THE EFFECT OF DROUGHT STRESS AND EXCESSIVE UV-B RADIATION ON THE MEMBRANE STATUS IN CUCUMBER COTYLEDONS AND LEAVES
DOI:10.30825/5.EJPAU.34.2017.20.4

Magdalena Rybus-Zając, Agata Korbas, Jan Kubiś
Department of Plant Physiology, Poznań University of Life Sciences, Poland

ABSTRACT

Crops grown in open ground are from early stages exposed to several stress factors acting simultaneously or in a sequence. One-week and three-week-old seedlings of cucumber plants (Cucumis sativus cv. Dar) were subjected to two abiotic stress factors, i.e. water deficit and UV-B radiation applied separately and in combination. The progressive stresses decreased the water content in cotyledons and leaves. The stresses caused membrane damage and an increase in the injury index (ID), which was the highest under drought and successively lower under UV-B/drought combination and UV-B on its own. An increase in lipoxygenase (LOX) activity and malondialdehyde (MDA) content was observed in cucumber seedlings. UV-B and water deficit caused more pronounced effect separately than in combination. Generally, the damage (injury index) observed in leaves was higher than in cotyledons and the increase in LOX activity was also slightly higher in leaves than in cotyledons. There was greater increase in the MDA content in leaves than in cotyledons.
In nature the abiotic stress  factors - water deficit, excessive radiation and heat, often occur simultaneously.  Plants respond to a combination of two different abiotic stresses in a unique manner and their response is different when stresses are applied individually. The research findings suggest that each of the stresses may alleviate the negative effect of the other stress factors. In the cotyledons and leaves of cucumber seedlings the water deficit and UV-B radiation applied together generally alleviated the effect of individual stresses within the cellular membrane, as measurements of the membrane damage, LOX activity and MDA content revealed.

Key words: abiotic stresses, cucumber, lipoxygenase, malondialdehyde, membrane damage.

INTRODUCTION

Cucumber is a vegetable, and as a thermophilic plant it is sown relatively late, at a higher temperature and under intensive solar radiation. This plant is from early stages exposed to a real risk of environmental stress factors – intensive solar radiation and soil drought. Because of a shallow root system, insufficient  and irregular rainfall, cucumbers grown in open ground often suffer the stress of water deficit as well. Recent studies revealed that the response of plants to a combination of two different abiotic stresses is unique and different from the response when each stress is applied individually. Each of the stresses may increase or alleviate the negative effect of the other stress [24]. The sequential or simultaneous effect of stress factors may result in cross-resistance. The negative effect of combined stresses is not so deleterious as additive effect or even as the effect of one of them alone [2]. There can be an antagonistic effect as well – decreased resistance to another factor acting at the same time [24]. Some plants’ responses to the combination of drought and UV-B were reviewed in detail by Bandurska et al. [8].

Drought is still a major limitation to crop productivity. Basic understanding of physiological, biochemical and gene regulatory network is essential for growing crops with enhanced tolerance to the water deficit stress [29]. In the last 20 years there has been a significant increase in the rate of transgenic or mutant plants tested for drought resistance [9]. In many agricultural areas drought is often accompanied by increased UV-B radiation [6].

Solar UV-B radiation (280–315 nm) accounts for less than 0.5% of total solar energy. Currently, solar ultraviolet-B radiation reaching the Earth`s surface is not far from the maximum because stratospheric ozone levels are near their lowest point [22, 23]. An increase in UV-B radiation may have adverse effects on crop production. According to An et al. [3], there are three potential targets for UV-B radiation in plant cells: the genetic system, photosynthetic system and membrane lipids. These authors suggested that little was known about the effect of UV-B radiation on microsomal membrane characteristics. To our knowledge, there have been too few studies concerning two or more abiotic stress factors interacting together in nature, and therefore there are very few results available. In view of this fact, we undertook the effort to study some physiological and biochemical changes within the cellular membrane of cucumber at two developmental stages – cotyledons/leaves under two abiotic stress factors, i.e. water deficit and UV-B radiation applied individually or in combination. The plant response was identified by estimation of the injury index calculated on the basis of membrane permeability and hydration status (relative water content), membrane lipid peroxidation (MDA formation) and lipoxygenase activity.

