The Peptide-Encoding CLE25 Gene Modulates Drought Response in Cotton

Abstract

CLAVATA3 (CLV3)/endosperm surrounding region (CLE) peptides have been reportedly involved in plant growth and development, as well as responses to abiotic stresses. However, the stress resilience of most CLE genes in cotton remains largely unknown. Here, induced expression pattern analysis showed that GhCLE25 was obviously responsive to osmotic and salt treatments, indicating that GhCLE25 was involved in abiotic stress tolerance. Furthermore, silencing GhCLE25 or the exogenous application of CLE25p effectively led to reduced and enhanced drought tolerance, respectively, as indicated by the activities of the plants’ POD, SOD, CAT, and MDA contents, as well as their height and fresh weight. We found that the knockdown of GhCLE25 promoted seedling growth and development, with a higher plant height and fresh weight in GhCLE25-silenced plants in comparison to control plants. In addition, a comparative transcriptome analysis of TRV:00 versus TRV:GhCLE25 and Mock versus CLE25p revealed that the CLE25-mediated signaling pathway is mainly involved in defense response and phytohormone signaling. Collectively, these findings indicate diverse roles of CLE25 in regulating plant growth and response to environmental stimuli and highlight the potential utilization of CLE25 to improve drought stress in modern agriculture via CLE25p spraying.

1. Introduction

As sessile organisms in nature, plants are frequently influenced by abiotic stressors, such as salinity, drought, and high/low temperatures. These environmental adversities significantly affect plant growth, development, and immune system function, ultimately leading to a severe reduction in the output of crops, for instance, leading to a 73% decline in cotton production worldwide [1,2,3]. To confront such severe sustaining conditions, plants have evolutionally developed multiple ways to mitigate the detrimental influences of environmental stresses during their whole life cycle, primarily through the modulation of plant phytohormone signaling pathways [2,3]. Plant polypeptide hormones, as important small-molecule signaling substances, are involved in controlling various processes, including regulating plant growth and development and stress resilience [4,5,6,7,8,9,10]. These small peptides are among the smallest members of the plant proteome, less than 100 amino acid residues in length, intricately fine-tuned for plant growth and development, and function similarly to phytohormones, facilitating necessary cell-to-cell exchanges [11,12,13,14]. Among them, the largest family of peptide hormones—the CLAVATA3 (CLV3)/ENDOSPERM SURROUNDING REGION CLE (ESR) (CLE) family of peptides—are plant-specific peptide signals that participate in various biological processes, including root architecture, shoot and vascular formation, embryogenesis, and stress responses [7,15,16,17].

Recent research underscores the essential roles played by CLE peptides in Arabidopsis and apple seedlings, including AtCLE9, AtCLE14, AtCLE25, and MdCLE4/5 [10,18,19,20], and especially CLE25 and CLE9, where they coordinate responses to drought stress in an ABA-dependent manner [10,19]. For example, the CLE25 peptide moves from the roots to the leaves to modulate the expression of 9-CISEPOXYCAROTENOID DIOXYGENASE 3 (NCED3) in the leaves under drought stress, thereby promoting abscisic acid (ABA) biosynthesis and increasing plant drought tolerance; this indicates that AtCLE25 plays an important role in the systemic signaling pathway during root water deficiency [10]. Exogenous CLE9 peptide application and its overexpression could induce stomatal closure and improve drought tolerance, demonstrating that AtCLE9 is involved in the drought stress response by regulating stomatal movement under water-deficient conditions [19]. Additionally, most apple CLE genes exhibited minor sensitivity, and only some CLE genes were slightly upregulated or downregulated under NaCl stress [20]. However, in Arabidopsis, the transcript abundance of AtCLE5, AtCLE9, AtCLE14, and AtCLE44 was upregulated after salt treatment [18,19,20]. It was reported that AtCLE14 can regulate leaf senescence by modulating the homeostasis of reactive oxygen species (ROS), and the overexpression of AtCLE14 or AtCLE14p application delayed leaf senescence under salt treatment, whereas cle14 mutants displayed an accelerated progression of leaf senescence under both normal and salt conditions [20]. Despite these extensively documented findings, the complicated molecular mechanisms underlying the perception, transduction, and downstream components of CLE peptide-mediated responses deserve comprehensive elucidation. Further investigations are needed to clarify these complexities and provide a deeper insight into the molecular mechanisms dominating CLE-mediated stress responses.

