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Arbuscular mycorrhizal fungi alter the content and composition of secondary metabolites in Bituminaria bituminosa L.

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Arbuscular mycorrhizal fungi alter the content and composition of secondary metabolites in Bituminaria bituminosa L.

Short title: B. bituminosa metabolites affected by AMF

Laura Pistelli 1,3, Virginia Ulivieri 2, Silvia Giovanelli 2, Luciano Avio4, Manuela Giovannetti 1,3, Luisa Pistelli 2,3 1Department of Agriculture, Food and Environment, University of Pisa, Via del Borghetto 80, 56124 Pisa, Italy 2 Department of Pharmacy, University of Pisa, Via Bonanno 6, 56126 Pisa, Italy

3 Interdepartmental Research Center Nutrafood – Nutraceuticals and Food for Health, University of Pisa, Via del

Borghetto 80, 56124 Pisa, Italy

4 Institute of Agricultural Biology and Biotechnology, CNR, Milan, Italy

corresponding author: Laura Pistelli Email: laura.pistelli@unipi.it Tel: +39 0502216536 Fax +39 0502216532 Keywords

Health-promoting compounds, pterocarpans, furanocoumarins, volatile organic compounds, mycorrhizal symbiosis Abstract

 Secondary metabolites may be affected by arbuscular mycorrhizal fungi (AMF), which are beneficial symbionts associated with the roots of most plant species. Bituminaria bituminosa (L.) C.H.Stirt is known as a source of several phytochemicals and therefore used in folk medicine as vulnerary, cicatrizing, disinfectant agent. Characteristic metabolites found in B. bituminosa are furanocoumarins and pterocarpans which are used in cosmetics and as chemotherapeutic agents. Here we address the question whether AMF inoculation may affect positively the synthesis of these phytochemicals.

B. bituminosa plants were inoculated with the different AMF and several metabolites were assessed during full vegetative stage and flowering phase. Pigments (chlorophylls and carotenoids), polyphenols, flavonoids were spectrophotometrically determined; specific isoflavones (genistein), furanocoumarins (psoralene and angelicin), pterocarpans (bitucarpin A and erybraedin C) and plicatin B were assessed by HPLC method; leaf volatile organic compounds were analysed by SPME and identified by GC-MS.

 During the vegetative stage, the inoculated plants showed a high amount of furanocoumarins (angelicin and psoralen) and pterocarpans (erybraedin C and bitucarpin A). The analysis of volatile organic compounds of inoculated plants showed different chemical composition compared with non mycorrhizal plants.

 Given the important potential role played by furanocoumarins and pterocarpans in pharmaceutic industry, AMF inoculation of B. bituminosa plants may represent a suitable biotechnological tool to obtain higher amounts of such metabolites for pharmaceutical and medicinal purposes.

Introduction 1

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Bituminaria bituminosa (L.) C.H.Stirt. (alias Psoralea bituminosa L., Arabian pea or pitch trefoil, Leguminosae family) is a perennial legume species from the Mediterranean region. In this environment it is used to provide hay or forage for livestock and it is suitable for low-input, sustainable production systems, due to its N2-fixation and drought tolerance (Walker et al. 2010). B. bituminosa, like other Psoralea spp. plants, is known as a source of several phytochemicals and therefore used in folk medicine as vulnerary, cicatrizing, disinfectant agent (Gutman et al. 2000).

Phytochemicals are plant secondary metabolites representing a rich source of natural compounds which play beneficial roles in human health. The most widely investigated chemical compounds involved in protective effects on human health are represented by phenolics - simple phenols and polyphenols - namely flavonoids (flavonols, flavones flavanols, flavanones, anthocyanidins, isoflavonoids) and non-flavonoids, such as stilbenes, phenolic acids, coumarins, tannins. Other phytochemicals belonging to carotenoids, alkaloids, and organosulfur compounds may interact with physiological and pathological processes. Phenolics have been studied for their role as scavengers of free radicals, quenchers of single oxygen formation and for their positive effects on different processes in mammalian cells, encompassing anti-carcinogenic and anti-atherogenic properties (Duthie 2000). Flavonoids like quercetin, kaempferol, myricetin, have been reported as bioactive compounds able to reduce the risk of cardiovascular diseases, to exert a chemioprotective action and to have a phytoestrogenic activity (Tham et al. 1998).

