A cis-regulatory element of the PHYTOCHROME A gene confers the submergence escape capacity for amphibious plants

Yang, Chuanwei; Liu, Wenbo; Qiu, Meiquan; Xie, Chenshu; Lu, Huanhuan; Liang, Wenjing; Qiu, Ruiyi; Li, Dongming; Li, Lin; Yang, Ji

doi:10.1038/s41467-025-66078-z

Download PDF

Article
Open access
Published: 16 December 2025

A cis-regulatory element of the PHYTOCHROME A gene confers the submergence escape capacity for amphibious plants

Nature Communications volume 16, Article number: 11167 (2025) Cite this article

2053 Accesses
5 Altmetric
Metrics details

Subjects

Abstract

Amphibious plants escape submergence by plastically accelerating shoot growth, yet the mechanism underlying this convergent strategy remains unclear. Here, we demonstrate that PHYTOCHROME A (PHYA), a light photoreceptor, acts as a central regulator of the contrasting submergence escape capacities between amphibious Alternanthera philoxeroides and its terrestrial congener A. pungens. PHYA is distinctively regulated upon submergence in two congeners and negatively regulates submergence-induced elongation. Crucially, we find a cis-regulatory element (CRE) variant in the PHYA promoter between two congeners, which governs EIN3-dependent transcriptional repression and drives species-specific PHYA expression. Expanded investigation unveils phyletic retention of this CRE in other amphibious species, providing preliminary evidence for convergent evolution in the submergence escape capacity through independent co-option of the same CREs. Our findings demonstrate a role of PHYA in mediating submergence responses, deepen the understanding of the molecular basis for plant secondary aquatic adaptation, and inform flood-resilient crops engineering via cis-regulatory manipulation.

A chromosome-level genome assembly for the amphibious plant Rorippa aquatica reveals its allotetraploid origin and mechanisms of heterophylly upon submergence

Article Open access 18 April 2024

The structure of Arabidopsis phytochrome A reveals topological and functional diversification among the plant photoreceptor isoforms

Article 08 June 2023

Structure of cryptophyte photosystem II–light-harvesting antennae supercomplex

Article Open access 12 June 2024

Introduction

Submergence/flooding stress constitutes a critical environmental factor influencing plant growth and development. With increasing frequency of extreme flooding events driven by global climate anomalies, this abiotic stress has emerged as a major threat to both natural ecosystems and agricultural systems¹. Amphibious plants, a specialized group thriving in both aquatic and terrestrial environments, predominantly inhabit shallow waters or periodically flooded zones. Living in areas prone to submergence, amphibious plants have evolved a distinctive escape strategy characterized by accelerated shoot elongation and aerenchyma formation, which enables them to restore contact with the aerial environment and facilitate internal gas diffusion^2,3,4,5,6,7. The repeated emergence of this escape strategy across multiple terrestrial plant lineages provides an exceptional model for investigating evolutionary convergence in stress adaptation mechanisms⁸. Comparative approach between amphibious plants and their non-amphibious relatives will undoubtedly contribute to the elucidation of the molecular mechanism underlying the convergent acquisition of the submergence escape capacity.

Knowledge about the molecular mechanisms underlying phenotypically plastic responses to submergence has greatly advanced in recent decades, largely due to the finding of group VII ethylene-responsive transcription factors (ERF-VIIs)-mediated signaling and hypoxia tolerance^{9,10,11,12,13}. The conserved oxygen-sensing mechanism mediated by the N-degron pathway is a central signaling cascade in plant responses to submergence-induced hypoxia. Specifically, the protein stability of ERF-VIIs is tightly regulated by the oxygen-dependent N-end rule pathway of proteolysis under submergence. Furthermore, the accumulation of ethylene due to reduced gas exchange under submergence prompts the activation of ethylene-signaling master transcription factor Ethylene insensitive 3 (EIN3), which upregulates genes associated with cell elongation, facilitating escape growth^12,14. In deepwater rice, an EIN3-like gene, OsEIL1b, triggers the expression of SNORKEL1/2, enhancing gibberellin biosynthesis for internode elongation under submergence stress¹².

In addition to restricted gas exchange, which leads to hypoxia and an ethylene hormone burst, submerged plants are often subjected to varying degrees of low-light stress, depending on submergence depth and turbidity^15,16. Moreover, light quality, such as the red/far-red light ratios, shifts dramatically underwater due to water’s spectral filtering effects¹⁷. Growing evidence suggests that responses to submergence are essentially responses to low light intensity and quality^{7,15,16,18,19}. Significant progress has been made in understanding the effects of submergence-induced decreases in light intensity and changes in the spectral composition of light on photosynthetic performance^20,21,22. Emerging evidence positions light signaling as a critical modulator of submergence responses. Phytochrome-interacting factors (PIFs) positively regulate submergence-induced cell elongation¹⁴, while CONSTITUTIVELY PHOTOMORPHOGENIC1(COP1)-MYB30 participates in regulating post-submergence recovery¹⁶. These findings establish light signaling as a key architect of adaptation strategies to submergence, though mechanistic details remain incomplete.

Alternanthera philoxeroides and A. pungens are congeneric species, and both are native to South America but have now spread to different regions of the world. A. philoxeroides dominates hydrologically variable habitats and can escape from complete submergence by faster internode elongation, while A. pungens exhibits much less degree of internode elongation than A. philoxeroides upon submergence, mainly occupying dry habitats^4,23. The amphibious A. philoxeroides and its terricolous congener A. pungens thus offer a valuable comparative system to unravel genetic drivers of the contrasting escape capacity.

Here, we showed that PHYA functions as a negative regulator of escape elongation in A. philoxeroides, which was divergently expressed between A. philoxeroides and A. pungens upon submergence. Importantly, we identified a cis-regulatory motif in the PHYA promoter that is targeted by the ethylene signaling component EIN3, leading to the repression of PHYA transcription in A. philoxeroides but not in A. pungens due to the absence of the EIN3-binding motif. The elevated PHYA level restrains plastic internode elongation in A. pungens, incapable of underwater escaping. Expanding the investigation to the amphibious species and their non-amphibious relatives from other lineages unveiled phyletic retention of this cis-regulatory element in amphibious species, providing preliminary evidence that convergent regulatory evolution is associated with convergent evolution in the submergence escape capacity.

Results

A. philoxeroides and A. pungens exhibit distinct plastic responses to submergence

To show differences in growth and development between two species in response to contrasting hydrological conditions, and collect materials for exploring the genetic and molecular mechanisms underlying submergence escape capacity, the plants of A. philoxeroides (Aph) and A. pungens (Apu) were subjected to terrestrial and submergence treatments, respectively. Following 3-day and 6-day submergence treatments, the invasive amphibious species Aph exhibited higher internode elongation than its terrestrial congener Apu (3-day: 2.65-fold vs. 1.53-fold, p < 0.0001; 6-day: 3.89-fold vs. 2.33-fold, p < 0.0001; Fig. 1a, b). This morphological advantage was accompanied by reduced leaf structural damage, including cell rupture and disruption of leaf lamina integrity (Fig. 1c), lower ROS accumulation (Fig. 1d), and higher chlorophyll retention (3-day: 0.68-fold vs. 0.39-fold, p < 0.0001; 6-day: 0.52-fold vs. 0.23-fold, p < 0.0001; Fig. 1e). Two-way ANOVA revealed that both environmental conditions and species background significantly affected internode elongation and chlorophyll retention (p < 0.001; Fig. 1b, e). These results together suggest that Aph displayed greater adaptive plasticity than its terrestrial congener Apu under submergence stress.

