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ArabidopsisRABgeranylgeranyltransferaseβ‐subunitmutantisconstitutivelyphotomorphogenic,andhasshootgrowthandgravitropicdefects

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Arabidopsis RAB geranylgeranyl transferase b-subunitmutant is constitutively photomorphogenic, and hasshoot growth and gravitropic defects

Michal Hala1, Hana Soukupova1, Lukas Synek1 and Viktor Zarsky1,2*

1Institute of Experimental Botany, Academy of Sciences of the Czech Republic, Rozvojova 263, 165 02 Prague 6, Czech Republic,

and2Department of Plant Physiology, Faculty of Science, Charles University, Vinicna 5, 128 44 Prague 2, Czech Republic

Received 18 September 2009; revised 26 January 2010; accepted 4 February 2010; published online 23 March 2010.*For correspondence (fax +420 225106461; e-mail zarsky@ueb.cas.cz).

SUMMARY

RAB GTPases are important directional regulators of intracellular vesicle transport. Membrane localization of

RAB GTPases is mediated by C-terminal double geranylgeranylation. This post-translational modification is

catalyzed by the a-b-heterodimer catalytic core of RAB geranylgeranyl transferase (RAB-GGT), which

cooperates with the RAB escort protein (REP) that presents a nascent RAB. Here, we show that RAB-

geranylgeranylation activity is significantly reduced in two homozygous mutants of the major Arabidopsis

b-subunit of RAB-GGT (AtRGTB1), resulting in unprenylated RAB GTPases accumulation in the cytoplasm.

Both endocytosis and exocytosis are downregulated in rgtb1 homozygotes defective in shoot growth and

morphogenesis. Root gravitropism is normal in rgtb1 roots, but is significantly compromised in shoots.

Mutants are defective in etiolation and show constitutive photomorphogenic phenotypes that cannot be

rescued by brassinosteroid treatment, similarly to the det3 mutant that is also defective in the secretory

pathway. Transcriptomic analysis revealed an upregulation of specific RAB GTPases in etiolated wild-type

plants. Taken together, these data suggest that the downregulation of the secretory pathway is interpreted as

a photomorphogenic signal in Arabidopsis.

Keywords: RAB GTPases, cell expansion, photomorphogenesis, vesicle trafficking, Arabidopsis thaliana,

prenylation.

INTRODUCTION

RAB proteins represent the largest subset of the Ras

superfamily of small GTPases in yeast and animals, as well

as in plants (Pereira-Leal and Seabra, 2001; Rutherford and

Moore, 2002). Although only some members of the RAB

family were characterized on the functional level, it seems

that most RABs are involved in the regulation of various

steps of vesicular transport within the secretion and

endocytotic pathways, including vesicle budding, transport

along the cytoskeleton and vesicle fusion with target

membranes (Deneka et al., 2003; Novick et al., 2006;

Fukuda, 2008; Woollard and Moore, 2008). The number of

distinct RABs varies considerably among species: 11 in

Saccharomyces cerevisiae, 29 in Caenorhabditis elegans,

40–60 in angiosperm plants and more than 60 in mammals

(Pereira-Leal and Seabra, 2001; Woollard and Moore,

2008).

Membrane localization of hydrophilic RAB proteins is

achieved by their post-translational hydrophobic modifica-

tion catalyzed by the RAB geranylgeranyl transferase (RAB-

GGT), one of three protein prenyl transferases present in

eukaryotic cells (Maurer-Stroh et al., 2003). Most RABs

contain a C-terminal motif with two cysteine residues

(usually -XXCC or -XCXC), to which geranylgeranyl moieties

are covalently attached via a thioether bond. Although RAB-

GGT prenylates exclusively RAB GTPases, it remains unclear

whether it is the only enzyme geranylgeranylating RABs

in vivo, as several members of the RAB family (e.g. the

mammalian Rab8 containing the -CXXX C-terminal motif)

have also been shown to serve as a substrate for geranyl-

geranyl transferase I (GGTI) in vitro (Wilson et al., 1998). In

plants, a novel type of RABs lacking the C-terminal preny-

lation motif was reported, and was shown to associate with

ª 2010 The Authors 615Journal compilation ª 2010 Blackwell Publishing Ltd

The Plant Journal (2010) 62, 615–627 doi: 10.1111/j.1365-313X.2010.04172.x

membranes via N-terminal myristoylation and palmitoyla-

tion (Bolte et al., 2000; Ueda et al., 2001). The functional

cycle of these RABs seems to be RAB-GDI-independent

(Ueda et al., 2001).

Similarly to other protein prenyl transferases, RAB-GGT is

a heterodimer of tightly associated a- and b-subunits

(Maurer-Stroh et al., 2003). However, in contrast to farnesyl

transferase and GGTI, it does not recognize its protein

substrate directly, but only in a complex with an accessory

RAB escort protein (REP). The mechanism of the reaction

has been well described in mammals and yeast (Anant et al.,

1998; Thoma et al., 2001a,b). Nascent RAB GTPase and REP

form a cytoplasmic complex which then binds to RAB-GGT

in a prenyl-substrate-dependent manner. Recently, Baron

and Seabra (2008) proposed that an alternative scenario

might be prevalent in vivo: first, REP binds to a catalytic

a-b heterodimer, and then nascent RAB is loaded into this

pre-assembled ternary complex. Geranylgeranylation pro-

ceeds step by step so that mono-geranylgeranylated inter-

mediates can be isolated (Shen and Seabra, 1996; Thoma

et al., 2001a). The use of mutant RAB GTPases partially or

completely lacking a prenylation motif inhibits the activity of

RAB-GGT, as shown for the plant RAB GTPase LeRab1 by

Loraine et al. (1996).

The interface of the REP-RAB-GGT complex is limited to a

relatively small area, and involves a combination of hydro-

phobic and charge interactions. It was shown that the

a-subunit of RAB-GGT undergoes a series of conformational

changes preceded by the formation of the a-b dimer in the

presence of a phosphoisoprenoid substrate. These changes

also force the REP-binding domains into the right confor-

mation for binding the REP-RAB complex (Pylypenko et al.,

2003).

Loss-of-function mutations in RAB-GGT subunits in yeast

are lethal (http://www.yeastgenome.org). In animals, the

gunmetal mouse, carrying a mutant allele of the RAB-GGT

a-subunit with mutated splicing site, displays reduced RAB

geranylgeranylation activity, resulting in a weak phenotype

characterized by platelets malformation (Detter et al., 2000).

