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. 2019 Jun 26;9(7):287. doi: 10.1007/s13205-019-1798-1

Identification and in silico characterization of cis-acting elements of genes involved in carotenoid biosynthesis in tomato

Archana Koul 1, Deepak Sharma 1, Sanjana Kaul 1, Manoj K Dhar 1,✉
PMCID: PMC6595038  PMID: 31297303

Abstract

Carotenoids, the widespread and structurally diverse class of pigments, accumulate in the fruits of tomato plants in a tissue specific manner. The carotenoid biosynthetic pathway genes have been cloned and characterized in tomato and other plants, however, its regulation is still obscure. We collected and analyzed forty different accessions of tomato for the present study. HPLC analysis revealed differential accumulation of major carotenoids (lycopene and ß-carotene) in the ripe fruit tissue. In order to understand the underlying regulatory mechanisms in carotenoid biosynthesis and accumulation, we sequenced the cis-acting elements i.e. promoter, 5′ and 3′ untranslated regions of the carotenoid pathway genes, in all accessions, followed by their in silico validation. Major differences observed in the CAAT Box, Opaque-2 Box and L-box in the promoters of carotenoid isomerase and lycopene-beta cyclase genes, respectively, along with the variations in musashi binding element of 5′ untranslated regions of the carotenoid isomerase gene, suggest their differential role in regulating the carotenogenesis process in tomato. The binding sites for various transcription factors namely RIN, AGAMOUS, CRY, RAP2.2 and PIF1 on the promoters of important carotenoid pathway genes were predicted in silico. We propose that expression of carotenoid genes and also the formation of protein product in ripe tomato fruits, is regulated efficiently by the binding of these transcription factors at selected sites in the promoter region. Finally, the differential expression of the above-mentioned genes in different developmental tissues supports the possible involvement of promoters and untranslated regions in carotenoid biosynthesis and accumulation process. The present study has generated significant information concerning regulatory players involved in the carotenoid biosynthesis in tomato.

Electronic supplementary material

The online version of this article (10.1007/s13205-019-1798-1) contains supplementary material, which is available to authorized users.

Keywords: Cis-acting elements, Carotenoids, BioMart, PlantCARE, Tomato, Gene regulation

Introduction

Tomato (Solanum lycopersicum L.) is the most important model system to understand the carotenoid biosynthetic pathway in plants (Dhar et al. 2014). The latter represents one of the most important secondary metabolic pathways in plants and the maximum accumulation of carotenoids occur in tomato fruits. Carotenoids especially, lycopene and ß-carotene are beneficial for the humans as they act as anti-oxidant, anti-cancerous, photoprotective, immune-stimulatory agents (Krinsky and Johnson 2005; Tanaka et al. 2012).

Genes that encode most of the enzymes of this pathway have been cloned and characterized in several plants such as in tomato, pepper, Arabidopsis, etc. (Chen et al. 2010; Ruiz-Sola and Rodriguez-Concepcion 2012; Su et al. 2015); however, reports on the characterization of cis-acting elements i.e. promoters and 5′ and 3′ untranslated regions (UTRs) present on the carotenoid genes is still in its infancy that needs to be elucidated. Therefore, it is important to understand the whole genomic biology/regulation of carotenoid biosynthetic pathway in order to develop a versatile and novel genotype of tomato with higher carotenoid content.

