High temperatures on root growth and lignification of transgenic glyphosate–resistant soybean
Altas temperaturas, crecimiento y lignificación de soya transgénica resistente al glifosato
P. da Costa Zonetti1*, L. Sayuri Suzuki2, E. Aparecida Bonini3, M. L. Lucio Ferrarese3, Osvaldo Ferrarese–Filho3
1 Federal University of Paraná, UFPR, Campus Palotina. Av. Pioneiro, 2153, 85.950–000 Palotina–PR, Brazil. Fax: +55 44 3649 6616. * Author for correspondence (patriciazonetti@ufpr.br).
2 Embrapa Dairy Cattle. Rua Eugenio do Nascimento, 610–Dom Bosco, 36038–330, Juiz de Fora–MG, Brazil. (lsuzuki@cnpgl.embrapa.br).
]]> 3 State University of Maringá. Av. Colombo, 5790, Jardim Universitario, 87020–900, Maringá–PR, Brazil, (oferrarese@uem.br).
Received: Noviembre, 2011.
Approved: Julio, 2012.
Abstract
Glyphosate resistant transgenic soybean [Glycine max (L.) Mettill] expresses a glyphosate insensitive EPSPS (5–enolpyruvylshikimate–3–phosphate synthase). This enzyme is involved in important secondary metabolism pathways, including lignin biosynthesis. Thus, differences in lignin content and growth between susceptible (OC14) and glyphosate–resistant soybean may be observed. The objective of this study was to evaluate differences between growth and lignin content of roots in transgenic and OC14 soybean cultivated at high temperatures. Seeds from the OC14 soybean and its transgenic cultivar, CD213RR, were germinated at 25, 27.5, 30 and 32.5 °C. After 3 d, seedlings were cultivated in Hoagland half–strength nutrient solution during 12 h photoperiods and at the same germination temperature. After 4 d, roots' relative length, fresh and dry biomass, and lignin content were determined. Increasing temperatures promoted root growth. There was decreased growth and higher lignin content in roots in CD213RR soybean as compared to OC14. The transgenic soybean may present a different lignin metabolism since it showed higher lignification independently of temperatures, which is important because can be associated to a higher tolerance to drought and heat, but also with impaired growth and higher susceptibility to breakage of the stem.
Key–words: glyphosate, abiotic stress, Glycine max, plants, climatic changes.
Resumen
]]> La soya transgénica [Glycine max (L.) Merrill] resistente al glifosato presenta glifosato EPSPS (5–enolpiruvilsiquimato–3–fosfato sintasa) insensible. Esta enzima participa en procesos importantes del metabolismo secundario, como la biosíntesis de la lignina. Así, se pueden observar diferencias en el contenido de lignina y crecimiento entre la soya susceptible (OC14) y la resistente al glifosato. El objetivo de este estudio fue evaluar las diferencias entre el crecimiento y el contenido de lignina de las raíces de la soya transgénica y la OC14, cultivadas con temperaturas altas. Las semillas de la soya OC14 y su cultivo transgénico, CD213RR, germinaron a 25, 27.5, 30 y 32.5 °C. Después de 3 d, las plántulas se cultivaron en una solución de nutrientes Hoagland de fuerza media, durante fotoperíodos de 12 h y a la misma temperatura de germinación. Después de 4 d se determinó la longitud relativa de las raíces, la biomasa fresca y seca, y el contenido de lignina. Las temperaturas cada vez más altas promovieron el crecimiento radicular. Hubo una disminución de crecimiento y mayor contenido de lignina en las raíces de la soya CD213RR en comparación con OC14. La soya transgénica puede presentar un metabolismo de la lignina diferente, ya que mostró lignificación mayor independientemente de la temperatura, lo cual es importante porque puede estar relacionado con la tolerancia mayor a la sequía y al calor, pero también con el retraso del crecimiento y mayor susceptibilidad a la rotura del tallo.Palabras clave: glifosato, estrés abiótico, Glycine max, plantas, cambios climáticos.
INTRODUCTION
Soybean [Glycine max (L.) Merrill] crops are prominent in Brazil, the second largest soybean producer after the US. For the 2010/2011 harvest, there were 23.6 million ha with soybean, the production was 75.3 million t with an average productivity of 3050 kg ha–1 (Embrapa, 2010; Conab, 2011).
The patterns of seed germination and plant development are genetically programmed and this expression can be changed by environmental factors during cultivation (Santos et al., 1992). Some soybean cultivars show their optimum growth between 25 and 30 °C (Miyasaka and Medina, 1981) because temperatures higher than 30 °C can modify cell membranes stability affecting several metabolic processes, particularly photosynthesis and cellular respiration (Nagarajan et al., 2005). Besides, modifications in the average temperature or rainfall may impact growth and development (Challinor and Wheeler, 2008). Abiotic stress changes primary and secondary metabolism of the plants as a protection response, whereas changes in the secondary metabolism include phenylpropanoids pathway, the main route for synthesizing phenolic compounds and lignin (Dixon and Paiva, 1995; Cabane et al., 2012).
