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Adaptability of Moringa oleifera Lam. under different water holding capacities
SAJB-02521; No of Pages 5 South African Journal of Botany xxx (2019) xxx
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Adaptability of Moringa oleifera Lam. under different water holding capacities S. Azam a, W. Nouman a,⁎, Ubaid-ur Rehman a, U. Ahmed a, T. Gull b, M. Shaheen a a b
Department of Forestry & Range Management, Bahauddin Zakariya University, Multan, Pakistan Department of Chemistry, University of Central Punjab, Lahore, Pakistan
a r t i c l e
i n f o
Article history: Received 12 March 2019 Received in revised form 12 June 2019 Accepted 5 August 2019 Available online xxxx Edited by B Ncube Keywords: Ascorbic acid Benzyl amino purine Hydrogen peroxide Salicylic acid Thiourea
a b s t r a c t Growth performance and enzymatic-antioxidant activities of Moringa oleifera were investigated under simultaneous effect of water availability and plant growth regulators. Plant growth regulators i.e., cytokinin (50 mg L−1), thiourea (5 mM), benzyl amino purine (BAP @ 50 mg L−1), salicylic acid (50 mg L−1), hydrogen peroxide (120 μM) and ascorbic acid (50 mg L−1) were exogenously applied to moringa plants at three water holding capacity levels, 100, 70, and 40% in a completely randomized design with three replications. A decrease in shoot length, leaf score and number of branches was recorded at 100 and 40% water holding capacity as the plants respond well at 70% water holding capacity. BAP, cytokinin and salicylic acid were found as effective plant growth regulators in improving drought tolerance in moringa plants even at 40% water holding capacity. On the basis of these findings, moringa can be suggested to cultivate as a fodder crop under water deficit conditions and its tolerance can be further improved by foliar application of benzyl amino purine and ascorbic acid. © 2019 SAAB. Published by Elsevier B.V. All rights reserved.
1. Introduction Morphological changes and physiological interactions in plants are greatly influenced by light intenstity, temperature, soil texture and its water holding capacity. Of these factors, water availability is very important factor which is imperative for plant growth and development as water affects crop yield, and its quality. Limited water availability may result in retarded plant growth, reduced yield and poor quality as cell division and elongation are the most sensitive plant functions which are sensitive to water availability. Cell sensitivity might be attributed to the reduction in turgor pressure and interruption of water flow from the xylem to the surrounding elongating cells (Hussain et al., 2008; Farooq et al., 2009). Beside these, reduced leaf area, root expansion and elongation in search of water, metabolic changes and oxidative damage are also caused by drought resulting in poor plant growth (Reddy et al., 2004; Farooq et al., 2009). The fodder crops are not exception and these also affected by drought conditions which result in affecting livestock fodder requirement. A number of studies have been carried out on exploring drought resistant fodder plants which can be cultivated under limited water availability. For this, various drought tolerant plants and cultivars of agricultural crops have been identified and are being grown in water deficit areas (Farooq et al., 2009). The use of plant growth regulators ⁎ Corresponding author. E-mail address: [email protected] (W. Nouman).
like ascorbic acid, benzyl aminopurine (BAP) and cytokinins is crucial to mitigate drought stress (Senaratna et al., 2000; Farooq et al., 2009). Plant growth regulators have been used in different ways like exogenous application (foliar/root), seed priming and soil fertigation to mitigate drought stress. Foliar application of plant growth regulators like ascorbic acid (Basra et al., 2006; Farooq et al., 2006), salicylic acid (Borsani et al., 2001) and BAP (Amin et al., 2007) manifested significant results on different crops and vegetables in terms of improving growth performance under water deficit conditions. Moringa oleifera is not an exception as being cultivated in seasonally dry tropical and subtropical regions due to its fast growth and uses as livestock fodder, plant growth enhancer, therapeutic effects, etc. Moringa oleifera is a potential fodder crop, which can also be grown as hedges, fences and tree orchard. Its foliage being rich in nutrients, antioxidants and phenolic compounds, is being used as animal fodder in different parts of the world especially in African countries. Recently, many research studies reported the importance of moringa as a fodder crop with good nutritional quality which can be a good alternate to other fodder crops (Nouman et al., 2013, 2014). Moringa is a hardy plant that can be grown under diverse soil conditions and saline lands (Nouman et al., 2013). This report supports the idea that moringa can be grown under drought conditions. For this, morphological changes, physiological adaptations and antioxidant activities which support the plant to withstand under stress conditions should be studied. A very limited studies are available which demonstrate moringa adaptation under saline conditions especially focusing on nutritional quality and
https://doi.org/10.1016/j.sajb.2019.08.020 0254-6299/© 2019 SAAB. Published by Elsevier B.V. All rights reserved.
Please cite this article as: S. Azam, W. Nouman, U. Rehman, et al., Adaptability of Moringa oleifera Lam. under different water holding capacities, South African Journal of Botany, https://doi.org/10.1016/j.sajb.2019.08.020
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S. Azam et al. / South African Journal of Botany xxx (2019) xxx
antioxidant activities (Santos et al., 2011; Nouman et al., 2012a, 2012b, 2014). Antioxidant system serves as plant's defensive mechanism which support it to withstand under stress conditions. Water deficit results in incomplete reduction of oxygen resulting in the formation of reactive oxygen species (ROS) like singlet oxygen, superoxide anion radical, and hydroxyl radical which affect plant vigor and lower its nutritional quality (Mittler, 2002; Munne-Bosch and Penuelas, 2003). A few scientists are with opinion that ROS are essential for normal cellular processes but too low or too high ROS levels impair plant growth and development (Mittler, 2017). Antioxidant system enables the plants to come over such oxidative damage through a number of enzymatic antioxidants. Damaging impact of stress can be minimized through enzymatic antioxidants like SOD, POD, CAT, etc. which improves plant tolerance to mitigate stress conditions (Athar et al., 2008; Munns and Tester, 2008). As it has been reported that moringa can survive under moderate saline conditions, it is still to investigate that how moringa growth is affected under limited water availability, is there any way to induce/ improve tolerance in moringa plants to withstand under drought conditions? If moringa can grow under water deficit conditions, drought affected areas can be brought under moringa cultivation which can support our livestock as moringa is a potential fodder crop being a rich source of nutrients. The present study was designed aiming at investigating the response of moringa under limited water availability including changes in its growth and activities of enzymatic antioxidants.
