How CLE Peptides Control Root and Nodule Development
Explore the ScienceBeneath the soil surface, an intricate chemical conversation is constantly occurring between plant roots and their environment.
While plants may seem stationary and silent to human observers, they have evolved a sophisticated molecular language that allows them to coordinate development, form beneficial partnerships, and optimize their growth. At the heart of this communication system are CLE peptides—tiny protein fragments that act as crucial messengers in cell-to-cell signaling.
These peptides enable plants to maintain stem cells, form beneficial relationships with nitrogen-fixing bacteria, and fine-tune their root systems according to environmental conditions. Recent discoveries have revealed that understanding this hidden language could revolutionize agricultural practices and lead to more sustainable crop production methods. This article explores the fascinating world of CLE signaling peptides and their pivotal role in shaping root architecture and nodule development.
CLE peptides help shape root architecture for optimal nutrient uptake.
They regulate partnerships with nitrogen-fixing bacteria.
Plants use CLE signals to balance energy costs and benefits.
CLAVATA3/EMBRYO SURROUNDING REGION-RELATED (CLE) peptides are a family of small signaling molecules that plants use to transmit information between cells. The model plant Arabidopsis thaliana contains 32 different CLE genes in its genome, each potentially encoding a unique peptide messenger 1 . These peptides are initially produced as larger precursor proteins containing three key regions: an N-terminal signal peptide that directs the protein for secretion, a variable middle region, and a highly conserved CLE domain at the C-terminal end that contains the active peptide 1 .
Before these peptides can function as messengers, they undergo precise processing. The precursor protein is cleaved by various enzymes in the endoplasmic reticulum and Golgi apparatus, resulting in a mature peptide only 12-14 amino acids long 1 . For some CLE peptides, this processing includes the addition of sugar molecules (arabinosylation) through the action of Hyp O-arabinosyltransferases (HPATs), which can be critical for their function 1 .
CLE peptides function as ligand molecules that are secreted into the space between cells (apoplast). Once secreted, they bind to specific receptors on the surface of neighboring cells—typically leucine-rich repeat receptor-like kinases (LRR-RLKs) located in the plasma membrane 1 7 . This binding triggers an intracellular signaling cascade that ultimately changes the activity of genes within the target cell, modifying its development and function.
The best-characterized CLE signaling pathway involves the CLV3 peptide in the shoot apical meristem (the growing tip of plants). CLV3 is produced by stem cells and binds to CLV1/CLV2 receptor complexes on adjacent cells. This binding suppresses the expression of the WUSCHEL (WUS) transcription factor that promotes stem cell identity, thereby maintaining the proper balance between stem cell maintenance and cell differentiation . Similar CLE-receptor modules operate in root meristems and other plant tissues, allowing for precise control of development throughout the plant.
CLE peptides act as mobile signals that allow plant cells to communicate with each other, coordinating growth and development across different tissues and organs.
Legume plants (such as soybeans, peas, and medics) have formed a remarkable symbiotic relationship with nitrogen-fixing bacteria called rhizobia. This partnership begins when the plant secretes chemical signals that attract rhizobia to its roots. The bacteria then infect the plant roots and stimulate the formation of specialized organs called nodules, which provide an ideal environment for the bacteria to convert atmospheric nitrogen into ammonia that the plant can use 1 4 . In return, the plant supplies the bacteria with carbohydrates produced through photosynthesis.
While this symbiotic relationship is mutually beneficial, it comes at a significant energy cost for the plant. The process of supporting nitrogen-fixing bacteria consumes substantial photosynthetic resources that could otherwise be used for growth and reproduction. Therefore, plants must carefully control the number of nodules they form to ensure that the benefits outweigh the costs—a classic biological example of resource allocation trade-offs.
To prevent excessive nodulation, legumes have evolved a sophisticated control system called autoregulation of nodulation (AON), and CLE peptides serve as the key mobile signals in this process 1 . When rhizobia infect plant roots, they trigger the activation of specific CLE genes. In the model legume Lotus japonicus, these include LjCLE-RS1, LjCLE-RS2, and LjCLE-RS3, while in Medicago truncatula, MtCLE12 and MtCLE13 perform similar functions 1 6 .
The induction of these CLE genes requires NODULE INCEPTION (NIN), a transcription factor that is essential for multiple aspects of rhizobial symbiosis 1 4 . NIN directly binds to the promoters of CLE genes and activates their expression in response to rhizobial infection 4 .
Once produced, the CLE peptides are modified by specific enzymes and transported to the shoot through the xylem 1 . In the shoot, these peptides are perceived by receptor complexes that include proteins such as HAR1 in Lotus japonicus or SUNN in Medicago truncatula 1 6 . This recognition triggers a downstream signaling pathway that ultimately suppresses further nodulation on the roots.
| Peptide Name | Plant Species | Expression Trigger | Primary Function |
|---|---|---|---|
| CLV3 | Arabidopsis thaliana | Developmental program | Shoot and root meristem maintenance |
| CLE40 | Arabidopsis thaliana | Developmental program | Root meristem maintenance |
| LjCLE-RS1/2 | Lotus japonicus | Rhizobial infection | Suppress nodulation through AON |
| MtCLE12/13 | Medicago truncatula | Rhizobial infection | Suppress nodulation through AON |
| GmRIC1/2 | Glycine max (soybean) | Rhizobial infection | Suppress nodulation through AON |
| CLE1/3/4/7 | Arabidopsis thaliana | Low nitrogen conditions | Inhibit lateral root development |
The CLE-mediated AON pathway allows plants to integrate information about their nitrogen status with nodulation activity. When nitrogen is plentiful, plants reduce nodulation to conserve carbon resources. When nitrogen is scarce, they allow more nodules to form. This regulation is remarkably sophisticated—the same NIN transcription factor that activates CLE expression also activates CEP (C-terminally encoded peptide) genes that promote nodulation, creating a balanced system that fine-tunes the plant's response to both nitrogen availability and rhizobial infection 4 .
