ALA inhibits pear pollen tube growth through regulation of vesicle trafficking

5-Aminolevunic acid, a natural plant growth regulator, can thin pear fruits by inhibiting pollen tube growth through Ca2+-ATPase-mediated Ca2+ efflux. Here, using a non-invasive micromeasurement technology, we detected the occurrence of an ALA-induced Ca2+ efflux. Transmission electron microscope observations showed that a disruption of the intracellular Ca pool, including endoplasmic reticulum and mitochondria, was also linked to ALA-decreased Ca2+ in pollen tubes. Then we further explored the mechanisms how ALA-decreased Ca2+ inhibits pollen tube growth. We observed that ALA-inhibited pollen tube growth was accompanied by tube swelling and disruption in an ALA concentration-dependent manner. Ultrastructural studies further showed that ALA increased the number of vesicles, and the vesicle aggregation resulted in the morphological abnormality of pollen tubes. Confocal laser scanning microscopy using the fluorescent marker FM 4-64 confirmed the increasing number of vesicles induced by ALA in the pollen tubes. Ultrastructural analysis of pollen tubes treated by ALA with exogenous CaCl2 or/ and EGTA not only indicated that a Ca2+ signal plays a crucial role in ALA-mediated increase of vesicles, but also suggested that destruction of endoplasmic reticulum and Golgi bodies is one of the vesicle sources. Confocal microscopy of FM 4-64 further demonstrated that Ca2+ signal involves in ALA-mediated increase of vesicles also through promoting endocytosis and inhibiting exocytosis. In addition, treatment of taxol, a microtubule stabilizer, partially rescued the effects of ALA on pollen tube growth and FM 4-64 staining, indicating that microtubules are involved in ALA-regulated pollen tube growth. Taken together, we conclude that ALA-decreased Ca2+ disrupts endoplasmic reticulum and Golgi apparatus, enhances endocytosis, suppresses exocytosis, impairs vesicle transport, and finally leads to vesicle accumulation and abnormality of pollen tubes. Our findings provide new information on how ALA regulates pollen tube growth.