Plant Physiology and Biochemistry 89 (2015) 100e106
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Research article
Enhanced production of steviol glycosides in mycorrhizal plants: A concerted effect of arbuscular mycorrhizal symbiosis on transcription of biosynthetic genes Shantanu Mandal, Shivangi Upadhyay, Ved Pal Singh, Rupam Kapoor* Department of Botany, University of Delhi, Delhi 110007, India
a r t i c l e i n f o
a b s t r a c t
Article history: Received 23 December 2014 Accepted 18 February 2015 Available online 19 February 2015
Stevia rebaudiana (Bertoni) produces steviol glycosides (SGs) e stevioside (stev) and rebaudioside-A (reb-A) that are valued as low calorie sweeteners. Inoculation with arbuscular mycorrhizal fungi (AMF) augments SGs production, though the effect of this interaction on SGs biosynthesis has not been studied at molecular level. In this study transcription profiles of eleven key genes grouped under three stages of the SGs biosynthesis pathway were compared. The transcript analysis showed upregulation of genes encoding 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway enzymes viz.,1-deoxy-D-xylulose 5-phospate synthase (DXS), 1-deoxy-D-xylulose 5-phospate reductoisomerase (DXR) and 2-C-methyl-Derytrithol 2,4-cyclodiphosphate synthase (MDS) in mycorrhizal (M) plants. Zn and Mn are imperative for the expression of MDS and their enhanced uptake in M plants could be responsible for the increased transcription of MDS. Furthermore, in the second stage of SGs biosynthesis pathway, mycorrhization enhanced the transcription of copalyl diphosphate synthase (CPPS) and kaurenoic acid hydroxylase (KAH). Their expression is decisive for SGs biosynthesis as CPPS regulates flow of metabolites towards synthesis of kaurenoid precursors and KAH directs these towards steviol synthesis instead of gibberellins. In the third stage glucosylation of steviol to reb-A by four specific uridine diphosphate (UDP)-dependent glycosyltransferases (UGTs) occurs. While higher transcription of all the three characterized UGTs in M plants explains augmented production of SGs; higher transcript levels of UGT76G1, specifically improved reb-A to stev ratio implying increased sweetness. The work signifies that AM symbiosis upregulates the transcription of all eleven SGs biosynthesis genes as a result of improved nutrition and enhanced sugar concentration due to increased photosynthesis in M plants. © 2015 Published by Elsevier Masson SAS.
Keywords: Arbuscular mycorrhizal fungi Stevia rebaudiana Steviol glycosides biosynthesis genes Gene transcription Methyl erythritol phosphate pathway Glucosylation
1. Introduction Stevia rebaudiana (Bertoni) finds an unparalleled use in the medicinal world due to singular properties of its diterpenoid steviol glycosides (SGs) d stevioside (stev) and rebaudioside-A (reb-A) that are nearly 300 times sweeter than sucrose (Lemus-Mondaca et al., 2011). These have been used for the treatment of diabetes, dental maladies, obesity, hypertension and cancer (Geuns, 2003) hence, many efforts have been propounded to increase the concentration of SGs. While certain physiological parameters such as photoperiodism are known to affect the concentration of SGs (Ceunen et al., 2012; Ceunen and Geuns, 2013; Yang et al., 2015) on
* Corresponding author. E-mail address: [email protected] (R. Kapoor). http://dx.doi.org/10.1016/j.plaphy.2015.02.010 0981-9428/© 2015 Published by Elsevier Masson SAS.
the other hand, mutualistic associations such as arbuscular mycorrhizal (AM) symbiosis offer a potent, economically conducive approach to encounter the growing demand of SGs (Mandal et al., 2013). Despite of several studies connoting the potential of AMF to augment the secondary metabolite production in commercially important medicinal plants (Kapoor et al., 2002a,b, 2004, 2007; Copetta et al., 2006); their role in manipulating the biosynthesis pathways of these metabolites have not been dissected at molecular level. In a first of such attempts, it was recently reported that AM symbiosis upregulates the expression of crucial artemisinin biosynthesis genes (Mandal et al., 2014). However, more studies need to be done to optimize the mechanism based on which AM symbiosis functions to increase the secondary metabolite production. Biosynthesis of SGs takes place in leaves (Brandle et al., 1998) in a seventeen steps process that can be categorized under three
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stages. SGs biosynthesis commences with the conversion of pyruvate and glyceraldehyde-3-phosphate to isopentyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) in eight sequential reactions forming the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway (Wanke et al., 2001; McGarvey and Croteau, 1995). 1deoxy-D-xylulose 5-phospate synthase (DXS) and 1-deoxy-D-xylulose 5-phospate reductoisomerase (DXR) play a central role in regulation of MEP pathway (Cordoba et al., 2009). These enzymes catalyse the condensation of pyruvate and glyceraldehyde-3phosphate into MEP (Kuzuyama et al., 2000; Takahasi et al., 1998). 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (MDS) is an important control point in SGs biosynthesis (Kumar et al., 2012b) as it could bind to intermediates of the pathway and control the metabolite flux by feedback regulation (Kemp et al., 2005). The second stage of the SGs biosynthesis pathway begins with a condensation reaction catalysed by important branch point enzyme geranylgeranyl diphosphate synthase (GGDPS) to form geranylgeranyl diphosphate (GGDP) which is a common precursor for the biosynthesis of several diterpenoids (Okada et al., 2007). GGDP is converted to steviol by consecutive action of 4 enzymes viz., copalyl diphosphate synthase (CPPS), kaurene synthase (KS), kaurene oxidase (KO) and kaurenoic acid hydroxylase (KAH) (Brandle and Telmer, 2007). Biosynthesis pathways of SGs and gibberellins share the same kaurenoid precursor and GGDPS, CPPS, KS and KO are all involved in the biosynthesis of both gibberellins and SGs (Kumar et al., 2012a; Richman et al., 2005). Since, CPPS is crucial for the conversion of GGDP to copalyl diphosphate (CDP) it is therefore, believed to be a control point for the flow of metabolites (Silverstone et al., 1997). Kaurene is produced from CDP in an ionization dependent cyclization by KS (Brandle and Telmer, 2007). It is important for the synthesis of SGs since KS is present in duplicates only in S. rebaudiana (Richman et al., 1999). KO catalyzes oxidation of kaurene to form kaurenoic acid (Humphrey et al., 2006). The divergence of steviol biosynthesis from gibberellin biosynthesis occurs with the hydroxylation of kaurenoic acid into steviol catalysed by KAH (Hanson and white, 1968; Kim et al., 1996). This is the first specific step towards SGs biosynthesis (Brandle and Telmer, 2007). In the third stage glucosylation of steviol to reb-A by four specific uridine diphosphate (UDP)-dependent glycosyltransferases (UGTs) occurs. However, in S. rebaudiana only three have been identified so far, namely, UGT85C2, UGT74G1 and UGT76G1 (Brandle and Telmer, 2007). These UGTs transfer sugar molecules from activated donor to an acceptor molecule and thus sugar concentration influences the glucosylation of ent-kaurene that form the SGs (Richman et al., 2005). Mohamed et al. (2011) suggested that glucosylation of steviol by UGT85C2 is a rate limiting step. Both UGT74G1 and UGT76G1 are necessary for the synthesis of stev and reb-A, respectively (Humphrey et al., 2006). With the knowledge of above, effect of AMF inoculation on transcript profiles of eleven enzymes crucial for SGs biosynthesis was studied. The present work is complementary to the previous study where it was established that AM symbiosis augments the production of SGs through nutritional and non-nutritional mechanisms (Mandal et al., 2013). 2. Material and methods 2.1. Experimental design Stem cuttings of S. rebaudiana var. Cimmeethi, a high yielding variety, were procured from Central Institute of Medicinal and Aromatic Plants (CIMAP), Pantnagar, India. Root induction in stem cuttings was achieved by hormone treatment in autoclaved sterile
