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Morpholoical and enzymatic responses to waterlogging in three Prunus species
Scientia Horticulturae 221 (2017) 62–67
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Morpholoical and enzymatic responses to waterlogging in three Prunus species
MARK
Chenping Zhoua,b,c, Tao Baia,b,c, Yi Wanga,b,c, Ting Wua,b,c, Xinzhong Zhanga,b,c, Xuefeng Xua,b,c, ⁎ Zhenhai Hana,b,c, a
Institute of Horticultural Plants, College of Horticulture, China Agricultural University, Beijing 100193, PR China Key Laboratory of Stress Physiology and Molecular Biology for Fruit Trees in Beijing Municipality, China Agricultural University, Beijing 100193, PR China c Key Laboratory of Biology and Genetic Improvement of Horticultural Crops (Nutrition and Physiology), Ministry of Agriculture, China Agricultural University, Beijing 100193, PR China b
A R T I C L E I N F O
A B S T R A C T
Keywords: Prunus Waterlogging Anaerobic respiration Enzymatic activities Plant growth
Anaerobic respiration is an important mechanism for plants to address energy deficiency under waterlogged (WL) conditions, when aerobic respiration is limited. Seedlings of three Prunus species—Prunus mira Koehne, Prunus persica (L.) Batsch, and Prunus amygdalus (L.)—were irrigated at 60% water (control) or waterlogged (100%) daily for 27 days, to investigate root morphological parameters and dynamics of anaerobic respiration enzymes. Both P. mira and P. persica had significantly increased the leaf number and plant height than P. amygdalus under WL. Additionally, WL decreased leaf chlorophyll content in P. mira and P. amygdalus significantly more than in P. persica. Root pyruvate carboxylase (PDC), alcohol dehydrogenase (ADH), and lactate dehydrogenase (LDH) activities first increased before decreasing under WL treatments. In P. persica, root PDC activity was significantly higher than in P. mira and P. amygdalus at 9 d and 21 d, whereas ADH activity was higher at 15 d, 21 d, and 27 d. Moreover, throughout the experiment, LDH activity was significantly higher in P. persica than in P. mira and P. amygdalus roots under WL treatments. The activities of all three measured enzymes were positively and significantly correlated. We suggest that, because of interspecific variation in root metabolic response to different environmental conditions, P. mira and P. persica may generate more energy anaerobically through EMP than through lactic-acid metabolism, whereas P. amygdalus was the reverse. We conclude that WL tolerance differed across the three Prunus, with P. persica being the most tolerant, followed by P. mira, and finally P. amygdalus.
1. Introduction Waterlogging (WL) is a severe threat to crop production worldwide (Wei et al., 2013; Y. Zhang et al., 2016), caused by multiple factors including improper irrigation and global warming (Bansal and Srivastava, 2015). Waterlogging subjects plant roots to an anoxic environment (Wang et al., 2002), limiting mitochondrial aerobic respiration and causing energy loss (Mustroph and Albrecht, 2007; Setter and Waters, 2003); this restricts plant growth and survival (Gibberd and Cocks, 1997; Gibberd et al., 2001). In response to hypoxia stress, plants can temporarily compensate with anaerobic respiration. However, this process produces harmful metabolic by-products that can cause plant death. Specifically, three highly expressed enzymes involved in anaerobic metabolic pathways—pyruvate dehydrogenase
(PDH), alcohol dehydrogenase (ADH), and lactate dehydrogenase (LDH) (Subbaiah and Sachs, 2003)—respectively produce acetaldehyde, ethanol, and lactic acid. Although ethanol is harmless, acetaldehyde accumulation is thought to be harmful to plant cells (An et al., 2016; Arismendi et al., 2015; Braun et al., 1995; Perata and Alpi, 1993), whereas lactic acid is a major cause of WL-stress-related death through its induction of cytoplasmic acidification and pH reduction. Hence, measuring the activity dynamics of anaerobic-respiration enzymes should improve our understanding of WL-induced damage in plants (Roberts et al., 1984). Woody fruit trees are economically important but do not have high tolerance to WL stress. In numerous fruit trees (e.g., apple, mango, avocado, carambola, cranberry, and peach), WL causes water loss in stems and leaves, lowers photosynthesis, decreases carbohydrate accu-
Abbreviations: WL, waterlogging; ADH, alcohol dehydrogenase; LDH, lactate dehydrogenase; PDC, pyruvate decarboxylase; EMP, ethanol metabolism pathway ⁎ Corresponding author at: Institute for Horticultural Plants, College of Horticulture, China Agricultural University, No. 2 Yuanmingyuan West Road, Haidian District, Beijing 100193, PR China. E-mail address: [email protected] (Z. Han). http://dx.doi.org/10.1016/j.scienta.2017.03.054 Received 28 November 2016; Received in revised form 8 March 2017; Accepted 21 March 2017 0304-4238/ © 2017 Elsevier B.V. All rights reserved.
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mulation (hindering new leaf formation), accelerates leaf abscission, and reduces branch growth (Amador et al., 2012; Malik et al., 2002; Schaffer et al., 1992). Peach trees are among the most WL sensitive because their roots exhibit stronger respiration and higher oxygen consumption than other fruit trees (Pimentel et al., 2014; Pinto, 2015; Schaffer et al., 1992). Peach cultivation is severely hampered during the rainy season, when WL deteriorates soil oxygen supply and thereby lowers fruit quality through negative effects on peach physiology (Lara et al., 2011; Sairam et al., 2008). Thus, the development of a WLtolerant cultivar is critical for the expansion of peach cultivation, particularly in areas with frequent and high precipitation. In China, common peach species differ in their degree of WL tolerance. The most widely cultivated is the endemic Prunus persica (L.) Batsch, which has stronger WL resistance than other Prunus spp. Prunus mira Koehne is endemic to the Tibetan plateau and among the most widely distributed wild fruit trees there (Bai et al., 2009; Guan et al., 2014; Hao et al., 2012). Although it grows robustly in riparian and rain-fed regions, previous attempts to quantify WL resistance in P. mira were confounded by soil or other environmental factors (Hork et al., 2006; Khabaz-Saberi et al., 2006). Finally, P. amygdalus (L.) is a drought-resistant shrub unique to the Gobi Desert of Central Asia; little data are available on its WL resistance (Guo et al., 2015). However, a previous comparative study indicated that the three species differ in their relative WL tolerance (Chen and Liu, 2003). Existing research on WL stress in woody fruit trees typically examined changes to plant morphology. Thus, little is known about the physiological mechanisms of WL stress response in Prunus species with differing WL sensitivity. In this study, we investigated WL’s effects on biological stress indicators and the dynamics of key enzymes related to root respiration in P. mira, P. persica, and P. amygdalus plants. Our objectives were to understand how anaerobic respiration varied across three Prunus under WL stress and how that variation may be related to interspecific differences in WL sensitivity.
