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TOMATO AGAMOUS-LIKE 1 is a component of the fruitripening 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 [email protected]).Present address: Interfaculty Institute for Biochemistry, Eberhard-Karls-University Tubingen, Hoppe-Seyler-Strasse 4, D-72076 Tubingen, Germany.

SUMMARY

After fertilization, the expanding carpel of fleshy 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-expressingTAGL1

as a chimeric repressorsuggested 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 TAGL1accumulated more lycopene, and their sepals were swollen, accumulated high

levels of the yellowflavonoid 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 rinmutant 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 fleshy 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

identified.

To date, most studies regarding the regulatory and

signalling pathways of ripening have been performed in

tomato, largely through investigation of ripening-deficient

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 deficient in their ethylene

pathway or downstream signal transduction. Other mutants,

including ripening inhibitor (rin), non-ripening (nor) and

colourless non-ripening (Cnr), exhibit altered transcription

factor activity (Thompsonet al., 1999; Vrebalovet al., 2002;

Manninget 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) (Slateret al.,

1985; Hamilton et al., 1990; Bleecker and Kende, 2000). In

tomato, bothACSand ACOare part of multi-gene families,

with nine copies ofACS(Zarembinski and Theologis, 1994)

and three copies of ACO (Bouzayen et al., 1993) so far

identified in the tomato genome. Two of the ACSgenes

2009 Weizmann Institute of Science 1081Journal compilation 2009 Blackwell Publishing Ltd

The Plant Journal (2009) 60, 10811095 doi: 10.1111/j.1365-313X.2009.04064.x

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(ACS2and ACS4) have been shown to be up-regulated by

ethylene and play an important role in tomato fruit ripening

(Olsonet al., 1991; Lincolnet 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 ACS2and ACS4 initiateethylene production during advanced ripening stages

(termed system 2), while ACS1A and ACS6participate 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

(Yokotaniet 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, Itoet al.

(2008) showed that RIN might regulate ACS2, as it binds to

its promoter.

MADS box transcription factors have been shown to play

a significant role in the development of reproductive organs,

including dry and fleshy 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 fleshy 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

(Pnueliet al., 1994).

Two MADS box genes of the C-type PLENA lineage,

SHATTERPROOF 1 and 2(AtSHP1and AtSHP2), have been

shown to control valve separation during Arabidopsis fruit

dehiscence (Ferrandiz et al., 1999, 2000; Liljegren et al.,

2000). A ripening-regulated peachSHPhomologue (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, includingRIN and JOINTLESS (Leseberget al., 2008).

A screen 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

flavonoid 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 1gene 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-specific 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 toTAG1may have been silenced in the

first VIGS assay.

Phylogenetic analysis of tomato MADS box transcription

factors that are closely related toTAG1suggestedTOMATO

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 specific fragment, resulted in altered fruit

1082 Maxim Itkinet al.

2009 Weizmann Institute of ScienceJournal compilation 2009 Blackwell Publishing Ltd, The Plant Journal, (2009), 60, 10811095

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pigmentation (Figure 1e,f,h,i). These results suggest that the

phenotype was due to silencing of theTAGL1gene.

TAGL1expression 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, flesh and seeds) from various developmental

stages (immature green, IG; mature green, MG; breaker,

Br; orange, Or; ripe) and in buds and flowers, but expres-

sion was not detected in leaves, roots and pollen. No sig-

nificant difference inTAGL1 transcript levels was observed

between peel and flesh 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 ofTAGL1on

fruit pigmentation.

(af) Mature green stage fruit of plants infected

with vectors containing no insert (Ev); specific

PHYTOENE DESATURASEsequence (PDS); full

TAG1 sequence (TAG1-Fu); specific TAG1 se-

quence (TAG1-Spe); full TAGL1 sequence(TAGL1-Fu); specific TAGL1 sequence (TAGL1-

Spe). Altered fruit sections are indicated by

arrows.

