The Plant Journal (2016) 86, 443–457 doi: 10.1111/tpj.

13188

Auxin-mediated lamina growth in tomato leaves is restricted

by two parallel mechanisms
Hadas Ben-Gera1, Asaf Dafna1, John Paul Alvarez2,3, Maya Bar1, Mareike Mauerer1,† and Naomi Ori1,*
1
The Robert H. Smith Institute of Plant Sciences and Genetics in Agriculture and The Otto Warburg Minerva Center for
Agricultural Biotechnology, Hebrew University, P.O. Box 12, Rehovot 76100 Israel
2
Department of Plant Sciences, Weizmann Institute of Science, Rehovot 76100 Israel and
3
School of Biological Sciences, Monash University, Clayton Campus, Melbourne, Vic. 3800 Australia

Received 1 December 2015; revised 5 April 2016; accepted 6 April 2016; published online 28 April 2016.
*For correspondence (e-mail naomi.ori@mail.huji.ac.il)
†
Present address: Albrecht Daniel Thaer-Institute of Agricultural and Horticultural Sciences, Humboldt University, Berlin 10115, Germany.

SUMMARY
In the development of tomato compound leaves, local auxin maxima points, separated by the expression of
the Aux/IAA protein SlIAA9/ENTIRE (E), direct the formation of discrete leaflets along the leaf margin. The
local auxin maxima promote leaflet initiation, while E acts between leaflets to inhibit auxin response and
lamina growth, enabling leaflet separation. Here, we show that a group of auxin response factors (ARFs),
which are targeted by miR160, antagonizes auxin response and lamina growth in conjunction with E. In
wild-type leaf primordia, the miR160-targeted ARFs SlARF10A and SlARF17 are expressed in leaflets, and
SlmiR160 is expressed in provascular tissues. Leaf overexpression of the miR160-targeted ARFs SlARF10A,
SlARF10B or SlARF17, led to reduced lamina and increased leaf complexity, and suppressed auxin response
in young leaves. In agreement, leaf overexpression of miR160 resulted in simplified leaves due to ectopic
lamina growth between leaflets, reminiscent of e leaves. Genetic interactions suggest that E and miR160-
targeted ARFs act partially redundantly but are both required for local inhibition of lamina growth between
initiating leaflets. These results show that different types of auxin signal antagonists act cooperatively to
ensure leaflet separation in tomato leaf margins.

Keywords: miR160, Solanum lycopersicum, auxin response factor, auxin, tomato, ENTIRE, compound leaf,
leaf development, GOBLET ARF10.

INTRODUCTION
Leaves are the main photosynthetic organs of most plants.
Diverse leaf shapes and sizes are produced by different
plant species, within the same species and by individual
plants. Compound leaves are composed of multiple sub-
units termed leaflets (Figure 1a). Tomato (Solanum lycop-
ersicum) plants have compound leaves with one or two
orders of leaflets. Tomato shows a wide diversity of leaf
sizes and shapes, flexibly responding to environmental
changes (Holtan and Hake, 2003; Menda et al., 2004; Brand
et al., 2007; Chitwood et al., 2012, 2013). The elaboration
of compound leaves depends on prolonged organogenic
activity and delayed maturation of the developing leaf,
mainly in its margin. Patterning at the leaf margin has
been shown to involve differential spatial and temporal
lamina growth and to be regulated by an array of transcrip-
tion factors and hormones (Hagemann and Gleissberg,
1996; Byrne et al., 2000; Dengler and Tsukaya, 2001;
Kaplan, 2001; Barkoulas et al., 2007; Blein et al., 2010;
Efroni et al., 2010; Floyd and Bowman, 2010; Koenig and
Sinha, 2010; Moon and Hake, 2010; Bar and Ori, 2015; Sluis
and Hake, 2015).
The plant hormone auxin has been shown to be
involved in the patterning of leaf margins in species with
both simple and compound leaves (Barkoulas et al., 2008;
DeMason and Polowicky, 2009; Koenig et al., 2009; Blein
et al., 2010; Bilsborough et al., 2011; Peng and Chen, 2011;
Zhou et al., 2011; Ben-Gera et al., 2012). The most studied
auxin response pathway involves receptors from the TIR1/
AFB family, auxin response inhibitors from the Aux/IAA
family, and ARF transcription factors (Salehin et al., 2015).
ARF proteins are transcriptional activators or repressors
that regulate the expression of auxin-responsive genes by
binding to auxin response elements (AuxREs) in their pro-
moters (Ulmasov et al., 1997; Tiwari et al., 2003; Chandler,

© 2016 The Authors 443

The Plant Journal © 2016 John Wiley & Sons Ltd
444 Hadas Ben-Gera et al.
(a) (b)
(c) (d) (e)
(f) (g) (h)
2015). Aux/IAA proteins bind activator ARFs and repress
their function. The TIR/AFB receptors are F-box proteins
that promote the degradation of Aux/IAAs (Gray et al.,
2001; Dharmasiri et al., 2005). TIR/AFBs and Aux/IAAs act
as co-receptor complexes (Calderon Villalobos et al., 2012).
In the absence of auxin, Aux/IAAs bind activator ARFs and
repress their activity, together with co-repressor proteins
such as TOPLESS (Szemenyei et al., 2008). Repression by
Aux/IAAs involves recruitment of a histone deacetylase
complex as well as prevention of the recruitment of SWI/
SNF chromatin-remodeling ATPases by activating ARFs
(Wu et al., 2015). Auxin binding to the co-receptor complex
leads to the degradation of the Aux/IAA and the resulting
activation of the ARF (Kepinski and Leyser, 2004; Weijers
and Jurgens, 2004; Dharmasiri et al., 2005). While this
basic model of auxin response is compatible with the activ-
ity of activator ARFs, the mode of activity of repressing
ARFs is still not fully understood, and most repressor ARFs
do not bind Aux/IAAs in yeast two-hybrid assays (Tiwari
et al., 2003; Vernoux et al., 2011; Guilfoyle and Hagen,
2012). TIR1/AFBs, Aux/IAA and ARFs belong to gene fami-
lies, enabling diverse auxin outputs (Okushima et al., 2005;
The Plant Journal © 2016 John Wiley & Sons Ltd, The Plant Journal, (2016), 86, 443–457
Figure 1. SlARF10A, SlARF10B and SlARF17 regu-
late lamina width and leaf complexity in tomato.
(a) Illustration of a mature tomato leaf, indicating
leaflet orders.
(b–h) Mature fifth leaves of the genotypes indicated
at the bottom left corner of each panel. Scale bars:
2 cm.
Kieffer et al., 2010; Rademacher et al., 2011, 2012; Vernoux
et al., 2011). Most ARF proteins contain a DNA-binding
domain (DBD), a middle region (MR) that acts as an activa-
tion (AD) or a repression domain (RD), and a carboxy-term-
inal dimerization domain (CTD), which is involved in
protein–protein interactions (Guilfoyle and Hagen, 2007).
Recently, a dimerization domain was identified within the
DBD (Boer et al., 2014). ARF proteins show diverse struc-
tures, for example, several ARFs lack the CTD. Five of the
23 Arabidopsis ARFs have been shown in protoplast
assays to act as transcriptional activators (Ulmasov et al.,
1999; Tiwari et al., 2003). The remaining ARFs are thought
to function as repressors, but this has been shown only for
a few of them. In tomato, a recent study identified eight
ARFs as repressors and five as activators (Zouine et al.,
2014).
In developing compound leaf primordia, the juxtaposi-
tion of an auxin maxima and a region with low auxin
response is thought to promote leaflet initiation and sepa-
ration. The tomato Aux/IAA gene ENTIRE (E) is expressed
between initiating leaflets, where it inhibits auxin response
and promotes leaflet separation. Mutant e leaves are
© 2016 The Authors

