Over expression of a novel gene responsible for Unstable factor for orange1 phenotypes in maize causes widespread stress and alters flavonoid metabolism
Open Access
- Author:
- Wittmeyer, Kameron Thomas
- Graduate Program:
- Plant Biology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- December 12, 2017
- Committee Members:
- Surinder Chopra, Dissertation Advisor/Co-Advisor
Surinder Chopra, Committee Chair/Co-Chair
Dawn Luthe, Committee Member
Majid R Foolad, Committee Member
Michael Axtell, Outside Member - Keywords:
- Ufo1
Ufo1-1
Unstable factor for orange1
Maize genetics
RNA-seq
maize epigenetics
paramutation
p1
pericarp color1
P1-wr - Abstract:
- Transcriptional gene silencing occurs when the chromatin of regulatory elements in genes changes from accessible euchromatin to dense heterochromatin. Changes in chromatin state are directed by reversible modifications of histone tails and DNA bases. Because these modifications or marks are heritable they are termed epigenetic, factors beyond the DNA sequence which can be passed through mitosis or meiosis. This transcriptional gene silencing plays important roles in normal development, environmental responses, plasticity, and phenotypic diversity among other things. The maize pericarp color1 (p1) pigment gene has served as a useful model for studying transcriptional gene silencing. P1 is an R2R3 MYB transcription factor which activates the expression of enzymes needed for production of dark red flavonoid pigments called phlobaphenes in floral tissues. There is astounding diversity of tissue specific expression patterns in different p1 alleles which has been accounted for by differential transcriptional gene silencing of regulatory elements. Some alleles of p1 also participate in the epigenetic phenomenon paramutation, where transcriptional gene silencing and the ability to cause transcriptional gene silencing is passed from one allele to another allele of the same gene in trans. That is when a silent paramutagenic allele is heterozygous with an expressing paramutable allele, the expressing allele is turned into a silent paramutagenic allele. Paramutation is quite interesting because it is a violation of Mendel’s first law that alleles segregate unchanged. An unlinked dominant mutation in maize, Unstable factor for orange1 (Ufo1), releases the transcriptional gene silencing of several p1 alleles including paramutagenic ones. The interaction of Ufo1 and p1 alleles is notable for the accumulation of ectopic phlobaphene pigments in vegetative tissues causing plants to appear red or orange. However, the only known allele, Ufo1-1, has a high degree of instability, frequent sectoring, incomplete penetrance, and poor expressivity. Additionally, Ufo1-1 plants display a range of pleiotropic defects such as reduced growth, bending, and abnormal leaf development. The mechanisms involved in Ufo1-1 phenotypes and interactions with p1 are not well understood. In chapter 1, I describe the identification of a candidate gene for Ufo1, GRMZM2G053177 (B73 RefGenV3, 5b+). Using RNA-seq data from three tissues of Ufo1-1 it was found that the candidate gene, located in the mapping region for Ufo1, was expressed more than 50-250-fold over the wild type controls. Additionally, we find that the candidate has a novel CACTA transposon insertion in the first intron that is not found in other inbred lines. The CACTA appears to act as an enhancer of expression, but is also subject to transcriptional gene silencing. Taken together these molecular features support the genetic behaviors of Ufo1-1 such as dominance, incomplete penetrance, and poor expressivity, making GRMZM2G053177 the most likely causal gene. GRMZM2G053177 is unique to a few grass species and appears to be rapidly diverging. It also lacks any sequence homology based annotations making its function hard to determine. I discuss some possible roles based on protein structural homology from protein modeling software. I also show that thousands of genes are misregulated in Ufo1-1 seedlings, leaves, and pericarps. Tests for GO enrichment reveal a few broad categories of genes affected in Ufo1-1 seedlings and leaves. Stress response genes are broadly upregulated, while DNA replication and ribosome biogenesis are downregulated. These are markers of a response to stress, however it is unclear whether Ufo1-1 plants are under stress or if Ufo1-1 directly regulates or interferes with normal stress responses. The metabolic costs of constitutively activating stress responses and downregulating normal processes needed for growth likely explains the reduced height of Ufo1-1 plants. The involvement of siRNAs and Ufo1-1 in regulating paramutagenic and non-paramutagenic alleles of p1 was studied in chapter 2. First, we find production of siRNAs from sequences required for paramutation in both a paramutagenic and a non-paramutagenic allele in leaves, immature tassels, immature cobs, and pericarps. This indicates that siRNAs alone are not sufficient to initiate paramutation at p1. Second, we show that Ufo1-1 does not alter global accumulation of siRNAs and its effects on siRNAs in different p1 alleles cannot explain its effects on DNA methylation at these alleles. In another set of experiments it was found that Ufo1-1 is not required for establishing paramutation at either P1-rr or B-I. Additionally, we find that double mutants of Ufo1-1 and siRNA biogenesis mutant gene mediator of paramutation1 (mop1) have increased B’ expression compared to mop1 mutants alone. This indicates that Ufo1-1 does not function via a siRNA mechanism like mop1 does. These results indicate that Ufo1-1 does not primarily regulate p1 through an epigenetic mechanism involving siRNA. Alternate mechanisms may involve direct or indirect changes in DNA methylation via enzymes regulated by Ufo1. While the exact mechanism remains unknown, here we have generated hypotheses about stress regulation of flavonoid genes and also identified the likely causal gene of Ufo1 allowing for future studies of this interesting locus.