TOMATO AGAMOUS-LIKE 1 is a component of the fruit

ripening regulatory network

Maxim Itkin, Heike Seybold

, Dario Breitel, Ilana Rogachev, Sagit Meir and Asaph Aharoni

*
Department of Plant Sciences, Weizmann Institute of Science, PO Box 26, Rehovot 76100, Israel
Received 15 September 2009; revised 14 October 2009; accepted 19 October 2009.
*
For correspondence (fax +972 8 934 4181; e-mail asaph.aharoni@weizmann.ac.il).

Present address: Interfaculty Institute for Biochemistry, Eberhard-Karls-University Tu bingen, Hoppe-Seyler-Strasse 4, D-72076 Tu bingen, Germany.
SUMMARY
After fertilization, the expanding carpel of eshy fruit goes through a phase change to ripening. Although the
role of ethylene signalling in mediating climacteric ripening has been established, knowledge regarding the
regulation of ethylene biosynthesis and its association with fruit developmental programs is still lacking. A
functional screen of tomato transcription factors showed that silencing of the TOMATO AGAMOUS-LIKE 1
(TAGL1) MADS box gene results in altered fruit pigmentation. Over-expressing TAGL1 as a chimeric repressor
suggested a role in controlling ripening, as transgenic tomato fruit showed reduced carotenoid and ethylene
levels, suppressed chlorophyll breakdown, and down-regulation of ripening-associated genes. Moreover,
fruits over-expressing TAGL1 accumulated more lycopene, and their sepals were swollen, accumulated high
levels of the yellowavonoid naringenin chalcone and contained lycopene. Transient promoter-binding assays
indicated that part of the TAGL1 activity in ripening is executed through direct activation of ACS2, an ethylene
biosynthesis gene that has recently been reported to be a target of the RIN MADS box factor. Examination of
the TAGL1 transcript and its over-expression in the rin mutant background suggested that RIN does not
regulate TAGL1 or vice versa. The results also indicated RIN-dependent and -independent processes that are
regulated by TAGL1. We also noted that fruit of TAGL1 loss-of-function lines had a thin pericarp layer,
indicating an additional role for TAGL1 in carpel expansion prior to ripening. The results add a new component
to the current model of the regulatory network that controls eshy fruit ripening and its association with the
ethylene biosynthesis pathway.
Keywords: ripening, fruit, regulation, ethylene, tomato, transcriptome.
INTRODUCTION
The switch to ripening involves the combined action of
hormonal signal transduction cascades, regulatory circuits
and environmental cues (Fei et al., 2004; Srivastava and
Handa, 2005; Carrari and Fernie, 2006; Seymour et al., 2008).
This integrated process triggers a phase change in fruit
development, typically characterized by dramatic shifts in
primary and secondary metabolism. Although many efforts
have been made in order to understand the mechanism
behind ripening, the core set of genetic components
required for activation of this process have not been yet
identied.
To date, most studies regarding the regulatory and
signalling pathways of ripening have been performed in
tomato, largely through investigation of ripening-decient
mutants. Several of these mutants, such as Never-ripe (Nr;
Lanahan et al., 1994; Wilkinson et al., 1995; Chen et al., 2004)
and Green ripe (Gr)/Never-ripe2 (Nr2; Kerr, 1981, 1982; Barry
and Giovannoni, 2006) were decient in their ethylene
pathway or downstreamsignal transduction. Other mutants,
including ripening inhibitor (rin), non-ripening (nor) and
colourless non-ripening (Cnr), exhibit altered transcription
factor activity (Thompson et al., 1999; Vrebalov et al., 2002;
Manning et al., 2006).
Ethylene is a fundamental signal in climacteric fruit
maturation (Yang and Hoffman, 1984; Alba et al., 2005).
S-adenosyl-L-methionine (SAM) is converted into 1-amino-
cyclopropane-1-carboxylic acid (ACC) by ACC synthase
(ACS) (Sato and Theologis, 1989), and ACC is further
converted into ethylene by ACC oxidase (ACO) (Slater et al.,
1985; Hamilton et al., 1990; Bleecker and Kende, 2000). In
tomato, both ACS and ACO are part of multi-gene families,
with nine copies of ACS (Zarembinski and Theologis, 1994)
and three copies of ACO (Bouzayen et al., 1993) so far
identied in the tomato genome. Two of the ACS genes
2009 Weizmann Institute of Science 1081
Journal compilation 2009 Blackwell Publishing Ltd
The Plant Journal (2009) 60, 10811095 doi: 10.1111/j.1365-313X.2009.04064.x
(ACS2 and ACS4) have been shown to be up-regulated by
ethylene and play an important role in tomato fruit ripening
(Olson et al., 1991; Lincoln et al., 1993).
An early model for ethylene biosynthesis by McMurchie
et al. (1972) was later extended (Nakatsuka et al., 1998;
Barry et al., 2000) to propose that ACS2 and ACS4 initiate
ethylene production during advanced ripening stages
(termed system 2), while ACS1A and ACS6 participate in
ethylene production in green tissues (termed system 1).
A recent study suggested that tomato fruit can initiate
ethylene system 2 independently of the cumulative effects
of system 1, providing evidence that ripening-associated
ethylene biosynthesis is regulated by both an auto-catalytic
system and by ethylene-independent developmental factors
(Yokotani et al., 2009).
Several transcription factors have been reported to act as
regulators of tomato ethylene biosynthesis. For example,
the homeobox protein HB-1 was recently reported to regu-
late ACO1 expression (Lin et al., 2008). Mutation in the
MADS box factor RIPENING INHIBITOR (RIN) (Vrebalov
et al., 2002) stops the characteristic processes associated
with ripening of tomato fruit, including auto-catalytic ethyl-
ene production (Herner and Sink, 1973). Recently, Ito et al.
(2008) showed that RIN might regulate ACS2, as it binds to
its promoter.
MADS box transcription factors have been shown to play
a signicant role in the development of reproductive organs,
including dry and eshy fruit (Becker et al., 2002; Becker and
Theissen, 2003; Balanza et al., 2006; Seymour et al., 2008). In
addition to the tomato MADS box factor RIN, which belongs
to the SEPALLATA (SEP) clade (Vrebalov et al., 2002;
Hileman et al., 2006), members of the C-type MADS box
group have also been associated with eshy fruit develop-
ment and ripening (Diaz-Riquelme et al., 2009; Tadiello
et al., 2009).
The tomato AGAMOUS (AG) orthologue, TAG1, has been
implicated in tomato fruit ripening in the AGAMOUS C-type
lineage. Ishida et al. (1998) showed that sepals of tomato
plants ectopically over-expressing TAG1 swell and ripen
in vivo, when induced by cold temperature (Bartley and
Ishida, 2003, 2007). Without cold induction, sepals of tomato
plants over-expressing TAG1 were swollen but did not ripen
(Pnueli et al., 1994).
Two MADS box genes of the C-type PLENA lineage,
SHATTERPROOF 1 and 2 (AtSHP1 and AtSHP2), have been
shown to control valve separation during Arabidopsis fruit
dehiscence (Ferrandiz et al., 1999, 2000; Liljegren et al.,
2000). A ripening-regulated peach SHP homologue (PpPLE-
NA) was ectopically expressed in tomato (Tani et al., 2007;
Tadiello et al., 2009). The sepals of the transgenic fruit
developed into carpel-like structures and ripened. Further-
more, PpPLENA-expressing fruit showed accelerated ripen-
ing as evidenced by induction of expression of characteristic
ripening genes. The SHP-like gene from tomato, TOMATO
AGAMOUS-LIKE 1 (TAGL1), was initially reported to be
expressed during early stages of fruit development (Busi
