A multisite gateway-based toolkit for targeted ... - Wiley Online Library

Plant Biotechnology Journal (2009) 7, pp. 298–309

doi: 10.1111/j.1467-7652.2009.00402.x

A multisite gateway-based toolkit for targeted gene expression and hairpin RNA silencing in tomato fruits Leandro Original Multisite Article Hueso gateway Estornell fruit expression et al. toolkit Blackwell Oxford, Plant PBI © 1467-7652 1467-7644 XXX 2009 Biotechnology UK Blackwell Publishing Publishing Journal Ltd Ltd

Leandro Hueso Estornell, Diego Orzáez*, Lucas López-Peña, Benito Pineda, María Teresa Antón, Vicente Moreno and Antonio Granell Instituto de Biología Molecular y Celular de Plantas, Consejo Superior de Investigaciones Científicas, Universidad Politécnica de Valencia, Ingeniero Fausto Elio s/n, 46022 Valencia, Spain

Received 8 October 2008; revised 16 December 2008; accepted 18 December 2008. *Correspondence (fax 34 963977859; e-mail [email protected])

Summary A collection of fruit promoters, reporter genes and protein tags has been constructed in a triple-gateway format, a recombination-based cloning system that facilitates the tandem assembly of three DNA fragments into plant expression vectors. The new pENFRUIT collection includes, among others, the classical tomato-ripening promoters E8 and 2A11 and a set of six new tomato promoters. The new promoter activities were characterized in both transient assays and stable transgenic plants. The range of expression of the new promoters comprises strong (PNH, PLI), medium (PLE, PFF, PHD) and weak (PSN) promoters driving gene expression preferentially in the fruit, and covering a wide range of tissues and developmental stages. Together, a total of 78 possible combinations for the expression of a gene of interest in the fruit, plus a set of five reporters for new promoter analysis, was made available in the current collection. Moreover, the pENFRUIT promoter collection is adaptable to hairpin RNA strategies aimed at tissue/organ-specific gene silencing with only an additional cloning step. The pENFRUIT toolkit broadens the spectrum of promoter activities available for fruit biotechnology and fundamental research, and bypasses technical

Keywords: fruit promoter, gateway,

difficulties of current ligase-dependent cloning techniques in the construction of fruit

hairpin RNA, recombinant protein,

expression cassettes. The pENFRUIT vector collection is available for the research community

tomato.

in a plasmid repository, facilitating its accessibility.

Introduction The engineering of fruits by genetic modification has multiple applications in plant fundamental research and biotechnology. Fruit-specific transgene expression is important for the confirmation of the physiological bases of previously defined quantitative trait loci (QTLs), and may be used as a first strategy in confirming/supporting candidate genes for important crop traits. As a biotechnological tool, transgenic expression in fruits has been used for the improvement of post-harvest characteristics (Sheehy et al., 1988; Picton et al., 1995), agronomic quality (Ficcadenti et al., 1999; Carmi et al., 2003), nutritional value (Muir et al., 2001; Mehta et al., 2002; Liu et al., 2004), organoleptic features (Davidovich-Rikanati et al., 2007) and even therapeutic value (Jani et al., 2002; Ma et al., 2003; Walmsley et al., 2003; Ramirez et al., 2007). Despite this, the biotechnological tools available for genetic modification

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of fruit-bearing plants are rather rudimentary, based on a handful of well-characterized fruit promoters which are often single-selected for a certain strategy following a rational approach. Indeed, most currently used promoters for fruit biotechnology are strong ethylene-responsive promoters which drive gene expression during ripening phase IV, such as E8 (Deikman et al., 1992) and PG (Montgomery et al., 1993; Nicholass et al., 1995) among others, with only a few examples of promoters directing gene expression in the fruit during fruit cell division (phase II) (Barg et al., 2005) or cell expansion (phase III) (Gillaspy et al., 1993) phases. There have been few comparative analyses of the different promoters available for fruit biotechnology, although it has been demonstrated that the correct choice of the spatial–temporal control of transgene expression is paramount in fruit genetic/ metabolic engineering. As an example, the phenotype obtained by DET1 RNAi suppression in tomato fruit strongly depends © 2009 The Authors Journal compilation © 2009 Blackwell Publishing Ltd

Multisite gateway fruit expression toolkit 299

on the fruit promoter used to direct hairpin RNA (hpRNA) expression (Davuluri et al., 2004, 2005). In this study, promoters operating at early developmental stages successfully yielded fruits with high antioxidant content, but showing marked differences in their respective antioxidant profiles; the same approach using ethylene-responsive promoters, however, was unsuccessful, probably because of the requirement for an early increase in hpRNA expression. Ideally, a robust experimental design should include the testing of several expression cassettes, e.g. controlled by different promoters, each providing specific spatial–temporal features; however, the lack of choice and, most importantly, the technical hurdles imposed as a result of the large size of the expression cassettes used in plant transformation often limit the number of constructs that can be tested.

