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Physiological changes of purslane (Portulaca oleracea L.) after progressive drought stress and rehydration
Scientia Horticulturae 194 (2015) 215–221
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Physiological changes of purslane (Portulaca oleracea L.) after progressive drought stress and rehydration Rui Jin a,b , Haitao Shi a , Chunyu Han a,b , Bao Zhong a,b , Qing Wang a , Zhulong Chan a,∗ a Key Laboratory of Plant Germplasm Enhancement and Specialty Agriculture, Wuhan Botanical Garden, Chinese Academy of Sciences, Wuhan 430074, China b University of Chinese Academy of Sciences, Beijing 100039, China
a r t i c l e
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Article history: Received 20 March 2015 Received in revised form 17 July 2015 Accepted 13 August 2015 Available online 28 August 2015 Keywords: Purslane Drought stress Rehydration ROS Antioxidant enzyme
a b s t r a c t Purslane (Portulaca oleracea L.) is a herbaceous fleshy plant, which is relatively more tolerant to drought than other species. So far, there is little information on the combined response of this plant to drought and rehydration. In this study, physiological changes were measured under drought stress and rehydration conditions in purslane. Soil water content (SWC), leaf water content (LWC), malondialdehyde (MDA), proline, electrolyte leakage (EL), chlorophyll content, O2 • − , SOD and POD activities were determined at designated time periods (5 d, 10 d, 15 d, 22 d after drought and 3 h, 1 d, 3 d after rehydration). Progressive drought treatment for 10 d significantly increased MDA, proline, EL, O2 • − , and activities of SOD and POD, while rehydration for 1–3 d caused decline of these parameters. Conversely, drought stress decreased LWC and chlorophyll content, while rehydration increased LWC quickly and chlorophyll content gradually. The present study indicated that the purslane has a great capability to cope with drought stress and activate many physiological mechanisms, which allow more efficient recovery during rehydration. These results provided the first physiological evidences that the harvest time for purslane was within 5–10 d of drought or 1–3 d after rehydration. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Drought is defined as the shortage of rain in plant environment (Rahdari and Hoseini, 2012), which leads to the damage of plants. The growth and development of plants were severely inhibited under drought stress condition.. To date, the effects of drought stress on plants have been well-documented in many plants, but the responses to rehydration after drought stress and related mechanism are still insufficient (Liu et al., 2010; Luo et al., 2014; Xu et al., 2010). The water availability is spatially and temporally heterogeneous, especially in arid and semi-arid ecosystems (Huxman et al., 2004). Consequently, plants are exposed to drought repeatedly during their life cycle. It is important to decipher the mechanisms that trigger off physiological responses to drought stress and rehydration. It is known that the vegetative growth of stressed plants can ˜ et al., 2005). The responses of recover after rewatering (Ortuno
Abbreviations: MDA, malondialdehyde; O2 • − , superoxide radical; POD, peroxidase; ROS, reactive oxygen species; SOD, superoxide dismutase; CAT, catalase; SWC, soil water content; LWC, leaf water content; EL, electrolyte leakage. ∗ Corresponding author. Fax: +86 27 87510251. E-mail address: [email protected] (Z. Chan). http://dx.doi.org/10.1016/j.scienta.2015.08.023 0304-4238/© 2015 Elsevier B.V. All rights reserved.
several crops, trees, mosses and cut flowers to drought and rehydration were partially documented (Filippou et al., 2011; Liu et al., 2010, 2013; Luo et al., 2011). Most of these plants demonstrated increasing sensitivity to drought induced oxidative damage but this procedure was reversed following rehydration. Purslane (Portulaca oleracea L.) is an annual herb with succulent and fleshy stems that may grow erect or prostrate depending on light availability (Chauhan and Johnson, 2009). Purslane belongs to family Portulacaceae, which is widely distributed all over the world, and grows well in diverse geographical environments (D’Andrea et al., 2014). Purslane is listed as one of the most useful medicinal plants and named “Global Panacea” by World Health Organization (Samy et al., 2005). As one of the valuable medical plants and vegetable, it is enrich with antioxidant vitamins and omega-3 fatty acids (Rahdari et al., 2012; Simopoulos et al., 1992). Omega-3 fatty acids are useful in preventing numerous cardiovascular diseases and maintaining a healthy immune system (Simopoulos, 2004; Uddin et al., 2014). All of the above characteristics made purslane flourisher than many other crops. Alam et al. (2014) screened the most salt-tolerant purslane accessions prioritizing the utilization as a resource of vegetable nutrients and commercial cultivation for saline agriculture and sustainable development. Rahdari et al. (2012) reported
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the effect of drought on germination, proline, sugar, lipid, protein and chlorophyll content in purslane, and the results indicated that purslane was tolerant to drought stress and could be used in arid and semi-arid regions. Yang et al. (2012) investigated the mechanisms underlying purslane’s tolerance to high temperature and high humidity stresses, and the results suggested that purslane deployed the multiple strategies to cope with combined stresses. In this study, we addressed a brief insight into the combined effects of drought and rehydration. Progressive drought stress treatment was applied on purslane by gradually decreasing soil water content (SWC). The results showed that drought treatment resulted in decreased LWC and chlorophyll content and increased malondialdehyde (MDA), proline, electrolyte leakage (EL), O2 •− , and activities of superoxide dismutase (SOD) and peroxidase (POD). Purslane leaves kept high water content under stressed condition and recovered from rehydration through increase of leaf water content (LWC) and chlorophyll content and decrease of other parameters. All these results indicated that purslane had great elasticity to cope with drought and rehydration.
