Piriformospora indica affects growth, tropane alkaloids production and gene expression in Atropa belladonna L. plantlets Homa Noora 1 , Saleh Shahabivand 1,* , Farokh Karimi 1 , Ahmad Aghaee 1 , Ali Asghar Aliloo 2 1 Department of Biology, Faculty of Science, University of Maragheh, Maragheh, Iran 2 Department of Agronomy, Faculty of Agriculture, University of Maragheh, Maragheh, Iran *E-mail: [email protected]; [email protected]University of Maragheh, Madar Square, Golshahr, Maragheh, Iran, Postal Code: 83111-55181, Tel: +98 41 37276068, Fax: +98 41 37276060 Abstract: Atropa belladonna is a perennial herbaceous plant and most importantly commercial source to acquiring pharmaceutical tropane alkaloids such as hyoscyamine and scopolamine that are anticholinergic drugs. The effect of Piriformospora indica, a plant growth-promoting root endophyte fungus, on growth, tropane alkaloids production and expression of two rate-limiting enzyme genes including Putrescine N-methyltransferase (pmt) and Hyoscyamine 6--hydroxylase (h6h) were studied in 1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
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Piriformospora indica affects growth, tropane alkaloids production and gene expression
in Atropa belladonna L. plantlets
Homa Noora1, Saleh Shahabivand1,*, Farokh Karimi1, Ahmad Aghaee1, Ali Asghar Aliloo2
1 Department of Biology, Faculty of Science, University of Maragheh, Maragheh, Iran
2 Department of Agronomy, Faculty of Agriculture, University of Maragheh, Maragheh, Iran
Plant secondary metabolites are used extensively in the food and pharmaceutical industries
and commonly, extracting these metabolites from whole plants is only economical method
for the production of pharmaceuticals. However, the low endogenous content of these
chemicals remains as the limitation for mass production in natural plants (Liu et al., 2010).
The tropane alkaloids such as scopolamine (hyoscine) and its precursor hyoscyamine are
among the most famous secondary metabolites with pharmacological activity (Palazón et al.,
2008). The both hyoscyamine and scopolamine which use as antagonists of acetylcholine in
the autonomic and central nervous system derived from Atropa, Duboisia, Datura,
Hyoscyamus and Scopolia species (Liu et al., 2010; Ziegler and Facchini, 2008).
Atropa belladonna, a member of the Solanaceae family and commonly known as deadly
nightshades, is a perennial herbaceous plant and most importantly commercial source of
bioactive tropane alkaloids. The principal place of alkaloid biosynthesis is the roots but
leaves can also accumulate significant quantities of these compounds (Palazón et al., 2008).
The production of these bioactive compounds is often low (less than 1% dry weight) and
depends greatly on the physiological and developmental stage of plant (Ramakrishna and
Ravishankar, 2011).
Five functional genes involvement in tropane alkaloids biosynthesis have been reported that
among of them, pmt and h6h are generally considered to be the rate-limiting-enzyme genes
(Yang et al., 2011). Putrescine N-methyltransferase (PMT; EC 2.1.1.53) catalyzes the S-
adenosylmethionine (SAM)-dependent N-methylation of putrescine and is the first enzyme
committed for the biosynthesis of tropane alkaloids (Rothe et al., 2003). Hyoscyamine 6-𝛽-
hydroxylase (H6H; EC 1.14.11.11) catalyzes the hydroxylation of hyoscyamine to 6-𝛽-
hydroxyhyoscyamine, as well as the epoxidation of 6-𝛽-hydroxyhyoscyamine to
scopolamine.
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There are diverse methods to increase the yield of secondary products through in vitro
cultures using biotic and abiotic elicitors. Treatment with elicitors such as fungi is one of the
most effective approaches for enhancing secondary metabolite production in plantlet cultures
and has newly found commercial application.
Piriformospora indica, a cultivable root endophyte, is a member of the Basidiomycetous
order Sebacinales and can be used as a biotic elicitor. P. indica is easily cultivable, lacks host
specificity and colonizes roots of many different plants which increases nutrient uptake and
allows plants to survive under biotic and abiotic stresses. The symbiotic fungus promoted the
overall growth and development of all the medicinal plants tested so far. It also promoted
enhancement of secondary metabolites contents in medicinal plants (Das et al., 2013; Varma
et al., 2012).