et al., 2003), and Hileman et al. (2006) later showed that
TAGL1 is also highly expressed in later stages of develop-
ment. Yeast two-hybrid (Y2H) assays implied that TAGL1
might interact with various MADS box proteins, including
RIN and JOINTLESS (Leseberg et al., 2008).
Ascreen using virus-induced gene silencing indicated that
silencing of the tomato TAGL1 MADS box transcription
factor results in altered fruit pigmentation. Loss of function
resulted in reduced carotenoid (e.g. lycopene) and ethylene
levels, suppressed chlorophyll breakdown, and down-regu-
lation of a set of ripening-associated genes. The fruit of
tomato plants over-expressing TAGL1 exhibited higher
lycopene levels, and their sepals were swollen and showed
ectopic lycopene production and accumulation of the yellow
avonoid naringenin chalcone. It appears that part of the
TAGL1 activity in ripening is executed through regulation
of the ACS2 ethylene biosynthesis gene. Examination of
TAGL1 over-expression in the rin mutant background
highlighted RIN-dependent and -independent ripening
processes. Finally, we provide evidence that TAGL1 is also
important for expansion of the carpel prior to ripening.
RESULTS
Silencing of the TOMATO AGAMOUS-LIKE 1 gene alters
fruit pigmentation
In order to identify regulatory genes that are associated with
tomato fruit ripening, we screened a set of tomato tran-
scription factors using the virus-induced gene silencing
(VIGS) method (Liu et al., 2002). Overall, cDNAs corre-
sponding to 114 putative transcription factors selected
based on their differential expression during fruit develop-
ment (Alba et al., 2005; Mintz-Oron et al., 2008) were used
for the infection, and fruit were screened for phenotypes.
The most obvious phenotype was detected in a plant
silenced with the tomato MADS box transcription factor
TOMATO AGAMOUS 1 (TAG1) cDNA, which had fruit that
exhibited dark green regions at the mature green stage
(Figure 1c). The same fruit showed yelloworange sectors
upon fruit maturation (Figure S1a). As the fragment used
corresponded to the full-length transcript, we performed
another VIGS assay using a TAG1-specic fragment (Fig-
ures 1d and S1b). However, this assay did not result in any
effect on fruit pigmentation. Thus, we hypothesized that a
gene closely related to TAG1 may have been silenced in the
rst VIGS assay.
Phylogenetic analysis of tomato MADS box transcription
factors that are closely related to TAG1 suggested TOMATO
AGAMOUS-LIKE 1 (TAGL1; SGN-U581068) as the best can-
didate for co-silencing with TAG1 in our original screen
(Figure 1j). Indeed, TAGL1 silencing, using either the full-
length cDNA or a specic fragment, resulted in altered fruit
1082 Maxim Itkin et al.
2009 Weizmann Institute of Science
Journal compilation 2009 Blackwell Publishing Ltd, The Plant Journal, (2009), 60, 10811095
pigmentation (Figure 1e,f,h,i). These results suggest that the
phenotype was due to silencing of the TAGL1 gene.
TAGL1 expression is ripening-regulated and altered in
1-MCP-treated fruit, but is not changed in the rin mutant
or by exposure to ethylene
The phenotype obtained through TAGL1 silencing sug-
gested that TAGL1 may be involved in the regulation of
ripening in tomato fruit. To examine whether TAGL1
expression correlates with a possible function in fruit
maturation, we measured its transcript levels in various
tomato organs (Figure 2a). TAGL1 is expressed in fruit tis-
sues (peel, esh and seeds) from various developmental
stages (immature green, IG; mature green, MG; breaker,
Br; orange, Or; ripe) and in buds and owers, but expres-
sion was not detected in leaves, roots and pollen. No sig-
nicant difference in TAGL1 transcript levels was observed
between peel and esh tissues, in which its expression
increases during ripening, peaking at the Or stage of fruit
development.
Sl-TAGL11
At-SHP1
At-SHP2
Pp-PLENA
1000
Sl-TAGL1
868
Pp-FAR
Fa-AG
At-AG
Sl-TAG1
981
809
911
956
985
Sl-MC
At-AP1
Sl-MADS-RIN
At-SEP4
At-SEP1
At-SEP2
At-SEP3
1000
733
968
1000
1000
(j)
1000
AG
SPH
0.05
(a) (c)
(d)
(b)
(i) (h)
(e)
(g)
Ev
Ev
PDS TAG1-Fu
TAG1-Spe TAGL1-Fu TAGL1-Spe
TAGL1-Fu TAGL1-Spe
(f)
Figure 1. A virus-induced gene silencing (VIGS)
screen in tomato reveals the effect of TAGL1 on
fruit pigmentation.
(af) Mature green stage fruit of plants infected
with vectors containing no insert (Ev); specic
PHYTOENE DESATURASE sequence (PDS); full
TAG1 sequence (TAG1-Fu); specic TAG1 se-
quence (TAG1-Spe); full TAGL1 sequence
(TAGL1-Fu); specic TAGL1 sequence (TAGL1-
Spe). Altered fruit sections are indicated by
arrows.
(gi) Ripe fruit of plants infected with Ev, TAGL1-
Fu and TAGL1-Spe.
(j) Phylogenetic analysis of selected MADS box
gene products indicated that TAGL1 is the closest
TAG1 paralogue. Full names and identiers of
these proteins are listed in Table S3.
TOMATO AGAMOUS-LIKE 1 and fruit ripening 1083
2009 Weizmann Institute of Science
Journal compilation 2009 Blackwell Publishing Ltd, The Plant Journal, (2009), 60, 10811095
The chemical 1-methylcyclopropene (1-MCP) is an inhib-
itor of ethylene perception and therefore interferes with fruit
ripening (Yokotani et al., 2009). To examine whether block-
ing the ethylene receptors alters TAGL1 expression, fruits of
three developmental stages were incubated with 1-MCP,
and TAGL1 expression was measured 24 h post-treatment.
The results revealed that TAGL1 is signicantly induced
when 1-MCP is applied to MG fruit compared to non-treated
fruit (Figure 2b). TAGL1 expression was also examined in
whole fruit tissues of the rin mutant (Figure 2c). No signif-
icant difference was observed between rin and wild-type
(WT), indicating that TAGL1 expression is not regulated by
RIN. We next examined whether TAGL1 is induced by the
application of exogenous ethylene. Although expression of
the ACS4, which served as a positive control, was signi-
cantly up-regulated, no signicant alteration in TAGL1
transcript levels was observed upon exposure to ethylene
(Figure 2d).
Thus, TAGL1 expression is ripening-regulated but is not
changed in the rin mutant, and is altered in fruit upon
inhibition of ethylene perception but not upon exposure to
ethylene.
Over-expression of TAGL1 as a dominant repressor results
in a loss-of-function phenotype that includes altered fruit
carotenoids and ethylene levels
A chimeric transcription factor fused to the EAR (ERF-asso-
ciated amphiphilic repression) motif functions as a domi-
nant repressor in the presence of both endogenous and
functionally redundant transcription factors for a target gene
(Takase et al., 2007). This strategy has been shown to be
valuable for obtaining loss-of-function phenotypes (Hiratsu
et al., 2003; Matsui et al., 2004; Takase et al., 2007), and was
therefore used in this study. More than 10 independent
transgenic lines that express TAGL1 as a dominant repressor
(termed TAGL1SRDX) under the control of the tomato
ethylene- and ripening-induced E8 gene promoter were
generated. Mature fruit of transgenic plants expressing
TAGL1SRDX did not turn red upon ripening and exhibited
an orange colour (Figure 3a). Expression analysis of the
TAGL1SRDX transcript showed high expression in the
transgenic fruit (data not shown), which also exhibited
signicant down-regulation of the endogenous TAGL1
transcript (Figure 3b).
Ripening stage Ripening stage
MG Br Or
3
2.5.
2
1.5
1
0.5
0
*
3.5
Untreated
1-MCP
T
A
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L
1

