Year of Publication

2020

Degree Name

Doctor of Philosophy (PhD)

Document Type

Doctoral Dissertation

College

Agriculture, Food and Environment

Department

Plant and Soil Sciences

First Advisor

Dr. Sharyn E. Perry

Abstract

Seeds make up approximately 70% of the human diet directly so understanding regulatory mechanisms to generate a seed and the embryo it contains is fundamentally important. The LAFL genes encode transcription factors (TF) that are critical for seed development. Three members of LAFL, LEAFY COTYLEDON2 (LEC2), ABSCISIC ACID3 (ABI3) and FUSCA3 (FUS3), are B3 domain factors that bind DNA motifs called RY motifs. While LEC2 is expressed earlier in embryo development, ABI3 and FUS3 are expressed during later development. All three of these TFs can induce embryo-specific programs after completion of germination to different extents up to and including formation of embryos in a process called somatic embryogenesis (SE). ABI3 also contributes to the abscisic acid (ABA) response. Prior work determined direct and indirect targets of FUS3. Here we report on chromatin immunoprecipitation-tiling array experiments to globally map binding sites for ABI3. We also assessed transcriptomes in response to ABI3 by comparing developing abi3-5 and wild type seeds and combine this information to ascertain direct and indirect responsive ABI3 target genes. ABI3 can directly induce and repress its target genes’ transcript accumulation and some intriguing differences exist in cis motifs between these groups of genes. Directly regulated targets reflect ABI3’s roles in seed maturation, desiccation tolerance, entry into a quiescent state and longevity. Interestingly, ABI3 directly represses a gene encoding a microRNA (MIR160B) that targets AUXIN RESPONSE FACTOR (ARF)10 and ARF16 that are involved in establishment of dormancy. In addition, ABI3, like FUS3, regulates genes encoding MIR156 but while FUS3 only induces genes encoding this product, ABI3 induces these genes during early stages of seed development, but represses these genes during late development. The interplay between ABI3, the other LAFL genes, and the VP1/ABI3-LIKE (VAL) genes that are involved in the transition to seedling development are examined and reveal complex interactions controlling development.

ABI3 directly regulates all five DUF1264 (Domain of Unknown Function 1264) members in Arabidopsis, while two other seed transcription factors FUS3 and AGL15 directly control subsets of genes in this family. Arabidopsis genes designated as DUF1264s appear to be expressed specifically within seeds and the encoded protein include a domain of unknown function that is highly conserved in various plants. Here, the direct association of the TFs with these genes and effect on transcript accumulation is verified. Also, higher order mutants were generated. Quadruple duf1264 mutant shows a reduction in SE compared to wild type control. Pentuple duf1264 mutant shows a hypersensitive response in seedlings to ABA compared to WT.

In an associated project, an Arabidopsis protein called SIN3A associated polypeptide 18 (AtSAP18) was investigated. SAP18 is a transcriptional co-regulator that is a component of histone deacetylase (HDAC) complexes which interacts with a TF of interest in the lab, AGL15 to control embryogenesis. A new phenotype of a loss-of-function mutant sap18 was documented in that the mutant is hypersensitive to ABA treatment compared to Columbia (Col) wild type (WT), suggesting an important role of SAP18 in modulation of ABA response.

Finally, the global targets of AGL15 were identified by combining previous RNA microarrays and ChIP microarrays with RNA-seq and ChIP-seq in this study. Also, some of these regulatory networks were investigated in the important crop plant, Glycine max.

Digital Object Identifier (DOI)

https://doi.org/10.13023/etd.2020.316

Funding Information

This work was supported by the National Science Foundation grants IOS-0922845 and IOS-1656380 from 2016 to 2020, by the Kentucky Science and Engineering Foundation KSEF-3524-RDE-019 from 2016 to 2020, by the National Institute of Food and Agriculture, U.S. Department of Agriculture, Hatch project under accession number 1013409 from 2015 to 2020, and by the Department of Plant and Soil Sciences from 2015 to 2020.

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