The role of endomembrane-localized VHA-c in plant growth

ABSTRACT

In plant cells, the vacuolar-type H⁺-ATPase (V-ATPase), a large multis`ubunit endomembrane proton pump, plays an important role in acidification of subcellular organelles, pH and ion homeostasis, and endocytic and secretory trafficking. V-ATPase subunit c (VHA-c) is essential for V-ATPase assembly, and is directly responsible for binding and transmembrane transport of protons. In previous studies, we identified a PutVHA-c gene from Puccinellia tenuiflora, and investigated its function in plant growth. Subcellular localization revealed that PutVHA-c is mainly localized in endosomal compartments. Overexpression of PutVHA-c enhanced V-ATPase activity and promoted plant growth in transgenic Arabidopsis. Furthermore, the activity of V-ATPase affected intracellular transport of the Golgi-derived endosomes. Our results showed that endomembrane localized-VHA-c contributes to plant growth by influencing V-ATPase-dependent endosomal trafficking. Here, we discuss these recent findings and speculate on the VHA-c mediated molecular mechanisms involved in plant growth, providing a better understanding of the functions of VHA-c and V-ATPase.

KEYWORDS:

• Activity
• endosomal trafficking
• overexpression
• plant growth
• subunit c
• V-ATPase

V-ATPase is a conserved proton pump found in eukaryotes, including yeasts, plants, and animals. V-ATPase consists of the cytosolic ATP-hydrolyzing V₁ domain (subunits A to H) and the membrane-bound proton-translocating V₀ domain (subunits a, c, c', c', d, and e).¹ The VHA-a containing three different isoforms is responsible for targeting V-ATPase to different subcellular sites in Arabidopsis.² V-ATPase complexes containing VHA-a1 are localized to the trans-Golgi network/early endosome (TGN/EE), whereas holoenzymes containing VHA-a2 or VHA-a3 are targeted to the tonoplast.³ V-ATPase activity in the TGN/EE is required for endocytic and secretory trafficking, whereas V-ATPase activity at the tonoplast is required for vacuolar ion homeostasis and efficient nutrient storage.^4,5

The V-ATPase c subunit (VHA-c), which includes four transmembrane helices, is a highly hydrophobic protein in the V₀ domain.¹ In a previous study, we identified a highly conserved VHA-c gene (PutVHA-c) from Puccinellia tenuiflora. Compared to the wild-type, transgenic Arabidopsis plants overexpressing PutVHA-c exhibit better growth phenotypes in root length, fresh weight, plant height, and silique number under normal conditions due to noticeably higher V-ATPase activity. Confocal and immunogold electron microscopy assays demonstrate that PutVHA-c is mainly localized to the endosomal compartments. Concanamycin A (ConcA) is a membrane-permeable macrolide antibiotic that specifically binds to VHA-c and inhibits proton transport. ConcA treatment resulted in accumulation of endosomes containing PutVHA-c.⁶ Previous studies showed that ConcA induces the aggregation of large Golgi-derived endosomes.³ These findings suggest that PutVHA-c-containing endosomes may be derived from the Golgi apparatus, and that endosomal trafficking was affected by V-ATPase activity. In addition, we found that endosomes containing PutVHA-c can fuse with each other (Fig. 1A, B), and can simultaneously transport and fuse with the plasma membrane, tonoplast, and cell plate (Fig. 1C, D), suggesting that PutVHA-c is distributed throughout the secretory pathway. Phenotype experiments showed that overexpression of PutVHA-c can promote cell expansion in yeast and Arabidopsis seedling roots (Fig. 1E, F). Membranes for cell growth, membrane proteins, extracellular proteins, and components of the cell wall, which are necessary for plant cell growth, are transported to target sites via the endosome-mediated trafficking.^5,7,8 Direct evidence shows that the reduced V-ATPase activity in the TGN/EE causes hypocotyl cell expansion inhibition by affecting the synthesis and trafficking of cell wall components.⁹ Therefore, the endosome-mediated transport of the components necessary for cell growth is likely related to plant cell expansion. As such, we hypothesized that overexpression of PutVHA-c may accelerate endosomal trafficking by enhancing V-ATPase activity, thereby promoting plant cell expansion (Fig. 1G). In the future, isolation of PutVHA-c localized-endosomes, and further identification of the components in endosomes will be essential.

Figure 1. Expression and localization of PutVHA-c in Arabidopsis and yeast cells. (A) PutVHA-c colocalizes with FM4-64 in the endosomal compartment. (B and C) Endosomes containing PutVHA-c fuse with each other (B) and with the plasma membrane (C). (D) PutVHA-c partially localized to the tonoplast (indicated with an arrow, in the left plate) and to the stump of the cell plate (indicated with an arrow, in the right plate) labeled by FM4-64. The asterisk indicates the vacuole. (E and F) Expression of PutVHA-c promotes cell expansion in yeast (E) and Arabidopsis root (F). (G) A summary of the localization of PutVHA-c in plant cells. TGN/EE, trans-Golgi network/early endosome; LE/MVB/PVC, late endosomes/multivesicular body/prevacuolar compartment. PM, plasma membrane. ConcA, Concanamycin A.

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Additional information

Funding

China Postdoctoral Science Foundation (2016M601409); Young talent Project of Northeast Agricultural University of China (16QC06); Natural Science Foundation of Heilongjiang Province of China (C2016024).