Plant growth and development involves temporal and spatial expression of the specific gene subsets which are influenced by various external and internal stimuli. Besides, the strength of cis-acting elements present either in the promoters or UTR’s (5′ and 3′) regions, plays a significant role in controlling specific gene expression in all the developmental tissues of a plant. Gene promoters; their cis sequences and different copy number of their motifs, allow differential expression of pathway genes and ultimately differential protein/metabolite production in the plant system (Biłas et al. 2016). Cis regulatory sequences/motifs are located directly in the transcribed DNA strand i.e. promoters or may be active during post-transcriptional modifications like 5’cap, poly-A tail and signal sequences present in the UTRs (Vaughn et al. 2012). However, cis sequences mediate the gene regulation by forming active complexes with the trans elements (Venter and Botha 2010; Porto et al. 2014) mainly transcription factors (TFs) which enhances the gene transcription and its further expression in different plant organs. TFs bind to a specific sequence; near CAAT box of promoter and recruit RNA polymerase for transcription and its further processing. The activity of TFs depends upon the sequence pattern of the specific gene promoter. The TFs in collaboration with promoters have been shown to effect carotenoid pathway (Dhar et al. 2014).

Some promoters of the carotenoid biosynthetic pathway genes have been functionally validated in plants. For example, Dalal et al. (2010) isolate and characterize the chromoplastic lyc-b promoter from Solanum habrochaites. Deletion fragment assay and its expression in fruto as well as in stable transgenic tomato revealed the tissue specific regulation of lyc-b promoter and its up-regulation in chromoplast-rich flowers and fruits. Similarly, UTR mediated regulation of carotenoid pathway genes has been observed and verified in plants. UTRs are known to be post-transcriptional regulators which effect the expression of each gene of a pathway in a different manner. Alvarez et al. (2016) showed regulation of carotenogenesis by 5′-UTR mediated translation of phytoene synthase splice variants in Arabidopsis thaliana. Two alternative splice variants (ASV) were found in A. thaliana with different length and exon/intron retention of their 5′-UTRs. ASV1 was found to contain a long 5′-UTR and predominantly involved in developmentally regulated carotenogenesis process whereas ASV2 has a short 5′-UTR and is induced when an immediate increase in carotenoid flux is required like under various kinds of stress (salt, water etc.) or different light intensities (Alvarez et al. 2016).

In a recent study, our group has identified notable regulators i.e. microRNAs (miRNAs), that are known to play a very significant role in the regulation of carotenogenesis process in tomato (Koul et al. 2016). In the latter publication, we have reported miRNAs that mediate possible regulation of carotenoid pathway genes at different developmental stages of tomato; negative correlation of miRNA and carotenoid genes was observed, with some exceptions.

From the above studies it has become clear that within a cell, an interlinked network of several regulatory mechanisms e.g. promoters and UTRs driven regulation, miRNA mediated gene silencing (PTGS), transcriptional expression and regulation by TFs exist, which contribute towards the carotenoid biosynthesis and accumulation in plants. The promoters and UTRs associated with the regulation of carotenogenesis process have not been characterized so far. The main aim of the present work was the in silico retrieval of the promoters and 5′ and 3′ UTR sequences, designing specific primers, sequencing and their characterization using various computational tools. Also, putative transcription factor binding sites on carotenoid gene promoters and their interactions was analysed. Therefore, the present study provides an insight into the coordinated role of promoters, UTRs and TFs in the regulation of carotenoid biosynthesis. These regulatory players can be employed to manipulate the pathway genes, through the process of metabolic engineering which may lead to the enhanced production of carotenoids specially lycopene and ß-carotene in tomatoes.

Materials and methods

Plant material

Seeds of forty different tomato accessions (Table S1) were collected from various areas of Jammu and Kashmir (J&K) and some other parts of the country. The plants were maintained in the Greenhouse at School of Biotechnology, University of Jammu under controlled conditions (Temperature; Day: 26 °C ± 2 and night: 16 °C ± 2, Photoperiod: 16 h). Mature (red-ripe) fruits were harvested and stored at − 80 °C for further use.

Profiling of carotenoids

For carotenoid profiling, tomato fruits were collected at red-ripe (RR) stage and plunged in liquid N2 and stored at − 80 °C for further extraction. Extraction of carotenoids was carried out in triplicates using the protocol of Nicolle et al. (2004). Separation and quantification of carotenoids (lycopene and ß-carotene) was done by employing the protocol of Koul et al. (2016). Carotenoids were identified using their retention times and absorption spectra of authentic standards (Sigma, USA). The concentration of each carotenoid was expressed as microgram per gram fresh weight (FW) and the mean of the data from triplicate samples of the same tomato accession.

