The RAB GTPase RABA1e localizes to the cell plate and shows distinct subcellular behavior from RABA2a under Endosidin 7 treatment

Abstract

Cytokinesis in plants requires the activity of RAB GTPases to regulate vesicle-mediated contribution of material to the developing cell plate. While some plant RAB GTPases have been shown to be involved in cell plate formation, many still await functional assignment. Here, we report cell plate localization for YFP-RABA1e in Arabidopsis thaliana and use the cytokinesis inhibitor Endosidin 7 to provide a detailed description of its localization compared to YFP-RABA2a. Differences between YFP-RABA2a and YFP-RABA1e were observed in late-stage cell plates under DMSO control treatment, and became more apparent under Endosidin 7 treatment. Taken together, our results suggest that individual RAB GTPases might make different contributions to cell plate formation and further demonstrates the utility of ES7 probe to dissect them.

Abbreviations

GTPase

guanosine triphosphatase

GDP

guanine diphosphate

GTP

guanine triphosphate

ES7

Endosidin 7

Vesicular trafficking in eukaryotes is a complex process involving the coordinated budding, targeting, and fusion of vesicles between distinct membrane-bound compartments. Precise mechanisms for vesicle sorting and targeting must be in place for each compartment to retain both its identity and function, especially during fundamental developmental processes such as cytokinesis.^1,2 Rab proteins are key regulators of vesicular trafficking and constitute the largest family of small Ras-like GTPases. They are peripheral membrane proteins that act as molecular switches, cycling between active and inactive states, to integrate intracellular signaling. Rab proteins are involved in several aspects of vesicular trafficking including: vesicle budding, delivery, tethering, and fusion with the target membrane.^3-5

Distinct sets of Rab GTPases exist in yeast, plants, and animals.⁶ Yeasts encode 7–11 Rab GTPases,⁶ whereas plants and animals encode approximately 60.^7,8 The increased number of Rab GTPase isoforms in plants and animals likely reflects their increased complexity and suggests that they have evolved new Rab GTPases to meet specific vesicle trafficking demands. Phylogenic analysis reveals several key differences between plant and animal Rab GTPases, including the substantial expansion and diversification of the RABA clade in plants compared to animals.^8,9 The functional roles of RABA proteins, and their potential involvement in plant-specific processes is a fascinating and highly active area of research.^4,10-15

Cell plate formation during plant cytokinesis requires the contribution of multiple vesicle populations to deliver membrane and cargo material in a time-dependent manner.^1,2,16 The cell plate forms along the plane of cell division, expands centrifugally and is further stabilized with polysaccharide deposition to partition the cytoplasm into two daughter cells.^16,17 This process follows 4 distinct developmental stages: (1) the initial vesicular fusion and fission events (fusion of Golgi-derived vesicles stage, FVS), (2) the formation of a tubulo vesicular network (TVN), (3) morphological transition to form a tubular network (TN) accompanied by callose deposition, and (4) the formation of a fenestrated sheet that fuses with the parental plasma membrane.¹⁶ Here, we refer to stage 1 as early-stage and stages 2, 3, and 4 as late-stage cell plate formation.¹⁷

It has been suggested that callose, a (1, 3)-β-glucan, stabilizes the delicate tubular network until the deposition of cellulose increases its rigidity.¹⁶ Callose accumulation is transient, with the polymer being removed once the cell plate has matured.^16,18 Although genetic studies have indicated the role of callose during cell plate maturation, they are hampered by the lethality associated with callose synthase mutants.^19-22

Endosidin 7 (ES7) is a small molecule that arrests cytokinesis by specifically inhibiting callose biosynthesis during cell plate formation.^17,23 By allowing precise and reversible control over callose biosynthesis at the cell plate, ES7 overcomes the challenge of lethality and is therefore a valuable tool for investigating callose deposition and cell plate maturation.^17,23

A vast amount of proteins, including those involved in vesicle trafficking, participate in cell plate formation.¹ Previous studies have reported that the RAB GTPases RABA2/3, RABA1c, RABA1d, and RABA1e are recruited to the cell plate during cytokinesis in Arabidopsis root tips.^24-27 Whether other RABA proteins localize to the cell plate is currently unknown. We have recently used ES7 to characterize the temporal accumulation of RABA2a during cytokinesis with respect to callose deposition.¹⁷ Here, we use ES7 to study the behavior of RABA1e and compare its localization pattern with RABA2a during cytokinesis.

RABA1e was observed at the cell plate during cytokinesis, corroborating its previously reported localization.²⁷ In early-stage cell plates, both YFP-RABA2a and YFP-RABA1e consistently localized to a disk-shaped structure at the center of the dividing cell (Fig. 1A and C). Furthermore, ES7 treatment did not alter the localization of either protein in early-stage cell plates (Fig. 1B and D). These results are consistent with previous reports of RABA2a localization^17,24 and confirm our previous observations that ES7 does not affect early-stage cell plate formation.¹⁷

Figure 1.

YFP-RABA2a and YFP-RABA1e localize to disk-shaped structures in early stage cell plates. Confocal micrographs show disk-shaped localizations for YFP-RABA2a (A, B) and YFP-RABA1e (C, D) in early-stage cell plates. There were no discernable differences between YFP-RABA2a (A) and YFP-RABA1e (C) under DMSO treatment. ES7 treatment did not affect the localization of YFP-RABA2a (B) or YFP-RABA1e (D). The fluorescent fusion proteins are indicated by green, and FM4-64 counterstaining of the plasma membrane is indicated by red. Graphs showing the intensities of the fusion proteins (green, solid line) and FM4-64 dye (red, dotted line) appear to the right of the corresponding micrograph. Plants were grown for 3 d on ¼ MS + 1% sucrose plates and treated for 3 h in ¼ MS liquid media containing 50 μM ES7 or a corresponding volume of DMSO prior to visualization. Scale bar = 10 μm.