2. Materials and methods 2.1. Seed material Moringa oleifera seeds were collected in June, 2015 from 5 years old tree grown in the research area of the Department of Forestry& Range Management, Bahauddin Zakariya University, Multan-Pakistan. Freshly collected seeds were immediately processed for the experiment as it has been reported that fresh moringa seeds have good germination which goes down with the passage of time (Nouman et al., 2012b). Before sowing, non-infected and vigorous seeds were sorted out and hydroprimed for 8 h to improve their tolerance under stress conditions (Nouman et al., 2014).
2.2. Plant vigor evaluation The experiment was conducted in glasshouse of the Department of Forestry & Range Management, Bahauddin Zakariya University, Multan-Pakistan where temperature was maintained at 33 ± 2 °C, and daylight/ night conditions were 16 and 8 h, respectively. Moringa seeds were sown in earthen pots filled with 3.7 kg of soil (clay loam), sand and green manure (1,1:1) in completely randomized design (CRD) with two factor factorial (water holding capacity and foliar application) arrangement and three replications. Five seeds in each pot were sown and it was noted that 83.3% seeds emerged, on average. After emergence, three plants were maintained in each pot; one pot was considered as one replication. When plants reached an age of one month, three water holding capacity levels (100, 70 and 40%) were applied in three replications and maintained for next two months. Water holding capacity i.e., 100% was considered as half of the soil saturation capacity. Beside these, exogenous foliar application of cytokinin (50 mg L−1), thiourea (5 mM), bezyl amino purine (BAP @50 mg L−1), salicylic acid (50 mg L−1), hydrogen peroxide (H2O2@120 μM) and ascorbic acid (50 mg L−1) were applied beside one control (no spray) and water to investigate the response of moringa plants under above mentioned water holding capacities. The growth performance of moringa plants was noted on the day of harvest (two months after the emergence). Shoot length, root length, number of leaves (leaf score) and roots (root score) were recorded.
2.3. Antioxidants assay For analyzing activities of enzymatic antioxidants i.e., superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT), fresh moringa leaves were plucked at dawn time and stored at −70 °C to avoid heat and light effect on enzymatic antioxidants. Superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) activities were noted at 560, 470 and 240 nm, respectively by using UV spectrophotometer (UV-4000, O.R.I. Germany) according to procedure described by Giannopolitis and Ries (1977) and Chance and Maehly (1955). 2.4. Statistical analysis The replicated data was analyzed in software (Statistix 8.1) and a CRD two factor factorial arrangement was applied for analysis of variance (Gomez and Gomez, 1984). Significance of variance (p b .05) was found for determining difference among treatments, water holding capacity levels and interaction of water holding capacity levels and foliar application of plant growth regulators. Least significance difference test (LSD @ 5% probability level) was used to compute the differences among the mean values (Steel et al., 1997). 3. Results In present investigation, growth characteristics of moringa seedlings and antioxidant activities were significantly (p b .05) affected by water availability and plant growth regulators. It was noted that moringa plants exhibited maximum shoot length, leaf score and number of branches at 70 followed by 100% water holding capacity while maximum root length was found at 70 followed by 40% water holding capacity while plant growth regulators were also found effective in improving moringa tolerance under limited water availability. Maximum shoot length was observed at 70% water holding capacity when moringa plants were exogenously sprayed with BAP followed by thiourea and cytokinin (22.4, 21.7 and 20.6 cm, respectively) while the least shoot length was recorded at 40% water holding capacity when moringa plants were not sprayed with any plant growth regulator (Table 1). A likewise trend was recorded in moringa branches and leaves where maximum branches and leaves were found at 70% water holding capacity while a significant reduction was recorded at 40% water holding capacity due to less water availability which was mitigated effectively by cytokinin, thiourea and BAP (Tables 2, 3). In general, moringa plants grew best under less water availability, perhaps as a result, the plants grew less in height under 100% water holding capacity. The response of moringa roots were found a bit different from shoot length. Cytokinin maximally improved root length of moringa plants at 70% water holding capacity followed by thiourea and BAP at same water holding capacity level (19.7, 16.5 and 15.4 cm, respectively) (Table 4). It was noted that moringa plants exhibited 11.2 cm root length at 40% water holding capacity when moringa plants were not
Table 1 Effect of plant growth regulators as foliar application on the shoot length (cm) of M. oleifera seedlings under different water holding capacities. Treatments
No spray Water Cytokinin Thiourea BAP Salicylic acid H2O2 Ascorbic acid
Water holding capacity 100%
70%
40%
17.8 c–h 17.1 h 18.1 c–h 17.3 f–h 19.2 c 17.8 d–h 18.2 c–h 18.0 c–h
18.9 cd 18.7 c–f 20.6 b 21.7 ab 22.4 a 18.8 c–e 17.6 d–h 18.6 c–g
16.8 h 17.4 f–h 18.1 c–h 17.3 f–h 17.3 f–h 17.3 f–h 17.2 gh 17.5 e–h
Means not showing same letters in a column differ significantly at 5% probability level, LSD (5%) value for Shoot Length: 1.39.
