Relationship between manganese toxicity and waterlogging tolerance in Zea mays L . cv .

The objective of this study was to evaluate the relationship between excess manganese and waterlogging tolerance in 18 selection cycle Zea mays L. cv. Saracura plants. Maize plants were transferred to plastic receptacles containing solutions with different concentrations of manganese. Leaves and roots were evaluated at the beginning of treatment and after 7, 14, and 21 days for chlorophyll content, biomass production and antioxidant metabolism. Mn was strongly translocated from the roots to the leaves, triggering a decrease in chlorophyll content. Excess Mn promoted an increase in reactive oxygen species that was accompanied by higher levels of antioxidative enzyme activity and lipid peroxidation. Zea mays L. cv. Saracura efficiently sequestered relatively large amounts of Mn in the leaves, with a significant impact on metabolism; however, we did not observe visual symptoms or a large decrease in biomass production.


Introduction
Soil waterlogging is a major abiotic stress affecting maize (Zea mays L.) grain yields (Yu et al., 2015).Waterlogging is typically caused by poor soil drainage combined with high levels of precipitation (Visser, Voesenek, Vartapetian, & Jackson, 2003) and is becoming increasingly frequent in many regions due to the changing climate.Long periods of soil inundation can have serious economic consequences for maize producers, inhibiting plant growth and resulting in severe yield loss (Bailey-Serres et al., 2012).
When exposed to prolonged low-oxygen stress, plants typically overproduce reactive oxygen species (ROS), which can cause oxidative damage to plant cells at high concentrations (Shabala, 2011).This damage is the result of ROS reacting with macromolecules, such as proteins, lipids and nucleic acids, leading to a loss of enzyme activity, altered membrane fluidity and genomic damage (Mittler et al., 2004).Efficient antioxidant systems that involve both nonenzymatic and enzymatic molecules can provide some protection against the deleterious effects of ROS (Mittler et al., 2004).For example, superoxide dismutases (SODs) are (uniquely) capable of scavenging O 2 -, producing H 2 O 2 .Catalase (CAT) degrades H 2 O 2 without any reducing power, providing plants with an energy-efficient way to remove this compound.However, catalase is active only at relatively high concentrations of H 2 O 2 .At lower concentrations, H 2 O 2 molecules are eliminated by ascorbate peroxidase (APX) and other peroxidases with the aid of various reductants, such as ascorbate and glutathione (Gechev, Van Breusegem, Stone, Denev, & Laloi, 2006).
In addition to reducing oxygen availability, water inundation leads a progressive decrease in soil redox potential (Zengin, 2013).Many metal oxides, including iron oxide III and manganese oxide IV, are utilized as alternative electron acceptors.Thus, the concentration of iron oxide II and manganese oxide II increases beyond that required by plants ( Khabaz-Saberi & Rengel, 2010).Under these conditions, Mn is easily taken up by the roots, reducing plant growth and altering various physiological processes (Hauck, Paul, Gross, & Raubuch, 2003).For example, a significant increase in iron and manganese concentrations was reported in maize leaves growing in sandy loam soil subjected to 34 days of waterlogging (Ashraf & Rehman, 1999).Excess exposure to metals also leads to chlorophyll degradation, probably as a consequence of the action of ROS on cell membranes (Zengin, 2013).Excess Mn-induced Fe deficiency may also cause reduced chlorophyll concentrations in plants, as Fe is essential for chlorophyll biosynthesis (El-Jaoual & Cox, 1998).Fe is required for the conversion of protoporphyrin IX to protochlorophyllide in chlorophyll biosynthesis (Beale, 1999).
Zea mays L. is one of the most sensitive cultivated species to hypoxia, restricting its production to areas that are not subject to waterlogging.In 1997, the Embrapa (Brazilian Agricultural Research Agency) Maize and Sorghum program launched (after nine cycles of selection) the Maize variety BRS 4154, commonly known as "Saracura".The main characteristic of this cultivar is its high tolerance to waterlogging, principally due to the enhanced development of aerenchyma, which increases oxygen availability to the plant (Alves et al., 2002).Saracura is the result of plant breeding for waterlogging tolerance, which has traditionally targeted traits that increase oxygen availability, prevent oxygen loss from root tissues or improve oxygen transport and storage in the roots (Jackson, & Armstrong, 1999).In contrast, the impacts of ion toxicity caused by waterlogging stress have rarely been studied despite their demonstrated importance (Shabala, 2011).
Improving waterlogging tolerance by targeting plant tolerance to ion toxicities has yet to be fully accepted in the plant breeding community (Huang et al., 2015).Nevertheless, evidence is accumulating that this could be an effective strategy.For example, wheat genotypes with an improved ability to remediate the toxic effects of ions, such as Mn 2+ , performed better than control genotypes in waterlogged soils (Khabaz-Saberi, Barker, & Rengel, 2012).To our knowledge, there have been no studies showing the adverse effects of the greater availability of manganese due to waterlogging in Zea mays L. cultivar Saracura.To address this research gap, we exposed maize plants (Saracura cultivar) to excess manganese and hypoxia and evaluated changes in biomass production, levels of photosynthetic pigments and antioxidant defense systems.

