DOI: 10.1007/s10535-015-0540-z BIOLOGIA PLANTARUM 59 (4): 783-787, 2015 BRIEF COMMUNICATION Gene expression and enzyme activities of the D-mannose/L-galactose pathway influence L-ascorbic acid content in Myrciaria dubia J.C. CASTRO1*, M. COBOS2, J.D. MADDOX3, S.A. IMÁN4, A. EGOAVIL1, J. TORRES1, and F. GUTIERREZ1 Unidad Especializada de Biotecnología, Centro de Investigaciones de Recursos Naturales de la Amazonía, Universidad Nacional de la Amazonía Peruana, 16006, Iquitos, Perú1 Laboratorio de Biotecnología y Bioenergética,Universidad Científica del Perú, 16006, Iquitos, Perú2 The Field Museum of Natural History, 60605, Chicago, IL, USA3 Instituto Nacional de Innovación Agraria, 16006, Iquitos, Perú4 Abstract The aim of this work was to elucidate the molecular and biochemical mechanisms that control L-ascorbic acid (AsA) content variation in Myrciaria dubia. The AsA was quantified by high-performance liquid chromatography, gene expression by real-time quantitative PCR, and enzyme activities by spectrophotometric methods from leaves and immature fruits of two genotypes (Md-60,06 and Md-02,04) with pronounced (about 2 times) differences in the AsA content. In either genotype, the fruit peel had ~ 1.5 times more AsA than the fruit pulp and ~ 15.0 times more than the leaf. All tissues examined demonstrated the capability for AsA biosynthesis through the D-mannose/L-galactose pathway because mRNAs of the six key genes [GDP-D-mannose pyrophosphorylase (GMP), GDP-D-mannose-3ꞌ,5ꞌ- epimerase (GME), GDP-L-galactose phosphorylase (GGP), L-galactose-1-phosphate phosphatase (GPP), L-galactose dehydrogenase (GDH), and L-galactono-1-4-lactone dehydrogenase (GLDH)] and catalytic activities of the corresponding enzymes (GMP, GDH, and GLDH) were detected. The differential expressions of genes and enzyme activities mostly correlated with the respective AsA content. Thus, the expression of several genes of the D-mannose/ L-galactose pathway determined the AsA content variation in tissues of M. dubia. Additional key words: GDP-D-mannose-3ꞌ,5ꞌ-epimerase; GDP-D-mannose pyrophosphorylase; GDP-L-galactose phosphorylase; L-galactono-1-4-lactone dehydrogenase; L-galactose dehydrogenase; L-galactose-1-phosphate phosphatase.  Myrciaria dubia (Kunth) McVaugh (common name Several studies have shown that a variety of genetic camu-camu) is an Amazonian fruit shrub that produces and environmental factors influence variation in AsA several bioactive phytochemicals, such as anthocyanins content in plant tissues (Davey et al. 2006, Roselló (Zanatta et al. 2005), ellagic acid derivatives, and other et al. 2011). These factors, directly or indirectly, phenolics (Fracassetti et al. 2013). However, the most influence the metabolic pathways of AsA biosynthesis valuable is its high L-ascorbic acid (AsA; i.e., vitamin C) (Conklin et al. 2013). Although a combination of radio- content in fruits which can be as much as 2 g of AsA per labelling, mutant analysis, and transgenic manipulation 100 g of pulp (Imán et al. 2011). It is also interesting the provides evidence for multiple pathways of AsA large variation in AsA pool size both among different biosynthesis in plants, the D-mannose/L-galactose tissue types of the same individual and among individuals (Smirnoff-Wheeler) pathway is generally considered the (Castro et al. 2013a). most important (Valpuesta and Botella 2004, Wheeler  Submitted 4 March 2015, last revision 27 April 2015, accepted 29 April 2015. Abbreviations: AsA - L-ascorbic acid; DTT - dithiothreitol; EDTA - ethylenediaminetetraacetic acid; f.m. - fresh mass; GDH - L-galactose dehydrogenase; GLDH - L-galactono-1,4-lactone dehydrogenase; GME – GDP-D-mannose-3ꞌ,5ꞌ-epimerase; GMP - GDP-D-mannose pyrophosphorylase; GPP - L-galactose-1-phosphate phosphatase; PMSF - phenylmethylsulfonyl fluoride; PP2A - serine/threonine protein phosphatase; PVP - polyvinylpyrrolidone. Acknowledgements: This research was financially supported by the Consejo Nacional de Ciencia, Tecnología e Innovación Tecnológica (CONCYTEC) from Perú (Contract N° 280-2010-CONCYTEC-OAJ), and the UNAP (R.R. 2012-UNAP). * Corresponding autor; fax: (+51) 065 263569, e-mail: juanccgomez@yahoo.es 783 J.C. CASTRO et al. et al. 1998). MuLV reverse transcriptase and oligo(dT)16 following the The factors that affect the variation in AsA content manufacturer’s instructions (Applied Biosystems, Foster among tissues and genotypes of M. dubia are largely City, CA, USA). unknown. Therefore, an objective in our study was to The expression profiles of the D-mannose/L-galactose understand the molecular and biochemical mechanisms pathway genes in leaves, fruit pulp, and fruit peel were that control AsA pool size variation by testing the determined by real-time quantitative PCR using SYBR hypothesis that differential gene expression and enzyme Green I technology on a Mastercycler ep Gradient