Please cite this article as: S. Azam, W. Nouman, U. Rehman, et al., Adaptability of Moringa oleifera Lam. under different water holding capacities, South African Journal of Botany, https://doi.org/10.1016/j.sajb.2019.08.020
S. Azam et al. / South African Journal of Botany xxx (2019) xxx Table 2 Effect of plant growth regulators as foliar application on the branches of M. oleifera seedlings under different water holding capacities. Treatments
No spray Water Cytokinin Thiourea BAP Salicylic acid H2O2 Ascorbic acid
Water holding capacity
Table 4 Effect of plant growth regulators as foliar application on the root length (cm) of M. oleifera seedlings under different water holding capacities. Treatments
100%
70%
40%
6.0 b–d 5.0 d–f 7.3 ab 6.0 b–d 5.0 d–f 7.3 ab 5.3 c–e 6.0 b–d
6.7 a–c 7.3 ab 8.0 a 8.0 a 7.0 ab 7.7 a 6.0 b–d 7.0 ab
2.7 g 4.3 ef 5.0 d–f 4.7 d–f 6.0 b–d 4.0 e–g 3.7 fg 6.0 b–d
3
No spray Water Cytokinin Thiourea BAP Salicylic acid H2O2 Ascorbic acid
Water holding capacity 100%
70%
40%
10.9 h–j 10.2 ij 10.0 d–g 11.5 f–j 11.9 e–i 14.2 cd 10.5 ij 10.9 h–j
12.9 d–g 12.3 e–h 19.7 a 16.5 b 15.4 bc 16.5 d–f 11.8 e–j 14.4 cd
11.2 h–j 14.2 cd 15.0 bc 10.2 j 15.2 bc 11.3 g–j 13.3 de 14.5 cd
Means not showing same letters in a column differ significantly at 5% probability level, LSD (5%) value for number of branches: 1.65.
Means not showing same letters in a column differ significantly at 5% probability level, LSD (5%) value for root length: 1.73.
sprayed with any plant growth regulator which is more than root length recorded in control plants at 100% water holding capacity while 35.7 and 33.9% increase in root length was recorded at 40% water holding capacity when BAP and cytokinin were exogenously applied to moringa plants which is even more than root lengths recorded at 100% water holding capacity (Table 4). Moringa adoption under water stress conditions might be due to antioxidant system. For this, activities of enzymatic antioxidants were also investigated. It was noted that antioxidant enzymes (SOD, POD and CAT) were significantly affected by water availability and foliar application of plant growth regulators. An increase in CAT activity was observed in moringa leaves at 70% water holding capacity in comparison to 100 while it was damaged at 40% (99.1, 64.8 and 41.8 unit mg−1 protein, respectively). Salicylic acid, cytokinin and BAP were statistically at par with each other sustaining and improving catalase activity (81.7, 80.9 and 77.7 unit mg−1 protein, respectively, on average) (Table 5). Salicylic acid and ascorbic acid maximally improved peroxidase activity in moringa plants exhibiting 693.4 and 684.5 unit mg−1 protein, respectively followed by thiourea and BAP. The least peroxidase activity (452.9 and 448.5 unit mg−1 protein, on average) were noted in control and water sprayed moringa plants, respectively. Moringa plants did not exhibit better POD activities at 100% water holding capacity while salicylic acid and ascorbic acid are more effective in sustaining peroxidase activities in moringa plants even at 40% water holding capacity (Table 6). Salicylic acid was found effective on sustaining and improving SOD activity of moringa leaves under limited water availability. Beside salicylic acid, foliar application of ascorbic acid and BAP were also found effective improving SOD activity resulted in mitigating drought stress in moringa plants (Table 7). The least SOD activity was recorded at 40% water holding capacity while the maximum was found when moringa plants were subjected to 70% water holding capacity. Foliar application of salicylic acid and ascorbic acid were at par with each other in improving SOD activities at 70% water holding capacity (354.9, 308.9 unit mg−1 protein). No doubt, severe decline was recorded in SOD activity at 40% water holding capacity (84.2 unit mg−1 protein) which was 38.2% less than
SOD activity at 70% water holding capacity without any foliar application, it was further improved by salicylic acid and ascorbic acid foliar application (Table 7).
During growth and development phases, plants face several stress conditions like salinity, heat, cold and drought which ultimately affect plant growth, yield and physiology. The impact of drought conditions on plant growth and development is well known but being a complex mechanism, and it is rather difficult to understand the complexities. In present study, the response of M. oleifera to water availability and stress mitigation through plant growth regulators were studied. As mentioned above, moringa plants performed best under at 70% water holding capacity while at 100 and 40% water holding capacity, shorter plant height was recorded. A significant improvement in shoot length, leaf score and number of branches was recorded with the assistance of exogenously applied plant growth regulators especially BAP and cytokine into mitigate drought conditions (Tables 1–3). A likewise trend was recorded in root length of moringa plants. It has been reported that cell division and elongation, growth and development determining physiological processes are drought sensitive (Farooq et al., 2009). Secondly, moringa plants thrived better at low water availability. For this, the plants showed less height at 100% water holding capacity. Increase in root length at 70 and 40% water holding capacitymight be attributed to lower osmotic pressure at stress conditions which trigger root proliferation in search of water and nutrients (Hsiao and Xu, 2000). Root expansion and proliferation might be due to ethylene production as reported by Dantas et al. (2007). Similar response of moringa roots under saline conditions have been previously reported by Nouman et al., 2012a, Nouman et al., 2014). It is well understood that reduction in shoot length and leaf score and increase in root length are survival strategies of plants to withstand under stress conditions. As in present study, leaf score decreased while root length increased at 40% water holding capacity, it might be due to plant survival mechanism reducing
Table 3 Effect of plant growth regulators as foliar application on leaf score of M. oleifera seedlings under different water holding capacities.
Table 5 Effect of plant growth regulators as foliar application on the catalase activity (unit mg−1 protein) of M. oleifera seedlings under different water holding capacities.
