ABSTRACT
Moringa (Moringa oleifera), which is a semi-tropical plant, is used as food and for the production of medicines and oil products, because of a large amount of various nutrients including ascorbic acid (AsA). Although Moringa leaf has a high AsA content, the molecular mechanisms of AsA accumulation in Moringa have received little attention. In this study, we isolated Moringa cDNAs for enzymes, belonging to the major AsA biosynthesis pathway (Smirnoff-Wheeler pathway) in higher plants. The predicted amino acid sequences showed 70% or more similarity to those of Arabidopsis. Quantitative RT-PCR indicated that Moringa GDP-L-galactose phosphorylase (GGP) is most highly expressed in Moringa during leaf development and light exposure. A significant high promoter activity of the Moringa GGP gene was detected by promoter assay in Arabidopsis protoplast.
Key words: Moringa oleifera, ascorbic acid, biosynthesis enzymes, gene expression.
Ascorbic acid (AsA), vitamin C is reported as the sum of AsA and dehydroascorbic acid (DHA), is an essential human nutrient. Vitamin C, however, cannot be endogenously synthesized in the human body due to the absence of the last enzyme in the AsA biosynthesis pathway (Chatterjee, 1973), and must instead be obtained from fruit and vegetables in the diet. AsA has a variety of physiological roles (Smirnoff and Wheeler, 2000). For example, through its antioxidant properties, AsA scavenges reactive oxygen species (ROS) that are produced by abiotic stresses such as light (Asada, 2006), high and low temperature (Suzuki and Mittler, 2006), and drought (Helena and Carvalho, 2008). AsA also plays major roles in cell growth, photosynthesis, and control of anthesis (Barth et al., 2006; Mano et al., 2004). Scrutiny of AsA biosynthesis has led to several proposed synthesis pathways in plants, and one major pathway is the Smirnoff-Wheeler pathway, in which AsA is synthesized via D-mannnose and L-galactose (Kanter et al., 2005; Radzio et al., 2003; Wheeler et al., 1998; Wolucka et al., 2001; Zhang et al., 2008). The Smirnoff-Wheeler pathway has been characterized, and involves eight AsA biosynthesis enzymes, namely, phosphomannose isomerase, phosphomannomutase, GDP-d-mannose pyrophosphorylase (GMP), GDP-d-mannose-3',5'-epimerase (GME), GDP-l-galactose phosphorylase (GGP), l-galactose-1-phosphate phosphatase (GPP), l-galactose dehydrogenase (GDH), and l-galactono-1,4-lactone dehydrogenase (GalLDH) (Wheeler et al., 1998). The vtc1 mutant of Arabidopsis, which is deficient in GMP gene, has 25% of wild-type AsA, and the vtc2 Arabidopsis mutant, which is deficient in GGP gene, has 10 to 20% of wild-type AsA (Conklin et al., 1999; Dowdle et al., 2007). Although other AsA biosynthesis pathways, such as the galacturonate and myo-inositol pathways, have been found to function in plant AsA biosynthesis (Agius et al., 2003; Lorence et al., 2004), the Smirnoff-Wheeler pathway appears to be predominant in higher plants.
Moringa (Moringa oleifera Lam), which is native to northwest India, is an important multi-purpose tree that is used as food and for the production of medicines and oil products (Morton, 1991). The many potential uses of Moringa have led to the recent publication of a number of reports discussing the use of seed- and leaf-powders and extracts for, among others, water purification, nutrition, and medicine (Anselme et al., 1995; Anwar and Bhanger, 2003; Bhuptawat et al., 2007). Moringa can grow rapidly in tropical areas as well as in soils with relatively low nutrients and low humidity (Morton 1991), and has a high leaf AsA content (Sreelatha and Padma, 2009), suggesting that it could serve as a valuable dietary source of vitamin C for populations in less-developed countries. However, an increase in AsA content is required to optimally utilize the already high production capacity in Moringa. To date, the molecular mechanisms through which AsA accumulates in Moringa have not been determined, and elucidation of these molecular mechanisms is essential for the future improvement of AsA content in Moringa leaf. In this study, Moringa cDNAs for AsA biosynthesis enzymes were identified and used to evaluate gene expression. Quantitative RT-PCR analysis indicated that MoGGP was most highly expressed in Moringa among biosynthesis gene of the six investigated. The 5'-upstream region of the MoGGP gene was determined, allowing investigation of cis-element(s) enhanced promoter activity using Arabidopsis protoplasts.
