Additional file 2: Figure S2. of Quantitative proteomics of the tobacco pollen tube secretome identifies novel pollen tube guidance proteins important for fertilization

Assessment of pollen tube integrity, secretome purity using an alcohol-dehydrogenase (ADH) assay, and secretion activities in pollen tubes. a Top panels: pollen tube penetration (left) and exit (right) through the transmitting tract of the style visualized with aniline blue. Bottom panels: excised stylar ends visualized by DAPI stain marking cell nuclei (asterisk, left) and by toluidine blue (right) outlining cell wall boundaries. Excised ends of pistils are marked with the dashed line; red and white arrowheads mark the upper border of pistil broken cells following excision; and yellow arrowheads mark the upper and lower borders of intact cells. Scale bars = 5 μM. b Frequency of viable and non-viable semi-in vivo pollen tubes as well as integrity following 48 h growth through stylar tissues and in vitro. Error bars represent ± standard error; asterisks indicate statistical significance by Student’s t-test with p values indicated. c Purity assessment using an ADH assay of the secretome samples. ADH activities were calculated as nmole/min/ml. SIV-PS1–3 SIV-pollen tube secretome samples 1–3. d–f Live-cell imaging of endocytic tracer FM4-64 uptake and recycling in tobacco in vitro germinated pollen tubes co-localized with ER tracer in the absence (d) or presence (e, f) of brefeldin A (BFA), an inhibitor of protein secretion. Addition of BFA resulted in aggregation of endocytic vesicles and formation of BFA compartments (arrows), evidence of a defective conventional protein secretion pathway. BFA-treated pollen tubes showed specific defects only in FM4-64 labeled vesicles and not in the ER network. Scale bars = 5 μM. (TIF 1554 kb