2.4. Determination of PDC, ADH, and LDH activities
2. Materials and methods
Under WL treatment, leaves did not fall in any species from 0 d to 15 d. After 15 d, old leaves on the stem base of P. amygdalus began to fall; as WL increased, the amount of leaves shed also increased, reaching seven leaves at 27 d. Additionally, P. mira leaves began to fall at 24 d under WL treatment. These data indicate that WL differentially affected leaf shedding across the three Prunus: P. amygdalus shed the largest number of leaves and P. mira shed significantly less, while P. persica shed none (Fig. 1). Plant heights increased in all three species during WL treatment, with no significant difference from control during 0 d to 3 d (Table 1). Subsequently, plant growth rates began to differ significantly on separate days (P. amygdalus on 6 d, P. mira on 9 d, and P. persica on 15 d), indicating that P. amygdalus was the first to experience negative effects under WL stress. Growth rates of WL-stressed P. mira, P. persica,
Enzymatic activity was determined with methods from Mustroph and Albrecht (2003) and Voesenek et al. (2006). Briefly, 0.2 g of the root’s central region was added with 1.6 mL precooled Tris-HCl (pH 6.8) to a mortar and ground in ice, then centrifuged at 12,000 × g and 4 °C for 20 min. The supernatant was the crude enzyme extraction. The reaction mixture for measuring PDC activities comprised: 50 mmol L−1 MES (pH 6.8), 25 mmol L−1 NaCl, 1 mmol L−1 MgCl2, 0.5 mmol L−1 TPP, 3 mmol L−1 DTT, 0.2 mmol L−1 NADH, 50 mmol L−1 sodium oxalate, 10 U ADH, and the extraction, 10 mmol L−1 pyruvate was added to start the reaction. The ADH reaction mixture comprised 50 mmol L−1 TES (pH 7.5), 2.5 μmol L−1 NADH, and the extraction, with the addition of 0.25 mmol L−1 acetaldehyde to start the reaction. The LDH reaction mixture included 0.1 mol L−1 phosphoric acid (pH 7.0), 4 μmol L−1 NADH, 0.25 mmol L−1 pyruvate, and the reaction was started with the extraction addition. Activities of PDC, ADH, and LDH were measured at 340 nm using an ultraviolet-visible spectrophotometer; the protein content was measured following the Bradford method (1976). 2.5. Statistical analysis This experiment was a randomized block design conducted on a single plot with six replicates per treatment. All data are presented as the mean ± SE of each treatment and tested with repeated measures ANOVA, followed by least significant difference (LSD) tests. Significance was set at P < 0.05. 3. Results 3.1. Plant growth
2.1. Plant culture, treatment, and sampling This study was conducted at China Agriculture University (40°01′26.51″N, 116°16′36.06″E). The seeds of wild P. mira, P. persica, and P. amygdalus were purchased from Tibet Nyingchi, Beijing, and Inner Mongolia Alashan League, respectively. Seeds were sown in 7 cm × 7 cm pots containing nutrient soil (peat:vermiculite:soil = 1:1:2). After the cotyledons spread, same-age seedlings from each Prunus species were selected; one seedling was transplanted to a pot with non-enriched soil and placed in a greenhouse. Water was added to each pot until a height of over 3 cm and either replenished as necessary to maintain water levels (WL treatment) or irrigated normally without flooding (control), for 27 days. Roots and leaves were collected at 0, 3, 6, 9, 15, 21, and 27 d during WL treatment, immediately frozen in liquid nitrogen, and stored at −80 °C, for subsequent determination of enzymatic activities and chlorophyll content. 2.2. Determination of growth parameters Plant height was measured using a tapeline, and numbers of total leaf and leaf shed of each plant were measured at 0, 3, 6, 9, 15, 21, and 27 d during WL treatment. 2.3. Measurement of leaf chlorophyll Leaf chlorophyll content was assayed following Holm-Hansen and Riemann (1978). Briefly, chlorophyll was extracted with 80% (v/v) acetone, and the content was determined at 652 nm using ultravioletvisible spectrophotometer (Shimadzu, China).
Fig. 1. Number of shed leaves in three Prunus species [P. mira Koehne, P. persica (L.) Batsch, and P. amygdalus (L.)] subjected to waterlogging treatment. “*” and “**” indicate significant difference at P < 0.05 and P < 0.01, respectively. Means ± SE, n = 6.
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Table 1 Plant total height (cm) under waterlogging treatments. Means ± SE, n = 6. CK: Control; WL: Waterlogging treatment. Species
Treatment time (d) 0
3
6
9
15
21
27
P. mira
CK WL
15.36 ± 0.94 15.36 ± 0.94
17.34 ± 1.12 16.93 ± 1.03
18.24 ± 1.39 17.84 ± 1.34
19.76 ± 1.76 18.60 ± 1.56*
24.43 ± 2.95 20.83 ± 2.15*
27.93 ± 4.98 23.43 ± 3.68*
34.23 ± 5.13 27.33 ± 4.93*
P. persica
CK WL
12.83 ± 1.06 12.83 ± 1.06
14.97 ± 1.11 14.87 ± 1.21
16.83 ± 1.39 16.43 ± 1.42
19.42 ± 1.78 18.94 ± 1.69
25.57 ± 2.92 23.67 ± 2.56*
31.63 ± 3.62 28.83 ± 3.39*
34.73 ± 4.57 30.33 ± 4.87*
P. amygdalus
CK WL
10.17 ± 1.13 10.17 ± 1.25
11.63 ± 1.64 11.23 ± 1.7
12.69 ± 2.31 11.83 ± 2.02*
13.86 ± 2.16 12.13 ± 2.28*
14.98 ± 1.91 12.30 ± 2.31*
16.11 ± 2.19 12.40 ± 2.21*
18.37 ± 2.51 13.00 ± 1.47*
* Indicates significant differences at P < 0.05, compared to the control plants.