Plant materials
Moringa plants were grown at 25°C in 16 h of light (light intensity; 55.6 μmol/s/m2) and 8 h of dark in a greenhouse. To compare the AsA contents and mRNA expression levels of AsA biosynthesis enzymes in the leaves of Moringa and Arabidopsis, the small (leaf length; <10 mm), medium (leaf length; 10 to 15 mm) and large leaves (leaf length; >15 mm) were prepared from Moringa plant. To test the effects of light on mRNA expression levels of AsA biosynthesis enzymes, leaf discs (8 mm diameter) were prepared from Moringa small leaves using a biopsy punch (8.0 mm, Kai industries). Leaf discs were floated on water in a petri dish, incubated in the dark overnight, and were then exposed to continuous light treatment (100 μmol/s/m2), or were left to continuous darkness (0 μmol/s/m2) at 25°C for 24 h. After light and dark treatment, discs were assayed for AsA content and mRNA expression levels of AsA biosynthesis gene.
Arabidopsis thaliana cv. Columbia seeds were placed on soil in a pot (8 cm wide by 7.5 cm high). The seedlings were soil-cultivated in a plant growth incubator at 25°C in 16 h of light (66.7 μmol/s/m2) and 8 h of dark for 3 weeks. To measure AsA contents and mRNA expression levels AsA biosynthesis enzymes, rosette leaves were used.
Determination of ascorbic acid content
Total vitamin C content, as ascorbic acid content, was determined using an ascorbate oxidase method. Moringa and Arabidopsis leaves were harvested and ground in liquid nitrogen. The powdered tissues (100 mg) were homogenized in 1.0 ml of cold 6.0% (v/v) perchloric acid and centrifuged at 12,000 × g for 10 min at 4°C. To determine total vitamin C content, supernatants (350 μl) were combined with 110 μl of 1.25 M K2CO3 and the volume brought to 525 μl with distilled water. The extract (420 μl) was combined with 4 μl of 1 M dithiothreitol, incubated in the dark at 30°C for 30 min, and then centrifuged at 12,000 × g for 10 min. The supernatant (50 μl) was then combined with 446 μl of 200 mM succinate buffer and the absorbance was immediately measured at 265 nm. The absorbance at 265 nm was remeasured 30 s after the addition of 2.5 U of ascorbate oxidase. Total vitamin C content was calculated using a standard curve.
Reverse transcription-polymerase chain reaction
Total RNA was isolated from Moringa leaves using an RNeasy Plant Mini Kit with DNase I (Qiagen) according to the manufacturer’s protocol. First-strand cDNA was synthesized from total RNA using a ReverTra Ace kit (Toyobo) and an oligo(dT)20 primer. The cDNA was used as a template for polymerase chain reaction (PCR) using KOD Dash (Toyobo). Primers for AsA biosynthesis genes were designed based on the conserved amino acid sequences of plant AsA biosynthesis enzymes. The sequences for primers were used from the cDNA sequences of Arabidopsis thaliana AsA biosynthesis enzymes, and are listed in Supplementary Table 1. After initial denaturation for 2 min at 94°C, 40 cycles of amplification were carried out with 10 s denaturation at 98°C, 30 s annealing at 64°C for GalLDH, 56°C for GDH, GPP, GGP and GME or 55°C for GMP, and 60 s extension at 72°C. The primary PCR products were used as templates for nested PCR with additional gene-specific primers. Nested PCR was performed using KOD Dash, with the following thermocycler conditions: initial denaturation for 2 min at 94°C, followed by 40 cycles of 10 s denaturation at 98°C, 30 s annealing at 58°C, and 60 s extension at 72°C. PCR products were subcloned into the pGEM-T Easy vector (Promega, Madison, WI) and sequenced using an Applied Biosystems 3130xl Genetic Analyzer (Applied Biosystems) with a BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems), according to the manufacturer’s protocol.
