First Report About the Identification and Preliminary Analysis of a Partial Sequence of Dihydropyrimidine Dehydrogenase (NADP+)
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Acta Physiol Plant (2017) 39:159 DOI 10.1007/s11738-017-2458-x ORIGINAL ARTICLE First report about the identification and preliminary analysis of a partial sequence of dihydropyrimidine dehydrogenase (NADP+) in Thermopsis turcica during floral development using degenerate primers 1 2 1 D. Tekdal • S. J. Lucas • S. Cetiner Received: 6 October 2016 / Revised: 19 June 2017 / Accepted: 3 July 2017 Ó Franciszek Go´rski Institute of Plant Physiology, Polish Academy of Sciences, Krako´w 2017 Abstract Thermopsis turcica Kit Tan, Vural & Ku¨c¸u¨ko¨- step in pyrimidine degradation, which is essential for the du¨k is an herbaceous perennial, endemic and listed as biosynthesis of beta-alanine and pantothenates in plants; it endangered species by IUCN in Turkey. This plant is noted has also been shown to be required for normal seed for its unusual floral structure characterized by a 2–4 development in Arabidopsis. Expression of Tt-DPD was carpellary ovary. In this study, a DPD (NADP?)-like gene monitored by both endpoint and real-time RT-PCR. High was partially sequenced for the first time in T. turcica. expression of the identified gene was observed in the mid- Since there is no previous molecular genetic information developmental stage of the pistil of T. turcica. The findings available for T. turcica, RT-PCR was performed using presented here provide a starting point for understanding degenerate primers targeted to conserved sequences of the roles of this gene in pyrimidine catabolism in T. WUS protein homologues from related legume species. turcica. Amplified cDNAs of the expected size were sequenced and analyzed using bioinformatics tools. The analysis strongly Keywords Degenerate primer Á DPD (NADP?) Á suggested that a 283 bp PCR product was part of a dihy- Pyrimidine catabolism Á Gene identification Á Sequencing Á dropyrimidine dehydrogenase (NADP?)-like coding Thermopsis turcica sequence with a Flavin mononucleotide binding domain. The putative gene was named Tt-DPD and the partial CDS Abbreviations submitted to the NCBI database (accession number Aa Amino acid(s) KT182937). This gene has not been identified previously in AGE Agarose gel electrophoresis T. turcica. The DPD (NADP?) enzyme is the rate-limiting BLAST Basic local alignment search tool bp Base pair C. arietinum Cicer arietinum cDNA Complementary deoxyribonucleic acid CDS Coding sequence Communicated by P. K. Nagar. DPD Dihydropyrimidine dehydrogenase 5-FU 5-Fluorouracil Electronic supplementary material The online version of this L. japonicus Lotus japonicus article (doi:10.1007/s11738-017-2458-x) contains supplementary material, which is available to authorized users. M. truncatula Medicago truncatula NADP Nicotinamide-adenine dinucleotide & D. Tekdal phosphate [email protected] NCBI National Center for Biotechnology Information 1 Biological Sciences and Bioengineering Program, Faculty of Engineering and Natural Sciences, Sabanci University, NGBG Nezahat Go¨kyig˘it Botanical Garden Orhanli-Tuzla, 34956 Istanbul, Turkey ORF Open reading frame 2 Nanotechnology Research and Application Centre, Sabanci PCR Polymerase chain reaction University, Orhanli-Tuzla, 34956 Istanbul, Turkey P. vulgaris Phaseolus vulgaris 123 159 Page 2 of 10 Acta Physiol Plant (2017) 39:159 RT-PCR Reverse transcription polymerase catabolism (Zrenner et al. 2006). Specifically, uracil and chain reaction thymine are converted to dihydrouracil and dihy- TBE Tris–boric acid–EDTA buffer drothymine, respectively, by DPD (Raman and Rathi- T. turcica Thermopsis turcica nasabapathi 2004). The pyrimidine degradation pathway is well characterized in certain bacterial species [Escherichia coli (Hidese et al. 2011); Alcaligenes eutrophus (Schmitt Introduction et al. 1996)] and animals [pig (Rosenbaum et al. 1997; Hagen et al. 2000); cow (Lu et al. 1993); and rat (Harris Thermopsis turcica Kit Tan, Vural & Ku¨c¸u¨ko¨du¨kisa et al. 1988; Lu et al. 1993)], but has not been clearly single endemic plant species belonging to the Fabaceae described in plants; however, it is thought to be the reverse family and found only in Turkey that is marked out by its function of the de novo biosynthetic pathway. The first polycarpellary morphology. It has been announced as a study of pyrimidine catabolic metabolism in plants was critically endangered (CR status) species in the Red Data announced in pea seedlings (Mazus and Buchowicz 1968). Books of Turkish plants, according to IUCN threat cate- The intracellular localization of a dihydropyrimidinase (EC gories (Ekim et al. 2000;O¨ zhayat et al. 2005; IUCN 2009). 3.5.2.2), the enzyme downstream of DPD, has been studied While 25 other species belonging to the genus Thermopsis in tomato cell suspension culture and Euglena gracilis are naturally found mainly in mountainous areas of Asia (Tintemann et al. 1987), and the secondary products gen- and North America, T. turcica is the only endemic species erated by dihydropyrimidinase examined in Albizia julib- of this genus in Turkey. T. turcica is unique in the Papil- rissin (Brown and Turan 1995; Turan and Sinan 2005). ionoideae subfamily due to its carpel polymerization Products of pyrimidine catabolism can be used in the (Cenkci et al. 2008, 2009; Ozdemir et al. 2008; Tekdal and biosynthesis of b-alanine and pantothenates (Zrenner et al. Cetiner 2014). 