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(b)
TAGL1 ACS4
Gene
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0
2.5
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1.5
1
0.5
*
3
2
1
0
4
MG Br Or Ripe
wt
rin
T
A
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1

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x
p
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(c)
(d) Untreated
+ ethylene
P
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s
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h
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d
s
P
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h
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s
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l
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a
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B
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s
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Tissue
7
6
5
4
3
2
0
1
(a)
T
A
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r
e
l
a
t
i
v
e

e
x
p
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e
s
s
i
o
n
M
a
t
u
r
e

l
e
a
v
e
s
IG MG Br Or Ripe
Figure 2. Expression pattern of TAGL1 in fruit of
wild-type and the rin mutant and fruit treated
with 1-MCP or ethylene.
(ac) Quantitative real-time PCR expression anal-
ysis of TAGL1 in (a) fruit of cv. MicroTom wild-
type and (b) fruit of cv. Ailsa Craig wild-type 24 h
after treatment with 1-methylcyclopropane (1-
MCP), and in (c) rin mutant fruit (cv. Ailsa Craig).
(d) Quantitative real-time PCR analysis of TAGL1
and ACS4 in fruit treated with ethylene. IG,
immature green; MG, mature green; Br, breaker;
Or, orange. The asterisk indicates a P value
< 0.01 (Students t-test, n = 3; n = 2 in wild-type
MG fruit), when comparing data for each geno-
type treatment versus the WT or untreated fruit.
1084 Maxim Itkin et al.
2009 Weizmann Institute of Science
Journal compilation 2009 Blackwell Publishing Ltd, The Plant Journal, (2009), 60, 10811095
To obtain a better insight into the ripening-associated
phenotype of the transgenic TAGL1SRDXfruit, we analysed
the levels of various isoprenoids (i.e. carotenoids, chloro-
phylls and tocopherols) at the Or stage (Figure 3c,d). Among
the carotenoids, lycopene and two of its derivatives, phy-
touene and phytoene, were reduced to trace levels in the
transgenic fruit. In contrast, the levels of chlorophyll a and b,
and their degradation product pheophytin a, were signi-
cantly increased in the TAGL1SRDX-expressing fruit.
To determine whether the phenotype observed in the
TAGL1SRDX-expressing fruit was the result of altered
climacteric ripening, we measured ethylene and CO
2
(an
indicator for respiration) emission from these fruit after the
Br stage (Figures 3e and S2). The results showed that
TAGL1SRDX-expressing fruit do not show the increases
in both ethylene and respiration (CO
2
) levels that are typical
of WT fruit shortly after the Br stage.
Transcriptome analysis detects down-regulated expression
of a large set of ripening-associated transcripts in
TAGL1SRDX-expressing fruit
Microarray analysis was performed in order to examine the
effect on gene expression in TAGL1SRDX-expressing fruit
(Br stage). Compared to WT, 37 and 21 genes showed sig-
nicant down- or up-regulated expression, respectively, in
TAGL1SRDX-expressing fruit (Table S1). The down-reg-
ulated transcripts in TAGL1SRDX-expressing fruit were
enriched (26 in total, approximately 70%) in genes previ-
ously found to be related to fruit ripening in tomato (or
detected as exhibiting ripening-regulated expression in
publicly available array data; Table 1).
Among the ripening-associated down-regulated tran-
scripts in TAGL1SRDX-expressing fruit were those encod-
ing key enzymes in ethylene (ACS4) and carotenoid
(PHYTOENE SYNTHASE 1, PSY1) biosynthesis, as well as
cell-wall enzymes (b-GALACTOSIDASE II isoforms 1 and 2
and MANNAN ENDO-1,4-b-MANNOSIDASE). Expression of
three genes putatively encoding enzymes associated with
the activity of the TCA cycle (ISOCITRATE DEHYDROGE-
NASE, PHOSPHOENOLPYRUVATE CARBOXYLASE 1 and its
phosphorylating enzyme PHOSPHOENOLPYRUVATE CAR-
BOXYLASE KINASE 2) was reduced in the TAGL1SRDX-
expressing fruit. ETHYLENE RESPONSE FACTOR 1 (ERF1),
which putatively takes part in the ethylene signal transduc-
tion during tomato fruit ripening, also exhibited reduced
transcript levels.
We performed quantitative real-time PCR in order to
corroborate the array data and evaluate the expression of
additional genes associated with carotenoids (Figure 4a),
ethylene (Figure 4b) and other ripening-related processes
(Figure 4c). The expression of two out of 10 examined
carotenoid pathway genes was signicantly altered. These
included PSY1, which was down-regulated in TAGL1SRDX-
expressing fruit, and LCY-e, which was up-regulated.
(e)
C
2
H
4

e
m
i
s
s
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(
m
l
/
(
k
g

f
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r
)
*
*
*
1 2 3 8 9 10 11
Days post breaker
*
8
6
5
4
3
1
7
0
2
wt
TAGL1-SRDX
L
y
c
o
p
e
n
e

d
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r
.

2
L
y
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P
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a
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(
x
1
0
3
)
Isoprenoid
C
h
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o
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o
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h
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b
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T
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(d)
*
*
*
*
*
*
*
1000
800
700
600
500
300
900
200
100
0
400
wt
TAGL1-SRDX
2000
1600
1200
800
400
P
e
a
k

