Arabidopsis RABA1 GTPases are involved in transport between the trans -Golgi network and the plasma...

Arabidopsis RABA1 GTPases are involved in transportbetween the trans-Golgi network and the plasma membrane,and are required for salinity stress tolerance

Rin Asaoka1, Tomohiro Uemura1, Jun Ito2,†, Masaru Fujimoto1, Emi Ito1, Takashi Ueda1,3 and Akihiko Nakano1,2,*1Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Bunkyo-ku, Tokyo, 113-0033,

Japan,2Molecular Membrane Biology Laboratory, RIKEN Advanced Science Institute, Wako, Saitama, 351-0198, Japan, and3PRESTO, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi, Saitama, 332-0012, Japan

Received 29 June 2012; revised 31 August 2012; accepted 10 September 2012; published online 23 November 2012.

*For correspondence (e-mail [email protected]).†Present address: Division of Cell Biology, Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Nara, 630-0192, Japan.

SUMMARY

RAB GTPases are key regulators of membrane traffic. Among them, RAB11, a widely conserved sub-group,

has evolved in a unique way in plants; plant RAB11 members show notable diversity, whereas yeast and ani-

mals have only a few RAB11 members. Fifty-seven RAB GTPases are encoded in the Arabidopsis thaliana gen-

ome, 26 of which are classified in the RAB11 group (further divided into RABA1–RABA6 sub-groups).

Although several plant RAB11 members have been shown to play pivotal roles in plant-unique developmental

processes, including cytokinesis and tip growth, molecular and physiological functions of the majority of

RAB11 members remain unknown. To reveal precise functions of plant RAB11, we investigated the subcellular

localization and dynamics of the largest sub-group of Arabidopsis RAB11, RABA1, which has nine members.

RABA1 members reside on mobile punctate structures adjacent to the trans-Golgi network and co-localized

with VAMP721/722, R-SNARE proteins that operate in the secretory pathway. In addition, the constitutive-

active mutant of RABA1b, RABA1bQ72L , was present on the plasma membrane. The RABA1b -containing

membrane structures showed actin-dependent dynamic motion . Vesicles labeled by GFP–RABA1b moved

dynamically, forming queues along actin filaments. Interestingly, Arabidopsis plants whose four major RABA1

members were knocked out, and those expressing the dominant-negative mutant of RABA1B, exhibited

hypersensitivity to salinity stress. Altogether, these results indicate that RABA1 members mediate transport

between the trans-Golgi network and the plasma membrane, and are required for salinity stress tolerance.

Keywords: RABA1, Rab11, salinity stress, post-Golgi traffic, Arabidopsis thaliana, actin filaments.

INTRODUCTION

Membrane traffic is essential for a variety of cellular

phenomena from housekeeping to development, and is

coordinately regulated by elaborate molecular machiner-

ies. RAB GTPases, molecular switches that act through the

conformational change between the GTP- and GDP-bound

forms, control docking, fusion and, in some cases, fission

events during vesicle trafficking (Miserey-Lenkei et al.,

2010; Valente et al., 2010). The RAB family has a large

number of members: for example, 60 in Homo sapiens and

57 in Arabidopsis thaliana. In order to maintain proper traf-

fic, each RAB member is considered to regulate a specific

step in the complicated network of membrane traffic.

In plants, RAB members consist of eight groups: RABA,

RABB, RABC, RABD, RABE, RABF, RABG and RABH, which

correspond to animal RAB11, RAB2, RAB18, RAB1, RAB8,

RAB5, RAB7 and RAB6, respectively (Rutherford and Moore,

2002). Despite of the lack of several RAB groups conserved

in animals, some plant RAB proteins have undergone

unique diversification. In particular, the RABA (RAB11)

group exhibits remarkable expansion in higher plants. In

A. thaliana, the RABA group comprises as many as 26

members out of the total of 57 RABs. However, animals and

yeast possess only a few corresponding members. In non-

polarized mammalian cells, RAB11a localizes to the mem-

brane of recycling endosomes (Ullrich et al., 1996; Green

et al., 1997), and regulates transport from the sorting endo-

somes to the recycling compartments (Ullrich et al., 1996).

In polarized epithelial cells, RAB11a, RAB11b and RAB25

© 2012 The AuthorsThe Plant Journal © 2012 Blackwell Publishing Ltd

240

The Plant Journal (2013) 73, 240–249 doi: 10.1111/tpj.12023

localize to the apical recycling endosomes (Casanova et al.,

1999). The RAB11 and RAB25 sub-classes appear to co-

localize but are suggested to control distinct transport

routes between the recycling endosome and the Golgi or

the plasma membrane (Casanova et al., 1999; Somsel Rod-

man and Wandinger-Ness, 2000). The RAB11 counterpart in

Saccharomyces cerevisiae, Ypt31/32, has been implicated in

export from the late Golgi compartment to the pre-vacuo-

lar/endosomal compartment and the plasma membrane

(Benli et al., 1996; Segev, 2001; Ortiz et al., 2002).

