auxin-mediated ribosomal biogenesis regulates vacuolar

Auxin-Mediated Ribosomal Biogenesis Regulates VacuolarTrafficking in Arabidopsis W

AbelRosado,a Eun JuSohn,a,1 GeorgiaDrakakaki,a Songqin Pan,a Alexandra Swidergal,a YuqingXiong,b Byung-HoKang,b Ray A. Bressan,c and Natasha V. Raikhela,2

a Department of Botany and Plant Sciences and Center for Plant Cell Biology, University of California, Riverside, California 92521b Electron Microscopy and Bioimaging Lab, Interdisciplinary Center for Biotechnology Research, University of Florida,

Gainesville, Florida 32611c Department of Horticulture and Landscape Architecture, Purdue University, West Lafayette, Indiana 47907

In plants, the mechanisms that regulate the transit of vacuolar soluble proteins containing C-terminal and N-terminal

vacuolar sorting determinants (VSDs) to the vacuole are largely unknown. In a screen for Arabidopsis thaliana mutants

affected in the trafficking of C-terminal VSD containing proteins, we isolated the ribosomal biogenesis mutant rpl4a

characterized by its partial secretion of vacuolar targeted proteins and a plethora of developmental phenotypes derived

from its aberrant auxin responses. In this study, we show that ribosomal biogenesis can be directly regulated by auxins and

that the exogenous application of auxins to wild-type plants results in vacuolar trafficking defects similar to those observed

in rpl4a mutants. We propose that the influence of auxin on ribosomal biogenesis acts as a regulatory mechanism for auxin-

mediated developmental processes, and we demonstrate the involvement of this regulatory mechanism in the sorting of

vacuolar targeted proteins in Arabidopsis.

INTRODUCTION

A functional vacuole and intact protein trafficking system are

necessary for plant cell viability and function. Perturbations of the

trafficking machinery often affect vital cellular processes, such

as plant hormone responses, cytokinesis, and the development

of tissue specificity (Surpin and Raikhel, 2004). In the classical

view, many soluble plant vacuolar proteins are sorted away from

proteins destined for secretion at the trans-Golgi network, a

process that requires the presence of positive sorting signals in

the primary sequence of vacuolar proteins (Matsuoka and

Nakamura, 1999; Vitale and Raikhel, 1999; Robinson et al.,

2005). Two of these sorting signals, an N-terminal propeptide

(NTPP) and a C-terminal propeptide (CTPP), are directed to the

vacuole by distinct pathways that converge at the prevacuolar

compartment (PVC) (Miao et al., 2008). The NTPP pathway is

believed to be common to plants and yeast, and several com-

ponents of the machinery involved in the sorting of NTPP-type

cargoes have been characterized (Zheng et al., 1999; Ahmed

et al., 2000; Bassham and Raikhel, 2000). The CTPP pathway is

believed to be unique to plants, and different genetic approaches

have been used to identify components that are specific for that

pathway (Sanmartın et al., 2007; Sohn et al., 2007). To isolate

newcomponents of the plant-specificCTPP sortingmachinery in

Arabidopsis thaliana, we used a T-DNA–mutagenized population

in the Vac2 background (Vac2 T-DNA). The Vac2 line contains a

genetically engineered CLAVATA3 (CLV3) fused to the barley

(Hordeum vulgare) lectin C-terminal vacuolar sorting signal

(CLV3:CTPPBL) in the clv3-2 mutant background (Figure 1A).

Previous studies using genetic crosses and ethyl methanesulfo-

nate mutagenesis of the Vac2 line have been successfully used

for the identification of components involved in the specific

sorting of CTPP proteins (Sanmartın et al., 2007; Sohn et al.,

2007). In this report, the use of the Vac2 T-DNA screen allowed

us to identify a previously unknown component of the auxin-

mediated vacuolar sorting machinery, the cytosolic ribosomal

protein (r-protein) L4/L1 (RPL4A).

Although protein trafficking defects of ribosomal mutants have

not been evaluated, the association of ribosomal mutations with

deficient auxin perception and distribution has been widely

reported in the literature. The pointed first leaf 1 (pfl1)/r-protein

S18 (rps18a), pfl2/rps13b, and the semidominant Arabidopsis

minute-like1 (aml1)/rps5a mutants display auxin-related devel-

opmental defects, including growth retardation, narrow leaves

with reductions in the palisade mesophyll layer, and cotyledon

vascular pattern defects (Van Lijsebettens et al., 1994; Ito et al.,

2000; Weijers et al., 2001). Mutations in the short valve1 (stv1)/

r-protein L24 (rpl24b) result in an apical-basal patterning defect

of the gynoecium by influencing the translation of the auxin

response factors ARF3 and ARF5 (Nishimura et al., 2005). STV1

together with the proteins RPL28A and RPL5A has been shown

to have important roles in specifying leaf adaxial identity (Yao

et al., 2008), andRPL5A, RPL10A, andRPL9 have been shown to

modulate leaf patterning mechanisms via the ribosome-mediated

translational regulation of genes in theHD-ZIPIII-KANADI pathway

1Current address: Division of Molecular and Life Sciences, PohangUniversity of Science and Technology, Pohang 790-784, Korea.2 Address correspondence to [email protected] 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: Natasha V. Raikhel([email protected]).WOnline version contains Web-only data.www.plantcell.org/cgi/doi/10.1105/tpc.109.068320

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(Pinon et al., 2008). Finally, mutations in the nucleolar protein

PARL1, involved in ribosomal biogenesis, cause similar auxin

related developmental defects, suggesting that auxin regulation

depends on protein turnover and ribosome biogenesis in areas of

growth (Petricka and Nelson, 2007).

In bacteria, the RPL4 protein has been shown to be crucial for

the maintenance of ribosomal translational efficiency and fidelity

(O’Connor et al., 2004), but aside from structural roles within the

ribosome, no other specific function has yet been assigned in

plants. In this study, we analyze the implications of themutations

in theArabidopsisRPL4 family for protein trafficking aswell as for

hormonal regulation leading to the altered sorting of vacuolar

targeted proteins.

The Arabidopsis cytosolic r-protein RPL4 family is composed

of two transcriptionally active genes (RPL4A and RPL4D) and

two nonexpressed pseudogenes (RPL4B and RPL4C) (Barakat

et al., 2001). Our analysis of the two transcriptionally active

members of the RPL4 family in Arabidopsis suggests that the

RPL4A and RPL4D proteins have equivalent functions and are

coexpressed. Mutations in either RPL4 gene cause a similar

auxin-related developmental defect, and both proteins are in-

volved in the delivery of vacuolar targeted proteins to the

vacuole. Moreover, our results suggest that the sorting defects

in rpl4mutants are due to problems in protein turnover and auxin

perception in metabolically active tissues. We propose that

ribosomal biogenesis influenced by auxins is a high level regu-

latory mechanism in metabolically active tissues that regulates

the vacuolar delivery of not only CTPP, but also NTPP and

recycled proteins.

Figure 1. Identification and Genetic Analysis of rpl4a Using a Visual

Screen for Mutants with Altered Trafficking to the Vacuole.

(A) Screening strategy. The Arabidopsis CLV3 protein (green) is synthe-

sized in the shoot apical meristem layers L1 and L2 and secreted to the

apoplasm. There, it activates the CLV1/2 LRR kinase receptor (black).

Plants lacking CLV3 protein (clv3-2) have uncontrolled growth at the

shoot apical meristem. The Vac2 reporter line targets the CLV3:CTPPBL

fusion protein to the vacuole (V) in the clv3-2 background. T-DNA plants

mutated in components of the vacuolar trafficking machinery shunt

CLV3:T7:CTPPBL to the default secretion pathway, thereby comple-

menting the clv3-2 phenotype.

(B) Floral meristems from wild-type Landsberg, clv3-2, Vac2, and the

21-4 (rpl4a-1) mutant isolated in the screen.

