Vacuolar trafficking in pollen tube growth and guidance

ABSTRACT

Vacuoles are versatile organelles in plant cells, critical for growth and responses to environmental cues. Vacuoles are dynamic tubular structures in pollen tubes, the male gametophytes. Mutations at vacuolar fusion machinery caused male gametophytic lethality by affecting pollen tube growth and guidance, which are critical steps leading to angiosperm reproduction. In comparison, the role of vacuolar trafficking and its cargoes in this process is less understood. In this mini-review, we summarize old and recent findings that indicate the involvement of vacuolar trafficking in pollen tube growth and guidance. We also point at future studies that would provide insights into a key role of vacuolar trafficking and its cargos in pollen tube growth and guidance.

Double fertilization of angiosperms depends on polar and directional growth of pollen tubes to deliver immotile sperm into female gametophyte, i.e. the embryo sac. Growth is restricted at a single polarity patch on male gametophyte, the pollen grain, leading to a tubular extension, i.e. the pollen tube, in which two sperm cells are enclosed.¹ Due to the structural complexity of pistils in most angiosperms, successful delivery of sperm cells requires rapid elongation of pollen tubes and their directional change upon female cues. In the past years, breakthroughs on key female and male components of the growth-guidance machinery have been reported.¹ Small secreted peptides such as LURE are generated by female gametophytes to attract pollen tubes^{2, 3} whereas cell-surface receptors at pollen tubes such as MALE DISCOVERER1 (MDIS1/2)-MDIS1-INTERACTING RECEPTOR LIKE KINASE1 (MIK1/2) and POLLEN RECEPTOR KINASE6 (PRK6)^4-6 translate female guidance cues into directional growth.

Plant vacuoles are versatile organelles, critical for cell growth, ion homeostasis, secondary metabolite storage, and responses to environmental stresses.⁷ Proper function of vacuoles requires proteins localized at the tonoplast, i.e. vacuolar membrane, and in vacuolar lumen. Vacuolar proteins are synthesized at the endoplasmic reticulum (ER) and delivered through vesicular trafficking.^7-10 Four vesicular trafficking routes are currently reported in plant cells.^{7, 10} The first involves sequential activation of small GTPases RAB5 and RAB7, through trans-Golgi nework/early endosomes (TGN/EE) to prevacuolar compartment/multivesicular bodies (PVC/MVB), finally to vacuoles.^11-13 Protein sorting at the TGN/EE also requires the activity of adaptor protein complex-1 (AP-1).^{14, 15} The second is mediated by AP-3 at the Golgi apparatus.^16-20 These two routes are conserved during evolution.^{7, 10} The third pathway is an evolutionarily distinct diversion from the first one such that it requires RAB5 but is independent of RAB7.^{12, 21} Tonoplast-associated component of vacuolar ATPases, VHA-a3, and the SNARE protein SYP22 take this route to target the tonoplast.^{12, 21} Finally, vacuolar proteins may be directly delivered from the ER although regulators of this route are yet to be characterized.²²

Pollen tube growth involves dynamic vacuolar organization and distribution.^{23, 24} Unlike in other cells, vacuoles in pollen tubes are tubular structures excluded from the apex.²³ Mutations at Arabidopsis VACUOLELESS1 (VCL1), a gene essential for vacuole formation and morphogenesis, caused a reduction of male gametophytic transmission.²³ However, it is unclear whether pollen tube growth and guidance of vcl1 is affected. Recently, we reported that mutations at AP-3 significantly reduced pollen tube growth.²⁵ Further investigation showed that AP-3 mediates pollen tube growth through vacuolar targeting of Arabidopsis PROTEIN S-ACYL TRANSFERASE10 (PAT10).^{20, 25} PAT10 is essential for S-acylation-dependent tonoplast association of CALCINEURIN B-LIKE2 (CBL2) and CBL3,^{26, 27} two Ca²⁺ sensors. Indeed, the tip-focused Ca²⁺ gradient in ap-3 pollen tubes was significantly reduced, which leads to reduced pollen tube growth.²⁵

Whether and how vacuolar trafficking is involved in the directional growth of pollen tubes is obscure. Mutations at VPS41, a component of the homotypic fusion and vacuolar protein sorting complex (HOPS), resulted in male gametophytic lethality. ²⁸ VPS41 loss-of-function resulted in abnormally shaped vacuoles in pollen tubes²⁰ likely due to failed fusion of vesicles with the tonoplast. Both pollen tube growth and guidance was compromised by VPS41 loss-of-function,²⁸ suggesting a key role of vacuolar delivery in pollen tube guidance. Interestingly, MDIS1, the recently identified LURE receptor at the pollen tubes, undergoes LURE-induced endocytosis,²⁹ suggesting the involvement of vacuolar trafficking in directional growth of pollen tubes upon sensing the female cues. However, no direct evidence has been shown to demonstrate an essential role of its endocytosis for pollen tube growth and guidance.

To provide further evidence that vacuolar trafficking and its key cargos play essential roles in pollen tube growth and guidance, genetic approaches shall be taken in the future. Because RAB5 mediates two vacuolar trafficking routes in plant cells, functional loss of RAB5 would be used to determine whether the two routes are involved in male gametophytic function. Another small GTPases RAB7 is encoded by a small gene family in Arabidopsis.^11-13 Either hierarchy mutants or mutants of guanine nucleotide exchange factors for RAB7 (Rab7) shall be analyzed regarding pollen tube growth and guidance. It will also be interesting to test whether MDIS1 is directed to vacuoles after internalization and the biological relevance of vacuolar trafficking in ligand-induced internalization of receptors.

Acknowledgments

Works in Y.Z's laboratory is partially supported by Tai-Shan Scholar Program by Shandong Provincial Government.