Amplification and in silico characterization of promoters

Out of different accessions, twelve were selected for the characterization purpose. Based on the HPLC data, these accessions were divided into three groups; maximum (SOL1, SOL21, SOL22, SOL26), intermediate (SOL6, SOL7, SOL11, SOL18) and minimum (SOL4, SOL5, SOL15, SOL16) (Table S1) carotenoid containing accessions. As 5′-UTR lie adjacent to the promoter region; therefore, the primers were designed in such a way that it amplified both the promoter as well as 5′-UTRs. The carotenoid genes whose promoters and UTRs were studied during the present case have been given in Table 1. For the amplification of carotenoid gene promoter regions from selected tomato accessions, specific primers were designed using IDT primer quest tool (Table S2) on the basis of nucleotide sequences which were retrieved from the Ensemble Plants genome browser. The nucleotide start site of the promoters was determined using transcription start site (TSSP) program of the Softberry software (Shahmuradov et al. 2003).

Table 1.

Carotenoid biosynthetic pathway genes whose promoters and UTRs were studied in tomato

Gene name Gene symbol
Geranyl geranyl pyrophosphate synthase 1 ggpps1
Phytoene synthase 1 psy1
Phytoene synthase 2 psy2
Phytoene desaturase pds
Zeta-carotene desaturase zds
Carotenoid isomerase crtISO
Lycopene-beta cyclase lyc-b
Lycopene-epsilon cyclase lyc-e
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Genomic DNA was isolated from the leaf tissues of the different accessions using Cetyl Trimethyl Ammonium Bromide (CTAB) protocol according to the protocol described by Doyle (1991). Extracted DNA was used for the amplification of the eight carotenoid gene promoter regions. Amplified PCR products were purified, sequenced and analysed. Analysis of sequencing data was done using various computational tools like BLAST and CLUSTAL W to identify the sequence similarities among different accessions and previously submitted sequences in the sequence databases. Two bioinformatic tools, PlantCARE and PLACE were used for the characterization of different regulatory factors present in carotenoid gene promoter regions in the studied tomato accessions (Higo et al. 1998; Lescot et al. 2002). Differences were calculated in terms of number and functionality of the motifs present in the promoter sequences.

Prediction of potential transcription factor binding sites (TFBS)

Potential binding sites of different TFs on the promoters of carotenoid genes were predicted using in-built weight matrices of MatInspector software (Cartharius et al. 2005).

Amplification and in silico characterization of 5′ and 3′ UTRs

The whole genome of tomato was accessed using the Ensembl Plants Genome browser in order to retrieve the transcript ID of the carotenoid gene sequences. The transcript ID was further used for extracting the UTR sequences using a data mining tool; BioMart (Smedley et al. 2015). After getting full length UTR sequences, specific primers were designed (Table S3) and synthesized. Amplification of the UTRs was attempted from the genomic DNA of above selected accessions. Amplified PCR products were purified, sequenced and analysed. Analysis of both 5′ and 3′ UTRs sequences was done using BLAST and CLUSTAL W. UTRs were further analysed using UTRdb and UTRScan softwares (Mignone et al. 2005). UTRScan algorithm was used for signal/motif searches whereas description of the signals was retrieved using UTRsite tool of the UTRdb.