Treatments
No spray Water Cytokinin Thiourea BAP Salicylic acid H2O2 Ascorbic acid
Water holding capacity
4. Discussion
Treatments
100%
70%
40%
39.3 gh 37.3 g–i 50.7 de 41.0 gh 43.7 e–g 42.0 gh 44.0 e–g 43.3 fg
53.0 cd 59.7 bc 61.0 b 52.3 d 69.7 a 52.0 d 70.0 a 61.7 b
21.0 k 35.0 hi 31.0 ij 41.7 gh 32.0 ij 27.0 jk 23.7 k 50.0 d–f
Means not showing same letters in a column differ significantly at 5% probability level, LSD (5%) value for leaf score: 7.28.
No spray Water Cytokinin Thiourea BAP Salicylic acid H2O2 Ascorbic acid
Water holding capacity 100%
70%
40%
49.7 gh 54.7 fg 77.2 d 63.9 ef 65.4 d–f 68.3 de 72.4 de 67.3 de
68.9 de 71.2 de 114.1 b 95.6 c 112.7 b 128.6 a 103.4 bc 98.0 c
35.9 ij 36.8 ij 51.4 gh 39.1 hi 54.9 fg 48.1 g–i 26.6 j 41.7 hi
Means not showing same letters in a column differ significantly at 5% probability level, LSD (5%) value for catalase activity: 12.28.
Please cite this article as: S. Azam, W. Nouman, U. Rehman, et al., Adaptability of Moringa oleifera Lam. under different water holding capacities, South African Journal of Botany, https://doi.org/10.1016/j.sajb.2019.08.020
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S. Azam et al. / South African Journal of Botany xxx (2019) xxx
Table 6 Effect of plant growth regulators as foliar application on the peroxidase activity (unit mg−1 protein) of M. oleifera seedlings under different water holding capacities. Treatments
No spray Water Cytokinin Thiourea BAP Salicylic acid H2O2 Ascorbic acid
Water holding capacity 100%
70%
40%
389.4 k 400.7 k 525.8 f–h 646.0 de 554.7 fg 540.8 f–h 601.8 ef 566.8 e–g
500.1 g–j 514.0 g–i 695.5 d 805.9 c 827.0 c 1002.8 a 819.9 c 916.0 b
469.2 h–k 431.0 i–k 541.1 f–h 465.9 h–k 526.0 f–h 536.8 f–h 422.1 jk 570.7 e–g
Means not showing same letters in a column differ significantly at 5% probability level, LSD (5%) value for peroxidase activity: 84.60.
water loss through transpiration and more water uptake by proliferated roots (Houle et al., 2001). Prolific and expanded root growth is an adaptation to water deficit allowing plants to uptake water and nutrients from deep soil (Nguyen et al., 1997; Kavar et al., 2007). Root extension and proliferation has been previously noted in moringa seedlings under saline condition while tea, onion and cotton plants also exhibited this phenomenon under drought stress (Farooq et al., 2009; Nouman et al., 2014). It was noted in the present study that foliar application of plant growth regulators improved moringa tolerance under drought conditions. Among these, cytokinin, BAP and ascorbic acid were found as effective agents inducing tolerance in moringa plants under limited water availability as stated earlier. Plant survival under stress conditions is not only through morphological changes while physiological or enzymatic changes also contribute to tolerate drought stress. Plants produce reactive oxygen species under water deficit, which affect plant vigor and nutritional quality. Enzymatic antioxidants enable plants to resist oxidative damage as antioxidants assist buffer environmental challenges, which help in maintaining plant vigor under stress conditions (Lum et al., 2014). Different enzymatic antioxidants like superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) serve as defensive antioxidant system to inhibit or limit generation of reactive oxygen species (Sgherri et al., 2004; Soliman et al., 2011, 2012). The incomplete reduction of oxygen under water deficit conditions triggers the formation of reactive oxygen species. ROS formation is considered as an oxidative damage which is a threat to cell membrane affecting protein, deoxyribonucleic acid, lipid molecules and the formation of protease-resistant cross-linked aggregates (Berlett and Stadtman, 1997). For this, Plants' tolerance to water deficit might be correlated to the efficacy of antioxidant system to detoxify ROS (Athar et al., 2008). Previously, higher antioxidant activities have been reported in drought tolerant plants in comparison to intolerant ones (Lum et al., 2014). Likewise, higher antioxidant activities at water deficit conditions indicated better antioxidant enzymes' production in present investigation. Better activities of SOD, POD and CAT were recorded at 70% water holding capacity with Table 7 Effect of plant growth regulators as foliar application on superoxide dismutase (unit mg−1 protein) activity of M. oleifera seedlings under different water holding capacities. Treatments
No spray Water Cytokinin Thiourea BAP Salicylic acid H2O2 Ascorbic acid
Water holding capacity 100%
70%
40%
90.4 j–l 88.6 kl 200.7 ef 155.9 f–i 141.2 hi 174.4 e–h 180.4 e–h 213.2 de
136.3 h–j 151.3 g–i 253.2 cd 220.2 de 191.1 e–g 354.9 a 286.6 bc 308.9 ab
84.2 kl 54.3 l 116.5 i–k 68.2 l 126.3 i–k 159. f–i 84.9 kl 153.9 g-i
Means not showing same letters in a column differ significantly at 5% probability level, LSD (5%) value for superoxide dismutase activity: 46.08.
