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Interaction of drought and 5-aminolevulinic acid on growth and drought resistance of Leymus chinensis seedlings
Acta Ecologica Sinica 36 (2016) 180–188
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Interaction of drought and 5-aminolevulinic acid on growth and drought resistance of Leymus chinensis seedlings Meiru Liu, Jinhuan Li, Jianhang Niu, Ran Wang, Jixuan Song, Jun Lv, Xuefeng Zong, Sangen Wang a b c
College of Agronomy and Biotechnology, Southwest University, Chongqing 400715, China Key Laboratory of Eco-environments in Three Gorges Reservoir Region, Ministry of Education, Chongqing 400715, China Engineering Research Center of South Upland Agriculture, Ministry of Education, Chongqing 400715, China
a r t i c l e
i n f o
Article history: Received 6 January 2015 Received in revised form 9 May 2015 Accepted 14 July 2015 Keywords: Leymus chinensis Drought stress 5-Aminolevulinic acid (5-ALA) Interaction effect
a b s t r a c t Leymus chinensis is a dominant grass species in the Inner Mongolia steppes owing to its high vegetative productivity, good palatability for cattle, and abundant horizontally creeping rhizomes. Drought is generally regarded as one of the main environmental issues that is becoming a daunting challenge to the growth of plants, and ultimately results in land degradation. Drought stress influences plant growth and development by modulating physiological and biochemical events; however, some growth substances such as 5-aminolevulinic acid (5ALA) show potential to ameliorate the damaging effects of drought. The objective of this study was to evaluate the response and mechanism of L. chinensis seedlings under drought stress, the effects of 5-ALA application, and the interaction of drought stress and 5-ALA application. Healthy and uniform-size seeds of L. chinensis were collected in a natural community of the Ecological Experimental Station in the Xilingole grassland. A potting experiment was carried out to determine the influence of exogenously applied 5-ALA at various concentrations (10 mg/L, 50 mg/L, and 100 mg/L) under different soil water regimes (50% and 80% soil relative water content) on the morphological and physiological attributes of L. chinensis plants from June to November 2014. The seeds were grown in a biochemical incubator, and then the seedlings were transferred to pots. Water and Hoagland nutrient solution was applied to ensure an adequate nutrient supply at 5-day intervals. When seedlings attained a height of 18–21 cm, 5-ALA was applied at different concentrations. Water spray was applied to L. chinensis plants as a control treatment. A second and third spray of 5-ALA was applied at 1-day intervals to exploit the full potential of 5-ALA application. Simultaneously, drought stress treatment was imposed using 50% soil relative water content; 80% soil relative water content was treated as the control. Therefore, there were eight treatments with three replications established in a random complete block design. The sampling for morphological, physiological, and biochemical attributes was conducted after 15 days of drought stress, respectively. The results showed that 5-ALA could promote the growth of L. chinensis seedlings, including plant height, leaf area, plant water content, biomass, root activity, and photosynthetic pigments (chlorophyll a, chlorophyll b, and carotenoid), under a soil relative water content of 80%. Among all concentrations, 10 mg/L 5-ALA proved to have the best effect on growth. Drought stress (50% soil relative water content) hampered plant growth. However, treatments of 5-ALA ameliorated the damaging effect of drought stress on seedlings, and improved the morphological index, biomass, plant water content, root activity, photosynthetic pigments, osmotic adjustments, and antioxidase activities viz. peroxidase (POD), superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), and ascorbate (APX), but reduced malondialdehyde (MDA) content and electrical conductivity. Among all concentrations, 50 mg/L 5-ALA proved to have the best effect under drought stress. In summary, 5-ALA application improved the performance of L. chinensis by modulating growth and other morphological and physiological traits. However, the effect of 5-ALA on L. chinensis was concentration-dependent. Furthermore, significant interactions between drought stress and 5–ALA treatment (at a given spraying concentration) were observed with respect to leaf area, leaf width, fresh weight, root activity, chlorophyll b, carotenoids, MDA, proline, soluble protein, SOD, APX, and GR. © 2016 Elsevier B.V. All rights reserved.
1. Introduction As a worldwide problem, drought stress is one of the most important environmental stresses that limits plant growth and reduces grain yield E-mail address: [email protected] (S. Wang).
http://dx.doi.org/10.1016/j.chnaes.2016.04.004 1872-2032/© 2016 Elsevier B.V. All rights reserved.
by changing the morphological and physiological indices and gene expression [1,2].The negative effect of drought stress on plant is losing water and disequilibrating water balance. Also drought stress influence the normal activities of plant by damaging the photosynthetic organ and restraining the photosynthesis [3]. Drought stress can also inhibit the expansion of the blade, speed up the leaf aging and cause lipid
M. Liu et al. / Acta Ecologica Sinica 36 (2016) 180–188
membrane peroxidation, leading to the accumulation of active oxygen in plant, and followed by causing oxidative stress to plant [4,5]. Currently, grassland of China has been deteriorated along with the serious of drought stress and salinization, making the grassland productivity greatly reduced [6]. Leymus chinensis (Trin.) Tzvel is a perennial gramineous plant that widely spreads in Songnen plains, the west Liaohe plain and Hulunbuir and Xilingol steppe of Inner Mongolia. L. chinensis has the good palatability for cattle and forage value for its high quality of nutrition [7]. However, drought stress is threatening the improvement of yield and quality of L. chinensis plant, and then influences the development of local animal husbandry [8]. 5-Aminolevulinic acid (5-ALA), also known as D-amino levulinic acid, D-amino ketone acid, is a kind of hydrocarbon that contains oxygen and nitrogen. It is a key precursor in the biosynthesis of all porphyrin compounds that can promote the synthesis of chlorophyll, regulate plant growth and development [9]. As biological metabolic intermediate, 5-ALA not only can improve the photosynthesis of plants and the quality of plant [10,11], but also have similar physiological function of plant hormones and can regulate physiological activity of protective enzyme system and osmotic regulation substance content to improve the performance of plants from adversity [11]. Studies have proved that low concentration of 5-ALA can improve the cold resistance [12,13] and salt resistance [14,15] of plant. However, the promotive effect of 5-ALA on different crops is crop-dependent [16]. Per our knowledge, no study has been carried out so far to increase drought resistance of L. chinensis through exogenous application of 5-ALA, and any research about the interaction effect of 5-ALA and drought stress. This study revealed the growth and resistance characteristics of L. chinensis under drought stress, different concentrations of 5-ALA and interaction of both treatments. This study will provide reference in relieving the damage of drought stress through application of 5-ALA and theoretical direction for high productivity of grassland.
2. Materials and experimental design 2.1. Experimental material Seeds of L. chinensis were collected for Chinese Inner Mongolia Ecological Experimental Station of L. chinensis natural distribution community in November 2013. Collected seed was dried at room temperature and sealed in bags and stored at 4 °C. Pot experiment was conducted in growth incubator during June to November 2014. The seeds were grown in incubator, after several days the seedlings were transferred to the pot that the diameter is 30 cm (each pot contains 40 seedlings). Each pot contains 10 kg sand culture (sand: humus = 3:1). Water was applied every five days according to the nature of treatment and each time adequate nutrient supply was ensured by applying 50 mL 1/4 Hoagland's nutrient solution. When seedlings attained the height of 18–21 cm, foliar applications of 5-ALA solution were carried out on L. chinensis seedlings with concentrations of 10, 50 and 100 mg/L and spray was applied continuous every day, in total 3 times. Distilled water was sprayed on seedlings as control treatment for comparison. Then drought stress was imposed. Reference Hsiao [17] hierarchies of plant water stress as well as other related researches on L. chinensis under drought stress [18–20]. In control soil relative water content was kept at 80% while at 50% of soil relative water content was used to impose drought stress. Soil was weighed daily to know the moisture consumed and water was applied as needed to ensure the adequate moisture content in each pot according to the nature of treatment [21]. There are 8 treatments and each treatment was replicated three times, in total 24 pots. Morphological and physiological attributes of L. chinensis were recorded 15 days after treatment (see Table 1).