(C) Genetic analysis of the clv3-2/CLV3:CTPPBL/rpl4a-1 segregant pop-

ulations. The top images show representative inflorescences, and the

bottom images show representative floral meristems for each genetic

background. The rpl4a-1 mutation is unable to bypass genetically the

clv3-2 phenotypes (plants rpl4a-1/clv3) unless the CLV3:CTPPBL marker

is present (plants rpl4a-1/ CLV3:CTPPBL/clv3). The rpl4a-1/CLV3:

CTPPBL plants have terminated floral meristems (arrows) likely due to

the secretion of the CLV3:CTPPBL marker to the apoplasm.

(D) The RFP:CTPPpha fluorescent marker is localized in the vacuoles of

wild-type plants (left panels, stars), but it is mis-sorted and partially

secreted to the apoplast in metabolically active tissues, such as primor-

dial leaves and young hypocotyls, in the 21-4 (rpl4a-1) background (right

panels, stars). Notice that the expression level of the RFP:CTPPpha

marker decreases in rpl4a-1 root meristems. For each position, the

images shown were acquired using the same microscopy settings.

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RESULTS

rpl4a-1 Has Altered Protein Sorting to the Vacuole

rpl4a-1 is a mutant characterized from a T-DNA–mutagenized

population in the Arabidopsis Vac2 background (Rojo et al.,

2002). The Vac2 line was transformed with the pSKI015 plasmid,

and ;8000 lines were divided into 144 independent pools

(Koiwa et al., 2006). The pools were screened for complemen-

tation of the clv3-2 shoot meristem phenotype (Sanmartın et al.,

2007; Sohn et al., 2007; Zouhar et al.,2009) (Figure 1A). By this

approach, we identified amutant designated 21-4 that displayed

reduced floral meristem size (Figure 1B). Genetic analyses

showed that the 21-4 line harbored one functional T-DNA inser-

tion that caused a recessive mutation in a single nuclear locus

(see Supplemental Table 1 online). Thermal asymmetric inter-

laced PCR was used to determine the T-DNA insertion position

and the genomic sequence flanking the T-DNA left border. As a

result, a T-DNA insertion located 283 bp downstream of the ATG

translation start site in the first exon of the At3g09630 locus was

identified. The At3g09630 gene was annotated as the cytosolic

L4/L1 protein of the large 60S ribosomal complex subunit

(RPL4A). Genetic linkage analysis confirmed that the reduced

meristems, the T-DNA insertion in RPL4A, and the developmen-

tal phenotypes observed in different rpl4a alleles (Figure 2) were

linked within a genetic distance of 0.5 centimorgans (see Sup-

plemental Table 1 online). Upon identification of the mutation in

RPL4A, we performed phenotypic and genetic analyses to test

whether the mutation acted as a bypass suppressor of clv3-2.

The analysis of populations in which rpl4a-1, clv3-2, and CLV3:

CTPPBL were segregating indicated that rpl4a-1 mutation was

unable to genetically bypass the clv3 phenotypes (plants rpl4a-1/

clv3) unless CLV3:CTPPBL was present (plants rpl4a-1/clv3/

CLV3:CTPPBL) (Figure 1C). Interestingly, the combination rpl4a-

1/CLV3:CTPPBL caused terminated meristems that mimicked

the phenotypes observed in CLV3-overexpressing lines (Rojo

et al., 2002) (Figure 1C). Based on these results, we concluded

that the reduced floral meristem phenotype in 21-4 (rpl4a-1/clv3/

CLV3:CTPPBL) was due to the secretion of the CLV3:CTPPBL

fusion protein to the apoplasm and that rpl4a-1was not a bypass

mutation of clv3.

To confirm further that rpl4a-1 causes the secretion of vac-

uolar targeted proteins at the cellular level, we crossed the

rpl4a-1 mutant line with two lines expressing fluorescent vac-

uolar markers. The first marker contained the vacuolar sorting

signal of proricin appended to the N terminus of the monomeric

red fluorescent protein RFP1 (NTPPpro:RFP) (Hunter et al.,

2007). The secondmarker contained the vacuolar sorting signal

of phaseolin fused to the C terminus of RFP1 (RFP:CTPPpha)

(Hunter et al., 2007). Both markers included the signal peptide

from sporamine fused to the N-terminal end of RFP1 that

caused the reporter to be directed into the default secretion

pathway when the vacuolar sorting signals were not recognized

(Craddock et al., 2008). Homozygous lines from the F2 gener-

ation were obtained, and the localization of the RFP fusion

proteins in different tissues and developmental stages using

scanning fluorescent confocal microscopy was determined. As

shown in Figure 1D, wild-type plants displayed vacuolar local-

ization of the RFP:CTPPpha in all tissues, whereas secretion in

the first pair of true leaves and lower hypocotyls and decreased

expression and partial secretion in root meristems were ob-

served in the rpl4a-1 line. Similar analyses were performed with

the NTPPpro:RFP marker, which presented similar vacuolar

localization in wild-type plants, but the secretion of NTPPpro:

RFP in different tissues from rpl4a-1 mutants was not as

dramatic as in the case of RFP:CTPPpha (see Supplemental

Figure 1 online). The differential results obtained with the

different vacuolar markers might be partially explained by the

lower expression of NTPPpro:RFP in both wild-type and rpl4a

backgrounds that, in turn, decreased the marker secretion

signal (see Supplemental Figure 1 online). The results obtained

from the vacuolar fluorescent markers analyses validate the

genetic screen for vacuolar trafficking mutants and suggest

that RPL4A is required for the proper sorting of vacuolar-

targeted proteins.

rpl4a Displays Aberrant Auxin-Related

Developmental Phenotypes

Early in its development, the rpl4a-1 mutant displays very

characteristic narrow pointed first leaves (pfl) and retarded

growth (Figure 2A). The pfl phenotype in rpl4a-1 mutants re-

sembled the phenotypes of the r-protein knockout mutants

rps13b and rps18a (Van Lijsebettens et al., 1994; Ito et al., 2000)

and was also similar to the phenotypes observed in RPL23-

silenced lines (Degenhardt and Bonham-Smith, 2008). To con-

firmwhethermutations in theRPL4A genewere the cause of the

pfl phenotypes, two additional T-DNA alleles in the Columbia-0

(Col-0) genetic background, rpl4a-2 (SALK_130595) and rpl4a-3

(SALK_063782), were isolated. Homozygous rpl4a-2 and rpl4a-3

seedlings exhibited similar developmental phenotypes to those

of rpl4a in a Landsberg background (Figure 2A). In early stages

of development, it was also observed that rpl4a-1 displayed

altered cotyledon architecture associated with aberrant distri-

butions of auxin maxima sites (Figure 2B). The vascular struc-

ture in the first leaves of rpl4a-1 deviated from the closed and

reticulate venation of controls. rpl4a-1 presented substantially

reduced venation, few or no tertiary or quaternary veins, and

aberrant anastamosis close to the hypocotyl-petiole junction

(Figure 2C). Other physiological and developmental conse-

quences of the rpl4a-1 mutation included reduced root elon-

gation, altered root gravitropic responses, delayed transition to

reproductive phase, and decreased hypocotyl elongation in

etiolated seedlings (Figures 2D to 2F). Impaired auxin homeo-

stasis may explain these phenotypic traits, which were remi-

niscent of mutants with altered ribosome biogenesis and auxin

responses (for references, see Supplemental Table 2 online).

However, the severely altered auxin responses observed in

rpl4a-1 did not influence the subcellular localization, polarity,

and tissue expression of auxin transporters, such as AUX1,

PIN2, and PIN7, in light-incubated rpl4a seedlings (see Sup-

plemental Figure 2 online).

