Shared and distinct functions of the pseudokinase CORYNE (CRN) in shoot and root stem cell maintenance of Arabidopsis

Abstract

Stem cell maintenance in plants depends on the activity of small secreted signaling peptides of the CLAVATA3/EMBRYO SURROUNDING REGION (CLE) family, which, in the shoot, act through at least three kinds of receptor complexes, CLAVATA1 (CLV1) homomers, CLAVATA2 (CLV2) / CORYNE (CRN) heteromers, and CLV1/CLV2/CRN multimers. In the root, the CLV2/CRN receptor complexes function in the proximal meristem to transmit signals from the CLE peptide CLE40. While CLV1 consists of an extracellular receptor domain and an intracellular kinase domain, CLV2, a leucine-rich repeat (LRR) receptor-like protein, and CRN, a protein kinase, have to interact to form a receptor-kinase complex. The kinase domain of CRN has been reported to be catalytically inactive, and it is not yet known how the CLV2/CRN complex can relay the perceived signal into the cells, and whether the kinase domain is necessary for signal transduction at all. In this study we show that the kinase domain of CRN is actively involved in CLV3 signal transduction in the shoot apical meristem of Arabidopsis, but it is dispensable for CRN protein function in root meristem maintenance. Hence, we provide an example of a catalytically inactive pseudokinase that is involved in two homologous pathways, but functions in distinctively different ways in each of them.

Keywords: Arabidopsis thaliana; CLAVATA; CORYNE; peptide signaling; pseudokinase; stem cell maintenance..

Fig. 1.

Schematic representations of the CRN protein variants. (A) CRN (wild-type); (B) crn-1; (C) CRΔEC; (D) CRΔEC1; (E) CRΔEC2; (F) crn-3; (G) CRΔKi; (H) CR(SD) and CR(SA); (I) CR (<>C1Ki). Light orange = signal peptide (SP); Blue = extracellular domain (EC); Yellow = transmembrane domain (TMD); Green = kinase domain; Olive = CLV1 kinase domain. aa = amino acids. The red arrowheads in (B), (F) and (H) indicate the positions of amino acid exchanges.

Fig. 2.

Carpel and root phenotypes of crn and clv2 mutants. (A) Quantification of the carpel phenotype. The scale (1.75–3.0) shows the number of carpels per silique in the different lines. (B, C) Representative siliques (B) and seedlings (C) of the different transgenic and reference lines. The seedlings in the root assay were grown on GM-medium containing 500nM CLV3 peptide. The reference lengths are indicated by the dotted lines: white = start of root; green = wild-type; red = mutant (crn or clv2). For the CRN(SA) variant, three independent transgenic lines were analyzed. For all other variants, between 7 and 11 independent transgenic lines were analyzed. Between 10 and 15 siliques per plant were analyzed from at least five different plants from each line. (D) Quantification of the root phenotype. The scale (0.5–2.25) is root length in cm. The different lines are: Col = Wild-type; crn = crn-3; clv2 = clv2-gk. The following variants are expressed in the crn-3 background: CRN, CRΔEC, CRΔEC1, CRΔEC2, CR(C1Ki), C2(CRKi), CRΔKi, CR(SD), and CR(SA). The following variants are expressed in the clv2-gk background: CLV2, C2(CRKi), C2(708), and C2(RA). * Indicates values significantly different from the mutant. ^# Indicates an enhanced mutant phenotype significantly different from the mutant. Bars in the graphs are standard error. Scale bars in the images are 0.5cm. For the CRN(SA) variant, two independent transgenic lines were analyzed. For all other variants, between 7 and 11 independent transgenic lines were analyzed. Between 33 and 122 roots were measured from each line.

Fig. 3.

CLV2 and CRN expressions patterns in the root of Arabidopsis.CRN::mCherry-H2B (A) and CLV2::Venus-H2B (B) expression in a Col-0 root. For better differentiation between the tissues, the QC is encircled in turquoise. The epidermis is encircled in blue, the cortex in green, and the endodermis in pink in the right half of the roots.

Fig. 4.