2. Materials and methods 2.1. Plant materials and growth conditions The purslane seeds were sown in 10 cm diameter and 10 cm deep plastic pots with vermiculite after stratified at 4 ◦ C in the dark for 3 d. Plants were grown in growth room with controlled temperature at 28 ± 1 ◦ C, 65–75% relative humidity, 300 mol s−1 m−2 light intensity, and 16 h light and 8 h dark cycles. The plastic pots were rotated daily to minimize the influence of environments. To determine drought tolerance, 21 d-old seedlings were subjected to progressive drought stress. The control group was irrigated regularly. Leaf samples were harvested at specific time points (5 d, 10 d, 15 d and 22 d after drought stress imposition). When the leaves of purslane appeared wilting at 22 d after drought treatment, rehydration was applied. Leaves were sampled after 0 h, 3 h, 1 d and 3 d of rehydration. Samples were frozen in liquid nitrogen and stored at −80 ◦ C for physiological analyses. Experiments were executed in triplicates and the top leaf tissues were collected from a minimum of 10 independent plants.
2.2. EL assay EL was determined as stated by Shi et al. (2012a) with slight modifications. Briefly, five intact leaves were incubated in 20 mL deionized water, and shaken for 6 h at room temperature. The initial conductivity (Ci ) was measured by conductivity meter (LeiciDDS-307A, Shanghai, China). The samples then were boiled for 20 min to completely induce all electrolytes. After cooling to room temperature, the conductivity (Cmax ) was determined. Relative EL(%) = (Ci /Cmax ) × 100.
2.3. Determination of SWC and LWC To assess soil water status, SWC was detected by soil moisture temperature recorder (L99-TWS-1, Fotel Precise Instrument Co., Ltd., Shanghai, China) at designated time intervals. For measurement of LWC, leaf samples were harvested at 0 d, 5 d, 10 d, 15 d and 22 d after drought stress, and 3 h, 1 d and 3 d after rehydration. Fresh weight (FW) was measured after harvest immediately, and the dry weight (DW) was measured after 16 h incubation at 80 ◦ C oven. LWC (%) was calculated as (FW–DW)/FW × 100%.
2.4. The measurement of MDA and proline contents MDA was extracted with the trichloroacetic acid (TBA) reaction and determined at 450 nm, 532 nm and 600 nm wavelength as described previously (Shi et al., 2012a,b) using Multiskan MK3 (Thermo Scientific, USA). Proline was determined as follows. Briefly, 0.5 g leaf sample was totally extracted in 3% (w/v) sulphosalicylic acid, then 2 mL of ninhydrin regent and 2 mL of glacial acetic acid were added before boiled for 30 min at 100 ◦ C. After cooling to room temperature, the mixture was centrifuged at 12,000 × g for 5 min. The proline content in the sample was calculated at 520 nm absorbance. Simultaneously, standard curve was measured using purified proline (Shi et al., 2012a,b, 2014). 2.5. Determination of O2 •− , antioxidant enzyme activities and chlorophyll content O2 •− content was measured by the SOA Assay Kit (Elisa, Shanghai, China). Based on the antibody–antigen reaction, O2 •− content was determined by measuring the absorbance of TMB substrate at 450 nm. SOD activity was assayed with the SOD Assay Kit (Beyotime, Shanghai, China). 2-(4-Iodophenyl)-3-(4-nitrophenyl)-5(2,4-disulfophenyl)-2H-tetrazolium (WST-1) was used in this assay, which couples with xanthine oxidase (XO) to generate O2 •− and formazan dye, This reaction is inhibited by SOD which catalyzing O2 •− into O2 and H2 O2 . Therefore, SOD activity can be measured by the absorbance of formazan dye at 450 nm. To determine the POD activity, Plant POD activity Assay Kit (Jiancheng, Nanjing, China) was used. Based on the guaiacol oxidation, POD activity was calculated by measuring the absorbance of reaction buffer at 340 nm. Chlorophyll content in leaf was determined as previously described with slight modification (Frank et al., 2005). Briefly, leaf samples were treated for 30 min at 95 ◦ C. After that, the leaf samples were dried to constant weight at 80 ◦ C. Then the samples were thoroughly homogenized and chlorophyll was totally extracted with 96% (v/v) ethanol in the dark at 4 ◦ C. For chlorophyll quantification, the supernatant of ethanol extract was measured at 649 nm and 665 nm wavelengths. 2.6. Statistic analysis Data shown are mean ± standard deviation (SD). Statistical analysis was performed using LSD test (P < 0.05). Asterisk symbols above the points in the figures indicate significant differences with control at the 0.05 level. 3. Results 3.1. Effects of drought and rehydration on purslane phenotype After the progressive drought stress treatment, purslane showed dwarfed phenotype and leaf area was significantly reduced. At 22 d under drought stress, the leaves appeared obvious wilting. After 3 d of rehydration, purslane achieved completely recovery and no wilting phenomenon was observed in treated purslane (Fig. 1). 3.2. Changes in SWC and LWC after drought and rehydration treatments To determine purslane drought tolerance, 21 d-old seedlings were subjected to a progressive drought stress and then followed with rehydration. During drought periods, SWC sharply declined from 95 to 20%. During the rehydration, SWC restored immediately, and achieved complete recovery at 3 h after rehydration (Fig. 2A).
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Fig. 1. Effects of drought and rehydration on purslane phenotype. Representative pictures were shown here. (A) Control and drought for 22 d and (B) control and 3 d after rehydration.