the assistance of exogenous foliar application of PGRs especially BAP and ascorbic acid (Tables 5–7). Previously, an increase in antioxidants' activities in moringa plants under saline conditions have been reported by Nouman et al. (2014). Drought tolerance in wheat, rice, pigeon pea and black gram is also attributed to better antioxidant activities (Pratap and Sharma, 2010; Lum et al., 2014). 5. Conclusion Keeping in view these findings, it can be concluded here that suitable water holding capacity for better moringa growth is 70% at which it produced maximum shoot length and leaf score while in areas where there is more water shortage, foliar application of plant growth regulators especially BAP and ascorbic acid can be useful mitigating drought stress. References Amin, A.A., Rashad, E.M., Hassanein, M.S., Zaki, M.N., 2007. Response of some white maize hybrids to foliar spray with benzyl adenine. Res. J. Agric. Biol. Sci. 3, 648–656. Athar, H., Khan, A., Ashraf, M., 2008. 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Please cite this article as: S. Azam, W. Nouman, U. Rehman, et al., Adaptability of Moringa oleifera Lam. under different water holding capacities, South African Journal of Botany, https://doi.org/10.1016/j.sajb.2019.08.020
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Adaptability of Moringa oleifera Lam. under different water holding capacities S. Azam a, W. Nouman a,⁎, Ubaid-ur Rehman a, U. Ahmed a, T. Gull b, M. Shaheen a a b
Department of Forestry & Range Management, Bahauddin Zakariya University, Multan, Pakistan Department of Chemistry, University of Central Punjab, Lahore, Pakistan
a r t i c l e
i n f o
Article history: Received 12 March 2019 Received in revised form 12 June 2019 Accepted 5 August 2019 Available online xxxx Edited by B Ncube Keywords: Ascorbic acid Benzyl amino purine Hydrogen peroxide Salicylic acid Thiourea
a b s t r a c t Growth performance and enzymatic-antioxidant activities of Moringa oleifera were investigated under simultaneous effect of water availability and plant growth regulators. Plant growth regulators i.e., cytokinin (50 mg L−1), thiourea (5 mM), benzyl amino purine (BAP @ 50 mg L−1), salicylic acid (50 mg L−1), hydrogen peroxide (120 μM) and ascorbic acid (50 mg L−1) were exogenously applied to moringa plants at three water holding capacity levels, 100, 70, and 40% in a completely randomized design with three replications. A decrease in shoot length, leaf score and number of branches was recorded at 100 and 40% water holding capacity as the plants respond well at 70% water holding capacity. BAP, cytokinin and salicylic acid were found as effective plant growth regulators in improving drought tolerance in moringa plants even at 40% water holding capacity. On the basis of these findings, moringa can be suggested to cultivate as a fodder crop under water deficit conditions and its tolerance can be further improved by foliar application of benzyl amino purine and ascorbic acid. © 2019 SAAB. Published by Elsevier B.V. All rights reserved.
1. Introduction Morphological changes and physiological interactions in plants are greatly influenced by light intenstity, temperature, soil texture and its water holding capacity. Of these factors, water availability is very important factor which is imperative for plant growth and development as water affects crop yield, and its quality. Limited water availability may result in retarded plant growth, reduced yield and poor quality as cell division and elongation are the most sensitive plant functions which are sensitive to water availability. Cell sensitivity might be attributed to the reduction in turgor pressure and interruption of water flow from the xylem to the surrounding elongating cells (Hussain et al., 2008; Farooq et al., 2009). Beside these, reduced leaf area, root expansion and elongation in search of water, metabolic changes and oxidative damage are also caused by drought resulting in poor plant growth (Reddy et al., 2004; Farooq et al., 2009). The fodder crops are not exception and these also affected by drought conditions which result in affecting livestock fodder requirement. A number of studies have been carried out on exploring drought resistant fodder plants which can be cultivated under limited water availability. For this, various drought tolerant plants and cultivars of agricultural crops have been identified and are being grown in water deficit areas (Farooq et al., 2009). The use of plant growth regulators ⁎ Corresponding author. E-mail address: [email protected] (W. Nouman).
like ascorbic acid, benzyl aminopurine (BAP) and cytokinins is crucial to mitigate drought stress (Senaratna et al., 2000; Farooq et al., 2009). Plant growth regulators have been used in different ways like exogenous application (foliar/root), seed priming and soil fertigation to mitigate drought stress. Foliar application of plant growth regulators like ascorbic acid (Basra et al., 2006; Farooq et al., 2006), salicylic acid (Borsani et al., 2001) and BAP (Amin et al., 2007) manifested significant results on different crops and vegetables in terms of improving growth performance under water deficit conditions. Moringa oleifera is not an exception as being cultivated in seasonally dry tropical and subtropical regions due to its fast growth and uses as livestock fodder, plant growth enhancer, therapeutic effects, etc. Moringa oleifera is a potential fodder crop, which can also be grown as hedges, fences and tree orchard. Its foliage being rich in nutrients, antioxidants and phenolic compounds, is being used as animal fodder in different parts of the world especially in African countries. Recently, many research studies reported the importance of moringa as a fodder crop with good nutritional quality which can be a good alternate to other fodder crops (Nouman et al., 2013, 2014). Moringa is a hardy plant that can be grown under diverse soil conditions and saline lands (Nouman et al., 2013). This report supports the idea that moringa can be grown under drought conditions. For this, morphological changes, physiological adaptations and antioxidant activities which support the plant to withstand under stress conditions should be studied. A very limited studies are available which demonstrate moringa adaptation under saline conditions especially focusing on nutritional quality and
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Please cite this article as: S. Azam, W. Nouman, U. Rehman, et al., Adaptability of Moringa oleifera Lam. under different water holding capacities, South African Journal of Botany, https://doi.org/10.1016/j.sajb.2019.08.020
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S. Azam et al. / South African Journal of Botany xxx (2019) xxx
antioxidant activities (Santos et al., 2011; Nouman et al., 2012a, 2012b, 2014). Antioxidant system serves as plant's defensive mechanism which support it to withstand under stress conditions. Water deficit results in incomplete reduction of oxygen resulting in the formation of reactive oxygen species (ROS) like singlet oxygen, superoxide anion radical, and hydroxyl radical which affect plant vigor and lower its nutritional quality (Mittler, 2002; Munne-Bosch and Penuelas, 2003). A few scientists are with opinion that ROS are essential for normal cellular processes but too low or too high ROS levels impair plant growth and development (Mittler, 2017). Antioxidant system enables the plants to come over such oxidative damage through a number of enzymatic antioxidants. Damaging impact of stress can be minimized through enzymatic antioxidants like SOD, POD, CAT, etc. which improves plant tolerance to mitigate stress conditions (Athar et al., 2008; Munns and Tester, 2008). As it has been reported that moringa can survive under moderate saline conditions, it is still to investigate that how moringa growth is affected under limited water availability, is there any way to induce/ improve tolerance in moringa plants to withstand under drought conditions? If moringa can grow under water deficit conditions, drought affected areas can be brought under moringa cultivation which can support our livestock as moringa is a potential fodder crop being a rich source of nutrients. The present study was designed aiming at investigating the response of moringa under limited water availability including changes in its growth and activities of enzymatic antioxidants.