In plants, the RABA group is further divided into six sub-

classes (RABA1–RABA6). Previous studies have shown

roles for RABA members in a wide range of cellular events.

NtRAB11b, a RABA1 member in Nicotiana tabacum, has

been shown to play a crucial role in pollen tube growth

(de Graaf et al., 2005). Arabidopsis RABA2 and RABA3,

which show high similarity to mammalian RAB11 and

yeast Ypt31/32, have been reported to localize on a novel

post-Golgi membrane domain, that partly overlaps with

the trans-Golgi network (TGN) in root tip cells. During cyto-

kinesis, RABA2 and RABA3 proteins localize precisely to

the growing margins of the cell plate, suggesting impor-

tant roles in cytokinesis via regulation of polarized secre-

tion (Chow et al., 2008). The involvement of RABA1a in

auxin signaling has also been reported (Koh et al., 2009).

Preuss et al. (2004, 2006) have shown that RABA4b func-

tions in polarized secretion in root hair cells, in cooperation

with its effector phosphatidylinositol 4-OH kinase beta 1

(PI-4Kb1). Szumlanski and Nielsen (2009) reported that

RABA4d is expressed in a pollen-specific manner and is

required for proper development of pollen tubes.

In A. thaliana, RABA1 is the largest sub-group of RABA,

with nine members (RABA1a–RABA1i). However, the physio-

logical roles of this sub-group remain unexploited. In this

study, we have focused on the RABA1 sub-group, particu-

larly RABA1a, RABA1b, RABA1c and RABA1d, whose expres-

sion is abundant in the whole plant body. We have shown

that these RABA1 proteins localize on a compartment adja-

cent to the TGN. Several lines of evidence further indicate

that RABA1 regulates transport between the TGN and the

plasma membrane. Moreover, we have found that RABA1

members redundantly function in salinity stress tolerance.

RESULTS

Expression patterns of RABA1 members

In A. thaliana, the RABA1 sub-group has nine members

(RABA1a–RABA1i). First, we compared the expression pro-

files of these members from the Arabidopsis Information

Resource database (http://www.arabidopsis.org/, Expression-

Set:1006710873), usingDART to access andorganize themicro-

array data (http://dandelion.liveholonics.com/dart/index.php).

RABA1a, RABA1b, RABA1c and RABA1d are abundant in

the whole plant body, whereas RABA1e is mainly

expressed in roots. The other four members (RABA1f,

RABA1g, RABA1h and RABA1i) are mainly expressed in

flowers (Figure S1). Next, we generated transgenic plants

expressing GFP/Venus-tagged RABA1a, RABA1b, RABA1c,

RABA1d, RABA1e and RABA1f proteins under the control of

their native regulatory elements and examined their expres-

sion patterns. RABA1a, RABA1b, RABA1c and RABA1d were

observed in all parts of the plant body, but their expression

patterns were slightly different. RABA1a, RABA1b and

RABA1c were abundant in the division zones of shoots and

roots. Expression of RABA1d was high in the stele and low

in other areas. RABA1e was detected only in roots (Fig-

ure 1a), especially in root hair cells (Figure 1b, arrowheads).

Consistent with the TAIR microarray data, RABA1f was

detected only in pollen tubes (Figure 1c). Thus, RABA1a,

RABA1b, RABA1c and RABA1d are major members of the

RABA1 sub-group that are expressed throughout all tissues,

although the TAIR data predicted modest expression of

RABA1f and RABA1g in vegetative parts as well (Figure S1).

Detailed observation of growing root hairs showed char-

acteristic accumulation of GFP–RABA1b, GFP–RABA1c and

GFP–RABA1e in the tip region of root hairs (Figures 1a and

2a). The pollen-specific member RABA1f also accumulated

in the tip region of growing pollen tubes (Figure 1a). The

amino acid sequence similarities among RABA1 proteins

are considerably high, approximately 70–80%. These

results imply that the five RABA1 (RABA1a, RABA1b,

RABA1c, RABA1d and RABA1e) members, and perhaps

other members as well, are involved in related transport

processes, even though they are expressed in different

tissues. In this report, we analyzed RABA1b as the repre-

sentative of the RABA1 sub-group, because it is the most

abundant in major tissues (Figure 1a).

