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Summary: Style penetration by pollen tubes is essential for reproductive success, a process requiring canonical Rab5s in Arabidopsis. However, functional loss of Arabidopsis Vps9a, the gene encoding for guanine nucleotide exchange factor (GEF) of Rab5s, did not affect male transmission, implying the presence of a compensation program or redundancy.By combining genetic, cytological, and molecular approaches, we report that Arabidopsis Vps9b is a pollen‐preferential gene, redundantly mediating pollen tube penetration of style with Vps9a.Vps9b is functionally interchangeable with Vps9a, whose functional distinction results from distinct expression profiles.Functional loss of Vps9a and Vps9b results in the mis‐targeting of Rab5‐dependent tonoplast proteins, defective vacuolar biogenesis, disturbed distribution of post‐Golgi vesicles, increased cellular turgor, cytosolic acidification, and disrupted organization of actin microfilaments (MF) in pollen tubes, which collectively lead to the failure of pollen tubes to grow through style. [ABSTRACT FROM AUTHOR]

Summary: Pollen tubes have dynamic tubular vacuoles. Functional loss of AP‐3, a regulator of one vacuolar trafficking route, reduces pollen tube growth. However, the role of canonical Rab5 GTPases that are responsible for two other vacuolar trafficking routes in Arabidopsis pollen tubes is obscure.By using genomic editing, confocal microscopy, pollen tube growth assays, and transmission electron microscopy, we demonstrate that functional loss of canonical Rab5s in Arabidopsis, RHA1 and ARA7, causes the failure of pollen tubes to grow through style and thus impairs male transmission.Functional loss of canonical Rab5s compromises vacuolar trafficking of tonoplast proteins, vacuolar biogenesis, and turgor regulation. However, rha1;ara7 pollen tubes are comparable to those of wild‐type in growing through narrow passages by microfluidic assays.We demonstrate that functional loss of canonical Rab5s compromises endocytic and secretory trafficking at the plasma membrane (PM), whereas the targeting of PM‐associated ATPases is largely unaffected. Despite that, rha1;ara7 pollen tubes contain a reduced cytosolic pH and disrupted actin microfilaments, correlating with the mis‐targeting of vacuolar ATPases (VHA). These results imply a key role of vacuoles in maintaining cytoplasmic proton homeostasis and in pollen tube penetrative growth through style. [ABSTRACT FROM AUTHOR]