EXOGENOUS NITRIC OXIDE PROMOTES WATERLOGGING TOLERANCE AS RELATED TO THE ACTIVITIES OF ANTIOXIDANT ENZYMES
IN CUCUMBER SEEDLINGS1 © 2014 H. F. Fan, C. X. Du, L. Ding, Y. L. Xu
School of Agriculture and Food Science, Zhejiang Agriculture & Forestry University, Lin'an, Zhejiang, China
Received December 10, 2012
We investigated the effects of exogenous sodium nitroprusside (SNP), a nitric oxide (NO) donor, on growth of cucumber (Cucumis sativus L., cv. Jinyou No. 1) seedlings and antioxidant enzyme activities in cucumber leaves under waterlogging stress. The growth of cucumber seedlings was significantly inhibited when plants were exposed to waterlogging, whereas shoot spraying with SNP significantly alleviated the inhibition of growth from this type of stress: height, fresh and dry weights of the flooded plants increased obviously. Waterlogging also caused the activation of the antioxidant enzymes (superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and ascorbate peroxidase (APX)), the reduction of the chlorophyll content, and the accumulation of MDA and protein in leaves. It was found that SNP treatment further potentiated the anti-oxidant enzyme activities and maintained the chlorophyll and protein content during the entire waterlogging period; however, it reduced the MDA content. Thus, NO protects plants from oxidative damage and promotes growth by activation of antioxidant enzymes in leaves in an extent sufficient for the alleviation of membrane injury. However, exogenous NO had no significant effects on cucumber seedlings growth and antioxi-dant enzyme activities under nonstress conditions.
Keywords: Cucumis sativus — nitric oxide — waterlogging — growth — antioxidant enzymes
Waterlogging is defined as the saturation of the soil with water around the roots . Due to the increased frequency of extreme climate events, waterlogging has become an important constraint to crop production globally, which causes depletion of soil oxygen and results in the diminished yield of crop plants, and finally increases plant mortality . For instance, waterlogging has been shown to severely reduce grain yield of wheat in the United Kingdom, North America, and Australia  by about 20 to 50%. In Asia, more than 22 million ha of rain-fed lowlands are affected by flashfloods , and these floods are highly unpredictable. Yield losses due to these flashfloods can range from 10 to 100% depending on the water depth, dura-
1 This text was submitted by the authors in English.
Abbreviations'. APX — ascorbate peroxidase; CAT — catalase; O^ — superoxide radical; ONOO- — peroxynitrite; POD — peroxidase; SNP — sodium nitroprusside; SOD — superoxide dismutase. Corresponding author. Chang-Xia Du. School of Agriculture and Food Science, Zhejiang Agriculture & Forestry University, Lin'an, Zhejiang 311300, China. Fax. +86 571 63741276; e-mail. email@example.com
tion of submergence, temperature, turbidity of water, soil fertility, light intensity, and age of the crop .
Higher plants have an absolute requirement for oxygen to sustain metabolism and growth. Oxygen deprivation is the primary stress factor in waterlogging soils. In most cases, oxygen shortage affects directly the roots and indirectly the shoots. When tissues are hypoxic or anoxic, the oxygen-dependent pathways are suppressed, especially the energy-generating system. The biochemical consequence of oxygen shortage for plant cell metabolism is the inhibition of respiratory ATP synthesis. The absence of an energy source in the roots disturbs physiological processes in the shoots. Both physiological and biochemical responses of plants to waterlogging have been extensively investigated. Impacts of waterlogging stress include an inhibition of growth and development, stomatal closure, ion absorption and transport, and a decrease in the rate of photosynthesis and altered distribution of pho-tosynthates. The susceptibility of plants to environmental stresses, including waterlogging, has been considered to be associated with antioxidant ability. Yor-danova and Popova  demonstrated that root oxygen deficiency caused photooxidative damage to barley
(Hordeum vulgare) leaves via an increased generation of reactive oxygen species (ROS), such as superoxide
radical (O2-), hydrogen peroxide (H2O2), and hy-
droxyl radical (OH). These ROS can cause severe oxidative damage to membranes, DNA, and proteins.
In order to maintain intense growth and grain yield under waterlogging conditions, numerous attempts have been made to improve the waterlogging crop tolerance by both traditional breeding programs and bio-technological methods, although only few practical results have been obtained to date. Exogenous chemical treatment is a convenient and effective approach for enhancing the stress tolerance of crops and eventually improving crop productivity. Kubis  and Yiu et al.  reported that exogenous polyamines, such as the spermidine and putrescine, effectively activated ROS-scavenging enzymes, thus effectively alleviating damages caused by waterlogging. Yiu et al.  reported that exogenous catechin increased antioxidant enzyme activities and promoted flooding tolerance of tomato (Solanum lycopersicum).
