Arabidopsis Vacuolar Sorting Mutants (green ﬂuorescent ... · Green Fluorescent Protein in Their Seeds W OA ... b Graduate School of Agriculture, Kyoto University, Gokasho, Uji,

Arabidopsis Vacuolar Sorting Mutants (green fluorescent seed)Can Be Identified Efficiently by Secretion of Vacuole-TargetedGreen Fluorescent Protein in Their Seeds W OA

Kentaro Fuji,a Tomoo Shimada,a Hideyuki Takahashi,a Kentaro Tamura,a Yasuko Koumoto,a Shigeru Utsumi,b

Keito Nishizawa,b Nobuyuki Maruyama,b and Ikuko Hara-Nishimuraa,1

a Department of Botany, Graduate School of Science, Kyoto University, Kyoto 606-8502, Japanb Graduate School of Agriculture, Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan

Two Arabidopsis thaliana genes have been shown to function in vacuolar sorting of seed storage proteins: a vacuolar

sorting receptor, VSR1/ATELP1, and a retromer component, MAIGO1 (MAG1)/VPS29. Here, we show an efficient and simple

method for isolating vacuolar sorting mutants of Arabidopsis. The method was based on two findings in this study. First,

VSR1 functioned as a sorting receptor for b-conglycinin by recognizing the vacuolar targeting signal. Second, when green

fluorescent protein (GFP) fusion with the signal (GFP-CT24) was expressed in vsr1, mag1/vps29, and wild-type seeds, both

vsr1and mag1/vps29 gave strongly fluorescent seeds but the wild type did not, suggesting that a defect in vacuolar sorting

provided fluorescent seeds by the secretion of GFP-CT24 out of the cells. We mutagenized transformant seeds expressing

GFP-CT24. From ;3,000,000 lines of M2 seeds, we obtained >100 fluorescent seeds and designated them green fluorescent

seed (gfs) mutants. We report 10 gfs mutants, all of which caused missorting of storage proteins. We mapped gfs1 to VSR1,

gfs2 to KAM2/GRV2, gfs10 to the At4g35870 gene encoding a novel membrane protein, and the others to different loci. This

method should provide valuable insights into the complex molecular mechanisms underlying vacuolar sorting of storage

proteins.

INTRODUCTION

Higher plants synthesize large amounts of storage proteins that

account for almost 80% of the total protein in seeds during seed

maturation. In maturing seeds, most storage proteins are syn-

thesized in the endoplasmic reticulum (ER) as precursors, which

are transported into protein storage vacuoles (PSVs), where they

are converted into mature forms (Hara-Nishimura et al., 1993;

Hara-Nishimura and Maeshima, 2000; Shimada et al., 2003b).

Multiple pathways are used for the transport of storage proteins

into PSVs (Vitale and Hinz, 2005). One of the pathways is an

aggregation sorting that is mediated by precursor-accumulating

(PAC) vesicles that are involved in the mass transport of storage

proteins to PSVs (Hara-Nishimura et al., 2004). A type I mem-

brane protein, PV72, was found on the membrane of the PAC

(Shimada et al., 1997, 2002). Arabidopsis thaliana has seven

homologs of PV72, which we designated At VSR1 to At VSR7 (for

vacuolar sorting receptor of Arabidopsis) (Shimada et al., 2003a).

The homolog most closely related to PV72 was designated

At VSR1, which is also referred to as ATELP (Ahmed et al., 1997).

We demonstrated that VSR1 functions as a receptor to sort seed

storage proteins to PSVs (Shimada et al., 2003a). vsr1 missorts

storage proteins by secreting them from cells, resulting in an

enlarged and electron-dense extracellular space in the seeds.

The vsr1 seeds abnormally accumulate the precursors of two

major storage proteins, 12S globulin and 2S albumin. These

findings show a receptor-mediated transport of seed storage

proteins to PSVs in higher plants.

Sorting of vacuolar proteins requires the interaction between a

vacuolar targeting signal and a sorting receptor that recognizes

the signal (Kirsch et al., 1994, 1996). Despite many articles that

discuss the interactions between VSR and a vacuolar targeting

signal (Kirsch et al., 1994; Sanderfoot et al., 1998; Ahmed et al.,

2000; Laval et al., 2003), there have been only a few in vivo

demonstrations of the interaction between VSR and the de-

termined vacuolar targeting signals: between VSR1 and an

N-terminal propeptide of either aleurain or sporamin (Ahmed

et al., 2000; Watanabe et al., 2004) and between pea (Pisum

sativum) BP-80 and the N-terminal propeptide of sporamin

(daSilva et al., 2005). Vacuolar targeting signals for PSVs have

been found in soybean (Glycine max) b-conglycinin (Nishizawa

et al., 2003, 2004), common bean (Phaseolus vulgaris) phaseolin

(Frigerio et al., 1998, 2001b), 2S albumin of Brazil nut (Saalbach

et al., 1996), barley (Hordeum vulgare) lectin (Muntz, 1998), and

castor bean (Ricinus communis) ricin (Frigerio et al., 2001a).

These signals exhibit no consensus amino acid sequence. It has

been shown that VSR binds to 2S albumin signals (Shimada

et al., 2003a; Jolliffe et al., 2004) and the ricin signal (Jolliffe et al.,

2004). The question raised is whether VSR binds to these

1 To whom correspondence should be addressed. E-mail [email protected]; fax 81-75-753-4142.The author responsible for distribution of materials integral to thefindings presented in this article in accordance with the policy describedin the Instructions for Authors (www.plantcell.org) is: Ikuko Hara-Nishimura ([email protected]).W Online version contains Web-only data.OA Open Access articles can be viewed online without a subscription.www.plantcell.org/cgi/doi/10.1105/tpc.106.045997

The Plant Cell, Vol. 19: 597–609, February 2007, www.plantcell.org ª 2007 American Society of Plant Biologists

different signals. It was also reported that At RMR1 (for Arabi-

dopsis receptor homology region transmembrane domain ring

H2 motif protein1) interacts with the C-terminal sorting domain of

phaseolin, which also belongs to the 7S globulin family and is a

homolog of b-conglycinin (Park et al., 2005). A reverse genetic

analysis with an RMR1-deficient mutant is necessary to demon-

strate that RMR1 functions as a sorting receptor for 7S globulins.

Both VSR1 and PV72 bind to peptides derived from storage

proteins, including 2S albumin (Watanabe et al., 2002) and 12S

globulin (Shimada et al., 2003a), in a Ca2þ-dependent manner.

Both VSRs have a binding ability not only to the storage protein

peptides but also to a vacuolar targeting signal for lytic vacuoles

(Watanabe et al., 2002; Shimada et al., 2003a). Overexpression

of the ER-localized soluble form of a seed-specific VSR (PV72)

interfered with the transport of a lytic vacuolar protein (ALEU) in

Arabidopsis leaves (Watanabe et al., 2004). This means that both

VSR for PSVs and VSR for lytic vacuoles share a similar mech-

anism for recognizing a vacuolar targeting signal.

