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Article

Physiological, Cellular, and Transcriptomic Analyses Provide Insights into the Tolerance Response of Arundo donax to Waterlogging Stress

by
Dandan Wu
1,†,
Zhaoran Tian
1,†,
Jialin Guo
1,
Zhengqing Xie
1,
Baoming Tian
1,
Ziqi Liu
1,
Weiwei Chen
1,
Gangqiang Cao
1,
Luyue Zhang
1,
Tian Yang
2,
Fang Wei
1,* and
Gongyao Shi
1,*
1
Henan International Joint Laboratory of Crop Gene Resources and Improvements, School of Agricultural Sciences, Zhengzhou University, Zhengzhou 450001, China
2
College of Life Sciences, Zhengzhou University, Zhengzhou 450001, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Horticulturae 2024, 10(7), 717; https://doi.org/10.3390/horticulturae10070717
Submission received: 29 April 2024 / Revised: 7 June 2024 / Accepted: 4 July 2024 / Published: 5 July 2024
(This article belongs to the Special Issue Advancements in Enhancing Environmental Stress Tolerance of Specialty Crops in Horticulture)
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Abstract

:
Arundo donax is widely used as an ornamental plant in landscape gardening because of its adaptability to varying degrees of waterlogged conditions. However, to date, little information is available about the adaptive mechanism of A. donax under waterlogging stress. The results showed that long-term mild waterlogging efficiently induced the formation of adventitious roots (ARs) and further promoted root aerenchyma development, and that the activity of antioxidant enzymes (SOD, POD, and CAT) in Ars also was greatly enhanced after waterlogging. At the transcriptomic level, the expression of genes related to apoptosis, the regulation of cell division, ethylene biosynthesis, alginate synthesis, auxin signaling pathways, and anaerobic respiration was mostly up-regulated after the occurrence of waterlogging stress but genes involved in the abscisic acid signaling pathways were partly down-regulated, which indicated a preferential and favorable transcriptional response in regulating adventitious root development. Taken together, this study definitely advances our knowledge of the morphological, physiological, and transcriptomic responses of A. donax under waterlogging stress and sheds new lights on its adaptive mechanisms.