In late-stage cell plates under DMSO control conditions, differences were observed between YFP-RABA2a and YFP-RABA1e (Fig. 2A, D, G, Table 1). YFP-RABA2a localized to ring-shaped structures spanning the plane of cell division (98%) whereas YFP-RABA1e localized to both ring-shaped structures (80%) and disk-shaped structures (20%), demonstrating an overall different pattern between the localization of the two proteins (p = 0.0019, Fisher's Exact Test).

Figure 2.

YFP-RABA2a and YFP-RABA2e show different subcellular behaviors under ES7 treatment in late stage cell plates. Confocal micrographs show YFP-RABA2a and YFP-RABA1e localizations in late-stage cell plates under DMSO and ES7 treatments. Under DMSO treatment, YFP-RABA2a (A) localized to ring-shaped structures, whereas YFP-RABA1e localized to both ring (D) and disk (G) shaped structures. Under ES7 treatment, YFP-RABA2a (B, C) localized primarily to cell plate gaps whereas YFP-RABA1e localized to cell plate gaps (E, F) and irregular aggregates (H, I) with similar frequencies (Table 1). (C, F, and I) represent 3D renderings. The fluorescent fusion proteins are indicated by green, and FM4-64 counterstaining of the plasma membrane is indicated by red. Graphs showing the intensities of the fusion proteins (green, solid line) and FM4-64 dye (red, dotted line) appear to the right of the corresponding micrograph. Plants were grown for 3 d on ¼ MS + 1% sucrose plates and treated for 3 h in ¼ MS liquid media containing 50 μM ES7 or a corresponding volume of DMSO prior to visualization. Scale bar = 10 μm.

Table 1.

Localization Patterns Observed for YFP-RABA2a and YFP-RABA1e in Late-Stage Cell Plates

	DMSO			Endosidin 7
Protein	Ring	Disk	n	Gaps	Aggregates	n
YFP-RABA2a	98%	2%	64	79%	21%	148
YFP-RABA1e	80%	20%	40	42%	58%	113

The localizations of YFP-RABA2a and YFP-RABA1e in late-stage cell plates were quantified under DMSO (left) and ES7 (right) treatments. Significant differences in the localization patterns of YFP-RABA2a and YFP-RABA1e were observed under both DMSO (p = 0.0019) and ES7 (P < 0.0001) conditions (Fisher's Exact Test). Plants were grown for 3 d on ¼ MS + 1% sucrose plates and treated for 3 h in ¼ MS liquid media containing 50 μM ES7 or a corresponding volume of DMSO prior to visualization.

In late-stage cell plates under ES7 treatment, differences between YFP-RABA2a and YFP-RABA1e became even more apparent (Fig. 2B, C, E, F, H, I, Table 1). YFP-RABA2a localized primarily to cell plate gaps (79%) whereas YFP-RABA1e localized primarily to irregular aggregates (58%). The differences in YFP-RABA2a and YFP-RABA1e localization patterns were statistically significant under ES7 treatment (P < 0.0001, Fisher's Exact Test).

In this study, ES7 was used to analyze the subcellular behavior of YFP-RABA1e at the cell plate in comparison to YFP-RABA2a. Using this probe, we showed that while YFP-RABA2a and YFP-RABA1e share many similarities, they are not completely identical (Fig. 2, Table 1). Although we present evidence that RABA1e and RABA2a display differences in cell plate localization, it remains unknown whether they have different functions during its formation. The observation that differences between YFP-RABA2a and YFP-RABA1e were enhanced under ES7 treatment (Table 1), further demonstrates the utility of ES7 in dissecting vesicle populations at the cell plate.

Functional characterization of RABA2a has shown that this protein is necessary for the delivery of trans-Golgi network (TGN)-derived vesicles to the leading edge of the cell plate.²⁴ Similar studies have shown cytokinesis defects in RABA1c dominant negative mutants,²⁶ but no functional characterization of RABA1e during cytokinesis exists. GFP-RABA1e is highly expressed in root hairs,²⁸ which, like dividing cells, require very high levels of vesicular trafficking. A recent study has shown that RABA1d is involved in oscillatory root hair growth and cell plate formation, suggesting a role in vesicular trafficking.²⁹ It is therefore tempting to speculate that RABA1e similarly plays a role in vesicle-mediated cargo delivery during cytokinesis and root hair elongation. With the increasing number of RAB proteins implicated in cell plate formation,^24-27 it will be important for future research to determine which proteins, or sets of proteins, have distinct functions. Vesicle proteomics can offer valuable insights into the cargo that vesicles carry during cytokinesis.^30,31 Functional studies to uncover what materials RAB proteins traffic to the cell plate, the timing of delivery, and which RABs reside on common vesicles will help to fully understand their roles in cell plate formation.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgements

We thank Dr. I Moore (U. Oxford) and Dr. N Geldner (U. of Lausanne) for providing seed stocks. We thank the members of Drakakaki lab for stimulating discussions.

Funding

This work was funded by the NSF-IOS-1258135 grant to G D.