MATERIAL AND METHODS

Plant material
Seeds of cucumber plants (Cucumis sativus cv. Dar) were sown in perlite (finally 5 per 1.0 l pot) and allowed to germinate and develop in a growth chamber under the following conditions: fluorescent light intensity of 120 μmol m^-2 s^-1, photon flux density of 400–700 nm supplied by Osram LUMILUX L18/840 lamps, at a photoperiod of 14/10 h and temperature of 25/20ºC day/night and 60–70% relative humidity. PAR intensity was measured with a phytophotometer FF-01 (Sonopan). The water content was maintained at 60% of total capacity. Seedlings were divided into four groups, a control sample and three groups subjected to stress conditions: UV-B radiation, water deficit, and a combination of water deficit and UV-B radiation. The control plants were well watered and kept under PAR only.

Cotyledons: 7-day-old seedlings were subjected to the stresses for 3–10 days. Leaves: 21-day-old seedlings were subjected to the stress conditions for 9 days.

Stresses: UV-B irradiation, supplied by Philips TL 20 W/01 RS lamps, with max. 315 nm at an intensity of 16 kJ m^-2 day^-1 for 8 h per day (3.25 μmol m^-2 s^-1 – photon flux density), for 8 h per day (3.25 μmol m^-2 s^-1 photon flux density), during a 14 h light period. Water deficit was applied by withholding the water supply, reducing the water content to about 40% of the total capacity of perlite. The two stresses were applied individually or in combination for 9 days. The cotyledons or third fully expanded leaf of each seedling were used for analysis. Each time a dose sample contained 25 plants (5 pots, 5 plants per pot).

Relative water content (RWC), indicating the level of water in leaves during dehydration, was estimated according to Weatherley [30], and calculated according to the formula [(fresh weight – dry weight) / (weight at full turgor – dry weight)] · 100%.

Injury index, determined by the conductivity method, was used as a measure of injury. The amounts of electrolytes released from the stressed or control tissues were compared with total electrolyte amounts released after boiling. The injury index was calculated according to the formula by Flint et al. [14]:

I_D = (L_D – L₀) / (100 – L₀) · 100%,

where I_D – injury index, L₀ – percentage of electrolyte leakage from the control tissue in the total electrolyte content, L_D – percentage of electrolyte leakage from the stressed tissue in the total electrolyte content.

Lipid peroxidation was measured as the amount of malondialdehyde (MDA) in nmol mg^-1 proteins, determined as thiobarbituric acid reactive substance (TBARS), as described by Dhindsa and Matowe [12]. The tissue (300 mg) was homogenised in 2 ml 0.25% thiobarbituric acid (TBA) and in 10% trichloroacetic acid (TCA). The mixture was heated for 15 min in a boiling water bath and then cooled and centrifuged for 10 min at 10,000 g. The absorbance of supernatant was determined at 532 and 600 nm. A reagent blank was run simultaneously and consisted of the extract treated only with 10% TCA. The results were given in nmol mg^-1 proteins.

Lipoxygenase activity (EC 1.13.11.12) was measured according to Borrell et al. [10]. Leaves (500 mg fresh weight) were homogenised in a pre-chilled mortar with 5 ml 50 mM K-phosphate buffer pH 7.0. The homogenates were centrifuged at 17,000 g for 20 min at 4°C. The supernatant was used as the enzyme extract. The substrate was prepared by intensive stirring of 25 ml 100 mM tetraborate buffer pH 10.0, 0.1% (v/v) Tween-20 and 100 ml of linoleic acid. The enzyme activity was estimated spectrophotometrically after the addition of 25 ml of the substrate and 25 ml of the enzyme extract to 2.95 ml of 100 mM tetraborate buffer pH 7.6. The increase in absorbance was monitored at a wavelength of 234 nm. An absorbance increase of 0.001 was taken as one unit of LOX activity and expressed as µg^-1 proteins.

The protein content in the extracts was determined according to Bradford [11]. There were 3 replicates.