There are no reports on the effect of P. indica in tropane alkaloid content in different plants.
Due to the large medicinal activities of tropane alkaloids, the aim of this work was to
investigate the effects of this novel fungus of the Sebacinales on growth, the alkaloid
(hyoscyamine and scopolamine) content and h6h and pmt genes expression levels in A.
belladonna.
Materials and Methods
Plant Material
A. belladonna plantlets were obtained by in vitro micropropagation of plantlets derived from
the National Institute of Genetic Engineering and Biotechnology in Tehran, Iran. The primary
plantlets were used for explant (a node with auxiliary buds) preparation. The explants were
cultured in Murashige and Skoog (MS) solid medium supplemented with 3% (w:v) sucrose.
These cultures were maintained at 25±1°C with a daily 16 h photoperiod. At the end of the
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experiment, shoots and roots of the propagated plantlets (4-weeks-old) were harvested
separately and their fresh weights and lengths were measured. Next, they were frozen in the
liquid nitrogen and then transferred to -80°C refrigerator for biochemical and molecular
analysis.
Preparation and addition of elicitor
P. indica was cultured in Petri dishes on a Hill & Kafer medium (Hill and Kafer, 2001). The
plates were placed in a growth chamber in the dark at 29±1°C for 2 weeks. For in vitro co‐cultivation of the two symbionts, one fungal plug of 5 mm in diameter was placed at a
distance of 1 cm from the roots of 2-weeks-old plantlet. The plantlets were incubated at
25±1°C for 14 days. At the end of cultivation, the plantlets were analyzed for the effect of
fungal elicitors on growth and product accumulation. Before analyze, root samples were
checked for P. indica colonization.
Root staining and measurement of root colonization
Root segments were detached from the plant stems and located in 50% ethanol until staining.
Root staining to determine the colonization of endophytic fungus followed using the
procedure of Philips and Hayman (1970). The roots of the sampled plants were heated for 5
min in a 10% KOH solution and then washed under running tap water three times. Root
samples were acidified with 1% HCl for 1 min and then immersed in 20% trypan blue
staining solution and were heated for a further 10 min. From the stained samples, 30 root
segments (1 cm long) per plant were cut and observed with a light microscope (Olympus BH-
2) at 20×. Root colonization was determined according to the gridline intersection method
described by Giovanetti and Mosse (1980). In this technique, the percentage of root
colonization per plant was determined by dividing the total number of colonized root
fragments by the total number of root pieces examined × 100.
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Total alkaloids extraction and HPLC analysis of tropane alkaloids
Total alkaloids in the plant roots and shoots (0.5 g) were extracted as described by Kamada et
al. (1986) with slight modification. Briefly, each samples were extracted with
CHCl3:MeOH:NH4OH (15:5:1, v/v/v). After homogenization and incubation for 1 h at 25ºC,
the extract was filtered (through Whatman No.1 filter paper) and washed two times with 1 ml
of chloroform. The filtrate was evaporated to dryness under air and at that time 5 ml of
chloroform and 2 ml of 0.5 M sulfuric acid were added to the remainder. The aqueous part
was adjusted to pH 10 with 28% ammonium hydroxide, and alkaloids were extracted two
times with 1 ml of chloroform. This mixture was then vaporized to dryness and dissolved in
methanol. Total alkaloids were measured by a spectrophotometer (Shimadzu UV visible-
1601 PC) at 258 nm according to the scopolamine sulfate standard curve. Then, methanolic
extracts analyzed by Knauer HPLC GmbH system: mobile phase was isocratic mixture of
water and acetonitrile at a ratio 65:35. The flow rate was 1 ml per minute. The detecting
wavelength was 210 nm. The temperature of column (250 mm × 4.6 mm) was 25ºC. The
sample solution of injection was 20 μl every time. The standard solutions of tropane alkaloids
hyoscyamine and scopolamine (Sigma, USA) were prepared in methanol at a final
concentration of 1000 μg/ml and diluted into 500, 250, 100, 50, 25 μg/ml. The alkaloids were
quantified as mg/g FW of plant tissues.
