Advertisement
Browse Subject Areas
?

Click through the PLOS taxonomy to find articles in your field.

For more information about PLOS Subject Areas, click here.

  • Loading metrics

Open Access

Peer-reviewed

Research Article

Regulation of the Fruit-Specific PEP Carboxylase SlPPC2 Promoter at Early Stages of Tomato Fruit Development

  • Carine Guillet,

    Affiliations Unité Mixte de Recherche 1332 Biologie du Fruit et Pathologie, Institut National de la Recherche Agronomique, Villenave d’Ornon, France, Unité Mixte de Recherche 1332 Biologie du Fruit et Pathologie, Université Bordeaux, Villenave d’Ornon, France

    ⨯
  • Mourad A. M. Aboul-Soud ,

    maboulsoud@ksu.edu.sa

    Affiliations Unité Mixte de Recherche 1332 Biologie du Fruit et Pathologie, Institut National de la Recherche Agronomique, Villenave d’Ornon, France, Unité Mixte de Recherche 1332 Biologie du Fruit et Pathologie, Université Bordeaux, Villenave d’Ornon, France, Biochemistry Department, Faculty of Agriculture, Cairo University, Giza, Egypt, Chair of Medical and Molecular Genetics Research, Department of Clinical Laboratory Sciences, College of Applied Medical Sciences, King Saud University, Riyadh, Saudi Arabia

    ⨯
  • Aline Le Menn,

    Affiliation Unité de Génétique et d’Amélioration des Fruits et Légumes, Institut National de la Recherche Agronomique, Montfavet, France

    ⨯
  • Nicolas Viron,

    Affiliations Unité Mixte de Recherche 1332 Biologie du Fruit et Pathologie, Institut National de la Recherche Agronomique, Villenave d’Ornon, France, Unité Mixte de Recherche 1332 Biologie du Fruit et Pathologie, Université Bordeaux, Villenave d’Ornon, France

    ⨯
  • Anne Pribat,

    Affiliations Unité Mixte de Recherche 1332 Biologie du Fruit et Pathologie, Institut National de la Recherche Agronomique, Villenave d’Ornon, France, Unité Mixte de Recherche 1332 Biologie du Fruit et Pathologie, Université Bordeaux, Villenave d’Ornon, France

    ⨯
  • Véronique Germain,

    Affiliations Unité Mixte de Recherche 1332 Biologie du Fruit et Pathologie, Institut National de la Recherche Agronomique, Villenave d’Ornon, France, Unité Mixte de Recherche 1332 Biologie du Fruit et Pathologie, Université Bordeaux, Villenave d’Ornon, France

    ⨯
  • Daniel Just,

    Affiliations Unité Mixte de Recherche 1332 Biologie du Fruit et Pathologie, Institut National de la Recherche Agronomique, Villenave d’Ornon, France, Unité Mixte de Recherche 1332 Biologie du Fruit et Pathologie, Université Bordeaux, Villenave d’Ornon, France

    ⨯
  • Pierre Baldet,

    Affiliations Unité Mixte de Recherche 1332 Biologie du Fruit et Pathologie, Institut National de la Recherche Agronomique, Villenave d’Ornon, France, Unité Mixte de Recherche 1332 Biologie du Fruit et Pathologie, Université Bordeaux, Villenave d’Ornon, France

    ⨯
  • Patrick Rousselle,

    Affiliation Unité de Génétique et d’Amélioration des Fruits et Légumes, Institut National de la Recherche Agronomique, Montfavet, France

    ⨯
  • Martine Lemaire-Chamley,

    Affiliations Unité Mixte de Recherche 1332 Biologie du Fruit et Pathologie, Institut National de la Recherche Agronomique, Villenave d’Ornon, France, Unité Mixte de Recherche 1332 Biologie du Fruit et Pathologie, Université Bordeaux, Villenave d’Ornon, France

    ⨯
  • Christophe Rothan

    Affiliations Unité Mixte de Recherche 1332 Biologie du Fruit et Pathologie, Institut National de la Recherche Agronomique, Villenave d’Ornon, France, Unité Mixte de Recherche 1332 Biologie du Fruit et Pathologie, Université Bordeaux, Villenave d’Ornon, France

    ⨯

Abstract

The SlPPC2 phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) gene from tomato (Solanum lycopersicum) is differentially and specifically expressed in expanding tissues of developing tomato fruit. We recently showed that a 1966 bp DNA fragment located upstream of the ATG codon of the SlPPC2 gene (GenBank AJ313434) confers appropriate fruit-specificity in transgenic tomato. In this study, we further investigated the regulation of the SlPPC2 promoter gene by analysing the SlPPC2 cis-regulating region fused to either the firefly luciferase (LUC) or the β-glucuronidase (GUS) reporter gene, using stable genetic transformation and biolistic transient expression assays in the fruit. Biolistic analyses of 5′ SlPPC2 promoter deletions fused to LUC in fruits at the 8th day after anthesis revealed that positive regulatory regions are mostly located in the distal region of the promoter. In addition, a 5′ UTR leader intron present in the 1966 bp fragment contributes to the proper temporal regulation of LUC activity during fruit development. Interestingly, the SlPPC2 promoter responds to hormones (ethylene) and metabolites (sugars) regulating fruit growth and metabolism. When tested by transient expression assays, the chimeric promoter:LUC fusion constructs allowed gene expression in both fruit and leaf, suggesting that integration into the chromatin is required for fruit-specificity. These results clearly demonstrate that SlPPC2 gene is under tight transcriptional regulation in the developing fruit and that its promoter can be employed to drive transgene expression specifically during the cell expansion stage of tomato fruit. Taken together, the SlPPC2 promoter offers great potential as a candidate for driving transgene expression specifically in developing tomato fruit from various tomato cultivars.

Citation: Guillet C, Aboul-Soud MAM, Le Menn A, Viron N, Pribat A, Germain V, et al. (2012) Regulation of the Fruit-Specific PEP Carboxylase SlPPC2 Promoter at Early Stages of Tomato Fruit Development. PLoS ONE 7(5): e36795. https://doi.org/10.1371/journal.pone.0036795

Editor: Frederik Börnke, Friedrich-Alexander-University Erlangen-Nurenberg, Germany

Received: December 19, 2011; Accepted: April 6, 2012; Published: May 17, 2012

Copyright: © 2012 Guillet et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: This work was supported by the Institut National de la Recherche Agronomique (INRA), by the Région Aquitaine, and by a grant from EUSOL European Integrated Project [FOOD-CT-2006-016214 to NV]. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Tomato (Solanum lycopersicum) is currently the plant model for the study of fleshy fruit development. Several national and international initiatives such as the SOL consortium have contributed to develop new genomic resources in tomato, including the sequencing of tomato genome, the generation of large scale EST and full-length cDNA collections [1], [2] and the expression profiling of developing fruit tissues [3]–[6]. Mining available tomato genomic resources has now produced a wealth of candidate genes with potential roles in the regulation of early fruit development and metabolism [7]. One of the methods of choice for analysing their functional role in the fruit or for bioengineering fruit quality is the generation of stable transgenic lines in which the expression of the candidate gene is specifically modulated in the tissue or at the developmental stage of interest 8,9. In this context, the use of fruit-specific promoters instead of constitutive promoters which may trigger non-specific alterations at whole plant level is usually preferable. To this end, new tomato transformation vectors integrating fruit-specific promoters have recently been crafted for the study of Solanaceae genes [10], [11]. One of these tool kits includes the promoter from the SlPPC2 tomato fruit-specific carboxylase gene previously isolated in our group [12]. In the MicroTom cultivar, the SlPPC2 promoter can be used to direct the mis-expression or silencing of genes-of-interest specifically in the expanding cells from developing tomato fruit [10]. These findings open new ways for the study of the cell expansion phase, which follows the cell division stage and precedes the onset of fruit ripening [13]. This period is crucial not only for fruit growth but also for the acquisition of other fleshy fruit attributes such as the accumulation of water, organic acids, starch and secondary metabolites of high nutritional and sensorial value. As an example of the use of the SlPPC2 promoter, the specific modulation of the cell cycle-related CDK inhibitor KRP in enlarging tomato fruit cells recently led to the demonstration that growth of tomato fruit cells could be uncoupled from cell ploidy level [14]. Such original result was not achieved previously by using the CaMV35S promoter to direct the expression of the CSCS52 endoreduplication-related gene [15], thus demonstrating the power of this approach. Additional insights into the regulation of the SlPPC2 promoter are now needed to delineate more precisely its mode of action in the various cell types of the fruit pericarp.

