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Article

Synergistic Response Mechanism and Gene Regulatory Network of Arundo donax Leaf Under Multiple Stresses

by
Yixin Huangfu
†,
Yibo Sun
†,
Weiwei Chen
,
Gongyao Shi
,
Baoming Tian
,
Gangqiang Cao
,
Luyue Zhang
,
Jialin Guo
,
Fang Wei
* and
Zhengqing Xie
*
Henan International Joint Laboratory of Crop Gene Resources and Improvements, School of Agricultural Sciences, Zhengzhou University, Zhengzhou 450001, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Horticulturae 2025, 11(8), 985; https://doi.org/10.3390/horticulturae11080985
Submission received: 4 July 2025 / Revised: 5 August 2025 / Accepted: 16 August 2025 / Published: 19 August 2025
(This article belongs to the Topic Innovative Strategies for Enhancing Plant Tolerance to Abiotic and Biotic Stresses and Ensuring Food Safety in Changing Climates)
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Versions Notes

Abstract

Arundo donax exhibits strong comprehensive stress resistance and high levels of crude protein and crude fiber, making it an ideal perennial forage crop. It adapts to various abiotic stresses and serves as a new model for studying plant stress response mechanisms. A. donax frequently encounters diverse environmental stresses during agricultural production, including drought, waterlogging, and temperature extremes. However, the response mechanisms of A. donax to multiple stresses remains elusive. By analyzing publicly available transcriptome data, we identified 9089, 19,272, and 8585 differentially expressed genes (DEGs) and 742 DEGs shared in the leaves of A. donax under drought, waterlogging, and cold conditions. The data showed that A. donax exhibits differential activation patterns in endogenous hormone signaling (jasmonate/gibberellin), energy metabolism (UDP-glucosyltransferase), and nitrogen metabolism pathways (acyltransferase) under these stresses. DEGs involved in the nitrogen metabolism and phenylpropanoid metabolism pathways were significantly enriched, while the gene expression patterns of these pathways varied among the drought, waterlogging, and cold stress conditions. Different stresses could affect the nitrogen accumulation in A. donax leaves. In addition, pairwise DEG comparisons indicated active roles of antioxidant defense and photosynthetic system in multiple stress responses. Physiological measurements validated these transcriptional changes: the activities of antioxidant enzymes (catalase (CAT), superoxide dismutase (SOD), and peroxidase (POD)) increased significantly, minimizing oxidative damage. Meanwhile, the photosynthetic pigments content also decreased in response to the three stresses. Soluble sugars, pyruvate, malate, and citrate, which are involved in energy metabolism in the leaves of A. donax, accumulated to sustain themaintenance of the plant’s own energy metabolism. In conclusion, our study revealed the transcriptome-based regulatory network related with synergistic response mechanisms of A. donax leaves under multiple stress conditions.

1. Introduction

In recent years, global climate change has intensified, with the frequent occurrence of droughts, waterlogging, and extreme temperatures, leading to a deterioration of the growth environment for plants. Thus, this situation has seriously threatened the normal growth and metabolism of plants [1,2]. Relevant studies have shown that extreme weather has a significant impact on the planting area and yield of crops [3,4]. Moreover, it can lead to a reduction in soil biodiversity [5] and an increase in the degree of land desertification [6]. In response to the current environmental challenges, breeding plants with multiple functions regarding stress resistance and ecological benefits has become an important breakthrough in solving these problems.
Arundo donax L. is a fast-growing and widely distributed perennial gramineous plant [7,8]. Due to its high biomass [9] and strong adaptability to different soil conditions [10], it is highly favored by researchers. A. donax has a wide range of applications and possesses ecological and economic value in multiple aspects. A. donax L. can serve as a renewable energy source with low pollutant emissions [11,12]. It can also serve as a raw material for papermaking [13], construction [14], and feed [15], and it possesses certain medicinal value [16]. Furthermore, in terms of environmental remediation, A. donax L. plays a crucial role in absorbing heavy metals, decomposing organic pollutants, preventing soil erosion, and alleviating water eutrophication [17,18,19].
A. donax not only has a wide range of applications but also has the ability to adapt to various abiotic stresses [20]. Under drought stress, the stomatal conductance and mesophyll conductance of A. donax decreased, but its photosynthetic rate was not severely affected [21]. Research by Haworth et al. [22] further confirmed its adaptability. A. donax can regulate stomata, maintain photosynthesis and biomass accumulation, and has a strong recovery ability after re-watering [23]. Under waterlogging stress, A. donax can adapt to the waterlogged environment by regulating stomatal behavior, the activity of osmoregulatory substances, and its antioxidant enzyme system [24]. Pompeiano et al. [25] found that, under waterlogging stress, the stomatal conductance of A. donax decreases significantly to reduce oxygen consumption and prevent the accumulation of toxic substances produced by anaerobic respiration. Cold stress affects the growth and distribution of plants. As a warm-season plant, A. donax has relatively weak tolerance to cold stress. However, research shows that appropriate acclimation can enhance the tolerance of A. donax [26]. Pompeiano et al. [27] found that, under cold stress, A. donax accumulates osmoregulatory substances to lower the freezing point of cells. The activity of antioxidant enzymes increases, but the photosynthetic rate drops significantly. Moreover, physiological parameters such as relative water content and leaf area of A. donax are also affected [28].
However, research on the stress resistance mechanisms of A. donax has long been hampered by limitations stemming from its asexual propagation characteristics [29], which result in underdeveloped rapid propagation systems, and by the early lack of complete genome sequences. Nevertheless, the recent rapid advancements in functional genomics and transcriptomics have provided avenues to overcome this bottleneck: by integrating public databases and laboratory-generated transcriptome data, researchers [30,31] have successfully conducted transcriptome analyses on root and leaf tissues of A. donax subjected to various stresses, including waterlogging, drought, and high nitrogen, identifying thousands of differentially expressed genes (DEGs). These DEGs are implicated in key pathways, such as nitrogen metabolism, phenylpropanoid biosynthesis, ROS scavenging, and water balance regulation. Particularly, the recent release of the A. donax genome [32] has further promoted the generation and analysis of transcriptome profiles in its leaves and roots under diverse stress conditions, laying the foundation for a systematic understanding of its coordinated stress resistance mechanisms [30]. In this experiment, we investigated the transcriptional regulatory network of A. donax leaves under drought, flooding and cold stress to reveal its response mechanism under different stresses.

