Arabinogalactan protein motif-containing receptor-like kinases are likely to play the negative feedback factor to maintain proper root hair length

ABSTRACT

Coordination of the events between cytoplasm and cell wall is necessary for the proper cellular activity of plants. Cell wall-associated receptor kinases are likely to play the interface for the extra-to-internal signaling process. Arabidopsis ROOT HAIR SPECIFIC 10 (RHS10), belonging to the proline-rich extensin-like receptor kinase (PERK) family, is a Ser/Thr protein kinase with arabinogalactan protein (AGP) motifs in its extracellular domain (ECD). RHS10 and other angiosperm PERK homologs are inhibitory in root hair tip growth. The ECD deletion analysis of RHS10 indicates that proline residues, including AGP motifs, in the ECD are required for the root hair inhibition. The kinase domain of RHS10 physically interacts with an RNase (RNS2), and both RHS10 and RNS2 show the consistent genetic interaction in terms of root hair phenotype and root RNA levels. The root hair-inhibitory function of the cell wall-associated receptor kinase RHS10 may provide a negative feedback tool between cell wall and cytoplasm for the determination of proper length of the root hair.

Abbreviations

AGP

arabinogalactan protein

ECD

extracellular domain

PERK

proline-rich extensin-like receptor kinase

RHS10

ROOT HAIR SPECIFIC 10

RNS2

ribonuclease 2

The plant cell wall appears to be simple rigid wall surrounding the plasma membrane (PM). However, the cell wall plays as the interface to mediate signals not only from the neighboring cells and the environment but also from the cell itself that they are surrounding. These primary extra and internal signals could affect cell-wall integrity, which subsequently leads to generation of secondary signals from the cell wall. PM-localized receptor-like kinases (RLKs) have been proposed to mediate extracellular signals.¹ In particular, cell wall-associated RLKs have been the ideal candidates sensing the changes in cell-wall integrity.² PM-localized RLKs generally consist of an extracellular domain (ECD), a transmembrane domain, and a cytoplasmic kinase domain.¹ Among known ECDs of cell wall-associated RLKs are the epidermal growth factor-like domain for wall-associated kinases (WAKs), the lectin-like domain for lectin receptor kinases (LecRKs), the RGD-binding motif for THESEUS1, and the extensin-like domain for proline-rich extensin-like receptor kinases (PERKs).² Although they are thought to play important roles in coordinating various cellular events, such as division, expansion, and differentiation, with the dynamics of cell wall integrity, the action mechanism of these cell wall-associated RLKs has been poorly characterized.

PERKs, inhibitors in root hair growth

Recently, we have reported a study about the role of Arabidopsis ROOT HAIR SPECIFIC 10 (RHS10)/PERK13 in root hair growth.³ Originally, we identified RHS10 in the course of screening root hair-specifically expressed genes in Arabidopsis.⁴ While most characterized root hair-related genes positively regulate root hair growth, RHS10 displays a negative function in root hair growth. The loss-of-function rhs10 mutant grows more than 30% longer hairs than wild type Arabidopsis plants, and RHS10 overexpression almost completely inhibits root hair growth.^3,4 Many other PERK homologs from Arabidopsis and other angiosperms also showed a similar inhibitory effect on root hair growth, suggesting that PERKs have a similar function in root hair cells and PERK-mediated root hair inhibition is general in angiosperms.³ While other RHS-kinases are implicated in regulation of root hair polarity such as waving and branching of root hairs, RHS10 is only related with root hair tip growth.⁴

Arabinogalactan protein (AGP) motifs of the RHS10 ECD

The unique root hair-inhibitory function of PERKs raised several questions on the molecular mechanism of root hair tip growth. How does the ECD affect the intracellular kinase domain to inhibit root hair growth? What are the downstream targets of the kinase domain? Is PERK-mediated root hair inhibition general in hair-growing plants?

PERKs were named due to the extensin-like motif in their ECD.⁵ Because extensins function as structural proteins in the cell wall,^6,7 it is conceivable that the extensin-like motifs of PERKs may associate with cell-wall components and sense the changes in cell-wall integrity. The ECD of RHS10 includes 7 extensin-like motifs in its N-terminal half. However, the deletion of all the 7 extensin-like motifs did not much affect the root hair-inhibitory function of RHS10,³ raising 2 questions; extensin-like motif would not function as typical extensin, and other motifs could be implicated in the inhibitory function. Although the ECD of PERKs has extensin-like motifs (SPx, x≥3 ), it lacks the tyrosine residue that is required for crosslinking of extensin monomers, suggesting that the extensin-like motif of PERKs may not function as canonical extensin. In addition to the proline residues in the extensin-like motifs, much more proline residues spread throughout the ECD of PERKs, and many of them are integrated as arabinogalactan protein (AGP) motifs (PA/AP/SP/TP). The ECD of most PERKs include several dozens of AGP motifs. The deletion analysis of the RHS10 ECD demonstrated that the 47-residue-long ECD region, including only 2 AGP motifs or 8 proline residues, is sufficient to considerably inhibit root hair growth and that the deletion including the last AGP motif significantly hampered RHS10s root hair-inhibitory capability.³ The study did not directly show that AGP motifs are implicated in RHS10-mediated root hair inhibition. Nonetheless, because AGPs are instrumental for cell-wall association and extracellular signaling,^2,8,9 AGP motifs of the PERK ECD are conceivable factors that are required for PERK-mediated root-hair inhibition.