a
r
e
a

(
x
1
0
3
)
Lycopene
(c)
*
0
wt
TAGL1-SRDX
(b)
Plant
T
A
G
L
1

r
e
l
a
t
i
v
e

e
x
p
r
e
s
s
i
o
n
0
0.4
0.8
1.2
wt
TAGL1-SRDX
*
wt TAGL1-
SRDX
(a)
Figure 3. Over-expression of TAGL1 as a chime-
ric repressor (TAGL1SRDX) induced a non-rip-
ening phenotype.
(a) TAGL1SRDX-expressing fruit do not change
from orange to red, in contrast to wild-type (WT)
fruit.
(b) Quantitative real-time PCR analysis of the
endogenous TAGL1 transcript in Br fruit of
TAGL1SRDX-expressing plants and WT.
(c, d) Levels of lycopene and other isoprenoids,
respectively, in TAGL1SRDX-expressing and
WT fruit (Or stage).
(e) Ethylene emissions from TAGL1SRDX-
expressing and WT fruit. Asterisks indicate
P value < 0.05 (Students t-test, n = 3), when
comparing data for each measurement between
the TAGL1-SRDX and WT. der, derivative.
TOMATO AGAMOUS-LIKE 1 and fruit ripening 1085
2009 Weizmann Institute of Science
Journal compilation 2009 Blackwell Publishing Ltd, The Plant Journal, (2009), 60, 10811095
Among the ethylene-related genes, expression of ACS4 and
ERF1 was signicantly reduced in TAGL1SRDX-expressing
fruit, as revealed by the array results. The expression of
ACS2 was also signicantly down-regulated in TAGL1
SRDX-expressing fruit. However, no signicant alteration
in gene expression was observed in the case of ACO1 and
S-adenosyl methionine synthase 1 (SAMS1), both of which
are involved in ethylene biosynthesis.
Among the ripening-related genes, no signicant alter-
ation in transcript levels was detected for the RIN, TDR8,
ACID INVERTASE (WIV-1) and LYPOXYGENASE (LOXC)
genes. However, PECTATE LYASE (PL), which is associated
with fruit softening (Marin-Rodriguez et al., 2002), was
strongly down-regulated.
TAGL1 over-expression induces swelling and ripening
of sepals
TAGL1 was subsequently over-expressed under the control
of the 35S CaMV promoter in tomato (TAGL1oe lines). The
increased expression of TAGL1 resulted in sepal swelling,
yellowing and the appearance of red regions, indicating the
presence of lycopene (Figures 5e, S3c,e,f and S7). HPLC
analysis of TAGL1oe ripe fruit sepals conrmed the presence
of the red carotenoid, lycopene, which does not accumulate
in WT sepals (Figure 6a). However, apart from lycopene, no
alteration in the accumulation of other isoprenoids was de-
tected (Figure S4). These results suggest that the yellowing
of the sepals is not caused by accumulation of carotenoids.
Tomato fruit, particularly its peel, typically accumulates high
levels of the yellow avonoid naringenin chalcone (NarCh).
We therefore examined NarCh levels in TAGL1oe sepals,
and detected a dramatic increase in its levels compared to
WT sepals (Figure 6b).
Expression analysis of ripening-related genes was subse-
quently performed in young sepals of TAGL1oe fruit (IG
stage; Figure 6c). Over-expression of TAGL1 had opposite
effects on two key genes from the ethylene biosynthetic
pathway. While ACS4 was signicantly up-regulated, the
Table 1 Ripening-induced genes that were down-regulated in breaker fruit of TAGL1SRDX compared to their expression in wild-type fruit
Genbank ID Putative protein Possible function
Fold
change
b
Reference
BM413158 Anthranilate synthase (ASA) Tryptophan biosynthesis )4.3 Mintz-Oron et al., 2008
X71900.1 Histidine decarboxylase (HDC) Histidine metabolism )2.7 Picton et al., 1993
AW223528
a
Phytoene synthase 1 (PSY1) Carotenoid biosynthesis )2.4 Bartley et al., 1992
AF020390.2 b-galactosidase II (TBG4) Cell-wall metabolism )4.4 Smith et al., 1998
AY034075.1 Mannan endo-1,4-b-mannosidase (MAN4) Cell-wall metabolism )3.1 Carrington et al., 2002
AF154421.1 b-galactosidase (TBG3) Cell-wall metabolism )2.1 Smith and Gross, 2000
BT014190.1
a
Pectate lyase (PL) Cell-wall metabolism )9.6 Mintz-Oron et al., 2008
CN550618 Replication licensing factor DNA replication )2.4 Mintz-Oron et al., 2008
M63490.1
a
1-aminocyclopropane-1-carboxylate synthase
(ACS4)
Ethylene biosynthesis )7.2 Lincoln et al., 1993
AY077626.1
a
Ethylene response factor 1 (ERF1) Ethylene signal transduction )2.7 Li et al., 2007
BG123587 b-ketoacyl CoA synthase (CER6) Fatty acid biosynthesis )2.5 Leide et al., 2007
BT014299.1 ULTRAPETALA1 (ULT1) Floral determination )2.0 Mintz-Oron et al., 2008
BE437087 L-allo-threonine aldolase Glycine biosynthesis )2.1 Mintz-Oron et al., 2008
CD002771 Peroxiredoxin Redox signaling )2.6 Mintz-Oron et al., 2008
BG643920 Cinnamoyl CoA reductase (CCR) Phenylpropanoid biosynthesis )3.9 Mintz-Oron et al., 2008
BT013266.1 Dehydroquinate dehydratase/shikimate
dehydrogenase (DHQD3)
AAA biosynthesis )3.7 Mintz-Oron et al., 2008
AW223174 Phosphoenolpyruvate carboxylase 1 (PEPC1) Replenishment of TCA cycle )2.1 Mintz-Oron et al., 2008
AY187634.1 Phosphoenolpyruvate carboxylase kinase 2
(PPCK2)
Replenishment of TCA cycle )5.1 Marsh et al., 2003
BI207393 Isocitrate dehydrogenase (IDH) TCA cycle )2.8 Mintz-Oron et al., 2008
BG627658 High leaf temperature 1 Regulation of stomata
movement
)3.7 Mintz-Oron et al., 2008
BM535639 Triacylglycerol lipase Lipid metabolism )2.3 Alba et al., 2005
BT012806.1 Rab GTPase Signal transduction )2.4 Mintz-Oron et al., 2008
BM410663 Soluble starch synthase (SStS) Starch biosynthesis )2.2 Mintz-Oron et al., 2008
BG626714 NSHF Unknown )2.1 Mintz-Oron et al., 2008
AW738056 NSHF Unknown )2.1 Mintz-Oron et al., 2008
BI208323 WRKY transcription factor Unknown )2.0 Mintz-Oron et al., 2008
a
Validated by quantitative real-time PCR (see also Figure 4).
b
Fold change for TAGL1SRDX versus wild-type.
NSHF, no signicant homology found; AAA, aromatic amino acid.
1086 Maxim Itkin et al.
2009 Weizmann Institute of Science
Journal compilation 2009 Blackwell Publishing Ltd, The Plant Journal, (2009), 60, 10811095
ACO1 gene was signicantly down-regulated in sepals of the
transgenic plants. Expression of PSY1 was also signicantly
down-regulated in young sepals of the TAGL1oe fruit. In
accordance with the detected increase in NarCh accumula-
tion, TAGL1oe sepals showed a signicant induction of
CHALCONE SYNTHASE (CHS), which encodes the enzyme
that catalyses biosynthesis of this avonoid.
Effect of TAGL1 over-expression on sepals in the rin mutant
background
To examine the effect of over-expressing TAGL1 on pheno-
types observed in the rin mutant, we transformed rin plants
with the same TAGL1 over-expression construct. This
experiment was designed to provide evidence for processes
that are RIN-dependent and those that can be executed by
TAGL1 in the absence of RIN. TAGL1 over-expression was
found to induce sepal swelling and ripening in the rin
background (Figures 5k and S3d). NarCh levels in rin/TA-
GL1oe sepals showed a sharp increase compared to rin
sepals (Figure 6d). The gene expression changes in the rin/
TAGL1oe sepals resembled those detected in TAGL1oe, with
ACS4 and CHS expression being induced and PSY1
expression being reduced (Figure 6e). However, no signi-
cant change in ACO1 expression was detected in the rin/
TAGL1oe sepals.
HPLC analysis of rin/TAGL1oe sepals revealed a signi-
cant reduction in the levels of all the isoprenoids examined
(i.e. neoxanthin, a-tocopherol, violaxanthin, chlorophyll a
and b, lutein, b-carotene and zeaxanthin) (Figure 6f). How-
ever, we did not detect the lycopene-accumulating regions
in rin/TAGL1oe sepals that were observed in TAGL1oe
sepals (Figures 5e and S3c,e,f), suggesting that carotenoid
accumulation could not be induced by TAGL1 independently
of RIN activity.
Effect of TAGL1 over-expression on fruit in the wild-type
and rin mutant backgrounds
Fruit pigmentation in the TAGL1oe plants appeared to be
more intense in comparison with WT fruit at the same
developmental stage (Figure 5d,e). HPLC analysis indicated
that ripe TAGL1oe fruit showed a mild increase in lycopene
levels (n = 3, P = 0.055) compared to those of the WT (Fig-
ure 7a). In addition, TAGL1oe fruit showed a signicant
increase in phytoene and phytouene levels (Figure 7b).
This indicated that TAGL1 over-expression induces part of
the fruit carotenoid biosynthesis pathway.
We also examined the fruit of plants over-expressing
TAGL1 in the rin mutant background, in which chlorophylls
are normally accumulated due to inhibition of ripening.
Isoprenoid analysis in ripe rin/TAGL1oe fruit revealed
*
Gene (carotenoid pathway)
R
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R
F
1
wt
TAGL1-SRDX
3
2
1.5
1
0.5
0
2.5
Gene (ethylene-related) Gene (ripening-related)
Figure 4. Ripening-associated gene expression
in TAGL1SRDX-expressing fruit.
Relative transcript levels in Br stage fruit for
genes related to: (a) the carotenoid pathway, (b)
the ethylene pathway and signaling, and (c) other
ripening processes. Asterisks indicate P value
< 0.05 (Students t-test, n = 3), when comparing
data for each measurement between the TAGL1-
SRDX and WT. Genes that were also found to
be signicantly down-regulated by microarray
experiments are underlined. The abbreviations
of gene names are dened in Appendix S1.
TOMATO AGAMOUS-LIKE 1 and fruit ripening 1087
2009 Weizmann Institute of Science
Journal compilation 2009 Blackwell Publishing Ltd, The Plant Journal, (2009), 60, 10811095
reduced accumulation of chlorophylls a and b and lutein, but
no accumulation of lycopene (Figure 7c). Thus, TAGL1 over-
expression can induce chlorophyll breakdown in the rin
mutant background. We also noted that the yellow colour
of mature rin/TAGL1oe fruit was more intense than that of a
typical rin fruit (Figure 5j,k). As we did not detect increased
accumulation of NarCh in the rin/TAGL1oe fruit (in contrast
to sepals; data not shown), this suggests that the degrada-
tion of chlorophylls (Figure 7c) exposed the yellow colour of