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Table 1 Experiment scheme for interaction of drought and 5-ALA. Soil relative water content
80% (normal water content, T) 50% (drought stress, HT)
5-ALA concentration (mg/L) 0
10
50
100
T0(CK) HT0
T1 HT1
T2 HT2
T3 HT3
2.2. Measuring method 2.2.1. Morphological indices, water content and dry matter content The seedlings were removed carefully from the pot and were separated aboveground and underground parts for the determination of morphological indices, such as plant fresh weight, plant height. Seedlings were rinsed with water 2–3 times and then dried with filter paper. Fresh weight and seedling length were measured. Seedlings were dried in oven at 105 °C for 15 min followed by drying at 65 °C till constant weight to determine the seedling dry weight. Plant water content was determined based on the formula of Yang et al. [22].
Watercontent ¼
Freshweight=Dry weight Freshweight
The root shoot ratio is the ratio of fresh weight (dry weight) underground parts and aboveground parts. Leaf area, leaf length and leaf width were measured with MSD-971 scanner. 2.2.2. Determination of physiological and biochemical indices The chlorophyll a, chlorophyll b, total chlorophyll and carotenoid contents were measured by the method of Arnon [23]. Select 0.1 g fresh leaves fully expanded and cut into filaments. Extract with 20 mL 95% ethanol to completely lose the green leaf and then measured using different wavelengths under 665, 649, 652 and 470 nm with an ultraviolet spectrophotometer. Selection of fresh leaves fully expanded 0.2 g and the soluble protein content was measured using Coomassie brilliant blue G-250 method [24]. Determination of soluble sugar was done by anthrone color method [25]. The malondialdehye (MDA) content was assayed using thiobarbituric acid (TBA) assay [26]. Selection of fresh leaves fully expanded 0.4 g and proline content was measured using the ninhydrin method [27]. Root samples (0.25 g) (including the root and rhizome) were weighted and root activity was measured by using the triphenyltetrazolium chloride (TTC) method of Higa et al. [28]. The leaf electrical conductivity was determined using a conductivity meter [29] which can represent the degree of cell membrane damage under stress, and the relative electric conductivity is the ratio of the electric conductivity boiled and not boiled. The method of extract of these antioxidant enzymes is refer to the method of Li et al. [30] and 0.5 g fresh leaves fully expanded were used to measure several antioxidant enzymes. Superoxide dismutase (SOD) was measured with the method of NBT-illumination method, catalase (CAT) was measured with ultraviolet absorption method, peroxidase (POD) was measured with Guaiacol oxidation method, ascorbate peroxidase (APX) was measured with ultraviolet absorption method and glutathione reductase (GR) activity was measured using the method of Khatuna [31]. 2.3. Statistical analysis Data were subjected to two-way analysis of variance (ANOVA), analyses of drought stress, different concentrations of 5-ALA and the interaction of both treatments on L. chinensis seedling growth and physiological and biochemical characteristics. Average between multiple comparisons was measured using LSD (least significant difference) test. All the data were analyzed using SPSS 17.0 statistical software. Graphics are done using Excel.
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3. Results and discussion 3.1. Effect of interaction of drought stress and varying concentrations of 5aminolevulinic acid (5-ALA) on morphological traits, water content and root activity of L. chinensis (Trin.) seedlings 5-ALA application promoted the growth of L. chinensis, but it had concentration effect. The result of variance analysis of drought stress and 5-ALA treatment showed that drought stress and 5-ALA treatment had interactive effects. Application of 10 mg/L 5-ALA proved to be most effective for it's promoting the growth of L. chinensis drastically under the condition of normal soil moisture (T). Drought stress (HT0) reduced the growth of L. chinensis drastically, and application of 5-ALA could alleviate the drought stress injury, application of 50 mg/L 5-ALA (HT2) proved to be most effective. However, high concentration of 5ALA did not alleviate drought stress injury in L. chinensis effectively when compared with controls (Table 2 and Fig. 1). Drought stress reduced the plant height of L. chinensis plants drastically when compared with control and had significant difference (P b 0.05). Application of 5-ALA could improve the plant height to some extent compared with control (T0) under normal soil water condition (T), but the concentration of 100 mg/L treatment did not prove to be much effective compared with control. Maximum amounts of plant height were recorder in the application of 10 mg/L 5-ALA and increased by 11.14% than the control treatment (T0) (Fig. 1a). Drought stress and 5-ALA treatment had interactive effect and application of 5-ALA could effective relieve the injury of L. chinensis on plant height under drought stress. The treatment of 50 mg/L 5-ALA had a significant effect (P b 0.05) (Table 2 and Fig. 1b) and the plant height was 1.2 times high as drought stress treatment, even added 6.7% more than the control (T0). Drought stress (HT0) drastically reduced the leaf area of L. chinensis when compared with control (T0) and had significant difference (P b 0.05) (Fig. 1b). In the normal soil water condition (T), application of 5-ALA could improve the leaf area to some extent compared with control (T0) and proved to be much effective, except that the concentration of 100 mg/L treatment. Maximum amounts of leaf area were recorder in the application of 10 mg/L 5-ALA and increased by 20.17% than the control treatment (T0). Exogenous application of 5-ALA could alleviate the injury under drought stress and was found had a significant effect at the concentration of 50 mg/L. The leaf area is 151.0% larger as drought stress (HT0). The interaction of drought stress and 5-ALA treatment could relieve the inhibition of leaf area to some extend (Table 2 and Fig. 1b). Fresh weight of L. chinensis plants was drastically reduced by drought stress (HT0), but had insignificant effect when compared with control (T0) (Fig. 1c). In the normal soil water condition (T), application of 5-ALA could improve the fresh weight to some extent compared with control (T0), except the concentration of 100 mg/L treatment. Application of 10 mg/L 5-ALA had significant difference compared with control (T0), and the fresh weight increased 31.03% compared with control (T0). Exogenous application of 5-ALA considerably alleviated the injury of drought stress, and had a significant effect (P b 0.05) at the concentration of 50 mg/L and the fresh weight was 145.0% weight as drought stress 125.9% weight as control (HT0).
However, the highest concentration (100 mg/L) had no significant effect on the control treatment. Also, the water content, root activity and other morphological indices all had the similar result and had concentration effect under drought stress, 5-ALA treatment and the interaction of both treatment (Table 2 and Fig. 1d and e). 3.2. Effect of interaction of drought stress and varying concentrations of 5-aminolevulinic acid (5-ALA) on photosynthetic pigment of L. chinensis (Trin.) seedlings The results of variance analysis of photosynthetic pigment content of L. chinensis seedlings under drought stress and 5-ALA treatment showed that proper concentration of 5-ALA could promote the photosynthetic pigments accumulating and had the best effect at 10 mg/L 5-ALA treatment. Drought stress (HT0) considerably inhibited the synthesis of photosynthetic pigments and application of 5-ALA could alleviate the injury to some extent. Application of 50 mg/L 5-ALA had notable significant ability to relieve the drought stress injury (Table 3 and Fig. 2). Chlorophyll a contents were appreciably enhanced with 5-ALA application under normal soil water content (T)(Fig. 2a), and a significant increase in chlorophyll a contents was observed in 10 mg/L 5-ALA treatment relative to the control. Drought stress (HT0) drastically reduced the chlorophyll a contents of L. chinensis plants, and application of 5-ALA could alleviate the drought stress injury on the contents of chlorophyll a, especially application of 50 mg/L 5-ALA had obvious effect. Furthermore, drought stress and 5-ALA had interaction effect and concentration effect (Table 3 and Fig. 2a). Also, the other photosynthetic pigments had the similar results under drought stress, 5-ALA treatment and the interaction of both treatments (Table 3 and Fig. 2). 3.3. Effect of interaction of drought stress and varying concentrations of 5-aminolevulinic acid (5-ALA) on MDA, osmotic adjustment substances and leaf electrical conductivity of L. chinensis (Trin.) seedlings The results of variance analysis of osmotic adjustment substances of L. chinensis seedlings under drought stress and 5-ALA treatment showed that both treatments had interaction effect (Table 4 and Fig. 3). Application of 5-ALA under normal soil water treatment modulated the levels of MDA and was observed to be significantly increased first then decreased. However, 5-ALA application at high concentration led to inhibit the growth and development (Fig. 3a). MDA content was observed to be significantly increased under drought stress (HT0) and increased 9.47% compared with control (T0). MDA content was observed to be significantly lower upon application of 5-ALA, and application of 50 mg/L 5-ALA proved to be much effective and the MDA content decreased 28.87% compared with drought stress (HT0) even less than control (T0) (Table 4 and Fig. 3). Drought stress and 5-ALA had the interaction effect and had significant effect on the content of MDA (P b 0.05) (Table 4). Application of 5-ALA modulated the contents of free proline and had an insignificant difference when compared with control (T0) under normal soil water condition (Fig. 3b). Drought stress exaggerated contents
Table 2 Two-way ANOVA results on the effects of soil moisture and 5-ALA concentration on morphological index, biomass and root activity of Leymus chinensis seedling. F-value Variation sources
Plant height
Leaf area
Leaf length
Leaf width
Fresh weight
Dry weight
Water content
Vigor of roots
Soil water Concentration of 5-ALA Water × concentration of 5-ALA
1.013⁎⁎⁎ 6.793⁎ 1.263⁎⁎⁎
0.749⁎⁎⁎ 21.97⁎⁎ 5.896⁎
0.261⁎⁎⁎ 8.363⁎⁎ 1.572⁎⁎⁎
8.194⁎ 11.972⁎⁎ 6.466⁎
2.303⁎⁎⁎ 12.767⁎⁎ 3.692⁎
4.485⁎ 4.416⁎ 2.323⁎⁎⁎
1.168⁎⁎⁎ 3.069⁎⁎⁎ 1.362⁎⁎⁎
1.494⁎⁎⁎ 4.259⁎ 3.651⁎
⁎ Indicates significant difference (P b 0.05). ⁎⁎ Means highly significant difference (P b 0.01). ⁎⁎⁎ Means no significant difference (P N 0.05).
