Changes in Amino Acid Profile in Roots of Glyphosate Resistant and

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Changes in amino acid profile in roots of glyphosate resistant and susceptible soybean (Glycine max) induced by foliar glyphosate application Carlos Alberto Moldes, Miguel Angel Cantarelli, José Manuel Camiña, Siu Mui Tsai, and Ricardo A Azevedo J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b03676 • Publication Date (Web): 18 Sep 2017 Downloaded from http://pubs.acs.org on September 21, 2017

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Journal of Agricultural and Food Chemistry

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Changes in amino acid profile in roots of glyphosate resistant and

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susceptible soybean (Glycine max) induced by foliar glyphosate

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application

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Carlos Alberto Moldesa,b*, Miguel Angel Cantarellia, José Manuel

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Camiñaa,b*, Siu Mui Tsaic and Ricardo Antunes Azevedod

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a

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Uruguay 151, CP 6300, Santa Rosa, La Pampa, Argentina.

Facultad de Ciencias Exactas y Naturales. Universidad Nacional de La Pampa. Av.

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b

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Uruguay 151, CP 6300 Santa Rosa, La Pampa, Argentina.

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c

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303, CEP 13400-970, Piracicaba, Brazil.

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d

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Universidade de São Paulo, Av. Pádua Dias, 11, Piracicaba, SP, CEP 13418-900,

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Piracicaba, Brazil.

Instituto de las Ciencias de la Tierra y Ambientales de La Pampa (INCITAP), Av.

Centro de Energia Nuclear na Agricultura, Universidade de São Paulo, Av. Centenário

Departamento de Genética, Escola Superior de Agricultura “Luiz de Queiroz”,

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* Corresponding authors: C.A. Moldes: e-mail [email protected]. Tel: +54-2954-

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2455220/30

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[email protected]. Tel: +54-2954-2455220/30 Int 7433. Fax: +54-2954-432535.

Int

7427

Fax:

+54-2954-432535.

1 ACS Paragon Plus Environment

J.M.

Camiña:

e-mail:


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Abstract

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Amino acid profiles are useful to analyze the responses to glyphosate in susceptible and

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resistant soybean lines. Comparisons of profiles for 10 amino acids (Asp, Asn, Glu,

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Gln, Ser, His, Gly, Thr, Tyr, Leu) by HPLC in soybean roots were performed in two

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near isogenic pairs (four varieties). Foliar application of glyphosate was made to

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soybean plants after 5 weeks of seeding. Roots of four varieties were collected at 0 and

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72 h after glyphosate application (AGA) for amino acids analysis by HPLC. Univariate

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analysis showed a significant increase of several amino acids in susceptible as well as

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resistant soybean lines; however, amino acids from the major pathways of carbon (C)

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and nitrogen (N) metabolism, such as Asp, Asn, Glu and Gln, and Ser, increased

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significantly in susceptible varieties at 72 h AGA. Multivariate analysis using principal

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components analysis (2D-PCA and 3D.PCA) allowed to identified and discriminated

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different groups based on the soybean genetic origin, showing the amino acid responses

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on susceptible and resistant varieties. Based on the results, it is possible to infer that the

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increase of Asn, Asp, Glu, Gln, and Ser in susceptible varieties would be related to the

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deregulation of C and N metabolism, as well as changes in the growth mechanisms

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regulated by Ser.

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Keywords: glyphosate; amino acid profile; soybean; transgenic; multivariate analysis.

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1. Introduction

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The analysis of free amino acids involved in connected metabolic pathways (i.e.

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amino acids from N assimilation such as Asp, Asn, Glu and Gln) can be useful to

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understand how it extends the effect of stressors in metabolism, including the

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comparison of conventional and transgenic counterparts of plant species after herbicide

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application.

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Glyphosate is a broad spectrum herbicide whose mode of action is the inhibition

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of 5-enolpyruvylshikimate 3-phosphate synthase (EPSPS) from shikimate pathway,

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responsible for the synthesis of aromatic amino acids (phenylalanine, tryptophan and

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tyrosine)1-4 . Although the mode of action of glyphosate does not involve other targets

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in metabolism than EPSPS, it application can induce increases in the major of

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individual amino acids4,5 especially when dealing with susceptible varieties6,7.

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Multivariate methods can explain complex relationships among big data

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collections, which are difficult to reveal by univariate analysis8; these tools have been

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used for a wide spectrum of analysis to obtain genotypical classification6,9,10.

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Furthermore, chemometrics appears as appropriate statistical tool, considering the

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complex nature of biochemical responses to stress and their interrelationships11.

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Chemometrics has been successfully applied to biological systems in order to analyze

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several type of interactions and effects: a) in the stress response using data of

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antioxidant enzymes activity in cotton plants fertilized by silicon12; b) to show

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differential antioxidant responses in wheat under pathogenic fungus incidence8; c) to

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obtain indicators for changes in processed and stored vegetables13 and d) to elucidate

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the metabolic pathways participants in development of plant pathogenicity14.



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The present work aims to study the free amino acid contents in roots of soybean

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lines resistant and susceptible to glyphosate focusing on amino acids profiles responses

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after glyphosate applications for the discrimination of soybean lines. Furthermore, it

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intends to explore the use of chemometric tools for interpretation of metabolic

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consequences of glyphosate application, describing the behavior and transport of amino

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acids from N assimilation, as well as the signaling of serine in soybean roots.

