CIPK15-mediated inhibition of NH₄⁺ transport protects Arabidopsis from submergence

Abstract

Ammonium (NH₄⁺) serves as a vital nitrogen source for plants, but it can turn toxic when it accumulates in excessive amounts. Toxicity is aggravated under hypoxic/anaerobic conditions, e.g., during flooding or submergence, due to a lower assimilation capacity. AMT1; 1 mediates NH₄⁺ uptake into roots. Under conditions of oxygen-deficiency, i.e., submergence, the CBL-interacting protein kinase OsCIPK15 has been shown to trigger SnRK1A signaling, promoting starch mobilization, thereby the increasing availability of ATP, reduction equivalents and acceptors for NH₄⁺ assimilation in rice. Our previous study in Arabidopsis demonstrates that AtCIPK15 phosphorylates AMT1; 1 whose activity is under allosteric feedback control by phosphorylation of T460 in the cytosolic C-terminus. Here we show that submergence cause higher NH₄⁺ accumulation in wild-type, plant but not of nitrate, nor in a quadruple amt knock-out mutant. In addition, submergence triggers rapid accumulation of AtAMT1;1 and AtCIPK15 transcripts as well as AMT1 phosphorylation. Significantly, cipk15 knock-out mutants do not exhibit an increase in AMT1 phosphorylation; however, they do display heightened sensitivity to submergence. These findings suggest that CIPK15 suppresses AMT activity, resulting in decreased NH₄⁺ accumulation during submergence, a period when NH₄⁺ assimilation capacity may be impaired.

1. Introduction

Nitrogen is a fundamental nutrient for all living organisms, as it plays a crucial role in various vital macromolecules, including nucleic acids, amino acids, and proteins. In the context of plants, nitrogen availability can significantly influence growth, development, and crop yield, either by being excessive or insufficient, outside the optimum range. In most soils, nitrogen is mainly present in inorganic forms (as nitrate, NO₃⁻, and as the ammonium ion, NH₄⁺). NH₄⁺ serves as a crucial nitrogen source for bacteria, fungi, and plants. However, it can become toxic when its concentration exceeds certain levels. In many conditions, plants preferentially take up NH₄⁺ over NO₃⁻ [[1], [2], [3], [4], [5]]. Average NH₄⁺ levels in the soil are often 10 to 1000 times lower than NO₃⁻ levels [2]. NH₄⁺ availability and distribution vary both spatially and temporally. Elevated NH₄⁺ levels can be toxic, although the sensitivity to NH4+ varies among different plant species [6]. Soil conditions also affect the nitrogen pool and nitrogen availability.

Under hypoxic conditions, such as flooding or submergence, the conversion of NO₃⁻ to NH₄⁺ (nitrification) is blocked, leading to NH₄⁺ accumulation in the soil [7,8]. At the same time, flooded roots are deprived of oxygen, limiting ATP production and nitrogen assimilation, which then impairs the conversion of NH₄⁺ to glutamine, aggravating NH₄⁺ toxicity. Ethylene plays a crucial role in acclimation to flooding which causes hypoxia [9]. Interestingly, ethylene and NH₄⁺ toxicity appear to be tightly linked, as seen when treatment of tomatoes grown in solution with ethylene inhibitors prevents NH₄⁺ toxicity [10]. NH₄⁺ accumulation and ethylene biosynthesis appear to be indicators of stress and may play roles in the development of nutritional stress symptoms. Ethylene evolution is preceded by NH₄⁺ accumulation but is detected before or at the same time as toxicity symptoms.

Plants utilize specific transporters to uptake NH₄⁺. The transport of NH₄⁺ is electrogenic and relies on high-affinity NH₄⁺ transporters from the AMT/MEP/Rhesus protein superfamily [[11], [12], [13], [14]]. Within the Arabidopsis genome, six AMT paralogs exist, with four of them (AMT1; 1-1; 5) playing an essential role in NH₄⁺ uptake [15,16]. Besides their function as transporters, AMTs also act as receptors, influencing root growth and development, similar to the yeast MEP2 transceptor that regulates pseudohyphal growth based on NH₄⁺ concentrations [17,18]. The activity of AMTs is under strict regulation, subject to feedback inhibition to prevent NH₄⁺ accumulation to toxic levels [19,20]. Previous studies have demonstrated that NH₄⁺-triggered phosphorylation of a critical threonine (T460) in the cytosolic C-terminus of AMT1; 1 leads to transport inhibition through allosteric regulation within the trimeric transporter complex [21,22]. This C-terminal domain of AMT1; 1, acting as an allosteric switch, is highly conserved among AMT homologs across species, from bacteria to higher plants [12,13,23]. Utilizing this allosteric regulation mechanism of AMT1; 1 for feedback control enables plants to rapidly and efficiently block NH₄⁺ uptake before reaching toxic levels [21,22]. However, the complete pathway responsible for NH₄⁺-dependent phosphorylation of AMTs remains incompletely understood. It is speculated that specific kinases may be activated during submergence to restrict NH₄⁺ accumulation.