the rin peel.
TAGL1 co-suppression inhibits ripening and alters fruit
isoprenoid levels
Several TAGL1oe and rin/TAGL1oe lines showed a different
fruit phenotype compared to the typical over-expressing
ones, suggesting the possibility of TAGL1 co-suppression
(TAGL1co-sup and rin/TAGL1co-sup lines). This was con-
rmed by TAGL1 expression analysis in fruit (Figure S5).
Fruit of TAGL1co-sup plants were dark green and rm at an
early stage of development (Figure 5c). Later in develop-
ment (ripe stage), TAGL1co-sup fruit showed patchy yellow
regions on a red background, suggesting that only these
yellow parts exhibited co-suppression (Figure 5f). Sepals of
TAGL1co-sup fruit appeared normal, but stayed green and
rm after fruit ripening (Figure 5f). HPLC analysis of
TAGL1co-sup ripe fruit revealed a signicant increase in
a-tocopherol, lutein and b-carotene, as well as phytoene
and phytouene levels (Figure 7b). Lycopene levels did not
change in this fruit.
The rin/TAGL1co-sup lines were dark green compared to
the rin fruit (at the MG stage) (Figure 5i,g), and resembled
the fruit of co-suppression lines in the WT background
(Figure 5c). Upon maturation, rin/TAGL1co-sup fruit showed
a greenyellow colour, had a thin pericarp and remained
small in size (Figure 5l). Isoprenoid analysis showed a
signicant increase in the levels of chlorophyll b, a-tocoph-
TAGL1oe wt TAGL1co-sup
(c)
(f) (e) (d)
(a) (b)
rin rin/TAGL1co-sup rin/TAGL1oe
TAGL1oe TAGL1co-sup wt
(i) (g) (h)
(l) (j)
rin rin/TAGL1co-sup rin/TAGL1oe
(k)
Figure 5. Over-expression and co-suppression
of TAGL1 in the wild-type and rin mutant back-
grounds.
TAGL1 over-expression (TAGL1oe) and co-sup-
pression (TAGL1co-sup) in the WT (af) and rin
mutant background (gl) (cv. MicroTom) in
mature green (MG) fruit (ac, gi) and ripe fruit
(df, jl). The ripe fruit of the TAGL1oe and rin/
TAGL1oe plants have swollen ripe sepals [indi-
cated by arrows in (e) and (k)]. The pericarp of rin/
TAGL1co-sup fruit (l) is thin (arrow), and resem-
bles the thin pericarp of TAGL1 fruit silenced by
VIGS (see Figure 1e,f,i).
1088 Maxim Itkin et al.
2009 Weizmann Institute of Science
Journal compilation 2009 Blackwell Publishing Ltd, The Plant Journal, (2009), 60, 10811095
erol and lutein in the rin/TAGL1co-sup ripe fruit compared to
control rin fruit (Figure 7c).
TAGL1 activates the ACS2 promoter in a transient
expression system
The down-regulation of both ACS2 and ACS4 in the
TAGL1SRDX-expressing fruit suggested that TAGL1 might
induce ripening through direct activation of this pair of
ethylene biosynthesis genes. We therefore used a lucifer-
ase-based transient expression system (Hellens et al., 2005)
to evaluate activation of the ACS gene promoter regions by
the TAGL1 transcription factor (Figure 8), and found that
TAGL1 is able to activate the promoter of ACS2 but not that
of ACS4.
(b)
1
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0.1
100
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1
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*
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wt
TAGL1oe
Ripe sepals
(e)
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Genotype
Figure 6. Sepals of fruit over-expressing TAGL1
in the WT and rin backgrounds exhibit altered
isoprenoid composition, naringenin chalcone
levels, and ripening-related gene expression.
(a, b) Lycopene and naringenin chalcone (NarCh)
levels, respectively, in ripe TAGL1oe sepals in the
wild-type (WT) background.
(c) Gene expression in immature green (IG) stage
TAGL1oe sepals in the WT background.
(d) NarCh levels in ripe stage TAGL1oe sepals in
the rin background (rin/TAGLoe).
(e) Gene expression in immature green (IG) stage
rin/TAGLoe sepals.
(f) Isoprenoid levels in ripe stage rin/TAGLoe
sepals. Asterisks indicate P value < 0.05
(Students t-test, n = 3), when comparing data
for each measurement between each genotype
and WT. The abbreviations of gene names are
dened in Appendix S1.
P
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TAGL1oe
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wt
TAGL1oe
C
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rin
rin/TAGL1oe
(c)
Figure 7. TAGL1 over-expression (TAGL1oe)
and co-suppression (TAGL1co-sup) in the wild-
type (WT) and rin backgrounds have major
effects on the isoprenoid composition of ripe
fruit.
(a) Lycopene levels in ripe fruit of TAGL1oe,
TAGL1co-sup and WT.
(b) Isoprenoid levels in ripe fruit of TAGL1oe and
TAGL1co-sup in the WT background.
(c) Isoprenoid levels in ripe fruit of TAGL1oe and
TAGL1co-sup in the rin background. Lettering
above the bars (ac) denotes signicant differ-
ences in metabolite levels calculated by the
Students t-test (P < 0.05, n = 3). der, derivative.
TOMATO AGAMOUS-LIKE 1 and fruit ripening 1089
2009 Weizmann Institute of Science
Journal compilation 2009 Blackwell Publishing Ltd, The Plant Journal, (2009), 60, 10811095
DISCUSSION
TAGL1 participates in the developmental regulation
of tomato fruit ripening
The gaseous hormone ethylene is a major factor in climac-
teric fruit ripening. Switching on the ethylene biosynthetic
pathway in a temporal and spatial manner is crucial for ini-
tiating the ripening process during climacteric fruit devel-
opment. Several transcription factors belonging to various
gene families have been proposed to link developmental
programs and ethylene biosynthesis in fruit. Characteriza-
tion of plants with various mutations of these regulators
indicated that additional factors are required for the full
execution of climacteric ripening (Vrebalov et al., 2002;
Giovannoni et al., 2004; Manning et al., 2006). Moreover,
some of these proteins, for example those belonging to the
MADS domain family, function as components of larger
multimeric complexes. In this study, we have identied
the TOMATO AGAMOUS-LIKE 1 MADS box protein as an
additional element of the regulatory network that mediates
between fruit development and activation of ethylene bio-
synthesis for triggering ripening.
Several lines of evidence conrm the role of TAGL1 in the
ripening process. The most signicant of these are the
effects of its modication on the typical metabolism and
gene expression associated with ripening. For example,
over-expressing TAGL1 as a chimeric repressor resulted in a
dramatic decrease in the levels of several carotenoids,
including phytoene, phytouene, lycopene and two of its
derivatives. The same fruit also contained high levels of
chlorophylls (suggesting decelerated chlorophyll degrada-
tion) and did not show climacteric elevated ethylene synthe-
sis. Approximately 70% of the genes that showed reduced
expression in these fruit were associated with ripening. The
putative functions of the down-regulated genes indicated a
major effect on cell-wall metabolism associated with fruit
softening, on ethylene biosynthesis and signalling (ACS2,
ACS4 and ERF1), and on biosynthesis of secondary meta-
bolites and their central/primary metabolite precursors.
The association between ethylene biosynthesis during
ripening and TAGL1 activity
Direct activation of key genes in the ethylene biosynthetic
pathway is a straightforward route for inducing ethylene
production and triggering expression of the downstream
ethylene-dependent ripening genes. Assays using the ACS2
and ACO1 gene promoters showed that they were bound by
the RIN and HB-1 proteins, respectively (Ito et al., 2008; Lin
et al., 2008). Down-regulated expression of ACS2 and ACS4
in TAGL1SRDX-expressing fruit and the absence of the
ripening ethylene peak suggested that TAGL1 exerts its
effect on ripening (or at least part of it) by regulating ACS
expression. ACO1 transcript levels were not altered in
TAGL1SRDX-expressing fruit, and are therefore not likely to
be controlled by TAGL1.
Using transient expression assays in Nicotiana benthami-
ana leaves, we examined the capacity of TAGL1 to activate
the promoters of ACS2 and ACS4. This assay showed that
TAGL1 could activate the promoter of ACS2 but not that of
ACS4. It could be that the signicant changes in ACS4
expression in both the TAGL1SRDX-expressing fruit and
the swollen/ripening sepals (in both the WT and rin back-
grounds) resulted from feedback prompted by the changes
in ethylene levels in these two organs. Nevertheless, due to
the limitation of this transient assay (a heterologous
system), the possibility cannot be ruled out that ACS4
expression is directly controlled by TAGL1.
The association between TAGL1 and ethylene is similar to
that observed for other known developmental ripening
regulators in aspects additional to those described above.
As detected previously in the case of RIN (Ito et al., 2008),
although TAGL1 is likely to activate ethylene biosynthesis,
its expression is not signicantly induced by exposure of
fruit to ethylene. Moreover, application of exogenous ethyl-
ene to the rin, nor and Cnr mutants does not restore fruit
ripening (Herner and Sink, 1973; Tigchelaar et al., 1978;
Thompson et al., 1999), and the same result was observed
here when TAGL1SRDX-expressing fruit were exposed to
ethylene (data not shown). This indicated that TAGL1 is
likely to control both ethylene-dependent and -independent
ripening pathways. Adams-Phillips et al. (2004) suggested
that the latter mode of control might represent conserved
mechanisms of ripening between climacteric and non-
climacteric fruit. However, TAGL1 was responsive to the
ethylene perception inhibitor 1-MCP, as its expression was
induced in MG stage fruit treated with 1-MCP.
The rst target genes identied for plant MADS box
transcription factors, e.g. DEFIECIENS, GLOBOSA, PISTIL-
30
20
10
R
a
t
i
o

