Ethylene signaling involves in seeds germination upon submergence and antioxidant response elicited confers submergence tolerance to rice seedlings
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Flooding has negative impact on agriculture. The plant hormone ethylene is involved in plant growth and stress responses, which are important role in tolerance and adaptation regulatory mechanisms during submergence stress. Ethylene signaling crosstalk with gibberellin signaling enhances tolerance in lowland rice (Flood Resistant 13A) through a quiescence strategy or in deepwater rice through an escape strategy when rice is submerged. Information regarding ethylene-mediated priming in submergence stress tolerance in rice is scant. Here, we used 1-aminocyclopropane-1-carboxylic acid, an ethylene precursor, to evaluate the response in submerged rice seedlings.
The germination rate and mean germination times of rice seeds was higher in seedlings under submergence only when ethylene signaling was inhibited by supplemented with silver nitrate (AgNO3). Reduced leaf chlorophyll contents and induced senescence-associated genes in rice seedlings under submergence were relieved by pretreatment with an ethylene precursor. The ethylene-mediated priming by pretreatment with an ethylene precursor enhanced the survival rate and hydrogen peroxide (H2O2) and superoxide (O2−) anion accumulation and affected antioxidant response in rice seedlings.
Pretreatment with an ethylene precursor leads to reactive oxygen species generation, which in turn triggered the antioxidant response system, thus improving the tolerance of rice seedlings to complete submergence stress. Thus, H2O2 signaling may contribute to ethylene-mediated priming to submergence stress tolerance in rice seedlings.
KeywordsRice Submergence Ethylene Reactive oxygen species Antioxidant enzyme activity
Reactive oxygen species
Reverse transcriptase-polymerase chain reaction
Severe climate-related disasters include flooding due to increased frequency of heavy rain. Flooding affects agriculture causing outright crop yield losses. The term flooding comprises both waterlogging and submergence. The pore space in the soil is filled with water when soil is under excess water stress; this decreases soil oxygen levels, limits gas diffusion and soil nutrient effusion, and impairs plant growth and development (Nishiuchi et al. 2012). With increased submergence duration, tiller number, green leaves number, and dry weight of rice decreases. The survival and growth of rice are severely affected by submergence (Reddy and Mittra 1985; Gautam et al. 2017; Wu and Yang 2016).
Unfavorable conditions activate phytohormonal signals in plants, in turn enhancing their tolerance to environmental stress. The gaseous plant hormone ethylene mediated developmental processes and stress tolerance, such as seed germination, senescence, and stress responses (Yu et al. 2017; Xia et al. 2015; El-Maarouf-Bouteau et al. 2015). Ethylene signaling-induced mitogen-activated protein kinase (MAPK) cascades can be activated with the application of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) in Medicago and Arabidopsis (Ouaked et al. 2003). Under salt stress, ethylene can activate the MAPK cascade and enhance reactive oxygen species (ROS) generation (Teige et al. 2004). The interplay of ethylene signaling and ROS production activates the antioxidant defense system for flooding responses in rice (Steffens 2014; Yang and Hong 2015).
Several studies have reported that ethylene is crucial against hypoxia signal-inducing flooding stress. Aerenchyma formation can be induced in maize roots by applying ethylene in flooded conditions (Rajhi et al. 2011). Ethylene signaling triggers the process of programmed cell death resulting in ethylene-responsive lysigenous aerenchyma formation (Guo et al. 2015; Muhlenbock et al. 2007; Chen et al. 2002). Two key ethylene biosynthesis enzymes, 1-aminocyclopropane-1-carboxylic acid synthase (ACS) and 1-aminocyclopropane-1-carboxylic acid oxidase (ACO), are involved in plant response to hypoxia stress. ACS converts S-adenosylmethionine (AdoMet) into ACC and the byproduct 5′-methylthioadenosine; then, ACO converts ACC to ethylene, thus increasing the ethylene levels (Rzewuski and Sauter 2008; Adams and Yang 1979). In this study, to further clarify the role of ethylene signaling during submergence, we used ACC, an ethylene precursor, to evaluate the response in submerged rice seedlings.