Because rpl4a-1 shared common features with a plethora of

previously reported ribosomal mutants, we investigated whether

other common phenotypes found in different r-protein mutants

were present in rpl4a-1 (see Supplemental Table 2 online). Leaf

Ribosomal Regulation of Vacuolar Sorting 145

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patterning organization and adaxial-abaxial polarity were dis-

rupted at the cellular level in rpl4a-1. Epidermal cells of the

adaxial lamina surface of the first leaf of rpl4a-1were altered and

presented angular lobes as opposed to the jigsawpatterning and

smooth lobes in the wild type. Abaxial surfaces in rpl4a-1 were

also different and displayed a mosaic pattern of normal abaxial

patches mixed with outgrowths and heterogeneous cell sizes

(Figure 2G). The stained transverse sections of first leaf cells in

rpl4a-1 presentedmany enlarged cells and intercellular spaces in

the adaxial palisade region, and the number of subepidermal

palisade cells was fewer than that of the wild type (Figure 2H).

Finally, the rpl4a-1 root meristem cells displayed more disor-

ganized nucleolar structures with an abundance of large nu-

cleolar vacuoles (71% of rpl4a nuclei n = 44) compared with

controls (10% of Vac2 nuclei n = 39), suggesting that ribosomal

biogenesis could be affected in that tissue (Figure 2I). The

extensive phenotypic similarities of rpl4a-1 with previously

reported r-protein mutants involved in ribosomal biogenesis,

together with the aberrant nucleolar structures in rpl4a-1,

support the hypothesis that the ribosome per se, and not

r-proteins acting independently, regulates auxin-mediated de-

velopmental processes.

Figure 2. The rpl4 Family Mutants Display Aberrant Auxin-Related Developmental Defects.

(A) Independent rpl4a alleles display narrow pointed first leaves. The original rpl4a-1mutant was compared with the wild-type Landsberg, whereas the

rpl4a-2 (SALK_130595) and rpl4a-3 (SALK_063782) alleles were compared with the wild-type Col-0.

(B) rpl4a-1 cotyledon structures and auxin maxima localizations. In the far right panel, GUS staining of rpl4a-1 cotyledons expressing the auxin reporter

DR5pro:GUS. The right cotyledon shows aberrant auxin maxima localizations (black stars), and the left cotyledon shows the expected apical auxin

maxima localization (white star).

(C) to (F) Auxin-related phenotypes in rpl4a-1 include incomplete vascular development (C), reduced root elongation and altered root gravitropic

responses (D), delayed transition to reproductive phase (E), and reduced hypocotyl elongation in etiolated seedlings (F).

(G) Scanning electron micrographs of the adaxial and abaxial surfaces of late rosette leaves from 28-d-old plants of the wild type (top) and rpl4a-1

(bottom).

(H) Stained transverse sections of first leaf cells in rpl4a-1 presented many enlarged cells and intercellular spaces in the adaxial palisade region and

fewer subepidermal palisade cells than did the wild type.

(I) Transmission electron microscopy of root apical meristem cells shows aberrant nucleolar structures in the rpl4a-1 mutant. NUC, nucleolus; NP,

nucleoplasma; NV, nuclear vacuole.

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Arabidopsis Has Heterogeneous RPL4-Containing

Ribosomes in Specific Tissues

The RPL4 family in Arabidopsis comprises two nearly identical

expressed members, RPL4A and RPL4D (Barakat et al., 2001;

Carroll et al., 2008) (Figure 3A). RPL4A is a basic protein (pI =

11.04) of 406 amino acids that shares an overall 95.1% amino

acid identity with RPL4D. The T-DNA mutants in the RPL4A

(rpl4a-2 and rpl4a-3) and the RPL4D (rpl4d-1 and rpl4d-2) genes

in the Col-0 background were isolated from the SALK collection

and their mRNA expressions analyzed by RT-PCR. As shown in

Figure 3B, the rpl4a-2 mutant (hereafter referred to as rpl4a)

showed reducedRPL4A expression, whereas the rpl4a-3, rpl4d-1

(hereafter referred to as rpl4d), and rpl4d-2 mutants showed no

discernible transcripts after 35 PCR cycles.

When the rpl4a and rpl4d mutants were grown side by side,

they were indistinguishable and displayed similar auxin-related

phenotypes (see Supplemental Figure 3 online). The high degree

of amino acid identity between RPL4A and RPL4D, together with

the similar phenotypes observed in the rpl4a and rpl4dmutants,

suggested that both proteins have similar functions. To evaluate

further their spatial and temporal equivalence, their respective

promoter:b-glucuronidase (GUS) expression patterns were an-

alyzed, and no significant differences between RPL4Apro:GUS

and RPL4Dpro:GUS expression patterns in our experimental

conditions were observed (Figures 6A to 6F; see Supplemental

Figure 4D online). Because a given ribosomal complex contains

only one RPL4 family member at a time (Ban et al., 2000), these

results suggested that distinct ribosomal populations (RPL4A

and RPL4D ribosomal complexes) simultaneously exist in wild-

type plants. This conclusion is supported by previous proteomic

studies that demonstrated the presence of ribosomal heteroge-

neity in Arabidopsis (Chang et al., 2005; Giavalisco et al., 2005;

Carroll et al., 2008).

The RPL4 Family Requires at Least Two Functional Gene

Copies for Plant Viability

We attempted to enhance the phenotypes observed in rpl4

mutants by eliminating both RPL4 proteins or, in case of lethality,

to determine the minimum RPL4 gene dosage required for plant

viability. For that purpose, we successively eliminated RPL4

gene function by performing reciprocal crosses between the

Figure 3. Genetic Analysis of the F2 Population from the Cross rpl4a 3 rpl4d.

(A) Exon-intron organization and schematic representation of the locations of the T-DNA insertions in the RPL4A and RPL4D genes. Orange, exon;

purple, intron; red, untranslated regions.

(B) RPL4 transcripts detected in 2-week-old seedlings in wild-type (Col-0) and rpl4a and rpl4d mutant alleles using RT-PCR. The positions of the

oligonucleotides used for the RT-PCR (F and R) are indicated with arrows in (A).

(C) Genotypes and segregation analysis of the F2 individuals from the cross rpl4a-23 rpl4d-1 (rpl4a3 rpl4d). The possible genotypic F2 combinations

derived from the rpl4a3 rpl4d cross are represented with green and red bars. Green bars indicate RPL4 genomic copies without T-DNA insertions. Red

bars with black triangles indicate RPL4 genomic copies containing T-DNA insertions.


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rpl4a and rpl4dmutants. Surprisingly, F1 plants from the rpl4a3rpl4d cross that were heterozygous for both recessive mutations

displayed similar morphological defects as the single mutants,

with one exception: cleared siliques of either of the single rpl4

mutants were normal, but the double heterozygous plants

showed empty spaces in the siliques, indicating embryo lethality

of;30% of expected seeds (see Supplemental Figure 5 online).

To confirm this result, phenotypic and genotypic analyses of 144

segregant F2 plants from the rpl4a 3 rpl4d cross without pfl

phenotype and 144 segregant F2 plants with pfl phenotype were

completed (Figure 3C). The analysis of the plants without phe-

notype resulted in 36 wild-type plants and 108 plants with a

T-DNA insertion in one of the four RPL4 copies, providing the

expected segregation ratio. However, all 144 plants that dis-

played the pfl phenotype had T-DNA mutations in two RPL4

copies and significant segregation distortions (x2 >16.75, P <

0.005) were observed. In this random population, we did not

identify plants with insertions inmore than twoRPL4 copies. This

result suggests that two active copies of RPL4, independent of

their identities, define the minimum threshold for plant viability.

