased only slightly after WS + CS. The concentration of GA₁ increased only after WS + CS. ABA concentration significantly decreased following the WS treatment. It is concluded that the mobility of water molecules and water content in cotyledons and endosperm is increased following WS + CS treatments. The occurrence of C-13 hydroxylated GAs suggests that the early C-13 hydroxylation pathway, → GA₅₃ → GA₄₄ → GA₁₉ → GA₂₀ → GA₁ → GA₈, is a major biosynthetic pathway in Styrax seeds.