Different phenylpropanoids are produced by B. bituminosa, such as flavonoids, furanocoumarins, represented by psoralen and its angular isomer angelicin, and isoflavones (daizein and genistein) (Boardley et al. 1986; Innocenti et al. 1997). The pterocarpans erybraedin C and bitucarpin A, together with the cinnamic ester plicatin B, were isolated and identified from B. bituminosa aerial parts (Pistelli et al. 2003). Such pterocarpans showed cytotoxicity in different human cancer cell lines (Cottiglia et al. 2005) and other effects, such as apoptosis induction in colon carcinoma cell lines and anti-clastogenic activity in lymphocytes (Maurich et al. 2004, 2006). Moreover, erybraedin C showed inhibitory activity on human topoisomerase I (Tesauro et al. 2010).

Several works showed that the content and composition of phytochemicals may be greatly affected by diverse factors, such as plant genotype, harvest season, cultivation site and techniques, soil quality, and by agronomic practices, i.e. quantity and quality of available nutrients and light, irrigation, use of pesticides and chemical fertilizers and conventional/organic management (Sbrana et al. 2014). One of the most promising factors affecting plant secondary metabolic pathway is represented by arbuscular mycorrhizal (AM) fungi (AMF), belonging to Glomeromycotina (Spatafora et.al. 2016), an important ecological and economical group of beneficial soil microorganisms living in association with the roots of most plants growing in natural and agricultural ecosystems (Rouphael et al. 2015). AMF affect the content and composition of secondary metabolites, such as polyphenols, flavonoids, carotenoids, phytoestrogens, and the activity of several antioxidant enzymes, both in crop and medicinal plants (Bruisson et al. 2016; Sbrana et al. 2014). In addition, they differentially affect the qualitative and quantitative accumulation of essential oils, volatile compounds (Hart et al. 2015; Welling et al. 2016) and glucosinolates (Cosme et al. 2014). The effect of AMF inoculation in medicinal and aromatic plants was reported in more than 50 plant species, including some endangered herbs and plants used for food additives (Zeng et al. 2013). For example, the inoculation of Glomus mosseae and Glomus versiforme transiently enhanced the levels of transcripts encoding phenylalanine ammonia-lyase in Medicago truncatula roots, while G. versiforme and Rhizophagus intraradices (formerly Glomus intraradices) increased chalcone synthase (CHS) transcripts accumulation in M. truncatula roots (Harrison and Dixon 1993; Bonanomi et al. 2001). The levels of antioxidant compounds, such as rosmarinic acid and caffeic acid, were enhanced in Ocimum basilicum after inoculation with various Glomus species (Copetta et al. 2006; Toussaint et al. 2007). A recent study confirmed the role 42 43

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of R. intraradices as a trigger of the overexpression of tyrosin amino-transferase in rosmarinic acid biosynthetic pathway (Battini et al. 2016). Mycorrhizal Hypericum perforatum plants showed enhanced concentrations of anthraquinone derivatives, such as hypericin and pseudohypericin (Zubek et al. 2012). A multidisciplinary work showed that tomato fruits produced by mycorrhizal plants contained higher levels (18.5%) of lycopene compared with controls and that their extracts - both hydrophilic and lipophilic fractions - strongly inhibited 17-β-oestradiol-human oestrogen receptor binding (Giovannetti et al. 2012).

In the present work, a multidisciplinary study was carried out to gain knowledge on the phytochemicals produced by B. bituminosa plants inoculated with the beneficial mycorrhizal symbiont R. intraradices or with native endophytes. To this aim, the establishment of mycorrhizal symbiosis was assessed, and biochemical parameters were evaluated at the vegetative and flowering stages. In particular, the production of the most important leaf secondary metabolites, such as flavonoids, furanocoumarins and pterocarpans was determined, together with leaf volatile organic compounds (VOCs) emission.

Materials and methods

Plant material

B. bituminosa plants were obtained by germination of seeds harvested from wild plants growing in Porto Azzurro, Elba Island, Livorno, Italy. Before germination the seeds were scarified by immersion in pure sulfuric acid for 50 minutes (Bouque et al. 1998), then rinsed with distilled water, and further sterilized with a 20% (v/v) sodium hypochlorite solution for 20 minutes to avoid microbial contamination. After the final washing with sterile distilled water, the seeds were incubated in sterile water in the dark at 25 °C for 48h, transferred on a rockwool substrate (Grodan® Pro Plug, Roermond, Netherlands) and fed with sterile Hoagland solution (Sigma Aldrich, Palo Alto CA, USA) for 25 days. Germination and seedling growth were performed in a growth chamber, with the following growing conditions: 22±1 °C temperature, with a 16/8 h photoperiod, irradiance 85 µmol m-2 s-1.