Cotton (Gossypium spp.) is one of the most important fiber crops globally and a valuable source of seed oil and protein. Previously, 55 CLE genes were extracted from wild diploid cotton Gossypium raimondii [21]. Recently, 93 CLE genes were characterized as GhCLE gene family members based on the presence of a conserved CLE motif of 13 amino acids in Gossypium hirsutum [22], and GhCLV3 was identified and functionally characterized into the cotton orthologs of the highly conserved CLV3-WUS meristem maintenance circuit in cotton [23]. However, the candidate CLE peptides involved in abiotic stresses in cotton are still unknown. In this study, to explore the function of drought tolerance-related GhCLE25 homologous to AtCLE25, we carried out gene expression analysis, virus-induced gene silencing (VIGS), exogenous spraying assays, and comparative RNA-seq analysis. Our results suggest a positive role for GhCLE25 in drought tolerance.

2. Materials and Methods

2.1. Plant Materials and Growth Conditions

The induced expression patterns of GhCLE25 were analyzed in upland TM-1 stored in our laboratory. Seedlings grown in the greenhouse under conditions of 60–65% humidity, an illumination intensity of 800 mol/m²/s, and a 16 h/8 h light/dark cycle (28 °C/25 °C) were treated at the two-true-leaf-and-one-heart stage with 15% PEG6000 at 99% purity (Beijing Baiao Leibo Technology Co., Ltd., Beijing, China) and 200 mM NaCl. The root tissues were sampled at 0, 3, 6, 9, 12, and 24 h post-treatment. The collected samples were frozen and stored at −70 °C for subsequent analysis.

2.2. qRT-PCR Analysis

Total RNA was extracted from cotton seedling roots and leaves (100–200 mg) using a plant RNA extraction kit (Tiangen Biotech, Beijing, China). Then, the first-strand cDNA was synthesized using the TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix and the HiScript III RT SuperMix (Vazyme Inc., Nanjing, China). The primers used are listed in Table S1. qRT-PCR analysis was carried out on a BIO-RAD CFX96TM real-time PCR system (BIO-RAD, Hercules, CA, USA) using ChamQ SYBR qPCR Master Mix (Vazyme Inc., Nanjing, China), with Cotton histone 3 (AF024716) [24] as the internal reference. The relative expression values were calculated using the 2^−ΔCT method [25] from three biological replicates.

2.3. Virus-Induced Gene Silencing

A VIGS target sequence was designed within the coding sequence of GhCLE25 to construct pTRV2 vectors. The primers used are listed in Table S1. An empty vector (TRV:00) was used as a negative control, and CLA1 (KJ123647, TRV:CLA1) encoding 1-deoxyxylulose 5-phosphatesyn-thase was used as an indicator displaying the albino phenotype in leaves upon silencing. The remaining steps were performed according to our previous study [24].

2.4. Drought Stress Treatment

After 2 weeks, when the VIGS seedlings grown in the greenhouse under conditions of 60–65% humidity, an 800 mol/m²/s illumination intensity, and a 16 h/8 h light/dark cycle (28 °C/25 °C) had reached the two-true-leaf stage, 24 seedlings in each group with similar growth vigor were selected for natural drought treatment. Well-watered plants were included in a Mock group. The natural drought treatment was performed by withholding water continuously for 14 days and observing obvious phenotypic changes to investigate the plants’ drought tolerance. The phenotype was examined and photographed with a digital camera.