The rpl4Mutants Have Altered Ribosomal Functions

In Escherichia coli, multiple defects in translation associatedwith

altered ribosomal protein L4 activity (the homolog of the Arabi-

dopsis RPL4s) have been shown (O’Connor et al., 2004). In

Arabidopsis, r-protein mutants, such as stv1/rpl24b (Nishimura

et al., 2005), rpl5a, and rpl9 (Yao et al., 2008), and the three

piggybackmutants (Pinon et al., 2008) have translational defects

due to their aberrant ribosomal function. To test whether the

Arabidopsis rpl4mutations compromised ribosomal function in a

similar way to their bacterial counterpart and other Arabidopsis

r-proteins, we used an in vivo protoplast transient expression

assay. In this assay, various green fluorescent protein (GFP)

fusion proteins under the control of the 35S cauliflower mosaic

virus promoter were expressed in protoplasts isolated fromwild-

type and rpl4 seedlings, and the levels of accumulation of

different GFP-fused proteins were evaluated temporally using

anti-GFP antibodies.

As shown in Figure 4A, the levels of GFP accumulation using a

cytosolic 35S:GFP construct were reduced in rpl4a and rpl4d

when compared with the wild-type protoplast 6 h after transfor-

mation. Those differences were reduced when longer incubation

times were analyzed (9 and 12 h), until near-wild-type levels of

accumulationwere observed after 18 h in rpl4a. In our conditions,

rpl4d showed a slightly lower GFP accumulation than rpl4a in all

time points likely due to the residual RPL4A expression of the

rpl4a mutant allele (Figure 2B).

Because we determined that the cytosolic GFP levels after 6 h

were different in wild-type and rpl4 protoplasts, we tested

whether this reduction was a general effect or specific for

cytosolic proteins. As shown in Figure 4B, the levels of accumu-

lation of different GFP fusion proteins, which included proteins

destined for different compartments such as the ER, cytoplasm,

chloroplast, and vacuole, were reduced in both rpl4 mutants

compared with the control. These results suggest that indepen-

dently of their subcellular localization, the rate of protein synthe-

sis was reduced in rpl4 protoplasts; hence, it takes longer to

reach the same protein levels in rpl4 mutants relative to the wild

type. However, we cannot exclude the possibility that the differ-

ential protein accumulation observed after 6 h was due to

transcriptional differences or enhanced degradation of newly

synthesized polypeptides in rpl4 backgrounds.

Once we determined that rpl4 mutations cause protein syn-

thesis defects, we evaluated whether the altered rpl4 ribosomal

function was due to either a structural defect in fully assembled

ribosomes or to the lack of ribosomes. For that purpose, we

analyzed rpl4 responses against an array of antibiotics with

known ribosomal targeting locations in prokaryotes (see Sup-

plemental Table 3 online). The rpl4 mutants did not display

increased sensitivity to any tested compounds, including the

eukaryotic protein synthesis inhibitor cycloheximide, which

binds the eukaryotic ribosomal complexes in a stoichiometic

manner (Oleinick, 1977). Moreover, rpl4 mutants displayed mild

resistance to chloramphenicol and erythromycin in shoots (Fig-

ure 4C) and streptomycin in roots and isolated protoplasts

(Figures 4D and 4E). The lack of general resistance or hypersen-

sitivity to antibiotics or cycloheximide in rpl4 suggested that the

total number of ribosomeswas similar to that in thewild type, and

the resistance for specific antibiotics suggested that the protein

synthesis problems in the rpl4 background were due to the

presence of an aberrant population of ribosomes unable to bind

properly to specific antibiotics. Interestingly, the presence of

aberrant ribosomes in rpl4 did not trigger an enhanced unfolded

protein response as demonstrated by the similar expression of

ER resident chaperones at the transcriptional and translational

levels (see Supplemental Figure 6 online) and the similar sensi-

tivity to tunicamycin (see Supplemental Table 3 online) of the rpl4

mutants compared with wild-type plants.

The rpl4Mutants Activate Compensatory Mechanisms at

the Protein Level

Because the RPL4 family is composed of two coexpressed

members, we hypothesized that the general protein accumula-

tion defects in rpl4 might be partially buffered by compensatory

mechanisms at the protein level. To test this hypothesis, we

checked the levels of total RPL4 protein in both rpl4a and rpl4d

single mutant backgrounds compared with the wild type. In this

assay, the RPL4 proteins were indistinguishable due to high

homology at the amino acid sequence level, so the observed

signals using human antiRPL4-specific antibodies accounted for

the contributions of both members. As shown in Figure 5A,

upregulation of the combined RPL4 proteins was observed in

both rpl4a and rpl4d mutants compared with the wild type.

Because our mutant lines were knockdowns, it was possible that

the lack of one member increased the expression of the other

family member to compensate functionally. To test this hypoth-

esis, liquid chromatography/tandem mass spectrometry (LC/

MS/MS) analysis was performed using protein extracts from

rpl4a and rpl4d 2-week-old seedlings. As shown in Figures 5B

and 5C, specific peptides for RPL4A and RPL4D were identified

by LC/MS/MS, and the ion chromatograms for the correspond-

ing peptides were quantified. Figure 5D shows the quantification

of the RPL4A- and RPL4D-specific peptides, which indicated

that the rpl4a mutant expressed more RPL4D protein and vice

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Figure 4. The rpl4 Mutations Reduce the Rate of Protein Synthesis and Cause Resistance to Specific Antibiotics.

(A) Immunoblots showing the GFP protein accumulation in wild-type and rpl4 backgrounds at different time points. Protoplasts were transformed with a

35S:GFP construct divided in four independent tubes and incubated at 228C in the light for different time periods (6 to18 h). Transformation efficiencies

were evaluated, and total proteins were separated by SDS-PAGE and normalized using Coomassie blue staining (load control lanes) prior to the

immunoblot analysis.

(B) Immunoblots showing the GFP protein accumulation in wild-type and rpl4 backgrounds 6 h after transformation. The protoplasts were transformed

with constructs containing proteins with different organelle specificity fused with GFP and normalized as in (A).


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versa. This compensation at the protein level in rpl4 backgrounds

correlated with an increased transcriptional activity of the alter-

native RPL4 protein and an increased transcriptional activity of

the r-protein S6 (see Supplemental Figure 6 online), which is

an important regulator of ribosome biogenesis in eukaryotes

(Volarevic et al., 2000).

Although the compensatory mechanisms in planta cannot fully

fulfill the requirements of RPL4protein dosage in specific tissues,

this mechanism could explain several observations derived from

our study: the relatively mild phenotypes observed in the adult

rpl4 plants (Figure 2E), the specificity of the secretion patterns for

certain tissues (Figure 1D), and the lack of differences between

the wild type and rpl4a when the protein levels of the vacuolar

trafficking machinery in planta were analyzed (see Supplemental

Figure 6 online).

RPL4A Transcription Is Regulated by Auxins

A model including a general protein accumulation defect in

metabolically active tissues is inadequate to explain the speci-

ficity of the auxin phenotypes in rpl4a. To link ribosomal function

and specific auxin responses, we analyzed whether auxins

mediate transcriptional regulation of theRPL4 promoters. Based

on the phenotypes observed in Figure 2, we expected that the

RPL4 promoter activity would be correlated with patterns of free

auxin distribution. To confirm our hypothesis, we characterized

theGUS staining patterns ofArabidopsis plants transformedwith

the RPL4Apro:GUS and RPL4Dpro:GUS reporter gene con-

structs. As shown in Figure 6A and Supplemental Figures 4A to

4D online, plants transformed with the RPL4Apro:GUS and

RPL4Dpro:GUS constructs showed very similar staining distri-

bution that strongly resembled the auxin distribution patterns

throughout the Arabidopsis development described by Teale

et al. (2006). Briefly, RPL4Apro:GUS expression was strong

during early stages of primordia development, and leaf apical

dominance was evident in the elongating tip (Figure 6A, primor-

dial leaf 1 and leaf 2). In later stages of leaf development, GUS

expression progressed basipetally along the margins (Figure 6A,

leaf 3), until its final appearance in the central regions of the

lamina (Figure 6A, leaf 4), and in a more diffuse fashion in mature

leaf mesophyll cells (Figure 6A, leaf 5). In main and lateral roots,

RPL4Apro:GUS expression also correlated with auxin distribu-

tion patterns. In themain root,RPL4Apro:GUS stainingwasmore

intense toward the root tip and more diffuse in the epidermis

(Figure 6B). In lateral roots, theGUSactivity started from the stele

to the new root tip and then continued through the epidermis

(Figure 6C). Finally, GUS staining was also observed in second-

ary sites of free auxin production, such as the seed endosperm

cap, stigma, stamen, and pollen (Figure 6D to 6F). These results

suggested that ribosome biogenesis and auxin biosynthesis and

transport are tightly linked processes. To confirm further that this

linkage exists, we tested whether exogenously applied auxins

alter the expression of the RPL4Apro:GUS lines. As shown in

Supplemental Figure 4E online, exogenous applications of

auxins at physiological levels (10 mM concentrations for 24 h)