**Fig. 1: *Alternanthera philoxeroides* (*Aph*) and *A. pungens* (*Apu*) exhibit distinct plastic responses to submergence.**

PHYA mediates distinct submergence responses between A. philoxeroides and A. pungens

Transcriptional profiling revealed divergent regulation of the far-red light photoreceptor PHYTOCHROME A (PHYA) between two species upon submergence (SRP312818). The expression of ApuPHYA was rapidly and strongly induced when plants grown under terrestrial continuous light conditions were subjected to submergence, whereas AphPHYA exhibited only a transient and weaker up-regulation (Fig. 2a). The ApuPHYA mRNA levels gradually returned to pretreatment levels during post-submergence recovery (Fig. 2a). The PHYA protein levels at different time points under submergence and recovery conditions were assessed by immunoblot analysis using a specific anti-PHYA endogenous antibody (Supplementary Fig. 1a). In agreement with the mRNA levels, submergence promoted significantly higher accumulation of PHYA protein in Apu than in Aph, which gradually returned to pretreatment levels during post-submergence recovery (Fig. 2b and Supplementary Fig. 1b). To assess the generality of submergence-induced dynamic expression of PHYA, the same treatments were applied to Arabidopsis. Similar patterns were observed in Arabidopsis, where submergence triggered PHYA accumulation, followed by rapid degradation during post-submergence recovery (Supplementary Fig. 1c, d). These results collectively indicated that PHYA is involved in plastic submergence responses.

**Fig. 2: *PHYA* expression mediates the differential responses of *A. philoxeroides* (*Aph*) and *A. pungens* (*Apu*) to submergence.**

To directly link PHYA expression divergence to interspecific phenotypic differences, we developed a genetic transformation system for Aph and generated transgenic lines overexpressing PHYA under the constitutive 35S promoter (Supplementary Fig. 2a). We cloned a paralog of ApuPHYA (ApuPHYA-B2, Chr19.g2095), which is the most significantly induced gene by submergence (Supplementary Fig. 2b), and generated 35S::ApuPHYA-B2-GFP/Aph transgenic lines. Western blot (WB) results showed that two independent 35S::ApuPHYA-B2-GFP/Aph lines (#2 and #3) had higher ApuPHYA-B2-GFP protein levels (Fig. 2c and Supplementary Fig. 2c). Two transgenic lines also exhibited Apu-like plastic phenotypes (Fig. 2d–h and Supplementary Fig. 2d–f), with shorter elongation internode lengths (Aph vs. line2 = 2.07-fold vs. 1.67-fold, p < 0.05; Aph vs. line3 = 2.07-fold vs. 1.66-fold, p < 0.05; Fig. 2d–f). This impaired escape plasticity was accompanied by elevated ROS accumulation (Fig. 2g and Supplementary Fig. 2f) and lower chlorophyll retention (Aph vs. line2 = 0.56-fold vs. 0.40-fold, p < 0.0001; Aph vs. line3 = 0.56-fold vs. 0.46-fold, p < 0.01; Fig. 2h). Two-way ANOVA revealed that genotype background significantly affected internode elongation and chlorophyll retention (p < 0.001; Fig. 2f, h), suggesting that differential PHYA transcriptional regulation is a primary driver of distinct elongation growth patterns and other submergence responses between Aph and Apu.

PHYA negatively regulates submergence-induced elongation growth

The phyA-related mutants of Arabidopsis were used to investigate how PHYA affects plant growth in response to submergence. When 2-week-old wild-type (Col-0) and loss-of-function atphyA-211 (mutagenized with gamma rays)²⁴ plants grown under terrestrial continuous light conditions ( ~ 10–15 μmol m⁻² s⁻¹) were subjected to submergence for 3 days (Supplementary Fig. 3a), we observed that the atphyA-211 mutant exhibited greater submergence-induced petiole elongation (Col-0 vs. atphyA-211 = 1.84-fold vs. 2.20-fold, p < 0.0001; Fig. 3a, b). The 35S::AtPHYA-YFP/atphyA-211 overexpression line (AtPHYAox) restored the enhanced petiole length phenotype of atphyA-211 under submergence (Fig. 3a, b). Moreover, two loss-of-function atphyA mutants in Ler background, atphyA-201 and atphyA-202 (mutagenized with ethyl methanesulfonate)²⁴, also displayed higher submergence-induced petiole elongation compared to the wild-type Ler (Ler vs. atphyA-201 = 1.53-fold vs. 2.29-fold, p < 0.0001; Ler vs. atphyA-202 = 1.53-fold vs. 2.18-fold, p < 0.0001; Fig. 3c, d). Two-way ANOVA revealed that genotype background significantly affected petiole elongation in Arabidopsis (p < 0.001; Fig. 3b, d).

**Fig. 3: PHYA negatively regulates the submergence-induced growth responses.**

We further generated PHYA knockout mutants in rice using the CRISPR-Cas9 system in the japonica variety Zhonghua 11 (ZH11) background. Two homozygous mutant lines were obtained with distinct mutations: osphyA-1 with a 1-base deletion in the first exon and osphyA-2 with a 1-base insertion in the first exon (Fig. 3e). Phenotypic analysis revealed that the PHYA knockout rice plants exhibited enhanced internode elongation (two-way ANOVA, p < 0.01; Fig. 3f) and increased plant height following submergence treatment (two-way ANOVA, p < 0.001; Fig. 3g). When 6-leaf-stage plants were subjected to 7-day submergence followed by 4-day recovery (Supplementary Fig. 3b), both osphyA-1 and osphyA-2 mutants showed greater plant height (ZH11 vs. osphyA-1 = 1.28-fold vs. 1.49-fold, p < 0.001; ZH11 vs. osphyA-2 = 1.28-fold vs. 1.51-fold, p < 0.01; two-way ANOVA, p < 0.05; Fig. 3h, i) and higher chlorophyll retention (ZH11 vs. osphyA-1 = 0.16-fold vs. 0.46-fold, p < 0.001; ZH11 vs. osphyA-2 = 0.16-fold vs. 0.37-fold, p < 0.01; two-way ANOVA, p < 0.05; Fig. 3j), confirming the inhibitory role of PHYA on submergence-induced elongation growth.

RNA-seq analyses were performed on Col-0 and atphyA-211 to explore the role of PHYA in submergence-induced transcription (Supplementary Fig. 3c). Considering the important role of ethylene signaling components AtEIN3/EIL1 in regulating submergence-induced elongation growth¹⁴, we also used the atein3eil1 mutant as a control for RNA-seq analysis. In Col-0, 3748 submergence-induced genes (fold change >2, p < 0.01; Supplementary Fig. 3c and Supplementary Data 1) showed compromised induction in atein3eil1 (p < 2.26e⁻¹⁶, Supplementary Fig. 3d, e). However, inducible expression of these genes was enhanced in atphyA-211 (p < 2.26e⁻¹⁶) (Supplementary Fig. 3d, e), indicating that AtPHYA and AtEIN3/EIL1 had opposite regulatory effects on this gene cluster. We further identified 4485 genes co-regulated by AtPHYA & AtEIN3/EIL1 (Supplementary Data 2). GO analysis revealed significant enrichment in growth-related terms, including “response to light stimulus”, “response to brassinosteroid”, “plant−type cell wall organization or biogenesis”, and “ethylene-activated signaling pathway” (Supplementary Fig. 3f and Supplementary Data 3). Representative targeted genes such as AtEBF2 and AtPIF3 were validated by qRT-PCR (Supplementary Fig. 3g). These results indicated that PHYA negatively regulates plant response to submergence stress by repressing the expression of submergence-induced genes.

Functional conservation of AphPHYA and ApuPHYA in regulating submergence-induced elongation growth

Five and four PHYA paralogs were identified in Aph and Apu, respectively. All sequence data are available in the China National GeneBank DataBase (CNGBdb; https://db.cngb.org/) under project accession numbers CNP0006773 and CNP0006794. These paralogs exhibited striking sequence conservation, with amino acid identities ranging from 92.87% to 99.55% (Supplementary Fig. 4a). Similar to Arabidopsis, all paralogs contained six canonical phytochrome domains and critical functional residues, including the chromophore binding site (C323)²⁵, ubiquitination sites (K65, K92, K143, K206, K603)^26,27, and functional sites (R279, G367, P632, C716, G727, G768, A893)²⁸ (Supplementary Fig. 4b).