In our recent comprehensive investigations using Xenopus oocytes, we have discovered the Arabidopsis protein kinase CIPK15 as a direct interactor that exerts a negative influence on AMT1 activity, thereby serving as an AMT regulator [24]. This inhibitory impact of CIPK15 on AMT1 activity was further validated using an NH4+ transporter activity sensor, AmTryoshka1; 3 LS-F138I, a genetically encoded biosensor with ratiometric capabilities in yeast [25]. It is worth noting that members of the CIPK kinase family are well-known for their ability to regulate the activities of various transporter types [22,[26], [27], [28], [29], [30], [31]]. CIPK15 from rice (also named PKS3/SnRK3.1) has been proposed to be a key regulator of the sensing cascade for successful rice germination under flooding [32]. Under oxygen-deprived conditions, OsCIPK15 triggers both accumulation of the Snf1-related protein kinase 1, SnRK1A, and SnRK1A-dependent signaling to promote starch degradation [[32], [33], [34]]. CIPK15 also appears to play a role in abscisic acid (ABA) signaling, which is also linked to sugar signaling [[35], [36], [37], [38]]. In this study, we demonstrate that Arabidopsis CIPK15, a close homolog of OsCIPK15 functions as a negative regulator of AMT1 activity during submergence conditions. The NH₄⁺ content in Col-0 but not qko mutant, which is the quadruple AMT1 transporter mutant, was largely increased under submergence condition. The transcripts of CIPK15 and AMT1;1 were largely increased under submergence condition. cipk15 knock-out mutants were hypersensitive to submergence condition as showing the NH₄⁺ toxicity symptoms. Furthermore, we observed a significant increase in the phosphorylation level of AMT1 under submergence conditions, but this effect was absent in cipk15 mutants. Collectively, these findings strongly suggest that during submergence, CIPK15 actively suppresses AMT activity to hinder excessive NH₄⁺ accumulation when the NH₄⁺ assimilation capacity is compromised.

2. STAR methods

Plant Material and Treatments.Arabidopsis thaliana ecotype Columbia-0 was used in this study, and T-DNA insertion lines for CIPK15, namely cipk15-1 (SALK_203,150) and cipk15-2 (GK604B06), were acquired from the Arabidopsis Biological Resource Center (http://www.arabidopsis.org/abrc/). The identification of AMT-qko, the quadruple AMT knock out line, and cipk15 knock-out line has been described [24,39]. Wild-type plants were Arabidopsis thaliana ecotype Columbia-0. Plant growth conditions for qRT-PCR analyses and protein blot assay have been described previously, here used with modifications [22] for submergence. Arabidopsis seeds underwent surface sterilization and were germinated on 0.5% modified Murashige Skoog medium (MS), nitrogen-free salts (Phytotechlab, M407), with 5 mM KNO₃ as the exclusive nitrogen source. The medium also contained 0.5% [w/v] sucrose and 1% [w/v] agar, with a pH of 5.8 [KOH], and the plates were kept in a vertical orientation. Seedlings were incubated in a 16-/8-h light/dark period at 22 °C. For submergence experiments, we followed the protocol from the Bailey-Serres's lab [40] with modifications. Briefly, seedlings grown in vertical orientation for 7 d on solid 0.5% MS medium containing 0.5% (w/v) sucrose were submerged under 1 mM NH₄Cl or 1 mM KNO₃ medium in a dark, 22 °C climate room. After 13 h of submergence, sample of seedlings or roots was collected after the indicated treatments and frozen in liquid nitrogen for qRT-PCR, protein blot experiments, and NH₄⁺ level measurements, or seedlings were returned to aerobic conditions on agar plates at pH 6 with 1 mM NH₄Cl or 1 mM KNO₃ as the sole nitrogen source to observe post submergence recovery and for NH₄⁺ level measurements and image scan. Seedlings were scanned on a flatbed scanner.

