GENE-39707; No. of pages: 6; 4C: Gene xxx (2014) xxx–xxx
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Molecular cloning and expression analysis of the sucrose transporter gene family from Theobroma cacao L. Fupeng Li, Baoduo Wu, Xiaowei Qin, Lin Yan, Chaoyun Hao, Lehe Tan, Jianxiong Lai ⁎ Spice and Beverage Research Institute, Chinese Academy of Tropical Agricultural Sciences, Wanning 571533, PR China Key Laboratory of Genetic Resources Utilization of Spice and Beverage Crops, Ministry of Agriculture, Wanning 571533, PR China Hainan Provincial Key Laboratory of Genetic Improvement and Quality Regulation for Tropical Spice and Beverage Crops, Wanning 571533, PR China
a r t i c l e
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Article history: Received 21 February 2014 Received in revised form 30 April 2014 Accepted 23 May 2014 Available online xxxx Keywords: Sucrose transporter Theobroma cacao Expression pattern Cloning
a b s t r a c t In this study, we performed cloning and expression analysis of six putative sucrose transporter genes, designated TcSUT1, TcSUT2, TcSUT3, TcSUT4, TcSUT5 and TcSUT6, from the cacao genotype ‘TAS-R8’. The combination of cDNA and genomic DNA sequences revealed that the cacao SUT genes contained exon numbers ranging from 1 to 14. The average molecular mass of all six deduced proteins was approximately 56 kDa (range 52 to 66 kDa). All six proteins were predicted to exhibit typical features of sucrose transporters with 12 trans-membrane spanning domains. Phylogenetic analysis revealed that TcSUT2 and TcSUT4 belonged to Group 2 SUT and Group 4 SUT, respectively, and the other TcSUT proteins were belonging to Group 1 SUT. Real-time PCR was conducted to investigate the expression pattern of each member of the SUT family in cacao. Our experiment showed that TcSUT1 was expressed dominantly in pods and that, TcSUT3 and TcSUT4 were highly expressed in both pods and in bark with phloem. Within pods, TcSUT1 and TcSUT4 were expressed more in the seed coat and seed from the pod enlargement stage to the ripening stage. TcSUT5 expression sharply increased to its highest expression level in the seed coat during the ripening stage. Expression pattern analysis indicated that TcSUT genes may be associated with photoassimilate transport into developing seeds and may, therefore, have an impact on seed production. © 2014 Elsevier B.V. All rights reserved.
1. Introduction In most plants, sucrose is the main form of carbohydrate for longdistance transport and is the major photosynthetic product in source (autotrophic) organs. Plant sucrose transporters (SUTs) mediate the transport of sucrose into and out of the sieve element-companion cell complex (SE-CCC). SUTs belong to the major facilitator superfamily (MFS) and have a common structure with 12 predicted transmembrane domains for sucrose transport (Pao et al., 1998). Since the first plant sucrose carrier was isolated from spinach (Spinacia oleracea L.) (Riesmeier et al., 1992), scores of SUT genes have been observed in both monocot and dicot plant species (Sauer, 2007). For many species, two or more SUT genes have been recorded. In Arabidopsis, nine SUT genes have been identified; seven genes are characterised as functional, and two are characterised as nonfunctional sucrose carriers (Sauer et al., 2004). In rice and Populus, five and six SUT genes with different characteristics Abbreviations: cDNA, DNA complementary to RNA; DEPC, diethylpyrocarbonate; DNase, deoxyribonuclease; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; pI, isoelectric point; KDa, kiloDalton(s); μL, microliter; nM, nanomolar; RT-PCR, reverse transcription-PCR; s, second; SD, standard deviation; SUT, Sucrose transporter; TcSUT, Theobroma cacao Sucrose transporter. ⁎ Corresponding author at: Spice and Beverage Research Institute, Chinese Academy of Tropical Agricultural Sciences, Wanning 571533, PR China. E-mail address: [email protected] (J. Lai).
in sucrose transport have been identified respectively (Aoki et al., 2003; Payyavula et al., 2011). Based on phylogenetic analyses of deduced peptide sequences, plant SUTs have been classified into five groups: SUT1 (dicot-specific), SUT3 and SUT5 (monocot-specific), and SUT2 and SUT4 (monocot and dicot) (Kühn and Grof, 2010). The SUT1 members are expressed in the plasma membranes of sieve elements (SE) and/or companion cells (CC) and exhibit apparent Km values between 0.07 and 2.0 mM sucrose (Knop et al., 2004; Kühn et al., 1997; Stadler et al., 1995). The SUT2 and SUT4 members demonstrate a lower affinity for sucrose, and have reported Km values of 4–20 mM; SUT2 members have been localised to the SE plasma membranes (Barker et al., 2000; Meyer et al., 2004), while SUT4 members have been identified mainly in the chloroplast fraction and the vacuole (Endler et al., 2006; Rolland et al., 2003). The SUT3 members have been immunolocalised to the SE and CC plasma membrane, and exhibit apparent Km values of 2–8 mM sucrose (Rae et al., 2005; Reinders et al., 2006; Scofield et al., 2007). Features of the SUT5 members have not been characterised. The SUT family is responsible for phloem loading, transporting and unloading of sucrose in many species (Braun and Slewinski, 2009). SUTs are expressed in various tissues of the transport pathway and sink cells (Sauer et al., 2004; Sivitz et al., 2008; Tang et al., 2010). The expression pattern of SUTs is regulated by cis-regulatory elements of the promoter regions (Ibraheem et al., 2010), sugar signalling, and light conditions (Matsukura et al., 2000;
http://dx.doi.org/10.1016/j.gene.2014.05.056 0378-1119/© 2014 Elsevier B.V. All rights reserved.