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Fig. 1. Changes in height, leaf area, fresh weight, water content and root activity in Leymus chinensis seedlings under drought and 5-ALA treatment (means ± S.E., n = 3).
of free proline which may explain that the L. chinensis plants had the drought resistance capability. Free proline contents were observed to be exaggerated continually upon application of 5-ALA and were 3.14 times compared with control (T0) at the higher concentration (50 mg/L). Drought stress and 5-ALA had the interaction effect and much effective on the levels of free proline (P b 0.01). Results showed that the interaction effect could relieve the drought stress injury on
L. chinensis plants. Other osmolytes such as soluble protein and sugar contents all had the similar results (Table 4 and Fig. 3). Exacerbated levels of electrical conductivity of L. chinensis plants were occasioned under drought stress. Application of 5-ALA lowered levels of electrical conductivity and minimum at the concentration of 10 mg/L, had significant difference when compared with control (T0) (P b 0.05) (Fig. 3e). Electrical conductivity was observed to be
Table 3 Two-way ANOVA results on the effects of soil moisture and 5-ALA concentration on photosynthetic pigment of Leymus chinensis seedlings. F-value Variation sources
Chlorophyll a
Chlorophyll b
Chlorophyll a/b
Carotenoid
Total chlorophyll
Soil water Concentration of 5-ALA Water × concentration of 5-ALA
1.393⁎⁎⁎ 4.131⁎ 2.125⁎⁎⁎
5.472⁎ 11.624⁎⁎ 6.475⁎
4.606⁎ 9.953⁎⁎ 3.777⁎
2.183⁎⁎⁎ 6.041⁎⁎⁎ 5.393⁎⁎⁎
1.395⁎⁎⁎ 3.098⁎⁎⁎ 1.137⁎⁎⁎
⁎ Indicates significant difference (P b 0.05). ⁎⁎ Means highly significant difference (P b 0.01). ⁎⁎⁎ Means no significant difference (P N 0.05).
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Fig. 2. Changes in contents of photosynthetic pigment in Leymus chinensis seedlings under drought and 5-ALA treatment (means ± S.E., n = 3).
significantly lowered upon application of 50 mg/L 5-ALA and only the 66.9% when compared with control (T0). 3.4. Effect of interaction of drought stress and varying concentrations of 5aminolevulinic acid (5-ALA) on antioxidant enzyme activity of L. chinensis (Trin.) seedlings The result of variance analysis of enzymatic antioxidants of L. chinensis seedlings under drought stress and 5-ALA treatment showed that both treatments had interaction effect (Table 5 and Fig. 4). The data pertaining to activity of different antioxidant enzymes showed that drought stress considerably improved antioxidant enzyme activity compared with control (T0). Application of 5-ALA boosted the biosynthesis and activity of antioxidant enzymes under normal soil water treatment and a significant increase in activities of all these antioxidant enzymes was observed in 10 mg/L 5-ALA treatment relative to control. Furthermore, drought stress and 5-ALA interaction effect boosted the biosynthesis and activity of antioxidant enzymes to some extend and proved to be much effective at the concentration of 50 mg/L (HT2) (Table 5 and Fig. 4). The SOD activity was drastically exaggerated under drought stress compared with control (P b 0.05) (Fig. 4b). In the normal soil water
condition (T), the activity of SOD increased drastically (P b 0.05) as a result of application of 5-ALA compared with control (T0), but the highest concentration (100 mg/L) had an insignificant effect on the control treatment. Drought stress and 5-ALA interaction effect boosted the biosynthesis and activity of SOD to some extend and reached the highest at the concentration of 50 mg/L. Also the other enzymatic antioxidants such as POD, CAT, GR and APX activity all had the similar results (Table 5 and Fig. 4). 4. Discussion Previous study revealed that lower 5-ALA concentration promotes rice, wheat, and barley increasing grain yields and maturing, at the same time, increasing the ability of cold resistance and salt resistant of cotton. However, higher 5-ALA concentration may cause lethal injury [14]. Our study suggests that lower 5-ALA concentration could promote the growth and development of L. chinensis plants. Drought stress will lead to plant short, growth slowly and biomass decreased, photosynthesis is blocked [32,33]. Leaf water content reflects the degree of water deficit in plant, with the higher water content, the stronger leaf water retention and drought resistance [34]. As an important index in plant growth, the root activity affects the growth of the ground part, nutrition
Table 4 Two-way ANOVA results on the effects of soil moisture and 5-ALA concentration on MDA, osmotic adjustment substances and leaf electrical conductivity of Leymus chinensis seedlings. F-value Variation sources
MDA
Proline
Soluble protein
Soluble sugars
Leaf electrical conductivity
Soil water Concentration of 5-ALA Water × concentration of 5-ALA
1.711⁎⁎⁎ 20.51⁎ 7.589⁎
4.982⁎ 10.621⁎⁎ 9.664⁎⁎
116.844⁎⁎ 7.176⁎ 4.233⁎
38.275⁎⁎ 2.392⁎⁎⁎ 0.662⁎⁎⁎
5.939⁎ 6.349⁎ 1.613⁎⁎⁎
⁎ Indicates significant difference (P b 0.05). ⁎⁎ Means highly significant difference (P b 0.01). ⁎⁎⁎ Means no significant difference (P N 0.05).
