Transcript BlaisdellSpr10x

Phenotypic Characterization of lrb Mutants in Arabidopsis thaliana.
Brandon D.
1
Blaisdell ,
Derek J.
1
Gingerich
of Biology, University of Wisconsin-Eau Claire, Eau Claire, WI
2Department of Genetics, University of Wisconsin-Madison, Madison, WI
Red light sensitivity in lrb1/lrb2 mutants is
PhyB
dependent
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Hypocotyl length (cm)
In plants as well as all living organisms the selective
degradation of cellular proteins is important for growth and
development. This degradation occurs when a cell no longer
has a need for an individual protein, either because some
change occurs in the environment or in response to
developmental cues. Selective protein degradation occurs by
activity of the ubiquitin (Ub)/26S proteasome system. In this
pathway, proteins to be degraded are tagged with multiple
Ubs through the action of three specific enzymes (E1, E2, and
E3). The E3 Ub-ligase is the final enzyme in this process as it
binds to the target and catalyzes the attachment of the Ub
tag to the protein. This tag is then recognized by the 26S
Figure 1. BTB/CUL3 E3 UbiquitinLigase Complex Structure.
proteasome and the protein is degraded (Smalle, 2004).
Hypocotyl length (cm)
An LRB1 transgene rescues the lrb1/lrb2
red light hypersensitive phenotype
This mutant analysis showed that LRB1 and LRB2 play a role in the regulation of
the phytochrome-mediated red light signaling pathway, however whether there is a
role in this pathway for the third family member (LRB3) in this pathway was unclear.
lrb1/lrb2 double mutants display hypersensitivity to red light. We created a number
of different Arabidopsis mutants containing various combinations of lrb and phy
(phytochrome photoreceptor) mutations in order to determine the relative
contributions of the LRB genes to red light signaling and to determine which
phytochromes they act downstream of. We present detailed phenotypic analysis of
the mutant’s responses to different fluence levels of red light, focusing particularly
on hypocotyl elongation and cotyledon expansion. Thus far our data suggests that
the LRB3 gene does not act in red light signaling.
WT
lrb1-1/lrb2-1
Figure 2. Examples of WT and lrb1-1/2-1 seedlings subjected to red light
treatment for 3 days. The mutant lrb1-1/2-1 red light hypersensitive short
hypocotyl phenotype shown.
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Figure 5. A transgene encoding an epitope-tagged WT version of LRB1 partially
rescues the red light hypersensitive phenotype of lrb1-1/lrb2-1.
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B
Hypocotyl length (cm)
LRB1 and LRB2, but not LRB3, act in the
red
light
signaling
pathway
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Hypocotyl length (cm)
 WT , lrb, and phy mutant seeds were sterilized and
plated on a nutrient media.
 These seeds were cold treated for 3 days in the dark,
then the plates were moved into white light for 8 hours to
induce germination
 This was followed by a 16 hour dark treatment at room
temperature.
 Seedlings were then grown under continuous red light
(670nm) at various fluence levels for 3 days. At the end of
the light treatments hypocotyl lengths or cotyledon areas
were measured.
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Figure 4. The lrb1/lrb2 red light hypersensitive phenotype is dependant on an active
phytochrome B photoreceptor. The phyB-9 single mutant and the phyB-9/lrb1-1/2-1
mutant exhibit similar hypocotyl elongation under red light (100 μmole m-2 sec-1).
The Cullin (CUL)-based Ub-ligases are one superfamily of E3s in both plants
and animals. In these complexes, the BTB (Bric-a-Brac, Tramtrack, and Broad
Complex) domain-containing proteins act as the target adapters, selecting for the
proteins to be ubiquitinated (and subsequently degraded) by directly binding to
them (Gingerich et al., 2005)(Figure 1). There are a total of 80 BTB proteins
encoded in the genome of Arabidopsis (Gingerich et al. 2005). LRB1, LRB2, and
LRB3 are members of a small gene family within the superfamily. We identified
Arabidopsis thaliana individuals with T-DNA mutations of LRB1, LRB2 and LRB3 in
order to determine their role(s) in plant growth and development. Under normal
growing conditions, plants with these mutations do not show obvious
phenotypes.
Experimental Design
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lrb1/lrb2 mutants have increased cotyledon
expansion in response to red light, and this
response is dependent on PhyB
Cotyledon Area (mm2)
Introduction
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Matthew Christians
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Figure 6. A. lrb1-1/2-1 show increased cotyledon expansion under red light. The lrb11/2-1 mutant and lrb1-1/2-1/3-1 exhibit similar cotyledon area under red light (10
μmole m-2 sec-1), again suggesting that LRB3 does not contribute to red light responses.
B. The phyB-9 single mutant and phyB-9/lrb1-1/2-1 triple mutant exhibit similar
cotyledon expansion under red light (100 μmole m-2 sec-1).
Conclusions
 lrb1/lrb2 mutants are hypersensitive to red light, as assayed by hypoctyl elongation and
cotyledon expansion.
 LRB3 does not appear to be involved in red light regulation of hypoctotyl elongation.
 LRB3 may be involved in red light regulation of cotyledon area, however an lrb3
mutation does not increase red sensitivity when added to other lrb mutations.
 LRB1 and LRB2 action in red light signaling is dependant on an active phytochrome B red
light receptor.
 An LRB1 transgene can partially rescue the lrb1/lrb2 red light phenotype, confirming
LRB1’s role in this pathway.
 Overall, this data suggests that LRB1 and LRB2 are involved in a signaling cascade that
perceives red light via phyB. Our data suggests that LRB3 probably is not involved in red
light signaling, however more analysis is required to confirm this.
References
Gingerich, D.J., Gagne, J.M., Salter, D.W., Hellmann, H., Estelle, M., Ma, L., and Vierstra, R.D.
(2005). Cullins 3a and 3b assemble with members of the broad complex/tramtrack/bric-abrac (BTB) protein family to form essential ubiquitin-protein ligases (E3s) in Arabidopsis. J.
Biol. Chem. 280, 18810-18821
Smalle, J., and Vierstra, R.D. (2004). The ubiquitin 26s proteasome protolytic pathway.
Annu. Rev. Plant Biol. 55, 55-590
Funding
Figure 3. A. Under dark conditions all genotypes show similar hypocotyl elongation,
demonstrating that these mutants are not generally defective in elongation. B. Plants
containing the lrb1-1 and lrb2-1 mutations have significantly shorter hypocotyls when
treated with 10μmole m-2 sec-1 red light (indicative of increased sensitivity).The
addition of the lrb3-1 mutation (lrb1-1/lrb2-1/lrb3-1) does not increase red light
sensitivity.
Brandon has received funding from a UWEC Office of Research and Sponsored Programs
Summer 2008/2009 Research Experience for Undergraduates grant and a Fall 2008/Spring
2009 Faculty/Student Collaborative Research grant.
This work was partially funded by a National Science Foundation-Research in
Undergraduate Institutions grant (#0919678)