Phenotypic Characterization of lrb Mutants in Arabidopsis thaliana. Brandon D. Blaisdell1, Matthew Christians 2, Derek J. Gingerich1

1Department of Biology, University of Wisconsin-Eau Claire, Eau Claire, WI2Department of Genetics, University of Wisconsin-Madison, Madison, WI

Introduction 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 proteasome and the protein is degraded (Smalle, 2004). 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.

Figure 1. BTB/CUL3 E3 Ubiquitin-Ligase Complex Structure.

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-a-brac (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

FundingBrandon 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)

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

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 , 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.

LRB1 and LRB2, but not LRB3, act in the red light signaling pathway

Figure 5. A transgene encoding an epitope-tagged WT version of LRB1 partially rescues the red light hypersensitive phenotype of lrb1-1/lrb2-1.

Experimental Design

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lrb1/lrb2 mutants have increased cotyledon expansion in response to red light, and this

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Red light sensitivity in lrb1/lrb2 mutants is PhyB dependent

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Figure 6. A. lrb1-1/2-1 show increased cotyledon expansion under red light. The lrb1-1/2-1 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).

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 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.

An LRB1 transgene rescues the lrb1/lrb2 red light hypersensitive phenotype

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).