simplified in comparison with wild-type leaves due to
expanded auxin response and ectopic lamina growth
between leaflets (Koenig et al., 2009; Ben-Gera et al.,
2012).
NAM transcription factors were also shown to be
involved in marginal patterning in several species (Niko-
vics et al., 2006; Blein et al., 2008; Bilsborough et al., 2011;
Cheng et al., 2012). The tomato NAM transcription factor
GOBLET (GOB) is expressed in leaflet boundaries and pro-
motes leaflet specification and separation (Brand et al.,
2007; Berger et al., 2009; Blein et al., 2010). NAM proteins,
E and auxin interact to pattern the leaf margin by complex
feedback interactions, the details of which are species
specific (Hasson et al., 2010; Bilsborough et al., 2011; Ben-
Gera et al., 2012).
Ectopic expression of the tomato ARF protein SlARF10A,
which is targeted by miR160, was recently shown to result
in very narrow leaflets (Hendelman et al., 2012). The
related group of miR160-targeted ARFs includes three
genes in Arabidopsis, ARF10, ARF16 and ARF17, which
have been shown to be involved in an array of develop-
mental processes (Mallory et al., 2005; Wang et al., 2005;
Liu et al., 2007, 2010, 2013). In soybean, proper regulation
of miR160 on ARF10, ARF16 and ARF17 was found to be
necessary for nodule formation (Nizampatnam et al.,
2015). In tomato, five related genes are predicted to con-
tain a miR160-binding site, SlARF10A, SlARF10B, SlARF17,
SlARF16A, SlARF16B (nomenclature according to Zouine
et al., 2014). SlARF10A and SlARF10B contain all the ARF
domains, while SlARF17 and SlARF16B lack the CTD.
SlARF10A and SlARF17 were recently shown to act as
repressors in protoplast activity assays, using the DR5 pro-
moter fused to GFP as the reporter (Zouine et al., 2014).
However, Arabidopsis ARF17 and ARF16 were shown to
positively regulate the auxin response sensor DR5 in pollen
and root cap, respectively (Wang et al., 2005; Yang et al.,
2013), and ARF10 and ARF16 were shown to positively reg-
ulate ABI3, although not directly (Liu et al., 2013). It is
therefore still unclear whether miR160-targeted ARFs act
as activators, repressors or both in different developmental
contexts.
Here, we investigated the role of miR160-targeted ARFs
in tomato leaf development by ectopic expression of either
pre-AtmiR160A, or miR160-resistant forms of miR160-tar-
geted ARFs. We show that leaves that overexpress
miR160-resistant forms of SlARF10A, SlARF10B and
SlARF17 are more complex and their leaflets have reduced
lamina. Furthermore, ectopic expression of miR160 in
tomato leaves leads to an expanded auxin response and
simpler leaves than the wild-type. Genetic and molecular
analyses of the interaction between SlARF10A and E indi-
cate that they act through parallel genetic pathways and
do not interact physically, but also depend on each other
for their function in lamina growth and leaflet separation.
© 2016 The Authors
The Plant Journal © 2016 John Wiley & Sons Ltd, The Plant Journal, (2016), 86, 443–457
miR160-targeted ARFs promote leaflet separation 445
Double mutant analysis shows that when GOBLET is not
active SlARF10A activity fails to promote leaflet separation.
RESULTS
Overexpression of miR160-targeted ARFs inhibits lamina
growth and promotes leaf complexity
Previously, ubiquitous overexpression of a SlARF10A form
with a mutation in the miR160-binding site was shown to
lead to substantially reduced leaflet lamina in tomato
(Hendelman et al., 2012). Examination of available tran-
scriptome databases (Park et al., 2011; http://tomato-
lab.cshl.edu/efp/cgi-bin/efpWeb.cgi) indicated that of the
five miR160-targeted ARFs, SlARF10A has the highest
expression in the vegetative meristem and young leaf pri-
mordia, while SlARF10B, SlARF16A and SlARF17 have
lower expression, and SlARF16B is not expressed in this
tissue. To assess the specific effect of SlARF10A on leaf
development, and the differential function of additional
miR160-targeted ARFs, we ectopically overexpressed
SlARF10A, as well as SlARF10B and SlARF17 with muta-
tions in the miR160-binding site (SlARF10Am, SlARF10Bm
and SlARF17m, respectively) (Figure S1) specifically at dif-
ferent stages of tomato leaf development using the trans-
activation system (Moore et al., 1998). The Arabidopsis
pFIL promoter drives expression throughout young devel-
oping leaves, and the pBLS promoter drives expression in
developing leaflets from the P4 stage. We have previously
used these promoters to investigate leaf development (Lif-
schitz et al., 2006; Shani et al., 2009). Leaf overexpression
of SlARF10Am, SlARF10Bm and SlARF17m under the control
of the pFIL promoter or the pBLS promoter led to reduced
leaf lamina and increased leaf complexity (Figures 1b–h,
S2 and S3a). To quantify the reduction in lamina width, we
calculated the ratio between the lamina area and the leaf
perimeter in the fifth leaf of the different genotypes.
pFIL>>SlARF10Am, pFIL>>SlARF10Bm pFIL>>SlARF17m and
pBLS>>SlARF10Am leaves had an approximately 50%
reduction in this ratio (Figure S3b), and a reduction of
approximately 28% was found in pBLS>>SlARF10Bm and
pBLS>>SlARF17m (Figure S3b). Quantification of leaf com-
plexity showed that in comparison with wild-type leaves,
leaves of plants that overexpressed SlARF10Am,
SlARF10Bm or SlARF17m had more secondary and inter-
calary leaflets and, unlike the wild-type, all had tertiary
leaflets. pFIL>>SlARF10Am leaves occasionally also had a
fourth order of leaflets (Figure S3c). The phenotypes
caused by expression with the pFIL promoter were more
severe than those caused by the expression with the pBLS
promoter (Figures 1 and S3). SlARF10Am overexpression
showed the strongest influence on lamina width and leaf
complexity. Analysis of the endogenous expression of
SlARF10A mRNA during wild-type leaf development
showed dynamic expression, with elevated expression at
446 Hadas Ben-Gera et al.

the P4 and P9 stages, possibly coinciding with primary and
secondary leaflet initiation (Figure S4). These results sug-
gest that SlARF10A, SlARF10B and SlARF17 can all affect
lamina width and leaf complexity when overexpressed
ectopically in developing leaves. The differences in their
overexpression phenotypes suggested that they have
somewhat differential activities and expression. To analyze
the spatial distribution of SlARF10A and SlARF17 in wild-
type leaf primordia, we performed in situ hybridization
experiments. SlARF10A and SlARF17 were found to be
expressed in the margins of developing leaf primordia,
with strong expression in young leaflets. Expression is
observed throughout very young initiating leaflets, and
later becomes restricted to the leaflet margin (Figure 2a–f).
SlARF10A expression is expanded in pFIL>>SlARF10Am
leaf primordia (Figure S5b). The expression dynamics of
miR160-targeted ARFs, together with the phenotypes
caused by their ectopic expression, suggests that miR160-
targeted ARFs act in a somewhat quantitative manner to
restrict lamina growth during early leaf development.
SlARF10A and SlARF17 suppress the effect of auxin on
lamina growth
Auxin was found to be a positive regulator of lamina
expansion (Barkoulas et al., 2008; Koenig et al., 2009; Ben-
Gera et al., 2012). Belonging to the ARF family, SlARF10A
and SlARF17 are putative mediators of the auxin signal. To
examine the involvement of SlARF10A and SlARF17 in the
regulation of auxin response, we analyzed the sensitivity
of pFIL>>SlARF10Am and pFIL>>SlARF17m to auxin. Wild-

(a) (b) (c) (d)

(e) (f) (g) (h)

Figure 2. Spatial expression of SlARF10A, SlARF17 and SlmiR160 in wild type leaf primordia.
In situ hybridization with SlARF10A probe (a–d), SlARF17 (e, f) and SlmiR160 probe (g, h), on longitudinal sections of a wild-type SAM (a, g) or leaf primordia
(b–f, h). Scale bars: 0.1 mm.

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 miR160-targeted ARFs promote leaflet separation 447

type and pFIL>>SlARF10Am plants were grown on MS
medium with or without 2,4-dichlorophenoxyacetic acid
(2,4-D). As shown previously, external 2,4-D application to
wild-type plants resulted in a simplified leaf form, with
wider and smoother leaflets and increased occurrence of
ectopic lamina, compared with untreated plants (Figure 3a,
c). In pFIL>>SlARF10Am plants the effect of auxin was
much milder than in the wild-type, as the treated leaves
essentially maintained the narrow lamina and deep lobing
phenotype of the pFIL>>ARF10Am leaves (Figure 3b,d).
This finding suggests that SlARF10A is involved in the reg-
ulation of auxin response in the leaf, but that additional
players may also regulate this process. To test the effect of
endogenous auxin, we generated plants that co-express
the bacterial auxin biosynthesis gene tryptophan
monooxygenase (iaaM) and SlARF17m under the control of
the FIL promoter. iaaM overexpression causes an increase
in endogenous free IAA levels (Romano et al., 1995).
pFIL>>iaaM leaves are simpler than wild-type leaves,
showing mainly primary leaflets and severe reduction in
secondary leaflets (Figure 3g) (Ben-Gera et al., 2012).
Co-expression of SlARF17m substantially suppressed the
iaaM phenotype, with the co-expressing plants having
narrower lamina and more complex leaves due to sec-
ondary leaflet formation and deep lobes (Figure 3h). Thus,
leaves that overexpress SlARF10Am or SlARF17m are less
sensitive to auxin than wild-type leaves, indicating that
these ARFs repress auxin response in leaf patterning. In
addition, SlARF10A and SlARF17 act antagonistically to
auxin as they have an opposite effect on lamina expansion
and leaf complexity.
miR160-targeted ARFs spatially inhibit auxin response and
promote leaflet separation
To further understand the role of miR160-targeted ARFs in
leaf development, we overexpressed the Arabidopsis pre-
miR160A in leaf primordia. As expected, qRT-PCR expres-
sion analysis showed reduced expression of SlARF10A,
SlARF16A and SlARF17 mRNA in leaves of pFIL>>miR160
plants (Figure S5). pFIL>>miR160 leaves were simpler than
wild-type leaves, as they had only primary leaflets that
were often fused due to ectopic lamina growth between
them. In addition, the margins of the leaflets were smooth
(Figure 4a,b). Examination of early stages of leaf develop-
ment of pFIL>>miR160 showed lamina growth in the termi-
nal leaflet and between leaflets, leading to improper leaflet

(a) (b) (c) (d)

(e) (f) (g) (h)

Figure 3. SlARF10A and SlARF17 act antagonistically to auxin in leaf elaboration.

