The RGI1–BAK1 module acts as the main receptor–coreceptor pair for regulating primary root gravitropism and meristem activity in response to RGF1 peptide in Arabidopsis

ABSTRACT

ROOT MERISTEM GROWTH FACTOR1 (RGF1) and its receptors RGF1 INSENSITIVEs (RGIs), a group of leucine-rich repeat receptor kinases, promote primary root meristem activity via a mitogen-activated protein kinase (MPK) signaling cascade and control root gravitropism in Arabidopsis. Genetic analyses and in vitro binding assays have indicated that among five RGIs identified in Arabidopsis, RGI1, RGI2, and RGI3 recognize RGF1 peptides. However, it remains unclear whether the RGF1 peptide is redundantly recognized by these RGIs or mainly by a single RGI in the regulation of primary root meristem activity. In the present study, we analyzed root meristem growth of the rgi1, rgi2, and rgi3 single mutants in response to RGF1 treatment and observed a significantly decreased sensitivity in meristem growth of rgi1 and complete insensitivity in rgi1 rgi2 rgi3 triple mutant compared with the wild type but not in the rgi1 and rgi2 single mutants. We also observed that both root gravitropism and meristem growth in the BRASSINOSTEROID INSENSITIVE1-ASSOCIATED RECEPTOR KINASE 1 (bak1) single mutant were insensitive to RGF1 peptide treatment, whereas other serk mutants, such as serk1, serk2, and serk4, were fully sensitive to RGF1 peptide like the wild type. These mutant analyses suggest that RGI1–BAK1 pair acts as the main receptor–coreceptor pair for regulating primary root gravitropism and meristem activity in response to RGF1 peptide in Arabidopsis.

KEYWORDS:

• Signaling peptide
• root meristem activity
• root gravitropism
• RGI1
• RGF1
• Arabidopsis
• leucine-rich repeat receptor kinase

Plant signaling peptides act like phytohormones and regulate a wide range of biological and developmental processes and biotic and abiotic stress adaptation responses.^1–6 Many signaling peptides are generated from larger precursor proteins. The precursor proteins are processed into small mature peptides by specific proteolysis and chemical modifications. Small mature peptides are then secreted from source cells and move to target cells. In target cells, leucine-rich repeat (LRR) receptor-like kinases (RLKs) that are embedded in the plasma membrane recognize these mature peptides and trigger cellular signaling to coordinate biological and developmental processes.^1,2 LRR-RLKs mostly associate with the coreceptors SOMATIC EMBRYOGENESIS RECEPTOR KINASEs (SERKs) to mediate peptide signaling.^1,2

ROOT MERISTEM GROWTH FACTORs (RGFs), also known as GOLVEN and CLAVATA3/EMBRYO-SURROUNDING REGION-RELATED-LIKE, control the primary root meristem activity through the receptors RGF1 INSENSITIVEs (RGIs), a group of LRR-RLKs.^7,8 In Arabidopsis, the RGF peptide gene family consists of 11 members. The mature RGF1 peptide is formed after proteolytic processing of the precursor proteins, tyrosine sulfation, and proline hydroxylation.⁷ Five RGIs have been identified in Arabidopsis.^9–11 An in vitro binding assay and structural analysis showed that RGI1 recognizes the RGF1 peptide.¹¹ Genetic and biochemical analyses have indicated that among five RGIs identified in Arabidopsis, RGI1, RGI2, and RGI3 act as receptors of RGF1 and that SERKs redundantly act as co-receptors of RGIs. These findings suggest that multiple RGIs can perceive RGF1 involving SERKs as co-receptors to promote primary root growth.^10,11 However, it remains unclear whether the RGF1 peptide is redundantly recognized by these RGIs or mainly by a single RGI in regulating primary root meristem activity.

The rgi quintuple mutants exhibited an extremely short root phenotype with a highly reduced number of meristematic cortex cells that was about 20% of that of the wild type.⁹ The number of meristematic cortex cells in the rgi1 rgi2 rgi3 mutant was about 55% of that of the wild type, and meristem activity was insensitive to exogenous treatment of the RGF1 peptide.¹⁰ However, the meristem activity of rgi single mutants in response to RGF1 peptide treatment has not yet been sufficiently analyzed. We have previously shown that the RGF1/RGI1 pair negatively regulates lateral root development.¹² We used a more sensitive meristem growth assay to assess the contribution of each RGI to primary root meristem growth in the presence of RGF1 peptide with the rgi1, rgi2, and rgi3 single mutants.¹³ To this end, we incubated 3-day-old rgi1–2, rgi2–1, and rgi3–1 single mutants¹², the rgi1 rgi2 rgi3 triple mutant¹⁰, and wild-type plants with or without 10 nM RGF1 for 1 day, after which we counted the cell number of meristematic cortex (Figure 1(a)). In wild-type plants, the cell number of meristematic cortex was significantly enhanced by RGF1 treatment, but this number was greatly reduced in the rgi1–2 mutant. However, the rgi2–1 and rgi3–1 mutants were fully sensitive to RGF1 peptide treatment like the wild type (Figure 1(b)). The meristematic cortex cell number was not altered in the rgi1 rgi2 rgi3 mutant by RGF1 treatment (Figure 1(c-d)). This result indicates that RGI1 plays a main role in RGF1-induced root meristem growth, whereas RGI2 and RGI3 play a minor role in this response. To identify a crucial SERK coreceptor of the RGF1–RGI1 module, we analyzed the root gravitropism and meristem activity of single serk mutants in response to RGF1. Root gravitropism of the BRASSINOSTEROID INSENSITIVE1-ASSOCIATED RECEPTOR KINASE 1 (bak1–4) single mutant was insensitive to RGF1 peptide treatment, whereas other serk single mutants, serk1–1, serk2–2, and serk4–1, were sensitive to RGF1 peptide like the wild type (Figure 2(a)).¹² Compared with that in the wild type, root meristem growth in bak1–4 was also relatively insensitive to the RGF1 peptide, whereas other serk single mutants were fully sensitive to RGF1 (Figure 2(b,c)). It has been shown that root gravitropism of the rgi1 mutants was insensitive to RGF1 peptide treatment, but other rgi single mutants were sensitive to RGF1 peptide treatment.¹² These results suggest that the RGI1/BAK1 pair acts as the main receptor-coreceptor pair for regulating primary root gravitropism and meristem activity in response to RGF1 peptide in Arabidopsis.