2006). Degenerate PCR primers were designed to amplify Pyrimidine nucleotides are required in a wide range of genes which govern possible mechanisms of gynoecium processes such as pollen tube growth, germination, and multiplication including WUSCHEL (WUS) and its nega- flowering (Cooke et al. 2001; Kafer et al. 2004; Raman and tive regulators, CLAVATA (CLV) and FASCIATA (FAS) Rathinasabapathi 2004). Control of these processes through (Weigel and Meyerowitz 1994;Ba¨urle and Laux regulation of cellular free pyrimidine levels is thought to be 2003, 2005; Choob and Sinyushin 2012; Endress 2014), a major role of the pyrimidine catabolic pathway (Cor- with the aim of providing information on the mechanism of nelius et al. 2011). gynoecium polymerization in T. turcica. Real-time RT-PCR permits the analysis of multiple Degenerate primers consist of a mixture of similar genes simultaneously both in specific organs and during oligonucleotides with variable bases at some positions that development (Song et al. 2011). In this study, qRT-PCR are used to amplify unknown DNA sequences. In particu- was used to monitor the expression of the isolated partial lar, this method is used to amplify gene-coding sequences, homologues of Tt-DPD in pistils from different develop- where genomic data are unavailable, based on previously mental stages of T. turcica during floral development with sequenced orthologous genes from different organisms the expectation that Tt-DPD expression might be increased (Rose et al. 1998; Song 2005; Walker 2011). In the same during flower bud initiation; in fact, Tt-DPD expression way, we recently used degenerate primers to isolate was greatest in pistils at mid-developmental stages in T. homologous genes in T. turcica, and identified five partial turcica, which might indicate involvement in pistil sequences of the target genes WUS, FAS, and CLV; how- enlargement. ever, they are not the subject of the present study. Herein, we report that unexpected sequences were also amplified using the degenerate primers, due to their potential to hybridize to more than one sequence. In this study, a partial Materials and methods sequence of an oxidoreductase family protein from T. turcica was identified. Sequence analysis indicates that the Plant materials ORF translates into the N-terminal 74 amino acids of a dihydropyrimidine dehydrogenase (DPD) enzyme, so it Thermopsis turcica plants, which are being maintained in was labelled Tt-DPD. This protein is involved in the beta- Nezahat Gokyigit Botanical Garden (NGBG) in Istanbul, alanine biosynthesis pathway (Kafer et al. 2004), and this is Turkey, were used as the main source of plant material. In the first time that it has been described in T. turcica. In addition, monocarpellary Phaseolus vulgaris (which other species, DPD is synthesized as a precursor containing belongs to the same taxonomic family), provided by local a signal peptide and acts with other enzymes in pyrimidine breeders in Turkey, was used for comparison. Flower buds 123 Acta Physiol Plant (2017) 39:159 Page 3 of 10 159 were collected from different growth stages for each spe- First-strand cDNA synthesis cies (T. turcica and P. vulgaris). Pistil samples at early- stage (1–3 mm in length), mid-stage (10–15 mm), and late- The first-strand cDNA was synthesized using the Tetro stage (20–25 mm) were removed and frozen immediately cDNA Synthesis Kit (Bioline, London, UK) with oligo-dT in liquid nitrogen, and stored at -80 °C for molecular primers according to the manufacturer’s protocol, and used analysis. as a template for RT-PCR and quantitative real-time RT- PCR analysis. Degenerate primer design RT-PCR analysis, agarose gel electrophoresis, To find possible orthologs of the WUS gene, protein and gel extraction sequences of legumes (Medicago truncatula, Cicer ariet- inum, and P. vulgaris) were retrieved from the NCBI For endpoint RT-PCR, thermal cycler conditions were as database. Degenerate primers of 16–24 nucleotides were follows: initial denaturation at 95 °C for 10 min followed designed based on sequences conserved between these by 35 cycles of: denaturation at 95 °C for 30 s, annealing species (Table 1). The WUS mRNA sequences identified in at 50 °C for 30 s, and extension at 72 °C for 1 min. These the selected species were first aligned using Clustal Omega cycles were followed by a final extension step at 72 °C for (Sievers et al. 2011) to identify conserved sequence seg- 7 min. ments, and degenerate primers were designed manually PCR products were separated and visualized on agarose (Fig. 1) predicated on minimum codon degeneracy. The gels (2%). cDNA samples mixed with loading dye were specificity of designed primers was checked using the loaded on the gel, which was run at 100 V for 45 min in NCBI’s Primer-BLAST tool.