Fig. 2. Effect of waterlogging on plant height (A), leaf number (B), and chlorophyll content (C) in three Prunus species [P. mira Koehne, P. persica (L.) Batsch, P. amygdalus (L.)]. Waterlogging inhibited rates of plant and leaf growth relative to control (B). The effects of waterlogging on the dynamics of anaerobic respiration enzymes (PDC, pyruvate carboxylase; alcohol dehydrogenase, ADH; and lactate dehydrogenase, LDH) are also shown (D, E and F). “*” indicates significant difference at P < 0.05. Means ± SE, n = 6.
3.2. Chlorophyll content
and P. amygdalus were 76.5%, 87.3%, and 67.2% higher, respectively, than the control (P < 0.05, Fig. 2A). Thus, WL stress decreased the growth potentials of all species, but P. mira was less tolerant than P. persica and more tolerant than P. amygdalus. Leaf numbers increased in all species under both control and WL; however, leaf growth rates decreased under WL stress, beginning on 9 d, 15 d, and 21 d for P. amygdalus, P. mira, and P. persica, respectively (Table 2 and Fig. 2B). After 27 d of WL, leaf growth rates of P. mira and P. persica were 80.5% and 86.7% higher than control, and significantly higher than the 73.3% rate of P. amygdalus. Thus, WL stress inhibited leaf growth, with P. mira and P. persica being similarly affected, although both exhibited higher rates than P. amygdalus.
Chlorophyll content in P. mira and P. amygdalus decreased slowly as WL stress duration increased, reaching the lowest values on 27 d (Fig. 2C). Thus, WL treatment induced leaf chlorophyll decomposition in P. mira and P. amygdalus seedlings, leading to leaf yellowing (data not shown). However, P. persica leaves did not significantly change in chlorophyll content during WL treatment. 3.3. Activities of PDC, ADH, and LDH in Prunus roots In all species, PDC activity rose first before falling; in particular, P. amygdalus root PDC activity increased dramaticallyafter 3 d of WL treatment (Fig. 2D). As WL treatment continued, P. amygdalus root PDC activity decreased rapidly at 21 d. In P. mira and P. persica, root PDC 64
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Table 2 Leaf number of three peaches under waterlogging treatments. CK: Control; WL: Waterlogging treatment. Means ± SE, n = 6. Species
Treatment time (d) 0
3
6
9
15
21
27
P. mira
CK WL
10.1 ± 2.0 10.1 ± 2.0
11.5 ± 1.4 11.3 ± 1.9
13.1 ± 1.1 12.9 ± 2.1
14.7 ± 1.9 14.1 ± 2.6
18.5 ± 2.1 16.0 ± 3.1*
22.6 ± 3.3 18.6 ± 4.5*
25.9 ± 5.1 20.9 ± 6.5*
P. persica
CK WL
13.7 ± 0.6 13.7 ± 0.6
14.9 ± 1.1 14.7 ± 1.2
16.8 ± 0.9 16.3 ± 0.6
18.8 ± 0.8 18.0 ± 0.6
23.7 ± 1.7 22.7 ± 1.6
29.7 ± 3.1 26.7 ± 4.7*
33.6 ± 6.2 29.3 ± 8.4*
P. amygdalus
CK WL
15.0 ± 2.6 15.0 ± 2.6
16.3 ± 3.1 15.7 ± 3.1
17.8 ± 3.6 17.0 ± 3.6
19.0 ± 3.5 17.3 ± 3.5*
22.1 ± 3.2 18.3 ± 3.2*
23.9 ± 2.6 18.7 ± 2.6*
25.8 ± 4.0 18.9 ± 4.0*
* Indicates significant differences at P < 0.05, compared to the control plants.
activities were lower than in P. amygdalus roots early during WL treatment, but became significantly higher in the later stages except at 15 d. Specifically, P. mira PDC activity was higher than activities in the other two species at 9 d, whereas P. persica PDC activity became highest at 21 d. Overall, WL’s effects on PDC activity were strongest in P. amygdalus roots, causing rapid and dramatic fluctuations early and late in treatment. Under WL stress, root ADH activities in all species increased first before decreasing, and from 0 to 9 d, no significant differences were observable. However, at 0.641 μmol mg−1 protein min−1 after 21 d, P. persica root ADH activity was 18.4 times higher than its activity at 0 d; it was also significantly higher than activity in P. mira or P. amygdalus during 15–27 d. In the latter two species, ADH activities dramatically increased during 0–6 d and 0–9 d, respectively, but were relatively low at later treatment stages (Fig. 2E). After WL for 6 d, LDH activities in all three peaches also rose first before decreasing (Fig. 2F). Root LDH activities of in P. mira and P. persica were significantly lower than activity in P. amygdalus. Moreover, this significant difference was sustained from 9 d to 27 d of WL treatment (Fig. 2F). Our data suggest that the lactic-acid pathway in P. amygdalus seedlings was regulated under WL, resulting in lactic acid over-accumulation during the early experimental stage and inducing cytoplasmic acidification. Root PDC and ADH activities were positively correlated with each other, as well as with LDH activity (Fig. 3).
4. Discussion Previous reports have indicated a negative correlation between leaf damage and the capacity of WL tolerance in plants (Tuo et al., 2015). In this study, P. amygdalus leaves began to drop after 18 d of WL, P. mira to a lesser extent after 24 d, whereas P. persica did not lose leaves (Fig. 1). Furthermore, WL decreased the chlorophyll content in all peach leaves except those of P. persica, but with P. mira having consistently higher chlorophyll content than P. amygdalus (Fig. 2C). Together, these data indicate that P. persica was the most tolerant to WL, followed by P. mira and P. amygdalus. Under WL stress, fruit trees grow more slowly and exhibit dwarf morphology, as well as decreased root dry weight (Schaffer et al., 1992). Our results corroborate those previous findings: under WL treatment, the more WL-tolerant P. mira and P. persica had similar morphologies, with significantly higher growth rates and rates of increase in leaf number than P. amygdalus (Tables 1 and 2). Because oxygen diffusion rates in water is ten thousand times lower than in air (Armstrong and Drew, 2002), plants experience insufficient oxygen supply and severely weakened aerobic respiration under WL conditions, leading to lowered ATP and NAD+ production (Drew, 1997). Thus, WL-related plant damage is indirect (Dennis et al., 2000), rather than direct damage from excess water (Voesenek et al., 2006). Plants respond to WL stress through a series of adaptation mechanisms, including the activation of anaerobic respiration enzymes
Fig. 3. Correlations between pyruvate decarboxylase (PDC), alcohol dehydrogenase (ADH), and lactate dehydrogenase (LDH) activities in Prunus roots.