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soil. Plants of uniform size were transplanted to pots (140 X 1300 ) with 13 kg of autoclaved soil in each. Before transplantation the soil was autoclaved twice for 1 h at 121 C and 15 psi on alternate days to kill resilient spores, if any. The soil used was a sandy loam soil (sand 14.7%, silt 35.5% and clay 22.8%) with the following chemical characteristics: pH (H2O) 7.6, EC 0.12 Sm1 at 32 C; organic C 1.12%; total N 0.49%; and available P, K, Na, Mg, Ca, Zn, and Cu were 11.1, 55, 61.3, 45, 150, 55, and 22 mg kg1, respectively. The experiment comprised of two treatments viz., plants without mycorrhizal inoculation represented non-mycorrhizal (NM) control plants, and plants inoculated with AMF Rhizophagus intraradices (N.C. Schenck& G.S. Sm.) C. Walker & A. Schüßler represented the mycorrhizal (M) plants. Each treatment consisted of two plants per pot replicated ten times. The experiment consisted of 20 pots arranged in a completely randomized block design. Plants were grown in the months of February to July under natural conditions of growth at Delhi India. The mean temperature at the time of experiment ranged between 25 C and 40 C; and relative humidity between 50% and 70%. Soil moisture was maintained by watering the plants twice every week with normal tap water. Estimation of all the parameters was carried out at full maturity i.e. 120 days post inoculation when the concentration of SGs is reported to be maximum (Brandle et al., 1998). 2.2. Inoculation and colonization by AMF The starter inoculum of R. intraradices (accession number CMCCWep319) was procured from The Energy and Resources Institute (TERI, New Delhi, India). Details of mass cultivation procedure have been published previously (Kapoor et al., 2007). Approximately 10 g of AMF inoculum containing soil and chopped roots was applied just below the roots of M plants at the time of transplantation. Roots of S. rebaudiana were cleared in KOH and stained with trypan blue (Phillips and Hayman, 1970) to confirm mycorrhization. Percent root colonization (87%) was estimated microscopically following Biermann and Linderman (1981). 2.3. Mineral nutrient analysis Analysis of mineral nutrients (P, Mg, K, Cu, Zn, Mn, and Fe) was carried out using oven dried leaf samples. Dried leaves were ground and approximately 0.2 g of each sample was digested in tri-acid mixture. The acid digest was allowed to cool for some time and diluted to 50 ml using double distilled water. A reagent blank was prepared by following the whole extraction procedure without a sample. Phosphorus concentration from the digested samples was determined following molybdate blue method (Allen, 1989) at 700 nm using a UVevis spectrophotometer (Beckman Coulter DU®730). The concentrations of Mg, K, Cu, Zn, Mn, and Fe in the samples were measured following Allen (1989) using atomic absorption spectrophotometer (Shimadzu AA-130). 2.4. Estimation of net photosynthetic rate and total sugars Measurement of net photosynthetic rate (PN) was performed on sunny days at 11:00 a.m. with fully expanded leaves using IRGA (Infra Red Gas Analyzer, LI-COR 6400 Portable Photosynthesis System, Lincoln, Nebraska, USA). IRGA was calibrated prior to carrying out measurements and also approximately after every 30 min during the measurement period. Each leaf was enclosed in the gas exchange chamber for 60 s. PN measured by IRGA was recorded six times for each treatment. Concentration of total sugars in the fresh leaves of S. rebaudiana was determined following the protocol described by Yemm and
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Willis (1954), using a UVevis spectrophotometer with wavelength set at 630 nm. Standard curve of glucose prepared at the same wavelength was used for calculating the concentration of sugars in samples.
Package for Social Sciences version 14 (SPSS, USA) was used to carry out the statistical analysis.
2.5. Extraction and analysis of steviol glycosides
The present work puts forth AM symbiosis as a certain approach to enhance SGs concentration in S. rebaudiana by evidencing that AMF upregulates the transcription of key SGs biosynthesis genes. This increase in concentration of SGs in response to AMF colonization is in accordance with the previous study (Mandal et al., 2013). However, the heightened response observed in this study can be explained by different combination of symbionts viz., Cimmeethi, a high yielding variety of S. rebaudiana and AMF R. intraradices.
Kumar et al. (2012a) reported that SGs is maximum in 3rd node leaf starting from apical meristem. Therefore, extraction of SGs was carried out from dried 3rd node leaves following the protocol of Kolb et al. (2001). Further HPLC analysis and validation were carried out following the methods described by Mandal et al. (2013). 2.6. Quantitative analysis of gene transcription RNA was extracted from 100 mg of fresh leaves (harvested from 3rd node) using RNAeasy Plant mini kit (Qiagen, USA) following manufacturer's instructions. RNA Quantification was done using NanoDrop TM 8000 Spectrophotometer (Thermo Scientific, USA). Genomic DNA contamination was removed by treating the RNA with DNase I (Fermentas, St Leo-Roth, Germany). cDNA was synthesized from 2 mg of purified RNA using cDNA reverse transcription kit (Ambion, USA). Amplification of cDNA was checked using 26S rRNA primer pair through endpoint PCR reaction. The synthesized cDNA was used for quantitative real time polymerase chain reaction (qRT-PCR) using gene specific primer pairs (Table 1) designed with the help of primer3 software (http://biotools. umassmed.edu/bioapps/primer3_www.cgi) and synthesized by Sigma, India. The PCR was carried out in Mx 5000P qPCR equipment (Stratagene, India). In a 25 mL reaction mixture, 1 mL of template (synthesized cDNA), 12.5 ml Power SYBR Green PCR master mix (Roche), 1 mL of 0.3 mmol of each gene specific primer pair and 9.5 mL of nuclease free water were added. Thermal cycles of qRT-PCR included one cycle at 95 C for 10 min, followed by 40 cycles at 95 C (15 s), 55 C (1 min), and 72 C (30 s). The analysis was performed in three biological repeats. Each repeat consisted of six leaves pooled from three individual plants. Relative fold change in gene transcription was analyzed by DDCT method using MxPro analysis software (Agilent technologies, USA). 2.7. Statistical analysis The data for nutrients analysis, estimation of sugar concentration and net photosynthetic rate is represented as mean of six replicates while data for SGs concentration and transcription analysis is represented as mean of three biological replicates. Numerical data obtained from the experiments was analyzed using Student's-test. An asterisk (*) in the tables and figures indicates significant difference between the means at p < 0.05. Statistical
3. Result and discussion
3.1. Mineral nutrient uptake AMF improves plant's nutritional status largely due to its extensive hyphal network that spans greater surface area and aids plant roots to absorb mineral nutrients that have low availability and mobility (Parniske, 2008). Concentration of P, K, Mg, Cu, Fe, Mn and Zn were recorded to be 215.7%, 6.95%, 42.47%, 298%, 87.22%, 203.09% and 105.12% respectively, higher in M plants (Table 2). Roles of mineral nutrients that hold significance to the study have been dealt subsequently. NM plants served as control in all the experiments as they showed no AMF colonization. 3.2. Net photosynthetic rate and total sugars An increase of 1.83 folds in PN was observed in M plants in comparison with NM plants (Table 3). Augmented nutritional status of the plant can be accredited for this, as mineral nutrients like Mg, Fe and Cu have crucial structural and functional roles to play in the process of photosynthesis (Taiz and Zeiger, 1998). An increase by 1.62 folds in total sugars was observed in M plants in comparison with NM plants (Table 3). There are many studies in congruence with these findings (Smith et al., 2010; Wu et al., 2011; Zubek et al., 2012; Mandal et al., 2013). To sustain its functionality AMF generates a strong sink for photosynthetically fixed carbon from the plant (Smith and Read, 2008) however, this increased carbon demand is compensated by increase in photosynthesis and carbon assimilation (Wright et al., 1998). 3.3. Concentration of steviol glycosides The concentration of stev and reb-A increased 1.68 and 3.4 folds respectively, upon mycorrhization (Table 4). Owing to increased photosynthesis in M plants, there was a consequent increase in carbon pool available for secondary metabolite production. Also,
Table 1 List of gene specific primers used in qRT-PCR analysis. Name
Left primer
Right primer
26S rRNA DXS (1-deoxy-D-xylulose 5-phospate synthase) DXR (1-deoxy-D-xylulose 5-phospate reductoisomerase) MDS (2-C-methyl-D-erytrithol 2,4-cyclodiphosphate synthase) GGDPS (geranylgeranyl diphosphate Synthase) CPPS (copalyl diphosphate synthase) KS (kaurene synthase) KO (kaurene oxidase) KAH: kaurenoic acid hydroxylase UGT85C2 UGT74G1 UGT76G1
50 -CACAATGATAGGAAGAGCCGAC-30 50 GCTTCAAGCCCATAACGTGT-30 50 -TGGTCCGTTTGTTCTTCCTC-30 50 -GCCTGGATACCCTCTCATCA-30 50 -CGTTTCCTCGTCTTCTTTGC-30 50 -ACAGCTGGTCGTTGGGTATC-30 50 -CTCCGGTGGACAGTTTCATT-30 50 -TCTTCACAGTCTCGGTGGTG-30 50 -CCTATAGAGAGGCCCTTGTGG-30 50 -GTTTCTCACAGTCCGGAAGC-30 50 -GGTAGCCTGGTGAAACATGG-30 50 -GACGCGAACTGGAACTGTTG-30
50 -CAAGGGAACGGGCTTGGCAGAATC-30 50 -CAATCGAGCCTTCCTCTACG 50 -CAAAGCACCTTCAGGAAAG-30 C 50 -ATTTGCCCGATGTCAGGTAG-30 50 -GGTGTTTCGAGATGGGTTTG-30 50 -CGAGTCTACCGTCGTGGAAT-30 50 -CCACCATGTGAAGTTGATGC-30 50 -GGTGGTGTCGGTTTATCCTG-30 50 -TAGCCTCGTCCCTTTGTGTC-30 50 -AACCGACAAGAACCCATCTG-30 50 -CTGGGAGCTTTCCCTCTTCT-30 50 -AGCCGTCGGAGGTTAAGACT-30
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Table 2 Effect of R. intraradices inoculation on concentration of nutrients in shoots of S. rebaudiana. Treatments
Non mycorrhizal Mycorrhizal
Nutrient (mg g1dry weight) P
K
Mg
Cu
Fe
Mn
Zn
260 ± 96 821 ± 398*
1380 ± 198 1476 ± 140*
1928 ± 224 2747 ± 434*
187 ± 56 746 ± 179*
1268 ± 695 2374 ± 880*
55 ± 27 166.7 ± 68*
54.54 ± 15 110.77 ± 0.81*
Means within a column followed by the asterisk (*) are significantly different at p < 0.05 between non mycorrhizal and mycorrhizal treatments. The data shown are mean of six replicates ± SD determined by unpaired equal student's t-test.