Characterization of the RABA1b compartment

In all the cells we observed, RABA1b was localized on small

punctate structures of various size that show dynamic

motion (Figure 2a and Movie S1). In the division zone of

roots, many dot-like RABA1b-containing compartments

were seen in the cytoplasm, especially near the plasma

membrane (Figure 2a). In the differentiation zone, fluores-

cent signals near the plasma membrane were reduced, and

distinct signals appeared along lines (Figure 2a). These

aligned signals are the fluorescent trajectories of high-

speed movement of RABA1b-containing vesicles along the

actin cytoskeleton (see Figure 7). RABA1b also accumulated

on cell plates in dividing cells and in the tip region of grow-

ing root hair cells (Figure 2a, arrow). Similar localization

patterns were observed in cells expressing fluorescence-

tagged RABA1a and RABA1c (Figure 2b). Transgenic

plants co-expressing Venus–RABA1a and mRFP–RABA1b,

GFP–RABA1b and mRFP–RABA1b, and GFP–RABA1c and

mRFP–RABA1b revealed that each pair of proteins

co-localize in the division zone of roots (Figure 2b). The

© 2012 The AuthorsThe Plant Journal © 2012 Blackwell Publishing Ltd, The Plant Journal, (2013), 73, 240–249

Analysis of RABA1 GTPases in A. thaliana 241

GFP–RABA1b and mRFP–RABA1b pair was used as a posi-

tive control of co-localization. We also performed quantita-

tive analysis of the co-localization using a macro written in

Metamorph software, which semi-automatically measures

the distance from the center of each RFP signal to the cen-

ter of the nearest GFP or Venus signal in acquired images

(Figure S4A) (Ito et al., 2012). GFP–RABA1d and GFP–

RABA1e were not detected in root tips, because their

expression is spatially restricted to the stele and root hair

cells, respectively. Hereafter, we refer to the compartment

labeled by GFP–RABA1b as the RABA1b compartment.

Next, the RABA1b compartment was tested for the pres-

ence of known organelle markers of post-Golgi traffic by

high-resolution confocal laser microscopy. As shown in

Figure 3(a–e), the RABA1 compartment showed partial

association with organelles labeled by mRFP-tagged

syntaxin of plants 43 (mRFP–SYP43, TGN marker) (Uemura

et al., 2004) and mRFP-tagged vacuolar proton ATPase

a1 (VHA-a1–mRFP, TGN marker) (Dettmer et al., 2006),

and less with sialyl transferase-fused mRFP (ST–mRFP,

Golgi marker) (Munro, 1995; Boevink et al., 1998), mRFP-

tagged arabidopsis rab GTPase homolog 7 (mRFP–ARA7,

endosome marker) (Ueda et al., 2001) or mRFP-tagged

arabidopsis rab GTPase homolog 6 (ARA6–mRFP, endo-

some marker) (Ueda et al., 2001). However, the co-localiza-

tion with VAMP721, R- soluble N-ethyl-maleimide sensitive

factor attachment protein receptor (R-SNARE), which is

another key regulator of membrane traffic, was remarkable

(Figure 3f). The comparison of the RABA1b compartment

with the TGN was interesting, because they seemed to be

frequently adjacent and sometimes associated (Figure 3a,b).

However, this association appeared to be not as tight and

stable as that of the Golgi and the TGN (Figure 3g). We

sometimes observed the RABA1b compartment adjacent

to the ARA6 compartment, but rarely adjacent to the ARA7

compartment. Quantitative analysis of the co-localization

clarified the tendency of the markers to co-localize with or

be adjacent to the RABA1b compartment (Figure S4B).

To characterize the RABA1b compartment in more detail,

we examined whether inhibitors of post-Golgi traffic affect

the RABA1b fluorescence pattern. Brefeldin A (BFA) inhibits

budding of vesicles and aggregates Golgi and post-

Golgi compartments, such as the TGN and endosomes

(Grebe et al., 2003; Dettmer et al., 2006). The RABA1b com-

partment also aggregated upon BFA treatment, although

some GFP–RABA1b signals remained on large bead-like

(a)

(b) (c)

Figure 1. Expression patterns of Arabidopsis

RABA1 members.

(a) Distribution patterns of RABA1 members.

Left, bright field. Right, fluorescence of GFP/

Venus-tagged RABA1s driven by their own

promoters. Venus–RABA1a, GFP–RABA1b, GFP

–RABA1c, GFP–RABA1d and GFP–RABA1e were

observed using a stereoscopic microscope.

Scale bars = 1 mm.

(b) Z-stack image of GFP–RABA1e in epidermal

cells with growing root hairs. Arrowheads indi-

cate root hair cells. Arrows indicate root hairs.

Fluorescence was not detected in non-root hair

cells. The image was obtained by confocal laser

microscopy. The distance between adjacent

Z-stack images was 0.58 m, and 100 planes are

stacked. Scale bar = 10 lm.

(c) Confocal image of Venus–RABA1f in grow-

ing pollen tubes. Scale bar = 10 lm.