The transcripts from the SlPPC2 gene encoding a fruit-specific phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) are among the most abundant transcripts found in expanding tomato fruit [12]. One of the functions fulfilled by PEPC is the replenishment of the TCA cycle with oxaloacetate by catalyzing the PEP to oxaloacetate conversion [16]. PEPC appears therefore as a key enzyme in the synthesis of malic and citric acids [16], the two major organic acids accumulated in most fleshy fruits. Regulation of fruit PEPCs is however poorly known. In addition to the tight control of PEPC activity exerted at post-translational level [17], [18], [19], evidence for coarse transcriptional and translational control of PEPC has been presented [16], [20]–[24]. In the C4-type maize, transcription of PEPC has been shown to be regulated by development, light, glucose and acetate [25]. In addition, elements of the tissue-specific and light-regulated control of expression of C4 PEPCs have been identified [24], [26], [27]. In contrast, much less data are available on non-photosynthetic PEPCs, which include the SlPPC2 fruit PEPC [12], though recent advances have shed new light on their regulation and functions [28].

To gain further insights onto the transcriptional regulation of SlPPC2 during the cell expansion stage, and to evaluate the potential use of SlPPC2 promoter for driving gene expression in various genetic or environmental contexts in tomato, we studied the regulation of SlPPC2 promoter in the early developing fruit. Combination of transient expression assays by particle bombardment of pericarp discs and of studies on transgenic tomato plants confirmed that the SlPPC2 promoter is able to confer a proper developmental regulation in the fruit. Strikingly, the fruit-specific expression of SlPPC2 promoter, observed in stable transgenic lines, was lost in transient expression assays, suggesting the need for chromatin integration for appropriate transcriptional regulation in the plant. This study also emphasizes the role of the leader intron located in the 5′UTR of the gene as a negative regulator of SlPPC2 and highlights the possible role of hormones (ethylene) and metabolites (sugars) in its regulation.

Materials and Methods

Ethics Statement

N/A.

Plant Material

Transgenic tomato plants (Solanum lycopersicum, cv. «Ferum») expressing GUS reporter gene under the control of CaMV35S or SlPPC2 promoters were grown in greenhouse as previously described [29], [30]. Plant tissues (seedling, leaflet and flower) and fruits were collected at the indicated stages of development for GUS staining. Biolistic transient expression assays were carried out using cherry tomato fruits (Solanum lycopersicum, cv. «WVa 106») cultivated under growth chamber conditions: cycles of 15 h (25°C) day and 9 h (20°C) night; light intensity of 400 µmole m−2 s−1. Number of inflorescence was limited to 3 per plant. Flowers were tagged on the plant at anthesis and fruits were harvested at the indicated stage, from 6 to 35 days after anthesis (daa), according to age and diameter. The mature green (30 daa) and orange (35 daa) fruits were further selected according to color. Leaflets of young leaves were collected from the same plants.

Isolation of SlPPC2 and Analysis of its Promoter Sequence

A genomic SlPPC2 clone with an insert size of 15 kb was obtained after screening a λ EMBL-3 tomato genomic library (var. «VFN8») (Clontech) with a 566-bp fragment PCR-amplified from the SlPPC2 cDNA clone [12] and sequenced (GenBank AJ313434). The genomic SlPPC2 insert isolated contained the entire coding region (5470 bp) plus 5 kb of sequence upstream the coding region and 4 kb downstream. The transcription start point of the SlPPC2 gene was determined by primer extension analysis using a reverse primer 5PEPC2AC (5′-GAACCCAGAGATGAAGAAAGG-3′) located 57 to 78 bp upstream of the translation initiation ATG codon. The extension reaction was performed at 37°C for 90 min with 100 units of M-MLV reverse transcriptase (Invitrogen) and 50 µM each of dCTP, dTTP and dGTP, and 50 µM of α-[33P]-ATP. The resulting DNA fragment was analyzed on a 6% polyacrylamide gel and was mapped by comparison to a sequence ladder produced from the SlPPC2 promoter using 5PEPC2AC primer to determine transcription start point. The SlPPC2 promoter was analyzed using PLACE [31], PlantCARE [32] and MAR Finder [33].

Reporter Gene Constructs for Biolistic Assays

For biolistic transient expression assays, the plasmid pRTL2-GUS consisting of the CAMV 35S promoter upstream of the tobacco etch virus leader fused to the GUSA gene of E. coli (here referred to as 35S-GUS) was used as a reference construct. A series of five promoter:LUC fusion plasmids were prepared for gene-expression analysis of the SlPPC2 promoter. The promoter fragments (−1528 to +439 [pPPC2pro1:LUC], −980 to +439 [pPPC2pro2:LUC], −430 to +439 [pPPC2pro3:LUC], −70 to +439 [pPPC2pro4:LUC] and −1528 to +195 [pPPC2pro5:LUC]) were PCR-amplified from the pCR-Script-SlPPC2 plasmid as template using the SlPPC2-specific primers designed with either a SacI or a NotI site at their 5′ end. They were further cloned into SacI/NotI sites of pGreen 0000SK LR [34] and sequenced. The LUC gene-nos 3′ terminator cassette from RBCS2-LUC was excised by NheI and EcoRI and ligated using XbaI and EcoRI sites into the five pGreen 0000SK LR plasmids containing the promoter fragments. All constructs were confirmed by sequencing.

Reporter Gene Construct and Generation of Tomato Transgenic Plants

A −1528 to +439 bp 5′ fragment relative to the transcription start site was cloned into the plant transformation vector pGreen 2 K vector at XhoI (5′) and EcoRI (3′) sites with GUS as reporter gene. This SlPPC2 promoteur:GUS construct was introduced into «Ferum» tomato (a medium fruit-sized greenhouse type cultivar) by Agrobacterium tumefaciens strain GV3101 according to a published protocol [35]. Regenerated plantlets were further checked for ploidy level by flow-cytometry analysis and polyploid plants were discarded. Up to twelve independent plants were generated and screened for GUS staining. Control plants corresponding to plants transformed with 35S-GUS or an empty vector were analyzed in parallel. Results presented are from a representative GUS staining experiment. Cherry «WVa106» cultivar was transformed with a SlPPC2 promoter:GFP-GUS fusion generated by cloning a 1972 bp SlPPC2 promoter fragment (including the 5′UTR and leader intron) into the pKGWFS7 vector [36].

Particle Bombardment

Experimental conditions were essentially as previously defined [37] for biolistic transient-expression assays in developing tomato fruit, with modifications. Each tomato fruit was cut into three thin slices (0.5 to 1.0 mm thickness) and soaked for 5 min in CPW 12 [38] supplemented with 12% (w/v) mannitol, 20 mM MES, pH 6.0. Young leaves were cut into pieces of approximately 1 cm2. When indicated, sugars (sucrose [5 to 100 mM], 3-0-methylglucose [50 mM], 2-deoxyglucose (50 mM), fructose (50 mM), glucose (50 mM)] or hormones [GA3 (5 µM), 2,4-D (0.5 to 500 µM), Kinetin (5 µM), ABA (50 µM), ACC (20 and 200 µM)] were added to the CPW 12 medium. In the experiments designed to inhibit ethylene action, fruit tissues were incubated for 2.5 min before osmotic treatment with 4 M silver thiosulfate (AgTS) or with 4 M sodium thiosulfate (control) as previously described [39]. All compounds were dissolved in water or dimethyl sulfoxide and the aqueous solutions were filter-sterilized before use.