multisite scaffold, so that they can be shuffled in a combinatorial manner with a certain gene of interest (GOI). Moreover, the ‘multisite gateway PRO’ variant uses attL1-L2 destination vectors, which are available from several collections. Therefore, the applicability of the multisite system is broadened by the already developed vectors. In this paper, we describe a set of six new promoters designed for transgenic expression in tomato fruits. The transcriptional strength and specificity conferred by each promoter are described in both stable and transient transformation experiments using β-glucuronidase (GUS) and green fluorescent protein (GFP) reporter genes. To provide a flexible toolkit for expression of a GOI in the fruit, all six promoters were incorporated into a multisite PRO triple recombination gateway system. The fruit expression toolkit was completed with the addition of seven

Gateway cloning systems, based on homologous recombination (Hartley et al., 2000), have become a popular alternative to traditional cloning methods based on T4 ligase for the construction of plant expression cassettes. Gateway is a ligasefree cloning system with high efficiency and versatility, which is easily adapted for high-throughput approaches. In order to be suitable for gateway cloning, traditional plant binary vectors need to be adapted with the addition of the so-called ‘gateway cassette’, which incorporates attR1 and attR2 recombination sites and the ccd4 gene for positive selection. Once converted into a ‘destination vector’ by incorporation of a gateway cassette, the plant vector becomes a recipient for any DNA fragment which is flanked by attL1 and attL2 sites following a single one-step recombination reaction catalysed by the enzyme LR clonase. There are currently several collections of plant destination vectors available for the research community (see Karimi et al., 2002; Curtis and Grossniklaus, 2003; Karimi et al., 2007 as examples). In each collection, a number of plant

previously described promoters and several reporter genes, transcriptional terminators and N-terminal fusion tags. Together, we provide a framework for 78 possible ways to express a GOI in tomato fruit.

destination vectors are constructed from a basic vector frame, which incorporates an array of additional elements flanking the gateway cassette (e.g. regulatory elements, reporter genes, fusion tags, etc.), therefore providing à la carte tools for almost any plant experiment (promoter analysis, localization studies, gene over-expression, RNAi, etc.). A further elaboration to the recombinatory cloning strategies is provided by the multisite gateway system. This methodology is based on the incorporation of unique attL and attR sites flanking each DNA fragment to be cloned. This refinement allows the simultaneous introduction of up to four DNA fragments within a destination vector frame. In multisite gateway, all recombinant DNA fragments are directionally incorporated in tandem following a unique LR reaction, which is carried out in a single tube. In this way, a collection of DNA (regulatory) elements can be built up in the different positions of the

exclusively in the fruit, were also abundant at earlier fruit developmental stages. We report the isolation and characterization of the promoter regions from six of these candidates. Three (corresponding to unigenes SGN-U212902, SGN-U144410 and SGN-U143318) responded to a classical ripening-related profile, albeit showing significant levels also in developing fruits. The three additional unigenes (SGN-U149666, SGNU212930 and SGNU-212704) were preferentially abundant in fruits at non-ripening stages. A gene walking strategy was followed for the cloning of 5′ regulatory regions from each candidate gene. For genespecific primer (GSP) design, unigene sequences from the SOL database were used. GSPs were designed, annealing as close as possible to the translational start considering primer melting temperature ( T m) constraints, and therefore incorporating possible 5′ untransformed (regulatory) regions

Results Isolation of new fruit promoters Digital expression databases (Fei et al., 2004) provided a first set of candidate theoretical contigs (TCs) preferentially expressed in fruits, which were later analysed using in-house microarray datasets and confirmed by reverse transcriptase-polymerase chain reaction (RT-PCR) (Figure S1, see Supporting information). It was observed that genes showing highest fruit specificity were those expressed only at ripening stages. As most currently available promoters are of this type, the list was enriched in candidates that, being expressed preferentially but not

© 2009 The Authors Journal compilation © 2009 Blackwell Publishing Ltd, Plant Biotechnology Journal, 7, 298–309

300 Leandro Hueso Estornell et al.

Table 1 Elements included in the pENFRUIT collection of ENTRY clones Name

Element

Size (bp)

Goal

SGN-U

Distance ATG

pEF 1-PNH-4

PNH

3675

Promoter

U212704

–8

pEF 1-PSN-4

PSN

592

Promoter

U149666

–15

pEF 1-PFF-4

PFF

3232

Promoter

U212902

–9

pEF 1-PLE-4

PLE

1411

Promoter

U212930

–92

pEF 1-PLI-4

PLI

1293

Promoter

U144410

–55

pEF 1-PHD-4

PHD

1499

Promoter

U143318

–377

pEF 1-2A11-4

2A11

4034

Promoter

U212581

–3

pEF 1-E8-4

E8

2226

Promoter

U212804

–1

pEF 1-35S-4

CaMV 35S

1049

Promoter

–

–1

pEF 1-SAG12-4

SAG12

1516

Promoter

–

–1

pEF 1-2x35S-4

2 × 35S

750

Promoter

–

–1

pEF 1-INO-4

INO

2175

Promoter

–

–1

pEF 1-DefH9-4

DefH9

3480

Promoter

–

–1

pEF1-4 promoter collection

pEF4r-3r collection pEF 4r-YFP-3r

YFP

737

Reporter gene

pEF 4r-GUS-3r

GUS

1809

Reporter gene

pEF 4r-GFPGUS-3r

GFP-GUS

2532

Reporter fusion

pEF 4r-DsRed-3r

DsRed

678

Reporter gene

pEFS 4r-3r

Intron (p1)

257

RNA silencing

pEF 3-Tnos-2

Tnos

250

Terminator

pEF 3-6HTnos-2

6H-Tnos

287

pEF 3-G6HTnos-2

GFP-6H-Tnos

1052

Terminator + 2 × tag

pEF 3-GUS-2

GUS

1809

Targeting

pEF 3-CFP-2

CFP

831

Targeting

pEF 3-YFP-2

YFP

737

Targeting

pEFS 3-2

Intron (p2)

245

RNA silencing

pEF3-2 collection

(UTRs), except for the HD candidate, where GSP was designed mostly upstream of the SGN-U143318 400-bp-long 5′ UTR. Following this approach, positive PCR bands (> 600 bp) were obtained from all six candidate genes, and subsequently cloned and confirmed by sequencing. Information about the new genes whose putative promoter regions were cloned is shown in the first six rows of Table 1.