2. Materials and Methods

2.1. Experimental Design

A. donax pre-cultivated in the laboratory was used as the research material. It was planted at the Agricultural College Campus of Zhengzhou University, using asexual reproduction. The budding stems of A. donax were selected as hydroponic explants, with internodes 3 cm downwards and 10 cm upwards, and stem nodes were excised.
To examine the physiological responses of A. donax leaves to abiotic stress, uniform seedlings (matched for plant height and leaf size) were acclimatized for one week prior to experimental treatments. Plants were randomly allocated into four treatment groups (n = 9 per group). In the cold treatment group, A. donax was placed in a 4 °C refrigerator. The drought stress group received 150 mL of 10% polyethylene glycol (PEG) 6000 solution every 3 days. The waterlogging treatment group underwent complete submergence (plants were placed in a container with water flooding to 10 cm above the top leaf). All plants were cultivated in loess substrate under controlled conditions. Physiological parameters were quantified following 15 days of continuous stress exposure [30].
Following this 15 days of stress exposure, key physiological indicators in A. donax leaves were quantified using standardized assays. Measurements included soluble sugar content, pyruvic acid concentration, cell wall composition, reactive oxygen species (ROS) levels, activities of ROS-scavenging enzymes, photosynthetic pigment content, concentrations of tricarboxylic acid (TCA) cycle intermediates, photosynthesis-related parameters, and plant nitrogen content. Subsequently, transcriptome data analysis was performed. All experimental measurements included at least three biological replicates.

2.2. Observation of Leaf Phenotype

At 5, 10, and 15 days after the different treatments of A. donax, uniformly growing plants were selected for phenotype imaging. Three to five plants were chosen to assess the changes in the leaf phenotypes of A. donax, including indicators such as the degree of yellowing, curling, proportion of withered leaves, leaf thickness, and leaf area.

2.3. Determination of Energy Metabolism Substances

Soluble Sugar Content, using anthrone colorimetry [33]. Homogenize 0.1 g of leaves in 0.8 mL of 80% ethanol in an ice bath. Make up the volume of the crude extract to 1.5 mL, incubate it in a 50 °C water bath for 20 min, and then centrifuge at 12,000 rpm for 10 min. Add the samples to be tested, blank controls, and reagents successively into the microplate, and measure the absorbance at a wavelength of 620 nm.
The contents of pyruvate, citric acid, and malic acid were determined according to the protocol provided by Grace Biotechnology Co., Ltd. (G0807F, G0684W, G0862W48, Suzhou Grace Biotechnology Co., Ltd., Suzhou, China). Following 15 days of treatment (blank control, drought, waterlogging, and cold stress), all parameters were quantified using three randomly selected plants per treatment group.

2.4. Determination of the Contents of Reactive Oxygen Species (ROS) and ROS-Scavenging Enzyme Systems

Commercial kits (G0116W, G0168W, Suzhou Grace Biotechnology Co., Ltd.) were used to evaluate the levels of superoxide anion (O2−) and hydrogen peroxide (H2O2), followed by examination using a spectrophotometer (IMPLEN, Westlake Village, CA, USA) [34].
The activities of reactive oxygen species-scavenging enzymes, including superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT), were determined using commercial kits (G0108F, G0103F, G0106F, Suzhou Grace Biotechnology Co., Ltd.). Briefly, weigh 0.1 g of tissue, add 1 mL of extraction solution, homogenize it in an ice bath, and then centrifuge at 10,000 rpm at 4 °C for 10 min. Take the supernatant as the sample to be tested. Add the sample to be tested, blank control, and reagents successively into the microplate, and then measure the absorbance at 560 nm for SOD, 470 nm for POD, and 240 nm for CAT. Detection was carried out using a spectrophotometer (UV-1900, Shimadzu, Kyoto, Japan) [35].

2.5. Determination of Photosynthetic Pigment Content and Photosynthesis-Related Parameters

The content of plant carotenoids was determined using a commercial kit (BC4330, Beijing Solarbio Science and Technology Co., Ltd., Beijing, China), and the content of plant chlorophyll was also determined using a commercial kit (G0301F, Suzhou Grace Biotechnology Co., Ltd.).
Determination of Photosynthesis-related Parameters: The photosynthetic rate was measured using a portable photosynthesis system. Fully expanded green leaves were randomly selected for the measurement. Six plants were selected per treatment, and the measurements were conducted at 10:00 a.m. The saturated light intensity was set at 400 μmol m−2 s−1, the CO2 concentration at 450 μmol mol−1, and the leaf temperature was maintained at 25 °C.