activities of the D-mannose/L-galactose pathway realplexS (Eppendorf, NY, USA) thermal cycler. The influence AsA pool size in M. dubia. To test this expression stability of candidate reference genes was hypothesis, we measured and compared the AsA content, evaluated in our sample set with actin, glyceraldehyde-3- gene expression, and activities of key enzymes of the phosphate dehydrogenase (GAPDH), and serine/threonine D-mannose/L-galactose metabolic pathway in different protein phosphatase (PP2A) standards using the tissues (i.e., leaves, fruit pulp, and peel) of two plant NormFinder software in R (Andersen et al. 2004). All genotypes characterized by their low and high AsA PCR reactions were performed following the content in fruit pulp. manufacturer’s instructions of a SYBR Green PCR core Leaves and unripe fruits (65 d after anthesis) were kit (Applied Biosystems) and using specific primers collected from Myrciaria dubia (Kunth) McVaugh (Table 1 Suppl.). A melting curve analysis ranging from genotypes Md-02,04 and Md-60,06 belonging to the 55 °C to 95 °C with incremental steps of +0.5 °C s-1 was M. dubia germplasm bank at the Instituto Nacional de used to confirm specificity of amplifications. Reaction Innovación Agraria of Peru. These plants were previously efficiency and CT were determined in our sample set characterized by a low [53 ± 4 mol(AsA) g-1(f.m.)] and using the LinRegPCR software (Ruijter et al. 2009). In high [122 ± 10 mol(AsA) g-1(f.m.)] AsA content, each run, three technical replications were performed for respectively (Castro et al. 2013b). The AsA content was each of the biological samples, and the relative measured using high-performance liquid chromatography quantification of expression was performed using the (HP 1100, Elite La Chrome, Waldbronn, Germany) comparative CT method (Simon 2003). All expression according to Ledezma-Gairaud (1993). data were calculated as expression ratio relative to PP2A For determination of GDP-mannose pyrophospho- which was the most stable reference gene. Means, rylase (GMP), L-galactose dehydrogenase (GDH), and standard deviations, and statistical significance of L-galactono-1,4-lactone dehydrogenase (GLDH) activi- differences were performed with one-way ANOVA, the ties, tissue extracts were prepared by grinding each Tukey's HSD-test, and Student’s t-test. Differences at sample (1 g) in a mortar and pestle in 5 cm3 of ice cold P < 0.05 were considered significant. 100 mM Tris-HCl (pH 7.5) containing 1 mM MgCl2, 2 mM Na2EDTA, 3 mM dithiothreitol (DTT), 1 mM benzamidine hydrochloride, 1 mM aminocaproic acid, 1 mM phenylmethylsulfonyl fluoride (PMSF), 3 % (m/v) polyvinylpyrrolidone, 0.2 % (m/v) Triton X-100, and 20 % (v/v) glycerol (Gatzek et al. 2002, and Hancock et al. 2003, Conklin et al. 2006). Cell debris was removed by centrifugation at 20 000 g and 4 °C for 15 min, and the supernatant was desalted by dialysis (membrane MWCO 6000-8000, FisherBrand, PA, USA) in 1 dm3 of a dialysis buffer (50 mM Tris-HCl, pH 7.5, 1 mM MgCl2, 0.1 mM EDTA, 1 mM DTT, 0.1 mM benzamidine hydrochloride, 0.1 mM aminocaproic acid, and 0.1 mM PMSF) at 4 °C for 6 h. Denatured proteins were removed by centrifugation at 20 000 g and 4 °C for 10 min. The supernatant was used for the assays to determine the activities of GMP, GDH, and GLDH according to Davis et al. (2004), Gatzek et al. (2002), and Hancock et al. (2003), respectively. Total RNA was isolated from leaves, fruits pulp, and Fig. 1. The AsA content in leaves, fruit pulp, and fruit peel of peel using the cetyltrimethylammonium bromide (CTAB) M. dubia. Means  SD, n = 3. Different letters above columns method, solvent extractions, and DNase treatment as indicate statistically significant differences (P < 0.001). described previously (Castro et al. 2013b). The RNA quality and quantity were assessed by standard All possible comparisons of AsA pool size both measurement of absorbance at 230, 260, and 280 nm among tissues types of the same individual and among (Fig. 1 Suppl.) and by formaldehyde denaturing gel individuals of the same tissue types showed a electrophoresis (Sambrook et al. 1989). Single-stranded significantly different AsA content (Fig. 1). The fruit peel cDNA was obtained from 1.5 µg of total RNA using had ~ 1.5× more AsA than the fruit pulp and ~ 15.0× than 784 D-MANNOSE/L-GALACTOSE PATHWAY the leaves. Similarly, the fruit pulp AsA content was correlation with the AsA content in the respective tissues. ~ 10× higher that the leaf AsA content. Finally, Md-60,06 The other genes, such as GMP, GGP, and GPP, exhibited had on average a 2× higher AsA content than Md-02,04. a weak positive correlation among the expressions and The experimental data indicate that the leaves, fruit AsA content. In contrast, the expression of GDH did not pulp, and fruit peel of M. dubia possessed the capability correspond to the AsA content