1. Introduction

Arundo donax L., commonly known as a giant reed, is a non-food, tall, rhizomatous, spontaneous perennial grass belonging to the Poaceae family [1]. The giant reed may grow directly in water or in a surrounding riparian area; is commonly found near rivers, lakes, ponds, and marshes; and is widely used in wetland conservation, landscaping, wastewater treatment, and ecological restoration [2].
Due to global climate change, waterlogging events are becoming more frequent [3,4]. Extended submersion can result in hypoxia and the suppression of aerobic respiration in a root system [5,6], affecting plant morphological characteristics and growth [7]. Furthermore, many toxic substances, such as ethanol and lactic acid, accumulate during anaerobic respiration [8], leading to plant death. However, after a long period of evolution and adaptation, plants can respond to waterlogging stress through morphological changes, changes in physiological and biochemical responses, and changes in gene expression, thereby reducing the adverse effects on their growth and development under waterlogging [9].
One key component of plants’ ability to withstand waterlogging is the formation of adventitious roots (ARs) and aerenchyma [10]. Many plants such as rice [11], cucumber [12], and maize [13] are capable of generating ARs from a flooded zone at the base of their stems [14]. These newly formed ARs can replace the root system that died under waterlogging [12], shortening the distance required for oxygen transportation to the root tip, increasing the oxygen concentration at the root tip, and thus enhancing gas exchange, and water and nutrient uptake in the plant; and therefore greatly improving plant tolerance to low aeration soils [15]. In addition to the production of ARs, the formation of aerenchyma within the roots, stems, and leaves of a plant, which are individually connected to each other to form a strong aeration structure, is a common strategy for plants to respond to waterlogging stress. Generally, aerenchyma can be divided into two types: cleavage and lysogenic aerenchyma. Cleavage aerenchyma are formed by the separation and expansion of cells, which thus form a gas space without cell death, but lysogenic aerenchyma are formed by the dissolution of some cells after death [16]. Plants under waterlogging stress produce lysogenic aerenchyma, for example, in the root cortex of wet plants such as reeds [17] and in the root cortex of gramineous plants such as rice, maize [18], and wheat [19]. The pith cells of many wet and aquatic plant stems tend to undergo programmed cell death (PCD) during development, forming hollow structures that specialize into aerenchyma [20], and forming pith cavities that are used for aeration or resistance to the mechanical stresses of an aquatic environment. Hypoxia due to prolonged submersion is the main reason for inducing the formation of ventilated tissues and promotes the PCD process in plants [21].
In recent years, physiological and molecular regulatory frameworks have been meticulously scrutinized in plants demonstrating waterlogging tolerance [7,22]. At the physiological level, waterlogging stress induces osmotic substance accumulation [23] and reactive oxygen species [24] (ROS) production but reduces the photosynthetic capacity of leaves [25]. Therefore, plants activate their antioxidant defense mechanisms to scavenge excess ROS. Genes and proteins involved in hormone signaling pathways and carbohydrate and energy metabolism have been reported to play important roles in the waterlogging stress response at the molecular level [26]. In the presence of ethylene, the imposition of waterlogging stress augments the expression of the respiratory burst oxidase homolog (RBOH), hereby engendering escalated concentrations of ROS and hydrogen peroxide (H2O2) [12]. ROS, functioning as pivotal intracellular secondary messengers, incite the programmed death of root cells and the formation of aerenchyma under stress [27]. Alcohol dehydrogenase (ADH) and pyruvate decarboxylase (PDC) activity play key roles in the ethanol fermentation process, and are deemed indispensable for the perpetuation of plant vitality during waterlogging conditions. Flood-tolerant plants can enhance the rate of anaerobic respiration and promote ATP production by regulating the expression of other related enzyme genes, such as ADH and PDC, to provide energy for plant growth under waterlogging [28]. In addition to this, transcription factors (TFs) are crucial in the plant response to waterlogging stress, and the main TFs reported to be involved in the plant response to waterlogging stress include bZIP (basic leucine zipper), NAC (NAM, ATAF1/2 and CUC2), WRKY, MYB (v-myb avian myeloblastosis viral), ERF (ethylene responsive factor), and the bHLH (basic helix-loop-helix) transcription factor family [29,30,31,32,33]. For example, it was found that in waterlogged soils, the WRKY and MYB transcription factors in rice roots were able to promote the formation of a barrier to oxygen radial loss by participating in the formation of corky plasmodesmata, making rice more suitable for aquatic environments [31]. The family of ethylene (ET) response factor (ERF) plays an important role in the plant tolerance to waterlogging or hypoxia stress, and the rice waterlogging tolerance genes SNORKEL1/2 (SK1/2) and Submergence1A (Sub1A) are transcription factors of the ethylene response factor class [33]. The rice Sub1A gene can regulate inhibitors of the gibberellin (GA) signaling pathway, and effectively inhibit the meristematic growth of stem tip tissues, slowing down the growth rate of a plant and reducing its energy consumption until the end of the waterlogging stress injury [34].
The giant reed is a fast-growing plant adapted to different climatic and soil conditions [35]. A. donax is also considered a leading plant for ecological governance or biomass production on marginal and degraded lands under different adverse conditions such as drought [36], salt [37], and heavy metal pollution [38]. Although several studies assessed the agronomic performance of A. donax on diverse abiotic stress, information on waterlogging tolerance is still limited. Since A. donax is one of the most common landscaping plants and can grow in wetlands for a long period of time, studies of the effect of waterlogging on A. donax become crucial to evaluate the most tolerant plants that can be used in landscaping, especially in areas with abundant rainfall. In this study, in addition to investigating the morphological, physiological, and biochemical changes in A donax, transcriptome profiling was performed to reveal the genes and regulatory networks potentially involved in the response to waterlogging stress.

2. Methods

2.1. Waterlogging Stress Treatment

Well-established A. donax with axillary were collected from an experimental base in Xingyang, China (34°36′–34°58′ N, 113°09′–113°28′ E) and used as the materials for this study. The A. donax were cut into 15 cm long segments for cultivation. The cuttings were placed in 34 cm × 30 cm (diameter × depth) pots with the branches facing upward. Each pot was filled with soil containing equal amounts of cacao peat, sand, organic matter and black soil. After that, the cuttings were placed in a greenhouse at Zhengzhou University for 180 d of acclimation growth under the same conditions.
Six-month-old cuttings were selected and classified into control and treatment groups. Each treatment was replicated three times with 20 pots of plants per replication. The waterlogging treatment group was placed in a water tank, flooded with water to 25 cm above the soil surface, and kept at a constant level for 45 days. The control group was placed next to the water tank and watered normally. The plants were maintained under a 16/8 h light/dark cycle at a controlled temperature (25/18 °C; day/night) in a well-controlled glasshouse. Daylight LEDs provided lighting. The light intensity was 200 μmol/m2/s, and the relative humidity of the air was about 70%.

2.2. Morphological Characterization and Determination of Morphological Indicators

The waterlogging treatment lasted 45 days. On day 5, 15, 30, and 45 of the experiment, underground roots were observed for their phenotype and photographed, and the plant height and number of ARs in nodes were measured. The morphological changes in the treated and untreated plants, such as the underground roots, plant height, and number of ARs in nodes, were determined for each time point.

2.3. Determination of the Physicochemical Indexes

At 5, 15, 30, and 45 days, the roots of the treated and untreated plants were collected for biochemical assays. The malondialdehyde content was determined using the thiobarbituric acid method, the proline content was determined through the sulfosalicylic acid method, and the soluble protein content was determined via Coomassie G-250 staining [39]. The superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) activities were determined according to the procedures detailed [40]. Hydrogen peroxide (H2O2), superoxide anions (O2−), and hydroxyl radicals (OH−) were determined following a spectrophotofluorometric (IMPLEN, Westlake Village, CA, USA) method [41].

