Connecticut State University
1998 University Research Grant Proposal Cover Sheet

Name: Ross E. Koning

University: Eastern CSU           Department: Biology

Campus Address: Goddard Hall, Willimantic, CT 06226

E-Mail:           Phone: 465-5327

Is this a joint proposal submitted with (an)other faculty member(s)? No

If YES, who are the other proposers? NA

Project Title: Construction of Transgenic Plants and Comparison of the Expression of Three Leaf Development Genes in Nicotiana sylvestris.

Amount Requested: $4000

Abstract (limit to 100 words):

The proposed research involves the molecular biology of plant leaf formation. A cloned cDNA homologous to the PHAN gene required for leaf blade formation in Antirrhinum will be used construct Nicotiana transgenic plants through Agrobacterium-mediated transformation. The resulting plants will be examined for complementation through histology of the developing leaf blades, probing with an antibody to PHAN protein, and antisense RNA silencing. If PHAN, LAM-1, and NS620 are alleles, I will examine how they differ in timing of gene expression with an inducible promoter. If not, I will construct double mutants to examine epistatic interaction among these genes.

____X_:Does your research involve human subjects and/or vertebrate animals? (See section 4 of guidelines)
______:If "YES" above, have you sought approval from the appropriate review committee on your campus?
______:If "YES" above, on what date did you, or do you expect to, receive approval from the review committee?_____________________

NOTE: Proposals cannot be returned to applicants. Funded proposals may be made available for evaluation by interested parties. Attach proposal narrative, proposed budget & curriculum vitae.



Historical Significance. Much of my career and most of my publication record deals with the development of the parts of flowers to achieve the three-dimensional display of those parts for effective pollination. This work was done using the biochemical and developmental biology techniques of the 1970s and early 1980s. Now sixteen years since my last formal research training, and now returning to normal faculty status after serving my department for six years as assistant department chairman, it is time to take my scientific work to a new level. To do this, I need to learn new techniques in molecular biology as they pertain to development of plant organs.

I have applied for my first sabbatic leave and trust that I will be released for that in Fall 1998. During the Spring 1998 semester, I plan to focus my efforts on reading and gaining the financial support needed to begin this new training in molecular plant physiology. The proposed laboratory work will then be done during Summer 1998 and Fall 1998 in the Genetics Laboratory of the Connecticut Agricultural Experiment Station.

A Golden Opportunity. Drs. Neil McHale and Neil Schultes at the Connecticut Agricultural Experiment Station have invited me to join them in a study of genes involved in the development of leaf blades in Nicotiana sylvestris (an ornamental tobacco). This work involves learning techniques new to me that I can bring back to ECSU and use in understanding the development of stamens (a possibly-homologous plant organ) in my career research project. This future work can also involve ECSU students in independent study projects and small parts of such work can be incorporated into my teaching as well. Drs. McHale and Schultes have also turned me toward a funding program with the US Department of Agriculture that specifically targets sabbatic leave projects that involve collaboration between primarily-undergraduate schools (ECSU) and USDA agencies (such as the CT Ag Expt St) in just certain states (CT is one of these). With the help of the funding requested here as a match, I hope to win additional funding for this project through the USDA-CSREES-NRICGP-Career Enhancement Award program. The USDA grant proposal is due February 15, 1998. Thus the funding through our AAUP/CSU program can help me gain even further federal support for this work.

The Gene Sources and Research Question. Neil McHale at the Connecticut Agricultural Experiment Station has cloned a gene in Nicotiana tabacum which is homologous to the PHAN gene of Antirrhinum. The PHAN gene is required for normal leaf development in snapdragon. He and coworkers have also isolated and thoroughly characterized a bladeless mutant called lam1. Neil Schultes at the Station has also isolated the mutant ns620 which causes polarity defects in blade formation. The proposed research draws these three genes together in a project studying the molecular biology of blade formation in Nicotiana sylvestris. As the three genes control similar parts of leaf development it seems reasonable to try to determine whether they are alleles or epistatic genes.

Constructing Transgenic Plants. The Nicotiana tabacum gene PHAN will be cloned into a kanamycin-resistant Ti plasmid of the bacterium Agrobacterium tumiefaciens by enzymatic trimming and splicing of the two DNA molecules. The vector plasmids will be returned to the bacterial cells and moved into lam-1 and ns620 Nicotiana sylvestris plants by the usual bacterial inoculation methods. Inside the tobacco cells, the Ti plasmids will transform the tobacco DNA by recombination. From the resulting transformed cells, whole Nicotiana sylvestris transgenic plants can be regenerated.

