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# Polyploidization leads to salt stress resilience via ethylene signaling in citrus plants

Xin Song, Miao Zhang, Ting-ting Wang, Yao-yuan Duan, Jie Ren, Hu Gao, Yan-jie Fan, Qiang-ming Xia, Hui-xiang Cao, Kai-dong Xie, et al.

► To cite this version:

Xin Song, Miao Zhang, Ting-ting Wang, Yao-yuan Duan, Jie Ren, et al. Polyploidization leads to salt stress resilience via ethylene signaling in citrus plants. New Phytologist, 2025, 246 (1), pp.176-191. 10.1111/nph.20428 . hal-05463743

HAL Id: hal-05463743

https://hal.science/hal-05463743v1

Submitted on 17 Jan 2026

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Distributed under a Creative Commons CC BY 4.0 - Attribution - International License
1 **Polyploidization Leads to Salt Stress Resilience via Ethylene Signaling in Citrus**
2 Plants

3

4 Xin Song<sup>1, 5#</sup>, Miao Zhang<sup>1, 3#</sup>, Ting-Ting Wang<sup>1</sup>, Yao-Yuan Duan<sup>1</sup>, Jie Ren<sup>1</sup>, Hu Gao<sup>1</sup>, Yan-Jie Fan<sup>1</sup>,

5 Qiang-Ming Xia<sup>1, 5</sup>, Hui-Xiang Cao<sup>1</sup>, Kai-Dong Xie<sup>1</sup>, Xiao-Meng Wu<sup>1</sup>, Fei Zhang<sup>1, 2</sup>, Si-Qi Zhang<sup>3</sup>,

6 Ying Huang<sup>4</sup>, Adnane Boualem<sup>3</sup>, Abdelhafid Bendahmane<sup>3</sup>, Feng-Quan Tan<sup>1, 3*</sup>, Wen-Wu Guo<sup>1, 2*</sup>

7 <sup>1</sup> National Key Laboratory for Germplasm Innovation & Utilization of Horticultural Crops, College of

8 Horticulture & Forestry Sciences, Huazhong Agricultural University, Wuhan 430070 China

9 <sup>2</sup> Hubei Hongshan Laboratory, Wuhan 430070, China

10 <sup>3</sup> Université Paris-Saclay, CNRS, INRAE, Université Evry, Institute of Plant Sciences Paris-Saclay

11 (IPS2); 91190 Gif sur Yvette, France

12 <sup>4</sup> Institute of Science and Technology (IST), 3400 Klosterneuburg, Austria

13 <sup>5</sup> Hubei Key Laboratory of Germplasm Innovation and Utilization of Fruit Trees, Institute of Fruit and
14 Tea, Hubei Academy of Agricultural Science, Wuhan, China

15 # these two authors contributed equally to this work

16 * Feng-Quan Tan; Wen-Wu Guo

17 **Email**: fengquan.tan@universite-paris-saclay.fr; **guoww@mail.hzau.edu.cn**

18

19 **Keywords**: Polyploid, Salt tolerance, Root architecture, Ethylene, Perennial woody plant

20

21 **Abstract**

22 Polyploidization is a common occurrence in the evolutionary history of flowering plants, significantly
23 contributing to their adaptability and diversity. However, the molecular mechanisms behind these
24 adaptive advantages are not well understood. Using apomictic *Citrus* and *Poncirus* species as model
25 systems, we generated tetraploid plants and investigated their resilience to salt stress. Tetraploid plants
26 developed compacted root system that promotes salt stress tolerance, a trait that is transmitted through
27 grafting to susceptible scions. Chromatin and transcriptomic analyses revealed elevated root ethylene
28 production in tetraploid plants, driven by increased expression of 1-aminocyclopropane-1-carboxylate
29 oxidase 1 (*ACOI*) ethylene biosynthesis gene. This increased ethylene production activates the
30 transcription of reactive oxygen species (ROS) scavenging genes and stress-related hormone
31 biosynthesis genes. As a result, tetraploid plants maintained superior root functionality under salt stress,
32 exhibiting improved cytosolic K<sup>+</sup>/Na<sup>+</sup> homeostasis. Our research elucidates the role of polyploidization
33 in plant resilience opening new avenues in plant breeding.
34

34
**Introduction**

Polyploidization, the process of genome doubling, has been pivotal in the evolution of flowering plants, greatly contributing to their adaptability, diversity, and survival<sup>1,2</sup>. Polyploid plants frequently display enhanced genetic variation, increased cell size, and new traits that offer a selective advantage under various environmental stresses<sup>3-8</sup>. However, the mechanisms by which genome doubling, without altering genes, can reprogram gene expression to prime resilience remain unclear.

Salinity presents a severe abiotic stress in soil environments, impacting over 7.5% of the global land area<sup>9</sup> and significantly curtailing the growth, productivity, and survival of major cereal<sup>10</sup> and horticultural crops<sup>11</sup>. Salt stress, especially from high sodium chloride (NaCl) levels, disturbs the sodium (Na<sup>+</sup>) and potassium (K<sup>+</sup>) ions homeostasis within plant cells, which is essential for maintaining cellular functions<sup>12</sup>. When this balance is disturbed, it triggers a cascade of harmful effects, one of the most detrimental being the excessive accumulation of reactive oxygen species (ROS)<sup>13,14</sup>.

Roots are the first to detect and respond to salt stress<sup>15</sup>. Their ability to adjust architecture and maintain functionality in response to salt stress has been harnessed to improve crop resilience<sup>16-19</sup>. Although the diversity in root system types suggests an evolutionary trend towards enhanced flexibility and adaptability under environmental pressures such as soil salinity<sup>20,21</sup>, how polyploidization affect root resilience is unclear.

Ethylene, a gaseous plant hormone, has a central role in root development, yet its role in resilience to salt stress is not well understood<sup>22-25</sup>. Ethylene biosynthesis proceeds from SAM through two dedicated enzymes: 1-aminocyclopropane-1-carboxylic (ACC) synthase (ACS) and ACC oxidase (ACO)<sup>26</sup>. While ACS is considered the rate-limiting step in ethylene biosynthesis, several studies suggest that ACO also serves as a control point under specific developmental and stress conditions in various plant species<sup>27,28</sup>.

Additionally, genes involved in ethylene biosynthesis and signaling appear to be preferentially retained after natural polyploidization, highlighting their critical role in plant adaptation<sup>29,30</sup>.

Annual plants have evolved various strategies to tolerate or avoid soil environmental stresses through rapid growth and reproduction<sup>31,32</sup>. In contrast, perennial plants must adapt their root systems to cope with various soil challenges, leading to different evolutionary trends in root systems between perennial and annual plants<sup>33,34</sup>. Annual plants typically have a fibrous root system with thin, thread-like roots forming a dense network near the soil surface, whereas perennial trees often exhibit a taproot system with thicker roots penetrating deep into the soil<sup>35,36</sup>. While the impacts of polyploidization on root systems and plant adaptability remain unclear, it is evident that genome doubling influences root system development differently in perennial woody plants compared to herbaceous annuals.

