Each multicellular organism, much like the communities they belong to, is not a discrete biological unit, but an union of elements that require constant communication in order to sustain growth. In both animals and plants, small peptide hormones serve as one such means of intercellular correspondence, typically secreted into the apoplastic space to be perceived by receptors in neighbouring cells. These molecular messengers form intricate feedback pathways that allow connective tissue layers to coordinate their diverse roles and functions.

A large family of signaling molecules called CLAVATA3/EMBRYO SURROUNDING REGION-RELATED (CLE) is conserved throughout the evolution of the land plant lineage. Its members encode prepropeptides that are processed into small 12-13 amino acid ligands, whose functions are implicated in diverse developmental processes, including cell fate acquisition, cell proliferation, and cell division plane orientation. The founding member of the CLE family is CLAVATA3 (CLV3), which is expressed at the shoot apex of the mustard plant Arabidopsis thaliana, to mediate activity of the stem cell niche there, also called the shoot apical meristem (SAM). The CLV3 signal is perceived by multiple receptor complexes in the underlying tissue layers, triggering a pathway that targets the homeodomain transcription factor WUSCHEL (WUS). WUS is a mobile protein that promotes stem cell identity within its area of effect, while also directly activating CLV3 expression. In turn, CLV3 signaling represses WUS expression and confines its domain to a few cells at the center of the SAM. The ensuing feedback loop between CLV3 and WUS thus maintains a small but stable stem cell population at the shoot tip, which proliferates to supply building materials for various growth processes aboveground, including vertical extension and generation of organs such as leaves, flowers, and axillary branches.

In this study, I characterized the biological functions of three genes related to CLV3, CLE16, CLE17, and CLE27, all of which are also expressed at the shoot apex of Arabidopsis. By themselves, CLE16, CLE17, and CLE27 appear to have no significant impact on stem cell activity at the SAM and overall plant architecture. However, via analyzing higher order mutants, I demonstrated that in the loss of endogenous CLV3 activity, the CLE16 and CLE17 peptides can independently act in its place to restrict stem cell accumulation, starting at the embryonic stage and throughout the entirety of the life cycle, ending at flower production. In contrast, CLE27 does not compensate for the loss of CLV3 signaling at least in the final stage of floral development. The ability of CLE16 and CLE17 to partially, but not fully, replace CLV3 function may stem from their binding affinity to a subset of CLV3’s cognate receptors. In addition, while the CLE16 and CLE17 signaling pathways appear to target WUS in a similar manner to CLV3, I provided evidence that both may have WUS-independent functions that are specific to the development of axillary branches. Together, these observations describe a complex interrelation that both expand upon the known CLV-WUS module, and reveal novel aspects in the regulation of shoot stem cell activity by CLE peptides.

Finally, I characterized functions of two other CLE genes in Arabidopsis leaves, CLE5 and CLE6. Both genes are expressed at the base of developing leaves, and appear to have a minor impact on overall leaf shape. Their expression levels are responsive to various transcription factors involved in leaf patterning, as well as the phytohormones auxin. This study provides the first instance of CLE peptides regulating leaf morphology, and thus opens up a new avenue for investigating the genetic regulation of leaf development in land plants.

ABSTRACT

Expression analysis of the CLE signaling gene family in Arabidopsis thaliana and functional

characterization of CLE8 in seed development

by Elisa Fiume

Doctor of Philosophy in Plant Biology

University of California, Berkeley

Professor Jennifer C. Fletcher, Chair

In plants, as in all multicellular organisms, cell-to-cell communication is fundamental for coordinating growth and differentiation. Although non-peptide hormones have been long known to act as signaling molecules in plants, the sequencing of the Arabidopsis genome revealed the presence of many potential ligand-encoding genes and receptors suggesting that plants widely use them in different signaling pathways. In the past decade, signaling mediated by peptides in plants has become an emerging area of research and has been found to be utilized in a broad range of developmental processes. However, very few receptors and ligands have been functionally characterized. The Arabidopsis CLAVATA3/EMBRYO SURROUNDING REGION (ESR)-related (CLE) small polypeptide family is currently the best studied, nonetheless only few members have been assigned a function. To unveil possible developmental processes regulated by these putative signaling molecules, I undertook a systematic expression analysis of its members. These studies showed that all Arabidopsis tissues express one or more CLE gene, suggesting that CLE-mediated signaling regulates a wide range of developmental processes. Furthermore, no two CLE genes showed the same expression pattern, indicating that specificity of function is in part achieved at the regulatory level. On the other hand, many CLE genes have overlapping expression patterns, suggesting that there could be a a high degree of redundancy among the family members.

The putative functional redundancy of the CLE genes has been used to explain the lack of cle single mutant phenotypes, however, many other factors could also contribute. For example, CLE genes encode small proteins and generally lack introns making it difficult to obtain knock-out mutants using T-DNA insertional mutagenesis. Alternatively, the CLE genes might be regulating processes that when impaired, produce phenotypes that are overlooked in conventional screens because they are subtle or conditional to environmental stimuli. In recent years, it has also been discovered that the Arabidopsis genome contains hundreds of essential genes, and because null mutations in these genes cause seed lethality, their functions are largely unknown. A number of signaling molecules has been implicated in regulating seed development, but no putative peptide ligand has been involved thus far.

In my research, I have isolated and studied a hypomorphic mutation in CLE8, the only member of the CLE family whose expression is limited to the young embryo and the endopserm. My phenotypic characterization of the mutant, together with molecular studies, have led me to discover that CLE8 is necessary for proper seed development, indicating that most likely it is an essential gene. I demonstrate that CLE8 is involved in patterning of the embryo basal domain, in elongation and patterning of suspensor cells, in endosperm maturation and thus ultimately regulates seed size. I found CLE8 to be expressed in the embryo-proper and the endosperm and to have a non-cell-autonomous effect on suspensor cells. I showed that CLE8 positively regulates the expression of the putative transcription factor WOX8 both in the suspensor cells and in the endosperm, and that it likely evolved to specifically fulfill its role during seed development and diverged from other members of the family to solely interact with its receptor/s.

Furthermore, I have analyzed a partial loss-of-function allele of another essential gene: EMB1611. EMB1611 encodes a large, novel protein with an N-terminal coiled-coil domain and two trans-membrane domains, but whose molecular function still remains elusive. From expression, phenotypic and molecular analysis I discovered that EMB1611 functions to maintain cells in growing tissues in a proliferative or uncommitted state through maintaining the cellular organization of the shoot apical meristem and directly promoting stem cell fate. EMB1611 null alleles are lethal at early stages of embryogenesis; thus, as for CLE8, the availability of a hypomorphic allele has been essential for studying the role of EMB1611 in plant development.