2.4. Histological Analysis

The fresh roots were fixed in formalin-acetic acid-alcohol (FAA) for 24 h. The samples were dehydrated across an ethanol series (75, 85, 90, 95, and 100%) and transferred to a chloroform solution. Finally, the prepared samples were embedded in the melted paraffin wax. The solidified wax blocks were placed on a microtome to obtain 15 μm sections. The slices were placed on a slide and stained with fancy red and solid green [42]. After dehydration and transparency, a neutral gum tablet was added to seal the tablet. The sections were examined using a fluorescence microscope (Olympus BX43, Tokyo, Honshu, Japan). The number of vessels, the thickness of the exodermis, the length of the aerenchyma, and the area of the pericycle were measured with ImageJ (v1.8.0.345) [43], and the proportion of vessel area to pericycle area was calculated.
The morphological changes in the cell nuclei in the root cells were observed with DAPI staining. Transverse paraffin sections of selected roots were deparaffinized and rinsed (see the above steps). Staining was performed with 1 mg/mL 40,6-diamidino-2- phenylindole (DAPI, Sigma, Shanghai, China) for 20 min at room temperature, protected from light, and washed three times with deionized water. The fluorescence signal was observed using a laser scanning confocal microscope (LSM880, ZEISS, Oberkochen, Germany).

2.5. Transcriptome Sequencing and Annotation

Total RNA was isolated from the unstressed and stressed roots after 30 and 45 days of incubation. Samples were collected in triplicate. Twelve cDNA libraries were sequenced with the Illumina Novaseq 6000 sequencing platform (Majorbio Bio-Pharm Technology Co., Ltd., Shanghai, China). The RNA-seq data were deposited in the sequence read archive (SRA) of the NCBI database (accession number: PRJNA1069878). For the transcriptome studies without reference genomes, all clean data were assembled de novo using Trinity and evaluated for reference sequences for a subsequent analysis. The BLAST2GO [44] program was used to obtain the GO annotations of unique assembled transcripts. A metabolic pathway analysis was performed using the Kyoto Encyclopedia of Genes and Genomes (KEGG) [45].

2.6. Differential Expression Analysis and Functional Enrichment

RSEM [46] was used to quantify the gene abundance. The software used for differential expression was DESeq2 (v1.24.0), |log2FC| ≥ 1 and DEG with FDR ≤ 0.05 (DESeq2) [47] was considered significantly differentially expressed genes (DEGs). GO functional enrichment and KEGG pathway analysis were completed using Goatools (v0.6.5) and KOBAS [48], respectively.

2.7. Real-Time Validation of Selected DEGs Using qRT-PCR

In order to verify the accuracy of RNA-Seq, seven candidate genes were randomly selected for qRT-PCR. The cDNA used the same RNA samples as those used in the RNA-Seq. Three biological replicates were used for each sample. Primer Premier 5.0 was used to design gene-specific primers and these primer sequences are listed in Table S1. The 2−ΔΔCT method was utilized to calculate the relative gene expression [36,49]. All the genes were normalized with the putative A. donax actin protein with the highest homology to the sorghum AC1 gene [36].

2.8. Statistical Analysis

All the experiments were run in triplicate or more, unless otherwise stated, and the results reported in this study are presented as the mean ± SD. All morphological and biochemical data were recorded and analyzed using one-way ANOVA in GraphPad Prism 7.0. The significance levels are * p < 0.05, ** p < 0.01.

3. Results

3.1. Long-Term Waterlogging Induces AR Formation in the Nodes of A. donax

After 5 days of waterlogging, a small number of ARs emerged from the soil surface, accompanied by the emergence of shoots from the nodes of the stem. At 15 d, shoots started to develop roots, and by the 45th day, well-developed ARs were clearly observed (Figure 1B). It is noteworthy that the control group, depicted in Figure 1A, did not exhibit any formation of ARs. In addition, morphological indicators of A. donax were counted. Although there was a discernible difference in the plant height of A. donax in the treated and untreated groups, the disparity did not reach a statistical significance (Figure 1D). Figure 1C showed the formation process of ARs in the nodes of the stem, and the number and length of the ARs (Figure 1E,F). Intriguingly, the underground root system appeared to have incurred damage as a result of waterlogging (Figure S1), but newly formed ARs that emerged from the new sprout formation seemed to have replaced the damaged original root system.

3.2. Effects of Waterlogging on Physicochemical Indexes of A. donax

Waterlogging affected the malondialdehyde (MDA), soluble protein, and proline content in the roots of A. donax. After 15 days of waterlogging, the MDA content was higher than that in the control. However, the content of MDA at 30 d was lower than that at 15 d (Figure 2A). Similarly, the soluble protein and proline contents were significantly higher than those of the control at 45 d (Figure 2B,C).
On day 5, for the O2−, no difference was found in the OH− and H2O2 contents of waterlogging (Figure 2G–I). However, after 15 days of waterlogging, the contents of O2− and OH− were significantly higher than those of the control. At a later stage, the contents of the O2− and OH− radicals decreased, while a significant increase in the SOD and POD activities for waterlogged plants was observed on day 30 (Figure 2D−F).