Characterization of new tomato promoters To obtain information on the gene expression patterns conferred by the newly cloned promoters, they were incorporated into GFP/GUS fusion reporter constructs and tested in plants. Promoter functionality and relative strength in fruits were initially analysed by transient gene expression using agroinjection, a technique that allows the rapid assay of gene expression in the placenta, gel and inner pericarp tissues of the fruit in a few days. To serve as references in the comparative analysis, promoters E8, cauliflower mosaic virus (CaMV) 35S and 2A11 were included in parallel transformation experiments. All fruits were co-agroinjected with plasmid

Terminator + tag

pBDsRed expressing DsRed fluorescent protein driven by CaMV 35S as an internal standard. Fruit slices were analysed in a fluorescence scanner, and the green/red fluorescence ratio was used as an indication of the relative strength of each promoter in the fruit (Figure 1a). CaMV 35S and PNH were the strongest promoters in transient assays. Most promoters showed higher expression levels in green fruits, except for E8 and LI, which were stronger in ripening slices. Promoters PSN and PHD showed expression levels below the background, and therefore their relative expression levels could not be established using this technique (Figure 1b). To obtain more information on the expression patterns provided by each promoter, stable transformations were conducted. At least 10 independent TG1 plants per construct were grown to maturity. After preliminary GUS activity tests in fruits, two plants per line showing representative activity levels were selected for detailed analysis. For each selected line, a quantitative GUS analysis was performed on different organs to establish the degree of fruit specificity. The following developmental points for GUS analysis were established: fruits at late phase II [5–7 days post-anthesis (dpa)], fruits at



is induced in potato stolon tips during tuberization stages (Taylor et al., 1992). PNH was selected as a candidate promoter to confer high expression during the expansion phase, based on its relative abundance in digital expression databases. Quantitative GUS analysis from two independent TG1 plants confirmed the expected expression profiles, with expression levels close to that of the CaMV 35S promoter, but with a much more fruit-specific profile. PNH drives gene expression in the developing fruit, reaching maximum levels at the end of phase III (Figure 2a). Most interesting, PNH promoter activity is distributed almost uniformly throughout all fruit tissues, including all three pericarp layers, placenta, columella and gel (see Figure 3a,b). The combination of uniform tissue distribution and high expression levels makes PNH ideal for strategies aimed at high product accumulation, such as molecular farming approaches.

early phase III (12–18 dpa), pericarp at late phase III (30– 35 dpa), pericarp at early phase IV (breaker) and pericarp at late phase IV (red/red-ripen), according to Gillaspy et al. (1993). To complete the picture on the tissue specificity of each promoter within the fruit, GUS histochemical localization was also performed.

PLI promoter expression starts in the outer pericarp and moves inwards with ripening A 1293-bp DNA fragment (PLI) corresponding to the promoter region of tomato SGN-U144410 was isolated by gene walking. SGN-U144410 encodes a 191-amino-acid plastidial early light-inducible protein (ELIP), with a putative function in chloroplast to chromoplast transitions (Bruno and Wetzel, 2004). PLI-directed GUS expression is negligible during phase II, and increases steadily thereafter, reaching a maximum at the red stage. The LI 1293-bp promoter region drives reporter expression predominantly in the outer pericarp at immature stages (Figure 3a,b). As the fruit reaches maturity, PLI-driven expression spreads to the whole fruit, reaching the highest expression levels at the red stage. We found very low PLIdriven GUS expression in non-fruit organs in the two transgenic lines analysed (Figure 2a). ELIP mRNA was found in flowers by Northern analysis (Bruno and Wetzel, 2004). SGN-U144410 expressed sequence tags (ESTs) are not present in flower cDNA libraries; however, ESTs from the highly homologous SGN-U314409 unigene are relatively abundant in flower libraries. Therefore, it is likely that the band observed by Bruno and Wetzel (2004) by Northern blotting was a result of probe cross-hybridization with SGN-U314409.