caused a general decrease in the RPL4A promoter activity in the

root meristem. Although this result suggested that auxins regu-

late RPL4A expression, no strong conclusions about the relative

sensitivity of the RPL4A promoter to different auxins could be

reached. To determine whether the different auxin treatments

were equivalent, exogenous applications of higher hormonal

concentrations (50 mM concentrations for 4 h) were used. As

shown in Figure 6G, the application of the natural auxin indole-3-

acetic acid (IAA), as well as the same concentration of the auxin

transport inhibitor 1-naphthylphthalamic acid (NPA), completely

abolished the RPL4A promoter activity in the root meristem and

root elongation zone without modifying the expression patterns

in the vasculature of fully developed roots. The same concentra-

tions and incubation times using 1-naphthaleneacetic acid (NAA),

2,4-D, and the auxin transport inhibitor 2,3,5-triiodobenzoic acid

caused a moderate decrease in GUS activity in root meristems,

suggesting that the promoter response to those treatments was

weaker (Figure 6G). As a positive control of induction, we evalu-

ated the effect of the similar treatments using the auxin inducible

DR5pro:GUS transgenic line (Ulmasov et al., 1997). As shown in

Figure 6H, DR5pro:GUS expression was induced by auxins;

however, no differences in induction were observed among the

NAA, IAA or 2,4-D treatments. In our conditions, no repression of

DR5pro:GUS was observed in the 2,3,5-triiodobenzoic acid and

NPA treatments compared with nontreated plants. Based on

these results, we concluded that auxins regulate ribosomal bio-

genesis in root meristems and that the RPL4 promoter is more

sensitive to IAA and NPA treatments.

Different Vacuolar Sorting Pathways Are Affected byAuxins

and rpl4a

Since IAA and NPA treatments (50 mM concentrations for 4 h)

were able to completely abolish the RPL4A promoter activity in

root meristems, we hypothesized that those treatments applied

to wild-type plants might mimic the vacuolar sorting defects

caused by the rpl4amutation. As shown in Figures 7A and 7B, the

IAA and NPA treatments caused large amounts of secretion of

both RFP:CTPPpha and NTPPpro:RFP fluorescent markers

Figure 4. (continued).

(C) rpl4mutants shoots are resistant to erythromycin and chloramphenicol. Wild-type and rpl4 seeds were surface sterilized and directly germinated on

plates with or without antibiotic. Plates were incubated horizontally in a growth chamber under long-day conditions. An independent kanamycin-

resistant line SALK_069239 was used as a negative control to evaluate antibiotic cross-resistance.

(D) rpl4mutants roots are resistant to streptomycin. The assay was performed as in (C), but the plates were incubated vertically and pictures were taken

7 d after sowing.

(E) Transient expression assay in streptomycin-treated protoplasts. The GFP protein was expressed in protoplasts for 6 h, followed by a treatment with

0 and 600 mg/mL of antibiotic for 12 additional hours.

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restricted to the root meristems and the elongation zone of the

root. Since the hormonal treatments were not specific for either

RFP:CTPPpha or NTPPpro:RFP markers, we hypothesized that

RPL4A acts in a regulatory mechanism that affects vacuolar

trafficking generally, not just markers containing C- or N-terminal

propeptides. To test that hypothesis, we analyzed the vacuolar

transport of the auxin-efflux carrier PIN2, a protein without

vacuolar sorting signals that is targeted for vacuolar degradation

after darkness treatments (Kleine-Vehn et al., 2008). As shown in

Figure 7C, dark-incubated wild-type PIN2:GFP plants displayed

fluorescent signals in the plasma membrane, endosomes, and

vacuole; however, the combined treatment of darkness and IAA

in wild-type plants restricted the PIN2:GFP fluorescent signal to

the plasma membrane (Figure 7D). This result is in accordance

with previous studies that demonstrated that PIN2 endocytosis,

and, therefore, the delivery of the PIN2:GFP marker to the

Figure 5. The rpl4 Mutants Increase the Expression of the Alternative RPL4 Protein.

(A) Immunoblot of RPL4 using human RPL4 polyclonal antibodies. Total protein from 2-week-old seedlings were extracted, separated by SDS-PAGE,

and normalized using Coomassie blue staining (load control lanes). Immunoblots with BiP in the same samples were used to confirm equal loading.

(B) Two proteotypic peptides used for the differentiation of the RPL4A and RPL4D proteins. The amino acid differences between RPL4A and RPL4D

peptides are indicated with a larger font.

(C) Mass spectra of two proteotypic peptides used in the differentiation of RPL4A and RPL4D proteins.

(D) LC/MS quantitative analysis of RPL4A and RPL4D proteotypic peptides showing the relative abundance of the two proteins in wild-type and rpl4

backgrounds. Light-gray shading, rpl4a mutant; dark-gray shading, rpl4d mutant; no shading, Col.


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vacuole, was inhibited by treatments with NAA (Paciorek et al.,

2005). Once we confirmed that IAA was also able to inhibit the

release of the PIN2:GFP marker to the vacuole, we crossed the

PIN2:GFP line with rpl4a and analyzed the vacuolar transport

of PIN2:GFP in rpl4a backgrounds and isogenic wild-type lines.

As shown in Figure 7E, the PIN2:GFP fluorescent signal of

dark-treated wild-type plants grown in solid media appeared in

plasma membrane, endosomes, and vacuole. However, the

PIN2:GFP signal of isogenic rpl4a plants was similar at the

plasma membrane but showed a marked decrease in the vac-

uole (Figure 7F). This result suggested that rpl4a mimics the

effect of exogenously applied IAA by partially inhibiting the

vacuolar delivery of PIN2:GFP. The partial inhibition of the PIN2:

GFP vacuolar delivery in rpl4a correlates with the partial secre-

tion of the RFP:CTPPpha and NTPPpro:RFP markers and sug-

gests that the trafficking of vacuolar targeted proteins is delayed

but not totally abolished in rpl4a. Finally, to analyze directly the

kinetics of trafficking to the vacuole, we analyzed the vacuolar

processing of a marker containing the Barley lectin vacuolar

propeptide fused with GFP (GFP:CTPPBL) in transformed wild-

type and rpl4a protoplasts. As shown in Figure 7G, 24 h after

transformation, a shift in the molecular weight of the GFP:

CTPPBL marker was observed in wild-type but not rpl4a proto-

plasts. This shift is due to the vacuolar processing of the BL

propeptide in wild-type plants. Forty-eight hours after transfor-

mation, rpl4a protoplasts started to process the marker; how-

ever, the ratio of nonprocessed versus processed GFP:CTPPBL

was higher in rpl4a than in wild-type protoplasts (Figure 7H). This

result confirms that trafficking toward the vacuole was strongly

delayed in rpl4a.

