The Arabidopsis SUMO E3 ligase SIZ1 controls

phosphate deficiency responses

Kenji Miura*, Ana Rus*, Altanbadralt Sharkhuu*, Shuji Yokoi*, Athikkattuvalasu S. Karthikeyan†,
Kashchandra G. Raghothama†, Dongwon Baek‡, Yoon Duck Koo‡, Jing Bo Jin*, Ray A. Bressan*,
Dae-Jin Yun*‡§, and Paul M. Hasegawa*
*Center for Plant Environmental Stress Physiology and †Department of Horticulture and Landscape Architecture, Purdue University,
West Lafayette, IN 47907; and ‡Environmental Biotechnology National Core Research Center and Division of Applied Life Science (BK21 program),
Graduate School of Gyeongsang National University, Jinju 660-701, Korea

Edited by Maarten J. Chrispeels, University of California at San Diego, La Jolla, CA, and approved April 5, 2005 (received for review February 2, 2005)

Plants sense phosphate (Pi) deficiency and initiate signaling that Pht2;1 transports Pi into chloroplasts and facilitates in planta Pi
controls adaptive responses necessary for Pi acquisition. Herein, distribution (12).
evidence establishes that AtSIZ1 is a plant small ubiquitin-like The molecular mechanisms controlling Pi starvation sensing,
modifier (SUMO) E3 ligase and is a focal controller of Pi starvation- signaling, and associated target gene activation that result in
dependent responses. T-DNA insertional mutated alleles of AtSIZ1 induced metabolic and phenotypic changes remain largely un-
(At5g60410) cause Arabidopsis to exhibit exaggerated prototypical resolved. The MYB transcription factor PHR1 is the only known
Pi starvation responses, including cessation of primary root molecular determinant required for Pi starvation-dependent
growth, extensive lateral root and root hair development, increase responses (7). However, PHR1 controls a small subset of Pi
in root兾shoot mass ratio, and greater anthocyanin accumulation, starvation responses that apparently includes only a few genes
even though intracellular Pi levels in siz1 plants were similar to wild whose expression is activated by Pi limitation. Genes controlled
type. AtSIZ1 has SUMO E3 ligase activity in vitro, and immunoblot by PHR1 genes include AtACP5 (acid phosphatase), AtIPS1兾3,
analysis revealed that the protein sumoylation profile is impaired At4, and RNS1 (7, 13, 14). PHR1 binds to the P1BS (PHR1-
in siz1 plants. AtSIZ1-GFP was localized to nuclear foci. Steady- binding sequence) element that exists in the promoters of these
state transcript abundances of Pi starvation-responsive genes genes (7). To a small extent, phr1 plants respond to Pi limitation
AtPT2, AtPS2, and AtPS3 were moderate but clearly greater in siz1 by accumulating less anthocyanin, fresh weight mass, and intra-
seedlings than in wild type, where Pi is sufficient. Pi starvation cellular Pi, and they exhibit a lower root兾shoot ratio than the wild
induced the expression of these genes to the same extent in siz1 type (7). Other root architecture modifications typically associ-
and wild-type seedlings. However, two other Pi starvation-respon- ated with Pi deficiency, such as increased root hair number and
sive genes, AtIPS1 and AtRNS1, are induced more slowly in siz1 length, are unaffected by the phr1 mutation (7). Together, the
seedlings by Pi limitation. PHR1, a MYB transcriptional activator of available data indicate that Pi starvation sensing and signal
AtIPS1 and AtRNS1, is an AtSIZ1 sumoylation target. These results control are complex and involve numerous molecular determi-
indicate that AtSIZ1 is a SUMO E3 ligase and that sumoylation is a nants, many of which are not part of the PHR1 regulon and
control mechanism that acts both negatively and positively on remain to be discovered.
different Pi deficiency responses. Herein, we establish that mutations in AtSIZ1 enhance sen-
sitivity of Arabidopsis plants to Pi deprivation based on mor-
phosphate starvation response 兩 phosphate starvation signaling 兩 phological responses, including reduction in primary root elon-
sumoylation 兩 phosphorous gation and enhanced lateral root development, root兾shoot mass
ratio, and root hair number and development. Furthermore,
transcript abundance of some Pi starvation-responsive genes is
P hosphorous is a component of many important biological
compounds, and it is an essential macronutrient for all
organisms. However, acquisition by plants is problematic be-
greater in siz1 [SAP (scaffold attachment factor, acinus, protein
inhibitor of activated signal transducer and activator of tran-
scription) and Miz1 (Msx2-interacting zinc finger), SIZ] plants
cause phosphate (Pi) is unevenly distributed and relatively
than in the wild type under Pi sufficient conditions. Interestingly,
immobile in soils (1). Plants react to Pi limitation by activating
the induction rates of two genes (AtIPS1 and AtRNS1), whose
numerous adaptive responses that presumably facilitate acqui-
responses to Pi starvation are controlled by PHR1, are reduced
sition of this essential nutrient (2). These responses include
in siz1 plants compared with the wild type. Heat shock-induced
biochemical processes that limit metabolic requirements for Pi,
sumoylation is less in siz1 plants, and recombinant AtSIZ1 can
secretion of organic acids to the apoplast, and synthesis of
function as a small ubiquitin-like modifier (SUMO) E3 ligase to
enzymes that enable access to phosphorus contained in intra-
mediate sumoylation in vitro. SUMO E3 ligases transfer the
cellular organic molecules, insoluble complexes, and organo-
SUMO peptide to the substrate in vivo (15) through functions
phosphates in the soil. Pi limitation also causes morphological that increase the affinity of the conjugating enzyme (E2) subunit
responses that are presumed to be adaptive, including attenuated for a specific target, stabilize E2–substrate interaction, orient the
primary root growth, increased lateral root development, root兾 substrate acceptor K in the sumoylation motif (⌿KXE), and
shoot mass ratio, lateral root number and length (3), and contribute mechanistically to conjugation (15–17). Conjugation
root兾hair production (2). Pi deficiency also induces the expres- of the SUMO superfamily of proteins to a substrate (sumoyla-
sion of genes that facilitate Pi uptake into roots, distribution in tion) in both yeast and animals affects both positive and negative
planta, and acquisition from organic sources (2).
Several Arabidopsis genetic loci appear to function in Pi
signaling and acquisition. Mutations in the PHO3, PSR1, PDR2, This paper was submitted directly (Track II) to the PNAS office.
and PHR1 genes impair Pi starvation signaling (4–7), whereas Abbreviations: MS, Murashige and Skoog; Pi, phosphate; PIAS, protein inhibitor of acti-
pho1, pho2, and pup1 mutations attenuate Pi uptake and distri- vated signal transducer and activator of transcription; Piint, intracellular Pi; SUMO, small
bution within tissues (8–10). The transporters AtPT1 (Pht1;1) ubiquitin-like modifier.
and AtPT2 (Pht1;4) facilitate Pi uptake in environments with §To whom correspondence should be addressed. E-mail: [email protected].
low (2 ␮M) and high (500 ␮M) levels of Pi, respectively (11). © 2005 by The National Academy of Sciences of the USA