In this study, we used three woody perennial crops, of *Citrus* and *Poncirus* genera, as model systems to investigate how polyploidization contributes to salt stress resilience in woody plants. Utilizing the apomictic reproduction system of these species, which can result in spontaneous tetraploidization, we generated plants that are genetically identical clones but differ in ploidy levels<sup>37,38</sup>. We assessed their root architecture, salt stress resilience, and the underlying molecular mechanisms. Our research highlights the role of chromatin remodeling following polyploidization in driving developmental and physiological changes associated with salt stress resilience.

**Results**

**Tetraploidization enhances salt stress resilience in the *Citrus* and *Poncirus* genera**

Apomictic species of *Citrus* and *Poncirus* genera, can undergo spontaneous tetraploidization<sup>37,38</sup>. We used this property to identify spontaneous tetraploid plants from three species, namely *Citrus junos*,
Citrus reticulata and Poncirus trifoliata (Figures 1A and 1B). The isolated tetraploid plants exhibited larger flowers, and seeds compared to the diploid progenitors (Figures 1A; Supplementary Figure 1). Roots are the primary interface between the plant and various biotic and abiotic stresses in the soil environment. We analyzed the root morphology of tetraploid and diploid plants. Tetraploid plants across the three genotypes displayed fewer lateral roots, reduced total root length, and increased root diameter (Figures 1C and 1D). We tested whether the root morphological changes in tetraploid plants can confer resilience to underground stresses. Thirty-day-old diploid P. trifoliata plants exposed to 50 mM, 100 mM, and 200 mM NaCl, showed leaf injuries after 68, 37, and 15 days, respectively (Supplementary Figure 2 and Figure 2C). Similar leaf injuries were observed in diploid C. junos and C. reticulata seedlings treated with 200 mM NaCl (Figures 2A and 2B). In contrast, tetraploid plants displayed fewer leaves with injuries, higher chlorophyll content and lower hydrogen peroxide ($H_2O_2$) levels, pointing to reduced sensitivity to salt stress (Figures 2A-2F).

The ability to retain $K^+$ and limit $Na^+$ accumulation, thereby maintaining a higher $K^+/Na^+$ ratio in the cytosol is a critical parameter for salt stress resilience³⁹,⁴⁰. We found tetraploid C. reticulata and P. trifoliata displaying higher $K^+$ uptake and a higher $K^+/Na^+$ ratio in both roots and shoots under salt stress conditions (Supplementary Figure 3). Under control conditions, tetraploid C. reticulata and P. trifoliata plants also displayed higher $K^+$ uptake in shoots (Supplementary Figure 3).

Given the superior performance of tetraploid root systems in maintaining $K^+/Na^+$ homeostasis under salt stress (Supplementary Figure 3), we tested whether their use as rootstocks could also confer salt tolerance to susceptible scions. Diploid scions were grafted onto both tetraploid and diploid rootstocks and subjected as above to 200 mM NaCl. Across the three genotypes, plants with tetraploid rootstocks consistently exhibited fewer injured leaves, higher chlorophyll content and lower $H_2O_2$ compared to those grafted onto diploid rootstocks (Figures 2G and 2H; Supplementary Figure 4). These findings highlight the role of polyploidization in shaping root development to confer resilience to salt stress.

## Tetraploid roots exhibit strong transcription of ethylene and ROS scavenging genes

To explore the molecular basis driving resilience to salt stress following genome doubling, we analyzed the roots transcriptome of tetraploid and diploid plants under control and salt stress conditions. Roots were sampled at 24 hours post treatment, in three biological replicates, at stage where the early salt-responsive genes, STZ1, WRKY33, and WRKY40³⁹,⁴¹,⁴² are highly expressed (Supplementary Figure 5 and 6; Supplementary Table 1).

Under salt stress, we found 3,585, 2,805, and 4,349 genes upregulated (>1.5-fold, FDR < 0.05), and 3,012, 1,537 and 3,782 genes downregulated (>1.5-fold, FDR < 0.05) in the roots of tetraploid C. junos, C. reticulata and P. trifoliata, compared to their diploid progenitors, respectively (Figure 3A; Supplementary Tables 2-4). Despite the divergence of the investigated genotypes, we found under salt treatment, 20% to 30% of upregulated genes (847 genes) and 18% to 28% of downregulated genes (245 genes) common across all the three tetraploid genotypes (Figure 3B; Supplementary Table 5). Under control conditions, despite comparable numbers of differentially expressed genes (Supplementary Figure 6 and Supplementary Tables 6-8), only 5% to 8% (55 genes) of upregulated, and 3% to 10% (64 genes) of downregulated genes, were common (Supplementary Figure 6; Supplementary Table 9).

Gene-Ontology (GO) analysis on shared 847 upregulated genes revealed enrichment for GO terms related to response to stimuli (295 genes) and stress (188 genes) (Figure 3C). Among the 188 stress response genes, six encode glutathione S-transferase (GSTUs), three encode peroxidase superfamily proteins (PRX52, PER16, and PRX25), one encodes a cupredoxin superfamily protein (SC2), and three
encode alternative oxidase (AOX1a, AOX1d-1 and AOX1d-2) (Figure 3D), key proteins implicated in the modulation of ROS homeostasis under salt stress<sup>43-46</sup>. Consistent with this, we found under salt stress conditions, roots of tetraploid plants significantly accumulating less H<sub>2</sub>O<sub>2</sub> (Figure 3E). Under control conditions, we also found an enrichment of stress response genes, pointing to a priming for stress resilience (Supplementary Figure 6). Alongside stress response genes, we found an enrichment in GO terms related to hormone with the largest number involved in the ethylene pathway, followed by jasmonic acid (JA) and abscisic acid (ABA) (Figures 3C and 3D). Specifically, 20 genes were involved in ethylene biosynthesis and signaling, including three ethylene biosynthesis-related genes: *ACO1*, *ACS1*, and *ACS6*, along with 15 ethylene response factors (*ERFs*) (Figure 3D). Notably, *ACO1* was highly upregulated in tetraploid roots under both salt stress and control conditions. (Figure 3F; Supplementary Figure 6). Beside ethylene, 12 genes involved in JA and six in ABA biosynthesis and signaling were also induced (Figure 3D), pointing to an enhanced hormonal cross-talk in roots of tetraploids.

We also investigated the downstream genes regulated by these hormones by examining motif enrichment in the promoters of the 874 up-regulated genes. The top three most enriched motifs were WRKY [TTGAC(C/T)], CAMTA1 (Calmodulin-binding transcription activator 1) (CGCGT), and the ERF binding motif (GCC box) (Supplementary Figure 7). Approximately 30% of the upregulated genes contained the GCC box motif in their promoters (P < 10<sup>-12</sup>), suggesting that genes responsive to ethylene play a major role in salt stress resilience of tetraploid.