2.6. Determination of Cell Wall Components

The leaf samples were dried, ground, and passed through a 40-mesh sieve for determination cell wall components. Commercial kits (G0715W48, G0708W48, Suzhou Grace Biotechnology Co., Ltd.) were used to measure cellulose and lignin contents, respectively. The anthrone colorimetric method and acetylation method were employed for the determination at 620 nm and 280 nm, respectively, and detection was performed using a microplate reader (Spark, Tecan, Männedorf, Switzerland).

2.7. Determination of Nitrogen in Plants

The nitrogen content was determined by the Kjeldahl method. First, the samples were digested using a digestion apparatus. Pre-digestion was carried out at 220 °C for 40 min, followed by digestion at 420 °C for 2 h until the solution in the digestion tube turned clear blue–green. Subsequently, the liquid in the digestion tube was measured using a Kjeldahl nitrogen analyzer [36].

2.8. Obtaining and Processing of Transcriptome Data

Transcriptome data were downloaded from NCBI’s Sequence Read Archive (www.ncbi.nlm.nih.gov/sra, accessed on 1 July 2024) using Xshell 7 software, including those from previous studies [31], guided by specific SRA numbers via the prefetch command (Table S1). Subsequently, the SRA Toolkit was utilized to convert the raw data from SRA format to FASTQ format. Quality assessment of the high-throughput sequencing data in FASTQ format was conducted using FastQC (Version 0.12.0). For data cleaning, Trimmomatic was employed to filter the sequencing data. Differential gene expression analysis was performed using RStudio (version 4.4.2). The gene expression datasets obtained under three distinct abiotic stress treatments were systematically organized and summarized to facilitate comprehensive transcriptome data analysis.

2.9. Gene Expression Analysis and Functional Enrichment

Salmon tool (version 0.13.1) [37] was used for rapid transcript quantification of RNA-seq data. Gene expression data under different treatments were obtained via quantitative analysis. Differentially expression gene (DEG) analysis was performed using the DESeq2 package (version 1.44.0) [38], with analyses conducted on the control group (CK) and three different treatment groups separately to screen for significant differentially expressed genes (DEGs) with |log2FC| > 1 and p-value < 0.05. GO (Gene Ontology) functional enrichment and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analyses were performed using Goatools (version 1.3.1) and Python scipy (version 1.11.1), respectively.

2.10. Statistical Analysis

All experiments were conducted with at least 3 biological replicates. Results reported in this study are expressed as mean ± SD. All physiological and biochemical data were statistically analyzed using GraphPad Prism software (version 10.4.1). The significance of differences between treatments was determined by one-way ANOVA. Significance levels were set as follows: * p < 0.05, ** p < 0.01, and ns indicates non-significant.

3. Results

3.1. Identification of DEGs by RNA-Seq Under Three Different Stresses

In order to investigate the mechanism of synergistic transcriptional regulation in A. donax leaves under drought, waterlogging, and cold stress, we used transcriptome sequencing data from previous studies of waterlogging- and cold-stressed A. donax leaves [31], and downloaded the RNA-Seq dataset of drought-stressed A. donax leaves from the NCBI SRA database. After quality control, a total of 444.4673 M mass-filtered pure reads were obtained, with Q20 and Q30 values above 97.52% and 92.31%, respectively. In addition, the mean value of guanine-cytosine (GC) was 49.96% (Table S2).
By analyzing the differentially expressed genes (DEGs) for the three abiotic stresses (|log2FC| ≥ 1, p-value < 0.05), we identified 9089 DEGs under drought stress, 19,272 DEGs under waterlogging stress, and 8585 DEGs under cold stress. The Venn diagram analysis was constructed based on the DEGs in drought, waterlogging, and cold treatments (Figure 1). The analysis revealed that most DEGs were uniquely regulated by a single type of abiotic stress: 5253 DEGs were specifically regulated by drought stress, 8753 DEGs by waterlogging stress, and 5388 DEGs by cold stress. In addition, there were DEGs overlapping between two or three stress types (referred to herein as common DEGs) that respond to multiple stresses. For example, there were 2950 common DEGs between drought and waterlogging stress. The lowest number of common DEGs, 1628, was observed between drought and cold stress. On the contrary, as many as 2311 DEGs were found between waterlogging and cold stresses. These results indicate that more common DEGs exist between waterlogging and drought stress. Furthermore, 742 DEGs were found to be responsive to all stresses.

3.2. Analysis of DEGs Under Single Stress of Drought, Waterlogging and Cold in A. donax

In order to explore the distinct response mechanisms of A. donax leaves to drought, waterlogging and cold stress, GO enrichment analyses were performed on DEGs specifically regulated by a single abiotic stress type to determine the biological functions of each stress-specific response gene. Specifically, drought stress-specific induced DEGs were mainly enriched in processes associated with plant endogenous hormone-related pathways, such as the jasmonate signaling pathway (GO:2000022) and gibberellin signaling pathway (GO:0009938) (Figure 2A). Similarly, DEGs particularly relevant to waterlogging stress were primarily enriched in GO terms significantly associated with energy metabolism-related pathways, including UDP-glucosyltransferase activity (GO:0035251) and carbohydrate metabolism processes (GO:0005975) (Figure 2B). In addition, DEGs uniquely responsive to cold stress were mainly enriched in nitrogen metabolism-related pathways such, as acyltransferase activity (GO:0016747) and nitrate reductase (NADPH) activity (GO:0050464) (Figure 2C). These findings suggest that nitrogen metabolism pathways in A. donax leaves may be altered under different environmental stresses such as drought, waterlogging and cold, which may underlie different response mechanisms and adaptation strategies.

