Does mechanical damage on soybean induces the production of flavonoids ?

The response of plants to grazing includes the production of chemical defense compounds such as proteases inhibitors and secondary metabolites as flavonoids, which makes them less palatable to feeding and negatively affecting the physiology of insects. The aim of this study was to evaluate the phytochemical response of soybean cultivars (Glycine max (L.) Merrill) resistant (IAC-17, IAC-24) and susceptible (IAC-P1) to insects after mechanical damage. These cultivars were mechanically injured, and after 24 hours samples of these plants were analyzed by HPLC to identify and quantify flavonoids. The flavonoids daidzein, quercetin, and rutin were quantified, with the highest concentration of daidzin in soybean cultivars after mechanical damage. Rutin was biosynthesized by IAC-24. The cultivars IAC-PL1, IAC-17, and IAC24 did not show a flavonoid response to mechanical damage. The soybean cultivars are not dependent on mechanical damage to produce flavonoids.


INTRODUCTION
Secondary metabolites are chemical compounds that confer plant resistance to insects.Host plant resistance is an effect strategy used in pest control programs to minimize economic damage (Hoffmann-Campo et al. 2006, Pilon et al. 2009).These metabolites can also be produced by Minthostachys mollis, Ocimum minimum in response to mechanical damage (Zabaras and Wyllie 2001, Banchio et al. 2005, Piesik et al. 2006).
Plant response to insect attack can occur directly and/or indirectly.The direct defense against herbivory is the negative effect of secondary metabolites on insect physiology.The indirect defense induces the production of volatile compounds to attract predators (Arimura et al. 2005).The herbivores triggers a chemical signaling to plants to biosynthesize secondary metabolites PAULO LUIZ DA SILVA et al. through the shikimate pathway and acetylcoenzyme A (Winkel-Shirley 2001, Mello andSilva-Filho 2002).
Mechanically injured plants produce volatile compounds as part of the defense system that can also attract natural enemies.The concentration of volatile compounds like eugenol, linalool, menthone, pulegone, and sesquiterpens increases in mechanically injury M. mollis and O. minimum (Zabaras and Wyllie 2001, Banchio et al. 2005, Piesik et al. 2006).
The increased expression of volatile compounds in plants injured are being studied, but the phytochemical flavonoid response in soybean plants needs to be better understood.Therefore, this study objected to investigate the phytochemical flavonoids response in soybean Glycine max (L.) Merrill cultivars resistant and susceptible to insects, after being undergo mechanical injuries.

MATERIALS AND METHODS
The experiments were performed at the Laboratory of Enzymology, Biochemistry of Proteins and Peptides, Instituto de Biotecnologia Aplicada a Agropecuária (BIOAGRO) and in the Laboratory of Biodiversity, Department of Biochemistry and Molecular Biology of the Universidade Federal de Viçosa, Minas Gerais State, Brazil.

SOYBEAN CULTIVARS AND PHYTOCHEMICAL RESPONSE TO MECHANICAL DAMAGE
Seeds of the soybean cultivars IAC-PL1 (susceptible to insects) and IAC-17 and IAC-24 (resistant to insects) were obtained from the Instituto Agronômico de Campinas (IAC) of São Paulo State, Brazil, and cultivated until the V3 stage.
Three soybean plants were growner in pots with 4.0 kg of soil in the green house (temperature 25 °C + 5, humidity 70% + 10) and injured or not with a paper punch (6 holes of 0.5 mm per leaflet).The plants in V3 stage of development control and injured were cultured independently in plastic pots.After 24 hours, nine leaflets were removed from nine plants and were immediately frozen -80 o C to phytochemical analysis.

OBTAINING THE LEAF EXTRACT
The extract of soybean leaves was prepared at 4 °C.The leaves were weighed (500 mg) and pulverized in liquid nitrogen with a mortar and pestle.The powder obtained was transferred to 15 ml plastic tubes with 5.0 ml of 80% methanol, kept in an ultrasound bath for 20 min, and centrifuged at 3.000 g for 5 min at 4 °C.The supernatants obtained were evaporatedina speed vacuum concentrator and the extracts dry were resuspended with 2 the test of ANOVA and Tukey test were applied.
The generalized linear model (GLM) was used to make the analysis ANOVA.Simplifying model was used with GLM.