Gene expression studies using qRT-PCR

Total RNA was isolated from different developmental tissues of the SOL21 and SOL5 accessions (having maximum and minimum carotenoid content, respectively) using TRIzol® reagent (Life Technologies, Carlsbad, CA). cDNA synthesis was done using RT enzyme (Fermentas, Canada). qRT-PCR of important carotenoid genes i.e. crtISO and lyc-β gene was performed using SYBR green Applied Biosystems 7500 Sequence Detection System. Actin was used as an internal control. Reaction included specific forward and reverse primers (10 mM) (Table S4). Reaction was carried out at 95 °C for 10 min, followed by 30 cycles of 95 °C for 15 s and 60 °C for 1 min and then by a thermal denaturing step to generate dissociation curves. Three technical replicates were used during the study. The gene expression was calculated using ∆∆CT method (Livak and Schmittgen 2001). Standard error was calculated from three technical replicates.

Results and discussion

Measurement of carotenoids

Lycopene and β-carotene content were determined by HPLC at red ripe (RR) stage of the tomato fruits. Values represent the mean of three replicates. Forty accessions used during the present study showed differential carotenoid accumulation during ripening stage (Table S1, Figure S1). Highest lycopene content of 40.8 ± 0.65 µg/g of fresh weight (FW) was observed in accession SOL 21 (SKUAST-J), while the β-carotene content was 7.6 ± 0.91 µg/g of FW. Similarly, least lycopene and β-carotene content of 5.52 ± 0.77 µg/g and 0.76 ± 0.68 µg/g of FW, respectively was observed in accession SOL 15 (Nougran, Jammu). Three internationally known varieties namely ArkaVikas, PUSA Ruby and PUSA Dwarf were also used as reference varieties. Among reference varieties, Arka Vikas showed highest lycopene and β-carotene accumulation (58 ± 0.31 µg/g of FW and 10.4 ± 0.11 µg/g of FW, respectively). Gupta et al. (2015) also reported the similar content of lycopene and β-carotene in ArkaVikas where as the mutants having down regulated carotenoid genes showed very minute amount of the respective carotenoids in the fruit tissue. Similar study was carried out by Rosati et al. (2000) in wild-type and transgenic tomato fruit tissues. This indicates that the metabolic flux of carotenoids gets drastically altered as the spatio-temporal expression of the carotenoid biosynthetic genes and other regulatory molecules, change in response to various natural as well as metabolically engineered stimuli (Rosati et al. 2000).

Comparative analysis of promoters and their cis-sequences

Regulation of gene expression is a very complex process and can occur at various levels i.e. during transcription, mRNA processing followed by translation and post-translation modifications. However, regulation primarily occurs at the gene transcriptional level with the coordinated action of cis-acting motifs present in the promoters and trans-acting factors. The cis-acting motifs are DNA sequences in both the coding and non-coding regions which are present in the genome. Epigenomic studies have demonstrated the role of cis-acting elements in the chromatin remodelling and its modifications to generate an accessible region for binding of trans-factors in order to initiate transcription of a particular gene (Riechmann 2002). Organization of all the eukaryotic genomes is universally similar and almost all the regulatory elements are same. However, difference lies in the copy number of regulatory motifs present in the particular tissues/organs (Twyman 2003; Venter and Botha 2010). Plant promoters have attracted increasing attention because of their irreplaceable role in modulating the spatio-temporal expression of genes by interacting with transcription factors (TFs) (Liu et al. 2013). In total, promoters of eight carotenoid genes namely ggpps1, psy1, psy2, pds, zds, crtISO, lyc-b and lyc-e (Table 1), were amplified from the twelve accessions on the basis of HPLC profiles mentioned above. PCR resulted in the amplification of full length promoters (Figure S2). Promoter sequences of carotenoid genes were analyzed using PlantCARE and PLACE databases. Analysis showed that all promoters contain motifs necessary for their function, whereas some of the promoters also contain motifs essential for the carotenogenesis process to occur (Table S5). The promoter sequences were also characterized using BLAST and Clustal W software (Altschul et al. 1990; Larkin et al. 2007). Clustal W analysis showed that the promoters of carotenoid genes i.e. ggpps1, psy2, pds, zds amplified from the twelve accessions were 100% similar to each other, respectively and the reference sequences retrieved from various databases. This suggests that the promoters of these genes play uniform role in carotenogenesis process in different tomato accessions. Variations were observed in the promoters of psy1, crtISO and lyc-b genes. Promoter sequences of psy1 gene showed 98–99% sequence similarity to one another however; the differences in terms of motifs and functionality were not significant. On the other hand, significant differences were observed in the promoter sequences of crtISO and lyc-b genes amplified from twelve accessions, which showed 97–98% and 98–99% sequence similarity to one another, respectively.