Given that Aph barely produces viable seeds and instead relies primarily on vegetative propagation for reproduction²⁹, generating PHYA-deficient mutants is challenging. We therefore cloned the submergence-induced CDS sequences of AphPHYA-B1 (Chr01.g428) and ApuPHYA-B2 (Chr19.g2095), and constructed overexpression lines in the atphyA-211 mutant background. Both AphPHYA-B1ox/atphyA-211 and ApuPHYA-B2ox/atphyA-211 transgenic lines rescued the enhanced petiole length of atphyA-211, restoring wild-type levels, when plants grown under continuous light conditions ( ~10–15 μmol m⁻² s⁻¹, 22 °C) were subjected to submergence for 3 days (Fig. 4a, b).

**Fig. 4: Functional conservation of AphPHYA and ApuPHYA in submergence-induced growth responses.**

The photoreceptor PHYA primarily functions in shade (low R:FR) and far-red (FR) environments^24,30, so we also examined the hypocotyl phenotypes of AphPHYA-B1ox/atphyA-211 and ApuPHYA-B2ox/atphyA-211 under FR-enriched conditions. Both lines rescued the long hypocotyl phenotype of atphyA-211 under shade (Supplementary Fig. 4c–e) and FR conditions (Supplementary Fig. 4f, g), demonstrating cross-species functional conservation of AphPHYA and ApuPHYA.

The expression patterns of submergence-responsive marker genes AtRAP2.2 and AtRAP2.3, the ethylene-related gene AtEBF2, and the elongation-regulating transcription factor gene AtPIF3 under terrestrial and submergence conditions were further examined and compared between Col-0, atphyA-211, AphPHYA-B1ox/atphyA-211 and ApuPHYA-B2ox/atphyA-211 using qRT-PCR. The results showed that the submergence-induced elevated expression of these genes in atphyA-211 was restored to wild-type levels in both AphPHYA-B1ox/atphyA-211 and ApuPHYA-B2ox/atphyA-211 (Fig. 4c–f). The phenotypic and qRT-PCR data together demonstrated that PHYA has a conserved role across species in response to submergence.

The cis-regulatory motif (^A / _C)CAGCT mediates AphPHYA promoter activity under submergence

To identify cis-regulatory motifs responsible for the divergent PHYA expression between Aph and Apu under submergence, we analyzed a 3-kb genomic region upstream of the PHYA locus in both species. Using a luciferase (LUC) reporter fusion with 3 kb of the sequence upstream of the start codon of both AphPHYA and ApuPHYA, we assessed promoter activity in Arabidopsis and tobacco in response to submergence. The Pro_ApuPHYA::LUC exhibited a significant increase in LUC activity in response to submergence, whereas the Pro_AphPHYA::LUC construct displayed only a modest level of LUC activity (Fig. 5a–c). The inactivation of AphPHYA under submergence suggested the possible presence of cis-acting elements that affect the AphPHYA expression.

**Fig. 5: The (^A/_C)*CAGCT* motif mediates *AphPHYA* promoter activity under submergence.**

To identify the functional elements, we made a series of 5’ deletions of the AphPHYA promoter (Fig. 5d, e). The results showed that a 600 bp region (from −2.4 kb to −1.8 kb) is required for AphPHYA expression under submergence. Comparative promoter analysis revealed a striking divergence: all five AphPHYA paralogs harbored one or two (^A/_C)CAGCT motifs, previously linked to EIN3-mediated repression in Arabidopsis³¹, while none were detected in ApuPHYA paralogs (Fig. 5f).

To validate the function of the (^A/_C)CAGCT motif, we engineered AphPHYA and ApuPHYA promoter-LUC constructs. Deletion of the (^A/_C)CAGCT motif from AphPHYA-B1 promoter (Pro_{AphPHYA-B1-Δ}::LUC) increased reporter activity by 2.5-fold in tobacco leaves (p < 0.0001; Fig. 5g), whereas insertion of the motif into ApuPHYA-B2 (Pro_{ApuPHYA-B2-add}::LUC) reduced activity by 44% (p < 0.0001; Fig. 5h). To further validate the functional role of the PHYA cis-regulatory motif in A. philoxeroides, we transiently transfected these plasmids into Aph leaves. Pro_{AphPHYA-B1-Δ}::LUC significantly enhanced submergence-induced Pro_AphPHYA-B1::LUC activity in Aph leaves (Fig. 5i), whereas Pro_{ApuPHYA-B2-add}::LUC significantly reduced Pro_ApuPHYA-B2::LUC activity under submergence (Fig. 5j).

We then constructed plasmids encoding AphPHYA-B1 fused to GFP driven by different promoter variants: Pro_AphPHYA-B1::AphPHYA-B1-GFP, Pro_{AphPHYA-B1-Δ}::AphPHYA-B1-GFP (with the motif deleted), and Pro_ApuPHYA-B2::AphPHYA-B1-GFP (driven by the ApuPHYA promoter). Using the A. philoxeroides system, we assessed AphPHYA-B1-GFP protein levels after submergence treatment, with 35S::GFP as a control (Supplementary Fig. 5a). Compared to the wild-type Pro_AphPHYA-B1::AphPHYA-B1-GFP, Pro_{AphPHYA-B1-Δ}::AphPHYA-B1-GFP (deletion of the motif) resulted in significantly increased submergence-induced AphPHYA-B1-GFP protein accumulation (Fig. 5k and Supplementary Fig. 5b, c). Similarly, AphPHYA-B1-GFP driven by the ApuPHYA promoter (Pro_ApuPHYA-B2::AphPHYA-B1-GFP) showed a marked trend toward increased submergence-induced protein accumulation compared to Pro_AphPHYA-B1::AphPHYA-B1-GFP (Fig. 5k and Supplementary Fig. 5b, c). These results clearly indicated that the (^A/_C)CAGCT motif was involved in the transcriptional repression of AphPHYA under submergence.

EIN3 modulates PHYA expression through the (^A / _C)CAGCT motif

Based on the published AtEIN3 ChIP-seq data (SRA063695)³², we found that ethylene could trigger the binding of AtEIN3 to the AtPHYA promoter, inhibiting its expression (SRA063695; Supplementary Fig. 6a, b). Our DAP-seq data further demonstrated that AphEIN3 can directly bind to the promoters of AphPHYA-A3 and AphPHYA-B1 (Callpeak was used macs2 with a cutoff q value < 0.05, GSE249660, Fig. 6a). To know whether EIN3 bound to PHYA promoter differentially in Aph and Apu, we assessed EIN3 occupancy at PHYA promoter in two species by ChIP-PCR. AphEIN3 exhibited strong promoter occupancy in Aph after submergence treatment, whereas ApuEIN3 binding in Apu was lower than in Aph (enriched input%: 0.038% vs. 0.014%, p < 0.01; Fig. 6b). We examined changes in the transcript levels of AphEIN3 and ApuEIN3 under submergence. We found that the transcription levels of AphEIN3 and ApuEIN3 were not significantly regulated by submergence, and there were no significant differences in the transcript levels of EIN3 between the two species (Supplementary Fig. 6c), indicating that the divergent interaction patterns are not attributed to interspecific variation in the EIN3 mRNA levels. Dual-luciferase assays confirmed that AphEIN3 suppresses AphPHYA-A3 and AphPHYA-B1 transcription in tobacco leaves (Fig. 6c). These results demonstrate that under submergence, AphEIN3 directly targets the (^A/_C)CAGCT motif to repress AphPHYA transcription, a regulatory interaction absent in Apu due to loss of the motif.