Please cite this article as: Li, F., et al., Molecular cloning and expression analysis of the sucrose transporter gene family from Theobroma cacao L., Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.05.056
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Wright et al., 2003). Mutation and antisense transformation studies have revealed that SUTs are responsible for pollen germination, restraining plant growth, and fruit size reduction (Hackel et al., 2006; Sivitz et al., 2008; Srivastava et al., 2009). Overexpression transformations showed lower sucrose concentration in leaves and increased growth rates of pea cotyledon (Leggewie et al., 2003; Rosche et al., 2002). Sucrose is the major sugar in the embryo during seed development (Bucheli et al., 2001), and its concentration is limiting for the rapid synthesis of lipids during seed filling in cacao (Pence, 1992). Increased lipid synthesis may be an effect of sucrose unloading (Weber et al., 2005). However, few studies on SUT in Theobroma cacao have been performed. T. cacao is a member of the Malvaceae family and is a neotropical species that originated in southern and central Mexico. It is widely grown in more than 50 countries throughout the humid tropical regions. Cacao beans are used for the chocolate, confectionery and beverage (Pritchett and Pritchett, 2012). Cacao production is essential to the livelihoods of 40 to 50 million people worldwide (WCF), and harvested areas are about 10 million ha (FAO). Based on morphological traits and geographical origins three major genetic groups have traditionally been defined within cacao: Criollo, Forastro, and Trinitario. Trinitario has been recognised as a ‘Criollo × Forastro’ hybrid (Cheesman, 1944). Recently, molecular markers and chromosome analyses have been used to classify cacao germplasm into 10 major groups: Amelonado, Contamana, Curaray, Guiana, Iquitos, Maraňón, Nanay, Purús, Criollo, and Nacional (Motamayor et al., 2008; Utro et al., 2012). The genomes of Criollo and Amelonado have been sequenced and cover 76% and 92% of the estimated genome size, respectively (Argout et al., 2011; Motamayor et al., 2013). These genomes are important tools for gene characterisation investigations. Cacao pods (fruits) grow mainly on the tree trunk. Photoassimilates are translocated from source organs (leaves) to sink organs (pods) through a long-distance phloem pathway. In the present study, our goal was to provide a comprehensive description of SUT function in cacao trees. By comparing translated sequences of the cacao genome with known SUT sequences, six genes were designated as encoding putative SUT proteins. Deduced amino acid sequences and gene structure of the SUT family of cacao were compared and expression patterns were characterised. 2. Materials and methods 2.1. Plant materials Unless otherwise noted, ‘TAS-R8’ cacao trees (T. cacao) were used in this study. They were cultivated at the experimental plantation of the Spice and Beverage Research Institute of Chinese Academy of Tropical Agricultural Science (Wanning, Hainan, China). ‘TAS-R8’ has a relatively larger pod weight, belongs to the Trinitario group, and has been planted for more than 30 years. Nineteen organs/tissues were sampled from adult tree, including leaf, bark with phloem, flower bud, pod, pod shell, seed coat, and seed. All organs/tissues were frozen in liquid nitrogen and stored at −80 °C until use. 2.2. RNA extraction and cDNA synthesis Total RNA was extracted from three replicates of each organ/tissue by using a Plant RNA Kit (OMEGA, Norcross, USA). The RNA was treated with DNase I, according to the manufacturer's instructions (TaKaRa, Dailian, China). An aliquot of RNA was electrophoretically separated on a 1.0% agarose gel to verify integrity. Final concentration was adjusted to 1 μg·μL−1 by using double-distilled water treated with 0.1% DEPC. Single-stranded cDNA was synthesised from 1.0 μg of total RNAs by using M-MLV reverse transcriptase (Fermentas, Ontario, Canada), according to the manufacturer's instructions. Three reverse transcriptions of each organ/tissue were carried out independently. The resultant first-
strand cDNA mixtures were diluted 1:20 with double-distilled water and stored at −20 °C before being used as a template for cloning and real-time PCR. 2.3. Cloning of sucrose transporter genes SUT gene models were identified from the sequence of cacao genome (Argout et al., 2011) by using BlastP search with nine Arabidopsis and five Populus SUT genes. Gene-specific primers (listed in Supplemental file 1) were developed for PCR amplification of TcSUT genes, according to the T. cacao genome sequences (v1.1) hosted at CATIE (http://cocoagendb.cirad.fr/) and the Cacao Genome Database (http://www.cacaogenomedb.org). The primers were designed using Primer Premier 5.0 software and synthesised by Sangon Biotech (Shanghai, China). PCR products were purified using the Gel Extraction Kit (OMEGA, Norcross, USA) and cloned into the pMD18-T vector by using the TA cloning kit (TaKaRa, Dailian, China). Transformed clones were selected for sequencing fully from both directions. The sequences were deposited in GenBank under accession numbers KF776545–KF776553. 2.4. Sequence analysis Sequence alignment was performed using DNAMAN 6.0 (version 5.0, Lynnon Biosoft). Characterisation and secondary structure of the proteins were analysed using ExPASy (http://expasy.org/tools/). Transmembrane regions were predicted using the TMHMM server (http://www.cbs.dtu.dk/services/TMHMM/). Selected plant sucrose transporter sequences were found using the BLAST algorithm (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Phylogenetic analyses were performed using MEGA 4.1 software (http://www.megasoftware.net/ index.html) with the neighbour-joining method, followed by phylogeny test options of 1000 bootstrap replicates. 2.5. Real-time PCR analysis Real-time PCR analysis was performed on a BIO-RAD CFX-96 realtime detection system with the SYBR Green Realtime PCR Master Mix (TOYOBO, Osaka, Japan). Specific primers (Supplemental file 1) were designed in the 5′ or 3′ region of TcSUT genes using the Primer Premier 5.0 software and were synthesised by Sangon Biotech (Shanghai, China). PCR reactions contained 400 nM of both forward and reverse gene-specific primers and 8.4 μL of the 50-fold diluted reverse transcription (RT) product in a final volume of 20 μL. The thermal cycling protocol was followed by DNA polymerase activation at 95 °C for 3 min. PCR amplification was carried out with 45 cycles of denaturation at 95 °C for 10 s, primer annealing at 60 °C for 15 s, and extension at 72 °C for 30 s. Optical data were acquired after the extension step, and the PCR reactions were subjected to a melting curve analysis beginning from 65 °C to 95 °C at 0.1 °C s−1. GAPDH was chosen as the reference gene on the basis of the study by Pinheiro et al. (2011). The data are presented as an average ± SD of three independently produced RT preparations used for PCR runs, each having at least three replicates. Expression data were analysed using Bio-Rad CFX Manager software with the 2− ΔCT method. Gene relative expression levels were drawn by Microsoft Excel 2010. 3. Results 3.1. Cloning of the cacao SUT gene family Six SUT genes were initially identified in the T. cacao (B97-61/B2) genome v1.1 (Argout et al., 2011). Full-length coding sequences for all except TcSUT6 were cloned from cacao by RT-PCR using a cDNA template mixture of leaf, bark with phloem, flower bud, and pod. Although repeated attempts to clone TcSUT6 from both a range of organ/tissue
Please cite this article as: Li, F., et al., Molecular cloning and expression analysis of the sucrose transporter gene family from Theobroma cacao L., Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.05.056
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cDNAs and genomic DNA, TcSUT6 target product was only amplified from genomic DNA. Based on sequence results, TcSUT1, TcSUT2 and TcSUT3 produced multiple transcripts, and the longest transcripts were selected for follow-up studies. TcSUT6 contains only one exon. TcSUT2 contains fourteen exons, about three times the number of the other four SUT genes, which have four or five exons (Fig. 1). The predicted molecular mass of TcSUT2 is approximately 66 kDa versus 52–57 kDa for the other proteins. TcSUT2 has acidic theoretical pI b 7, while other SUTs have alkaline theoretical pIs N 8.5 (Table 1).
