Transcript HelminiakSpr15x

Creation and Characterization of LRB (Light-Response BTB) /PIF (PhytochromeInteracting Factor) Mutant Lines in Arabidopsis thaliana
Luke T. Helminiak, Kari J. Carothers, Derek J. Gingerich
Department of Biology, University of Wisconsin-Eau Claire
Introduction
PCR Genotyping of Mutants
Light is vital to plant survival and thus plants have developed
sophisticated light-sensing and response pathways to respond properly to
their environments. Plants have the ability to sense, via photoreceptors,
specific wavelengths of light. One family of photoreceptors are the red
(R)/far-red (FR) absorbing phytochromes (phys). Absorption of red light
activates the phys, which causes translocation from the cytosol to the
nucleus where they then modulate gene expression. They do so by
regulating the activity and levels of a family of transcription factors called
Phytochrome-Interacting Factors (PIFs). In response to red light the active
phys cause PIFs to be degraded, which activates expression of PIFrepressed genes (Mathews, 2006).
Recently published reports have implicated two genes (called LightResponse BTB 1 and 2 [LRB1 and LRB 2]) as critical regulators of the
phy/PIF light-response pathway (Christians et al. 2012, Ni. et al. 2014).
LRB1 and LRB2 encode BTB (Bric-a-Brac, Tramtrack, Broad Complex)
domain-containing proteins that act as target adapters in E3 ubiquitinligase complexes. E3 ligases are enzymes that covalently attach a small
protein called ubiquitin to target proteins, which often leads to degradation
of the target. Plants with disruptions of the LRB genes have reduced lightdependent degradation of phys and, like plants with disruptions of PIF
genes, exhibit hypersensitivity to red light (Christians et al. 2012). The
mechanism by which the LRBs modulate phy levels is not entirely clear,
however a report published this past summer showed the LRBs can bind
to a complex of a PIF protein (PIF3) and a phy (phyB), leading to
ubiquitylation and degradation of both PIF3 and phyB (Ni et al. 2014)
(Figure 1).
One way to better understand how the LRB and PIF genes work is
to create plants with disruptions of both LRB and PIF genes. We are
working to create pif lrb mutants in the model flowering plant
Arabidopsis thaliana. Study of the phenotypes of these plants may
shed light on how these two families of genes work together to regulate
red light responses.
Genotyping the lrb/pif mutant lines involves PCR reactions, which amplify specific regions
of DNA using genomic DNA from individual plants as template. The products of the PCR
reactions are analyzed by gel electrophoresis. The presence or absence of specific
products tells us the genotypes of the individual plants. The general strategy for
genotyping the T-DNA mutants is shown below, along with representative gels.
Genotyping for the pif3-3 deletion mutation involves a different strategy (not shown).
Creation of pif lrb Mutants
In order to create pif/lrb mutants
we crossed Arabidopsis thaliana
plants homozygous for
mutations in the LRB genes
(lrb1-1 lrb2-1) with plants
homozygous for mutations in
PIF7 and PIF3 or PIF4 (pif3-3
pif7-1 and pif4-2 pif7-1).The F1
offspring from these crosses
were heterozygous for all four
genes. Self-crossing these F1
plants produce F2 populations of
plants with, theoretically, all
possible mutant combinations.
F+R
F+R
T-DNA + R
T-DNA + R
B
Figure 3: Representative PCR genotyping reactions. A) Gel electrophoresis results for
five individuals (lanes 2-6) being genotyped for the pif7-1 insertion. Col-WT, pif4-2 pif7-1,
and no template controls are shown in lanes 7, 8, and 9 respectively. The individual in
lane 2 is homozygous mutant, the remainder are heterozygous. B) Gel electrophoresis
results for eight individuals (lanes 2-9) being genotyped for the lrb1-1 insertion. lrb1-1/21, Col-WT, and no template controls are shown in lanes 10, 11, and 12 respectively. The
individuals in lanes 2-4 are homozygous mutant, the individuals in lanes 5-8 are wildtype, and the individual in lane 9 is heterozygous.
LRB2
PIF4
PIF7
A
1.4
Figure 2: Structure of LRB1, LRB2, PIF3, PIF4, and PIF7 genes with locations of the lrb11, lrb2-1, pif4-2, and pif7-1 T-DNA insertions and the pif3-3 deletion indicated. Boxes
indicate exons and lines indicate introns. Yellow indicates coding region and gold
represents 5’ and 3’ UTRs. Triangles indicate locations of T-DNA insertions. Bracket
indicates portion of PIF3 gene deleted.
Conclusions
Col-WT
WT
1.2
lrb1-1 lrb2-1
lrb1-1/2-1
1
lrb1-1 lrb2-1
lrb1-1/2-1
pif 7-1pif7-1
0.8
0.6
0.4
0.2
0
0
0.05
0.1
1
10
•
We have successfully generated lrb1 lrb2 pif7 triple mutants.