3.3. Transcriptome Data Analysis

To examine the transcriptional differences of A. donax under control and waterlogged conditions, twelve RNA-seq libraries were sequenced. The RNA-Seq results are presented in Tables S2 and S3. Figure 3A illustrates the number of DEGs in the roots of A. donax under different comparisons. Venn diagrams of the DEGs mentioned in R_CK_30d_vs_R_WL_30d and R_CK_45d_vs_R_WL_45d were constructed, and 18,551 (12,856 up-regulated and 5275 down-regulated) DEGs were found in common (Figure 3B).
To further explore the regulatory network of A. donax under waterlogging, 12,856 and 5275 identified DEGs were analyzed for GO and KEGG enrichment, respectively. Obviously, a significant enrichment of DEGs was found in the apoptosis-related GO, such as the regulation of apoptosis (GO:0042981), the regulation of cytokinesis (GO:0032465), and the negative regulation of apoptosis (GO:0043066). The down-regulated genes were mainly enriched in the regulation of hydrogen peroxide metabolism processes (GO:0010310) (Figure 4A,B; Tables S4 and S5). A further KEGG analysis revealed the significant enrichment of the pentose phosphate pathway, citric acid cycle (TCA cycle), and hormone-related pathways (Figure 4C,D; Tables S6 and S7).

3.4. Waterlogging Promotes the Formation of Well-Developed Aerenchyma in A. donax Roots

Under hypoxic conditions, root cortical cells die and fuse to form aerenchyma. At 30 and 45 days of waterlogging, genes related to apoptosis and cell division regulation in A. donax roots were significantly up-regulated (Figure 5A,B). In order to further verify the formation of aerenchyma as PCD, DAPI staining was performed. The nuclei of the root cells of fully watered A. donax were completely round, and the fluorescent staining gave off a uniform bright blue color. The nuclei of the waterlogged underground roots and the newly formed ARs were concentrated and crescent shaped, which are the typical characteristics of PCD (Figure 5C). In addition, ARs formed under waterlogging had significantly more well-developed aerenchyma, the shape of the vessels was regular and round compared with those of the control, and the aerenchyma of the root system were more uniform after waterlogging (Figure 5D). The number of vessels was higher in waterlogged underground roots and new ARs (Figure 5E), the length of the aerenchyma was longer (Figure 5F), the exodermis was thicker (Figure 5G), and vessels accounted for a higher proportion of the pericycle (Figure 5H) to provide oxygen for waterlogging. This is necessary to facilitate long-distance oxygen transport by activating a strategy to avoid anaerobiosis.

3.5. Ethylene Synthesis, Energy Metabolism, and ROS Homeostasis of A. donax under Waterlogging Stress

Ethylene is a major plant hormone that promotes aerenchyma and AR formation under waterlogging [50]. Studies have shown that the increased synthesis of ACC in roots under waterlogging can induce a large amount of ethylene accumulation [51]. Simultaneously, 13 DEGs were found to be related to ethylene synthesis in waterlogged roots at different stages (30 and 45 days). The overall expression of ACS and ACO genes was significantly up-regulated, suggesting that these genes may be involved in the ethylene synthesis of A. donax after waterlogging (Figure 6A).
Trehalose and anaerobic respiration are the main energy supply pathways of plants under waterlogging. Trehalose synthesis is catalyzed by TPS and TPP to produce trehalose, which is degraded into glucose under the action of TRE [52]. A total of 18 DEGs were associated with trehalose synthesis, 10 TPS genes were up-regulated and 8 TPS genes were down-regulated after 30 and 45 days of waterlogging (Figure 6B). The respiration pattern of plant roots gradually changes from being aerobic to anaerobic under waterlogging [5]. Plants produce energy mainly through ethanol fermentation, lactic fermentation, and alanine fermentation under low-oxygen conditions. The RNA-seq results showed 28 DEGs in the control and waterlogged A. donax. Genes associated with energy metabolism were up-regulated in response to waterlogging, i.e., 18 ALDH genes and 1 LDH gene on days 30 and 45, while ADH1 was down-regulated at 30 days and up-regulated at 45 days (Figure 6C).
NADPH oxidase, also known as respiratory burst oxidase homolog (RBOH), regulates the production of ROS in cells. Nine RBOH in the roots of Arundo after 30 and 45 days of waterlogging were down-regulated (Figure 6D). In order to alleviate waterlogging stress, the plant activated the antioxidant protection system to eliminate or reduce the damage of ROS. SOD, CAT, and GPX were up-regulated under waterlogging conditions (Figure 6E). These results indicate that waterlogging A. donax activates an antioxidant enzyme system to eliminate the accumulation of ROS.