(
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C
/
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N
)
0
T
A
G
L
1
T
A
G
L
1
+

p
A
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S
2
T
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1
+

p
A
C
S
4
E
v

+

p
A
C
S
2
E
v

+

p
A
C
S
4
*
Figure 8. TAGL1 is able to activate the promoter of ACS2.
The ASC2 and ACS4 promoters fused to a luciferase reporter were co-
inltrated with a plasmid containing TAGL1 fused to the 35S CaMV promoter.
The TAGL1 plasmid alone or plasmids containing either the ASC2 or ACS4
promoter co-inltrated with an empty vector (Ev) were used as negative
controls. The asterisk indicates P value < 0.01 (Students t-test, n = 8), when
comparing data between measurements derived from TAGL1 pACS2/
pACS4 co-inltrated plants versus controls (TAGL1, Ev pACS2,
Ev pACS4).
1090 Maxim Itkin et al.
2009 Weizmann Institute of Science
Journal compilation 2009 Blackwell Publishing Ltd, The Plant Journal, (2009), 60, 10811095
LATA, AGAMOUS and APETALA3, were the MADS genes
themselves (Schwarz-Sommer et al., 1992; Trobner et al.,
1992; Jack et al., 1994; Chen et al., 2000; Gomez-Mena et al.,
2005). Such positive auto-regulatory loops are widespread
mechanisms that maintain expression patterns of genes (de
Folter and Angenent, 2006). Analysis of the TAGL1SRDX-
expressing fruit showed that the level of endogenous TAGL1
transcript was signicantly reduced. This suggested that
TAGL1 might bind and activate its own promoter, and
therefore down-regulates its own transcript when over-
expressed as a chimeric repressor.
TAGL1 and RIN may interact in fruit to activate ethylene
biosynthesis but have separate roles in the regulation of
other ripening processes
In order to position the TAGL1 protein in the tomato ripening
regulatory network, we examined its relationship to the RIN
protein. Examination of RIN transcripts in the TAGL1SRDX-
expressing fruit and TAGL1 expression in the rin mutant
background suggested that they do not regulate one
another. The similar expression pattern of both genes dur-
ing fruit development and their association with the ACS2
promoter supports the possibility of interaction between
these two MADS box proteins through cooperative binding
of the ACS2 gene upstream region. Further support for such
putative interaction was provided recently by a two-hybrid
assay in yeast (Leseberg et al., 2008).
The rin mutant displays strong inhibition of ripening,
including altered carotenoid prole (primarily decreased
lycopene levels), reduced fruit softening and avour pro-
duction, suppressed climacteric respiration and lack of the
characteristic ethylene production prole (Herner and Sink,
1973). Over-expression of TAGL1 in the rin mutant back-
ground provided us with additional information regarding
the relationship between these two proteins, and the level
and points of overlap in controlling ripening. The results of
this experiment indicated that TAGL1 requires RIN activity
for the induction of lycopene accumulation in fruit and in the
swollen/ripening sepals.
In contrast to lycopene accumulation, chlorophyll break-
down during the transition from the MG to the Br stage of
fruit development appears to be independent of RIN activity
and could be activated by TAGL1 only. Over-expression of
TAGL1 in the rin background resulted in reduced chlorophyll
levels in both fruit and swollen/ripening sepals compared to
their levels in rin plants. Moreover, fruit over-expressing
TAGL1SRDX accumulated higher levels of chlorophylls.
In addition, the fruit of plants in which co-suppression
occurred showed enhancement of the rin phenotype, with a
darker green appearance (compared to the yellowish colour
of rin fruit), higher chlorophyll levels, and smaller size.
Interestingly, the fruit of rin/TAGL1oe plants were softer and
their peel could be removed more easily than that of rin fruit,
suggesting that the activity of genes encoding enzymes
responsible for cell-wall degradation and fruit softening
might also be regulated by TAGL1 in a RIN-independent
manner.
The phenylpropanoid/avonoid pathway and its regulation
by TAGL1
The avonoid NarCh provides a yellow appearance to
tomato fruit peel, accumulating to approximately 1% of the
cuticular layer in the Or stage before decreasing in the
ripe fruit. Recently, Mintz-Oron et al. (2008) demonstrated
co-expression of genes associated with the biosynthesis of
NarCh and its precursors during the Br and Or stages of fruit
development. While tomato fruit avonoids have been
investigated to a reasonable extent, knowledge regarding
their association with the ripening program is limited. The
accumulation of NarCh in the swollen/ripening sepals of
fruit over-expressing TAGL1 was therefore intriguing. The
accumulation of NarCh in the swollen/ripening sepals of
rinTAGL1oe plants suggests that, at least in tomato sepals,
the avonoid pathway could be activated by TAGL1, with no
involvement of RIN activity. This was further supported by
the signicant induction of CHS expression (encoding the
enzyme catalysing NarCh biosynthesis) in the swollen/
ripening sepals.
In contrast to Saladie et al. (2007), we detected a severe
reduction in NarCh levels in rin fruit (Figure S6). Both Bargel
and Neinhuis (2004) and Saladie et al. (2007) reported that
isolated cuticles of the nor mutant display reduced pig-
mentation. Gene expression analysis of the rin mutant and
treatment of tomato fruit with 1-MCP showed down-regula-
tion of multiple genes associated with avonoid biosyn-
thesis (A. Aharoni and A. Adato, Department of Plant
Sciences, Weizmann Institute of Science, Israel, unpublished
results). In contrast to the results described above, we did
not observe accumulation of NarCh in fruit of plants over-
expressing TAGL1 or down-regulation of avonoid biosyn-
thesis genes in the array assay of TAGL1SRDX-expressing
fruit. It therefore remains to be examined in detail whether
avonoids are part of the biochemical processes controlled
by TAGL1 and other developmental regulators, or alterna-
tively are independent of climacteric ethylene.
Functional conservation of TAGL1 and its homologues
in dry and eshy fruit development
In Arabidopsis, a number of MADS box genes are required
for proper development of the dehiscence zone and normal
cell division, expansion and differentiation during silique
morphogenesis (Becker and Theissen, 2003). Characteriza-
tion of RIN and TAGL1 in this study suggests that the func-
tion of genes associated with dry fruit development evolved
to regulate eshy fruit formation and ripening. Thus, even
though a basic similarity exists in the function of these
homologous genes with regard to dry and eshy fruit for-
mation, it remains to be examined whether mutants such as
TOMATO AGAMOUS-LIKE 1 and fruit ripening 1091
2009 Weizmann Institute of Science
Journal compilation 2009 Blackwell Publishing Ltd, The Plant Journal, (2009), 60, 10811095
SHP1/2 (the Arabidopsis homologues of TAGL1) could be
fully complemented by TAGL1. Combined changes in the
number, expression pattern and interaction of such key
regulatory factors most likely facilitated the alteration of
their function in fruit development.
Among eshy fruit species, functional conservation