Real-Time qRT-PCR analyses. Real-time qRT-PCR was performed as described [24,27]. In brief, primers were designed to achieve a melting temperature (Tm) of approximately 60 °C and generate PCR products of around 200−400 base pairs. For each experiment, expression levels were initially standardized by comparing them to the expression of Ubiquitin10, measured from the same cDNA samples. The primer sequences used were as follows: AMT1;1, forward primer: 5′- ACGACATTATCAGTCGC; reverse primer: 5′-CTGTCCTGTGTAGATTAACG; and CIPK15, forward primer: 5′-GGCTACGCATCTGACT; reverse primer: 5′-CGTGCAAGCGACTATC; Ubiquitin10, forward primer: 5′-CTTCGTCAAGACTTTGACCG; reverse primer: 5′-CTTCTTAAGCATAACAGAGACGAG.

Extraction of membrane fractions and protein gel blot analyses. For plasma membrane extraction, roots were ground in liquid nitrogen and then resuspended in a buffer containing 250 mM Tris-Cl (pH 8.5), 290 mM sucrose, 25 mM EDTA, 5 mM β-mercaptoethanol, 2 mM DTT, 1 mM phenylmethylsulfonyl fluoride (PMSF), 0.53 mM Complete Protease Inhibitor Cocktail (Sigma-Aldrich), and 0.53 mM PhosStop Phosphatase Inhibitor Cocktail (Roche Applied Science). The samples were centrifuged at 10,000×g for 15 min, and the resulting supernatants were filtered through Miracloth (Calbiochem) and recentrifuged at 100,000×g for 45 min. The microsome-containing sediment was resuspended in a storage buffer consisting of 400 mM mannitol, 10% glycerol, 6 mM MES/Tris (pH 8), 4 mM DTT, 2 mM PMSF, and 13 mM phosphatase inhibitor cocktails 1 and 2 (Sigma-Aldrich). To denature the proteins, they were incubated in loading buffer (62.5 mM Tris-HCl, pH 6.8, 10% [v/v] glycerol, 2% [w/v] SDS, 0.01% [w/v] bromophenol blue, and 1% PMSF) at 37 °C for 30 min, with or without 2.5% [v/v] β-mercaptoethanol at 0 °C. Electrophoresis was then performed using 8–20% SDS polyacrylamide gels (Invitrogen), and the proteins were subsequently transferred to polyvinylidene fluoride membranes. Detection of proteins was accomplished using either the anti-AMT1; 1 antibody or the anti-P-AMT1 T460 antibody [22]. Blots were developed using an ECL Advance Western Blotting Detection Kit (Amersham). Protein and phosphorylation levels were quantified using ImageJ software.

3. Results

3.1. Submergence causes NH₄⁺ accumulation

NH₄⁺ accumulation has been observed in a variety of plants. To test whether submergence also causes NH₄⁺ accumulation in Arabidopsis, wild-type Col-0 7-day-old seedlings were submerged under medium in the presence of either NH₄⁺ or NO₃⁻ for 13 h in dark. Submergence led to a significant increase in NH₄⁺ levels in wild-type roots when NH₄⁺, but not NO₃⁻, was present (Fig. 1A). To evaluate if NH₄⁺ accumulation under submergence involved only endogenous accumulation due to reduced assimilation, or if NH₄⁺ uptake from the medium also played a role, NH₄⁺ levels in seedlings of a quadruple knock-out AMT mutant (qko), which carries T-DNA insertions in AMT1;1, 1;2, 1;3, and 2;1, were measured. Submergence did not cause an increase of NH₄⁺ levels in the qko mutant in the NH₄⁺ condition (Fig. 1B), implicating AMT-mediated NH₄⁺-uptake in the elevation of NH₄⁺ levels during submergence.

Fig. 1.

Submergence increased NH₄⁺accumulation in Col-0. NH₄⁺ concentrations were measured in Col-0 (A) roots under submergence condition in 1 mM NH₄⁺ or 1 mM NO₃⁻ and in qko (B) roots under submergence in 1 mM NH₄⁺. Comparable results were obtained in six independent experiments (each experiment n > 12–15, total n > 80). * Indicates p < 0.01 compared to Col-0 before submergence (Student's t-test).

