Steven E. Clark

The CLAVATA1 gene encodes a putative receptor kinase that controls shoot and floral meristem size in arabidopsis

The shoot apical meristem is responsible for above-ground organ initiation in higher plants, accomplishing continuous organogenesis by maintaining a pool of undifferentiated cells and directing descendant cells toward organ formation. Normally, proliferation and differentiation are balanced, so that the structure and size of the shoot meristem is maintained. However, Arabidopsis plants homozygous for mutations at the CLAVATA1 (CLV1) locus accumulate excess undifferentiated cells. We describe the molecular cloning and expression pattern of the CLV1 gene. It encodes a putative receptor kinase, suggesting a role in signal transduction. The extracellular domain is composed of 21 tandem leucine-rich repeats that resemble leucine-rich repeats found in animal hormone receptors. We provide evidence that CLV1 expression in the inflorescence is specifically associated with meristematic activity.

The Arabidopsis CLAVATA2 gene encodes a receptor-like protein required for the stability of the CLAVATA1 receptor-like kinase

The CLAVATA2 (CLV2) gene regulates both meristem and organ development in Arabidopsis. We isolated the CLV2 gene and found that it encodes a receptor-like protein (RLP), with a presumed extracellular domain composed of leucine-rich repeats similar to those found in plant and animal receptors, but with a very short predicted cytoplasmic tail. RLPs lacking cytoplasmic signaling domains have not been previously shown to regulate development in plants. Our prior work has demonstrated that the CLV1 receptor-like kinase (RLK) is present as a disulfide-linked multimer in vivo. We report that CLV2 is required for the normal accumulation of CLV1 protein and its assembly into protein complexes, indicating that CLV2 may form a heterodimer with CLV1 to transduce extracellular signals. Sequence analysis suggests that the charged residue in the predicted transmembrane domain of CLV2 may be a common feature of plant RLPs and RLKs. In addition, the chromosomal region in which CLV2 is located contains an extremely high rate of polymorphism, with 50 nucleotide and 15 amino acid differences between Landsberg erecta and Columbia ecotypes within the CLV2 coding sequence.

Class III Homeodomain-Leucine Zipper Gene Family Members Have Overlapping, Antagonistic, and Distinct Roles in Arabidopsis Development

The Arabidopsis thaliana genome contains five class III homeodomain-leucine zipper genes. We have isolated loss-of-function alleles for each family member for use in genetic analysis. This gene family regulates apical embryo patterning, embryonic shoot meristem formation, organ polarity, vascular development, and meristem function. Genetic analyses revealed a complex pattern of overlapping functions, some of which are not readily inferred by phylogenetic relationships or by gene expression patterns. The PHABULOSA and PHAVOLUTA genes perform overlapping functions with REVOLUTA, whereas the PHABULOSA, PHAVOLUTA, and CORONA/ATHB15 genes perform overlapping functions distinct from REVOLUTA. Furthermore, ATHB8 and CORONA encode functions that are both antagonistic to those of REVOLUTA within certain tissues and overlapping with REVOLUTA in other tissues. Differences in expression patterns explain some of these genetic interactions, whereas other interactions are likely attributable to differences in protein function as indicated by cross-complementation studies.

The CLAVATA1 Receptor-like Kinase Requires CLAVATA3 for Its Assembly into a Signaling Complex That Includes KAPP and a Rho-Related Protein

The CLAVATA1 (CLV1) and CLAVATA3 (CLV3) genes are required to maintain the balance between cell proliferation and organ formation at the Arabidopsis shoot and flower meristems. CLV1 encodes a receptor-like protein kinase. We have found that CLV1 is present in two protein complexes in vivo. One is ~185 kD, and the other is ~450 kD. In each complex, CLV1 is part of a disulfide-linked multimer of ~185 kD. The 450-kD complex contains the protein phosphatase KAPP, which is a negative regulator of CLV1 signaling, and a Rho GTPase–related protein. In clv1 and clv3 mutants, CLV1 is found primarily in the 185-kD complex. We propose that CLV1 is present as an inactive disulfide-linked heterodimer and that CLV3 functions to promote the assembly of the active 450-kD complex, which then relays signal transduction through a Rho GTPase.

LRR-containing receptors regulating plant development and defense

Despite the presence of more than 400 genes that encode receptor-like kinases (RLKs) in the Arabidopsis thaliana genome, very little is known about the range of biological processes that they control, or the mechanisms by which they function. This review focuses on the most recent findings from studies of several leucine-rich-repeat (LRR) class RLKs in A. thaliana, and their implications for our understanding of plant receptor function and signaling. We compare the biological functions of plant and animal LRR-containing receptors, and the potential commonalities in the signaling mechanisms employed.