IDENTIFICATION OF FLAVONOIDS IN SOYBEAN CULTIVARS
Soybean cultivars resistant  and susceptible (IAC-PL1) analyzed by liquid chromatography showed three peaks, identified as the compounds daidzein, quercetin, and rutin (Figure 1).
The flavonoids in the soybean leaf extracts were evaluated quantitatively by comparison with purchased standards (Sigma ® ) by injections of kaempferol, daidzein, daidzein, genistein, genistin, quercetin, and rutin in the HPLC.Calibration curves were made with injection concentrations of daidzein (1.2, 2.4, 3.6, 4.8 mg), quercetin (0.8, 1.6, 2.4, 3.2 mg), and rutin (0.8, 1.6, 2.4, 3.2 mg).Linear regression functions were obtained from the responses of these compounds, measured as peak area to calculate the concentration of flavonoids in the samples.

STATISTICAL ANALYSES
The experiment was conducted in a completely randomized design and the data analyzed with the R statistical program, considering 5% error.The experiment had treatments per cultivar, with three replications.The concentration of flavonoid were analyzed for normality with the Shapiro test, and

DISCUSSION
Flavonoids have diverse biological functions protecting plant against abiotic and biotic factors (Pourcel et al. 2007) including damage by ultraviolet light (Takahashi and Ohnishi 2004) and against microorganisms and insects and they are also responsible for flower colors (Yao et al. 2004, Griesbach 2005).
The concentration of daidzein in the cultivar IAC-24 was approximately 6.3 times higher than that of the quercetin and rutin, which is not observed in the IAC-17.These compounds can affect the physiology of insects, although the mode of action of this phytoestrogens needs to be better studied.Soybean seeds injured by stink bug produced a higher quantity of daidzein and genistein isoflavones, which reduced food quality for insects (Piubelli et al. 2003).
The concentration of flavonoids rutin was 2.5 times higher in the IAC-24 cultivar than in the IAC-17 for (F, p = 0.027).The concentration of daidzein was 5.7 times higher than the lowest level of cultivar IAC-PL1, susceptible to insects.These concentrations of flavonoids can give rise in these cultivars to increased resistance to insects because these compounds affect the physiology of these organisms (Boué andRaina 2003, Jadhav et al. 2012).Furthermore, it has been shown that in genetically modified tobacco plants increased synthesis of rutin has an insecticidal effect on larvae Spodoptera litura Fabricius, 1775 (Lepidoptera: Noctuidae) and Helicoverpa armigera Hübner, The cultivars IAC-PL1, IAC-17, and IAC-24 did not show a phytochemical response related to increased flavonoids after 24h of mechanical damage.However, mechanically damaged plants tend to express a phytochemical response (Zabaras and Wyllie 2001, Banchio et al. 2005, Piesik et al. 2006), as observed for wheat leaves, where there was an increase in the release of the secondary metabolites linalool (3,7-dimethyl-1,6-octadien-3-ol) and linalool oxide (5-ethenyltetrahydro-2furanmethanol) (Piesik et al. 2006).Synthesized compounds due to mechanical damage tend to have lower levels after a certain time (Banchio et al. 2005).The absence of a chemical plant response of plants by producing flavonoids after mechanical damage may be related to the absence of the elicitors ß-glucosidase and volicitin from insects (Alborn et al. 1997).These compounds in contact with the cell membrane of plant can trigger a cascade of signaling and consequently activating ion flows in the plasma membrane, changes in the cytoplasmic calcium concentration, active oxygen generation, and protein phosphorylation (De Bruxelles and Robert 2001).It also induces the jasmonic acid production, responsible for triggering genes in the shikimate and acetyl coenzyme A metabolic pathways, both synthesizers of secondary metabolites (Winkel-Shirley 2001, Mello andSilva-Filho 2002).These metabolites, such as volatiles and terpenes, are produced by mosses in response to herbivory by Coleoptera (Piesik et al. 2011a(Piesik et al. , 2014)).Microorganisms also induce volatile production incorn roots and leaves (Piesik et al. 2011b).
The production of chemical compounds in soybean leaves indicates the need of further studies to understand the flavonoid biosynthesis in these plants.The physiological response or its absence in soybean plants after abiotic stress confirms the importance of studying the potential of flavonoids as part of plant defense against herbivory.

Figure 2 -
Figure 2 -Flavonoid concentrations in leaf extracts of the insect resistant soybean cultivars IAC-17 (a) and IAC-24 (b) mechanically damaged.Means followed by the same letter are similar between them by ANOVA (IAC-17 p<0.001 and IAC-24 p<0.001).

ACKNOWLEDGMENTS
Thank you for Proof reading the correction work and IAC -Instituto Agronômico de Campinas by the soybean seeds.We thank the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG), and Instituto Nacional de Ciência e Tecnologia em Interações Planta-Praga (INCT-IPP), Brazil by the financial support.

TABLE I Concentration of flavonoids rutin, daidzein and quercetin (mg/g) in insect resistant and susceptible soybeans with and without mechanical damage.
: without, wi 2 : with.The values did not differ by ANOVA, p>0.05. 1