**Fig. 6: AphEIN3 modulates *AphPHYA* expression through the (^A/_C)*CAGCT* motif.**

To further clarify whether EIN3 is involved in regulating submergence-induced elongation, we measured petiole lengths in wild-type (Col-0), atein3eil1 mutants, and AtEIN3ox/atein3eil1 overexpression lines after the submergence treatment. When 2-week-old plants grown under continuous light were subjected to submergence for 3 days, the atein3eil1 mutant exhibited significantly reduced submergence-induced petiole elongation compared to Col-0 (2.05-fold vs. 1.58-fold, p < 0.001; Fig. 6d, e). The AtEIN3ox/atein3eil1 lines restored the atein3eil1 phenotype to wild-type levels following submergence (Fig. 6d, e). Two-way ANOVA revealed that genotype background significantly affected petiole elongation (p < 0.001; Fig. 6e), confirming the positive role of EIN3 in submergence-induced growth responses.

Reoccurrence of the (^A / _C)CAGCT motif in the PHYA promoters of other amphibious plants

To know whether the EIN3-PHYA regulatory module is evolutionarily conserved in amphibious plants, we first analyzed EIN3 and PHYA gene copy numbers across 61 angiosperm species spanning diverse life forms (17 amphibious plants, 8 floating plants, 8 submerged plants, and 28 terrestrial relatives). No significant differences in PHYA copy numbers were detected between groups (Fig. 7a, Supplementary Fig. 7a and Supplementary Data 4). However, comparative analysis of the PHYA promoters revealed striking divergence in cis-regulatory sequences, with amphibious species exhibiting a higher frequency of presence of the (^A/_C)CAGCT motif than their terrestrial relatives (4.3-fold, p < 0.001; Fig. 7b and Supplementary Fig. 7b), suggesting a conserved mechanism for submergence escape based on fine-tuning of PHYA expression.

**Fig. 7: The (^A/_C)*CAGCT* motif of *PHYA* is associated with the escape response to submergence in amphibious plants.**

To test this hypothesis, we examined the number of the (^A/_C)CAGCT motif, the expression level of PHYA, and associated submergence-induced phenotypic variations between amphibious and non-amphibious species pairs from different families. We found that the content of the (^A/_C)CAGCT motif in the PHYA promoter of Nasturtium officinale (Nof) from Brassicaceae was higher than that in its terrestrial relative Cardamine enshiensis (Cen) (Fig. 7c). The induction of PHYA by submergence was lower in Nof than in Cen (Fig. 7d and Supplementary Fig. 7c). Additionally, amphibious Nof exhibited superior escape phenotypes, including greater stem elongation (Cen vs. Nof = 1.21-fold vs. 2.15-fold, p < 0.0001) and higher chlorophyll retention (Cen vs. Nof = 0.09-fold vs. 0.65-fold, p < 0.001; Fig. 7e, f). Two-way ANOVA revealed that species background significantly affected plant height (p < 0.001; Fig. 7f) and chlorophyll retention (p < 0.05; Fig. 7f). By comparing the amphibious plant Oenanthe javanica (Oja) with its terrestrial relative Daucus carota (Dca) from Umbelliferae, we observed a lower accumulation level of the PHYA protein in response to submergence in Oja than in Dca (Supplementary Fig. 7d), and enhanced growth responses in Oja (Supplementary Fig. 7e, f).

Then, we compared amphibious species Spartina alterniflora (Sal) and Phragmites australis (Pau), with their terrestrial relatives Zoysia japonica (Zja) and Panicum virgatum (Pvi). Again, higher densities of the (^A/_C)CAGCT cis-element in PHYA promoters were detected in amphibious Sal and Pau (Sal: 1.0 motif/copy; Pau: 1.9 motif/copy) compared to terrestrial Zja and Pvi, which completely lacked the motif (Fig. 7c). This cis-regulatory divergence was most likely associated with the suppressed PHYA induction (Fig. 7g, h and Supplementary Fig. 7g, h) and enhanced submergence-induced growth responses in amphibious species, which showed longer plant height (two-way ANOVA: Zja vs. Sal, p < 0.001; Pvi vs. Pau, p < 0.001) and higher chlorophyll retention contents (two-way ANOVA: Zja vs. Sal, p < 0.001; Pvi vs. Pau, p < 0.05) (Fig. 7i, j). These findings together demonstrated that the cis-regulatory module mediated by the (^A/_C)CAGCT motif within the PHYA promoters is conserved, associated with the convergent acquisition of the submergence escape capacity in phylogenetically distant amphibious plants (Fig. 8 and Supplementary Data 5).

**Fig. 8: Working model for determining of *cis*-regulatory variation in *PHYA* between amphibious and terrestrial relatives.**

Discussion

In this study, we delineated a cis-regulatory framework driving the convergent evolution of the submergence escape strategy in amphibious plants (Fig. 8). Comparative analyses between the amphibious species Aph and its terrestrial congener Apu revealed that divergence in PHYA promoter, specifically the presence of an EIN3-bound (^A/_C)CAGCT motif, enabled repression of PHYA under submergence, thereby activating stem elongation programs. In contrast, its terrestrial relative Apu lacking this motif exhibited constitutive PHYA accumulation, which constrained escape plasticity. The retention of the (^A/_C)CAGCT motif in phylogenetically distant amphibious plants underscored its adaptive significance in specific ecological niches (Fig. 8). It is vital for plants to respond to abrupt changes of water regime in habitats.

Regulatory evolution can drive phenotypic innovation by altering non-coding regulatory sequences rather than protein-coding regions^33,34. For example, by altering non-coding alleles in the oxygen-sensing gene RAP2.12, plant populations can adapt to a range of environmental conditions, including drought and flooding³⁵. Cis-regulatory element (CRE) changes, including the gain or loss of a whole CRE (normally called “CRE turnover”), have emerged as a critical mechanism for rapid adaptation, enabling tissue- or condition-specific modulation of gene expression without pleiotropic costs^33,34. In this study, we showed clearly that the contrasting capacities of submergence escape between amphibious and non-amphibious plants were associated with the evolutionary dynamics of CRE turnover in PHYA.

PHYA, functioning as a photoreceptor, mediates both the very low-fluence response (VLFR) and far-red light-induced responses^36,37,38. The reduced light intensity and altered red-to-far-red light ratio in submerged environments create the precise conditions required for PHYA to execute its biological function. Light acts as a pivotal environmental signal in regulating submergence responses, integrating with physiological and molecular pathways to enable adaptive plasticity in dynamic underwater environments³⁹. Its biological significance encompasses multiple key aspects: (1) serving as a cue to sense submergence depth and duration, exemplified by diatom phytochromes (DPH) that function as key detectors of underwater light cues, mediating depth-dependent physiological adjustments even in R/FR-poor marine environments⁴⁰; (2) regulating energy metabolism and photosynthetic acclimation²²; and (3) inducing photomorphogenetic and developmental changes^14,19,41.

There is evidence that the light signal cooperates with hypoxic^7,16,18 and ethylene signals^42,43 to regulate plastic growth. We here demonstrated a previously unknown interaction between the ethylene signaling transcription factor EIN3 and the light signaling component PHYA to regulate the submergence escape behavior. This finding revealed how plants integrate external light signals and endogenous ethylene signaling to coordinate submergence escape strategies, thereby promoting secondary aquatic adaptation in amphibious plants.