PNH promoter drives high expression levels during fruit expansion and ripening The promoter region named PNH expands 3675 bp upstream from the translation start from tomato unigene SGNU212704. Its transcript contains an open reading frame (ORF) of 949 bp encoding an acidic 15-kDa hypothetical protein with no known functional domains. The PNH ORF shows high homology with TUB8, a cDNA of unknown function, which

PFF-driven expression shows a biphasic expression pattern Tomato unigene SGN-U212902 encodes a putative vacuolar invertase. Fruit (acid) vacuolar invertases are involved in sink– source relationships, and their activity has been linked to osmotic control of cell expansion. The promoter region described here (PFF) comprises 3232 bp upstream of the theoretical translational start. A 3.6 kb region was previously reported from the same gene (Elliott et al., 1993). PFF-driven expression is low

Figure 1 Transient promoter analysis. Promoter–β-glucuronidase/green fluorescent protein (GUS/GFP) fusions were co-agroinjected with a cauliflower mosaic virus (CaMV) 35S:DsRed construct into MicroTom fruits. Slices from agroinjected fruits were scanned and the green/red fluorescence ratios were scored. (a) Example of a scanning image of a fruit agroinjected with 35SDsRed. (b) Green/red fluorescence ratios for all eight promoters under analysis in agroinjected tomatoes harvested at mature green (green columns) and breaker (red columns) stages. Bars represent average ratios ± standard deviation.



Figure 2 Promoter activity in stably transformed tomato plants. Promoter activity of newly isolated promoters is shown as quantitative β-glucuronidase (GUS) activity from two independent TG1 plants per construct. For the graphical display, those promoters with a similar activity range were grouped together. (a) Activity conferred by strong promoters PNH (open bars) and PLI (filled bars). (b) Promoters with medium activity: PLE (open bars); PFF (filled bars); PHD (grey bars). (c) Two representative plants from the weak promoter PSN (depicted as black and white bars, respectively) in which a sample from ripened columella (CO) was also included. (d) GUS activities from ‘classical’ promoters CaMV 35S (filled bars), E8 (open bars) and 2A11 (grey bars). FL, flowers; L, leaves; S, stems; II, fruits at 5–7 days post-anthesis (dpa); IIIa, fruits at 12–18 dpa; IIIb, mature green fruits between 30 and 35 dpa; IVa, breaker fruits (between 35 and 40 dpa); IVb, red fruits (between 45 and 50 dpa). (e) Promoter strength relative to CaMV 35S at the above-defined stages: II (dotted bars), IIIa (dark grey bars), IIIb (open bars), IVa (filled bars) and IVb (light grey bars). Each bar represents the average values from three organs per plant measured in triplicate ± standard deviation.

at phase II, increasing sharply at early phase III. Pericarp GUS levels are relatively low at the mature-green stage, but increase during ripening (Figure 2b). As can be observed in Figure 3a,b, expression in early phase II is localized to the central columella. Expression in this area and in the rest of the fruit decreases as the fruit expands, reaching a minimum as expansion phase III comes to an end. Later, at phase IV, PFF resumes activity, starting at the central columella and gradually expanding to the rest of the fruit (data not shown). By the time the fruit turns red, PFF-driven expression has reached a second maximum, this time also affecting the pericarp. PSN and PHD are highly specific fruit promoters The DNA region named PSN corresponds to the upstream region of SGN-U149666, a unigene built from a few ESTs exclusively isolated from fruit tissues. This unigene contains a 441-bp ORF encoding a protein sharing 74% homology with the Capsicum annuum Sn-1 gene, previously reported as a fruit-specific gene whose protein product is localized in vacuoles (Meyer

et al., 1996). SGN-U149666 belongs to the Bet-VI superfamily, which includes strong allergens and pathogenesis-related genes. The isolated SGN-U149666 promoter region expands only 592 bp upstream of the putative ATG, and confers the highest specificity for fruit expression detected in our comparative analysis. No detectable GUS levels were found in any organ except the fruit (Figure 2c). Within the fruit, GUS staining was localized to the central columella, placenta and surrounding vascular tissue, and only in ripening fruits (Figure 3a,b). Quantitative GUS analysis confirmed 5-bromo-4-chloro-3indoyl-β-D-glucuronide (X-gluc) expression in and around the central columella (Figure 2c). SGN-U149666 is a low-abundant unigene in expression databases, in accordance with the low GUS activity found in PSN-GUS transgenic plants. PSN also corresponds to the smallest promoter region isolated in this study, and therefore the possibility that additional putative upstream regulatory regions have been missed cannot be discarded. HD promoter isolation was initially designed on the basis of SGN-U143318, a unigene with homology to histidine



Figure 3 Histochemical analysis of βglucuronidase (GUS) expression patterns conferred by tomato promoters in the fruit. Patterns of promoter activity during fruit development were determined by GUS histochemical analysis. Fruit slices from transgenic plants were incubated at 37 °C for 10 min (PNH, E8, 35S, 2A11), 1 h (PLI, PHD) or overnight (PFF, PSN, PLE) with GUS substrate. (a) General view of GUS staining at four developmental stages as described in Figure 1. Scale bars represent 5 mm. (b) Close-ups illustrating most representative features for new promoters. All close-ups represent GUS staining, except for PNH, where the general distribution of green fluorescent protein (GFP) in the pericarp is illustrated. Scale bars represent 2 mm.