Mutation in human REP1 causes choroideremia disease, an

X-chromosome-linked retinal dystrophy (Cremers et al.,

1992; Merry et al., 1992; Seabra et al., 1992). Surprisingly,

it seems that the mutation of the single C. elegans REP

selectively affects only specific RABs, whereas others are

correctly geranylated and localized in the supposed absence

of wild-type REP (Tanaka et al., 2008).

Protein prenylation was first documented in plants by

Randall et al. (1993). The Arabidopsis farnesyl transferase

has been found to be involved in abscisic acid-dependent

plant developmental processes, as demonstrated by the

phenotype of era1, a mutant in the farnesyl transferase

b-subunit (Cutler et al., 1996), or in the a-subunit mutant plp

(Running et al., 2004). Also, the ggb mutant in the Arabid-

opsis geranylgeranyl transferase I b-subunit is compro-

mised in some aspects of ABA and auxin responses;

interestingly, the phenotype of a double mutant combining

ggb and era1 lesions is identical to that of the plp mutant

(Johnson et al., 2005). Functional analysis of the Arabidop-

sis CaaX processing enzymes also using, among others,

RNAi suppression of isoprenyl cysteine methyltransferase,

showed that phenotypic deviations of these RNAi plants are

somewhat similar to those in the era1 mutant phenotype

(Bracha-Drori et al., 2008).

Biermann et al. (1996) characterized several prenylated

GTPases in plants, and also described the RAB-GGT activity

prenylating recombinant Rab1 and Rab2 in tobacco extracts.

Yalovsky et al. (1996) reported a wide substrate specificity of

different plant RAB-GGTs in total extracts, a preference for

geranylgeranyl pyrophosphate (GGPP) and an inhibitory

effect of RAB GTPases lacking C-terminal cysteine motifs.

Loraine et al. (1996) observed an ability of tomato Rab1 to

undergo geranylgeranylation in the yeast extract in vitro,

and to complement the yeast ypt1 mutant. We have previ-

ously characterized a single Arabidopsis RAB escort protein,

and demonstrated its plant-specific structural feature in the

RAB-GGT interaction with REP: site-directed amino acid

substitution into the Opisthokontian consensus sequence

allowed plant REP to complement mrs6, the yeast REP

mutant (Hala et al., 2005). Recently, the plant-specific plas-

tidial pathway of prenylation precursors biosynthesis was

shown to be crucial for protein geranylgeranylation in plant

cells (Gerber et al., 2009).

In this paper, we report that a mutation in the major

b-subunit of Arabidopsis RAB-GGT results in RAB geranyl-

geranylation deficiency, causes distinct phenotypic changes

and affects many physiological processes, such as shoot

gravitropism and cell elongation. Surprisingly, rgtb1

mutants also exhibit a constitutive photomorphogenesis

phenotype.

RESULTS

RAB geranylgeranyl transferase in the Arabidopsis genome

A search of the Arabidopsis genome revealed the presence

of two genes encoding RAB-GGT a-subunits (AtRGTA1, lo-

cus At4g24490; AtRGTA2, locus At5g41820), as well as two

genes encoding RAB-GGT b-subunits (AtRGTB1, locus

At5g12210; AtRGTB2, locus At3g12070).

The AtRGTA1 protein shares 27% sequence identity and

41% similarity with the human RAB-GGT a-subunit;

AtRGTB1 shares 56% identity and 71% similarity with the

human RAB-GGT b-subunit. Although AtRGTA2 and

AtRGTA1 subunits share 66% identity and 74% similarity

with each other, AtRGTB1 and AtRGTB2 are more similar to

each other (83% identity and 89% similarity).

The presence of two RAB-GGT subunits in the Arabidop-

sis genome is unusual when compared with other non-plant

eukaryotes. To test whether this is a common plant feature,

616 Michal Hala et al.

ª 2010 The AuthorsJournal compilation ª 2010 Blackwell Publishing Ltd, The Plant Journal, (2010), 62, 615–627

we constructed a phylogenetic tree of protein prenyl trans-

ferase b-subunits (Figure S1). Both b-subunits of Arabidop-

sis thaliana, RGTB1 and RGTB2, branch together, but

separately from orthologous couples in Vitis vinifera, Physc-

omitrella patens, and Selaginella moellendorffii. This obser-

vation implies independent recent duplication events.

Comparison of expression profiles for all RAB-GGT genes

and REP using Genevestigator (Zimmermann et al., 2004)

revealed the relatively low level of AtREP expression in all

tissues examined (also in Hala et al., 2005; Wojtas et al.,

2007). AtRGTB1 mRNA is generally 10 times more abundant

than AtRGTB2 mRNA in sporophytes, whereas levels of both

mRNAs are comparable in male gametophytes. AtRGTA1 is

expressed in all tissues, in contrast to AtRGTA2, which is

only significantly expressed in pollen (Figure S2).

Disruption of Arabidopsis RGTB1 results in pleiotropic

growth and development defects

Two independent T-DNA insertional mutants in the

AtRGTB1 gene, rgtb1-1 and rgtb1-2, were obtained from the

SALK collection (Appendix S1). After germination the pop-

ulation started to segregate, with one-quarter producing

dwarf plants, both in vitro and in soil growth conditions.

These plants were confirmed by PCR genotyping to be

homozygous mutants.

Both mutant lines exhibited an identical phenotype: root-

tip organization, cell differentiation in the elongation zone,

total length of the primary root, as well as root-hair

formation were unaffected, and so were similar to that of

the wild-type (WT) plants, although a weak tendency

towards longer primary roots was observed in 1-week-old

etiolated mutants (see below). However, detailed study

revealed that differences between rgtb1 homozygotes and

WT plants are prominent in the shoot. The leaves of the

homozygotes became epinastic and smaller than wild-type

leaves (Figure 1a). The number of rgtb1-1 and rgtb1-2

rosette leaves is statistically indistinguishable from the

number in WT plants, but the diameter of rosettes is much

lower in homozygous mutants (Figure 1a,b).

Homozygous rgtb1-1 and rgtb1-2 plants produce multiple

inflorescence stems, indicating a loss of apical dominance

(Figure 1c). Although the number of floral organs remains

constant, mutant flowers never open fully, featuring distinct

pistil protuberance (Figure 1d). Flowering in homozygous

rgtb1-1 and rgtb1-2 plants started at the same time as in the

WT, but the siliques remained mostly sterile. Reciprocal

crossing with WT plants, however, resulted in fertilization,

regardless of whether rgtb1-1 or rgtb1-2 were pollen donors

or recipients. In all cases, the progeny was comprised of a

(a)

(b)

(c)

(d)

Figure 1. Phenotype of homozygous rgtb1

mutants.