Intracellular localization of the different CRN ECD variants. The different CRN variants are tagged with GFP, and CLV2 is tagged with Cerulean. In the red channel, either the membrane dye FM4-64 (A–D) or ER-localized RTNLB2-mCherry (A′–D′) is shown. Wild-type CRN is shown in (A) and (A′), CRΔEC in (B) / (B′), CRΔEC1 in (C) / (C′), and CRΔEC2 in (D) / (D′). In the PM focal plane (A–D), the signal is homogenous along the PM for CRN, CRΔEC1, and CRΔEC2, co-localizing with FM4-64 (A, C, D), and patchy along the PM for CRΔEC (B). In the ER focal plane (A′-D′), there is signal visible in all CRN variants tested; however, the pattern is distinct from the net-like ER-structure exhibited by RTNLB2-mCherry in (A′), (C′), and (D′). Supplementary Fig. S4 shows the same cells with switched focal planes: the FM4-64 marked cell is focused on the ER, the RTNLB2-marked cell is focused on the PM. Scale bar = 10 µm.

Fig. 5.

Intracellular localization of the different CLV2 variants. The different CLV2 variants are tagged with GFP, and CRN is tagged with Cerulean. In the red channel, either the membrane dye FM4-64 (A–D) or ER-localized RTNLB2-mCherry (A′–D′) is shown. Wild-type CLV2 is shown in (A) / (A′), C2(708) in (B) / (B′), C2(RA) in (C), (C′) and C2(CRKi) in (D) / (D′). In the PM focal plane (A–D), all four protein variants co-localize with FM4-64 (A–D). In the ER focal plane (A′-D′), there is signal visible in all CLV2 variants tested, but different from the distinct net-like ER-structure exhibited by RTNLB2-mCherry. This net-like structure is also visible in the C2(CRKi) protein (D′), indicating impaired, but not completely blocked ER-export of this CLV2 variant. Supplementary Fig. S5 shows the same cells with switched focal planes: the FM4-64 marked cell is focused on the ER, the RTNLB2-marked cell is focused on the PM. Scale bar = 10 µm.

Fig. 6.

Schematic representations of the CLV2 protein variants. (A) CLV2, (B) C2(RA), (C) C2(708), (D) C2(CRKi). Light orange = signal peptide (SP); Red = LRR receptor domain; Yellow = transmembrane domain (T); Green = CRN kinase domain. aa = amino acids. The red arrowhead indicates the position of the putative RxR motif.

Fig. 7.

Intracellular localization of the different CRN kinase variants. The different CRN kinase variants are tagged with GFP, and CLV2 is tagged with Cerulean. In the red channel, either the membrane dye FM4-64 (A–E) or ER-localized RTNLB2-mCherry (A′-E′) is shown. CRNΔKi is shown in (A) / (A′), crn-3 in (B) / (B′), CR(C1Ki) in (C) / (C′), CR(SD) in (D) / (D′) and CR(SA) in € / (E′). In the PM focal plane (A–D), all protein variants co-localize with FM4-64 (A–E), In the ER focal plane (A′-E′), there is signal visible in all CLV2 variants tested, but different from the distinct net-like ER-structure exhibited by RTNLB2-mCherry. This net-like structure is only also visible for the crn-3 protein (B′), indicating impaired, but not completely blocked ER-export of this CRN variant. Supplementary Fig. S6 shows the same cells with switched focal planes: the FM4-64 marked cell is focused on the ER, the RTNLB2-marked cell is focused on the PM. Scale bar = 10 µm.

Fig. 8.

Model of the different receptor complexes involved in CLE signaling in the shoot and proximal root meristem. In the shoot, two CLV1 proteins (blue) form signaling-active homomers that can bind CLV3 (black) via their receptor domains and autophosphorylate (black arrows and ‘P’) on their kinase domains. The CLV2 (red) / CRN (green) heteromers interact either with CLV1 or an unknown receptor ‘X’ (light grey) to form multimers. The interacting RLKs may bind CLV3 together with CLV2, and transphosphorylate (red arrows) the CRN kinase domain. Following phosphorylation of the kinases, different effector proteins interact with the kinase domains of either CLV1 (‘A’, orange) or CRN (‘B’, yellow) and Protein ‘X’ (‘C’, purple). In the proximal root meristem the CLV2 (red) / CRN (green) heteromers function independently of CLV1, but possibly together with an unknown receptor ‘X’ (light grey). Here, receptor ‘X’ perceives a CLE peptide signal together with CLV2. Downstream signaling is independent of the CRN kinase, possibly due to autophosphorylation of the kinase domain of receptor ‘X’ and its effector protein ‘C’ (purple).