Figure 1. Analysis of primary root meristem activity in the wild type (WT), rgi1–2, rgi2–1, rgi3–1 single mutants, and rgi1 rgi2 rgi3 (rgi1,2,3) triple mutant in response to RGF1 peptide treatment. (a) Confocal images of propidium iodide (PI)-stained roots of 4-day-old light-grown WT, rgi1–2, rgi2–1, and rgi3–1 plants treated with mock or 10 nM RGF1 for additional 24 h. PI staining is red. White arrowheads indicate the junction between the meristematic and elongation zones. White bars indicate 100 μm. (b) Quantitative analysis of the number of meristematic cortex cells of the WT, rgi1–2, rgi2–1, and rgi3–1 mutants. Mean ± SD values were determined from at least 21 seedlings. Asterisks indicate significant differences compared with the mock treatment as determined by a Student’s t-test and heteroscedastic analysis (*P < 0.05; ***P < 0.001). (c) Confocal images of PI-stained roots of 4-day-old light-grown WT and rgi1,2,3 plants treated with mock or 10 nM RGF1 for 24 h. (d) Quantitative analysis of the number of meristematic cortex cells of the WT and rgi1,2,3 plants. Mean ± SD values were determined from at least 17 seedlings. Asterisks indicate significant differences compared with the mock treatment as determined by a Student’s t-test and heteroscedastic analysis. ns, no statistical difference.

Figure 2. Analysis of primary root meristem activity and gravitropism in the WT and various serk mutants in response to RGF1 peptide treatment. (a) Representative seedlings of the WT, serk1–1, serk2–1, bak1–4, and serk4–1 mutants, grown for 7 days after germination and treated with mock or 10 nM RGF1. (b) Confocal images of PI-stained roots of 4-day-old light-grown WT, serk1–1, serk2–1, bak1–4, and serk4–1 plants treated with mock or 10 nM RGF1 for 24 h. White bars indicate 100 μm. (c) Quantitative analysis of the number of meristematic cortex cells of the WT, serk1–1, serk2–1, bak1–4, and serk4–1 plants treated with mock or 10 nM RGF1 for 24 h. Mean ± SD values were determined from at least 20 seedlings. Asterisks indicate significant differences compared with the mock treatment as determined by a Student’s t-test and heteroscedastic analysis (***P < 0.001).

The insensitivity of the rgi1 rgi2 rgi3 mutant to RGF1 peptide treatment indicates that RGI4 and RGI5 are not involved in RGF1-induced growth of the primary root meristem, but they may be involved in root meristem growth mediated by other RGF peptides. It has been previously shown that the RGF1/RGI1 pair inhibits lateral root development.¹² Therefore, RGI1 acts as the main receptor for RGF1 in regulating Arabidopsis root development. It is intriguing that root gravitropism, meristem growth in the primary root, and lateral root development are coordinately regulated by RGF1. In the previous study, it has been shown that overexpression of various members of RGF genes in Arabidopsis causes decreased gravitropic index, enhancement of root meristem size, and lower lateral root density, indicating that there is a strong correlation between these root phenotypes regulated by RGF peptides.^7,14,15 The deregulation of the RGF function impairs the formation of auxin gradients by modulating the trafficking dynamics of the auxin efflux carrier PINFORMED2 involved in root tropic responses and meristem organization.¹⁴ A recent work further demonstrated that root tropic responses, but not root apical meristem maintenance, are regulated by asymmetric RGF-RGI signaling in the lateral root cap.¹⁶

An RGF1-RGI signaling cascade has been identified, which involves RGF1-INDUCIBLE TRANSCRIPTION FACTOR1 enhancing the stability of PLETHORA 2 (PLT2), an essential regulator of quiescent center specification and stem cell activity.¹³ YODA–MAP kinase kinase 4/5–MAP kinase 3/6 signaling cascade was shown to function downstream of the RGF1–RGI1 module to control PLT1 and PLT2 expression in the maintenance of root stem cell niche.^17,18 Whether other RGF–RGI pairs also share these signaling cascades or utilize other signaling components remains to be investigated. Other RGIs may control root development in response to other members of the RGF peptide family. Because multiple RGIs can bind to multiple RGF peptide family members but with different affinity in vitro,¹⁰ further investigations are necessary to understand which RGIs recognize which RGF peptide members in vivo.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This research was supported by grants from the Basic Research Lab Program (2020R1A42002901 to JK) and Basic Science Research Program (2021R1I1A1A01056814 to BWJ) through the National Research Foundation, funded by the Ministry of Science and ICT of Korea.