242 Rin Asaoka et al.

structures (Figure 4a, arrowheads, and Figure S5). Wort-

mannin, an inhibitor of phosphatidylinositol 3-kinase

(PI3K), exaggerates the different characteristics of the

RABA1b compartment and the TGN. Wortmannin caused

no morphological change to the TGN labeled by mRFP–

SYP43. However, GFP–RABA1b signals increased on the

plasma membrane upon wortmannin treatment, while dot-

like signals of GFP–RABA1b in the cytoplasm decreased

(Figure 4b and Figure S5). Treatment with dimethylsulfox-

ide (DMSO) had no effect on these compartments (Figure

S2). These results indicate that the RABA1b compartment

has a property that is different from the TGN represented

by SYP43, and that shows a tendency to associate with the

plasma membrane upon wortmannin treatment.

RABA1 members are involved in transport between the

TGN and the plasma membrane

VAMP721 and VAMP722 are R-SNARE proteins that

operate redundantly in the secretory pathway (Kwon et al.,

2008). Double staining of RABA1b and VAMP721 revealed

that the two proteins largely co-localize on punctate

structures (Figure 3f). Most mRFP–VAMP721 signals were

(a)

(b)

Figure 2. Subcellular localization of GFP–RABA1 members.

(a) Confocal images of GFP–RABA1b in actively dividing cells in the root tip,

differentiated cells in the root, and root hairs. Characteristic accumulation in

the cell plate (arrow) in the root tip was observed. Scale bars = 10 lm.

(b) Subcellular localization of Venus–RABA1a, GFP–RABA1b and GFP–RABA1c. The regions enclosed by white squares are magnified and shown

in the right column. Green, GFP/Venus- tagged RABA1 proteins; magenta,

mRFP–RABA1b. Scale bars = 10 lm.

(a)

(b)

(c)

(d)

(e)

(f)

(g)

Figure 3. The RABA1b compartment associates with the TGN.

(a–f) Co-expression of GFP–RABA1b and mRFP-tagged organelle markers:

mRFP–SYP43, VHA-a1–mRFP, ST–mRFP, mRFP–ARA7, ARA6–mRFP and

mRFP–VAMP721 in root tip cells. Green, GFP–RABA1b; magenta, mRFP-

tagged markers. The regions enclosed by white squares are magnified and

shown in the right column. Scale bars = 10 lm.

(g) Co-expression of GFP–RABA1b, ST-Venus and mRFP–SYP43 in root tip

cells. The region enclosed by a white square is magnified and shown in the

bottom panels. Scale bar = 10 lm.



accompanied by GFP–RABA1b, but some GFP–RABA1b

signals were not associated with mRFP–VAMP721. GFP–

RABA1b also co-localized with mRFP–VAMP722 (Figure

S2). mRFP–VAMP722 showed similar responses to GFP–

RABA1b upon treatment with wortmannin and BFA. Thus

the RABA1b compartment appeared to be closely related

to the compartment to which VAMP721 and VAMP722

localize, suggesting a role for RABA1b in transport toward

the plasma membrane.

To examine the involvement of RABA1b in transport

between the TGN and the plasma membrane, we gener-

ated transgenic plants expressing a mutant form of

RABA1b, either GTP-fixed (GFP–RABA1bQ72L) or GDP-fixed

(GFP–RABA1bS27N). GTP-fixed RAB GTPases are constitu-

tively active, and are thought to accumulate on the target

membrane. However, GDP-fixed RAB GTPases act in a

dominant-negative way by trapping the guanine nucleotide

exchange factor (Burstein et al., 1992; Olkkonen and

Stenmark, 1997; Feig, 1999). Significant localization of GFP–

RABA1bQ72L to the plasma membrane was observed in

root tip cells (Figure 5a, upper panel, arrowhead, and

Figure S6) in addition to the punctate structures (arrow).

Such relocation was not evident in differentiated cells

(Figure 5a, lower panel). The punctate structures of GFP–

RABA1bQ72L co-localized with mRFP–VAMP721 almost

completely (Figure 5d and Figure S7), and also overlapped

well with the TGN labeled by mRFP–SYP43 and VHA-a1–

mRFP (Figure 5b,c and Figure S7). RABA1bQ72L showed

increased cytosolic labeling compared to the wild-type. In

contrast to RABA1bQ72L, GFP–RABA1bS27N was localized to

(a)

(b)

Figure 4. Effects of BFA and wortmannin on the RABA1b compartment.

(a) Root tip cells expressing GFP–RABA1b and mRFP–SYP43 (above) or

mRFP–VAMP722 (below) treated with 50 lM brefeldin A for 1 h. Green, GFP

–RABA1b; magenta, mRFP-tagged markers. Arrowheads indicate large

bead-like structures that remain separate from aggregates. Scale

bar = 10 lm.