AMF inocula

In mycorrhizal treatments, two different kinds of inocula were applied. The first one was represented by the AM fungal species R. intraradices (N. C. Schenck & G. S. Sm.) C. Walker & Schussler (formerly known as G. intraradices) (isolate IMA5), obtained from Trifolium alexandrinum and Medicago sativa pot cultures maintained in the collection of the Soil Microbiology Laboratory of the Department of Agriculture, Food and Environment, University of Pisa, Italy (hereafter IMA5). The second inoculum was represented by the soil excavated from the immediate vicinity (within 10 cm) of B. bituminosa plants occurring in its natural habitat (Porto Azzurro, Elba Island, Livorno, Italy), and which were colonized by native AMF as assessed by staining of root tissue and identification of arbuscules (hereafter ELBA).

Aliquots of 25 g of crude inoculum (mycorrhizal roots and soil containing spores and extraradical mycelium) were used to inoculated individual plants. Plants used as control were not inoculated (hereafter NM).

Experimental design

The 25-days old rooted seedlings were transplanted into 1 L (10x10x12 cm) plastic pots (one seedling per pot) containing a mixture (1:1, by vol.) of soil (sandy loam collected near S. Piero (Pisa, Italy) and Terragreen (calcinated clay; OILDRI, Chicago, IL, USA) The soil mixture was steam-sterilised three times (121°C, 40 min interval) to kill 83

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naturally occurring AMF (Giovannetti et al. 2012). Physical and chemical characteristics of soil were as follows: water pH 7.7, total N 1.38 g kg-1 (Kjeldahl method), conductivity 48 µs, clay 18.1%, silt 19.5%, sand 62.4%, available P 11.4 mg kg-1 (Olsen method), organic matter 2.1%.

The experiment was setup as a 3 x 2 completely randomized factorial design inoculum type (NM, IMA5 and ELBA) and growth stage (first sampling vegetative stage, second sampling flowering stage) as main factors. Six plants for each inoculum type were used for the first sampling and 9 plants for each inoculum type for the second sampling, for a global number of 45 plants.

Each seedling received 50 mL of a bacterial filtrate to ensure a common microflora to all treatments. The bacterial filtrate was prepared as follows: 100g of native soil and 100g of crude inoculum were mixed and then suspended in 700 mL deionised water, filtered through 50µm pore diameter sieve and Whatman paper no. 1 (Whatman International Ltd, Maidstone, Kent, UK). Inoculated and control plants were grown in the growth chamber at the same growing condition (22±1°C temperature, 16/8 h photoperiod, irradiance 85 µmol m-2 s-1) and supplied with tap water and with a weekly fertilization of half-strength Hoagland’s solution (10 mL per pot).

Ninety days after inoculation all plants were transferred into the greenhouse under ambient conditions in spring (average Temperature 15-20°C, daylights 14 h, average humidity 79±6%) to complete their cycle. The first sampling (vegetative stage) was carried out 10 days after the transfer into the greenhouse. The second sampling (full flowering stage) was carried out 60 days after the first harvest. Each sample was obtained by collecting leaves from 2 or 3 plants (first and second sampling, respectively) producing a total of three homogenous samples for each treatment. Leaves were used fresh or after storage at -80°C. Dry weight was assessed at the vegetative and flowering stages.

Analysis of mycorrhizal colonization

Root samples from B. bituminosa plants collected at vegetative stage were washed in tap water, cleared with 10% KOH in water bath at 80 °C for 15 min, neutralised in 2% aqueous HCl and stained with 0.05% Trypan blue in lactic acid. Percentage of mycorrhizal root length was assessed on each root sample under a dissecting microscope (Wild, Leica, Milano, Italy) at x25 or x40 magnification, by the gridline intersect method (Giovannetti and Mosse 1980). Colonized roots were selected, mounted on microscope slides and observed under a Reichert–Jung (Wien, Austria) Polyvar light microscope to detect intraradical fungal structures.

Biochemical analyses

One hundred mg of fresh leaves per homogenous samples were used for the determination of pigments (chlorophyll and carotenoids), total polyphenols and 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) antiradical activity, according to the protocols reported in Pistelli et al. (2017). Total flavonoid content was determined using the colorimetric method of Kim et al. (2003). The presented data are the mean of three replicates from three independent and homogenous samples. HPLC analyses

A modified extraction procedure was followed in this work to analyse specific phytochemicals, avoid any acidic treatment to exclude the alteration of secondary metabolites present in the homogenous samples, according to D’Angiolillo et al. (2014). Leaves were extracted with chloroform (2 x 0.25 L, one week). The extracts were filtered through filter paper, and then dried by rotatory evaporator, and kept at -20°C until analysed. The dried chloroformic extracts (10 or 20 mg) were dried and dissolved in methanol (5 or 10 mL) to obtain known concentration, than filtered through a cartridge-type sample filtration unit with a polytetrafluoroethylene filter (PTFE, 0.45 μm, 25 mm) before 124