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Table 1 Characteristics of the cacao sucrose transporter proteins as deduced from cDNA sequences. Size
TcSUT1 TcSUT2 TcSUT3 TcSUT4 TcSUT5 TcSUT6
pI
a.a.
kDa
536 616 494 513 491 488
57.0 65.7 52.7 55.5 52.2 52.0
9.29 6.82 9.13 9.22 8.54 8.54
% similarity (n.t./a.a.) to SUT2
SUT3
SUT4
SUT5
SUT6
60/42 –
70/64 63/45 –
61/52 65/48 63/51 –
69/65 62/46 81/79 63/53 –
69/65 63/46 81/80 64/53 94/91 –
3.2. Sequence alignment and phylogenetic analysis Analysis of the deduced amino acid sequences of the putative SUT genes revealed that all six SUTs contained 12 transmembrane domains (Supplemental file 2) with both N- and C-termini on the cytoplasmic side, as seen in other sugar transporters (Lalonde et al., 2004). The conserved histidine residue, which is responsible for sucrose binding in transport processes by site-directed mutagenesis of the AtSUC1 protein (Lu and Bush, 1998), was present in all putative SUT peptides in the extracellular loop between the first and second membrane spanning helices (Fig. 2). Sequence alignments revealed that TcSUT5 and TcSUT6 showed high similarity (N90%) with distinct gene structure. When compared to TcSUT3, both TcSUT5 and TcSUT6 showed approximately 80% similarity, which amino acid sequence of the other three TcSUTs b64% similarity. Plant SUT proteins were recently classified into five phylogenetic groups (Berthier et al., 2009; Kühn and Grof, 2010). A phylogenetic analysis of SUT orthologs from perennial and herbaceous species was carried out and is shown as an unrooted dendrogram in Fig. 3. Group 3 and Group 5 represent the monocot-specific branches and Group 1 representing dicot-specific branch. TcSUT1, TcSUT3, TcSUT5, and TcSUT6 belonged to Group 1 and clustered with SUT orthologs from other perennial species (e.g. Populus, Juglans) and distinct herbaceous species of the Arabidopsis- and legume-specific subclades. TcSUT3, TcSUT5, and TcSUT6 formed a single subclade in this group. TcSUT2 and TcSUT4 fall into subclades of Group 2 and Group 4 respectively, which both form monocot- and dicot-specific subclades. 3.3. Expression pattern of cacao SUT family Spatial and developmental expression profiling of the entire cacao SUT family was assessed by quantitative real-time PCR (Fig. 4A). TcSUT1 transcripts were most abundant in developing pods and very low in other organs/tissues. Expression of TcSUT2 was similar in mature leaves, bark with phloem, and developing pods, and lower in flower buds. TcSUT3 and TcSUT4 were highly expressed in photoassimilate transport organs/tissues, with higher levels in bark with phloem and developing pods. TcSUT5 expression was detected mostly in flower buds and developing pods. Transcript levels of the highly similar TcSUT5 isoform, TcSUT6, were not detected in any tested organs/tissues. Therefore, TcSUT6 was not investigated in the subsequent study. For distinct developmental stage of leaves, TcSUT1 transcripts accumulated mainly in sink and transit leaves (transition from ‘sink’ to ‘source’), with minimal levels in mature leaves. TcSUT2, TcSUT3, and TcSUT4 transcripts showed higher expression levels in mature leaves, and TcSUT4 showed an equivalent expression level in transit leaves and mature leaves. Compared
Fig. 1. Structure of six cacao SUT genes. The grey bars and black lines denote exons and introns in the coding domain sequence.
with the other SUT genes, TcSUT5 expression was minimal in leaves (Fig. 4B). TcSUT4 transcripts were higher in buds just before bud break (3–4 mm in length) than in other bud developmental stages (Fig. 4C). TcSUT5 transcripts increased with bud developmental, reaching its highest level at bud break. TcSUT1, TcSUT2, and TcSUT3 expression levels were overall lower than TcSUT4 and TcSUT5 in buds. To investigate the expression pattern of cacao SUT genes in diverse organs/tissues of various pod developmental stages, pod husk, seed coat, and seed of enlarging, stereotype (pod volume reach maximum) and ripening pods were investigated respectively (Fig. 5). TcSUT1 transcripts were found mostly in seeds and increased with seed maturation. TcSUT1 transcripts were found in low levels in pod husks. TcSUT2 expression was stable in pod husks and seed coats, but increased in seeds from pod enlargement to pod ripening. TcSUT3 expression also increased with seed maturation, but remained constant in pod husks and seed coats. Expression of TcSUT4 was constant in the three organs/ tissues of pod enlargement and stereotype stages. At pod ripening stage, TcSUT4 increased in pod husks while decreased in seed coats. TcSUT5 expression was highest in seed coats, and expression levels increased in pod husks and seed coats with pod maturation. 4. Discussion The draft sequence of T. cacao genome has provided investigators with a powerful tool that enables searches for gene families of known protein. By searching the genome sequence, we found six putative SUT genes, TcSUT1, TcSUT2, TcSUT3, TcSUT4, TcSUT5, and TcSUT6, comprising the SUT family in the cacao genome. However, it should be noted that the genomes are not fully sequenced and that one or more SUT may exist in the remaining 8% of the Amelonado genome and 24% of the Criollo genome. Phylogenetic analysis revealed that the plant SUT family can be divided into five distinct clades: Groups 1–5 (Kühn and Grof, 2010). Proton-coupled Group 1 (dicot) and Group 2 (monocot and dicot) SUTs are in the plasmalemma. They mediate apoplastic phloem loading and are essential for normal plant growth (Hackel et al., 2006; Srivastava et al., 2008). Some Group 4 (monocot and dicot) membrane SUTs, such as OsSUT2 (Eom et al., 2011) and PtaSUT4 (Frost et al., 2012), have been localised to the vacuolar membrane through proteomic and/or GFP fusion analyses. The function of Group 5 (monocot) SUTs has not been characterised. TcSUT1, TcSUT3, and TcSUT5, which are in Group 1, consist of the same number of exons. Their similarity in gene structure suggests that they diverged from a prototype gene, presumably by gene duplications. TcSUT6 showed high similarity with
Fig. 2. Sequence alignments of cacao SUT proteins with the AtSUC1. Putative transmembrane domains of the SUT peptide are underlined. The functionally important and conserved histidine residue is shown in bold.