To test whether the activation of ethylene pathway is associated to ethylene production, we measured ethylene levels in roots under both control and salt stress conditions. As expected, tetraploid roots had significantly higher ethylene levels under both conditions (Figure 3G). The citrus genome contains nine putative *ACO* genes (Supplementary Figure 8), among which only *ACO1* and *ACO5* were highly expressed in the roots (Supplementary Figure 8). *ACO1* showed the highest expression in the roots compared to other plant tissues (Supplementary Figure 8), indicating its significant role in ethylene production in roots. Several transcription factors known to enhance resistance to abiotic stresses were also among the 847 upregulated genes (Figure 3D).

In altogether, our transcriptomic analysis suggests that polyploidization-mediated salt stress resilience likely arises from a comprehensive reprogramming of the expression of genes related to ethylene biosynthesis and signaling, stress-responsive transcription factors and ROS scavenging genes.

**Ethylene treatment in diploids replicates the root phenotype and salt stress resilience in tetraploids**

Increased ethylene production is known to inhibit primary root elongation by affecting both cell division and cell elongation processes, and it also reduces the formation of lateral roots<sup>47-49</sup>. Since tetraploid plants are characterized by shorter, thicker primary roots with a reduced number of lateral roots, we hypothesized that elevated ethylene levels in tetraploids could be responsible for driving these changes. To explore this, we treated diploid seedlings with 1-aminocyclopropane-1-carboxylic acid (ACC), an ethylene precursor, to artificially elevate ethylene levels. After 45 days of treatment with 20 µM ACC, the plants exhibited thicker, shorter primary roots with fewer lateral roots, closely resembling the root architecture seen in tetraploid plants (Supplementary Figure 9; Figures 1C and 1D). Similar root modifications were also observed at lower ACC concentrations (10 µM and 5 µM; Supplementary Figure 9).

To determine whether the ethylene-induced root phenotype also contributes to enhanced salt stress resilience, we grew ACC-treated diploid plants under salt stress conditions. Compared to the control plants, the ACC-treated plants demonstrated fewer wilted and chlorotic leaves, maintained higher
164 chlorophyll content, and exhibited lower levels of H₂O₂, indicative of reduced oxidative stress (Supplementary Figure 9). These findings suggest that exogenous ethylene treatment mimics the effects of genome doubling on root architecture and improves salt stress resilience.
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166

167 **Analysis of ACO1 overexpression and knockout lines under salt stress**

168 The increased expression of the *ACO1* gene in tetraploid plants, along with the results from the ethylene treatment experiment, underscore the pivotal role of *ACO1* in enhancing salt stress resilience following genome doubling (Figures 3F; Supplementary Figure 9). To functionally validate this role, we generated both *ACO1* overexpression and knockout lines in diploid citrus plants. The overexpression lines were developed by inducing root-specific expression of *ACO1* using Agrobacterium-mediated hairy root transformation (Figure 4A; Supplementary Figures 10). The *ACO1* knockout lines were created using the CRISPR-Cas9 gene-editing tool (Figure 4B; Supplementary Figure 10). In total, four *ACO1* overexpression lines and five knockout mutants were produced, including two homozygous lines (KO4 and KO5) and one heterozygous line (KO2) with an 81.3% allele mutation rate (Figure 4B; Supplementary Figure 10). As expected, the *ACO1* overexpression (*CjACO1*-OE) lines displayed significantly higher ethylene levels in the roots, while the *ACO1* knockout (KO) lines showed a marked reduction in ethylene content (Figure 4C). Phenotypically, the *CjACO1*-OE lines developed shorter total root lengths, fewer lateral roots, and increased root diameter, closely mimicking the root architecture observed in tetraploids, and in diploid plants treated with ethylene (Figure 4D; Figures 1C and 1D; Supplementary Figure 9). Conversely, the KO2 and KO5 lines showed longer and thinner roots, further confirming *ACO1*'s effect on root development (Figure 4D). To further explore whether the enhanced salt stress resilience of tetraploid plants is driven by upregulated *ACO1* expression, we subjected both *CjACO1*-OE and KO plants to 200 mM NaCl salt stress, for 14 and 7 days, respectively. The *CjACO1*-OE lines showed significantly improved salt tolerance, evidenced by fewer wilted and chlorotic leaves, higher chlorophyll content, and lower H₂O₂ levels compared to non-transformed control plants (Figures 4E and 4G). In contrast, the KO lines were severely affected by the salt stress, exhibiting extensive wilting and bleaching of the shoots (Figures 4F and 4G). The KO lines also accumulated much lower levels of chlorophyll and higher levels of H₂O₂ than the non-transformant control plants (Figure 4G).
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193 **Increased ACO1 expression in root enhances ROS scavenging and flavonol biosynthesis**

194 Considering that enhanced salt tolerance is linked to elevated *ACO1* expression, and that knocking out *ACO1* disrupts this resilience, we conducted a detailed analysis of the root transcriptomes of *CjACO1* overexpression lines (*CjACO1*-OE) (Supplementary Figure 11; Supplementary Table 1). We identified 1,206 genes that were significantly upregulated (>1.5-fold, FDR < 0.05), and 2,168 genes that were significantly downregulated (>1.5-fold, FDR < 0.05), in the roots of *CjACO1*-OE, compared to the untransformed control line (Figures 5A and 5B; Supplementary Table 10). Similar to the root transcriptomes of tetraploids, the upregulated genes in *CjACO1*-OE roots were enriched in pathways associated with ethylene biosynthesis, ROS scavenging, and metabolic processes of phenylpropanoids and flavonoids. This includes 34 genes involved in ethylene biosynthesis and signaling, such as *ACS8*, *EIN3*, and 10 ERFs (Figures 5C and 5D). Additionally, several ROS scavenging-related genes, such as *APX2* (*ASCORBATE PEROXIDASE 2*), *SRO5* (*SIMILAR TO RCD ONE 5*), and *NDA1* (*ALTERNATIVE NAD(P)H DEHYDROGENASE 1*), were also upregulated (Figure 5D). These genes are vital for mitigating oxidative stress under abiotic stress conditions, as documented in previous studies⁴¹,⁴⁵,⁵⁰. Key
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enzymes that regulate phenylpropanoid and flavonoid biosynthesis<sup>51</sup>, including PAL1 (PHENYLALANINE AMMONIA LYASE), C4H (CINNAMATE 4-HYDROXYLASE), FLS (FLAVONOL SYNTHASE), and the transcription factor *MYB12* (a master regulator of the R2R3-MYB family), were also upregulated in *CjACO1*-OE roots (Figure 5D). Consistent with these transcriptomic changes, both *CjACO1*-OE and ACC-treated plants exhibited lower H<sub>2</sub>O<sub>2</sub> levels and higher flavonol concentrations in their roots (Supplementary Figure 11). The increased flavonol biosynthesis is likely a key factor contributing to the enhanced salt stress resilience, observed in *CjACO1*-OE, ACC-treated, and tetraploid plants, as flavonols are known substrates for peroxidases involved in ROS scavenging, particularly of H<sub>2</sub>O<sub>2</sub><sup>52</sup>.