in all the three tissue for AsA biosynthesis through the D-mannose/L-galactose types. pathway because the six genes and the corresponding With regard to the enzyme activities, it is evident that enzymes (GMP, GDH, and GLDH) of this metabolic the three enzyme activities evaluated here were higher in pathway were detected in all three tissues analyzed. the fruit pulp and peel in comparison with the leaves However, in all the tissues, these genes and enzymes (Fig. 2). The enzyme activities of GMP and GLDH were showed differential expression and activities, higher in genotype Md-60,06 than Md-02,04 for all the respectively. three tissue types. Furthermore, there was a strong The expressions of all six genes were higher in the positive correlation between the GLDH activity and AsA fruit pulp and peel in comparison with the leaves (Fig. 2). content. The other enzymes evaluated showed a weak but Also, the expressions of these genes were higher in positive correlation (GMP) or no correlation (GDH) with genotype Md-60,06 in comparison with genotype the AsA content. Finally, it is evident that GLDH showed Md-02,04 in the leaves (GME, GLDH), fruit pulp (GMP, positive correlations among the gene expression, enzyme GME, GGP, GPP, and GLDH) and peel (GMP, GME, activities, and AsA content (Fig. 1). GGP, and GLDH). Additionally, in both the genotypes, Our results indicate that the leaves, fruit pulp, and the genes GME and GLDH showed a strong positive peel of M. dubia had the capability for AsA biosynthesis, Fig. 2. The relative gene expressions and enzyme activities of the D-mannose/L-galactose pathway in leaves, fruit pulp, and fruit peel of M. dubia. Means  SD, n = 3. Different letters above columns indicate statistically significant differences (P < 0.05). 785 J.C. CASTRO et al. because all the genes and the corresponding enzymes by ascorbate peroxidase (Dunajska-Ordak et al. 2014), (GMP, GDH, and GLDH) of the D-mannose/L-galactose 2) utilization as enzyme cofactor in anabolic pathways for pathway were detected (Fig. 2). These findings are phytohormones, anthocyanins, and hydroxyprolin rich similar to previous reports in other plant species, such as proteins biosynthesis (Valpuesta and Botella 2004, Gallie Malpighia glabra (Badejo et al. 2009), Actinidia spp. (Li 2013), and 3) catabolism to threonate, oxalate, and et al. 2010), and Vitis vinifera (Melino et al. 2009), tartrate (Smirnoff and Wheeler 2000). The relative confirming that fruits have capability for in situ contributions of these biochemical processes, therefore, biosynthesis of AsA via the D-mannose/L-galactose were likely to influence the AsA content in tissues of pathway. M. dubia more than any single process, though this needs These results also demonstrate that the AsA content be tested empirically. variation in the leaves, fruit pulp, and fruit peel in The experimental data show that the differential gene M. dubia was due, in part, to differential gene expressions expressions and enzyme activities of the D-mannose/ and enzyme activities of the D-mannose/L-galactose L-galactose pathway were responsible for the AsA pool pathway. Although this biosynthetic pathway is size variation in the tissues of M. dubia. These findings considered most important for AsA biosynthesis in plants are consistent with reports from Bulley et al. (2009) who (Wheeler et al. 1998), several studies have shown that showed that fruits of Actinidia eriantha with a high plants possess other biosynthetic pathways, such as myo- expression of genes GME and GGP have a 3 to 16× inositol pathway (Lorence et al. 2004), L-gulose pathway higher AsA content than genotypes with their low (Wolucka and Van Montagu 2003), galacturonic acid expression. Likewise, Yang et al. (2011) showed that a pathway, and animal-like pathway (Valpuesta and Botella higher AsA accumulation in fruit pulp of Citrus sinensis 2004). It is thus likely that the AsA pool size variation may be related to a higher expression of GME, GPP, among these tissues was caused by differential GDH, and GLDH. However, Mellidou et al. (2012) contributions of these metabolic pathways. showed that only the expression of one ortologue of GGP An AsA content in fruit pulp can be increased by correlates with a high AsA content found in a tomato three processes: 1) ex situ biosynthesis (leaf, fruit peel, cultivar characterized by fruits with a high AsA content. phloem, etc.) and import by long distance transport to In conclusion, our data clearly show that the fruit pulp (Franceschi and Tarlyn 2002, Tedone et al. expression of several genes of the D-mannose/ 2004), 2) in situ biosynthesis (Li et al. 2010), and L-galactose pathway correlated with the AsA pool size 3) recycling through the ascorbate-glutathione cycle variation in the tissues of M. dubia, and that AsA (Gest et al. 2013). 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