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2. Material and Methods

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2.1. Reagents

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Amino acid standards and o-phthaldialdehyde (OPA) were acquired from Sigma

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(St. Louis, MO). Methanol, sodium acetate, disodium phosphate, acetic acid and

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tetrahydrofuran were obtained from Merck (Darmstadt, Germany). Ultrapure water was

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obtained from Millipore Milli-Q Integral Water Purification System (ultrapure water-

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18.2 MΩ cm) (Billerica, MA).

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2.2. Instrumental

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Amino acid profiles were obtained from AKTA Purifier FPLC (Amersham

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Pharmacia Biotech, Piscataway, NJ). Separation of amino acids was carried out on a

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Spherisorb ODS-2 C18 column (5 µm, 4 x 250 mm). Amino acids were detected by a

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fluorescence detector Shimadzu, model RF350 (Kyoto, Japan) operating with an

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excitation wavelength of 250 nm and an emission wavelength of 480 nm.

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2.3. Plant samples


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Two pairs of nearly isogenic of soybean varieties glyphosate susceptible and

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resistant were used: pair DM48-DM4800RG obtained from INTA Marcos Juarez

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(Argentina) and pair Msoy7501-Msoy7575RR from CENA, USP (Brazil).

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2.4. Experimental design and treatments

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The presence of CP4 EPSPS gene was determined in seeds of the varieties

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mentioned above by PCR as described by Moldes et al.4 to verify the characteristic of

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glyphosate susceptibility-resistance, with positive results for DM4800RG and

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MSoy7575RR , and negative ones for DM48 and MSoy7501. Seeds of each soybean

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variety were superficially sterilized using hypochlorite solution (2%) soaked in water

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and placed in sterile plates. Pre-germination was carried out in the dark at 30ºC for 48 h.

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Pre-germinated seeds were sown in sterile sand:vermiculite (3:1) in 3000 cm3 pots.

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Three seedlings per pot were grown in a glasshouse at 15–30ºC, 30–60% humidity,

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under a natural light regime. Plants were supplied twice a week with 100 cm3 of nutrient

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solution without the addition of vitamins15. No insecticide and fungicide application

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was made during all the experiment. Glyphosate (Agrisato 480 CS manufactured by

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ALKAGRO) was sprayed on 5-week-old plants in an application chamber. The

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herbicide was diluted in water at 2:100 rates and applied on the foliar surface using a

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precision sprayer device, equipped with continuous-deposition tips (XR110015) to 0.50

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m from the pot’s upper surface. A pressure of 200 kPa allowed an intake corresponding

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to 200 dm3 ha-1 of solution, which is the dose of glyphosate recommended by

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manufacturers for field applications. Plants were harvested at 0 h and 72 h after

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glyphosate application (AGA), frozen in liquid nitrogen and stored at -80ºC.

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Experiments were carrying out in a complete randomized design with four replications.



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For simplify names of conventional (C) and resistant (R) varieties were called DM48 as

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DC; DM4800RG as DR; Msoy7501 as MC; Msoy7575RR as MR. Treatments at 0 and

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72 AGA were called for variety DM48 at 0 h AGA as DC0; DM48 at 72 h AGA as

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DC72; DM4800RG at 0 h AGA as DR0; DM4800RG at 72 h AGA as DR72;

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Msoy7501 at 0 h AGA as MC0; Msoy7501 at 72 h AGA as MC72; Msoy7575RR at 0 h

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AGA as MR0; Msoy7575RR at 72 h AGA as MR72.

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2.5. Extraction of amino acids and HPLC quantification

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Amino acids were obtained by MCW extraction solution (12 cm3 methanol, 5 cm3

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chloroform and 3 cm3 water). Frozen root tissue (0.100 g) was homogenized in 2 cm3 of

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MCW solution and centrifuged at 2,500 g for 20 min at 4ºC. The supernatant was

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collected and added to 0.5 cm3 chloroform and 0.75 cm3 Milli-Q water to immiscible

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phase separation. The water-soluble phase was used for further analysis. Three replicate

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samples of each plant were analyzed by HPLC as o-phthaldialdehyde (OPA) derivatives

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as described by Puiatti and Sodek16 (1999). Eluent solution was performed with solution

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of 65 % methanol and phosphate buffer pH 7.25 (50 mmol dm-3 sodium acetate, 50

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mmol dm-3 disodium phosphate, 1.5 cm3 acetic acid, 20 cm3 tetrahydrofuran, 20 cm3

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methanol in 1000 cm3 ultrapure water) increasing the proportion of 65 % methanol from

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20 to 60 % between 0 and 25 min, 60 to 75 % from 25 to 31 min and 75 to 100 % from

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31 to 50 min at a 1 cm3 min-1 flow rate. Data obtained from 10 amino acids detected by

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HPLC (Asp, Glu, Asn, Ser, Gln, His, Gly, Thr, Tyr and Leu) were expressed as ηmol

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amino acid g-1 root fresh weight.

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2.6. Statistical analysis


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Data analysis was performed by ANOVA and significant differences between the

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responses of cultivars to glyphosate were determined by Tukey’s multiple range test

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using the SAS statistical program (SAS Institute Inc., Cary, NC, USA, 1999) (P

Changes in Amino Acid Profile in Roots of Glyphosate Resistant and

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