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Fig. 3. Changes in MDA, osmotic adjustment substances and leaf electrical conductivity in Leymus chinensis seedlings under drought and 5-ALA treatment (means ± S.E., n = 3).
and yield directly [35]. Drought stress inhibited the growth of plant, makes the biomass, plant height, leaf area and root activity decreased drastically (Table 2). Application of 5-ALA promoted the growth of plant and boosted many index. Drought stress and 5-ALA had
Table 5 Two-way ANOVA results on the effects of soil moisture and 5-ALA concentration on antioxidant enzyme activity of Leymus chinensis seedlings. Variation sources
F-value POD
SOD
APX
CAT
GR
Soil water 7.086⁎ 328.406⁎⁎ 498.047⁎⁎ 157.354⁎⁎ 197.436⁎⁎ Concentration of 5-ALA 1.168⁎⁎⁎ 62.24⁎⁎ 42.544⁎⁎ 7.72⁎ 8.481⁎ Water × concentration ⁎⁎⁎ ⁎⁎ ⁎⁎ ⁎⁎⁎ 0.511 25.13 43.767 0.305 3.722⁎ of 5-ALA ⁎ Indicates significant difference (P b 0.05). ⁎⁎ Means highly significant difference (P b 0.01). ⁎⁎⁎ Means no significant difference (P N 0.05).
interaction effect. Application of 5-ALA relieved the injury of drought stress and had best effect at the concentration of 50 mg/L. The content of chlorophyll determines the photosynthetic efficiency and closely connected with the attenuation of photosynthetic efficiency [36,37]. The results of our study showed that drought stress severely impacted the chlorophyll a, b and total chlorophyll contents, which may have relationship with chlorophyll breakdown, and inducing leaf senescence, reducing effective leaf area, destroying the plant photosynthesis. At the same time, the chlorophyll a/b ratio was decreased drastically and had greater influence on chlorophyll b. Application of 5-ALA could increase photosynthetic pigment content and highest at the concentration of 10 mg/L. Drought stress and 5-ALA treatment had interaction effect, and 5-ALA application enlivened the photosynthetic pigments under stress. When the concentration of 5-ALA was 50 mg/L, photosynthetic pigment content was proper and chlorophyll b content and chlorophyll a/b ratio were significantly affected. Drought stress results in membrane lipid peroxidation and osmotic adjustment changing [38]. Malondialdehyde (MDA) is one of the main
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Fig. 4. Changes in antioxidant enzyme activity in Leymus chinensis seedlings under drought and 5-ALA treatment (means ± S.E., n = 3).
products of membrane lipid peroxidation, the cumulative amount can be used as the degree index of membrane lipid peroxidation [39], which has relationship with the degree of plasma membrane damage [15]. What's more, the plasma membrane permeability increased is the main damage of drought stress. The cell electrolyte leakage increased drastically and the leaf conductivity increases or did not become the important indices for evaluation of drought resistance [40]. Results from present study revealed that under drought stressed conditions there was increased production and accumulation of MDA and higher electric conductivity of L. chinensis plants. The treatment of 5-ALA to plants has been found to have no significant difference as compared to control, and at the concentration of 10 mg/L MDA content and electric conductivity was lower. Application of 5-ALA could relieve the injury of drought stress and MDA content and electric conductivity was minimum at the concentration of 50 mg/L. The L. chinensis plant can resist drought stress by regulation osmotic substances [41]. Free proline and soluble proteins and sugars are the main osmotic adjustment. The accumulation of these osmotic
adjustments can decrease the cell water potential and help to hold water of cell or tissue, relieve the injury of water deficit and enhance drought stress resistance [42–45]. Our study showed that there was enhanced production and accumulation of free proline and soluble proteins and sugars in L. chinensis under drought stress as compared with control, which was similar to the other reports that the L. chinensis plant has the ability to resist drought stress [8,46]. Application of 5-ALA changed the contents of free proline and soluble proteins and sugars, but were not significant. Application of 5-ALA could boost the content of free proline and soluble proteins and sugar under drought stress, and reach the maximum at the concentration of 50 mg/L. This result showed that application of 5-ALA could enhance the drought stress resistance by increasing the osmotic regulation substances to some extent. There was enhanced accumulation of reactive oxygen species (ROS) under drought stress thus induce oxidative damage [47]. Plant can scavenge of ROS by antioxidants and osmotic adjustments effectively in the evolution [48]. Antioxidant system is consist of many enzymes
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and reduced substance and SOD, POD, CAT, APX, and GR are the key enzymes of this system [49], which can scavenge the ROS to keep them below the level that causes oxidative stress. SOD is the pivotal enzyme of antioxidant system that can scavenge the ROS and the level of SOD has positive correlation with stress resistance of plant [50]. Our study showed that L. chinensis had drought resistance because SOD activity enhanced under drought stress. Application of 5-ALA could boost the activity SOD under normal soil water condition, and reach the highest at the concentration of 10 mg/L. Application of 50 mg/L 5-ALA could make the SOD activity maximum under the interaction of drought stress and 5-ALA. This showed that proper concentration of 5-ALA had the best effect to relieve drought stress. POD and CAT not only can remove but also break down the H2O2 that disproportionate by SOD and protect the plant tissue from the damage of H2O2 and •O2 [51–53]. Ascorbate– glutathione cycle plays an important role in the antioxidant system and which has two important enzymes, such as APX and GR [53]. Our study showed that POD activity enhanced under drought stress. Application of 5-ALA could boost the activity POD and reach the highest at the concentration of 10 mg/L. Application of 50 mg/L 5-ALA could make the POD activity maximum under the interaction of drought stress and 5-ALA. However, the activity of APX, CAT and GR was increased first and decreased then by application of 5-ALA, and reaches the highest at the concentration of 50 mg/L. We could know that these five antioxidant enzymes had the similar tendency on resistance of drought stress. In conclusion, application of 5-ALA could boost the growth of L. chinensis plant under normal soil water condition and had the best effect at the concentration of 10 mg/L. Drought stress inhibited the growth of L. chinensis plant, and application of proper concentration 5-ALA could relieve the injury of drought stress. The treatment with 50 mg/L 5-ALA had the best effect, which was relate to decreasing of membrane peroxidation product MDA content, increasing osmotic adjustments such as free proline and soluble proteins and sugars, and enhancing the activity of enzymatic antioxidants. Drought stress and 5-ALA had the interaction effect. The leaf area, plant width, fresh weight, root activity, chlorophyll b, carotenoid, MDA content, free proline, soluble proteins, SOD, APX, GR of L. chinensis plant were all influenced by the interaction of drought stress and 5-ALA. This study was mainly on the seedling stage of L. chinensis, it needs to make further investigation for understanding the mechanism after the seedling stage. Acknowledgments The authors are grateful to National Key Basic Research Program of China (2014CB138806) along with Crop Germplasm Resources Utilization and Innovation Base Program of the 111 project of China (104510-205001). References [1] J.S. Boyer, Plant productivity and environment, Science 218 (4571) (1982) 443–448. [2] H. Upadhyaya, S.K. Panda, B.K. Dutta, Variation of physiological and antioxidative responses in tea cultivars subjected to elevated water stress followed by rehydration recovery, Acta Physiol. Plant. 30 (4) (2008) 457–468. [3] Y.P. Guo, F.G. Mi, L.J. Yan, Y.X. Ren, S.J. Lv, B.Z. Fu, Physiological response to drought stresses and drought resistances evaluation of different Kentucky bluegrass varieties, Acta Prataculturae Sin. 23 (4) (2014) 220–228. [4] X.Y. Liu, X.Y. Xu, W.Q. Chen, Research on photosynthetic bacteria strain to biological format ion of 5-aminolevulinic acid, J. Zhejiang Univ. Sci. 29 (3) (2002) 336–340. [5] J.K. Guo, Y.R. Bi, H.Y. Li, H.G. Liang, Inhibitory effect of exogenous narciclasine on biosynthesis of δ-aminolevulinic acid and relationship to the actions of 6-BA and ABA in etiolated plants, Acta Phytophysiol. Sin. 26 (5) (2000) 437–440. [6] S.L. Fan, Z.H. Yuan, L.J. Feng, X.H. Wang, X.M. Ding, H.L. Zhen, Daily variation characteristics of photosynthesis and chlorophyll fluorescence in dahlia leaves under water stress, Acta Botan. Boreali-Occiden. Sin. 31 (6) (2011) 1223–1228. [7] T.C. Zhu, Leymus chinensis Biological Ecology, Changchun Science & Technology Press, Jilin, 2004 177–191. [8] D.F. Han, Y.J. Hu, J.X. Guo, H.L. Li, The effection of brassinolide for the drought resistance of L. chinensis, J. Changchun Norm. Univ. Nat. Sci. 26 (6) (2007) 51–53. [9] D. Von Wettstein, S. Gough, C.G. Kannangara, Chlorophyll biosynthesis, Plant Cell 7 (1995) 1039–1057.