between these regulatory genes is expected to be higher,
as described recently by Tadiello et al. (2009) for the
TAGL1 homologue of peach (i.e. PpPLENA). Ectopic
expression of the ripening-regulated PpPLENA gene in
tomato resulted in the transformation of sepals into eshy
and ripening carpel-like structures, and fruit exhibiting
accelerated ripening. The authors suggested that PpPLENA
interfered with the endogenous activity of TAGL1. This
possibility is further corroborated by this work in which
similar phenotypes were obtained upon modulation of
TAGL1 expression.
The results from both studies implicate functioning of
these two C-type MADS box proteins (i.e. TAGL1 and
PpPLENA) not only in ripening, the ultimate step of carpel
development, but also in the preceding phases in which
the carpel is formed and expands to almost its nal size.
Swelling of the sepals (or calyx) by TAGL1 over-expression
may involve interaction with JOINTLESS, the closest
homologue of the MPF2 protein that is essential for the
inated calyx syndrome in Physalis pubescens (Mao et al.,
2000; He and Saedler, 2005). Interestingly, Y2H assays
recently showed that JOINTLESS may interact with TAGL1
and TAG1 (Leseberg et al., 2008), while over-expressing
TAG1 results in sepal swelling and ripening in vivo (Bartley
and Ishida, 2003). In both cases (i.e. TAG1 and TAGL1),
sepal swelling and ripening could be a result of either
direct activation of the ripening process or homeotic
conversion of the sepal to a carpeloid organ that expands
and ripens. Future work may nd that the interaction
between these two MADS box factors is signicant for
tomato carpel expansion.
This study provides evidence for the involvement of
TAGL1 in controlling tomato carpel ripening and perhaps
its expansion earlier during its development. A scheme
representing the proposed model for TAGL1 function in the
network of regulatory factors controlling fruit ripening is
presented in Figure 9. It is apparent that many additional
genetic and environmental components of the comprehen-
sive network that mediates eshy fruit development and
ripening await discovery.
EXPERIMENTAL PROCEDURES
Plant material
Tomato plants (Solanum lycopersicum) cv. Ailsa Craig (AC)
(obtained from the Tomato Genetics Resource Center; http://
tgrc.ucdavis.edu) and cv. MicroTom (obtained from Avi Levy, Plant
Sciences Department, Weizmann Institute of Science, Isreal) were
grown in a climate-controlled greenhouse at 24C during the day
and 18C during the night, with natural light. The fruit stages used
were immature green (IG), mature green (MG), breaker (Br), orange
(Or) and ripe, which were picked on average 10, 35, 38, 41 and
44 days post-anthesis, respectively. Sepals were collected based on
the fruit ripening stage.
Generation of transgenic tomato plants
The 35S::TAGL1 construct was generated by cloning of the TAGL1
cDNA (using NcoI and BamHI sites) into pAA100-35S between the
35S CaMV promoter and a nopaline synthase (NOS) terminator,
extracting the 35S::TAGL1::tNOS cassette (using PacI and AscI
sites), and cloning into pBIN-PLUS (van Engelen et al., 1995). A
dominant repressor construct was created by generating a transla-
tional fusion between the EAR repression domain (SRDX; Hiratsu
et al., 2003) and the 3 end of the TAGL1 cDNA, introducing TAGL1
SRDX into pAA100 (using NcoI and SacI sites) containing the E8
promoter (the 35S CaMV in pAA100 was replaced previously by the
E8 promoter through BamHI and NcoI cloning), and transfer of the
E8::TAGL1-SRDX cassette to pBIN-PLUS (using PacI and AscI sites).
Constructs were transformed into cv. MicroTom as described by
Meissner et al. (1997, 2000). Oligonucleotides used in this study as
listed in Table S2.
Virus-induced gene silencing (VIGS)
ESTs putatively corresponding to 114 transcription factors that were
found to be differentially expressed during tomato fruit maturation
were generously provided by the Tomato Molecular Resource Dis-
tribution Center (Boyce Thompson Institute, Cornell University,
TAGL1
RIN
HB1
CNR
NOR
SAM
ACC
Ethylene
ACO1
ACS2
ACS4
?
Primary metabolism (e.g. TCA cycle)
Various phenylpropanoids/flavonoids (e.g.
naringenin chalcone)
Carotenoids (e.g. lycopene)
Cell wall metabolism
Chlorophyll breakdown
?
Figure 9. Scheme representing the proposed model for TAGL1 function in the
network of regulatory factors controlling fruit ripening.
1092 Maxim Itkin et al.
2009 Weizmann Institute of Science
Journal compilation 2009 Blackwell Publishing Ltd, The Plant Journal, (2009), 60, 10811095
Ithaca, NY). ESTs were individually amplied, cloned (AscI and NotI)
into pENTR/D-TOPO (Invitrogen, http://www.invitrogen.com/) and
introduced into pTRV2-AttR1-AttR2 (Liu et al., 2002) using the
Gateway LR clonase enzyme kit (Invitrogen). The pTRV2-AttR1-
AttR2 plasmid was transformed into Escherichia coli, and the
insertion sequence was veried, and subsequently transferred to
Agrobacterium tumefaciens strain AGLO. A PHYTOENE DESAT-
URASE (PDS) gene fragment was introduced into pTRV2-AttR1-
AttR2 to serve as a positive control. For plant inoculation, Agro-
bacterium containing pTRV1 and pTRV2 (empty or containing the
insert) was grown as described by Liu et al. (2002), and when the
bacteria reached an absorbance of 1, they were mixed in a 1:1 ratio,
shaken for 4 h, and concentrated to an absorbance of 10, before
inoculation of 3-week-old seedlings by stabbing using a wooden
toothpick in three or four places along the stem. Fruit were exam-
ined visually several times during ripening, and positive clones
were re-tested three times.
Ethylene and CO
2
measurements
Br stage fruit were kept for 1 day in 250 ml asks at room tem-
perature (pool of 37 fruit in each of the three biological repli-
cates), asks were sealed for 4 h, ethylene and CO
2
were
measured in the headspace by sampling using a syringe through
a septum in the ask lid), and the asks were left open for 20 h
each day (11 days). Measurements were performed as described
by Fallik et al. (2003) with slight modications (for details, see
Appendix S1).
Ethylene and 1-MCP treatments
Fruit (cv. Ailsa Craig) at the MG, Br and Or stages were incubated
with 1 ppm of 1-MCP for 19 h, moved to open air for 24 h, and
subsequently frozen. MG fruit (cv. MicroTom) were incubated in
40 ppm ethylene for 16 h, followed by 8 h of aeration at room
temperature before snap freezing. Control fruit were incubated in air
instead of ethylene or 1-MCP.
Isoprenoid and avonoid extraction and analyses
Isoprenoid extraction was performed as described by Bino et al.
(2005). Analysis was performed using an HPLC-PDA detector
(Waters, http://www.waters.com) and an YMC C30 column (YMC Co.
Ltd., http://www.ymc.co.jp/en/) as described by Fraser et al. (2000).
Flavonoids were extracted and proled by UPLC-QTOF-MS as
described previously by Mintz-Oron et al. (2008). Peak areas of the
compounds were determined according to the spectral character-
istic, wavelength and the retention time given in Table S4.
Microarray and bioinformatics analysis
Total RNA was extracted from three pools of ve or six Br fruits
using the hot phenol method (Verwoerd et al., 1989), and treated
with DNase I (Sigma, http://www.sigmaaldrich.com/). Biotinylated
cRNA was fragmented and hybridized to the Affymetrix GeneChip