decarboxylases. This unigene was later reorganized into five different unigenes. The promoter region cloned here corresponds to SGN-U312404. According to EST data, SGNU312404, which is present only in fruit libraries, is expressed at lower levels than is the major histidine decarboxylase unigene. Consequently, the 1499-bp promoter region isolated as PHD was found to confer moderate GUS expression levels in fruits, and very low levels in the remaining tissues (Figure 2b). GUS expression reaches all fruit layers, and is especially strong in vascular tissues (Figure 3a,b). It should be noted that SGN-U312404 contains a Kozak consensus sequence 93 bp upstream of the ATG proposed for histidine decarboxylase. This Kozak sequence is not present in the most abundant HDC unigene (because of a single G insertion), suggesting a possible regulatory role for this region. Thus, the utilization of a PHD promoter containing its full 5′ UTR, including the early Kozak motif, could lead to

the formation of a fusion peptide of 32 amino acids at the N-terminal end of any GOI. To prevent this possibility from occurring, the 5′ UTR from the PHD promoter was not included in our final construct. PLE promoter is preferentially expressed during phase III SGN-U314285, encoding a late embryogenesis-like protein, was used as template to clone the PLE 1411-bp promoter. SGN-U314285 ESTs are found in several EST libraries, but its presence is particularly abundant in those containing green fruit material. As expected from its relative abundance, the transgenic lines analysed here showed high late embryogenesisabundant (LEA)-driven GUS expression in green fruits, peaking at early or late expansion phase depending on the line (Figures 2b and 3a,b). The LEA promoter did not show high fruit specificity, and particularly high GUS levels were found in flowers (Figure 2b).



CaMV 35S, E8 and 2A11 promoters were integrated into the same reporter vector as the rest of the promoters to serve as reference in a comparative analysis. All three promoters showed expression patterns consistent with those reported previously (Van Haaren and Houck, 1991, 1993; Deikman et al., 1992). GUS activity levels of a representative transgenic line from each construct are depicted in Figure 2d. The CaMV 35S promoter yielded the highest GUS activity in this study in all fruit stages, except breaker and red, where its strength was comparable with ripening-specific promoters. CaMV 35S was used as a reference for the comparative analysis of promoter strength, as shown in Figure 2e. The E8 promoter (2226 bp) drove almost ripening-specific expression in fruits, although vascular tissue staining was also observed in previous developmental stages (Figure 3a). The 2A11 promoter showed a sharp increase in pericarp at late phase III (Figure 2d). Interestingly, 2A11 yielded relatively low levels in placenta and gel (Figure 3a). Given that agroinjectionmediated expression takes place mainly in placenta and gel, this could explain the relatively low ratios observed for 2A11 transient expression when compared with that of other strong promoters (see Figure 1b).

Generation of multisite gateway fruit expression kit To facilitate the construction of expression cassettes, all the promoters described above were adapted to the modular multisite gateway PRO system. The multisite gateway allows the incorporation of two, three or four fragments in tandem, depending on the set-up of choice. With the purpose of constructing expression cassettes, the three-fragment version was chosen. This version allows the incorporation of the promoter regions as a 5′ end fragment and the GOI as a central fragment, leaving a 3′ end fragment position for the introduction of C-terminal tags, terminator sequences, or a combination of both (Figure 4). All six newly isolated promoters were PCR amplified using specific primers that incorporated attB1 and attB4 sequences, and were cloned into the pDONR1-4 vector by BP recombination. Furthermore, to expand the collection, additional promoters with activity in seeds/fruits were incorporated into pDONR1-4: the double 35S promoter, the early ovule-specific promoter from Antirrhinum majus DefH9 (Ficcadenti et al., 1999), the Arabidopsis ovule-specific INO promoter (Meister et al., 2004) and the senescence-specific promoter SAG12 (Swartzberg et al., 2006; Carbonell J. et al., pers. commun.). The collection, named pENFRUIT1-4, currently comprises a total of 13 promoters that, together, provide an unprecedented coverage of developmental stages, tissue specificities and

Figure 4 Triple recombination scheme. In a typical cloning strategy for the recombinant expression of a gene of interest (GOI) in the fruit, the target gene is PCR amplified with attB3r/4r extensions and BP cloned into pDONR4r3r vector. The fruit promoter is selected from the promoter collection (cloned in pEF1-4 entry vectors), whereas 3′ additions are chosen from the pEF3-2 vector collection, which contains transcription terminators and/or protein tags. Following a triple LR reaction, all three elements are tandemly incorporated in a plant destination vector containing attR1-attR2 sites, generating a fruit expression vector. Alternatively, the same system can be used for promoter analysis incorporating any of the pEF4r-3r cloned reporter genes [β-glucuronidase/green fluorescent protein (GUS/GFP), GUS, yellow fluorescent protein (YFP), red fluorescent protein (DsRed)]. In this case, new promoter sequences need to be BP cloned into pDONR 1-4 vector prior to incorporation in an expression vector by triple gateway recombination. An indicative chronogram for the cloning procedures is provided on the left side of the figure.

activity ranges in tomato fruit (pEF1-4 promoter collection in Table 1). To facilitate new promoter analysis, yellow fluorescent protein (YFP), red fluorescent protein (DsRed) and a GFP–GUS fusion were BP cloned into pDONR4-3 vector (pEF4r-3r collection in Table 1). Finally, six 3′ end modules were incorporated into terminal pDONR3-2 vector, including a nopaline synthase terminator (Tnos) and several modules for C-terminal tagging of proteins of interest: 6 × His-Tnos, GFP-6 × His-Tnos, cyan fluorescent protein (CFP), YFP and GUS (pEF3-2 collection in Table 1). The practicability of triple gateway LR recombination using the current promoter collection has been repeatedly confirmed in our laboratory with specific GOIs (data not shown).