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Acta Ecologica Sinica journal homepage: www.elsevier.com/locate/chnaes
Interaction of drought and 5-aminolevulinic acid on growth and drought resistance of Leymus chinensis seedlings Meiru Liu, Jinhuan Li, Jianhang Niu, Ran Wang, Jixuan Song, Jun Lv, Xuefeng Zong, Sangen Wang a b c
College of Agronomy and Biotechnology, Southwest University, Chongqing 400715, China Key Laboratory of Eco-environments in Three Gorges Reservoir Region, Ministry of Education, Chongqing 400715, China Engineering Research Center of South Upland Agriculture, Ministry of Education, Chongqing 400715, China
a r t i c l e
i n f o
Article history: Received 6 January 2015 Received in revised form 9 May 2015 Accepted 14 July 2015 Keywords: Leymus chinensis Drought stress 5-Aminolevulinic acid (5-ALA) Interaction effect
a b s t r a c t Leymus chinensis is a dominant grass species in the Inner Mongolia steppes owing to its high vegetative productivity, good palatability for cattle, and abundant horizontally creeping rhizomes. Drought is generally regarded as one of the main environmental issues that is becoming a daunting challenge to the growth of plants, and ultimately results in land degradation. Drought stress influences plant growth and development by modulating physiological and biochemical events; however, some growth substances such as 5-aminolevulinic acid (5ALA) show potential to ameliorate the damaging effects of drought. The objective of this study was to evaluate the response and mechanism of L. chinensis seedlings under drought stress, the effects of 5-ALA application, and the interaction of drought stress and 5-ALA application. Healthy and uniform-size seeds of L. chinensis were collected in a natural community of the Ecological Experimental Station in the Xilingole grassland. A potting experiment was carried out to determine the influence of exogenously applied 5-ALA at various concentrations (10 mg/L, 50 mg/L, and 100 mg/L) under different soil water regimes (50% and 80% soil relative water content) on the morphological and physiological attributes of L. chinensis plants from June to November 2014. The seeds were grown in a biochemical incubator, and then the seedlings were transferred to pots. Water and Hoagland nutrient solution was applied to ensure an adequate nutrient supply at 5-day intervals. When seedlings attained a height of 18–21 cm, 5-ALA was applied at different concentrations. Water spray was applied to L. chinensis plants as a control treatment. A second and third spray of 5-ALA was applied at 1-day intervals to exploit the full potential of 5-ALA application. Simultaneously, drought stress treatment was imposed using 50% soil relative water content; 80% soil relative water content was treated as the control. Therefore, there were eight treatments with three replications established in a random complete block design. The sampling for morphological, physiological, and biochemical attributes was conducted after 15 days of drought stress, respectively. The results showed that 5-ALA could promote the growth of L. chinensis seedlings, including plant height, leaf area, plant water content, biomass, root activity, and photosynthetic pigments (chlorophyll a, chlorophyll b, and carotenoid), under a soil relative water content of 80%. Among all concentrations, 10 mg/L 5-ALA proved to have the best effect on growth. Drought stress (50% soil relative water content) hampered plant growth. However, treatments of 5-ALA ameliorated the damaging effect of drought stress on seedlings, and improved the morphological index, biomass, plant water content, root activity, photosynthetic pigments, osmotic adjustments, and antioxidase activities viz. peroxidase (POD), superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), and ascorbate (APX), but reduced malondialdehyde (MDA) content and electrical conductivity. Among all concentrations, 50 mg/L 5-ALA proved to have the best effect under drought stress. In summary, 5-ALA application improved the performance of L. chinensis by modulating growth and other morphological and physiological traits. However, the effect of 5-ALA on L. chinensis was concentration-dependent. Furthermore, significant interactions between drought stress and 5–ALA treatment (at a given spraying concentration) were observed with respect to leaf area, leaf width, fresh weight, root activity, chlorophyll b, carotenoids, MDA, proline, soluble protein, SOD, APX, and GR. © 2016 Elsevier B.V. All rights reserved.
1. Introduction As a worldwide problem, drought stress is one of the most important environmental stresses that limits plant growth and reduces grain yield E-mail address: [email protected] (S. Wang).
http://dx.doi.org/10.1016/j.chnaes.2016.04.004 1872-2032/© 2016 Elsevier B.V. All rights reserved.
by changing the morphological and physiological indices and gene expression [1,2].The negative effect of drought stress on plant is losing water and disequilibrating water balance. Also drought stress influence the normal activities of plant by damaging the photosynthetic organ and restraining the photosynthesis [3]. Drought stress can also inhibit the expansion of the blade, speed up the leaf aging and cause lipid
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membrane peroxidation, leading to the accumulation of active oxygen in plant, and followed by causing oxidative stress to plant [4,5]. Currently, grassland of China has been deteriorated along with the serious of drought stress and salinization, making the grassland productivity greatly reduced [6]. Leymus chinensis (Trin.) Tzvel is a perennial gramineous plant that widely spreads in Songnen plains, the west Liaohe plain and Hulunbuir and Xilingol steppe of Inner Mongolia. L. chinensis has the good palatability for cattle and forage value for its high quality of nutrition [7]. However, drought stress is threatening the improvement of yield and quality of L. chinensis plant, and then influences the development of local animal husbandry [8]. 5-Aminolevulinic acid (5-ALA), also known as D-amino levulinic acid, D-amino ketone acid, is a kind of hydrocarbon that contains oxygen and nitrogen. It is a key precursor in the biosynthesis of all porphyrin compounds that can promote the synthesis of chlorophyll, regulate plant growth and development [9]. As biological metabolic intermediate, 5-ALA not only can improve the photosynthesis of plants and the quality of plant [10,11], but also have similar physiological function of plant hormones and can regulate physiological activity of protective enzyme system and osmotic regulation substance content to improve the performance of plants from adversity [11]. Studies have proved that low concentration of 5-ALA can improve the cold resistance [12,13] and salt resistance [14,15] of plant. However, the promotive effect of 5-ALA on different crops is crop-dependent [16]. Per our knowledge, no study has been carried out so far to increase drought resistance of L. chinensis through exogenous application of 5-ALA, and any research about the interaction effect of 5-ALA and drought stress. This study revealed the growth and resistance characteristics of L. chinensis under drought stress, different concentrations of 5-ALA and interaction of both treatments. This study will provide reference in relieving the damage of drought stress through application of 5-ALA and theoretical direction for high productivity of grassland.
2. Materials and experimental design 2.1. Experimental material Seeds of L. chinensis were collected for Chinese Inner Mongolia Ecological Experimental Station of L. chinensis natural distribution community in November 2013. Collected seed was dried at room temperature and sealed in bags and stored at 4 °C. Pot experiment was conducted in growth incubator during June to November 2014. The seeds were grown in incubator, after several days the seedlings were transferred to the pot that the diameter is 30 cm (each pot contains 40 seedlings). Each pot contains 10 kg sand culture (sand: humus = 3:1). Water was applied every five days according to the nature of treatment and each time adequate nutrient supply was ensured by applying 50 mL 1/4 Hoagland's nutrient solution. When seedlings attained the height of 18–21 cm, foliar applications of 5-ALA solution were carried out on L. chinensis seedlings with concentrations of 10, 50 and 100 mg/L and spray was applied continuous every day, in total 3 times. Distilled water was sprayed on seedlings as control treatment for comparison. Then drought stress was imposed. Reference Hsiao [17] hierarchies of plant water stress as well as other related researches on L. chinensis under drought stress [18–20]. In control soil relative water content was kept at 80% while at 50% of soil relative water content was used to impose drought stress. Soil was weighed daily to know the moisture consumed and water was applied as needed to ensure the adequate moisture content in each pot according to the nature of treatment [21]. There are 8 treatments and each treatment was replicated three times, in total 24 pots. Morphological and physiological attributes of L. chinensis were recorded 15 days after treatment (see Table 1).