3.3. Coordinated Regulation Analysis of DEGs in Response to Drought and Waterlogging Stress in A. donax

To gain deeper insights into the common and differential mechanisms by which A. donax responds to various stresses, we first analyzed the 2950 common DEGs in A. donax under drought and waterlogging stress (Figure 3A). To explore the shared response mechanisms of A. donax to drought and waterlogging stress, we examined the KEGG enrichment of the commonly regulated DEGs under these two stress conditions. The DEGs were mainly enriched in the plant hormone signal transduction pathway (ko04075), followed by carbohydrate metabolism (ko00520) and energy metabolism (ko00910) (Figure 3C). It is worth noting that the majority of the DEGs enriched in these pathways were up-regulated under drought stress, while they are down-regulated under waterlogging stress. In order to further explore the common regulatory mechanisms of A. donax in response to drought and waterlogging stress, GO enrichment analysis on these DEGs was also conducted (Figure 3B). The results indicated that these DEGs were enriched in pathways related to the phenylpropanoid metabolic process, such as phenylalanine ammonia–lyase activity (GO:0045548) and cinnamic acid biosynthetic process (GO:0009800). Moreover, heatmap analysis revealed that genes associated with the glycolysis pathway in energy metabolism were also significantly up-regulated (Figure 3D). These results suggest that A. donax adapts to drought and waterlogging stress by up-regulating phenylpropanoid metabolism and accumulating energy-metabolizing substances.

3.4. Coordinated Regulation Analysis of DEGs in Response to Drought and Cold Stress in A. donax

Subsequently, 1628 common DEGs between drought stress and cold stress were analyzed to explore the common transcriptional regulatory mechanisms of A. donax in response to drought and cold stress (Figure 4A). We conducted the KEGG enrichment analysis on the commonly regulated DEGs to investigate differences in transcriptional regulation (Figure 4C). These DEGs were predominantly enriched in signal transduction (ko04016), lipid metabolism (ko00592), and metabolism of cofactors and vitamins (ko00740). Most of the DEGs in these pathways were up-regulated under drought stress, while they were down-regulated under cold stress. Next, in order to explore the common regulatory mechanisms of A. donax in response to drought and cold stress, we performed GO enrichment analysis on DEGs with consistent expression patterns (Figure 4B). Apparently, DEGs were significantly enriched in gibberellin (GA) related GO terms, such as the negative regulation of the gibberellin-mediated signaling pathway (GO:0009938) and response to gibberellin (GO:0009739). In addition, the carotenoid catabolic process (GO:0016121) and the carotenoid dioxygenase activity (GO:0010436) related to photosynthetic pigments also showed a significant enrichment of DEGs. Furthermore, DEGs were significantly enriched in terms related to cell wall components, such as cell wall biogenesis (GO:0042546) and xylan metabolic process (GO:0010411). The analysis of the expression patterns of genes involved in carotenoids and plant cell wall components revealed that carotenoid-related genes were significantly down-regulated under both drought and cold stress, while most genes related to plant cell wall composition were up-regulated (Figure 4D). This indicates that these two processes are important components of the common response mechanism of A. donax to drought and cold stress.

3.5. Coordinated Regulation Analysis of DEGs in Response to Waterlogging and Cold Stress in A. donax

Next, an analysis was conducted on the 2311 common DEGs under waterlogging and cold stress to elucidate the common transcriptional regulatory mechanisms employed by A. donax in responding to these two stresses (Figure 5A). By conducting KEGG enrichment analysis on the commonly regulated DEGs, we analyzed the synergistic mechanisms by which A. donax responds to waterlogging and cold stress. The DEGs were mainly enriched in carbohydrate metabolism (ko00500), energy metabolism (ko00910), and signal transduction (ko04016) (Figure 5C). Most of the DEGs in these pathways were up-regulated under waterlogging stress, while they were down-regulated under cold stress. Subsequently, to further investigate common regulatory mechanisms of A. donax in adapting to waterlogging and cold stress, GO enrichment analysis was performed on the DEGs with consistent expression patterns under these two stresses. The results showed that the pathways related to the scavenging of reactive oxygen species (ROS) were enriched with DEGs, specifically including response to reactive oxygen species (GO:0000302) and the regulation of the response to oxidative stress (GO:1902882). In addition, DEGs were significantly enriched in GO terms related to nitrogen cycle regulation, such as nitrate reductase (NADPH) activity (GO:0050464), nitrate assimilation (GO:0042128), and nitric oxide biosynthetic process (GO:0006809) (Figure 5B). By analyzing the expression patterns of genes involved in reactive oxygen species (ROS) scavenging and the nitrogen cycle regulation, it was found that, under waterlogging stress, the nitrogen cycle was inhibited, and the genes responding to ROS were up-regulated. Under cold stress, the nitrogen cycle was enhanced, while the response to ROS was inhibited (Figure 5D). These findings indicate that the scavenging of ROS in the leaves and the maintenance of nitrogen steady state play important roles in the process of A. donax in resisting waterlogging and cold stress.