The most abundant class of phenolic compounds in plants is the derivative from phenylalanine. Through the elimination of an ammonia molecule, phenylalanine is converted into cinnamic acid and this reaction is catalyzed by phenylalanine ammonia lyase (PAL) which operates at a branching point between the primary and secondary metabolisms. Production of lignin, coumarins, benzoic acid derivatives, anthocyanins, isoflavones, tannins and other flavonoids starts with phenylalanine (Vogt, 2010). Lignins are complex phenolic polymers deposited in cell walls, making them rigid and impervious. In addition to developmentally programmed deposition of lignin, its biosynthesis can also be induced by various biotic and abiotic stress conditions (Vanholme et al., 2010; Cabane et al., 2012). The incorporation of lignin in cell walls leads to structural rigidity and resistance of plant tissues (Polle et al., 1994; Boudet et al., 2003), because the thickening mechanism decreases the extensibility of the cell wall due to the formation of diphenyl bridges between wall polymers due to peroxidase enzymes (Sanchez et al., 1996). Increasing soil temperatures close to 45 °C might enhance lignin biosynthesis (Brenbrook, 2001).
Genetically modified soybean, resistant to glyphosate (Roundup ReadyTM or RR cultivar), expresses a variant of EPSPS (5–enolpyruvylshikimate–3–phosphate synthase) with low affinity to glyphosate, giving the plant resistance to this herbicide (Harrison et al., 1996; Von Pinho, 2002). This enzyme acts in the shikimic acid pathway leading to synthesis of lignin, phenylalanine and tyrosine (Steinrucken and Amrhein, 1980).
High temperatures may affect plants by increasing deposition of lignin in cell walls and decreasing synthesis of essential aromatic amino acids, thus reducing plant growth. In this context, evaluating growth and lignin content in roots of transgenic RR and conventional soybean at high temperatures may show differences in their metabolism. This information may explain differential developmental responses of these cultivars in crop systems.
Considering that RR soybean has a variant of an EPSPS belonging to the shikimate pathway which is active in plants under adverse temperature conditions, the hypothesis of this study was that susceptible (OC14) and transgenic (CD213RR) soybean cultivars grown at high temperatures would differ in root growth and lignin content. The objective of this study was to evaluate the growth and lignification of CD213RR and OC14 soybean roots, grown under high temperatures.
]]>MATERIALS AND METHODS
All experiments were conducted in B.O.D chambers (Tecnal TE 400, Brasil), at the Plant Biochemistry Laboratory, State University of Maringá, Paraná, Brazil. Soybean seeds of CD213RR (glyphosate–resistant) and OC14 cultivars were supplied by COODETEC (Cascavel, Brazil); presence of CP4EPSPS gene in CD213RR was analyzed by PCR.
Soybean seeds were surface–sterilized with 2 % sodium hypochlorite for 3 min and rinsed with deionized water. Seeds were dark–germinated in rolls of three sheets of moistened germination paper and conditioned in flasks containing enough water to maintain moisture. Rolls were kept in a dark chamber (Tecnal TE 400, Brasil) at 80 % relative humidity and 25 °C (control), 27.5, 30 or 32.5 °C. After 3 d of germination seedlings were placed in a system with 25 three–day–old seedlings of uniform size, on an adjustable acrylic plate placed into a glass container (10x16 cm) filled with 200 mL of half–strength Hoagland's solution (Ferrarese et al., 2000; Santos et al., 2004). This nutrient solution was treated with 17 mM potassium buffer, adjusted to pH 6.0 and monitored over time. The container was kept in a growth chamber, at 80 % relative humidity, 12 h light photoperiod, 280 mmol m–2 s–1 (PAR) irradiance at 25, 27.5, 30 or 32.5 °C. Cultivation lasted 96 h and the nutrient solution was replaced daily.
After cultivation, tap roots relative length, fresh and dry roots biomass and root lignin content were evaluated. Tap roots relative length was obtained by the difference between roots length before and after seedlings cultivation in each temperature tested. Next, roots were cut, carefully blotted with an absorbent paper and fresh weight was measured. Dry root weight was determined after oven–drying at 80 °C until reaching constant weight. Lignin content of roots was determined by the thioglycolic method (Chen et al., 2000; Ferrarese et al., 2002): cell wall extraction, acidic digestion, lignothioglycolic acid (LTGA) extraction and using UV spectrophotometer.