Although VSR is unique to plants, VSR has a short cytosolic tail

containing the sequence YMPL, which is similar to Tyr-based

motifs (YXXF, in which X represents any amino acid and F

represents a hydrophobic residue with a bulky side chain) of

yeast VPS10 (Cooper and Stevens, 1996) and mammalian man-

nose 6-phosphate receptor (Honing et al., 1997). The Tyr-based

motif is recognized by the adapter complex of clathrin-coated

vesicles in yeast (Deloche et al., 2001) and animals (Klumperman

et al., 1998). Recently, the Tyr-based motif of BP-80 was

reported to be required for the transport of vacuolar proteins

(daSilva et al., 2006).

We recently isolated a mutant (maigo1 [mag1]) that missorts

storage proteins by secreting them out of the seed cells and

identified the responsible gene as At3g47810, which encodes a

homolog of the yeast retromer component VPS29 (Shimada

et al., 2006). MAG1/VPS29 might be involved in recycling VSR1

for the efficient sorting of seed storage proteins. VSR1 and

MAG1/VPS29 are the only factors that have been demonstrated

in planta to function in vacuolar sorting of seed storage proteins,

although VACUOLELESS (Rojo et al., 2001) and DRP2A (ADL6)

(Jin et al., 2001) were reported to be involved in trafficking to lytic

vacuoles. Forward genetics should be a useful approach to fully

clarify the sorting machinery for PSVs. We have isolated vacuolar

sorting mutants (mag) that accumulate storage protein precur-

sors in seeds (Shimada et al., 2006; Li et al., 2006). In this study,

we established a more efficient and easier method (green fluo-

rescent seed [gfs] method) of isolating vacuolar sorting mutants

and identified factors for vacuolar sorting of storage proteins.

RESULTS

VSR1 Is Involved in Vacuolar Sorting of the Exogenously

Expressed a9-Subunit of b-Conglycinin by Recognizing the

C-Terminal Vacuolar Targeting Signal in Arabidopsis Seeds

Two vacuolar targeting signals have been identified in 7S glob-

ulins. One is the 10–amino acid C-terminal peptide (CT10; Figure

1A) of the a9-subunit of b-conglycinin of soybean (Nishizawa

et al., 2003), and the other is the 4–amino acid C-terminal peptide

of phaseolin of common bean (Frigerio et al., 1998). In Arabi-

dopsis seeds, CT10 functions as a necessary and sufficient

vacuolar targeting signal (Nishizawa et al., 2003). To determine

whether VSR binds to the CT10 of b-conglycinin, we applied

microsomal proteins from Arabidopsis siliques, which contained

VSR1, to an affinity column conjugated with CT10. VSR1 bound

to CT10 in the presence of 1 mM CaCl2 and was eluted with CT10

or EGTA (Figure 1B), indicating that VSR1 binds to the vacuolar

targeting signal of b-conglycinin in a Ca2þ-dependent manner.

This means that VSR1 binds to the targeting signal of

b-conglycinin, as it binds to the signal of the storage protein 2S

albumin (Shimada et al., 2003a), even though the targeting

signals of these storage proteins have no consensus amino

acid sequences.

To determine whether VSR is required for vacuolar sorting of

b-conglycinin in Arabidopsis seeds, we expressed b-conglycinin

exogenously under the control of a seed-specific promoter in the

wild type (WT; Columbia) to produce the transformed plant

WT/conglycinin and in two vsr1 mutants to produce the trans-

formed plants vsr1-1/conglycinin and vsr1-2/conglycinin. WT/

conglycinin seeds gave several bands on an immunoblot with

anti-b-conglycinin antibody (Figure 2A, lane 2), although non-

transformant seeds gave no signal on the blot (Figure 2A, lane 1).

b-Conglycinin, when expressed in Arabidopsis seeds, was

shown to be transported to PSVs and to be partially degraded

to give several bands on the immunoblot, probably because it is

less stable in Arabidopsis PSVs than in soybean PSVs (Nishizawa

Figure 1. VSR1 Can Bind a Vacuolar Targeting Signal of a Soybean

Seed Storage Protein (b-Conglycinin) in a Calcium-Dependent Manner.

(A) Scheme of the preproprotein precursor of the soybean b-conglycinin

a9-subunit, which contains a signal peptide (SP) at the N terminus and a

vacuolar targeting signal composed of 10 amino acids (CT10) at the C

terminus.

(B) Affinity chromatography with a CT10-conjugated column. Micro-

somes were prepared from Arabidopsis siliques including maturing

seeds and were applied to the affinity column in the presence of 1 mM

CaCl2. The bound VSR1 was eluted competitively with the CT10 peptide

and was also eluted with EGTA. Total microsomes, the flow-through

fraction, the wash fraction, and the eluted fractions (fractions 1, 3, 5, and

7) were subjected to immunoblot analysis with anti-VSR1 antibody.

Molecular masses are given at left.

598 The Plant Cell

et al., 2003). The level of the nonprocessed form of b-conglycinin

was higher in both vsr1-1 and vsr1-2 seeds than in wild-type

seeds, although the protein profiles of b-conglycinin in the

mutants and the wild type were similar (Figure 2A).

The extracellular space of the vsr1-1/conglycinin seeds was

abnormally enlarged and filled with electron-dense material

(Figure 2C). To determine the localization of b-conglycinin, we

performed immunocytochemistry with an anti-b-conglycinin an-

tibody that does not give any signal on nontransformant seeds

(Nishizawa et al., 2003). b-Conglycinin was distributed in the

electron-dense region of the extracellular space in the vsr1-1/

conglycinin seeds (Figure 2E), whereas b-conglycinin was local-

ized in the aggregates within PSVs of WT/conglycinin seeds

(Figure 2D). We previously showed that vsr1 mutants missort

the storage proteins 12S globulin and 2S albumin by secreting

them out of seed cells (Shimada et al., 2003a). Our findings

revealed that b-conglycinin, like authentic storage proteins, is

abnormally secreted in vsr1 seeds, indicating that VSR is in-

volved in the vacuolar sorting of b-conglycinin in maturing seeds

of Arabidopsis.

vsr1 Generates Green Fluorescent Seeds by Secreting the

Green Fluorescent Protein Fusion Protein with the Vacuolar

Targeting Signal CT24 of b-Conglycinin

Previously, we generated an Arabidopsis transformant (WT/

GFP-CT24) that expressed SP-GFP-CT24, which was com-

posed of a signal peptide and green fluorescent protein (GFP)

followed by the b-conglycinin C-terminal 24 amino acids (CT24),

including CT10, under the control of a seed-specific promoter

(Nishizawa et al., 2003). WT/GFP-CT24 gave GFP fluorescence

that was completely merged with the red autofluorescence of

PSVs in seeds, showing that GFP-CT24 was transported into

PSVs (Figure 3A).

