Morphoanatomical and Biochemical Changes in Zeyheria Tuberculosa Exposed to Glyphosate Drift
Total Page：16
File Type：pdf, Size：1020Kb
Botany Morphoanatomical and biochemical changes in Zeyheria tuberculosa exposed to glyphosate drift Journal: Botany Manuscript ID cjb-2020-0150.R3 Manuscript Type: Article Date Submitted by the 28-Sep-2020 Author: Complete List of Authors: Freitas-Silva, Larisse; Universidade Federal do Reconcavo da Bahia Castro, Naila; Universidade Federal de Viçosa Campos da Silva, Luzimar; Universidade Federal de Viçosa Herbicide, Oxidative stress, Plant anatomy, Scanning electron Keyword: Draft microscopy Is the invited manuscript for consideration in a Special Not applicable (regular submission) Issue? : © The Author(s) or their Institution(s) Page 1 of 23 Botany Morphoanatomical and biochemical changes in Zeyheria tuberculosa exposed to glyphosate drift Larisse de Freitas-Silva1,2, Naila Diniz e Castro1, Luzimar Campos da Silva1* 1 Departamento de Biologia Vegetal, Universidade Federal de Viçosa, 36570-900 Viçosa, MG, Brazil. 2 Universidade Federal do Recôncavo da Bahia, 44380-000 Cruz das Almas, BA, Brazil. *Dr. Luzimar Campos da Silva - Corresponding author, email: [email protected]. Universidade Federal de Viçosa, MG, Brazil. Phone number: +55 31 3891 1666 Dr. Larisse de Freitas Silva, email: [email protected] Naila Diniz e Castro, email: [email protected] 1 © The Author(s) or their Institution(s) Botany Page 2 of 23 Abstract – During glyphosate application, a portion of the herbicide can reach adjacent vegetation and impact the natural plant community structure and diversity over the long term. This study evaluated the response of leaves of Zeyheria tuberculosa (Vell.) Bureau ex Verl. (Bignoniaceae) to the herbicide glyphosate. Plants were exposed to aerial applications of the herbicide at concentrations of 0, 360, 720, 1080 and 1440 g a. e. ha-1. The shikimic acid concentrations in leaves of herbicide-treated plants were always higher than the control. Visual symptoms became apparent 4 DAA from 720 g a. e. ha-1. Glyphosate induced an increase in malondialdehyde in Z. tuberculosa leaves. The lowest values of chlorophyll a content were found for the three last applied doses and protein content decreased with the glyphosate treatment. Necrosis was observed on the epidermis and in the mesophyll. Glandular trichomes were also plasmolyzed. On the midrib there was plasmolysis of non-lignifiedDraft cells. Micromorphologically, there were cell plasmolysis and rupture of glandular trichomes Glyphosate is phytotoxic to Z. tuberculosa by promoting biochemical, anatomical and morphological alterations. The morphoanatomical injuries found on Z. tuberculosa are severe, suggesting that the presence of glyphosate can impact this species irreversibly and compromise its survival. Keywords: Herbicide, Oxidative stress, Plant anatomy, Scanning electron microscopy 2 © The Author(s) or their Institution(s) Page 3 of 23 Botany Introduction Glyphosate N- (phosphonomethyl glycine) is the most widely used herbicide in the world and is marketed in more than 119 countries (Dupont et al. 2018). It acts by inhibiting the chloroplast enzyme 5-enolpyruvyl-chiquimate-3-phosphate synthase (EPSPS EC 2.5.1.19), acting on the shikimic acid pathway (Mobin et al. 2014), and is post-emergent and systemic, which makes it a nonselective herbicide that is efficient for many weeds (Beltrano et al. 2013). Inhibition of the shikimic acid pathway promotes the accumulation of shikimic acid (Gomes et al. 2017) and, consequently, the aromatic amino acid pools of tryptophan, phenylalanine and tyrosine are depleted (Yanniccari et al. 2012). These amino acids are essential for protein synthesis and cell division, in addition to participating in the formation of secondaryDraft metabolites (Mobin et al. 2014). A decrease in their production compromises the plant metabolism, causing dysfunction in important cellular processes of plants and even causing plant death (Piola et al. 2013). Herbicides are xenobiotic and cause oxidative stress in plants from the generation of an excess of reactive oxygen species (ROS) that react with lipids, proteins, pigments and nucleic acids and cause lipid peroxidation, membrane damage and inactivation of enzymes, thus affecting cell viability (Gomes et al. 2014; Freitas- Silva et al. 2017; Freitas-Silva et al. 2020). Although not its first action target, glyphosate can also cause changes in photosynthesis by impairing key processes, such as carbon metabolism and chlorophyll biosynthesis or degradation, causing dysfunction in important cellular processes of plants (Gomes et al. 2016; Gomes et al. 2017; Vital et al. 2017). Alterations in cell biochemistry can also culminate in changes in plant morphology and anatomy. Chlorosis, necrosis, cell plasmolysis, an increase or decrease in the thickness of leaf blades, collapse of parenchyma cells, and rupture of the 3 © The Author(s) or their Institution(s) Botany Page 4 of 23 epidermis are damage caused by glyphosate (Silva et al. 2016; Rezende-Silva et al. 2019; Freitas-Silva et al. 2020). Drift may occur during glyphosate application (Christofoletti 