Previous studies have shown that the red-light receptor PHYB directly interacted with EIN3 and stimulated EIN3 degradation by associating with SCF^EBF1/EBF2 E3 ligases⁴³. Although it remains to be elucidated whether PHYA interacts with EIN3 in a similar way, to negatively regulate plastic shoot elongation upon submergence by impairing the stability of the EIN3 protein, it is obvious that the occurrence of the (^A/_C)CAGCT motif in the promoter region of PHYA would lead to a decline in the PHYA-mediated EIN3 degradation rate by suppressing PHYA expression, forming a PHYA-EIN3 mutually negative regulatory loop. Functioning of this mutually inhibitory loop may result in elevated accumulation of the EIN3 protein, which subsequently promotes internode elongation through intrinsic transcriptional activation of downstream plastic growth-related genes. Such mutually negative feedback loops have been observed in various biological systems: the PHYB-PIF interaction optimizes seedling growth under canopy shade⁴⁴; the mutually inhibitory interaction between auxin and cytokinin specifies vascular pattern in roots⁴⁵; mutually inhibitory Ras-PI(3,4)P2 feedback loops mediate cell migration⁴⁶ and the mutually inhibitory feedback loop between the 20S proteasome and its regulator NQO1 coordinates protein levels with the metabolic status⁴⁷. Further research is required to determine the mutually inhibitory relationship between PHYA and EIN3.

A. philoxeroides is a globally invasive weed with strong phenotypic plasticity. The heritable nature of plastic capacity has long been recognized⁴⁸, yet its molecular basis remains elusive. By integrating comparative genomics, cis-regulatory analysis, and phenotypic profiling, we establish PHYA as a key modulator of submergence-induced plasticity. The divergent cis-regulatory activity of PHYA between Aph and Apu likely reflects adaptation to distinct hydrological habitats, with high plasticity conferring invasive advantages in heterogeneous habitats⁴⁹. Furthermore, the identification of cis-regulatory elements provides a promising target for developing climate-resilient crop varieties. Cis-regulatory editing of the (^A/_C)CAGCT motif could selectively suppress PHYA expression during flooding events, aligning with the pressing need for “plasticity-enhanced” crops that can adapt to yield losses under increasing hydrological extremes.

Methods

Genetic material

Columbia (Col-0), Landsberg (Ler) ecotype, atphyA mutants (atphyA-211, atphyA-201, atphyA-202), AtPHYAox overexpression lines (35S::AtPHYA-YFP/atphyA-211) and atein3eil1 have been previously described^30,43. The tobacco plant sources, Alternanthera philoxeroides and Alternanthera pungens, are preserved in the laboratory⁴.

For the AphPHYA-B1ox/atphyA-211 and ApuPHYA-B2ox/atphyA-211 transgenic lines, full-length AphPHYA-B1 and ApuPHYA-B2 cDNAs were amplified using PCR to generate the AphPHYA-B1-GFP and ApuPHYA-B2-GFP constructs. Their sequence data are available in the China National GeneBank DataBase (CNGBdb; https://db.cngb.org/) under project accession numbers CNP0006773 and CNP0006794. These constructs were subsequently transformed into Agrobacterium tumefaciens strain GV3101 (WEIDI, Shanghai, China). Transgenic plants were screened on half-strength Murashige and Skoog (1/2 MS) nutrient medium (Duchefa Biochemie, Haarlem, Netherlands) containing Basta and confirmed by immunoblot analysis. For the AtEIN3ox/atein3eil1 transgenic lines, full-length AtEIN3 cDNA was amplified via PCR to generate AtEIN3-Flag constructs. These constructs were subsequently introduced into Agrobacterium tumefaciens strain GV3101, which was subsequently used to infect atein3eil1 mutants. The osphyA-1 and osphyA-2 mutants were generated in the Oryza sativa (ZH11) background using CRISPR/Cas9 technology with the single guide RNA sequence GCAGCAAGCTCGTTCTGAGA. Their identities were verified by PCR genotyping (Supplementary Data 6).

Phenotypic evaluation

For the A. philoxeroides submergence treatment shown in Figs. 1 and 2, three-week-old terrestrial-grown (LD, 16/8 h light/dark cycles, ~60 μmol m⁻² s⁻¹, 22 °C) plants were either maintained under terrestrial conditions or subjected to submergence. At least 12 plants were used for monitoring the changing trends in stem internode length. The chlorophyll contents of detached leaves were incubated in darkness for 12 h in a 95% acetone/ethanol solution (2:1, v/v) containing 5% (v/v) ddH₂O, and the chlorophyll contents were calculated according to the formula (20.23 × A₆₄₅ + 8.023 × A₆₆₃) per gram fresh weight. To evaluate ROS accumulation, leaves were placed in 1 mg ml⁻¹ DAB (3,3’-diaminobenzidine) (Boster, Wuhan, China) solution (pH 3.8) for 3 h at room temperature in the dark and then transferred to boiling ethanol (75%) until the samples were cleared. ImageJ software was used to quantify the ROS levels.

The changes in petiole length in Arabidopsis are shown in Figs. 3a–d and 4a, b. Two-week-old plants grown under terrestrial continuous light conditions ( ~ 10–15 μmol m⁻² s⁻¹, 22 °C) were either maintained under terrestrial continuous light conditions or subjected to submergence for 3 days (Supplementary Fig. 3a). Under submergence, the light intensity experienced by plants was approximately 4–6 μmol m⁻² s⁻¹. At least 12 plants were used for monitoring the changing trends in petiole length.

With respect to Oryza sativa, Cardamine enshiensis, Nasturtium officinale, Zoysia japonica, Spartina alterniflora, Phragmites australis, Panicum virgatum, Daucus carota, and Oenanthe javanica, the plants in the submergence treatment shown in Figs. 3i, j and 7f, j, six-leaf-stage terrestrial-grown (LD, 16/8 h light/dark cycles, ~60 μmol m⁻² s⁻¹, 22 °C) plants were either maintained under terrestrial conditions or subjected to 7 days of submergence, followed by 4 days of recovery (Supplementary Fig. 3b).

For the Arabidopsis phenotype under shaded conditions shown in Supplementary Fig. 4c–e, seeds were sown on 1/2 Murashige and Skoog (MS) medium. After stratification for 3 days, the seedlings were transferred into a growth chamber with white light (LED light, R: ~20 μmol m⁻² s⁻¹; B: ~20 μmol m⁻² s⁻¹; FR: ~5 μmol m⁻² s⁻¹; R:FR = 4). After being grown for 3 days under this white light, the seedlings were either maintained under the same white light or transferred to the shade (R: ~20 μmol m⁻² s⁻¹; B: ~20 μmol m⁻² s⁻¹; FR: ~60 μmol m⁻² s⁻¹; R:FR = 0.3) for 4 days. The hypocotyl length and cotyledon petiole length were subsequently measured and analyzed. To determine the Arabidopsis phenotype under different far-red light intensities (Supplementary Fig. 4f, g), the plants were sown on 1/2 MS medium. After stratification for 3 days, the seedlings were subjected to 4 days of treatment with a series of far-red light intensities.

Genetic transformation of A. philoxeroides

The cDNA of ApuPHYA-B2 was cloned, and the 35S::ApuPHYA-B2-GFP plasmid was constructed. The plasmid containing the target gene was transferred into Agrobacterium GV1301, and aseptic A. philoxeroides seedlings were infected with Agrobacterium at the growth stage. After infection, callus induction was carried out, and callus screening was carried out on a medium containing resistance. Positive calli were screened, differentiation and regeneration of positive calli were induced, and positive seedlings were obtained. The positive seedlings were identified by PCR and Western blotting, and the expression of the target gene was determined for subsequent phenotype analysis.

Immunoblot assays

For the analysis of the AphPHYA and ApuPHYA protein levels shown in Fig. 2b and Supplementary Fig. 1b, 2-week-old A. philoxeroides and A. pungens plants grown under terrestrial continuous light conditions ( ~ 60 μmol m⁻² s⁻¹, 22 °C) were subjected to submergence, followed by recovery under terrestrial continuous light conditions for the indicated durations. Young tissues, including the 2nd internodes and leaves from the apex, were collected across species for Western blot analysis. An anti-Tubulin was used as a loading control to normalize the PHYA protein levels. To analyze protein expression levels in plants, total proteins were extracted with the extraction buffer [125 mM Tris-HCl (pH 8.0), 375 mM NaCl, 2.5 mM EDTA, 1% SDS, and 1% β-mercaptoethanol]. The proteins were separated on SDS‒PAGE gels, followed by wet transfer to nitrocellulose membranes. The signals were detected using a Thermo Scientific ECL kit (Thermo Fisher Scientific). Anti-PHYA and anti-Tubulin antibodies were used for analysis. For quantification of the Western blot results, ImageJ software was used to calculate the mean grayscale values of the bands.