Total cellular RNA extraction and RT-PCR analysis of h6h and pmt genes
100 mg of root tissue was ground thoroughly in liquid nitrogen using a pre-chilled mortar and
pestle. Total RNA was extracted from roots of A. belladonna plant treated with P. indica
using Plant Total RNA Extraction Kit (Roche). The concentration of the RNA was
determined using spectrophotometer NanoDrop (Thermo Fisher Scientific, USA) at 260 nm.
Quality of the RNA was checked by both gel and NanoDrop at the 260/280 ratio. Five
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micrograms of isolated RNA were used as a template for reverse transcriptase-polymerase
chain reaction assay (RT-PCR) to investigate the expression patterns of h6h and pmt in
plantlets of A. belladonna. The accession numbers of sequences used for primer design were
AB018570.1 and AB018571.1 for pmt, and JN415637.1 for h6h. First strand cDNA was
synthesized using an oligo-d (T) primer. PCR amplification was carried out in a 25 μl total
volume containing 2.5 μl of 10×PCR buffer, 10 pmoles of each oligonucleotide primers
including h6h primers (F: 5'-AATGATGTTCCTTTAGG-3' and R: 5’-
ATGGTGCAAGAATAATCA-3') and pmt specific primers (F: 5'-
GCTTTCAACAACTTCTTCAG-3' and R: 5'-GGGGCGTAGGAGGAAAGC-3'), 1.5 mM
MgCl2, 2.5 mM dNTPs, 1μg of template cDNA, and 1.5 units of Taq DNA polymerase
(fermentas). Meanwhile the RT-PCR reaction for the house-keeping gene (α-tubulin gene)
using specific primers (F: 5'-GCTTTCAACAACTTCTTCAG-3' and R: 5'-
GGGGCGTAGGAGGAAAGC-3') was designed according to the conserved regions of
tubulin genes. The house-keeping gene was used as an internal control to estimate whether
equal amounts of RNA were used among samples. The PCR was performed in a thermal
cycler system (Eppendorf, Germany) with the following program: predenaturation for 5 min
at 95 °C, followed by 36 cycles of 45 s at 95°C, 45 s at 55°C, and 50 s at 72 °C, and a final 7
min extension was performed at 72 °C. PCR products were detected on 1% agarose gels by
ethidium bromide staining. Quantification of the amplified bands was done by the software
GelQuant.NET.
Statistical Analysis
Statistical analysis data were expressed as mean ± SE for the plant growth parameters and the
alkaloids contents. Results were analyzed with IBM SPSS version 19 software. For the
expression levels of h6h and pmt mRNA, differences in the measured parameters across the
different groups were statistically assessed using repeated measures ANOVA followed by
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Fisher’s protected least significant difference, multiple range test P<0.05 was considered
statistically significant.
Results
Growth changes
The symbiosis with P. indica significantly (P<0.05) increased the shoot length and fresh
weight of root and shoot in treated plants compared to the control. As shown in Fig. 1a, b, A.
belladonna plantlets inoculated with P. indica showed a 1.9-, 1.86- and 1.83-fold increase in
shoot length, root fresh weight and shoot fresh weight, respectively, in comparison with
controls.
Root colonization
Treated roots were colonized by the fungus and produced chlamydospores and cellular
hyphae (Fig. 2). The spores were observed at maturity. The results from this study showed
that root colonization percentage was about 76% in treated roots of A. belladonna.
Total alkaloid contents
Total alkaloid contents were considerably increased (P<0.05) in aerial parts (stem and leaf)
of treated plantlets by P. indica (Fig. 3). Total alkaloids in treated stem and leaf were found
to be significantly increased by 1.97- and 3.52-fold, respectively, compared to control. The
highest level of total alkaloids (30.22 mg/g FW) was observed in leaf of treated plantlet. The
total alkaloid content was approximately constant in roots of treated and untreated plantlets.