PLACE and Plant Care analysis of the promoters of crtISO showed differences in the number of CAAT box and Opaque-2 box (O2). For example, SOL 21 had 17 CAAT-box and one O2-box whereas; SOL 5 had 14 CAAT-box and no O2-box (Fig. 1). CAAT-Box is the essential enhancer element present in the promoter, which is one of the driving elements for enhancing the expression of a particular gene. CAAT box is required by the pathway genes for higher expression which ultimately results in the increased production of proteins inside the cell e.g. the CAAT box in association with neighboring elements and bending by NF-Y is considered to be a major regulatory mechanism for the transcription activation (Mantovani 1999). Dai et al. (1992) characterized the functional role of CAAT box present in the nopaline synthase (nos) promoter by mutational analysis. Point mutation in the CAAT box region reduced the promoter activity in the transgenic tobacco plants. Similarly, in our case, CAAT box represents one of the major regulatory motifs present in the promoter region that possibly enhances the transcription/expression of crtISO gene, which ultimately leads to the enhanced biosynthesis and accumulation of lycopene in the red-ripe fruits as confirmed by HPLC data. However, in vivo elucidation needs to be carried out in order to explore the actual involvement of the CAAT boxes in the enhancement of gene transcription. The second difference in the promoters of crtISO lied in the O2 (Opaque-2) box. Promoter of crtISO gene in SOL 21 has an O2 box whereas SOL 5 does not contain such motif. O2 box is a bZIP type transcriptional activator (Wu et al. 1998) which enhances transcription of a particular gene. Wu et al. (1998) explained that o2 mutant had significantly reduced contents of the product as compared to the wild-type. Similarly, Aukerman et al. (1991) demonstrated that an arginine to lysine substitution by ethyl methanesulfonate (EMS) mutation in the o2 mutant reduced the transcriptional expression of the storage protein, zein which shows the importance of this motif for the enhanced expression of a gene. Presence or absence of the O2 motif in the promoters of crtISO gene, may be responsible for the differential expression of this gene, and possibly differential amount of lycopene in the tomato fruits of various accessions. Similarly, differences were observed in the promoter sequences of lyc-b gene. Clustal W analysis of promoter of lyc-b revealed SNPs, which were further confirmed using PLANT CARE and PLACE software. For example, In SOL 21, extra light responsive motifs namely L-box, AAGAA and TCCACCT were present, which play essential roles in enhancing the carotenogenesis process (Fig. 2). The last two motifs are cis-elements involved in seed-specific expression and developmental processes. The major difference lied in L-box which is a light responsive element. In SOL 21, extra light responsive motif i.e. L-box was present which was absent from the promoter of lyc-b gene of SOL 5. It has been established that light has a major impact as a regulator of transcriptional control in higher plants (Meier et al. 2011), especially carotenoids. Similarly, Meier et al. (2011) showed that the expression of many carotenoid genes is suppressed in dark grown plants and activated by photoactivated phytochromes. Light induced synthesis of carotenoids is characterized by an increase in the expression of psy and MEP pathway genes.

Fig. 1.

Fig. 1

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Depiction of presence of O2 motif in the promoter of crtISO gene amplified from SOL 21 (represented by red arrow) while as it is absent in SOL 5, detected using Plant Care algorithm

Fig. 2.