7760 –7765 兩 PNAS 兩 May 24, 2005 兩 vol. 102 兩 no. 21 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0500778102
regulation by modulating protein–protein and protein–DNA Results
interactions and intracellular targeting and controlling ubiquiti- T-DNA Insertional Mutations in AtSIZ1 Suppress the Salt-Sensitive
nation and other covalent modifications of proteins. Recently, Phenotype of sos3-1 and Cause Hyperresponsiveness to Pi Limitation.
functional evidence for a SUMO conjugation pathway in plants T2 lines from a T-DNA insertion population (pSKI015) in the
has been reported (18–20). Here, we determined that PHR1, Arabidopsis Col-0 gl1 sos3-1 background were screened to iden-
which is the only presently identified transcriptional modulator tify second-site mutations that enhance NaCl tolerance (21). The
of low Pi-induced responses, is sumoylated by an AtSIZ1- siz1-1 mutation suppressed NaCl sensitivity of sos3-1 seedlings,
dependent process. Considering these findings and its function but to a lesser extent than did hkt1 alleles (data not shown and
as a SUMO E3 ligase with multiple targets, AtSIZ1 appears to ref. 21). Decreasing the macronutrient concentration in the MS
be an important regulator of Pi starvation responses in plants. salt formulation to 1兾20⫻ resulted in a substantial reduction in
primary root growth of sos3-1 siz1-1 seedlings (data not shown).
Materials and Methods Normal root growth was restored by inclusion in the medium of
Plant Materials and Growth Conditions. The Arabidopsis T-DNA 1.25 mM PO43⫺, either as KH2PO4 or NaH2PO4, but not by
population in the Col-0 gl1 sos3-1 background and identification inclusion of any other macronutrient at the concentration in the
of mutations that suppress sos3-1 Na⫹ hypersensitivity were full-strength MS salt formulation, e.g., 20 mM KCl, 19 mM
described in ref. 21. Unless indicated otherwise, 3-day-old KNO3, 3 mM CaCl2, or 1.6 mM MgSO4 (data not shown).
seedlings were transferred to basal medium containing 1⫻ sos3-1 siz1-1 seedlings exhibited substantially more pro-
Murashige and Skoog (MS) micronutrients, 1兾5⫻ or 1兾20⫻ nounced prototypical Pi starvation root architecture responses
than sos3-1 or wild-type seedlings (Fig. 1A). On Pi-limited
macronutrients without KH2PO4, 3% sucrose, 2.5 mM Mes, B5
medium, sos3-1 siz1-1 seedlings exhibited an inhibition of pri-
vitamins, 1.2% agar, and without or with various amounts of
mary root growth (Fig. 1 A; see also Fig. 6A, which is published
KH2PO4 or NaH2PO4. The Pi content of the basal medium
as supporting information on the PNAS web site), an increase in
without a Pi supplement was 10.6 ⫾ 0.75 ␮M. lateral root development and length (Figs. 1A and 6 B–D), an
Three-week-old plants were removed from Metro-mix (Scotts, increase in root hair number and length (Fig. 1B), and higher
Marysville, OH), roots were carefully washed with water, and root兾shoot fresh weight ratio (Table 1, which is published as
plants were transferred to modified Hoagland’s solution (with- supporting information on the PNAS web site) than wild type.
out aeration) containing 1 mM KH2PO4 (22). After 4 days of sos3-1 siz1-1 plants grown on Pi-deficient medium for 25 days
adaptation to solution culture, plants were transferred to a also accumulated more anthocyanin in leaves than did the wild
modified Hoagland’s solution with 0.01 or 1 mM KH2PO4 (22). type, and many of the siz1 leaves were necrotic (data not shown).
sos3-1 siz1-1 plants grown in the greenhouse or growth chamber
Molecular Genetic Analysis of siz1 T-DNA Insertion Alleles. The always exhibited reduced shoot and root biomass relative to the