(b) Root tip cells expressing GFP–RABA1b and mRFP–SYP43 (above) or

mRFP–VAMP722 (below) treated with 33 lM wortmannin for 1 h. Green,

GFP–RABA1b; magenta, mRFP-tagged markers. Scale bar = 10 lm.

(a)

(b)

(c)

(d)

(e)

Figure 5. RABA1b mediates transport between the TGN and the plasma

membrane.

(a) Subcellular localization of GFP–RABA1b (WT), GFP–RABA1bQ72L (QL) and

GFP–RABA1bS27N (SN) in root tip cells (above) and root differentiated cells

(below). The arrow indicates a dot-like signal for GFP–RABA1bQ72L and the

arrowhead indicates the signal for GFP–RABA1bQ72L on the plasmamembrane.

(b–e) Co-expression of GFP–RABA1bQ72L or GFP–RABA1bS27N and mRFP-

tagged markers in root tip cells. The regions enclosed by white squares are

magnified and shown in the right column. Scale bars = 10 lm.



larger punctate structures (Figure 5a and Figure S6). These

structures co-localized well with ARA6–mRFP (Figure 5e and

Figure S7), suggesting recolation of RABA1bS27N to multi-

vesicular endosomes (Ebine et al., 2011). Interestingly, the

size of the ARA6–mRFP compartments appeared larger than

in the control (Figure 3e) when co-expressed with GFP

–RABA1bS27N.

The co-localization of RABA1b and VAMP721/722 and the

localization of GFP–RABA1bQ72L on the plasma membrane

support the idea that RABA1b may function in transport

from the TGN to the plasma membrane. To test whether

RABA1b plays any role in endocytosis, we examined the

effect of GFP–RABA1bQ72L or GFP–RABA1bS27N expression

on the uptake of FM4-64, a lipophilic dye used to trace endo-

cytosis. As shown in Figure 6, no significant difference was

observed in the kinetics of FM4-64 internalization.

Dynamic motion of RABA1b vesicles is actin-dependent

As shown in Figure 2, which was taken with a long

exposure time (1 sec), localization of GFP–RABA1b

appeared to occur along distinct lines. To increase the time

resolution, we applied variable incidence angle fluorescent

microscopy (VIAFM), a technique that is related to total

internal reflection fluorescence microscopy (TIRFM), which

has a high signal-to-noise ratio and can take pictures at 10

frames per sec. High-speed movies clearly showed that

these aligned signals were the fluorescent trajectories of

GFP–RABA1b-containing vesicles moving along filamen-

tous structures (Movie S2). To reveal the nature of these

structures, we studied transgenic plants co-expressing Life-

act–Venus (actin marker, Era et al., 2009) and GFP–

RABA1b. The results indicated that vesicles labeled by GFP

–RABA1b form queues along actin filaments (Figure 7a).

Such aligned signals were dispersed when actin filaments

were depolymerized by latrunculin B. No apparent change

was observed in response to treatment with oryzalin, a

microtubule-depolymerization agent (Figure 7b and Movie

S3). Thus the dynamic motion of RABA1b vesicles is

actin-dependent.

RABA1 members function in salinity stress tolerance

All pieces of evidence obtained so far suggest that the

major function of RABA1b is in the secretory pathway, at

the transport step from the TGN to the plasma membrane.

Figure 6. RABA1b mutants do not affect endo-

cytosis.

Endocytosis in wild-type cells (Control) and

transgenic cells expressing GFP–RABA1b, GFP–RABA1bQ72L or GFP–RABA1bS27N was visualized

using FM4-64. Images were captured at 10, 30,

60, 90, 120 and 180 min after staining. Scale

bar = 10 lm.

(a)

(b)

Figure 7. Dynamic motion of the RABA1b compartment is actin-dependent.

(a) Co-expression of GFP–RABA1b (green) and Lifeact–Venus (magenta) in

root differentiated cells. Scale bar = 10 lm.

(b) Root differentiated cells expressing GFP–RABA1b treated with 2 lM la-

trunculin B (upper) or 10 lM oryzalin (lower) for 3 h. Scale bar = 10 lm.



To further understand possible physiological roles of

RABA1 members, we obtained four T-DNA-inserted mutant

lines, which were completely knocked out for expression

of RABA1A, RABA1B, RABA1C and RABA1D (Figure 8a).

Single mutants showed no macroscopically abnormal

phenotypes. We therefore constructed multiple mutated

plants by cross-pollination. The quadruple mutant raba1a

raba1b raba1c raba1d exhibited only a marginal phenotype

under ordinary growth conditions, with slightly shorter

roots than the wild-type (Figure 8b,c). Transgenic plants

(a)

(b)

(c)

(d) (e)

Figure 8. RABA1 members are required for salinity stress tolerance.

(a) Schematic structures of RABA1 genes and the positions of T-DNA insertions.