2.5. Synthesis of Peptide CLE25p and Spraying Assay

Peptide (CLE25p, RRVPNGPDPIHN) synthesis was carried out on Wang Resin (0.44 mmol/g, 0.2 mmol scale). The C-terminal residue (Asn) was coupled using Fmoc-Asn(Trt)-OH (8 equiv.), HCTU (8 equiv.), and DIEA (16 equiv.) for 50 min (2 cycles) at room temperature. The remaining amino acids were coupled via standard Fmoc-SPPS on the synthesizer to complete peptide synthesis using Fmoc-AA (6 equiv.), DIC (6 equiv.), and Oxyma (5 equiv.) at 30 °C for 30 min. Then, the peptide–resin mixture was treated for cleavage using a cocktail of TFA/trisopropylsilane/water (95:2.5:2.5) to obtain a crude peptide. The progress of the synthesis was monitored using LC-MS, and the crude peptide was purified using RP-HPLC to obtain pure peptide with a purity of >95% (146 mg, 52% yield). The peptides were dissolved in deionized water and diluted into different concentrations using deionized water. After the seedlings had grown in the greenhouse with 60–65% humidity and 800 mol/m²/s illumination intensity under the conditions of 16 h/8 h light/dark cycle (28 °C/25 °C), they were subjected to natural drought to the point where the soil was nearly dry. During pretreatment, different concentrations of CLE25p (0, 2.5, 5.0, 7.5, and 10 μM) were used for exogenous spraying, with concentrations between 2.5 and 5.0 μM and between 7.5 and 10 μM showing similar effectiveness. Therefore, water (Mock), 2.5, and 10 μM were used for subsequent experiments. The leaf surface of seedlings under drought treatment was sprayed using water, 2.5 μM, or 10 μM CLE25p solution every 2 days.

2.6. Determination of Physiological Indexes and Phenotypic Parameters

Fresh leaves of TRV:00 vs. TRV:GhCLE25 and Mock (H₂O) vs. CLE25p-treated plants were collected for the measurement of physiological indexes under drought and Mock treatments. The activities of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and malonaldehyde (MDA) were determined using the corresponding analytical kits (Nanjing Jiancheng Bioengineering Research Institute, Nanjing, China). The phenotypes were photographed, and the plant height and fresh weight were measured. Each experiment was carried out with at least three biological replicates for each treatment.

2.7. RNA-Seq Analysis

To identify the differentially expressed genes (DEGs) between the TRV:00 vs. TRV:GhCLE25 and Mock (H₂O) vs. CLE25p-treated plants, leaf tissues from each treatment group were collected for total RNA extraction, with three biological repeats. RNA sequencing libraries were prepared, and differential gene expression analysis was carried out as previously described [24]. Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) analyses were carried out to identify the enrichment of DEGs in metabolic pathways and GO terms. The default parameters were used for all bioinformatics software.

2.8. Statistical Analyses

For all experiments, data are shown as the mean ± standard deviation (SD). Statistical analyses were conducted using a two-sided Student’s t-test in Excel. * and ** represent a significant difference at the p < 0.05 and p < 0.01 levels, respectively.

3. Results

3.1. GhCLE25 Is Involved in the Response to Drought and Salt Stresses

CLE family peptides are plant-specific signaling peptides that act as mediators of cell-to-cell communication during the response to various stresses [15,16]. Therefore, we wanted to understand whether cotton CLE25 participated in the response to abiotic stresses. First, to detect the expression pattern of GhCLE25 under drought and salt stresses, we carried out qRT-PCR analysis using salt and PEG6000-treated TM-1 seedlings. The results show that GhCLE25 was significantly downregulated (p < 0.01) by osmotic stress from 3 to 24 h and induced by salt treatment in root tissues, especially at 3 h and 24 h after salt treatment (Figure 1A,B), indicating that GhCLE25 is involved in plant abiotic stress tolerance.

3.2. The Downregulation of GhCLE25 Reduces Drought Tolerance in Cotton

Subsequently, we investigated the physiological roles of GhCLE25 in plants by silencing GhCLE25 expression in G. hirsutum acc. TM-1 via the virus-induced gene silencing (VIGS) technique. The specific sequences of the GhCLE25 and GhCLA1 genes were independently inserted into a TRV2 vector to generate TRV:GhCLE25 and TRV:GhCLA1 constructs. Plants infected with TRV:GhCLA1 were used as a biological indicator to determine the moment when TRV:GhCLA1-filtrated plants demonstrated a photo-bleaching phenotype in their true leaves (Figure 2A). Then, the phenotypes of the plants infected by TRV:00 and TRV:GhCLE25 were investigated, and the leaves of the treated plants were sampled to analyze the silencing efficiency of VIGS and the expression level of GhCLE25. As expected, the expression of GhCLE25 was drastically decreased in the TRV:GhCLE25-infected plants (Figure 2B). Then, the TRV:GhCLE25-infected plants and TRV:00 plants were tested using natural drought treatment. After 14 days, the TRV:GhCLE25-infected plants showed more severe wilting and yellow leaves than the TRV:00 plants under drought stress (Figure 2C). Moreover, the height and fresh weight (FW) of the plants incubated with TRV:GhCLE25 was significantly higher than the TRV control plants under normal watering conditions; however, the height and fresh weight (FW) of the plants incubated with TRV:GhCLE25 was significantly lower than those of the TRV:00 plants under drought conditions (Figure 2D). In addition, the activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) were significantly lower, and malonaldehyde (MDA) content was significantly higher in the TRV:GhCLE25-infected plants than in the TRV:00 plants after drought stress (Figure 2E–G). Thus, the knockdown of GhCLE25 significantly reduced drought tolerance in cotton.