improved P nutrition due to AM symbiosis (Table 2) can be accredited as P is crucial for the synthesis of both primary and secondary metabolites (Marschner, 2002) and it is discernible that P is an important constituent of various intermediates of MEP pathway such as deoxy-D-xylulose 5-phosphate (DXP) and 2-CMethyl-D-erythritol 2,4-cyclodiphosphate (cMEPP). As a consequence of glucosylation pattern and sugar moiety, the organoleptic properties of all the SGs are different (DuBois and Stepehenson, 1985). Stev is 143 times sweeter than sucrose whereas, reb-A is 242 times sweeter (Kasai et al., 1981). Reb-A has a better taste quality than stev, as it is sweeter (DuBois and Stepehenson, 1985). Thus ratio of reb-A to stev governs the sweetening property of S. rebaudiana (Brandle et al., 1998). In the present study ratio increased from 0.29 in NM plants to 0.60 in M plants, suggesting that sweetening properties of S. rebaudiana enhanced as a result of AM symbiosis. 3.4. Transcription studies of steviol biosynthetic genes To enable a better understanding, transcription profile of genes under three stages of the SGs biosynthesis pathway were studied viz., MEP pathway as the first stage, that results in a pool of IPP and DMAP, which serve as precursors to a number of terpenoids; second stage comprises of five sequential reactions catalyzing the synthesis of steviol; and the third stage, often referred to as glucosylation pathway that is committed to SGs biosynthesis. 3.4.1. First stage/MEP pathway Upon mycorrhization the transcript levels of DXS, DXR increased to 1.7 and 1.8 folds respectively, in comparison with the NM plants (Fig. 1A and B). Induction of MEP pathway due to upregulation of DXS and DXR has been particularly studied at root level where the plant and fungus interact mainly (Walter et al., 2000; Hans et al., 2004; Strack and Fester, 2006; Flob et al., 2008). However, it is known that AM symbiosis greatly influences the physiology and metabolism of aerial parts of the plant as well (Toussaint, 2007) and yet there have been few studies that explore the molecular basis of interaction between mycorrhiza and MEP pathway genes at the shoot level (Mandal et al., 2014). Hsieh and Goodman (2005) suggested that increased sugar concentration augments the expression of several genes of MEP pathway. Also, Cordoba et al. (2009) reported that increased carbohydrates alter the expression profile of DXS and DXR; and so probably enhanced sugars reported in the
Table 3 Effect of R. intraradices inoculation on net photosynthetic rate and concentration of total carbohydrates in shoots of S. rebaudiana. Treatments
Net photosynthetic rate (mmol CO2 m2 s1)
Total carbohydrates (% fresh weight)
Non mycorrhizal Mycorrhizal
10.08 ± 1.12 18.53 ± 1.63*
18.43 ± 3.34 30.03 ± 3.68*
Means within a column followed by the asterisk (*) are significantly different at p < 0.05 between non mycorrhizal and mycorrhizal treatments. The data shown are mean of six replicates ± SD determined by unpaired equal student's t-test.
present study might have influenced the transcription of these genes. The upregulation of DXS and DXR propelled by AMF perhaps has a cascading effect on downstream genes of the pathway. AMF affects the biosynthesis of several terpenoids (Asensio et al., 2012) and upregulation of the key MEP pathway genes establishes an elementary mechanism to enhance the pool of IPP and DMAP from which a broad range of terpenoids and terpenoid-derived compounds are produced through several different pathways. Transcript level of MDS also increased to 1.84 folds in M treatments in comparison with NM treatments (Fig. 1C). The expression of MDS may be regulated by Zn and Mn concentrations as the homo-trimeric MDS protein requires Zn2þ and Mn2þ for its activity (Richard et al., 2002; Steinbacher et al., 2002). Incidentally, an increase of 105.12% and 203.09% in Zn and Mn level respectively, was observed in M plants. Therefore, it can be conjectured that increased concentrations of Zn and Mn as a result of the nutritional benefits of AM symbiosis, might have resulted in upregulation of MDS. 3.4.2. Second stage The transcript levels of all the five second stage genes of the SGs biosynthesis pathway viz., GGDPS, CPPS, KS, KO and KAH increased upon mycorrhization. The transcript levels of GGDPS, CPPS and KO increased to 1.42, 1.48 and 6.05 folds respectively, in M plants in comparison with NM plants (Fig. 1D, E, and G), suggesting a similar trend in their upregulation. However, the comparative transcription profile of KS suggested an increase of 11.38 folds in M plants (Fig. 1F). Given that KS is present in duplicates in S. rebaudiana (Richman et al., 1999) provides a plausible explanation for an increase of such magnitude. Transcript level of KAH increased to1.35 folds in M plants (Fig. 1H). KAH represents a branch point from the gibberellins biosynthesis considering its capacity to direct the flow of metabolites specifically towards the biosynthesis of SGs (Humphrey et al., 2006). Thus, increase in its transcription implies enhanced steviol production in M plants. 3.4.3. Third stage/glucosylation pathway The metabolic channelling between KAH and UGT85C2, and among all the UGTs is specific to SGs biosynthesis (Guleria et al., 2014) implying that upregulation of these ensures higher concentration of SGs. The expression of UGT85C2 has been considered critical for the accumulation of SGs as it catalyses a rate limiting
Table 4 Effect of R. intraradices inoculation on concentration of rebaudioside A, stevioside and the ratio of rebaudioside A/stevioside in shoots of S. rebaudiana. Treatments
Rebaudioside-A concentration (% dry wt.)
Stevioside concentration (% dry wt.)
Rebaudioside A/stevioside
Non mycorrhizal Mycorrhizal
2.36 ± 0.13 8.14 ± 0.1*
8.08 ± 0.18 13.51 ± 0.25*
0.29 ± 0.01 0.60 ± 0.02*
Means within a column followed by the asterisk (*) are significantly different at p < 0.05 between non mycorrhizal and mycorrhizal treatments. The data shown are mean of three replicates ± SD determined by unpaired equal student's t-test.