Tungsten particles (7 mg, 1.1 µm diameter, Bio-Rad) were coated with either 15 µg reporter plasmid or a 1∶1 ratio of reporter and reference plasmid (15 µg each) in order to obtain 10 cartridges for the helium-driven Gene Gun Helios System (Bio-Rad). Each fruit slice was placed on plate and bombarded with DNA-coated tungsten particles from one cartridge. The Gene Gun was perpendicular to the fruit or leaf surface, its spacer touched the target area and a helium pressure of 210 psi was used. Fruit slices were bombarded a second time after flipping the slices on the plate. The bombarded fruit tissues were placed on 0.8% agar in CPW 4 (4% [w/v] mannitol) supplemented or not with the various hormones and metabolites as indicated above and were incubated for 20 h under growth chamber conditions (22°C, light). The bombarded leaves were placed on 0.8% agar in H2O and incubated for 20 h under growth chamber conditions (22°C, light). These conditions differ from those previously described [37] and were found to be the best adapted to the plant material (cherry tomato fruit, cv. «WVa106») and the biolistic system (BioRad Gene Gun) used. For each construct analyzed and for a given tissue or developmental stage, 9 to 15 tissue samples were independently bombarded and analyzed for Luciferase and GUS activities.

Luciferase and GUS Assays

For biolistic transient expression assays, the fruit or leaf discs were weighed, frozen in liquid nitrogen, ground in a mortar, and homogenized with a 1 ml Tenbroeck Tissue Grinder (Wheaton Millville) in lysis buffer (0.3 M Tris-phosphate pH 7.8, 2 mM dithiotreitol, 2 mM diaminocyclohexane tetracetic acid, 10% glycerol, 1% Triton ×-100) [40], using 2 mL buffer g−1 plant tissue. The extract was cleared by centrifugation (15 000×g for 10 min). Protein concentration was determined with the Coomassie plus protein assay reagent (Pierce) adapted for the MR5000 microplate reader (Dynatech) using BSA as a standard. Luciferase activity was determined immediately after extract preparation using the Promega Luciferase Assay System (Promega) as previously described [41]. Light emission was measured for 1 min in a 1254 Luminova luminometer (Bio-Orbit Oy). Statistical comparisons between results within a given experiment were made using a Student’s t-test. All differences were significant to at least a value of P<5%. GUS activity was determined by adding 20 µL of supernatant to 2 mL GUS buffer (50 mM NaH2PO4 pH7, 10 mM Na2EDTA, 10 mM β-mercaptoethanol, 0.1% Sarcosyl, 0.1% Triton ×100) containing 0.75 g/mL of 4-methyl umbelliferyl β-D-glucuronide (MUG). After incubation at 37°C, 400 µL of reaction mixture was collected at 0, 30 and 90 min and mixed with 2 mL stop buffer (200 mM Na2CO3). Fluorescence was measured at 455 nm after excitation at 365 nm (Hitachi spectrofluorimeter). For GUS staining, plant tissues were soaked in 0.15 M phosphate buffer pH 7, vacuum-infiltrated and incubated for 1 hour at 37°C in GUS staining solution (0.5 mM 3-indolyl glucuronide, 0.15 M NaH2PO4 pH 7, 2 mM K3Fe(CN)6, 2 mM K4Fe(CN)6, 0.05% Triton ×100).

Extraction and Determination of Sugars and Organic Acids

Slices from 8 daa tomato fruit were incubated in the various conditions as described for biolistic transient expression assays, weighed, frozen in liquid nitrogen and ground in a mortar. Briefly, soluble sugars and starch were extracted using alcoholic extraction method and starch converted to glucose as previously described [42]. Soluble sugars were then measured using a MR 5000 microplate reader (Dynatech) microassay. Citric and malic acids were extracted as previously described [12] and enzymatically measured following instructions of the Boehringer’s kit adapted for MR5000 reader micro assay.

Results and Discussion

Features of the SlPPC2 Promoter

Plant PEPCs show a highly conserved structure and amino acid sequence [16]. Like most other plant PEPC genes, the SlPPC2 gene is formed of 10 exons interrupted by nine introns located at conserved positions (Figure S1). Comparison of SlPPC2 genomic sequence and 5′ UTR sequence of SlPPC2 cDNA also revealed the presence of an additional intron in the 5′ leader sequence of SlPPC2. Its location and size (200 bp) is close to that of the leader intron found in the well-studied C4 PEPC ppcA1 gene from dicot F. trinervia ppcA1 gene (177 bp), which is expressed in mesophyll cells and fulfils very different roles [12], [21], [23], [43].

A SlPPC2 promoter fragment including the 5′ untranslated region (UTR) of the gene (–1969 to −3 bp from the translation start site) was obtained by PCR amplification and restriction. The putative transcriptional start point determined by primer extension analysis was located 442 nucleotides upstream of the translational start codon ATG (Figure S2). A putative TATA box is located at nucleotide –20 relative to the transcriptional start point. Analysis with PLACE [31] and PlantCARE [32] unravelled putative cis-regulating elements known to play a role in the regulation of transcription. In addition, several motifs identified as binding sites for transcription factors (MADS domain factors, TCP, WRKY) were also found upstream of transcription start (Figure 1). Of particular interest is the −1500 to −900 region where motifs putatively involved in the binding of MADS domain protein (CArG box) [44] and in signalling pathways for auxin and brassinosteroid (ARFAT) [45], gibberellin (GADOWNAT and GARE) [46], [47], abscisic acid and calcium (ABRE-like motif) [48], [49] and ethylene (ERE) [50] were found. In plants, regulatory elements usually tend to be highly clustered in the vicinity of the core-promoter elements, but can also be found all along the promoter [51], [52]. In tomato, the distal 5′ flanking regions are crucial for the regulation of at least two genes, the ripening-associated tomato polygalacturonase (PG) and the E8 gene [53]–[55].

thumbnail
Download:
Figure 1. Localization of putative cis-acting elements in SlPPC2 promoter sequence.

Sequence was analyzed using PLACE and PlantCARE databases.

https://doi.org/10.1371/journal.pone.0036795.g001

Several hormones play a prominent role in the regulation of early fruit growth. Among these are the auxins and brassinosteroids, which have a synergistic effect on cell elongation in plants [56], and may control the expansion of the fruit mesocarp cells in which SlPPC2 is expressed [5], [12], [57], [58]. The fruit ripening hormone ethylene can also be implicated in early fruit growth in tomato, owing to its role in the control of endoreduplication and cell expansion in various plant species and organs [59]–[61]. Other elements identified in light or circadian-regulated genes are scattered along the SlPPC2 promoter sequence (I box, GT1 element, Evening Element EE and Z box) [50], [62]–[65]. Though there is no evidence for light or circadian clock regulation of PEPC in fruit, the light involvement in the regulation of fruit development and metabolism is now well established [9] and recent results indicate that a sugar-metabolism gene, the LIN6 invertase, is regulated by diurnal rhythm in tomato fruit [66]. A SURE motif [67] and two G boxes separated by 17 bp including ACT [68] can also indicate the involvement of sugars in SlPPC2 regulation. The search for fruit-specific elements identified in other plant species [69], [70] remained unsuccessful.

The SlPPC2 Promoter Confers Proper Developmental Regulation in Developing Tomato Fruit

The miniature MicroTom tomato previously used to monitor SlPPC2 promoter activity in tomato [10] is likely mutated in the brassinosteroid dwarf gene and may thus display altered hormonal and developmental regulations. To investigate whether the organ-specificity and developmental patterns observed in MicroTom were conserved in other tomato genotypes, transgenic tomato plants were generated with SlPPC2 promoter:GUS or SlPPC2 promoter:GFP-GUS transcriptional fusions, using two different tomato cultivars. The cultivars used were «Ferum», a cultivated greenhouse tomato variety with medium-sized fruits, and «Wva106», a cherry-type tomato well adapted to the study of early fruit development [14], [15], [71]. In «Ferum», the SlPPC2 promoter:GUS primary transformants showed consistently (>10 independent transformants) GUS staining in expanding fruit tissues but exhibited no staining of young seedlings, leaves or flowers (except for faint staining of stamens) (Figure 2A). A representative GUS staining of T2 homozygous fruits (single copy insertion line) is shown in Figure 2B. Time-course analysis of SlPPC2 promoter activity along fruit development indicated that SlPPC2 promoter activity peaked between 25 and 40 daa, i.e. during the cell expansion phase which lasts from ∼10 to 40 daa in the «Ferum» cultivar [71]. During fruit development, the staining progressed from the placental tissue, which differentiates early, to the outer pericarp. No staining was observed during the early stages of cell division while residual GUS activity was seen in ripe fruit. In contrast, 35S-driven GUS activity was high in all plant organs and fruit stages analyzed. Similar results were obtained in the cherry tomato «WVa 106» using SlPPC2 promoter:GFP-GUS fusion (pKGWFS7 vector, data not shown). These results are consistent with the pattern of SlPPC2 transcript accumulation in the plant [12] and with previous results obtained in MicroTom transgenics [10], thereby confirming that the SlPPC2 promoter is specifically active in the fruit during the cell expansion phase.

thumbnail
Download:
Figure 2. GUS activity in tomato (cv. «Ferum») stably transformed with SlPPC2 promoter:GUS transgenes.