(gi) Ripe fruit of plants infected with Ev, TAGL1-

Fu andTAGL1-Spe.

(j) Phylogenetic analysis of selected MADS box

gene products indicated that TAGL1 isthe closest

TAG1 paralogue. Full names and identifiers of

these proteins are listed in Table S3.

TOMATO AGAMOUS-LIKE 1 and fruit ripening 1083


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The chemical 1-methylcyclopropene (1-MCP) is an inhib-

itor of ethylene perception and therefore interferes with fruit

ripening (Yokotaniet al., 2009). To examine whether block-

ing the ethylene receptors alters TAGL1expression, fruits of

three developmental stages were incubated with 1-MCP,

andTAGL1 expression was measured 24 h post-treatment.

The results revealed that TAGL1 is significantly 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 rinmutant (Figure 2c). No signif-

icant difference was observed between rinand wild-type

(WT), indicating thatTAGL1 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 signifi-

cantly up-regulated, no significant 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 TAGL1as 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; Matsuiet al., 2004; Takaseet 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

TAGL1SRDXdid 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

significant 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

TAGL1relativeexpression

(b)

TAGL1 ACS4

Gene

Relativeexpression

0

2.5

2

1.5

1

0.5

*3

2

1

0

4

MG Br Or Ripe

wt

rin


(c) (d) Untreated+ ethylene

Peel

Flesh

Seeds

Peel

Flesh

Seeds

Peel

Flesh

Seeds

Peel

Flesh

Seeds

Peel

Flesh

Seeds

Youngleaves

Buds

Flowers

Roots

Pollen

Tissue

7

6

5

4

3

2

0

1

(a)


Matureleaves

IG MG Br Or Ripe

Figure 2. Expression pattern ofTAGL1in 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)rinmutant fruit (cv. Ailsa Craig).(d) Quantitative real-time PCR analysis ofTAGL1

and ACS4 in fruit treated with ethylene. IG,

immature green; MG, mature green; Br, breaker;

Or, orange. The asterisk indicates a Pvalue

< 0.01 (Studentst-test,n= 3; n= 2 in wild-type

MG fruit), when comparing data for each geno-

type treatment versus the WT or untreated fruit.



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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). Amongthe carotenoids, lycopene and two of its derivatives, phy-

tofluene and phytoene, were reduced to trace levels in the

transgenic fruit. In contrast, the levels of chlorophyll aand b,

and their degradation product pheophytin a, were signifi-

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 CO2 (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 (CO2) 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-

nificant 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

andMANNAN 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 significantly altered. These

included PSY1, which was down-regulated in TAGL1SRDX-

expressing fruit, and LCY-e, which was up-regulated.

(e)

C2

H4emission(ml/(kgfruitx

hour)

**

*

1 2 3 8 9 10 11

Days post breaker

*8

6

5

4

3

1

7

0

2

wt

TAGL1-SRDX

Lycopeneder.2

Lycopeneder.1

Peakarea(x103)

IsoprenoidChlorophyllb

a-Tochopherol

Chlorophylla

Lutein

Phytoene

Phytofluene

Pheophytina

b-Carotene

(d)

*

*

*

*

*

**

1000

800700

600500

300

900

200100

0

400

wt

TAGL1-SRDX

2000

1600

1200

800

400Peakarea(x10

3)

Lycopene

(c)

*

0

wt

TAGL1-SRDX

(b)

PlantTAGL1relativeexpression

0

0.4

0.8

1.2

wt

TAGL1-SRDX

*

wt TAGL1-

SRDX

(a)Figure 3. Over-expression ofTAGL1as 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 ofTAGL1SRDX-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

Pvalue < 0.05 (Students t-test, n = 3), when

comparing data for each measurement between

theTAGL1-SRDXand WT. der, derivative.