3.6. Coordinated Regulation Analysis of DEGs in Response to Three Stresses in A. donax

Finally, we analyzed 742 DEGs shared among the three stress conditions to elucidate the common transcriptional regulatory mechanisms employed by A. donax in response to these stresses (Figure 6A). KEGG enrichment analysis of these commonly regulated DEGs revealed significant enrichment in signaling transduction (ko04016), carbohydrate metabolism (ko00500), and lipid metabolism (ko00592) pathways (Figure 6C). To further investigate the coordinated regulatory mechanisms of A. donax under the three stresses, we performed GO enrichment analysis on DEGs showing consistent expression patterns across all stress conditions. The results indicated notable enrichment of DEGs in pathways related to gibberellin signaling, including negative regulation of gibberellin-mediated signaling pathway (GO:0009938) and response to gibberellin (GO:0009739). Additionally, significant enrichment of DEGs was observed in GO terms associated with carbohydrate metabolism regulation, such as triphosphate phosphatase activity (GO:0004805), dehalogenation involved in carbohydrate biosynthesis (GO:0005992), and carbohydrate metabolic process in response to stress (GO:0070413) (Figure 6B). These findings collectively highlight the critical roles of the gibberellin signaling pathway and carbohydrate metabolism in A. donax leaves during its adaptation to these three stresses.

3.7. Scavenging of ROS and Adjustment of Cell Wall Components Are Involved in the Response of A. donax Leaves to the Three Stresses

The above results indicate that scavenging of reactive oxygen species, modulation of cell wall components, and accumulation of energy metabolites—common mechanisms for coping with drought, waterlogging, and cold stress—play a crucial role in the response of A. donax to multiple stresses. To further verify this, A. donax was subjected to drought, waterlogging, and cold stress treatments. Observations of the plant phenotypes revealed that plant growth was inhibited under different stresses (Figure S1). The contents of superoxide anion (O2−) and hydrogen peroxide (H2O2) increased significantly under drought, waterlogging, and cold stress (Figure 7D,E). Regarding antioxidant enzymes, the activities of superoxide dismutase (SOD) and catalase (CAT) increased significantly under all three stresses, while the activity of peroxidase (POD) increased significantly only under drought and cold stress (Figure 7A–C). In addition, carotenoid content decreased significantly (Figure 7H). Compared with the control group (CK), the contents of four energy metabolism regulating substances—soluble sugars, pyruvic acid, malic acid, and citric acid—also increased significantly under drought, waterlogging, and cold stress (Figure 7I–L). Finally, cell wall components in A. donax leaves exhibited changes under these three stresses (Figure 7M,N). Specifically, the lignin content increased significantly under drought stress, while it decreased remarkably under waterlogging and cold stress. Meanwhile, cellulose content decreased significantly under all three stresses.
These results indicate that drought, waterlogging, and cold stress can cause oxidative damage to A. donax leaves. By increasing the activities of antioxidant enzymes and antioxidants contents, A. donax can maintain its redox balance. Meanwhile, A. donax also adjusts its cell wall components and accumulate energy metabolites to maintain sufficient energy and ionic balance internally.
Meanwhile, these physiological measurements are highly consistent with the molecular regulatory characteristics of specific DEG pathways under various stresses, as revealed by transcriptome analysis in the previous section, forming a systematic association from gene expression changes to physiological functional responses. Specifically, transcriptome analysis showed significant upregulation of DEGs related to ROS clearance—a molecular feature confirmed by physiological measurements demonstrating a marked increase in O2− and H2O2 contents under stress, along with enhanced activities of antioxidant enzymes such as SOD and CAT—directly validating the activation of ROS clearance mechanisms. The differential expression of genes involved in cell wall synthesis and degradation in the transcriptome is fully consistent with physiological-level phenotypic changes: lignin accumulation and cellulose reduction under drought stress, and decreased contents of both under waterlogging and cold stress, confirming the molecular basis for the dynamic regulation of cell wall components. Additionally, the enrichment pattern of DEGs in energy metabolism pathways is highly synchronized with the accumulation trends of soluble sugars, pyruvic acid, and other metabolites observed in physiological experiments, revealing the molecular regulatory logic underlying energy homeostasis maintenance. In summary, the molecular pathway characteristics derived from transcriptome data and the metabolic/phenotypic responses reflected by physiological indicators together form a multi-level, mutually verifiable evidence chain, systematically elucidating the molecular–physiological synergistic mechanisms by which A. donax responds to abiotic stress.

4. Discussion

As a model plant with extreme adaptability, A. donax is a perennial grass plant with multiple ecological and economic values. Its high cellulose content, rapid growth rate, and broad adaptability confer strong stress resistance [39]. However, due to the underdeveloped rapid propagation system and the lack of corresponding genomic data for A. donax, research on its stress resistance has been severely limited. In recent years, with the continuous in-depth research on the functions and transcriptomes of A. donax, it has become feasible to study the transcriptome data of A. donax leaves under various stresses. In this study, leaves of polyploid A. donax were used as the research material. By collecting, evaluating, filtering and analyzing transcriptome data of A. donax under various abiotic stresses (drought, waterlogging and cold), the physiological and molecular mechanisms underlying A. donax ’s response to different stresses were revealed through physiological and molecular experiments.