Fig. 2

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Pictorial representation of important motifs present in the promoter of lyc-b gene detected using Plant Care software. In SOL 21, extra light responsive motif i.e. L-box, AAGAA and TCCACCT (represented by red arrows) are present which is absent from SOL 5

Prediction of potential transcription factor binding sites (TFBS)

Transcription factors represent the trans-factors which in coordination with cis-regulatory elements lead to the enhanced gene expression and ultimately enhanced protein/metabolite accumulation. Potential binding sites of different TFs on the promoters of carotenoid pathway genes were predicted using MatInspector software using in-built weight matrices. TFBS were found on the promoters of the three important carotenoid genes; psy1, crtISO and lyc-b. Three TFs; RAP2.2 (light responsive motif), PIF1 and RIN were predicted to bind the promoter of psy1 gene (Fig. 3) which were assigned to the matrix family P$LREM. Similarly, PIF1 and RIN were assigned to the matrix family P$MYCL and P$AHBP, respectively. TF AGAMOUS (MADS-box) was predicted to bind the promoter of crtISO gene (Fig. 3) which was assigned to the matrix family P$MADS. Three TFs; AGAMOUS, CRY and RAP2.1 were predicted to bind the promoter of lyc-b gene (Fig. 3). CRY and RAP2.1 were observed to be assigned to the matrix families P$IBOX and P$DREB, respectively. These TFs are known to play essential roles in modulating the expression of promoters belonging to the carotenoid gene family using various mechanisms in plants (Dhar et al. 2014). A transcription factor may regulate multitude of genes individually or in a coordinated manner by binding with cis-regulatory element for the precise expression patterns (Srivastava and Kumar 2019). Therefore, various individual and coordinated gene regulatory networks have to be designed using bioinformatics tools followed by in vivo elucidation, in order to unravel the regulatory roles of cis-acting elements along with trans-factors, which ultimately lead to the enhanced protein production inside the cell in a particular organ/tissue of a plant.

Fig. 3.

Fig. 3

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Binding of the different motifs on the promoters of a psy1, b crtISO and c lyc-b genes which are assigned by the different matrix families. Binding of the TFs; RAP2.2, PIF, RIN, AGAMOUS, CRY and RAP2.1 have been shown by the arrows and different colors assigned to the different matrix families P$LREM, P$MYCL and P$AHBP, P$MADS, P$IBOX and P$DREB, respectively

5′ and 3′ UTRs analysis

The UTR region represents a segment of mRNA transcript, encoded in DNA strand and acts during transcription and not the translation process, however, it actually regulates the latter process (Barrett et al. 2012). In the present study, PCR amplification and in silico characterization of 5′ and 3′-UTRs of carotenoid genes was conducted. PCR amplification of 5′ and 3′-UTRs of carotenoid genes i.e. ggpps1, psy1, psy2, pds, zds, crtISO, lyc-b and lyc-e from twelve accessions was attempted using genomic DNA as template. PCR resulted in the amplification of full length UTRs (Figures S2, S3). 5′-UTRs were represented as the nucleotide sequence upstream of the start codon whereas 3′-UTRs as the nucleotide sequence downstream of the stop codon to the poly (A) tail according to Aoki et al. (2010). Further start and stop nucleotide sequence site of the promoter and 5′-UTR were differentiated using the TSSP and UTRdb softwares. Accession numbers of the promoter and UTR sequences submitted to the GenBank are from KY100933 to KY100975.