We generated vsr1-1/GFP-CT24 and vsr1-2/GFP-CT24 by

crossing each of two vsr1 mutants (vsr1-1 and vsr1-2) with WT/

GFP-CT24, respectively. Figure 3B shows that vsr1-1/GFP-CT24

gave a much higher intensity of GFP fluorescence in the extra-

cellular space than WT/GFP-CT24, suggesting that most of the

GFP-CT24 molecules were secreted out of the vsr1 seed cells.

Immunocytochemistry revealed that GFP-CT24 (Figures 3C and

3D) and 12S globulin of the authentic storage proteins (Figure 3E)

were detected in the electron-dense and enlarged extracellular

space of vsr1-1 seeds. vsr1-1 missorted GFP-CT24 by secreting

it out of the seed cells together with the authentic storage

proteins. vsr1-2 gave the same result as vsr1-1 (data not shown).

These results indicate that VSR1 sorts and delivers GFP-CT24 to

PSVs by recognizing CT24 of b-conglycinin. Considering that

VSR1 binds to CT10 of b-conglycinin in vitro (Figure 1B), CT10

might be sufficient to be recognized by VSR1.

Figure 4A shows an immunoblot of the transformant seeds

with anti-GFP antibody to evaluate the amount of GFP. The

seeds gave two bands on the blots, which might correspond to

28-kD GFP and 30-kD GFP-CT24. We found that both vsr1-1/

GFP-CT24 and vsr1-2/GFP-CT24 accumulated much higher

amounts of GFP in their seeds than WT/CT24 (Figure 4A). It is

possible that GFP-CT24 is more stable in the extracellular space

than in PSVs of Arabidopsis seeds. Interestingly, the high accu-

mulation of GFP in the extracellular space of vsr1 caused the

seeds to be fluorescent. Figures 4D and 4E show that all of the

seeds of both vsr1-1/GFP-CT24 and vsr1-2/GFP-CT24 exhibited

strong GFP fluorescence. On the contrary, the seeds of WT/GFP-

CT24, in which GFP was accumulated in PSVs, exhibited ex-

tremely low levels of GFP fluorescence (Figure 4C).

Figure 2. b-Conglycinin Is Missorted by Secreting Out of the Cells When

It Is Expressed in vsr1 Seeds.

(A) b-Conglycinin was expressed in both the wild type (Columbia) and

vsr1 mutants under the control of a seed-specific promoter. Seeds of

nontransformant (lane 1), WT/conglycinin (lane 2), vsr1-1/conglycinin

(lane 3), and vsr1-2/conglycinin (lane 4) plants were subjected to immu-

noblot analysis with either anti-VSR1 antibody or anti-b-conglycinin

antibody. Molecular masses are given at left.

(B) and (C) Ultrastructures of the seeds of both WT/conglycinin (B) and

vsr1-1/conglycinin (C) plants. The extracellular space of the transformed

vsr1-1 seeds was abnormally enlarged and filled with electron-dense

material. Arrowheads indicate the extracellular spaces. Bars ¼ 5 mm.

(D) and (E) Immunoelectron micrographs of WT/conglycinin (D) and

vsr1-1/conglycinin (E) seeds with anti-b-conglycinin antibody. Exoge-

nously expressed b-conglycinin was localized in the electron-dense

extracellular space (ECS) of vsr1-1/conglycinin seeds (white arrowheads

in [E]), whereas it was localized in PSVs of WT/conglycinin seeds (white

arrowheads in [D]). Bars ¼ 1 mm.

Vacuolar Sorting Mutant gfs 599

An Easy Method for Isolating Vacuolar

Sorting–Deficient Mutants (gfs)

We recently identified another factor, MAG1, for storage protein

sorting to PSVs. We found that the mag1 mutant missorts

storage proteins by secreting them out of the Arabidopsis seed

cells and that MAG1 is a homolog of the yeast retromer com-

ponent VPS29 (Shimada et al., 2006). To determine whether

GFP-CT24 was secreted in mag1/vps29 seeds, we crossed

each of two mag1/vps29 alleles (mag1-1/vps29-1 and mag1-2/

vps29-2) with WT/GFP-CT24 to produce mag1-1/vps29-1/GFP-

CT24 and mag1-2/vps29-2/GFP-CT24, respectively. Figure 5A

shows that some seeds of the F2 population fluoresced green.

Highly fluorescent seeds of mag1-1/vps29-1/GFP-CT24 and

Figure 3. A GFP Fusion Protein with a Vacuolar Targeting Signal Is

Secreted out of the Cells When Expressed in vsr1 Seeds but Is Localized

in PSVs When Expressed in Wild-Type Seeds.

(A) and (B) GFP-CT24 was expressed in the wild type (WT/GFP-CT24;

[A]) and the vsr1-1 mutant (vsr1-1/GFP-CT24; [B]). The seeds were

inspected with a confocal laser scanning microscope to obtain GFP

fluorescent images showing the localization of GFP-CT24 (GFP) and

autofluorescent image of PSVs. Both images were then merged. GFP

fluorescence was detected in PSVs of WT/GFP-CT24 seeds (A), whereas

a higher intensity of GFP fluorescence was detected in the extracellular

space of vsr1-1/GFP-CT24 seeds (B). Bars ¼ 5 mm.

(C) Immunoelectron micrograph of vsr1-1/GFP-CT24 seeds with anti-

GFP antibody, showing that the GFP fusion was localized in the extra-

cellular space (ECS; arrowheads). Bar ¼ 2 mm.

(D) Enlarged image of an immunoelectron micrograph of vsr1-1/GFP-

CT24 seeds with anti-GFP antibody, showing the distribution of the GFP

fusion in the extracellular space (ECS; arrowhead). Bar ¼ 1 mm.

(E) Immunoelectron micrograph of vsr1-1/GFP-CT24 seeds with anti-

12S globulin antibody, showing the secretion of 12S globulin in the vsr1-1

seeds (ECS; arrowhead). Bar ¼ 1 mm.

Figure 4. vsr1 Mutants That Express GFP-CT24 Produce Green Fluo-

rescent Seeds by Accumulating a Much Greater Amount of GFP-CT24 in

Seeds Than Do Wild-Type Plants That Express GFP-CT24.

(A) Expression levels of GFP-CT24 in the transformed seeds. Seeds of

nontransformant (lane 1), WT/GFP-CT24 (lane 2), vsr1-1/GFP-CT24 (lane

3), and vsr1-2/GFP-CT24 (lane 4) plants were subjected to immunoblot

analysis with either anti-VSR1 antibody or anti-GFP antibody. Molecular

masses are given at left.

(B) to (F) Seeds of wild-type (B), WT/GFP-CT24 (C), vsr1-1/GFP-CT24

(D), and vsr1-2/GFP-CT24 (E) plants were inspected with a binocular

microscope (top panels) and a fluorescence microscope (bottom

panels). For comparison of the intensities of fluorescence, the photo-

graphs were taken under the same conditions. Both vsr1-1/GFP-CT24

and vsr1-2/GFP-CT24 produced green fluorescent seeds.