(a) Two-week-old plants. Scale bars: 5 mm.

(b) Three-week-old plants.

(c) Four-week-old plants.

(d) Flowers of 4-week-old plants.

RAB prenylation and photomorphogenesis 617

ª 2010 The AuthorsJournal compilation ª 2010 Blackwell Publishing Ltd, The Plant Journal, (2010), 62, 615–627

uniform population of heterozygotes, suggesting that

mutant pollen is functional.

Although wild-type plants are typically more than 24 cm

high 30 days after germination, rgtb1-1 and rgtb1-2 homo-

zygotes reach only approximately 6 cm in height (Figure 1c).

In addition, the onset of senescence in rgtb1-1 and rgtb1-2

plants is delayed by 2 months, compared with wild-type

plants, in our growth conditions.

Biochemical characterization of rgtb1 mutants

Using RT-PCR, we analyzed homozygous rgtb1-1 and rgtb1-

2 mutants as well as WT plants for the expression of mRNA

encoding the RGTB1 subunit. It turned out that the full-

length mRNA of the RGTB1 subunit was absent in the

homozygous mutants tested, whereas actin (ACT2) and

RGTB2 were expressed at the normal level (see Figure S3).

Next, we tested the possible presence of unprenylated

RAB GTPases, substrates of the RAB-GGT, in rgtb1-1 and

rgtb1-2 homozygotes. Western blot analysis using the anti-

AtRAB-A2a antibody (provided by I. Moore, University of

Oxford) revealed a band of AtRAB-A2a in the WT. However,

in the case of both rgtb1-1 and rgtb1-2 homozygotes, this

band was accompanied by other weaker bands of higher

molecular weight, probably representing unprenylated

AtRAB-A2a (Figure 2a). This shift in mobility might be

caused by the well characterized more effective SDS binding

to the prenyl-modified proteins under conditions of SDS-

PAGE (Sanford et al., 1995). No accumulation of such

possibly unprenylated RAB GTPases was observed in the

WT. To further address the subcellular localization of these

putatively unprenylated RAB GTPases, we prepared micro-

somal and cytosolic fractions from etiolated hypocotyls of

WT and rgtb1-1 homozygotes. Although a large pool of

AtRAB-A2a was still attached to the membranes, a signifi-

cant accumulation of a higher molecular weight second

band of possibly unprenylated RAB occured in the cytosolic

fraction of rgtb1-1 homozygote hypocotyls (Figure S4).

In order to compare the activity of RAB-GGT in vitro,

extracts from 3-week-old light-grown plantlets were pre-

pared, and the specific RAB-GGT activity was tested by the

addition of recombinant AtRAB-A2a as a substrate.

Figure 2(b) shows that the RAB geranylgeranylation activity

in both rgtb1-1 and rgtb1-2 homozygous mutants was

reduced to �25% of the WT level (similar results were seen

using a few other recombinant RABs as substrates; data not

shown). As both rgtb1-1 and rgtb1-2 exhibit identical

phenotypes and biochemical characteristics mentioned

above, we used only rgtb1-1 for further experiments.

Arabidopsis rgtb1 mutants show gravitropic defect in the

shoot

To test for a possible defect in gravitropic response, often

associated with mutations in vesicle transport organizers

(e.g. SNARE proteins; Kato et al., 2002; Yano et al., 2003; for

a review see Bassham and Blatt, 2008), we set up a gravi-

tropic assay on roots in a light-grown segregating popula-

tion of the rbtg1-1/RGTB1 progeny. Ten days after

germination, the vertical agar plates were rotated by 90�.Roots of all plants responded to the change in gravity vector

equally (Figure 3a). Next, pots containing 3-week-old WT

plants or rgtb1-1 mutants were rotated by 90�, and shoot

gravitropism was tested. Plants were kept in the dark for

12 h before images were taken. Shoots of all WT plants re-

sponded to the new gravity direction, whereas no response

was observed in rgtb1-1 homozygotes (Figure 3b).

Arabidopsis rgtb1 mutants exhibit secretory defects

RAB GTPases are important regulators of vesicle trafficking

in the cell. Deficiency in RAB geranylgeranylation activity in

the rgtb1-1 mutants, as documented above, should result in

the defective functioning of the secretory machinery, there-

by leading to impaired cell elongation. To test this hypoth-

esis, we crossed rgtb1-1 mutants with plants transformed

with the secretory GFP construct (secGFP, provided by I.

Moore). Under normal conditions, secGFP is secreted out of

the cell, and the environment outside the cell (low pH and

(a)

(b)

Figure 2. Biochemical characteristics of homozygous rgtb1 mutants.

(a) Western blot analysis in extracts from rgtb1-1, rgtb1-2 and wild-type (WT)

plants using rabbit polyclonal anti-AtRAB-A2a antibody (1:1000). Two differ-

ent forms of AtRAB-A2a are labeled: unprenylated AtRAB-A2a and prenylated

AtRAB-A2a-GG.

(b) RAB geranylgeranylation activities detected after the addition of 4 lg of

recombinant AtRAB-A4a in cell extracts from rgtb1-1 and rgtb1-2 homozy-

gous mutants. Each column represents three independent measurements;

bars correspond to standard deviations.

618 Michal Hala et al.

ª 2010 The AuthorsJournal compilation ª 2010 Blackwell Publishing Ltd, The Plant Journal, (2010), 62, 615–627

presence of proteases) quenches the fluorescence. When the

secretion is perturbed secGFP accumulates inside the cell,

and a fluorescent signal becomes visible (Batoko et al.,

2000).

We tested 1-week old dark-grown progeny of an rgtb1-1/

RGTB1 heterozygote expressing secGFP. We observed that

secGFP expression did not alter the rgtb1 phenotype.

Microscopic analysis revealed significantly higher accumu-

lation of secGFP inside the cells of rgtb1-1 hypocotyls

(Figure 4a,c), in contrast to the WT (Figure 4b,d). In rgtb1-1

cells, confocal microscopy showed that secGFP is largely

accumulated in diverse internal structures [such as the

endoplasmic reticulum (ER) and ER-like bodies], rather

than in the cytoplasm (Figure 4c). However, the strongly

increased overall fluorescence of rgtb1 mutant hypocotyls is

partly the result of increased non-specific autofluorescence.