Major variation was observed in the number and strength of signals of 5′ and 3′ UTRs of crtISO and psy2 genes, respectively. The UTR region is a regulatory player situated at the 5′ and 3′ ends of protein coding genes, which are transcribed into mRNA but are not translated into proteins (Barrett et al. 2012; Bilas et al. 2016). Barrett et al. (2012) discussed that how the presence of more number of regulatory motifs like IRES, uORF etc. in the UTR regions, impact the expression/transcription of the pathway genes. uORF has an essential role in the post-transcriptional regulation of gene expression, specifically translation (von Arnim et al. 2014). uORFs confer regulation of the metabolic pathways by transcription factor mediated feedback mechanisms e.g. uORFs act as post-transcriptional regulator of ascorbate (Vitamin C) biosynthesis in Arabidopsis (Laing et al. 2015). Here ascorbate biosynthesis pathway is controlled via post-transcriptional repression of GDP-l-galactose phosphorylase (GGP), a major check-point enzyme in the pathway under high ascorbate concentration. Disruption of the uORF in the UTRs leads to the increased ascorbate concentration in the leaves. Similar mechanism may be present during UTR mediated regulation of carotenoid genes in different tissues of a plant. UTRs of carotenoid genes showed differences in terms of number and hence strength of uORFs present within them. Highest number of uORFs was observed in the 3′ UTR of psy2 gene. Psy2 is actively expressed in the green-tissues (mainly leaves) which results in the biosynthesis of lutein and has very less participation in lycopene accumulation. Therefore, maximum number of uORFs in this gene may lead to feedback regulation of carotenoids in leaves. 5′ UTR region of ggpps and crtISO gene contains minimum number of uORF i.e. only one, confirming no feedback regulation and therefore, their active participation in the biosynthesis and accumulation of lycopene in the ripe fruit. Similarly, lyc-b and lyc-e (major catabolic genes) contain higher number of uORFs, indicating the feedback regulation of these genes, which possibly results in the increased accumulation of lycopene than its catabolic products i.e. β-carotene and lutein in the red-ripe fruit.

Another major difference was observed in the Musashi binding elements (MBE) present in SOL1, SOL21, SOL22 and SOL26 (having maximum carotenoid accumulation) accessions. MBE represents one of the most significant UTR motif observed in the 5′ UTRs of crtISO gene of different accessions which possibly leads to the early translational activation and therefore, increases the gene expression (Charlesworth et al. 2006; Arumugam et al. 2010). Significant differences observed in the 5′-UTRs of crtISO gene in terms of number of MBE hints towards its differential role in regulating carotenoid biosynthesis (mainly lycopene) and accumulation. The tomato accessions having medium and lowest amounts of lycopene had one MBE whereas the accessions having highest amounts of carotenoids contained two MBE. Musashi is known to be one of the post-transcriptional regulators which act in different signal transduction pathways and enhance the gene expression and its activity (Zearfoss et al. 2014). However, an in-depth analysis of UTR regions needs to be done using transgenic and functional characterization approach in order to prove that whether the difference in the carotenoid content in different accessions is due to the difference in the sequences present in the UTR regions of the carotenoid genes. Different functional motifs present in 5′ and 3′ UTR’s of carotenoid genes predicted by UTRScan software have been listed in Table S6. There are several reports on the role of 3′ UTR in enhancing the gene expression (Papadakis et al. 2004; Vignesh et al. 2013) which has been ascribed to presence of several regulatory factors or coordinated role of 3′ UTR and 5′ UTR during loop formation facilitating the translation initiation process. Also, many miRNAs are known to bind to the 3′ UTR and facilitate the enhanced expression of the pathway genes which ultimately leads to the enhanced product accumulation in different tissues of plants (Barrett et al. 2012).