(Armstrong and Drew, 2002). The anaerobic fermentation pathway is a short-term adjustment to temporarily produce enough ATP and NAD+ that allows maintaining plant cell function (Good and Crosby, 1989a, 1989b). Previous studies on hypoxia stress in plants showed that PDC, ADH, and LDH activities markedly increased in rice, wheat, maize, and Arabidopsis thaliana roots (Gonzali et al., 2005; Zhang et al., 2015). In this study, anaerobic respiration enzymes in all three tested species increase the activity at the beginning, decreasing later during the treatment period. Our data support the hypothesis that the activation of anaerobic respiration enzymes in plant roots is a positive mechanism to 65
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Fig. 4. Hypotetical pathways of anaerobic respiration under waterlogging conditions in P. persica (L.) Batsch (A) and P. amygdalus (L.) (B). ADH, alcohol dehydrogenase; LDH, lactate dehydrogenase; PDC, pyruvate decarboxylase.
significantly in wheat plants with weak anti-hypoxic capacity (Hossain and Uddin, 2011), and delays to peak LDH activity positively affects cherry-root response to WL stress (Cheng et al., 2008). This research is consistent with our findings: under early WL stress, P. amygdalus LDH activity increased rapidly and remained high throughout the experiment, likely leading to lactic acid accumulation and inducing earlier cytoplasmic acidification in P. amygdalus than in P. mira and P. persica. As a result, P. amygdalus quickly experienced the symptoms of WL damage, including shed leaves, slow plant growth, and chlorophyll degradation (Tables 1, 2 and Fig. 2). Our results illustrate that interspecific differences among Prunus germplasms were responsible for fluctuations in PDC, ADH, and LDH activities; which may lead to the discrepancies in secondary metabolite accumulation emerged across the three species, causing variation in WL tolerance. We suggest that P. persica may generate more energy anaerobically through EMP than through the lactic-acid metabolism pathway, whereas P. amygdalus was the reverse; this difference affected the latter species’ root PDC and ADH activities; and P. mira was intermediate. Our data are also consistent with the hypothesis that cytosolic pH shifts act as a switch from one metabolic route to the other in WL conditions. Under WL stress, pH may fall below 7 (LDH optimum), into the optimal range for PDC, where PDC can convert pyruvate to acetaldehyde, which is subsequently converted to ethanol by ADH (Dat et al., 2004; Xu et al., 2016). This hypothesized mechanism could explain the diverse behavior of Prunus germplasm to flooding conditions as summarized in Fig. 4.
mitigate energy deficiency under WL stress. The overexpression of PDC may enhance plant resistance to hypoxia stress, because PDC is a key enzyme that catalyzes pyruvate-toacetaldehyde conversion in the ethanol metabolism pathway (EMP) (Shiao et al., 2002; Kato-Noguchi and Morokuma, 2007; J.Y. Zhang et al., 2016). In this study, PDC activities in all tested plants roots were consistent during 0–15 d of WL treatment, but subsequently, P. amygdalus experienced a rapid decrease in root PDC activity, likely the result of damage to root cells. In contrast, PDC activities remained relatively high in P. mira and P. persica roots (Fig. 2D), suggesting WL damaged P. mira and P. persica roots less than P. amygdalus roots. Changes to ADH activity should also affect WL-tolerance potential in plants (Xu et al., 2014). In Arabidopsis thaliana and Zea mays L. adh1 mutants, shifts in ADH activity was essential for resistance to WL stress (Johnson et al., 1994). Furthermore, hypoxia-tolerant cucumber cultivars exhibited higher ADH activity than control (Kang et al., 2008), accelerating the transformation of acetaldehyde to ethanol. In Puccinellia stricta, Isolepis cernua, Samolus repens, Selliera radicans, and Triglochin striata, shoot and root ADH activity significantly increased in response to WL (Brownstein et al., 2013). Our results supported these previous findings suggesting that ADH increase is an adaptive response to anoxia stress, as we demonstrated that P. mira and P. persica ADH activities significantly increased in later stages of WL treatment. The underlying mechanism of benefits from ADH increase is probably linked to the heightened production of ATP and NAD+ for maintaining plant cellular function, as well as the catalysis of harmful acetaldehyde to ethanol (Geigenberger, 2003). Ethanol is harmless to plants because it easily diffuses through cell membrane’s lipid bilayer to the external environment (Perata and Alpi, 1993), which is a byproduct of ADH activity, high ethanol concentrations is a positive indicator of plant defense against oxygen deficiency (Bailey-Serres and Chang, 2005; Agarwal and Grover, 2006). In this study, ADH activity in P. amygdalus roots were low and did not fluctuate much (Fig. 2E). Some data suggest that ADH is not a major limiting factor for ethanol fermentation; instead, PDC activity is the key limiting factor (Mohanty and Ong, 2003; Catherine et al., 2006). In our study, ADH activity in P. persica roots was significantly higher than in the other two species at 15–27 d of WL treatment (Fig. 2E). Therefore, P. persica appeared to have higher WL tolerance than P. amygdalus and P. mira, as the former was more capable than the latter two of removing acetaldehyde under WL stress. The dynamics of ADH activity in the three species was consistent with their WL tolerance, suggesting that when PDC activity remains generally constant, ADH is important to the decrease of harmful metabolites in Prunus roots (Fig. 4). Under hypoxia stress, increased LDH activity leads to lactic acid accumulation and cytoplasmic acidification (Ismond et al., 2003). Under hypoxic conditions such as WL and flood damage, pH reduction is the main cause of plant death. Under WL, LDH activity increases
Acknowledgements This work was supported by a Special Fund for Agro-scientific Research in the Public Interest (grant number 201203075) and the Beijing Municipal Education Commission (CEFFPXM2017_014207_000043). We thank the Key Laboratory of Biology and Genetic Improvement of Horticultural Crop Nutrition and Physiology, from the Ministry of Agriculture in P. R. China for providing plant seeds. References Agarwal, S., Grover, A., 2006. Molecular biology，biotechnology andgenomics floodingassociated low O2 stress response in plants. Crit. Rev. Plant Sci. 25, 1–21. Amador, M.L., Sancho, S., Bielsa, B., Gomez-Aparisi, J., Rubio-Cabetas, M.J., 2012. Physiological and biochemical parameters controlling waterlogging stress tolerance in Prunus before and after drainage. Physiol. Plant. 144 (4), 357–368. An, Y., Qi, L., Wang, L., 2016. ALA pretreatment improves waterlogging tolerance of fig plants. PLoS One 11 (1), e0147202. Arismendi, M.J., Almada, R., Pimentel, P., Bastias, A., Salvatierra, A., Rojas, P., Hinrichsen, P., Pinto, M., Genova, A.D., Travisany, D., Maass, A., Sagredo, B.M., 2015. Transcriptome sequencing of Prunus sp. rootstocks roots to identify candidate genes involved in the response to root hypoxia. Tree Genet. Genom. 11 (1), 1–16. Armstrong, W., Drew, M.C., 2002. Root growth and metabolism under oxygen deficiency.