Fig. 2. (A) Soil water content and (B) leaf water content during the drought stress and rehydration for purslane. Dark grey shading indicates the duration of drought; light grey shading indicates rehydration duration. Results are shown as means standard deviation (SD).
LWC in stressed seedlings decreased slightly with ongoing soil drought stress and dropped to 91% after 22 d of drought stress treatment. LWC restored to the control level after 1 d of rehydration. The control seedlings showed slight changes in the LWC throughout the experiment, ranging from 95 to 96% (Fig. 2B).
stress while decrease after rehydration (Fig. 3C). These results indicated that drought stress for longer than 10 d had adverse effects on purslane growth, while purslane recovered quickly at 1 d after rehydration.
3.3. Effects of drought and rehydration on purslane MDA, proline and EL
3.4. Effect of drought and rehydration on purslane chlorophyll content
In this study, MDA and proline contents showed significant increase after 10 d of drought stress treatment and peaked at 22 d after drought stress. After rehydration, MDA and proline contents showed a significantdecrease after 3 h and almost equaled to the control level at 1 d (Fig. 3A and B). Change tendency of EL was in parallel with those of MDA and proline, showing increase after drought
Drought stress caused a significant reduction in chlorophyll content. At the early stage of rehydration, the chlorophyll content kept decreasing tendency. After 3 h of rehydration, chlorophyll content increased gradually but still lower than that in the control level (P < 0.05) at 3 d after rehydration, indicating photosynthesis of purslane recovered at this stage (Fig. 4).
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Fig. 3. (A) MDA content, (B) proline content and (C) EL during the drought stress and rehydration for purslane. Dark grey shading indicates the duration of drought; light grey shading indicates rehydration duration. Results are shown as means standard deviation (SD).
Fig. 4. Chlorophyll content during the drought stress and rehydration for purslane. Dark grey shading indicates the duration of drought; light grey shading indicates rehydration duration. Results are shown as means standard deviation (SD).
R. Jin et al. / Scientia Horticulturae 194 (2015) 215–221
3.5. Effect of drought and recovery on purslane ROS and antioxidant enzyme activities O2 •− content increased during the drought stress (Fig. 5A). Rehydration reversed this tendency, but O2 •− content in stressed plants was still higher than that in the control after 3 d of rehydration, which indicated that the purslane plants was not fully recovered in terms of ROS (Fig. 5A). Drought stress from 1 to 10 d had no significant effect on activities of SOD and POD. During 15–22 d of drought stress, activities of SOD and POD increased significantly. Rehydration resulted in significant decline but was still beyond the control (Fig. 5B and C).
4. Discussion Drought stress is the key factor which limits plant growth and development. In this study, we tested drought tolerance in purslane. As an important medicinal plant, purslane has a valuable
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place in a healthy diet and was reported to be drought tolerance (Rahdari et al., 2012). The results showed that LWC remained relatively stable in the control plants and modestly declined under drought stress condition. Purslane still held very high LWC (91%) although it was wilted at 22 d after drought stress. Under drought stress condition, stomata of purslane closed quickly which might have permitted high water potential (Ren et al., 2011). Moreover, Purslane is a dicotyledon and one of the C4 plants displays Kranz anatomy structure. The term “CAM-like” has been used to refer to purslane case (Lara et al., 2003). In addition to the bundle sheath and mesophyll cells, the leaves of purslane present large and vacuolated water storage cells contributing to the succulence of the leaves (Lara et al., 2003). CAM is a photosynthetic type that can be induced by abiotic and biotic stresses (Borland et al., 2006). The fundamental features of CAM are abilities to take carbon at night and to accumulate it in the vacuole as an organic acid (Silvera et al., 2010). Lara et al. (2004) reported purslane subjected to drought showed typical characteristics of CAM, such as signifi-
Fig. 5. (A) O2 • − , (B) POD, and (C) SOD activities during the drought stress and rehydration for purslane. Dark grey shading indicates the duration of drought; light grey shading indicates rehydration duration. Results are shown as means standard deviation (SD).