(A) Representative images of GUS activity in seedlings, leaf, flower and 6, 14 and 21 days after anthesis (daa) tomato fruit with the 35S:GUS and SlPPC2 promoter:GUS constructs. (B) Representative images of GUS activity in «Ferum» fruits at the various stages of fruit development (daa) as indicated.

https://doi.org/10.1371/journal.pone.0036795.g002

In order to analyse the regulation of SlPPC2 promoter by various metabolites and hormones in developing fruit, we next used transient expression assays. We preferred this technique over the use of whole transgenic fruits expressing GUS for several reasons. Because tomato fruit is a bulky organ, the penetration and transport of hormones and metabolites in the various tissues from whole transgenic fruits is very difficult to control. This may therefore strongly bias the results and affect their reproducibility. In contrast, the method of biolistic transformation of osmotically-treated tomato fruit tissues developed by Baum and co-authors [36], allows quantitative, systematic and reproducible measurements in fruit tissues. In this method, the use of luciferase as reporter gene allows studying the fine control of promoter activity whereas using GUS as reference allows normalization of the data and therefore comparison between multiple experiments. In a first step, we tested this technique by fusing 1966 bp of the 5′ flanking regions of the SlPPC2 gene (including 439 bp of the 5′ UTR and leader intron) to LUC (firefly Luciferase) reporter gene and by examining its expression in developing tomato fruit. Adaptation of this protocol to our conditions (see Materials and Methods) led to a consistent 35S promoter-driven luciferase activity that was about 600-fold over background activity in young green fruit from the “Wva106” cultivar (data not shown). Changing mannitol concentration in the incubation medium from 12% to 4% led to a further increase of 1.5 fold in the promoter activity. To take into account the possible light or circadian clock regulation of SlPPC2 (see above), all experiments were conducted with fruits collected early in the morning at the same time. Under these conditions, tissues from tomato fruit at various stages of development (from 6 daa to 30 daa mature green stage) were co-transformed with the pPPC2pro1:LUC (SlPPC2 promoter-LUC construct) and 35S:GUS constructs as internal controls. Results indicated that full-length SlPPC2 promoter was sufficient to drive a high reporter gene activity in the developing fruit, with a notable expression from 6 to 15 daa and peaking at 8 daa, consistent with the timing of the cell expansion phase and changes in SlPPC2 transcript abundance level in «WVA 106» fruit (Figure 3). Therefore, the 1966 bp of the 5′ flanking regions of the SlPPC2 gene studied contains all the information necessary to confer proper developmental regulation in tomato fruit.

thumbnail
Download:
Figure 3. Transient reporter-gene expression analysis of the SlPPC2 promoter in developing tomato fruit («WVa 106» cherry).

Fruit slices at the indicated stages of development from 6 to 30 days after anthesis (daa) were transformed by biolistic with a 35S:GUS plasmid co-delivered with a promoter:LUC fusion plasmid (promoter:LUC construct pPPC2pro1:LUC) that included the nucleotides –1528 to +439 of SlPPC2 (with respect to the transcription start site). The pGr (plasmid alone) and pLUC (promoterless LUC construct) plasmids were used as negative controls to transform 8 daa fruit slices. Data were normalized using the 35S:GUS construct as internal standard and are expressed as % of maximum activity (8 daa fruit). The mean values and SE of 6 to 12 independent transformations are shown. Insert represents the RT-PCR analysis of SlPPC2 expression during wild-type tomato fruit development.

https://doi.org/10.1371/journal.pone.0036795.g003

Deletion of the Leader Intron Increases SlPPC2 Promoter Activity but Affects its Developmental Pattern

To further analyze the role of the SlPPC2 5′ flanking regions and of the leader intron in the regulation of SlPPC2 gene expression, a set of 5′ deletions was produced (Figure 4) and their expression was analyzed in 8 daa fruit, when SlPPC2 full-length promoter activity (pPPC2pro1:LUC construct) is maximum (Figure 3). Deletion to position −980 (pPPC2pro2:LUC) reduced the activity relative to pPPC2pro1:LUC by about 28% in 8 daa fruit. Additional deletions to positions −430 and −70 further reduced the activity relative to pPPC2pro1:LUC by about 71% and 78%, respectively. Control (pLUC) only showed a marginal luciferase activity (4.6% of the activity of pPPC2pro1:LUC at 8 daa). These data suggest that the major cis-acting elements responsible for high level of SlPPC2 promoter activity in young fruit (8 daa) are located between positions −1528 to –430, a region which is particularly rich in putative regulatory elements (Figure 1).

thumbnail
Download:
Figure 4. Transient reporter-gene expression analysis of SlPPC2 promoter deletions in 8 daa tomato fruit.

Slices from 8 daa fruit («WVa 106» cherry) were transformed by biolistic with a 35S:GUS plasmid co-delivered with SlPPC2 promoter:LUC fusion plasmids (pPPC2pro1-4:LUC construct; sizes in nucleotides from the transcription start indicated; grey box indicates leader intron).The pLUC plasmid (promoterless LUC construct) was used as a negative control. Data were normalized using the 35S:GUS construct as internal standard and are expressed as % of maximum activity (pPPC2pro1:LUC construct). The mean values and SE of 6 to 12 independent transformations are shown.

https://doi.org/10.1371/journal.pone.0036795.g004

To test whether the 200-bp intron located in the 5′ UTR is important for the control of the developmental expression, we deleted the region spanning from +195 to +439 (pPPC2proΔ:LUC construct), which comprises both the leader intron and the 5′ UTR between the leader intron and the start codon. Deletion of the leader intron enhanced the transcriptional activity of the promoter by 1.8 to 9.6-fold, depending on the developmental stage of the fruit, and led to a loss of its proper regulation during fruit development (Figure 5). Contrary to the activity of the full length promoter (pPPC2pro1:LUC construct) and to the level of SlPPC2 transcripts (Figure 3), the activity of pPPC2proΔ:LUC was much higher at 6 daa, i.e. in mitotic cells, than in tissues undergoing cell differentiation and expansion. In this context, these data provide clear evidence that the first intron functions as a negative regulatory element that contributes to the developmental regulation of SlPPC2 expression in the fruit.

thumbnail
Download:
Figure 5. Transient reporter-gene expression analysis of the SlPPC2 promoter deleted from its leader intron in developing tomato fruit and in leaf.

(A) Details of the constructs. (B) Young leaf discs and slices of tomato fruit («WVa 106» cherry) at the indicated stages of development were transformed by biolistic with the –1528 to +439 construct (pPPC2pro1:LUC, in black) or with the –1528 to +195 construct (pPPC2proΔ:LUC, in grey) as indicated in Figure 1. Data are expressed as % of the pPPC2pro1:LUC activity at 8 daa. The mean values and SE of 10 independent transformations are shown.

https://doi.org/10.1371/journal.pone.0036795.g005

Both positive and negative roles for leader introns have been demonstrated in several plant genes including the sucrose synthase gene SUS3 [72] and the Arabidopsis cytochrome C oxidase gene COX5C in which the leader intron is essential to direct high-level and tissue-specific expression [73]. In contrast, recent work on the F. trinervia C4 isoform of PEPC suggested that the leader intron in this gene is not essential for achieving high mesophyll-specific expression [74]. A growing number of plant expression studies have also revealed that the presence of a leader intron within the 5′UTR may affect not only transcription but also post-transcriptional processes [73], [75]. Regardless of the control level of LUC activity exerted by the leader intron, the main conclusion is that the full-length promoter (including leader intron) is necessary to deliver mRNA/protein to fruit cells specifically during the cell expansion stage of tomato fruit development.

