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In Arabidopsis, the feedback regulatory loop between CLAVATA3 (CLV3) signaling pathway and transcription factor, WUSCHEL (WUS) plays a significant role in shoot apical meristems (SAM) maintenance. Previously, CLV1/CLV2 heterodimers were supposed to perceive and transmit CLV3 signaling. Recent genetic analysis isolated a novel receptor kinase, CORYNE (CRN), which was found to be involved in the CLV3 pathway. Therefore, new hypothesis was put forward that CRN probably acts with CLV2 to transmit CLV3 in parallel with CLV1 based on genetic analysis. In our recent work, we took advantage of firefly luciferase complementation imaging (LCI) assay to analyze the interactions among CLV1, CLV2 and CRN in both Arabidopsis thaliana protoplasts and Nicotiana benthamiana leaves. We identified the physical interaction between CLV2 and CRN in the absence of CLV3 and found some interesting phenomenon such as CLV1, CLV2 and CRN may form a complex, and that CRN was able to form homodimers. These new observations make the relationships among these three proteins more complex than that indicated in two-parallel pathway model. Combining current genetic and our new biochemical evidence, a more possible and detailed model for CLV3 pathway was developed.
The stem cells located in SAM constantly progress cell division and cell differentiation to generate all of the aboveground organs in plants. CLV3 signaling pathway negatively regulates the fate of stem cells and maintains the SAM size through repressing the homeodomain transcription factor, WUS, which promotes the stem cell differentiation.1–3 Current knowledge believes that CLV3 encodes a secreted extracellular peptide,4 and CLV1 encodes a leucine-rich-repeat receptor-like kinase (LRR-RLK); CLV2 encodes a receptor-like protein (RLP) lacking a kinase domain.5,6 Previous studies hypothesized that CLV1/CLV2 heterodimers function as receptors to transmit CLV3 ligand signaling.4,7
Recently, a new gene, CRN/SOL2, was isolated and predicted to encode membrane-associated kinase with a very short extracellular domain. Genetic evidences predicted that SOL2/CRN functions together with CLV2 in parallel with CLV1 in CLV3 signaling.8,9 Accordingly new two-parallel pathway model was raised that: one uses CLV1 homodimers and the other uses CRN-CLV2 heterodimers.8 In our recent work, we seek to analyze the interactions among these receptor proteins at biochemical level.
Owing to the specific and limited expression patterns of these receptor proteins, it would be difficult to conduct biochemical analysis only using SAM due to its slight amount. Therefore, we took advantage of transient expression systems including Arabidopsis protoplasts and N. benthamiana leaves to analyze the interactions among CLV1, CLV2 and CRN using the newly developed LCI method, which is believed to have high signal-tonoise ratio and be able to detect dynamic interactions including membrane protein interactions.10–12 Our results showed that CLV2 can directly interact with CRN in the absence of the CLV3 peptide, and CLV1 can weakly interact with CRN, but it cannot interact with CLV2. Interestingly, CLV1, CLV2 and CRN may bind together to form a complex. In addition, CRN (rather than CLV1 and CLV2) is able to form homodimers, although we are not sure whether CLV1 could form homodimers due to the low expression level of CLV1-NLuc proteins.13 Resently, the Simon group also obtained similar results such as CLV2-CRN physical interaction and CLV1-CLV2-CRN forming complexes in N. benthamiana leaves by fluorescence resonance energy transfer (FRET).14 Surprisingly, they observed that CLV2 and CRN can transport from ER to the plasma membrane (PM) when both proteins were co-expressed in N. benthamiana leaves, and CLV1 can form homodimers via FRET.14
Combining recent genetic and biochemical evidences, a possible model for CLV3 signaling pathway is depicted in Figure 1. It shows that at least three receptor complexes may participate to transmit CLV3 signaling. The first pathway is CLV1 monomer or CLV1 homodimer, since CLV3 can directly bind the extracellular LRR domain of CLV1.15 The second pathway is that CLV2-CRN heterodimers form a tetramer through CRN-CRN interaction as a functional receptor based on recent genetic evidence and our LCI results.8,13 However, the ligand-receptor interaction between CLV3 peptide and LRR of CLV2 should be verified in future. In addition, the third pathway consists of CLV1 and CLV2-CRN heterodimers. 13,14 Does CRN function as a bridge or co-receptor to help CLV1/CLV2 interaction? What the biological significance is for these three proteins binding together is worthy of in-depth study. Although all these three pathways have the potential capacity of mediating CLV3 and other CLE peptides signaling, they may not contribute to transmitting CLV3 signaling equally. It would be necessary to investigate which pathway is preferred to sense CLV3 during different stages of plant development.
Another confused phenomenon is that we failed to observe that exogenous 12-aa CLV3 peptide affected the interactions among CLV1, CLV2 and CRN by LCI assays in our systems. So far, we could not make conclusion that these interactions were ligand independent possibly because of transient overexpression in our systems and the limited sensitive of LCI assay. Moreover, the most probable reason may be that these semi-in vivo systems could not respond CLV3 peptide stimulation because only shoot apical meristems (SAM) and root apical meristems (RAM) were sensitive to CLV3 in Arabidopsis. Therefore, future study should verify these interaction results in vivo (SAM or RAM) with more sensitive analysis.
Single molecular techniques (SMT) have been widely applied and become powerful tools for biologists because they can provide complementary and better prospects for biological processes both at a molecular and systems-level.16 However, few studies on single molecular methods were reported in plants. Future study should take advantage of these advance approaches, such as single particle tracking (SPT), fluorescence correlation spectroscopy (FCS), and atomic force spectroscopy (AFM) to further investigate the dynamics and diffusions of CLV1, CLV2 and CRN receptors in living plant cells, the ligand-receptor interaction between CLV3 peptide and LRR of CLV2, and assembly of these receptor proteins.
This work was supported by the Ministry of Science and Technology of China (2006CB910606, 2007CB108703 and 2009CB119105), the key project of NSFC (30730009) and A Hundred Talents Program of Chinese Academy of Sciences.
Previously published online: www.landesbioscience.com/journals/psb/article/10790