Endocytosis is an essential process of the secretory

pathway, and is as important as exocytosis. Using confocal

microscopy, we visualized endocytic compartments by

vacuum infiltration of the styryl dye FM4-64 in etiolated

hypocotyls of 1-week-old seedlings. Whereas visible

FM-labeled moving particles (endocytic compartments)

soon appeared, reproducibly, inside the hypocotyl cells in

WT plants (Figure 4h–j), only a few of them were observed in

rgtb1-1 homozygotes up to 90 min (Figure 4e–g). Using

image analysis, we measured the fluorescence inside hypo-

cotyl cells cytoplasm. The average fluorescence was

17.5 � 2.0 relative units in WT cells and 7.3 � 4.3 relative

units in rgtb1 cells (n = 20; Student’s t-test P < 0.001).

Moreover, between 30–90 min after treatment with brefel-

din A (BFA), BFA compartments emerged in WT hypocotyl

cells, but not in rgtb1-1 hypocotyl cells. Taken together, we

conclude that the rgtb1-1 mutant has defects in the secretory

pathways.

We also observed roots of rgtb1 mutants in both exper-

iments. As expected, based on the WT-like root phenotypes

in both rgtb1 mutants, root cells secrete secGFP and perform

FM4-64 endocytosis like the WT (data not shown).

Arabidopsis rgtb1 mutant homozygotes show de-etiolated

phenotype in the dark

The dwarf phenotype of light-grown rgtb1 mutants led us to

test the ability of rgtb1 cells to elongate in the dark during

etiolation (skotomorphogenesis). We germinated a segre-

gating population of rgtb1-1/RGTB1 heterozygote progeny

in the dark. After 7 days we found a segregating subpopu-

lation of plantlets with short hypocotyls (Figures 5a and 6)

and opened upright cotyledons (Figure 6a). Mutant

hypocotyls consisted of irregularly patterned short cells

instead of the bundles of highly elongated cells typical for

the WT (Figure 5b), and possessed a portion of dead cells.

This subpopulation represented 26% of all plantlets

(n = 120), and following genotyping confirmed they were

rgtb1-1 homozygotes. At this stage, the rgtb1-1 phenotype is

very similar to the de-etiolated 3 (det3) phenotype, a repre-

sentative of a group of mutant plants showing de-etiolation

and constitutive photomorphogenesis obtained under the

same conditions (Figure 6).

To test for possible brassinosteroid dependence of the

rgtb1-1 phenotype, we grew etiolated seedlings on vertical

agar supplemented with 10)6, 10)7, 10)8 or 10)9M 24-

epibrassinolide. We saw no rescue of the rgtb1-1 phenotype

by external brassinosteroid addition for either concentration

(data not shown), as is the case for det3 (Szekeres et al.,

1996).

As the phenotype of dark-grown rgtb1-1 homozygotes

was similar to known det/cop mutants, we prolonged the

dark cultivation of plants for up to 3 weeks. Similarly to the

previous experiment, the population (n = 120) segregated

out 27% of plants exhibiting a constitutively photomorpho-

genic phenotype, which were proven by genotyping to be

rgtb1-1 homozygotes. These plants have fully opened

cotyledons with long petioles, the apical meristem protrud-

(a)

(b)

Figure 3. Gravitropic response of rgtb1-1 plants.

(a) Root gravitropism of the segregating population of rgtb1-1/RGTB1

progeny. Ten days after germination the plate was rotated by 90�. Plants are

labeled according to the later genotyping.

(b) Shoot gravitropism assay. Pots with either wild-type (WT) or rgtb1-1 plants

(3-weeks old) were reoriented and cultivated for 12 more hours. Arrows show

the gravity vector before (1.) and after (2.) reorientation.

RAB prenylation and photomorphogenesis 619

ª 2010 The AuthorsJournal compilation ª 2010 Blackwell Publishing Ltd, The Plant Journal, (2010), 62, 615–627

ing and are developing the first true leaves (Figure 6b).

Obviously, the phenotypic deviations of the 3-week-old det3

mutant grown under the same conditions are more pro-

nounced as compared with rgtb1 (e.g. higher number of

leaves and shorter petioles). The growth direction of dark-

grown rgtb1 plants was variable on vertical agar plates,

pointing again to the defect in shoot gravitropism.

Many RAB GTPases are upregulated during the etiolation

growth of Arabidopsis

The rapid elongation growth of etiolated Arabidopsis seed-

lings in the dark is supported by the activated exocytotic

machinery. We were interested whether expression levels of

RAB GTPases in dark-grown plantlets are generally

(a) (e) (f) (g)

(h) (i) (j)

(b)

(c)

(k) (l)

(d)

Figure 4. Vesicle trafficking defects in rgtb1-1 plants.

(a) Hypocotyls of the 1-week-old dark-grown rgtb1-1 mutant expressing secGFP. Scale bar: 100 lm.

(b) Hypocotyls of a 1-week-old dark-grown wild-type (WT) plant expressing secGFP. Scale bar: 100 lm.

(c) Confocal laser scanning microscope (CLSM) projection through an rgtb1-1 hypocotyl expressing secGFP. Scale bar: 50 lm.

(d) CLSM projection through a WT hypocotyl expressing secGFP. Scale bar: 50 lm.

(e, f, g) Endocytosis in hypocotyls of 1-week-old dark-grown rgtb1-1 plants visualized by FM4-64 dye: (e) immediately after vacuum infiltration, (f) 30 min later and (g)

90 min later. Scale bar: 20 lm.

(h, i, j) Endocytosis in hypocotyls of 1-week-old dark-grown WT plants visualized by FM4-64 dye: (e) immediately after vacuum infiltration, (f) 30 min later and (g)

90 min later. Scale bar: 20 lm.

(k) CLSM projection through a 1-week-old dark-grown rgtb1-1 hypocotyl treated with 50 lM brefeldin A (BFA) for 90 min. Diverse stable patches along the cell

periphery emitting autofluorescence were common in rgtb1-1 hypocotyls, in contrast to those of the WT. Scale bar: 50 lm.

(l) CLSM projection through a 1-week-old dark-grown WT hypocotyl treated with 50 lM BFA for 90 min. Scale bar: 50 lm.

620 Michal Hala et al.

ª 2010 The AuthorsJournal compilation ª 2010 Blackwell Publishing Ltd, The Plant Journal, (2010), 62, 615–627

increased, as predicted for two pea RAB-A proteins (Yoshida

et al., 1993; Nagano et al., 1995). Therefore, we performed

microarray analysis of gene expression on both light- and

dark-grown 7-day-old WT seedlings using the Arabidopsis

ATH1 chip (Table 1, Table S1 and Table S2).