Surprisingly, in transient expression assays, fruit-specificity was lost since pPPC2pro1:LUC and pPPC2proΔ:LUC activities were similar in leaf and in 6 daa or 8 daa fruit, respectively (Figure 5). This was also true for the various deletion constructs tested (data not shown). The above findings suggest that chromatin integration is essential to confer an appropriate pattern of expression in the plant, as previously found for the tomato fruit RBCS3A promoter [76].

Hormonal Regulation of the SlPPC2 Promoter

Hormones are known regulators of fruit set and early fruit development [13] and several putative hormone responsive elements were identified in the SlPPC2 5′ flanking region by in silico analysis (Figure 1). Therefore, the effects of auxins (2,4-D), cytokinins (kinetin), gibberellins (GA3), abscisic acid (ABA) and ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) on full-length SlPPC2 promoter activity were investigated using biolistic transient expression assay. Kinetin (5 µM), GA3 (5 µM) and ABA (50 µM) did not display any significant effect on SlPPC2 promoter activity (data not shown). By contrast, the synthetic auxin 2,4-D significantly increased the SlPPC2 transcriptional activity when applied at 50 µM (data not shown), whereas the 2,4-D non-functional analog 2,3-D failed to trigger any change in SlPPC2 promoter activity, at the same concentration. Auxin plays a major role in early fruit development in addition to its well known effect on fruit set [56], [77]–[79]. However, 2,4-D is usually physiologically active at much lower concentrations (<5 µM), suggesting that the 2,4-D effect on SlPPC2 promoter activity is indirect. In contrast, the ethylene precursor ACC, fed to pericarp discs, had a strong and significant effect on SlPPC2 transcriptional activity (Figure 6). Notably, ACC significantly increased SlPPC2 promoter activity at 20 µM and enhanced it by two-fold at 200 µM (Figure 6). In the presence of silver thiosulfate (AgTS), a known inhibitor of ethylene action [39], promoter activity was significantly reduced, even in the presence of ACC at 20 µM. In contrast, its inactive AgTS analogue failed to inhibit the action of ethylene produced by 20 µM ACC. Though ethylene is much better known for its coordination of fruit ripening [80], this hormone may control endoreduplication and cell expansion in various plant species and organs [59]–[61]. It is therefore likely that ethylene is involved in the regulation of the cell expansion phase in early developing fruit, as suggested by the analysis of the auxin mutant diageotropica [78].

thumbnail
Download:
Figure 6. Influence of ethylene (ACC) on SlPPC2 promoter activity.

Fruit slices from 8 daa fruit («WVa 106» cherry) were transformed by biolistic with a 35S:GUS plasmid co-delivered with SlPPC2 promoter:LUC fusion pPPC2pro1:LUC plasmid and incubated for 20 h on CPW4 medium supplemented with 1-aminocyclopropane-1-carboxylic acid (ACC, 20 µM and 200 µM), silver thiosulfate (AgTS), or ACC (20 µM) plus AgTS or NaTS. Control was CPW4 medium. Data were normalized using the 35S:GUS construct as internal standard and are expressed as % of the control. The mean values and SE of 12 independent transformations are shown. * indicates a significant statistical difference using a Student’s t-test (*: P<0.05; **: P<0.001).

https://doi.org/10.1371/journal.pone.0036795.g006

We further investigated whether the ethylene-modulated changes in SlPPC2 transcriptional activity were accompanied by variations in organic acid content in tomato fruit tissues. Significant effects were observed with 200 µM ACC, which increased L-citric acid and L-malic acid contents by 50% and 60%, respectively (Table 1). Conversely, addition of AgTS to pericarp discs fed with 20 µM ACC resulted in a slight but significant reduction in L-citric acid content of about 35% (Table 1). These results are consistent with transgenic experiments in which PEPC overexpression in tobacco redirects the metabolic flow, in particular towards malate synthesis [81], [82].

thumbnail
Download:
Table 1. Malic and citric acid contents of 8 daa tomato fruit slices.

https://doi.org/10.1371/journal.pone.0036795.t001

Metabolic Regulation of the SlPPC2 Promoter

Various metabolites such as sugar hexoses may regulate PEPC transcription [25]. In order to investigate the possible regulation of SlPPC2 by sugars, the activity of the SlPPC2 full-length promoter was determined on bombarded 8 daa tomato fruit slices incubated with various concentrations of sucrose, glucose, fructose, the glucose analogs 2-deoxyglucose (2-dG) and 3-O-methylglucose (3-OMG), or mannitol as an osmotic control. We first controlled that the sugars were taken up by the fruit tissues by measuring the concentration in various metabolic compounds (sucrose, glucose, fructose, starch, malic and citric acids) in fruit discs at the end of the incubation period (see Figure S3). Sugars could effectively enter the fruit slices and were metabolized, as evidenced by the cleavage of sucrose to glucose and fructose, the interconversion of glucose and fructose and the synthesis of starch and organic acids further accumulated in fruit tissues. This indicated that fruit slices are suitable for studying the regulation of SlPPC2 by sugars. While no significant alterations of SlPPC2 promoter activity were observed in sugar-supplemented fruit tissues, our data showed that high sucrose concentration (100 mM) resulted in a signification reduction in promoter activity (Figure 7). However, because mannitol was used at 50 mM in the control, a possible osmotic effect of 100 mM sucrose on SlPPC2 promoter activity cannot be excluded.

thumbnail
Download:
Figure 7. Influence of sugars on SlPPC2 promoter activity.

Fruit slices from 8 daa fruit («WVa 106» cherry) were transformed by biolistic with a 35S:GUS plasmid co-delivered with SlPPC2 promoter:LUC fusion pPPC2pro1:LUC plasmid and incubated for 20 h on CPW4 medium supplemented with sucrose concentrations ranging from 5 mM to 100 mM as indicated, 3-O methylglucose (3-OMG, 50 mM), 2-deoxyglucose (2-dG, 50 mM), glucose (50 mM) or fructose (50 mM). Control was CPW4 medium supplemented with mannitol (50 mM). Data were normalized using the 35S:GUS construct as internal standard and are expressed as % of the control. The mean values and SE of 12 independent transformations are shown. * indicates a significant statistical difference using a Student’s t-test (*: P<0.05; **: P<0.001).

https://doi.org/10.1371/journal.pone.0036795.g007

In contrast, SlPPC2 promoter activity was reduced by about one-third when the glucose analog 3-O-methylglucose (3-OMG) was supplied at 50 mM to the tissues, and reduced by two-thirds when 2-deoxyglucose (2-dG) was supplied at the same concentration (Figure 7). Malic acid content was significantly reduced only in 2-dG supplied tissues, in which SlPPC2 promoter activity was strongly affected, whereas sugar (sucrose, glucose and fructose) or starch contents were not significantly affected in the tissues supplied with either 3-OMG or 2-dG (Figure S3). Results obtained for 2-dG are consistent with previous observations showing that variation in PEPC transcription may lead to changes in malic acid content in plant [81], [82] and fruit tissues [83]. The 2-dG can be transported into the tomato fruit cells and phosphorylated by hexokinase, but the phosphorylated product 2-deoxyglucose 6-phosphate (2-dG-6p) cannot be further metabolized [84]. The 3-OMG is transported into the plant cells but is metabolized very slowly [85]. Thus, the repression caused by 3-OMG and 2-dG supports the hypothesis that sugar is required for regulation of SlPPC2 expression in the fruit. However, we cannot rule out that this result is achieved through a more general effect on fruit metabolism.