Adapting the promoter collection to hpRNA approaches To further expand the uses of our promoter collection, a simple strategy was developed to easily incorporate hpRNA constructs into the triple gateway set-up. For this purpose, two new entry vectors were engineered: pEFS4r-3r and pEFS32. Both vectors contain the same small polylinker but, in



Figure 5 Hairpin RNA (hpRNA) strategy using triple gateway promoter collection. The fruit promoter toolkit can be adapted to hpRNA work by generating two additional pENTR vectors, namely pEFS4r3r and pEFS32. Each plasmid incorporates approximately one-half of a tomato intron next to a small polylinker. The polylinker contains SacI, SmaI and XhoI sites and is set in inverted orientations in each plasmid. For hpRNA strategies, a fragment of the gene of interest (GOI) needs to be cloned into both pEFS4r3r and pEFS32 using two of the restriction enzymes of the polylinker. The GOI fragment is, in this way, inserted in inverted orientations in separate plasmids. On triple gateway recombination, an hpRNA construct driven by a user-defined promoter and containing a spliceable tomato intron is generated. As can be observed in the figure, the attB3 site is conveniently located inside the intron (grey triangle) and therefore will be spliced out on RNA maturation.

inverted orientations, where the gene fragments to be silenced can be inserted by traditional cloning. A fragment of a tomato spliceable intron is set next to each cloning site. The combination of a pEF1-4 tomato promoter with pEFS vectors in a triple gateway recombination generates a hairpin construct with the spliceable intron at its loop (Figure 5). These modules can be used for target silencing of a GOI in specific tissues, such as those driven by the different specificities of the promoters described above and listed in Table 1.

Discussion Recombination-based cloning and gateway-based plasmid collections simplify cloning procedures and facilitate mediumand high-throughput strategies. To date, gateway vector collections in the plant field have been devoted to general purposes, and therefore collections comprise mainly destination vectors incorporating regulatory/reporter elements aimed for general use. As a complementary approach, in this study, we provide a gateway toolkit devoted specifically to gene expression

(and silencing) in the fruit. Unavoidably, such a specific approach needs to provide a set of promoters operating at the organ/tissue of interest, which, in this case, comprises two constitutive (CaMV 35S and 2 × 35S), two ‘classical’ ripening-associated (E8 and 2A11), two ovule-specific (DefH9 and INO), a senescence-specific (SAG12) and six new fruit promoters, aimed at filling gaps in the spectrum of currently available promoters, particularly during phases II and III of tomato fruit development. As an initial comparative test, all new promoters, but also E8, 2A11 and CaMV 35S, were transiently assayed by fruit agroinjection and subsequent fluorescence scanning. Agroinjection-mediated gene expression is not homogeneous in all fruit tissues, and therefore only provides information on promoter functionality and relative strength in those tissues in which transient transformation is achieved efficiently (i.e. placenta, gel and endocarp). Despite this, we found a remarkable correlation between the results obtained in stable and transient experiments in all promoters under study. Indeed, weak promoters showing GUS activity levels below 2–3 nmol MU/min/mg in stable transgenic lines (namely PHD and PSN) fell below background levels in transient assays. It is likely that the use of luminescent instead of fluorescent reporter genes could increase the sensitivity of the method. Although the missing of additional regulatory regions cannot be discounted, the analysis of promoter:GUS transgenic plants confirmed, in general terms, the expression trends deduced from in-house microarray data (Table S2, see Supporting information) and from the prevalence of the parent unigenes in the EST databases. Close inspection of the transgenic plants allowed a more detailed description of their specificities and tissue distribution within the fruits. Most of the isolated promoters, despite being mainly active in the fruits, also showed detectable expression levels in some of the vegetative organs under study. This observation also follows the general trend observed in digital databases and microarray analysis, where strict fruit specificity was only observed in those mRNAs associated with ripening. Despite this, the specificity of promoters, such as PNH or PLI, showing more than 10-fold enrichment in fruits, should be sufficient for most applications requiring a certain degree of fruit-specific expression. Strict fruit specificity is indeed difficult to establish, as reputed ripeningspecific promoters, such as E8, have occasionally been detected in non-fruit tissues (Kneissl and Deikman, 1996). In addition, in our hands, detectable levels of E8-driven GUS expression were found in unexpected tissues (e.g. vascular tissues from immature fruits). All promoters in the collection were incorporated into a multisite gateway system to take full advantage of the tomato



