Investigation into the regulation of meristem formation and development by the REL2 family of transcriptional corepressors in Zea mays
Creators: name: Gregory, Jason R.
Publisher: name: No Publisher Supplied
Year: 2024

description: Maize (Zea mays L.) is a monoecious cereal crop that serves as one of the four pillars of human caloric sources. This foundational status is the result of several thousand years of artificial selection and breeding which turned a highly tillered progenitor with small ears of 5 – 12 encased kernels into a large, apically dominant plant with an ear of over 300 or more naked kernels. The genetic reprogramming of developmental pathways that yielded modern maize targeted the action and size regulation of meristems, organized structures containing plant stem cells. The post-embryonic formation of the plant body proceeds in a sequential manner through the action of meristems, and tightly coordinated meristematic regulation is required for proper development and reproductive success, eventually determining yield. A major goal of my research was to understand the role of transcriptional co-repression and related transcriptional machinery in regulating maize reproductive growth and in particular the development of the ear. I focused on the RAMOSA ENHANCER LOCUS2 (REL2) family of transcriptional corepressor proteins which includes four members, REL2, RELK1 (REL2-LIKE1), RELK2, and RELK3.In the first chapter of my thesis, my research focused on the molecular characterization of small-upright, a double mutant identified in an unbiased genetic screen for enhancers of the rel2 mutant that bore fewer and shorter internodes and enlarged female inflorescence meristems (IMs) carryingiiimutations in RELK1. I performed RNA-seq analysis on IM tips and showed that REL2 and RELK1 cooperatively regulate IM development by regulating genes involved in redox balance, hormone catabolism, and differentiation ultimately tipping the meristem toward a molecular environment favorable to expanded expression of ZmWUS1, encoding a stem-cell promoting transcription factor. To further investigate the function and relationship among the various family members CRISPR-Cas9 induced mutations in RELK2 and RELK3 were generated. My analysis shows that RELK genes have largely redundant functions in the development and maintenance of various meristem types through the plant life cycle. I also showed that the loss of one functional copy of REL2 can promote an increase in kernel row number across a diverse set of F1 hybrid combinations, indicating that tinkering with the REL2 corepressor family has the potential to increase seed yield. These data are currently incorporated in a bioRxiv preprint and a manuscript is under review for publication in Plant Physiology.In the second chapter of my thesis, I explored upstream cis-regulation, and downstream targets of REL2. Using ATAC-seq and DAP-seq data, I defined two cis-regulatory modules (CRMs) upstream of REL2. By employing CRISPR- Cas9 to induce edits, I found deletions in CRM1 and CRM2 that decreased expression levels of REL2. In order to identify direct gene targets of REL2 complexes, I designed and validated the specificity of an anti-REL2 C terminal antibody for use in ChIP-seq experiments in immature inflorescences to identify direct targets of REL2 regulation. One putative target of REL2 repression is CLE8, an uncharacterized CLE peptide which is upregulated in inflorescence meristem tips of rel2;relk1. To functionally characterize this peptide, I performed peptide feeding assays on embryos and germinating seedling. I found that CLE8 neither affected SAM width nor root length.In the third chapter of my thesis, we provided new insight into ZmWUS1 activity in the inflorescence meristem by performing single cell ASSAY FOR TRANSPOSASE-ACCESSIBLE CHROMATIN (ATAC)-seq analysis in inflorescence meristem tips of Bif3, a semidominant mutant caused by overexpression of WUS1. This works has been done in collaboration with Prof.iv Robert Schmitz and Sohyun Bang at the University of Georgia Athens, as part of a joint NSF grant. Enrichment of the homeodomain recognition CAATAATGC motif was found in genomic regions of increased chromatin accessibility suggesting that ZmWUS1 functions as an activator in the central zone. Conversely, genomic regions of decreased chromatin accessibility in Bif3 inflorescence meristem tips were often adjacent to AUXIN RESPONSE FACTOR (ARF) genes suggesting auxin signaling is repressed in the central zone of the inflorescence meristem. These data are incorporated into a manuscript on which I am a co-author and has been deposited on bioRxiv. My contribution to this publication was genotyping and harvesting of inflorescences for the scATAC-seq analysis performed by Sohyun Bang. At the same time, I have also collected rel2 and rel2;relk1 inflorescences for generating chromatin accessibility data. These data will be carefully analyzed in the future.In the fourth chapter of my thesis, we provided new insight into the transcription factor binding landscape of the maize genome. By employing DAP- seq, transcription factor binding events for 200 transcription factor belonging to 30 families were mapped in B73 and Mo17 inbreds. Comparative analysis of transcription factor binding events between the inbreds revealed changes in background gene expression is driven by structural variation and presence- absence variation in intergenic cis-regulatory regions/modules (CRMs). The biological relevance of our identified CRMs was validated by CRISPR-Cas9 editing of TSH1 and BIF2 CRMs. Mutations in CRMs altered gene expression and recapitulated reference mutant phenotypes. These data are incorporated into a manuscript on which I am a co-author and has been deposited on bioRxiv. My contribution to this publication was generating the TSH1 CRM1 and CRM2 CRISPR-Cas9 constructs, and genotyping and phenotyping of T0 mutants.Understanding the developmental biology of the ear inflorescence meristem has important implications for positively fine-tuning meristem size to achieve increases in yield. Given projected population growth, more food will need to be produced on the same amount of land in a rapidly changing climate, so achieving even small yield increases per plant can translate into majoragronomic gains. My research showed that basic understanding of the molecular mechanisms regulating maize meristems can provide new tools to face this daunting challenge.