DISCUSSION

The results provided by the characterization of the ribosomal

rpl4a mutant isolated in this screen demonstrate that the Vac2

T-DNA collection is an excellent tool to describe mechanisms of

transport toward the plant vacuole. RPL4A belongs to a totally

different group of proteins compared with the ones identified

using ethyl methanesulfonate mutagenesis or genetic crosses of

the Vac2 line (Sanmartın et al., 2007; Sohn et al., 2007), demon-

strating that the Vac2T-DNA collection can identify unknown com-

ponents of plant vacuolar trafficking pathways. The described

Figure 6. The RPL4 Expression Correlates with Patterns of Free Auxin

Distribution, and It Is Regulated by Auxins.

(A) to (F) GUS activity assay in transgenic plants harboring the RPL4A

promoter-GUS fusion.

(A) Whole plant showing GUS staining in leaves at different develop-

mental stages (1 to 5).

(B) Root tip and root vasculature.

(C) Water-embedded seeds.

(D) Lateral root.

(E) Anthers and pollen grains.

(F) Stigma and petals.

(G) GUS activity assay in the presence of auxin transport inhibitors and

auxins in RPL4Apro:GUS transgenic plants. TIBA, 2,3,5-triiodobenzoic

acid.

(H) The auxin-inducible DR5pro:GUS transgenic lines were used as a

control.

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function of RPL4A links vacuolar protein sorting with ribosomal

biogenesis and provides new insights into the auxin-mediated

regulation of vacuolar trafficking in metabolically active tissues.

The Role of Ribosomal Biogenesis in

Auxin-Related Responses

Ribosomes are responsible for protein translation in eukaryotic

cells, and their biogenesis is a complex process involving the

coordination of many nonribosomal and ribosomal proteins. Pre-

vious reports have described individual mutations in r-proteins

that cause specific auxin-related phenotypes, and different

models have been proposed to explain the specificity of the

observed phenotypes. It has been shown that mutations in

SHORT VALVE1 (STV1), which encodes an L24 r-protein and

causes carpel tissue patterning defects, regulates the translation

of the auxin response factors ETTIN and MONOPTEROS via

translation of short upstream open reading frames (Nishimura

et al., 2005). The defects in the adaxial-abaxial polarity in the

ribosomal piggyback mutants have been proposed to be due to

defects in the HD-ZIPIII-KANADI pathway in a process regulated

by the specific targeting of small RNAs to HD-ZIPIII- KANADI

elements (Pinon et al., 2008). Accordingly, a model of the

ribosome function in the leaf patterning regulatory network has

been proposed by Yao et al. (2008). In this model, the ribosome

genetically promotes the HD-ZIPIII–mediated pathway in the

adaxial domain of leaves or genetically represses the auxin-

responsive genes ARF3/4, KAN, or their downstream genes.

However, none of the models directly applied to individual

r-proteins properly address a broader question: How does the

protein synthesis machinery affect specific auxin responses? To

answer this question, it has been proposed that different iso-

forms and posttranslational modifications of r-proteins, resulting

in ribosome heterogeneity, may generate functionally distinct

ribosomes with target-transcript specificity (Giavalisco et al.,

2005; Carroll et al., 2008; Komili et al., 2007). Indeed, the similar

expression patterns of both RPL4 proteins found in this study

Figure 7. Different Vacuolar Sorting Pathways Are Affected by Auxins

and RPL4A.

(A) and (B) Exogenous applications of IAA and NPA cause secretion of

vacuolar markers containing C-terminal VSDs (A) and N-terminal VSDs

(B). Transgenic plants harboring the RFP:CTPPpha and NTPPpro:RFP

markers were incubated in liquid media (4 h) supplemented with DMSO

or 50 mM solutions of IAA or NPA. The RFP fluorescence was analyzed

using scanning fluorescent confocal microscopy. All the images shown

were acquired using the same settings.

(C) to (F) Vacuolar sorting of the PIN2:GFP marker in the dark.

(C) and (D) PIN2:GFP localization at the plasma membrane and vacuolar

compartments in dark-treated (6 h) wild-type epidermal root cells in the

absence (C) or presence (D) of IAA. IAA or DMSOwas added in darkness

2 h before the end of the 6-h dark treatments.

(E) and (F) PIN2:GFP localization at the plasma membrane and vacuolar

compartments in dark-treated (6 h) wild-type (E) or rpl4a (F) root

meristems. All the images shown were acquired using the same settings.

(G) Processing of the vacuolar targeted marker GFP:CTPPBL in wild-type

and rpl4a protoplasts. Protein extracts from GFP:CTPPBL transformed

protoplasts were prepared 24 or 48 h after transformation, and the

vacuolar processing was analyzed by immunoblots using anti-GFP

antibodies.


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support that heterogeneous ribosomal populations are present

in the same tissues, but also, the similarity of both rpl4 mutant

phenotypes suggest that RPL4A and RPL4D-containing ribo-

somes are equivalent and do not account for the target transcript

specificity. In accordance with the equivalence of RPL4 ribo-

somal types,RPL4 gene dosage, and not the identity of the RPL4

proteins, is the key element forArabidopsis survival. In our study,

50% RPL4 gene dosage reductions caused similar phenotypes,

independent of RPL4 identity, and 75% RPL4 gene dosage

reductions caused lethality. Furthermore, the activation of com-

pensatory mechanisms that increased the protein levels of the

alternate RPL4 protein in mutant backgrounds suggest that

RPL4 ribosomal heterogeneity does not confer auxin specificity,

but acts as a backup mechanism to maintain basic cellular

functions when RPL4 availability is limited.

To explain the auxin specificity of the r-protein mutant re-

sponses, our study uses a global analysis of the phenotypes

derived from ribosomal mutations (see Supplemental Table 2

online). Since most of the r-protein mutant phenotypes are

similar, we propose that those phenotypes are downstream

effects of a failure in a common regulatory mechanism for all

r-proteins, the auxin-mediated ribosomal biogenesis. At least

four independent lines of evidence were provided to support this

model. First, as indicated above, except for variations in the

severity of the phenotypes, and rare specific phenotypes that

could be associated with extraribosomal functions (Wool, 1996),

several independent r-protein mutants described in the literature

display very similar auxin-related phenotypes (see Supplemental

Table 2 online). Second, parl1, a mutant in a nucleolin that

globally affects ribosomal biogenesis (Petricka and Nelson,

2007) displays auxin-related phenotypes similar to those of rpl4

mutants. Third, the transcriptional levels of RPL4, and presum-

ably other coexpressed r-proteins, can be modified by direct

application of auxins and auxin transport inhibitors in certain

tissues. Finally, our general model predicts that alterations in the

auxin-mediated ribosomal biogenesis might regulate auxin-

related processes not previously reported, for example, the

sorting of vacuolar proteins (see below).

Role of Ribosomal Biogenesis in the Vacuolar Sorting

of Proteins

rpl4a was initially isolated in a screen for mutants with altered

protein sorting to the vacuole, and we hypothesize that the

mis-sorting is caused by an imbalance in the regulation of the

auxin-mediated ribosomal biogenesis. To illustrate the role of

ribosomal biogenesis in vacuolar trafficking in Arabidopsis, we

will use a process known to be regulated by auxins and vacuolar

trafficking elements, leaf vascular development. Leaf veins are

known to form by differentiation of vascular cells from ground

meristem cells in a process regulated by the polar flow of auxins

(Galweiler et al., 1998). In this context, the PVC and vacuoles

have recently been reported to function in the regulation of the

polarized transport and recycling of the auxin efflux carriers PIN1

and PIN2 (Kleine-Vehn et al., 2008; Laxmi et al., 2008; Shirakawa

et al., 2009; Spitzer et al., 2009). Severalmutants belonging to the

PVC-to-vacuole pathway, including vti11 vti12/+, vps9a, and

vam3, have been reported to have defects in protein trafficking

and vascular network formation (Shirakawa et al., 2009). VPS9A

is the homolog of yeast VPS9, which is involved in vacuolar

sorting, the vti11 vti12/+mutant has defects in the sorting of seed

storage protein (Goh et al., 2007, Sanmartın et al., 2007), and

vam3belongs to the vacuolar SNARE (solubleN-ethylmaleimide-

sensitive factor attachment protein receptor) complex and is

reported to be localized to the PVC and the vacuolar membrane

(Sato et al., 1997; Sanderfoot et al., 1999, 2001). Since we have

Figure 8. Model: Ribosomal Biogenesis Affects the Sorting of Vacuolar

Proteins in a Process Regulated by Auxins.

