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Dissertation
Characterization of Phytohormone signaling in Ceratopteris richardii: reproductive transition and meristematic activity
University of Iowa
Doctor of Philosophy (PhD), University of Iowa
Spring 2024
DOI: 10.25820/etd.007455
Abstract
Land plants utilize a diverse group of plant-specific signaling molecules to respond to their environment and direct growth. Over evolutionary time, the pathways producing and responding to these signals have diversified to control development of complex structures such as multicellular meristems, floral organs, and conductive vascular tissues found in many agriculturally and ecologically relevant plant species. As the sister clade to seed plants, ferns occupy an intermediate position in the land plant phylogenetic tree and exhibit traits shared with both bryophytes (mosses, hornworts, and liverworts) and the more recently evolved flowering plants. The fern Ceratopteris richardii is a powerful model system to interrogate the participation of signaling molecule pathway members on plant body plans.
The small peptide receptor EXCESS MICROSPOROCYTES1 (EMS1) is a crucial component of reproductive cell fate signaling in male reproductive development that occurs in the anther of flowering plants. EMS1 is conserved among all land plant clades, but the function of EMS1 in a plant that makes neither seeds nor flowers has yet to be determined. I examined the role of a Ceratopteris homolog of EMS1 (CrEMS1) in both the haploid and diploid life phases of the fern, using a combination of RNAi-mediated knockdown, image-processing based phenotyping, and in situ hybridization. I found that CrEMS1 inhibits the ectopic formation of sporangia on vegetative leaves and discovered a new role for an EMS1 gene in male gametangium initiation. My research uncovered multiple Ceratopteris EMS1 homologs, as in mosses, heterosporous ferns, and monocots, but not as in eudicots like the model Arabidopsis that has a single EMS1 gene. Future studies with Ceratopteris EMS1 genes will reveal the evolutionary history of this important reproductive gene family.
Auxin is an organic molecule used by plants to control response to the environment and developmental patterning. The localization of auxin determines its action in plant cells and plays an important role in response to wounding and regeneration. The Ceratopteris hermaphrodite gametophyte is a single layer of cells and possesses a multicellular meristem, serving as a simple and easy-to-manipulate system to understand the influences of auxin on meristem formation. Using a laser ablation technique, I discovered that loss of a single stem cell from the meristem triggers meristem arrest and regeneration of the meristem in a new location on the plant body. By application of various exogenous auxins and auxin inhibitors, I found that only indole-3-acetic acid (IAA) and inhibition of polar auxin transport by the chemical N-1-naphthylphthalamic acid (NPA) can influence position of the meristem during normal development. Furthermore, only IAA was able to disturb meristem regeneration, causing the position of new meristems to form distal to the spore. From these results, I proposed a model of auxin gradients during normal meristem development and regeneration that can be tested in the future by auxin-sensitive reporter systems and mutagenesis of the auxin biosynthesis genes identified.
CLAVATA3/EMBRYO SURROUNDING REGION (CLE) peptides are 12-13 amino acid-long signaling molecules that serve as positional signals during development. The core CLE signaling module consists of a CLE peptide and a leucine-rich repeat receptor-like kinase, but flowering plants have also incorporated WUSCHEL-RELATED HOMEOBOX (WOX) transcription factors to form negative feedback loops that regulate stem cell maintenance in the shoot and root. It is not known when WOX genes were co-opted into CLE signaling pathways, only that mosses and liverworts do not require WOX for CLE-regulated stem cell activities. I identified eleven CLE peptides in Ceratopteris, including one (CrCLV3) with homology to shoot meristem CLE peptide CLAVATA3. I performed the first functional characterization study of a fern CLE using techniques including RNAi knockdown, synthetic peptide dosage, and the first Agrobacterium-mediated leaf infiltration assay described in a fern system. I found that CrCLV3 promotes cell proliferation and stem cell identity in the gametophyte meristem. I also discovered evidence for CrCLV3 regulation of the WOX gene CrWOXA during the developmental stage when female gametangium formation begins. My discoveries provide a new avenue for CLE peptide research in fern systems and clarify the evolutionary timeline of CLE-WOX signaling in land plants.
Apogamy is the transition from haploid to diploid life phases without fertilization, a process up to 10% of ferns can do in nature. Environmental changes such as lack of water, continuous light, or exogenous carbohydrate sources can induce apogamy. Although Ceratopteris does not undergo apogamy in nature, it can be induced with sugar. Ceratopteris gametophytes exhibit a sharp increase in apogamy induction between 10- and 12- days exposure to sugar, a period accompanied by transcriptomic changes observed long before morphological evidence of apogamy. I performed a time-course RNA-Seq analysis covering this important developmental period to detect differentially expressed genes associated with sugar induction. I discovered six distinct clusters, enriched with genes predicted to function in cell wall modifications, hormone signaling, transcription factor activity, and stress responses. From these results, I proposed candidate transcription factors and associated transcriptional regulators that may control the initial switch from normal development to apogamy. Future characterization studies of these candidates may discover new genes applicable to agricultural clonal propagation tools such as apomixis and somatic embryogenesis.
Details
- Title: Subtitle
- Characterization of Phytohormone signaling in Ceratopteris richardii: reproductive transition and meristematic activity
- Creators
- Kelley A. Renninger
- Contributors
- Chi-Lien Cheng (Advisor)Erin E. Irish (Committee Member)Ana Llopart (Committee Member)Bryan Phillips (Committee Member)Noah Butler (Committee Member)
- Resource Type
- Dissertation
- Degree Awarded
- Doctor of Philosophy (PhD), University of Iowa
- Degree in
- Integrated Biology
- Date degree season
- Spring 2024
- Publisher
- University of Iowa
- DOI
- 10.25820/etd.007455
- Number of pages
- xx, 288 pages
- Copyright
- Copyright 2024 Kelley A. Renninger
- Language
- English
- Date submitted
- 04/19/2024
- Description illustrations
- Illustrations, tables, graphs, charts
- Description bibliographic
- Includes bibliographical references (pages 247-271).
- Public Abstract (ETD)
Plants control growth and development by using a combination of signaling hormones able to be transported between cells, including plant-specific organic molecules and small peptides. The functions of each hormone type have diversified among species that make different body structures, such as mosses and flowering plants. The model plant Ceratopteris richardii provides an opportunity to investigate how ferns use signaling hormones during proliferative and reproductive development, within a body plan consisting of independent photosynthetic haploid and diploid life stages unique to ferns. I discovered the role of a peptide hormone receptor EXCESS MICROSPOROCYTES1 in the reproductive transition of the fern, a function never described before. I also revealed the role of the small organic hormone auxin in positioning of the stem cell niche in the fern haploid life stage during normal development and in response to stem cell destruction. I pioneered new techniques in Ceratopteris to discover the function of the short peptide ligand CLAVATA3 (CrCLV3) in promoting stem cell identity and provided the first evidence of regulation between CrCLV3 and a WUSCHEL-RELATED HOMEOBOX transcription factor in a fern system, previously thought to only exist in seed plants. Lastly, I identified candidate genes that may promote the switch between haploid and diploid developmental programs in the absence of fertilization. These findings pave the way for future studies in fern cell-to-cell communication and expand our knowledge of signaling hormone functional diversity in plant growth and reproduction.
- Academic Unit
- Biology
- Record Identifier
- 9984647556202771
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