Please cite this article as: Li, F., et al., Molecular cloning and expression analysis of the sucrose transporter gene family from Theobroma cacao L., Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.05.056
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Fig. 3. A neighbour-joining tree with predicated SUT amino acid sequences from cacao and other plant species. Sequences from cacao are marked with grey circle. Sequence names and/or accession numbers are provided in Supplemental file 3.
TcSUT5, but without introns. This may suggest that TcSUT6 was produced from TcSUT5 by reverse transcription. TcSUT2 and TcSUT4 belong to Group 2 and Group 4 SUTs, respectively. They may be similar to the tonoplast-localised SUC4-type, which mediates tonoplast transmembrane transport of sucrose (Schneider et al., 2011). TcSUT6 target product was amplified from the genomic DNA template, rather than from the cDNA template mixture. TcSUT6 was not expressed in any tested organs/tissues. The cDNA sequence of TcSUT6 had been deposited in the Cacao Genome Database, indicating that TcSUT6 may be expressed in some organs/tissues not used in our study. The predicted TcSUT6 peptide also has the conserved histidine residue (Lu and Bush, 1998). A higher similarity of TcSUT6 with other functional SUTs showed that TcSUT6 may play an important role in sucrose transport. TcSUT1, TcSUT2, and TcSUT3 produce multiple transcripts, and only the longest transcripts were studied. TcSUT1 shows one possible alternative splicing at exon 2, which resulted in the absence of 9 amino acids. TcSUT2 and TcSUT3 could give rise to alternative splicing at intron 6 and intron 2, respectively, resulting in a frame shift within the coding region. Based on the sequence information in the GenBank database, TcSUT2 produces eight different transcripts. These data indicate that there may be more undetectable transcripts of SUTs in cacao. To validate the compositive expression patterns of cacao SUTs, the primers were developed to measure the all transcripts of each gene. Alternative splicing produces multiple mRNA transcripts from the same pre-mRNA, and occurs commonly in multicellular eukaryotes (Eckardt, 2013; Wang et al., 2014). Alternative splicing plays important roles in regulating gene expression and increasing transcriptome diversity and proteome complexity. Further research of SUT transcripts is needed for member cloning, expression regulation and function identification. Cacao pods are the most valuable sink organs of cacao trees producing cacao beans, and the number of seeds per pod and the mean seed weight are important traits involved in increasing production per tree (Cilas et al., 2010). Photoassimilates distributed to developing pods are essentially carbon skeletons used to synthesise major storage products, and show positive association with seed yield (Rawsthorne, 2002; Weber et al., 2005). The different expression patterns of TcSUT genes in cacao plants observed in this work suggest that the SUT gene family has many roles in both source and sink organs/tissues and in different developmental stages. During pod development, TcSUT1, TcSUT4, and TcSUT5 were expressed more in seed coats and seeds, with the highest expression of TcSUT5 in the seed coat of ripening pods. These results
Fig. 4. Expression of SUT genes in cacao tissues. (A) Expression in different organs, including mature leaf (ML), bark with phloem (BP), flower bud (FB) and developing pod (DP). (B) Expression in different developmental stages of leaf including sink leaf (SL), transit leaf (TL) and mature leaf (ML). (C) Expression in bud at various developmental stages including various sizes in bud length: b2 mm, 2–3 mm, 3–4 mm and blooming flower (BF). Error bars represent the SD of three technical replicates. Abbreviated names of tested organs/tissues were listed in Supplemental file 4.
suggest that these three genes may play a role in photoassimilate unloading to developing seeds. Expression of SUT in the valuable sink organs has been improved to influence seed/tuber growth rate and yield (Li et al., 2011; Rosche et al., 2002; Sun et al., 2011). The consistent
Please cite this article as: Li, F., et al., Molecular cloning and expression analysis of the sucrose transporter gene family from Theobroma cacao L., Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.05.056
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Fig. 5. Expression of cacao SUT genes in various pod tissues at different pod developmental stages. E-PH, E-SC, E-S represent pod husk, seed coat, and seed of enlargement pod; “S-” and “R-” represent corresponding tissues of stereotype and ripening pod respectively. Error bars represent the SD of three technical replicates. Abbreviated names of tested organs/tissues were listed in Supplemental file 4.
expression patterns indicated that TcSUT genes were associated with photoassimilate transport into seeds during development and resulted in higher seed yields. However, investigations of the relationship between TcSUT variation and pod/seed traits are necessary to validate this association. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.gene.2014.05.056. Conflict of interest We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in, or the review of, the manuscript entitled. Acknowledgments This work was financially supported by the National Science Foundation of China (31301389) and the Tropical Crop Germplasm Resources Protection Project of the Ministry of Agriculture (13RZZY-24). References Aoki, N., Hirose, T., Scofield, G.N., Whitfeld, P.R., Furbank, R.T., 2003. The sucrose transporter gene family in rice. Plant Cell Physiol. 44, 223–232. Argout, X., Salse, J., Aury, J.M., Guiltinan, M.J., Droc, G., Gouzy, J., et al., 2011. The genome of Theobroma cacao. Nat. Genet. 43, 101–108. Barker, L., Kühn, C., Weise, A., Schulz, A., Gebhardt, C., Hirner, B., et al., 2000. SUT2, a putative sucrose sensor in sieve elements. Plant Cell 12, 1153–1164. Berthier, A., Desclos, M., Amiard, V., Morvan-Bertrand, A., Demmig-Adams, B., Adams, W.W., et al., 2009. Activation of sucrose transport in defoliated Lolium perenne L.: an example of apoplastic phloem loading plasticity. Plant Cell Physiol. 50, 1329–1344. Braun, D.M., Slewinski, T.L., 2009. Genetic control of carbon partitioning in grasses: roles of sucrose transporters and tie-dyed loci in phloem loading. Plant Physiol. 149, 71–81. Bucheli, P., Rousseau, G., Alvarez, M., Laloi, M., McCarthy, J., 2001. Developmental variation of sugars, carboxylic acids, purine alkaloids, fatty acids, and endoproteinase activity during maturation of Theobroma cacao L. seeds. J. Agric. Food Chem. 49, 5046–5051. Cheesman, E.E., 1944. Notes on the nomenclature, classification and possible relationships of cocoa populations. Trop. Agric. 21, 144–159. Cilas, C., Machado, R., Motamayor, J.-C., 2010. Relations between several traits linked to sexual plant reproduction in Theobroma cacao L.: number of ovules per ovary, number of seeds per pod, and seed weight. Tree Genet. Genomes 6, 219–226. Eckardt, N.A., 2013. The plant cell reviews alternative splicing. Plant Cell 25, 3639. Endler, A., Meyer, S., Schelbert, S., Schneider, T., Weschke, W., Peters, S.W., et al., 2006. Identification of a vacuolar sucrose transporter in barley and Arabidopsis mesophyll cells by a tonoplast proteomic approach. Plant Physiol. 141, 196–207.