5'- and 3'- rapid amplification of cDNA ends
Gene-specific primers (Supplementary Table 1) were designed and used for 5'- and 3'-rapid amplification of cDNA ends (RACE) to determine the nucleotide sequences of partial cDNA fragments. For 3'-end amplification, single-stranded cDNA was synthesized from total RNA (500 ng) using an oligo (dT)17 adaptor primer and ReverTra Ace. PCR for 3'-RACE was performed using KOD FX (Toyobo) with an adaptor primer and gene-specific primers.
After initial denaturation for 2 min at 94°C, 35 cycles of amplification were carried out with 10 s denaturation at 98°C, 30 s annealing at 55°C and 60 s extension at 68°C. The primary PCR product was used as templates for nested PCR, using an adaptor primer and a gene-specific primer and the same thermocycler conditions for the primary PCR. The resultant PCR products were subcloned and sequenced as described above. For 5'-RACE, circularized cDNA was synthesized from total RNA. Total RNA (5 μg) was reverse-transcribed using ReverTra Ace and a 5'-end phosphorylated oligo(dT)17 primer. After hydrolysis of total RNA with RNase H (TaKaRa) at 30°C for 60 min, cDNA was circularized by ligation with T4 RNA Ligase (TaKaRa) at 15°C overnight. PCR was performed with KOD Dash and primers (GPP, oKT066 and oKT087; GGP, oKT064 and oKT065), using the circularized cDNA as templates. PCR conditions were as follows: 40 cycles of 30 s denaturation at 94°C, 10 s annealing at 55°C for GPP or 60°C for GGP, and 60 s at 72°C. PCR products were subcloned and sequenced as described above.
Cloning and sequencing of the Moringa GGP genomic sequence
The genome sequence of MoGGP was determined using gene-specific primers (Supplementary Table 1) designed against the MoGGP cDNA sequence. Genomic DNA was isolated from Moringa leaves using a DNeasy Plant Mini Kit (Qiagen) according to the manufacturer’s protocol. PCR was performed using KOD Dash with gene-specific primers, with 40 cycles of 30 s denaturation at 94°C, 10 s annealing at 60°C, and 60 s extension at 72°C. PCR products were subcloned and sequenced as described above. The 5'-upstream region of the MoGGP gene was isolated using the cassette-ligation mediated PCR method with an LA PCR in vitro Cloning Kit (TaKaRa). After digestion of genomic DNA with EcoRI or XbaI, the cleaved genomic DNA fragments were ligated to the appropriate double-stranded DNA cassette. Primary PCR was performed with a cassette-specific primer (C1) and a gene-specific primer, using KOD FX Neo reagents (Toyobo). PCR conditions were as follows: initial denaturation for 2 min at 94°C, 40 cycles of 10 s denaturation at 98°C, 30 s annealing at 64°C, 60 s extension at 68°C. Subsequent nested PCR used the primary PCR product as templates, alongside a cassette-specific primer (C2) and a gene-specific primer, and used the same thermocycler conditions as the primary PCR. PCR products were subcloned and sequenced as described above.
Real-time PCR
For internal reference of quantitative RT-PCR, partial cDNA fragment of Moringa rRNA was cloned by RT-PCR as described above. Primers (Supplementary Table 1) for Moringa rRNA were designed from the conserved sequences of plant rRNA; Arabidopsis (Acc. No. X52320), Brassica napus (Acc. No. D10840), Cercidiphyllum japonicum (Acc. No. AF274639) and Disanthus cercidifolius (Acc. No. AF274645). Reaction conditions were as follows: initial denaturation 30 s for 95°C, followed by 30 cycles of 5 s at 95°C and 10 s at 60°C. The resultant PCR products were subcloned and sequenced. The cDNA sequences are deposited in GeneBank under an accession number LC005430.