3.2. Submergence and NH₄⁺-induced CIPK15 mRNA accumulation

In roots, CIPK15 governs the NH4+ uptake activity of AMT1; 1 [24]. Based on the role of the rice homolog in hypoxia, we hypothesized that the Arabidopsis CIPK15 may also play a role under submergence conditions, and thus affect NH₄⁺ uptake. During submergence, CIPK15 could undergo regulation at the transcriptional level or activation, which enables it to control AMT activity. Using public database searches (eFP browser) and qRT-PCR analyses, we found that AMT1;1 transcript levels were elevated in Arabidopsis roots and leaves after flooding [36]. (Fig. 2 and Supplementary Figs. S1–3). In response to submergence, levels of CIPK15 transcript increased about five-fold in Col-0 and the qko mutant (Supplementary Fig. S2). The data strongly support the idea that both AMT1; 1 and CIPK15 are involved in the response to submergence.

Fig. 2.

Submergence-triggered CIPK15 mRNA accumulation. qRT-PCR analyses of AMT1;1 and CIPK15 mRNA levels in roots before and after submergence in 1 mM NH₄⁺. Levels were normalized to UBQ10 (mean ± SE for four independent experiments. (Each experiment n > 50, total n > 200). * Indicates p < 0.01 for mRNA levels of AMT1;1 and CIPK15 either after submergence compared to before submergence (Student's t-test).

3.3. CIPK15 is a key factor in flooding tolerance in arabidopsis

We show here that NH₄⁺ accumulates in seedlings during submergence (Fig. 1). To investigate whether CIPK15 regulates NH₄⁺ toxicity by modulating AMT1; 1 transport activity during submergence, cipk15 mutants and wild-type plants were assessed under submerged conditions. Following submergence, cipk15 mutant seedlings displayed heightened sensitivity to NH₄⁺ but not NO₃⁻, and exhibited reduced fresh weight compared to the wild-type (Fig. 3A–B and 3D). Additionally, NH₄⁺ levels were significantly higher in cipk15 mutants compared to wild-type controls under NH₄⁺ conditions, while no such difference was observed under NO₃⁻ conditions (Fig. 3C and E). These findings collectively suggest that CIPK15 is crucial for restricting NH₄⁺ accumulation during submergence.

Fig. 3.

cipk15 mutants are sensitive to submergence and high NH₄⁺accumulation under NH₄Cl treatment. Stratified seeds grown in the presence of 0.5% (w/v) sucrose on vertically oriented plates under a diurnal light cycle for 7 d and submerged into water in the present of NH₄⁺ (A–C) or NO₃⁻ (D–E) for up to 13 h. (A, D) Representative before (pre-submergence) and after (after-submergence) seedlings following recovery (2 days), (B) fresh weight, and (C, E) NH₄⁺ concentration in roots of seedlings before and after under submergence in the present of NH₄⁺ or NO₃⁻ (each experiment n > 15, total n > 80) in wild-type Col-0 and two cipk15 knock-out mutants. * Indicates significant change (p < 0.01) compared to wild-type (Student's t-test).

3.4. Upon submergence, CIPK15 induces the phosphorylation of AMT1

CIPK15 has been demonstrated to interact directly with AMT1; 1, inhibiting its activity through phosphorylation of T460 in the cytosolic C-terminus. To investigate whether the restriction of NH₄⁺ accumulation during submergence is a result of AMT1; 1 activity inhibition by CIPK15, the phosphorylation status of T460 in AMT1; 1 was examined in both wild-type Col-0 and cipk15 mutant plants. Protein gel blots were incubated with either anti-AMT1; 1 or anti-P-T460 antibody, which recognizes the phosphorylation status of T460 in vivo [22]. Following submergence and exposure to NH₄⁺, the protein levels of AMT1; 1 in both wild-type and cipk15 mutants remained similar compared to the pre-submergence condition. However, in wild-type Col-0, AMT1; 1 phosphorylation exhibited a significant increase, while this effect was not observed in cipk15 mutants (Fig. 4A–B). These findings provide strong evidence that submergence triggers CIPK15-mediated phosphorylation of AMT1; 1 at position T460, which subsequently hinders AMT1; 1 activity, preventing the accumulation of NH₄⁺ to toxic levels.

Fig. 4.

