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

*E-mail: shahabi70@yahoo.com ; shahabi@maragheh.ac.ir

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-𝛽-hydroxylase (h6h) were studied in micropropapated A. belladonna

plantlets. The results showed that inoculation of P. indica significantly increased the growth

parameters, total alkaloids, and hyoscyamine and scopolamine amounts recorded by HPLC

analysis in aerial parts of the plantlets. Furthermore, an increase in the gene expression of the

two enzymes i.e. pmt and h6h which play a distinct role in tropane alkaloids production was

observed in the roots of treated plantlets. The current study provides an effective approach for

commercially production of hyoscyamine and scopolamine by P. indica inoculation in A.

belladonna plants.

Keywords: Atropa belladonna; Elicitor; Hyoscyamine; Piriformospora indica; Scopolamine

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Introduction

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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Fig. 1. Effect of P. indica on the growth in A. belladonna plantlets. Changes in a)shoot length and b)shoot and

root fresh weight of A. belladonna plantlets treated with P. indica for 2 weeks. SL: shoot length; RFW: root

fresh weight; SFW: shoot fresh weight. All the values are the means of three biological replicates (± SE).

Fig. 2. Fungal structures in root cells. A. belladonna plants were inoculated with P. indica, for 2 weeks then

roots were stained with trypan blue and observed by light microscopy. a) magnification ⨯100, untreated root

(control), b) magnification ⨯400, showing intercellular hyphae, c) magnification ⨯1000, cells with coiled and

branched intracellular hyphae, d) magnification ⨯400, hyphae and chlamydospores of P. indica.

Fig. 3. Effects of P. indica on total alkaloid contents in root, stem and leaf of A. belladonna. Total alkaloids

were measured by spectrophotometer at 258 nm according to the scopolamine sulfate standard curve. All the

values are the means of three biological replicates (± SE).

Fig. 4. HPLC chromatograms of a) Untreated root and b) Treated root with P. indica in A. belladomma.

Fig 5. Effect of P.indica on a) hyoscyamine and b) scopolamine contents in root, stem and leaf of A.

belladonna. All the values are the means of three biological replicates (± SE).

Fig. 6. Semi-quantitative RT–PCR analysis of pmt mRNA expression in A. belladonna plantlets treated with P.

indica. Lane1: 1-kb DNA molecular weight marker (fermentase), lane 2, 3: PCR mixture without cDNA

template, lane 3: untreated root, lane 4: treated root.

Fig. 7. Relative expression levels of pmt gene in A. belladonna plantlets treated with P. indica. All the values

are the means of three biological replicates (± SE).

Fig. 8. Semi-quantitative RT–PCR analysis of h6h mRNA expression in A. belladonna plantlets treated with P.

indica. Lane 1: PCR mixture without cDNA template, lane 2: 1-kb DNA molecular weight marker (fermentase),

lane 3: untreated root, lane 4: treated root.

Fig. 9. Relative expression levels of h6h gene in A. belladonna plantlets treated with P. indica. All the values

are the means of three biological replicates (± SE).

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Control P. indica

SL SL

01234567

a

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t len

gth

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)

lortnoC lortnoC acidni .P acidni .PWFR WFS WFR WFS

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Fig. 1.

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Root Stem Leaf Root Stem LeafControl Control Control P.indica P.indica P.indica

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Root Stem Leaf Root Stem LeafControl Control Control P.indica P.indica P.indica

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