Morphoanatomical and Biochemical Changes in Zeyheria Tuberculosa Exposed to Glyphosate Drift

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? :

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]

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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

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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

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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

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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

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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).

Lipid peroxidation assay

The extent of lipid peroxidation in all leaves located at the third node of all the five repetitions was determined as described by Hodges et al. (1999). 150 mg of fresh leaf material was homogenized in 2 mL of TBA reagent (20% w/v trichloroacetic acid +

0.5% w/v thiobarbituric acid), heated to 95 °C for 30 min, cooled for 15 min, and

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centrifuged at 10000 xg for 15 min. The amount of MDA- TBA complex was measured

by its specific absorbance at 532 nm; the nonspecific absorbance at 600 nm was

subtracted from 532 nm, using a spectrophotometer (model Cary 100, Varian,

Maryland, EUA). The amount of MDA was calculated from an extinction coefficient of

155 mM-1 cm-1.

Metabolite assay

To determine the protein and chlorophyll contents, 150 mg of all leaves from the

third node were harvested at midday and the metabolites were extracted as described by

Gibon et al. (2004). The soluble fraction was used to determine the chlorophyll as

described by Porra et al. (1989). The absorbance was read at 665 nm to determine the

chlorophyll a concentration. To determineDraft the protein content, the insoluble fraction

was used, as described by Gibon et al. (2004), and the absorbance was read at 595 nm,

using a spectrophotometer (model Cary 100, Varian, Maryland, EUA).

Structural and ultrastructural characterization

Samples of leaf median and marginal regions from fully expanded leaves at the

third node, without visual symptoms of damage, were collected, fixed in Karnovsky

solution (Karnovsky 1965), dehydrated in an ethyl series and embedded in

methacrylate. Cross sections (5 m thick) were made using a rotary microtome (model

(RM2155, Leica Microsysems Inc. Deerfield, USA) and stained with 5% toluidine blue,

at pH 7.2 (O’Brien and McCully 1981). For each treatment, five repetitions were used,

and three glass slides were prepared for each repetition. For each slide, three sections

were randomly chosen for observation. Photographs were taken using a light

microscope coupled to a U-Photo Camera system (model AX70RF, Olympus Optical,

Japan).

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For the micromorphological characterization, samples were dehydrated in an ethyl series, critically point dried (model Baltec, CPD 030, Liechtenstein), mounted on stubs, and sputter coated with gold (Balzers, FDU 010, Liechtenstein). Examinations and photographs were made using a scanning electron microscope (model (1430 VP,

LEO, England) at the Núcleo de Microscopia e Microanálise of Universidade Federal de

Viçosa (UFV).

Statistical analyses

Statistical analyses were performed based on a completely randomized design with five treatments and five repetitions, one plant per treatment. Data were subjected to an analysis of variance (ANOVA) using the software SAEG – Sistema de Análises Estatísticas e Genéticas (StatisticalDraft and Genetic Analysis System) at UFV (Euclydes 2004). Treatment means were compared using Tukey’s test at 5% probability.

Results

Visual symptoms became apparent 4 DAA on the plants exposed to three highest doses. They were characterized by chlorotic and necrotic spots that began and remained more apparent at the edge of the leaf blade (fig. 1).

The shikimic acid concentrations in leaves of herbicide-treated plants were always higher than the control (fig. 2). In plants of Z. tuberculosa exposed to 1440 g a. e. ha-1, the shikimic acid concentration was up to 280 mol g-1 of the fresh weight (FW), which was 14- fold higher than that found in control plants (fig. 2).

Glyphosate induced an increase in malondialdehyde (MDA) in Z. tuberculosa leaves. A higher value was found for the 1440 g a. e. ha-1 treatment; the MDA concentration was 4.8-fold higher than that found in control plants (fig. 3a). The lowest

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values of chlorophyll a content were found on the three highest doses (fig. 3b), and

overall protein content decreased with the glyphosate treatment (fig. 3c).

The leaves of Z. tuberculosa are hypostomatic with an uni-layered epidermis and

have dorsiventral mesophyll (fig. 4c). Trichomes are on the both epidermis surface, but

higher in density on the abaxial surface (fig. 4c). There was severe damage on the

leaves exposed to glyphosate. On the midrib there was plasmolysis of non-lignified cells

(i.e., parenchyma cells and cells of phloem elements totally lost their conformation and

consequently their functionality) (fig. 4b). In the leaf blade, necrosis was mainly

characterized by the collapse and degradation of epidermal cells (fig. 4d). Glandular

trichomes were also plasmolyzed (fig. 4d). The mesophyll was totally necrotic and

deformed (fig 4d), making it impossible to delimit the cellular contours, which caused

the appearance of intracellular spacesDraft throughout the leaf blade (fig. 4d).