PLANT BIOLOGY
genomic sequence flanking the T-DNA left border in sos3-1 wild type that was not evident when these plants were grown in
siz1-1 plants (the Col-0 gl1 background) was determined by vitro. Despite this phenomenon, the relative increase in root兾
thermal asymmetric interlaced PCR analysis as described in ref. shoot biomass was greater for sos3-1 siz1-1 plants than the wild
21. siz1-2 and siz1-3 alleles in the Col-0 background were type after transfer to Pi-deficient medium in hydroponic solution
identified in the Salk Institute Genome Analysis Laboratory (Table 2, which is published as supporting information on the
database, and T3 seeds were provided by the Salk Institute PNAS web site). sos3-1 siz1-1 plants grown hydroponically began
laboratory through the Arabidopsis Biological Resource Center anthocyanin accumulation sooner than the wild type after
at Ohio State University. transfer to Pi-deficient medium (data not shown).
sos3-1 siz1-1 seedlings and plants grown in Pi-sufficient me-
Analysis of Sumoylation Profiles. Total protein from seedlings dium (Fig. 2A) and hydroponic solution (Fig. 2B), respectively,
incubated at 24°C or at 40°C for 30 min were extracted as accumulated more intracellular Pi (Piint) in shoots than did the
described in ref. 20 and separated by SDS兾PAGE. The gel blot wild type or sos3-1, but Piint in roots of these plants was similar.
was probed with the AtSUMO1 antibody (kindly provided by G. Symptoms of Pi toxicity were not detected in shoots of sos3-1
Coupland, Max Planck Institute, Cologne, Germany) and de- siz1-1 seedlings, as is the case in shoots of Pi hyperaccumulating
tected by using ECL plus (Amersham Pharmacia). pho2 (9) that was included as a control in this experiment (Fig.
2). Piint levels in sos3-1 siz1-1, sos3-1, and wild-type seedlings and
Purification of Proteins for the in Vitro SUMO Conjugation Assay. To plants were similar when grown in Pi-deficient medium or
construct pGST-SIZ1, a cDNA fragment of AtSIZ1 ORF was solution (Fig. 2) even though sos3-1 siz1-1 seedlings exhibited
inserted in-frame into the pGEX-2TK vector (Amersham Phar- responses to Pi limitation earlier and to a greater extent than the
wild type (Figs. 1 and 6).
macia). To construct PHR1-expressing plasmids, a cDNA frag-
ment encoding wild-type PHR1 (pT7-PHR1) or a mutated PHR1
AtSIZ1 Encodes a Putative SUMO E3 Ligase. Thermal asymmetric
[AA782A to AGA and AA1115A to AGA by site-directed mu- interlaced PCR analysis of sos3-1 siz1-1 plants identified a
tagenesis pT7-PHR1(2KR); ref. 23] was inserted into pET21a T-DNA insertion in the 13th exon of At5g60410 (GenBank
vector (Novagen). The wild-type protein is designated as T7- accession no. NM㛭125434) at ⫹986 bp from the translation
PHR1, and the variant is designated as T7-PHR1(2KR), har- initiation codon as determined by analysis of the full-length
boring a mutation (K261R and K372R) in each of the two cDNA sequence (R22197; Fig. 3A). At5g60410 is annotated as
predicted sumoylation motifs in PHR1. Recombinant protein AtSIZ1, and this mutant allele is designated as siz1-1. Using the
synthesis from pGST-SIZ1, pT7-PHR1, pT7-PHR1(2KR), or GenBank database, the AtSIZ1 product is predicted to be a
other expression vectors (kindly provided by Y. Kikuchi, Tokyo peptide of 833 aa, which is an apparent ortholog of Saccharo-
University, Tokyo) and the in vitro sumoylation assay were myces cervisiae Siz1 and Siz2 and human protein inhibitor of
performed as described in ref. 23. activated signal transducer and activator of transcription (PIAS)
proteins (Fig. 3B; see also Fig. 7, which is published as supporting
Supporting Information. For additional methods, see Supporting information on the PNAS web site) (19). AtSIZ1 contains five
Methods, which is published as supporting information on the predicted domains conserved in Siz兾PIAS SUMO E3 ligases.
PNAS web site. These domains include a SAP domain that is implicated in