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Short communication
Morpholoical and enzymatic responses to waterlogging in three Prunus species
MARK
Chenping Zhoua,b,c, Tao Baia,b,c, Yi Wanga,b,c, Ting Wua,b,c, Xinzhong Zhanga,b,c, Xuefeng Xua,b,c, ⁎ Zhenhai Hana,b,c, a
Institute of Horticultural Plants, College of Horticulture, China Agricultural University, Beijing 100193, PR China Key Laboratory of Stress Physiology and Molecular Biology for Fruit Trees in Beijing Municipality, China Agricultural University, Beijing 100193, PR China c Key Laboratory of Biology and Genetic Improvement of Horticultural Crops (Nutrition and Physiology), Ministry of Agriculture, China Agricultural University, Beijing 100193, PR China b
A R T I C L E I N F O
A B S T R A C T
Keywords: Prunus Waterlogging Anaerobic respiration Enzymatic activities Plant growth
Anaerobic respiration is an important mechanism for plants to address energy deficiency under waterlogged (WL) conditions, when aerobic respiration is limited. Seedlings of three Prunus species—Prunus mira Koehne, Prunus persica (L.) Batsch, and Prunus amygdalus (L.)—were irrigated at 60% water (control) or waterlogged (100%) daily for 27 days, to investigate root morphological parameters and dynamics of anaerobic respiration enzymes. Both P. mira and P. persica had significantly increased the leaf number and plant height than P. amygdalus under WL. Additionally, WL decreased leaf chlorophyll content in P. mira and P. amygdalus significantly more than in P. persica. Root pyruvate carboxylase (PDC), alcohol dehydrogenase (ADH), and lactate dehydrogenase (LDH) activities first increased before decreasing under WL treatments. In P. persica, root PDC activity was significantly higher than in P. mira and P. amygdalus at 9 d and 21 d, whereas ADH activity was higher at 15 d, 21 d, and 27 d. Moreover, throughout the experiment, LDH activity was significantly higher in P. persica than in P. mira and P. amygdalus roots under WL treatments. The activities of all three measured enzymes were positively and significantly correlated. We suggest that, because of interspecific variation in root metabolic response to different environmental conditions, P. mira and P. persica may generate more energy anaerobically through EMP than through lactic-acid metabolism, whereas P. amygdalus was the reverse. We conclude that WL tolerance differed across the three Prunus, with P. persica being the most tolerant, followed by P. mira, and finally P. amygdalus.
1. Introduction Waterlogging (WL) is a severe threat to crop production worldwide (Wei et al., 2013; Y. Zhang et al., 2016), caused by multiple factors including improper irrigation and global warming (Bansal and Srivastava, 2015). Waterlogging subjects plant roots to an anoxic environment (Wang et al., 2002), limiting mitochondrial aerobic respiration and causing energy loss (Mustroph and Albrecht, 2007; Setter and Waters, 2003); this restricts plant growth and survival (Gibberd and Cocks, 1997; Gibberd et al., 2001). In response to hypoxia stress, plants can temporarily compensate with anaerobic respiration. However, this process produces harmful metabolic by-products that can cause plant death. Specifically, three highly expressed enzymes involved in anaerobic metabolic pathways—pyruvate dehydrogenase
(PDH), alcohol dehydrogenase (ADH), and lactate dehydrogenase (LDH) (Subbaiah and Sachs, 2003)—respectively produce acetaldehyde, ethanol, and lactic acid. Although ethanol is harmless, acetaldehyde accumulation is thought to be harmful to plant cells (An et al., 2016; Arismendi et al., 2015; Braun et al., 1995; Perata and Alpi, 1993), whereas lactic acid is a major cause of WL-stress-related death through its induction of cytoplasmic acidification and pH reduction. Hence, measuring the activity dynamics of anaerobic-respiration enzymes should improve our understanding of WL-induced damage in plants (Roberts et al., 1984). Woody fruit trees are economically important but do not have high tolerance to WL stress. In numerous fruit trees (e.g., apple, mango, avocado, carambola, cranberry, and peach), WL causes water loss in stems and leaves, lowers photosynthesis, decreases carbohydrate accu-
Abbreviations: WL, waterlogging; ADH, alcohol dehydrogenase; LDH, lactate dehydrogenase; PDC, pyruvate decarboxylase; EMP, ethanol metabolism pathway ⁎ Corresponding author at: Institute for Horticultural Plants, College of Horticulture, China Agricultural University, No. 2 Yuanmingyuan West Road, Haidian District, Beijing 100193, PR China. E-mail address: [email protected] (Z. Han). http://dx.doi.org/10.1016/j.scienta.2017.03.054 Received 28 November 2016; Received in revised form 8 March 2017; Accepted 21 March 2017 0304-4238/ © 2017 Elsevier B.V. All rights reserved.