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cant daily malate fluctuation, net CO2 uptake in the dark and daily changes in the relative activity and regulatory properties of PEPC. Portulacaceae have evolved from CAM ancestors (Guralnick and Jackson, 2001). In the course of evolution, it is possible that over an exiting CAM tissue the C4 photosynthesis somehow occurred in purslane and that CAM now has a role only in particular stress conditions such as drought (Sage, 2002). The operation of a CAM-like metabolism in C4 purslane under drought would serve as a mechanism for water saving, evading carbon losses due to respiration and prolonging the life of the cell (Herrera, 2009). Cell membrane stability is vital for maintaining cell turgor pressure and physiological functions, particularly during drought stress. EL has been widely used to estimate cell membrane stability by means of measuring the extent of cell injury when subjected to all kinds of environmental stressors (Hu et al., 2010a,b; Zhao et al., 2011). Although drought treatment significantly (P < 0.05) increased EL, the maximum of EL at 22 d after drought treatment was only 11% (Fig. 3C), which was much lower than that in bermudagrass (Cynodon dactylon) assayed in the same lab (Shi et al., 2012a, 2013), indicating that succulent purslane plant might be more tolerant to drought stress than other species. Proline is accumulated as an osmoprotectant in response to adverse conditions and MDA is one of the indicators of lipid peroxidation under stress (Liu et al., 2014). Purslane under drought stress accumulated high level of proline (Fig. 3B), which was important to alleviate cellular hyperosmolarity and ion disequilibrium. Additionally, Drought stress significantly increased MDA content in purslane at 10 d after drought treatment (Fig. 3A). This result was consistent with the previous researches that drought stress induced membrane-lipid peroxidation (Filippou et al., 2011; Naya et al., 2007). Other plants such as bermudagrass and sorghum (Sorghum bicolor) showed similar pattern of lipid peroxidation under drought stress condition (Shi et al., 2012a; Zhang and Kirkham, 1996). MDA is formed by ROS induced decomposition of polyunsaturated lipids (Pryor and Stanley, 1975). Interestingly, O2 •− content increased significantly at 10 d after drought treatment (Fig. 5A), which was in line with MDA data. Facing adverse environment, plants have developed an efficient antioxidant defense system to scavenge ROS, which are the important signal molecules induced by various environmental stresses, including H2 O2 , O2 •− , hydroxyl radical and singlet oxygen (Smirnoff, 1993). The antioxidant defense system is comprised of enzymatic and nonenzymatic components. Enzymatic system produces antioxidants or antioxidant enzymes that directly react with ROS and scavenge it, including SOD, POD and CAT, while nonenzymatic system produces enzymes that regenerate antioxidants such as glutathione, glutathione reductase, ascorbate and ascorbate reductase (Smirnoff, 1993). In this study, drought stress significantly increased activities of SOD and POD after 15 d. After rehydration for 1–3 d, the activities of SOD and POD gradually decreased indicating that ROS metabolism returned to the control level. Drought stress inhibited the synthesis of chlorophyll in plants, and proteins involved in chloroplast synthesis were hampered. Moreover, the original chlorophyll was decomposed under severe drought stress condition (Parida et al., 2003). A rapid loss of the chlorophyll content during rehydration has been reported in Physcomitrella patens (Cui et al., 2012; Frank et al., 2005). The decrease of chlorophyll under drought stress in purslane indicated that drought stress affected purslane chlorophyll biosynthesis. Unlike other physiological parameters, chlorophyll content kept decreasing at 3 h after rehydration, and then showed increase trend (Fig. 4). At this time, MDA, proline, EL, O2 •− , and activities of SOD and POD showed reversed change tendency when compared to these after drought treatment. This result indicated that chloro-
Fig. 6. Model depicting drought and rehydration induced changes in purslane plant.
phyll biosynthesis was delayed during purslane plant recovery from drought stress induced damages. In summary, purslane was relatively drought tolerant and survived from drought stress for at least 22 d and recovered quickly after rehydration for 1–3 d in terms of physiological assays (Fig. 6). To minimize nutritional value loss, the harvest time could be determined with 5–10 d of drought and 3–5 d after rehydration. For field cultivation of purslane, timely watering and keep the soil moisture over 50% will not influence the regular growth of plants. Both unique nutritive quality and distinguished drought tolerance make purslane a promising candidate to be used as healthy diet and in stress response related researches in agriculture. Acknowledgments This research was supported by “the Hundred Talents Program”, the Knowledge Innovative Key Program of Chinese Academy of Sciences (Grant No. Y154761O01076 and No.Y329631O0263) to Zhulong Chan. References Alam, M.A., Juraimi, A.S., Rafii, M.Y., Hamid, A.A., Aslani, F., 2014. Screening of purslane (Portulaca oleracea L.) accessions for high salt tolerance. Sci. World J., 627–916. Borland, A., Elliott, S., Patterson, S., Taybi, T., Cushman, J., Pater, B., Barnes, J., 2006. Are the metabolic components of crassulacean acid metabolism up-regulated in response to an increase in oxidative burden? J. Exp. Bot. 57, 319–328. Cui, S., Hu, J., Guo, S., Wang, J., Cheng, Y., Dang, X., Wu, L., He, Y., 2012. Proteome analysis of Physcomitrella patens exposed to progressive dehydration and rehydration. J. Exp. Bot. 63, 711–726. D’Andrea, R.M., Andreo, C.S., Lara, M.V., 2014. Deciphering the mechanisms involved in Portulaca oleracea (C4 ) response to drought: metabolic changes including crassulacean acid-like metabolism induction and reversal upon re-watering. Physiol. Plant. 152, 414–430. Filippou, P., Antoniou, C., Fotopoulos, V., 2011. Effect of drought and rewatering on the cellular status and antioxidant response of Medicago truncatula plants. Plant Signal. Behav. 6, 270–277. Frank, W., Ratnadewi, D., Reski, R., 2005. Physcomitrella patens is highly tolerant against drought, salt and osmotic stress. Planta 220, 384–394. Guralnick, L.J., Jackson, M.D., 2001. The occurrence and phylogenetics of crassulacean acid metabolism in the Portulacaceae. Int. J. Plant Sci. 162, 257–262. Herrera, A., 2009. Crassulacean acid metabolism and fitness under water deficit stress: if not for carbon gain, what is facultative CAM good for? Ann. Bot. 103, 645–653. Hu, L., Wang, Z., Du, H., Huang, B., 2010a. Differential accumulation of dehydrins in response to water stress for hybrid and common bermudagrass genotypes differing in drought tolerance. J. Plant Physiol. 167, 103–109. Hu, L., Wang, Z., Huang, B., 2010b. Diffusion limitations and metabolic factors associated with inhibition and recovery of photosynthesis from drought stress in a C3 perennial grass species. Physiol. Plant. 139, 93–106. Huxman, T.E., Snyder, K.A., Tissue, D., Leffler, A.J., Ogle, K., Pockman, W.T., Sandquist, D.R., Potts, D.L., Schwinning, S., 2004. Precipitation pulses and carbon fluxes in semiarid and arid ecosystems. Oecologia 141, 254–268.