Miura et al. PNAS 兩 May 24, 2005 兩 vol. 102 兩 no. 21 兩 7761
Fig. 1. Arabidopsis plants harboring siz1 mutations are hyperresponsive to Pi limitation in root architecture development. (A) Photographs are of
representative wild-type [Col-0 or Col-0 gl1 (Col-gl1)], sos3-1, sos3-1 siz1-1, siz1-2, and siz1-3 seedlings 11 days after transfer onto medium containing 1兾20⫻ MS
macronutrients without or with KH2PO4 supplement. sos3-1 and sos3-1 siz1-1 are in the Col-0 gl1 background, and siz1-2 and siz1-3 are in the Col-0 background.
(B) Photographs are of wild-type and siz1-2 seedling roots 7 days after transfer onto medium containing 1兾20⫻ MS macronutrients without (⫺Pi) or with 1.25
mM KH2PO4 (⫹Pi).

chromatin organization, a Siz兾PIAS-RING domain that is nec- (25), and a putative nuclear localization sequence (Fig. 3B).
essary for SUMO E3 ligase activity, a ‘‘PINIT’’ motif for nuclear AtSIZ1 also has a plant homeodomain-finger (PHD) domain
retention (24), a ‘‘SXS’’ motif that promotes binding to SUMO that is not present in Siz兾PIAS proteins. The PHD domain is
associated with chromatin remodeling complexes (26) or func-
tions as an ubiquitin E3 ligase (27).
F1 and F2 analyses of progeny derived from a backcross with
sos3-1 (sos3-1 ⫻ sos3-1 siz1-1) indicated that siz1-1 is a mono-
genic recessive mutation [F2 analysis, n ⫽ 407 for, ␹2 ⫽ 1.25, P ⫽
0.26 for a 3:1 (normal:short primary root on Pi-deficient me-
dium) segregation ratio]. Seedlings harboring additional muta-
tions in AtSIZ1, siz1-2 (SA LK㛭065397), and siz1-3
(SALK㛭034008) (Fig. 3A) exhibited similar root architecture
responses to Pi deficiency as did sos3-1 siz1-1 seedlings, and these
responses were suppressed by the addition of either KH2PO4 or

Fig. 3. AtSIZ1 locus is At5g60410 as determined by thermal asymmetric

interlaced PCR and T-DNA insertion diagnostic PCR. (A) Gene organization of
Fig. 2. Piint in the shoot and the root of wild-type and siz1 seedlings and plants AtSIZ1 and schematic representation of T-DNA insertion alleles are illustrated
is comparable. Shown are seedlings grown for 11 days on medium containing (arrowheads). Filled boxes are exons, and open rectangles are 5⬘ or 3⬘ untrans-
1兾5⫻ MS macronutrients with 0.0125 or 1.25 mM KH2PO4 (A) or plants grown for lated regions. (B) Diagram illustrates the domain organization of AtSIZ1 (see
2 weeks in hydroponic solution with 0.01 or 1 mM KH2PO4 (B) were harvested, Results). (C) AtSIZ1 is expressed in the root (R) and the shoot (S) of the wild type
mean values ⫾ SE, n ⫽ 6. pho1 and pho2 seedlings were used as controls in A (8, 9). but not in these organs of siz1 seedlings.