Total RNA was isolated from Moringa leaves using an RNeasy Plant Mini Kit with DNase I (Qiagen). Single-stranded cDNA was synthesized from RNA (500 ng) using the ReverTra Ace qPCR RT Kit (Toyobo), according to the manufacturer’s protocol. Real-time PCR quantitation of transcripts of AsA biosynthesis enzymes was performed using Chrome4 (BioRad) and SsoFast EvaGreen Supermix (BioRad), according to the manufacturer’s protocol. Reaction conditions were as follows: initial denaturation 30 s for 95°C, followed by 30 cycles of 5 s at 95°C and 10 s at 60°C with respective gene-specific primers (Supplementary Table 1). From the sequences of Moringa (Acc. No. LC005430) and Arabidopsis (Acc. No. X52320) rRNA, gene-specific primers were designed and used as respective internal reference. Transcript levels were determined using standard curves generated with DNA samples of known concentration. Normalization was performed by dividing Moringa AsA biosynthesis gene transcript levels with those of rRNA. PCR specificity was assured by examining the melting curve between 65°C and 95°C every 2 s with increments of 0.2°C, and by use of agarose gel electrophoresis to check for a single amplifying band. Real-time PCR experiments were performed in triplicate.
Promoter assay
The 5'-upstream region of the MoGGP gene was amplified by PCR using primers designed for In-Fusion PCR cloning, according to the manufacturer’s instructions. For the Arabidopsis GGP promoter, 878 bp upstream of the AtGGP initiation codon was used. Amplified fragments were subcloned upstream of the Renilla luciferase coding sequence using In-Fusion PCR cloning. Preparation and transformation were performed using Arabidopsis protoplasts. Briefly, Arabidopsis protoplasts were prepared from rosette leaves of approximately 3-week-old Arabidopsis thaliana (ecotype Columbia) plants using cellulase “onozuka” R10 (Yakult Pharmaceutical Industry Co., Ltd.) and macerozyme R10 (Yakult Pharmaceutical Industry Co., Ltd.). Isolated protoplasts were suspended in MMg (0.4 M mannitol, 15 mM MgCl2, and 4 mM MES pH 5.7) and used for protoplast transformation, which was performed in a 96-well plate (96-well U-bottom plate, Thermo SCIENTIFIC) using polyethylene glycol. Plasmid (1 pmol) expressing full-length Renilla luciferase under the control of the MoGGP promoter was used to transfect Arabidopsis protoplasts (3.0 × 105 cells). Plasmid (0.5 pmol) expressing full-length click beetle red luciferase under the control of the cauliflower mosaic virus 35S promoter was co-transformed for use as an internal reference. Luciferase luminescence was measured using a microplate luminometer (ARVOx4 2030 Multilabel Reader, Perkin Elmer, MA). The luminescence signals in each well were integrated for 3 s, 20 min after adding ViviRen (Promega) in each well. Luciferase luminescence was normalized for transformation efficiency by dividing the relative light units (RLU) of Renilla luciferase by the RLU of beetle red luciferase. Beetle luciferase activity was assessed by measuring luminescence through a red filter (effect filter 106, Always CO., LTD), with a 3 s integration period after the addition of the substrate.
Isolation of cDNA clones encoding Moringa ascorbic acid biosynthesis enzymes
Reverse transcription (RT)-PCR was performed using primers designed from the cDNA sequences of Arabidopsis AsA biosynthesis enzymes. Partial cDNA fragments were isolated, and their nucleotide sequences were highly similar to those of the Arabidopsis AsA biosynthesis genes (data not shown). Rapid amplification of cDNA ends (RACE) methods were subsequently used to determine full-length cDNA sequences for Moringa GGP and GPP. The cDNA of Moringa GGP (MoGGP) has a 1,320 bp open reading frame (ORF) that is predicted to encode a protein of 440 amino acids with a calculated molecular mass of 48,963 Da (Supplementary Figure 1). Primary structure analysis using the Conserved Domain Database (CDD) (Marchler-Bauer et al., 2013)suggested that MoGGP contains a histidine triad motif found in Arabidopsis GGP (AtGGP). The cDNA of Moringa GPP (MoGPP) has an 804 bp ORF that is predicted to encode a protein of 268 amino acids with a calculated molecular mass of 28,954 Da (Supplementary Figure 2). Primary structure analysis using CDD suggested that MoGPP has related domains of inositol monophosphatase family. MoGGP and MoGPP have 75 and 81% identities, respectively, to the Arabidopsis proteins, as indicated in Supplementary Table 2. We successfully identified the 3'-downstream sequences of Moringa GMP, GME, GDH and GalLDH cDNAs using 3'-RACE, but in this study we were unable to determine the 5'-upstream sequences of cDNAs using 5'-RACE. Comparison of the sequences between the Arabidopsis AsA biosynthesis enzymes and the partial predicted Moringa proteins indicated that high levels of similarity exist at the amino acid sequence level (Supplementary Table 2). We therefore concluded that the partial cDNAs isolated in this study are those encoding the Smirnoff-Wheeler pathway AsA biosynthesis enzymes in Moringa. The cDNA sequences are deposited in GeneBank under the following accession numbers: AB924374 for MoGalLDH, AB924375 for MoGDH, AB924376 for MoGPP, AB924377 for MoGGP, AB924378 for MoGME, and AB924379 for MoGMP.
