Melatonin-ROS signal module regulates plant lateral root development

ABSTRACT

Lateral root (LR) branches from primary root. LR is vital for plants acquiring water and nutrients from soil, especially under stress conditions. LR development involves the complicated signaling network, which has not yet been fully understood. Melatonin, a novel endogenous plant regulator, plays a role in the regulation of LR development. However, we still have limited knowledge about melatonin-modulated signaling during LR development. Our recent study identifies that reactive oxygen species (ROS) acts as downstream signaling of melatonin to facilitate LR development. The recently identified receptor of melatonin in plants controls a signaling module involving G protein, ROS, and Ca²⁺. Based on these findings, we propose a novel signaling network for LR development controlled by melatonin.

Melatonin (N-acetyl-5-methoxytryptamine) is an endogenous biomolecule with various functions in regulating mammalian physiology. In recent years, more and more evidence suggests that melatonin also plays a role in the regulation of plant physiology.^1,2 Melatonin is emerging as an endogenous plant master regulator involving multiple physiological actions, such as growth and abiotic/biotic stress adaptation.³ Root branching, such as the formation of adventitious root (AR) and lateral root (LR), is important for plants’ acquisition of water and nutrients.⁴ Melatonin is a novel plant signal with the function of the modulation of root system architecture, e.g., primary root (PR) elongation, AR formation, and LR development, in many plant species.^5–8 Auxin signaling is the core regulatory node that controls root system architecture, including PR elongation and LR formation.⁹ The effect of melatonin at hormone level on PR elongation has been associated with the regulation of auxin signaling.¹⁰ However, the relationship between auxin signaling and melatonin-promoted LR formation seems to be elusive.

N‐acetylserotonin‐O‐methyltransferase (ASMT) is one of the key enzymes required for melatonin biosynthesis. Transgenic Arabidopsis overexpressing ASMT showed enhanced melatonin levels, accompanied by increased LR number and decreased indoleacetic acid (IAA) level in root, but it cannot evidence the direct link between melatonin and auxin during LR development.¹¹ In another study, melatonin fails to regulate typical auxin-responsive markers during the elicitation of root branching in Arabidopsis.¹² The results from RNA-seq analysis indicate that auxin-related genes show minimal expression differences during melatonin-promoted LR formation in cucumber seedlings.¹³ These findings suggest that melatonin regulates root branching in dicots, likely acting independently of auxin signaling. Then, what is the downstream signal of melatonin during LR formation? Understanding the mechanism for melatonin-regulated LR development is a long-standing question that needs to be answered.

Either auxin or reactive oxygen species (ROS) controls the process of lateral root primordia (LRP) initiation and formation, likely through independent pathways.¹⁴ Our recent publication demonstrates that melatonin stimulates LR formation by regulating ROS signaling.¹⁵ LR development starts from the initiation of LRP emergence from the outer layer cells of PR, which is a process of cell transformation determined by cell cycle regulatory genes (CCRGs), such as CDKA, CYCD, and KRP. Melatonin not only enhances LRP number but also accelerates LRP emergence, which is dependent on endogenous ROS signaling. This has been closely linked to the involvement of ROS in melatonin-modulated expression of CCRGs.¹⁵ Similarly, the transgenic plants with enhanced melatonin level showed increased LR formation as well as endogenous ROS concentration. Decreasing ROS level in these transgenic plants prohibited the formation of LRP and LR, accompanying with the decrease in the expression of several CCRGs.¹⁶ These findings confirm that ROS acts downstream of melatonin to facilitate LR formation.

H₂O₂ and O₂^•¯ are two typical ROS in plant cells. Polyamine oxidase (PAO) and respiratory burst oxidase homolog (RBOH) have been identified to produce H₂O₂ and O₂^•¯, respectively.¹⁷ In tomato roots, melatonin targets PAO1 and RBOH3/4 to induce the generation of these two kinds of ROS, further triggering the expression of CCRGs and LPR initiation.¹⁵ RBOHs are well-characterized plasma membrane-localized proteins contributing to ROS burst in apoplast.¹⁸ In our study, tomato PAO1 was also found to be localized to apoplast as well.¹⁵ Different kinds of ROS can transform each other in apoplast. For instance, the apoplastic superoxide dismutase (SOD) can convert O₂^•¯ into H₂O₂.¹⁹ Cell wall properties are important for LRP emergency from PR.²⁰ The apoplastic peroxidase (POD) can convert H₂O₂ into hydroxyl radical (OH^•). This process plays a role in loosening cell wall, leading to cell extension growth and LRP development.^21,22 Intriguingly, the upregulation of a set of POD genes has been found during melatonin-promoted LR formation in cucumber seedlings.¹³ Thus, melatonin seems to induce apoplast ROS burst to further trigger downstream signaling for LRP and LR formation. Then, what is the upstream of ROS in apoplast during melatonin-triggered LR development?