To further validate the role of *ACO1* in regulating ROS scavenging and flavonol biosynthesis, we transiently expressed *CjACO1* fused to a Red Fluorescent Protein (RFP) reporter in citrus leaf protoplasts. Subsequent qPCR analysis and ROS staining assays revealed that protoplasts expressing *CjACO1-RFP* exhibited activation of both ROS scavenging and flavonol biosynthesis-related genes, while the control protoplasts expressing RFP alone showed no significant changes (Figure 5E). Furthermore, protoplasts expressing *CjACO1-RFP* displayed significantly lower ROS levels compared to the controls (Figures 5F and 5G). These findings demonstrate that elevated *ACO1* expression enhances ROS scavenging and promotes flavonol biosynthesis, thereby contributing to improved stress tolerance. Interestingly, several JA biosynthesis genes were also upregulated in the roots of *CjACO1*-OE plants (Figure 5D) as well as in tetraploid plants (Figure 3D), suggesting that ethylene signaling may play a role in promoting JA biosynthesis, another hormone associated with resilience to stresses.

In summary, our findings highlight that increasing ethylene production in roots stimulates the transcription of genes involved in ROS scavenging, JA biosynthesis, and flavonol biosynthesis, all of which contribute to the plant's resilience to salt stress. These results also underscore the central role of *ACO1* expression in coordinating salt stress tolerance, particularly in tetraploid plants.

**Tetraploidization results in hypomethylation and enhanced chromatin accessibility of ACO1**

To explore how *ACO1* expression is regulated in the roots of tetraploid plants, we conducted a comprehensive analysis of genome-wide DNA methylation and chromatin accessibility. DNA methylation analysis was performed using whole-genome bisulfite sequencing (BS-seq) on the roots of tetraploids and their diploid progenitors under control conditions (Supplementary Table 1). The Pearson correlation coefficients among the three biological replicates exceeded 0.98, indicating high reproducibility of the methylome data (Supplementary Figure 12). To identify variations in DNA methylation, we looked for differentially methylated regions (DMRs) between tetraploid and diploid plants ($P \le 0.05$, $\Delta mC \ge 0.1$). Across the genomes of tetraploid *C. junos*, *C. reticulata*, and *P. trifoliata*, we found 8,194, 10,901, and 3,165 hypermethylated DMRs and 6,560, 7,518, and 3,165 hypomethylated DMRs, respectively (Supplementary Tables 11-13). Interestingly, no significant changes in CHG and CHH DNA methylation were observed in the *ACO1* promoter and gene body between tetraploid and diploid plants (Supplementary Figure 13). However, we identified hypomethylated DMRs in the CG context within the gene body of *ACO1* (Figure 6A; Supplementary Figure 14).

To further confirm the presence of these CG hypo-DMRs in the *ACO1* gene body, we employed bisulfite sequencing-PCR (BSP). We specifically focused on the region containing the identified CG hypo-DMR, which is located 3' downstream of the start codon, spanning 597 bp to 1,268 bp. This region exhibited significantly reduced CG methylation in tetraploid plants compared to diploid plants across all three
249 genotypes, validating our genome-wide methylation analysis (Figure 6B and 6C; Supplementary Figure 14).
250
251 We also assessed chromatin accessibility using ATAC-seq analysis on root chromatins from two
tetraploid genotypes, *C. reticulata* and *P. trifoliata*, as well as their diploid progenitors under control
252 conditions. (Supplementary Table 1). Genome-wide chromatin accessibility patterns in tetraploid and
253 diploid roots were strikingly similar, showing enrichment at transcription start and termination sites
254 (Figures 6D and 6E; Supplementary Figure 15). Nevertheless, *ACO1* showed significantly increased
255 chromatin accessibility (P < 0.05), corresponding with its upregulated expression under both control and
256 stress conditions in tetraploid plants (Figures 6F). This supports our finding that tetraploid roots have a
257 stronger transcriptional response to early-stage salt stress compared to diploid roots (Figures 3A and 3D).
258 Alongside *ACO1*, other ethylene biosynthesis and signaling genes, such as *ACS6*, *ERF1*, *ERF104* and
259 *ERF017*, as well as the flavonol biosynthesis genes *FLS1* and *PAL1*, and the ROS scavenging gene
260 *AOX1d*, which were upregulated in tetraploid roots under salt treatment, also showed greater chromatin
261 accessibility in tetraploid *C. reticulata* and *P. trifoliata* (Figure 3D; Supplementary Figure
262 16). Collectively, our findings emphasize that genome doubling drives hypomethylation and chromatin
263 remodeling of upregulated genes, such as *ACO1*, which is essential for activating pathways that enhance
264 resilience to salt stress.
265

266 Discussion

267 Throughout evolution, most flowering plants have undergone one or more independent genome-doubling
268 events, underscoring polyploidization as a key driver in the emergence of new species and adaptation to
269 changing environments⁴. However, it remains unclear whether genome doubling alone is sufficient to
270 mediate some of these adaptations, or if subsequent mutations leading to new gene functions are
271 necessary to confer genetic advantages. In this study, we leveraged the apomictic reproductive system of
272 citrus species within the Rutaceae family, which can spontaneously undergo tetraploidization, to generate
273 genetically identical clones differing only in ploidy levels, and comparative phenotyping to the diploid
274 progenitors for resilience. Our findings reveal that tetraploid clones exhibit a stronger root system and
275 improved functionality under salt stress, phenotypes that are orchestrated by chromatin remodeling of
276 the *ACO1* gene.

277 Our analysis also indicates that the elevated expression of *ACO1* in tetraploid plants does not stem from
278 changes in promoter DNA methylation in the CHG and CHH contexts, as no significant alterations were
279 observed (Supplementary Figure 13). Instead, we detected a decrease in CG DNA methylation within
280 the *ACO1* gene body across all tetraploid plants, which correlates with *ACO1* expression upregulation
281 (Figure 6B and 6C; Supplementary Figure 14). Although the precise function of intragenic methylation
282 remains unclear, similar decreases in gene body methylation have been observed in newly formed
283 tetraploid grapevines⁵³, suggesting a possible link between reduced methylation and increased gene
284 expression.

285 Significantly, we found a marked increase in chromatin accessibility at the transcription start site of
286 *ACO1* in tetraploid plants (Figure 6F). This increase in accessibility likely facilitates the elevated
287 expression of *ACO1*, which in turn drives higher ethylene production in tetraploid roots, contributing
288 significantly to their enhanced salt tolerance (Figure 7A). Supporting evidence includes experiments
289 where overexpressing *ACO1* in diploid plants' roots boosted ethylene production and activated resilience
290 metabolic pathways similar to those observed in tetraploids (Figure 4C; Figure 5D). Conversely,
291 knocking out *ACO1* compromised the expression of these pathways (Figure 4). Additionally, pretreating
292 the roots of diploid plants with ACC also enhanced salt tolerance (Supplementary Figure 9),
293 demonstrating that ethylene plays a critical role in the plant's stress response.