2. Materials and methods 2.1. Seed material Moringa oleifera seeds were collected in June, 2015 from 5 years old tree grown in the research area of the Department of Forestry& Range Management, Bahauddin Zakariya University, Multan-Pakistan. Freshly collected seeds were immediately processed for the experiment as it has been reported that fresh moringa seeds have good germination which goes down with the passage of time (Nouman et al., 2012b). Before sowing, non-infected and vigorous seeds were sorted out and hydroprimed for 8 h to improve their tolerance under stress conditions (Nouman et al., 2014).
2.2. Plant vigor evaluation The experiment was conducted in glasshouse of the Department of Forestry & Range Management, Bahauddin Zakariya University, Multan-Pakistan where temperature was maintained at 33 ± 2 °C, and daylight/ night conditions were 16 and 8 h, respectively. Moringa seeds were sown in earthen pots filled with 3.7 kg of soil (clay loam), sand and green manure (1,1:1) in completely randomized design (CRD) with two factor factorial (water holding capacity and foliar application) arrangement and three replications. Five seeds in each pot were sown and it was noted that 83.3% seeds emerged, on average. After emergence, three plants were maintained in each pot; one pot was considered as one replication. When plants reached an age of one month, three water holding capacity levels (100, 70 and 40%) were applied in three replications and maintained for next two months. Water holding capacity i.e., 100% was considered as half of the soil saturation capacity. Beside these, exogenous foliar application of cytokinin (50 mg L−1), thiourea (5 mM), bezyl amino purine (BAP @50 mg L−1), salicylic acid (50 mg L−1), hydrogen peroxide (H2O2@120 μM) and ascorbic acid (50 mg L−1) were applied beside one control (no spray) and water to investigate the response of moringa plants under above mentioned water holding capacities. The growth performance of moringa plants was noted on the day of harvest (two months after the emergence). Shoot length, root length, number of leaves (leaf score) and roots (root score) were recorded.
2.3. Antioxidants assay For analyzing activities of enzymatic antioxidants i.e., superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT), fresh moringa leaves were plucked at dawn time and stored at −70 °C to avoid heat and light effect on enzymatic antioxidants. Superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) activities were noted at 560, 470 and 240 nm, respectively by using UV spectrophotometer (UV-4000, O.R.I. Germany) according to procedure described by Giannopolitis and Ries (1977) and Chance and Maehly (1955). 2.4. Statistical analysis The replicated data was analyzed in software (Statistix 8.1) and a CRD two factor factorial arrangement was applied for analysis of variance (Gomez and Gomez, 1984). Significance of variance (p b .05) was found for determining difference among treatments, water holding capacity levels and interaction of water holding capacity levels and foliar application of plant growth regulators. Least significance difference test (LSD @ 5% probability level) was used to compute the differences among the mean values (Steel et al., 1997). 3. Results In present investigation, growth characteristics of moringa seedlings and antioxidant activities were significantly (p b .05) affected by water availability and plant growth regulators. It was noted that moringa plants exhibited maximum shoot length, leaf score and number of branches at 70 followed by 100% water holding capacity while maximum root length was found at 70 followed by 40% water holding capacity while plant growth regulators were also found effective in improving moringa tolerance under limited water availability. Maximum shoot length was observed at 70% water holding capacity when moringa plants were exogenously sprayed with BAP followed by thiourea and cytokinin (22.4, 21.7 and 20.6 cm, respectively) while the least shoot length was recorded at 40% water holding capacity when moringa plants were not sprayed with any plant growth regulator (Table 1). A likewise trend was recorded in moringa branches and leaves where maximum branches and leaves were found at 70% water holding capacity while a significant reduction was recorded at 40% water holding capacity due to less water availability which was mitigated effectively by cytokinin, thiourea and BAP (Tables 2, 3). In general, moringa plants grew best under less water availability, perhaps as a result, the plants grew less in height under 100% water holding capacity. The response of moringa roots were found a bit different from shoot length. Cytokinin maximally improved root length of moringa plants at 70% water holding capacity followed by thiourea and BAP at same water holding capacity level (19.7, 16.5 and 15.4 cm, respectively) (Table 4). It was noted that moringa plants exhibited 11.2 cm root length at 40% water holding capacity when moringa plants were not
Table 1 Effect of plant growth regulators as foliar application on the shoot length (cm) of M. oleifera seedlings under different water holding capacities. Treatments
No spray Water Cytokinin Thiourea BAP Salicylic acid H2O2 Ascorbic acid
Water holding capacity 100%
70%
40%
17.8 c–h 17.1 h 18.1 c–h 17.3 f–h 19.2 c 17.8 d–h 18.2 c–h 18.0 c–h
18.9 cd 18.7 c–f 20.6 b 21.7 ab 22.4 a 18.8 c–e 17.6 d–h 18.6 c–g
16.8 h 17.4 f–h 18.1 c–h 17.3 f–h 17.3 f–h 17.3 f–h 17.2 gh 17.5 e–h
Means not showing same letters in a column differ significantly at 5% probability level, LSD (5%) value for Shoot Length: 1.39.
