Foliar bioactive compounds in Amburana cearensis (Allemao) A.C. Smith seedlings: Increase of biosynthesis using mycorrhizal technology

Programa de Pós-Graduação em Biologia Celular e Molecular Aplicada, Instituto de Ciências Biológicas, Universidade de Pernambuco, Rua Arnóbio Marques, 310, Santo Amaro – 50100130 Recife, PE-Brasil. Laboratório de Fisiologia Vegetal, Instituto de Ciências Biológicas, Campus Santo Amaro, Universidade de Pernambuco, Rua Arnóbio Marques, 310, Santo Amaro – 50100130 Recife, PE-Brasil. Laboratório de Enzimologia e Fitoquímica Aplicada a Micologia, Universidade de Pernambuco, Campus Petrolina, 56328-900, Petrolina, PE-Brasil.


INTRODUCTION
Arbuscular mycorrhizal fungi (AMF) are inhabitants of the soil and belong to the Phylum Glomeromycota (Schubler et al., 2001).Such organisms are obligatory symbionts because they complete their life-cycle only in the presence of a host plant (Souza et al., 2008).After the fungus has established on the root, the AMF absorb water and nutrients from the soil and in exchange the phytobiont makes about 20% of carbon available for the development of the fungus (Smith and Read, 2008).
Various studies relate the benefits of mycorrhizal association with legumes and point out the increased vegetable growth and optimized production of primary (Manoharan et al., 2010) and secondary metabolites (Silva et al., 2014a;Nisha and Rajeshkumar, 2010;Kapoor et al., 2004).
The increase in the production of secondary compounds in plants associated with AMF may be due to the increased nutritional supply (Toussaint et al., 2007), hormonal changes, enzymatic activation (Zhang et al., 2013) and increased activity of plastidial and mitochondrial pathways (Lohse et al., 2005), however, the effects seem to be somatory and multifactorial (Toussaint et al., 2007).
The Caatinga is a biome that is rich in leguminous species with medicinal properties that are widely used by the local population as phytotherapeutic drugs (Agra et al., 2007(Agra et al., , 2008)).Amburana cearensis is found among the medicinal plants of the Caatinga, a legume that is used by the local population.Parts of this plant, such as the stem, the seeds and bark are used in the production of pastilles, syrups and teas for the treatment of various diseases due to their antioxidant (Leal et al., 2003), antiinflammatory (Leal et al., 2008), antifungal (Santos et al., 2009), antibacterial (Figueiredo et al., 2013) and antineoplastic (Costa-Lotufo et al., 2003) properties.Such therapeutic benefits have been attributed to the presence of secondary compounds, especially phenolic compounds (Canuto and Silveira, 2006;Bravo et al., 1999).However, it is unknown in mycorrhizal that inoculation influences the increase in the production of secondary metabolites in A. cearensis seedlings.Therefore, the following hypothesis was tested: inoculation with AMF increases the production of bioactive compounds in A. cearensis with the benefits depending on the fungus that was tested.The aim of this study was to examine the efficiency of the AMF in increasing the production of foliar bioactive compounds in A. cearensis seedlings.

Plant, AMF and experimental implementation
A. cearensis seeds were disinfected with 20% of NaClO (2% of active chlorine) for 2 min, washed in distilled water and put to germinate in plastic pots containing sterilized soil (autoclave at 121°C/30 min/2 consecutive days).
Plantlets with two definite leaves were transferred to the pots and inoculated at the root region with soil-inoculum of the tested AMF (200 glomerospores + colonized roots + hyphae).A. cearensis seedlings remained under experimental roofing for 160 days at the University of Pernambuco -Campus Petrolina, Brazil, under ambient temperature conditions (minimum: 21.7°C and maximum: 29.7°C), relative air humidity (42%) and an average global radiation (461.8 ly/day).

Evaluation of the experiment and preparation of the extract
The experiment was evaluated 160 days after inoculation.Chlorophylls (total, a and b) were tested in vivo, using the CFL1030 -an electronic chlorophyll level meter ClorofiLOG (Silva et al., 2014a).After examining chlorophyll, the aerial part was separated from the roots and dried (45°C) for 3 consecutive days to determine the dry matter of the aerial part.The subterranean part was removed from the substrate and the fine roots were separated from the stylopodium, washed and preserved in ethanol (50%) until examination.
Aliquots (100 mg) of the leaves were punctured and put in amber flasks containing 20 ml of ethanol (95% v/v) and maceration lasted 12 days at 25°C.After this period, the extract was filtered with gauze and refiltered with qualitative paper filter and stocked in amber flasks (-4°C) (Brito et al., 2008).The extract was used to quantify the biomolecules.