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HPLC injection. HPLC-DAD analyses were performed using a Waters 600E multisolvent delivery system, a Waters 717plus autosampler and a Waters 486 Tunable Absorbance Detector, equipped with Millennium32 Chromatography Manager software. Analyses were performed using a 250 x 4.60 mm, 5 µm Synergi Fusion RP-80 column (Phenomenex). For the determination of plicatin B, bitucarpin A and erybraedin C, each sample (5 or 20 µL) was analysed eluting in isocratic conditions with acetonitrile-water in 7:3 concentration (flow rate 1 mL min-1). Each sample was analysed in triplicate. For the determination of psoralen, angelicin and genistein 20 µL of each samples were analysed eluting with a mixture of acetonitrile (Solvent A) and water (Solvent B) in a linear gradient with the following conditions (flow rate: 1 mL min-1): initial conditions 50% A; from 0 to 10 min 50% A; from 10 to 15 min from 50% to 70% A; from 15 to 40 min 70% A; from 40 to 41 min back to 50% A, followed by a period of 10 min for column equilibration. The spectral data from the PDA detector were analysed during the whole run in the range 210-395 nm. The identification of each constituent was carried out by comparing the peaks obtained in the different chromatograms with the retention time and UV spectra of the standard compounds under the same chromatographic conditions. The authentic samples of bitucarpin A, erybraedin C, plicatin B, psoralen, angelicin, daidzein, and genistein were isolated and characterized by NMR and MS techniques in our laboratory (Pistelli et al. 2003) and their spectra were included in a home-made data base of natural compounds.

Calibration curves. The amount of the marker constituents was determined by the external standard method using five concentration levels for each compound. Standard calibration curves were prepared over a concentration range of 0.1- 0.5 mg mL-1. An aliquot (10 L) of each standard compound was analysed in triplicate under the same conditions used for the analyses of the extracts by HPLC-PDA-UV. The standard solutions of the authentic samples were dissolved in methanol. The retention times, the UV absorbance, the linear regression equations and the correlation coefficients of the external standards are show in Table 1.

Analysis of Volatile organic compounds (VOCs)

Volatile organic compounds (VOCs) spontaneously emitted by B. bituminosa leaves were analyzed according to the SPME (solid phase microextraction) method, Gas Chromatography with flame ionization detector (FID) - Gas Chromatography-Mass Spectrometry analysis reported in Pistelli et al. (2013). Identification of the constituents was based on comparison of their retention times with those of authentic samples, and on computer matching against commercial and home-made library mass spectra built from pure substances and MS literature data (Swigar and Silvestein 1981; Adams 1995; Davies 1990; NIST 1998). The relative proportions of the volatile constituents were percentages obtained by peak-area normalisation, and all relative response factors were taken as one.

Statistical analysis

Two-way ANOVA was performed on plant biochemical data, after assessing the homogeneity of variances by Levene test (SPSS 19.0 software, IBM Corp., Armon, NY Inc., USA), when needed data were transformed to homogenize variance. Means differences were determined using the Tukey procedure; when interactions were significant, differences among inoculum treatments within each growth stage, were assessed after one-way ANOVA. Genistein data were analysed by t-test procedure, furanocumarins data were analysed only for AMF plants. Percentage colonization data were arcsine-transformed before analysis.

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Mycorrhizal colonization and plant growth

R. intraradices IMA5 successfully established mycorrhizal symbioses with B. bituminosa plants, showing percentages of colonized root length ranging from 73 to 90% . The naturally occurring AMF (ELBA) showed lower mycorrhizal percentages, which ranged from 38 to 72%. No colonization was observed in control plants. Colonization pattern of both IMA5 and ELBA inoculated plants was very similar with arbuscules and many vesicles (Fig. 1). Dry weights of mycorrhizal and not mycorrhizal B. bituminosa plants did not differ at the vegetative stage, while ELBA inoculated plants showed a higher dry weight than IMA5 inoculated and control plants at the flowering stage (6.3±0.2 g vs 4.6±0.3 g and 3.9±0.8 g, respectively).

Metabolites analyses

Total chlorophyll content was determined in plants at two different ontogenetic stages. At the vegetative stage similar chlorophyll levels were detected in the three treatments, either without mycorrhiza (NM) or in presence of the inocula IMA5 and ELBA (Tab. 2). At the full flowering stage chlorophyll content was significantly higher in leaves originated from IMA5 inoculated plants, compared with the other two treatments (2.70, 1.95 and 1.72 mg g-1 fresh weight, in IMA5, ELBA, and NM, respectively). Similar results were obtained for carotenoids, whose content in leaves was significantly higher in IMA5 inoculated plants than in control and ELBA treatments, at the full flowering stage (Tab. 2). Total polyphenols increased in all treatments at the flowering stage in comparison to values of the vegetative stage. The lowest amount was observed in ELBA leaves during the vegetative stage, while no statistical differences were found among the three treatments at flowering stage (Tab. 2). Total flavonoid content and antioxidant activity were similar in the three treatments (Tab. 2).