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Fig. 1. Effect of R. intraradices inoculation on the transcript level of A) 1-deoxy-D-xylulose 5-phospate synthase (DXS), B) 1-deoxy-D-xylulose 5-phospate reductoisomerase (DXR), C) 2-Cmethyl-D-erytrithol 2,4-cyclodiphosphate synthase (MDS), D) geranylgeranyl diphosphate synthase (GGDPS), E) Copalyl diphosphate synthase (CPPS), F) kaurene synthase (KS), G) kaurene oxidase (KO), H) kaurenoic acid hydroxylase (KAH), and uridine diphosphate (UDP)-dependent glycosyltransferases (UGTs) I) UGT85C2, J) UGT74G1 and K) UGT76G1 in leaves of S. rebaudiana. Data is presented as mean ± SD (n ¼ 3). An asterisk (*) on the bar indicates significant differences between the means at p < 0.05 NM, non mycorrhizal; M, R. intraradices inoculation.
reaction in glucosylation pathway (Mohamed et al., 2011). The transcript level of UGT85C2 was 7.42 folds higher in M plants (Fig. 1I). The majority of SGs in leaves of S. rebaudiana are tri-glycoside stev and tetra-glycoside reb-A (Kinghorn and Soejarto, 1985). Transcription of UGT74G1 and UGT76G1 increased to 7.37 and 4.29 folds respectively, in M plants (Fig. 1J and K). Upregulation of UGT74G1 led to augmented concentration of stev and that of UGT76G1 led to higher concentration of reb-A in M plants (Table 4). Conversion of stev to reb-A is controlled by UGT76G1 (Richman et al., 2005) and its higher transcription ascertains increased ratio
of reb-A to stev in M plants. 4. Conclusion The present work signifies that AM symbiosis upregulates the transcription of important SGs biosynthesis genes at all stages of the pathway d a consequence of systemic effect of augmented sugar concentration due to increased photosynthesis and improved nutrition. The enhanced transcription of upstream genes of MEP pathway results in a greater pool of IPP and DMAP from which
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various terpenoids are produced through disparate pathways. However, AMF mediated enhanced transcription of downstream genes viz., KAH and UGTs insinuate a rather specific mechanism to enhance SGs production. The present work is first report on regulation of SGs biosynthesis genes in response to AMF. Authors' contribution S. Mandal and S. Upadhyay performed the experiments, analysed the data and wrote the manuscript; V.P. Singh edited the manuscript and helped in data analysis; R. Kapoor designed the experiments, helped in data analyses, and wrote the manuscript. Acknowledgements The authors wish to acknowledge the Council of Scientific and Industrial Research (CSIR) for the award of Research Fellowships. We are also thankful to the DR. J. P. Singh CIMAP Uttarakhand for providing the stem cuttings of S. rebaudiana. References Allen, S.E., 1989. Chemical Analysis of Ecological Materials, second ed. Blackwell Scientific Publishers, London. ~ uelas, J., 2012. AM fungi root colonization increases the Asensio, D., Rapparini, F., Pen production of essential isoprenoids vs. nonessential isoprenoids especially under drought stress conditions or after jasmonic acid application. Phytochemistry 77, 149e161. Biermann, B.J., Linderman, R.G., 1981. Quantifying vesicular arbuscular mycorrhizae: a proposed method towards standardization. New. Phytol. 89, 63e67. Brandle, J.E., Starratt, A.N., Gijzen, M., 1998. Stevia rebaudiana: its agricultural, biological, and chemical properties. Can. J. Plant Sci. 78, 527e536. Brandle, J.E., Telmer, P.J., 2007. Steviol glycoside biosynthesis. Phytochemistry 68, 1855e1863. Ceunen, S., Werbrouck, S., Geuns, J.M.C., 2012. Stimulation of steviol glycoside accumulation in Stevia rebaudiana by red LED light. J. Plant Physiol. 169, 749e752. Ceunen, S., Geuns, J.M.C., 2013. Influence of photoperiodism on the spatio-temporal accumulation of steviol glycosides in Stevia rebaudiana (Bertoni). Plant Sci. 198, 72e82. Copetta, A., Lingua, G., Berta, G., 2006. Effects of three AM fungi on growth, distribution of glandular hairs, and essential oil production in Ocimum basilicum L var. Genovese. Mycorrhiza 16, 485e494. Cordoba, E., Salmi, M., Leon, P., 2009. Unravelling the regulatory mechanisms that modulate the MEP pathway in higher plants. J. Exp. Bot. 60, 2933e2943. DuBois, G.E., Stephenson, R.A., 1985. Diterpenoid sweeteners. Synthesis and sensory evaluation of stevioside analogues with improved organoleptic properties. J. Med. Chem. 28, 93e98. Floß, D.S., Hause, B., Lange, P.R., Kuster, H., Strack, D., Walter, M.H., 2008. Knockdown of the MEP pathway isogene 1-deoxy-d-xylulose 5-phosphate synthase 2 inhibits formation of arbuscular mycorrhiza-induced apocarotenoids, and abolishes normal expression of mycorrhiza-specific plant marker genes. Plant J. 56, 86e100. Geuns, J.M.C., 2003. Stevioside. Phytochemistry 64, 913e921. Guleria, P., Masand, S., Yadav, S.K., 2014. Overexpression of SrUGT85C2 from Stevia reduced growth and yield of transgenic Arabidopsis by influencing plastidal MEP pathway. Gene 539, 250e257. Hanson, J.R., White, A.F., 1968. Studies in terpenoid biosynthesis - II. Biosynth. Steviol. Phytochem. 7, 595e597. Hans, J., Hause, B., Fester, T., Strack, D., Walter, M.H., 2004. Cloning, characterization and immunolocalization of a mycorrhizal-inducible 1-deoxy-D-xylulose 5phosphate isomerase in arbuscule-containing cells of maize. Plant Physiol. 134, 614e624. Hsieh, M.H., Goodman, H.M., 2005. The Arabidopsis IspH homolog is involved in the plastid Nonmevalonate pathway of isoprenoids biosynthesis. Plant Physiol. 138, 641e653. Humphrey, T.V., Richman, A.S., Menassa, R., Brandle, J.E., 2006. Spatial organisation of Four enzymes from Stevia rebaudiana that are involved in steviol glycoside synthesis. Plant Mol. Biol. 61, 47e62. Kapoor, R., Giri, B., Mukerji, K.G., 2002a. Glomus macrocarpum: a potential bioinoculant improves essential oil quality and concentration in Dill (Anethum graveolens L.) and Carum (Trachyspermum ammi Linn.) Sprague. World J. Microb. Biot. 18, 459e463. Kapoor, R., Giri, B., Mukerji, K.G., 2002b. Mycorrhization of coriander (Coriandrum sativumL) to enhance the concentration and quality of essential oil. J. Sci. Food Agric. 82, 339e342. Kapoor, R., Giri, B., Mukerji, K.G., 2004. Improved growth and essential oil yield and quality in Foeniculum vulgaremill on mycorrhizal inoculation supplemented
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Current status of stevioside as a sweetening agent forhuman use. In: Wagner, H., Hikino, H., Farnsworth, N.R. (Eds.), Economic and Medical Plant Research. Academic Press, London, UK. Kim, K.K., Sawa, Y., Shibata, H., 1996. Hydroxylation of (-)- kaurenoic acid to steviol in Stevia rebaudiana Bertoni - purification and partial characterization of the enzyme. Arch. Biochem. Biophys. 332, 223e230. Kolb, N., Herrera, J.L., Ferreyra, D.J., Uliana, R.F., 2001. Analysis of sweet diterpene glycosides from Stevia rebaudiana: improved HPLC method. J. Agric. Food Chem. 49, 4538e4541. Kumar, H., Kaul, K., Gupta-Bajpai, S., Kaul, V.K., Kumar, S., 2012a. A comprehensive analysis of fifteen genes of steviol glycosides biosynthesis pathway in Stevia rebaudiana (Bertoni). Gene 492, 276e284. Kumar, H., Singh, K., Sanjay Kumar, S., 2012b. 2C-methyl-D-erythritol 2,4cyclodiphosphate synthase from Stevia rebaudiana Bertoni is a functional gene. Mol. Biol. Rep. 39, 10971e10978. Kuzuyama, T., Takagi, M., Takahashi, S., Seto, H., 2000. Cloning and characterization of 1-deoxy-D-xylulose 5-phosphate synthase from Streptomyces sp strain CL190, which uses both the mevalonate and nonmevalonate pathways for isopentenyl diphosphate biosynthesis. J. Bacteriol. 182, 891e897. lvez, A., Zura-Bravo, L., Ah-Hen, K., 2011. Stevia Lemus-Mondaca, R., Vega-Ga rebaudiana Bertoni, source of a high-potency natural sweetener: a comprehensive review on the biochemical, nutritional and functional aspects. Food Chem. 132, 1121e1132. Mandal, S., Evelin, H., Giri, B., Singh, V.P., Kapoor, R., 2013. Arbuscular mycorrhiza enhances the production of stevioside and rebaudioside-A in Stevia rebaudiana via nutritional and non-nutritional mechanisms. Appl. Soil Ecol. l72, 187e194. Mandal, S., Upadhyay, S., Wajid, S., Ram, M., Jain, D.C., Singh, V.P., Abdin, M.Z., Kapoor, R., 2014. 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Plant Physiology and Biochemistry journal homepage: www.elsevier.com/locate/plaphy