(a–d) Exogenous auxin treatment: shown are third leaves of plants that were transferred 7 days after germination to MS medium without (a, b) or with (c, d)
1 lM 2,4-dichlorophenoxyacetic acid. Genotypes are indicated at the bottom left corner of each panel.
(e–h) Third leaves of the genotypes indicated at the bottom left corner of each panel. Scale bars: 2 cm.

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448 Hadas Ben-Gera et al.
(a) (b)
Figure 4. miR160-targeted ARFs inhibit lamina growth between leaflets.
(a–d) Mature fifth leaf of the indicated genotypes. Scale bars: 2 cm (a–d).
separation (Figure S6). pBLS>>miR160 leaves showed
round and smooth primary leaflets and no higher-order
leaflets. The primary leaflets were properly separated in
accordance with the pBLS expression domain (Figure 4c).
Interestingly, leaves that overexpressed miR160 showed
some phenotypic similarity to mutant e leaves. In plants
that co-expressed miR160 and SlARF10Am under the con-
trol of pFIL, SlARF10Am overexpression only partially sup-
pressed the miR160 overexpression phenotype (Figure 4d),
suggesting that additional miR160-targeted ARFs are nec-
essary for proper leaf patterning, or that the mutated
SlARF10A retains some miR160 sensitivity. The effects of
ectopic expression of miR160 and miR160-targeted ARFs in
leaves indicated that these ARFs promote leaflet separation
by inhibiting lamina growth between leaflets. Examination
of the spatial distribution of SlmiR160 in wild-type leaf pri-
mordia by in situ hybridization experiments revealed that
SlmiR160 expression is detected mainly in provascular tis-
sue (Figure 2g,h).
Auxin has been shown to be involved in the specifica-
tion of leaflets in tomato. Furthermore, the ectopic lamina
and lamina expansion seen in pFIL>>miR160 leaves was
similar to the phenotype caused by auxin application or
increased endogenous auxin in wild-type leaves. To assess
whether miR160 targeted ARFs affect auxin response,
we expressed pFIL>>SlARF10Am, pFIL>>SlARF17m or
pFIL>>miR160 in the background of the auxin response
sensor pDR5::VENUS (Figure 5) (Ulmasov et al., 1997;
Heisler et al., 2005; Ben-Gera et al., 2012). In wild-type
tomato leaves, pDR5::VENUS expression maxima marked
the sites of leaflet initiation at the leaf margin, and follow-
ing initiation its expression became restricted to the leaflet
The Plant Journal © 2016 John Wiley & Sons Ltd, The Plant Journal, (2016), 86, 443–457
(c) (d)
tip and the initiating vascular tissue (Ben-Gera et al., 2012;
Figure 5a). In pFIL>>SlARF10Am and pFIL>>SlARF17m
leaves, maxima of pDR5::VENUS signal preceded each
leaflet or lobe initiation and accumulated at their tips fol-
lowing initiation, in a similar pattern to wild-type (Fig-
ure 5b,c). In contrast, in miR160-overexpressing leaves,
pDR5::VENUS expression was expanded to throughout the
leaf margin (Figure 5d). These results suggest that the
miR160-targeted ARFs function to spatially restrict auxin
response and lamina generation between leaflets, enabling
the formation of distinct leaflets.
ENTIRE and SlARF10A jointly restrict lamina generation
between leaflets
The results presented above suggest that miR160-targeted
ARFs inhibit auxin response and lamina generation
between leaflets and in leaflet margins. A similar role was
shown for the Aux/IAA protein ENTIRE (E)/SlIAA9 (Koenig
et al., 2009; Ben-Gera et al., 2012). This suggests that, in
this case, Aux/IAAs and ARFs have similar rather than
antagonistic functions, and raises the question of the rela-
tionship between these factors in mediating the effect of
auxin in leaf patterning. We addressed this question genet-
ically by combining transgenes that affect E and SlARF10A
activity (Figure 6). Overexpression of a stabilized form of E
(EdIIm) in leaves alone, or as a fusion with the GUS repor-
ter, leads to smaller leaves with narrower lamina. Interest-
ingly, overexpression of EdIIm and SlARFm both lead to
lamina narrowing, but the general appearance of the leaf is
different (Koenig et al., 2009; Ben-Gera et al., 2012) (Fig-
ure 6b,c). In addition, EdIIm overexpression affects lamina
width strongly under both promoters, while SlARFm
© 2016 The Authors

(a) (b)
Figure 5. Auxin response is expended to throughout the leaf margin, including the intercalary space, in pFIL>>miR160 leaf primordia.
Confocal images showing pDR5::VENUS expression (green) in P5-staged leaf primordia of the genotypes indicated at the bottom left corner of each panel. Scale
bars: 250 lm.
overexpression leads to a stronger phenotype in early leaf
development under the control of the pFIL promoter, and
affects lamina width more mildly when expressed at later
stages of leaf development under the control of the pBLS
promoter. These differences correspond to the different
expression domains of these factors in developing leaf pri-
mordia, where E expression is stronger between initiating
leaflets (Ben-Gera et al., 2012), while SlARF10A and
SlARF17 expression is stronger in leaflets. Leaves that co-
expressed EdIIm-GUS and miR160 under the control of the
FIL promoter, or leaves that co-expressed EdIIm and miR160
under the control of the BLS promoter, had wider leaflets
than leaves that overexpressed only EdIIm and were more
compound than leaves that overexpressed only miR160
due to distinct primary and secondary leaflets formation
(Figures 4 and 6d,e). entire (e) loss-of-function mutants
have simplified leaves due to ectopic lamina growth, simi-
lar to miR160-overexpressing leaves (Koenig et al., 2009;
Ben-Gera et al., 2012; Figure 6a). The e phenotype is
almost completely epistatic to that of pFIL>>SlARF10Am
(Figure 6f). Thus, the effect of EdIIm is reduced in the back-
ground of miR160 and the effect of SlARF10A is sup-
pressed in the background of e. These results suggest that
both SlARF10A and ENTIRE partially depend on each other
for their function in restricting lamina generation between
leaflets. Nevertheless, overexpression of EdIIm partially
enabled distinct leaflet formation upon downregulation of
miR160-targeted ARFs expression, while SlARF10Am activ-
ity could not compensate for the reduction of E activity in
this process. Leaves that co-expressed EdIIm and SlAR-
F10Am show a slight enhancement of both phenotypes
(Figure 6g). Leaves that co-expressed e and miR160 show
© 2016 The Authors
The Plant Journal © 2016 John Wiley & Sons Ltd, The Plant Journal, (2016), 86, 443–457
miR160-targeted ARFs promote leaflet separation 449
(c) (d)
an enhanced phenotype of very simple leaves that are
composed of a single, continuous blade (Figure 6h), sug-
gesting that E and SlARF10A act via partially separate path-
ways. Inspection of early development of e pFIL>>miR160
shows continuous lamina forming throughout the leaf
margin and lack of initiation of distinct leaflets from very
early stages of leaf development (Figure 6i). Cumulatively,
these results suggest that E and miR160-targeted ARFs are
both required to restrict lamina generation, and act in this
process via partly separate pathways.
SlARF10A and ENTIRE do not interact in yeaste
We used yeast two-hybrid assays to examine possible
interactions between ENTIRE and miR160-targeted ARFs,
as well as among these ARFs. E showed strong interaction
with itself and SlARF10A showed a weaker interaction with
itself (Figure 7a,b). We did not observe interactions
between E and the examined miR160-targeted ARFs (Fig-
ure 7). This result is in agreement with previous research
suggesting that Aux/IAAs and repressing ARFs do not
interact directly in vitro (Vernoux et al., 2011; Guilfoyle and
Hagen, 2012). Therefore, while both E and miR160-targeted
ARFs repress auxin response and lamina growth between
leaflets, and depend on each other for their activity, they
likely do not directly interact, or interact via a third partner.
GOBLET mediates SlARF10A activity
Leaflet specification and separation in tomato was also
found to be regulated by the NAM transcription factor
GOBLET (GOB), which is expressed in leaflet boundaries
(Blein et al., 2008; Berger et al., 2009). To understand the
relationship between SlARF10A and GOB in leaf
450 Hadas Ben-Gera et al.
(a) (b) (c)
(d) (e) (f)
(g) (h) (i)
Figure 6. SlARF10A and ENTIRE depend on each other for their activity.
(a–h) Mature fifth leaves of the genotypes indicated on the bottom left cor-
ner of each panel. (i) A P4-staged leaf primordium of e pFIL>>miR160 cap-
tured by a stereoscope. Scale bars: 2 cm (a–h), 1 mm (i).
development, we examined genotypes with altered activity
of both GOB and SlARF10A. Leaves that overexpressed
miR164, the negative regulator of GOB, are simplified with
only primary leaflets and smooth margins (Berger et al.,
2009; Figure 6c). Strikingly, when SlARF10Am was overex-
pressed in the background of miR164 overexpression
under the control of pFIL, the miR164-overexpression phe-
notype was completely epistatic to overexpression of
SlARF10Am (Figure 8e), suggesting that GOB is required
for SlARF10A activity. Leaves that overexpressed both a
miR164-resistant GOB form (GOBm) and miR160 developed
separated primary and secondary round leaflets (Fig-
ure 8f), resembling in shape leaflets of GOBm-overexpres-
sing leaves (Figure 8d). Thus, the activity of GOBm was
The Plant Journal © 2016 John Wiley & Sons Ltd, The Plant Journal, (2016), 86, 443–457
(a)
(b)
(c)
Figure 7. ENTIRE does not interact with SlARF10A, SlARF10B and SlARF17
in yeast two-hybrid assays.
(a–c) Yeast two-hybrid experiments demonstrating the interaction between
ENTIRE, SlARF10A, SlARF10B and SlARF17. Each panel shows half a Petri
dish, divided into three sections, each representing a different protein com-
bination. Colony growth and blue colour indicate positive interaction. A
strong positive interaction is shown for ENTIRE 9 ENTIRE (a) and a weaker
interaction for SlARF10A 9 SlARF10A, as reflected by the relatively low
number of colonies and by the orange colour resulting from the lack of ade-
nine in the medium (b). All other protein combinations did not interact in
this assay.
sufficient to rescue the process of leaflet separation even
when the activity of miR160-targeted ARFs was down regu-
lated. Leaves that co-expressed GOBm and SlARF10Am
show suppression of both phenotypes, but retain charac-
teristics of GOBm overexpression (Figure 8g), and co-
expression of miR160 and miR164 led to variable pheno-
types that were similar or more enhanced than the miR160
overexpression phenotype (Figure 8h). Together, these
results suggest that GOB mediates the activity of SlARF10A
in leaflet patterning. The slightly enhanced phenotype of
co-expression of miR160 and miR164 implies that GOB can
also be a mediator of additional genetic pathways that reg-
ulates leaflet separation. To examine whether GOB is a tar-
get of SlARF10A, we used qRT-PCR to analyze its relative
© 2016 The Authors