4.1. The Leaves of A. donax Tolerate Typical Abiotic Stresses by Activating the Scavenging of ROS

Under abiotic stress, the level of ROS in plants increases rapidly. These active oxygen molecules can also act as messengers to activate signaling pathways such as the mitogen-activated protein kinase (MAPK) pathway and the calcium ions pathway. This induces changes in gene expression within plant cells, triggering the initiation of defense mechanisms. As a result, plants synthesize more antioxidant enzymes and stress-responsive proteins to cope with abiotic stress. A complex regulatory network is formed among ROS signals, calcium signals, and phytohormone signals. When plants encounter abiotic stresses such as drought and salt stress, these signaling molecules transmit information. During abscisic acid (ABA)-induced stomatal closure, ROS transmits signals to downstream regulatory elements [40]. Under salt stress, ROS regulate the expression of genes related to salt stress by activating the mitogen-activated protein kinase (MAPK) cascade reaction [41]. Under stressful conditions, plants produce relatively large amount of ROS, which causes oxidative stress to cells. In order to resist the detrimental effects of ROS stress, plants induce the expression of certain antioxidant enzymes, such as SOD, CAT, POD, to scavenge ROS [42]. They also synthesize antioxidant substances, such as ascorbic acid and GSH, to enhance the ability of plant cells to resist oxidative stress [43].
Our study also found that, when A. donax was subjected to abiotic stress, the content of reactive oxygen species in the leaves—including superoxide anion (O2−) and hydrogen peroxide (H2O2)—was significantly higher than that in the control group (CK) (Figure 7A–E). Among these, the increase range of superoxide anion (O2−) was the highest, followed by that of hydrogen peroxide (H2O2). Under the three stresses treatments, the activities of SOD and POD in A. donax leaves exhibited a relatively significant increase compared with those in CK. These two enzymes are key components of the antioxidant defense system, as they can catalyze the conversion and scavenging of reactive oxygen species, thereby protecting cells from oxidative damage. In addition, the activity of catalase (CAT) also increased significantly under the three stress treatments, with a relatively small magnitude of increase observed under cold stress.

4.2. The Leaves of A. donax Tolerate Typical Abiotic Stresses by Changing the Composition of the Cell Wall

The cell wall is mainly composed of cellulose, hemicellulose, pectin and lignin. Under drought stress, plants enhance their mechanical strength by increasing lignin deposition. Meanwhile, they reduce pectin methylation to decrease cell wall extensibility, thereby restricting water loss [44]. Salt stress induces callose synthesis, resulting in the thickening of the cell wall and restricting transmembrane transport of sodium ions [45]. Under low-temperature conditions, the expression of arabinogalactan proteins (AGPs) in the cell wall is up-regulated. By binding to calcium ions, AGPs regulate the connection between the cell wall and the plasma membrane, maintaining membrane stability [46]. As a second messenger, hydrogen peroxide (H2O2) activates the mitogen-activated protein kinase (MAPK) pathway, induces the expression of the cellulose synthase (CesA) genes, and promotes the orderly deposition of cellulose microfibrils [47]. Abscisic acid (ABA) phosphorylates the transcription factor NAC043 through the SnRK2 kinase, promoting the expression of genes related to lignin synthesis (such as phenylalanine ammonia-lyase, PAL; 4CL). Additionally, ethylene signaling inhibits the expression of pectin-degrading enzyme genes via ethylene-insensitive 3 protein (EIN3), maintaining cell wall integrity [48]. In recent years, proteomics techniques (e.g., iTRAQ) have been applied in legume research to identify multiple stress-responsive proteins. For example, the GmPRP (proline-rich protein) in soybean can enhance salt tolerance by regulating the extensibility of the cell wall [49].
Our study found that A. donax leaves exhibit differential responses to the three stresses (Figure 7M,N). Under drought stress, the lignin content in A. donax leaves increases, while cellulose content decreases. Under waterlogging and cold stress, both the lignin and cellulose contents in the leaves decrease significantly. This indicates that, when subjected to abiotic stress, A. donax may actively adjust the content or proportion of cell walls components in its leaves to mitigate damage to the cell interior caused by abiotic stress.

4.3. The Leaves of A. donax Tolerate Typical Abiotic Stresses by Accumulating Substances Involved in Energy Metabolism

Under abiotic stresses such as drought and extreme temperatures, plants maintain cellular osmotic balance, protect biological macromolecules, and regulate redox homeostasis by accumulating energy metabolites, such as soluble sugars, proline, and lipids [50]. Drought stress induces plants to synthesize soluble sugars, such as sucrose and trehalose. These soluble sugars maintain cell turgor pressure through osmotic regulation. At the same time, they serve as a carbon source to support the expression of stress-responsive genes [51,52]. Plants promote the conversion of glucose to pyruvate by activating pyruvate kinase (PK), and the latter can act as an antioxidant to scavenge reactive oxygen species [53]. The contents of citric acid and malic acid in plant leaves increase, and they participate in the drought resistance response as osmotic regulatory substances. For example, under drought stress, the activity of mitochondrial citrate synthase (CS) in maize is enhanced, promoting the condensation of acetyl-CoA and oxaloacetic to produce citric acid. Meanwhile, the activity of malate dehydrogenase (MDH) decreases, reducing the oxidation of malic acid and maintaining cytoplasmic pH homeostasis [54]. Under salt stress, the activity of sugar transporters (e.g., AtSWEET11) is enhanced, which promotes the transportation of sugars to the vacuole to reduce the concentration of Na+ in the cytoplasm [48]. Under salt stress, the activity of α-ketoglutarate dehydrogenase (KGDH) in Arabidopsis thaliana is inhibited, reducing the conversion of α-ketoglutarate to succinyl-CoA, which leads to the obstruction of amino acid metabolism. In contrast, the activity of succinate dehydrogenase (SDH) is enhanced, promoting succinate oxidation and providing reducing power for the electron transport chain [46]. In rice roots, phosphoenolpyruvate (PEP) content decreases, and carbon flux shifts toward alanine synthesis to maintain nitrogen metabolic balance [55]. Low temperature-induced accumulation of fructose-1,6-bisphosphate enhances cold tolerance by activating the CBF transcription factors. On the contrary, high temperature stress inhibits glycolytic enzymes activity, reducing pyruvate production and impairing mitochondrial respiratory chain function [56]. Under high temperature stress, the activity of mitochondrial isocitrate dehydrogenase (IDH) in tomatoes decreases, and isocitrate content increases, triggering the antioxidant enzyme system to scavenge excessive reactive oxygen species (ROS). Conversely, low temperature stress inhibits fumarase (FH) activity, reducing the conversion of fumarate to malic acid, which leads to a decrease in the mitochondrial membrane potential [44].
This study found that, when A. donax is subjected to abiotic stress, a series of energy metabolism-related products accumulate significantly in its leaves. For instance, metabolites such as pyruvate, malic acid, and citric acid accumulate (Figure 7I–L). It is likely that under stress, A. donax adjusts corresponding pathways to maintain energy metabolism, ensuring that its cells can sustain stable energy metabolism for life activities even under adverse stress conditions.