Inorganic nitrogen (NO3−, NH4+) and transported forms of amino acids in the fruit (glutamine and asparagine) were also tested but no significant effect on SlPPC2 promoter activity was detected. Thus, under the experimental conditions of the study, there is no conclusive evidence of the transcriptional control of SlPPC2 by metabolites other than sugar in the fruit.

Conclusion

This study demonstrated that a 1966 bp 5′ region of the SlPPC2 fruit PEPC gene including −1528 bp of promoter region plus 439 bp of 5′ untranslated leader region is able to confer appropriate fruit-specificity and developmental expression in tomato fruit. Transient expression assays further showed that the deletion of an intron in the 5′ untranslated leader region leads to loss of proper developmental regulation, suggesting that leader intron acts as a negative regulatory element. Though no correlation was found between promoter activity and sugar metabolites, sugar signaling may modulate SlPPC2 promoter activity, as indicated by the effects of 2-dG and 3-OMG. Noticeably, results indicate that SlPPC2 may be regulated by the plant hormone ethylene. While auxin is known for its role in the regulation of fruit growth [73], [74], the implication of ethylene and of cross-talks between auxin and ethylene for controlling early stages of fruit development has been poorly studied. The enhancement of SlPPC2 promoter activity by ethylene and the concomitant organic acid increase in fruit tissues are consistent with the hypothesis that the PEPC-mediated organic acid synthesis sustains osmotic potential to allow rapid fruit cell expansion [12], [28] and is under hormonal control in developing tomato fruit. In addition, this study opens the way for the use of SlPPC2 promoter for the functional study of candidate genes in the fruit and for the biotechnological improvement of fruit sensorial and nutritional quality.

Supporting Information

Figure S1.

Exon/intron organization of the tomato SlPPC2 gene. The tomato SlPPC2 gene (GenBank accession No. AJ313434) was compared to the Flaveria trinervia ppcA1 gene (Genbank accession No. AJ011844). Introns (grey boxes) are numbered from I to X and their sizes indicated above the diagrams.

https://doi.org/10.1371/journal.pone.0036795.s001

(TIF)

Figure S2.

Determination of the transcription start point of the SlPPC2 gene by primer extension analysis. Lane PE shows the extension product obtained after reverse transcription using a SlPPC2-specific oligonucleotide primer. The band, indicated by an arrow, corresponds to a G located 442 nucleotides upstream from the ATG codon. The sequencing ladder was generated using the same primer on a cloned fragment of the SlPPC2 genomic clone. Sequence upstream from the transcription start site is presented, showing location of putative TATA box.

https://doi.org/10.1371/journal.pone.0036795.s002

(TIF)

Figure S3.

Carbohydrate content of tomato fruit slices incubated on medium supplemented with various sugars. (A) Sucrose; (B) Glucose; (C) Fructose; (D) Starch; (E) Malic acid; (F) Citric acid. Eight (8) daa tomato fruit slices were incubated or not (no incubation) for 20 h on CPW4 medium containing 50 mM mannitol, 5 mM to 100 mM sucrose, 50 mM 3-OMG, 50 mM 2-dG, 50 mM glucose or 50 mM fructose. Data are means ± SE (n = 3).

https://doi.org/10.1371/journal.pone.0036795.s003

(TIF)

Acknowledgments

The pRTL2-GUS and RBCS2-LUC plasmids were kindly provided by I. Meier and K. Baum (Hamburg University, Germany). The pGreen 0000SK LR plasmid was kindly provided by Nicola Leyland and Phil Mulineaux (John Innes Center, Norwich, UK).

Author Contributions

Conceived and designed the experiments: CR. Performed the experiments: CG MAMA-S ALM NV AP VG ML-C DJ PB PR. Analyzed the data: CG PR CR. Contributed reagents/materials/analysis tools: DJ PB. Wrote the paper: CR.

