1. Abstract
References
rmr3 affects plant development and small RNA biogenesis in Zea mays
1. Hollick, Jay B. "Paramutation and Development." Annual Review of Cell and Developmental Biology 26.1 (2010): 557-79. Print.
2. Hollick JB, Kermicle JL, Parkinson SE. 2005. Rmr6 maintains meiotic inheritance of paramutant states in Zea mays. Genetics171:725–40
3. Stonaker, Jennifer Lynn. "Evolution of plant-specific Snf2 proteins and RNA polymerases and their function in maintaining paramutations in Zea mays." Thesis (2010).
4. Erhard, Karl F., et al. "Nascent Transcription Affected by RNA Polymerase IV in Zea Mays." Genetics 199.4 (2015): 1107-25. Print.
5. Erhard, Karl F., et al. "Maize RNA Polymerase IV Defines Trans-Generational Epigenetic Variation." The Plant Cell 25.3 (2013): 808-19. Print.
6. Erhard, K. F., Jr. (2012). Alternative polymerase genome regulation in Zea mays (Order No. 3527092). Available from ProQuest Dissertations & Theses A&I; ProQuest Dissertations & Theses Global.
(1081485351).
7. Parkinson, Susan E., Stephen M. Gross, and Jay B. Hollick. "Maize Sex Determination and Abaxial Leaf Fates are Canalized by a Factor that Maintains Repressed Epigenetic States." Developmental
biology 308.2 (2007): 462-73. Print.
8. Erhard, Karl F., et al. "Nascent Transcription Affected by RNA Polymerase IV in Zea Mays." Genetics 199.4 (2015): 1107-25. Print..
Fiigure 1. A.The Pl-Rhoades (Pl-Rh) allele is an
unstable allele that can spontaneously change from an
active state into a transcriptionally repressed state, Pl'.
When a homozygous Pl-Rh plant is crossed with a
heterozygous Pl-Rh/Pl' sibling all of the progeny exhibit
the repressed state.
BA
rmr3 affects 24nt sRNA biogenesis
Figure 3. A. rmr3 mutants (dark gray) show significant reduction in plant height and delayed flowering time compared to
WT. B. Similar to Pol IV, rmr3-1 mutants display disease lesion mimic phenotypes. C. Pol IV mutants display disease
lesion phenotype. D. rmr3-1 mutants show no difference in juvenile to adult phase change data (t-test for H0= there is no
difference; data set 1: n=24 and n=16, p= 0.059 and data set 2: n=21 and 20, p=0.859 for WT and mutant, respectively)
unlike rpd1/Pol IV mutants7).
rmr loci encode genes required for epigenetic silencing
rmr3 is necessary for normal plant development
B. Of the factors required to maintain repression of Pl'
state, some have been shown to be part of an
orthologous pathway in Arabidopsis thaliana that leads
to the biogenesis of 24nt RNAs that then lead to
downstream 5meC methylation.
Amiel Emerson1,2, Natalie C. Deans1, Janelle M. Gabriel1, Joy-El R.B. Talbot2,3 , Stacey A. Simon4, Blake C. Meyers4, and Jay B. Hollick1
1. Department of Molecular Genetics, The Ohio State University 2. Department of Biology, The University of the South 3.Department of Molecular and Cell Biology, University of California, Berkeley 4. Delaware
Biotechnology Institute, University of Delaware, Newark, Delaware 19711
ocl2 mRNA levels are unchanged in rmr3 mutants
Figure 2. small RNA sequencing libraries representing immature ears 24nt RNAs in heterozygous +/rmr3-1 and
homozygous rmr3-1/3-1 sibling plants.The levels of 22nt RNAs were used to normalize total abundance of RNAs
between samples.
rmr3 candidate gene models include some encoding
subunits of Pol IV
WT siblingrmr3-1 mutant
BA
D
Conclusions
• Rmr3 is required for normal plant development.
• rmr3-1 mutants, unlike Pol IV mutants, shows no effect on juvenile to
adult phase change.
• Preliminary data suggests ocl2 is not significantly upregulated in
rmr3-1 homozygous mutants, unlike in Pol IV mutants.
• Interesting candidate gene models include those encoding known
polymerase subunits and other proteins involved in nucleic acid
biology.
A. Candidate genes in a 20 Mb window predicted by whole genome sequence analysis. The blue dashes
represent SSR markers that were identified to narrow down the region of interest containing rmr3. Some
interesting gene models encode proteins involved in nucleic acid biology (red dashes). B. Schematic of how
simple sequence sepeat (SSR) markers could help narrow the region of interest in EMS induced rmr3-
1mutant in A619 background highly introgressed into the A632 background.
A
B Designing
SSR markers
There are specific haplotypes in plants that can undergo epigenetic alterations through genetic silencing that are
heritable1. One locus in particular in Zea mays, purple plant1 (pl1), has an allele Pl1-Rhoades, that exits in different
regulatory states; a transcriptionally active state (Pl-Rh) characterized by purple plant and anther color due to
anthocyanin pigment production, and a derived transcriptionally repressed state( Pl’) that shows a lightly-colored
phenotype. Pl’ alleles can silence Pl-Rh in Pl’/Pl-Rh heterozygotes such that only Pl’ alleles are transmitted to the
progeny2. Factors that repress the Pl’ state, encoded by required to maintain repression (rmr) loci, were identified
in a genetic screen3. These loci, encoding factors that maintain repression of Pl', affect 24nt siRNA biogenesis
related to a plant-specific RNA-directed DNA Methylation pathway (RdDM). Mutants of some of these loci have
effects on plant development (rpd1/Pol IV, and rmr12) while other rmr mutants show no effects on development4,5.
Here we show that rmr3 mutants have decreased levels of 24nt sRNAs, suggesting a role for rmr3 in their
biogenesis. While effects on development, such as stunted growth, delayed flower time, and disease lesion mimic
seen in rmr3-1 are comparable to similar defects in Pol IV mutants, transitions from juvenile to adult is unaffected
in rmr3-1 mutants confirming that they have unique effects on development6. In addition, preliminary data shows
that ocl2, a putative gene whose dysregulation could lead to the developmental phenotypes seen in Pol IV mutants,
does not show increased mRNA abundance in rmr3-1. Identification of the rmr3 encoded product could address
whether it acts as an accessory to Pol IV regulation of 24nt sRNAs or in a different manner affecting 24nt sRNA
levels; thus, candidate gene models were evaluated.
*Erhard 2012 (dissertation)
Example SSR marker
1
A
B
Figure 4. A. Global run-on sequencing reads representing Pol IV mutant and WT (normalized to reads per million
uniquely mapped) transcription over the outer cell layer 2 (ocl2) coding region8. B. Quantatative RT-PCR results
representing relative ocl2 mRNA levels of DCL3 and Pol IV WT and mutants. C. Pol IV-mediated repression of Pol II
transcription. D. RT PCR of rmr3-1 mutants and Mo17 WT comparing ocl2 mRNA levels in day 14 seedlings.
ocl2 expression in
rmr3-1 mutants
C Models of Pol IV based repression
Relative ocl2 mRNA levels in Pol IV and DCL3
mutants
D
Necrotic leaf phenotype
C
Pol IV mutants *Erdhard (2012) Thesis6
WT (no necrosis)
rmr3-1 mutants (necrosis)
3
3.5
4
4.5
5
5.5
6
No Necrosis
Necrosis
2
AveragePhaseChangeLeaf