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Among the ethylene-related genes, expression ofACS4and

ERF1was significantly reduced in TAGL1SRDX-expressing

fruit, as revealed by the array results. The expression of

ACS2 was also significantly down-regulated in TAGL1

SRDX-expressing fruit. However, no significant alteration

in gene expression was observed in the case ofACO1 and

S-adenosyl methionine synthase 1 (SAMS1), both of which

are involved in ethylene biosynthesis.

Among the ripening-related genes, no significant 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.

TAGL1over-expression induces swelling and ripening

of sepals

TAGL1was subsequently over-expressed under the control

of the35S CaMVpromoter in tomato (TAGL1oe lines). The

increased expression ofTAGL1 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 ofTAGL1oe ripe fruit sepals confirmed 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 flavonoid 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 ofTAGL1 had opposite

effects on two key genes from the ethylene biosynthetic

pathway. While ACS4was significantly up-regulated, the

Table 1Ripening-induced genes that were down-regulated in breaker fruit ofTAGL1SRDXcompared to their expression in wild-type fruit

Genbank ID Putative protein Possible function

Fold

changeb 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

AW223528a Phytoene synthase 1 (PSY1) Carotenoid biosynthesis )

2.4 Bartleyet 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

BT014190.1a 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

M63490.1a 1-aminocyclopropane-1-carboxylate synthase

(ACS4)

Ethylene biosynthesis )7.2 Lincoln et al., 1993

AY077626.1a 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 Leideet 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

aValidated by quantitative real-time PCR (see also Figure 4).bFold change for TAGL1SRDXversus wild-type.

NSHF, no significant homology found; AAA, aromatic amino acid.



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ACO1 gene wassignificantly down-regulated in sepals of the

transgenic plants. Expression ofPSY1was also significantlydown-regulated in young sepals of the TAGL1oe fruit. In

accordance with the detected increase in NarCh accumula-

tion, TAGL1oe sepals showed a significant induction of

CHALCONE SYNTHASE(CHS), which encodes the enzyme

that catalyses biosynthesis of this flavonoid.

Effect of TAGL1 over-expression on sepals in the rinmutant

background

To examine the effect of over-expressing TAGL1on pheno-

types observed in therinmutant, we transformedrinplants

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 signifi-

cant change in ACO1 expression was detected in the rin/

TAGL1oe sepals.

HPLC analysis of rin/TAGL1oe sepals revealed a signifi-

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 TAGL1over-expression on fruit in the wild-type

andrinmutant 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 ripeTAGL1oe 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 significant

increase in phytoene and phytofluene 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

TAGL1in therinmutant background, in which chlorophylls

are normally accumulated due to inhibition of ripening.

Isoprenoid analysis in ripe rin/TAGL1oe fruit revealed

*

Gene (carotenoid pathway)

Relativeexpression

CRTISO

CRTR

-b1

CRTR

-b2

LCY

-b

LCY

-e

NCED

PSY1

VDE

ZDS

PSY2

(a)wt

TAGL1-SRDX

3

2

1.5

1

0.5

0

2.5

*

*

PL

TDR8

RIN

WIV

-1

(c)

Relativeexpressio

n

wt

TAGL1-SRDX

3

2

1.5

1

0.5

0

2.5

LOXC

** *

Relativeexpression

(b)

SAMS1

ACS2

ACS4

ACO1

ETR1

ERF1

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

inTAGL1SRDX-expressing fruit.

Relative transcript levels in Br stage fruit for

genes related to: (a) the carotenoid pathway, (b)

theethylene pathway andsignaling,and (c)other

ripening processes. Asterisks indicate Pvalue

< 0.05 (Studentst-test, n= 3), when comparing

data for each measurement between the TAGL1-SRDX and WT. Genes that were also found to

be significantly down-regulated by microarray

experiments are underlined. The abbreviations

of gene names are defined in Appendix S1.



8/11/2019 tom pap

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reduced accumulation of chlorophylls aand bandlutein, 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 maturerin/TAGL1oe fruit was more intense than that of a

typicalrin 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

therinpeel.