To determine the PHYA protein levels of Col-0 and AtPHYA-YFP/atphyA-211 (Supplementary Fig. 1c, d), 7-day-old Arabidopsis plants grown under terrestrial continuous light were subjected to submergence, followed by recovery under continuous white light for the indicated durations. For the PHYA protein levels of Cen, Nof, Zja, Sal, Pau, Pvi, Dca, and Oja in Fig. 7 and Supplementary Fig. 7, six-leaf-stage plants under terrestrial continuous light ( ~ 60 μmol m⁻² s⁻¹) were either maintained under terrestrial conditions or subjected to submergence for 12 h. Young tissues, including the 2nd internodes and leaves from the apex, were collected across species for Western blot analysis, and an anti-Tubulin antibody was used as a loading control to normalize the PHYA protein levels. Antibodies used: anti-PHYA (PhytoAB, cat. no. PHY1907, 1:2000 dilution), anti-Tubulin (Abmart, cat. no. M30109, 1:4000 dilution), Anti-GFP (Abmart, cat. no. M20004, 1:3000 dilution), Anti-Mouse (Abmart, cat. no. M21001, 1:4000 dilution), Anti-Rabbit (Abmart, cat. no. M21002, 1:4000 dilution).

Promoter activity analysis

To analyze the promoter activity of AphPHYA and ApuPHYA in Arabidopsis, seedlings were subjected to submergence treatment as previously described with minor modifications¹⁴. Sterilized seeds were sown on 1/2 MS medium with 1.0% agar. Pre-germinated seeds grew in glass vessels (9 cm diameter × 3.0 cm height) for 6 days at ~60 μmol m⁻² s⁻¹ photon flux. Seedlings were then submerged in distilled water with a 2 cm water layer above the medium, either kept under terrestrial or submerged for 6 h. LUC activity was measured using a Synergy 2 multimode microplate luminometer (Bio-Tek, USA), and images were captured with a Tanon 5500 system (Tanon, China).

To analyze the promoter activity of AphPHYA and ApuPHYA in tobacco leaf cells, agrobacteria containing relevant plasmids were incubated in Luria–Bertani (LB) medium supplemented with 0.1% kanamycin and 0.1% rifampicin for 2 days. The cells were harvested by centrifugation at 4000 × g, and the pellets were resuspended in a solution containing 10 mM MES (2-(N-morpholino) ethanesulfonic acid), 10 mM MgCl₂, and 200 mM acetosyringone (AS) to a final optical density (OD₆₀₀) of 1.0. This Agrobacterium suspension was then injected into tobacco leaves. The plants were then subjected to 12 h of dark treatment followed by 2 days of continuous light and then either maintained under terrestrial conditions or submerged for 6 h.

For the analysis of CRE function in A. philoxeroides leaves shown in Fig. 5i–k and Supplementary Fig. 5, agrobacteria containing plasmids were incubated in LB medium supplemented with 0.1% kanamycin and 0.1% rifampicin for 2 days. The cells were harvested by centrifugation at 4000 × g, and the pellets were resuspended in a solution containing 10 mM MES (2-(N-morpholino) ethanesulfonic acid), 10 mM MgCl₂, and 200 mM acetosyringone (AS) to a final optical density (OD₆₀₀) of 1.0. This agrobacterium suspension was then injected into each fully expanded leaf of A. philoxeroides. The plants were then subjected to 2 days of dark treatment followed by 5 days of continuous light and harvested after 6 h or 12 h of submergence (Supplementary Fig. 5a).

Dual-LUC assay

To analyze the influence of EIN3 on the PHYA promoter in tobacco leaf cells, agrobacteria containing relevant plasmids (Pro_AphPHYA-A3::LUC, Pro_AphPHYA-B1::LUC, 35S::AphEIN3, and 35S:REN) were incubated in LB medium supplemented with 0.1% kanamycin and 0.1% rifampicin for 2 days. The cells were harvested by centrifugation at 4000 × g, and the pellets were resuspended in a solution containing 10 mM MES (2-(N-morpholino) ethanesulfonic acid), 10 mM MgCl₂, and 200 mM acetosyringone (AS) to a final optical density (OD₆₀₀) of 1.0. Reporter constructs (Pro_AphPHYA-A3::LUC or Pro_AphPHYA-B1::LUC) were cotransformed with empty vectors (negative control) or 35S::AphEIN3 in tobacco leaves. LUC expression was normalized to that of 35S::REN (internal transfection control).

RNA-seq and analysis

Terrestrial-grown plants were either maintained under terrestrial conditions or subjected to submergence for 6 h. Approximately 0.1–0.2 g of fresh plant material was harvested for standard RNA-seq library preparation and construction. Three replicates were prepared for each condition and each line. RNA was extracted using an ethanol precipitation protocol and CTAB-PBIOZOL reagent. RNA sequencing was conducted using the Illumina NovaSeq platform. Cufflinks methods were used to determine expression values. To identify DEGs, the expression level of each gene was calculated according to the fragments per kilobase per million reads (FPKM) method. Differential expression analysis was performed using DESeq2, and genes whose |log2(fold change)| was ≥1 and whose p was ≤ 0.01 were considered significant DEGs. Heatmap2 in the “gplots” package of the R program was used to construct heatmaps. The expression level of each gene was normalized by shifting the baseline median value to zero. The R package 3.0.2 was used to construct the boxplot graph.

RNA extraction and qRT‒PCR

To analyze the PHYA mRNA levels shown in Fig. 2a, 2-week-old A. philoxeroides and A. pungens plants grown under terrestrial continuous light conditions ( ~ 60 μmol m⁻² s⁻¹, 22 °C) were submerged, followed by recovery under terrestrial continuous light conditions for the indicated durations. To analyze AtRAP2.2, AtRAP2.3, AtEBF2, and AtPIF3 mRNA levels, as shown in Fig. 4c–f, 2-week-old Arabidopsis plants grown under terrestrial continuous light conditions ( ~ 10–15 μmol m⁻² s⁻¹, 22 °C) were either maintained under terrestrial continuous light or subjected to submergence for 6 h. To quantify the results of the PCR experiments, RNA was extracted using a TRIzol Kit (Promega). A 2-μg aliquot of RNA was used for first-strand cDNA synthesis with a FastQuant RT kit (Tiangen, KR118-02). Analysis was performed with a Real-Time System CFX96™ C1000 Thermal Cycler (Bio-Rad). The UBC10 gene was used as the internal control. The relative expression levels were calculated using the 2^−ΔCt method. The data are presented as the means ± s.d.s of three biological replicates normalized to the reference. The primers used for qRT‒PCR are listed in Supplementary Data 6.

Statistical analyses

Data are presented as mean ± s.d.s deviation with error bars. Two-sided Student’s t test implemented in GraphPad Prism 10 was used to compare differences between two groups, with p < 0.05 considered statistically significant. A two-way ANOVA implemented in GraphPad Prism 10 was used for main effects of Species (Spe) and Environment (Env), with statistically significance considered by p <0.05 (*), <0.01 (**), <0.001 (***).

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

All mutants and transgenic lines are available upon request from the corresponding authors. RNA-seq and DAP-seq data generated in this study have been deposited in the NCBI GEO database under accession codes GSE274369 and GSE249660, respectively.