3.6. Plant Hormone Signaling Pathways

In 18,551 DEGs, 485 TFs were identified from 28 TF families, including MYB, AP2/ERF, NAC, WRKY, bHLH, C2C2, bZIP, B3, and C3H (Figure S2A). Since KEGG analysis showed that some DEGs were highly enriched in plant hormone signaling pathways (ko04075), we paid special attention to hormone-related DEGs in A. donax under waterlogging and found seven TFs associated with hormone signaling pathways (Figure S2B and Table S8). Plants alter the balance of plant hormone synthesis and transport through complex signaling and regulate responses to waterlogging [53].
Auxin (IAA) plays an important role in plant growth and development [54]. The IAA signal transduction pathway consists of auxin receptor TIR1, transcription suppressor AUX/IAA, auxin response factor (ARF) and downstream target genes [55]. Under waterlogging, three AUX1 were down-regulated, eight AUXIAA (five down-regulated, three up-regulated), and five ARF were down-regulated, among which ARF (gene ID: TRINITY_DN8035_c0_g1) was a TF. There were nine GH3 genes (five down-regulated and four up-regulated) and seven SAUR genes (four down-regulated and three up-regulated) (Figure 7A).
PYR/PYL is a receptor of abscisic acid (ABA) signaling. PP2C plays a negative regulatory role in the process of ABA signaling. ABF (gene ID: TRINITY_DN11428_c0_g1, TRINITY_DN35519_c0_g1) belongs to the bZIP family and plays a positive regulatory role in ABA signaling. There were a total of four PYL, with two up-regulated at 30 days and four up-regulated at 45 days. PP2C, SnRK2, and ABF were down-regulated after 30 and 45 days of waterlogging (Figure 7B).
GA is an important plant hormone that regulates plant growth and development. Three TFs were found in the gibberellin signaling metabolic pathways (one PIF3, two PIF4). PIF3 was down-regulated after 30 and 45 days of waterlogging, while two PIF4 were up-regulated (Figure 7C).
ET can regulate plant growth and development and respond to biotic and abiotic stresses. ERF is a type of TF unique to plants. For the ET signal, one ERF1 (gene ID: TRINITY_DN3539_c0_g2) was detected and down-regulated after waterlogging (Figure 7D).

3.7. Quantitative RT-PCR Confirmation of the RNA-Seq Data

To validate the reliability of the RNA-Seq results, we further randomly selected seven DEGs and tested them with qRT-PCR. As shown in Figure S3, the results for the expression changes in DEGs were consistent with those of RNA-Seq, which proved that the RNA-Seq results were real and reliable.

4. Discussion

4.1. Effects of Waterlogging on the Morphology of A. donax

Under waterlogging stress, plants show a range of symptoms of damage, including leaf yellowing, wilting, and abscission, as well as the subterranean decay of their root systems [56]. Plants adapt to flooding through morphological changes, such as increasing root spaces, thickening stems, and forming ARs and aerenchyma [57]. In our study, waterlogging induced the sprouting of dormant buds at the nodes of A. donax stems and further formed a large number of ARs, which contained well-developed aerenchyma (Figure 1). These results are consistent with previous studies [58,59]. The formation of aerenchyma is crucial for plant tolerance to waterlogging, as it not only transports oxygen from unflooded tissues to the root system, but also releases carbon dioxide and toxic volatiles from flooded tissues [60]. Therefore, the newly formed ARs can replace the damaged roots.

4.2. Formation of ARs and Aerenchyma May Be Related to Plant Hormone Signaling

The formation of ARs and aerenchyma has been shown to be helpful in alleviating waterlogging stress in plants [61]. Plant hormones mediate complex signaling in response to waterlogging stress [26]. Ethylene is a major signal in response to waterlogging stress. Waterlogging induces a large accumulation of ethylene in the stem [51]. ACS and ACO were up-regulated after waterlogging (Figure 6A). Under hypoxic conditions, ethylene can induce the death of cortical cells in an ROS-mediated manner, thus contributing to the formation of aerenchyma. Genes related to the apoptotic process and the regulation of cell division were up-regulated after waterlogging. DAPI staining further confirmed that the nuclei of the roots of A. donax had typical PCD characteristics (Figure 5). Waterlogging induced PCD in the root cells of A. donax and led to the formation of cavities, ensuring normal gas exchange, which is an important mechanism for the morphological changes in A. donax under waterlogging stress. In addition, the results showed that the expression of AUX/IAA, GH3, and SAUR in the roots of A. donax after waterlogging were consistent with previous studies (Figure 7A) [62], suggesting that GH3 acts as an important component of the IAA signaling pathway along with ARF and AUX/IAA in regulating the growth and development of plant roots. ABA has been reported to negatively regulate waterlogging tolerance, and the formation of ARs and secondary aerenchyma requires a reduction in the free ABA concentration [63]. The expression levels of PP2C and ABF in the ABA signal transduction pathway were lower (Figure 7B), and these results are similar to those of a previous study on flooding-tolerant soybeans [64]. Moreover, a decrease in free ABA is essential for waterlogged-induced GA to promote shoot elongation. Exogenous GA has been reported to effectively reduce the MDA content of the leaves and roots of oilseed rape under waterlogging stress, and GA can promote ethylene-induced AR formation, thereby improving plant tolerance to waterlogging [4]. In our transcriptome data, the expression of GA-related PIF genes was generally up-regulated (Figure 7C), which may explain the ability of A. donax to adapt to long-term waterlogging. TFs play an important role in this process of the plant response to stress, and they usually activate or repress the expression of downstream genes under stress conditions [65]. ERF-VII promotes the formation of AR systems by negatively regulating ethylene under hypoxic conditions [66]. However, although ethylene biosynthesis genes were up-regulated, waterlogging stress did not affect the downstream genes in the ethylene pathway, such as SIMKK and ERF (Figure 7D). It is noteworthy that ERF1 was down-regulated at 30 and 45 days of waterlogging, and the down-regulation was greater at 45 days. However, the genes’ precise functions should be further elucidated in the waterlogging tolerance of A. donax.