TAGL1co-suppression inhibits ripening and alters fruit

isoprenoid levels

SeveralTAGL1oe andrin/TAGL1oe lines showed a different

fruit phenotype compared to the typical over-expressing

ones, suggesting the possibility ofTAGL1 co-suppression

(TAGL1co-sup and rin/TAGL1co-sup lines). This was con-

firmed by TAGL1 expression analysis in fruit (Figure S5).

Fruit ofTAGL1co-sup plants were dark green and firm 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

firm after fruit ripening (Figure 5f). HPLC analysis of

TAGL1co-sup ripe fruit revealed a significant increase in

a-tocopherol, lutein and b-carotene, as well as phytoene

and phytofluene levels (Figure 7b). Lycopene levels did not

change in this fruit.

Therin/TAGL1co-sup lines were dark green compared to

the rinfruit (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

significant increase in the levels of chlorophyll b,a-tocoph-

TAGL1oewt TAGL1co-sup

(c)

(f)(e)(d)

(a) (b)

rin rin/TAGL1co-suprin/TAGL1oe

TAGL1oe TAGL1co-supwt

(i)(g) (h)

(l)(j)

rin rin/TAGL1co-suprin/TAGL1oe

(k)

Figure 5. Over-expression and co-suppression

ofTAGL1 in the wild-type and rinmutant 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 arrowsin (e)and (k)]. Thepericarp ofrin/

TAGL1co-sup fruit (l) is thin (arrow), and resem-

bles the thin pericarp ofTAGL1fruit silenced by

VIGS (see Figure 1e,f,i).



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erol and lutein in the rin/TAGL1co-sup ripe fruit compared to

controlrinfruit (Figure 7c).

TAGL1 activates the ACS2promoter 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 (Hellenset al., 2005)

to evaluate activation of theACSgene promoter regions by

the TAGL1 transcription factor (Figure 8), and found that

TAGL1 is able to activate the promoter ofACS2but not that

ofACS4.

(b)

1

10

0.1

100

Peakarea

(Naringeninchalcone)

TAGL1oe

wt

Genotype

1000

10

1

Gene

*

Relat

iveexpression10000

0.1

*

*

ACO1

PSY1

ACS4

PL

CHS

TAGL1

* *

(c)

100

wt

TAGL1oe

Ripe sepals

(e)

** *

*

ACO1

PSY1

ACS4

PL

CHS

TAGL1

Gene

10000

100

10

1

1000

0.1Relativeexpression rin

rin/TAGL1oe

(d)

2

4

0

6

Peakarea

(Nar

ingeninchalcone)

rin

/TAGL1oe

rin

Genotype

100

10

1

1000

0.1

(f)

Chlorophyllb

Chlorophylla

a-Tocho

pherol

Lutein

Isoprenoid

b-Ca

rotene

Zeax

anthin

Violax

anthin

Neox

anthin

Peakarea(x10

3)

* * * * *

*

**

rin

rin/TAGL1oe

Ripe sepals

IG sepals Ripe sepals

IG sepals

*

(a)Ripe sepals

Peakarea

(x10

3)(Lycopene)

*100

1

1000

10

TAGL1oe

wt

Genotype

Figure 6. Sepals of fruit over-expressing TAGL1

in the WT and rinbackgrounds exhibit altered

isoprenoid composition, naringenin chalcone

levels, and ripening-related gene expression.

(a, b) Lycopene and naringenin chalcone (NarCh)

levels,respectively, in ripe TAGL1oesepalsin the

wild-type (WT) background.

(c)Gene expressionin immature green (IG) stageTAGL1oe sepals in the WT background.

(d) NarCh levels in ripe stageTAGL1oe sepals in

therinbackground (rin/TAGLoe).

(e)Gene expressionin 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

defined in Appendix S1.