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Molecular cloning and expression analysis of the sucrose transporter gene family from Theobroma cacao L. Fupeng Li, Baoduo Wu, Xiaowei Qin, Lin Yan, Chaoyun Hao, Lehe Tan, Jianxiong Lai ⁎ Spice and Beverage Research Institute, Chinese Academy of Tropical Agricultural Sciences, Wanning 571533, PR China Key Laboratory of Genetic Resources Utilization of Spice and Beverage Crops, Ministry of Agriculture, Wanning 571533, PR China Hainan Provincial Key Laboratory of Genetic Improvement and Quality Regulation for Tropical Spice and Beverage Crops, Wanning 571533, PR China
a r t i c l e
i n f o
Article history: Received 21 February 2014 Received in revised form 30 April 2014 Accepted 23 May 2014 Available online xxxx Keywords: Sucrose transporter Theobroma cacao Expression pattern Cloning
a b s t r a c t In this study, we performed cloning and expression analysis of six putative sucrose transporter genes, designated TcSUT1, TcSUT2, TcSUT3, TcSUT4, TcSUT5 and TcSUT6, from the cacao genotype ‘TAS-R8’. The combination of cDNA and genomic DNA sequences revealed that the cacao SUT genes contained exon numbers ranging from 1 to 14. The average molecular mass of all six deduced proteins was approximately 56 kDa (range 52 to 66 kDa). All six proteins were predicted to exhibit typical features of sucrose transporters with 12 trans-membrane spanning domains. Phylogenetic analysis revealed that TcSUT2 and TcSUT4 belonged to Group 2 SUT and Group 4 SUT, respectively, and the other TcSUT proteins were belonging to Group 1 SUT. Real-time PCR was conducted to investigate the expression pattern of each member of the SUT family in cacao. Our experiment showed that TcSUT1 was expressed dominantly in pods and that, TcSUT3 and TcSUT4 were highly expressed in both pods and in bark with phloem. Within pods, TcSUT1 and TcSUT4 were expressed more in the seed coat and seed from the pod enlargement stage to the ripening stage. TcSUT5 expression sharply increased to its highest expression level in the seed coat during the ripening stage. Expression pattern analysis indicated that TcSUT genes may be associated with photoassimilate transport into developing seeds and may, therefore, have an impact on seed production. © 2014 Elsevier B.V. All rights reserved.
1. Introduction In most plants, sucrose is the main form of carbohydrate for longdistance transport and is the major photosynthetic product in source (autotrophic) organs. Plant sucrose transporters (SUTs) mediate the transport of sucrose into and out of the sieve element-companion cell complex (SE-CCC). SUTs belong to the major facilitator superfamily (MFS) and have a common structure with 12 predicted transmembrane domains for sucrose transport (Pao et al., 1998). Since the first plant sucrose carrier was isolated from spinach (Spinacia oleracea L.) (Riesmeier et al., 1992), scores of SUT genes have been observed in both monocot and dicot plant species (Sauer, 2007). For many species, two or more SUT genes have been recorded. In Arabidopsis, nine SUT genes have been identified; seven genes are characterised as functional, and two are characterised as nonfunctional sucrose carriers (Sauer et al., 2004). In rice and Populus, five and six SUT genes with different characteristics Abbreviations: cDNA, DNA complementary to RNA; DEPC, diethylpyrocarbonate; DNase, deoxyribonuclease; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; pI, isoelectric point; KDa, kiloDalton(s); μL, microliter; nM, nanomolar; RT-PCR, reverse transcription-PCR; s, second; SD, standard deviation; SUT, Sucrose transporter; TcSUT, Theobroma cacao Sucrose transporter. ⁎ Corresponding author at: Spice and Beverage Research Institute, Chinese Academy of Tropical Agricultural Sciences, Wanning 571533, PR China. E-mail address: [email protected] (J. Lai).
in sucrose transport have been identified respectively (Aoki et al., 2003; Payyavula et al., 2011). Based on phylogenetic analyses of deduced peptide sequences, plant SUTs have been classified into five groups: SUT1 (dicot-specific), SUT3 and SUT5 (monocot-specific), and SUT2 and SUT4 (monocot and dicot) (Kühn and Grof, 2010). The SUT1 members are expressed in the plasma membranes of sieve elements (SE) and/or companion cells (CC) and exhibit apparent Km values between 0.07 and 2.0 mM sucrose (Knop et al., 2004; Kühn et al., 1997; Stadler et al., 1995). The SUT2 and SUT4 members demonstrate a lower affinity for sucrose, and have reported Km values of 4–20 mM; SUT2 members have been localised to the SE plasma membranes (Barker et al., 2000; Meyer et al., 2004), while SUT4 members have been identified mainly in the chloroplast fraction and the vacuole (Endler et al., 2006; Rolland et al., 2003). The SUT3 members have been immunolocalised to the SE and CC plasma membrane, and exhibit apparent Km values of 2–8 mM sucrose (Rae et al., 2005; Reinders et al., 2006; Scofield et al., 2007). Features of the SUT5 members have not been characterised. The SUT family is responsible for phloem loading, transporting and unloading of sucrose in many species (Braun and Slewinski, 2009). SUTs are expressed in various tissues of the transport pathway and sink cells (Sauer et al., 2004; Sivitz et al., 2008; Tang et al., 2010). The expression pattern of SUTs is regulated by cis-regulatory elements of the promoter regions (Ibraheem et al., 2010), sugar signalling, and light conditions (Matsukura et al., 2000;
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Please cite this article as: Li, F., et al., Molecular cloning and expression analysis of the sucrose transporter gene family from Theobroma cacao L., Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.05.056
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Wright et al., 2003). Mutation and antisense transformation studies have revealed that SUTs are responsible for pollen germination, restraining plant growth, and fruit size reduction (Hackel et al., 2006; Sivitz et al., 2008; Srivastava et al., 2009). Overexpression transformations showed lower sucrose concentration in leaves and increased growth rates of pea cotyledon (Leggewie et al., 2003; Rosche et al., 2002). Sucrose is the major sugar in the embryo during seed development (Bucheli et al., 2001), and its concentration is limiting for the rapid synthesis of lipids during seed filling in cacao (Pence, 1992). Increased lipid synthesis may be an effect of sucrose unloading (Weber et al., 2005). However, few studies on SUT in Theobroma cacao have been performed. T. cacao is a member of the Malvaceae family and is a neotropical species that originated in southern and central Mexico. It is widely grown in more than 50 countries throughout the humid tropical regions. Cacao beans are used for the chocolate, confectionery and beverage (Pritchett and Pritchett, 2012). Cacao production is essential to the livelihoods of 40 to 50 million people worldwide (WCF), and harvested areas are about 10 million