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Table 1 Experiment scheme for interaction of drought and 5-ALA. Soil relative water content
80% (normal water content, T) 50% (drought stress, HT)
5-ALA concentration (mg/L) 0
10
50
100
T0(CK) HT0
T1 HT1
T2 HT2
T3 HT3
2.2. Measuring method 2.2.1. Morphological indices, water content and dry matter content The seedlings were removed carefully from the pot and were separated aboveground and underground parts for the determination of morphological indices, such as plant fresh weight, plant height. Seedlings were rinsed with water 2–3 times and then dried with filter paper. Fresh weight and seedling length were measured. Seedlings were dried in oven at 105 °C for 15 min followed by drying at 65 °C till constant weight to determine the seedling dry weight. Plant water content was determined based on the formula of Yang et al. [22].
Watercontent ¼
Freshweight=Dry weight Freshweight
The root shoot ratio is the ratio of fresh weight (dry weight) underground parts and aboveground parts. Leaf area, leaf length and leaf width were measured with MSD-971 scanner. 2.2.2. Determination of physiological and biochemical indices The chlorophyll a, chlorophyll b, total chlorophyll and carotenoid contents were measured by the method of Arnon [23]. Select 0.1 g fresh leaves fully expanded and cut into filaments. Extract with 20 mL 95% ethanol to completely lose the green leaf and then measured using different wavelengths under 665, 649, 652 and 470 nm with an ultraviolet spectrophotometer. Selection of fresh leaves fully expanded 0.2 g and the soluble protein content was measured using Coomassie brilliant blue G-250 method [24]. Determination of soluble sugar was done by anthrone color method [25]. The malondialdehye (MDA) content was assayed using thiobarbituric acid (TBA) assay [26]. Selection of fresh leaves fully expanded 0.4 g and proline content was measured using the ninhydrin method [27]. Root samples (0.25 g) (including the root and rhizome) were weighted and root activity was measured by using the triphenyltetrazolium chloride (TTC) method of Higa et al. [28]. The leaf electrical conductivity was determined using a conductivity meter [29] which can represent the degree of cell membrane damage under stress, and the relative electric conductivity is the ratio of the electric conductivity boiled and not boiled. The method of extract of these antioxidant enzymes is refer to the method of Li et al. [30] and 0.5 g fresh leaves fully expanded were used to measure several antioxidant enzymes. Superoxide dismutase (SOD) was measured with the method of NBT-illumination method, catalase (CAT) was measured with ultraviolet absorption method, peroxidase (POD) was measured with Guaiacol oxidation method, ascorbate peroxidase (APX) was measured with ultraviolet absorption method and glutathione reductase (GR) activity was measured using the method of Khatuna [31]. 2.3. Statistical analysis Data were subjected to two-way analysis of variance (ANOVA), analyses of drought stress, different concentrations of 5-ALA and the interaction of both treatments on L. chinensis seedling growth and physiological and biochemical characteristics. Average between multiple comparisons was measured using LSD (least significant difference) test. All the data were analyzed using SPSS 17.0 statistical software. Graphics are done using Excel.
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3. Results and discussion 3.1. Effect of interaction of drought stress and varying concentrations of 5aminolevulinic acid (5-ALA) on morphological traits, water content and root activity of L. chinensis (Trin.) seedlings 5-ALA application promoted the growth of L. chinensis, but it had concentration effect. The result of variance analysis of drought stress and 5-ALA treatment showed that drought stress and 5-ALA treatment had interactive effects. Application of 10 mg/L 5-ALA proved to be most effective for it's promoting the growth of L. chinensis drastically under the condition of normal soil moisture (T). Drought stress (HT0) reduced the growth of L. chinensis drastically, and application of 5-ALA could alleviate the drought stress injury, application of 50 mg/L 5-ALA (HT2) proved to be most effective. However, high concentration of 5ALA did not alleviate drought stress injury in L. chinensis effectively when compared with controls (Table 2 and Fig. 1). Drought stress reduced the plant height of L. chinensis plants drastically when compared with control and had significant difference (P b 0.05). Application of 5-ALA could improve the plant height to some extent compared with control (T0) under normal soil water condition (T), but the concentration of 100 mg/L treatment did not prove to be much effective compared with control. Maximum amounts of plant height were recorder in the application of 10 mg/L 5-ALA and increased by 11.14% than the control treatment (T0) (Fig. 1a). Drought stress and 5-ALA treatment had interactive effect and application of 5-ALA could effective relieve the injury of L. chinensis on plant height under drought stress. The treatment of 50 mg/L 5-ALA had a significant effect (P b 0.05) (Table 2 and Fig. 1b) and the plant height was 1.2 times high as drought stress treatment, even added 6.7% more than the control (T0). Drought stress (HT0) drastically reduced the leaf area of L. chinensis when compared with control (T0) and had significant difference (P b 0.05) (Fig. 1b). In the normal soil water condition (T), application of 5-ALA could improve the leaf area to some extent compared with control (T0) and proved to be much effective, except that the concentration of 100 mg/L treatment. Maximum amounts of leaf area were recorder in the application of 10 mg/L 5-ALA and increased by 20.17% than the control treatment (T0). Exogenous application of 5-ALA could alleviate the injury under drought stress and was found had a significant effect at the concentration of 50 mg/L. The leaf area is 151.0% larger as drought stress (HT0). The interaction of drought stress and 5-ALA treatment could relieve the inhibition of leaf area to some extend (Table 2 and Fig. 1b). Fresh weight of L. chinensis plants was drastically reduced by drought stress (HT0), but had insignificant effect when compared with control (T0) (Fig. 1c). In the normal soil water condition (T), application of 5-ALA could improve the fresh weight to some extent compared with control (T0), except the concentration of 100 mg/L treatment. Application of 10 mg/L 5-ALA had significant difference compared with control (T0), and the fresh weight increased 31.03% compared with control (T0). Exogenous application of 5-ALA considerably alleviated the injury of drought stress, and had a significant effect (P b 0.05) at the concentration of 50 mg/L and the fresh weight was 145.0% weight as drought stress 125.9% weight as control (HT0).
However, the highest concentration (100 mg/L) had no significant effect on the control treatment. Also, the water content, root activity and other morphological indices all had the similar result and had concentration effect under drought stress, 5-ALA treatment and the interaction of both treatment (Table 2 and Fig. 1d and e). 3.2. Effect of interaction of drought stress and varying concentrations of 5-aminolevulinic acid (5-ALA) on photosynthetic pigment of L. chinensis (Trin.) seedlings The results of variance analysis of photosynthetic pigment content of L. chinensis seedlings under drought stress and 5-ALA treatment showed that proper concentration of 5-ALA could promote the photosynthetic pigments accumulating and had the best effect at 10 mg/L 5-ALA treatment. Drought stress (HT0) considerably inhibited the synthesis of photosynthetic pigments and application of 5-ALA could alleviate the injury to some extent. Application of 50 mg/L 5-ALA had notable significant ability to relieve the drought stress injury (Table 3 and Fig. 2). Chlorophyll a contents were appreciably enhanced with 5-ALA application under normal soil water content (T)(Fig. 2a), and a significant increase in chlorophyll a contents was observed in 10 mg/L 5-ALA treatment relative to the control. Drought stress (HT0) drastically reduced the chlorophyll a contents of L. chinensis plants, and application of 5-ALA could alleviate the drought stress injury on the contents of chlorophyll a, especially application of 50 mg/L 5-ALA had obvious effect. Furthermore, drought stress and 5-ALA had interaction effect and concentration effect (Table 3 and Fig. 2a). Also, the other photosynthetic pigments had the similar results under drought stress, 5-ALA treatment and the interaction of both treatments (Table 3 and Fig. 2). 3.3. Effect of interaction of drought stress and varying concentrations of 5-aminolevulinic acid (5-ALA) on MDA, osmotic adjustment substances and leaf electrical conductivity of L. chinensis (Trin.) seedlings The results of variance analysis of osmotic adjustment substances of L. chinensis seedlings under drought stress and 5-ALA treatment showed that both treatments had interaction effect (Table 4 and Fig. 3). Application of 5-ALA under normal soil water treatment modulated the levels of MDA and was observed to be significantly increased first then decreased. However, 5-ALA application at high concentration led to inhibit the growth and development (Fig. 3a). MDA content was observed to be significantly increased under drought stress (HT0) and increased 9.47% compared with control (T0). MDA content was observed to be significantly lower upon application of 5-ALA, and application of 50 mg/L 5-ALA proved to be much effective and the MDA content decreased 28.87% compared with drought stress (HT0) even less than control (T0) (Table 4 and Fig. 3). Drought stress and 5-ALA had the interaction effect and had significant effect on the content of MDA (P b 0.05) (Table 4). Application of 5-ALA modulated the contents of free proline and had an insignificant difference when compared with control (T0) under normal soil water condition (Fig. 3b). Drought stress exaggerated contents
Table 2 Two-way ANOVA results on the effects of soil moisture and 5-ALA concentration on morphological index, biomass and root activity of Leymus chinensis seedling. F-value Variation sources
Plant height
Leaf area
Leaf length
Leaf width
Fresh weight
Dry weight
Water content
Vigor of roots
Soil water Concentration of 5-ALA Water × concentration of 5-ALA
1.013⁎⁎⁎ 6.793⁎ 1.263⁎⁎⁎
0.749⁎⁎⁎ 21.97⁎⁎ 5.896⁎
0.261⁎⁎⁎ 8.363⁎⁎ 1.572⁎⁎⁎
8.194⁎ 11.972⁎⁎ 6.466⁎
2.303⁎⁎⁎ 12.767⁎⁎ 3.692⁎
4.485⁎ 4.416⁎ 2.323⁎⁎⁎
1.168⁎⁎⁎ 3.069⁎⁎⁎ 1.362⁎⁎⁎
1.494⁎⁎⁎ 4.259⁎ 3.651⁎
⁎ Indicates significant difference (P b 0.05). ⁎⁎ Means highly significant difference (P b 0.01). ⁎⁎⁎ Means no significant difference (P N 0.05).