promoter collection. In our approach, the PRO version of the multisite gateway system was selected. This version makes use of conventional and widely available attR1/attR2containing binary vectors as destination vectors. Therefore, the pENFRUIT collection is compatible with other plant attR1/ 2 gateway vector collections and contributes to expand their possibilities. In our experience, the most challenging step in gateway cloning is the generation of ENTRY vectors incorporating large A/T-rich promoter regions. With the generation of the pENFRUIT promoter collection, this limiting step is already accomplished, and the construction of a fruit-specific expression vector can be completed normally within five working days. As in ectopic expression, RNA silencing strategies often require fine-tune regulation, as the depletion of target RNA in different timings/locations can induce undesirable pleiotropic effects. The possibility of achieving regulated gene silencing by incorporating inducible (Wielopolska et al., 2005) or fruitspecific (Davuluri et al., 2005; Wang et al., 2008) promoters has been demonstrated previously. Despite this, most hpRNA plasmids for plant transformation are directed by constitutive promoters. A step forward in filling this gap has been elegantly provided by Karimi et al. (2007) by the incorporation of destination vectors adapted for a classical multisite gateway which facilitates regulated promoter strategies. This approach, however, requires users to undertake de novo cloning of promoter regions into the pENTR vector, which is a challenging step. As a fruit-specific alternative, the set-up presented here allows easy adaptation of the pENFRUIT collection to hpRNA strategies with the addition of two pENTR_RNAi plasmids to the collection. In this way, the pENFRUIT promoter collection is made fully usable for RNAi strategies with the addition of only one traditional cloning step. The combination of gateway and traditional cloning expands the possibilities of gateway systems, as demonstrated recently by Dubin et al. (2008). It is interesting to note that, with the incorporation of a tomato spliceable intron, hpRNA strategies in tomato are possible when the only non-tomato DNA sequences in the expression cassette (excluding border sequences) are attB sites. In total, 13 promoters with activity in tomato fruits have been included in the toolkit. It has not escaped our attention that this collection can be used in fruit-bearing plants other than tomato; however, the expression profiles conferred by each promoter should be established experimentally in each case. In tomato, the newly described promoters cover developmental stages, tissue specificities and activity ranges that were not covered by previously available promoters. Complete updated information on the collection will be displayed at http://www.ibmcp.upv/fruit. Plasmids will be made

available for the tomato research community at the Addgene plasmid repository (http://www.addgene.org).

Experimental procedures Promoter isolation and plant transformation Tomato genomic DNA was isolated from 7-day-old seedlings and digested separately overnight with EcoRV, DraI, PvuII and SspI digestion enzymes. Linker adaptors were ligated to the fragmented DNA using a GenomeWalker™ Universal Kit (Clontech, Mountain View, CA, USA), following the manufacturer’s instructions. Promoter regions for selected candidate genes were amplified in a doublenested PCR using linker-specific (AP1, AP2) and gene-specific (GSP1n, GSP2n) primers under the manufacturer’s recommended conditions. GSP2n primers were all designed to match the most downstream sequence possible within the 5′ UTR considering Tm constraints, except for the PHD sequence (unigene SGN-U143318), whose GSP2 was designed to match the 5′ end of the 457-bp 5′ UTR region. A list of GSP primers is provided in Table S1 (see Supporting information). DNA fragments resulting from double-nested PCR were resolved in agarose gels. Those above 0.6 kb were recovered, cloned into cloning vector pCR 2.1-TOPO (Invitrogen, Carlsbad, CA, USA) and confirmed by sequencing. Positive clones were identified as those whose sequence next to the GSP primer matched with the 5′ UTR of the corresponding unigene. CaMV 35S was isolated from a suitable plasmid DNA. E8 and 2A11 promoters were amplified from tomato genomic DNA. attB1 and attB2 extensions were then added to PNH, PSN, PFF and 2A11 by short PCR amplification with proofreading polymerase, and BP cloned using pDONR221 vector. For the remaining clones, (LE, LI and HD, CaMV 35S and E8), the BP reaction failed, and so direct cloning into pENTR™ Directional TOPO vector (Invitrogen) was followed as an alternative strategy. The resulting ENTRY clones were confirmed by restriction analysis and sequencing of the vector-insert joining regions, and were subsequently LR cloned next to a GFP/GUS translational fusion using plant destination vector pKGWFS7.0 (Plant Systems Biology, Ghent, Belgium). Plasmids were transferred to Agrobacterium tumefaciens LBA4404 strain by electroporation and used for tomato stable transformation (var. MoneyMaker), as described previously (Ellul et al., 2003).

Transient promoter analysis Plant expression plasmids were transferred to Agrobacterium tumefaciens strain C58 and assayed transiently in fruits, as described previously (Orzaez et al., 2006), except that bacterial cultures were infiltrated at a final optical density (OD) of 0.05. Briefly, overnight cultures were recovered by gentle centrifugation and diluted in infiltration medium to the above-mentioned OD. All GFP/GUScontaining cultures were mixed 1 : 9 (vol : vol) with an Agrobacterium strain containing a CaMV 35S:DsRed construct (culture OD = 0.05), which was used as internal standard. Mixtures were syringe-infiltrated into mature green tomatoes (var. MicroTom) as described previously (Orzaez et al., 2006). Tomatoes were harvested between 4 and 6 days after infiltration and separated into two groups (green or ripening) according to their developmental status. Tomato slices were simultaneously double-scanned (Typhoon TRIO Variable Mode



Imager, Amersham Biosciences, Piscataway, NJ, USA) using an excitation wavelength (λex) of 532 nm and 610-nm emission filters (λem) to detect DsRed, and λex = 488 nm and a λem = 520 nm filter for a second channel scan detecting GFP. For each tomato slice, four circular areas with radii of 10 pixels each, corresponding to placenta and/or gel areas showing DsRed fluorescence, were selected, and signal ratios between green and red fluorescence channels were calculated. At least six slices from four different tomatoes per construct were analysed. Background values, calculated as the average green to red ratio in tomatoes agroinjected with CaMV 35S:DsRED only, were subtracted from the final values for representation.

extraction buffer were then added to each well, followed by an additional round of shaking at 25 s–1 frequency. Plates were then centrifuged for 1 h at 3950 g at 4 °C. Cleared supernatant was recovered and used for quantitative GUS assay (Jefferson, 1987) and total protein quantification employing a 96-well plate spectrofluorimeter (GENios Pro™ Microtiter TECAN). Scanned plates were then analysed with XFLUOR™ (TECAN, Männedorf, Switzerland) software. Promoter sequences are deposited in GENBANK with accession numbers FJ586227–FJ586232. Collection ENTRY vectors are deposited in the Addgene repository with accession numbers 20083–20107. Requests for INO and DefH9 promoters should be addressed to Dr A. Dandekar, Department of Pomology, University of California, Davis, CA, USA.