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mulation (hindering new leaf formation), accelerates leaf abscission, and reduces branch growth (Amador et al., 2012; Malik et al., 2002; Schaffer et al., 1992). Peach trees are among the most WL sensitive because their roots exhibit stronger respiration and higher oxygen consumption than other fruit trees (Pimentel et al., 2014; Pinto, 2015; Schaffer et al., 1992). Peach cultivation is severely hampered during the rainy season, when WL deteriorates soil oxygen supply and thereby lowers fruit quality through negative effects on peach physiology (Lara et al., 2011; Sairam et al., 2008). Thus, the development of a WLtolerant cultivar is critical for the expansion of peach cultivation, particularly in areas with frequent and high precipitation. In China, common peach species differ in their degree of WL tolerance. The most widely cultivated is the endemic Prunus persica (L.) Batsch, which has stronger WL resistance than other Prunus spp. Prunus mira Koehne is endemic to the Tibetan plateau and among the most widely distributed wild fruit trees there (Bai et al., 2009; Guan et al., 2014; Hao et al., 2012). Although it grows robustly in riparian and rain-fed regions, previous attempts to quantify WL resistance in P. mira were confounded by soil or other environmental factors (Hork et al., 2006; Khabaz-Saberi et al., 2006). Finally, P. amygdalus (L.) is a drought-resistant shrub unique to the Gobi Desert of Central Asia; little data are available on its WL resistance (Guo et al., 2015). However, a previous comparative study indicated that the three species differ in their relative WL tolerance (Chen and Liu, 2003). Existing research on WL stress in woody fruit trees typically examined changes to plant morphology. Thus, little is known about the physiological mechanisms of WL stress response in Prunus species with differing WL sensitivity. In this study, we investigated WL’s effects on biological stress indicators and the dynamics of key enzymes related to root respiration in P. mira, P. persica, and P. amygdalus plants. Our objectives were to understand how anaerobic respiration varied across three Prunus under WL stress and how that variation may be related to interspecific differences in WL sensitivity.
2.4. Determination of PDC, ADH, and LDH activities
2. Materials and methods
Under WL treatment, leaves did not fall in any species from 0 d to 15 d. After 15 d, old leaves on the stem base of P. amygdalus began to fall; as WL increased, the amount of leaves shed also increased, reaching seven leaves at 27 d. Additionally, P. mira leaves began to fall at 24 d under WL treatment. These data indicate that WL differentially affected leaf shedding across the three Prunus: P. amygdalus shed the largest number of leaves and P. mira shed significantly less, while P. persica shed none (Fig. 1). Plant heights increased in all three species during WL treatment, with no significant difference from control during 0 d to 3 d (Table 1). Subsequently, plant growth rates began to differ significantly on separate days (P. amygdalus on 6 d, P. mira on 9 d, and P. persica on 15 d), indicating that P. amygdalus was the first to experience negative effects under WL stress. Growth rates of WL-stressed P. mira, P. persica,
Enzymatic activity was determined with methods from Mustroph and Albrecht (2003) and Voesenek et al. (2006). Briefly, 0.2 g of the root’s central region was added with 1.6 mL precooled Tris-HCl (pH 6.8) to a mortar and ground in ice, then centrifuged at 12,000 × g and 4 °C for 20 min. The supernatant was the crude enzyme extraction. The reaction mixture for measuring PDC activities comprised: 50 mmol L−1 MES (pH 6.8), 25 mmol L−1 NaCl, 1 mmol L−1 MgCl2, 0.5 mmol L−1 TPP, 3 mmol L−1 DTT, 0.2 mmol L−1 NADH, 50 mmol L−1 sodium oxalate, 10 U ADH, and the extraction, 10 mmol L−1 pyruvate was added to start the reaction. The ADH reaction mixture comprised 50 mmol L−1 TES (pH 7.5), 2.5 μmol L−1 NADH, and the extraction, with the addition of 0.25 mmol L−1 acetaldehyde to start the reaction. The LDH reaction mixture included 0.1 mol L−1 phosphoric acid (pH 7.0), 4 μmol L−1 NADH, 0.25 mmol L−1 pyruvate, and the reaction was started with the extraction addition. Activities of PDC, ADH, and LDH were measured at 340 nm using an ultraviolet-visible spectrophotometer; the protein content was measured following the Bradford method (1976). 2.5. Statistical analysis This experiment was a randomized block design conducted on a single plot with six replicates per treatment. All data are presented as the mean ± SE of each treatment and tested with repeated measures ANOVA, followed by least significant difference (LSD) tests. Significance was set at P < 0.05. 3. Results 3.1. Plant growth
2.1. Plant culture, treatment, and sampling This study was conducted at China Agriculture University (40°01′26.51″N, 116°16′36.06″E). The seeds of wild P. mira, P. persica, and P. amygdalus were purchased from Tibet Nyingchi, Beijing, and Inner Mongolia Alashan League, respectively. Seeds were sown in 7 cm × 7 cm pots containing nutrient soil (peat:vermiculite:soil = 1:1:2). After the cotyledons spread, same-age seedlings from each Prunus species were selected; one seedling was transplanted to a pot with non-enriched soil and placed in a greenhouse. Water was added to each pot until a height of over 3 cm and either replenished as necessary to maintain water levels (WL treatment) or irrigated normally without flooding (control), for 27 days. Roots and leaves were collected at 0, 3, 6, 9, 15, 21, and 27 d during WL treatment, immediately frozen in liquid nitrogen, and stored at −80 °C, for subsequent determination of enzymatic activities and chlorophyll content. 2.2. Determination of growth parameters Plant height was measured using a tapeline, and numbers of total leaf and leaf shed of each plant were measured at 0, 3, 6, 9, 15, 21, and 27 d during WL treatment. 2.3. Measurement of leaf chlorophyll Leaf chlorophyll content was assayed following Holm-Hansen and Riemann (1978). Briefly, chlorophyll was extracted with 80% (v/v) acetone, and the content was determined at 652 nm using ultravioletvisible spectrophotometer (Shimadzu, China).
Fig. 1. Number of shed leaves in three Prunus species [P. mira Koehne, P. persica (L.) Batsch, and P. amygdalus (L.)] subjected to waterlogging treatment. “*” and “**” indicate significant difference at P < 0.05 and P < 0.01, respectively. Means ± SE, n = 6.
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Table 1 Plant total height (cm) under waterlogging treatments. Means ± SE, n = 6. CK: Control; WL: Waterlogging treatment. Species
Treatment time (d) 0
3
6
9
15
21
27
P. mira
CK WL
15.36 ± 0.94 15.36 ± 0.94
17.34 ± 1.12 16.93 ± 1.03
18.24 ± 1.39 17.84 ± 1.34
19.76 ± 1.76 18.60 ± 1.56*
24.43 ± 2.95 20.83 ± 2.15*
27.93 ± 4.98 23.43 ± 3.68*
34.23 ± 5.13 27.33 ± 4.93*
P. persica
CK WL
12.83 ± 1.06 12.83 ± 1.06
14.97 ± 1.11 14.87 ± 1.21
16.83 ± 1.39 16.43 ± 1.42
19.42 ± 1.78 18.94 ± 1.69
25.57 ± 2.92 23.67 ± 2.56*
31.63 ± 3.62 28.83 ± 3.39*
34.73 ± 4.57 30.33 ± 4.87*
P. amygdalus
CK WL
10.17 ± 1.13 10.17 ± 1.25
11.63 ± 1.64 11.23 ± 1.7
12.69 ± 2.31 11.83 ± 2.02*
13.86 ± 2.16 12.13 ± 2.28*
14.98 ± 1.91 12.30 ± 2.31*
16.11 ± 2.19 12.40 ± 2.21*
18.37 ± 2.51 13.00 ± 1.47*
* Indicates significant differences at P < 0.05, compared to the control plants.