We found that one-third of Arabidopsis RAB GTPase

paralogs (especially in the B, D, E and F subfamilies) were at

least 50% upregulated in WT dark-grown plants when

compared with light-grown plants. Of these, RAB-G3b, -G2,

-B1c and -A5e showed at least a threefold increase in their

mRNA expression and RABGDI1 and 2 are also upregulated

(Tables 1 and 2). However, there was also one-tenth of

downregulated RAB GTPases, namely RAB-G3a, -G3c, -E1b

and -A1c in the dark-grown Arabidopsis plantlets. In any

case, these results are in accord with the data mentioned

above on the dark-induced upregulation of pea RABs, and

show the importance of the upregulation of RAB GTPases

for the implementation of the skotomorphogenic develop-

mental program.

DISCUSSION

Using phylogenetic analyses, we have found that different

angiosperm species acquired independently duplicated

loci encoding RAB-GGT subunits. As in Arabidopsis, two

paralogs of the b-subunit can also be found in the genomes

of V. vinifera, P. patens and S. moellendorffii, whereas in

(a)

(b)

Figure 5. Length of hypocotyls and roots and hypocotyl cell morphology of

dark-grown rgtb1-1 mutants.

(a) Length of hypocotyls (in white) and roots (in black) of rgtb1-1 and wild-type

(WT) plants. Scale bars correspond to standard deviations (n > 132 for each

column). For hypocotyl length, Student’s t-test P < 0.001; for root length,

Student’s t-test P < 0.001.

(b) Morphology of hypocotyl cells in dark-grown 1-week-old seedlings labeled

by 20 mM propidium iodide for 10 min (CLSM projections). Scale

bars: 50 lm.

(a)

(b)

Figure 6. Morphology of dark-grown rgtb1-1 seedlings.

(a) One-week-old rgtb1-1, det3 and wild-type (WT) seedlings grown in the

dark. Only the apical part of the WT is shown (the total hypocotyl length was

18 mm). Scale bars: 2 mm.

(b) Three-week-old rgtb1-1, det3 and WT seedlings grown in the dark. Only the

apical part of the WT is shown (the total hypocotyl length was 19 mm). Scale

bar: 2 mm.

RAB prenylation and photomorphogenesis 621

ª 2010 The AuthorsJournal compilation ª 2010 Blackwell Publishing Ltd, The Plant Journal, (2010), 62, 615–627

genomes of poplar (Populus trichocarpa) and grasses (Oryza

sativa and Sorghum bicolor) only one b-subunit is present

(Figure S1). The presence of two paralogs encoding RGTB

subunits in the genomes of Arabidopsis and V. vinifera are

thus probably the result of independent gene duplications.

Maurer-Stroh et al. (2003) showed that all human prenyl

transferase genes have multiple alternative splice variants,

although their role is unclear, and they may be derived from

mis-splicing rather than being real splice variants. Interest-

ingly, the level of mRNA for the AtRGTA1 a-subunit reaches

on average only 50–60% of the expression level of both

b-subunits. As the RAB-GGT heterodimeric catalytic core is

formed by a- and b-subunits in a 1:1 ratio, this points to

the additional post-transcriptional regulation of gene

expression.

In budding yeast, disruptions of BET2 or BET4 genes,

encoding RAB-GGT b- or a-subunits, respectively, are lethal

(http://www.yeastgenome.org). In animals, several muta-

tions accumulating unprenylated RAB GTPases were

described. A spontaneous point mutation changing

the splicing site of the RAB-GGT a-subunit was described

in the gunmetal (gm) mouse (Detter et al., 2000). Instead of

the expected properly spliced full-length mRNA, a novel

alternative splice product, utilizing cryptic donor and accep-

tor splice sites, was detected at low abundance. RAB

geranylgeranylation activity is reduced in gm homozygotes

to approximately 20% of the normal level. The phenotype of

this mutation is characterized by partial albinism, and by

defects in blood platelets and megacaryocytes; lysosomes

and lysosome-derived organelles are affected on a subcel-

lular level (Seabra et al., 2002). The accumulation of unpre-

nylated Rab27 in the cytoplasm of platelets (described in

Detter et al., 2000) is not the only effect of this mutation.

Eventually, all tissues are affected, accumulating unpreny-

lated forms of different RABs (Seabra et al., 2002; Zhang

et al., 2002). Similarly, unprenylated Rab27 accumulates in

the lymphoblast cells during choroideremia disease (Seabra

et al., 1995), although it can be potentially prenylated by the

REP2-RAB-GGT complex (Rak et al., 2004). The answer to

this discrepancy possibly lies in the different affinities of

RAB GTPases to REPs, varying by two orders of magnitude.

When REP-assisted prenylation is the rate-limiting step,

RABs with lowest affinity remain unprenylated, probably

also in rgtb1 mutants. Although ablation of the Arabidopsis

RGTB1 gene product also results in the reduction of RAB

geranylgeranylation activity to approximately 25% of the

wild-type level, phenotypic changes affect the growth of the

whole plant shoot, and not only specific tissues or organs,

suggesting a higher sensitivity/vulnerability of plants to

defects in secretion machinery throughout the development.

It is interesting, however, that root system development

looks normal in the rgtb1 mutant, including root hairs and

root gravitropism. Similarly as in the gm mouse (Detter

et al., 2000), some tissues (and respective sets of RABs

expressed there) might be more sensitive to a lower

geranylation level than others. This might be associated

with the above mentioned differences in the affinity of

different RABs to REP-RAB-GGTase machinery. Also,

depending on the type of cells, even a substantial decrease

in RAB-GGT activity (down to 20%) does not necessarily

Table 2 Expression levels of GGT, GDI and REP genes in light-grownand dark-grown 7-day-old seedlings of Arabidopsis

MISP code Gene name

Expression

In light In dark

At4g24490 RGTA1 83 66At5g41820 RGTA2 NS NSAt5g12210 RGTB1 193 197At3g12070 RGTB2 NS 10At2g44100 RABGDI1 736 997At3g59920 RABGDI2 664 808At5g09550 RABGDI3 NS 5At3g06540 REP 50 65

Note: these are average values from two biological replicates. NS,non-significant values at a significance level of 0.05 (typically very lowexpression values).