HPLC determination of specific metabolites

Characteristic secondary metabolites of B. bituminosa were investigated by HPLC-DAD analyses (Tab. 3). The isoflavone genistein was only detectable in AMF plants during the flowering stage, without any statistical difference (38.5 and 64.6 g g-1 dry extract for ELBA and IMA5 leaves respectively).

Within furanocoumarins, angelicin levels were higher than psoralen, at both plant developmental stages. AMF plants showed higher amounts of both compounds at the vegetative than at the flowering stage, independently of inoculum type. In control plants these metabolites were only detectable at flowering stage, when the concentration is significantly higher than AMF plants.

Pterocarpans (bitucarpin A and erybraidin C) and plicatin B metabolites are differently affected by inoculation treatments depending on growth stage as shown by the significant interaction (Tab. 3). Both bitucarpin A and erybraidin C levels were significantly higher in control leaves (1.25 and 5.86 g g-1 dry extract, respectively) than in mycorrhizal leaves at the flowering stage (Tab. 3). The cinnamic ester plicatin B was 7-times and 12-times higher in control plants than ELBA and IMA5 inoculated plants, at flowering stage. (Tab. 3).

VOCs analysis

The headspace analysis of the B. bituminosa leaves was performed at the flowering period (Tab. 4). VOCs composition was different between the treatments. Concerning the class of compounds, leaves from control plants showed high percentages of sesquiterpene hydrocarbons (sh, 42.7%), followed by non-terpenoids (nt, 22.7%) and monoterpene hydrocarbons (mh, 14.7%). The most representative components were E-β-farnesene (21.1%) and (Z)-3-hexenol acetate (12.4%). 206 207 208

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Leaves harvested from IMA5 inoculated plants showed the following amounts: sesquiterpene hydrocarbons (31.8%) > monoterpene hydrocarbons (31.0%) > non-terpenoids, (17.1%). E-β–farnesene (14.9%) was the most abundant compound together with tricyclene (11.1%), (Z)-3-hexenol acetate (10.8%) and camphene (6.8%).

ELBA mycorrhizal plants emitted VOCs with different percentages; non-terpenoids were the most representative components (71.3%), followed by sesquiterpene hydrocarbons (14.0%) and monoterpene hydrocarbons (7.8%). The leaves gave a great percentage of (Z)-3-hexenol acetate (63.8%), while (E)-β-farnesene was reduced to 5.3%.

Discussion

Here, for the first time, we reported that AMF affected the production of pterocarpans and furanocoumarins in B. bituminosa leaves, during both vegetative and reproductive developmental stages.

During the vegetative stage, mycorrhizal plants showed significantly higher amounts of angelicin and psoralen, compared with control plants, where these molecules were not detectable: such compounds represent the typical furanocoumarins in Fabaceae plants, in particular angelicin, which has been reported as the major component of furanocumarins in wild grown B. bituminosa leaves sampled in Elba island (D’Angiolillo et al. 2014). During the flowering stage, AMF inoculation decreased the production of bitucarpin A and erybraidin C, the latter representing the major component of pterocarpans in wild grown B. bituminosa leaves (D’Angiolillo et al. 2014). The only data available on the role of AMF in the development and growth of B. bituminosa plants concern its ability to survive in polluted sites, which increased when inoculated with AMF (Alguacil et al. 2011). However, in such a study no biochemical data were given on the influence of AMF on B. bituminosa phytochemical contents. The positive influence of AMF on the levels of furanocoumarins in the leaves of B. bituminosa, suggests that these beneficial symbionts may represent an important tool to obtain B. bituminosa metabolites of pharmaceutical importance, as the furanocoumarins psoralen and angelicin are currently used in cosmetics and as photochemoterapeutic agents (Bordin et al. 1991).