294 Under normal conditions, tetraploid plants also exhibit increased chromatin accessibility in ROS
295 scavenging genes including those encoding alternative oxidase, glutathione S-transferase (GSTUs), and
296 flavonol biosynthesis enzymes, that play a crucial role in protecting plants from oxidative stress⁴⁵,⁵⁰,⁵².

297 These genes help neutralize harmful ROS, which can accumulate under salinity stress⁴¹. Alternative
298 oxidases functions as a terminal oxidase in the mitochondrial electron transport chain, preventing over-
299 reduction of components and reducing ROS formation under plant stress conditions⁵⁴, while GSTUs
300 detoxify harmful compounds by conjugating them with glutathione⁴³. Flavonol biosynthesis, on the other
301 hand, produces flavonoids that act as antioxidants, further enhancing the plant's defense system⁵². This
302 increased accessibility allows transcriptional regulators to more readily bind and activate these genes
303 when the plants are exposed to salt stress, enabling a quicker and more robust transcriptional response
304 compared to diploid counterparts.

305 Genome doubling during evolution and adaptation often results in multiple gene paralogs, though most
306 duplicated genes are eventually lost⁵⁵. Some are retained through neofunctionalization,
307 subfunctionalization, or beneficial increases in gene dosage⁵⁶. In this context, ACO genes have multiple
308 homologs in most plants (e.g., 5 ACOs in *Arabidopsis thaliana* and 9 in the *C. sinensis* genome). Among
309 these, *ACO1* is the most highly expressed in citrus roots, and its overexpression leads to higher ethylene
310 production and enhanced salt tolerance. Conversely, loss of *ACO1* function results in lower ethylene
311 production and increased salt sensitivity, underscoring its key role in root resilience. We found the
312 expression of the other ACO genes does not change with genome doubling (Supplementary Tables 2-4),
313 indicating that they do not contribute to the salt stress resilience observed in tetraploid plants.

314 The elevated expression of *ACO1* in tetraploid genotypes results in thicker roots with reduced lateral
315 branching (Figures 1C and 1D), which may improve soil penetration, promote deeper rooting, and reduce
316 the metabolic costs associated with root construction¹⁷,¹⁹,⁵⁷. These structural changes in the roots likely
317 contribute to the increased survival and adaptability of newly formed polyploid plants in specific soil
318 conditions (Figure 7B). Moreover, increased *ACO1* expression or ethylene biosynthesis may also
319 promote early fruit maturation⁵⁸ and seed germination²⁷, aiding in seed dispersal and boosting the
320 reproductive capacity of newly formed polyploids.

321 Elevated *ACO1* expression has also been observed in colchicine-induced tetraploid grapevines⁵³ and in
322 tetraploid *C. junos* leaves⁵⁹, suggesting that gene dosage significantly influences *ACO1* function.
323 Interestingly, the *ctrl* (*constitutive triple response 1*) mutant of *Arabidopsis*, which exhibits increased
324 ethylene signaling, shows higher polyploidy levels in hypocotyls⁶⁰, further highlighting the complex
325 interplay between polyploidy and ethylene signaling in plant adaptation and evolution.

326 This study sheds new light on the role of polyploidization in enhancing plant resilience, revealing how
327 genome doubling can lead to significant adaptations that improve a plant's ability to withstand
328 environmental stresses. By understanding the molecular mechanisms behind this process, this research
329 not only deepens our knowledge of evolutionary biology but also opens up exciting new avenues in plant
330 breeding, offering the potential to develop crops with enhanced stress tolerance to changing
331 environmental conditions. This could lead to more sustainable agricultural practices and a mean to face
332 of global challenges such as climate change.

333 **Materials and Methods**

334 **Plant Materials and Growth Conditions**
We used diploid and tetraploid *Citrus* rootstocks in *C. reticulata* Blanco, *C. junos Sieb.* ex Tanaka and *P. trifoliata* L. Raf. backgrounds. Tetraploids were identified among a population of seedlings derived from the corresponding diploid genotypes at the National Citrus Breeding Center at Huazhong Agricultural University³⁷,⁵⁹. Diploid *C. junos* were used for genetic transformations. All plants were cultured in 0.5×Hoagland nutrient solution or commercial soil in a growth chamber at 25°C under a photoperiod of 16 h/8 h (light/dark).

### Tissue K⁺ and Na⁺ Quantification

Seedlings were cultured in a 0.5×Hoagland nutrient solution either with or without the addition of 200 mM NaCl for 15 d. Roots were washed three times with deionized water prior to harvest. Roots and shoots were separated and dried at 65°C for 3d. Dry samples were digested with a mixed solution of acids HNO₃/HClO₄ (85/15, vol/vol). The contents of K⁺ and Na⁺ were measured by inductively coupled plasma mass spectrometry (PerkinElmer NexION-300×, Waltham, MA).

### DAB staining assay and measurement of ROS accumulation and chlorophyll content

DAB staining was performed as previously described⁶¹. H₂O₂ content was measured using a H₂O₂ content assay kit (boxbio, Beijing, China) according to the manufacturer's instructions. Chlorophyll content was measured as previously described⁶². Three biological replicates were analyzed with each composed of tissue from three independent plants. One of the representative data sets are shown for each experiment.

### Fluorescence detection of ROS in citrus protoplast

Intracellular total ROS were detected using the Reactive Oxygen Species Assay Kit (50101ES01, Yisheng Biotechnology Co., Ltd., Shanghai, China) according to the manufacturer's protocol. Citrus callus protoplasts were transfected with 35S:CjACO1-RFP and 35S:RFP (control). The protoplasts were incubated with the WI solution (0.4 M mannitol, 20 mM KCl, 4 mM MES, pH 5.7) in the dark (28°C) for 16 h. The protoplasts were supplemented with 10 µM DCFH-DA staining for 20 min at 28°C in the darkness and supplemented with 100 µM Rosup for 10 min at 28°C in the darkness. Then the protoplasts were washed several times with the W5 solution (154 mM NaCl, 125 mM CaCl₂, 5 mM KCl, 2 mM MES, pH 5.7) to clear excess dye prior to imaging. The fluorescence images were acquired using a Leica TCS SP8 (Leica Microsystems) confocal laser scanning microscope with the following wavelength settings: DCF: Excitation = 488 nm, Emission = 505-550 nm; RFP: Excitation = 552 nm, Emission = 580-630 nm.