Peakarea(x104)

Isoprenoida-Tochopherol

Lycopeneder.2

Lutein

Phytoene

Phytofluene

b-Carotene

Lycopeneder.1

(b)

1000

100

10

1

a

bb

a

bb

a c

b

a

bb

a bab

aa

aa c

b600

500

400

300

200

Peak

area(x105)

Lycopene

(a)

100

0

a

a

a

TAGL1co-sup

wt

TAGL1oe

TAGL1co-sup

wt

TAGL1oe

Chlorophyllb

Chlorophylla

a-Tochophero

l

Lutein

1000

100

10

1

Isoprenoid

b-CaroteneP

eakarea(x104) a

bb

a

c

a

cb

a

b

aa a

b

a

rin/TAGL1co-sup

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 ofTAGL1oe and

TAGL1co-sup in the WT background.

(c) Isoprenoid levels in ripe fruit ofTAGL1oe and

TAGL1co-sup in the rinbackground. Lettering

above the bars (ac) denotes significant differ-

ences in metabolite levels calculated by the

Studentst-test (P< 0.05,n= 3). der, derivative.



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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 identified

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 confirm the role of TAGL1 in the

ripening process. The most significant of these are the

effects of its modification 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, phytofluene, 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,

ACS4and 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 theACS2

andACO1 gene promoters showed that they were bound by

the RIN and HB-1 proteins, respectively (Itoet al., 2008; Lin

et al., 2008). Down-regulated expression ofACS2and 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, andare therefore not likely to

be controlled by TAGL1.

Using transient expression assays inNicotiana benthami-

analeaves, we examined the capacity of TAGL1 to activate

the promoters ofACS2and ACS4. This assay showed that

TAGL1 could activate the promoter ofACS2but not that of

ACS4. It could be that the significant changes in ACS4

expression in both the TAGL1SRDX-expressing fruit and

the swollen/ripening sepals (in both the WT and rinback-

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 heterologoussystem), 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 significantly induced by exposure of

fruit to ethylene. Moreover, application of exogenous ethyl-

ene to the rin, norand Cnrmutants does not restore fruit

ripening (Herner and Sink, 1973; Tigchelaar et al., 1978;

Thompsonet 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 first target genes identified for plant MADS box

transcription factors, e.g. DEFIECIENS, GLOBOSA, PISTIL-

30

20

10

Ratio(LUC/REN)

0

TAGL1

TAGL1+p

ACS2

TAGL1+p

ACS4

Ev+p

ACS2

Ev+p

ACS4

*

Figure 8. TAGL1 is able to activate the promoter ofACS2.

The ASC2 and ACS4 promoters fused to a luciferase reporter were co-

infiltrated with a plasmid containing TAGL1 fused to the 35SCaMVpromoter.

The TAGL1 plasmid alone or plasmids containing either the ASC2or ACS4

promoter co-infiltrated with an empty vector (Ev) were used as negative

controls. The asterisk indicates Pvalue < 0.01 (Students t-test,n= 8), when

comparing data between measurements derived from TAGL1 pACS2/

pACS4 co-infiltrated plants versus controls (TAGL1, Ev pACS2,

Ev pACS4).



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LATA, AGAMOUSand 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 significantly 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 ofRINtranscripts 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 theACS2gene 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 profile (primarily decreased

lycopene levels), reduced fruit softening and flavour pro-

duction, suppressed climacteric respiration and lack of thecharacteristic ethylene production profile (Herner and Sink,

1973). Over-expression of TAGL1 in the rinmutant 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 rinbackground resulted in reduced chlorophyll

levels in both fruit and swollen/ripening sepals compared to

their levels in rinplants. Moreover, fruit over-expressing

TAGL1SRDX accumulated higher levels of chlorophylls.

In addition, the fruit of plants in which co-suppression

occurred showed enhancement of therinphenotype, with a

darker green appearance (compared to the yellowish colour

of rin fruit), higher chlorophyll levels, and smaller size.