AMT1;1-T460 phosphorylation is increased under submergence and is reduced in cipk15 mutant plants. Before and after submergence of Col-0 seedlings (A) and seedlings after submergence (B) in present of NH₄⁺, membrane proteins were probed with anti-AMT1; 1 serum and anti-AMT1-P serum [22]. Ponceau S staining serves as the loading control. Corresponding result as shown in Supplementary Fig. S5.

4. Discussion

Our observations reveal that under submergence conditions, the activity of the triple-barreled NH₄⁺ transporters (AMTs) can be allosterically regulated through phosphorylation by CIPK15. We found evidence that this regulatory system can serve as a safety mechanism to prevent accumulation of NH₄⁺ to toxic levels under submergence condition. The conserved threonine (T460) in the cytosolic C-terminus of AMTs can undergo phosphorylation by CIPK15, enabling allosteric regulation [22,24,41]. In this regulatory system, some components are yet unknown, including a hypothetical NH₄⁺ receptor, a protein kinase that phosphorylates AMTs under submergence conditions, and the regulatory network that triggers AMT inactivation in conditions that otherwise would lead to NH₄⁺ overaccumulation. Here we demonstrated the submergence-triggered inhibition of AMT activity by CIPK15 to prevent overaccumulation of NH₄⁺ to toxic level. Under submergence condition, we found that NH₄⁺ content was highly accumulated in Col-0 in the present of NH₄⁺ but not qko mutant or in the present of NO₃⁻. Following submergence and exposure to NH₄⁺, the transcript levels of both AMT1;1 and CIPK15 were significantly increased. In the presence of NH₄⁺, cipk15 knock-out mutants displayed heightened sensitivity to submergence, exhibiting reduced fresh weights and elevated NH₄⁺ content compared to Col-0, underscoring the essential role of CIPK15 in protecting against NH₄⁺ toxicity during submergence. Furthermore, as CIPK15 can directly interact with and phosphorylate AMTs [24], its role in regulating AMTs under submergence is further supported. This is evident from the observation that submergence-induced phosphorylation of T460 in AMT1; 1 is impaired in cipk15 knock-out mutants, emphasizing the involvement of CIPK15 in AMT regulation during submergence.

Bacteria, fungi, and plants that live in soil are exposed to dramatically varying environmental conditions, including drought, rain, and even flooding/submergence, a condition that limits oxygen availability, prevents efficient respiration, and thus reduces ATP and NADH availability. Both ATP and NADH are essential for efficient NH₄⁺ assimilation. Submergence has also been reported to modify soil structure and nutrient dynamics, leading to crop losses [42,43]. Submergence of plants affects the concentration of nitrogen and nitrogen metabolism to improve survival and recovery from hypoxia by reducing the potential accumulations of toxic products [42,43]. In submerged/flooded conditions or hypoxic soil, one of the main transformations of nitrogen in soil is the accumulation of NH₄⁺, whereas, almost all the nitrate disappears in short period of time [[44], [45], [46], [47], [48]]. Conversion of NH₄⁺ to nitrate by nitrifying bacteria in soil or to amino acids in plant cell is inhibited due to lack of oxygen with hypoxia. Cytosolic NH₄⁺ levels rise during hypoxia, thus similar to elevated NH₄⁺ conditions, survival under hypoxic condition might also require efficient feedback inhibition of NH₄⁺ uptake. Here we show that NH₄⁺ increased in Arabidopsis roots during submergence. This concept of preventing toxic amount of NH₄⁺ accumulation in plant with hypoxia is also supported by the increasing the activity of glutamate synthase, which is the key enzyme to transfer NH₄⁺ to amino acids [44]. The increased sensitivity of cipk15 mutants to submergence supports a role of CIPK15 in protection from hypoxia, possibly through the inhibition of AMT activity.