(b) Expression of RABA1 genes was not detected in the quadruple mutant by RT-PCR.

(c) Phenotypes of the RABA1 quadruple mutant under normal growth and salinity stress conditions. Plants were grown for 14 days. Scale bar = 1 cm.

(d) Phenotypes of transgenic plants expressing dominant-negative or constitutive-active mutant proteins under normal growth and salinity stress conditions.

Plants were grown for 14 days. Scale bar = 1 cm.

(e) Relative fresh weight of wild-type and mutant plants. Values are means ± standard errors normalized to the mean fresh weight of wild-type plants under

each condition.



expressing the dominant-negative RABA1bS27N mutant

showed a more prominent growth defect than the

quadruple mutant (Figure 8d), suggesting that other RABA1

members support loss of RABA1a–RABA1d but are domi-

nantly impaired by expression of RABA1bS27N. Next, we

focused on stress tolerance and investigated the

phenotypes of the quadruple mutants and the plants

expressing RABA1bS27N under biotic or abiotic stress. We

found that these plants exhibited prominent phenotypes

when subjected to salinity stress. Interestingly, the quadru-

ple mutant showed a very severe growth defect under salin-

ity stress conditions (Figure 8c). On one-tenth strength MS

medium containing 15 mM NaCl, the majority of the quadru-

ple mutants died during the cotyledon stage (Figure 8c). The

fresh weights of the quadruple mutant were fivefold lower

than those of the wild-type plants on average (Figure 8e).

This salt hypersensitivity of the quadruple mutant was

complemented by Venus–RABA1a, GFP–RABA1b or GFP–

RABA1c (Figure 8c). The plants expressing RABA1bS27N

were also hypersensitive to salinity stress (Figure 8d,e). Nei-

ther the quadruple knockout mutation nor expression of

RABA1bS27N affected growth sensitivity to 30 mM sorbitol

(Figure S3), indicating that the hypersensitivity was not due

to high osmolarity but to the high salt concentration. Taken

together, these results show that at least four members of

the RABA1 group, RABA1a, RABA1b, RABA1c and RABA1d,

act redundantly in tolerance to salinity stress.

DISCUSSION

Characteristics of the RABA1 compartment

In this study, we analyzed the subcellular localization of

RABA1b as a representative of the RABA1 group, and show

that its compartment overlaps with the TGN or lies in its

vicinity. The RABA1b compartment shows dynamic motion

in an actin-dependent fashion. Members of other RABA

sub-groups, RABA2 and RABA3, have been reported to

partially co-localize with structures labeled by VHA-a1–GFP

and FM4-64, but not with the Golgi apparatus or the

pre-vacuolar compartment labeled by ST–GFP and GFP–

BP-80 (Chow et al., 2008). The authors concluded that the

RABA2/3 compartment is an early endosomal TGN-associ-

ated membrane domain. In a collaborative study, Feraru

et al. (2012) showed that RABA1b and RABA2a co-localize.

Whether these sub-classes of RABA share membrane

traffic events around the TGN remains to be pursued.

RABA1b regulates the transport between the TGN

and the plasma membrane

RAB GTPases generally act as molecular switches through

conformational change between GTP- and GDP-bound

forms in order to control docking and fusion events. GTP-

bound Rab GTPases are considered to localize on the target

organelle (Chavrier and Goud, 1999). The fact that GTP-

fixed RABA1b largely localized to the plasma membrane is

likely to reflect the function of the RABA1b, suggesting a

role in the exocytic event. Dettmer et al. (2006) indicates

that the TGN functions as the early endosome in the endo-

cytic pathway. However, neither the GTP-fixed nor the

GDP-fixed mutant of RABA1b affected endocytosis of the

FM4-64 dye. A similar result was obtained in an indepen-

dent study on epidermal leaf cells of Nicotiana benthami-

ana (S. Choi, Department of Biological Sciences, Graduate

School of Science, The University of Tokyo, T. Tamaki,

Department of Biological Sciences, Graduate School of

Science, The University of Tokyo,

K. Ebine, Department of Biological Sciences, Graduate

School of Science, The University of Tokyo, T. Uemura, T.

Ueda and A. Nakano, unpublished results). A similar con-

clusion was also drawn on the basis of another indepen-

dent study on recycling of PIN proteins (Feraru et al., 2012).

Thus, we suggest that RABA1b, and probably other RABA1

members as well, act on the transport process from the

TGN-associated compartment to the plasma membrane.

We also observed that RABA1b shows partial associa-

tion with ARA6, which is more evident when RABA1b is

locked in the GDP form. ARA6 is a plant-unique RAB5

member that mediates transport from the multi-vesicular

endosomes to the plasma membrane (Ebine et al., 2011).

Expression of the RABA1b GDP mutant increased the size

of ARA6 endosomes. These results may provide an

indication of the molecular mechanisms of the complicated

post-Golgi traffic system in plants.