3.3. Exogenous CLE25p Application Enhances Drought Tolerance

The active forms of peptide signals are the mature peptides released from pro-proteins [9,26]. The CLE gene encodes a precursor protein with a full length of approximately 100 amino acids (aa), which is processed by an unknown protease to form a mature peptide with a length of 12 to 14 aa [7,27]. To determine whether the mature form of the CLE25 peptide is enough to function as a regulator of cotton responding to drought stress, we synthesized a 12 aa peptide (CLE25p, RRVPNGPDPIHN) derived from the CLE domain of the CLEL25 precursor. Cotton plants at the two-true-leaf-and-one-heart stage were sprayed with water (Mock) or 2.5 μM or 10 μM CLE25p every 2 days. Remarkably, time-course and peptide concentration analyses revealed that the exogenous application of CLE25p significantly increased the drought tolerance after recovery and acted in a concentration-dependent manner (Figure 3A). Moreover, lower levels of MDA and higher POD, SOD, and CAT activities were observed compared with the controls under both the 2.5 μM and 10 μM CLE25p treatments (Figure 3B–E). Furthermore, the exogenous spraying assay was performed using a larger number of plants, and the data show that the survival rate under the CLE25p treatment increased by 20% (p < 0.01) compared with that of the Mock treatment (Figure 4A,B). Together, our data demonstrate that the exogenous application of synthetic CLE25p is able to enhance drought tolerance in cotton.

3.4. Comparative Transcriptome Analysis Revealed the CLE25-Mediated Signaling Pathway

To better understand the mechanism by which GhCLE25 influenced seedling growth and the CLE25-mediated signaling pathway, we conducted a transcriptome analysis of leaves of TRV:00 vs. TRV:GhCLE25 and Mock vs. CLE25p, respectively. Compared with TRV:00, in total, 1,363 differentially expressed genes (DEGs) were identified in the TRV:GhCLE25 leaves (|log2(FC)| > 1, FDR < 0.05), of which 509 were upregulated and 854 were downregulated (Figure 5A and Supplemental Table S2). The Gene Ontology (GO) enrichment analysis of downregulated DEGs indicated that some entries involved in the “intrinsic component of membrane” and “membrane part” were enriched for cellular components; “chitin binding”, “chitinase activity”, and “catalytic activity” were enriched for molecular function; and “isoprenoid biosynthetic process”, “defense response to nematode”, and “negative regulation of peptidase activity” were enriched for biological processes (Figure 5B). Compared with the Mock treatment, a total of 2128 differentially expressed genes (DEGs) were identified in the CLE25p-sprayed leaves, of which 549 were upregulated and 1579 were downregulated (Figure 5C and Supplemental Table S3). The KEGG analysis showed that a variety of entries enriched from upregulated DEGs in CLE25p-sprayed leaves were mainly involved in “plant–pathogen interaction” and “MAPK signaling pathway—plant” (Figure 5D). In addition, two DEGs (GH_D08G2009 and GH_A10G0104) encoding Leucine-rich repeat (LRR) receptor-like kinase with higher similarity to BAM proteins from Arabidopsis were identified in the Mock vs. CLE25p combination. qRT-PCR analysis validated that GH_D08G2009 and GH_A10G0104 were significantly upregulated after CLE25p application (Figure 5E,F). Furthermore, we observed five genes related to upregulated DEGs in the CLE25p application group and downregulated DEGs in the TRV:GhCLE25 group (Figure 6A), namely GH_A05G1068 (P-loop containing nucleoside triphosphate hydrolase superfamily protein), GH_D12G1730 (PLATZ transcription factor family protein), GH_D02G1989 (folic acid binding/transferase), GH_D07G0187 (SAUR-like auxin-responsive protein family), and GH_D11G3404 (disease resistance protein (TIR-NBS-LRR class)) (Figure 6B), results which were further confirmed via qRT-PCR analysis (Figure 6C–G). These results indicate that defense response was the main CLE25-mediated signaling pathway.

