RabA2b Overexpression Alters the Plasma-Membrane Proteome and Improves Drought Tolerance in Arabidopsis.

Vivek Ambastha,Dafna Tidhar,Yehoram Leshem,Ifat Matityahu

doi:10.3389/fpls.2021.738694

Abstract

Rab proteins are small GTPases that are important in the regulation of vesicle trafficking. Through data mining, we identified RabA2b to be stress responsive, though little is known about the involvement of RabA in plant responses to abiotic stresses. Analysis of the RabA2b native promoter showed strong activity during osmotic stress, which required the stress hormone Abscisic acid (ABA) and was restricted to the vasculature. Sequence analysis of the promoter region identified predicted binding motifs for several ABA-responsive transcription factors. We cloned RabA2b and overexpressed it in Arabidopsis. The resulting transgenic plants were strikingly drought resistant. The reduced water loss observed in detached leaves of the transgenic plants could not be explained by stomatal aperture or density, which was similar in all the genotypes. Subcellular localization studies detected strong colocalization between RabA2b and the plasma membrane (PM) marker PIP2. Further studies of the PM showed, for the first time, a distinguished alteration in the PM proteome as a result of RabA2b overexpression. Proteomic analysis of isolated PM fractions showed enrichment of stress-coping proteins as well as cell wall/cuticle modifiers in the transgenic lines. Finally, the cuticle permeability of transgenic leaves was significantly reduced compared to the wild type, suggesting that it plays a role in its drought resistant properties. Overall, these data provide new insights into the roles and modes of action of RabA2b during water stresses, and indicate that increased RabA2b mediated PM trafficking can affect the PM proteome and increase drought tolerance.

Highlights

Ras-related proteins in brain (Rab) proteins consist of a large family of small GTPases (20–25 KDa) with more than 60 Rab members identified in the human genome
Examination of the region we cloned revealed that RabA2b promoter activity is induced by osmotic stresses as well as by the stress hormone Abscisic acid (ABA) (Figure 2)
The strong reduction in RabA2b promoter activity we observed during osmotic stress in the abi mutants background indicates that the activation of RabA2b during osmotic stress is ABA dependent (Figure 3)

Summary

Introduction

Ras-related proteins in brain (Rab) proteins consist of a large family of small GTPases (20–25 KDa) with more than 60 Rab members identified in the human genome. In plant genomes, the RabA sub-family, which is related to mammalian Rab, is highly diversified: in Arabidopsis, among the 57 identified Rab genes, 26 are members of the RabA sub-family (Rutherford and Moore, 2002; Woollard and Moore, 2008). Nielsen et al (2008) reviewed several RabA studies in plants and indicated that some RabA members play a role in secretion and/or cell wall recycling processes. This suggestion was further supported by Lunn et al (2013) who found that in several RabA mutants the profile of major cell wall components differed from the wild type (wt) profile. Several intracellular localization studies reported that several Arabidopsis RabA members operate along the trans-Golgi network (TGN) – plasma membrane (PM) pathway, during cytokinesis and tip growth in the root and elongating pollen tubes (Chow et al, 2008; Szumlanski and Nielsen, 2009; Asaoka et al, 2013a; Nielsen, 2020)

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