Research article
Enhanced production of steviol glycosides in mycorrhizal plants: A concerted effect of arbuscular mycorrhizal symbiosis on transcription of biosynthetic genes Shantanu Mandal, Shivangi Upadhyay, Ved Pal Singh, Rupam Kapoor* Department of Botany, University of Delhi, Delhi 110007, India
a r t i c l e i n f o
a b s t r a c t
Article history: Received 23 December 2014 Accepted 18 February 2015 Available online 19 February 2015
Stevia rebaudiana (Bertoni) produces steviol glycosides (SGs) e stevioside (stev) and rebaudioside-A (reb-A) that are valued as low calorie sweeteners. Inoculation with arbuscular mycorrhizal fungi (AMF) augments SGs production, though the effect of this interaction on SGs biosynthesis has not been studied at molecular level. In this study transcription profiles of eleven key genes grouped under three stages of the SGs biosynthesis pathway were compared. The transcript analysis showed upregulation of genes encoding 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway enzymes viz.,1-deoxy-D-xylulose 5-phospate synthase (DXS), 1-deoxy-D-xylulose 5-phospate reductoisomerase (DXR) and 2-C-methyl-Derytrithol 2,4-cyclodiphosphate synthase (MDS) in mycorrhizal (M) plants. Zn and Mn are imperative for the expression of MDS and their enhanced uptake in M plants could be responsible for the increased transcription of MDS. Furthermore, in the second stage of SGs biosynthesis pathway, mycorrhization enhanced the transcription of copalyl diphosphate synthase (CPPS) and kaurenoic acid hydroxylase (KAH). Their expression is decisive for SGs biosynthesis as CPPS regulates flow of metabolites towards synthesis of kaurenoid precursors and KAH directs these towards steviol synthesis instead of gibberellins. In the third stage glucosylation of steviol to reb-A by four specific uridine diphosphate (UDP)-dependent glycosyltransferases (UGTs) occurs. While higher transcription of all the three characterized UGTs in M plants explains augmented production of SGs; higher transcript levels of UGT76G1, specifically improved reb-A to stev ratio implying increased sweetness. The work signifies that AM symbiosis upregulates the transcription of all eleven SGs biosynthesis genes as a result of improved nutrition and enhanced sugar concentration due to increased photosynthesis in M plants. © 2015 Published by Elsevier Masson SAS.
Keywords: Arbuscular mycorrhizal fungi Stevia rebaudiana Steviol glycosides biosynthesis genes Gene transcription Methyl erythritol phosphate pathway Glucosylation
1. Introduction Stevia rebaudiana (Bertoni) finds an unparalleled use in the medicinal world due to singular properties of its diterpenoid steviol glycosides (SGs) d stevioside (stev) and rebaudioside-A (reb-A) that are nearly 300 times sweeter than sucrose (Lemus-Mondaca et al., 2011). These have been used for the treatment of diabetes, dental maladies, obesity, hypertension and cancer (Geuns, 2003) hence, many efforts have been propounded to increase the concentration of SGs. While certain physiological parameters such as photoperiodism are known to affect the concentration of SGs (Ceunen et al., 2012; Ceunen and Geuns, 2013; Yang et al., 2015) on
* Corresponding author. E-mail address: [email protected] (R. Kapoor). http://dx.doi.org/10.1016/j.plaphy.2015.02.010 0981-9428/© 2015 Published by Elsevier Masson SAS.
the other hand, mutualistic associations such as arbuscular mycorrhizal (AM) symbiosis offer a potent, economically conducive approach to encounter the growing demand of SGs (Mandal et al., 2013). Despite of several studies connoting the potential of AMF to augment the secondary metabolite production in commercially important medicinal plants (Kapoor et al., 2002a,b, 2004, 2007; Copetta et al., 2006); their role in manipulating the biosynthesis pathways of these metabolites have not been dissected at molecular level. In a first of such attempts, it was recently reported that AM symbiosis upregulates the expression of crucial artemisinin biosynthesis genes (Mandal et al., 2014). However, more studies need to be done to optimize the mechanism based on which AM symbiosis functions to increase the secondary metabolite production. Biosynthesis of SGs takes place in leaves (Brandle et al., 1998) in a seventeen steps process that can be categorized under three
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stages. SGs biosynthesis commences with the conversion of pyruvate and glyceraldehyde-3-phosphate to isopentyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) in eight sequential reactions forming the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway (Wanke et al., 2001; McGarvey and Croteau, 1995). 1deoxy-D-xylulose 5-phospate synthase (DXS) and 1-deoxy-D-xylulose 5-phospate reductoisomerase (DXR) play a central role in regulation of MEP pathway (Cordoba et al., 2009). These enzymes catalyse the condensation of pyruvate and glyceraldehyde-3phosphate into MEP (Kuzuyama et al., 2000; Takahasi et al., 1998). 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (MDS) is an important control point in SGs biosynthesis (Kumar et al., 2012b) as it could bind to intermediates of the pathway and control the metabolite flux by feedback regulation (Kemp et al., 2005). The second stage of the SGs biosynthesis pathway begins with a condensation reaction catalysed by important branch point enzyme geranylgeranyl diphosphate synthase (GGDPS) to form geranylgeranyl diphosphate (GGDP) which is a common precursor for the biosynthesis of several diterpenoids (Okada et al., 2007). GGDP is converted to steviol by consecutive action of 4 enzymes viz., copalyl diphosphate synthase (CPPS), kaurene synthase (KS), kaurene oxidase (KO) and kaurenoic acid hydroxylase (KAH) (Brandle and Telmer, 2007). Biosynthesis pathways of SGs and gibberellins share the same kaurenoid precursor and GGDPS, CPPS, KS and KO are all involved in the biosynthesis of both gibberellins and SGs (Kumar et al., 2012a; Richman et al., 2005). Since, CPPS is crucial for the conversion of GGDP to copalyl diphosphate (CDP) it is therefore, believed to be a control point for the flow of metabolites (Silverstone et al., 1997). Kaurene is produced from CDP in an ionization dependent cyclization by KS (Brandle and Telmer, 2007). It is important for the synthesis of SGs since KS is present in duplicates only in S. rebaudiana (Richman et al., 1999). KO catalyzes oxidation of kaurene to form kaurenoic acid (Humphrey et al., 2006). The divergence of steviol biosynthesis from gibberellin biosynthesis occurs with the hydroxylation of kaurenoic acid into steviol catalysed by KAH (Hanson and white, 1968; Kim et al., 1996). This is the first specific step towards SGs biosynthesis (Brandle and Telmer, 2007). In the third stage glucosylation of steviol to reb-A by four specific uridine diphosphate (UDP)-dependent glycosyltransferases (UGTs) occurs. However, in S. rebaudiana only three have been identified so far, namely, UGT85C2, UGT74G1 and UGT76G1 (Brandle and Telmer, 2007). These UGTs transfer sugar molecules from activated donor to an acceptor molecule and thus sugar concentration influences the glucosylation of ent-kaurene that form the SGs (Richman et al., 2005). Mohamed et al. (2011) suggested that glucosylation of steviol by UGT85C2 is a rate limiting step. Both UGT74G1 and UGT76G1 are necessary for the synthesis of stev and reb-A, respectively (Humphrey et al., 2006). With the knowledge of above, effect of AMF inoculation on transcript profiles of eleven enzymes crucial for SGs biosynthesis was studied. The present work is complementary to the previous study where it was established that AM symbiosis augments the production of SGs through nutritional and non-nutritional mechanisms (Mandal et al., 2013). 2. Material and methods 2.1. Experimental design Stem cuttings of S. rebaudiana var. Cimmeethi, a high yielding variety, were procured from Central Institute of Medicinal and Aromatic Plants (CIMAP), Pantnagar, India. Root induction in stem cuttings was achieved by hormone treatment in autoclaved sterile
