1. Introduction Luke T. Helminiak, Kari J. Carothers, Derek J. Gingerich Department of Biology, University of Wisconsin-Eau Claire Luke T. Helminiak, Kari J. Carothers, Derek J. Gingerich Department of Biology, University of Wisconsin-Eau Claire Creation and Characterization of LRB (Light-Response BTB) /PIF (Phytochrome- Interacting Factor) Mutant Lines in Arabidopsis thaliana 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 PIF- repressed genes (Mathews, 2006). Recently published reports have implicated two genes (called Light- Response 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 ubiquitin- ligase 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 light- dependent 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). The pif and lrb Mutations Creation of pif lrb Mutants lrb1 lrb2 pif7 Mutant Characterization of an lrb1 lrb2 pif7 Mutant Conclusions References Funding PCR Genotyping of Mutants 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. 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. 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). 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. 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 lrb1-1, lrb2-1, pif4-2, and pif7-1 mutations we are working with are T- DNA 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. Figure 2: Structure of LRB1, LRB2, PIF3, PIF4, and PIF7 genes with locations of the lrb1- 1, 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. 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). Disrupting the LRB1, LRB2, PIF4, and PIF7 Genes Together May Prevent Normal Development 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. 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. 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. 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. lrb1-1/2- 1, 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 wild- type, and the individual in lane 9 is heterozygous. LRB1 PIF4 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. A LRB2 PIF7 PIF3 B F+R T-DNA + R F+R T-DNA + R Col-WT lrb1-1 lrb2-1 lrb1-1 lrb2-1 pif7-1 Col-WT lrb1-1 lrb2-1 lrb1-1 lrb2-1 pif7-1 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 A B 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.