Local auxin accumulations cause the transcriptional repression of ribo-

somal proteins (1), followed by the translational repression of auxin-

responsive factors (such as HD-ZIPIII, KANADI, and ARF3/ARF4) (2).

r-protein mutations mimic the effect of exogenous auxin application by

decreasing the auxin-responsive factor synthesis rate (2). Whether the

decreased protein synthesis rate acts as a feedback mechanism that

represses auxin biosynthetic genes remain unclear (3). The translational

repression of auxin-responsive factors slows down the PVC-to-vacuole

delivery of vacuolar sorted markers, such as CTPP:RFPpha, NTPPpro:

RFP, NTPPBL:GFP, and PIN2:GFP, either directly or through PVC ele-

ments, such as VAM3, VPS9A, or VTI11 (4). The delayed delivery of the

CTPP:RFPpha and NTPPpro:RFP markers to the vacuole leads to their

partial secretion (5), whereas the delayed processing of PIN2:GFP (6)

likely modifies the auxin polar flows and causes the array of auxin-related

developmental phenotypes observed in rpl4 mutants (7). Black arrows:

normal delivery of CTPP:RFPpha, NTPPpro:RFP, and PIN2 to the vacuole

in wild-type Arabidopsis. Dashed arrows: effects of local auxin accumu-

lations or rpl4a backgrounds in vacuolar trafficking.

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shown that the delivery of vacuolar proteins is delayed in rpl4a, it

is not surprising that the vascular defects observed in mutants

with altered PVC-to-vacuole trafficking resemble the pheno-

types observed in many r-protein mutants and particularly rpl4.

Interestingly, the explanatory models are highly complementary.

From the vacuolar trafficking perspective, the defects in protein

trafficking caused by the PVC-to-vacuole mutants modify the

distribution of auxin. This is achieved by regulating the localiza-

tion of auxin carriers (PINs), which in turn, determines where

procambium cells are located in the leaf primordium (Shirakawa

et al., 2009). In our model (Figure 8), auxin-dependent ribosomal

biogenesis affects the sorting of vacuolar targeted proteins, as

demonstrated by the delayed processing of the vacuolar tar-

geted protein GFP:CTPPBL, the secretion of the RFP:CTPPpha

and NTPPpro:RFP markers, and the delayed processing of PIN2

in the rpl4 backgrounds. Thus, in specific tissues, auxin accu-

mulations cause the transcriptional repression of r-proteins,

followed by the translational repression of auxin-responsive

factors, such as HD-ZIPIII, KANADI, and ARF3/ARF4, regulated

by the r-proteins (Nishimura et al., 2005; Pinon et al., 2008; Yao

et al., 2008). As a consequence, downstream elements affecting

the sorting of vacuolar targeted proteins are repressed, and the

vacuolar delivery of the CTPP:RFPpha, NTPPpro:RFP, NTPPBL:

GFP, and PIN2:GFP markers is delayed. The delayed delivery of

theCTPP:RFPpha andNTPPpro:RFPmarkers to the vacuole leads

to their partial secretion; meanwhile, the delayed processing of

PIN2 likely modifies the auxin polar flow required for the forma-

tion of the vascular network. In this way, the delayed vacuolar

degradation of PIN2, or other PIN proteins, might explain the

vascular defects and other auxin-related developmental pheno-

types observed in the rpl4 mutants.

In conclusion, we speculate that auxin-dependent ribosomal

biogenesis is an active part of a previously unknown mechanism

regulating the proper delivery of vacuolar targeted proteins (such

as PIN2) to the vacuole. Using this mechanism, ribosomal

biogenesis might regulate auxin fluxes and influence auxin-

related developmental processes. Whether this regulation oc-

curs through general mechanisms dependent on protein and

lipid synthesis, for example, of membrane sterols (Pan et al.,

2009), or through the inhibition of specific PVC elements, such as

VPS9A, VAM3, or VTI11, as suggested by the similarities in the

vascular defects when compared with rpl4 mutants is still un-

clear, and it is the focus of our current research.

METHODS

Plant Materials, Insertion Identification, and Growth Conditions

The Arabidopsis thaliana Vac2 T-DNA insertion lines in the Landsberg

background used in the screen were a gift from Ray A. Bressan (Koiwa

et al., 2006). The mutant screen was performed as described (Sohn et al.,

2007), and the DNA flanking the left border of the inserted T-DNA was

isolated by thermal asymmetric interlaced-PCR (Liu et al., 1995; Koiwa

et al., 2002). The NTPPpro:RFP and RFP:CTPPpha vacuolar markers

(Hunter et al., 2007) were a gift from Lorenzo Frigerio. For histochemical

analysis of the RPL4pro:GUS expression, the DR5pro:GUS transgenic

line (Ulmasov et al., 1997) was used as a control. The Arabidopsis Col-0

T-DNA insertion mutants SALK_130595 (rpl4a-2), SALK_063782 (rpl4a-3),

SALK_029203 (rpl4d-1), and SALK_065625 (rpl4d-2) and the control line

SALK_069239 were obtained from the ABRC stock center. The amplified

fragments in the 21-4 mutant were sequenced, and genetic cosegrega-

tion analysis was performed using an F2 population created by back-

crossing 21-4 to Vac2. Primer sets used are shown in Supplemental Table

4 online. For growth assays, surface-sterilized and cold-stratified Arabi-

dopsis seeds were sown onto half-strength Murashige and Skoog

phytoagar medium (0.53 Murashige and Skoog salts, 10 g/L sucrose,

and 7 g/L phytoagar, pH 5.7). Phytoagar plates containing sterilized seed

were incubated in an environmental chamber that was set for long-day

lighting conditions (16 h light/8 h dark) and a temperature of 228C. For

antibiotic treatments, the appropriate amounts of filter sterilized antibiotic

stock solutions were added to cooled autoclaved growth media. The

seeds were directly germinated on the antibiotic-containing plates, and

the plates were incubated horizontally for shoot analysis or vertically for

root elongation assays for up to 21 d.

RNA Isolation and Transcript Analyses

Total RNAwas prepared from2-week-old Col-0, rpl4a-2, rpl4a-3, rpl4d-1,

and rpl4d-2 Arabidopsis plantlets grown in sterile conditions using the

RNeasy plant mini kit (Qiagen), and cDNA was synthesized with the

Superscript III cDNA synthesis kit (Invitrogen) applying anchored-oligo

(dT)18 primer with equalized amounts of template RNA. PCR (30, 35, and

40 cycles) were performed with the cDNAs and ExTaq polymerase

(TaKaRa), and products were visualized with ethidium bromide staining.

For quantitative PCRs, SYBRGreen Supermix (Bio-Rad) was used in

reaction volume of 25 mL. Reactions were run on an iQ5 thermocycler

(Bio-Rad). For relative quantification, ratios were calculated according to

the method of Pfaffl (2001). All Ct values were the average of three

replicates, and all treatments were subject to three biological replicates.

Primer sets used are shown in Supplemental Table 4 online.

RPL4pro:GUS Constructs and Histochemical Analysis

To obtain theRPL4A andRPL4Dpromoter:GUS-fusions, 1.7 and 1.5 kb of

the genomic sequence upstream of the RPL4A and RPL4D translation

start sites were cloned using the Gateway recombination cloning technol-

ogy (Invitrogen) into the binary vector PMDC162 (Curtis and Grossniklaus,

2003) and transformed into Agrobacterium tumefaciens strain GV3101.