4.3. ROS Homeostasis and Energy Metabolism in A. donax under Waterlogging Stress

Waterlogging stress usually leads to the accumulation of MDA and ROS [67]. Significant differences in MDA levels at different stages indicated that the MDA levels in A. donax were time-specific and began to decrease after 15 days of waterlogging, but the content of MDA in waterlogged roots was higher during the whole treatment period (Figure 2). It is important for plants to maintain a balance between ROS accumulation and removal in response to abiotic stresses. In this study, the increase in antioxidant enzyme activities in A. donax roots under waterlogging, along with decrease in the O2− and OH− contents (Figure 2) indicated that A. donax has an effective ROS scavenging system. Transcriptomic data showed that the RBOH involved in ROS production and the SOD, CAT, and GPX genes related to antioxidant systems involved in ROS clearance showed similar characteristics (Figure 6D,E). Although excessive ROS are harmful to plant cells, ROS can also act as signaling molecules in plant cells under stress [26]. The increased expression of AtrbohD under waterlogging conditions leads to increased H2O2 production and ADH1 gene expression in Arabidopsis thaliana, increased ethanol fermentation, and increased plant survival under waterlogging [68].
As an important osmoregulatory solute, under abiotic stress, plants rapidly accumulate trehalose to protect cell membranes and proteins, among others, from damage [69]. Our results were consistent with this (Figure 6B), suggesting that A. donax may respond to waterlogging stress by increasing trehalose synthesis. Plants need to obtain the necessary energy supply through glycolysis and ethanol fermentation in response to the energy shortage caused by waterlogging stress [70]. Our results showed that the expressions of ADH, LDH, and ALDH were up-regulated under waterlogging (Figure 6C), which suggests that flood-tolerant plants can increase the rate of ethanol fermentation by regulating the expression of ADH, PDC, and other related enzyme genes, which can temporarily provide energy for plant growth under waterlogging stress [28].

5. Conclusions

In conclusion, flooded A. donax exhibited abundant AR formation, well-developed root aerenchyma, enhanced antioxidant enzyme activities, and changes in the expression of waterlogging-related genes compared with non-stressed plants. Based on these results and previous studies, we hypothesized a model for the waterlogging response in A. donax (Figure 8). Waterlogging affects changes in plant morphology, physiology, antioxidant enzyme systems, energy metabolism, and AR germination. To respond and adapt to waterlogging, A. donax have evolved to rapidly elongate their adventitious roots, form aerenchyma, and activate their antioxidant defense system. Our data revealed transcriptional changes in the downstream components of hormone signaling and anaerobic metabolisms during waterlogging. Our findings provide insight into the complex molecular events involved in the response to the waterlogging stress in A. donax, which could help develop waterlogging-resilient crops for future climates.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/horticulturae10070717/s1, Figure S1: Morphological characteristics of roots in A. donax at different stages (0, 5, 15, 30, and 45 days) of waterlogging stress; Figure S2: Statistical analysis of DEGs and TFs in A. donax; Figure S3: qRT-PCR analysis of nine randomly selected genes. Table S1: List of primers used in qRT-PCR experiments; Table S2: Statistical analysis of transcriptome data quality; Table S3: Comparison efficiency statistics for clean reads; Table S4: GO enrichment analysis of 12856 up-regulated DEGs; Table S5: GO enrichment analysis of 5275 down-regulated DEGs; Table S6: KEGG enrichment analysis of 12856 up-regulated DEGs; Table S7: KEGG enrichment analysis of 5275 down-regulated DEGs; Table S8: TFs associated with hormone signaling pathways.

Author Contributions

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

Funding

This study was supported by the Zhengzhou Collaborative Innovation Project of Zhengzhou University (No. 22XTZX09030); the Henan Province science and technology research project (No. 242102111151); the Undergraduate Innovation Project of Zhengzhou University (No. 202310459164); and the Postdoctoral Research Funding Project of Henan Province (No. HN2024128).

Data Availability Statement

Data are contained within the article and Supplementary Materials.