600 The Plant Cell

mag1-2/vps29-2/GFP-CT24 exhibited GFP fluorescence in the

extracellular space (Figure 5B), as did vsr1/GFP-CT24, suggest-

ing that most of the GFP-CT24 molecules were secreted out of

mag1/vps29 seed cells. We selected highly green fluorescent

seeds (Figure 5C, lanes 3 to 5 and 9 to 11) and faintly fluorescent

seeds (Figure 5C, lanes 6 to 8 and 12 to 14) from the F2

population and subjected them to immunoblot analysis with

either anti-GFP antibody or anti-12S globulin antibody. The

intensity of GFP fluorescence of the seeds correlated not only

with the level of GFP accumulation but also with 12S globulin

precursor accumulation (Figure 5C). This finding suggested that

the intensity of green fluorescence of the seeds is a useful marker

for isolating vacuolar sorting mutant seeds.

Only two factors, VSR1 and MAG1/VPS29, have been shown

in planta to function in vacuolar sorting of storage proteins to

PSVs. The results in Figures 4 and 5 show that both vsr1 and

mag1/vps29 secreted a vacuole-targeted GFP, resulting in green

fluorescent seeds. This finding suggests that vacuolar sorting–

deficient mutants would produce green fluorescent seeds if

vacuole-targeted GFP was expressed in seeds. This provided an

idea for an efficient method for screening vacuolar sorting–

deficient mutants. Namely, a mutant seed can be easily identified

Figure 5. mag1/vps29 Mutants That Express GFP-CT24 Also Produce Green Fluorescent Seeds.

(A) mag1-1/vps29-1 was crossed with WT/GFP-CT24. The seeds of the F2 population were inspected with a binocular microscope (top panel) and a

fluorescence microscope (bottom panel). mag1/vps29/GFP-CT24 produced green fluorescent seeds (arrowheads).

(B) Highly fluorescent seeds of mag1-1/vps29-1/GFP-CT24 and mag1-2/vps29-2/GFP-CT24 were inspected with a confocal laser scanning

microscope. GFP fluorescence was detected out of the seed cells, showing missorting of GFP-CT24. The GFP fluorescence image was not merged

with the autofluorescent image (red) of PSVs. Each image of green and red fluorescence is shown in Supplemental Figure 1 online. Bars ¼ 10 mm.

(C) Selected highly green fluorescent seeds (lanes 3 to 5 and 9 to 11) and faintly fluorescent seeds (lanes 6 to 8 and 12 to 14) from the F2 population were

subjected to immunoblot analysis with anti-GFP and anti-12S globulin antibodies. The intensity of the GFP fluorescence of seeds correlated not only

with the accumulation levels of GFP but also with the accumulation of the 12S globulin precursor. p12S, 12S globulin precursor; 12S-a, a-subunit of

mature 12S globulin. Molecular masses are given at left.


by the strong green fluorescence that is caused by missorting of

GFP-CT24.

Based on this idea, we mutagenized the seeds of WT/GFP-

CT24 with ethyl methanesulfonate to obtain M2 seed populations.

Green fluorescent seeds were easily selected from a thousand of

the mutagenized M2 seeds with a fluorescence stereomicro-

scope, as shown in Figure 6A. We obtained >100 mutant lines

from ;3,000,000 lines of M2 seeds and designated them gfs

mutants. In this study, we describe 10 of these gfs mutants.

Four gfs1 Mutants Are vsr1 Alleles

Four of the mutants were named gfs1 because they were allelic

(discussed below). Figure 6B shows confocal laser scanning

microscopic images of seeds of three of them (gfs1-1, gfs1-2,

and gfs1-3). As expected, GFP fluorescence was observed out of

the cells in the seeds of these gfs mutants. An immunoblot

revealed that storage protein precursors, pro12S globulin and

pro2S albumin, were abnormally accumulated in the seeds of

these four allelic lines (gfs1-1 to gfs1-4), as in vsr1 seeds (Figure

6C). The different accumulations of pro2S albumin might be

attributable to different growing conditions of the plants and/or

different mutation sites. This result suggested that gfs1 mutants

have a defect in sorting of the storage proteins into PSVs.

The gene responsible for gfs1 mutants was roughly mapped to

a region close to the VSR1 gene (At3g52850). Immunoblot

analysis showed that VSR1 was not accumulated in the gfs1

seeds, as in the vsr1 seeds (Figure 7A). The faint signal on the blot

of these mutants might be attributable to the cross-reactivity of

the antibody with other homologs (VSR2 to VSR7). These results

suggested that the gfs1 mutants were allelic to vsr1. For a

complementation test, gfs1-1 was crossed with vsr1-2. Fourteen

F2 seeds were subjected to immunoblotting with anti-12S glob-

ulin antibody (Figure 7B). All of the F2 seeds accumulated 12S

globulin precursor, as did vsr1-2 and gfs1-1, suggesting that the

gfs1-1 mutant has a defect in VSR1. A complementation test of

gfs1-2 gave the same result (data not shown). Finally, we found

that each of the gfs1 mutants had a nucleotide substitution in the

VSR1 gene (At3g52850), as shown in Figure 7C. gfs1-1 had a

nucleotide substitution from GGA (Gly-214) to GAA (Glu). This

result suggests that the Gly-214 of gfs1-1 is essential for VSR1

function. gfs1-2 had a nucleotide substitution from CTTGga to

CTTGaa at the splicing donor site of the second intron. Both

gfs1-3 and gfs1-4 had the nucleotide substitutions from TGG

(Trp-105) to TGA (stop) and from TGG (Trp-297) to TGA (stop),

respectively. These results show that selecting green fluorescent

seeds among a large number of the seeds succeeded in isolating

vsr1 alleles.

Mapping of Other Mutations to Different Loci

We also analyzed six gfs mutants (gfs2, gfs3, gfs4, gfs5, gfs9, and

gfs10). All of them exhibited GFP fluorescence in the extracellular

matrix (Figure 8A), suggesting that they secrete vacuole-targeted

GFP out of the seed cells. The accumulation levels of GFP in

seeds of all of these mutants (data not shown) were similar to

those of vsr1/gfs1 (Figure 4A) and vps29/GFP-CT24 (Figure 5C).

An immunoblot revealed that these mutants abnormally

Figure 6. Selected gfs Mutants Have a Defect in Vacuolar Sorting of

Storage Proteins into PSVs.

(A) Seeds of the transformant (WT/GFP-CT24) were mutagenized with

ethyl methanesulfonate. The M2 seed populations were inspected with a

light microscope (left panel) and a fluorescence microscope (right panel)

to select green fluorescent seeds. Finally, ;100 fluorescent M2 lines

were isolated from 3,000,000 M2 seeds and were designated gfs

mutants.

(B) Seeds of the selected mutant lines were inspected with a confocal

laser scanning microscope. GFP fluorescence was detected out of the

seed cells of all gfs1 (gfs1-1, gfs1-2, and gfs1-3) mutants, showing

missorting of GFP-CT24. The GFP fluorescence image was not merged

with the autofluorescent image (red) of PSVs. Each image of green and

red fluorescence is shown in Supplemental Figure 1 online. Bars ¼10 mm.