4. Discussion

4.1. GhCLE25 Probably Played a Positive Role in the Response to Drought Stress in Cotton

Abiotic stress always has a negative effect on plant growth and can cause a substantial decline in both yield and quality in agriculture. In order to alleviate these abiotic stresses, plants respond via physiological and developmental adaptations. Among these adaptations, peptide hormones mediate intercellular communication to modulate the growth and development of plants and their response to various internal and external environmental factors [4,5]. As the largest group of peptides, CLE peptides play an essential role in modulating meristem activity in shoot and root tissues, as well as responding to abiotic environmental stimuli [27,28,29,30]. Under water deficiency, the CLE25 peptide moves from the roots to the leaves to promote NCED3 expression, leading to an increase in ABA and enhancing plant drought tolerance [10]. Exogenous CLE9 peptide application and the overexpression of AtCLE9 could induce stomatal closure and improve drought tolerance [19]. This indicates that AtCLE25 and AtCLE9 play positive roles in response to drought stress. In this study, we identified that a gene encoding the CLV3/ ESR (CLE) small peptide, GhCLE25, was related to drought and salt stress tolerance in cotton. The transcripts of GhCLE25 were substantially regulated by PEG and salt stresses (Figure 1A,B), and the silencing of GhCLE25 and application of the synthesized 12 aa peptide (CLE25p) also significantly reduced and increased drought tolerance in cotton, respectively (Figure 2 and Figure 3), as indicated by more severe wilting, yellow leaves, and decreased antioxidant activity in GhCLE25-silenced plants and increased antioxidant activity and a higher survival rate in CLE25p-sprayed plants, respectively. These data indicate that GhCLE25 positively regulates drought response in cotton.

In vivo and in vitro experiments have indicated that the CLE motif is the functional domain, and the functional 12 aa CLE small peptides come from their 14 aa CLE motifs [27,29,30]. The exogenous application of synthesized 12 aa CLE42p functionally stimulated the overexpression of CLE42 and delayed leaf senescence, indicating that peptides containing only the C-terminal CLE domain are enough to support CLE activities, which is consistent with previous findings [27,29,30]. In the present study, the exogenous spraying of CLE25p evidently enhanced cotton drought tolerance, demonstrating that the C-terminal CLE25 domain functions similar to CLE activities, which is consistent with previous findings [20]. Moreover, the SOD, POD, and CAT activities for ROS scavenging were lower and higher in GhCLE25-silenced and CLE25p-sprayed plants compared with that of TRV:00 plants and Mock-sprayed plants, respectively, suggesting GhCLE25 modulates the production of H₂O₂ to improve drought tolerance in cotton. Further studies are needed to elucidate the detailed mechanisms of the GhCLE25 protein.