Analysis of soluble carbohydrates and total proteins
Total proteins were quantified by a modification of the Bradford (1976): 50 µl of the extract was added to 2.5 ml of Bradford reagent and readings were taken with a spectrophotometer (595 nm) with a standard BSA curve (Bovine Serum Albumin).Total soluble carbohydrates were determined by a modification of the Dubois et al. (1956) method.The following was added to a test tube: 20 µl of the plant extract, 95 µl of distilled water, 50 µl of 80% phenol (w/v) and 2 ml of sulfuric acid.Readings were taken with a spectrophotometer (490 nm) and glucose was used to prepare the standard curve.

Analysis of phenols, flavonoids and total tannins
Total phenols were determined by a modification of the Folin-Ciocalteu method (Monteiro et al., 2006).The following was added to 100 ml volumetric balloons: 1 ml of the plant extract, 5 ml of the Folin-Ciocalteu reagent (10%, w/v) and 10 ml of sodium carbonate solution (7.5%, w/v) and the volume was completed with distilled water.Readings were taken with a spectrophotometer (760 nm) and tannic acid was used to prepare the standard curve.
Total flavonoids were quantified by a modification of the Araújo et al. (2008) method.The following was added to 25 ml flasks: 1 ml of the plant extract, 0.6 ml of glacial acetic acid, 10 ml of pyridinemethanol solution (2:8, v/v) and 2.5 ml of aluminum chlorate (5% w/v, in absolute methanol) and the volume was completed with distilled water.Readings were taken with a spectrophotometer (420 nm) and rutin was used to prepare the standard curve.Analysis of total tannins was carried out with a modification of the Monteiro et al. (2006) method: 3 ml of the plant extract was mixed with 0.5 g of casein and the mixture was kept under agitation for 3 h at 25°C (160 rpm).After this period, the material was filtered with qualitative paper filter and the resulting volume was transferred to 25 ml volumetric balloons and completed with distilled water.Analysis of the remaining phenols was carried out by the Folin-Ciocalteu method and the concentration of total tannins corresponded to the difference between the levels found in this analysis and those found during quantification of total phenols.

Experimental outline and statistical analysis
The experimental outline was entirely randomized with four inoculation treatments (AMF control, inoculated with G. albida, inoculated with A. longula or inoculated with C. etunicatum), with five repetitions, totaling 20 experimental units.The data were submitted for analysis of variance (ANOVA) and the means were compared by the Tukey test (5%) using the Assistat program (2013).