The primer sets used are shown in Supplemental Table 4 online. Wild-

type Col-0 plants were transformed using floral dipping, and primary

transformants were isolated using hygromycin as a selective marker.

Eight independent homozygous lines were isolated for each construct.

For hormonal treatments, the transgenic RPL4Apro:GUS and DR5pro:

GUS lines were incubated in liquid media supplemented with 50 mM

solutions of each compound for 4 h, andGUS activity was detected in situ

as described (Jefferson et al., 1987).

Microscopy

For scanning electron microscopy of adaxial and abaxial leaf surfaces,

fresh leaves were mounted on scanning electron microscopy stubs, flash

frozen in liquid air, and imaged in a Hitachi TM-1000 scanning electron

microscope at an accelerating voltage of 5 kV. Confocal images of water-

mounted seedlings were collected using a Leica TCS SP2/UV fitted with

320 or 363 water immersion objectives. A 543-nm line from a He-Ne

laser was used to excite RFP, and fluorescence was detected in the 560-

to 640-nm range. A 488-nm line from an argon laser was used to excite

GFP, and fluorescence was detected in the 500- to 530-nm range. For

analysis of leaf vasculature, leaf 5 of 28-d-old plants were fixed in 3%

glutaraldehyde, dehydrated through an ethanol series to 100% ethanol,

and embedded in JB4 resin (Agar Scientific). Embedded tissue was

sectioned at 3 mm and subsequently stained with 0.02% Toluidine Blue.


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Images were obtained using a Nikon E800 microscope. For transmission

electron microscopy, 5-d-old Vac2 and rpl4a-1 root tips were dissected

out, transferred to a B-type aluminum planchette (Technotrade Interna-

tional) containing a 150 mM sucrose solution, and frozen with a HPM 100

high-pressure freezer (Leica). The frozen root tipswere freeze-substituted

in acetone containing 2% OsO4 at 2808C for 4 d. After substitution, the

sample temperaturewas slowly warmed up to2208Cover 24 h, from220

to 48C over 12 h, and from 48C to room temperature over 4 h. The root tips

werewashed three timeswith anhydrous acetone and embedded in Epon

resin (Ted Pella). After polymerization, samples were sliced into 80-nm

sections and stained with uranylacetate solution (2% w/v) and subse-

quently with lead citrate solution (26 g/L lead nitrate and 35g/L sodium

citrate). Electron micrographs were captured with a Hitachi TEM H-7000

operated at 80 kV.

Proteomic Analysis

Total protein extracts purified from 2-week-old Arabidopsis seedlings

were separated by SDS-PAGE. The gels were blotted onto nitrocellulose,

and immunoblot analysis was performed using human RPL4-specific

antibodies (Proteintech Group). The protein amounts were normalized

using Coomassie Brilliant Blue staining and AntiBiP antibodies (Rose

Biotechnology). Protein bands corresponding to the immunoblot signal

were excised and prepared as previously described (Carter et al., 2004).

Proteotypic peptides ions for RPL4A and RPL4D were selected based on

the criteria described by Baerenfaller et al. (2008) for the inclusion list to

perform targeted data-dependent acquisition analysis using a nanoLC/

MS/MS system with combination of the nano-Acquity UPLC and Q-TOF

Premier (Waters). After peptide sequenceswere confirmed by the tandem

mass spectra, the relative abundance of RPL4A and RPL4D in different

backgrounds was evaluated using LC/MS quantitative analysis as pre-

viously described (Rojo et al., 2004).

Transient Expression Analysis and in Vivo Translational Assays

in Protoplasts

Protoplasts were isolated from 2-week-old seedlings grown on B5 agar

media, and the preparations were transformed with 10 mg of the BiP:GFP

(Kim et al., 2001), RbcS:GFP (Lee et al., 2002), AALP:GFP (Sohn et al.,

2003), and GFP constructs using a polyethylene glycol–mediated proce-

dure previously described (Jin et al., 2001). Equal transformation effi-

ciencies were ensured measuring the ratio transformed:nontransformed

protoplast using confocal microscopy. For the streptomycin-treated

protoplasts, the GFP protein was expressed in protoplasts for 6 h,

followed by a treatment with 0 and 600 mg/mL of antibiotic for 12

additional hours. Protein extracts from protoplasts derived from the

different assays were prepared at different time points after transforma-

tion as described (Sohn et al., 2003). Briefly, protoplast incubation media

was collected after centrifugation at 6000g for 5 min. One milligram of

BSA was added to the media, which was then precipitated with 10%

trichloroacetic acid and followed by centrifugation at 10,000g for 5 min.

Precipitated protein aggregates were dissolved in 0.1 M NaOH. Prior to

the immunoblot analysis, the protoplast preparations were normalized

measuring total protein contents. Immunoblots were developed using a

Living Colors GFPMonoclonal antibody (Clontech) and an ECL detection

kit (Pierce Biotechnology).

Accession Numbers

Sequence data from this article can be found in the Arabidopsis Genome

Initiative or GenBank/EMBL databases under the following accession

numbers: RPL4A, locus At3g09630 cDNA, NM_001035586; protein,

NP_001030663; RPL4D, locus At5g02870 cDNA NM_001125687; pro-

tein, NP_001119159. T-DNA insertion lines SALK_130595 (rpl4a-2),

SALK_063782 (rpl4a-3), SALK_029203 (rpl4d-1), SALK_065625 (rpl4d-

2), and SALK_069239 (negative control for the antibiotic assays).

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure 1. The Secretion of the Vacuolar Markers in

rpl4a-1 Depends on Their Expression Levels and Developmental

Stage.

Supplemental Figure 2. Cellular Localization of Auxin Transporters in

rpl4a-1.

Supplemental Figure 3. The rpl4a and rpl4d Mutants Display Similar

Developmental Phenotypes.

Supplemental Figure 4. Histochemical Analysis of RPL4pro:GUS

Transgenic Lines.

Supplemental Figure 5. Double Heterozygous rpl4a-rpl4d Plants

Have Similar Phenotypes as the Single Mutants.

Supplemental Figure 6. The rpl4a Mutation Does Not Cause En-

hanced Unfolded Protein Response or Translational Defects in Vac-

uolar Trafficking Machinery.

Supplemental Table 1. Genetic Linkage Analysis Indicates That the

21-4 Phenotypes and the Protein Trafficking Defects Are Linked to the

Mutation in RPL4A.

Supplemental Table 2. Auxin-Related Phenotypes in Arabidopsis

Mutants with Altered Ribosome Biogenesis.

Supplemental Table 3. Specificity of the Antibiotic Response in rpl4

Mutants.

Supplemental Table 4. Primers Used in This Study.

ACKNOWLEDGMENTS

We thank Glenn Hicks, Marci Surpin, Julia Bailey-Serres, and Patty

Springer (University of California, Riverside) for their critical reading of

the manuscript. We also thank Mien Van De Ven, April Agee, Latasha

Johnson, and Alexander Michkov for technical assistance, L. Frigerio

(University of Warwick, UK) for the gift of the NTPPpro:RFP and the RFP:

CTPPpha marker lines, and I. Hwang (Pohang University, Korea) for the

gift of the BiP:GFP, GFP, RbcS:GFP, and AALP:GFP constructs. We

thank the anonymous reviewers for their useful comments and sugges-

tions. This work was funded by Department of Energy, Division of

Energy Biosciences, Grant DE-FG03-02ER15295/A000 (to N.V.R.) and

by the Spanish Fulbright postdoctoral fellowship MEC-FU-0248-2006

(to A.R.).

Received April 29, 2009; revised December 7, 2009; accepted December

19, 2009; published January 8, 2010.

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