### RNA-Seq and qRT-PCR analysis

Three biological replicates were analyzed for each sample. Total RNA isolation, library construction and sequencing were performed in Novogene Bioinformatics Technology Co., Ltd. Sequencing was performed on an Illumina HiSeq 4000 platform. Over 73% of the high-quality reads were mapped to the reference genome of *C. sinensis* v1.1 (Phytozome) using HISAT2 (2.2.1) with default parameters⁶³ and normalized gene expression levels (fragments per kilobase of transcript per million mapped reads, FPKM) were calculated by Cufflinks (v.2.2.1). Genes with a p value <0.05 following Benjamini and Hochberg correction and an absolute value of log₂ fold change >0.58 were considered differentially expressed. Gene ontology (GO) enrichment analysis was carried out using AgriGO (http://systemsbiology.cau.edu.cn/agriGOv2/). Subsequently, RNA was converted to cDNA using HiScript II Q RT SuperMix for qPCR kit (Vazyme, R223-01, Nanjing, China). qRT-PCR was performed using a 2X Universal SYBR Green Fast qPCR Mix (ABclonal, RM21203). All data were normalized to
376 Actin (CsACT7) and relative expression levels were calculated using the 2⁻ΔΔCt method as previously described. Primer pairs are listed in Supplementary Table 14.
377

378 Bisulfite sequencing and data analysis

379 Roots were collected from the four-month-old seedling for WGBS. DNA extraction, bisulfite treatment, library preparation, and next generation sequencing on the DNBseq™ platform were conducted in BGI company (Shenzhen, China). The BS-seq raw reads were filtered using Trimmomatic software to remove low-quality reads and adapters. Clean reads were mapped to C. sinensis v3.0 or P. trifoliata v1.0 genome (http://citrus.hzau.edu.cn/) using BS-Seeker2 software. Reads aligned to a unique position on the genome were retained for subsequent analysis. DMRs were identified using CgmapTools with default parameter (P≤0.05, ΔmC≥0.1). DNA methylation levels were calculated using the ratio of methylated cytosines (mC) over total cytosines (mC + nonmC) in each bin, with the absolute methylation difference ≥0.1, and P ≤ 0.01 (T-test).

388 Bisulfite sequencing was applied to detect a given region's DNA methylation status. Genomic DNA from four-month-old seedling root was extracted with the cetyl trimethylammonium bromide (CTAB) method.
389 Bisulfite treatment was performed using 500 ng of DNA with the EZ DNA Methylation-Lightning Kit
390 (D5005; Zymo Research). The gene regions were amplified from the 40% bisulfite-treated root DNA
391 with primers listed in Supplementary Table 19. The amplified PCR fragments were cloned into pTOPO
392 cloning vector (Aidlab) and at least 12 subclones were selected for each sample to conduct Sanger
393 sequencing. Kismeth software (https://katahdin.girihlet.com/kismeth/revpage.pl) was used to obtain the
394 percentage of methylated sites.
395

396 ATAC-seq experiment and data analysis

397 The method of nuclei extraction was performed as before⁶⁴. After checking the nuclear integrity, the
398 nuclei extracted (~ 5,000 per reaction) were incubated with the Tn5 transposase and tagmentation buffer
399 at 37°C for 30 min (Vazyme Biotech, TD501-01). After tagmentation, the DNA is purified by PCR
400 purification kit (QIAGEN, 28,106). PCR was performed to amplify the library for 9-12 cycles using the
401 following PCR conditions: 72°C for 5 min; 98°C for 30 s; and thermocycling at 98°C for 15 s, 63°C for
402 30 s and 72°C for 40 s; following by 72°C for 5 s. After the PCR reaction, libraries were purified with
403 AMPure beads (Beckman, A63881). The library was sequenced using an Illumina Novaseq platform by
404 Annoroad Gene Technology. Paired-end read sequences were mapped using Bowtie2 (2.4.1) on the C.
405 sinensis v3.0 or P. trifoliata v1.0 genome using the following parameters: --no-mixed --no-discordant -
406 X 2000. Duplicate fragments mapped on the genome were removed using Picard MarkDuplicates. Peak
407 calling using MACS2 (2.2.7) was performed with the following settings: --keep-dup all -B --SPMR --
408 nomodel --shift -75 --extsize 150. Peak merging and identification of differential peaks were performed
409 using DiffBind (v2.14.0) and peaks with P-value < 0.05 and fold change > 1.5 were considered
410 differentially accessible region.
411

411 Plasmid construction and genetic transformation

412 The CDS of *CjACO1* were amplified from cDNA of diploid *C. junos* and cloned into pDONR221 and
413 then transferred into the binary vector pK7WG2D downstream of the CaMV35S promoter (p35S).
414 CRISPR/Cas9 genome editing vectors were produced as previously described⁶⁵. The mutation
415 frequencies of the transgenic lines were analyzed by Hi-Tom sequencing (http://www.hi-tom.net/hi-
tom/). All the primers used in this study are listed in Supplementary Table 14.
416

417 The pK7WG2D-CjACO1 and proYAO-hspCas9-NOS-CjACO1 binary vectors were introduced into
418 *agrobacterium rhizogenes* strain MSU440. Diploid *C. junos* plant transformation was performed as
419 previously described⁶⁶. GFP fluorescence in plants was inspected with a hand-held blue-green lamp
420 (LUYOR-3415).

421 Quantification of ethylene content

422 Root from four-month-old seedlings were incubated for 1 h at 25°C to allow wound-induced ethylene
423 content to subside and then transferred into a 5-ml Falcon tube with all the root submerged in water. The
424 tube was tightly sealed with a rubber cap for 12 h at 25°C. The ethylene that was emitted was
425 subsequently measured using a gas chromatography instrument⁶⁷,⁶⁸. Four to six biological replicates were
426 measured.

427 Quantification of total flavonol content

428 Total flavonol content was determined following the previously reported method⁶⁹. Roots were ground
429 to powder. 1.5 ml of 80% ethanol solution was added to 0.1 g powder for 1 h low-temperature sonication.
430 The flavonol was extracted for 12 h at 4°C, followed by centrifugation at 4,500 rpm for 20 min, and
431 liquid supernatant was retained for quantification.

432 Statistical analysis

433 Data are presented as mean values ± standard deviation. Statistically significant differences between the
434 two groups were determined using a two-tailed Student's t-test (*P* < 0.05, **P* < 0.01, ***P* < 0.001 and
435 ****P* < 0.0001). For multiple comparisons, statistically significant differences were determined using a
436 one-way ANOVA followed by Tukey's multiple range test or Duncan's tests (P < 0.05) or Kolmogorov-
437 Smirnov test. Statistical analyses were performed with GraphPad Prism 8.0 and Microsoft Excel 2016.

438 Data availability

439 All high-throughput sequencing data generated in this study have been deposited in the SRA database.
440 The accession for the RNA-seq data, the BS-seq data and the ATAC-seq data are PRJNA1127087.
441 Source data are provided with this paper.

442 Acknowledgments

443 We thank Prof. Qi Xie from Institute of Genetics and Development, Chinese Academy of Sciences for
444 providing the *YAO* promoter-driven CRISPR/Cas9 vector, and our colleague Dr. Robert M. Larkin from
445 Huazhong Agricultural University for critical reading of the manuscript.