The EPIP Peptide of INFLORESCENCE DEFICIENT IN ABSCISSION Is Sufficient to Induce Abscission in Arabidopsis through the Receptor-Like Kinases HAESA and HAESA-LIKE2

In Arabidopsis thaliana, the final step of floral organ abscission is regulated by INFLORESCENCE DEFICIENT IN ABSCISSION (IDA): ida mutants fail to abscise floral organs, and plants overexpressing IDA display earlier abscission. We show that five IDA-LIKE (IDL) genes are expressed in different tissues, but plants overexpressing these genes have phenotypes similar to IDA-overexpressing plants, suggesting functional redundancy. IDA/IDL proteins have N-terminal signal peptides and a C-terminal conserved motif (extended PIP [EPIP]) at the C terminus (EPIP-C). IDA can, similar to CLAVATA3, be processed by an activity from cauliflower meristems. The EPIP-C of IDA and IDL1 replaced IDA function in vivo, when the signal peptide was present. In addition, synthetic IDA and IDL1 EPIP peptides rescued ida and induced early floral abscission in wild-type flowers. The EPIP-C of the other IDL proteins could partially substitute for IDA function. Similarly to ida, a double mutant between the receptor-like kinases (RLKs) HAESA (HAE) and HAESA-LIKE2 (HSL2) displays nonabscising flowers. Neither overexpression of IDA nor synthetic EPIP or EPIP-C peptides could rescue the hae hsl2 abscission deficiency. We propose that IDA and the IDL proteins constitute a family of putative ligands that act through RLKs to regulate different events during plant development.

Cell signalling at the shoot meristem

The regulation of cell differentiation at meristems is crucial to developmental patterning in plants. Rapid progress has been made in identifying the genes that regulate differentiation and the receptor-mediated signalling events that have a key role in this process. In particular, we are now learning how the CLAVATA receptor kinase signalling pathway promotes stem cell differentiation in balance with the initiation of stem cells by the transcription factor WUSCHEL.

CLAVATA1 dominant-negative alleles reveal functional overlap between multiple receptor kinases that regulate meristem and organ development

The CLAVATA1 (CLV1) receptor kinase controls stem cell number and differentiation at the Arabidopsis shoot and flower meristems. Other components of the CLV1 signaling pathway include the secreted putative ligand CLV3 and the receptor-like protein CLV2. We report evidence indicating that all intermediate and strong clv1 alleles are dominant negative and likely interfere with the activity of unknown receptor kinase(s) that have functional overlap with CLV1. clv1 dominant-negative alleles show major differences from dominant-negative alleles characterized to date in animal receptor kinase signaling systems, including the lack of a dominant-negative effect of kinase domain truncation and the ability of missense mutations in the extracellular domain to act in a dominant-negative manner. We analyzed chimeric receptor kinases by fusing CLV1 and BRASSINOSTEROID INSENSITIVE1 (BRI1) coding sequences and expressing these in clv1 null backgrounds. Constructs containing the CLV1 extracellular domain and the BRI1 kinase domain were strongly dominant negative in the regulation of meristem development. Furthermore, we show that CLV1 expressed within the pedicel can partially replace the function of the ERECTA receptor kinase. We propose the presence of multiple receptors that regulate meristem development in a functionally related manner whose interactions are driven by the extracellular domains and whose activation requires the kinase domain.

The BAM1/BAM2 Receptor-Like Kinases Are Important Regulators of Arabidopsis Early Anther Development

Anther development involves the formation of several adjacent cell types required for normal male fertility. Only a few genes are known to be involved in early anther development, particularly in the establishment of these different cell layers. Arabidopsis thaliana BAM1 (for BARELY ANY MERISTEM) and BAM2 encode CLAVATA1-related Leu-rich repeat receptor-like kinases that appear to have redundant or overlapping functions. We characterized anther development in the bam1 bam2 flowers and found that bam1 bam2 anthers appear to be abnormal at a very early stage and lack the endothecium, middle, and tapetum layers. Analyses using molecular markers and cytological techniques of bam1 bam2 anthers revealed that cells interior to the epidermis acquire some characteristics of pollen mother cells (PMCs), suggesting defects in cell fate specification. The pollen mother-like cells degenerate before the completion of meiosis, suggesting that these cells are defective. In addition, the BAM1 and BAM2 expression pattern supports both an early role in promoting somatic cell fates and a subsequent function in the PMCs. Therefore, analysis of BAM1 and BAM2 revealed a cell–cell communication process important for early anther development, including aspects of cell division and differentiation. This finding may have implications for the evolution of multiple signaling pathways in specifying cell types for microsporogenesis.

Evidence for functional conservation, sufficiency, and proteolytic processing of the CLAVATA3 CLE domain.

Arabidopsis (Arabidopsis thaliana) CLAVATA3 (CLV3) is hypothesized to act as a ligand for the CLV1 receptor kinase in the regulation of stem cell specification at shoot and flower meristems. CLV3 is a secreted protein, with an amino-terminal signal sequence and a conserved C-terminal domain of 15 amino acids, termed the CLE (CLV3/ESR-related) domain, based on its similarity to a largely unstudied protein family broadly present in land plants. We have tested the function of 13 Arabidopsis CLEs in vivo and found a significant variability in the ability of CLEs to replace CLV3, ranging from complete to no complementation. The best rescuing CLE depends on CLV1 for function, while other CLEs act independently of CLV1. Domain-swap experiments indicate that differences in function can be traced to the CLE domain within these proteins. Indeed, when the CLE domain of CLV3 is placed downstream of an unrelated signal sequence, it is capable of fully replacing CLV3 function. Finally, we have detected proteolytic activity in extracts from cauliflower (Brassica oleracea) that process both CLV3 and CLE1 at their C termini. For CLV3, processing appears to occur at the absolutely conserved arginine-70 found at the beginning of the CLE domain. We propose that CLV3 and other CLEs are C-terminally processed to generate an active CLE peptide.