7762 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0500778102 Miura et al.

NaH2PO4 (Figs. 1 and 6). Furthermore, siz1-2 and siz1-3 seed-
lings and plants accumulated comparable Piint and developed a
higher root兾shoot mass ratio in response to Pi deprivation, as did
sos3-1 siz1-1 (Fig. 2 and Tables 1 and 2). These data establish that
the responses to Pi limitation exhibited by sos3-1 siz1-1 seedlings
are due to the siz1-1 mutation. Primary root growth analysis on
Pi-deficient medium of F1 progeny from the cross between siz1-2
and siz1-3 indicated that the mutations are allelic (data not
shown). AtSIZ1 mRNA was more abundant in roots than in
shoots of wild-type and sos3-1 seedlings (Fig. 3C) but was not
detected in sos3-1 siz1-1 seedlings (Fig. 3C) or siz1-2 or siz1-3
seedlings (data not shown).

In Vitro and in Vivo Data Indicate That AtSIZ1 Functions as a SUMO E3

Ligase. An immunoblot of total protein by using anti-AtSUMO1
(20) indicated no difference in accumulation of SUMO or
SUMO conjugation products in siz1-2 and wild-type seedlings
that are grown at 24°C (Fig. 4A). In contrast, esd4 seedlings
accumulate numerous sumoylated products, whereas the
amounts of free SUMO peptides are depleted (data not shown
and ref. 20). Heat shock increased SUMO conjugation in
wild-type plants (19), but the extent of heat shock-induced
sumoylation was substantially less in siz1-2 plants (Fig. 4A).
Together these results provide evidence that AtSIZ1 is involved
in the sumoylation of Arabidopsis proteins.
GST-AtSIZ1 could substitute for T7-ScSiz2-His to stimulate
sumoylation in an in vitro assay (23) that included S. cerevisiae E1
(GST-ScAos1 ⫹ GST-ScUba2), E2 (ScUbc9), SUMO substrate
(T7-ScCdc3-His), and SUMO (His-ScSmt3) and used anti-
ScSmt3 to detect sumoylated products (Fig. 4B Upper). GST-
AtSIZ1 (112 kDa) or ScSiz2 (82 kDa) was detected (Fig. 4B) on