5. Conclusions

In this study, three types of abiotic stresses—drought, waterlogging, and cold—were imposed on A. donax seedlings. Physiological and morphological changes, including leaf morphology, energy metabolites, photosynthetic pigments, cell wall components, reactive oxygen species (ROS), and their scavenging enzyme systems, were measured to comprehensively explore the commonalities and differences in the response mechanisms of A. donax under these three stresses. Meanwhile, for A. donax, a species lacking a reference genome, we collected the transcriptome data under these three abiotic stresses from our laboratory’s previous studies and the NCBI database. After performing normalization processing on the data, we carried out a joint analysis among the transcriptomes. This not only verified the reliability of the physiological data but also uncovered common pathways involved in abiotic stress responses, such as the nitrogen cycle and tricarboxylic acid cycle. Specifically, A. donax ensures its energy supply and maintains ion and osmotic balance by accumulating energy metabolites in leaves, adjusting cell wall components and nitrogen content, and scavenging ROS. These results not only provide a theoretical reference for studying the key pathways of A. donax in response to abiotic stresses but also lay a foundation for subsequent research on the key transcription factors involved in this response. This also strongly enriches our understanding of plant adaptation mechanisms to abiotic stresses.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/horticulturae11080985/s1, Figure S1: Phenotypic changes in the leaves of A. donax under three stresses; Table S1: Overview of NCBI SRA RNA-Seq accessions analyzed in this study; Table S2: Quality control of sequencing data under different stresses. And the original data files (including gene ID, GO&KEGG, and physicochemical indexes).

Author Contributions

Formal analysis, Y.H. and Y.S.; investigation, J.G., G.C. and B.T.; methodology, Y.H., Y.S. and F.W.; conceptualization, G.S. and F.W.; project administration, L.Z., W.C. and Z.X.; resources, B.T., G.S. and F.W.; data curation, Y.H. and Y.S.; writing—original draft, Y.H. and Y.S. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by the Zhengzhou Collaborative Innovation Project (Zhengzhou University) (No. 2024XTCX002), Henan Province Major Science and Technology Special Project (No. 231100320100), Henan Province Postdoctoral Research Funding Project (No. HN2024128) and Henan Province Science and Technology Research Project (No. 242102111151).

Data Availability Statement

Data are contained within the article and Supplementary Materials.

Acknowledgments

We are very grateful to the School of Agricultural Sciences, Zhengzhou University, and Henan International Joint Laboratory of Crop Gene Resources and Improvements for providing necessary support and facilities for research.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:
DEGsDifferentially expressed genes
GOGene Ontology
KEGGKyoto Encyclopedia of Genes and Genomes
CATcatalase
SODsuperoxide dismutase
PODperoxidase