ha (FAO). Based on morphological traits and geographical origins three major genetic groups have traditionally been defined within cacao: Criollo, Forastro, and Trinitario. Trinitario has been recognised as a ‘Criollo × Forastro’ hybrid (Cheesman, 1944). Recently, molecular markers and chromosome analyses have been used to classify cacao germplasm into 10 major groups: Amelonado, Contamana, Curaray, Guiana, Iquitos, Maraňón, Nanay, Purús, Criollo, and Nacional (Motamayor et al., 2008; Utro et al., 2012). The genomes of Criollo and Amelonado have been sequenced and cover 76% and 92% of the estimated genome size, respectively (Argout et al., 2011; Motamayor et al., 2013). These genomes are important tools for gene characterisation investigations. Cacao pods (fruits) grow mainly on the tree trunk. Photoassimilates are translocated from source organs (leaves) to sink organs (pods) through a long-distance phloem pathway. In the present study, our goal was to provide a comprehensive description of SUT function in cacao trees. By comparing translated sequences of the cacao genome with known SUT sequences, six genes were designated as encoding putative SUT proteins. Deduced amino acid sequences and gene structure of the SUT family of cacao were compared and expression patterns were characterised. 2. Materials and methods 2.1. Plant materials Unless otherwise noted, ‘TAS-R8’ cacao trees (T. cacao) were used in this study. They were cultivated at the experimental plantation of the Spice and Beverage Research Institute of Chinese Academy of Tropical Agricultural Science (Wanning, Hainan, China). ‘TAS-R8’ has a relatively larger pod weight, belongs to the Trinitario group, and has been planted for more than 30 years. Nineteen organs/tissues were sampled from adult tree, including leaf, bark with phloem, flower bud, pod, pod shell, seed coat, and seed. All organs/tissues were frozen in liquid nitrogen and stored at −80 °C until use. 2.2. RNA extraction and cDNA synthesis Total RNA was extracted from three replicates of each organ/tissue by using a Plant RNA Kit (OMEGA, Norcross, USA). The RNA was treated with DNase I, according to the manufacturer's instructions (TaKaRa, Dailian, China). An aliquot of RNA was electrophoretically separated on a 1.0% agarose gel to verify integrity. Final concentration was adjusted to 1 μg·μL−1 by using double-distilled water treated with 0.1% DEPC. Single-stranded cDNA was synthesised from 1.0 μg of total RNAs by using M-MLV reverse transcriptase (Fermentas, Ontario, Canada), according to the manufacturer's instructions. Three reverse transcriptions of each organ/tissue were carried out independently. The resultant first-
strand cDNA mixtures were diluted 1:20 with double-distilled water and stored at −20 °C before being used as a template for cloning and real-time PCR. 2.3. Cloning of sucrose transporter genes SUT gene models were identified from the sequence of cacao genome (Argout et al., 2011) by using BlastP search with nine Arabidopsis and five Populus SUT genes. Gene-specific primers (listed in Supplemental file 1) were developed for PCR amplification of TcSUT genes, according to the T. cacao genome sequences (v1.1) hosted at CATIE (http://cocoagendb.cirad.fr/) and the Cacao Genome Database (http://www.cacaogenomedb.org). The primers were designed using Primer Premier 5.0 software and synthesised by Sangon Biotech (Shanghai, China). PCR products were purified using the Gel Extraction Kit (OMEGA, Norcross, USA) and cloned into the pMD18-T vector by using the TA cloning kit (TaKaRa, Dailian, China). Transformed clones were selected for sequencing fully from both directions. The sequences were deposited in GenBank under accession numbers KF776545–KF776553. 2.4. Sequence analysis Sequence alignment was performed using DNAMAN 6.0 (version 5.0, Lynnon Biosoft). Characterisation and secondary structure of the proteins were analysed using ExPASy (http://expasy.org/tools/). Transmembrane regions were predicted using the TMHMM server (http://www.cbs.dtu.dk/services/TMHMM/). Selected plant sucrose transporter sequences were found using the BLAST algorithm (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Phylogenetic analyses were performed using MEGA 4.1 software (http://www.megasoftware.net/ index.html) with the neighbour-joining method, followed by phylogeny test options of 1000 bootstrap replicates. 2.5. Real-time PCR analysis Real-time PCR analysis was performed on a BIO-RAD CFX-96 realtime detection system with the SYBR Green Realtime PCR Master Mix (TOYOBO, Osaka, Japan). Specific primers (Supplemental file 1) were designed in the 5′ or 3′ region of TcSUT genes using the Primer Premier 5.0 software and were synthesised by Sangon Biotech (Shanghai, China). PCR reactions contained 400 nM of both forward and reverse gene-specific primers and 8.4 μL of the 50-fold diluted reverse transcription (RT) product in a final volume of 20 μL. The thermal cycling protocol was followed by DNA polymerase activation at 95 °C for 3 min. PCR amplification was carried out with 45 cycles of denaturation at 95 °C for 10 s, primer annealing at 60 °C for 15 s, and extension at 72 °C for 30 s. Optical data were acquired after the extension step, and the PCR reactions were subjected to a melting curve analysis beginning from 65 °C to 95 °C at 0.1 °C s−1. GAPDH was chosen as the reference gene on the basis of the study by Pinheiro et al. (2011). The data are presented as an average ± SD of three independently produced RT preparations used for PCR runs, each having at least three replicates. Expression data were analysed using Bio-Rad CFX Manager software with the 2− ΔCT method. Gene relative expression levels were drawn by Microsoft Excel 2010. 3. Results 3.1. Cloning of the cacao SUT gene family Six SUT genes were initially identified in the T. cacao (B97-61/B2) genome v1.1 (Argout et al., 2011). Full-length coding sequences for all except TcSUT6 were cloned from cacao by RT-PCR using a cDNA template mixture of leaf, bark with phloem, flower bud, and pod. Although repeated attempts to clone TcSUT6 from both a range of organ/tissue
Please cite this article as: Li, F., et al., Molecular cloning and expression analysis of the sucrose transporter gene family from Theobroma cacao L., Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.05.056
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cDNAs and genomic DNA, TcSUT6 target product was only amplified from genomic DNA. Based on sequence results, TcSUT1, TcSUT2 and TcSUT3 produced multiple transcripts, and the longest transcripts were selected for follow-up studies. TcSUT6 contains only one exon. TcSUT2 contains fourteen exons, about three times the number of the other four SUT genes, which have four or five exons (Fig. 1). The predicted molecular mass of TcSUT2 is approximately 66 kDa versus 52–57 kDa for the other proteins. TcSUT2 has acidic theoretical pI b 7, while other SUTs have alkaline theoretical pIs N 8.5 (Table 1).