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Scientia Horticulturae journal homepage: www.elsevier.com/locate/scihorti
Physiological changes of purslane (Portulaca oleracea L.) after progressive drought stress and rehydration Rui Jin a,b , Haitao Shi a , Chunyu Han a,b , Bao Zhong a,b , Qing Wang a , Zhulong Chan a,∗ a Key Laboratory of Plant Germplasm Enhancement and Specialty Agriculture, Wuhan Botanical Garden, Chinese Academy of Sciences, Wuhan 430074, China b University of Chinese Academy of Sciences, Beijing 100039, China
a r t i c l e
i n f o
Article history: Received 20 March 2015 Received in revised form 17 July 2015 Accepted 13 August 2015 Available online 28 August 2015 Keywords: Purslane Drought stress Rehydration ROS Antioxidant enzyme
a b s t r a c t Purslane (Portulaca oleracea L.) is a herbaceous fleshy plant, which is relatively more tolerant to drought than other species. So far, there is little information on the combined response of this plant to drought and rehydration. In this study, physiological changes were measured under drought stress and rehydration conditions in purslane. Soil water content (SWC), leaf water content (LWC), malondialdehyde (MDA), proline, electrolyte leakage (EL), chlorophyll content, O2 • − , SOD and POD activities were determined at designated time periods (5 d, 10 d, 15 d, 22 d after drought and 3 h, 1 d, 3 d after rehydration). Progressive drought treatment for 10 d significantly increased MDA, proline, EL, O2 • − , and activities of SOD and POD, while rehydration for 1–3 d caused decline of these parameters. Conversely, drought stress decreased LWC and chlorophyll content, while rehydration increased LWC quickly and chlorophyll content gradually. The present study indicated that the purslane has a great capability to cope with drought stress and activate many physiological mechanisms, which allow more efficient recovery during rehydration. These results provided the first physiological evidences that the harvest time for purslane was within 5–10 d of drought or 1–3 d after rehydration. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Drought is defined as the shortage of rain in plant environment (Rahdari and Hoseini, 2012), which leads to the damage of plants. The growth and development of plants were severely inhibited under drought stress condition.. To date, the effects of drought stress on plants have been well-documented in many plants, but the responses to rehydration after drought stress and related mechanism are still insufficient (Liu et al., 2010; Luo et al., 2014; Xu et al., 2010). The water availability is spatially and temporally heterogeneous, especially in arid and semi-arid ecosystems (Huxman et al., 2004). Consequently, plants are exposed to drought repeatedly during their life cycle. It is important to decipher the mechanisms that trigger off physiological responses to drought stress and rehydration. It is known that the vegetative growth of stressed plants can ˜ et al., 2005). The responses of recover after rewatering (Ortuno
Abbreviations: MDA, malondialdehyde; O2 • − , superoxide radical; POD, peroxidase; ROS, reactive oxygen species; SOD, superoxide dismutase; CAT, catalase; SWC, soil water content; LWC, leaf water content; EL, electrolyte leakage. ∗ Corresponding author. Fax: +86 27 87510251. E-mail address: [email protected] (Z. Chan). http://dx.doi.org/10.1016/j.scienta.2015.08.023 0304-4238/© 2015 Elsevier B.V. All rights reserved.
several crops, trees, mosses and cut flowers to drought and rehydration were partially documented (Filippou et al., 2011; Liu et al., 2010, 2013; Luo et al., 2011). Most of these plants demonstrated increasing sensitivity to drought induced oxidative damage but this procedure was reversed following rehydration. Purslane (Portulaca oleracea L.) is an annual herb with succulent and fleshy stems that may grow erect or prostrate depending on light availability (Chauhan and Johnson, 2009). Purslane belongs to family Portulacaceae, which is widely distributed all over the world, and grows well in diverse geographical environments (D’Andrea et al., 2014). Purslane is listed as one of the most useful medicinal plants and named “Global Panacea” by World Health Organization (Samy et al., 2005). As one of the valuable medical plants and vegetable, it is enrich with antioxidant vitamins and omega-3 fatty acids (Rahdari et al., 2012; Simopoulos et al., 1992). Omega-3 fatty acids are useful in preventing numerous cardiovascular diseases and maintaining a healthy immune system (Simopoulos, 2004; Uddin et al., 2014). All of the above characteristics made purslane flourisher than many other crops. Alam et al. (2014) screened the most salt-tolerant purslane accessions prioritizing the utilization as a resource of vegetable nutrients and commercial cultivation for saline agriculture and sustainable development. Rahdari et al. (2012) reported
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the effect of drought on germination, proline, sugar, lipid, protein and chlorophyll content in purslane, and the results indicated that purslane was tolerant to drought stress and could be used in arid and semi-arid regions. Yang et al. (2012) investigated the mechanisms underlying purslane’s tolerance to high temperature and high humidity stresses, and the results suggested that purslane deployed the multiple strategies to cope with combined stresses. In this study, we addressed a brief insight into the combined effects of drought and rehydration. Progressive drought stress treatment was applied on purslane by gradually decreasing soil water content (SWC). The results showed that drought treatment resulted in decreased LWC and chlorophyll content and increased malondialdehyde (MDA), proline, electrolyte leakage (EL), O2 •− , and activities of superoxide dismutase (SOD) and peroxidase (POD). Purslane leaves kept high water content under stressed condition and recovered from rehydration through increase of leaf water content (LWC) and chlorophyll content and decrease of other parameters. All these results indicated that purslane had great elasticity to cope with drought and rehydration.