Preliminary analysis of this line suggests that addition of the
pif7 mutation does not significantly alter the red light
hypersensitivity conferred by the lrb1 and lrb2 mutations.
We observed tiny individuals that grow very slowly and
eventually died in a population generated from a self-crossed
LRB1/lrb1-1 lrb2-1/lrb2-1 pif4-2/pif4-2 pif7-1/pif7-1 parent.
Genotyping is still in progress in these offspring, but
preliminary evidence suggests that disrupting all four of the
LRB1, LRB2, PIF4, and PIF7 genes may severely disrupt
Arabidopsis development.
Fluence Level (µmole/m2/sec)
B
References
1.2
col-o
wt
Col-WT
1
lrb1-1 lrb2-1
lrb1-1/2-1
0.8
lrb1-1 lrb2-1
lrb1-1/2-1
pif 7-1pif7-1
0.6
0.4
0.2
0
00
PIF3
Figure 6. Images of 2 of the severely stunted A1-10-7-17 offspring 40 days after
germination. Note the compact rosette structure and that the bottom leaves on the
second plant are chlorotic. Both of these plants eventually died.
•
Normalized hypocotyl length
(cm)
LRB1
Figure 5. Photograph showing A1-10-7-17 offspring #1-8 26 days after germination. Text
under each plant indicates if the plant lived or died. We were able to genotype
individuals #1-5 for the LRB1 gene; all turned out to homozygous wild-type for the gene.
We were able to generate an lrb1 lrb2 pif7 triple mutant. lrb1 lrb2 mutants
are hypersensitive to red light. To see if the addition of a pif7 mutations
alters that hypersensitivity, we exposed WT, lrb1 lrb2, and lrb1 lrb2 pif7
Arabidopsis plants to various levels of red light and analyzed elongation of
the hypocotyls (which are sensitive to red light).
The pif and lrb Mutations
The lrb1-1, lrb2-1, pif4-2, and pif7-1 mutations we are working with are TDNA insertion mutations. In T-DNA mutants a large piece of foreign DNA is
inserted in the gene, disrupting it’s function. The pif3-3 mutant has a large
deletion at the 5’ end of the gene, resulting in complete inactivation.
We were able to identify a plant that was homozygous
mutant for the LRB2, PIF4, and PIF7 genes and was
heterozygous for LRB1. This individual was allowed to
self-cross and the offspring were germinated and grown.
We noticed distinct phenotypes in the population.
Approximately ¼ of the offspring grew fairly normally, the
remaining ¾ were severely stunted and grew very slowly.
The majority of these turned chlorotic and died after the
emergence of only 2-10 leaves. One continues to survive,
but has yet to produce reproductive structures. Example
individuals from this population are shown below.
Characterization of an lrb1 lrb2
pif7 Mutant
Hypocotyl Length (cm)
Figure 1. Structure of LRB/CUL3 E3
ubiquitin ligase complex, with PIF3
and phyB targets shown. Figure is
based on model of LRB action
presented by Ni et. al. (2014).
A
Disrupting the LRB1, LRB2, PIF4,
and PIF7 Genes Together May
Prevent Normal Development
1
2
3
0.1
1.0
0.05
Fluence Level (µmole/m2/sec)
4
10
Figure 4 A) Hypocotyl length and B) Normalized hypocotyl length of wild-type, lrb1-1
lrb2-1, and lrb1-1 lrb2-1 pif7-1 4 day-old seedlings grown under various levels of red
light. At least 20 seedlings were measured for each genotype/light treatment. Error
bars indicate standard error of the mean. In (B) the lengths of each hypocotyl was
normalized to mean dark length for that genotype.
1. Christians, Matthew J.; Gingerich, Derek J.; Hua, Zhihua; Lauer, Timothy D.;
Vierstra, Richard D. (2012). The Light-Response BTB 1 and 2 Proteins Assemble
Nuclear Ubiquitin Ligases That Modify Phytochrome B and D signaling in
Arabidopsis. Plant Physiology. 160(1), 118-134
2. Mathews, Sarah. (2006). Phytochrome-mediated Development in Land Plants: Red
Light Sensing Evolves to Meet the Challenges of Changing Light Environments.
Molecular Ecology, 15, 3483-3503.
3. Ni, Weimin; Xu, Shou-Ling; Tepperman, James M.; Stanley, David J.; Maltby, Dave
A.; Gross, John D.; Burlingame, Alma L.; Wang, Zhi-Yong; Quail, Peter H. (2014) A
mutually assured destruction mechanism attenuates light signaling in Arabidopsis.
Science. 344(6188). 1160-1164
Funding
This work was/is funded by two National Science Foundation-Research in Undergraduate Institutions (RUI) grants
(#0919678 and #1354438), a National Science Foundation Arabidopsis 2010 Program Grant (MCB-0115870), a
National Institutes of Health Ruth L. Kirschstein Postdoctoral Fellowship (F32-GM68361), and University of
Wisconsin-Eau Claire Differential Tuition through the UWEC Office of Research and Sponsored Programs.