446 Author Contributions: W-W.G., X.S., and F-Q.T. designed the research; X.S., M.Z., T-T.W., J.R., Y-
447 J.F., Q-M.X., H-X.C., S-Q.Z. and K-D.X. performed the experiments; Y-Y.D., and M.Z. analyzed
448 RNA-Seq data; M.Z., and H.G. analyzed whole-genome bisulfite sequencing data; M.Z. analyzed
449 ATAC-Seq data. X.S., and F-Q. T. wrote the manuscript; A.B., A.B., Y.H., F.Z., and X-M.W.
450 improved the manuscript.

451 Competing Interest Statement: The authors declare that they have no conflicts of interest related to
452 this work.

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605
<figure><img src="image_3.png" /><figcaption>**Figure 1.** Tetraploidization leads to a compacted root system in the *Citrus* and *Poncirus* genera</figcaption></figure>

(A) Schematic diagram for identification of spontaneous tetraploids using flow cytometric analysis. Seedlings with greener and larger leaves, which have a higher probability of being tetraploids (4x), from diploid (2x) apomictic citrus genotypes were selected for analysis. Flowers and seeds of 4x and 2x *Citrus junos* were shown. Scale bars = 1 cm.

(B) Phylogenetic tree of representative citrus species. Three genotypes highlighted in red were used in this study. A phylogenetic tree was constructed using the maximum likelihood method. Single nucleotide polymorphisms (SNPs) in the regions of conserved single-copy genes from 11 representative accessions within the Rutaceae family were used.

(C) Comparison of root morphology between 4x and 2x plants. Scale bars = 1 cm.

(D) Statistical analysis of root parameters in 4x and 2x plants. Bar plots represent mean ± SD (n = 10-20). Statistically significant differences was tested by Student's t-test, *P*<0.05, **P**<0.01.
<figure><img src="image_4.png" /></figure>

C. junos, NaCl (15 d)

<figure><img src="image_5.png" /></figure>

C. junos-2x

<figure><img src="image_6.png" /></figure>

C. reticulata, NaCl (15 d)

<figure><img src="image_7.png" /></figure>

C. reticulata-4x

<figure><img src="image_8.png" /></figure>

P. trifoliata, NaCl (15 d)

<figure><img src="image_9.png" /></figure>

P. trifoliata-4x

<figure><img src="image_10.png" /></figure>

C. junos-2x/2x

C. junos-2x/4x

<figure><img src="image_11.png" /></figure>

C. junos-2x/2x

<caption>Figure 2. Tetraploidization enhances salt stress resilience in the *Citrus* and *Poncirus* genera</caption>

(A), (B), (C) Leaf injuries and ROS staining of 4x and 2x plants from three genotypes treated with NaCl. Four-month-old plants were treated with 200 mM NaCl for 15 days. Red arrows indicate wilted leaves. Leaves stained with DAB were used to assess the accumulation of H₂O₂. Scale bar = 2 cm.

(D), (E), (F) Statistical analysis of injured leaf number, leaf chlorophyll and H₂O₂ content in NaCl-treated 2x and 4x plants. Bars indicate mean values ± SD (n=3-4). Statistically significant differences were determined using a Student's t-test, **P<0.01, ***P<0.01, ****P<0.0001.

(G) Leaf injuries of grafted 2x/2x (scion/rootstock) and 2x/4x (scion/rootstock) *C. junos* plants. One-month-old plants were used for grafting, and after one month of recovery, the grafted plants were subjected to a 15-day treatment with 200 mM NaCl. Grafted plants of other genotypes are shown in supplementary figure 4. Red and white arrows indicate wilted leaves and grafting interface, respectively. Scale bar = 5 cm.

(H) Statistical analysis of injured leaf number, leaf chlorophyll and H₂O₂ content in grafted plants treated with NaCl. Bar plots represent mean ± SD (n = 4-6). Statistically significant differences was tested by Student's t-test (**P<0.01) or ANOVA (different letters).
<figure><img src="image_12.png" /><figcaption>Figure 3. Tetraploid roots exhibit stronger transcription of ethylene and ROS scavenging genes</figcaption></figure>

(A) Number of differentially expressed gene in roots between 2x and 4x plants across three genotypes under NaCl treatment. Entire roots after 24 hours of NaCl treatment (200mM) were used for transcriptomic analysis. Genes with FDR (False Discovery Rate) < 0.05 and FC (Fold Change) > 1.5 were considered differentially expressed.

(B) Venn diagrams showing up-regulated genes in 4x roots relative to 2x roots overlapping among three genotypes under NaCl treatment.

(C) GO enrichment analysis of 847 up-regulated genes in 4x roots relative to 2x roots shared among three genotypes under NaCl treatment. Top 10 GO terms were shown. *P*-values were calculated using Fisher's test.

(D) Heatmap showing expression levels of stress-related pathway genes between 2x and 4x plants under NaCl treatment.

(E) Statistical analysis of H₂O₂ content in roots of 4x and 2x plants under NaCl treatment. Bar plots indicate mean values ± SD (n=5). Statistically significant differences were determined using a Student's *t*-test, **P*<0.01.

(F) Expression levels of ethylene biosynthesis gene *ACO1* between 4x and 2x roots among three genotypes under NaCl treatment and normal conditions. Expression data was obtained from entire root transcriptomic analysis. Data represent mean values ± SD (n = 3,**P* < 0.01, Student's *t*-test).

(G) Statistical analysis of ethylene content in the roots of 4x and 2x plants under normal condition and NaCl treatment. Data represent mean values ± SD (n = 5,**P* < 0.01, Student's *t*-test).
<figure><img src="image_13.png" /><figcaption>Figure 4. Transgenic manipulation of ACO1 replicates the root phenotype and salt stress resilience observed in tetraploids</figcaption></figure>

(A) Plant phenotypes of wild type (WT) and *CjACO1* root-specific overexpression (OE) lines. *C. junos*-2x plants were used for Agrobacterium rhizogenes-mediated transformation. Two-month-old plants were shown. Scale bars = 5 cm.

(B) Plant phenotypes of WT and *CjACO1* knockout (KO) lines. Two gRNA target sites are indicated by red vertical lines (upper). Two-month-old plants were shown. Scale bars = 5 cm.

(C) Statistical analysis of ethylene content in the roots of WT, *CjACO1*-OE and *CjACO1*-KO plants. Bar plots indicate mean values ± SD (n = 4-6). *Statistically significant differences were tested by Student's t-test, **P*<0.01.

(D) Statistical analysis of root parameters in WT, *CjACO1*-OE and *CjACO1*-KO plants. Bar plots represent mean ± SD (n = 3-6). *Statistically significant differences were tested by Student's t-test, *P*<0.05, **P*<0.01, ns, no significant difference.