Table 1 RAB genes that exhibit at least 50% difference in expressionlevel in light-grown compared with dark-grown 7-day-old seedlingsof Arabidopsis

MISP code Gene name

Expression Regulation

In light In dark In dark

At1g06400 RAB-A1a 220 508 UpAt5g45750 RAB-A1c 219 102 DownAt1g09630 RAB-A2a 145 94 DownAt5g59150 RAB-A2d 122 208 UpAt5g47960 RAB-A4c 20 35 UpAt3g07410 RAB-A5b 59 35 DownAt2g43130 RAB-A5c 109 68 DownAt1g05810 RAB-A5e 46 141 UpAt1g73640 RAB-A6a 45 86 UpAt4g35860 RAB-B1b 90 250 UpAt4g17170 RAB-B1c 239 889 UpAt5g03530 RAB-C2a 29 55 UpAt1g02130 RAB-D2a 150 226 UpAt4g17530 RAB-D2c 319 497 UpAt3g53610 RAB-E1a 158 265 UpAt4g20360 RAB-E1b 3430 1240 DownAt3g46060 RAB-E1c 246 460 UpAt5g03520 RAB-E1d 288 519 UpAt3g54840 RAB-F1 204 423 UpAt5g45130 RAB-F2a 347 910 UpAt4g19640 RAB-F2b 499 1232 UpAt2g21880 RAB-G2 38 184 UpAt4g09720 RAB-G3a 342 194 DownAt1g22740 RAB-G3b 82 431 UpAt3g16100 RAB-G3c 163 58 Down

Note: these are average values from two biological replicates. Forexpression values of all RAB genes see Table S1.

622 Michal Hala et al.

ª 2010 The AuthorsJournal compilation ª 2010 Blackwell Publishing Ltd, The Plant Journal, (2010), 62, 615–627

cause a significant accumulation of unprenylated RABs (see

e.g. Seabra et al., 2002; Zhang et al., 2002). Genevestigator

data show that the expression level of RGTB2, the second

paralog, varies around 20% of RGTB1 expression on the

mRNA level, showing the potential to balance the lack of

RGTB1 activity.

Two major developmental abnormalities, apart from loss

of apical dominance and small stature, are typical for rgtb1

mutants: defective shoot gravitropic response (Figure 3) and

constitutive photomorphogenesis (Figure 6). Gravity sens-

ing is localized to the inflorescence stem endodermis in

Arabidopsis. At the tissue level, two opposite gradients of

auxin and jasmonate are established upon gravitropic

signaling, and these gradients are sufficient to start the

gravitropic response (Gutjahr et al., 2005). At the subcellular

level, it was shown that the normal gravitropic response

depends partially on vesicle transport from the trans-Golgi

network (TGN) to the vacuole – a process that might be

affected by the RGTB1 mutation along with the secretion.

The vacuolar t-SNARE protein AtVAM3 and the soluble

N-ethylmaleimide-sensitive factor AtVNI11 forming a

complex were shown to be necessary for shoot gravitro-

pism, directly affecting tonoplast dynamics and amyloplast

localization (Yano et al., 2003). Similarly, the GRAVITRO-

PISM DEFECTIVE 2 (GRV2) protein was shown to be local-

ized in the prevacuolar compartment, and to cooperate in

the final trafficking from this compartment to the vacuole

(Silady et al., 2008).

The observation of dark-grown mutant plants uncovered

the most interesting aspect of the rgtb1 mutant phenotype:

the de-etiolated (det) or constitutive photomorphogenesis

(cop) phenotypes. Many cop/det mutants were shown to be

brassinosteroid-deficient (reviewed in Schumacher and

Chory, 2000; Bishop and Koncz, 2002). Brassinosteroid-

deficient plants typically exhibit the development of primary

leaves in the dark, apical hook and cotyledons opening, and

thick short hypocotyls – phenotypes complemented by the

external addition of brassinosteroids (BRs) (Szekeres et al.,

1996). However, rgtb1 mutants seem to be most similar to

the det3 mutant. Szekeres et al. (1996) realized that in

contrast to most other cop/det mutants, det3 is insensitive

to complementation by BR treatment.

Arabidopsis det3 mutants grown in light lose apical

dominance, and are smaller than the WT (Cabrera y Poch

et al., 1993). When Schumacher et al. (1999) identified DET3

as the C-subunit of the vacuolar-type H+-ATPase (VHA), they

also found that its mutation leads to a defect in the execution

of the actual dark growth response, rather than in the

signaling pathways initiating it. Dettmer et al. (2006) and

Brux et al. (2008) proved that VHA function in plants is

essential for the operation of the secretory pathway – both

endocytosis and exocytosis via TGN (which serves as an

endosome in plants; Dettmer et al., 2006). Arabidopsis

mutants have shown that the VHA is essential for Golgi

function in pollen development, and during embryogenesis

(Dettmer et al., 2005; Strompen et al., 2005). All these results

imply that the growth inhibition observed in plants with

reduced VHA activity is caused by a defect in vesicle

trafficking, rather than by reduced turgor pressure attribut-

able to a lack of osmolyte transport into the vacuole (Dettmer

et al., 2005). Mutants in DET3, COP4 and SHY2/IAA3 also

exhibit a defective gravitropic response: shy2 and det3

exhibit a weaker defect in hypocotyls, whereas cop4 exhibits

the defect in both roots and hypocotyls (Hou et al., 1993; Kim

et al., 1998; Schumacher et al., 1999).

In all these aspects, we can see a remarkable similarity to

the rgtb1 mutant, which also exhibits a block in the secretory

pathway (both exocytosis and endocytosis), obviously via

insufficient prenylation of RAB GTPases. Already in the first

reports on plant RAB GTPases, the negative effect of light

(mediated by phytochrome) on RAB expression in etiolated

pea plants was recognized (Yoshida et al., 1993; Nagano

et al., 1995); likewise, the downregulation of a specific

RAB-A homolog in tobacco seems to induce hypocotyl

shortening in the dark (Kang et al., 2001). We have fully

corroborated these early observations in our Arabidopsis

transcriptome analysis, as we have found that group of RAB

GTPases, and RABGDI1 and RABGDI2 are upregulated in

dark-grown seedlings. Publicly available expression data

reveal that at least three RAB genes are significantly

deregulated in the det3 mutant under dark conditions

(Newman et al., 2004). Two of them also show the same

regulation in our expression analysis of the photomorpho-

genic WT: RAB-B1c is downregulated, whereas RAB-E1b is

upregulated. At least five RAB genes (RAB-A1f, -A2b, -A4a,

-A6a and -G3c) are also significantly deregulated in the cop1

mutant. Interestingly, the expression of RGTB1 is signifi-

cantly downregulated compared with the WT.

