Orosa-Puenteet al.,Science 362 , 1407–1410 (2018) 21 December 2018 3of4
ARF7 (WT) ARF7 (4*K/R)
0 min 20 min 0 min 20 min250KDa130
100250
130SUMO conjugated ARF7
anti-SUMO1
ARF7-GFP
anti-GFP
RubiscoARF7 (4*K/R)ARF7 (WT)SUMO1
SUMO1ARF7-GFP250KDa130
250
130Ponceau
+ NAA (15 min)01234Col-0 arf7-1 L1-7 L2-7 L10-10Lateral root density (LR/cm)b bac
bgARF7 (4*K/R) arf7-1% Air% ContactgARF7 (4*K/R) arf7-1(^0) L1-7 L2-7 L10-10
20
40
60
80
100
Col-0 arf7-1
Emerged LR %
bbba b
0
1
2
3
4
Col-0 arf7-1 L4-4 L5-3
Lateral root density (LR/cm)
gARF7 (WT) arf7-1
b b
a
% Air b
% Contact
gARF7 (WT) arf7-1
(^0) L4-4 L5-3
20
40
60
80
100
Col-0 arf7-1
Emerged LR %
a b a a
SUMO-HA
250
130
ARF7-YFP
250
130
KDa^ 4K/RWT K104
K104 K151 K282 K889
ARF7 DNA binding Gln-rich III IV
A
EF
CD
G
H
B
Fig. 2. ARF7 SUMOylation regulates hydropatterning and DNA
binding affinity.(A) Schematic of ARF7 domains and four predicted
SUMO sites K104, K151, K282, and K889. (B)ReplacingallARF7
SUMO site lysine with arginine residues in ARF7-GFP(4K/R) blocks
SUMOylation with HA-SUMO1 (but not WT ARF7 or single SUMO
K104) in transient expression assays. YFP, yellow fluorescent protein.
(CandD) Bioassays reveal that two independent transgenic lines
expressing WT gARF7 can rescuearf7-1hydropatterning (C) and
LR density defects (D).nLR = 196 (Col-0), 78 (arf7-1), 292 (L4-4), and
231 (L5-3);nplants = 7 (Col-0), 5 (arf7-1), 10 (L4-4), and 9 (L5-3).
(EandF) Bioassays reveal that three independent transgenic lines
expressing gARF7(4K/R) cannot rescuearf7-1hydropatterning (E) but
do restore LR density (F).nLR = 374 (Col-0), 268 (arf7-1), 198 (L1-7),
286 (L2-7), and 206 (L10-10);nplants = 12 (Col-0), 16 (arf7-1), 8 (L4-4),
11 (L5-3), and 8 (L10-10). Data are mean values ± SE, and statistics were
performed as in Fig. 1C. (G) Immunoprecipitation reveals that ARF7-GFP
[but not ARF7-GFP(4K/R)] is rapidly SUMOylated 15 min after
naphthaleneacetic acid (NAA) treatment. (H)Immunoprecipitationreveals
that ARF7-GFP [but not ARF7-GFP(4K/R)] is rapidly SUMOylated
20 min after seedlings were removed from their agar plates.
Fig. 3. SHY2 interacts with ARF7 in a SUMO-
dependent manner to control hydropatterning.
(A) Bioassay reveals thatIAA3/SHY2mutant allele
shy2-31does not exhibit a hydropatterning response.
Data shown are mean ± SE. Letters indicate a
significant difference compared with WT (Ler) roots
based on Student’sttest (P< 0.05).nLR = 208 (Ler)
and 604 (shy2-31);nplants = 7 (Ler) and 19 (shy2-
31). (B)TheIAA3(but notIAA14) sequence contains a
putative SIM, suggesting that IAA3 could bind
SUMOylated ARF7. (C) Transient expression of IAA3/
SHY2–HA (WT-SIM) or IAA3/SHY2–HA (SIM mutant)
with ARF7-GFP or ARF7-GFP(4K/R), followed by
immunoprecipitation and western analysis, revealed
that IAA3 interacts with ARF7 in a SIM- and SUMO-
dependent manner. (D) PhenotypingArabidopsis
seedlings expressingshy2-2± SIM by using the
endodermalCASP1promoter revealedCASP1:shy2-2
(WT) blocks LR branching (top), whereasCASP1:shy2-2
(non-SIM) branch normally (bottom). Seedlings are
from six independent lines termed SIM-containing
CASP1:shy2-2(WT L1, L2, and L3) and non–SIM-
containingCASP1:shy2-2(SIML1, L2, and L3).
(E) Schematic summarizing the SUMO-dependent
ARF7 model for hydropatterning, in which ARF7 is
SUMOylated on the air side of the root, resulting in an
interaction with IAA3 that inhibits LR initiation. On the
contact side of the root, ARF7 is not SUMOylated,
enabling the transcriptional factor to activate expression
of genes involved in LR initiation.
Wet ARF7
ARF7
SUMO
Dry
IAA3
Col-0 +SIM (L1) +SIM (L2) +SIM (L4)
Col-0 -SIM (L1) -SIM (L2) -SIM (L4)
pCASP1::shy2-2 WT-SIM
pCASP1::shy2-2 non-SIM
15
ARF7
anti-GFP
anti-HA
25
KDa
130
IAA3
250
IAA3 SIM mutant
WTGFP 4K/R WT 4K/R
IAA3 WT
Input
SIM prediction sites
IAA3
IAA14
% Contact % Air 0 100 200
0 20406080100
Ler
shy2-31
Emerged LR %
b
a
D
E
ABC
RESEARCH | REPORT
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