(E-F) Leaf injuries of WT, *CjACO1*-OE (E) and *CjACO1*-KO (F) plants treated with NaCl. Four-month-old plants were treated with 200mM NaCl for 15 days (E) or 7 days (F). Red arrows indicate injured leaves. Scale bar = 5 cm.

(G) Statistical analysis of injured leaf number, leaf chlorophyll and H₂O₂ content in WT, *CjACO1*-OE and *CjACO1*-KO plants. Bar plots represent mean ± SD (n = 4). *Statistically significant differences were tested by Student's t-test, *P*<0.05, **P*<0.01, ***P*<0.001
<figure><img src="image_14.png" /><figcaption>Figure 5. Increased ACO1 expression in root enhances ROS scavenging and flavonol biosynthesis</figcaption></figure>

(A) Average read count plots of gene expression changes in roots of *CjACO1*-OE versus wild-type. The x axis represents average gene expression level (FPKM), and the y axis represents log₂ FC(*CjACO1*-OE/WT). Significantly (FDR < 0.05 and 1.5-FC) upregulated and downregulated DEGs are indicated.

(B) FPKM values of *ACO1* in *CjACO1*-OE and wild-type (WT) roots. FPKM values were extracted from root transcriptomic data. Bar plots represent mean ± SD (n = 3). \*\* *P* < 0.01 (Student's *t*-test).

(C) GO enrichment analysis of up-regulated genes in *CjACO1*-OE plants. Top five GO terms are shown. *P*-values were calculated using Fisher's test.

(D) Heatmap showing expression levels of genes from GO terms in (C). Heatmap presents scaled values of normalized FPKM from transcriptomic data. Typical genes in corresponding term were indicated on the right.

(E) qRT-PCR analysis of genes in citrus leaf protoplasts expressed *CjACO1-RFP* or *RFP*. *CsUBL5* and *CsACT7* are control genes. Bar plots represent mean ± SD (n=3). \* *P* < 0.05, \*\* *P* < 0.01; ns, no significant difference (Student's *t*-test).

(F) DCFH-DA staining of citrus protoplasts expressed *CjACO1-RFP* or *RFP*. Scale bars = 10 µm. Total of 60 protoplasts were observed.

(G) Relative DCFH-DA fluorescence intensity in citrus protoplasts expressed *CjACO1-RFP* or *RFP*. Fluorescence (F488/F552 nm) was quantified using ImageJ. Bar plots represent mean ± SD (n = 60). \*\*\* *P* < 0.0001 (Kolmogorov-Smirnov test).
<figure><img src="image_15.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_16.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_17.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_18.png" /><figcaption>E</figcaption></figure>

<figure><img src="image_19.png" /><figcaption>**Figure 6. Proposed working model illustrating distinct root architecture and enhanced salt stress resistance in tetraploids**</figcaption></figure>

(A-B) Genome-wide distribution of ATAC-seq peaks detected in 4x and 2x roots of *C. reticulata* (A) and *P. trifoliata* (B). Entire roots under control conditions were used in ATAC-seq. Window size: gene body ± 3.0 kb.

(C) ATAC-seq profiles at *ACO1* gene locus. Dotted box indicated differentially accessible peaks between 4x and 2x roots. Black arrow indicating gene transcriptional direction.

(D) Molecular working model depicting the distinct root architecture and enhanced salt tolerance in tetraploids. Tetraploid roots exhibit elevated ethylene levels due to increased expression of *ACO1* and *ACS6*, consequently influencing the distinctive root architecture observed in tetraploid plants. Meanwhile, higher ethylene levels stimulate the transcription of genes associated with JA, ABA, and flavonol biosynthesis, as well as genes involved in ROS scavenging. This cascade enhances the root's capacity for ROS scavenging, ultimately contributing to the enhanced salt tolerance observed in tetraploids.

(E) Significance of root system remodeling by genome doubling in flowering plant evolution. After whole genome doubling, the root system of the neo-tetraploid was remodeled, resulting in stronger roots and enhanced salt tolerance, which facilitated neo-tetraploid survival and establishment in specific soil environments. Subsequent diploidization and speciation led to the evolution of the current flowering plants.
<figure><img src="image_20.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_21.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_22.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_23.png" /><figcaption>E</figcaption></figure>

<figure><img src="image_24.png" /><figcaption>F</figcaption></figure>

<figure><img src="image_25.png" /><figcaption>**Figure 6. Tetraploidization results in hypomethylation and enhanced chromatin accessibility of ACO1**</figcaption></figure>

(A) CG DNA methylation (mCG) levels across *ACO1* gene body in 4x and 2x roots of *C. reticulata* and *P. trifoliata*. Entire roots under control conditions were used in whole genome bisulfite sequencing. Arrow indicates gene transcriptional direction. Black line indicates the region for bisulfite sequencing-PCR analysis.

(B) Estimation of methylation levels across *ACO1* gene body in 4x and 2x roots of *C. reticulata* and *P. trifoliata*. The pattern is representative of analyses of three independent experiments with similar results were obtained for each. Bisulfite sequencing-PCR was performed for the region indicated in (A).

(C) Statistical analysis of methylation levels across *ACO1* gene body in 4x and 2x roots of *C. reticulata* and *P. trifoliata*. A total of 12 independent clones from each reaction were sequenced in bisulfite sequencing-PCR analysis. Values represent mean ± SD. \*P < 0.05, \*\*P < 0.01; ns, no significant difference (n = 3, Student's t-test).

(D-E) Genome-wide distribution of ATAC-seq peaks detected in 4x and 2x roots of *C. reticulata* (D) and *P. trifoliata* (E). Entire roots under control conditions were used in ATAC-seq. Window size: gene body ± 3.0 kb.

(F) ATAC-seq and RNA-seq profiles at *ACO1* gene locus. Dotted box indicated differentially accessible peaks between 4x and 2x roots. Black arrow indicating gene transcriptional direction.
A

<figure><img src="image_26.png" /><figcaption>**Figure 7. Proposed working model illustrating distinct root architecture and enhanced salt stress resistance in tetraploids**</figcaption></figure>

B

<figure><img src="image_27.png" /></figure>

(A) Molecular working model depicting the distinct root architecture and enhanced salt tolerance in tetraploids. Tetraploid roots exhibit elevated ethylene levels due to increased expression of *ACO1* and *ACS6*, consequently influencing the distinctive root architecture observed in tetraploid plants. Meanwhile, higher ethylene levels stimulate the transcription of genes associated with JA, ABA, and flavonol biosynthesis, as well as genes involved in ROS scavenging. This cascade enhances the root's capacity for ROS scavenging, ultimately contributing to the enhanced salt tolerance observed in tetraploids.

(B) Significance of root system remodeling by genome doubling in flowering plant evolution. After whole genome doubling, the root system of the neo-tetraploid was remodeled, resulting in stronger roots and enhanced salt tolerance, which facilitated neo-tetraploid survival and establishment in specific soil environments. Subsequent diploidization and speciation led to the evolution of the current flowering plants.