Multisite PRO plasmid construction For the construction of the multisite PRO toolkit, all promoter fragments cloned in pCR 2.1-TOPO were re-amplified with oligonucleotides containing attB1/attB4 recombination sites (Table S1, see Supporting information) and incorporated into pDONRP1-P4 by BPmediated recombination. SAG12, isolated from genomic DNA using gene-specific primers, and DefH9 and INO promoters, kindly provided by Dr A. Dandekar (UC Davis, CA, USA), were also incorporated into pDONRP1-P4. In parallel, YFP, GFP/GUS translational fusion and DsRed reporter sequences were amplified with primers incorporating attB4R and attB3R extensions. Finally, Tnos, 6 × His-Tnos and GFP-TNoscontaining DNA fragments were amplified from suitable plasmids using attB3 and attB2 extensions, and BP cloned into pDONRP3P2. As a result, three subcollections of entry clones were generated: (i) pENTRL1-Prom-L4 entry clones containing promoter regions; (ii) pENTRR4-REP-R3 entry clones containing reporter genes; and (iii) pENTRL3-TER-L2 containing 3′ tag sequences and terminators. Triple recombination was tested for selected pENTRL1-Prom-L4 clones, in combination with pENTRR4-GFP/GUS-R3 and pENTRL3-NosT-L2, by overnight incubation with LR clonase II following Invitrogen’s protocols, using pKGWFS7.0 (Plant Systems Biology) as destination binary plasmid. Positive clones were selected in spectinomycin-containing plates and confirmed by plasmid restriction analysis and by sequencing of junction regions. For hpRNAi ENTRY clone construction, the two halves of a tomato intron (SGN-U324070) were PCR amplified separately with specific primers incorporating opportune attB sites and Xho/SmaI/SacI polylinker extensions (Table S1, see Supporting information). The 5′ segment of the intron was BP cloned with pDNOR4r-3r (Invitrogen), generating pEFS4r-3r cloning plasmid. The 3′ segment of the intron was BP recombined with pDONR3-2 (Invitrogen), generating pEFS3-2 cloning plasmid for hpRNA constructs.

GUS assays Histochemical detection of GUS was performed on fruit slices from stably transformed tomatoes, as described by Jefferson (1987), but modified to include a fixation step (20 min on ice in 90% acetone) previous to enzyme detection. For quantitative GUS activity, 100-mg samples representing different organs were set in 96-well plates in triplicate. Unless otherwise stated, fruit samples from mature green to red stages correspond to pericarp sections of the fruit. Plates with samples were freeze-dried and stored at – 80 °C. Next, freeze-dried tissue was disrupted with glass beads by shaking twice for 3 min at 25 s–1 frequency in a plate homogenizer (Retsch Vibration Mill Type MM 300, Qiagen, Valencia, CA, USA). Six hundred microlitres of GUS

Acknowledgements We wish to thank the Functional Genomics Unit, Plant Systems Biology (VIB-Ghent University, Ghent, Belgium) for providing gateway plant expression vectors, Dr A. Dandekar for providing the INO and SAG12 clones, Asun Fernandez for sharing information on tomato introns and Rafael Martínez for skilled nursing of tomato plants. This work was supported by the Spanish Ministry of Science and Education (Ramón y Cajal programme and BIO2005-01015 project), the European Commission (programme I3P and EU-SOL project) and Fundación Genoma España (ESP-SOL project).

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Supporting information Additional Supporting information may be found in the online version of this article: Figure S1 Reverse transcriptase-polymerase chain reaction (RT-PCR) gene expression measurements. Relative expression levels in tomato seedlings (1), anthesis ovaries (2), fruits at 5–7 days post-anthesis (dpa) (3), pericarp of mature green fruits between 30 and 35 dpa (4) and pericarp of red fruits (between 45 and 50 dpa) (5) of NH (open bars), LI (filled bars), HD (horizontal stripes), FF (dotted bars), SN (bricks) and LE (vertical stripes) genes. All expression data were normalized to the expression levels found in leaves. Bars represent the logarithmic values of the ratios (fruit/leaf) of the means obtained from three replicates ± standard deviation.

Table S1 List of oligonucleotides used in the construction of the pENFRUIT collection. Table S2 Microarray (TOM1) expression data obtained from a duplicate (dye swap) direct match assay made between two pools, one consisting of vegetative parts (leaves, seedlings and roots) and the other consisting of fruit tissue of IIa, IVa and IVb fruits from three independent tomato plants. Data are ordered as a function of the mean log ratio (fruit/vegetative parts) values. 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.