References

  1. 1. Fei Z, Tang X, Alba RM, White JA, Ronning CM, et al. (2004) Comprehensive EST analysis of tomato and comparative genomics of fruit ripening. Plant J 40: 47–59.
  2. 2. Yamamoto N, Tsugane T, Watanabe M, Yano K, Maeda F, et al. (2005) Expressed sequence tags from the laboratory-grown miniature tomato (Lycopersicon esculentum) cultivar Micro-Tom and mining for single nucleotide polymorphisms and insertions/deletions in tomato cultivars. Gene 356: 127–134.
  3. 3. Carrari F, Baxter C, Usadel B, Urbanczyk-Wochniak E, Zanor MI, et al. (2006) Integrated analysis of metabolite and transcript levels reveals the metabolic shifts that underlie tomato fruit development and highlight regulatory aspects of metabolic network behavior. Plant Physiol 142: 1380–1396.
  4. 4. Vriezen WH, Feron R, Maretto F, Keijman J, Mariani C (2008) Changes in tomato ovary transcriptome demonstrate complex hormonal regulation of fruit set. New Phytol 177: 60–76.
  5. 5. Lemaire-Chamley M, Petit J, Garcia V, Just D, Baldet P, et al. (2005) Changes in transcriptional profiles are associated with early fruit tissue specialization in tomato. Plant Physiol 139: 750–769.
  6. 6. Matas AJ, Yeats TH, Buda GJ, Zheng Y, Chatterjee S, et al. (2011) Tissue- and cell-type specific transcriptome profiling of expanding tomato fruit provides insights into metabolic and regulatory specialization and cuticle formation. Plant Cell 23: 3893–910.
  7. 7. Mounet F, Moing A, Garcia V, Petit J, Maucourt M, et al. (2009) Gene and metabolite regulatory network analysis of early developing fruit tissues highlights new candidate genes for the control of tomato fruit composition and development. Plant Physiol 149: 1505–1528.
  8. 8. Davuluri GR, van Tuinen A, Fraser PD, Manfredonia A, Newman R, et al. (2005) Fruit-specific RNAi-mediated suppression of DET1 enhances carotenoid and flavonoid content in tomatoes. Nature Biotech 23: 890–895.
  9. 9. Enfissi EM, Barneche F, Ahmed I, Lichtlé C, Gerrish C, et al. (2010) Integrative transcript and metabolite analysis of nutritionally enhanced DE-ETIOLATED1 down-regulated tomato fruit. Plant Cell 22: 1190–1215.
  10. 10. Fernandez AI, Viron N, Alhagdow M, Karimi M, Jones M, et al. (2009) Flexible tools for gene expression and silencing in tomato. Plant Physiol 151: 1729–1740.
  11. 11. Estornell LH, Orzáez D, López-Peña L, Pineda B, Antón MT, et al. (2009) A multisite gateway-based toolkit for targeted gene expression and hairpin RNA silencing in tomato fruits. Plant Biotechnol J 7: 298–309.
  12. 12. Guillet C, Just D, Benard N, Destrac-Irvine A, Baldet P, et al. (2002) A fruit-specific phosphoenolpyruvate carboxylase is related to rapid growth of tomato fruit. Planta 214: 717–726.
  13. 13. Gillaspy G, Ben-David H, Gruissem W (1993) Fruits: a developmental perspective. Plant Cell 5: 1439–1451.
  14. 14. Nafati M, Cheniclet C, Hernould M, Do PT, Fernie AR, et al. (2011) The specific overexpression of a cyclin-dependent kinase inhibitor in tomato fruit mesocarp cells uncouples endoreduplication and cell growth. Plant J 65: 543–556.
  15. 15. Mathieu-Rivet E, Gévaudant F, Sicard A, Salar S, Do PT, et al. (2010) The functional analysis of the Anaphase Promoting Complex activator CCS52A highlights the crucial role of endoreduplication for fruit growth in tomato. Plant J 62: 727–41.
  16. 16. Chollet R, Vidal J, O’Leary MH (1996) Phosphoenolpyruvate carboxylase: a ubiquitous, highly regulated enzyme in plants. Annu Rev Plant Physiol 47: 273–98.
  17. 17. Izui K, Matsumura H, Furumoto T, Kai Y (2004) Phosphoenolpyruvate carboxylase: a new era of structural biology. Ann Rev Plant Biol 55: 69–84.
  18. 18. Hartwell J, Gill A, Nimmo GA, Wilkins MB, Jenkins GI, et al. (1999) Phosphoenolpyruvate carboxylase kinase is a novel protein kinase regulated at the level of expression. Plant J 20: 333–342.
  19. 19. Chen ZH, Jenkins GI, Nimmo HG (2008) pH and carbon supply control the expression of phosphoenolpyruvate carboxylase kinase genes in Arabidopsis thaliana. Plant Cell Environ 31: 1844–1850.
  20. 20. Jeanneau M, Vidal J, Gousset-Dupont A, Lebouteiller B, Hodges M, et al. (2002) Manipulating PEPC levels in plants. J Exp Bot 53: 1837–1845.
  21. 21. Gowik U, Burscheidt J, Akyildiz M, Schlue U, Koczor M, et al. (2004) Cis-Regulatory elements for mesophyll-specific gene expression in the C4 plant Flaveria trinervia, the promoter of the C4 phosphoenolpyruvate carboxylase gene. Plant Cell 16: 1077–1090.
  22. 22. Sánchez R, Flores A, Cejudo FJ (2006) Arabidopsis phosphoenolpyruvate carboxylase genes encode immunologically unrelated polypeptides and are differentially expressed in response to drought and salt stress. Planta 223: 901–909.
  23. 23. Akyildiz M, Gowik U, Engelmann S, Koczor M, Streubel M, et al. (2007) Evolution and function of a cis-regulatory module for mesophyll-specific gene expression in the C4 dicot Flaveria trinervia. Plant Cell 19: 3391–402.
  24. 24. Offermann S, Dreesen B, Horst I, Danker T, Jaskiewicz M, et al. (2008) Developmental and environmental signals induce distinct histone acetylation profiles on distal and proximal promoter elements of the C4-Pepc gene in maize. Genetics 179: 1891–1901.
  25. 25. Kausch AP, Owen TP , Zachwieja SJ, Flynn AR, Sheen J (2001) Mesophyll-specific, light and metabolic regulation of the C4 PPCZm1 promoter in transgenic maize. Plant Mol Biol 45: 1–15.
  26. 26. Offermann S, Danker T, Dreymüller D, Kalamajka R, Töpsch S, et al. (2006) Illumination is necessary and sufficient to induce histone acetylation independent of transcriptional activity at the C4-specific phosphoenolpyruvate carboxylase promoter in maize. Plant Physiol 141: 1078–1088.
  27. 27. Windhovel A, Hein I, Dabrowa R, Stockaus J (2001) Characterization of a novel class of plant homeodomain proteins that bind the C4 phosphoenolpyruvate carboxylase gene of Flaveria trinervia. Plant Mol Biol 45: 201–214.
  28. 28. O’Leary B, Park J, Plaxton WC (2011) The remarkable diversity of plant PEPC (phosphoenolpyruvate carboxylase): recent insights into the physiological functions and post-translational controls of non-photosynthetic PEPCs. Biochem J. 436: 15–34.
  29. 29. Aboul-Soud MAM, El-Shemy HA (2009) Identification and subcellular localisation of Sl;INT7: A novel tomato (Solanum lycopersicum Mill.) fruit ripening-related and stress-inducible gene. Plant Sci 176: 241–247.
  30. 30. Aboul-Soud MAM (2010) Exogenous nitric oxide has negative impacts on ethylene emissions from intact and fresh-cut tomato fruit. J Hortic Sci Biotech 85: 516–520.
  31. 31. Higo K, Ugawa Y, Iwamoto M, Korenaga T (1999) Plant cis-acting regulatory DNA elements (PLACE) database: 1999. Nucl Acids Res 27: 297–300.
  32. 32. Lescot M, Déhais P, Thijs G, Marchal K, Moreau Y, et al. (2002) PlantCARE, a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences. Nucl Acids Res 30: 325–327.
  33. 33. Singh GB, Kramer JA, Krawetz SA (1997) Mathematical model to predict regions of chromatin attachment to the nuclear matrix. Nucl Acids Res 25: 1419–1425.
  34. 34. Hellens RP, Edwards EA, Leyland NR, Bean S, Mullineaux PM (2000) pGreen: a versatile and flexible binary Ti vector for Agrobacterium-mediated plant transformation. Plant Mol Biol 42: 819–832.
  35. 35. Hamza S, Chupeau Y (1993) Re-evaluation of conditions of plant regeneration and Agrobacterium-mediated transformation from tomato (Lycopersicon esculentum). J Exp Bot 44: 1837–1845.
  36. 36. Karimi M, Inze D, Depicker A (2002) Gateway vectors for Agrobacterium-mediated plant transformation. Trends Plant Sci 7: 193–195.
  37. 37. Baum K, Groning B, Meier I (1997) Improved ballistic transient transformation conditions for tomato fruit allow identification of organ-specific contributions of I-box and G-box to the RBCS2 promoter activity. Plant J 12: 463–9.
  38. 38. Power JB, Chapman JV (1985) Isolation, culture and genetic manipulation of plant protoplasts. In: Dixon RA, ed. Plant cell culture: a practical approach. Oxford: IRL Press, 37–66:
  39. 39. Henstrand JM, Handa AK (1989) Effect of ethylene action inhibitors upon wound-induced gene expression in tomato pericarp. Plant Physiol 91: 157–162.
  40. 40. Manzara T, Tarchevskaya S, Narita J (1994) Optimization of luciferase activity in a tomato transient assay experiment. Plant Mol Biol Rep 12: 221–226.
  41. 41. Aboul-Soud MAM, Chen X, Kang J-G, Yun B-Y, Barton H, et al. (2009) Activation tagging of ADR2 conveys a spreading lesion phenotype and resistance to biotrophic pathogens. New Phytol 183: 1163–1175.
  42. 42. Devaux C, Baldet P, Joubès J, Dieuaide-Noubhani M, Just D, et al. (2003) Physiological, biochemical and molecular analysis of sugar-starvation responses in tomato roots. J Exp Bot 54: 1143–1151.