The majority of works assessing the production of secondary metabolites by mycorrhizal medicinal plants have considered the roots, and only few data are available on the influence of AMF in metabolite biosynthesis in leaves, flowers and fruits (Sbrana et al., 2014). For example, leaves and flower heads of globe artichoke plants showed large increases in total polyphenolic content and antioxidant activity after inoculation with G. mosseae and R. intraradices (Ceccarelli et al. 2010). In the leaves of Ocimum basilicum the content of antioxidant compounds, such as rosmarinic and caffeic acids, was enhanced by different AMF (Copetta et al. 2006; Toussaint et al. 2007), while an increased level of key enzymes for the biosynthesis of rosmarinic acid was recently reported (Battini et al. 2016). Interestingly, Schweiger et al. (2014b) defined ‘phytometabolome’ the leaf metabolites produced by some mycorrhizal plants, focusing their attention on primary metabolites - carbohydrates, organic acids, aminoacids -, and found that such phytometabolomes were either species- or taxon-specifically regulated. In our work, the content of specific secondary metabolites in B. bituminosa leaves was differentially affected by the developmental stage and the mycorrhizal status, as angelicin and psoralene levels were much higher at the vegetative than at the flowering stage in AMF inoculated plants. On the contrary, plicatin B content was much higher in control plants during the flowering stage. These findings confirm that the synthesis of phytochemicals in B. bituminosa is regulated by different factors, as suggested also by Walker et al. (2012), who reported that furanocoumarins in the leaves B. bituminosa wild plants varied depending on drought stress and season. Other factors affecting the production of phytochemicals were previously reported also by Larose et al. (2002), who demonstrated that flavonoid accumulation in M. sativa roots was dependent on the 248

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developmental stage of the symbiosis and on the identity of root-colonizing AMF. Recently Bruisson et al. (2016) reported the influence of AMF in the phenylpropanoid biosynthesis and stilbenoid production in grapevine leaves , affected by pathogen infections.

No differences were observed between control and mycorrhizal plants for other class of compounds (photosynthetic pigments, polyphenols, flavonoids) in B. bituminosa leaves during the vegetative stage, although a significantly higher concentration of total chlorophyll and carotenoids was detected during the full flowering period in particular in the mycorrhized plants. Our data differ from those reported by other authors on differential photosynthetic performance in mycorrhizal plant species during the vegetative stage (Schweiger et al. 2014a). Total flavonoids represent the large majority of total polyphenols detected in the vegetative stage, whereas polyphenols showed higher amounts in reproductive stage in comparison with flavonoids. So it could be hypothesized that in the flowering stage other phenolic compounds are biosynthesized.

All these results reported in table 2 and 3 let us to suggest a different distribution of metabolites in the flowering stage between the treatments. In fact, AMF plants exhibited a higher dry weight and a lower content of the typical secondary metabolites, while an opposite behavior was observed NM plants. The hypothesis is that the photoassimilates in AMF plants are addressed either to roots for the AMF nutrition or to increase the aerial biomass, with a consequent low production of secondary metabolites. On the contrary the NM plants showed different biochemical pathways, where the synthesis of plicatin B and furanocumarins were stimulated due to the absence of AMF. However the predicted assimilate distribution cannot be directly linked to the amount of photosynthetic pigments, so further studies related to the sugars content will be necessary to confirm this hypothesis.

In this work VOCs emission, monitored in the aerial part of B. bituminosa, showed that inoculated plants had a different chemical composition, compared with non mycorrhizal plants. In addition, changes in the composition of VOCs classes were found: leaves of control plants showed high percentages (42.7%) of sesquiterpene hydrocarbons, while leaves from ELBA-inoculated plants emitted mostly non-terpenoids (71.3%). In IMA5-inoculated plants, the most representative VOCs components in leaves were sesquiterpene hydrocarbons and monoterpene hydrocarbons (31.0%). Such data compare well with previous works reporting changes in terpenoid concentrations associated with mycorrhiza symbiosis, which can positively affect the terpenoid yield, depending on plant and fungal genotypes (Welling et al. 2016). Accordingly, the emission of limonene and artemisia ketone was stimulated by AMF inoculation, although VOCs emission and total terpene content of Artemisia annua was not affected (Rapparini et al. 2008). Overall, we cannot exclude that different distributions of terpenoids could be detected in relation to plant tissues and organs, as monoterpenes and sesquiterpenes were reported to accumulate in glandular trichomes, while diterpenes and carotenes were found in fruits, leaf or root tissues (Welling et al. 2016). The analysis of single components revealed some discrepancy from previous findings: IMA 5 and ELBA inoculated plants exhibited components that were absent in spontaneous plants grown in open field (Bertoli et al. 2004).

Conclusions

In this work plants of B. bituminosa inoculated with the AMF species R. intraradices or native endophytes showed high amounts of secondary metabolites as furanocumarins and pterocarpans at the vegetative stage. Mycorrhizal B. bituminosa plants may represent an important source of such metabolites for pharmaceutical and medicinal purpose, as 289

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the furanocoumarins psoralen and angelicin are currently used in cosmetics and as photochemoterapeutic agents, and the pterocarpan erybraedin C and bitucarpin A may induce apoptosis in colon carcinoma cell lines.