(a) (b)
(e) (f)
Figure 8. SlARF10A activity in leaf development is mediated by GOBLET.
(a–h) Mature fifth leaves of the indicated genotypes. Scale bars: 2 cm.
expression in genotypes that expressed different levels of
SlARF10A: pFIL>>miR160, wild-type, FIL>>SlARF10Am and
pFIL>>SlARF10Am-SRDX and pFIL>>SlARF10Am-VP16 (see
below) (Figure S7). GOB expression was similar among
these genotypes, indicating that although GOB is develop-
mentally required for SlARF10A activity in leaf patterning,
it is not a target of SlARF10A.
SlARF10A acts mainly as a transcriptional repressor in
tomato leaf patterning
Arabidopsis miR160-targeted ARFs have been classified as
transcriptional repressors based on the amino acids that
compose their MR (Tiwari et al., 2003; Guilfoyle and
Hagen, 2007), and tomato SlARF10A and SlARF17 have
been recently shown to inhibit the activating effect of auxin
on the expression of pDR5::GFP in co-transfection assays
in tobacco protoplasts (Zouine et al., 2014). To test geneti-
cally whether the activity of SlARF10A in inhibiting lamina
generation represents transcriptional activation or repres-
sion, we expressed fusions of SlARF10Am with either the
© 2016 The Authors
The Plant Journal © 2016 John Wiley & Sons Ltd, The Plant Journal, (2016), 86, 443–457
miR160-targeted ARFs promote leaflet separation 451
(c) (d)
(g) (h)
activation domain from herpes simplex virus protein VP16
(Sadowski et al., 1988; Busch et al., 1999), or the plant
repression domain SRDX (Hiratsu et al., 2003) in leaves
(Figure 9). The leaf phenotype of pFIL>>SlARF10Am-SRDX
and pBLS>>SlARF10Am-SRDX was similar to, but more
severe than, that of plants that overexpressed the unfused
SlARF10Am from the same promoters (Figure 9a,b).
pFIL>>SlARF10Am-VP16 and pBLS>>SlARF10Am-VP16
leaves showed a range of phenotypes but in general all
transgenic lines had similar or milder phenotypes in com-
parison with pFIL>>SlARF10Am and pBLS>>SlARF10Am,
respectively (Figures 9c,d and S8). These results suggest
that in the context of tomato leaf patterning, SlARF10A acts
mainly as a transcriptional repressor: fusion to the SRDX
enhanced its repressing effect, while the VP16 fusion par-
tially compromised the repression activity of SlARF10Am.
DISCUSSION
Leaf patterning involves precise spatial and temporal local-
ization of lamina growth aided by auxin signals. Here we
452 Hadas Ben-Gera et al.
(a) (b)
(c) (d)
(e) (f)
Figure 9. SlARF10A acts mainly as a transcriptional repressor in tomato leaf
patterning.
(a–f) Mature fifth leaves of the indicated genotypes. Scale bars: 2 cm.
show that miR160-targeted ARFs affect leaf patterning by
modulating lamina generation. Several recent reports have
suggested that mechanisms of leaflet separation involve
juxtaposition of regions with lamina growth and regions
where growth is inhibited, determined in part by dispersed
expression of growth regulators. In Arabidopsis leaf mar-
gins, local growth inhibition by specific expression of
KRP1, led to the formation of deep lobes (Malinowski
et al., 2011). The C. hirsuta homeodomain protein RCO is
expressed specifically in leaflet bases where it inhibits
growth and consequently promotes leaflet separation. RCO
is thought to have arisen via gene duplication and acquisi-
tion of specific expression in leaflet bases. It was lost in
Arabidopsis, which has simple leaves, while its paralogue
LMI1 exists in both species but is not expressed in leaflet
The Plant Journal © 2016 John Wiley & Sons Ltd, The Plant Journal, (2016), 86, 443–457
bases (Vlad et al., 2014). Variations in leaf lobing and serra-
tions within the genus Capsella (shepherd’s purse) was
found involve, differences in CrRCO-A alleles (Sicard et al.,
2014). Here, miR160-targeted ARFs are shown to promote
leaflet separation and leaf complexity. SlARF10A and
SlARF17 expression is not restricted to the leaflet bases
(Figure 2) (Hendelman et al., 2012). Therefore, its growth-
inhibiting effect probably depends on the level of expres-
sion and its interaction with additional factors that pattern
growth. Leaflet separation requires that auxin maxima at
the leaf margin will be separated by areas without auxin
(Barkoulas et al., 2008; DeMason and Polowicky, 2009;
Koenig et al., 2009; Bilsborough et al., 2011; Ben-Gera
et al., 2012). In Arabidopsis fruits, the transcription factor
INDEHISCENT (IND) is specifically expressed at the silique
valve margins, where it promotes local auxin depletion,
stimulating fruit opening (Sorefan et al., 2009). ENTIRE
and miR160-targeted ARFs may be involved in generating
a similar active local auxin response inhibition in the pro-
cess of leaflet separation.
Overexpression of SlARF10Am or SlARF17m in the leaf
under the control of the FIL promoter did not repress the
establishment of auxin maxima and leaflet initiation. This
result may suggest that the leaflet initiation sites and the
intercalary spaces represent two different ‘developmental
domains’ that utilize different mediators of auxin response,
and that SlARF10A activity is specific to the intercalary
space. Alternatively, SlARF10A may act only after leaflet
initiation, or act in a somewhat quantitative manner.
Expression of SlARF10Am and SlARF17m under the control
of native promoters may complete this picture.
Specification of boundaries between leaflets in tomato
leaves depends on the activity of the GOB gene, which
marks the sites of leaflet initiation in the leaf margin, and
properly spaced GOB expression was found to promote
leaflet separation (Blein et al., 2008; Berger et al., 2009).
Auxin was shown to mediate the activity of GOB in leaf
patterning, and E expression is affected by GOB (Ben-Gera
et al., 2012). Co-expression of miR164 almost completely
suppressed the SlARF10Am overexpression phenotype,
and miR164-resistant GOB substantially suppresses the
miR160 overexpression phenotype. Taken together, these
results suggest that, genetically, SlARF10A acts upstream
of GOB. As GOB and E were shown to redundantly affect
auxin response and leaflet separation (Ben-Gera et al.,
2012), and E and SlARF10A are shown here to act together
but partially redundantly in this process, these factors
appear to be involved in a complex feedback mechanism
(Figure 10). Comparison of the effects of altering the activ-
ity of GOB, E and miR160-targeted ARFs on leaf marginal
patterning and auxin response suggests that they affect
different aspects of this process. While in pFIL>>miR164
leaves the initiation of primary leaflets and auxin maxima
were not affected (Ben-Gera et al., 2012), in pFIL>>miR160
© 2016 The Authors
 miR160-targeted ARFs promote leaflet separation 453

Figure 10. A scheme showing the proposed activity of GOBLET, ENTIRE and SlARF10A in the inhibition of auxin response and lamina generation between leaf-
lets, enabling leaflet separation.
Specification of discrete leaflets requires local areas of growth adjacent to areas where growth is inhibited. In green is an illustration of longitudinal side view of
the leaf. Bulges represent leaflets and the gap between them represents the intercalary apace. Yellow circles indicate the auxin maxima located at the leaflets
initiation area, leading to lamina growth. In the intercalary space, GOBLET, ENTIRE and SlARF10A actively and locally repress auxin response, to promote leaflet
separation.