The experimental design was completely randomized with a factorial (2 cultivars x 4 temperatures of cultivation) arrangement of treatments with four replicates and each replicate contained 25 seedlings. Data were analyzed using ANOVA and means were compared with the Tukey test (p<0.05). Time factor was analyzed by a polynomial regression analysis for each cultivar. Sisvar software (Ferreira, 2006), was used for data analysis.
RESULTS AND DISCUSSION
Tap root relative length, roots biomass and lignin content showed significant differences between cultivars (Table 1). The relative length of tap roots was higher for OC14 soybean compared to CD213RR, only at 27.5 °C. Fresh and dry biomass at 27.5 and 30 °C and fresh biomass at 32.5 °C showed higher significant values for OC14. In all the temperatures, CD213RR soybean presented higher lignification than OC14. In general, the slower growth of the root system in CD213RR soybean may be due to the higher root lignification of this cultivar, at the four temperatures.
As the temperature increased there was a linear increase in fresh biomass of the root system of OC14 soybean: for each increase of 2.5 °C the increment was 0.0100 g (Figure 1). Besides, for dry biomass, the same cultivar showed a quadratic response and the maximum point of the fitted equation was 28.98 °C (Figure 2). In CD213RR soybean there was a decrease in growth of the fresh root system at 27.5 °C, with a minimum (0.064 g) at 28.02 °C, obtained by mathematical inference, and an increment at 30 and 32.5 °C (Figure 1). The same behavior was observed with the dry biomass of this cultivar, where the minimum point was 28.06 °C, obtained by mathematical inference, with an increment at 30 and 32.5 °C (Figure 2).
]]> Both cultivars presented better growth of the root system at temperatures close to 30 °C, which may be due to the fact that temperatures between 25 and 30 °C are optimal for growth and development of most soybean cultivars in Brazil (Embrapa, 2010). Soybean cultivars adapted to colder climates may present a different behavior, since a temperature increase from 26 to 30 °C throughout the growing season reduced seed size, and any stress related to evaporative demand in addition to high temperatures may be involved in this effect Tacarindua et al., 2012).The analysis of lignin content (Figure 3) in both cultivars showed a significant quadratic equation, and a higher lignin content when exposed to temperatures above 25 °C. Higher temperatures increase evaporative demand and may cause tissue thickening as a consequence of enhanced lignin biosynthesis. This may be observed when plants are exposed to abiotic stresses such as drought and excess UV–B radiation, which often are associated to high temperatures (Cabane et al., 2012; Tacarindua et al., 2012). There was increased lignin biosynthesis but reduced growth in Citrullus lanatus roots, and it was associated with drought resistance (Yoshimura et al., 2008).
The maximum lignin content was observed close to 30 °C, with 34.65 and 26.19 mg LTGA g–1 for CD213RR soybean and the conventional OC14 soybean. Lignin content at 30 °C in CD213RR was approximately 31 % higher than that of the CO14; besides, in warmer climates, lignin content of this soybean is around 20 % higher than in conventional soybean (Coghlam, 1999). Consequences of a higher lignification include more rigidity and strength of the stem; besides, lignin waterproofs the cell wall (Boerjan et al., 2003) and may generates plants more resistant to drought but also with a higher susceptibility to stem breakage.
Transgenic plants may show changes in lignin amount, composition, primary structure and phenotypic effects by altering the expression of a single gene (Zobiole et al., 2010). According to Padgette et al. (1995) and Facchini et al. (2000), transgenic and conventional soybeans presents differences in the composition of several metabolites, including aromatic amino acids; one of the transgenic soybean contained lower levels of phenylalanine than the conventional line. A similar situation was observed in corn regarding tyrosine (Sidhu et al., 2000). Phenylalanine and tyrosine are important for plant development as precursor molecules for the phenylpropanoids pathway and the main pathway for synthesis of phenolic compounds, including monolignols which are precursors of lignin (Strack, 1997).
Thus, it is reasonable to assume that differences in metabolites composition in transgenic soybeans could be associated to the expression of a variant of EPSPS, thus enhancing lignin content. This is an important feature as it can be associated to a higher drought and heat tolerance, but also with impaired growth and higher susceptibility to stem breakage.
CONCLUSIONS
The analysis of the results indicated that CD213RR and OC14 soybeans are adapted to higher temperatures but CD213RR showed reduced root growth and higher lignification, which could be associated to the expression of a variant of EPSPS and is an intrinsic feature of transgenic soybean.
ACKNOWLEDGEMENTS
]]> The authors are grateful to Coordenacao de aperfeicoamento de pessoal de nivel superior (CAPES) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for fellowships and financial support.
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