Expression profiling of crtISO and lyc-b gene

As the differences were observed in the promoters and UTRs of crtISO and lyc-b genes, transcript profiles of these genes were generated in both SOL 21 and  SOL  5 in vegetative as well as reproductive tissues. Transcript profiling allowed us to identify the differential expression of crtISO and lyc-b among different developmental tissues of tomato: leaves and other green tissues (stem, IG and MG fruits), flower, ripening fruits (BR and RR fruits) and roots gene in  SOL 21 and  SOL 5 accessions. Differential expression was observed in various tissues of the tomato plant (Fig. 4). CrtISO gene showed highest expression at red ripe stage (RR) in  SOL 21 as compared to  SOL 5 followed by root, stem, breaker stage (BS), immature green (IG), flower, mature green (MG) while leaf had the least. The expression increased during the ripening process and about 1 to twofold increased transcript level was detected in RR with lowest in MG (Fig. 4). On the other hand, highest expression of lyc-b gene was observed in  SOL 5 as compared to  SOL 21 with higher expression in stem followed by flower, leaf and root and less expression in ripening tissues i.e. RR, IG, MG and least in BS (Fig. 4). CrtISO gene was highly expressed in ripening fruits (BR and RR) indicating its major role in the carotenogenesis, especially biosynthesis of lycopene in fruits. As for the chromoplastic-specific cyclase, lcy-b seems to be highly expressed in flower, green tissues and in roots while as less expression was observed in the ripening stages of the fruit. This up-regulation of lcy-b in flower and green tissues is responsible for the accumulation of β-carotene and various other downstream carotenoids like zeaxanthin and lutein (Stigliani et al. 2011).

Fig. 4.

Fig. 4

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Relative transcript levels quantified by qRT-PCR in relative to the expression of actin (internal control), expressed as 2−ΔΔCt. Relative gene expression profile of a crtISO, b lcy-b in  SOL 21 and c crtISO, d lcy-b in  SOL 5, respectively

A pictorial representation showing the possible regulation of carotenoid biosynthesis in tomato by promoters, UTRs, cis sequences and TFs is shown as Fig. 5. However, many aspects like copy number of cis sequences and their diversity within the plant species still remain unexplained and need further in silico and in vivo research (Liu and Bao 2009). Construction of the novel gene cassettes using the above regulatory players can be an excellent strategy to increase the expression of carotenoid genes and ultimately the production and accumulation of carotenoids in plants. For example, Liu and Bao (2009) explained that the availability of TFs is a limiting factor for the transcription process to occur. Therefore, using the gene constructs with multiple motif copies definitely requires the sufficient quantity of TFs in the nucleus. Therefore, the major aim is to obtain stable, efficient and universal cassettes for driving the increased expression of the pathway genes.

Fig. 5.

Fig. 5

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A pictorial representation depicting the possible mechanisms responsible for the differential expression as well as differential accumulation of carotenoids in the different tomato accessions

Conclusion

The present study depicts the possible regulation of carotenoid biosynthetic pathway at the transcriptional as well as biochemical level. Carotenoid profiling of different accessions reveals differential accumulation of carotenoids (lycopene and ß-carotene) at red-ripe stage of tomato. Characterization of promoters, UTRs, their cis-motifs, TFs indicates the possible regulation of this pathway via. differential expression/regulation of crtISO and lyc-b gene. This was also validation using Real-time PCR at different developmental stages. Overall, the possible inter-relationship between gene promoters, UTRs and carotenoid content was predicted. This study has provided us an initial path towards understanding the cis-regulatory elements (promoters and UTR’s) of the carotenoid biosynthetic pathway which can be employed in future for the production of carotenoid rich tomatoes.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOCX 51 kb) (50.2KB, docx)
Supplementary material 2 (DOCX 557 kb) (557.3KB, docx)
Supplementary material 3 (DOCX 426 kb) (426.4KB, docx)
Supplementary material 4 (DOCX 18 kb) (18.4KB, docx)
Supplementary material 5 (DOCX 13 kb) (13.8KB, docx)
Supplementary material 6 (DOCX 12 kb) (12.4KB, docx)
Supplementary material 7 (DOCX 16 kb) (16.1KB, docx)
Supplementary material 8 (DOCX 14 kb) (14.6KB, docx)

Acknowledgements

This work was supported by the Department of Biotechnology (DBT), Govt. of India funded research project on tomato metabolomics. Authors thank Coordinator, Bioinformatics Centre (DBT BIF) for providing necessary facilities. Archana Koul is grateful to the Department of Science and Technology (DST), Govt. of India for INSPIRE Fellowship [xxxviii].

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

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