(C) Seeds of wild-type (lane 1), vsr1-1 (lane 2), vsr1-2 (lane 3), gfs1-1 (lane

4), gfs1-2 (lane 5), and gfs1-3 (lane 6) plants were subjected to immu-

noblot analysis with anti-12S globulin and anti-2S albumin antibodies to

determine the behavior of endogenous storage proteins in gfs1 seeds. All

gfs1 mutants abnormally accumulated precursors of storage proteins

in seeds, as did the vsr1 mutant. p12S, 12S globulin precursors; 12S-a,

a-subunit of mature 12S globulin; p2S, 2S albumin precursors. Molecular

masses are given at left.

602 The Plant Cell

accumulated storage protein precursor of 12S globulin in their

seeds (Figure 8B), suggesting that they have a defect in vacuolar

sorting of storage proteins into PSVs. A rough map-based cloning

analysis mapped the gfs mutations to different loci (Figure 8C).

In gfs2, further mapping and DNA sequencing revealed a

single base pair mutation from G to A in the At2g26890 gene of

gfs2-1, which might cause a nonsense mutation from TGG (Trp-

33) to TGA (stop codon) (Figure 9A). To confirm that At2g26890 is

the gene responsible for the phenotype conferred by gfs2, we

obtained a knockout mutant of the At2g26890 gene

(SALK_132563). The mutant, which had T-DNA inserted into an

exon of the At2g26890 gene, was named gfs2-4 (Figure 9A).

gfs2-4 accumulated precursors of storage proteins, both 12S

globulin and 2S albumin (data not shown). We crossed gfs2-4

with WT/GFP-CT24 to generate gfs2-4/GFP-CT24. Some of the

F2 seeds were fluoresced (Figure 9B), and gfs2-4/GFP-CT24

exhibited GFP fluorescence in the extracellular space (Figure

9C). This result indicates that the mutation in the At2g26890 gene

is responsible for the phenotype conferred by gfs2.

Figure 7. Four gfs1 Mutant Lines Have a Mutation in the VSR1 Gene.

(A) Seeds of wild-type, vsr1-1, vsr1-2, gfs1-1, gfs1-2, and gfs1-3 plants

were subjected to immunoblot analysis with anti-VSR1 antibody. Faint

signals detected on the blot of vsr1 indicate cross-reactivity of the anti-

VSR1 antibody with the other VSR homologs in seeds.

(B) We crossed gfs1-1 with vsr1-2 and obtained F2 lines. Seeds of 14 F2

lines (one grain per lane) were subjected to immunoblot analysis with

anti-12S globulin antibody. All of the F2 seeds abnormally accumulated

12S globulin precursor, as did vsr1-2 and gfs1-1.

(C) Map-based cloning reveled that four gfs1 mutants had a mutation in

the VSR1 gene. Each point mutation site of gfs1-1 to gfs1-4 is shown in a

scheme of the VSR1 gene. Closed boxes indicate coding regions, and

solid lines indicate introns. The T-DNA insertion sites of vsr1-1 and vsr1-2

are also shown.

Figure 8. Six Different Mutants (gfs2, gfs3, gfs4, gfs5, gfs9, and gfs10)

Other Than vsr1/gfs1 Alleles Were Isolated.

(A) Seeds of six gfs mutants (gfs2, gfs3, gfs4, gfs5, gfs9, and gfs10) were

inspected with a confocal laser scanning microscope. GFP fluorescence

was detected out of the cells of these mutant seeds, showing missorting

of GFP-CT24. The GFP fluorescence image was not merged with the

autofluorescent image (red) of PSVs. Each image of green and red

fluorescence is shown in Supplemental Figure 1 online. Bars ¼ 10 mm.

(B) Seeds of wild-type, gfs2, gfs3, gfs4, gfs5, gfs6, gfs9, and gfs10 plants

were subjected to immunoblot analysis with anti-12S globulin antibody

to determine the behavior of endogenous seed storage proteins in these

seeds. These gfs mutants abnormally accumulated 12S globulin in

precursors in seeds. p12S, 12S globulin precursors; 12S-a, a-subunit

of mature 12S globulin. Molecular masses are given at left.

(C) Mutations of gfs1, gfs2, gfs3, gfs4, gfs5, gfs9, and gfs10 were

mapped to different loci from each other: gfs1 to At3g52850 (VSR1), gfs2

to At2g26890 (KAM2/GRV2), and gfs10 to At4g35870.


The At2g26890 gene was reported to be responsible for the

gravitropism defective2 (grv2) mutant, which is deficient in shoot

gravitropism and phototropism (Silady et al., 2004). We recently

found that the At2g26890 gene is also responsible for an

Arabidopsis mutant, katamari2 (kam2), that had deformed endo-

somes involving other endosomal compartments and formed

abnormally large aggregates with various organelles in the cells

(Tamura et al., 2007). kam2 alleles missorted seed storage

proteins by secreting them from cells (Tamura et al., 2007),

indicating that GFS2/KAM2 is involved in the vacuolar sorting of

storage proteins into PSVs. The GFS2/KAM2 gene encodes a

homolog of a DnaJ domain–containing RME8 (for receptor-

mediated endocytosis8) that is required for endocytosis and the

organization of endosomes in Caenorhabditis elegans (Zhang

et al., 2001), Drosophila melanogaster (Chang et al., 2004), and

human (Girard et al., 2005). KAM2 protein localized on punctate

structures, which did not merge with any markers for the Golgi,

the trans-Golgi network, endosomes, or prevacuolar compart-

ments, suggesting that GFS2/KAM2 is localized in an unchar-

acterized endosomal compartment (Tamura et al., 2007). It is

possible that DnaJ domain–containing GFS2/KAM2/GRV2 is

involved in the folding of protein(s) needed for the proper forma-

tion of endosomes that mediate protein trafficking to PSVs and

the biogenesis of endosomal compartments, including PSVs.

In gfs10, fine-scale mapping and DNA sequencing revealed a

single base pair mutation from G to A in the At4g35870 gene of

gfs10-1, which might cause a nonsense mutation from TGG (Trp-

397) to TGA (stop codon) (Figure 10A). We obtained a T-DNA–

tagged knockout mutant of the At4g35870 gene (SALK_139226)

and named it gfs10-2 (Figure 10A). An immunoblot showed that

gfs10-2 accumulated 12S globulin precursors in seeds, as did

gfs10-1 (Figure 10C). We crossed gfs10-2 with gfs10-1 that

expressed GFP-CT24 to generate F1 seeds. The expression of

GFP in the F1 seeds indicated that the two lines were success-

fully crossed. Each F1 seed grain accumulated 12S globulin

precursor as did both parent seeds (Figure 10D) and exhibited

GFP fluorescence in the extracellular matrix of the seed cells

(Figure 10E). These results indicate that the mutation in the

At4g35870 gene is responsible for the phenotype conferred by

gfs10. The ultrastructure of gfs10-1 seeds revealed that the

extracellular space was enlarged and filled with electron-dense

materials, possibly as a result of the secretion of storage proteins

(Figure 10F). The phenotype is very similar to those of the

vacuolar sorting mutants vsr1 (Shimada et al., 2003a), mag1/

vps29 (Shimada et al., 2006), and gfs2/kam2 (Tamura et al.,

2007). The At4g35870 gene, which is an intronless gene, en-

codes a novel membrane protein of unknown function that has

10 putative transmembrane domains (Figure 10B). The protein is

specific to higher plants. Thus, plants appear to have a novel and

plant-specific mechanism for the vacuolar sorting of storage

proteins. Further identification of the responsible genes of the

other gfs mutants should provide valuable insights into the

complex molecular mechanisms underlying the vacuolar sorting

of storage proteins.