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Fig. 1. Changes in height, leaf area, fresh weight, water content and root activity in Leymus chinensis seedlings under drought and 5-ALA treatment (means ± S.E., n = 3).
of free proline which may explain that the L. chinensis plants had the drought resistance capability. Free proline contents were observed to be exaggerated continually upon application of 5-ALA and were 3.14 times compared with control (T0) at the higher concentration (50 mg/L). Drought stress and 5-ALA had the interaction effect and much effective on the levels of free proline (P b 0.01). Results showed that the interaction effect could relieve the drought stress injury on
L. chinensis plants. Other osmolytes such as soluble protein and sugar contents all had the similar results (Table 4 and Fig. 3). Exacerbated levels of electrical conductivity of L. chinensis plants were occasioned under drought stress. Application of 5-ALA lowered levels of electrical conductivity and minimum at the concentration of 10 mg/L, had significant difference when compared with control (T0) (P b 0.05) (Fig. 3e). Electrical conductivity was observed to be
Table 3 Two-way ANOVA results on the effects of soil moisture and 5-ALA concentration on photosynthetic pigment of Leymus chinensis seedlings. F-value Variation sources
Chlorophyll a
Chlorophyll b
Chlorophyll a/b
Carotenoid
Total chlorophyll
Soil water Concentration of 5-ALA Water × concentration of 5-ALA
1.393⁎⁎⁎ 4.131⁎ 2.125⁎⁎⁎
5.472⁎ 11.624⁎⁎ 6.475⁎
4.606⁎ 9.953⁎⁎ 3.777⁎
2.183⁎⁎⁎ 6.041⁎⁎⁎ 5.393⁎⁎⁎
1.395⁎⁎⁎ 3.098⁎⁎⁎ 1.137⁎⁎⁎
⁎ Indicates significant difference (P b 0.05). ⁎⁎ Means highly significant difference (P b 0.01). ⁎⁎⁎ Means no significant difference (P N 0.05).
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Fig. 2. Changes in contents of photosynthetic pigment in Leymus chinensis seedlings under drought and 5-ALA treatment (means ± S.E., n = 3).
significantly lowered upon application of 50 mg/L 5-ALA and only the 66.9% when compared with control (T0). 3.4. Effect of interaction of drought stress and varying concentrations of 5aminolevulinic acid (5-ALA) on antioxidant enzyme activity of L. chinensis (Trin.) seedlings The result of variance analysis of enzymatic antioxidants of L. chinensis seedlings under drought stress and 5-ALA treatment showed that both treatments had interaction effect (Table 5 and Fig. 4). The data pertaining to activity of different antioxidant enzymes showed that drought stress considerably improved antioxidant enzyme activity compared with control (T0). Application of 5-ALA boosted the biosynthesis and activity of antioxidant enzymes under normal soil water treatment and a significant increase in activities of all these antioxidant enzymes was observed in 10 mg/L 5-ALA treatment relative to control. Furthermore, drought stress and 5-ALA interaction effect boosted the biosynthesis and activity of antioxidant enzymes to some extend and proved to be much effective at the concentration of 50 mg/L (HT2) (Table 5 and Fig. 4). The SOD activity was drastically exaggerated under drought stress compared with control (P b 0.05) (Fig. 4b). In the normal soil water
condition (T), the activity of SOD increased drastically (P b 0.05) as a result of application of 5-ALA compared with control (T0), but the highest concentration (100 mg/L) had an insignificant effect on the control treatment. Drought stress and 5-ALA interaction effect boosted the biosynthesis and activity of SOD to some extend and reached the highest at the concentration of 50 mg/L. Also the other enzymatic antioxidants such as POD, CAT, GR and APX activity all had the similar results (Table 5 and Fig. 4). 4. Discussion Previous study revealed that lower 5-ALA concentration promotes rice, wheat, and barley increasing grain yields and maturing, at the same time, increasing the ability of cold resistance and salt resistant of cotton. However, higher 5-ALA concentration may cause lethal injury [14]. Our study suggests that lower 5-ALA concentration could promote the growth and development of L. chinensis plants. Drought stress will lead to plant short, growth slowly and biomass decreased, photosynthesis is blocked [32,33]. Leaf water content reflects the degree of water deficit in plant, with the higher water content, the stronger leaf water retention and drought resistance [34]. As an important index in plant growth, the root activity affects the growth of the ground part, nutrition
Table 4 Two-way ANOVA results on the effects of soil moisture and 5-ALA concentration on MDA, osmotic adjustment substances and leaf electrical conductivity of Leymus chinensis seedlings. F-value Variation sources
MDA
Proline
Soluble protein
Soluble sugars
Leaf electrical conductivity
Soil water Concentration of 5-ALA Water × concentration of 5-ALA
1.711⁎⁎⁎ 20.51⁎ 7.589⁎
4.982⁎ 10.621⁎⁎ 9.664⁎⁎
116.844⁎⁎ 7.176⁎ 4.233⁎
38.275⁎⁎ 2.392⁎⁎⁎ 0.662⁎⁎⁎
5.939⁎ 6.349⁎ 1.613⁎⁎⁎
⁎ Indicates significant difference (P b 0.05). ⁎⁎ Means highly significant difference (P b 0.01). ⁎⁎⁎ Means no significant difference (P N 0.05).