Mutant det3 and rgtb1 phenotypes fit very well into the

notion that re-programming and a boost of the secretory

pathway is necessary to support remarkable elongation

growth of etiolated plantlets. However, why do lesions in

secretory pathways result in the full-blown development of

the cop/det phenotype: i.e. why does compromised secre-

tion induce photomorphogenesis in the dark, and not just a

cessation of hypocotyl elongation? In this respect we can

only speculate. In the case of the conditional det3 mutant

phenotype, the involvement of oxylipin and ethylene in

mediating changes in gene expression in response to

cellulose deficiency were implicated in the development of

the cop/det phenotype (Brux et al., 2008). It might be

expected that the deficient secretory pathway of the Arabid-

opsis rgtb1 mutant could also result in a defective cell wall

composition, accompanied by a similar stress response as

described for the det3 mutant ultimately leading to the

cop/det phenotype. However, it is equally possible that a

direct signaling feedback relay from the secretory pathway

to plant morphogenic programs also exists, switching on

RAB prenylation and photomorphogenesis 623

ª 2010 The AuthorsJournal compilation ª 2010 Blackwell Publishing Ltd, The Plant Journal, (2010), 62, 615–627

photomorphogenesis when vesicle trafficking is downregu-

lated. This might obviously be the case in plants treated by

endosidin1 (ES1), a new anti-endocytosis drug, which

releases the ICR1 adaptor protein from ROP GTPases at the

tip of growing pollen tubes. ES1 inhibits secretion specifi-

cally via TGN-dependent endocytosis, and induces the cop/

det phenotype when applied to the whole dark-grown

seedlings (this cannot be relieved by the addition of BRs;

Robert et al., 2008). The report suggests that a compromised

secretory pathway might influence BR signaling by interfer-

ing with the BRI1 signaling endosome (Robert et al., 2008).

So there might be dark/light and BR signaling upstream of

the secretory pathway execution of etiolated growth, but

also the secretory pathway might reciprocally influence

signal transduction pathways regulating photomorphogen-

esis (e.g. BR signal transduction via a signalling endosome).

In any case, cell elongation growth supported by de novo

translated RAB GTPases is an important part for the imple-

mentation of the dark-induced morphogenetic programme

in Angiosperms, and the efficient geranylation of nascent

RAB GTPases is necessary for their function.

EXPERIMENTAL PROCEDURES

Plant material and growth conditions

Mutant lines of the Columbia-0 ecotype of Arabidopsis thaliana L.Heynh with T-DNA insertions were obtained from the SALK Institute(Alonso et al., 2003): rgtb1-1, SALK_015871; rgtb1-2, SALK_125416.The det3-1 mutant (At1g12840; Cabrera y Poch et al., 1993) waskindly provided by K. Schumacher (University of Heidelberg) andM. Campbell (University of Toronto). The location of each T-DNAinsertion within the RGTB1 gene (At5g12210) was verified bysequencing from each end of the insert (Appendix S1).

Arabidopsis seeds were surface-sterilized, stratified at 4�C for3–5 days and planted on vertical agar plates with growth media[half-strength MS salts, 2% (w/v) sucrose, vitamins and 1.6% agar]or soil. Light-grown plants were cultivated at 22�C under long-dayconditions (16 h of light per day). Plates with dark-grown plantswere wrapped into the aluminium foil after a light treatment for2–4 h to stimulate germination, and were cultivated at 22�C.

Preparation of plant extracts

For western blot analysis, either 100 mg of 7-day-old etiolatedplants or 100 mg of hypocotyls of these plants were ground inbuffer A [100 mM Tris, pH 7.8, 5 mM EGTA, 5 mM EDTA, 10 mM

beta-mercaptoethanol and 10% (w/v) glycerol] containing 1x plantprotease inhibitor cocktail and 1 mM phenylmethylsulfonyl fluoride(PMSF) (all from Sigma-Aldrich, http://www.sigmaaldrich.com) andcentrifuged for at 10 000 g for 5 min at 4�C. The supernatant waseither subjected to western analysis or further centrifuged at100 000 g for 1 h, at 4�C. The supernatant and pellet fractions wereprecipitated with trichloroacetic acid (TCA), the pellets were washedwith acetone and then dissolved in SDS/PAGE sample buffer.

For the geranylgeranylation assay, plant extracts were preparedaccording to the method described by Loraine et al. (1996). Freshplant material was frozen in liquid nitrogen and then ground on icein a mortar with acid-washed sand in buffer A (2 ml g)1 of cells).Lysates were centrifuged at 30 000 g for 30 min at 4�C, dialyzedagainst buffer B [50 mM Tris, pH 7.8, 10% (v/v) glycerol and 1 mM

DTT], aliquoted, frozen in liquid nitrogen and stored at )80�C untiluse.

The total protein concentration was measured using the Bio-RadDc Protein Assay according to the manufacturer’s protocol (Bio-RadLaboratories, http://www.bio-rad.com).

Preparation of recombinant fusion proteins

AtRAB-A2a was amplified from Arabidopsis pollen cDNA andcloned into the pET30a vector (Novagen, now part of Merck, http://www.merck-chemicals.com). Expression in Escherichia coli strainBL21 and purification of the recombinant N-terminal 6His-fusionprotein on Ni-NTA agarose (Qiagen, http://www.qiagen.com) wasperformed according to the Qiagen expression handbook.

Geranylgeranylation assay

The geranylgeranylation assay was performed as described byBenito-Moreno et al. (1994). Briefly, 200 lg of yeast or plant extractswere used in a total volume of 60 ll 1x geranylgeranylation buffer(50 mM phosphate buffer, pH 7.6, 10 mM MgCl2 and 5 mM DTT)with the addition of 0.5 lM [1-3H] all trans-geranylgeranyl pyro-phosphate (15–30 Ci mmol)1) (Amersham, now part of GE Health-care, http://www.gelifesciences.com) and 4 lg of recombinantAtRAB-A2a protein. The reaction mixture was then incubated at32�C for 40 min, and the reaction was stopped by the addition of12 ll of 6x loading buffer and boiled, and finally separated on 15%SDS-PAGE. Gels were boiled in 5% TCA solution for 5 min in thewater bath, and were briefly washed with distilled water. Acidictreatment was neutralized by 5 min of incubation with 100 mM TRISsolution at 25�C. Fluorographic intensification of the autoradio-graphic signal was achieved by incubation of the gel with 1 M

sodium salicylate. Gel was then dried on the gel drier and exposedto the X-Omat AR Kodak film or FOMA X-Ray film for 1 week at)80�C. The intensity of the bands occurring after the film develop-ment was measured by the IMAGEJ freeware program (http://rsb.info.nih.gov/ij/download.html).