2. Materials and methods 2.1. Plant materials and growth conditions The purslane seeds were sown in 10 cm diameter and 10 cm deep plastic pots with vermiculite after stratified at 4 ◦ C in the dark for 3 d. Plants were grown in growth room with controlled temperature at 28 ± 1 ◦ C, 65–75% relative humidity, 300 mol s−1 m−2 light intensity, and 16 h light and 8 h dark cycles. The plastic pots were rotated daily to minimize the influence of environments. To determine drought tolerance, 21 d-old seedlings were subjected to progressive drought stress. The control group was irrigated regularly. Leaf samples were harvested at specific time points (5 d, 10 d, 15 d and 22 d after drought stress imposition). When the leaves of purslane appeared wilting at 22 d after drought treatment, rehydration was applied. Leaves were sampled after 0 h, 3 h, 1 d and 3 d of rehydration. Samples were frozen in liquid nitrogen and stored at −80 ◦ C for physiological analyses. Experiments were executed in triplicates and the top leaf tissues were collected from a minimum of 10 independent plants.
2.2. EL assay EL was determined as stated by Shi et al. (2012a) with slight modifications. Briefly, five intact leaves were incubated in 20 mL deionized water, and shaken for 6 h at room temperature. The initial conductivity (Ci ) was measured by conductivity meter (LeiciDDS-307A, Shanghai, China). The samples then were boiled for 20 min to completely induce all electrolytes. After cooling to room temperature, the conductivity (Cmax ) was determined. Relative EL(%) = (Ci /Cmax ) × 100.
2.3. Determination of SWC and LWC To assess soil water status, SWC was detected by soil moisture temperature recorder (L99-TWS-1, Fotel Precise Instrument Co., Ltd., Shanghai, China) at designated time intervals. For measurement of LWC, leaf samples were harvested at 0 d, 5 d, 10 d, 15 d and 22 d after drought stress, and 3 h, 1 d and 3 d after rehydration. Fresh weight (FW) was measured after harvest immediately, and the dry weight (DW) was measured after 16 h incubation at 80 ◦ C oven. LWC (%) was calculated as (FW–DW)/FW × 100%.
2.4. The measurement of MDA and proline contents MDA was extracted with the trichloroacetic acid (TBA) reaction and determined at 450 nm, 532 nm and 600 nm wavelength as described previously (Shi et al., 2012a,b) using Multiskan MK3 (Thermo Scientific, USA). Proline was determined as follows. Briefly, 0.5 g leaf sample was totally extracted in 3% (w/v) sulphosalicylic acid, then 2 mL of ninhydrin regent and 2 mL of glacial acetic acid were added before boiled for 30 min at 100 ◦ C. After cooling to room temperature, the mixture was centrifuged at 12,000 × g for 5 min. The proline content in the sample was calculated at 520 nm absorbance. Simultaneously, standard curve was measured using purified proline (Shi et al., 2012a,b, 2014). 2.5. Determination of O2 •− , antioxidant enzyme activities and chlorophyll content O2 •− content was measured by the SOA Assay Kit (Elisa, Shanghai, China). Based on the antibody–antigen reaction, O2 •− content was determined by measuring the absorbance of TMB substrate at 450 nm. SOD activity was assayed with the SOD Assay Kit (Beyotime, Shanghai, China). 2-(4-Iodophenyl)-3-(4-nitrophenyl)-5(2,4-disulfophenyl)-2H-tetrazolium (WST-1) was used in this assay, which couples with xanthine oxidase (XO) to generate O2 •− and formazan dye, This reaction is inhibited by SOD which catalyzing O2 •− into O2 and H2 O2 . Therefore, SOD activity can be measured by the absorbance of formazan dye at 450 nm. To determine the POD activity, Plant POD activity Assay Kit (Jiancheng, Nanjing, China) was used. Based on the guaiacol oxidation, POD activity was calculated by measuring the absorbance of reaction buffer at 340 nm. Chlorophyll content in leaf was determined as previously described with slight modification (Frank et al., 2005). Briefly, leaf samples were treated for 30 min at 95 ◦ C. After that, the leaf samples were dried to constant weight at 80 ◦ C. Then the samples were thoroughly homogenized and chlorophyll was totally extracted with 96% (v/v) ethanol in the dark at 4 ◦ C. For chlorophyll quantification, the supernatant of ethanol extract was measured at 649 nm and 665 nm wavelengths. 2.6. Statistic analysis Data shown are mean ± standard deviation (SD). Statistical analysis was performed using LSD test (P < 0.05). Asterisk symbols above the points in the figures indicate significant differences with control at the 0.05 level. 3. Results 3.1. Effects of drought and rehydration on purslane phenotype After the progressive drought stress treatment, purslane showed dwarfed phenotype and leaf area was significantly reduced. At 22 d under drought stress, the leaves appeared obvious wilting. After 3 d of rehydration, purslane achieved completely recovery and no wilting phenomenon was observed in treated purslane (Fig. 1). 3.2. Changes in SWC and LWC after drought and rehydration treatments To determine purslane drought tolerance, 21 d-old seedlings were subjected to a progressive drought stress and then followed with rehydration. During drought periods, SWC sharply declined from 95 to 20%. During the rehydration, SWC restored immediately, and achieved complete recovery at 3 h after rehydration (Fig. 2A).
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Fig. 1. Effects of drought and rehydration on purslane phenotype. Representative pictures were shown here. (A) Control and drought for 22 d and (B) control and 3 d after rehydration.