PLANT BIOLOGY
the anti-ScSmt3 immunoblot because each protein is sumoy-
lated. Immunoblot analysis by using anti-T7 antibody (detecting
T7-ScCdc3-His) indicated that AtSIZ1 mediates ScSmt3 coju-
gation to ScCdc3, as does ScSiz2 (Fig. 4B Lower). ScCdc3
contains seven sumoylation sites, and these different products
are evident on the blot (Fig. 4B Lower and ref. 23). Together, the
immunoblot analysis of the in vivo sumoylation products and the
in vitro assay results establish that AtSIZ1 functions as a SUMO
E3 ligase in Arabidopsis.
AtSIZ1-GFP was detected specifically in the same compart-
ment as the red fluorescent protein control (ref. 28 and Fig. 8A, Fig. 4. In vitro and in vivo assays indicate that AtSIZ1 is a SUMO E3 ligase. (A)
which is published as supporting information on the PNAS web In planta sumoylation profiles of wild-type and siz1-2 seedlings that were
site), indicating that AtSIZ1 is localized to the nucleus. Higher grown for 10 days on 1⫻ MS medium at 24°C (24) and then exposed to a 30-min
magnification revealed that AtSIZ1 localizes predominantly to heat shock of 40°C (40). (B) Analysis of in vitro sumoylation profiles indicates
punctuate structures in the nucleus (Fig. 8B). Expression of that AtSIZ1 facilitates production of ScSmt3-ScCdc3 conjugates. The indicated
AtSIZ1-GFP suppressed both the hyperresponses of siz1-2 seed- amount of GST-AtSIZ1 (0, 2, 4, or 10 ␮g) or ScSIZ2 (0 or 2 ␮g) was added to the
reaction mixture and incubated with 10 mM ATP at 37°C for 0 (inc. ⫺) or 90 min
lings to Pi starvation and the reduced shoot and root biomass
(inc. ⫹) as described in ref. 23. Immunoblot analysis was performed with
phenotypes of siz1-2 plants (data not shown). These results anti-ScSmt3 (Upper) or anti-T7 (detecting T7-ScCdc3; Lower). Filled arrow-
indicate that AtSIZ1-GFP is functional and that AtSIZ1 com- heads indicate bands corresponding to ScSmt3-ScCdc3 conjugates. Open ar-
partmentalizes to nuclear speckles, as do PIAS proteins (29). rowheads identify the AtSIZ1-ScSmt3 and ScSiz2-ScSmt3 conjugates. An aster-
isk identifies the T7-ScSIZ2-His protein. Numerals identify the migration
siz1 Mutations Cause an Alteration in Pi Starvation-Dependent Sig- position and molecular mass of the markers (kilodaltons).
naling. AtPT2 (Pht1;4), AtPS2 (acid phosphatase; ref. 30), and
AtPS3 (glycerol-3-phosphate permease, Raghothama, unpub-
lished data), but not AtIPS1 or AtRNS1, transcript abundances by AtSIZ1 in Pi-sufficient conditions, and AtIPS1 and AtRNS1
were greater in siz1 than in wild-type seedlings when both were are positively regulated by AtSIZ1 during the initial stages of
grown in Pi-sufficient medium (Fig. 5A). However, Pi starvation induction by Pi limitation (for primer sequences for these genes,
induced the expression of AtPT2, AtPS2, and AtPS3 to a com- see Table 3, which is published as supporting information on the
parable level in both the wild-type and siz1 seedlings after 48 h PNAS web site).
in Pi-deficient medium (Fig. 5A). Pi starvation induction of AtIPS1 and AtRNS1 are members of the PHR1 regulon (7).
AtIPS1 and AtRNS1 transcript abundances occurred more slowly The MYB transcription factor PHR1, which binds to a P1BS
in siz1 than in wild-type seedlings (Fig. 5A) but eventually element in the promoter of AtIPS1 and is necessary for tran-
increased to levels similar to the wild type after an extended scription of AtIPS1 and AtRNS1 (7), contains two predicted
period of Pi limitation (i.e., 48 h or 72 h; data not shown). sumoylation sites. AtSIZ1 facilitated the sumoylation of PHR1
Together, these results indicate that the function of AtSIZ1 in Pi (Fig. 5B). Mutations to residues in the predicted sumoylation
starvation signaling is different for these two specific subsets of sites, K261R and K372R, in PHR1 prevented sumoylation of
target genes. AtPT2, AtPS2, and AtPS3 are negatively regulated PHR1 (Fig. 5B). These results further indicate that sumoylation