RESULTS AND DISCUSSION
The mycorrhizal treatments had no effect on the chlorophyll a content, on the concentration of total proteins and on the concentration and content of total carbohydrates (Table 1).
The dry matter of the aerial part (DMAP) increased when the seedlings were colonized by G. albida (52.70%) and C. etunicatum (78.37%), in relation to the noninoculated control treatment (Table 2), which means that the mycorrhization with G. albida and C. etunicatum was beneficial for the growth of A. cearensis.Similar results were found by Araim et al. (2009), Baslam et al. (2011) and Toussaint et al. (2007), for Echinacea purpurea, in varieties of Lactuta sativa and in Ocimum basilicum, respectively.
Increased values of mycorrhizal colonization were found in the roots of inoculated plants in relation to the control (Table 2), which supports the results obtained for other Leguminosae, such as Libidibia ferrea (Silva et al., 2014a).Inoculation with G. albida and C. etunicatum increased by 24.06 and 24.28% the concentration of total chlorophyll in relation to the non-inoculated control, respectively.Similar results were obtained for chlorophyll b (Table 2).On the other hand, the benefits of inoculation for chlorophyll a (Table 2) were not documented.As was suggested by Singh et al. (2012), the increase in chlorophyll content may be related to the increased nutrient absorption, taking into consideration that various studies indicate maximization in the production of photosynthetic pigments in terms of mycorrhization, which leads to an improvement of the nutritional status of the host (Selvaraj et al., 2009;Singh et al., 2012).
Mycorrhization did not alter the concentration and the total foliar protein content and soluble carbohydrates in A. cearenis (Table 3); on the other hand, there are situations in which inoculation with AMF favors the accumulation of proteins and plant sugars, as was documented by Ratti et al. (2010) and Baslam et al. (2011).There are situations in which the increase in the content of primary metabolites directs the synthesis of secondary compounds (Oliveira et al., 2013), a fact that has not been documented in this study (Tables 2, 3 and 4).
Mycorrhization with C. etunicatum increased in relation to the non-inoculated control, the production of total foliar phenolic compounds in the A. cearensis seedlings, both in content (198.92%) and concentration (47.82%) (Table 4).Levels of phenolic compounds also varied because of mycorrhizal inoculation, as was documented by Araim et al. (2009), Ceccarelli et al. (2010) and Singh et al. (2012), which makes the use of mycorrhizal technology an alternative to increase the production of such compounds with pharmacological importance.
The use of C. etunicatum maximized the content of total foliar flavonoids in relation to the non-inoculated control (Table 4).Possibly, mycorrhization lead to an increased absorption of nutrients, increasing the synthesis of production precursors of such compounds, such as the enzyme Chalcone synthase (Chs), which regulates the biosynthesis of this group of phenols (Zhang et al., 2013).An increase in the production of this group of phenolic compounds was also found in other situations (Antunes et al., 2006;Larose et al., 2002), as well as in other Leguminosae species in the Caatinga (Pedone-Bonfim et al., 2013).
Inoculation increased the production of total tannins when C. etunicatum was used (Table 5).Nisha and Rajeshkumar (2010) also observed an increase in the biosynthesis of tannins in Wedilla chinensis seedlings when inoculated with Glomus aggregatum.It is probable that intermediaries of biosynthetic pathways of the tannins, such as gallic acid have optimized the production through mycorrhization, as has been recently documented for the Leguminosae L. ferrea (Silva et al., 2014b).
Various mechanisms, nutritional and non-nutritional, have been suggested to explain the effects of mycorrhization on the increase in the biosynthesis of secondary compounds (Mandal et al., 2013;Zhang et al., 2013).Taking into consideration that mycorrhization did not alter the production of primary metabolites (Table 3), it is probable that the mechanisms that are involved in the  foliar phenols increase in A. cearensis are non-nutritional as was suggested by Toussaint et al. (2007).Such mechanisms involve an increase in the enzymatic activity, increase in the gene expression, maximized activation of the metabolic pathways and optimized biosynthesis of signaling in mycorrhizal plants (Walter et al., 2000;Lohse et al., 2005;Zhang et al., 2013).Furthermore, it is probable that the inoculated AMF increased the absorption of P, a fact that is well documented for mycorrhizal plants (Smith and Read, 2008), which is an important requirement for the biosynthetic pathways of phenolic compounds (Heldt, 2005).Benefits of the mycorrhizal technology for the production of bioactive compounds were found for other plants from the Caatinga, as was referred to by Pedone-Bonfim et al. (2013), Oliveira et al. (2013) and Silva et al. (2014a), for Anadenanthera colubrina, Myracrodruon urundeuva and L. ferrea, respectively.Such benefits were also observed for the first time in A. cearensis, which confirms the initial working hypothesis.
The mycorrhizal technology, employing selected AMF, favored the production of the phytomass of A. cearensis with an elevated concentration of bioactive compounds, which possess various therapeutic properties.Therefore, the fungus C. etunicatum is recommended as a biotechnological alternative to maximize the production of foliar bioactive compounds in A. cearensis seedlings.This way, a low cost biotechnological protocol was established to maximize the production of plant biomolecules that are important to the phytotherapeutic industry.Other experiments have to be carried out to elucidate the benefits under field conditions and to determine whether there is a specific increase of molecules that are of industrial interest, such as vanillic acid.

Table 1 .
Analysis of variance for the studied variables.

Table 2 .
Dry matter of the aerial part (DMAP), total chlorophyll a and b and mycorrhizal colonization (MC) in Amburana cearensis seedlings, inoculated or non-inoculated with arbuscular mycorrhizal fungi, 160 days after inoculation under experimental roofing.
a Means followed by the same letter do not differ from the Tukey test (5 %).FCI: Falker chlorophyll index.

Table 3 .
Concentration and content of total proteins and foliar soluble carbohydrates in Amburana cearensis seedlings, inoculated or non-inoculated with arbuscular mycorrhizal fungi, 160 days after inoculation under experimental roofing.
a Means followed by the same letter do not differ from the Tukey test (5%).

Table 4 .
Concentration of total foliar content of phenols and flavonoids in Amburana cearensis seedlings, inoculated or non-inoculated with arbuscular mycorrhizal fungi, 160 days after inoculation under experimental roofing.

Table 5 .
Concentration of total foliar content of tannins in Amburana cearensis seedlings, inoculated or non-inoculated with arbuscular mycorrhizal fungi, 160 days after inoculation under experimental roofing.
a Means followed by the same letter do not differ from the Tukey test (5%).