DISCUSSION

A VSR Is Involved in Vacuolar Sorting of Storage Proteins

into PSVs by Recognizing Their Vacuolar Targeting Signals

It has long been thought that VSR is a sorting receptor for lytic

vacuoles but not a sorting receptor for PSVs (Hinz et al., 1999; Li

et al., 2002), although a VSR homolog (PV72) was isolated from

PAC vesicles of maturing pumpkin (Cucurbita maxima) seeds

(Shimada et al., 1997). However, we reported the involvement of

VSR1 in the proper sorting of storage protein precursors into PSV

cells (Shimada et al., 2003a). Additionally, in this study, we

isolated four vsr1 alleles by applying our green fluorescent seed

method (Figure 7) and found that all of the vsr1 alleles missorted

storage protein precursors in their seeds (Figure 6). It is now clear

that VSR1 is involved in the sorting of storage proteins into PSVs.

Arabidopsis has seven members (VSR1 to VSR7) of the VSR

family. Previously, we isolated seven mutants, each being defi-

cient in a different VSR gene, and found that only vsr1 accumu-

lated storage protein precursors in the seeds, whereas vsr2 to

vsr7 accumulated no precursors in the seeds (Shimada et al.,

2003a). By screening >3,000,000 seeds, we isolated four alleles

of vsr1 but no alleles of vsr2 to vsr7. These results suggest that

VSR1 functions primarily in vacuolar sorting of storage proteins in

Figure 9. A gfs2 Mutant Has a Mutation in the GRV2 Gene (At2g26890),

Which Encodes a Homolog of Mammalian RME8.

(A) A point mutation site of gfs2-1 is shown in a scheme of the GRV2

gene. Closed boxes indicate coding regions, and solid lines indicate

introns. A T-DNA–tagged allele (gfs2-4; SALK_132563) is also shown.

(B) We crossed the T-DNA–tagged mutant gfs2-4 with the transformant

WT/GFP-CT24. The F2 seed population was inspected with a binocular

microscope (top panel) and a fluorescence microscope (bottom panel).

gfs2-4/GFP-CT24 produced green fluorescent seeds (arrowheads).

(C) The gfs2-4/GFP-CT24 seeds were inspected with a confocal laser-

scanning microscope. GFP fluorescence was detected out of the cells of

these mutant seeds, showing missorting of GFP-CT24. The GFP fluo-

rescence image was not merged with the autofluorescent image (red) of

PSVs. Each image of green and red fluorescence is shown in Supple-

mental Figure 1 online. Bar ¼ 10 mm.

604 The Plant Cell

maturing seeds. This finding is consistent with VSR1 being the

most abundant of the seven VSRs in seeds.

Examination of several plants revealed >10 vacuolar targeting

signals for different vacuolar proteins, about half of which are

storage proteins of various plants (Vitale and Hinz, 2005). It is

possible that VSR functions in the sorting of storage proteins into

PSVs by recognizing the signals in various maturing seeds. This

notion is supported by the findings that VSR1 is responsible for

the sorting of 2S albumin and 12S globulin into PSVs of

Arabidopsis (Shimada et al., 2003a) and that VSR-like receptors

are involved in the sorting of 2S albumin and ricin into PSVs in

castor bean (Jolliffe et al., 2004).

What about the third major storage protein, 7S globulin? The

C-terminal peptide CT10 of b-conglycinin, which belongs to the

7S globulin family, was reported to act as a necessary and

sufficient signal for vacuolar targeting, because (1) deletion of the

signal from b-conglycinin resulted in secretion of the protein and

(2) addition of the signal to a GFP reporter resulted in delivery of

the GFP into PSVs (Nishizawa et al., 2003). Here, we demon-

strate that the vacuolar targeting signal was recognized by VSR1

for vacuolar sorting of b-conglycinin in Arabidopsis seeds.

As described above, in vivo and in vitro analyses have shown

that VSRs have an ability to bind to the vacuolar targeting signals

of various storage proteins. It remains to be determined how

VSRs can recognize a variety of signal sequences, because

these targeting signals have no consensus sequences.

Multiple Transport Pathways to PSVs

All of the gfs mutants that we isolated accumulated both pre-

cursor and mature forms of storage proteins (Figures 6 and 8), as

did vsr1 (Shimada et al., 2003a) and mag1/vps29 (Shimada et al.,

2006). We also found that another vacuolar sorting mutant,

mag2, accumulated both precursor and mature forms of storage

proteins in seeds. MAG2 might bind to ER-localized t-SNAREs

and function in the transport of storage protein precursors

between the ER and the Golgi complex in plants (Li et al.,

2006). These mutants normally deliver a portion of storage

proteins into PSVs, even though they have a defect in vacuolar

sorting of storage proteins: vsr1, mag1/vps29, gfs2/kam2/grv2,

and gfs10 secreted part of the storage proteins, whereas mag2

developed precursor-accumulating bodies within the seeds

(Li et al., 2006). The incomplete secretion of storage proteins

in these mutants might not be attributable to the redundancy

of each responsible gene, because MAG1/VPS29, MAG2,

GFS2/KAM2, and GFS10 do not have closely related homologs

in the Arabidopsis genome. Vacuolar sorting mutants that

accumulate all storage proteins as only precursor forms have

not been isolated. Such mutants may be lethal because they

could be affected in all mechanisms of vacuolar sorting.

Another possibility is that plants use multiple transport pathways

to PSVs.

One of the pathways is aggregation sorting, which is an

efficient way of transporting large amounts of protein. Two

compartments have been shown to mediate aggregation sorting:

ER-derived PAC vesicles in pumpkin (Hara-Nishimura et al.,

1993, 1998; Mitsuhashi et al., 2001), castor bean (Hiraiwa et al.,

1993), soybean (Mori et al., 2004), and rice (Oryza sativa)

Figure 10. A gfs10 Mutant Has a Mutation in the At4g35870 Gene,

Which Encodes a Novel Membrane Protein.

(A) A point mutation site of gfs10-1 is shown in a scheme of the

At4g35870 gene, which is an intronless gene. A T-DNA–tagged mutant

allele (gfs10-2; SALK_139226) is also shown.

(B) Scheme of GFS10 with putative 10 transmembrane domains (green

boxes).

