Modified distribution in the polyphenolic profile of rosemary leaves induced by plant inoculation with an arbuscular mycorrhizal fungus Running title: Polyphenolic changes in rosemary leaves induced by mycorrhizas

BACKGROUND
Rosemary forms an arbuscular mycorrhizal (AM) symbiosis with a group of soilborne fungi belonging to the phylum Glomeromycota, which can modify the plant metabolome responsible for the antioxidant capacity and other health beneficial properties of rosemary.


RESULTS
The effect of inoculating rosemary plants with an AM fungus on their growth via their polyphenolic fingerprinting was evaluated after analyzing leaf extracts from non-inoculated and inoculated rosemary plants by ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS). Plant growth parameters indicated that mycorrhizal inoculation significantly increased plant height and biomass. Chemical modifications in the plant polyphenolic profile distribution were found after a principal components analysis (PCA) loading plots study. Four compounds hosting strong antioxidant properties - ferulic acid, asiatic acid, carnosol, and vanillin - were related to mycorrhizal rosemary plants while caffeic and chlorogenic acids had a higher influence on non-mycorrhizal plants.


CONCLUSION
Mycorrhization was found to stimulate growth to obtain a higher biomass of plant leaves in a short time, avoiding chemical fertilization, while analytical results demonstrate that there is an alteration in the distribution of polyphenols in plants colonized by the symbiotic fungus, which can be related to an improvement in nutritional properties with future industrial significance. © 2018 Society of Chemical Industry.


Introduction
Rosemary (Rosmarinus officinalis L.) shrubs are ubiquitous in Mediterranean environments and intensively cultivated in nurseries for revegetation of degraded land, sustainable landscaping, medicinal and culinary purposes. The species belongs to the Lamiaceae family and, among other herbs, it is a valuable plant due to its high content in active ingredients with therapeutic, aromatic and organoleptic properties. 1 Those characteristics make the crop very attractive for pharmacological applications, as well as for nutraceutical and food industries. The effects on human health of bioactive compounds from plants has been a major research topic in the last decades focusing on their functional skills. 2 Concerning rosemary, a high content in leaves of phenolic acids, flavonoids, essential oil, triterpenic acids and triterpenic alcohols can be responsible for stimulating the nervous and the circulatory systems 3 and for providing anticancer effects. 4 As most vascular plant species in all geographical terrestrial areas, 5 rosemary forms an arbuscular mycorrhizal (AM) symbiosis with a group of soilborne fungi belonging to the phylum Glomeromycota. 6 In this mutualistic association, the fungus benefits from the plant by acquiring photosynthates delivered by the host while it helps the plant absorbing nutrients from the soil through an extended root system via the fungal extraradical mycelium. The arbuscular mycorrhizal relationship, besides facilitating nutrient uptake by plants and significantly stimulating plant growth, 7 has proven to increase tolerance against abiotic stresses such as drought, salinity and soil toxicity, 8 and to outgrow the disease damage caused by plant pathogens. 7 The plant's physiology is positively altered by the symbiosis with quantified changes in root exudation 9 and in the composition of secondary metabolites, 10 often related to plant defense mechanisms. 11 The symbiosis forms spontaneously in natural ecosystems where native AM fungal propagules are present in undegraded soils. When plants are industrially produced in intensive nursery agronomic systems on free-soil substrates, they lack mycorrhizas in their roots unless they were artificially inoculated with selected AM fungi. Accordingly, the plant metabolomics composition in active compounds will most probably not be the same than the one expected to be found in a plant with a fully established mycorrhizal root system. An experimental set up was designed in order to confirm the latter hypothesis.
Polyphenols are aromatic secondary metabolites ubiquitously spread through the plant kingdom comprising more than eight thousand substances with highly diverse structures. The main reasons for the interest in polyphenols deals with the recognition of 4 their antioxidant properties, the great abundance in the diet, and the important role in the prevention of various diseases. 12-14 Furthermore, polyphenols, which also constitute the active substance found in many medicinal plants, modulate the activity of a wide range of enzymes and cell receptors. 15 They are also playing an important role in plant defense mechanisms being involved in the interaction between pathogens and the plants. 1,16,17 The analysis of polyphenols in plant materials is relatively complex due to the great variety of compounds that can be present, which differ in polarity and size (from simple phenolic acids to tannins), but also because many of these compounds are found at low concentration levels. Liquid chromatography coupled to mass spectrometry (LC-MS) and tandem mass spectrometry (LC-MS/MS) is the most effective technique for the structural characterization and determination of polyphenols in a great variety of sample matrices. 18,19 Recently, high resolution mass spectrometry (HRMS) and accurate mass measurements have gained popularity due to their great ability to provide more comprehensive information concerning the exact molecular mass, elemental composition and detailed molecular structure of a given compound, being today one of the better options when dealing with the characterization and determination of polyphenols in plant derived products. 19,20 In the present work, the polyphenolic profile in leaves of greenhouse grown mycorrhizal and non-mycorrhizal rosemary plants was evaluated by UHPLC-HRMS in a quadrupole-Orbitrap mass spectrometer. Rosemary plant leaves from both mycorrhizal and non-mycorrhizal rosemary plants were periodically collected at different time periods, and bioactive compounds extracted using methanol followed by a simple solidphase extraction (SPE) clean-up step with C18 cartridges. After UHPLC-HRMS analysis of the obtained plant extracts, a targeted polyphenolic approach using a target accurate mass database list comprising 55 characterized polyphenols was employed, and the obtained polyphenolic profiles were then subjected to exploratory principal component analysis (PCA) to establish patterns showing the effect of plant inoculation with an arbuscular mycorrhizal fungus on rosemary plant polyphenolic distribution.