Fig. 2. Effect of waterlogging on plant height (A), leaf number (B), and chlorophyll content (C) in three Prunus species [P. mira Koehne, P. persica (L.) Batsch, P. amygdalus (L.)]. Waterlogging inhibited rates of plant and leaf growth relative to control (B). The effects of waterlogging on the dynamics of anaerobic respiration enzymes (PDC, pyruvate carboxylase; alcohol dehydrogenase, ADH; and lactate dehydrogenase, LDH) are also shown (D, E and F). “*” indicates significant difference at P < 0.05. Means ± SE, n = 6.
3.2. Chlorophyll content
and P. amygdalus were 76.5%, 87.3%, and 67.2% higher, respectively, than the control (P < 0.05, Fig. 2A). Thus, WL stress decreased the growth potentials of all species, but P. mira was less tolerant than P. persica and more tolerant than P. amygdalus. Leaf numbers increased in all species under both control and WL; however, leaf growth rates decreased under WL stress, beginning on 9 d, 15 d, and 21 d for P. amygdalus, P. mira, and P. persica, respectively (Table 2 and Fig. 2B). After 27 d of WL, leaf growth rates of P. mira and P. persica were 80.5% and 86.7% higher than control, and significantly higher than the 73.3% rate of P. amygdalus. Thus, WL stress inhibited leaf growth, with P. mira and P. persica being similarly affected, although both exhibited higher rates than P. amygdalus.
Chlorophyll content in P. mira and P. amygdalus decreased slowly as WL stress duration increased, reaching the lowest values on 27 d (Fig. 2C). Thus, WL treatment induced leaf chlorophyll decomposition in P. mira and P. amygdalus seedlings, leading to leaf yellowing (data not shown). However, P. persica leaves did not significantly change in chlorophyll content during WL treatment. 3.3. Activities of PDC, ADH, and LDH in Prunus roots In all species, PDC activity rose first before falling; in particular, P. amygdalus root PDC activity increased dramaticallyafter 3 d of WL treatment (Fig. 2D). As WL treatment continued, P. amygdalus root PDC activity decreased rapidly at 21 d. In P. mira and P. persica, root PDC 64
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Table 2 Leaf number of three peaches under waterlogging treatments. CK: Control; WL: Waterlogging treatment. Means ± SE, n = 6. Species
Treatment time (d) 0
3
6
9
15
21
27
P. mira
CK WL
10.1 ± 2.0 10.1 ± 2.0
11.5 ± 1.4 11.3 ± 1.9
13.1 ± 1.1 12.9 ± 2.1
14.7 ± 1.9 14.1 ± 2.6
18.5 ± 2.1 16.0 ± 3.1*
22.6 ± 3.3 18.6 ± 4.5*
25.9 ± 5.1 20.9 ± 6.5*
P. persica
CK WL
13.7 ± 0.6 13.7 ± 0.6
14.9 ± 1.1 14.7 ± 1.2
16.8 ± 0.9 16.3 ± 0.6
18.8 ± 0.8 18.0 ± 0.6
23.7 ± 1.7 22.7 ± 1.6
29.7 ± 3.1 26.7 ± 4.7*
33.6 ± 6.2 29.3 ± 8.4*
P. amygdalus
CK WL
15.0 ± 2.6 15.0 ± 2.6
16.3 ± 3.1 15.7 ± 3.1
17.8 ± 3.6 17.0 ± 3.6
19.0 ± 3.5 17.3 ± 3.5*
22.1 ± 3.2 18.3 ± 3.2*
23.9 ± 2.6 18.7 ± 2.6*
25.8 ± 4.0 18.9 ± 4.0*
* Indicates significant differences at P < 0.05, compared to the control plants.
activities were lower than in P. amygdalus roots early during WL treatment, but became significantly higher in the later stages except at 15 d. Specifically, P. mira PDC activity was higher than activities in the other two species at 9 d, whereas P. persica PDC activity became highest at 21 d. Overall, WL’s effects on PDC activity were strongest in P. amygdalus roots, causing rapid and dramatic fluctuations early and late in treatment. Under WL stress, root ADH activities in all species increased first before decreasing, and from 0 to 9 d, no significant differences were observable. However, at 0.641 μmol mg−1 protein min−1 after 21 d, P. persica root ADH activity was 18.4 times higher than its activity at 0 d; it was also significantly higher than activity in P. mira or P. amygdalus during 15–27 d. In the latter two species, ADH activities dramatically increased during 0–6 d and 0–9 d, respectively, but were relatively low at later treatment stages (Fig. 2E). After WL for 6 d, LDH activities in all three peaches also rose first before decreasing (Fig. 2F). Root LDH activities of in P. mira and P. persica were significantly lower than activity in P. amygdalus. Moreover, this significant difference was sustained from 9 d to 27 d of WL treatment (Fig. 2F). Our data suggest that the lactic-acid pathway in P. amygdalus seedlings was regulated under WL, resulting in lactic acid over-accumulation during the early experimental stage and inducing cytoplasmic acidification. Root PDC and ADH activities were positively correlated with each other, as well as with LDH activity (Fig. 3).