(C) Seeds of gfs10 were subjected to immunoblot analysis with anti-12S

globulin. Two gfs10 alleles abnormally accumulated 12S globulin pre-

cursors. p12S, 12S globulin precursors; 12S-a, a-subunit of mature 12S

globulin. Molecular masses are given at left.

(D) We crossed gfs10-2 with gfs10-1 that expressed GFP-CT24. Six F1

seeds was subjected to immunoblot analysis with either anti-12S glob-

ulin antibody or anti-GFP antibody. The accumulation of GFP in each F1

seed grain indicated that crossing was successful. Each F1 seed

accumulated 12S globulin precursors, as did parent seeds.

(E) An F1 seed was inspected with a confocal laser scanning micro-

scope. GFP fluorescence was detected out of the cells, showing

missorting of GFP-CT24. The GFP fluorescence image was not merged

with the autofluorescent image (red) of PSVs. Each image of green and

red fluorescence is shown in Supplemental Figure 1 online. Bar¼ 10 mm.

(F) Electron micrograph of a gfs1-1 seed. The extracellular space of the

gfs1-1 seeds was abnormally enlarged and filled with electron-dense

materials (arrowheads). Bar ¼ 5 mm.


(Takahashi et al., 2005) and Golgi-derived dense vesicles in pea

(Hinz et al., 1999) and common bean (Chrispeels, 1983). PAC-like

vesicles were induced in transgenic soybean seeds that cosup-

pressed the a-subunit of b-conglycinin (Kinney et al., 2001) and

in transgenic Arabidopsis leaves that expressed a fusion protein

composed of a small subunit of 2S albumin and phosphinothricin

acetyltransferase (Hayashi et al., 1999). PAC vesicles can be

regarded as prevacuolar compartments or endosomes in ma-

turing seeds of various plant species.

We proposed a hypothetical model of the multiple pathways

that is composed of three steps. Storage proteins that are

actively synthesized form aggregates within the ER. As a result,

aggregates and free molecules of storage proteins exist together

in the ER lumen. This means that a storage protein species takes

two forms: an aggregate form and a free molecule form. Aggre-

gates develop to generate the PAC vesicles (first step), whereas

free molecules leave the ER for the Golgi complex, where they are

trapped by a VSR to be recruited to PAC vesicles (second step).

PAC vesicles are incorporated directly into PSVs (third step).

VSR1 and MAG1/VPS29 are involved in the second step. MAG2

might function in the exit from the ER in the first or second step.

Considering a subcellular localization of GFS2/KAM2/GRV2

in uncharacterized compartments (Tamura et al., 2007), a novel

pathway should be involved in vacuolar sorting. Further charac-

terization and subcellular localization of GFS10 will provide a clue

to the complex mechanism of vacuolar sorting.

A Powerful Tool for the Screening of Mutants Deficient

in Vacuolar Sorting

To understand the molecular mechanism underlying the multiple

pathways, forward genetics might be a useful approach. In this

study, we developed a simple method (the green fluorescent

seed method) to isolate vacuolar sorting mutants using GFP

fluorescence as an indicator. Using this method, we succeeded

in isolating four vsr1 alleles. We also identified GFS2/KAM2/

GRV2 and GFS10 as factors of the sorting of storage proteins to

PSVs. This technique must be one of the easiest and most

efficient for hunting vacuolar sorting mutants.

We found that mag1/vps29 plants that expressed GFP-CT24

exhibited green fluorescent seeds. MAG1/VPS29 is encoded by

a single-copy gene and is expressed ubiquitously in various

organs, including leaves, roots, flowers, and developing seeds

(Schmid et al., 2005). Thus, the MAG1/VPS29 gene may play an

indispensable role in various tissues of Arabidopsis. MAG1/

VPS29 might be required for vacuolar protein sorting into not only

PSVs but also lytic vacuoles. gfs2/kam2/grv2 mutants exhibit a

defect in the endomembrane organization of vegetative tissues

(Tamura et al., 2007), and both gfs4 and gfs5 mutants exhibit a

defect in the development of vegetative tissues (data not shown).

Considering these results, vacuolar sorting mutants for both

PSVs and lytic vacuoles could be isolated using our method.

The green fluorescent seed method makes it possible to

efficiently screen a large number of seeds and to easily isolate

mutant seeds. Selecting 3,000,000 seeds gave us >100 gfs

mutants, some of which might be allelic. Identification of the

responsible genes of these gfs mutants should provide a valu-

able insight into the complex molecular mechanisms in the

multiple pathways unique to higher plants, as do yeast sec and

vps mutants (Bonangelino et al., 2002). Further analysis of these

mutants will reveal not only the vacuolar sorting mechanism of

PSVs but also that of lytic vacuoles.

METHODS

Plant Materials and Growth Conditions

We used wild-type plants of Arabidopsis thaliana (ecotypes Columbia and

Wassilewskijia-2) and the Arabidopsis mutants vsr1-1 and vsr1-2 (Shimada

et al., 2003a) and mag1-1/vps29-1 and mag1-2/vps2-2 (Shimada et al.,

2006). We also used transgenic plants that were generated previously

(Nishizawa et al., 2003): Arabidopsis (ecotype Columbia) that expressed

soybean (Glycine max) b-conglycinin a9-subunit (WT/conglycinin) and

Arabidopsis (ecotype Columbia) that expressed GFP-CT24 (WT/GFP-

CT24). We crossed vsr1-1 and vsr1-2 with WT/conglycinin and WT/GFP-

CT24 to generate the transgenic plants vsr1-1/conglycinin and vsr1-2/

conglycinin and the transgenic plants vsr1-1/GFP-CT24 and vsr1-2/GFP-

CT24, respectively. We crossed vsr1-2 with gfs1-1 for complementation

tests. We crossed mag1-1/vps29-1 and mag1-2/vps29-2 with WT/GFP-

CT24 to generate two transgenic plants (mag1-1/vps29-1/GFP-CT24 and

mag1-2/vps29-2/GFP-CT24, respectively). We obtained T-DNA insertion

mutants (SALK_132563 and SALK_139226) from the ABRC at Ohio State

University. We crossed the SALK_132563 line (gfs2-4) with the trans-

formant WT/GFP-CT24 to produce gfs2-4/GFP-CT24. We further

crossed gfs10-1 with the SALK_139226 line (gfs10-2) to produce F1

seeds. Surface-sterilized seeds were sown onto 0.5% Gellan Gum

(Wako) with Murashige and Skoog medium supplemented with 1%

sucrose and were grown at 228C under continuous light.

Affinity Column Chromatography

We chemically synthesized the C-terminal peptide (CT10; PLSSILRAFY)

of soybean b-conglycinin a9-subunit and immobilized the peptide (5 mg)

to Aminolink coupling gel (2 mL of a bed volume; Pierce). Affinity column

chromatography was performed essentially as described (Watanabe

et al., 2002). Microsomes were prepared from young Arabidopsis siliques

and were applied to the column that had been equilibrated with 20 mM

HEPES-NaOH, pH 7.0, 150 mM NaCl, 1% CHAPS, and 1 mM CaCl2. The

bound proteins were eluted with either the CT10 peptide or 5 mM EGTA.