1999). Thus, lethal and sublethal herbicide concentrations can reach non-target plants (i.e., non-crop plants outside the crop area) (Dupont et al. 2018; Lucadamo et al. 2018), negatively impacting non-target species. Certain places, such as preservation areas with endangered species and forest fragments adjacent to cultivated areas, are especially sensitive to herbicide drift (Egan et al. 2014; Silva et al. 2016). Some authors argue that the dispersion of herbicides in the environment contributes to significant losses in neighboring forest patches (Boutin et al. 2014). Several studies describe the effect of glyphosate on non-target plants and evaluate the impact of this herbicideDraft on leaf morphology and anatomy (Silva et al. 2016), cell biochemistry (Freitas-Silva et al. 2020), photosynthesis (Gomes et al. 2017; Vital et al. 2017), flower, fruit and seed set of herbaceous plants (Boutin et al. 2014), and flower sterility (Londo et al. 2014). These studies have increased the evidence of the ecotoxicological effects of this herbicide on agroecosystem biodiversity (Florencia et al. 2017) and, therefore, the negative impacts on plant community structure and diversity over the long term (Londo et al. 2014). Although using glyphosate is a widespread practice in Brazil, there are few studies that elucidate the phytotoxic effects of this herbicide on non-agricultural species. Zeyheria tuberculosa (Vell.) Bureau ex Verl. (Bignoniaceae) (Ipê preto) is widely distributed throughout the country and a pioneer species that colonizes degraded areas. It is often found near agricultural areas and, therefore, under the potential effects of herbicides such as glyphosate. It also has high quality wood used in construction, fences and tools (CNC Flora 2020). Zeyheria tuberculosa is listed as vulnerable on the red list 4 © The Author(s) or their Institution(s) Page 5 of 23 Botany of threatened species of the Brazilian flora (Souza et al. 2016) and the International Union for Conservation of Nature (IUCN) Red List of Threatened Species. In consideration of the vulnerability of Z. tuberculosa, due to its occurrence near agricultural areas in Brazil and the wide use of glyphosate in this country, this study evaluated the response of Z. tuberculosa leaves to this herbicide. We tested the following hypothesis: glyphosate application on Z. tuberculosa will cause severe damage to the anatomy and metabolism of this species, compromising its development and survival. Materials and Methods Cultivation conditions and application treatment The experiment was conductedDraft in a greenhouse at the Universidade Federal de Viçosa (UFV), Brazil. Individuals of Z. tuberculosa (Vell.) Bureau ex Verl. that were eight months old were obtained from a nursery in the Sociedade de Investigações Florestais of the Departamento de Engenharia Florestal at UFV. The seedlings were cultivated in 4-liter pots (1 plant per pot) that contained substrate (Vivato®). They were irrigated every five days with Hoagland solution at half ionic strength, pH 5.5 (Hoagland and Arnon 1950), and watered every two days. They remained for 30 days under these conditions for acclimatization. Subsequently, healthy individuals (visually uniform based on height and number of leaves) were selected for the experiment. The plants (N=5), for each concentration including the control, were exposed to the herbicide RoundUp® Ultra (Monsanto Company, USA) containing 65% (w/w) glyphosate, N-phosphonomethyl glycine as the active ingredient at concentrations of 0, 360, 720, 1080 and 1440 g a. e. ha-1, which corresponds to 0%, 25%, 50%, 75% and 100% field application rate in Brazil. The herbicide was applied using a hand-back 5 © The Author(s) or their Institution(s) Botany Page 6 of 23 sprayer (Herbicat®, Catanduva, Brazil) with continuous pressure from compressed CO2, a rod coupled to four spray tips (Teejet, model XR11002VP) spaced 0.5 m apart, and a constant pressure regulating valve at 150 KPa. An application volume flow rate of 150 L ha-1 was used. Spraying was performed in the early morning directly over the plants. On the 1st, 4th and 7th days after glyphosate application (DAA), all the leaves were photographed with a digital camera to describe visible symptoms. Collections for the biochemical analyses were made 72 hours after the glyphosate application (HAA) and the collections for the anatomical analyses were made 7 DAA, in both cases, from five plants (N=5). Shikimic acid quantification The shikimic acid content wasDraft determined from all leaves located at the third node, of all the five repetitions, using 25 mg of fresh-frozen leaves ground in a mortar with 0.25 N hydrochloric acid. The homogenate was centrifuged at 15000 xg, at 4 °C for 25 min. 30 L of supernatant was mixed with 500 L of 1% periodic acid and the mixture was incubated at 37 ºC for 45 min. Then, 500 μL of 1 N sodium hydroxide and 300 μL of 0.1 M glycine were added. The absorbance of this mixture was measured at 380 nm using a spectrophotometer (model Cary 100, Varian, Maryland, EUA) and the shikimic acid content was determined using a molar extinction coefficient of 4.76 x 104 M-1 cm-1 (Singh and Shaner 1998).