Sorting (screening) the Transgenic Plants. Of course some regenerants will not have received or failed to recombine any portion of the Ti plasmid; these will be easily screened out by their sensitivity to kanamycin. The regenerants that are insensitive to kanamycin have at least recombined the resistance gene from the Ti plasmid and are worthy of additional study.

Of those transformed regenerants, some may show a normal leaf blade and this could be caused by recombination of the PHAN gene into the genome, restoring normal development. This complementation would indicate that PHAN and lam1 or ns620 are likely alleles, but it could also mean that PHAN is recombined elsewhere in the genome but is epistatic to lam1 or ns620. Close examination of their histogenesis and development will be needed to resolve this. The mutant lam1 will be more difficult to distinguish as a recessive in this regard, but ns620 is a semi-dominant and so complementation would be particularly interesting.

It is also possible that some regenerants will show lam-1 or ns620 phenotypes because they did not recombine the PHAN gene from the modified plasmid or because PHAN is neither allelic nor epistatic with respect to lam-1 or ns620. In this case, transgenics that are verified to have both PHAN and lam1 or ns620 will be interesting developmentally as the possible epistasis between these three genes can be investigated.

Traditional Complementation Testing. The various regenerants can be grown up to flowering, can be self-pollinated and the T1 progeny checked for appropriate ratios of normal and mutant phenotypes. This is a tedious process and does not represent a new technique for me and so is only a small part of the proposed research.

Gene Expression Analysis. The expression of the PHAN, lam1, and NS620 genes in heterozygous transgenes could be very revealing about how these three genes are expressed in the sequence of development. The production of the proteins from these three genes and be monitored by in-situ localization using an antibody against the wild-type and/or mutant proteins produced during the expression of these genes. Such a technique will reveal which cells are expressing the genes and at which times in the development of leaf primordia. It can also clear up questions about heterozygosity.

Another new approach to this work however is to construct 5' antisense DNAs for the genes including a constitutive promoter which can be cloned and moved into the PHAN/lam1 or PHAN/NS620 heterozygotes. The antisense mRNA being produced constitutively binds to any corresponding sense mRNA being produced by the normal genes in the heterozygotes. The resulting double-stranded RNA cannot be translated by ribosomes, so the genes of the heterozygotes can be "silenced". The introduction of the antisense gene for PHAN into the heterozygote, then, should restore the mutant (lam1 or NS620) phenotype even though PHAN might be allelic or epistatic.

A final, and even more powerful, technique I plan to learn is the use of an inducible promoter in these gene constructs. A new gene vector is available which features a copper-ion-inducible transcription factor. This will allow me to learn how to put the PHAN gene into heterozygotes with a bit of temporal control. The transformed regenerants will express the heterozygotic phenotype until I choose to treat them with copper ions. Then the overexpression of the PHAN gene will allow the interaction of these genes to be observed developmentally using the antibodies previously mentioned. I can then dissect the sequence of expression in these three genes and determine when in development their interaction is critical and for which layers of tissue in the developing leaves these interactions are essential.

Publication and Communication. It is hoped that my work at the CT Ag Expt Station will result in a publication on which I would be a junior author. I anticipate a pre-print of this work would be placed on my website ( well before that time. I suspect that I will be able to present the findings too at a national meeting of either the American Society of Plant Physiologists or Botanical Society of America.

Concluding Remarks. In summary, the proposed research meets two of the priorities of the University Grants Committee. The project establishes a new pathway of research for an established member, and it should improve my chances of external funding from the USDA for which I am submitting a proposal within the next two weeks.

Personally, however, the most important aspect of the proposed work is modernizing my own approach to the questions I am interested in for the next decade of my research career. I am hoping ultimately to be able to answer questions about the molecular aspects of development of flower parts. The proposed research will lead me in this direction with invaluable training in these modern techniques. A new dimension to my research skills and focus will make a tremendous addition to my research career.



Plant Culture Supplies
      Seeds, Filter Paper,
      Petri Dishes, Forceps, Media
$ 150
Recombinant DNA Techniques
      Chemical Reagents, Enzymes, Buffers$ 1500
      Supplies, disposable plasticware$ 250
Publication Costs
      Graphic Arts Films, Processing, Mounts$ 100
Personnel Stipend Support
      Ross E. Koning, (Attached Curriculum Vitae)
      (this will chiefly support the transportation costs
      in commuting to New Haven for Summer and Fall 1998)
$ 2,000

Go back to Ross Koning's Curriculum Vitae

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