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Figure 1. Differential expressed gene (DEG) analysis of A. donax roots under drought, waterlogging and cold stress.
Figure 1. Differential expressed gene (DEG) analysis of A. donax roots under drought, waterlogging and cold stress.
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Figure 2. GO annotation analysis of transcriptome data under three single stresses. (A) GO annotation analysis of transcriptome data under drought stress, (B) GO annotation analysis of transcriptome data under waterlogging stress, and (C) GO annotation analysis of transcriptome data under cold stress. The three stresses include drought, waterlogging, and cold stress.
Figure 2. GO annotation analysis of transcriptome data under three single stresses. (A) GO annotation analysis of transcriptome data under drought stress, (B) GO annotation analysis of transcriptome data under waterlogging stress, and (C) GO annotation analysis of transcriptome data under cold stress. The three stresses include drought, waterlogging, and cold stress.
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Figure 3. Analysis of common DEGs in A. donax leaves between drought and waterlogging stress. (A) Venn diagram of DEGs in A. donax leaves under drought and waterlogging stress, (B) GO enrichment analysis of co-expressed differential genes under drought and waterlogging stress, (C) Enrichment analysis of co-expressed differential gene KEGG under drought and waterlogging stress, and (D) Heatmap of entries related to phenylpropane metabolism and glycolysis.
Figure 3. Analysis of common DEGs in A. donax leaves between drought and waterlogging stress. (A) Venn diagram of DEGs in A. donax leaves under drought and waterlogging stress, (B) GO enrichment analysis of co-expressed differential genes under drought and waterlogging stress, (C) Enrichment analysis of co-expressed differential gene KEGG under drought and waterlogging stress, and (D) Heatmap of entries related to phenylpropane metabolism and glycolysis.
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Figure 4. Analysis of common DEGs in A. donax leaves between drought and cold stress. (A) Venn diagram of DEGs in A. donax leaves under drought and cold stress, (B) GO enrichment analysis of co-expressed differential genes under drought and cold stress, (C) Enrichment analysis of co-expressed differential gene KEGG under drought and cold stress, and (D) Heatmap of entries related to Carotenoid synthesis and Cell wall component.
Figure 4. Analysis of common DEGs in A. donax leaves between drought and cold stress. (A) Venn diagram of DEGs in A. donax leaves under drought and cold stress, (B) GO enrichment analysis of co-expressed differential genes under drought and cold stress, (C) Enrichment analysis of co-expressed differential gene KEGG under drought and cold stress, and (D) Heatmap of entries related to Carotenoid synthesis and Cell wall component.
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Figure 5. Analysis of common DEGs in A. donax leaves between cold and waterlogging stress. (A) Venn diagram of DEGs in A. donax leaves under cold and waterlogging stress, (B) GO enrichment analysis of co-expressed differential genes under cold and waterlogging stress, (C) Enrichment analysis of co-expressed differential gene KEGG under cold and waterlogging stress, and (D) Heatmap of entries related to Nitrogen cycle and Reactive oxygen response.
Figure 5. Analysis of common DEGs in A. donax leaves between cold and waterlogging stress. (A) Venn diagram of DEGs in A. donax leaves under cold and waterlogging stress, (B) GO enrichment analysis of co-expressed differential genes under cold and waterlogging stress, (C) Enrichment analysis of co-expressed differential gene KEGG under cold and waterlogging stress, and (D) Heatmap of entries related to Nitrogen cycle and Reactive oxygen response.
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Figure 6. Analysis of common DEGs in A. donax leaves among three stress. (A) Venn diagram of DEGs in A. donax leaves among three stress, (B) GO enrichment analysis of co-expressed differential genes among three stress, and (C) Enrichment analysis of co-expressed differential gene KEGG among three stress. The three stresses include drought, waterlogging and cold stress.
Figure 6. Analysis of common DEGs in A. donax leaves among three stress. (A) Venn diagram of DEGs in A. donax leaves among three stress, (B) GO enrichment analysis of co-expressed differential genes among three stress, and (C) Enrichment analysis of co-expressed differential gene KEGG among three stress. The three stresses include drought, waterlogging and cold stress.
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Figure 7. Determination of the physicochemical indices of A. donax leaves was conducted after 15 days of treatment under drought, waterlogging, and cold stress. (A–C) Reactive oxygen scavenging enzyme system content, (D,E) Active oxygen content, (F) Nitrogen content, (G,H) Photosynthetic pigment content, (I–L) Energy metabolite content, and (M,N) Cell wall component content. Data (mean ± SD, n = 3) were subjected to a one-way ANOVA. Different lowercase letters on the bars indicate significant differences (p < 0.05). The same letter present for different treatments indicates no significant difference among them. CK indicates the blank control group, while Drought, Waterlogging and Cold represent the treatment groups, respectively.
Figure 7. Determination of the physicochemical indices of A. donax leaves was conducted after 15 days of treatment under drought, waterlogging, and cold stress. (A–C) Reactive oxygen scavenging enzyme system content, (D,E) Active oxygen content, (F) Nitrogen content, (G,H) Photosynthetic pigment content, (I–L) Energy metabolite content, and (M,N) Cell wall component content. Data (mean ± SD, n = 3) were subjected to a one-way ANOVA. Different lowercase letters on the bars indicate significant differences (p < 0.05). The same letter present for different treatments indicates no significant difference among them. CK indicates the blank control group, while Drought, Waterlogging and Cold represent the treatment groups, respectively.
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MDPI and ACS Style

Huangfu, Y.; Sun, Y.; Chen, W.; Shi, G.; Tian, B.; Cao, G.; Zhang, L.; Guo, J.; Wei, F.; Xie, Z. Synergistic Response Mechanism and Gene Regulatory Network of Arundo donax Leaf Under Multiple Stresses. Horticulturae 2025, 11, 985. https://doi.org/10.3390/horticulturae11080985

AMA Style

Huangfu Y, Sun Y, Chen W, Shi G, Tian B, Cao G, Zhang L, Guo J, Wei F, Xie Z. Synergistic Response Mechanism and Gene Regulatory Network of Arundo donax Leaf Under Multiple Stresses. Horticulturae. 2025; 11(8):985. https://doi.org/10.3390/horticulturae11080985

Chicago/Turabian Style

Huangfu, Yixin, Yibo Sun, Weiwei Chen, Gongyao Shi, Baoming Tian, Gangqiang Cao, Luyue Zhang, Jialin Guo, Fang Wei, and Zhengqing Xie. 2025. "Synergistic Response Mechanism and Gene Regulatory Network of Arundo donax Leaf Under Multiple Stresses" Horticulturae 11, no. 8: 985. https://doi.org/10.3390/horticulturae11080985

APA Style

Huangfu, Y., Sun, Y., Chen, W., Shi, G., Tian, B., Cao, G., Zhang, L., Guo, J., Wei, F., & Xie, Z. (2025). Synergistic Response Mechanism and Gene Regulatory Network of Arundo donax Leaf Under Multiple Stresses. Horticulturae, 11(8), 985. https://doi.org/10.3390/horticulturae11080985

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