4.2. CLE25-Mediated Signaling Pathway Mainly Involved in Defense Response in Cotton

It was reported that CLE peptides regulate various biological processes by integrating a variety of phytohormone signaling pathways in plants [18]. For example, the overexpression of CLE14 or CLE20 impacts root system formation by reducing cytokinin content; CLE10 suppresses protoxylem formation by inhibiting the expression of ARR5 and ARR6, which are two negative regulators involved in cytokinin signaling; and CLE40 influences cytokinin signaling by repressing the expression of genes involved in signaling and biosynthesis [31,32,33]. Moreover, CLE26 influences the primary root protophloem by modulating auxin signaling [34], while the CLE25 peptide modulates ABA biosynthesis in the leaves to regulate stomatal closure under drought conditions [10]. CLE14 also delays leaf senescence by controlling H₂O₂ homeostasis, and the CLE42-PXY module regulates leaf senescence through the ethylene signaling pathway [20,35]. TaCLE24b interacts with TaCLV1 and enhances the binding affinity and phosphorylation ability of TaCLV1 to TaSG-D1, positively fine-tuning lateral root development and thereby improving drought stress tolerance in wheat [36]. In this study, by means of RNA-seq analysis, we found five genes common to upregulated DEGs in plants treated with CLE25p and downregulated DEGs in TRV:GhCLE25 plants (Figure 5A). Among them, GH_D07G0187 encodes a SAUR-like auxin-responsive protein, and it was reported that the SAUR39 gene modulates auxin levels by negatively regulating auxin synthesis and transport in rice [37,38]. GH_D12G1730, which encodes the PLATZ transcription factor family protein, played a role in plant growth, development, senescence, and stress response. Cotton PLATZ1 mediated the signaling pathways for ABA, gibberellin (GA), and ethylene and was involved in seed germination and seedling establishment in transgenic Arabidopsis [39]. GH_D02G1989, encoding folic acid binding/transferase and folates of its product or substrate, can fine-tune auxin sensitivity and influence auxin distribution during seedling development in Arabidopsis [40]. Therefore, we speculated that cotton CLE25 affects plant height, probably by modulating phytohormone signaling, such as auxin, ABA, GA, and ethylene signals. Two other genes, GH_A05G1068 (P-loop containing nucleoside triphosphate hydrolase superfamily protein) and GH_D11G3404 (TIR-NBS-LRR class), play an important role in pathogen recognition and disease resistance [41,42,43]. Moreover, the GO enrichment analysis of downregulated DEGs in TRV:GhCLE25 plants showed that “chitin binding” and “chitinase activity” were enriched, and the KEGG analysis showed that “plant–pathogen interaction” and “mitogen-activated-protein-kinase (MAPK) signaling pathway—plant” were enriched from upregulated DEGs in CLE25p-sprayed leaves. Currently, many reports have documented that MAPK cascades participate in stress signaling by activating downstream stress-responsive proteins to respond to abiotic stresses, such as drought and salinity, in plants [44,45]. Therefore, we postulated that CLE25p regulates drought response by activating the MAPK cascade pathway. Additionally, our RNA-seq data reveal the differential expression of genes associated with disease resistance proteins such as TNLs, as well as immune-related receptors like PRRs, and this observation may suggest that CLE25 mediates the potential cross-talk between abiotic stress and immune signaling pathways. Future studies focusing on how CLE25 perceives and transduces adaptive stress signals and disease resistance will contribute to a deeper insight into the multifunctionality of CLE proteins in cotton. All together, these data demonstrate that CLE25 possibly participates in cotton pathogen resistance. This requires further investigations.

Like phytohormones, small secreted CLE peptides participate in plant development and stress responses by stimulating intracellular signaling, usually via interacting with different membrane-localized Leucine-rich repeat receptor-like kinases (RLKs) and then triggering various downstream signaling events [46,47]. For example, CLE9/10 is involved in regulating stomatal development through interacting with receptor kinase HAESA-LIKE 1 (HSL1) [19]. The CLE25–BAM module probably acts as one of the signaling molecules for long-distance signaling transduction during dehydration response [10]. In our study, we also identified two BAM genes orthologous to Arabidopsis BAM, which appeared to be significantly upregulated after the application of CLE25p. Identifying the candidate BAMs interacting with GhCLE25, validating the interaction between them, identifying downstream components, and performing a function analysis of drought response in cotton all deserve further investigation. In addition, a CRISPR/Cas-based genome editing approach for knockout GhCLE25 will be explored in the near future in order to strengthen the genetic basis of these findings in cotton. Collectively, these research findings will contribute to stress management and crop improvement strategies in modern agriculture.

5. Conclusions

In this study, we analyzed the induced expression patterns of GhCLE25 under osmotic and salt treatments, finding that GhCLE25 was involved in abiotic stress tolerance. Silencing GhCLE25 or the exogenous application of CLE25p effectively led to reduced or enhanced drought tolerance, respectively. Furthermore, comparative transcriptome analysis revealed that the CLE25-mediated signaling pathways are mainly involved in defense response and phytohormone signaling. The current research provides a clue to further understanding the probable molecular mechanisms of GhCLEs and their potential utilization in improving drought stress in cotton, which opens up new opportunities for practical application in modern agriculture.