Samples and sample treatment
The effect of inoculating rosemary plants with AM fungi on the growth of rosemary plants via their polyphenolic fingerprinting was evaluated as follows: 5 Rosemary rooted cuttings were used in the experimental set up. Before planting, fifteen leaves from ten cuttings, chosen at random and labelled as RT0, were collected and refrigerated until their analysis. At the same time, one hundred cuttings were planted in 1.5 L containers filled with a substrate mixture of autoclaved sandy soil (120ºC, 1hour), quartz sand and sphagnum peat (3 Root samples were also excised from eight plants per treatment in order to estimate the mycorrhizal root colonization extent. Root samples were clarified and stained, 25 and the percentage of mycorrhizal root was measured. 24 Sample extraction was performed as follows: 0.1 g of leaf samples from ten plants were suspended in 6 mL of LC-MS grade methanol and mechanically extracted by employing Ultraturrax T25 basic (Ika-Werke, Staufen, Germany). The extract solution was then processed by solid-phase extraction (SPE) for clean-up using Discovery ® DSC-18 SPE (500 mg, 6 mL) cartridges obtained from Supelco (Darmstadt, Germany), that were previously activated-conditioned with LC-MS grade methanol. The first mL was discarded, and then a portion of 1.5-2 mL of extract solution was transferred into 2 mL injection vials. The extracts were then stored at -4 o C until their analysis with the proposed UHPLC-HRMS method.
Additionally, a chemometric quality control (QC) sample was prepared by mixing 50 µL of each sample extract.

Chemicals
Unless otherwise stated, all reagents were of analytical grade. Fifty-five polyphenolic standards belonging to different families (phenolic acids, benzoic acids, cinnamic acids, phenolic aldehydes, phenolic terpenes, flavones, flavanols, proanthocyanidins and stilbenes), all of them obtained from Sigma-Aldrich (Steinhein, Germany), were employed to build a user target accurate mass database for TraceFinder TM software. Stock standard solutions of studied polyphenolic standards (~1000 mg/L) were prepared in methanol in amber-glass vials. Intermediate working solutions were prepared weekly from these stock standard solutions by appropriate dilution with water. All stock solutions were stored at 4 o C for not more than one month.