and e mutants the auxin signal was expanded to through-
out the leaf margin and primary leaflets were fused. In addi-
tion, overexpression of GOB, E and miR160-targeted ARFs
led to distinct phenotypes. These findings may imply that
these factors regulate different aspects of auxin response.
This is in agreement with the expression patterns of these
genes: while GOB is expressed in narrow stripes in the
boundaries between leaflets and the leaf margin (Berger
et al., 2009), E is expressed throughout the region between
initiating leaflets (Ben-Gera et al., 2012), and the expression
of SlARF10A and SlARF17 is not specific to this region. In
Arabidopsis leaves, the GOB ortholog CUC2 was found to
facilitate the establishment of auxin maxima in serration
development by reorientation of the auxin efflux carrier
PINFORMED1 (PIN1) (Bilsborough et al., 2011). This raises
the possibility that GOB might be a mediator of auxin distri-
bution also in tomato leaves. While GOB activity was found
to be developmentally required for SlARF10 activity, GOB
expression did not change in pFIL>>miR160, pFIL>>SlAR-
F10Am, pFIL>>SlARF10Am-SRDX, indicating that GOB is not
a target of SlARF10A. miR164 overexpression was found to
suppress the phenotypes of several tomato mutants with
increased leaf complexity (Bar et al., 2015) and to enhance
the phenotype of several mutants with simplified leaves
(Busch et al., 2011; Ben-Gera et al., 2012). Therefore, GOB
emerges as a central regulator of leaflet separation, on
which the effects of many other regulators of leaf complex-
ity converge.
Comparison of the phenotypes caused by expression of
a miR160-resistant SlARF10A and its SRDX and VP16
fusions suggests that in the context of tomato leaf devel-
opment SlARF10A acts mainly as a transcriptional repres-
sor. The similar but milder phenotype of SlARF10Am-VP16
suggests that the transcriptional repression activity of
SlARF10Am competes with the activation activity of VP16,
as also reported previously (Ohta et al., 2001; Tiwari et al.,
2004; Pekker et al., 2005). Repressor domains of Aux/IAAs
were previously shown to dominate activator domains of
VP16 or ARF5 when combined in the same protein (Tiwari
et al., 2004). The repressor activity of miR160-targeted
ARFs is also supported by the expansion of the DR5 signal
in pFIL>>miR160 leaf primordia. Alternatively, SlARF10A
could act as both a transcriptional activator and repressor.
Such a dual role has been shown for some plant transcrip-
tion factors (Bonaccorso et al., 2012). It was also proposed
that the same ARF can act as transcriptional activator or
repressor depending on its interacting partners (Guilfoyle
et al., 1998). Loss of both induction and repression of gene
expression by auxin in several Arabidopsis ARF mutants
also indicates that ARF proteins can act as both activators
and repressors (Okushima et al., 2005; Lokerse and Wei-
jers, 2009). Arabidopsis ARF16 was shown to activate
auxin-responsive genes as well as DR5 in roots (Wang
et al., 2005), and complex results were reported with
respect to the activity of ARF17 (Mallory et al., 2005; Yang
et al., 2013). In Arabidopsis, the miR160-targeted ARFs
comprise a distinct subgroup of the ARFs proteins, and the
known repressing ARFs were found to be closer to the acti-
vating ARFs than to the miR160-targeted ARFs (Remington
et al., 2004). Furthermore, they do not contain known
repressor domains (Lokerse and Weijers, 2009). These find-
ings suggest that the miR160-targeted ARFs may represent
a specialized ARF subclass.
The basic model for the auxin signal transduction is com-
patible with the activity of activating ARFs, and the activity
of repressing ARFs in the context of this model is unclear.
Repressor ARFs were proposed to act by competition with
activating ARFs on binding to the promoters of auxin-

© 2016 The Authors

The Plant Journal © 2016 John Wiley & Sons Ltd, The Plant Journal, (2016), 86, 443–457
454 Hadas Ben-Gera et al.
responsive genes, direct repression of target genes, or
direct repression of activator ARFs (Tiwari et al., 2003; Guil-
foyle and Hagen, 2007; Lokerse and Weijers, 2009; Vernoux
et al., 2011; Pierre-Jerome et al., 2013). The interaction
between repressing ARFs and Aux/IAAs and consequently
their auxin-responsiveness is also unclear. Aux/IAAs and
repressor ARFs in general do not interact in yeast two-
hybrid assays (Vernoux et al., 2011; Guilfoyle and Hagen,
2012), and Aux/IAAs were so far shown to act only as nega-
tive regulators of auxin response. In agreement, we did not
detect an interaction between E and miR160-targeting ARFs
in yeast two-hybrid assays. E and miR160-targeted ARFs
both appear to inhibit auxin response and lamina growth
between leaflets, thus promoting leaflet separation. Fur-
thermore, E and SlARF10A depend on each other for their
activity. It thus seems that SlARF10A and E act coopera-
tively, rather than antagonistically, in leaf patterning. How
could they cooperate without physical interaction? One pos-
sibility is that they interact via a third partner. Alternatively,
they could affect the same targets independently. For exam-
ple, they may both inhibit the same activating ARF, or E
could inhibit the transcription of a target gene through inhi-
bition of an activating ARF, and SlARF10A could directly
inhibit the transcription of the same target. However, the
enhanced phenotype of the combined overexpression of
miR160 and the e mutant suggests that E and miR160-
targeted ARFs also act partially redundantly.
In conclusion, we show that leaflet formation depends
on the inhibition of auxin response between initiating leaf-
lets, by ENTIRE, miR160-targeted ARFs and GOBLET (Fig-
ure 10). This active mechanism is required for the
formation of distinct leaflets.
EXPERIMENTAL PROCEDURES
Plant material
Tomato seeds (Solanum lycopersicum cv. M82, sp) were sown in
a commercial nursery, and transferred to a greenhouse under nat-
ural daylight. For in situ hybridization and expression analyses,
plants were grown in a growth chamber under an 18 h/6 h light/
dark regimen. The previously described e-3 allele (Berger et al.,
2009; Ben-Gera et al., 2012) was used for the genetic analysis.
Transgenic genotypes were generated by the LhG4 transactivation
system (Moore et al., 1998). The Arabidopsis pFIL promoter drives
expression throughout young developing tomato leaves and other
lateral organs and the pBLS promoter drives expression at later
stages of leaf development, from the P4 stage, in developing
leaflets (Lifschitz et al., 2006; Shani et al., 2009). The following
transgenic genotypes were described previously: Tomato (M82,
sp) plants expressing pDR5::VENUS, pFIL>>iaaM, pFIL>>EdIIm-
GUS, pBLS>>EdIIm (Ben-Gera et al., 2012), pFIL>>GOBm and
pFIL>>miR164 (Berger et al., 2009).
Cloning and plant transformation
To generate op:SlARF10Am, op:SlARF10Bm and op:SlARF17m we
amplified each gene from cDNA (M82 background), and used
The Plant Journal © 2016 John Wiley & Sons Ltd, The Plant Journal, (2016), 86, 443–457
assembly PCR to introduce silent mutations (see Table S1) at the
miR160-recognition site of each gene, downstream to an Operator
array (Moore et al., 1998). At least five independent lines from
each transgene were crossed to the driver lines, examined, and a
representative line was selected for further analysis. In the case of
SlARF17m, different lines showed a range of phenotypes, shown
in Figure S2. To generate op:SlARF10Am-SRDX, complementary
single-stranded DNA fragments of the SRDX RD (see Table S1)
were mixed together for the annealing reaction (50 lM of each
fragment were mixed to make 1:1 ratio at final volume of 50 ll,
denaturation 10 min at 60°C, annealing 1 h at 25°C). The DNA
fragments of the SRDX sequence used for the annealing reaction
are indicated in Table S1. The VP16 activation domain was origi-
nally amplified from the pRG50 plasmid (Sadowski et al., 1988;
Parcy et al., 1998; Pekker et al., 2005). We used ligation reactions
in order to ligate the SRDX or the VP16 to the C-terminus of the
op:SlARF10Amsequence through the KpnI restriction site. To gen-
erate op:miR160, we amplified the miR160A (AT2G39175) from
Arabidopsis Ler genomic DNA using the primers shown in
Table S1. For yeast two-hybrid experiments, we amplified the
genes ENTIRE, SlARF10A, SlARF10B and SlARF17 from tomato
cDNA (M82 background, sp). Each of the amplified genes was
cloned into the entry vector pENTR D-TOPO of the Gateway sys-
tem (Invitrogen, Carlsbad, CA, USA). The clones were LR-crossed
into the gateway yeast two-hybrid destination vectors pDEST32
(BD; LEU) and pDEST22 (AD; Trp) (Invitrogen). The primers used
for cloning are indicated in Table S1.
Quantification of phenotypes and statistical analysis
Quantification of lamina narrowing: the fifth leaf of at least six
plants of each genotype was photographed and analyzed using
the ImageJ software, and the ratio between the lamina area to the
leaf perimeter of each leaf was calculated. Leaflets quantification:
Leaflets of the sixth leaf of 2-month-old plants were counted.
Averages and standard errors of each genotype were calculated
from six different plants. Statistical analysis was performed using
JMP software (SAS Institute, http://www.sas.com). The Student’s
t-test was used for comparison of means, which were deemed sig-
nificantly different at a P < 0.05.
Exogenous auxin application
Sterile wild-type and pFIL>>SlARF10Am seeds were sown on MS
medium [0.44% w/v MS (M-0222.0050; Duchefa, http://www.
duchefa.com), 1% sucrose w/v, 0.75% plant agar (Duchefa, Haar-
lem, the Netherlands), 1 mM Plant Preservative Mixture (Caisson
Laboratories, Smithfield, UT, USA)]. Seven-day-old seedlings were
cut 2 cm beneath the SAM, and vegetative shoot apices were
transplanted to MS medium containing 1 lM 2,4-D (Sigma-Aldrich
http://www.sigmaaldrich.com), or a mock treatment, for 4 weeks.
Tissue collection and RNA analysis
Second leaf primordia at the stage of P5 were collected and trans-
ferred directly into liquid nitrogen. At least three biological repeats
were collected for each genotype, each containing six leaves. RNA
was extracted using the Plant/Fungi Total RNA Purification Kit
(Norgen Biotek, Thorold, ON, Canada) according to the manufac-
turer’s instructions, including DNase treatment. cDNA synthesis
was performed using the Verso cDNA Kit (Thermo Scientific, Wal-
tham, MA, USA) with 1 lg of RNA. Tissue collection and RNA
extraction for dynamic expression of SlARF10A at successive
stages of wild-type leaf development was performed as described
(Shleizer-Burko et al., 2011).
© 2016 The Authors