Statistical analyses were performed on the basis of three to five replications and the data were presented as a mean ± standard deviation.  The experimental data were subjected to one-way ANOVA and significant differences between the means were determined by Tukey's multiple range test using STATISTICA program. The data which differed significantly from the respective control sample were marked with asterisks * P< 0.05, ** P< 0.01.

RESULTS

RWC
The water deficit and excessive UV-B radiation lowered the RWC in cucumber seedlings (Fig. 1).  At the end of the 9-day stress period of drought the water content in the cotyledons was considerably reduced to 72% (Fig. 1a). UV-B radiation decreased the water content to 88% but both stresses reduced it to 78%. In the leaves the lowest water content was observed on the 9th of drought stress period – 60% (Fig. 1b). Both stresses reduced the water content to 89%, whereas UV-B to 95%. As far as the organs of the seedlings are concerned, progressive stresses decreased the RWC in the leaves more than in the cotyledons.

INJURY INDEX
The stress factors induced gradual membrane permeability. The injury index increased and the highest values were noted on the 9th day (Fig. 2). As far as the cotyledons are concerned, the index values amounted to 9.7% – water deficit, 6.9% – UV-B and dehydration, 5.9% – UV-B (Fig. 2a). The injury index in the leaves increased to 10.2% - water deficit, 7.9% – UV-B and drought, 6.2% – UV-B (Fig. 2b). Generally, the membrane permeability in the leaves was higher than in the cotyledons.

MDA
Stress-induced lipid peroxidation within the cell membrane was reflected by the MDA content (Fig. 3). In cotyledons, the highest 75% increase of MDA content was observed on the 7th day of water deficit as compared to control (Fig. 3a). It was lower under supplemental UV-B radiation (30%), but remained at the same level as in the control sample (93%) when the stresses of drought and UV-B were applied together. In leaves the highest  MDA content was observed on 7th day, and was 121% higher than in control (Fig. 3b). It was lower under the water deficit (72%), but there was no increase (91%) under both stresses as compared to relative control. The intensity of lipid peroxidation in the leaves was higher than in the cotyledons.

LOX
The stress factors altered the activity of lipoxygenase activity (Fig. 4). In comparison with the control sample – 100%, in the cotyledons the highest level was observed on the 7th day of excessive UV-B 29% (Fig. 4a). It was lower under drought (18%), but the combination of drought and UV-B radiation resulted in the same level as in the control sample. The highest LOX activity in the leaves was observed on  9th day of stress and was 45% higher than in control but it was lower under drought (30%) and at the same level as in the control sample when both drought and UV-B were applied simultaneously (Fig. 4b). Generally, LOX activity measured in the leaves was higher than in the cotyledons.

DISCUSSION

Various abiotic stress factors lead to overproduction of reactive oxygen species (ROS) in plants which are highly reactive and cause damage to proteins, lipids and carbohydrates as well as DNA and membrane degradation [15, 16]. In nature the stresses hardly ever occur separately. Water deficit, excessive radiation and heat often appear simultaneously. The activation of antioxidant defence was observed in cucumber cotyledons and leaves exposed to the stress of water deficit and excessive UV-B radiation [20, 26]. Changes occurring within the cell membranes of cucumber plants exposed to a combination of soil drought and enhanced UV-B radiation are a matter of interest.

Progressive stress conditions slowly reduced the water content, being less pronounced in the cucumber cotyledons (Fig. 1a) and more pronounced in the leaves (Fig. 1b). Drought caused the highest dehydration, whereas the combination of UV-B and drought as well as UV-B on its own resulted in relatively lower dehydration. UV-induced ion permeability of the membranes, associated with specific loss of K+ from guard cells in ion channels caused a loss of stomatal conductance [25] and it may explain lower evaporation from cotyledons/leaves exposed to supplementary UV-B radiation under the drought condition. There were similar relations observed by Feng et al. [13] in three water-stressed, UV-B irradiated  wheat cultivars, where the RWC was significantly higher than under the exposure to drought only.