Tomato Genome Array as described in the Affymetrix technical

manual (available at http://www.affymetrix.com). Statistical analy-
sis of microarray data was performed using the Partek

Genomics
Suite (http://www.partek.com) and the robust microarray averaging
(RMA) algorithm (Irizarry et al., 2003). Changes in expression level
were determined by ANOVA analysis. The false discovery rate (FDR)
was applied to correct for multiple comparisons (Benjamini and
Hochberg, 1995). Differentially expressed genes were chosen based
on an FDR < 0.15 and a twofold change between genotypes and
signal above background in at least one microarray. Functional
annotation analysis was performed manually using publicly avail-
able databases.
Quantitative real-time PCR
RNA isolation from fruit (without placenta and seeds) was per-
formed by the hot phenol method (Verwoerd et al., 1989), from
seeds (cleaned of gel) as described by Ruuska and Ohlrogge
(2001), and from all other tissues by the Trizol method (Sigma).
DNase I-treated RNA was reverse-transcribed using a high-
capacity cDNA reverse transcription kit (Applied Biosystems,
http://www.appliedbiosystems.com/) and cDNA was used for real-
time PCR analysis performed as described by Mintz-Oron et al.
(2008). Gene-specic oligonucleotides were designed using
Primer Express 2 software (Applied Biosystems). The CAC gene
(Exposito-Rodriguez et al., 2008) was used as an endogenous
control.
Luciferase transient assay
The luciferase transient assay was performed as described by
Hellens et al. (2005), except for the inltration medium, which
was prepared as described by Voinnet et al. (2003).
ACKNOWLEDGEMENTS
We thank Danny Gamrasni (Fruit Storage Research Laboratory,
Kiryat Shmona, Israel) for the 1-MCP-treated fruit; Elazar Fallik and
Sharon Alkalai-Tuvia (Department of Postharvest Science, Agri-
cultural Research Organization, Volcani Center, Israel) for ethylene
measurements; Ester Feldmesser and Dena Leshkowitz (Bioinfor-
matics and Biological Computing Unit, Weizmann Institute of
Science, Israel) for array data analysis; James Giovannoni and
Ruth White Tomato Molecular Resource Distribution Center
(Boyce Thompson Institute, Cornell University, NY), for the EST
clones; Savithramma Dinesh-Kumar (Molecular, Cellular & Devel-
opmental Biology, Yale University, CT) for the pTRV vectors;
Alexander Vainstein and Marianna Ovadis (The Robert H. Smith
Institute of Plant Sciences and Genetics in Agriculture, The
Hebrew University of Jerusalem, Israel) for the help with VIGS;
Rivka Barg (Department of Vegetable Research, Agricultural Re-
search Organization, Volcani Center, Israel) for the E8 promoter;
Roger Hellens (HortResearch, Mt Albert Research Centre, New
Zealand) for the transient assay vector, and Arie Tishbee and Riri
Kramer (Department of Organic Chemistry, Weizmann Institute of
Science, Israel) for help with UPLCQTOFMS analysis. We also
thank Avital Adato for critical reading of the manuscript and
fruitful discussions. A.A. is the incumbent of the Adolpho and
Evelyn Blum Career Development Chair of Cancer Research. The
work in the A.A. laboratory was supported by the Minerva foun-
dation and the Benoziyo Institute.
SUPPORTING INFORMATION
Additional Supporting Information may be found in the online
version of this article:
Figure S1. Virus-induced gene silencing (VIGS) of TAG1 resulted in
yelloworange sectors upon fruit maturation.
Figure S2. Over-expression of TAGL1 as a chimeric repressor
(TAGL1SRDX) resulted in a reduction in CO
2
emission from fruit of
transgenic plants.
Figure S3. TAGL1 over-expression in fruit induces swelling and
ripening of sepals.
Figure S4. Over-expression of TAGL1 in the wild-type background
has no effect on the levels of most isoprenoids in sepals.
Figure S5. TAGL1 relative transcript levels in wild-type, rin mutant
and transgenic lines in the rin background.
Figure S6. Levels of naringenin chalcone (NarCh) in the peel of wild-
type and rin ripe fruit.
TOMATO AGAMOUS-LIKE 1 and fruit ripening 1093
2009 Weizmann Institute of Science
Journal compilation 2009 Blackwell Publishing Ltd, The Plant Journal, (2009), 60, 10811095
Figure S7. Sepals of TAGL1 over-expressing lines remain fused
throughout owering.
Table S1. List of genes showing altered expression in TAGL1SRDX-
expressing breaker fruit.
Table S2. Oligonucleotides that were used in this study.
Table S3. Full names and identiers of protein sequences used in
the phylogenetic analysis.
Table S4. Isoprenoids detected by HPLC analysis.
Appendix S1. Supplementary experimental procedures.
Please note: Wiley-Blackwell are not responsible for the content or
functionality of any supporting materials supplied by the authors.
Any queries (other than missing material) should be directed to the
corresponding author for the article.
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2009 Weizmann Institute of Science
Journal compilation 2009 Blackwell Publishing Ltd, The Plant Journal, (2009), 60, 10811095