RNA in situ hybridization was performed as described (Shani
et al., 2010). The SlARF10A and SlARF17 probes were synthesized
with UTP-DIG and expression of miR160 was detected using a
SlmiR160-LNA probe (Table S1) labeled with DIG at the 30 and 50
ends (Exiqon, Vedbaek, Denmark). Quantitative RT-PCR analysis
was performed using a Rotor-Gene Q device from Qiagen and
Absolute Blue qPCR SYBR Green ROX Master mix (ABgene,
Epsom, UK). Levels of mRNA were calculated relative to the gene
EXPRESSED as an internal control, which has been shown to be
expressed at a similar level throughout tomato development
(Exposito-Rodriguez et al., 2008).
Imaging, microscopy and GUS staining
Intact leaves were photographed using a Nikon D5200 camera.
Images of early leaf development were captured using an Olym-
pus SZX7 stereoscope microscope equipped with a Nikon
DXM1200 camera and ACT-1 software or by Nikon SMZ1270 stere-
oscope with a Nikon DS-Ri2 camera and NIS-element software.
For pDR5::VENUS expression, dissected whole-leaf primordia
were placed into drops of water on glass microscope slides and
covered with cover slips. The pattern of VENUS expression was
detected by a confocal laser scanning microscope (CLSM model
SP8; Leica, Mannheim, Germany), with the solid-state laser set at
514 nm for excitation and 530 nm for emission. Chlorophyll aut-
ofluorescence was detected at 488 nm for excitation and 700 nm
for emission. LAS AF and ImageJ software were used for analysis
of captured images. Scanning electron microscopy was performed
using a JEOL 5410 LV microscope as described (Brand et al.,
2007). Images were manipulated uniformly using Adobe Photo-
shop CS6 software (Adobe Systems Inc., San Jose, CA, USA).
Yeast two-hybrid assays
The PJ69-4 yeast strain was transformed following CLONTECH
small-scale LiAc manual (www.clontech.com/xxclt_ibcGetAttach-
ment.jsp?cItemId=17602). We first transformed the pDEST32, by
performing separate transformations for each of the genes: E,
SlARF10A, SlARF10B and SlARF17, and transformed yeast lines
were selected on Sabouraud Dextrose (SD) agar plates without
tryptophan. Each transformed yeast line was transformed with
each of the genes in the pDEST22 plasmid in order to have all
genes combinations (ExE, ExSlARF10, ExSlARF10B, ExSlARF17,
SlARF10AxSlARF10A, SlARF10AxSlARF10B, SlARF10AxSlARF17,
SlARF10BxSlARF10B, SlARF10BxSlARF17, SlARF17xSlARF17). As
a control, we co-introduced an empty pDEST32 vector with
each of the genes in pDEST22 vector. Transgenic yeast lines
containing both plasmids were selected on SD agar plates with-
out tryptophan and without leucine. Transgenic yeasts contain-
ing putatively interacting proteins were selected on SD agar
plates without histidine, leucine, and tryptophan (His/Leu/
Trp) and also on SD agar plates without adenine, histidine,
leucine, and tryptophan (Ade/His/Leu/Trp) for more strin-
gent selection of positive interactions. The SD agar plates also
contained b-galactosidase for the in vivo blue colour assay.
Positive interaction was considered only for transgenic lines
that grew on SD agar plates without adenine, histidine, leucine,
and tryptophan (Ade/His/Leu/Trp) and showed a blue or
light blue colour.
ACCESSION NUMBERS
Sequence data used in this study can be found in the Sol
Genomic Network (SGN) database under the following
accession numbers: SlARF10A (Solyc11g069500); SlARF10B
© 2016 The Authors
The Plant Journal © 2016 John Wiley & Sons Ltd, The Plant Journal, (2016), 86, 443–457
miR160-targeted ARFs promote leaflet separation 455
(Solyc06g075150); SlARF17 (Solyc11g013480 [Nter] + Soly-
c11g013470 [Cter]); AtmiR160A (AT2G39175); ENTIRE (Soly-
c04g076850); GOBLET (Solyc07g062840); EXPRESSED
(Solyc07g025390).
ACKNOWLEDGEMENTS
We would like to thank Yuval Eshed for the miR160 driver line
and, the VP16 construct, Tzahi Arazi and Yuval Eshed for critical
reading of the manuscript, Dario Breitel and Louise Maor for help-
ing with the yeast two-hybrid procedure, Shiri Goldental for tech-
nical assistance, and members of our groups for fruitful
discussions. This research was supported by grants from the
Israel Science Foundation (539/14), US–Israel Binational Agricul-
tural Research and Development Fund (IS 4531-12(c)), The Israeli
ministry of Agriculture (837-0140-14) and German–Israel Project
Cooperation Foundation (OR309/1-1; FE552/12-1).
SUPPORTING INFORMATION
Additional Supporting Information may be found in the online ver-
sion of this article.
Figure S1. Silent mutations at the miR160 recognition site of
SlARF10A, SlARF10B and SlARF17, as used for the transgenic
plants.
Figure S2. The phenotypic range in different transgenic lines over-
expressing SlARF17m.
Figure S3. Quantification of lamina narrowing and leaf complexity
in leaves overexpressing SlARF10A, SlARF10B and SlARF17.
Figure S4. Dynamic expression of SlARF10A mRNA during wild-
type leaf development.
Figure S5. Modulation of the expression of miR160-targeted ARFs
by miR160.
Figure S6. Phenotype of young developing leaf primordia overex-
pressing miR160.
Figure S7. GOBLET expression is not affected by SlARF10A.
Figure S8. Variation of leaf phenotypes between different lines of
SlARF10Am-VP16.
Table S1. Primers used in this study.
REFERENCES
Bar, M. and Ori, N. (2015) Compound leaf development in model plant spe-
cies. Curr. Opin. Plant Biol. 23, 61–69.
Bar, M., Ben-Herzel, O., Kohay, H., Shtein, I. and Ori, N. (2015) CLAUSA
restricts tomato leaf morphogenesis and GOBLET expression. Plant J.
83, 888–902.
Barkoulas, M., Galinha, C., Grigg, S.P. and Tsiantis, M. (2007) From genes
to shape: regulatory interactions in leaf development. Curr. Opin. Plant
Biol. 10, 660–666.
Barkoulas, M., Hay, A., Kougioumoutzi, E. and Tsiantis, M. (2008) A devel-
opmental framework for dissected leaf formation in the Arabidopsis rela-
tive Cardamine hirsuta. Nat. Genet. 40, 1136–1141.
Ben-Gera, H., Shwartz, I., Shao, M.R., Shani, E., Estelle, M. and Ori, N.
(2012) ENTIRE and GOBLET promote leaflet development in tomato by
modulating auxin response. Plant J. 70, 903–915.
Berger, Y., Harpaz-Saad, S., Brand, A., Melnik, H., Sirding, N., Alvarez, J.P.,
Zinder, M., Samach, A., Eshed, Y. and Ori, N. (2009) The NAC-domain
transcription factor GOBLET specifies leaflet boundaries in compound
tomato leaves. Development, 136, 823–832.
Bilsborough, G.D., Runions, A., Barkoulas, M., Jenkins, H.W., Hasson, A.,
Galinha, C., Laufs, P., Hay, A., Prusinkiewicz, P. and Tsiantis, M. (2011)
Model for the regulation of Arabidopsis thaliana leaf margin develop-
ment. Proc. Natl Acad. Sci. USA, 108, 3424–3429.
Blein, T., Pulido, A., Vialette-Guiraud, A., Nikovics, K., Morin, H., Hay, A.,
Johansen, I.E., Tsiantis, M. and Laufs, P. (2008) A conserved molecu-
lar framework for compound leaf development. Science, 322, 1835–
1839.
456 Hadas Ben-Gera et al.
Blein, T., Hasson, A. and Laufs, P. (2010) Leaf development: what it needs to
be complex. Curr. Opin. Plant Biol. 13, 75–82.
Boer, D.R., Freire-Rios, A., van den Berg, W.A. et al. (2014) Structural basis
for DNA binding specificity by the auxin-dependent ARF transcription
factors. Cell, 156, 577–589.
Bonaccorso, O., Lee, J.E., Puah, L., Scutt, C.P. and Golz, J.F. (2012) FILA-
MENTOUS FLOWER controls lateral organ development by acting as
both an activator and a repressor. BMC Plant Biol. 12, 176.
Brand, A., Shirding, N., Shleizer, S. and Ori, N. (2007) Meristem mainte-
nance and compound-leaf patterning utilize common genetic mecha-
nisms in tomato. Planta, 226, 941–951.
Busch, M.A., Bomblies, K. and Weigel, D. (1999) Activation of a floral
homeotic gene in Arabidopsis. Science, 285, 585–587.
Busch, B.L., Schmitz, G., Rossmann, S., Piron, F., Ding, J., Bendahmane, A.
and Theres, K. (2011) Shoot branching and leaf dissection in tomato are
regulated by homologous gene modules. Plant Cell, 23, 3595–3609.