Evolution of RABA1 members – redundancy and diversity

Here, we have demonstrated that RABA1 members

function redundantly. It is also possible that some mem-

bers of other RABA sub-groups share part of functions

with RABA1. Many members of the RABA family show

similar localization patterns, especially in relation to the

TGN. Furthermore, RABA4b actively accumulates in the tip

region of root hairs (Preuss et al., 2004), as for RABA1b

and RABA1e (Figures 1a and 2a). RABA4d, which has a

crucial role in the formation of pollen tubes (Szumlanski

and Nielsen, 2009), accumulates in the tip of pollen tubes,

as does RABA1f (Figure 1c). On the other hand, some

RABA members have been implicated in endocytic events,

unlike RABA1b (our unpublished results). Further extensive

analysis is necessary to understand the whole spectrum of

the RABA GTPase functions, which have shown multiplica-

tion and diversification during evolution.

Analysis of the quadruple raba1a raba1b raba1c raba1d

mutant has revealed that RABA1a–d proteins are required

for tolerance to salinity stress. This implies that these

RABA1 proteins regulate the localization of cell-surface

proteins, such as pumps and channels. The fact that

the GDP-fixed mutant of RABA1b causes even more severe

salt sensitivity indicates that other members of the RABA1



sub-family are also involved. For land plants, it is impera-

tive to adapt quickly and flexibly to a change in environ-

ment. The expansion and diversification of RABA

members may be advantageous to sense the emergence of

stress and respond to it. Determination of the cargo trans-

ported by RABA1 members and regulators of RABA1 is a

very important subject for future studies.

EXPERIMENTAL PROCEDURES

Plant materials and growth conditions

T-DNA insertion lines were derived from the Col-0 accession of A.thaliana. Both lines were backcrossed three times into the Col-0accession. Seeds for raba1a (SALK77747), raba1c (SALK145363)and raba1d (SALK88879) were obtained from the Arabidopsis Bio-logical Resource Center. Seeds for raba1b (SAIL875F08) wereobtained from the Nottingham Arabidopsis Stock Centre.

Arabidopsis seeds were sterilized and planted on 0.3% agarplates containing half-strength Murashige and Skoog medium, 1%w/v sucrose and vitamins (pH 5.7). Plants were grown in a climatechamber at 23°C under the continuous light.

RT-PCR

Total RNA was extracted from rosette leaves of 10-day-old wild-type and mutant plants using RNAqueous-4PCR (Life Technologies,http://www.lifetechnologies.com/), and cDNA was synthesizedusing SuperScript III reverse transcriptase (Life Technologies).PCR conditions were as follows: 95°C for 2 min, then 30 cycles of95°C for 30 s, 55°C for 30 sec and 72°C for 1 min, then 72°C for5 min.

Plasmids and transformation of plants

By fluorescent tagging of full-length proteins (Tian et al., 2004),fragments of each gene, including cDNA encoding GFP, Venus ormRFP in front of the start codon of each gene, were generated.The RABA1A fragment contains 3.3 kb of 5′ and 1.1 kb of 3′ flank-ing sequences. The RABA1B fragment contains 1.7 kb of 5′ and 1.0kb of 3′ flanking sequences. The RABA1C fragment contains 1.9 kbof 5′ and 1.2 kb of 3′ flanking sequences. The RABA1D fragmentcontains 3.2 kb of 5′ and 0.9 kb of 3′ flanking sequences. TheRABA1E fragment contains 2.4 kb of 5′ and 0.7 kb of 3′ flankingsequences. The RABA1F fragment contains 1.6 kb of 5′ and 0.8 kbof 3′ flanking sequences. Amplified chimeric fragments were sub-cloned into the binary vector pGWB1, a kind gift from Dr. T. Naka-gawa (Center for Integrated Research in Science, Shimane Univer-sity, Japan), which were used for transforming Arabidopsisplants. Transformation of Arabidopsis plants was performed byfloral dipping (Clough and Bent, 1998) using Agrobacterium tum-efaciens strain GV3101::pMP90.

Microscopy

Plants expressing GFP-, Venus- or mRFP-tagged proteins wereobserved under an LSM710 confocal microscope equipped with aMETA device (Carl Zeiss, http://corporate.zeiss.com/) for multi-color observations, and an Olympus (http://www.olympus-global.com/) BX51 microscope equipped with a confocal scanner unit(model CSU10; Yokogawa Electric, http://www.yokogawa.com/)for single-color observations, with a cooled CCD camera (modelORCA-AG; Hamamatsu Photonics, http://www.hamamatsu.com/index.html). The co-localization analysis was performed as

described previously (Ito et al., 2012). Images were captured usingan LSM710 confocal microscope with oil immersion lens (9 63,numerical aperture = 1.40). The line scan analysis was performedusing Metamorph software (Molecular Devices, http://www.mole-culardevices.com/).