References

Bailey-Serres, J., Lee, S. C. & Brinton, E. Waterproofing crops: effective flooding survival strategies. Plant Physiol. 160, 1698–1709 (2012).
Article PubMed PubMed Central CAS Google Scholar
Deng, X. B. et al. Time-course analysis and transcriptomic identification of key response strategies to complete submergence in Nelumbo nucifera. Hortic. Res. 9, uhac001 (2022).
Article PubMed PubMed Central CAS Google Scholar
Reynoso, M. A. et al. Evolutionary flexibility in flooding response circuitry in angiosperms. Science 365, 1291–1295 (2019).
Article PubMed PubMed Central ADS CAS Google Scholar
Gao, L. X., Geng, Y. P., Yang, H. X., Hu, Y. H. & Yang, J. Gene expression reaction norms unravel the molecular and cellular processes underpinning the plastic phenotypes of Alternanthera philoxeroides in contrasting hydrological conditions. Front. Plant Sci. 6, 991 (2015).
Article PubMed PubMed Central Google Scholar
Hattori, Y., Nagai, K. & Ashikari, M. Rice growth adapting to deepwater. Curr. Opin. Plant Biol. 14, 100–105 (2011).
Article PubMed Google Scholar
van Veen, H. et al. Two Rumex species from contrasting hydrological niches regulate flooding tolerance through distinct mechanisms. Plant Cell 25, 4691–4707 (2013).
Article PubMed PubMed Central Google Scholar
Mommer, L., de Kroon, H., Pierik, R., Bögemann, G. M. & Visser, E. J. W. A functional comparison of acclimation to shade and submergence in two terrestrial plant species. N. Phytol. 167, 197–206 (2005).
Article Google Scholar
Koga, H., Ikematsu, S. & Kimura, S. Diving into the water: amphibious plants as a model for investigating plant adaptations to aquatic environments. Annu. Rev. Plant Biol. 75, 579–604 (2024).
Article PubMed CAS Google Scholar
Gibbs, D. J. et al. Homeostatic response to hypoxia is regulated by the N-end rule pathway in plants. Nature 479, 415–418 (2011).
Article PubMed PubMed Central ADS CAS Google Scholar
Licausi, F. et al. Oxygen sensing in plants is mediated by an N-end rule pathway for protein destabilization. Nature 479, 419–422 (2011).
Article PubMed ADS CAS Google Scholar
Fukao, T. & Bailey-Serres, J. Submergence tolerance conferred by Sub1A is mediated by SLR1 and SLRL1 restriction of gibberellin responses in rice. Proc. Natl. Acad. Sci. USA 105, 16814–16819 (2008).
Article PubMed PubMed Central ADS CAS Google Scholar
Hattori, Y. et al. The ethylene response factors SNORKEL1 and SNORKEL2 allow rice to adapt to deep water. Nature 460, 1026–1030 (2009).
Article PubMed ADS CAS Google Scholar
Wang, L. N. et al. Survival mechanisms of plants under hypoxic stress: physiological acclimation and molecular regulation. J. Integr. Plant Biol. 67, 440–454 (2025).
Article PubMed Google Scholar
Wang, X. H. et al. Submergence stress-induced hypocotyl elongation through ethylene signaling-mediated regulation of cortical microtubules in Arabidopsis. J. Exp. Bot. 71, 1067–1077 (2020).
PubMed CAS Google Scholar
Das, K. K., Panda, D., Sarkar, R. K., Reddy, J. N. & Ismail, A. M. Submergence tolerance in relation to variable floodwater conditions in rice. Environ. Exp. Bot. 66, 425–434 (2009).
Article CAS Google Scholar
Xie, L. J. et al. MYB30 integrates light signals with antioxidant biosynthesis to regulate plant responses during postsubmergence recovery. N. Phytol. 237, 2238–2254 (2023).
Article CAS Google Scholar
Lin, X. W. et al. How do different growth forms of winter submerged macrophytes species respond to underwater light quality in a mesocosm study? Ecol. Evol. 14, e70441 (2024).
Article PubMed PubMed Central Google Scholar
Abbas, M. et al. Oxygen sensing coordinates photomorphogenesis to facilitate seedling survival. Curr. Biol. 25, 1483–1488 (2015).
Article PubMed PubMed Central CAS Google Scholar
van Veen, H. et al. Transcriptomes of eight Arabidopsis thaliana accessions reveal core conserved, genotype- and organ-specific responses to flooding stress. Plant Physiol. 172, 668–689 (2016).
PubMed PubMed Central Google Scholar
Mommer, L., Pons, T. L., Wolters-Arts, M., Venema, J. H. & Visser, E. J. W. Submergence-induced morphological, anatomical, and biochemical responses in a terrestrial species affect gas diffusion resistance and photosynthetic performance. Plant Physiol. 139, 497–508 (2005).
Article PubMed PubMed Central CAS Google Scholar
Vashisht, D. et al. Natural variation of submergence tolerance among Arabidopsis thaliana accessions. N. Phytol. 190, 299–310 (2011).
Article CAS Google Scholar
Sasidharan, R. et al. Signal dynamics and interactions during flooding stress. Plant Physiol. 176, 1106–1117 (2018).
Article PubMed CAS Google Scholar
Gao, L. X., Geng, Y. P., Li, B., Chen, J. K. & Yang, J. Genome-wide DNA methylation alterations of Alternanthera philoxeroides in natural and manipulated habitats: implications for epigenetic regulation of rapid responses to environmental fluctuation and phenotypic variation. Plant Cell Environ. 33, 1820–1827 (2010).
Article PubMed CAS Google Scholar
Reed, J. W., Nagatani, A., Elich, T. D., Fagan, M. & Chory, J. Phytochrome-A and Phytochrome-B have overlapping but distinct functions in Arabidopsis Development. Plant Physiol. 104, 1139–1149 (1994).
Article PubMed PubMed Central CAS Google Scholar
Zhang, Y. X. et al. Structural insights into plant phytochrome A as a highly sensitized photoreceptor. Cell Res. 33, 806–809 (2023).
Article PubMed PubMed Central CAS Google Scholar
Aguilar-Hernández, V. et al. Mass spectrometric analyses reveal a central role for ubiquitylation in remodeling the Arabidopsis proteome during photomorphogenesis. Mol. Plant 10, 846–865 (2017).
Article PubMed Google Scholar
Rattanapisit, K., Cho, M. H. & Bhoo, S. H. Lysine 206 in Arabidopsis phytochrome A is the major site for ubiquitin-dependent protein degradation. J. Biochem. 159, 161–169 (2016).
Article PubMed CAS Google Scholar
Yanovsky, M. J. et al. Missense mutation in the PAS2 domain of phytochrome A impairs subnuclear localization and a subset of responses. Plant Cell 14, 1591–1603 (2002).
Article PubMed PubMed Central CAS Google Scholar
Pan, X. Y. et al. Invasive Alternanthera philoxeroides: biology, ecology and management. J. Syst. Evol. 45, 884–900 (2007).
Article Google Scholar
Yang, C. W. et al. Phytochrome A negatively regulates the shade avoidance response by increasing auxin/indole acidic acid protein stability. Dev. Cell 44, 29–41.e24 (2018).
Article PubMed CAS Google Scholar
Wang, L. K., Ko, E. E., Tran, J. & Qiao, H. TREE1-EIN3-mediated transcriptional repression inhibits shoot growth in response to ethylene. Proc. Natl. Acad. Sci. USA 117, 29178–29189 (2020).
Article PubMed PubMed Central ADS CAS Google Scholar
Chang, K. N. et al. Temporal transcriptional response to ethylene gas drives growth hormone cross-regulation in Arabidopsis. Elife 2, e00675 (2013).
Article PubMed PubMed Central Google Scholar
Marand, A. P., Eveland, A. L., Kaufmann, K. & Springer, N. M. cis-regulatory elements in plant development, adaptation, and evolution. Annu. Rev. Plant Biol. 74, 111–137 (2023).
Article PubMed PubMed Central CAS Google Scholar
Prud’homme, B., Gompel, N. & Carroll, S. B. Emerging principles of regulatory evolution. Proc. Natl. Acad. Sci. USA 104, 8605–8612 (2007).
Article PubMed PubMed Central ADS Google Scholar
Lou, S. L. et al. Allelic shift in cis-elements of the transcription factor RAP2.12 underlies adaptation associated with humidity in Arabidopsis thaliana. Sci. Adv. 8, eabn8281 (2022).
Article PubMed PubMed Central ADS CAS Google Scholar
Chen, M. & Chory, J. Phytochrome signaling mechanisms and the control of plant development. Trends Cell Biol. 21, 664–671 (2011).
Article PubMed PubMed Central CAS Google Scholar
Legris, M., Ince, Y. Ç & Fankhauser, C. Molecular mechanisms underlying phytochrome-controlled morphogenesis in plants. Nat. Commun. 10, 5219 (2019).
Article PubMed PubMed Central ADS CAS Google Scholar
Cheng, M. C., Kathare, P. K., Paik, I. & Huq, E. Phytochrome signaling networks. Annu. Rev. Plant Biol. 72, 217–244 (2021).
Article PubMed PubMed Central CAS Google Scholar
Ragni, M. & D’Alcalà, M. R. Light as an information carrier underwater. J. Plankton Res. 26, 433–443 (2004).
Article Google Scholar
Duchêne, C. et al. Diatom phytochromes integrate the underwater light spectrum to sense depth. Nature 637, 691–697 (2025).
Article PubMed ADS Google Scholar
Cho, H. Y., Chou, M. Y., Ho, H. Y., Chen, W. C. & Shih, M. C. Ethylene modulates translation dynamics in Arabidopsis under submergence via GCN2 and EIN2. Sci. Adv. 8, eabm7863 (2022).
Article PubMed PubMed Central ADS CAS Google Scholar
Shi, H. et al. Seedlings transduce the depth and mechanical pressure of covering soil using COP1 and ethylene to regulate EBF1/EBF2 for soil emergence. Curr. Biol. 26, 139–149 (2016).
Article PubMed CAS Google Scholar
Shi, H. et al. The red light receptor phytochrome B directly enhances substrate-E3 ligase interactions to attenuate ethylene responses. Dev. Cell 39, 597–610 (2016).
Article PubMed PubMed Central CAS Google Scholar
Leivar, P., Monte, E., Cohn, M. M. & Quail, P. H. Phytochrome signaling in green Arabidopsis seedlings: impact assessment of a mutually negative phyB-PIF feedback loop. Mol. Plant 5, 734–749 (2012).
Article PubMed Google Scholar
Bishopp, A. et al. A mutually inhibitory interaction between auxin and cytokinin specifies vascular pattern in roots. Curr. Biol. 21, 917–926 (2011).
Article PubMed CAS Google Scholar
Li, X. G. et al. Mutually inhibitory Ras-PI(3,4)P2 feedback loops mediate cell migration. Proc. Natl. Acad. Sci. USA 115, E9125–E9134 (2018).
Article PubMed PubMed Central CAS Google Scholar
Moscovitz, O. et al. A mutually inhibitory feedback loop between the 20S proteasome and its regulator, NQO1. Mol. Cell 47, 76–86 (2012).
Article PubMed CAS Google Scholar
Schlichting, C. D. & Levin, D. A. Phenotypic plasticity: an evolving plant character. Biol. J. Linn. Soc. 29, 37–47 (1986).
Article Google Scholar
Hulme, P. E. & Bernard-Verdier, M. Comparing traits of native and alien plants: can we do better? Funct. Ecol. 32, 117–125 (2018).
Article Google Scholar