Please cite this article as: S. Azam, W. Nouman, U. Rehman, et al., Adaptability of Moringa oleifera Lam. under different water holding capacities, South African Journal of Botany, https://doi.org/10.1016/j.sajb.2019.08.020
S. Azam et al. / South African Journal of Botany xxx (2019) xxx Table 2 Effect of plant growth regulators as foliar application on the branches of M. oleifera seedlings under different water holding capacities. Treatments
No spray Water Cytokinin Thiourea BAP Salicylic acid H2O2 Ascorbic acid
Water holding capacity
Table 4 Effect of plant growth regulators as foliar application on the root length (cm) of M. oleifera seedlings under different water holding capacities. Treatments
100%
70%
40%
6.0 b–d 5.0 d–f 7.3 ab 6.0 b–d 5.0 d–f 7.3 ab 5.3 c–e 6.0 b–d
6.7 a–c 7.3 ab 8.0 a 8.0 a 7.0 ab 7.7 a 6.0 b–d 7.0 ab
2.7 g 4.3 ef 5.0 d–f 4.7 d–f 6.0 b–d 4.0 e–g 3.7 fg 6.0 b–d
3
No spray Water Cytokinin Thiourea BAP Salicylic acid H2O2 Ascorbic acid
Water holding capacity 100%
70%
40%
10.9 h–j 10.2 ij 10.0 d–g 11.5 f–j 11.9 e–i 14.2 cd 10.5 ij 10.9 h–j
12.9 d–g 12.3 e–h 19.7 a 16.5 b 15.4 bc 16.5 d–f 11.8 e–j 14.4 cd
11.2 h–j 14.2 cd 15.0 bc 10.2 j 15.2 bc 11.3 g–j 13.3 de 14.5 cd
Means not showing same letters in a column differ significantly at 5% probability level, LSD (5%) value for number of branches: 1.65.
Means not showing same letters in a column differ significantly at 5% probability level, LSD (5%) value for root length: 1.73.
sprayed with any plant growth regulator which is more than root length recorded in control plants at 100% water holding capacity while 35.7 and 33.9% increase in root length was recorded at 40% water holding capacity when BAP and cytokinin were exogenously applied to moringa plants which is even more than root lengths recorded at 100% water holding capacity (Table 4). Moringa adoption under water stress conditions might be due to antioxidant system. For this, activities of enzymatic antioxidants were also investigated. It was noted that antioxidant enzymes (SOD, POD and CAT) were significantly affected by water availability and foliar application of plant growth regulators. An increase in CAT activity was observed in moringa leaves at 70% water holding capacity in comparison to 100 while it was damaged at 40% (99.1, 64.8 and 41.8 unit mg−1 protein, respectively). Salicylic acid, cytokinin and BAP were statistically at par with each other sustaining and improving catalase activity (81.7, 80.9 and 77.7 unit mg−1 protein, respectively, on average) (Table 5). Salicylic acid and ascorbic acid maximally improved peroxidase activity in moringa plants exhibiting 693.4 and 684.5 unit mg−1 protein, respectively followed by thiourea and BAP. The least peroxidase activity (452.9 and 448.5 unit mg−1 protein, on average) were noted in control and water sprayed moringa plants, respectively. Moringa plants did not exhibit better POD activities at 100% water holding capacity while salicylic acid and ascorbic acid are more effective in sustaining peroxidase activities in moringa plants even at 40% water holding capacity (Table 6). Salicylic acid was found effective on sustaining and improving SOD activity of moringa leaves under limited water availability. Beside salicylic acid, foliar application of ascorbic acid and BAP were also found effective improving SOD activity resulted in mitigating drought stress in moringa plants (Table 7). The least SOD activity was recorded at 40% water holding capacity while the maximum was found when moringa plants were subjected to 70% water holding capacity. Foliar application of salicylic acid and ascorbic acid were at par with each other in improving SOD activities at 70% water holding capacity (354.9, 308.9 unit mg−1 protein). No doubt, severe decline was recorded in SOD activity at 40% water holding capacity (84.2 unit mg−1 protein) which was 38.2% less than
SOD activity at 70% water holding capacity without any foliar application, it was further improved by salicylic acid and ascorbic acid foliar application (Table 7).
During growth and development phases, plants face several stress conditions like salinity, heat, cold and drought which ultimately affect plant growth, yield and physiology. The impact of drought conditions on plant growth and development is well known but being a complex mechanism, and it is rather difficult to understand the complexities. In present study, the response of M. oleifera to water availability and stress mitigation through plant growth regulators were studied. As mentioned above, moringa plants performed best under at 70% water holding capacity while at 100 and 40% water holding capacity, shorter plant height was recorded. A significant improvement in shoot length, leaf score and number of branches was recorded with the assistance of exogenously applied plant growth regulators especially BAP and cytokine into mitigate drought conditions (Tables 1–3). A likewise trend was recorded in root length of moringa plants. It has been reported that cell division and elongation, growth and development determining physiological processes are drought sensitive (Farooq et al., 2009). Secondly, moringa plants thrived better at low water availability. For this, the plants showed less height at 100% water holding capacity. Increase in root length at 70 and 40% water holding capacitymight be attributed to lower osmotic pressure at stress conditions which trigger root proliferation in search of water and nutrients (Hsiao and Xu, 2000). Root expansion and proliferation might be due to ethylene production as reported by Dantas et al. (2007). Similar response of moringa roots under saline conditions have been previously reported by Nouman et al., 2012a, Nouman et al., 2014). It is well understood that reduction in shoot length and leaf score and increase in root length are survival strategies of plants to withstand under stress conditions. As in present study, leaf score decreased while root length increased at 40% water holding capacity, it might be due to plant survival mechanism reducing
Table 3 Effect of plant growth regulators as foliar application on leaf score of M. oleifera seedlings under different water holding capacities.
Table 5 Effect of plant growth regulators as foliar application on the catalase activity (unit mg−1 protein) of M. oleifera seedlings under different water holding capacities.