Interestingly, the fruit ofrin/TAGL1oe plants were softer and

their peel could be removed more easily than that ofrinfruit,

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/flavonoid pathway and its regulation

by TAGL1

The flavonoid 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 flavonoids 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 flavonoid pathway could be activated by TAGL1, with no

involvement of RIN activity. This was further supported by

the significant induction ofCHS 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 rinfruit (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 rinmutant and

treatment of tomato fruit with 1-MCP showed down-regula-

tion of multiple genes associated with flavonoid 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-

expressingTAGL1 or down-regulation of flavonoid biosyn-

thesis genes in the array assay ofTAGL1SRDX-expressing

fruit. It therefore remains to be examined in detail whether

flavonoids 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 fleshy 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 fleshy fruit formation and ripening. Thus, even

though a basic similarity exists in the function of these

homologous genes with regard to dry and fleshy fruit for-

mation, it remains to be examined whether mutants such as



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SHP1/2(the Arabidopsis homologues ofTAGL1) 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 fleshy fruit species, functional conservationbetween 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 fleshy

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 final size.

Swelling of the sepals (or calyx) by TAGL1over-expression

may involve interaction with JOINTLESS, the closest

homologue of the MPF2 protein that is essential for the

inflated 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

TAG1results 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 find that the interaction

between these two MADS box factors is significant 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 fleshy 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 usedwere 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 pAA100-35Sbetween 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 3end of the TAGL1cDNA, introducingTAGL1

SRDX into pAA100 (using NcoI and SacI sites) containing the E8promoter (the35S CaMVin pAA100 was replaced previously by the

E8promoter throughBamHI andNcoI cloning), and transfer of the

E8::TAGL1-SRDXcassette to pBIN-PLUS (using PacI andAscI sites).

Constructs were transformed into cv. MicroTom as described by

Meissneret al. (1997, 2000). Oligonucleotides used in this study as

listed in Table S2.

Virus-induced gene silencing (VIGS)

ESTsputatively corresponding to 114 transcription factorsthat 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. Schemerepresentingthe proposedmodelfor TAGL1 functionin the

network of regulatory factors controlling fruit ripening.



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Ithaca,NY). ESTswere individuallyamplified, cloned (AscIand 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 verified, 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 CO2 measurements

Br stage fruit were kept for 1 day in 250 ml flasks at room tem-

perature (pool of 37 fruit in each of the three biological repli-

cates), flasks were sealed for 4 h, ethylene and CO2 weremeasured in the headspace by sampling using a syringe through

a septum in the flask lid), and the flasks were left open for 20 h

each day (11 days). Measurements were performed as described

by Fallik et al. (2003) with slight modifications (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 snapfreezing. Control fruit wereincubated in air

instead of ethylene or 1-MCP.

Isoprenoid and flavonoid 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 YMCC30 column (YMC Co.

Ltd., http://www.ymc.co.jp/en/) as described by Fraseret al. (2000).

Flavonoids were extracted and profiled by UPLC-QTOF-MS as

described previously by Mintz-Oronet 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 five 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 ANOVAanalysis. 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-specific 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 infiltration medium, which

was prepared as described by Voinnetet 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) ofTAG1resulted in

yelloworange sectors upon fruit maturation.

Figure S2. Over-expression of TAGL1 as a chimeric repressor

(TAGL1SRDX) resulted in a reduction in CO2emission from fruit of

transgenic plants.

Figure S3. TAGL1 over-expression in fruit induces swelling and

ripening of sepals.

Figure S4. Over-expression ofTAGL1 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,rinmutant

and transgenic lines in therinbackground.

Figure S6. Levels of naringenin chalcone (NarCh) in the peel of wild-

type andrinripe fruit.



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Figure S7. Sepals of TAGL1 over-expressing lines remain fused

throughout flowering.

Table S1. Listof genes showing alteredexpressionin TAGL1SRDX-

expressing breaker fruit.

Table S2.Oligonucleotides that were used in this study.

Table S3. Full names and identifiers 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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