4. Discussion Previous reports have indicated a negative correlation between leaf damage and the capacity of WL tolerance in plants (Tuo et al., 2015). In this study, P. amygdalus leaves began to drop after 18 d of WL, P. mira to a lesser extent after 24 d, whereas P. persica did not lose leaves (Fig. 1). Furthermore, WL decreased the chlorophyll content in all peach leaves except those of P. persica, but with P. mira having consistently higher chlorophyll content than P. amygdalus (Fig. 2C). Together, these data indicate that P. persica was the most tolerant to WL, followed by P. mira and P. amygdalus. Under WL stress, fruit trees grow more slowly and exhibit dwarf morphology, as well as decreased root dry weight (Schaffer et al., 1992). Our results corroborate those previous findings: under WL treatment, the more WL-tolerant P. mira and P. persica had similar morphologies, with significantly higher growth rates and rates of increase in leaf number than P. amygdalus (Tables 1 and 2). Because oxygen diffusion rates in water is ten thousand times lower than in air (Armstrong and Drew, 2002), plants experience insufficient oxygen supply and severely weakened aerobic respiration under WL conditions, leading to lowered ATP and NAD+ production (Drew, 1997). Thus, WL-related plant damage is indirect (Dennis et al., 2000), rather than direct damage from excess water (Voesenek et al., 2006). Plants respond to WL stress through a series of adaptation mechanisms, including the activation of anaerobic respiration enzymes
Fig. 3. Correlations between pyruvate decarboxylase (PDC), alcohol dehydrogenase (ADH), and lactate dehydrogenase (LDH) activities in Prunus roots.
(Armstrong and Drew, 2002). The anaerobic fermentation pathway is a short-term adjustment to temporarily produce enough ATP and NAD+ that allows maintaining plant cell function (Good and Crosby, 1989a, 1989b). Previous studies on hypoxia stress in plants showed that PDC, ADH, and LDH activities markedly increased in rice, wheat, maize, and Arabidopsis thaliana roots (Gonzali et al., 2005; Zhang et al., 2015). In this study, anaerobic respiration enzymes in all three tested species increase the activity at the beginning, decreasing later during the treatment period. Our data support the hypothesis that the activation of anaerobic respiration enzymes in plant roots is a positive mechanism to 65
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Fig. 4. Hypotetical pathways of anaerobic respiration under waterlogging conditions in P. persica (L.) Batsch (A) and P. amygdalus (L.) (B). ADH, alcohol dehydrogenase; LDH, lactate dehydrogenase; PDC, pyruvate decarboxylase.
significantly in wheat plants with weak anti-hypoxic capacity (Hossain and Uddin, 2011), and delays to peak LDH activity positively affects cherry-root response to WL stress (Cheng et al., 2008). This research is consistent with our findings: under early WL stress, P. amygdalus LDH activity increased rapidly and remained high throughout the experiment, likely leading to lactic acid accumulation and inducing earlier cytoplasmic acidification in P. amygdalus than in P. mira and P. persica. As a result, P. amygdalus quickly experienced the symptoms of WL damage, including shed leaves, slow plant growth, and chlorophyll degradation (Tables 1, 2 and Fig. 2). Our results illustrate that interspecific differences among Prunus germplasms were responsible for fluctuations in PDC, ADH, and LDH activities; which may lead to the discrepancies in secondary metabolite accumulation emerged across the three species, causing variation in WL tolerance. We suggest that P. persica may generate more energy anaerobically through EMP than through the lactic-acid metabolism pathway, whereas P. amygdalus was the reverse; this difference affected the latter species’ root PDC and ADH activities; and P. mira was intermediate. Our data are also consistent with the hypothesis that cytosolic pH shifts act as a switch from one metabolic route to the other in WL conditions. Under WL stress, pH may fall below 7 (LDH optimum), into the optimal range for PDC, where PDC can convert pyruvate to acetaldehyde, which is subsequently converted to ethanol by ADH (Dat et al., 2004; Xu et al., 2016). This hypothesized mechanism could explain the diverse behavior of Prunus germplasm to flooding conditions as summarized in Fig. 4.
mitigate energy deficiency under WL stress. The overexpression of PDC may enhance plant resistance to hypoxia stress, because PDC is a key enzyme that catalyzes pyruvate-toacetaldehyde conversion in the ethanol metabolism pathway (EMP) (Shiao et al., 2002; Kato-Noguchi and Morokuma, 2007; J.Y. Zhang et al., 2016). In this study, PDC activities in all tested plants roots were consistent during 0–15 d of WL treatment, but subsequently, P. amygdalus experienced a rapid decrease in root PDC activity, likely the result of damage to root cells. In contrast, PDC activities remained relatively high in P. mira and P. persica roots (Fig. 2D), suggesting WL damaged P. mira and P. persica roots less than P. amygdalus roots. Changes to ADH activity should also affect WL-tolerance potential in plants (Xu et al., 2014). In Arabidopsis thaliana and Zea mays L. adh1 mutants, shifts in ADH activity was essential for resistance to WL stress (Johnson et al., 1994). Furthermore, hypoxia-tolerant cucumber cultivars exhibited higher ADH activity than control (Kang et al., 2008), accelerating the transformation of acetaldehyde to ethanol. In Puccinellia stricta, Isolepis cernua, Samolus repens, Selliera radicans, and Triglochin striata, shoot and root ADH activity significantly increased in response to WL (Brownstein et al., 2013). Our results supported these previous findings suggesting that ADH increase is an adaptive response to anoxia stress, as we demonstrated that P. mira and P. persica ADH activities significantly increased in later stages of WL treatment. The underlying mechanism of benefits from ADH increase is probably linked to the heightened production of ATP and NAD+ for maintaining plant cellular function, as well as the catalysis of harmful acetaldehyde to ethanol (Geigenberger, 2003). Ethanol is harmless to plants because it easily diffuses through cell membrane’s lipid bilayer to the external environment (Perata and Alpi, 1993), which is a byproduct of ADH activity, high ethanol concentrations is a positive indicator of plant defense against oxygen deficiency (Bailey-Serres and Chang, 2005; Agarwal and Grover, 2006). In this study, ADH activity in P. amygdalus roots were low and did not fluctuate much (Fig. 2E). Some data suggest that ADH is not a major limiting factor for ethanol fermentation; instead, PDC activity is the key limiting factor (Mohanty and Ong, 2003; Catherine et al., 2006). In our study, ADH activity in P. persica roots was significantly higher than in the other two species at 15–27 d of WL treatment (Fig. 2E). Therefore, P. persica appeared to have higher WL tolerance than P. amygdalus and P. mira, as the former was more capable than the latter two of removing acetaldehyde under WL stress. The dynamics of ADH activity in the three species was consistent with their WL tolerance, suggesting that when PDC activity remains generally constant, ADH is important to the decrease of harmful metabolites in Prunus roots (Fig. 4). Under hypoxia stress, increased LDH activity leads to lactic acid accumulation and cytoplasmic acidification (Ismond et al., 2003). Under hypoxic conditions such as WL and flood damage, pH reduction is the main cause of plant death. Under WL, LDH activity increases
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