Miura et al. PNAS 兩 May 24, 2005 兩 vol. 102 兩 no. 21 兩 7763
 results indicate that AtSIZ1 is a plant member of the Siz兾PIAS
category of SUMO E3.
AtSIZ1 is annotated as the only member of a single-gene
family in Arabidopsis (19); however, three independent dysfunc-
tional siz1 T-DNA insertion alleles are not lethal. Apparently, an
alternative SUMO E3 performs the necessary functions of
AtSIZ1, or AtSIZ1 is not essential for survival, even though
sumoylation through AtSIZ1 controls critical processes in plants,
as established by the results presented herein. Mutations in both
ScSiz genes that are present in yeast also are not lethal (16).
Although no additional gene encoding a Siz兾PIAS E3 is evident
in the Arabidopsis genome based on database analysis, it should
be noted that ESD4 was not predicted to be a SUMO protease
by bioinformatics (19, 20), further suggesting that there are
unidentified functional Arabidopsis loci that can substitute for
the function of AtSIZ1. Alternatively, two other categories of
SUMO E3 are known to exist in animals: RanBP2 (Ran binding
protein 2) and Pc2 (polycomb protein 2). RanBP2 functions as
an E3 that attaches SUMO to RanGAP, a process that is
involved in nucleocytoplasmic trafficking (15). At3g18610 and
At2g34150 encoding AtRanGAP1 and AtRanGAP2, respec-
tively, are likely to be sumoylated by a yet to be identified
RanBP2-like E3 ligase (31). The Pc2-type SUMO E3 is a
member of the polycomb group (PcG) of proteins that form large
multimeric corepressor complexes (PcG bodies) that facilitate
gene silencing (15, 32). Mammalian Pc2 contains a CHROMO
(chromatin organization modifier) domain, and sumoylation is a
Fig. 5. Pi-responsive transcript abundance is altered in siz1 plants, and
control process involved in the function of PcG complexes (32).
AtSIZ1 facilitates sumoylation of PHR1. (A) Pi starvation-responsive gene
mRNA levels in Col-0 (wild type, open bars) and siz1-2 (filled bars) seedlings
Arabidopsis LHP1兾TFL2 has a CHROMO domain and functions
were determined by quantitative PCR. Seedlings were grown in liquid medium in the PcG complex to repress floral homeotic genes and
containing 1兾5⫻ MS macronutrients and 1⫻ MS micronutrients with 1.25 mM modulate plant development (33). LHP1 is also localized to
KH2PO4 for 7 days and then transferred to medium without KH2PO4. Samples discrete rounded subnuclear foci throughout the nucleoplasm
were collected immediately after transfer to medium without Pi (0 h) or after similar to Pc2 in Drosophila (33).
various time points in this medium (6, 12, or 48 h). (B) AtSIZ1 mediates in vitro
sumoylation of PHR1. T7-PHR1 or T7-PHR1(2KR) were used as substrates for AtSIZ1 Is a Repressor of the Low Pi-Induced Responses. siz1 seedlings
sumoylation in the reaction mixture containing ScAos1 (E1), ScUba2 (E1), and plants exhibit exaggerated symptoms that are associated
ScUbc9 (E2), ScSmt3 (SUMO, 23), and AtSIZ1 (E3) proteins. PHR1 proteins were
with Pi deficiency, including reduced primary root growth and
detected with anti-T7 antibody. Arrowheads indicate the position of sumoy-
lated PHR1 proteins.
increased lateral root and root hair number and length, root兾
shoot mass ratio, and anthocyanin accumulation (Figs. 1 and 6
and refs. 34 and 35). Pi limitation reduces primary root growth
by attenuating cell division and promoting lateral root density
of PHR1 positively controls the expression of AtIPS1 and and length, root architecture changes that are considered to
AtRNS1. facilitate Pi acquisition (3, 34). These results indicate that
AtSIZ1 is a negative regulator of Pi starvation-dependent sig-
Discussion naling that controls root architecture and anthocyanin accumu-
Genetic, physiological, molecular, and biochemical data presented lation. Interestingly, these hyperresponses to low extracellular Pi
here establish an important function for the plant SUMO E3 occur even though Pint is somewhat greater (sufficient medium)
AtSIZ1 in the signaling system that coordinates plant responses to or equivalent (deficient medium) in the shoots of siz1 seedlings
Pi starvation. More than any other identified locus or molecular and plants compared with the wild type (Fig. 2). However, shoot
genetic determinant yet described, AtSIZ1 influences a greater Piint accumulation (sufficient medium) in siz1 seedlings is not
number of processes that are considered to be adaptation responses equivalent to pho2 plants that hyperaccumulate Pi and exhibit
to low Pi availability, including root architecture changes, gene symptoms of Pi toxicity (9).
expression modulation, and anthocyanin accumulation (4–12). Our Transcript abundances of the Pi starvation responsive genes
results establish that the MYB transcription factor PHR1, the only AtPT2, AtPS2, and AtPS3 are greater in siz1 compared with the
defined molecular controller of Pi deficiency responses (7), is a wild-type seedlings grown under Pi sufficient conditions (Fig.
5A). However, Pi deprivation still increases the abundances of
sumoylation target of AtSIZ1. However, AtSIZ1 affects several Pi
transcripts of these genes in siz1 seedlings to the same extent as
starvation responses that are not controlled by PHR1, indicating
observed in the wild type. This capacity for siz1 seedlings and
that sumoylation is a more encompassing process in the control of plants to still respond to Pi limitation by increasing the expres-
adaptation to Pi deficiency. sion of these genes, by changing root architecture, and by
AtSIZ1 can replace ScSiz2 to sumoylate the substrate ScCdc3 accumulating anthocyanin is indicative that the function of
in an in vitro assay, providing evidence of its SUMO E3 ligase AtSIZ1 may be downstream of the Pi starvation sensing mech-
activity (Fig. 4B and ref. 23). Reduction of heat shock-induced anism (Fig. 5). Other genetic loci, including PHR1, PDR2,
SUMO conjugation in seedlings with a defective AtSIZ1 gene PHO1-3, Pht1;1, Pht1;4, and PSR1 (4–12), are not individually
demonstrates that AtSIZ1 facilitates sumoylation in planta (Fig. required to regulate all of the Pi starvation responses that are
4A). AtSIZ1 localizes to nuclear speckles (Fig. 8), as do mam- affected by AtSIZ1, indicating that AtSIZ1 may be a critical
malian Siz兾PIAS proteins, which are in structures that resemble regulator that is upstream of these other determinants.
the promyelocytic leukemia nuclear bodies (29). Together these Because coregulation of transcriptional activators is a major