Fig. 2. (A) Soil water content and (B) leaf water content during the drought stress and rehydration for purslane. Dark grey shading indicates the duration of drought; light grey shading indicates rehydration duration. Results are shown as means standard deviation (SD).
LWC in stressed seedlings decreased slightly with ongoing soil drought stress and dropped to 91% after 22 d of drought stress treatment. LWC restored to the control level after 1 d of rehydration. The control seedlings showed slight changes in the LWC throughout the experiment, ranging from 95 to 96% (Fig. 2B).
stress while decrease after rehydration (Fig. 3C). These results indicated that drought stress for longer than 10 d had adverse effects on purslane growth, while purslane recovered quickly at 1 d after rehydration.
3.3. Effects of drought and rehydration on purslane MDA, proline and EL
3.4. Effect of drought and rehydration on purslane chlorophyll content
In this study, MDA and proline contents showed significant increase after 10 d of drought stress treatment and peaked at 22 d after drought stress. After rehydration, MDA and proline contents showed a significantdecrease after 3 h and almost equaled to the control level at 1 d (Fig. 3A and B). Change tendency of EL was in parallel with those of MDA and proline, showing increase after drought
Drought stress caused a significant reduction in chlorophyll content. At the early stage of rehydration, the chlorophyll content kept decreasing tendency. After 3 h of rehydration, chlorophyll content increased gradually but still lower than that in the control level (P < 0.05) at 3 d after rehydration, indicating photosynthesis of purslane recovered at this stage (Fig. 4).
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Fig. 3. (A) MDA content, (B) proline content and (C) EL during the drought stress and rehydration for purslane. Dark grey shading indicates the duration of drought; light grey shading indicates rehydration duration. Results are shown as means standard deviation (SD).
Fig. 4. Chlorophyll content during the drought stress and rehydration for purslane. Dark grey shading indicates the duration of drought; light grey shading indicates rehydration duration. Results are shown as means standard deviation (SD).
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3.5. Effect of drought and recovery on purslane ROS and antioxidant enzyme activities O2 •− content increased during the drought stress (Fig. 5A). Rehydration reversed this tendency, but O2 •− content in stressed plants was still higher than that in the control after 3 d of rehydration, which indicated that the purslane plants was not fully recovered in terms of ROS (Fig. 5A). Drought stress from 1 to 10 d had no significant effect on activities of SOD and POD. During 15–22 d of drought stress, activities of SOD and POD increased significantly. Rehydration resulted in significant decline but was still beyond the control (Fig. 5B and C).
4. Discussion Drought stress is the key factor which limits plant growth and development. In this study, we tested drought tolerance in purslane. As an important medicinal plant, purslane has a valuable
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place in a healthy diet and was reported to be drought tolerance (Rahdari et al., 2012). The results showed that LWC remained relatively stable in the control plants and modestly declined under drought stress condition. Purslane still held very high LWC (91%) although it was wilted at 22 d after drought stress. Under drought stress condition, stomata of purslane closed quickly which might have permitted high water potential (Ren et al., 2011). Moreover, Purslane is a dicotyledon and one of the C4 plants displays Kranz anatomy structure. The term “CAM-like” has been used to refer to purslane case (Lara et al., 2003). In addition to the bundle sheath and mesophyll cells, the leaves of purslane present large and vacuolated water storage cells contributing to the succulence of the leaves (Lara et al., 2003). CAM is a photosynthetic type that can be induced by abiotic and biotic stresses (Borland et al., 2006). The fundamental features of CAM are abilities to take carbon at night and to accumulate it in the vacuole as an organic acid (Silvera et al., 2010). Lara et al. (2004) reported purslane subjected to drought showed typical characteristics of CAM, such as signifi-
Fig. 5. (A) O2 • − , (B) POD, and (C) SOD activities during the drought stress and rehydration for purslane. Dark grey shading indicates the duration of drought; light grey shading indicates rehydration duration. Results are shown as means standard deviation (SD).