Acknowledgments

This work was funded by the University of Pisa (Fondi di Ateneo).

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Figure 1: Mycorrhizal colonization of Bituminaria bituminosa plants. A, B) Light micrograph of IMA5 colonized roots of B. bituminosa at vegetative stage showing intraradical hyphae, vesicles (A, scale bar: 25m) and arbuscules (B,, scale bar: 250m). C) percentage of mycorrhizal root length of ELBA plants inoculated with native mycorrhizal endophytes from Elba island and IMA5 plants inoculated with Rhizophagus intraradices IMA5 (mean ± SEM; n=6). Different letters above the bars indicate statistically significant differences (p<0.05).

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Table 1 HPLC-DAD analysis of the target compounds used as external standards: retention time, linear regression equations and correlation coefficients

compound Rt ( min ) λmax

Concentration range ( ppm ) correlation coefficients linear regression equation Genistein 6.2 259.5 0-3200 0.999 y = 23348x Plicatin B 6.7 232-315 0.992 y =10106x Psoralen 8 244-291 0-3000 0.987 y = 15798x Angelicin 8.5 246-298 0-3000 0.994 y = 27463x Erybraidin C 10.7 287 0-4000 0.981 y = 2945x Bitucarpin A 18 284 0-4000 0.994 y = 30045x 466

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Table 2. Determination of foliar metabolites (mg g−1 FW) and the DPPH free radical scavenging activity of B. bituminosa plants collected during two growing stages (GS) in different inoculum type (NM, ELBA; IMA5, see the material and methods). Mean values were obtained from 3 independent replicates ± SD and were analysed by two way analysis of variance. Means followed by the same letters within columns are not significantly different by Tukey’s test at 5% level. When interactions are significant letters indicate statistically different values among inoculum treatment within each growth stage after one-way ANOVA.

Inoculum type Total

chlorophyll (mg g-1 FW) Total carotenoids (µg g-1 FW) Total polyphenols (mg g-1 FW) Total flavonoids (mg g-1 FW) DPPH (IC50 mg mL-1) vegetative stage NM 1.33 ± 0.43 a 98.07 a 3.28 ± 0.53 b 4.06 ± 0.20a 2.03 ± 0.20 a ELBA 1.64 ± 0.44 a 120.62 a 2.57 ± 0.27 a 3.54 ± 0.98 a 3.37 ± 0.78 a IMA5 1.20 ± 0.91 a 85.52 a 3.25 ± 0.38 b 3.28 ± 0.53 a 2.73 ± 0.7 a flowering stage NM 1.72 ± 0.61 a 88.54 ± 18.63 a 8.58 ± 0.93 a 4.22 ± 0.11 a 1.19 ± 0.27 a ELBA 1.95 ± 0.52 ab 105.85 ± 14.84 ab 9.80 ± 0.86 a 4.75 ± 1.01 a 1.29 ± 0.23 a IMA5 2.70 ± 0.21 b 123.60 ± 8.64 b 9.03±1.36 a 4.09 ± 0.47 a 1.67 ± 0.12 a ANOVA p value Inoculum type 0.15 0.023 0.749 0.244 <0.001 Growth stage (GS) <0.001 0.412 <0.001 0.05 0.412 Inoculum type x GS 0.021 0.002 0.013 0.201 0.03 470

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Table 3. Amount of foliar metabolites of B. bituminosa plants collected during as affected by growing stages (GS) and inoculum type (NM. ELBA; IMA5). Mean values were obtained from 3 independent replicates ± SD.

Isoflavones Furanocoumarins Pterocarpans Cinnamic esters

Inoculum type Genistein g g-1 dry extract Psoralene mg g-1 dry extract Angelicin mg g-1 dry extract Bitucarpin A g g-1 dry extract Erybraidin C g g-1 dry extract Plicatin B g g-1 dry extract vegetative stage NM n.d. n.d. n.d. 2.56 ± 1.18 a 5.49 ± 2.04 a 12.78 ± 6.99 a ELBA n.d. 0.78 ± 0.71 14.70 ± 9.37 2.22 ± 1.10 a 4.04 ± 3.38 a 9.66 ± 9.01 a IMA5 n.d. 0.31 ± 0.10 6.91 ± 4.07 2.95 ± 0.42 a 9.93 ± 0.19 a 14.83 ± 9.45 a flowering stage NM n.d. 0.18 ± 0.07 1.40 ± 0.11 1.25 ± 0.76 a 5.86 ± 2.33 a 22.62 ±7.19 a ELBA 38.5 ± 5.0 0.06 ± 0.06 0.33 ± 0.25 0.13 ± 0.04 b 1.17 ± 0.34 b 3.32 ± 1.36 b IMA5 64.6 ± 24.7 0.01 ± 0.004 0.16 ± 0.07 0.07 ± 0.03 b 0.75 ± 0.25 b 1.85 ± 0.77 b ANOVA p value Inoculum type Genistein

