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What is the ancestral role of a master regulator of flowering?
Functional analysis of LEAFY Homologs in the Fern Ceratopteris richardii
Jonathan Yee and Verónica Di Stilio. Department of Biology, University of Washington, Seattle
Abstract: The purpose of this research is to explore the ancestral role of a master regulator of flower development. The transcription factor LEAFY (LFY) acts as a flower meristem identity
gene in flowering plants (angiosperms), turning on downstream genes that contribute to the development of floral organs, rather than leaves. Outside of angiosperms, LEAFY homologs
have been studied functionally in moss only (a member of the one of the first lineages of land plants) where it plays a fundamental role in promoting the first cell divisions of the zygote
(the sporophyte). Given the immense phylogenetic distance between mosses and angiosperms, we set out to explore the role of homologs of this master regulator of flowering in the
model fern Ceratopteris richardii, a member of an intermediate lineage of non-flowering vascular plants.
Introduction
Goals
 LEAFY is a flower meristem identity gene in angiosperms.
 Study the role of LEAFY in the evolution and development
of land plants by investigating its function in an
intermediate lineage of land plants.
 LEAFY regulates development of the zygote (the young
sporophyte) in moss. ljlkjlkj
LEAFY is a master regulator of flower
development in angiosperms (by F. Parcy,
2002 and D. Weigel, 1992). (A) Arabidopsis
thaliana wildtype flower with petals (pe), sepals
(se), stamens (st) and carpels (ca). (B) In a leafy
mutant, flowers turn into shoots, with leaves
instead of floral organs.
LEAFY is a master regulator of
zygote (sporophyte) development
in moss (by D. Cove, 2009). Wild
type
Physcomitrella
patens
sporophytes. In a leafy mutant,
sporophytes do no develop.
Results
Selection of Transgenic
Gametophytes
As expected, wildtype spores
germinated to produce healthy
gametophytes
in
non-selective
media (A). No spores germinated in
selective media (B) . Transgenic
spores successfully germinated in
non-selective media (C). Reduced
germination occurs because only
spores carrying the RNAi LEAFY
constructs carry the Hygromycin
resistance gene (D).
 Silence the two homologs of LEAFY, CrLFY1 and CrLFY2
(individually and together), in the fern C. richardii using
loss of function transgenic spores (RNAi constructs)
Hypothesis
 LEAFY has a role in sporophyte development: most likely
not as early as in moss, but perhaps at a later stage during
its reproduction.
Methods
 Functional studies of LEAFY have only been carried at the
two ends of the land plant evolutionary tree.
(mosses)
Selection of Transgenic Sporophytes. Wildtype sporophytes did not grow in selection media, this shows that
the selection process using Hyg B is effective (A). Not all sporophytes from transgenic spores transplanted from
control media survive (B), not all sporophytes carry the transgenic constructs (notice dead ones), but a high
percentage do (green ones). Sporophytes that successfully grew from fertilized gametophytes under antibiotic
selection continued to grow well after transplantation (C), LEAFY homologs do not affect sexual reproduction
of the gametophytes, a novel finding in this research, the published protocol (Plackett et al 2014) showed
that sexual reproduction did not occur under antibiotic selection.
(ferns et al)
Conclusions and Discussion
(angiosperms)
Main lineages of land plants (Freeman, 2012). Highlighted in yellow are plants where LEAFY has
been studied functionally. Highlighted red are plants under study here.
Ac k nowl edg ments
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My principal investigator and mentor Verónica Di Stilio
The Di Stilio Lab (Jesus Martinez-Gomez , Valeria Soza, Kelsey Galimba, and Tatiana Arias)
Andy Plackett and Jane Langdale, University of Oxford Plant Sciences.
Lisa Peterson, Greg Diggs-Yang, and contributors of GenOM ALVA
Funding: University of Washington GenOM Project – NIH 5R25HG007153-03
Donations from Anne Dinning and Michael Woolf
Reconstruction of Ceratopteris richardii life cycle from live images (photos by J. Yee). Spores
were sterilized then sowed onto Carolina © C-Fern media to germinate and grow into young
gametophytes. Gametophytes can be hermaphrodite or male; after 10 days after sowing
gametophytes were flooded with 5ml of miliQ water to fertilize hermaphroditic gametophytes
with swimming sperm. Sporophytes grew on the hermaphroditic gametophytes and were
transplanted onto antibiotic selective media after 25 days. Sporophytes will be transplanted into
soil to grow and mature, then quantify sporangia (spore sacs) and spore production.
References
Maizel, A. "The Floral Regulator LEAFY Evolves by Substitutions in the DNA Binding
Domain." Science 308.5719 (2005): 260-63.
Plackett, A et al. "High-Efficiency Stable Transformation of the Model Fern Species
Ceratopteris Richardii via Microparticle Bombardment." Plant Phys 165 (2014): 3-14.
University of Washington GenOM Project: ALVA 2014
C. richardii gametophytes reproduced sexually and young
sporophytes developed normally under antibiotic selection for
RNAi-LEAFY silencing constructs. The zygote is able to grow in the
absence of LEAFY. This finding is different from the role of LEAFY in
moss and will be pursued further.
Future Directions
To further research LEAFY’s effects in C.richardii we plan to:
• Grow the fern sporophytes and note phenotypes related to
growth and reproduction.
• Characterize sporangia and quantify spores from mature ferns.
• Perform molecular validation of transgenic lines by PCR and test
for reduced expression of LEAFY in transgenic sporophyte.