Byrne, M.E., Barley, R., Curtis, M., Arroyo, J.M., Dunham, M., Hudson, A.
and Martienssen, R.A. (2000) Asymmetric leaves1 mediates leaf pattern-
ing and stem cell function in Arabidopsis. Nature, 408, 967–971.
Calderon Villalobos, L.I., Lee, S., De Oliveira, C. et al. (2012) A combinatorial
TIR1/AFB-Aux/IAA co-receptor system for differential sensing of auxin.
Nat. Chem. Biol. 8, 477–485.
Chandler, J.W. (2016) Auxin response factors. Plant, Cell Environ. 39, 1014–
1028.
Cheng, X., Peng, J., Ma, J., Tang, Y., Chen, R., Mysore, K.S. and Wen, J. (2012)
NO APICAL MERISTEM (MtNAM) regulates floral organ identity and lateral
organ separation in Medicago truncatula. New Phytol. 195, 71–84.
Chitwood, D.H., Headland, L.R., Filiault, D.L., Kumar, R., Jimenez-Gomez,
J.M., Schrager, A.V., Park, D.S., Peng, J., Sinha, N.R. and Maloof, J.N.
(2012) Native environment modulates leaf size and response to simu-
lated foliar shade across wild tomato species. PLoS ONE, 7, e29570.
Chitwood, D.H., Kumar, R., Headland, L.R. et al. (2013) A quantitative
genetic basis for leaf morphology in a set of precisely defined tomato
introgression lines. Plant Cell, 25, 2465–2481.
DeMason, D.A. and Polowicky, P.L. (2009) Patterns of DR5:GUS expression
in organs of Pea (Pisum sativum). Int. J. Plant Sci. 170, 1–11.
Dengler, N.G. and Tsukaya, H. (2001) Leaf morphogenesis in dicotyledons:
current issues. Int. J. Plant Sci. 162, 459–464.
Dharmasiri, N., Dharmasiri, S. and Estelle, M. (2005) The F-box protein TIR1
is an auxin receptor. Nature, 435, 441–445.
Efroni, I., Eshed, Y. and Lifschitz, E. (2010) Morphogenesis of simple and
compound leaves: a critical review. Plant Cell, 22, 1019–1032.
Exposito-Rodriguez, M., Borges, A.A., Borges-Perez, A. and Perez, J.A.
(2008) Selection of internal control genes for quantitative real-time RT-
PCR studies during tomato development process. BMC Plant Biol. 8, 131.
Floyd, S.K. and Bowman, J.L. (2010) Gene expression patterns in seed plant
shoot meristems and leaves: homoplasy or homology? J. Plant. Res.
123, 43–55.
Gray, W.M., Kepinski, S., Rouse, D., Leyser, O. and Estelle, M. (2001) Auxin
regulates SCF(TIR1)-dependent degradation of AUX/IAA proteins. Nature,
414, 271–276.
Guilfoyle, T.J. and Hagen, G. (2007) Auxin response factors. Curr. Opin.
Plant Biol. 10, 453–460.
Guilfoyle, T.J. and Hagen, G. (2012) Getting a grasp on domain III/IV respon-
sible for Auxin Response Factor-IAA protein interactions. Plant Sci. 190,
82–88.
Guilfoyle, T.J., Ulmasov, T. and Hagen, G. (1998) The ARF family of tran-
scription factors and their role in plant hormone-responsive transcrip-
tion. Cell. Mol. Life Sci. 54, 619–627.
Hagemann, W. and Gleissberg, S. (1996) Organogenetic capacity of leaves:
the significance of marginal blastozones in angiosperms. Plant Syst.
Evol. 199, 121–152.
Hasson, A., Blein, T. and Laufs, P. (2010) Leaving the meristem behind: the
genetic and molecular control of leaf patterning and morphogenesis. C R
Biol. 333, 350–360.
Heisler, M.G., Ohno, C., Das, P., Sieber, P., Reddy, G.V., Long, J.A. and
Meyerowitz, E.M. (2005) Patterns of auxin transport and gene expression
during primordium development revealed by live imaging of the Ara-
bidopsis inflorescence meristem. Curr. Biol. 15, 1899–1911.
Hendelman, A., Buxdorf, K., Stav, R., Kravchik, M. and Arazi, T. (2012) Inhi-
bition of lamina outgrowth following Solanum lycopersicum AUXIN
The Plant Journal © 2016 John Wiley & Sons Ltd, The Plant Journal, (2016), 86, 443–457
RESPONSE FACTOR 10 (SlARF10) derepression. Plant Mol. Biol. 78, 561–
576.
Hiratsu, K., Matsui, K., Koyama, T. and Ohme-Takagi, M. (2003) Dominant
repression of target genes by chimeric repressors that include the EAR
motif, a repression domain, in Arabidopsis. Plant J. 34, 733–739.
Holtan, H.E. and Hake, S. (2003) Quantitative trait locus analysis of leaf dis-
section in tomato using Lycopersicon pennellii segmental introgression
lines. Genetics, 165, 1541–1550.
Kaplan, D.R. (2001) Fundamental concepts of leaf morphology and morpho-
genesis: a contribution to the interpretation of molecular genetic
mutants. Int. J. Plant Sci. 162, 465–474.
Kepinski, S. and Leyser, O. (2004) Auxin-induced SCFTIR1-Aux/IAA interac-
tion involves stable modification of the SCFTIR1 complex. Proc. Natl
Acad. Sci. USA, 101, 12381–12386.
Kieffer, M., Neve, J. and Kepinski, S. (2010) Defining auxin response con-
texts in plant development. Curr. Opin. Plant Biol. 13, 12–20.
Koenig, D. and Sinha, N. (2010) Evolution of leaf shape: a pattern emerges.
Curr. Top. Dev. Biol. 91, 169–183.
Koenig, D., Bayer, E., Kang, J., Kuhlemeier, C. and Sinha, N. (2009) Auxin
patterns Solanum lycopersicum leaf morphogenesis. Development, 136,
2997–3006.
Lifschitz, E., Eviatar, T., Rozman, A., Shalit, A., Goldshmidt, A., Amsellem,
Z., Alvarez, J.P. and Eshed, Y. (2006) The tomato FT ortholog triggers
systemic signals that regulate growth and flowering and substitute for
diverse environmental stimuli. Proc. Natl Acad. Sci. USA, 103, 6398–
6403.
Liu, P.P., Montgomery, T.A., Fahlgren, N., Kasschau, K.D., Nonogaki, H. and
Carrington, J.C. (2007) Repression of AUXIN RESPONSE FACTOR10 by
microRNA160 is critical for seed germination and post-germination
stages. Plant J. 52, 133–146.
Liu, X., Huang, J., Wang, Y., Khanna, K., Xie, Z., Owen, H.A. and Zhao, D.
(2010) The role of floral organs in carpels, an Arabidopsis loss-of-func-
tion mutation in MicroRNA160a, in organogenesis and the mechanism
regulating its expression. Plant J. 62, 416–428.
Liu, X., Zhang, H., Zhao, Y., Feng, Z., Li, Q., Yang, H.Q., Luan, S., Li, J. and
He, Z.H. (2013) Auxin controls seed dormancy through stimulation of
abscisic acid signaling by inducing ARF-mediated ABI3 activation in Ara-
bidopsis. Proc. Natl Acad. Sci. USA, 110, 15485–15490.
Lokerse, A.S. and Weijers, D. (2009) Auxin enters the matrix – assembly of
response machineries for specific outputs. Curr. Opin. Plant Biol. 12,
520–526.
Malinowski, R., Kasprzewska, A. and Fleming, A.J. (2011) Targeted manipu-
lation of leaf form via local growth repression. Plant J. 66, 941–952.
Mallory, A.C., Bartel, D.P. and Bartel, B. (2005) MicroRNA-directed regula-
tion of Arabidopsis AUXIN RESPONSE FACTOR17 is essential for proper
development and modulates expression of early auxin response genes.
Plant Cell, 17, 1360–1375.
Menda, N., Semel, Y., Peled, D., Eshed, Y. and Zamir, D. (2004) In silico
screening of a saturated mutation library of tomato. Plant J. 38, 861–872.
Moon, J. and Hake, S. (2010) How a leaf gets its shape. Curr. Opin. Plant
Biol. 14, 24–30.
Moore, I., Galweiler, L., Grosskopf, D., Schell, J. and Palme, K. (1998) A tran-
scription activation system for regulated gene expression in transgenic
plants. Proc. Natl Acad. Sci. USA, 95, 376–381.
Nikovics, K., Blein, T., Peaucelle, A., Ishida, T., Morin, H., Aida, M. and
Laufs, P. (2006) The balance between the MIR164A and CUC2 genes
controls leaf margin serration in Arabidopsis. Plant Cell, 18, 2929–
2945.
Nizampatnam, N.R., Schreier, S.J., Damodaran, S., Adhikari, S. and Subra-
manian, S. (2015) microRNA160 dictates stage-specific auxin and cytoki-
nin sensitivities and directs soybean nodule development. Plant J. 84,
140–153.
Ohta, M., Matsui, K., Hiratsu, K., Shinshi, H. and Ohme-Takagi, M. (2001)
Repression domains of class II ERF transcriptional repressors share an
essential motif for active repression. Plant Cell, 13, 1959–1968.
Okushima, Y., Overvoorde, P.J., Arima, K. et al. (2005) Functional genomic
analysis of the AUXIN RESPONSE FACTOR gene family members in Ara-
bidopsis thaliana: unique and overlapping functions of ARF7 and ARF19.
Plant Cell, 17, 444–463.
Parcy, F., Nilsson, O., Busch, M.A., Lee, I. and Weigel, D. (1998) A genetic
framework for floral patterning. Nature, 395, 561–566.
© 2016 The Authors