Each fraction was subjected to immunoblotting with anti-VSR1 antibody.

Specific Antibodies and Immunoblot Analysis

Immunoblot analysis of Arabidopsis seeds was performed essentially as

described previously (Shimada et al., 2002), except that dilutions of the

antibodies were as follows: anti-VSR1 (1:5000) (Yamada et al., 2005),

anti-b-conglycinin (1:5000) (Nishizawa et al., 2003), anti-2S albumin

(1:10,000), anti-12S globulin a-subunit (1:10,000) (Shimada et al.,

2003b), and anti-GFP (1:5000) (Tamura et al., 2003).

Immunoelectron Microscopy

Dry seeds were cut into slices with a razor blade and then fixed with 4%

(w/v) paraformaldehyde, 1% (v/v) glutaraldehyde, 0.05 M cacodylate

buffer, pH 7.4, and 10% DMSO. Procedures for immunoelectron micro-

scopic analysis were essentially as described previously (Hara-Nishimura

et al., 1993). Ultrathin sections were treated with antibodies against Arabi-

dopsis 2S albumin (dilution, 1:50), Arabidopsis 12S globulin a-subunit

(1:50), b-conglycinin (1:50), and GFP (1:50). Sections were examined with

a transmission electron microscope (model 120WX; JEOL) at 80 kV.

606 The Plant Cell

Confocal Laser Scanning Microscopy

Dry seeds were pressed in glycerol between glass slides and cover strips

to push out cotyledons. The cotyledons were inspected with a confocal

laser scanning microscope (LSM510 META; Carl Zeiss) using the 488-nm

line of a 40-mW Ar/Kr laser and an emission filter (BP 530-505) for GFP

fluorescence and the 544-nm line of a 1-mW He/Ne laser and an emission

filter (LP 560) for autofluorescence of PSVs with a 40 3 0.75 numerical

aperture dry objective. All images were obtained in multitrack mode and

analyzed using LSM image-examiner software (Carl Zeiss). The data were

exported as eight-bit TIFF files and processed using Adobe Photoshop

5.5 (Adobe Systems).

Isolation of the gfs Mutants

Seeds of the transformed plant that expressed GFP-CT24 (WT/GFP-

CT24) were mutagenized by soaking them for 16 h in 0.2 or 0.25% (v/v)

methanesulfonic acid ethyl ester (Sigma-Aldrich) and then washed for 11

h in running water to obtain M1 seeds. The M1 seeds were grown after

self-fertilization to generate the M2 seed populations. These seeds were

inspected with a fluorescence stereomicroscope (MZ-APO; Leica Micro-

systems), and images were acquired using a digital camera (DS-5ML1;

Nikon).

Map-Based Cloning

Each homozygous mutant (background, ecotype Columbia) was crossed

with Landsberg erecta wild-type plants to generate a mapping popula-

tion. Genomic DNA was isolated from leaves of each mutant that was

selected from the F2 progeny. The polymorphism between Columbia and

Landsberg erecta was analyzed using a combination of cleaved amplified

polymorphic sequences and simple sequence length polymorphism

markers with data obtained from The Arabidopsis Information Resource

(http://www.Arabidopsis.org).

According to our green fluorescent seed method, the mutant seeds can

be quickly and easily selected from the F2 progeny. For rough mapping,

we selected ;10 to 20 recombinants of the F2 progeny of each gfs

mutant. As a result, all four gfs1 mutations were roughly mapped near the

VSR1 gene on chromosome 3 and the other mutations (gfs2, gfs3, gfs4,

gfs5, gfs9, and gfs10) were roughly mapped as shown in Figure 8C. For

identification of the responsible genes of gfs1 mutants, we subjected the

seeds of the four gfs mutants to immunoblot analysis with anti-VSR1

antibody to see no accumulation of VSR1, crossed each gfs1 mutant with

vsr1-2 to confirm the responsible gene to be VSR1, and sequenced the

VSR1 gene of each gfs1 mutant to determine the mutation site of the

gene, as shown in Figure 7. We further performed fine-scale mapping for

gfs2 and gfs10 mutants. We selected 120 mutants from the F2 progeny for

gfs2 and 100 mutants from the F2 progeny for gfs10 by the green

fluorescent seed method and prepared genomic DNAs from the leaves of

each mutant for fine-scale mapping. We sequenced the respective genes

to determine the mutation sites of gfs2-1 and gfs10-1. Finally, to deter-

mine the responsible genes of gfs2-1 and gfs10-1, we obtained T-DNA–

tagged lines that had a defect of each gene to examine the phenotype. For

gfs2, we had six mutant alleles, all of which exhibited the vacuolar sorting

defect (Tamura et al., 2007). For further confirmation of the responsible

gene of gfs10-1, we crossed gfs10-1 with gfs10-2, as shown in Figure 10D.

Nucleotide sequences were determined from both strands using an ABI

Prism Big Dye Terminator Cycle sequence reaction kit (Applied Biosystems)

and a DNA sequencer (Prism 3100; Applied Biosystems).

Accession Numbers

Arabidopsis Genome Initiative locus identifiers for the genes mentioned in

this article are as follows: GFS1/VSR1 (At3g52850), MAG1/VPS29

(At3g7810), GFS2/KAM2/GRV2 (At2g26890), and GFS10 (At4g35870).

Supplemental Data

The following material is available in the online version of this article.

Supplemental Figure 1. Green Fluorescent and Red Fluorescent

Images of Seeds Are Shown Separately for All Transformants in This

Study.

ACKNOWLEDGMENTS

This work was supported by the Core Research for Evolutional Science

and Technology program of the Japan Science and Technology Cor-

poration and by Grants-in-Aid for Scientific Research (Grants 16044224,

16085203, and 17107002) and for 21st Century Centers of Excellence

Research, Kyoto University (Grant A14), from the Ministry of Education,

Culture, Sports, Science, and Technology of Japan. We are grateful to

the ABRC at Ohio State University for providing the seeds of Arabi-

dopsis T-DNA insertion mutants (SALK_132563 and SALK_139226).

Received July 18, 2006; revised December 19, 2006; accepted January

22, 2007; published February 9, 2007.

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DOI 10.1105/tpc.106.045997; originally published online February 9, 2007; 2007;19;597-609Plant Cell

Utsumi, Keito Nishizawa, Nobuyuki Maruyama and Ikuko Hara-NishimuraKentaro Fuji, Tomoo Shimada, Hideyuki Takahashi, Kentaro Tamura, Yasuko Koumoto, Shigeru

Secretion of Vacuole-Targeted Green Fluorescent Protein in Their Seeds) Can Be Identified Efficiently bygreen fluorescent seed Vacuolar Sorting Mutants (Arabidopsis

This information is current as of March 30, 2019

Supplemental Data /content/suppl/2007/01/22/tpc.106.045997.DC1.html

References /content/19/2/597.full.html#ref-list-1

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