Micromorphologically, the leaf epidermis of Z. tuberculosa has a high density

of glandular and non-glandular trichomes on the abaxial surface (fig. 4e, 4f) and a lower

density of trichomes on the adaxial surface (fig. 4g, 4h). On abaxial epidermis of leaves

exposed to glyphosate, there was rupture of glandular trichomes (fig. 4f) in relation to

control treatment (fig. 4e). On the adaxial leaf surface, there was plasmolysis of

glandular trichomes (fig. 4g) and of the ordinary epidermis cells, which resulted in a

loss of the contour of the surface of these cells and, consequently, the leaf relief (fig.

4h).

Discussion

At the application rates used in this study, glyphosate affected the leaf

morphology, anatomy, and biochemistry of Z. tuberculosa plants. The damage occurred

a few days after the application of the herbicide and was severe. Shikimic acid

accumulation indicated that glyphosate can promote inhibition in EPSPS activity in Z.

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tuberculosa leaves (Schrübbers et al. 2014; Silva et al. 2016). This accumulation is proof that the internal glyphosate concentration was high enough to cause a biological response at a physiological level. Therefore, the intracellular accumulation of shikimate can be used as a sensitive physiological biomarker for glyphosate toxicity in plant tissues (Schrübbers et al. 2014; Freitas-Silva et al. 2020), including for Z. tuberculosa.

The resistance or sensitivity of plant species to different xenobiotics is related to different intrinsic factors of the plant, such as leaf architecture (Barroso et al. 2015).

Thus, leaf morphology is one of the factors that influence the differential response of plants to herbicides (Costa et al. 2020; Freitas-Silva et al. 2020). In this study, the presence of trichomes on the epidermis of Z. tuberculosa allowed the herbicide drops to adhere more easily to the leaves. Thus, the herbicide stayed in contact with the leaf for a longer time, which probably increasedDraft its penetration.

The chemical interactions between the cuticle and the herbicide drop disrupts the integrity of epidermal cells, leading to localized chlorosis and necrosis (Lima et al.

2017) prior to the manifestation of the biochemical effects. Therefore, glyphosate is also capable of promoting damage to the application site before its translocation.

Once absorbed, glyphosate is translocated throughout the plant to metabolic sinks (Beltrano et al. 2013) and acts systemically by promoting an increase in ROS production (Gomes and Juneau 2016; Freitas-Silva et al. 2017). The strategies that plants adopt against oxidative stress are species-specific (Bussotti et al. 2005).

However, when these strategies are not enough to decrease the overproduction of ROS, oxidative damage to membrane lipids promotes loss of turgor in the cells and then a variety of anatomical and ultrastructural changes occur, such as protoplast retraction, cell apoptosis and cell collapse (Silva et al. 2016; Freitas-Silva et al. 2020). In our work,

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all these events culminate with anatomically severe changes that compromised the

viability of Z. tuberculosa leaves.

Necrosis can occur at the contact site between the leaf surface and herbicide

droplets (Mahakhode and Somkuwar 2012; Lima et al. 2017). In the leaves of Z.

tuberculosa there was complete collapse of the leaf blade in some regions, caused by

tissue death. The necrosis that occurred in the midrib promoted the mechanical

limitation of photoassimilate flow through the plant body (Orcaray et al. 2010).

Additionally, the necrosis exposed the leaf mesophyll, which promoted injuries

(i.e., cell plasmolysis and meatus formation) that contributed to photosynthesis

disturbances by altering the gas diffusion dynamics in the chlorenchyma (Freitas-Silva

et al. 2020). Another impacting factor would be the collapse and degradation of

epidermal cells. This can increaseDraft the entry of glyphosate into the foliar tissues,

contributing to severe injury in the mesophyll (Lima et al. 2017; Freitas-Silva et al.

2020).

Although it is not the first site of action of glyphosate, photosynthesis is affected

by the application of the herbicide (Gomes et al. 2017). Lower chlorophyll a

biosynthesis is one of the biochemical limitations that can occur because glyphosate is a

strong chelator and affects mineral nutrition of the plant by promoting the

immobilization and translocation of essential ions, such as magnesium that is an

essential element in the formation of the chlorophyll molecule (Cakmak et al. 2009;

Zobiole et al. 2011).