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soil. Plants of uniform size were transplanted to pots (140 X 1300 ) with 13 kg of autoclaved soil in each. Before transplantation the soil was autoclaved twice for 1 h at 121 C and 15 psi on alternate days to kill resilient spores, if any. The soil used was a sandy loam soil (sand 14.7%, silt 35.5% and clay 22.8%) with the following chemical characteristics: pH (H2O) 7.6, EC 0.12 Sm1 at 32 C; organic C 1.12%; total N 0.49%; and available P, K, Na, Mg, Ca, Zn, and Cu were 11.1, 55, 61.3, 45, 150, 55, and 22 mg kg1, respectively. The experiment comprised of two treatments viz., plants without mycorrhizal inoculation represented non-mycorrhizal (NM) control plants, and plants inoculated with AMF Rhizophagus intraradices (N.C. Schenck& G.S. Sm.) C. Walker & A. Schüßler represented the mycorrhizal (M) plants. Each treatment consisted of two plants per pot replicated ten times. The experiment consisted of 20 pots arranged in a completely randomized block design. Plants were grown in the months of February to July under natural conditions of growth at Delhi India. The mean temperature at the time of experiment ranged between 25 C and 40 C; and relative humidity between 50% and 70%. Soil moisture was maintained by watering the plants twice every week with normal tap water. Estimation of all the parameters was carried out at full maturity i.e. 120 days post inoculation when the concentration of SGs is reported to be maximum (Brandle et al., 1998). 2.2. Inoculation and colonization by AMF The starter inoculum of R. intraradices (accession number CMCCWep319) was procured from The Energy and Resources Institute (TERI, New Delhi, India). Details of mass cultivation procedure have been published previously (Kapoor et al., 2007). Approximately 10 g of AMF inoculum containing soil and chopped roots was applied just below the roots of M plants at the time of transplantation. Roots of S. rebaudiana were cleared in KOH and stained with trypan blue (Phillips and Hayman, 1970) to confirm mycorrhization. Percent root colonization (87%) was estimated microscopically following Biermann and Linderman (1981). 2.3. Mineral nutrient analysis Analysis of mineral nutrients (P, Mg, K, Cu, Zn, Mn, and Fe) was carried out using oven dried leaf samples. Dried leaves were ground and approximately 0.2 g of each sample was digested in tri-acid mixture. The acid digest was allowed to cool for some time and diluted to 50 ml using double distilled water. A reagent blank was prepared by following the whole extraction procedure without a sample. Phosphorus concentration from the digested samples was determined following molybdate blue method (Allen, 1989) at 700 nm using a UVevis spectrophotometer (Beckman Coulter DU®730). The concentrations of Mg, K, Cu, Zn, Mn, and Fe in the samples were measured following Allen (1989) using atomic absorption spectrophotometer (Shimadzu AA-130). 2.4. Estimation of net photosynthetic rate and total sugars Measurement of net photosynthetic rate (PN) was performed on sunny days at 11:00 a.m. with fully expanded leaves using IRGA (Infra Red Gas Analyzer, LI-COR 6400 Portable Photosynthesis System, Lincoln, Nebraska, USA). IRGA was calibrated prior to carrying out measurements and also approximately after every 30 min during the measurement period. Each leaf was enclosed in the gas exchange chamber for 60 s. PN measured by IRGA was recorded six times for each treatment. Concentration of total sugars in the fresh leaves of S. rebaudiana was determined following the protocol described by Yemm and
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Willis (1954), using a UVevis spectrophotometer with wavelength set at 630 nm. Standard curve of glucose prepared at the same wavelength was used for calculating the concentration of sugars in samples.
Package for Social Sciences version 14 (SPSS, USA) was used to carry out the statistical analysis.
2.5. Extraction and analysis of steviol glycosides
The present work puts forth AM symbiosis as a certain approach to enhance SGs concentration in S. rebaudiana by evidencing that AMF upregulates the transcription of key SGs biosynthesis genes. This increase in concentration of SGs in response to AMF colonization is in accordance with the previous study (Mandal et al., 2013). However, the heightened response observed in this study can be explained by different combination of symbionts viz., Cimmeethi, a high yielding variety of S. rebaudiana and AMF R. intraradices.
Kumar et al. (2012a) reported that SGs is maximum in 3rd node leaf starting from apical meristem. Therefore, extraction of SGs was carried out from dried 3rd node leaves following the protocol of Kolb et al. (2001). Further HPLC analysis and validation were carried out following the methods described by Mandal et al. (2013). 2.6. Quantitative analysis of gene transcription RNA was extracted from 100 mg of fresh leaves (harvested from 3rd node) using RNAeasy Plant mini kit (Qiagen, USA) following manufacturer's instructions. RNA Quantification was done using NanoDrop TM 8000 Spectrophotometer (Thermo Scientific, USA). Genomic DNA contamination was removed by treating the RNA with DNase I (Fermentas, St Leo-Roth, Germany). cDNA was synthesized from 2 mg of purified RNA using cDNA reverse transcription kit (Ambion, USA). Amplification of cDNA was checked using 26S rRNA primer pair through endpoint PCR reaction. The synthesized cDNA was used for quantitative real time polymerase chain reaction (qRT-PCR) using gene specific primer pairs (Table 1) designed with the help of primer3 software (http://biotools. umassmed.edu/bioapps/primer3_www.cgi) and synthesized by Sigma, India. The PCR was carried out in Mx 5000P qPCR equipment (Stratagene, India). In a 25 mL reaction mixture, 1 mL of template (synthesized cDNA), 12.5 ml Power SYBR Green PCR master mix (Roche), 1 mL of 0.3 mmol of each gene specific primer pair and 9.5 mL of nuclease free water were added. Thermal cycles of qRT-PCR included one cycle at 95 C for 10 min, followed by 40 cycles at 95 C (15 s), 55 C (1 min), and 72 C (30 s). The analysis was performed in three biological repeats. Each repeat consisted of six leaves pooled from three individual plants. Relative fold change in gene transcription was analyzed by DDCT method using MxPro analysis software (Agilent technologies, USA). 2.7. Statistical analysis The data for nutrients analysis, estimation of sugar concentration and net photosynthetic rate is represented as mean of six replicates while data for SGs concentration and transcription analysis is represented as mean of three biological replicates. Numerical data obtained from the experiments was analyzed using Student's-test. An asterisk (*) in the tables and figures indicates significant difference between the means at p < 0.05. Statistical
3. Result and discussion
3.1. Mineral nutrient uptake AMF improves plant's nutritional status largely due to its extensive hyphal network that spans greater surface area and aids plant roots to absorb mineral nutrients that have low availability and mobility (Parniske, 2008). Concentration of P, K, Mg, Cu, Fe, Mn and Zn were recorded to be 215.7%, 6.95%, 42.47%, 298%, 87.22%, 203.09% and 105.12% respectively, higher in M plants (Table 2). Roles of mineral nutrients that hold significance to the study have been dealt subsequently. NM plants served as control in all the experiments as they showed no AMF colonization. 3.2. Net photosynthetic rate and total sugars An increase of 1.83 folds in PN was observed in M plants in comparison with NM plants (Table 3). Augmented nutritional status of the plant can be accredited for this, as mineral nutrients like Mg, Fe and Cu have crucial structural and functional roles to play in the process of photosynthesis (Taiz and Zeiger, 1998). An increase by 1.62 folds in total sugars was observed in M plants in comparison with NM plants (Table 3). There are many studies in congruence with these findings (Smith et al., 2010; Wu et al., 2011; Zubek et al., 2012; Mandal et al., 2013). To sustain its functionality AMF generates a strong sink for photosynthetically fixed carbon from the plant (Smith and Read, 2008) however, this increased carbon demand is compensated by increase in photosynthesis and carbon assimilation (Wright et al., 1998). 3.3. Concentration of steviol glycosides The concentration of stev and reb-A increased 1.68 and 3.4 folds respectively, upon mycorrhization (Table 4). Owing to increased photosynthesis in M plants, there was a consequent increase in carbon pool available for secondary metabolite production. Also,