Calcium (Ca²⁺) signals, widely recognized as a key regulatory signal for many processes in plant cells including plant adaptive responses to the environments, in cytosol is involved in triggering the hypoxia response [49]. Hypoxia causes a rapid elevation of the cellular Ca²⁺ concentration in many plants, including maize, rice, wheat, and Arabidopsis [50,51]. Moreover, elevated Ca²⁺ levels significantly influence the nitrogen metabolism and acquisition in hypoxia-stressed roots [44,52,53]. Recently, the Ca²⁺-sensing protein kinase OsCIPK15 was shown to play a central role in flooding tolerance in rice. CIPK15 activates the kinase SnRK1A, thereby connecting anaerobic signaling to SnRK1-mediated sugar-signaling. Activation of sugar metabolism would provide more carbon skeletons and more ATP, both of which are limited under hypoxic conditions [32,54,55]. CIPKs are known to affect the activity of many transporters in a reconstituted heterologous system. In our previous study, we found that the Arabidopsis CIPK15 is able to interact with and phosphorylate several AMT paralogs and that coexpression of CIPK15 with the AmTryoshka1; 3 LS-F138I biosensor and AMT1; 1 leads to inactivation of both the sensor and transporter. AMT phosphorylation was triggered by exposure to elevated levels of NH₄⁺ [24]. Here, we showed that Arabidopsis cipk15 mutants were more sensitive to NH₄⁺ under submergence condition compared to wild-type, implicating the kinase in general protection from NH₄⁺ toxicity, and were more sensitive to submergence.

Many interesting questions remain: (i) if CIPK15-mediated AMT phosphorylation requires ethylene or if the cytosolic NH₄⁺ and/or Ca²⁺ elevation during submergence are sufficient; (ii) if AMTs serve as NH₄⁺ sensors to help trigger phosphorylation during submergence; and (iii) if AtCIPK15 triggers SnRK activation to simultaneously increase availability of carbon skeletons and ATP to improve NH₄⁺ assimilation.

Another interesting question is if rice varieties that grow in flooded paddies and use NH₄⁺ as the predominant form of nitrogen also use CIPK15 for tolerance to submergence and NH₄⁺ toxicity. In Arabidopsis, CIPK15 can interact with CBL1/4 [56], while in rice, CIPK15 can interact with CBLs (CBL3-8) [38,57]. Although Arabidopsis CIPK15 is closely related to rice CIPK15, in the same clade [58], whether the two CIPK15 homologs are orthologs or participate in the same biotic and abiotic stress responses or use the same regulatory mechanisms is still unclear and will need to be further addressed. CIPK15 in rice promotes starch mobilization, increases availability of ATP, affects NH₄⁺ assimilation [32,54,55]. CIPK15 in rice interacts with the ethylene response transcription factor Sub1A in response to flooding in rice [59]. Thus, it will interesting and important to study the sugar catabolism pathway with CIPK15 on the regulation of ammonium assimilation in submergence in the future. CIPK may also be an important component of other regulatory networks by phosphorylating proteins that mediate signal transduction during potassium uptake, pollen germination, and sugar-, hormone-, and/or ROS-signaling [[60], [61], [62]]. It will be interesting to explore the physiological connections between these processes under conditions that lead to NH₄⁺ accumulation. For instance, increased potassium uptake to counteract the mechanisms of NH₄⁺ toxicity may lead to reduced NH₄⁺ uptake and accumulation, thereby preventing the improper binding of NH₄⁺ in metabolic processes that require potassium [63].

Exploring the interaction between CIPK15 and CIPK23 would be intriguing, especially considering that CIPK23 has also been demonstrated to phosphorylate AMTs [30]. CIPK23 works with the calcium sensor CBL1 to inhibit the transport activities of AMTs. The transcript of CIPK23 was not changed after submergence (Fig. S4). Thus the CBL-CIPK network or even calcium may act upstream to inhibit NH₄⁺ transport during submergence. It will be interesting to test if CIPK23 also participates in submergence tolerance.

This work provides a new perspective on the role of allosteric feedback regulation of NH₄⁺ transport during submergence and has demonstrated a new component of the regulatory circuit, the kinase CIPK15 during submergence. It is worth be noted that AMT1;1 is a NH₄⁺-induced gene [24], here we showed that the transcript of AMT1;1 also trigged under submergence; therefore, a different rate of transcription or factor might involve in the regulation of AMT1;1 transcript in NH₄⁺ signaling or submergence conditions. It will be interested to study the submergence responses of AMT1 overexpression plants in different environments, e.g., light and time, in the future. A fuller understanding of the response to submergence may provide new ways to engineer crops with higher flooding tolerance.

Author contribution statement

Yen-Ning Chen: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data. </p>;

Cheng-Hsun Ho: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data; Wrote the paper. </p>

Data availability statement

Data included in article/supp. material/referenced in article.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary data

The following is/are the supplementary data to this article.