HPLC analysis
Some HPLC chromatograms were shown in Fig. 4a, b. The amount of hyoscyamine and
scopolamine were significantly (P<0.05) increased in leaves and stems of P. indica-treated
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plantlets compared to control (Fig.5 a, b). Inoculation with P. indica resulted in a significant
increase in stem and leaf hyoscyamine by 22.2- and 5.2-fold, respectively, in comparison
with control plantlets. Under similar conditions, stem and leaf scopolamine were found to be
increased by 6.69- and 9.56-fold, respectively. The highest levels of hyoscyamine and
scopolamine contents (15.71 and 7.75 mg/g FW, respectively) were observed in leaf of the
plantlets inoculated with P. indica. Based on the results, reduced hyoscyamine and
scopolamine contents were observed in the root of inoculated plantlets in comparison to
untreated control.
Differential Expression of h6h and pmt
The presence of transcripts for h6h and pmt was determined by semi-quantitative RT-PCR.
Amplification of the cDNA with the h6h and pmt primers described in the materials and
methods section resulted, as expected, in a product of 815 bp and 1216 bp for h6h and pmt
genes, respectively (Fig. 6, 8). The quantitative expression level analysis showed that P.
indica-treated roots significantly (P<0.05) elevated levels of h6h and pmt transcripts (1.6-
and 1.85-fold, respectively) compared with control (Fig. 7, 9).
Discussion
In this experiment, we showed that the root endophytic fungus P. indica strongly interacted
with the roots of A. belladonna, resulting in efficient colonization (Fig. 2). The hyphae and
spores were detected around the roots, and in the extracellular space and within root cells.
The earlier researches have shown that P. indica interacted symbiotically with plants and
stimulated growth of many plants including agricultural and medicinal crops. In this study,
growth of A. belladonna plantlets, co-cultivated with P. indica was strongly promoted
compared to non-colonized plantlets. The percent increase in shoot height, and shoot and root
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fresh weight of fungus treated plantlets were 90%, 83% and 86%, respectively, as compared
to control. The obtained results from growth changes in our experiment are in agreement with
those of Vadassery et al. (2009), who reported that P. indica promotes growth of Arabidopsis
seedlings. This increase in biomass indicates the mycorrhiza-like growth-promoting activity
of P. indica. Shahabivand et al. (2012) reported that the endophytic fungus P. indica may
increase host fitness and competitive abilities by increasing growth rate through evolving
biochemical pathways to produce plant growth hormones such as indole-3-acetic acid and
cytokinins or enhance the uptake of nutrients especially P and N by the host plant. Recently, a
gene encoding a phosphate transporter (PiPT) from P. indica was reported that is actively
involved in the phosphate transportation and as a result P. indica helps to improve the
nutritional status of the host plant (Yadav et al., 2010).
The results also revealed that the secondary metabolites were strongly influenced by fungus
interactions. Hyoscyamine and scopolamine are widely used in medicine and they possess
anticholinergic, mydriatic, antispasmodic and sedative properties. Due to the relative
complexity of chemical structure in hyoscyamine and scopolamine and their rather long
biosynthetic pathway, the synthetic production of these compounds is more expensive than
their extraction from plant materials (Huang et al., 2005). Therefore, our findings could be
effective way to reduce these costs. It has been extensively studied that P. indica enhanced
bioactive compounds of the medicinal and economically important plants by forming
association with their roots. The data from this study showed a noticeable increase in the total
alkaloids, hyoscyamine and scopolamine amounts in the shoots of treated plantlets by P.
indica in comparison with untreated control. Similar cases of enhancement of active
ingredients, in treated plants by this fungus, were also showed in other studies such as
podophyllotoxins in Linum album (Baldi et al., 2009), saponin from Chlorophytum sp. (Gosal
et al., 2010), essential oils in Thymus vulgaris (Dolatabadi et al., 2011), asiaticoside from
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Centella asiatica (Satheesan et al., 2012) and withaferin A in Withania somnifera (Ahlawat
et al., 2016). Recently, Kumar et al. (2016) reported the positive effect of P. indica on
triterpenoids synthesis including ursolic acid, oleanolic acid and betulinic acid in Lantans
camara suspension cultures. They concluded that P. indica has the potential of acting as a
good source of elicitor in enhancing the production of useful secondary metabolites in plant
cell cultures. The enhancement in these bioactive compounds production could be due to
elicitation of plant defense in response to fungal elicitors like lipopolysaccharides and
glycoproteins formed by the action of plant-derived hydrolases secreted in response to
endophyte colonization (Gao et al., 2010). Improved plant defense responses correlated with
endophytic colonization are associated with the increased requisition for energy-reducing
equivalents and carbon skeletons provided by primary metabolic pathways (Bolton, 2009;
Gao et al., 2010).