| Receptor Name | Plant Species | CLE Ligand | Biological Function |
|---|---|---|---|
| CLV1 | Arabidopsis thaliana | CLV3 | Meristem maintenance |
| CLV2/CRN | Arabidopsis thaliana | Multiple CLEs | Coreceptors for various CLE peptides |
| HAR1 | Lotus japonicus | LjCLE-RS1/2 | AON signaling in shoots |
| SUNN | Medicago truncatula | MtCLE12/13 | AON signaling in shoots |
| CIKs | Arabidopsis thaliana | CLV3 | Coreceptors for CLV3 signaling |
One of the most compelling experiments demonstrating the role of CLE peptides in nodulation regulation was published by Okamoto et al. in 2013, with findings summarized in more recent reviews 1 . The researchers sought to identify the mobile signal responsible for communicating from roots to shoots in the AON pathway.
Researchers first identified candidate CLE genes (LjCLE-RS1, LjCLE-RS2, and LjCLE-RS3) that were strongly induced in Lotus japonicus roots following rhizobial infection.
Using transgenic approaches, the team constitutively expressed these CLE genes in hairy roots and observed that nodulation was suppressed not only on the transformed roots but also on untransformed roots, suggesting a systemic effect.
To directly demonstrate that the CLE peptides could travel from roots to shoots, the researchers collected xylem sap from the hypocotyls of plants expressing LjCLE-RS2 and detected the presence of the mature LjCLE-RS2 peptide using advanced biochemical methods.
Through mass spectrometry, they determined that the mature LjCLE-RS2 peptide was a 13-amino-acid peptide with a hydroxyproline residue at position 7 that was modified with three arabinose molecules—a modification critical for its activity.
Using binding assays, the team demonstrated that the arabinosylated LjCLE-RS2 peptide could directly bind to the HAR1 receptor kinase, providing a direct molecular link between the mobile signal and its receptor.
This elegant series of experiments provided compelling evidence that root-derived CLE peptides serve as the long-distance signals that travel from roots to shoots to regulate nodule number. The discovery that these peptides require specific post-translational modifications (arabinosylation) for activity explained why earlier attempts to identify the AON signal had failed and highlighted the importance of these modifications in peptide function.
Furthermore, the direct demonstration of CLE peptide binding to the HAR1 receptor established a complete signaling pathway from the site of peptide production (roots) to the site of perception (shoots) and back to the site of action (roots). This work has profound implications for understanding how plants integrate local and long-distance signaling to optimize their associations with beneficial microbes.
| Enzyme Name | Location | Function in CLE Processing |
|---|---|---|
| Signal Peptide Peptidase | Endoplasmic Reticulum | Removes signal peptide from prepropeptide |
| Prolyl-4-Hydroxylases | Endoplasmic Reticulum | Adds hydroxy groups to proline residues |
| Hyp O-arabinosyltransferases (HPATs) | Golgi Apparatus | Add arabinose chains to hydroxyprolines |
| Subtilases | Extracellular Space | Proteolytically cleave propeptides to mature forms |
The Okamoto et al. experiment provided the first direct evidence that CLE peptides travel from roots to shoots as long-distance signals, establishing a complete molecular pathway for autoregulation of nodulation.
Studying the intricate functions of CLE peptides requires a specialized set of research tools and reagents.
Here are some of the key materials that plant biologists use to unravel the mysteries of peptide signaling:
Chemically synthesized versions of native CLE peptides and their modified variants are essential for testing peptide function. These are typically produced using solid-phase peptide synthesis methods, which allow for precise control over amino acid sequence and post-translational modifications 7 .
To track the movement and localization of CLE peptides within plant tissues, researchers create versions tagged with fluorescent markers such as TAMRA (5-carboxytetramethylrhodamine) or FITC (fluorescein isothiocyanate) 7 . These tags allow visualization of peptides using confocal microscopy.
Various in vitro binding assays using isolated receptor extracellular domains help determine binding affinities between CLE peptides and their candidate receptors 7 . These assays provide crucial evidence for establishing ligand-receptor relationships.
Mutant plants with defects in CLE genes, receptors, or processing enzymes are indispensable for determining gene function. Additionally, plants with reporter genes (such as GUS or GFP) driven by CLE promoters reveal when and where these genes are expressed.
For legumes that are difficult to transform, scientists use Agrobacterium rhizogenes-mediated hairy root transformation to create composite plants with transgenic roots and wild-type shoots, allowing rapid testing of gene function in roots 6 .
Advanced mass spectrometry techniques enable researchers to detect and characterize endogenous CLE peptides, including their specific post-translational modifications, directly from plant tissues 1 .
The discovery of CLE peptides as key regulators of root and nodule development has opened exciting new avenues for plant research.
These tiny signaling molecules exemplify the sophistication of plant communication systems and highlight how much we still have to learn about the molecular dialogues that shape plant growth and development.
As we deepen our understanding of CLE signaling, we move closer to practical applications in agriculture. By manipulating these signaling pathways, we might develop crops that better regulate their root architecture to explore soil more efficiently or optimize their symbiotic relationships to reduce the need for nitrogen fertilizers. These applications could significantly enhance the sustainability of agricultural systems and reduce environmental impacts.
The study of CLE peptides reminds us that plants are not passive organisms simply responding to environmental cues. Instead, they are active participants in their environments, constantly communicating through a complex chemical language that we are only beginning to decipher. As research continues, we will undoubtedly discover more fascinating aspects of this hidden world of plant communication, potentially revolutionizing how we grow our food and manage our natural ecosystems.