Transcriptional levels of AsA biosynthesis enzymes in Moringa leaves
Genetic and biochemical studies indicate that the Smirnoff-Wheeler pathway is both ubiquitously expressed and is the dominant AsA biosynthesis pathway in higher plants (Conklin, 2001; Conklin et al., 2000; Wheeler et al., 1998; Wolucka and Van Montagu, 2007). Figure 1A shows that Moringa small (< 10 mm), medium (10 to 15 mm) and large (> 15 mm) leaves contained 10.9, 5.2 and 4.1 µmol/gFW (gram fresh weight) AsA, respectively. AsA content in Arabidopsis rosette leaf was 3.6 µmol/gFW. Sreelatha and Padma reported that Moringa leaf contained about 30.5 µmol/gFW AsA (Sreelatha and Padma, 2009). The Moringa plants used in this study were grown under fluorescent at 25°C in a green house. The comparative-low AsA contents in Moringa leaves used in this study, may be due to growth condition, such as light intensity, humidity or temperature, because AsA content in plant is subject to ambient growth condition (Davey et al., 2000). To evaluate AsA biosynthesis at transcriptional levels in Moringa leaves, mRNA levels of AsA biosynthesis enzymes isolated in this study were measured in small, medium and large leaves using quantitative RT-PCR (Figure 1B). The mRNAs encoding all six Moringa AsA biosynthesis enzymes were expressed during Moringa leaf development, indicating that the Smirnoff-Wheeler pathway is likely functional during Moringa leaf development. As indicated in Figure 1B and 1C, the transcriptional patterns of the Moringa and Arabidopsis genes tested in this study were different from each other. Of the biosynthesis genes tested, the MoGGP mRNA was expressed at the highest levels at all developmental stages. The Arabidopsis vtc2 mutant is defective in the production of GGP, and contains only 10 to 25% of the AsA of a wild-type plant (Conklin et al., 1999; Dowdle et al., 2007). On the other hand, estrogen inducible transiently overexpression of AtGGP resulted in an increase in AsA contents (Yoshimura et al., 2014). This indicates that AtGGP is a key enzyme in Arabidopsis AsA biosynthesis. In acerola, which contains extremely abundant AsA, expression levels of GGP mRNA were the highest of the AsA biosynthesis mRNAs tested (Badejo et al., 2009). The acerola data, alongside our findings here, suggest that MoGGP may be the predominant enzymes contributing to AsA accumulation in Moringa leaves.
Effects of light exposure on mRNA levels of AsA biosynthesis enzymes in Moringa leaf
Exposure of plants to light causes an increase in AsA levels (Cruz-Rus et al., 2010; Fukunaga et al., 2010; Li et al., 2009; Yabuta et al., 2007). Multiple AsA biosynthesis enzymes are thought to participate in light-triggered AsA biosynthesis in plants. We evaluated the effects of light on AsA production in Moringa by measuring mRNA levels in leaf discs treated with continuous light exposure. As indicated in Figure 2A, AsA content was significantly higher in leaf discs illuminated at 100 μmol/s/m2 light than in those incubated in the dark (0 μmol/s/m2). Quantitative RT-PCR analysis revealed that MoGGP was the only AsA biosynthesis gene to display significantly increased expression on exposure to light treatment (Figure 2B). In Arabidopsis transiently overexpressing AtGGP, the increase in AsA levels was enhanced under continuous light (Yoshimura et al., 2014). The finding that only the MoGGP mRNA levels increase with light exposure suggests that this is the dominant enzyme involved in light-triggered AsA biosynthesis in Moringa.