RHS10 targets

PERK-mediated root-hair inhibition seems to potentially overwrite any other positive influence of root hair-enhancing factors. Auxin and ethylene are the strong positive factors that stimulate root-hair tip growth at the downstream step of root hair development.^10,11 In Arabidopsis, the MYB-WD40-bHLH complex maintains the expression of GLABRA2 (GL2, a homeodomain transcription factor) in the non-hair-cell position where GL2 inhibits ROOT HAIR DEFECTIVE 6 (RHD6, a bHLH) expression.¹² Conversely, the lack of GL2 in the hair-cell position leads to the expression RHD6 that regulates downstream transcription factors for root-hair morphogenesis.¹² RHS genes, including RHS10, have the common root hair-specific cis-element (RHE) on their promoter region and are thought to play morphogenetic roles at the far downstream such as signaling, polarity, and cell wall dynamics for root hair formation.^4,13 Auxin and ethylene act downstream of RHD6, as they restore root hair growth of the rhd6 mutant.^10,11 On the other hand, these hormones act upstream of RHSs so that they induce the expression of RHS genes.¹⁴ Consistently with this mechanistic hierarchy, auxin and ethylene are not able to rescue RHS10- and other PERK-suppressed root hair.³ Although this complete suppression of root hair growth by PERKs seems due to overexpression of those PERKs, these results suggest that PERKs basically can overwrite other positive downstream processes for root hair growth.

Here, a question is what could be the RHS10 targets that play critical roles for root hair growth. In an effort to find such RHS10 kinase targets, we have found that an RNase (RNS2) interacts with the RHS10 kinase domain in yeast 2-hybrid assay.³ Consistently with this, the loss-of-function rns2 mutant grows longer root hairs and RNS2-overexpressing transformants grow shorter root hairs than wild type. Furthermore, RNA levels in the root are increased in the rhs10 mutant and decreased in the RHS10-overexpressing transformant. These results suggest that RHS10 positively regulates RNS2 to inhibit root hair growth. However, because RNS2-mediated root hair inhibition is not as strong as RHS10-medaited inhibition,³ RNS2 should be only a part of RHS10 targets. In the in vitro kinase assay, the RHS10 kinase domain itself is not able to phosphorylate RNS2, although it autophosphorylates.³ The observation that the RHS10 kinase domain without the ECD is unable to inhibit root hair growth implies that the signaling process via the ECD is necessary for the RHS10 kinase domain to phosphorylate the substrate.³

Recently, Humphrey et al.¹⁵ also showed that the kinase domain of PERKs, including RHS10/PERK13, interacts with AGC-family protein kinases; AGC1-9 and kinesin-like calmodulin-binding protein-interacting protein kinases. Although these PERK-interacting AGC kinases have not been studied in root hair growth, several other AGC kinases are known to regulate root hair development. RHS3/AGC1-6 affects root hair polarity,⁴ and its close 2 paralogs, AGC1-5 and AGC1-7, also regulate pollen tube polarity.¹⁶ AGC2-1 and INCOMPLETE ROOT HAIR ELONGATION (IRE) are implicated in root hair tip growth.^17,18

RHS10 is a native suppressor of root hair growth as shown by the longer root hair phenotype of the rhs10 mutant than that of wild type. The long root hair phenotype of rhs10 mutant results from its longer hair-growing period than wild type.³ RHS10 is one of RHS genes whose expression is induced by a common RHE-binding transcription factor. Although most RHS gene products positively function for root hair growth, at least RHS10 and RHS1 (Calmodulin-like 1, CML1) play the negative role in root hair growth.⁴ Co-expression of both positive and negative RHS genes in the root hair cell implies that they cooperate to maintain a certain length of root hair by forming a negative feedback loop (Fig. 1).

Figure 1.

(A)model illustrating the RHS10-mediated negative feedback control of root hair growth. The growing root-hair cell generates turgor pressure so as to cause the change in cell-wall integrity. The change in cell-wall integrity is sensed by AGP motifs of the RHS10 ECD which then transduces the signal to the cytoplasmic kinase domain of RHS10 for autophosphorylation. Activated RHS10 phosphorylates downstream targets that would inhibit the machinery for root hair growth. AGP, arabinogalactan protein; ECD, extracellular domain; P, phosphate; PM, plasma membrane; TM, transmembrane domain.

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Funding

This research was supported by the grants from the Next-Generation BioGreen 21 program (The Agricultural Genome Center PJ011195) of the Rural Development Administration and the Mid-career Researcher Program (2015002633) of the National Research Foundation.