Instrumental conditions
UHPLC separation was performed on an Accela liquid chromatography system (Thermo Fisher Scientific, San José, CA, USA), equipped with a quaternary pump, an autosampler and a column oven. An Accucore C18 reversed-phase (150 × 2.1 mm, 2.6 µm particle size) fused-core column (Thermo Fisher Scientific) was used for the proposed 7 method. Gradient separation was created from solvent A (0.1 % formic acid aqueous solution) and solvent B (acetonitrile) as follows: 0-1 min, isocratic elution at 10% B; 1-20 min, linear gradient elution from 10 to 95% B; 20-27 min, at 95% B; 27-28 min, back to initial conditions; and from 28 to 33 min, at 10% B for column re-equilibration. The mobile phase flow rate was 300 µL/min. Column was kept at room temperature and the injection volume used was 2 µL.
The UHPLC instrument was coupled to a Q-Exactive The data was acquired using data-dependent scan, using a full scan followed by a product ion scan for those ions above a threshold intensity value of 10 5 . The selected ions were

Data analysis
Polyphenolic fingerprints for each analyzed rosemary plant were obtained as follows: HRMS raw data was processed using TraceFinder TM 3.3 software (Thermo Fisher

Results and discussion
Growth parameters measured indicated that mycorrhizal inoculation with R.
irregulare significantly increased plant height and shoot biomass (Figures 1 and 2). RTM plants were already significantly higher than RTC plants six weeks after inoculation. By the end of the experiment, thirty weeks after mycorrhizal inoculation, the root colonization extent achieved by the symbiotic fungus in RTM plants was 89 + 4 % (mean + SD) while no mycorrhizal colonization was observed in RTC plants. Previous results already demonstrated growth stimulation due to mycorrhizal colonization of rosemary plants under greenhouse and field conditions as well as a higher production of essential oils. 28 Those results obtained after inoculation with an AM fungus may be related to chemical variations in the plant metabolomics, which can be associated to modifications in the distribution as well as the contents of plant bioactive compounds such as polyphenols.

UHPLC-HRMS Polyphenolic Fingerprinting
. In the present work, the polyphenolic fingerprints of control rosemary plants (non-inoculated) as well as those inoculated with R. irregulare were studied at different time periods while the plants were growing after the inoculation process.As an example, Figure 3 shows the total ion chromatogram (TIC) for sample RT0-5. For polyphenolic fingerprinting, a threshold signal of 10 5 was set in the screening software to consider that a compound could be present in a sample, and several confirmation parameters such as accurate mass measurements (mass error lower than 5 ppm) and isotope pattern fit (higher than 85%) were used to confirm the presence of the compound. Additionally, chromatographic retention times and product ion spectra were employed to ensure the identity among the 55 native polyphenols studied. After raw data processing with TraceFinder TM a report was provided for each rosemary plant extract analyzed (see Table   1 as an example).. It should be commented that TraceFinder TM software is only assigning a match when an expected m/z value within the accurate mass database list is found in the sample, but isobaric compounds are not differentiated. In those cases, and when standards are available, assignment was also performed from the chromatographic retention time.
As an example, Figure 3 shows

Exploratory principal component analysis
The data matrix to be analyzed included the rosemary plant polyphenolic profiles from the peak areas provided by TraceFinder TM software at the different retention times assigned (native polyphenols and their derivatives). The dimension of the data set was 123 samples × 194 variables. This matrix was then subjected to a non-supervised exploratory PCA method. Data was autoscaled with respect to the overall polyphenolic signal to provide similar weights to all the samples. PCA results showed that 4 PCs allowed to explain most of the data variance observed within the analyzed samples. Figure   4 shows the score plots of PC1 vs PC2. As can be seen, QCs appeared grouped in the center of the plot, demonstrating the good repeatability and robustness of the chemometric method employed and the feasibility of the PCA results.   samples. However, many of these variables were not native polyphenolic compounds but derivatives or isomeric forms that were not completely identified (not the aim of the present contribution). In Figure 6, the names of the variables that are identified and confirmed from chemical standards available are given. From this study, it was concluded that several polyphenolic compounds hosting strong antioxidant properties such as ferulic acid, 29 asiatic acid, 30 carnosol, 31

Conclusions
The process of artificially inoculating rosemary plants with effective arbuscular mycorrhizal fungi in nurseries can lead to several benefits according to the results exposed. Mycorrhization will clearly stimulate growth in order to obtain a higher biomass