Sequence and activity of the Moringa GGP promoter
The MoGGP gene sequence (Acc. No. AB924665) was determined through amplification of genomic DNA using primers designed from the MoGGP cDNA. The MoGGP gene structure was determined by comparing the genomic and cDNA sequences, and was found to comprise seven exons (Figure 3A).
The genomic and cDNA sequences were compared between MoGGP and AtGGP, which indicated that both exon number and length were well conserved. This suggests that the GGP gene likely arose prior to the divergence of Arabidopsis and Moringa. We also sequenced ~0.8 kb of the 5'-region upstream of the MoGGP initiation codon (Figure 3B). The 215 bp directly upstream of the initiation codon was identical to the 5'-UTR of the MoGGP cDNA, indicating that the transcription start site (TSS) of the MoGGP gene is more than 200 bp upstream of the initial ATG. The 5'-upstream region was searched for transcription factor recognition sites using the PLACE program (Higo et al., 1999). Several consensus elements were found, including two SORLIP1AT (GCCAC; Hudson and Quail, 2003) and IBOX (GATAAG; Giuliano et al., 1988) motifs within a 600 bp promoter region. Expression of MoGGP was induced by light exposure, suggesting that these sequences are likely to function as light responsible cis-elements. We could not, however, find TATA or CAAT boxes upstream of the MoGGP gene, and the core promoter was a minimum promoter region capable of initiation basal transcription. By contrast, the PLACE program located two TATA boxes at 292 and 344 bp upstream of the Arabidopsis GGP TSS (data not shown), suggesting that different transcription factors are involved in GPP promoter activation in the two species. We tested the cis-element promoter activity of MoGGP with a transient expression assay using Arabidopsis protoplasts. Protoplasts were isolated from Arabidopsis leaves and transiently transfected with MoGGP-LUC reporter construct. Protoplasts transfected with MoGGP-LUC exhibited 2-fold higher luminescence intensities than mock-treated protoplasts (Figure 3C). Although TATA boxes do not appear to be present in the MoGGP promoter region determined in this study, an unknown MoGGP promoter element may be driving expression. Further investigation of the promoter regions upstream of MoGGP is necessary to address this question.
In this study, we wished to examine the biosynthesis pathways underlying the high levels of AsA found in Moringa leaves. We identified six novel Moringa genes putatively encoding AsA biosynthesis enzymes in the Smirnoff-Wheeler pathway, and suggest that these genes play a major role in AsA biosynthesis during leaf development and under light conditions. Unlike acerola, in which several enzymes are important for AsA synthesis, MoGGP seems to be the major important enzyme regulating AsA content in Moringa. No TATA boxes were found in the 5'-region upstream of MoGGP. We found that the putative cis-elements may be involved in the light response, but we were unable to provide direct evidence of cis-element(s) enhanced gene expression in this study. Further investigation of more distal 5'-regions is necessary to determine the elements through which MoGGP expression is regulated, providing valuable information concerning the mechanisms underlying GGP transcription in Moringa.
AsA, Ascorbic acid; GalLDH, l-galactono-1,4-lactone dehydrogenase; GDH, l-galactose dehydrogenase; GGP, GDP-l-galactose phosphorylase; GME, GDP-d-mannose-3',5'-epimerase; GMP, GDP-d-mannose pyrophosphorylase; GPP, l-galactose-1-phosphate phosphatase; RACE, rapid amplification of cDNA ends; RLU, relative light units; ROS, reactive oxygen species; TSS, transcription start site.
The author(s) have not declared any conflict of interest.
This research was supported by the MEXT project “Practical Research on Sustainable Development in Central American and Caribbean Countries”. The sequence analysis was carried out at the Analysis Center of Life Science, Natural Science Center for Basic Research and Development, Hiroshima University.
SUPPLEMENTARY FIGURES AND TABLES
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