Fig. 2: PA composition analysis in 7 DAP siliques of the A. thaliana ans-4 mutant.

a–c HPLC-QqQ chromatograms for characterization of the soluble PA fraction in 7 DAP siliques of the A. thaliana ans-4 mutant and wild-type (WT) plants. a MRM transition of m/z (289 → 123) showing that trace amounts of (−)-epicatechin remained in the ans-4 mutant. b MRM transition of m/z (577 → 407) showing that the ans-4 mutant has lost B-type procyanidin dimer. c MRM transition of m/z (865 → 407) showing that the ans-4 mutant has lost B-type procyanidin trimer. d Visualization of the presence of PA extension units in 7 DAP siliques of ans-4 mutant and WT by the butanolysis method. The upper panel shows the non-lyophilized soluble PA and the residues containing insoluble PA and total PA before applying butanolysis. Soluble PA was extracted with acetone/water followed by chloroform extraction to remove chlorophyll. Insoluble PA remained in the residues after soluble PA extraction. The residues containing total PA were obtained as the ground tissue only with chloroform extraction to remove chlorophyll. The lower panel shows the butanolysis supernatant of the lyophilized soluble PA and the residues containing insoluble PA and total PA after reaction. Procyanidin B3 (0.25 mM) standard (sd) served as the positive control. e Quantification of total PA extension units in 7 DAP siliques of ans-4 mutant and WT. PA extension levels were determined by the butanol-HCl method and expressed as procyanidin B3 (PB3) equivalents. FW: fresh weight. Data are shown as the mean ± SD (for n = 3 biologically independent samples; *P < 0.05, two-tailed unpaired Student’s t tests). Source data of Fig. 2e are provided as a Source Data file.