Table 1 List of gene specific primers used in qRT-PCR analysis. Name
Left primer
Right primer
26S rRNA DXS (1-deoxy-D-xylulose 5-phospate synthase) DXR (1-deoxy-D-xylulose 5-phospate reductoisomerase) MDS (2-C-methyl-D-erytrithol 2,4-cyclodiphosphate synthase) GGDPS (geranylgeranyl diphosphate Synthase) CPPS (copalyl diphosphate synthase) KS (kaurene synthase) KO (kaurene oxidase) KAH: kaurenoic acid hydroxylase UGT85C2 UGT74G1 UGT76G1
50 -CACAATGATAGGAAGAGCCGAC-30 50 GCTTCAAGCCCATAACGTGT-30 50 -TGGTCCGTTTGTTCTTCCTC-30 50 -GCCTGGATACCCTCTCATCA-30 50 -CGTTTCCTCGTCTTCTTTGC-30 50 -ACAGCTGGTCGTTGGGTATC-30 50 -CTCCGGTGGACAGTTTCATT-30 50 -TCTTCACAGTCTCGGTGGTG-30 50 -CCTATAGAGAGGCCCTTGTGG-30 50 -GTTTCTCACAGTCCGGAAGC-30 50 -GGTAGCCTGGTGAAACATGG-30 50 -GACGCGAACTGGAACTGTTG-30
50 -CAAGGGAACGGGCTTGGCAGAATC-30 50 -CAATCGAGCCTTCCTCTACG 50 -CAAAGCACCTTCAGGAAAG-30 C 50 -ATTTGCCCGATGTCAGGTAG-30 50 -GGTGTTTCGAGATGGGTTTG-30 50 -CGAGTCTACCGTCGTGGAAT-30 50 -CCACCATGTGAAGTTGATGC-30 50 -GGTGGTGTCGGTTTATCCTG-30 50 -TAGCCTCGTCCCTTTGTGTC-30 50 -AACCGACAAGAACCCATCTG-30 50 -CTGGGAGCTTTCCCTCTTCT-30 50 -AGCCGTCGGAGGTTAAGACT-30
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Table 2 Effect of R. intraradices inoculation on concentration of nutrients in shoots of S. rebaudiana. Treatments
Non mycorrhizal Mycorrhizal
Nutrient (mg g1dry weight) P
K
Mg
Cu
Fe
Mn
Zn
260 ± 96 821 ± 398*
1380 ± 198 1476 ± 140*
1928 ± 224 2747 ± 434*
187 ± 56 746 ± 179*
1268 ± 695 2374 ± 880*
55 ± 27 166.7 ± 68*
54.54 ± 15 110.77 ± 0.81*
Means within a column followed by the asterisk (*) are significantly different at p < 0.05 between non mycorrhizal and mycorrhizal treatments. The data shown are mean of six replicates ± SD determined by unpaired equal student's t-test.
improved P nutrition due to AM symbiosis (Table 2) can be accredited as P is crucial for the synthesis of both primary and secondary metabolites (Marschner, 2002) and it is discernible that P is an important constituent of various intermediates of MEP pathway such as deoxy-D-xylulose 5-phosphate (DXP) and 2-CMethyl-D-erythritol 2,4-cyclodiphosphate (cMEPP). As a consequence of glucosylation pattern and sugar moiety, the organoleptic properties of all the SGs are different (DuBois and Stepehenson, 1985). Stev is 143 times sweeter than sucrose whereas, reb-A is 242 times sweeter (Kasai et al., 1981). Reb-A has a better taste quality than stev, as it is sweeter (DuBois and Stepehenson, 1985). Thus ratio of reb-A to stev governs the sweetening property of S. rebaudiana (Brandle et al., 1998). In the present study ratio increased from 0.29 in NM plants to 0.60 in M plants, suggesting that sweetening properties of S. rebaudiana enhanced as a result of AM symbiosis. 3.4. Transcription studies of steviol biosynthetic genes To enable a better understanding, transcription profile of genes under three stages of the SGs biosynthesis pathway were studied viz., MEP pathway as the first stage, that results in a pool of IPP and DMAP, which serve as precursors to a number of terpenoids; second stage comprises of five sequential reactions catalyzing the synthesis of steviol; and the third stage, often referred to as glucosylation pathway that is committed to SGs biosynthesis. 3.4.1. First stage/MEP pathway Upon mycorrhization the transcript levels of DXS, DXR increased to 1.7 and 1.8 folds respectively, in comparison with the NM plants (Fig. 1A and B). Induction of MEP pathway due to upregulation of DXS and DXR has been particularly studied at root level where the plant and fungus interact mainly (Walter et al., 2000; Hans et al., 2004; Strack and Fester, 2006; Flob et al., 2008). However, it is known that AM symbiosis greatly influences the physiology and metabolism of aerial parts of the plant as well (Toussaint, 2007) and yet there have been few studies that explore the molecular basis of interaction between mycorrhiza and MEP pathway genes at the shoot level (Mandal et al., 2014). Hsieh and Goodman (2005) suggested that increased sugar concentration augments the expression of several genes of MEP pathway. Also, Cordoba et al. (2009) reported that increased carbohydrates alter the expression profile of DXS and DXR; and so probably enhanced sugars reported in the
Table 3 Effect of R. intraradices inoculation on net photosynthetic rate and concentration of total carbohydrates in shoots of S. rebaudiana. Treatments
Net photosynthetic rate (mmol CO2 m2 s1)
Total carbohydrates (% fresh weight)
Non mycorrhizal Mycorrhizal
10.08 ± 1.12 18.53 ± 1.63*
18.43 ± 3.34 30.03 ± 3.68*
Means within a column followed by the asterisk (*) are significantly different at p < 0.05 between non mycorrhizal and mycorrhizal treatments. The data shown are mean of six replicates ± SD determined by unpaired equal student's t-test.
present study might have influenced the transcription of these genes. The upregulation of DXS and DXR propelled by AMF perhaps has a cascading effect on downstream genes of the pathway. AMF affects the biosynthesis of several terpenoids (Asensio et al., 2012) and upregulation of the key MEP pathway genes establishes an elementary mechanism to enhance the pool of IPP and DMAP from which a broad range of terpenoids and terpenoid-derived compounds are produced through several different pathways. Transcript level of MDS also increased to 1.84 folds in M treatments in comparison with NM treatments (Fig. 1C). The expression of MDS may be regulated by Zn and Mn concentrations as the homo-trimeric MDS protein requires Zn2þ and Mn2þ for its activity (Richard et al., 2002; Steinbacher et al., 2002). Incidentally, an increase of 105.12% and 203.09% in Zn and Mn level respectively, was observed in M plants. Therefore, it can be conjectured that increased concentrations of Zn and Mn as a result of the nutritional benefits of AM symbiosis, might have resulted in upregulation of MDS. 3.4.2. Second stage The transcript levels of all the five second stage genes of the SGs biosynthesis pathway viz., GGDPS, CPPS, KS, KO and KAH increased upon mycorrhization. The transcript levels of GGDPS, CPPS and KO increased to 1.42, 1.48 and 6.05 folds respectively, in M plants in comparison with NM plants (Fig. 1D, E, and G), suggesting a similar trend in their upregulation. However, the comparative transcription profile of KS suggested an increase of 11.38 folds in M plants (Fig. 1F). Given that KS is present in duplicates in S. rebaudiana (Richman et al., 1999) provides a plausible explanation for an increase of such magnitude. Transcript level of KAH increased to1.35 folds in M plants (Fig. 1H). KAH represents a branch point from the gibberellins biosynthesis considering its capacity to direct the flow of metabolites specifically towards the biosynthesis of SGs (Humphrey et al., 2006). Thus, increase in its transcription implies enhanced steviol production in M plants. 3.4.3. Third stage/glucosylation pathway The metabolic channelling between KAH and UGT85C2, and among all the UGTs is specific to SGs biosynthesis (Guleria et al., 2014) implying that upregulation of these ensures higher concentration of SGs. The expression of UGT85C2 has been considered critical for the accumulation of SGs as it catalyses a rate limiting
Table 4 Effect of R. intraradices inoculation on concentration of rebaudioside A, stevioside and the ratio of rebaudioside A/stevioside in shoots of S. rebaudiana. Treatments
Rebaudioside-A concentration (% dry wt.)
