An ancient RAB5 governs the formation of additional vacuoles and cell shape in petunia petals.

Shuangjiang Li,Michiel Vandenbussche,Lara Reale,Ronald Koes,Mattijs Bliek,Pamela Strazzer,Yanbang Li,Cornelis Spelt,Francesca M Quattrocchio,Biao Lai,Martina Cerri,Enric Martínez-Calvó

doi:10.1016/j.celrep.2021.109749

Abstract

Homologous ("canonical") RAB5 proteins regulate endosomal trafficking to lysosomes in animals and to the central vacuole in plants. Epidermal petal cells contain small vacuoles (vacuolinos) that serve as intermediate stations for proteins on their way to the central vacuole. Here, we show that transcription factors required for vacuolino formation in petunia induce expression of RAB5a. RAB5a defines a previously unrecognized clade of canonical RAB5s that is evolutionarily and functionally distinct from ARA7-type RAB5s, which act in trafficking to the vacuole. Loss of RAB5a reduces cell height and abolishes vacuolino formation, which cannot be rescued by the ARA7 homologs, whereas constitutive RAB5a (over)expression alters the conical cell shape and promotes homotypic vacuolino fusion, resulting in oversized vacuolinos. These findings provide a rare example of how gene duplication and neofunctionalization increased the complexity of membrane trafficking during evolution and suggest a mechanism by which cells may form multiple vacuoles with distinct content and function.

Highlights

In all eukaryotes, membrane traffic is an essential process that delivers proteins and other compounds to distinct cellular compartments by membrane vesicles that bud off from donor membranes to fuse with specific target compartments
Identification of three canonical RAB5 homologs in petunia Examination of RNA sequencing (RNA-seq) data indicated that petunia petals express three canonical RAB5 genes, namely, RAB5a, RAB5a1, and RAB5a2 (Figure 1B; Figure S1A)
To examine the relationship of these petunia proteins with the canonical Arabidopsis RAB5s (ARA7 and RHA1) and AtARA6, which belongs to a distinct plant-specific clade of RAB5-like proteins, we retrieved highly similar proteins from genome databases and performed phylogenetic analyses (Figure 1E)

Summary

Introduction

Membrane traffic is an essential process that delivers proteins and other compounds to distinct cellular compartments by membrane vesicles that bud off from donor membranes to fuse with specific target compartments. SNARE proteins and RAB GTPases are major factors in determining the identity of vesicles/endosomes and their fusion with membranes from specific target compartments. SNAREs, RABs, and other trafficking regulators are encoded by gene families that existed in early eukaryotes. Since the separation of animals and plants, these families have expanded by independent gene duplications in both lineages, which is thought to have been important for the increased tissue specificity and complexity of trafficking systems during evolution. Secretion is a major trafficking pathway that delivers proteins to the plasma membrane or, when the protein has a specific vacuolar sorting domain, to the vacuole or lysosome. These cells contain a large central vacuole (CV) filled with anthocyanin pigments, as well as numerous small vacuolar compartments, called vacuolinos (Figure 1A), that lack anthocyanins (Faraco et al, 2017)

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