  43. 43. Ernst K, Westhoff P (1997) The phosphoenolpyruvate carboxylase (ppc) gene family of Flaveria trinervia (C4) and F. pringlei (C3): molecular characterization and expression analysis of the ppcB and ppcC genes. Plant Mol Biol 34: 427–443.
  44. 44. De Folter S, Angenent GC (2006) Trans meets cis in MADS science. Trends Plant Sci 11: 224–31.
  45. 45. Ulmasov T, Liu ZB, Hagen G, Guilfoyle TJ (1995) Composite structure of auxin response elements. Plant Cell 7: 1611–1623.
  46. 46. Ogawa M, Hanada A, Yamauchi Y, Kuwahara A, Kamiya Y, et al. (2003) Gibberellin biosynthesis and response during Arabidopsis seed germination. Plant Cell 15: 1591–1604.
  47. 47. Sutoh K, Yamauchi D (2003) Two cis-acting elements necessary and sufficient for gibberellin-upregulated proteinase expression in rice seeds. Plant J 34: 635–645.
  48. 48. Kaplan B, Davydov O, Knight H, Galon Y, Knight MR, et al. (2006) Rapid transcriptome changes induced by cytosolic Ca2+ transients reveal ABRE-related sequences as Ca2+-responsive cis elements in Arabidopsis. Plant Cell 18: 2733–2748.
  49. 49. Aboul-Soud MAM, Aboul-Enein AM, Loake GJ (2009) Nitric oxide triggers specific and dose-dependent cytosolic calcium transients in Arabidopsis. Plant Signal Behav 4: 191–196.
  50. 50. Rawat R, Xu ZF, Yao KM, Chye ML (2005) Identification of cis-elements for ethylene and circadian regulation of the Solanum melongena gene encoding cysteine proteinase. Plant Mol Biol 57: 629–643.
  51. 51. Yamamoto YY, Ichida H, Matsui M, Obokata J, Sakurai T, et al. (2007) Identification of plant promoter constituents by analysis of local distribution of short sequences. BMC Genomics 8: 67.
  52. 52. Mahmoud EA, Mohei El-Din SM, Aboul-Soud MAM, Aboul-Enein AM, Sobhy GA, et al. (2009) Cloning of a novel antifungal promoter from Phaseolus vulgaris and the determination of its activity in stably transformed Nicotiana tabacum plants. Curr Iss Mol Biol 11 (Suppl. 1): i55–63.
  53. 53. Deikman J, Xu R, Kneissl ML, Ciardi JA, Kim KN, et al. (1998) Separation of cis elements responsive to ethylene, fruit development, and ripening in the 5′-flanking region of the ripening-related E8 gene. Plant Mol Biol 37: 1001–1011.
  54. 54. Nicholass FJ, Smith CJ, Schuch W, Bird CR, Grierson D (1995) High levels of ripening-specific reporter gene expression directed by tomato fruit polygalacturonase gene-flanking regions. Plant Mol Biol 28: 423–435.
  55. 55. Zhao L, Lu L, Zhang L, Wang A, Wang N, et al. (2009) Molecular evolution of the E8 promoter in tomato and some of its relative wild species. J Biosci 34: 71–83.
  56. 56. Nemhauser JL, Mockler TC, Chory J (2004) Interdependency of brassinosteroid and auxin signaling in Arabidopsis. PLoS Biol 2: E258.
  57. 57. Montoya T, Nomura T, Yokota T, Farrar K, Harrison K, et al. (2005) Patterns of Dwarf expression and brassinosteroid accumulation in tomato reveal the importance of brassinosteroid synthesis during fruit development. Plant J 42: 262–269.
  58. 58. Wang H, Jones B, Li Z, Frasse P, Delalande C, et al. (2005) The tomato Aux/IAA transcription factor IAA9 is involved in fruit development and leaf morphogenesis. Plant Cell 17: 2676–2692.
  59. 59. Polko JK, van Zanten M, van Rooij JA, Marée AF, Voesenek LA, et al. (2011) Ethylene-induced differential petiole growth in Arabidopsis thaliana involves local microtubule reorientation and cell expansion. New Phytol 193: 339–348.
  60. 60. Gendreau E, Orbovic V, Höfte H, Traas J (1999) Gibberellin and ethylene control endoreduplication levels in the Arabidopsis thaliana hypocotyl. Planta 209: 513–516.
  61. 61. Barry CS, Fox EA, Yen H-C, Lee S, Ying T-J, et al. (2001) Analysis of the ethylene response in the epinastic (epi) mutant of tomato. Plant Physiol 127: 58–66.
  62. 62. Terzaghi WB, Cashmore AR (1995) Light-regulated transcription. Ann Rev Plant Physiol Plant Mol Biol 46: 445–474.
  63. 63. Zhou DX (1999) Regulatory mechanism of plant gene transcription by GT-elements and GT-factors. Trends Plant Sci 4: 210–214.
  64. 64. Harmer SL, Hogenesch JB, Straume M, Chang HS, Han B, et al. (2000) Orchestrated transcription of key pathways in Arabidopsis by the circadian clock. Science 290: 2110–2113.
  65. 65. Yadav V, Mallappa C, Gangappa SN, Bhatia S, Chattopadhyay S (2005) A basic helix-loop-helix transcription factor in Arabidopsis, MYC2, acts as a repressor of blue light-mediated. Plant Cell 17: 1953–1966.
  66. 66. Proels RK, Roitsch T (2009) Extracellular invertase LIN6 of tomato: a pivotal enzyme for integration of metabolic, hormonal, and stress signals is regulated by a diurnal rhythm. J Exp Bot 60: 1555–1567.
  67. 67. Grierson C, Du JS, de Torres Zabala M, Beggs K, Smith C, et al. (1994) Separate cis sequences and trans factors direct metabolic and developmental regulation of a potato tuber storage protein gene. Plant J 5: 815–826.
  68. 68. Urwin NA, Jenkins GI (1997) A sucrose repression element in the Phaseolus vulgaris rbcS2 gene promoter resembles elements responsible for sugar stimulation of plant and mammalian genes. Plant Mol Biol 35: 929–942.
  69. 69. Yamagata H, Yonesu K, Hirata A, Aizono Y (2002) TGTCACA motif is a novel cis-regulatory enhancer element involved in fruit-specific expression of the cucumisin gene. J Biol Chem 277: 11582–11590.
  70. 70. Yin T, Wu H, Zhang S, Lu H, Zhang L, et al. (2009) Two negative cis-regulatory regions involved in fruit-specific promoter activity from watermelon (Citrullus vulgaris S.). J Exp Bot 60: 169–185.
  71. 71. Lemaire-Chamley M, Petit J, Garcia V, Just D, Baldet P, et al. (2005) Changes in transcriptional profiles are associated with early fruit tissue specialization in tomato. Plant Physiol 139: 750–769.
  72. 72. Fu H, Kim SY, Park WD (1995) A potato Sus3 sucrose synthase gene contains a context-dependent 3′ element and a leader intron with both positive and negative tissue-specific effects. Plant Cell 7: 1395–1403.
  73. 73. Curi GC, Chan RL, Gonzalez DH (2005) The leader intron of Arabidopsis thaliana genes encoding cytochrome c oxidase subunit 5c promotes high-level expression by increasing transcript abundance and translation efficiency. J Exp Bot 56: 2563–2571.
  74. 74. Engelmann S, Zogel C, Koczor M, Schlue U, Streubel M, et al. (2008) Evolution of the C4 phosphoenolpyruvate carboxylase promoter of the C4 species Flaveria trinervia: the role of the proximal promoter region. BMC Plant Biol 8: 4.
  75. 75. Hu WW, Gong H, Pua EC (2005) The pivotal roles of the plant S-adenosylmethionine decarboxylase 5′ untranslated leader sequence in regulation of gene expression at the transcriptional and posttranscriptional levels. Plant Physiol 138: 276–286.
  76. 76. Meier I, Callan KL, Fleming AJ, Gruissem W (1995) Organ-specific differential regulation of a promoter subfamily for the Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase small subunit genes in tomato. Plant Physiol 107: 1105–1118.
  77. 77. Catalá C, Rose JK, Bennett AB (2000) Auxin-regulated genes encoding cell wall-modifying proteins are expressed during early tomato fruit growth. Plant Physiol 122: 527–534.
  78. 78. Balbi V, Lomax TL (2003) Regulation of early tomato fruit development by the diageotropica gene. Plant Physiol 131: 186–187.
  79. 79. de Jong M, Wolters-Arts M, Feron R, Mariani C, Vriezen WH (2009) The Solanum lycopersicum auxin response factor 7 (SlARF7) regulates auxin signaling during tomato fruit set and development. Plant J 57: 160–170.
  80. 80. Giovannoni JJ (2007) Fruit ripening mutants yield insights into ripening control. Curr Opin Plant Biol 10: 283–289.
  81. 81. Jeanneau M, Vidal J, Gousset-Dupont A, Lebouteiller B, Hodges M, et al. (2002) Manipulating PEPC levels in plants. J Exp Bot 53: 1837–1845.
  82. 82. Rademacher T, Häusler RE, Hirsch HJ, Zhang L, Lipka V, et al. (2002) An engineered phosphoenolpyruvate carboxylase redirects carbon and nitrogen flow in transgenic potato plants. Plant J 32: 25–39.
  83. 83. Neelam A, Cassol T, Mehta RA, Abdul-Baki AA, Sobolev AP, et al. (2008) A field-grown transgenic tomato line expressing higher levels of polyamines reveals legume cover crop mulch-specific perturbations in fruit phenotype at the levels of metabolite profiles, gene expression, and agronomic characteristics. J Exp Bot 59: 2337–2346.
  84. 84. Pego JV, Weisbeek PJ, Smeekens SC (1999) Mannose inhibits Arabidopsis germination via a hexokinase-mediated step. Plant Physiol 119: 1017–1023.
  85. 85. Cortès S, Gromova M, Evrard A, Roby C, Heyraud A, et al. (2003) In plants, 3-o-methylglucose is phosphorylated by hexokinase but not perceived as a sugar. Plant Physiol 131: 824–837.