Progress 08/15/11 to 08/14/13
Outputs
Target Audience: The target audiences are maize researchers and maize farmers, both individual and industrial scale. Developmental biologists with an interest in crop plants and an interest in learning the role that key developmental genes play during leaf, ear and tassel growth and differentiation. Additionally, maize scientists interested in the overlap between plant form and function may find this work of importance. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Through the NIFA fellows program I was able to meet a fellow maize researcher whose focus is on root development (while my focus is on the development of above ground organs), Christopher Topp. Through this program we were able to meet and discover some areas of common interest and research overlap leading to a collaborative study to examine root growth in two mutant backgrounds. We have been studying these mutants due to a leaf phenotype and Dr. Topp has worked to characterize the root phenotype. I am hopeful that we will be able to submit and joint manuscript in the future. Additionally, creation of the LG1 antibody has been received by the maize research community with great interest. When presenting these results at the annual Maize Conference, I was able to meet and initiate collaborative studies with David Jackson's lab at Cold Spring Harbor. This collaboration has already resulted in the submission of one publication and the initiation of a long term collaboration between myself and David Jackson's lab to determine all of the promoter sites to which the LG1 protein can bind. How have the results been disseminated to communities of interest? At professional meetings and through publication in peer reveiwed journals. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Creation of a marker for the LIGULELESS1 (LG1) protein (the LG1 antibody) has revealed a role for LG1 in reproductive development as well as leaf development. LG1 is specifically expressed at the adaxial junction of developing tassel branches. Anaylsis of a new LG1 point mutation reveals a decreased tassel branch number phenotype as well as decreased tassel branch angle phenotype for those branches that are generated. Ongoing work to identify and characterize the protein affected by the Wavy auricle in Blade (Wab) phenotype has also demonstrated a role for WAB in tassel branch development. Loss of function WAB mutant plants have decreased tassel branch number and angle and the expression of LG1 is lost in the tassel that does develop. This suggests that induction of LG1 by WAB is required for proper tassel branch development. Ectopic expression of WAB in the leaf blade, where is is normally not expressed, is sufficient for ectopic expression of LG1 in the blade of leaves resulting in the Wab leaf phenotype. Using ZmPIN1a marker lines crossed into the lg1 loss of function background we have found that expression of the ZmPIN1a marker requires differentiation of the preligule band and is loss when preligule band development is blocked (i.e. in the lg1 mutant background). This suggests that ZmPIN1a expression is guided by the differentiation of the ligule structure. Therefore, differentiation of the ligule is not patterned by the expression of ZmPIN1a and likely occurs independent of auxin concentration. Preliminary evidence of physical interaction between LIGULELESS2, LIGULELESS NARROW and SISTER of LIGULELESS NARROW are currently being validated using co-immunoprecipitation procedures.
Publications
- Type:
Journal Articles
Status:
Submitted
Year Published:
2013
Citation:
Eveland AL, Goldshmidt A, Pautler M, Morohashi K, Liseron-Monfils C, Lewis MW, Kumari S, Yang F, Hiraga S, Unger-Wallace E, Olson A, Stanfield S, Hake S, Schimdt RJ, Vollbrecht E, Grotewold E, Ware D and Jackson D. Defining the regulatory networks controlling maize inflorescence architecture Submitted to Nature Genetics
- Type:
Journal Articles
Status:
Other
Year Published:
2013
Citation:
Lewis MW, Bolduc N, Hake K, Htike Y, Candela H, Hay A, Hake S. "Regulation of Liguleless1 by Wavy Auricle in blade during maize vegetative and reproductive development." Manuscript in preparation.
- Type:
Other
Status:
Published
Year Published:
2012
Citation:
Bolduc N, OConnor D, Moon J, Lewis M and S Hake. How to Pattern a Leaf in Cold Spring Harbor Symposia on Quantitative Biology, Vol XXVII. Cold Spring Harbor Laboratory Press. 2012.
