Stem Cell Signaling in Arabidopsis Requires CRN to Localize CLV2 to the Plasma Membrane

Abstract

Stem cell number in shoot and floral meristems of Arabidopsis (Arabidopsis thaliana) is regulated by the CLAVATA3 (CLV3) signaling pathway. Perception of the CLV3 peptide requires the receptor kinase CLV1, the receptor-like protein CLV2, and the kinase CORYNE (CRN). Genetic analysis suggested that CLV2 and CRN act together and in parallel with CLV1. We studied the intracellular localization of receptor fusions with fluorescent protein tags and their capacities for interaction via efficiency of fluorescence resonance energy transfer. We found that CLV2 and CRN require each other for export from the endoplasmic reticulum and localization to the plasma membrane (PM). CRN readily forms homomers and interacts with CLV2 through the transmembrane domain and adjacent juxtamembrane sequences. CLV1 forms homomers independently of CLV2 and CRN at the PM. We propose that the CLV3 signal is perceived by a tetrameric CLV2/CRN complex and a CLV1 homodimer that localize to the PM and can interact via CRN.In Arabidopsis (Arabidopsis thaliana), the stem cell number in the shoot apical meristem is regulated by negative feedback regulation. Stem cell induction and maintenance are controlled by the homeodomain protein WUSCHEL (WUS), and WUS expression is in turn repressed by CLAVATA3 (CLV3; Brand et al., 2000;Schoof et al., 2000), which encodes a 13-amino acid arabinosylated glycopeptide that is secreted from stem cells (Ohyama et al., 2009). Three genes have been identified that encode receptors for CLV3 signaling. Mutations in CLV1 (Clark et al., 1997), encoding a leucine-rich repeat (LRR) receptor kinase, CLV2, encoding a LRR receptor-like protein (Jeong et al., 1999), and CORYNE (CRN), encoding a receptor-like kinase, disrupt CLV3 signaling and allow the stem cell domain to expand (Sablowski, 2007;Mü ller et al., 2008). Binding of CLV3 to the LRR domains of CLV1 was recently shown (Ogawa et al., 2008).A simple readout for CLV3 signaling is carpel number. Stem cells of floral meristems are normally consumed with the production of two central carpels. Any reduction in CLV3 signaling, which results in increased WUS expression and production of more stem cells, causes an increase in carpel number. Mutations in CLV1, CLV2, or CRN showed an intermediate carpel number phenotype and reduced CLV3 signaling (Mü ller et al., 2008). Double mutants of clv2 with crn were epistatic, but double mutants of clv1 with clv2 or crn were synergistic and abolished CLV3 signaling. This indicated that CLV1 acts independently from, and in parallel with, CLV2 and CRN to transmit the CLV3 signal. Furthermore, clv2 and crn mutants showed additional phenotypes, such as elongated pedicels and defects in stamen development, suggesting that CLV2 and CRN act in a common pathway (Mü ller et al., 2008). Both CRN and CLV2 were proposed to be membrane localized and may physically interact via their transmembrane domains or immediately adjacent sequences (Fig. 1A). A loss-of-function mutation of CRN, crn-1, is caused by an amino acid exchange within the predicted tra...

Protein Disulfide Isomerase-2 of Arabidopsis Mediates Protein Folding and Localizes to Both the Secretory Pathway and Nucleus, Where It Interacts with Maternal Effect Embryo Arrest Factor

“…The values represent FRET efficiencies calculated on 10 cells averaged ± SD. A FRET efficiency above 4% is considered to indicate close interaction between proteins for the GFP and mCherry pair (Bleckmann et al, 2010).…”

“…5B). A FRET efficiency above 4% is considered to indicate close interaction between proteins for the GFP and mCherry pair (Bleckmann et al, 2010). Therefore, FRET efficiency results indicated that PDI2 and MEE8 interacted in vivo.…”

“…Photobleaching was performed by scanning nuclei with a 543 nm laser (laser power 90%) 20 times. FRET efficiency of the nucleus was calculated as the percentage change of the GFP intensity before and after bleaching with the equation of E FRET = (GFP after -GFP before )/GFP after X100 (Bleckmann et al 2010). We were able to fully separate GFP and mCherry spectra from chloroplast autofluorescence using the TCS SP5 microscope.…”

New insights into receptor-like protein functions in Arabidopsis

“…1 For instance, it has been shown that CLV2 was functionally associated with CRN in a receptor complex to perceive the CLV3 signal. 22 Likewise, TMM, forming a receptor complex with ERECTA family members, was found to regulate the proper stomatal distribution and differentiation. 23 Recently, SUPPRESSOR OF BIR1-1 (SOBIR1) was found to associate specifically with various RLPs that either function in plant development or in plant immunity.…”

Mystery in genetics: PUB4 gives a clue to the complex mechanism of CLV signaling pathway in the shoot apical meristem

“…7 Genetic approach has demonstrated that these 3 receptor complexes function downstream of CLV3 basically, independently of each other, however, it remains unclear whether they are functionally redundant or each has distinct functions. [6][7][8]15 Previously, we have shown that a synthetic CLV3 peptide, MCLV3 could be a useful tool for investigating downstream components of the CLV signaling pathway. 7,9 Here we report the defects of SAM in a recently identified MCLV3 resistant mutant, pub4, 10 and discuss the possible relationship of its causal gene with the CLV signaling pathway.…”