Submergence-induced germination inhibition was alleviated after ethylene signaling was blocked
Survival rate was enhanced after pretreatment with ethylene precursor in rice seedlings subjected to submergence stress
Reduced leaf chlorophyll contents and induced senescence-associated genes in rice seedlings under submergence were alleviated after ethylene precursor pretreatment
Pretreatment with an ethylene precursor lead to hydrogen peroxide and superoxide ion accumulation and affected antioxidant response during submergence
The activities of antioxidative enzymes, namely catalase (CAT), ascorbate peroxidase (APX), superoxide dismutase (SOD), and total peroxidase (POX), were then determined after complete submergence with or without ACC pretreatment. The CAT, SOD, and APX activities decreased under submergence stress with or without ACC pretreatment. However, SOD activity significantly decreased after ACC pretreatment under submergence. POX activity increased under submergence stress; it was particularly higher under submergence after ACC pretreatment (Fig. 5b). Thus, pretreatment with an ethylene precursor may affect intracellular redox homeostasis and antioxidant systems under submergence stress.
The plant hormone ethylene plays important roles in plant adaptation to submergence stress. It is the principal factor initiating fast underwater elongation of leaves or stems—the so-called escape strategy in deepwater rice (Hattori et al. 2011). In lowland rice Flood Resistant 13A, it is elicits a quiescence strategy based on suppression of elongation to avoid energy consumption during flash flooding (Manzur et al. 2009). Studies presented that some QTLs (quantitative trait loci) associated with tolerance of flooding during germination have been identified that revealed ABA and GA involved in submergence tolerance during germination (Miro and Ismail, 2013). The role of ethylene in priming during submergence stress remains ambiguous. To further understand the effects of ethylene-mediated priming on tolerance and antioxidant response to submergence stress, we used an ethylene precursor ACC to investigate change in physiological and molecular responses in rice seedlings under submergence stress. Studies have revealed the roles of ethylene in the release of primary and secondary dormancy and the germination of nondormant seeds under normal and stressed conditions in many plant species (Petruzzelli et al. 2000; Kepczynski and Kepczynska 1997); poor germination is a feature of the Arabidopsis ethylene-insensitive mutant (Johnson and Ecker 1998). Waterlogging or submergence causes a rapid decline in dissolved oxygen concentrations in the soil water, thus resulting in seed germination failure and lengthening the germination time in pea, oak, and lupin seeds (Sarlistyaningsih et al. 1995; Perez-Ramos and Maranon 2009; Jackson and Hall 1987). In the current study, the germination rate of TK9 rice seeds considerably decreased but their MGT considerably increased under the Sub condition compare with that under the Nor condition (Fig.1 a and b). However, germination inhibition was partially disrupted under submergence in Sub + AgNO3-treated seedlings because ethylene signaling was inhibited through blocking ethylene perception with silver ions (Fig. 1 a and b). Therefore, the results imply that in addition to ethylene signaling, other pathways are involved in the regulation of seed germination under submergence stress. Studies indicated that silver ions affects not only ethylene signaling may also auxin efflux to affect root elongation (Strander et al., 2009). Whether the auxin signaling involved in the regulation of seed germination under submergence stress need further research.
Complete submergence of rice plants can severely delay physiological responses, retard growth and development, reduce yield, and even cause death (Jackson and Ram 2003; Yang et al. 2017). We demonstrated that the survival rate of rice seedlings can be improved under complete submergence stress through ACC pretreatment (Fig. 2). Moreover, under submergence, chlorophylls b and total chlorophyll contents could be maintained by ACC pretreatment (Fig. 3). Under complete submergence, ACC-pretreated seedlings demonstrated lower SAG mRNA expression than did untreated seedlings (Fig. 4). Thus, ethylene-mediated priming has senescence inhibition-associated positive effects on plant submergence tolerance.
ROS plays a key role in signal transduction in cells (Mittler et al. 2011). Homeostatic regulation of ROS–antioxidant interactions in plant cells confers increased to environmental stress tolerance to the plants. Cellular antioxidants influence plant growth and development by modulating processes from cell division and cell elongation to senescence and death (Foyer and Noctor 2005). In plants, complex intracellular mechanisms regulate the ROS production and scavenging, particularly under stress. In our study, H2O2 accumulation and POX activity increased under submergence with ACC pretreatment compared with that under submergence alone (Fig. 5). These results may imply that H2O2 signaling contributes to ethylene-mediated priming on submergence tolerance in rice seedlings. Taken together, this study demonstrated that ACC pretreatment trigger positive priming mechanism to increase plant tolerance to submergence.
In conclusion, our results demonstrated that the germination rates of rice seeds under submergence partially increased after ethylene signaling was inhibited. In rice seedlings, ethylene-mediated priming through pretreatment with an ethylene precursor modulated leaf chlorophyll content and SAG expression, enhanced survival, increased H2O2 and O2− accumulation, and reduced antioxidant response was affected by. Thus, seed germination and rice seedling tolerance can be improved under complete submergence by modulating ethylene signaling because ethylene-mediated priming affects senescence induction and ROS and antioxidant response conferring submergence tolerance to rice. Whether this regulatory mechanism can crosstalk with other pathways remains unclear and merits further study.
