Date of Award
5-2023
Document Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Department
Genetics and Biochemistry
Committee Chair/Advisor
Dr. Julia Frugoli
Committee Member
Dr. Amy Lawton-Rauh
Committee Member
Dr. Hong Luo
Committee Member
Dr. Rajadeep Sekhon
Abstract
The unique evolutionary adaptation of legumes for nitrogen-fixing symbiosis leading to nodulation is tightly regulated by the host plant. One pathway negatively regulates the number of nodules formed in response to the metabolic status of the shoot (carbon) and root (nitrogen); this long-distance systemic regulation is the autoregulation of nodulation (AON) pathway. AON is a root to shoot pathway that allows the plant to limit the number of nodules formed on roots. Central to the receptor complex in the shoots is a leucine-rich-repeat receptor-like kinase (LRR-RLK) called MtSUNN mutation of which results in plants with too many nodules. SUNN has a high sequence similarity to CLAVATA1 (CLV1), a LRR-RLK in Arabidopsis involved in regulating stem cell populations in the root and shoot. And SUNN uses a CLV1-like regulatory model to control nodule number. Another member of this class of receptors is the Arabidopsis BARELY ANY MERISTEM family (BAM1, BAM2, BAM3), which bind to CLE peptides to regulate meristem development in the CLV1 pathway, I reasoned that the AON and CLV pathways could share similar proteins, so I investigated if the BAMs identified in M. truncatula function in AON through a similar multi-protein complex to regulate nodule numbers. I was part of a team utilizing laser capture microdissection to generate a spatiotemporal transcriptomic profile of nodulating Medicago truncatula roots during critical time points of nodule formation. Using this root tissue specific transcriptome, I determined all five MtBAMs were expressed in the roots during nodule formation, but only MtBAM1 and MtBAM2 are highly expressed in the nodules 48hours after inoculation. I isolated plants homozygous for mutations in each BAM from a Tnt-1 insertion library and tested them for phenotypes. Like Atbams which were isolated as suppressors of clv1, no nodule number or root length phenotype was observed in single mutants or plants containing mutation in more than one BAM gene. However, Mtbam2 crossed to a sunn-5 suppressed the sunn-5 hypernodulation phenotype and bam2 can partially rescue the short root length phenotype of sunn-5. Grafting experiments suggest that bam2 suppresses supernodulation from the roots regardless of SUNN status of the root. Overexpression of MtBAM2 rescues the sunn-5 hypernodulation phenotype to wild type levels, while overexpression of MtBAM2 in wild type plants increases nodule numbers. I tested the relative expression of five nodule specific transcription factors (MtWOX5 and MtPLETHORA1-4) downstream of the putative bam2 sunn-5 complex for altered expression that would indicate disruption of signaling; MtWOX5 expression is inhibited in the bam2 sunn-5 mutant differently than in the bam2 mutant and this appears to inhibit the expression of the downstream PLETHORA genes. I propose a genetic model in which the root interactions of MtBAM2 and SUNN are critical for signaling in stem cell homeostasis in the nodules of M. truncatula. In this model the disruption of a multi-protein receptor complex containing both BAM2 and SUNN inhibits the transfer of a signal to halt nodulation, leading to the downregulation of WOX5 in the central nodule vasculature, which in turn inhibits WOX5 regulation of PLTs in their unique nodule domains.
Recommended Citation
Thomas, Jacklyn, "A Multi-Pronged Investigation to Identify Genes Affecting the Autoregulation of Nodulation: Lasercapture Microdissection and the Barely Any Meristem Gene Family in Medicago Truncatula" (2023). All Dissertations. 3349.
https://tigerprints.clemson.edu/all_dissertations/3349