In addition, the rupture of membranes caused by excess ROS harmed the anchoring

of proteins and pigments (e.g., chlorophyll) in the organelle membranes, impairing the

photosynthetic rate (Gomes et al. 2017). This can also cause anatomical changes due to

the occurrence of cell plasmolysis and the death of plant tissues observed anatomically and visually as necrosis (Lima et al. 2017; Freitas-Silva et al. 2020).

Overproduction of ROS degrades the pigments of the antenna complex, which influences the decrease in chlorophyll a (Gomes et al. 2017). The action of the herbicide on chlorophyll a may still have occurred as result of the degradation of chloroplasts, as reported by Zobiole et al. (2009) and Mateos-Naranjo and Perez-Martin (2013). This provides evidence for the loss of the photosynthetic apparatus and changes in photosynthetic ability, resulting in reduced carbon assimilation, growth, survival and reproduction.

Considering that the first site of action of glyphosate is on the shikimate pathway, decreasing the synthesis of aromatic amino acids (Orcaray et al. 2010), a depletion in the pool of proteins Draft commonly occurs. This can lead to decreased cell division, which results in decreased plant growth and deficiency in the production of secondary metabolites such as anthocyanins and lignins (Rabello et al. 2012). Non- target plants are impacted by the herbicide glyphosate. Plant diversity resources may be at significant ecotoxicological risk from herbicides used to control weeds in nearby crop fields.

Conclusion

Our results demonstrate that glyphosate promotes an increase in shikimic acid and lipid peroxidation, and a decrease in chlorophyll a and protein content in Z. tuberculosa leaves. The morphoanatomical injuries found on the leaves of Z. tuberculosa such as: collapse and degradation of epidermal cells, cellular plasmolysis, necrotic areas in the mesophyll, rupture of glandular trichomes are severe, suggesting that the presence of glyphosate can impact this species irreversibly and compromise its survival.

Acknowledgments

This study was partially financed by the Coordenação de Aperfeiçoamento de Pessoal

de Nível Superior- Brasil CAPES- Finance code 001 and Projeto Floresta Escola. L. C.

Silva thanks the Conselho Nacional de Desenvolvimento Científico e Tecnológico

(CNPq) for the Research Productivity Scholarship 309308/2018-6. The authors also

thank the Núcleo de Microscopia e Microanálise of Universidade Federal de Viçosa for

helping with the scanning electron microscopy.

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Draft

Figure captions:

Figure 1- Visual change of Zeyheria tuberculosa leaves subjected to 1440 g a. e. ha-1 of the herbicide glyphosate. T0 (A), immediately before application of the herbicide, 4

DAA (B) and 7 DAA (C) B- Thick arrow and thin arrow indicates chlorotic and necrotic points on the leaf margin, respectively. C- Thin arrow indicates presence of necrotic points on the entire surface of’leaf blade. Bars: 5mm.

Figure 2- Shikimic acid content in leaves of Zeyheria tuberculosa 72 hours after application of herbicide glyphosate. Vertical bars indicate the standard error of the means of the treatments. Means with the same letters do not differ by Tukey´s test at 5% probability. (N = 5).

Figure 3- Lipid peroxidation, chlorophyllDraft and protein content in leaves of Zeyheria tuberculosa 72 hours after application herbicide glyphosate. Vertical bars indicate the standard error of the means of the treatments. Means with the same letters do not differ by Tukey´s test at 5% probability. (N = 5).

Figure 4- Anatomical change in Zeyheria tuberculosa leaves 7 days after application of different doses of the herbicide glyphosate. Transverse section observed under light microscopy. Control (A, C); leaves subjected to 1440 g a.e. ha-1 (B) and 1080 g a. e. ha-1

(D). Scanning electron microscopy of abaxial leaf surface (E, F) and adaxial leaf surface

(G, H). Control (E); leaves subjected to 1080 g a.e. ha-1 (F-H). Co: collenchyma, Xy: xylem, Ph: phloem, Pa: parenchyma, N: necrosis, Ead: epidermis of the leaf adaxial surface, Eab: epidermis of the leaf abaxial surface, Pp: palisade parenchyma, Sp: spongy parenchyma, Vb: vascular bundle, Ng: non-glandular trichome, Gt: glandular trichome.

In B: details reveal collapse of non-lignified cells, thin arrow: necrosis, asterisks: meatus,

square: plasmolyzed cells, thick arrow: rupture of Gt, circle: plasmolyzed Gt, arrowhead:

cell plasmolysis. A–D bars: 100 m. I–H bars: 50m.

Draft