Materials and methods
Plant materials and growth conditions
TK9 rice (Oryza sativa Japonica) was used in this study. Rice seeds were sterilized by dipping in 3% sodium hypochlorite solution for 30 min, followed by gentle washing with distilled water for at least four to five times. The sterilized seeds were subsequently placed on a wet filter paper for 3 days at 28 °C under a 16-h light–8-h dark cycle in a growth chamber. The germinated seeds were transferred onto a metal grid placed over a 500-mL beaker containing Kimura B medium for growth. For the seed germination assay, data were collected from 30 seeds for each treatment in three independent experiments. Seeds were placed under Nor, in 4.5-cm-deep water in a water tank (Sub), or in 4.5-cm-deep water containing 10 μM AgNO3 in a water tank (Sub + AgNO3) for pretreatment for 2 days; then, the seeds were transferred onto a wet filter paper for germination. Germination was confirmed when the radicles were 1 mm long. Germination percentage was recorded every 24 h for 7 days. The number of germinated seeds was expressed as a percentage of the total number of seeds plated for the indicated periods. MGTs were calculated to assess the time required for germination (Matthews and Khajeh-Hosseini 2007).
Seedlings submergence, ACC treatment, and survival rate determination
For submergence treatment, 8-day-old seedlings were completely submerged with or without 10 μM ACC pretreatment for 2 days, followed by transferring into a water tank (40 × 40 × 60 cm3) with 55-cm-deep water for 4, 6, 8, and 10 days under a 16-h light–8-h dark cycle. Under Nor, seedlings were placed under normal condition for the indicated periods. The water was drained out for subsequent 10-day recovery. The ability to grow new leaves after 10-day recovery was considered the measure of survival. Experiments were repeated three times, and at least 30 seedlings were measured independently each time. After each treatment, samples tissues were immediately frozen in liquid nitrogen and stored at − 80 °C for further assay.
Plant chlorophyll content measurements and qRT-PCR analyses
For chlorophyll content assay, the 8-day-old seedlings were treated under Nor and Sub with and without 10 μM ACC pretreatment for 2 days followed by submergence for 6, 8, and 10 days. Above-ground tissue (50 mg) was collected and ground in 2 mL of sodium phosphate buffer (50 mM, pH 6.8); 40 μL of this solution was added to 960 mL of 99% ethanol and incubated for 30 min at room temperature in the dark with gentle shaking. After centrifugation at 4 °C for 15 min at 1000 g, the absorbance of the supernatant was measured at 665 and 649 nm using a spectrophotometer (Metertec SP8001) for determining chlorophyll a and b and total chlorophyll contents. The values were collected from three biologically independent experiments.
Primers used for quantitative RT-PCR experiments
Osl85 - forward
5′-catgggcaaaggagttactgaagag − 3′
Osl85 - reverse
5′-ggatttggcaagaacatggctg − 3’
OsRCCR1 - forward
5′-gcaccttctcactgacagcaatac − 3’
OsRCCR1 - reverse
5′-accacgcactatctcttccaagg − 3’
Osubiquitin - forward
Osubiquitin - reverse
OsCAO1 - forward
OsCAO1 - reverse
OsHEMA1 - forward
OsHEMA1 - reverse
OsNYC1 - forward
OsNYC1 - reverse
OsNOL - forward
OsNOL - reverse
Histochemical staining and antioxidative enzyme activity assay
The detached leaves of 8-day-old rice seedlings were treated under Nor, Sub, and submergence supplemented with 10 μM ACC (Pre ACC + Sub) for 2 days and then submerged for 4 days. O2− and H2O2 accumulation in cells was observed through the NBT and DAB staining methods, as previously described (Yang and Hong 2015). The results were obtained from three independent experiments. For the antioxidative enzyme assay, shoot tissue (50 mg) was excised and immediately used for enzyme extraction. The levels of CAT, APX, POX, and SOD activity were analyzed as previously described (Wu and Yang 2016). Each experiment was repeated three times.
This manuscript was edited by Wallace Academic Editing.
This work was supported by a National Science Council grant (NSC 101–2311-B-005-001) to Chin-Ying Yang.
Availability of data and materials
All data generated or analysed during this study are included in this published article.
Y-C Huang and T-H Yeh conducted experiments and analyzed the data. Dr. C-Y Yang conceived, designed research, and wrote the manuscript. All authors read and approved the final manuscript.
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Consent for publication
The authors declare that they have no competing interests.
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