RELATIONSHIP BETWEEN NITRIC OXIDE, ENZYMATIC ANTIOXIDANT SYSTEM AND ABA IN MAIZE UNDER LONG-TERM DROUGHT

- The relationship between nitric oxide (NO) and up-regulation of the antioxidant system ABA-mediated in maize was evaluated under long-term drought. Two maize genotypes with contrasting drought tolerance (BRS1010 – sensitive and DKB390 - tolerant) were exposed to two soil water levels, field capacity (FC) and water deficit (WD) at pre-flowering. After 12 days under these conditions, plants were irrigated and soil water level kept at FC until harvest. The plants BRS1010 under water deficit (WD) showed lower catalase activity and reduced levels of NO, whereas ABA and malondialdehyde (MDA) levels increased compared to plants under FC. Although, DKB390 plants under WD did not present changes on the activity of enzymatic antioxidant system and ABA compared to plants at FC, and cellular damage remain unaltered; this fact was associated with the increase in NO levels. As a result, DKB390 plants under WD showed harvest index values 50% higher than BRS1010 under the same condition.

Maize (Zea mays L.) is a species that presents high water consumption, and stomatal closure is one of the first responses under water deficit (WD) condition, which limits the leaf gas exchange and, consequently, productivity (Souza et al., 2013). This fact occurs because reduction in soil water potential with the occurrence of WD induces the biosynthesis of signaling molecules in dehydrated roots, especially abscisic acid (ABA).
This sign is emitted and transmitted to leaves where its accumulation prevents decrease in leaf water status via stomatal closure (Benesová et al., 2012), and thus induces the increase of the activity of enzymes of the antioxidant system (Souza et al.

2014).
Analysis of early-term drought in maize by Souza et al. (2014) showed that stomatal conductance to water vapor (g s ) declined with the decrease of water status, which invariably coincided with the decrease in the photosynthetic rate (A) and transpiration (E). Furthermore, these authors proposed that A improvements in maize genotypes tolerant to WD occur, in a greater extent, by controlling the oxidative stress by increasing the activity of enzymes of the antioxidant system in leaves, being this process induced by the increase in ABA levels.
As drought progress, the levels of reactive oxygen species (ROS) potentially able to cause lipid peroxidation (Chugh et al., 2011) increases, impairing A and yield due to the decline in the enzyme activity of the antioxidant system and ABA levels (Souza et al. 2014). Several evidences suggest that nitric oxide (NO) is also an important signaling molecule involved in mechanisms of defense in response to long-term drought, by inducing ABA biosynthesis (Zhao et al., 2001;Xing et al., 2004;Zhang et al, 2011) and reducing damage at membrane level and mesophyll cells (Jie et al, 2012;Zhuk & Musiyenko, 2012).

Recent studies have suggested that NO
is also involved in enhancing the enzyme activity mediated by ABA antioxidant system such as superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX) (Zhou et al., 2005;Shi et al., 2014). However, it has been observed the action of NO inhibiting the expression and activity of these enzymes (Clark et al., 2000;Murgia et al., 2004). Further evidences are required to find out the involvement of NO in the increasing of the activity of enzymes of the antioxidant system ABA-mediated.
Apparently, increases in ABA levels when plants are exposed to long-term drought occur at the expense of basal levels depletion of NO in plant tissues (Lozano-Juste & Leon, 2010), or in the absence of other tolerance mechanism.
The objective of this study was to evaluate the relationship between NO and the up-regulation of enzymes of the antioxidant system ABA-mediated in maize under long-term drought.

Plant material, cultivation conditions and experimental design
The experiment was carried out in a greenhouse at National Maize and Sorghum Scott-Knott test at 5% probability was used to test any contrast.

Enzymatic assays
The activity of the enzymes of the antioxidant system, named dismutase superoxide (SOD, EC 1.15.1.1), catalase (CAT, EC 1.11.1.6) and ascorbate peroxidase (APX, EC 1.11.1.11), was determined from the plant material extracted in a medium containing potassium phosphate buffer 0.1M (pH 6.8), 0.1 mM EDTA, 1 mM DTT, 1 mM PMSF and 1% PVPP (w/v). Total SOD activity was determined by measuring the ability to inhibit the photochemical reduction of p-nitro-blue-tetrazolium chloride at 560 nm. The activity of CAT was estimated by measuring the rate of decomposition of H 2 O 2 at 240 nm, while total APX activity was determined by monitoring the decline in absorbance at 290 nm.
The levels of ABA quantification was performed using immunoenzymatic assay kits (Phytodetec

Photosynthetic gas exchange measurements
The photosynthetic rate (A), stomatal conductance to water vapor (g s ), substomatal CO 2 concentrations (C i ) and transpiration rate (E) were measured simultaneously with chlorophyll a fluorescence parameters from 10:00 a.m. to 1:00 p.m., when A is at its maximum, under artificial PPFD of 1500 µmol photons m -2 s -1 at the leaf level, 400 mol CO 2 mol air -1 and 21% O 2 . During the measurements, the leaf-to-air vapor pressure deficit was ca 1.0 kPa and leaf temperature of 25ºC. The equipment was programmed to make curves A/C i varying sequentially CO 2 partial pressure: 40,30,20,10,5,40,60,80,100,120, 140 and 160 Pa. Estimations of g m were performed using the combined gas exchange/fluorescence data (Harley et al., 1992). A-C i curves were converted into A-C c curves to estimate the maximum rate of carboxylation of Ribulose 1,5 bisphosphate (Rubisco, Photographs were obtained using a stereoscopic microscope and the ImageJ program was used to calculate the ratio between the areas of the endosperm and the embryo.

Results and Discussion
The activity of SOD was not significantly different between the genotypes and soil water levels (data not shown). With exposure to WD, APX activity also remained unchanged, while CAT decreased only in BRS1010 genotype; in this genotype the decrease in CAT activity was followed by significant increase