|
Progress 08/15/11 to 08/14/12
Outputs
OUTPUTS: I am using biochemical methods to investigate the nature and role of the putative transmembrane kinases Liguleless narrow and Sister of Liguleless narrow. Lgn was identified by its mutant phenotype and Sln is implicated because its RNA expression is massively unregulated in a lgn mutant background. It is possible that the mutant LGN protein may exhibit different phosphorylation capabilities; different protein-protein interaction potential or both. To investigate potential physical interactions between these proteins, I have performed directed yeast two hybrid experiments as described in the project narrative. I have had preliminary success with a protocol for co-immunoprecipitation of LGN and LG2. I plan to expand these studies to look at all combinations suggested: LGN:LGN, LGN:SLN, LG2:LGN and LG2:SLN. I am currently validating a custom antibody generated against LG2 for aid in this study. These are currently high priority experiments. In order to determine if expression of auxin markers guide or are guided by preligule band formation, I have begun introducing the markers into mutant backgrounds in which ligule and auricle development is different from wild-type. If patterning of the auxin markers precedes preligule development then marker expression will be unchanged in mutant backgrounds. If presence of these markers requires the mutated genes, this may suggest that changes in expression of the auxin markers is caused by loss of preligule development. So far this year, two conferences have been especially useful to my professional development. The International Maize meeting was held in Portland, Oregon March 15-18 2012. There I was able to meet with standing collaborator's as well as enjoy new connections leading to my implementation of a key control and new protocol in my biochemistry experiments mentioned above. The second event was the AFRI NIFA fellows PD meeting in Washington DC running August 16-17 2012. I am excited to explore a new collaboration with another AFRI NIFA fellow with whom I hope to explore a connection between the Lgn-r mutant and root development. I have actively mentored two undergraduate students on a daily basis. Kristen Valanoski joined my efforts for the summer via the Environmental Leadership Pathway program run by the UC Berkeley College of natural resources. Jessica Channick, a pre-med honor's student, has been working with me for over a year and will complete an Honors thesis this spring in her final semester. For use in biochemistry and expression studies, I have created antisera raised against LG1 and LG2. These tools provide a unique resource for my biochemical tests but may prove just as important for an experiment to which they were to serve a redundant purpose. The antibodies raised against LG1 and LG2 may prove to be useful for determining the subcellular and tissue specific localization of these proteins using immunolocalization techniques. The results from this work and other projects have been presented at the events described above. Additionally, we have standing collaborations with the lab of Anne Sylvester at the University of Wyoming and Michael Scanlon at Cornell University. PARTICIPANTS: I have actively mentored two undergraduate students, encouraging their training in genetics and biochemistry as well as fostering professional development. Kristen Valanoski joined my efforts for the summer via the Environmental Leadership Pathway program run by the UC Berkeley College of natural resources. She currently attends City college of San Francisco and plans to continue her studies in that schools' molecular biology Masters program. Jessica Channick, a pre-med honor's student, has been working with me for over a year and will complete an Honors thesis this spring in her final semester. Her focus is on the yeast two hybrid experiments and the search for novel proteins interacting with LGN and SLN. Both joined through a university affiliated program and volunteer their time. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
My broader goals are to dissect the role of Liguleless1 (Lg1), Liguleless2 (Lg2), Liguleless narrow (Lgn) and Sister of Liguleless narrow (SLN) in the regulation of ligule development. I have found that the kinase domain of SLN but not LGN can be transphosphorylated by the kinase domain of BRASSINOSTEROID INSENSITIVE1 (BRI1). Unlike BRI1, the kinase domains of LGN and SLN cannot phosphorylate the synthetic substrate Myelin basic protein. Taken together with the lack of a large extra-cellular domain, these results suggest that LGN and SLN may function as co-receptors, perhaps in a larger protein complex regulating ligule differentiation. Fusions between full length ORF and C-Terminal YFP tags were introduced into tobacco leaf epidermal cells via agrobacterium mediated transformation. SLN:YFP expression suggests plasma membrane (PM) localization but similar experiments with a LGN:YFP construct reveal an intercellular and punctate expression. If the expression pattern is correct, further investigation using co-localization with markers to known endosomal locations could pin point the subcellular localization of LGN. LG1 can be phosphorylated, encodes a putative transcription factor is localized in the nucleus. However, the known factors in the pathway that could potentially phosphosphorylate LG1 are LGN and SLN that do not localize to the nucleus. This suggests they do not interact with LG1 and therefore an unknown factor capable of regulating LG1 phosphorylation may exist. In pilot experiments, I found evidence for a LG2 and SLN interaction in a yeast system. Building on these experiments, an undergraduate honors student has found that LGN and SLN can homodimerize as well as heterodimerize with each other. We have also confirmed the interaction between LG2 and SLN as well as finding evidence for interaction between LG2 and LGN in this system. Expression analysis of LG2:YFP constructs transiently in tobacco epidermal cells shows LG2 appearing cytoplasmic or pressed against the PM. LG2 expression in T1 Arabidopsis roots appears to be either at the PM or juxta-membrane. These cells are more uniformly square than the puzzle-piece like tobacco epidermal cells making it easier to discern subcellular expression and to see patterns within a field of cells. These results suggest that LG2 may be available within the cytoplasm to interact with SLN. Sln expression is massively upregulated in the lgn mutant background suggesting the expression change of SLN may be causative or resultant in relation to the lgn mutant phenotype. If SLN expression is the causative agent for the lgn mutant phenotype then we would expect that a loss of Sln in the lgn mutant background would restore a wild-type phenotype. To perform these experiments, I have obtained 4 putative mutator insertions within the Sln genomic region from Pioneer. I am currently working to validate these mutator insertion lines to confirm location of transposition and gene expression in the four backgrounds. SLN mutator lines that show a reduction in endogenous SLN expression will be introduced into the lgn mutant background and scored for phenotype.
Publications
- No publications reported this period
|
|