$

Psoralene* Angelicin* Bitucarpin A** Erybraidin C** Plicatin B** Inoculum type 0.256 0.163 0.172 0.038 0.022 0.019 Growth stage (GS) 0.004 <0.001 <0.001 0.001 0.157 Inoculum type x GS 0.595 0.280 0.022 0.003 0.028

$ t test analysis performed on data of ELBA and IMA5 treatments

*Two way analysis performed on data of ELBA and IMA5 treatments. after square root transformation

**Two way analysis performed on square root transformed data. Different letters indicate statistically different values among inoculum treatment within each growth stage after one-way ANOVA.

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Table 4: Main volatile compounds and classes of compounds (%) emitted by B. bituminosa leaves in the flowering stages. Data are shown as mean ± standard deviation (SD). SD below 0.09 is not reported. Legend: l.r.i. = linear retention indices (DB-5 column); mh = monoterpene hydrocarbons; om = oxygenated monoterpenes; sh = sesquiterpene hydrocarbons; os = oxygenated sesquiterpenes; nt = non-terpenoid substances; tr = percentage.

compound l.r.i. NM ELBA IMA5

nt 1,3 butanediol 769 tr nt (E)- 3-hexen-1-ol 851 2.9 mh santoline triene 908 1.9 0.7 4.6 mh tricyclene 926 4.8 11.1 mh α-pinene 939 2.5 mh camphene 953 3 1.4 6.8 mh sabinene 976 0.3 0.4 0.6 mh myrcene 991 2.1 1.9 3.3 nt (Z)- 3-hexenol acetate 1016 12.4 63.8 10.8 mh limonene 1031 1.7 0.6 1.9 mh (E)-β-ocimene 1050 0.9 0.3 2.7 nt 1-octanol 1070 0.1 nt n-undecane ( C11 ) 1100 0.3 nt n-nonanal 1102 2.3 0.2 0.3

nt phenyl ethyl alcohol (phenetol ) 1110 0.2 0.4 0.2

nt (Z)-3-hexenyl isobutyrate ( isobutanoat) 1145 0.1 0.1

nt (Z)-3-hexenyl butyrate 1186 1.6 nt methyl salycilate 1190 0.1 nt n-dodecane 1199 0.6 0.3 0.3 nt n-decanal 1204 1.1 0.3 0.8 om geraniol 1255 0.2 nt heptadecene 0.1 nt n-tridecane (cis) 1299 0.7 0.4 1

om trans-carvyl acetate (methylanthranylate) 1337 0.1 0.4

sh cyclosativene 1368 0.5 0.2 sh α-copaene 1376 2.5 1.4 1.6 om geranyl acetate 1383 0.8 0.7 0.4 sh β-elemene 1391 1 0.4 1 nt ethyl-decanoate 1394 1.9 0.5 1.4 nt n-tetradecane 1399 0.7 0.3 0.7 nt dodecanal 1407 0.6 0.1 0.4 sh β-caryophyllene 1418 8.9 2.2 4.3 sh β-copaene 1429 0.3 0.2 0.3 sh α-humulene 1454 2.3 0.5 1.2 sh (E)-β-farnesene 1454 21.1 5.3 14.9 sh γ-muurulene 1477 0.7 0.3 0.5 sh germacrene D 1481 2.8 2.5 5.4 sh bicyclogermacrene 1494 0.3 0.3 0.5 sh α-muurulene 1499 0.8 0.3 0.8 sh (E.E)-α-farnesene 1508 0.8 0.2 0.4 sh δ-cadinene 1524 0.7 0.2 0.9 nt (Z)-3-hexenyl benzoate 1570 0.6 0.1 0.4 os germacrene D-4-ol 1574 0.3 0.1 0.2 os caryophyllene oxide 1581 1.7 0.3 0.7 nt n-hexadecane ( C16 ) 1600 1.1 0.2 0.7 TOTAL 82.9 94.5 81.6 Monoterpene hydrocarbons (mh) 14.7 7.8 31

Oxygenated monoterpenes (om) 0.8 1 0.8

Sesquiterpene hydrocarbons (sh) 42.7 14 31.8

Oxygenated sesquiterpenes (os) 2 0.4 0.9

Non-terpenoid substances (nt) 22.7 71.3 17.1 488 489

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82.9 94.5 81.6

494

Riferimenti

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