Western blot analysis

An appropriate volume of the sample was loaded on 13.5% SDS-PAGE. Proteins were transferred on polyvinylidene fluoride (PVDF)membrane, blocked overnight at 4�C with 5% non-fat dry milk inTris-buffered saline (TBS), and incubated with the rabbit polyclonalanti-RAB-A2a antibody (diluted 1:1000 in 5% non-fat dry milk in TBSsupplemented with 0.5% Tween 20; kindly provided by I. Moore) for1 h. Secondary anti-rabbit alkaline-phosphatase-conjugated anti-body (1:5000; Sigma-Aldrich) was applied for 30 min, followed bycolour detection (Western Blue Stabilized Substrate; Promega,http://www.promega.com).

Phylogenetic analysis

Using the BLAST server and databases held at the National Centerfor Biotechnology Information (http://www.ncbi.nlm.nih.gov) andDOE Joint Genome Institute (http://genome.jgi-psf.org; http://www.phytozome.net), we searched for genes encoding b-subunitsof protein prenyl transferases in genomes of the following organ-isms: A. thaliana, Candida albicans, Caenorhabditis elegans,Drosophila melanogaster, Entomoeba histolytica, Giardia lamblia,Homo sapiens, Mus musculus, Neurospora crassa, O. sativa, Plas-modium falciparum, Physcomitrella patens, Populus trichocarpa,Saccharomyces cerevisiae, Schizosaccharomyces pombe,Selaginella moellendorffii, Sorghum bicolor, Volvox carteri andVitis vinifera.

624 Michal Hala et al.

ª 2010 The AuthorsJournal compilation ª 2010 Blackwell Publishing Ltd, The Plant Journal, (2010), 62, 615–627

Multiple alignments of protein sequences were constructed usingthe CLUSTALX program with the default settings (Thompson et al.,1997) and manually adjusted. For phylogenetic analysis onlyreliably aligned regions were included: poorly conserved and gap-containing regions were removed (the alignment is available uponrequest). The phylogenetic tree was constructed using the maxi-mum-likelihood (ML) method, as implemented in the PHYML pro-gram (Guindon and Gascuel, 2003) with the JTT amino acidsubstitution matrix, discrete approximation to a C distribution (fourrate categories) and taking into account invariable sites (JTT+C+Imodel). Bacterial prenyl transferase from Mycobacterium tubercu-losis (MtPT) (Maurer-Stroh et al., 2003) was used as a root. Allmodel parameters were estimated from the data. ML bootstrapanalysis was performed on 100 bootstrap replicates (the JTT+C+Imodel with the same parameters as for the original data set), usingthe PHYML online execution server (http://atgc.lirmm.fr/phyml).

Microscopy

Styryl dye FM4-64 in a 15 lM concentration (diluted in 0.5x MSliquid medium) was vacuum infiltrated for 8 min into etiolated7-day-old plantlets. Quantification of endocytic compartments wasperformed as a measuring of the fluorescence inside hypocotylcells. The analyzed images were normalized by the signal of thecytoplasmic membranes, and the background was subtracted.Seedlings treated with 50 lM BFA were labeled with FM4-64 shortlybefore observation. Imaging of FM4-64 as well as secGFP was per-formed at different times using a confocal laser scanning micro-scope (LSM 510; Zeiss, http://www.zeiss.com) and a fluorescencemicroscope (BX51; Olympus, http://www.olympus-global.com)(Figure 4a,b).

Expression analysis

Seven-day-old seedlings grown in both continuous light and darkwere harvested from vertical agar plates to liquid nitrogen (200 mgof tissue per sample) and then ground. RNA was extracted in twoparallels for each set of conditions using an RNeasy� Mini kit(Qiagen) following the manufacturer’s instructions for plant mate-rial. Samples were sent on dry ice to NASC’s InternationalAffymetrix Service for standard processing on Affymetrix ATH1chips (http://affymetrix.arabidopsis.info). Values at a significancelevel of 0.05 were selected and average values for correspondingreplicas were calculated.

ACKNOWLEDGEMENTS

We thank I. Moore (University of Oxford) for help with the analysisof RAB prenylation, M. Potocky (IEB ASCR) for help with phyloge-netic analysis and for comments on the manuscript, andK. Schumacher (University of Heidelberg) and M. Campbell (Uni-versity of Toronto) for seeds of the det3-1 mutant. The work wassupported by the Grant Agency of the Czech Republic (204/06/P0457), the Ministry of Education, Youth and Sports of the CzechRepublic (MSMT LC06034) and part of VZ’s income is covered byMSM0021620858. Affymetrix data were supported by the GrantAgency of the Academy of Sciences of the Czech Republic (projectKJB600380802).

SUPPORTING INFORMATION

Additional Supporting Information may be found in the onlineversion of this article:Figure S1. Phylogenetic analysis of prenyl transferases b-subunits.Figure S2. Expression profiles of RAB-GGT subunits and AtREP.Figure S3. Semi-quantitative RT-PCR screening of AtRGTB1 andAtRGTB2 expression in the wild type and in rgtb1 mutants.

Figure S4. Biochemical characteristics of homozygous rgtb1mutants.Table S1. Expression levels of RAB, RAB-GDI, RAB-GGT and REPgenes in 7-day-old Arabidopsis seedlings (Col-0) in light or darkconditions.Appendix S1. Verification of T-DNA positions in rgtb1-1 and rgtb1-2insertion lines.Please note: As a service to our authors and readers, this journalprovides supporting information supplied by the authors. Suchmaterials are peer-reviewed and may be re-organized for onlinedelivery, but are not copy-edited or typeset. Technical supportissues arising from supporting information (other than missingfiles) should be addressed to the authors.

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Accession numbers: Sequence data from this article can be found in the EMBL/GenBank data libraries under the following accession numbers(AGI codes): AtRGTB1, At5g12210; AtRGTB2, At3g12070; AtRGTA1, At4g24490; AtRGTA2, At5g41820. Seed stocks from the SALK Institute:rgtb1-1, SALK_015871; rgtb1-2, SALK_125416.

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