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Table 1 Characteristics of the cacao sucrose transporter proteins as deduced from cDNA sequences. Size
TcSUT1 TcSUT2 TcSUT3 TcSUT4 TcSUT5 TcSUT6
pI
a.a.
kDa
536 616 494 513 491 488
57.0 65.7 52.7 55.5 52.2 52.0
9.29 6.82 9.13 9.22 8.54 8.54
% similarity (n.t./a.a.) to SUT2
SUT3
SUT4
SUT5
SUT6
60/42 –
70/64 63/45 –
61/52 65/48 63/51 –
69/65 62/46 81/79 63/53 –
69/65 63/46 81/80 64/53 94/91 –
3.2. Sequence alignment and phylogenetic analysis Analysis of the deduced amino acid sequences of the putative SUT genes revealed that all six SUTs contained 12 transmembrane domains (Supplemental file 2) with both N- and C-termini on the cytoplasmic side, as seen in other sugar transporters (Lalonde et al., 2004). The conserved histidine residue, which is responsible for sucrose binding in transport processes by site-directed mutagenesis of the AtSUC1 protein (Lu and Bush, 1998), was present in all putative SUT peptides in the extracellular loop between the first and second membrane spanning helices (Fig. 2). Sequence alignments revealed that TcSUT5 and TcSUT6 showed high similarity (N90%) with distinct gene structure. When compared to TcSUT3, both TcSUT5 and TcSUT6 showed approximately 80% similarity, which amino acid sequence of the other three TcSUTs b64% similarity. Plant SUT proteins were recently classified into five phylogenetic groups (Berthier et al., 2009; Kühn and Grof, 2010). A phylogenetic analysis of SUT orthologs from perennial and herbaceous species was carried out and is shown as an unrooted dendrogram in Fig. 3. Group 3 and Group 5 represent the monocot-specific branches and Group 1 representing dicot-specific branch. TcSUT1, TcSUT3, TcSUT5, and TcSUT6 belonged to Group 1 and clustered with SUT orthologs from other perennial species (e.g. Populus, Juglans) and distinct herbaceous species of the Arabidopsis- and legume-specific subclades. TcSUT3, TcSUT5, and TcSUT6 formed a single subclade in this group. TcSUT2 and TcSUT4 fall into subclades of Group 2 and Group 4 respectively, which both form monocot- and dicot-specific subclades. 3.3. Expression pattern of cacao SUT family Spatial and developmental expression profiling of the entire cacao SUT family was assessed by quantitative real-time PCR (Fig. 4A). TcSUT1 transcripts were most abundant in developing pods and very low in other organs/tissues. Expression of TcSUT2 was similar in mature leaves, bark with phloem, and developing pods, and lower in flower buds. TcSUT3 and TcSUT4 were highly expressed in photoassimilate transport organs/tissues, with higher levels in bark with phloem and developing pods. TcSUT5 expression was detected mostly in flower buds and developing pods. Transcript levels of the highly similar TcSUT5 isoform, TcSUT6, were not detected in any tested organs/tissues. Therefore, TcSUT6 was not investigated in the subsequent study. For distinct developmental stage of leaves, TcSUT1 transcripts accumulated mainly in sink and transit leaves (transition from ‘sink’ to ‘source’), with minimal levels in mature leaves. TcSUT2, TcSUT3, and TcSUT4 transcripts showed higher expression levels in mature leaves, and TcSUT4 showed an equivalent expression level in transit leaves and mature leaves. Compared
Fig. 1. Structure of six cacao SUT genes. The grey bars and black lines denote exons and introns in the coding domain sequence.
with the other SUT genes, TcSUT5 expression was minimal in leaves (Fig. 4B). TcSUT4 transcripts were higher in buds just before bud break (3–4 mm in length) than in other bud developmental stages (Fig. 4C). TcSUT5 transcripts increased with bud developmental, reaching its highest level at bud break. TcSUT1, TcSUT2, and TcSUT3 expression levels were overall lower than TcSUT4 and TcSUT5 in buds. To investigate the expression pattern of cacao SUT genes in diverse organs/tissues of various pod developmental stages, pod husk, seed coat, and seed of enlarging, stereotype (pod volume reach maximum) and ripening pods were investigated respectively (Fig. 5). TcSUT1 transcripts were found mostly in seeds and increased with seed maturation. TcSUT1 transcripts were found in low levels in pod husks. TcSUT2 expression was stable in pod husks and seed coats, but increased in seeds from pod enlargement to pod ripening. TcSUT3 expression also increased with seed maturation, but remained constant in pod husks and seed coats. Expression of TcSUT4 was constant in the three organs/ tissues of pod enlargement and stereotype stages. At pod ripening stage, TcSUT4 increased in pod husks while decreased in seed coats. TcSUT5 expression was highest in seed coats, and expression levels increased in pod husks and seed coats with pod maturation. 4. Discussion The draft sequence of T. cacao genome has provided investigators with a powerful tool that enables searches for gene families of known protein. By searching the genome sequence, we found six putative SUT genes, TcSUT1, TcSUT2, TcSUT3, TcSUT4, TcSUT5, and TcSUT6, comprising the SUT family in the cacao genome. However, it should be noted that the genomes are not fully sequenced and that one or more SUT may exist in the remaining 8% of the Amelonado genome and 24% of the Criollo genome. Phylogenetic analysis revealed that the plant SUT family can be divided into five distinct clades: Groups 1–5 (Kühn and Grof, 2010). Proton-coupled Group 1 (dicot) and Group 2 (monocot and dicot) SUTs are in the plasmalemma. They mediate apoplastic phloem loading and are essential for normal plant growth (Hackel et al., 2006; Srivastava et al., 2008). Some Group 4 (monocot and dicot) membrane SUTs, such as OsSUT2 (Eom et al., 2011) and PtaSUT4 (Frost et al., 2012), have been localised to the vacuolar membrane through proteomic and/or GFP fusion analyses. The function of Group 5 (monocot) SUTs has not been characterised. TcSUT1, TcSUT3, and TcSUT5, which are in Group 1, consist of the same number of exons. Their similarity in gene structure suggests that they diverged from a prototype gene, presumably by gene duplications. TcSUT6 showed high similarity with
Fig. 2. Sequence alignments of cacao SUT proteins with the AtSUC1. Putative transmembrane domains of the SUT peptide are underlined. The functionally important and conserved histidine residue is shown in bold.
Please cite this article as: Li, F., et al., Molecular cloning and expression analysis of the sucrose transporter gene family from Theobroma cacao L., Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.05.056
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Fig. 3. A neighbour-joining tree with predicated SUT amino acid sequences from cacao and other plant species. Sequences from cacao are marked with grey circle. Sequence names and/or accession numbers are provided in Supplemental file 3.