7764 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0500778102 Miura et al.

function of mammalian PIAS family proteins (17, 29), repression PHO3 (4–6). The pdr2 mutation causes inhibition of primary
of low Pi-induced root architecture and anthocyanin accumula- root cell division and subsequent loss of meristematic activity
tion, and AtPT2, AtPS2, and AtPS3 expression responses that are (6). PDR2 may function in modulating meristematic activity and
controlled by AtSIZ1 likely involves negative regulation of controlling root architecture when Pi is limiting. However, the
transcription factor(s) through AtSIZ1-mediated sumoylation. sequences of these genes have not been identified, and, there-
Two regions of the AtPT2 promoter bind nuclear protein factors fore, it is not possible to establish by more direct evidence
from Pi-sufficient but not Pi-deficient plants (36), indicating that whether sumoylation is involved in their functions.
these factors may play a role in the repression of AtPT2 expres- Taken together, our results confirm that multiple signaling
sion. However, these regulatory factors have yet to be identified, pathways are involved in the control of Pi starvation responses
so direct evidence that sumoylation is necessary for the function (Fig. 5). AtIPS1 and AtRNS1 are positively controlled by AtSIZ1
of a repressor complex that controls low Pi signaling cannot be through sumoylation of the transcription factor PHR1. However,
assessed. PHR1 and its sumoylation apparently do not have a major effect
on low Pi-induced phenotypic responses or on the expression of
AtSIZ1 Acts also as a Positive Regulator of Some Pi Starvation AtPT2, AtPS2, and AtPS3, which are involved in adaptation and
Responses. Pi starvation-dependent transcript accumulation of
are negatively controlled by AtSIZ1. Alternatively, PHR1 func-
AtIPS1 and AtRNS1 occurs at a reduced rate in siz1 seedlings tion in Arabidopsis may be redundant. In any case, it is clear that
compared with wild type (Fig. 5A). Transcriptional activation of
a major function AtSIZ1 in Pi-starvation responses involves the
these genes has been attributed to the interaction of the MYB
control of a repressor system that attenuates gene expression and
transcription factor PHR1 with the P1BS element in the pro-
developmental responses that are only derepressed (activated)
moter of these genes (7). Recently, additional evidence based on
experimentation by using HvPht1;1 has supported the possibility by Pi starvation. The role of AtSIZ1 as an important regulator
that the P1BS element has a transcriptional activation function of plant adaptation to Pi deficiency constitutes a previously
(37). Our determination that AtSIZ1 participates in the sumoy- uncharacterized function of sumoylation. It is clear that further
lation of PHR1 (Fig. 5B) indicates that sumoylation may activate research is necessary to define all of the mechanisms by which
PHR1, which provides a mechanism for reduced Pi starvation AtSIZ1 controls Pi deficiency responses.
induction of AtIPS1 and AtRNS1 expression in siz1 seedlings.
Sumoylation functions to activate transcription factors in ani- We thank Ms. Wanda Hunter and Ms. Tomoko Miura for assistance and
the Arabidopsis Biological Research Center and the Salk Institute for
mals, including heat shock factors (38). After prolonged Pi
T-DNA-tagged lines and a cDNA clone. This work was supported by
deprivation, AtIPS1 and AtRNS1 transcript abundances are National Science Foundation Plant Genome Award DBI-98-13360,
similar in siz1 and wild type (Fig. 5A for AtIPS1 and data not grants from the Biogreen 21 program, the Environmental Biotechnology
shown for AtRNS1), indicating that AtSIZ1 has a transient National Core Research Center (R15-2003-012-01002-0), the Plant

PLANT BIOLOGY
function in this process. However, this mechanism does not Diversity Research Center, the Ministry of Sports and Technology
explain how AtSIZ1 affects PHR1 modulation of anthocyanin or (PF0330401-00), and the BK21 program, and JSPS-Junior Scientist
Pi accumulation or root兾shoot mass ratio. Other genetic loci are Research Fellowship 3117. This work is Purdue University Agricultural
implicated in Pi starvation signaling, such as PDR2, PSR1, and Research Program Paper 2005-17626.

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Miura et al. PNAS 兩 May 24, 2005 兩 vol. 102 兩 no. 21 兩 7765