Transcript Factors affecting flowering in the biennial

Abstract
An exciting avenue to explore in the post-arabidopsis genome world is the application
of tools and information developed in arabidopsis to other plants with unique attributes.
To this end, our lab is analyzing the signal transduction pathway regulating flowering in
biennials. As a model, we have chosen the obligate biennial crucifer Barbarea verna.
Like other obligate biennials, B. verna requires an extended cold treatment to flower.
We have shown that B. verna is unresponsive to vernalization treatment until it has
grown vegetatively for five or more weeks. The vernalization treatment itself must be at
least five weeks long to be effective. In addition to the cold treatment, we have
determined that B. verna requires long-days for flowering, producing an abortive
inflorescence under short days. The long-day requirement for flowering can be replaced
by gibberellic acid treatment.
Drawing an analogy to work done in arabidopsis and other species, we are asking
whether the cold treatment leads to changes in DNA methylation. Treatments with the
demethylating agent 5-azacytidine did not lead to early flowering. Additional
demethylating agents are currently being tested, and the degree of DNA methylation in
cold- and chemical-treated plants is being measured directly.
Additionally, we are generating an Agrobacterium-based transformation protocol for B.
verna, using both floral dip and root regeneration protocols. Our goal is transform B.
verna with constructs known to induce early flowering in Arabidopsis thaliana to
analyze the effects of overexpression of these genes in a biennial plant. We hope to use
these experiments to “map” the cold requirement in the signal transduction pathway for
flowering in the biennial B. verna.
Factors Affecting Flowering in the Biennial Crucifer
Barbarea verna
Brian W. Tague, Kendrah O. Kidd, Brian J. Ferguson, Rebecca W. Todd,
Maryn E. Whittles and Erin Davis
Department of Biology, Wake Forest University
Winston-Salem NC 27109
Introduction
Our lab is applying the tools and information developed for
Arabidopsis thaliana to related crucifers with attributes not found in
arabidopsis. We are currently analyzing the signal transduction
pathway regulating flowering in biennials. The biennial habit is an
important characteristic of many crop and horticultural plants and
could be a target for genetic modification. As a model, we have
chosen the obligate biennial Barbarea verna (Brassicaceae).
Questions:
Must B. verna reach a minimum age before flowering?
How long is the vernalization period needed for flowering?
What is the effect of photoperiod on flowering?
Does the level of genomic DNA methylation influence flowering?
Can B. verna be transformed?
“Flowering” in these studies is defined as the conversion of a
vegetative meristem to a floral meristem, observed at
the macroscopic level.
Barbarea verna as a model biennial
Barbarea verna
B. verna (also known as Upland Cress or creasy greens) is a
monocarpic perennial that requires vernalization to flower. It grows
as a rosette, with characteristics similar to A. thaliana.
•Relatively small
•Rosette diameter:
•Inflorescence height
•Generation time (in lab):
•Large seed count
•Easy to grow in soil or plates
•Self-fertile
~20 cm
~40 cm
25-30 weeks
~200/plant
Barbarea verna must be five weeks old
to respond to vernalization
Barbarea verna must receive
5 weeks of vernalization to flower
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Age of plants before vernalization (weeks)
Vegetative apex
Floral apex
Short days delay flowering in B. verna
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Length of vernalization (weeks)
B. verna seeds were germinated in 4-6” pots and grown in a greenhouse (t = 20-26C). Plants
from 1 to 9 weeks old (N >30 for each age) were vernalized for 5 weeks (t = 4C). Flowering
was scored by the macroscopic appearance of floral buds; plants were observed for 3 months
after vernalization.
B. verna seeds were grown as above. 5 week old plants (N >30 for each age) were vernalized
from 1 to 10 weeks (t = 4C). Flowering was scored as described. Unvernalized plants had not
flowered after 12 months. Longer vernalization leads to a higher percent of the plants flowering.
Long days promote inflorescence elongation
Gibberellic acid cannot replace vernalization in B. verna....
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Cold treatment:
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24 h
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Day length:
8h
Cold treatment:
Five week old plants vernalized for 5 or 10 weeks were subjected to 8 h light/16 hr dark or 24 hr
light photoperiods and then scored for flowering. Response of greenhouse grown plants (>12
hours of light) is shown for comparison
Treatment
10 wks veg
10 wks veg
5 wks veg, 5 wks
cold, 8 hrs lt
5 wks veg, 5 wks
cold, 8 hrs lt
Mock
100 mg/L GA
Mock
% Inflorescence
elongated
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100 mg/L GA
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5 weeks
8h
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10 weeks
Greenhouse (>12 h)
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10 w
Data derived as in previous figure. In the case of plants that were vernalized for five weeks and
grown with eight hours of light, 65% of the plants showed conversion to a floral meristem;
only 25% subsequently bolted.
...but GA can replace long photoperiod induction of
inflorescence elongation...
Plants
24 h
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Measuring cytosine methylation
by thin layer chromatography
... and continued exposure leads to “GA-overdose”
Isolate genomic DNA
dmCMP
Restrict with MspI or HpaII
dCMP
End-label with g-32P-ATP
Digest to nucleotides
Untreated
GA-treated
Separate C and
mC
by TLC
Autoradiography
5-azacytidine treatment can cause cytosine demethylation
In certain A. thaliana ecotypes and other plant species that
respond to vernalization, chemical demethylation using 5-azaC
can induce non-vernalized plants to flower earlier than untreated
controls.
MspI
(CCGG,
CmCGG)
HpaII
(CCGG)
5-azaC treatment of B. verna does not induce flowering
Conditions tested:
Application of 5-azacytidine, 10-100 mM
Application to >5 week old plants
Application to germinating seedlings
Treatments led to dwarfing and/or necrosis but no flowering
Control
50mM 5-azaC
Control
10 mm
5-azaC
GA-treated
Ten week old B. verna plants (N >20) were sprayed with a 100 mg/L (0.29 mM) solution of GA3
until dripping, every day for 9 weeks. No conversion to a floral meristem was observed, although
GA-treated plants were greener and had elongated petioles.
Proportion of cytosine methylation during vernalization
Methylation can be measured using methylation-insensitive restriction enzymes
Greener, longer petioles
Shorter inflorescence than
normal
Longer inflorescence,
abortive flowers
Mock treated
100 mm
5-azaC
Proportion of cytosines methylated
Day length:
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Length of vernalization (weeks)
B. verna plants were vernalized and transferred to the greenhouse. DNA was isolated from
newly emerged leaves and analyzed for cytosine methylation by TLC. Individual spots from the
TLC plate were quantified in a scintillation counter. Data shown is average of three
experiments; bars show standard error. Gray trend line indicates little change in methylation.
Conclusions
 B. verna: is a suitable model system for studying biennialism
must be at least 5 weeks old to respond to vernalization
requires 5 weeks of vernalization to flower
 Long days can promote inflorescence elongation
 GA does not induce flowering in B. verna
 Preliminary experiments indicate no role for methylation in the
vernalization of B. verna
 Meristem conversion and inflorescence development can be
uncoupled
Ongoing
 Transformation:
Floral dipping has not been successful
Have regenerated plants from root tissue culture
Expression of A. thaliana floral pathway genes in B. verna?
 Demethylation:
Measuring methylation in 5-azaC treated plants
HPLC analysis of cytosine methylation