Stevioside concentration (% dry wt.)
Rebaudioside A/stevioside
Non mycorrhizal Mycorrhizal
2.36 ± 0.13 8.14 ± 0.1*
8.08 ± 0.18 13.51 ± 0.25*
0.29 ± 0.01 0.60 ± 0.02*
Means within a column followed by the asterisk (*) are significantly different at p < 0.05 between non mycorrhizal and mycorrhizal treatments. The data shown are mean of three replicates ± SD determined by unpaired equal student's t-test.
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Fig. 1. Effect of R. intraradices inoculation on the transcript level of A) 1-deoxy-D-xylulose 5-phospate synthase (DXS), B) 1-deoxy-D-xylulose 5-phospate reductoisomerase (DXR), C) 2-Cmethyl-D-erytrithol 2,4-cyclodiphosphate synthase (MDS), D) geranylgeranyl diphosphate synthase (GGDPS), E) Copalyl diphosphate synthase (CPPS), F) kaurene synthase (KS), G) kaurene oxidase (KO), H) kaurenoic acid hydroxylase (KAH), and uridine diphosphate (UDP)-dependent glycosyltransferases (UGTs) I) UGT85C2, J) UGT74G1 and K) UGT76G1 in leaves of S. rebaudiana. Data is presented as mean ± SD (n ¼ 3). An asterisk (*) on the bar indicates significant differences between the means at p < 0.05 NM, non mycorrhizal; M, R. intraradices inoculation.
reaction in glucosylation pathway (Mohamed et al., 2011). The transcript level of UGT85C2 was 7.42 folds higher in M plants (Fig. 1I). The majority of SGs in leaves of S. rebaudiana are tri-glycoside stev and tetra-glycoside reb-A (Kinghorn and Soejarto, 1985). Transcription of UGT74G1 and UGT76G1 increased to 7.37 and 4.29 folds respectively, in M plants (Fig. 1J and K). Upregulation of UGT74G1 led to augmented concentration of stev and that of UGT76G1 led to higher concentration of reb-A in M plants (Table 4). Conversion of stev to reb-A is controlled by UGT76G1 (Richman et al., 2005) and its higher transcription ascertains increased ratio
of reb-A to stev in M plants. 4. Conclusion The present work signifies that AM symbiosis upregulates the transcription of important SGs biosynthesis genes at all stages of the pathway d a consequence of systemic effect of augmented sugar concentration due to increased photosynthesis and improved nutrition. The enhanced transcription of upstream genes of MEP pathway results in a greater pool of IPP and DMAP from which
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various terpenoids are produced through disparate pathways. However, AMF mediated enhanced transcription of downstream genes viz., KAH and UGTs insinuate a rather specific mechanism to enhance SGs production. The present work is first report on regulation of SGs biosynthesis genes in response to AMF. Authors' contribution S. Mandal and S. Upadhyay performed the experiments, analysed the data and wrote the manuscript; V.P. Singh edited the manuscript and helped in data analysis; R. Kapoor designed the experiments, helped in data analyses, and wrote the manuscript. Acknowledgements The authors wish to acknowledge the Council of Scientific and Industrial Research (CSIR) for the award of Research Fellowships. We are also thankful to the DR. J. P. Singh CIMAP Uttarakhand for providing the stem cuttings of S. rebaudiana. References Allen, S.E., 1989. Chemical Analysis of Ecological Materials, second ed. Blackwell Scientific Publishers, London. ~ uelas, J., 2012. AM fungi root colonization increases the Asensio, D., Rapparini, F., Pen production of essential isoprenoids vs. nonessential isoprenoids especially under drought stress conditions or after jasmonic acid application. Phytochemistry 77, 149e161. Biermann, B.J., Linderman, R.G., 1981. Quantifying vesicular arbuscular mycorrhizae: a proposed method towards standardization. New. Phytol. 89, 63e67. Brandle, J.E., Starratt, A.N., Gijzen, M., 1998. Stevia rebaudiana: its agricultural, biological, and chemical properties. Can. J. Plant Sci. 78, 527e536. Brandle, J.E., Telmer, P.J., 2007. Steviol glycoside biosynthesis. Phytochemistry 68, 1855e1863. Ceunen, S., Werbrouck, S., Geuns, J.M.C., 2012. Stimulation of steviol glycoside accumulation in Stevia rebaudiana by red LED light. J. Plant Physiol. 169, 749e752. Ceunen, S., Geuns, J.M.C., 2013. Influence of photoperiodism on the spatio-temporal accumulation of steviol glycosides in Stevia rebaudiana (Bertoni). Plant Sci. 198, 72e82. Copetta, A., Lingua, G., Berta, G., 2006. Effects of three AM fungi on growth, distribution of glandular hairs, and essential oil production in Ocimum basilicum L var. Genovese. Mycorrhiza 16, 485e494. Cordoba, E., Salmi, M., Leon, P., 2009. Unravelling the regulatory mechanisms that modulate the MEP pathway in higher plants. J. Exp. Bot. 60, 2933e2943. DuBois, G.E., Stephenson, R.A., 1985. Diterpenoid sweeteners. Synthesis and sensory evaluation of stevioside analogues with improved organoleptic properties. J. Med. Chem. 28, 93e98. Floß, D.S., Hause, B., Lange, P.R., Kuster, H., Strack, D., Walter, M.H., 2008. Knockdown of the MEP pathway isogene 1-deoxy-d-xylulose 5-phosphate synthase 2 inhibits formation of arbuscular mycorrhiza-induced apocarotenoids, and abolishes normal expression of mycorrhiza-specific plant marker genes. Plant J. 56, 86e100. Geuns, J.M.C., 2003. Stevioside. Phytochemistry 64, 913e921. Guleria, P., Masand, S., Yadav, S.K., 2014. Overexpression of SrUGT85C2 from Stevia reduced growth and yield of transgenic Arabidopsis by influencing plastidal MEP pathway. Gene 539, 250e257. Hanson, J.R., White, A.F., 1968. Studies in terpenoid biosynthesis - II. Biosynth. Steviol. Phytochem. 7, 595e597. Hans, J., Hause, B., Fester, T., Strack, D., Walter, M.H., 2004. Cloning, characterization and immunolocalization of a mycorrhizal-inducible 1-deoxy-D-xylulose 5phosphate isomerase in arbuscule-containing cells of maize. Plant Physiol. 134, 614e624. Hsieh, M.H., Goodman, H.M., 2005. The Arabidopsis IspH homolog is involved in the plastid Nonmevalonate pathway of isoprenoids biosynthesis. Plant Physiol. 138, 641e653. Humphrey, T.V., Richman, A.S., Menassa, R., Brandle, J.E., 2006. Spatial organisation of Four enzymes from Stevia rebaudiana that are involved in steviol glycoside synthesis. Plant Mol. Biol. 61, 47e62. Kapoor, R., Giri, B., Mukerji, K.G., 2002a. Glomus macrocarpum: a potential bioinoculant improves essential oil quality and concentration in Dill (Anethum graveolens L.) and Carum (Trachyspermum ammi Linn.) Sprague. World J. Microb. Biot. 18, 459e463. Kapoor, R., Giri, B., Mukerji, K.G., 2002b. Mycorrhization of coriander (Coriandrum sativumL) to enhance the concentration and quality of essential oil. J. Sci. Food Agric. 82, 339e342. Kapoor, R., Giri, B., Mukerji, K.G., 2004. Improved growth and essential oil yield and quality in Foeniculum vulgaremill on mycorrhizal inoculation supplemented
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