TcSUT5, but without introns. This may suggest that TcSUT6 was produced from TcSUT5 by reverse transcription. TcSUT2 and TcSUT4 belong to Group 2 and Group 4 SUTs, respectively. They may be similar to the tonoplast-localised SUC4-type, which mediates tonoplast transmembrane transport of sucrose (Schneider et al., 2011). TcSUT6 target product was amplified from the genomic DNA template, rather than from the cDNA template mixture. TcSUT6 was not expressed in any tested organs/tissues. The cDNA sequence of TcSUT6 had been deposited in the Cacao Genome Database, indicating that TcSUT6 may be expressed in some organs/tissues not used in our study. The predicted TcSUT6 peptide also has the conserved histidine residue (Lu and Bush, 1998). A higher similarity of TcSUT6 with other functional SUTs showed that TcSUT6 may play an important role in sucrose transport. TcSUT1, TcSUT2, and TcSUT3 produce multiple transcripts, and only the longest transcripts were studied. TcSUT1 shows one possible alternative splicing at exon 2, which resulted in the absence of 9 amino acids. TcSUT2 and TcSUT3 could give rise to alternative splicing at intron 6 and intron 2, respectively, resulting in a frame shift within the coding region. Based on the sequence information in the GenBank database, TcSUT2 produces eight different transcripts. These data indicate that there may be more undetectable transcripts of SUTs in cacao. To validate the compositive expression patterns of cacao SUTs, the primers were developed to measure the all transcripts of each gene. Alternative splicing produces multiple mRNA transcripts from the same pre-mRNA, and occurs commonly in multicellular eukaryotes (Eckardt, 2013; Wang et al., 2014). Alternative splicing plays important roles in regulating gene expression and increasing transcriptome diversity and proteome complexity. Further research of SUT transcripts is needed for member cloning, expression regulation and function identification. Cacao pods are the most valuable sink organs of cacao trees producing cacao beans, and the number of seeds per pod and the mean seed weight are important traits involved in increasing production per tree (Cilas et al., 2010). Photoassimilates distributed to developing pods are essentially carbon skeletons used to synthesise major storage products, and show positive association with seed yield (Rawsthorne, 2002; Weber et al., 2005). The different expression patterns of TcSUT genes in cacao plants observed in this work suggest that the SUT gene family has many roles in both source and sink organs/tissues and in different developmental stages. During pod development, TcSUT1, TcSUT4, and TcSUT5 were expressed more in seed coats and seeds, with the highest expression of TcSUT5 in the seed coat of ripening pods. These results
Fig. 4. Expression of SUT genes in cacao tissues. (A) Expression in different organs, including mature leaf (ML), bark with phloem (BP), flower bud (FB) and developing pod (DP). (B) Expression in different developmental stages of leaf including sink leaf (SL), transit leaf (TL) and mature leaf (ML). (C) Expression in bud at various developmental stages including various sizes in bud length: b2 mm, 2–3 mm, 3–4 mm and blooming flower (BF). Error bars represent the SD of three technical replicates. Abbreviated names of tested organs/tissues were listed in Supplemental file 4.
suggest that these three genes may play a role in photoassimilate unloading to developing seeds. Expression of SUT in the valuable sink organs has been improved to influence seed/tuber growth rate and yield (Li et al., 2011; Rosche et al., 2002; Sun et al., 2011). The consistent
Please cite this article as: Li, F., et al., Molecular cloning and expression analysis of the sucrose transporter gene family from Theobroma cacao L., Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.05.056
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Fig. 5. Expression of cacao SUT genes in various pod tissues at different pod developmental stages. E-PH, E-SC, E-S represent pod husk, seed coat, and seed of enlargement pod; “S-” and “R-” represent corresponding tissues of stereotype and ripening pod respectively. Error bars represent the SD of three technical replicates. Abbreviated names of tested organs/tissues were listed in Supplemental file 4.
expression patterns indicated that TcSUT genes were associated with photoassimilate transport into seeds during development and resulted in higher seed yields. However, investigations of the relationship between TcSUT variation and pod/seed traits are necessary to validate this association. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.gene.2014.05.056. Conflict of interest We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in, or the review of, the manuscript entitled. Acknowledgments This work was financially supported by the National Science Foundation of China (31301389) and the Tropical Crop Germplasm Resources Protection Project of the Ministry of Agriculture (13RZZY-24). References Aoki, N., Hirose, T., Scofield, G.N., Whitfeld, P.R., Furbank, R.T., 2003. The sucrose transporter gene family in rice. Plant Cell Physiol. 44, 223–232. Argout, X., Salse, J., Aury, J.M., Guiltinan, M.J., Droc, G., Gouzy, J., et al., 2011. The genome of Theobroma cacao. Nat. Genet. 43, 101–108. Barker, L., Kühn, C., Weise, A., Schulz, A., Gebhardt, C., Hirner, B., et al., 2000. SUT2, a putative sucrose sensor in sieve elements. Plant Cell 12, 1153–1164. Berthier, A., Desclos, M., Amiard, V., Morvan-Bertrand, A., Demmig-Adams, B., Adams, W.W., et al., 2009. Activation of sucrose transport in defoliated Lolium perenne L.: an example of apoplastic phloem loading plasticity. Plant Cell Physiol. 50, 1329–1344. Braun, D.M., Slewinski, T.L., 2009. Genetic control of carbon partitioning in grasses: roles of sucrose transporters and tie-dyed loci in phloem loading. Plant Physiol. 149, 71–81. Bucheli, P., Rousseau, G., Alvarez, M., Laloi, M., McCarthy, J., 2001. Developmental variation of sugars, carboxylic acids, purine alkaloids, fatty acids, and endoproteinase activity during maturation of Theobroma cacao L. seeds. J. Agric. Food Chem. 49, 5046–5051. Cheesman, E.E., 1944. Notes on the nomenclature, classification and possible relationships of cocoa populations. Trop. Agric. 21, 144–159. Cilas, C., Machado, R., Motamayor, J.-C., 2010. Relations between several traits linked to sexual plant reproduction in Theobroma cacao L.: number of ovules per ovary, number of seeds per pod, and seed weight. Tree Genet. Genomes 6, 219–226. Eckardt, N.A., 2013. The plant cell reviews alternative splicing. Plant Cell 25, 3639. Endler, A., Meyer, S., Schelbert, S., Schneider, T., Weschke, W., Peters, S.W., et al., 2006. Identification of a vacuolar sucrose transporter in barley and Arabidopsis mesophyll cells by a tonoplast proteomic approach. Plant Physiol. 141, 196–207.
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