Our experiment showed that in root of un-inoculated plants, the hyoscyamine and
scopolamine contents were higher in comparison with amounts of these alkaloids in stem and
leaf (Fig. 5). It has been demonstrated that main site in biosynthesis of tropane alkaloids is
the root and these active compounds are translocated from root to shoot of the plants. Also,
activities of two enzymes PMT and H6H have been shown to be high in the root but very low
in the shoot. The mechanism of hyoscyamine and scopolamine translocation from the root to
the shoot is still a matter of controversy. Recently, h6h expression has been reported in the
shoots of some Solanaceae plants and the h6h transcript was presented in both roots and
shoots (Palazon et al., 2008; Vakili et al., 2012). These results indicate that biosynthesis site
of tropane alkaloids may diverge remarkably in different plant species.
The first and last steps in the biosynthetic pathway of hyoscyamine and scopolamine were
carried out by PMT and H6H enzymes (Ziegler and Facchini, 2008). It has been previously
demonstrated that the expression of the pmt gene was pericycle-specific and H6H was
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localized in the root pericycle. According to this cause, we examined the expression of pmt
and h6h genes in the roots of A. belladonna. In treated root with P. indica, we observed
enhancement on the levels of pmt and h6h transcripts compared to control. Saxena et al.
(2016) observed that in hairy root cultures of Withania somnifara the expression of all the
genes of withanolide biosynthetic pathway was upregulated in presence of P. indica comared
to control. It has been shown that pmt gene is regulated by some of the various factors such as
plant hormones, light and elicitors like jasmonates and their strong expression is primarily in
the cultured roots (Ghosh, 2000). The colonization by P. indica was also shown to alter
expression patterns of genes involved in salicylic acid, jasmonate and ethylene signalling in
barley (Schafer et al., 2009). These results suggest that this endophytic fungus may affect the
expression of pmt and h6h genes, and can act as an elicitor for tropane alkaloid metabolism
under in vitro-culture condition.
In the roots, the expression profiles of h6h and pmt were not similar to the pattern of
hyoscyamine and scopolamine accumulation (treated roots had higher levels of pmt and h6h
transcripts but lower contents of hyoscyamine and scopolamine). It is likely that P. indica
stimulates hyoscyamine and scopolamine production through the activation of biosynthetic
enzymes or the induction of their gene expression in the roots and then translocates these
compounds via an unknown mechanism to the shoots (P. indica inoculation diminished
hyoscyamine and scopolamine amounts in root but increased them in the stem and leaf). The
detailed mechanism of the effect of P. indica on hyoscyamine and scopolamine production
and expression of key enzymes has to be further investigated.
The elicitation is considered to involve the second messenger Ca2+. Vadassery et al. (2009)
reported that P. indica was able to promote plant growth in Arabidopsis and induces a
cytoplasmic Ca2+ increase in root cells. In potato, P. indica increased transcript expression of
the two Ca2+-dependent proteins i.e. calmodulin and calcium-dependent protein kinase
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(Upadhyaya et al., 2013). On the other hand, as respect to effect of external source of Ca2+ on
the induction of alkaloid biosynthesis (Facchini, 2001), it may be concluded that Ca2+ and
perhaps calmodulin participate in the signal transduction pathway of tropane alkaloid
biosynthesis mediated by P. indica. Further studies on P. indica-mediated signal transduction
pathways will lead to better understanding of tropane alkaloids metabolism under P. indica
treatment in different plant species.
In conclusion, our experiment, for the first time, showed that presence of P. indica as a biotic
elicitor had a positive effect on the growth, hyoscyamine and scopolamine production and the
expression of h6h and pmt genes in A. belladonna plantlets. The stimulatory influence of this
fungus on tropane alkaloids production is at least partly due to the induction of genes
expression of biosynthetic pathway enzymes and/or the activation these enzymes. Also
p.indica enhanced translocation of the secondary metabolites from root to upper parts
especially to the leaf. We suggest the consideration of this fungus as a tool for commercially
large-scale production of hyoscyamine and scopolamine in A. belladonna.
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