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cant daily malate fluctuation, net CO2 uptake in the dark and daily changes in the relative activity and regulatory properties of PEPC. Portulacaceae have evolved from CAM ancestors (Guralnick and Jackson, 2001). In the course of evolution, it is possible that over an exiting CAM tissue the C4 photosynthesis somehow occurred in purslane and that CAM now has a role only in particular stress conditions such as drought (Sage, 2002). The operation of a CAM-like metabolism in C4 purslane under drought would serve as a mechanism for water saving, evading carbon losses due to respiration and prolonging the life of the cell (Herrera, 2009). Cell membrane stability is vital for maintaining cell turgor pressure and physiological functions, particularly during drought stress. EL has been widely used to estimate cell membrane stability by means of measuring the extent of cell injury when subjected to all kinds of environmental stressors (Hu et al., 2010a,b; Zhao et al., 2011). Although drought treatment significantly (P < 0.05) increased EL, the maximum of EL at 22 d after drought treatment was only 11% (Fig. 3C), which was much lower than that in bermudagrass (Cynodon dactylon) assayed in the same lab (Shi et al., 2012a, 2013), indicating that succulent purslane plant might be more tolerant to drought stress than other species. Proline is accumulated as an osmoprotectant in response to adverse conditions and MDA is one of the indicators of lipid peroxidation under stress (Liu et al., 2014). Purslane under drought stress accumulated high level of proline (Fig. 3B), which was important to alleviate cellular hyperosmolarity and ion disequilibrium. Additionally, Drought stress significantly increased MDA content in purslane at 10 d after drought treatment (Fig. 3A). This result was consistent with the previous researches that drought stress induced membrane-lipid peroxidation (Filippou et al., 2011; Naya et al., 2007). Other plants such as bermudagrass and sorghum (Sorghum bicolor) showed similar pattern of lipid peroxidation under drought stress condition (Shi et al., 2012a; Zhang and Kirkham, 1996). MDA is formed by ROS induced decomposition of polyunsaturated lipids (Pryor and Stanley, 1975). Interestingly, O2 •− content increased significantly at 10 d after drought treatment (Fig. 5A), which was in line with MDA data. Facing adverse environment, plants have developed an efficient antioxidant defense system to scavenge ROS, which are the important signal molecules induced by various environmental stresses, including H2 O2 , O2 •− , hydroxyl radical and singlet oxygen (Smirnoff, 1993). The antioxidant defense system is comprised of enzymatic and nonenzymatic components. Enzymatic system produces antioxidants or antioxidant enzymes that directly react with ROS and scavenge it, including SOD, POD and CAT, while nonenzymatic system produces enzymes that regenerate antioxidants such as glutathione, glutathione reductase, ascorbate and ascorbate reductase (Smirnoff, 1993). In this study, drought stress significantly increased activities of SOD and POD after 15 d. After rehydration for 1–3 d, the activities of SOD and POD gradually decreased indicating that ROS metabolism returned to the control level. Drought stress inhibited the synthesis of chlorophyll in plants, and proteins involved in chloroplast synthesis were hampered. Moreover, the original chlorophyll was decomposed under severe drought stress condition (Parida et al., 2003). A rapid loss of the chlorophyll content during rehydration has been reported in Physcomitrella patens (Cui et al., 2012; Frank et al., 2005). The decrease of chlorophyll under drought stress in purslane indicated that drought stress affected purslane chlorophyll biosynthesis. Unlike other physiological parameters, chlorophyll content kept decreasing at 3 h after rehydration, and then showed increase trend (Fig. 4). At this time, MDA, proline, EL, O2 •− , and activities of SOD and POD showed reversed change tendency when compared to these after drought treatment. This result indicated that chloro-
Fig. 6. Model depicting drought and rehydration induced changes in purslane plant.
phyll biosynthesis was delayed during purslane plant recovery from drought stress induced damages. In summary, purslane was relatively drought tolerant and survived from drought stress for at least 22 d and recovered quickly after rehydration for 1–3 d in terms of physiological assays (Fig. 6). To minimize nutritional value loss, the harvest time could be determined with 5–10 d of drought and 3–5 d after rehydration. For field cultivation of purslane, timely watering and keep the soil moisture over 50% will not influence the regular growth of plants. Both unique nutritive quality and distinguished drought tolerance make purslane a promising candidate to be used as healthy diet and in stress response related researches in agriculture. Acknowledgments This research was supported by “the Hundred Talents Program”, the Knowledge Innovative Key Program of Chinese Academy of Sciences (Grant No. Y154761O01076 and No.Y329631O0263) to Zhulong Chan. References Alam, M.A., Juraimi, A.S., Rafii, M.Y., Hamid, A.A., Aslani, F., 2014. Screening of purslane (Portulaca oleracea L.) accessions for high salt tolerance. Sci. World J., 627–916. Borland, A., Elliott, S., Patterson, S., Taybi, T., Cushman, J., Pater, B., Barnes, J., 2006. Are the metabolic components of crassulacean acid metabolism up-regulated in response to an increase in oxidative burden? J. Exp. Bot. 57, 319–328. Cui, S., Hu, J., Guo, S., Wang, J., Cheng, Y., Dang, X., Wu, L., He, Y., 2012. Proteome analysis of Physcomitrella patens exposed to progressive dehydration and rehydration. J. Exp. Bot. 63, 711–726. D’Andrea, R.M., Andreo, C.S., Lara, M.V., 2014. Deciphering the mechanisms involved in Portulaca oleracea (C4 ) response to drought: metabolic changes including crassulacean acid-like metabolism induction and reversal upon re-watering. Physiol. Plant. 152, 414–430. Filippou, P., Antoniou, C., Fotopoulos, V., 2011. Effect of drought and rewatering on the cellular status and antioxidant response of Medicago truncatula plants. Plant Signal. Behav. 6, 270–277. Frank, W., Ratnadewi, D., Reski, R., 2005. Physcomitrella patens is highly tolerant against drought, salt and osmotic stress. Planta 220, 384–394. Guralnick, L.J., Jackson, M.D., 2001. The occurrence and phylogenetics of crassulacean acid metabolism in the Portulacaceae. Int. J. Plant Sci. 162, 257–262. Herrera, A., 2009. Crassulacean acid metabolism and fitness under water deficit stress: if not for carbon gain, what is facultative CAM good for? Ann. Bot. 103, 645–653. Hu, L., Wang, Z., Du, H., Huang, B., 2010a. Differential accumulation of dehydrins in response to water stress for hybrid and common bermudagrass genotypes differing in drought tolerance. J. Plant Physiol. 167, 103–109. Hu, L., Wang, Z., Huang, B., 2010b. Diffusion limitations and metabolic factors associated with inhibition and recovery of photosynthesis from drought stress in a C3 perennial grass species. Physiol. Plant. 139, 93–106. Huxman, T.E., Snyder, K.A., Tissue, D., Leffler, A.J., Ogle, K., Pockman, W.T., Sandquist, D.R., Potts, D.L., Schwinning, S., 2004. Precipitation pulses and carbon fluxes in semiarid and arid ecosystems. Oecologia 141, 254–268.
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