Roots of plants after 5 days of culture were observed. Forobservation of root hairs, seeds were germinated on thin agarmedium (compositionally the same as described above) in aglass-bottomed dish and observed without any preparation after10 days of culture.

Variable incidence angle fluorescent microscopy

Roots of transgenic A. thaliana seedlings were placed on glassslides (76 9 26 mm; Matsunami, http://www.matsunami-glass.co.jp/), covered with a 0.12–0.17 mm thick cover slip (24 9 60 mm;Matsunami), and root epidermal cells were observed under a fluo-rescence microscope (Nikon Eclipse TE2000-E and a CFI ApoTIRF 9 100 H/1.49 numerical aperture objective, http://www.nikon.com/) equipped with a Nikon TIRF2 system. GFP was excited usingan 488 nm laser. All images were acquired using an Andor iXo-nEM electron multiplying charge coupled device (EMCCD) camera(http://www.andor.com/).

Chemical treatments

To visualize the process of endocytosis, seedlings were mountedin half-strength MS liquid with 16.5 lM FM4-64 (Invitrogen/Molec-ular Probes; T13320; diluted from a 16.5 mM stock in water) on ice,then washed out and incubated at 23°C. For BFA treatment, seed-lings were incubated in half-strength MS liquid containing 50 lMBFA diluted from a 50 mM stock in DMSO, and then mounted onthe slides in the presence of BFA. Wortmannin was used at 33 lMdiluted from a 10 mM stock in DMSO, and the treatment wasperformed in a similar way as BFA treatment. Latrunculin B andoryzalin treatments were also performed in similar ways. Controltreatment was performed with DMSO in place of each chemical.

Salinity stress assay

The salinity stress assay was performed as described by Krebset al. (2010). Surface-sterilized seeds were sown on 1% agar platescontaining one-tenth strength MS medium, 0.5% w/v sucrose and10 mM MES/KOH (pH5.8) with or without 15 mM sodium chlorideor 30 mM sorbitol (as an osmotic control). Fresh weight was mea-sured after 14 days of culture.

ACCESSION NUMBERS

The Arabidopsis Genome Initiative locus identifiers for the

genes referred to in this paper are At1g06400 (RABA1A),

At1g16920 (RABA1B), At5g45750 (RABA1C), At4g18800

(RABA1D), At4g18430 (RABA1E), At5g60860 (RABA1F),

At3g15060 (RABA1G), At2g33870 (RABA1H), and At1g28550

(RABA1I).

ACKNOWLEDGEMENTS

We thank the Arabidopsis Biological Resource Center and Notting-ham Arabidopsis Stock Centre for providing T-DNA insertionmutants of Arabidopsis. We also thank T. Nakagawa (Center forIntegrated Research in Science, Shimane University, Japan) andM. Sato (Graduate School of Biostudies, Kyoto Prefectural Univer-sity, Japan) for sharing materials, N. Tsutsumi (Graduate Schoolof Agricultural and Life Sciences, University of Tokyo, Japan) for



variable incidence angle fluorescent microscopy and K. Ebine(National Institute of Infectious Diseases, Tokyo, Japan) forvaluable advice. This work was supported by a Grant-in-Aid forSpecially Promoted Research from the Ministry of Education,Culture, Sports, Science and Technology of Japan, and by fundsfrom the Extreme Photonics and Cellular Systems Biology Pro-jects of RIKEN. R.A. is the recipient of a Research Fellowship forYoung Scientists from the Japan Society for the Promotion ofScience.

SUPPORTING INFORMATION

Additional Supporting Information may be found in the online ver-sion of this article.Figure S1. Expression patterns of RABA1 members from theonline microarray data.

Figure S2. Control treatment with DMSO.

Figure S3. Osmotic stress assay as a control.

Figure S4. Quantitative analysis of the co-localization betweenRABA1b and other RABA1 members or organelle markers.

Figure S5. Line scan analysis of GFP–RABA1b, mRFP–SYP43 andmRFP–VAMP722 after BFA or wortmannin treatment.

Figure S6. Line scan analysis of GFP–RABA1b, GFP–RABA1bQ72L

and GFP–RABA1bS27N.

Figure S7. Line scan analysis of GFP–RABA1b, GFP–RABA1bQ72L,GFP–RABA1bS27N and mRFP-tagged organelle markers.

Movie S1. GFP–RABA1b-containing compartments in differen-tiated cells of roots observed by confocal microscopy.

Movie S2. GFP–RABA1b-containing compartments in root differ-entiated cells by variable incidence angle fluorescent microscopy.

Movie S3. RABA1 compartments treated with 2 lM latrunculin B or10 lM oryzalin.

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