Park, S.J., Jiang, K., Schatz, M.C. and Lippman, Z.B. (2011) Rate of meris-
tem maturation determines inflorescence architecture in tomato. Proc.
Natl Acad. Sci. USA, 109, 639–644.
Pekker, I., Alvarez, J.P. and Eshed, Y. (2005) Auxin response factors mediate
Arabidopsis organ asymmetry via modulation of KANADI activity. Plant
Cell, 17, 2899–2910.
Peng, J. and Chen, R. (2011) Auxin efflux transporter MtPIN10 regulates
compound leaf and flower development in Medicago truncatula. Plant
Signal Behav. 6, 1537–1544.
Pierre-Jerome, E., Moss, B.L. and Nemhauser, J.L. (2013) Tuning the auxin
transcriptional response. J. Exp. Bot. 64, 2557–2563.
Rademacher, E.H., Moller, B., Lokerse, A.S., Llavata-Peris, C.I., van den
Berg, W. and Weijers, D. (2011) A cellular expression map of the Ara-
bidopsis AUXIN RESPONSE FACTOR gene family. Plant J. 68, 597–606.
Rademacher, E.H., Lokerse, A.S., Schlereth, A. et al. (2012) Different auxin
response machineries control distinct cell fates in the early plant embryo.
Dev. Cell, 22, 211–222.
Remington, D.L., Vision, T.J., Guilfoyle, T.J. and Reed, J.W. (2004) Contrast-
ing modes of diversification in the Aux/IAA and ARF gene families. Plant
Physiol. 135, 1738–1752.
Romano, C.P., Robson, P.R., Smith, H., Estelle, M. and Klee, H. (1995) Trans-
gene-mediated auxin overproduction in Arabidopsis: hypocotyl elonga-
tion phenotype and interactions with the hy6-1 hypocotyl elongation and
axr1 auxin-resistant mutants. Plant Mol. Biol. 27, 1071–1083.
Sadowski, I., Ma, J., Triezenberg, S. and Ptashne, M. (1988) GAL4-VP16 is
an unusually potent transcriptional activator. Nature, 335, 563–564.
Salehin, M., Bagchi, R. and Estelle, M. (2015) SCFTIR1/AFB-based auxin per-
ception: mechanism and role in plant growth and development. Plant
Cell, 27, 9–19.
Shani, E., Burko, Y., Ben-Yaakov, L., Berger, Y., Amsellem, Z., Goldshmidt,
A., Sharon, E. and Ori, N. (2009) Stage-specific regulation of Solanum
lycopersicum leaf maturation by class 1 KNOTTED1-LIKE HOMEOBOX
proteins. Plant Cell, 21, 3078–3092.
Shani, E., Ben-Gera, H., Shleizer-Burko, S., Burk, Y., Weiss, D. and Ori, N.
(2010) Cytokinin regulates compound leaf development in tomato. Plant
Cell, 22, 3206–3217.
Shleizer-Burko, S., Burko, Y., Ben-Herzel, O. and Ori, N. (2011) Dynamic
growth program regulated by LANCEOLATE enables flexible leaf pattern-
ing. Development, 138, 695–704.
Sicard, A., Thamm, A., Marona, C., Lee, Y.W., Wahl, V., Stinchcombe, J.R.,
Wright, S.I., Kappel, C. and Lenhard, M. (2014) Repeated evolutionary
changes of leaf morphology caused by mutations to a homeobox gene.
Curr. Biol. 24, 1880–1886.
Sluis, A. and Hake, S. (2015) Organogenesis in plants: initiation and elabora-
tion of leaves. Trends Genet. 31, 300–306.
© 2016 The Authors
The Plant Journal © 2016 John Wiley & Sons Ltd, The Plant Journal, (2016), 86, 443–457
miR160-targeted ARFs promote leaflet separation 457
Sorefan, K., Girin, T., Liljegren, S.J., Ljung, K., Robles, P., Galvan-Ampudia,
C.S., Offringa, R., Friml, J., Yanofsky, M.F. and Ostergaard, L. (2009) A
regulated auxin minimum is required for seed dispersal in Arabidopsis.
Nature, 459, 583–586.
Szemenyei, H., Hannon, M. and Long, J.A. (2008) TOPLESS mediates auxin-
dependent transcriptional repression during Arabidopsis embryogenesis.
Science, 319, 1384–1386.
Tiwari, S.B., Hagen, G. and Guilfoyle, T. (2003) The roles of auxin
response factor domains in auxin-responsive transcription. Plant Cell,
15, 533–543.
Tiwari, S.B., Hagen, G. and Guilfoyle, T.J. (2004) Aux/IAA proteins contain a
potent transcriptional repression domain. Plant Cell, 16, 533–543.
Ulmasov, T., Murfett, J., Hagen, G. and Guilfoyle, T.J. (1997) Aux/IAA
proteins repress expression of reporter genes containing natural and
highly active synthetic auxin response elements. Plant Cell, 9, 1963–
1971.
Ulmasov, T., Hagen, G. and Guilfoyle, T.J. (1999) Activation and repression
of transcription by auxin-response factors. Proc. Natl Acad. Sci. USA, 96,
5844–5849.
Vernoux, T., Brunoud, G., Farcot, E. et al. (2011) The auxin signalling net-
work translates dynamic input into robust patterning at the shoot apex.
Mol. Syst. Biol. 7, 508.
Vlad, D., Kierzkowski, D., Rast, M.I. et al. (2014) Leaf shape evolution
through duplication, regulatory diversification, and loss of a homeobox
gene. Science, 343, 780–783.
Wang, J.W., Wang, L.J., Mao, Y.B., Cai, W.J., Xue, H.W. and Chen, X.Y.
(2005) Control of root cap formation by MicroRNA-targeted auxin
response factors in Arabidopsis. Plant Cell, 17, 2204–2216.
Weijers, D. and Jurgens, G. (2004) Funneling auxin action: specificity in sig-
nal transduction. Curr. Opin. Plant Biol. 7, 687–693.
Wu, M.F., Yamaguchi, N., Xiao, J., Bargmann, B., Estelle, M., Sang, Y. and
Wagner, D. (2015) Auxin-regulated chromatin switch directs acquisition
of flower primordium founder fate. eLife, 4, e09269.
Yang, J., Tian, L., Sun, M.X., Huang, X.Y., Zhu, J., Guan, Y.F., Jia, Q.S.
and Yang, Z.N. (2013) AUXIN RESPONSE FACTOR17 is essential for
pollen wall pattern formation in Arabidopsis. Plant Physiol. 162, 720–
731.
Zhou, C., Han, L., Hou, C., Metelli, A., Qi, L., Tadege, M., Mysore, K.S. and
Wang, Z.Y. (2011) Developmental analysis of a Medicago truncatula
smooth leaf margin1 mutant reveals context-dependent effects on com-
pound leaf development. Plant Cell, 23, 2106–2124.
Zouine, M., Fu, Y., Chateigner-Boutin, A.L., Mila, I., Frasse, P., Wang, H.,
Audran, C., Roustan, J.P. and Bouzayen, M. (2014) Characterization
of the tomato ARF gene family uncovers a multi-levels post-transcrip-
tional regulation including alternative splicing. PLoS ONE, 9, e84203.