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Expression analysis of the CLE signaling gene family in Arabidopsis thaliana and functional characterization of CLE8 in seed development



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.

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