Conflicts of Interest

The authors declare no conflicts of interest.

References

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Figure 1. Observations of phenotypes of A. donax after 5, 15, 30, and 45 days of waterlogging. (A) No waterlogging (CK); (B) A. donax under waterlogging stress at different stages (WL); dashed line indicates the height of waterlogging; bar: 5 cm; the dashed line indicates the height of the waterlogging; (C) the number of adventitious roots; (D) the plant height; (E) the adventitious root formation, bar: 2 cm; (F) the adventitious root length, with data presented as means ± standard deviation.
Figure 1. Observations of phenotypes of A. donax after 5, 15, 30, and 45 days of waterlogging. (A) No waterlogging (CK); (B) A. donax under waterlogging stress at different stages (WL); dashed line indicates the height of waterlogging; bar: 5 cm; the dashed line indicates the height of the waterlogging; (C) the number of adventitious roots; (D) the plant height; (E) the adventitious root formation, bar: 2 cm; (F) the adventitious root length, with data presented as means ± standard deviation.
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Figure 2. The effects of waterlogging on physiological indices in roots of A. donax. (A) Malondialdehyde, (B) soluble protein, and (C) proline contents; (D) POD, (E) SOD, and (F) CAT activities; and (G) O2−, (H) OH−, and (I) H2O2 contents. Data are presented as means ± standard deviation, * p < 0.05, ** p < 0.01.CK, control; WL, waterlogging.
Figure 2. The effects of waterlogging on physiological indices in roots of A. donax. (A) Malondialdehyde, (B) soluble protein, and (C) proline contents; (D) POD, (E) SOD, and (F) CAT activities; and (G) O2−, (H) OH−, and (I) H2O2 contents. Data are presented as means ± standard deviation, * p < 0.05, ** p < 0.01.CK, control; WL, waterlogging.
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Figure 3. An analysis of DEGs in the roots of A. donax under waterlogging stress. (A) the distribution of gene expression abundance in A. donax; (B) a statistical plot of DEGs. R, root; CK_30d, after 30 days of being well-watered; WL_30d, 30 days of waterlogging; CK_45d, after 45 days of being well-watered; WL_45d, 45 days of waterlogging.
Figure 3. An analysis of DEGs in the roots of A. donax under waterlogging stress. (A) the distribution of gene expression abundance in A. donax; (B) a statistical plot of DEGs. R, root; CK_30d, after 30 days of being well-watered; WL_30d, 30 days of waterlogging; CK_45d, after 45 days of being well-watered; WL_45d, 45 days of waterlogging.
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Figure 4. GO and KEGG analysis of DEGs’ GO enrichment of up-regulated (A) and down-regulated (B) genes in co-expressed DEGs; KEGG enrichment of up-regulated (C) and down-regulated (D) genes in co-expressed DEGs.
Figure 4. GO and KEGG analysis of DEGs’ GO enrichment of up-regulated (A) and down-regulated (B) genes in co-expressed DEGs; KEGG enrichment of up-regulated (C) and down-regulated (D) genes in co-expressed DEGs.
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Figure 5. Anatomical structure of A. donax roots after waterlogging stress. (A) Expression patterns of genes related to the apoptosis process; (B) expression patterns of genes related to cell division regulation; (C) DAPI staining; scale bar: 100 µm; (D) anatomical observation of the roots of A. donax; Ac: aerenchyma; Mv: metaxylem vessels; Ex: exodermis; Pe: pericycle; (E) the total number of vessels in the root cells; (F) the length of the air cavity in the root cells; (G) the thickness of the exodermis; (H) the proportion of vessels in root cells to the pericycle, * p < 0.05, ** p < 0.01.
Figure 5. Anatomical structure of A. donax roots after waterlogging stress. (A) Expression patterns of genes related to the apoptosis process; (B) expression patterns of genes related to cell division regulation; (C) DAPI staining; scale bar: 100 µm; (D) anatomical observation of the roots of A. donax; Ac: aerenchyma; Mv: metaxylem vessels; Ex: exodermis; Pe: pericycle; (E) the total number of vessels in the root cells; (F) the length of the air cavity in the root cells; (G) the thickness of the exodermis; (H) the proportion of vessels in root cells to the pericycle, * p < 0.05, ** p < 0.01.
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Figure 6. DEGs related to ethylene synthesis, energy metabolism, and ROS homeostasis under waterlogging in A. donax. (A) The ethylene synthesis pathway and expression patterns of related genes; (B) the trehalose synthesis pathway and expression patterns of related genes; (C) expression patterns of genes associated with anaerobic respiration; (D) expression patterns of genes associated with reactive oxygen species’ production; (E) expression patterns of genes related to antioxidant enzymes. SAM, S-adenosyl-L-methionine; ACC, 1-aminocyclopropane-1-carboxylic acid; ACS, ACC synthase; ACO, aconitate hydratase; TPS, Trehalose-6-phosphate synthases; TPP, Trehalose-6-phosphate phosphatases; TRE, trehalase; LDH, Lactate dehydrogenase; PDC, pyruvate decarboxylase; ADH, alcohol dehydrogenase; ALDH, aldehyde dehydrogenase.
Figure 6. DEGs related to ethylene synthesis, energy metabolism, and ROS homeostasis under waterlogging in A. donax. (A) The ethylene synthesis pathway and expression patterns of related genes; (B) the trehalose synthesis pathway and expression patterns of related genes; (C) expression patterns of genes associated with anaerobic respiration; (D) expression patterns of genes associated with reactive oxygen species’ production; (E) expression patterns of genes related to antioxidant enzymes. SAM, S-adenosyl-L-methionine; ACC, 1-aminocyclopropane-1-carboxylic acid; ACS, ACC synthase; ACO, aconitate hydratase; TPS, Trehalose-6-phosphate synthases; TPP, Trehalose-6-phosphate phosphatases; TRE, trehalase; LDH, Lactate dehydrogenase; PDC, pyruvate decarboxylase; ADH, alcohol dehydrogenase; ALDH, aldehyde dehydrogenase.
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Figure 7. Plant hormone signaling pathways. (A) Auxin signaling pathways; (B) abscisic acid signaling pathways; (C) gibberellin signaling pathways; (D) ethylene signaling pathways. IAA, auxin; ABA, abscisic acid; GA, Gibberellin; ET, ethylene; TIR1, transport inhibitor protein 1; AUX/IAA, auxin-responsive; ARF, auxin response factor; PYR/PYL, pyrabactin resistance 1, PYR1-like; PP2C, protein phosphatase 2C; SNRK2, serine/threonine-protein kinase SRK2; GID1, gibberellin insensitive dwarf1; ETR, Ethylene receptor; CTR1, constitutive triple response 1; EIN2, ethylene insensitive 2; EIN3/EIL, ethylene insensitive 3/EIN3-Like.
Figure 7. Plant hormone signaling pathways. (A) Auxin signaling pathways; (B) abscisic acid signaling pathways; (C) gibberellin signaling pathways; (D) ethylene signaling pathways. IAA, auxin; ABA, abscisic acid; GA, Gibberellin; ET, ethylene; TIR1, transport inhibitor protein 1; AUX/IAA, auxin-responsive; ARF, auxin response factor; PYR/PYL, pyrabactin resistance 1, PYR1-like; PP2C, protein phosphatase 2C; SNRK2, serine/threonine-protein kinase SRK2; GID1, gibberellin insensitive dwarf1; ETR, Ethylene receptor; CTR1, constitutive triple response 1; EIN2, ethylene insensitive 2; EIN3/EIL, ethylene insensitive 3/EIN3-Like.
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Figure 8. A hypothesis model of the waterlogging stress response in A. donax. The arrows indicate the activation of the pathway, while the blunt end arrows indicate the inhibition of the pathway. Red and blue arrows indicate up- and down-regulation induced by waterlogging priming under waterlogging stress, respectively.
Figure 8. A hypothesis model of the waterlogging stress response in A. donax. The arrows indicate the activation of the pathway, while the blunt end arrows indicate the inhibition of the pathway. Red and blue arrows indicate up- and down-regulation induced by waterlogging priming under waterlogging stress, respectively.
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MDPI and ACS Style