Apoplast is the intercellular space between cell wall and plasma membrane. Apoplast is important for plant cells to sense outside signals, followed by transducing them quickly through receptor-like proteins localized in plasma membrane.²³ A kind of G-protein-coupled receptor localized in plasma membrane has been identified as the first phytomelatonin receptor (PMTR1) involving G-protein (e.g., Gα and Gγβ dimer) signaling. The binding of melatonin to PMTR1 results in the dissociation of Gα and Gγβ dimer. Then, the released Gα triggers downstream signaling.²⁴ Gα regulates LR development positively based on the evidence that Gα loss-of-function mutant has fewer LR, an effect of Gα on the modulation of root cell proliferation and division.^25,26 Interestingly, Gα activated by melatonin-PMTR1 can trigger RBOH-dependent ROS signaling.²⁴ Therefore, it can be deduced that PMTR1-triggered Gα may act upstream of ROS during melatonin-stimulated LR development.

Besides ROS, calcium (Ca²⁺) is another signal triggered by melatonin-PMTR1-Gα module.²⁴ Ca²⁺ can directly bind to the N-terminal EF-hand domains of ROBH to enhance its activity for ROS production. Ca²⁺ plays a role in the regulation of CCRGs and LR development.^27–29 LRP initiation is closely related to the increased cytosolic Ca²⁺ level in the founder cell in PR.³⁰ Therefore, Ca²⁺ may act upstream of ROS to regulate LR development. In mammals, Gα interacts with Ca²⁺ channels localized in plasma membrane to activate Ca²⁺ influx.³¹ In plants, it still needs further studies to understand which Ca²⁺ channels can be regulated by Gα. In plant root cells, H₂O₂ activates Ca²⁺ channel at either extracellular or intracellular membrane face in order to promote Ca²⁺ influx.^32,33 Therefore, H₂O₂ may also act as upstream of Ca²⁺ to form a possible H₂O₂-Ca²⁺ feedback loop to amplify the signal. However, further studies are needed to elucidate the role of this feedback loop in the downstream of melatonin-PMTR1-Gα module during LR development.

In sum, we propose a schematic model for the signal transduction of melatonin-regulated LR development (Figure 1). Melatonin binds to its receptor PTMR1 to trigger the disassociation between Gα and Gγꞵ, resulting in the release of Gα. Then, Gα interacts with Ca²⁺ channel to promote Ca²⁺ influx. Ca²⁺ binds to the EF-hand domain (located in the inner side of the membrane) of RBOH to activate its activity to produce O₂^•¯ in apoplast. The O₂^•¯ can be transformed to H₂O₂ with automatic protonation or by SOD-mediated dismutation. Melatonin can induce PAO-dependent H₂O₂ production in apoplast. Then, H₂O₂ and Ca²⁺ cross the membrane to the intracellular space (cytosol) to trigger downstream signal transduction to induce the expression of CCRGs, further resulting in the modulation of cell proliferation and division to initiate LR development. In this signaling pathway, H₂O₂ may also directly activate the Ca²⁺ channel to promote Ca²⁺ influx to form a feedback loop. It is also possible that OH^• produced from H₂O₂ (a POD-meditated process in apoplast) can help loosen cell wall to facilitate cell extension and LR formation. We deduce a schematic model for the role of melatonin-ROS in regulating LR development, but several questions still need to be answered. Can Gα and H₂O₂ interact with the same or different Ca²⁺ channels? Gγꞵ can also regulate Ca²⁺ channels in mammalian cells.³⁴ Dose Gγꞵ play a role in the downstream of melatonin for the regulation of Ca²⁺-ROS signaling? What is the mechanism for melatonin-triggered PAO-dependent H₂O₂ production? What is the mechanism to balance the Ca²⁺-H₂O₂ feedback loop? Which downstream signaling pathways of Ca²⁺-ROS are associated with melatonin-induced LR development. The answers to these questions would help us understand the signaling transduction of melatonin-induced LR development in detail, which may be applicable to modulate plant root development and nutrient acquisition. Melatonin is an emerging endogenous phytohormone with multiple physiological functions. Melatonin tends to eliminate ROS in plants upon abiotic stress conditions,^35,36 but here, we find that melatonin stimulates ROS signal to regulate LR development under normal growth conditions. Thus, melatonin may fine-tune ROS signal in plants upon different environmental conditions, leading to enhanced understanding of the biology of melatonin in plants.

Figure 1.

Schematic model for the signaling transduction of melatonin-regulated lateral root development

In root cells, phytomelatonin receptor (PMTR) located in plasma membrane senses and binds with melatonin to release Gα that further activates Ca²⁺ influx. Then, the increased cytosolic Ca²⁺ activates respiratory burst oxidase homolog (ROBH) in the inner face of the plasma membrane to produce superoxide radical (O₂^•¯) that can be transformed into hydrogen peroxide (H₂O₂) in apoplast. Melatonin may also induce polyamine (PAO)-dependent H₂O₂ production in apoplast. Then, H₂O₂ crosses plasma membrane and works with Ca²⁺ to trigger a set of cytosol signaling to induce the expression of cell cycle regulatory genes (CCRGs) in order to promote lateral root development. In this signaling network, H₂O₂ may also activate Ca²⁺ influx to form a feedback loop. And melatonin may activate apoplastic peroxidase (POD) to produce a hydroxyl radical (OH^•), leading to the cell wall relaxation and LR formation.

Funding Statement

This work was supported by the National Natural Science Foundation of China under Grant 31771705 and Jiangsu Agricultural Science and Technology Innovation Fund under Grant CX(20)1011.

Disclosure of Potential Conflicts of Interest

The authors declare no conflict of interest.