Material and methods
Zea mays L. (cv.Saracura) seeds were germinated on germination paper in a growth chamber (B.O.D. type) for ten days.The paper was first moistened with distilled water, another paper was placed on top, and both papers were made into a roll.The amount of distilled water was determined according Maia et al. (2012) in relation to the weight of the paper (2.5 mL g -1 paper).After selection for uniformity in size and vigor, plants were transferred to 10 L plastic containers (33x31x38 -WxHxD) containing a nutrient solution (Hoaglang, & Arnon, 1950).Plants were acclimated for 28 days, after which solutions with increasing concentrations were added in the following order: ¼ strength for 7 days, ½ strength for 7 days, and full strength for 14 days.Plants were then subjected to hypoxic conditions and two treatments: control and excess manganese.The original concentration of the nutrient solution was used for the both the control (2 μM Mn) and the excess Mn treatment, with the latter also exposed to manganese (500 μM Mn).The volume of the nutrient solution was replenished with deionized water on a daily basis.The pH of the solution was also adjusted daily to 5.5 ± 0.5 with NaOH solution (1 mol L -1 ), and solutions were completely replaced on a weekly basis.All plants were maintained under hypoxia, by aeration suspension, throughout the experimental period.
Evaluations were performed on leaves and roots at the beginning of the experiment and after 7, 14 and 21 days.The experimental design was completely randomized (CRD) using a 2 x 4 factorial scheme: two treatments (control and excess Mn) and four time periods (0, 7, 14, and 21 days), for a total of 8 treatments with five replications.Each experimental plot consisted of five seedlings.Simova-Stoilova, Stoyanova, Holzer, & Feller, 2004;Shi et al., 2005).
ROS generation and detoxification are well regulated under normal conditions.However, when plants are exposed to excess metals, they can overproduce ROS, leading to oxidative stress and an imbalance in cellular antioxidants (Sharma & Dubey, 2007).Excess Mn has been shown to induce oxidative stress in many plant species and alter the activity of antioxidative enzymes (Demirevska-Kepova et al., 2004;Boojar, & Goodarzi, 2008).
ROS are scavenged enzymatically by a variety of antioxidant enzymes (Apel, & Hirt, 2004).Among the antioxidative enzymes, superoxide dismutase (SOD) is responsible for the conversion of superoxide radical into hydrogen peroxide and water.H 2 O 2 is the substrate of the enzyme catalase.In this way, a positive correlation is anticipated between the activities of these enzymes.If there is an overproduction of H 2 O 2 followed by poor neutralization by antioxidant systems, this will result in damage to cell membranes.Under this scenario, SOD activity must also be correlated with MDA levels.However, in our study, these correlations were only observed in the roots.This is because H 2 O 2 in the leaves can be produced by other pathways in addition to dismutation of superoxide radicals.Indeed, there is increasing evidence that, at least partially, metal toxicity is due to oxidative damage (Xiong, Fu, Tao, & Zhu, 2010).
Increased SOD activity in response to Mn toxicity suggests induction of a protective mechanism against oxidative damage in Mn-stressed plants caused by O 2 .-.Similar increases in SOD activity have been observed in Cucumis sativus L. and Lycopersicon esculentum Mill when exposed to excess Mn (Shi et al., 2005).The earlier SOD activity observed in roots was probably because the root is the first plant organ to come into contact with the excess Mn.Conversely, SOD activity was altered in leaves slightly later in response to Mn being translocated from the roots.
CAT and APX are involved in the metabolism of H 2 O 2 produced in the cells (Apel, & Hirt, 2004).CAT has a poor affinity for H 2 O 2 because two molecules of H 2 O 2 must simultaneously enter the same active site.Therefore, its action occurs predominantly under high concentrations of the substrate.This is exactly what happens in the leaves, where higher concentrations of H 2 O 2 cause high CAT activity under excess Mn.In contrast, APX has a much higher affinity for H 2 O 2 than CAT and consequently functions in sites with low concentrations of its substrate (Ahmad, 2014).In the present study, lower H 2 O 2 generation and higher APX activity were observed in the roots.

Conclusion
Zea mays L. cv.Saracura is adapted to low oxygen availability in soil through the formation of aerenchyma (Alves et al., 2002).In addition, this cultivar efficiently sequesters relatively large amounts of Mn in the leaves, although there is a significant impact on metabolism.Further work is necessary to determine the mechanisms underlying the increased shoot accumulation of Mn in this cultivar.Although there are relatively large amounts of Mn in leaves, there were no visual symptoms and only a moderate decrease in biomass production.
H 2 O 2 and MDA (0.80 for leaves and 0.82 for roots), as well as a negative correlation between the concentrations of H 2 O 2 and chlorophyll a (-0.70).These results support the negative impact of ROS on cellular components and photosynthetic pigments.Similarly, excess Mn elevated H 2 O 2 levels and induced oxidative stress in Hordeum vulgare L. and Cucumis sativus plants (Demirevska-Kepova, gá, v. 39, n. 1, p., n. 1, p. 75-82, J gá, v. 39, n. 1, p.