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Fig. 3. Changes in MDA, osmotic adjustment substances and leaf electrical conductivity in Leymus chinensis seedlings under drought and 5-ALA treatment (means ± S.E., n = 3).
and yield directly [35]. Drought stress inhibited the growth of plant, makes the biomass, plant height, leaf area and root activity decreased drastically (Table 2). Application of 5-ALA promoted the growth of plant and boosted many index. Drought stress and 5-ALA had
Table 5 Two-way ANOVA results on the effects of soil moisture and 5-ALA concentration on antioxidant enzyme activity of Leymus chinensis seedlings. Variation sources
F-value POD
SOD
APX
CAT
GR
Soil water 7.086⁎ 328.406⁎⁎ 498.047⁎⁎ 157.354⁎⁎ 197.436⁎⁎ Concentration of 5-ALA 1.168⁎⁎⁎ 62.24⁎⁎ 42.544⁎⁎ 7.72⁎ 8.481⁎ Water × concentration ⁎⁎⁎ ⁎⁎ ⁎⁎ ⁎⁎⁎ 0.511 25.13 43.767 0.305 3.722⁎ of 5-ALA ⁎ Indicates significant difference (P b 0.05). ⁎⁎ Means highly significant difference (P b 0.01). ⁎⁎⁎ Means no significant difference (P N 0.05).
interaction effect. Application of 5-ALA relieved the injury of drought stress and had best effect at the concentration of 50 mg/L. The content of chlorophyll determines the photosynthetic efficiency and closely connected with the attenuation of photosynthetic efficiency [36,37]. The results of our study showed that drought stress severely impacted the chlorophyll a, b and total chlorophyll contents, which may have relationship with chlorophyll breakdown, and inducing leaf senescence, reducing effective leaf area, destroying the plant photosynthesis. At the same time, the chlorophyll a/b ratio was decreased drastically and had greater influence on chlorophyll b. Application of 5-ALA could increase photosynthetic pigment content and highest at the concentration of 10 mg/L. Drought stress and 5-ALA treatment had interaction effect, and 5-ALA application enlivened the photosynthetic pigments under stress. When the concentration of 5-ALA was 50 mg/L, photosynthetic pigment content was proper and chlorophyll b content and chlorophyll a/b ratio were significantly affected. Drought stress results in membrane lipid peroxidation and osmotic adjustment changing [38]. Malondialdehyde (MDA) is one of the main
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Fig. 4. Changes in antioxidant enzyme activity in Leymus chinensis seedlings under drought and 5-ALA treatment (means ± S.E., n = 3).
products of membrane lipid peroxidation, the cumulative amount can be used as the degree index of membrane lipid peroxidation [39], which has relationship with the degree of plasma membrane damage [15]. What's more, the plasma membrane permeability increased is the main damage of drought stress. The cell electrolyte leakage increased drastically and the leaf conductivity increases or did not become the important indices for evaluation of drought resistance [40]. Results from present study revealed that under drought stressed conditions there was increased production and accumulation of MDA and higher electric conductivity of L. chinensis plants. The treatment of 5-ALA to plants has been found to have no significant difference as compared to control, and at the concentration of 10 mg/L MDA content and electric conductivity was lower. Application of 5-ALA could relieve the injury of drought stress and MDA content and electric conductivity was minimum at the concentration of 50 mg/L. The L. chinensis plant can resist drought stress by regulation osmotic substances [41]. Free proline and soluble proteins and sugars are the main osmotic adjustment. The accumulation of these osmotic
adjustments can decrease the cell water potential and help to hold water of cell or tissue, relieve the injury of water deficit and enhance drought stress resistance [42–45]. Our study showed that there was enhanced production and accumulation of free proline and soluble proteins and sugars in L. chinensis under drought stress as compared with control, which was similar to the other reports that the L. chinensis plant has the ability to resist drought stress [8,46]. Application of 5-ALA changed the contents of free proline and soluble proteins and sugars, but were not significant. Application of 5-ALA could boost the content of free proline and soluble proteins and sugar under drought stress, and reach the maximum at the concentration of 50 mg/L. This result showed that application of 5-ALA could enhance the drought stress resistance by increasing the osmotic regulation substances to some extent. There was enhanced accumulation of reactive oxygen species (ROS) under drought stress thus induce oxidative damage [47]. Plant can scavenge of ROS by antioxidants and osmotic adjustments effectively in the evolution [48]. Antioxidant system is consist of many enzymes
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and reduced substance and SOD, POD, CAT, APX, and GR are the key enzymes of this system [49], which can scavenge the ROS to keep them below the level that causes oxidative stress. SOD is the pivotal enzyme of antioxidant system that can scavenge the ROS and the level of SOD has positive correlation with stress resistance of plant [50]. Our study showed that L. chinensis had drought resistance because SOD activity enhanced under drought stress. Application of 5-ALA could boost the activity SOD under normal soil water condition, and reach the highest at the concentration of 10 mg/L. Application of 50 mg/L 5-ALA could make the SOD activity maximum under the interaction of drought stress and 5-ALA. This showed that proper concentration of 5-ALA had the best effect to relieve drought stress. POD and CAT not only can remove but also break down the H2O2 that disproportionate by SOD and protect the plant tissue from the damage of H2O2 and •O2 [51–53]. Ascorbate– glutathione cycle plays an important role in the antioxidant system and which has two important enzymes, such as APX and GR [53]. Our study showed that POD activity enhanced under drought stress. Application of 5-ALA could boost the activity POD and reach the highest at the concentration of 10 mg/L. Application of 50 mg/L 5-ALA could make the POD activity maximum under the interaction of drought stress and 5-ALA. However, the activity of APX, CAT and GR was increased first and decreased then by application of 5-ALA, and reaches the highest at the concentration of 50 mg/L. We could know that these five antioxidant enzymes had the similar tendency on resistance of drought stress. In conclusion, application of 5-ALA could boost the growth of L. chinensis plant under normal soil water condition and had the best effect at the concentration of 10 mg/L. Drought stress inhibited the growth of L. chinensis plant, and application of proper concentration 5-ALA could relieve the injury of drought stress. The treatment with 50 mg/L 5-ALA had the best effect, which was relate to decreasing of membrane peroxidation product MDA content, increasing osmotic adjustments such as free proline and soluble proteins and sugars, and enhancing the activity of enzymatic antioxidants. Drought stress and 5-ALA had the interaction effect. The leaf area, plant width, fresh weight, root activity, chlorophyll b, carotenoid, MDA content, free proline, soluble proteins, SOD, APX, GR of L. chinensis plant were all influenced by the interaction of drought stress and 5-ALA. This study was mainly on the seedling stage of L. chinensis, it needs to make further investigation for understanding the mechanism after the seedling stage. Acknowledgments The authors are grateful to National Key Basic Research Program of China (2014CB138806) along with Crop Germplasm Resources Utilization and Innovation Base Program of the 111 project of China (104510-205001). References [1] J.S. Boyer, Plant productivity and environment, Science 218 (4571) (1982) 443–448. [2] H. Upadhyaya, S.K. Panda, B.K. Dutta, Variation of physiological and antioxidative responses in tea cultivars subjected to elevated water stress followed by rehydration recovery, Acta Physiol. Plant. 30 (4) (2008) 457–468. [3] Y.P. Guo, F.G. Mi, L.J. Yan, Y.X. Ren, S.J. Lv, B.Z. Fu, Physiological response to drought stresses and drought resistances evaluation of different Kentucky bluegrass varieties, Acta Prataculturae Sin. 23 (4) (2014) 220–228. [4] X.Y. Liu, X.Y. Xu, W.Q. Chen, Research on photosynthetic bacteria strain to biological format ion of 5-aminolevulinic acid, J. Zhejiang Univ. Sci. 29 (3) (2002) 336–340. [5] J.K. Guo, Y.R. Bi, H.Y. Li, H.G. Liang, Inhibitory effect of exogenous narciclasine on biosynthesis of δ-aminolevulinic acid and relationship to the actions of 6-BA and ABA in etiolated plants, Acta Phytophysiol. Sin. 26 (5) (2000) 437–440. [6] S.L. Fan, Z.H. Yuan, L.J. Feng, X.H. Wang, X.M. Ding, H.L. Zhen, Daily variation characteristics of photosynthesis and chlorophyll fluorescence in dahlia leaves under water stress, Acta Botan. Boreali-Occiden. Sin. 31 (6) (2011) 1223–1228. [7] T.C. Zhu, Leymus chinensis Biological Ecology, Changchun Science & Technology Press, Jilin, 2004 177–191. [8] D.F. Han, Y.J. Hu, J.X. Guo, H.L. Li, The effection of brassinolide for the drought resistance of L. chinensis, J. Changchun Norm. Univ. Nat. Sci. 26 (6) (2007) 51–53. [9] D. Von Wettstein, S. Gough, C.G. Kannangara, Chlorophyll biosynthesis, Plant Cell 7 (1995) 1039–1057.
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