Author ORCID Identifier

https://orcid.org/0000-0003-0971-2058

Year of Publication

2022

Degree Name

Doctor of Philosophy (PhD)

Document Type

Doctoral Dissertation

College

Agriculture, Food and Environment

Department/School/Program

Plant and Soil Sciences

First Advisor

Dr. Sharyn E. Perry

Abstract

Plants have amazing regeneration properties with single somatic cells, or groups of cells able to give rise to fully formed plants. One means of regeneration is Somatic Embryogenesis (SE), by which an embryonic structure is formed that “converts” into a plantlet. Somatic embryogenesis has been used as a model for zygotic processes that are buried within layers of maternal tissues. Understanding mechanisms of somatic embryo induction and development are important as a more accessible model for seed development. We rely on seed development not only for most of our caloric intake, but also as a delivery system for engineered crops to meet agricultural challenges. Regeneration of transformed cells is needed for this applied work as well as basic research to understand gene function. Here we focus on a MADS-domain transcription factor, AGAMOUS-Like15 (AGL15) that shows a positive correlation between accumulation levels and capacity for SE. SE is a valuable and unique biotechnological tool used for plant improvement, micropropagation, and clonal regeneration to meet food demands or, at a basic level, to test gene product functions. AGL15 can directly induce and repress target gene expression, for which promotion of SE is positively correlated with accumulation. An ethylene-responsive element binding factor-associated amphiphilic repression (EAR) motif of the form LxLxL within the carboxyl-terminal domain of AGL15 was shown to be involved in repression of gene expression. We examine whether AGL15′s ability to repress gene expression is needed to promote SE. While a form of AGL15 where the LxLxL is changed to AxAxA can still promote SE, another form with a strong transcriptional activator at the carboxy-terminal end does not promote SE and, in fact, is detrimental to SE development. Select target genes were examined for response to the different forms of AGL15.Furthermore, in an associated project, one of the intriguing proteins with which AGL15 interacts in yeast 2-hybrid assays is LBD40. LBD40 encodes a LATERAL ORGAN BOUNDARIES (LOB)-domain TF that is unique to plants, is specifically expressed during seed development and has a role in supporting SE. In planta protein interaction of AGL15 and LBD40 has now been documented using co-immunoprecipitation. SE tissue with epitope-tagged transgenes were used for Chromatin Immunoprecipitation (ChIP) that allows one to determine where TFs bind to DNA in vivo, a step necessary to understanding genes directly controlled by a TF. More than four hundred binding regions for LBD40 were found genome-wide in three biological replications of ChIP-sequencing. RNA-seq results of 7-8 days old seeds from an lbd40/41 mutant line compared to wild-type seeds showed more than seven hundred genes had increased transcript accumulation in the mutant, while 2086 genes were downregulated. Lbd40 along with close redundant gene lbd41, showed a significant reduction in one form of SE, Shoot Apical Meristem Somatic Embryogenesis (SAM SE). Using RNA-seq data for the SAM SE system, we found a total of 2426 genes showed significantly increased transcript accumulation whereas 1145 genes showed decreased transcript accumulation. The Gene Ontology (GO) enrichment analysis of these regulated genes showed an overrepresentation of biological processes that are associated with SE, further indicating the importance of LBD40 in SE.

Digital Object Identifier (DOI)

https://doi.org/10.13023/etd.2022.62

Available for download on Wednesday, April 19, 2023

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