Nitric oxide (NO) is a small, highly diffusible, gaseous free radical and an ubiquitous bioactive molecule that plays a key role as an intra- and intercellular messenger inducing various processes via either redox or additive chemistry in plants. It is able to scavenge ROS as an antioxidant agent and alter antioxidant gene expression as a signaling molecule, thus protecting plant cells from oxidative damage. In many studies, it was found that NO could counteract oxidative damage and had protective effect against various stressful conditions. NO increased the antioxidant enzyme activities of superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX) in mushroom (Agaricus bisporus) throughout the storage period . Furthermore, NO could be one of the prime signals during flooding as suggested by Dordas et al. .
The importance of NO in the plant response to hy-poxia has recently been demonstrated. The spatial accumulation of NO correlated with the initiation of cell death in alfalfa (Medicago sativa) roots exposed to hy-poxia conditions . In germinating pea (Pisum sati-vum) seeds, it was revealed that NO could regulate the respiratory oxygen consumption, so the seeds maintained some oxygen in order to prevent themselves from encountering complete anoxia . Benamar et al.  also suggested a nitrite—NO cycle to occur under hypoxia. Igamberdiev et al.  and Igamberdi-ev and Hill  reported that NO produced in mitochondria is oxidized by non-symbiotic hemoglobins,
and the resulting N O- becomes available as a substrate for nitrate reductase. This cyclic process helps to generate ATP during oxygen-deprivation conditions.
However, to date, data in explaining the relationship between waterlogging, NO, and oxidative stress are still scarce. Cucumber is an important vegetable in the world, which likes the humid environment but sensitive to waterlogging. In Asia, especially in southeast China, excessive rainfall rains often disturb cucumber production during the hot summer season. Therefore, in this study, we investigated the impact of exogenous NO on the ROS-scavenging system in cucumber seedlings under waterlogging stress.
MATERIALS AND METHODS
Plant materials and treatments. The experiments were carried out in the environment-controlled greenhouse. Cucumber (Cucumis sativus L., cv. Jinyou No. 1) seeds were placed in sterile Petri plates on filter paper moistened with distilled water. They were allowed to germinate in the dark in a thermostatically-controlled chamber at 29 ± 1°C for approximately 30 h. The germinated seeds were sown in 50 cells plug trays containing peat moss and perlite (1 : 1, v/v) at 1200 ^mol/(m2 s) photosynthetic photo flux density and 14/10 h (25-30/16-20°C) day/night regime. Relative humidity fluctuated between 60 and 75%. At the two-leaf stage, uniformly growing seedlings were transplanted to 8 cm tall and 10 cm in diameter plastic pots filled with the same compost. Plants were watered with half-strength Hoagland nutrient solution every other day to maintain optimal irrigation. At the three-leaf stage, the plants were waterlogged for 8 days by placing pots in 40 x 30 x 13-cm plastic containers filled with sufficient water to raise levels to 2 cm above the substrate surface. The seedlings were treated as follows: (1) control (CK), plants remained well-watered, and the shoots were sprayed with distilled water once each day during the period of the experiment; (2) control + NO (CK + NO), plants remained well-watered, and the shoots were sprayed with 100 ^M sodium ni-troprusside (SNP) once each day during the period of the experiment; (3) waterlogging (Waterlogging), the level of water remained 2 cm above the surface of the substrate, and the shoots were sprayed with distilled water once each day during the period of the experiment; (4) waterlogging + SNP (Waterlogging + NO), the level of water remained 2 cm above the surface of substrate, and the shoots were sprayed with 100 ^M SNP once each day during the period of the experiment. Each treatment has nine containers, and each container includes 6 plants, providing a total of 54 plants per treatment. Tween 20 (0.5%, v/v) was used as the surfactant. Leaf samples were harvested in triplicate at 0, 2, 4, 6, and 8 days after the treatment initiation and immediately analyzed. After 8 days of treatment, 15 plants per treatment were collected for
the determination of plant height, stem thickness, fresh weight, and dry weight.
Growth characteristic. Plant height was measured by a ruler with an accuracy of 1 mm from cotyledons to the apical point. Stem thickness was measured by a vern
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