Download references

Acknowledgements

We thank Dr. Hongwei Guo (Southern University of Science and Technology, China) and Dr. Zhiping Song (Fudan University, China) for providing constructive suggestions. We thank Dr. Shangwei Zhong (Peking University, China) for providing the atein3eil1 mutant seeds. We thank Dr. Yonghong Hu (Shanghai Chenshan Botanical Garden, Shanghai, China) for providing the Zoysia japonica and Panicum virgatum plants. This research was supported by the National Key R&D Program of China (2021YFC2600102), the grant from Science and Technology Commission of Shanghai Municipality (14DZ2260400) and the Faculty Resources Project of College of Life Sciences, Inner Mongolia University (2022-105) to J.Y., and sponsored by Natural Science Foundation of Shanghai (25ZR1402028), State Key Laboratory of Wetland Conservation and Restoration-Wetland Young Scientist Program (SDGZ-QN2025-02) and Young Elite Scientists Sponsorship Program (2022QNRC001) to C.Y.

Author information

Authors and Affiliations

State Key Laboratory of Wetland Conservation and Restoration, National Observations and Research Station for Wetland Ecosystems of the Yangtze Estuary, Ministry of Education Key Laboratory for Biodiversity Science and Ecological Engineering, and Institute of Eco-Chongming, School of Life Sciences, Fudan University, Shanghai, China
Chuanwei Yang, Wenbo Liu, Meiquan Qiu, Chenshu Xie, Ruiyi Qiu & Ji Yang
State Key Laboratory of Genetics and Development of Complex Phenotypes, Fudan University, Shanghai, China
Huanhuan Lu, Wenjing Liang & Lin Li
Key Laboratory of Herbage & Endemic Crop Biology of Ministry of Education, School of Life Sciences, Inner Mongolia University, Hohhot, China
Dongming Li
Shanghai Key Laboratory of Plant Functional Genomics and Resources, Shanghai Chenshan Botanical Garden, Shanghai, China
Ji Yang

Authors

Chuanwei Yang
View author publications
Search author on:PubMed Google Scholar
Wenbo Liu
View author publications
Search author on:PubMed Google Scholar
Meiquan Qiu
View author publications
Search author on:PubMed Google Scholar
Chenshu Xie
View author publications
Search author on:PubMed Google Scholar
Huanhuan Lu
View author publications
Search author on:PubMed Google Scholar
Wenjing Liang
View author publications
Search author on:PubMed Google Scholar
Ruiyi Qiu
View author publications
Search author on:PubMed Google Scholar
Dongming Li
View author publications
Search author on:PubMed Google Scholar
Lin Li
View author publications
Search author on:PubMed Google Scholar
Ji Yang
View author publications
Search author on:PubMed Google Scholar

Contributions

J.Y. and C.Y. designed the experiments. C.Y. and W. Liu performed most of the experiments. W. Liang generated the AtEIN3ox/atein3eil1 transgenic plants. M.Q., C.X., H.L., R.Q., and D.L. provided technical assistance and participated in project discussions. C.Y. and J.Y. wrote the manuscript. J.Y., C.Y., and L.L. conceived and supervised the study.

Corresponding authors

Correspondence to Chuanwei Yang, Lin Li or Ji Yang.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Nature Communications thanks Eirini Kaiserli, Seisuke Kimura and the other anonymous reviewer(s) for their contribution to the peer review of this work. A peer review file is available.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Description of Additional Supplementary Files

Supplementary Data 1

Supplementary Data 2

Supplementary Data 3

Supplementary Data 4

Supplementary Data 5

Supplementary Data 6

Reporting Summary

Transparent Peer Review file

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.

Reprints and permissions

About this article

Cite this article

Yang, C., Liu, W., Qiu, M. et al. A cis-regulatory element of the PHYTOCHROME A gene confers the submergence escape capacity for amphibious plants. Nat Commun 16, 11167 (2025). https://doi.org/10.1038/s41467-025-66078-z

Download citation

Received: 11 April 2025
Accepted: 29 October 2025
Published: 16 December 2025
Version of record: 16 December 2025
DOI: https://doi.org/10.1038/s41467-025-66078-z