Wu, D.; Tian, Z.; Guo, J.; Xie, Z.; Tian, B.; Liu, Z.; Chen, W.; Cao, G.; Zhang, L.; Yang, T.; et al. Physiological, Cellular, and Transcriptomic Analyses Provide Insights into the Tolerance Response of Arundo donax to Waterlogging Stress. Horticulturae 2024, 10, 717. https://doi.org/10.3390/horticulturae10070717

AMA Style

Wu D, Tian Z, Guo J, Xie Z, Tian B, Liu Z, Chen W, Cao G, Zhang L, Yang T, et al. Physiological, Cellular, and Transcriptomic Analyses Provide Insights into the Tolerance Response of Arundo donax to Waterlogging Stress. Horticulturae. 2024; 10(7):717. https://doi.org/10.3390/horticulturae10070717

Chicago/Turabian Style

Wu, Dandan, Zhaoran Tian, Jialin Guo, Zhengqing Xie, Baoming Tian, Ziqi Liu, Weiwei Chen, Gangqiang Cao, Luyue Zhang, Tian Yang, and et al. 2024. "Physiological, Cellular, and Transcriptomic Analyses Provide Insights into the Tolerance Response of Arundo donax to Waterlogging Stress" Horticulturae 10, no. 7: 717. https://doi.org/10.3390/horticulturae10070717

APA Style

Wu, D., Tian, Z., Guo, J., Xie, Z., Tian, B., Liu, Z., Chen, W., Cao, G., Zhang, L., Yang, T., Wei, F., & Shi, G. (2024). Physiological, Cellular, and Transcriptomic Analyses Provide Insights into the Tolerance Response of Arundo donax to Waterlogging Stress. Horticulturae, 10(7), 717. https://doi.org/10.3390/horticulturae10070717

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