Treatments
No spray Water Cytokinin Thiourea BAP Salicylic acid H2O2 Ascorbic acid
Water holding capacity
4. Discussion
Treatments
100%
70%
40%
39.3 gh 37.3 g–i 50.7 de 41.0 gh 43.7 e–g 42.0 gh 44.0 e–g 43.3 fg
53.0 cd 59.7 bc 61.0 b 52.3 d 69.7 a 52.0 d 70.0 a 61.7 b
21.0 k 35.0 hi 31.0 ij 41.7 gh 32.0 ij 27.0 jk 23.7 k 50.0 d–f
Means not showing same letters in a column differ significantly at 5% probability level, LSD (5%) value for leaf score: 7.28.
No spray Water Cytokinin Thiourea BAP Salicylic acid H2O2 Ascorbic acid
Water holding capacity 100%
70%
40%
49.7 gh 54.7 fg 77.2 d 63.9 ef 65.4 d–f 68.3 de 72.4 de 67.3 de
68.9 de 71.2 de 114.1 b 95.6 c 112.7 b 128.6 a 103.4 bc 98.0 c
35.9 ij 36.8 ij 51.4 gh 39.1 hi 54.9 fg 48.1 g–i 26.6 j 41.7 hi
Means not showing same letters in a column differ significantly at 5% probability level, LSD (5%) value for catalase activity: 12.28.
Please cite this article as: S. Azam, W. Nouman, U. Rehman, et al., Adaptability of Moringa oleifera Lam. under different water holding capacities, South African Journal of Botany, https://doi.org/10.1016/j.sajb.2019.08.020
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S. Azam et al. / South African Journal of Botany xxx (2019) xxx
Table 6 Effect of plant growth regulators as foliar application on the peroxidase activity (unit mg−1 protein) of M. oleifera seedlings under different water holding capacities. Treatments
No spray Water Cytokinin Thiourea BAP Salicylic acid H2O2 Ascorbic acid
Water holding capacity 100%
70%
40%
389.4 k 400.7 k 525.8 f–h 646.0 de 554.7 fg 540.8 f–h 601.8 ef 566.8 e–g
500.1 g–j 514.0 g–i 695.5 d 805.9 c 827.0 c 1002.8 a 819.9 c 916.0 b
469.2 h–k 431.0 i–k 541.1 f–h 465.9 h–k 526.0 f–h 536.8 f–h 422.1 jk 570.7 e–g
Means not showing same letters in a column differ significantly at 5% probability level, LSD (5%) value for peroxidase activity: 84.60.
water loss through transpiration and more water uptake by proliferated roots (Houle et al., 2001). Prolific and expanded root growth is an adaptation to water deficit allowing plants to uptake water and nutrients from deep soil (Nguyen et al., 1997; Kavar et al., 2007). Root extension and proliferation has been previously noted in moringa seedlings under saline condition while tea, onion and cotton plants also exhibited this phenomenon under drought stress (Farooq et al., 2009; Nouman et al., 2014). It was noted in the present study that foliar application of plant growth regulators improved moringa tolerance under drought conditions. Among these, cytokinin, BAP and ascorbic acid were found as effective agents inducing tolerance in moringa plants under limited water availability as stated earlier. Plant survival under stress conditions is not only through morphological changes while physiological or enzymatic changes also contribute to tolerate drought stress. Plants produce reactive oxygen species under water deficit, which affect plant vigor and nutritional quality. Enzymatic antioxidants enable plants to resist oxidative damage as antioxidants assist buffer environmental challenges, which help in maintaining plant vigor under stress conditions (Lum et al., 2014). Different enzymatic antioxidants like superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) serve as defensive antioxidant system to inhibit or limit generation of reactive oxygen species (Sgherri et al., 2004; Soliman et al., 2011, 2012). The incomplete reduction of oxygen under water deficit conditions triggers the formation of reactive oxygen species. ROS formation is considered as an oxidative damage which is a threat to cell membrane affecting protein, deoxyribonucleic acid, lipid molecules and the formation of protease-resistant cross-linked aggregates (Berlett and Stadtman, 1997). For this, Plants' tolerance to water deficit might be correlated to the efficacy of antioxidant system to detoxify ROS (Athar et al., 2008). Previously, higher antioxidant activities have been reported in drought tolerant plants in comparison to intolerant ones (Lum et al., 2014). Likewise, higher antioxidant activities at water deficit conditions indicated better antioxidant enzymes' production in present investigation. Better activities of SOD, POD and CAT were recorded at 70% water holding capacity with Table 7 Effect of plant growth regulators as foliar application on superoxide dismutase (unit mg−1 protein) activity of M. oleifera seedlings under different water holding capacities. Treatments
No spray Water Cytokinin Thiourea BAP Salicylic acid H2O2 Ascorbic acid
Water holding capacity 100%
70%
40%
90.4 j–l 88.6 kl 200.7 ef 155.9 f–i 141.2 hi 174.4 e–h 180.4 e–h 213.2 de
136.3 h–j 151.3 g–i 253.2 cd 220.2 de 191.1 e–g 354.9 a 286.6 bc 308.9 ab
84.2 kl 54.3 l 116.5 i–k 68.2 l 126.3 i–k 159. f–i 84.9 kl 153.9 g-i
Means not showing same letters in a column differ significantly at 5% probability level, LSD (5%) value for superoxide dismutase activity: 46.08.
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Please cite this article as: S. Azam, W. Nouman, U. Rehman, et al., Adaptability of Moringa oleifera Lam. under different water holding capacities, South African Journal of Botany, https://doi.org/10.1016/j.sajb.2019.08.020
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Please cite this article as: S. Azam, W. Nouman, U. Rehman, et al., Adaptability of Moringa oleifera Lam. under different water holding capacities, South African Journal of Botany, https://doi.org/10.1016/j.sajb.2019.08.020