The stresses caused membrane damage and an increase in the injury index (Fig. 2) calculated on the basis of ion permeability measurements. It was the highest under drought and successively lower under the UV-B/drought combination and UV-B on its own. Generally, there was greater damage observed in the leaves (Fig. 2b) than in the cotyledons (Fig. 2b). An increase in ion permeability was observed as an early consequence of UV-B radiation [17]. An et al. [3] suggested that UV-B radiation may cause changes in the structural complexity and function of microsomal membranes in spring wheat leaves. Alexieva et al. [1] noted the opposite relation in pea and wheat seedlings, i.e. greater membrane damage under UV-B than under drought stress. However, under different experimental conditions there was higher energy of UV-B and the RWC was less reduced by drought. In wheat cultivars [13] membrane permeability was significantly enhanced by UV-B and soil water stress. UV-B (13 kJ m^-2 day^-1) had greater effect than the water stress or the combination of UV-B and water stress. Membrane damage was also observed in barley and cucumber leaves under water deficit [5, 19]. Damage to lipids caused by enhanced generation of ROS was also a response to water deficit and UV-B applied individually or in combination [8]. Enhanced production of ROS may activate LOX-specific isozymes [28]. In this study the increase in LOX activity (Fig. 4) was generally a little higher in the leaves (Fig. 4b) than in the cotyledons (Fig. 4a). UV-B and water deficit caused a more pronounced effect individually than in combination. In pea and wheat seedlings there was higher LOX activity noted under higher UV-B energy supplementation [1]. This result was observed in olive leaves grown at high temperature and irradiance and subjected to controlled water deficit [28]. It was also observed in wheat exposed to UV-B irradiation only [3] and in cucumber leaves and roots exposed to water stress [4, 21]. The peroxidation of lipids is considered the most damaging process. One of the products formed from polyunsaturated precursors is malondialdehyde. It is often used as a suitable biomarker for lipid peroxidation [15, 16]. As results from changes in the LOX activity (Fig. 4), the increase in the MDA content was greater in cucumber leaves (Fig. 3b) than in cotyledons (Fig. 3a) subjected to individual stress conditions. The combination of UV-B and water deficit caused lower MDA accumulation. An increase in the MDA content was observed in olive leaves subjected to combined stresses [28]. There were similar relations observed in barley roots, but they were opposite in leaves exposed to UV-B and water deficit separately and in combination [7]. In wheat cultivars the MDA content was significantly enhanced by UV-B and soil water stress. Less intense UV-B than in this study had greater effect on lipid peroxidation than water stress or the combination of UV-B and water stress [13]. Water deficit also caused increased lipid peroxidation in barley [19] and in cucumber leaves and roots [4, 21], whereas enhanced UV-B radiation caused this effect in spring wheat leaves [3], radish [27] and oat [31].

Biomembranes are regarded as potential targets of UV-B [3] and water deficit [19, 21] but the response of plants to combined stresses is unique [24]. To summarise the results, when UV-B and water deficit were applied to cucumber cotyledons/leaves individually, they caused more pronounced effects within membranes than the combination of the stresses. Kakani et al. [18] reported that the pre-application of either UV-B or drought stress reduced the damage caused by subsequent application of other stresses. Hideg et al. [16] hypothesised that low UV-B doses, at an ambient level, caused positive effects and that stimuli-specific signalling pathways predisposed plants to a state of low alertness  and activation of antioxidant defence, which was earlier also observed in cucumber cotyledons [26] and leaves [20].  However, in this study the two environmental stress factors acted synergistically and induced protective mechanisms in the cell membrane region because one of the stresses reduced the damage caused by the other stress.

CONCLUSION

1. Both stresses caused membrane damage. It was the greatest under drought and, successively, lesser under the UV-B/drought combination and UV-B on its own. In general, the stresses caused greater damage to the leaves than to the cotyledons.
2. The increase in LOX activity was generally a little higher in the leaves than in the cotyledons. When UV-B and water deficit were applied individually, they caused a more pronounced effect than in combination.
3. The increase in the MDA content was greater in the cucumber leaves than in the cotyledons subjected to individual stress conditions. The MDA accumulation was less pronounced under the combination of UV-B and water deficit.
4. The stresses of UV-B and water deficit applied to cucumber cotyledons/leaves individually caused more pronounced effects within membranes, whereas the combination of the stresses acted synergistically and induced protective mechanisms in the cell membrane region.

REFERENCES

Accepted for print: 21.12.2017

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