UNITED STATES DEPARTMENT OF AGRICULTURE
COOPERATIVE STATE RESEARCH, EDUCATION, AND EXTENSION SERVICE
FOR CSREES USE ONLY APPLICATION FOR FUNDING OMB Approved 0524-0022
PROGRAM AREA CODE
 
PROPOSAL CODE Expires 5/31/98
1. LEGAL NAME OF ORGANIZATION TO WHICH AWARD SHOULD BE MADE

      Eastern Connecticut State University

3. NAME OF AUTHORIZED ORGANIZATIONAL REPRESENTATIVE
Dimitrios S. Pachis
Vice President for Academic Affairs
4. a. PHONE NUMBER (Include Area Code)
(860)-465-5246
    b. FAX NUMBER
(860)-465-4485
    c. INTERNET ADDRESS
pachis@ecsu.ctstateu.edu
2. ADDRESS (Give complete mailing address and Zip Code-including County)
      83 Windham Street
      Willimantic, CT 06226
      Windham County
5. ADDRESS OF AUTHORIZED ORGANIZATIONAL REPRESENTATIVE
      (If different from Item 2.)
6. TITLE OF PROPOSED PROJECT (80-character Maximum, including spaces)

      Expression of Leaf Development Genes in Transgenic Nicotiana sylvestris

7. PROGRAM TO WHICH YOU ARE APPLYING
(Refer to Federal Register Announcement where applicable)

USDA:CSREES:NRICGP:AREA:
8. PROGRAM AREA AND NUMBER (Refer to Federal Register Announcement where applicable)
AREA: Research Career Enhancement Awards: Sabbatical Awards 80.1
9. IRS NO.
061277820
10. CONGRESSIONAL DISTRICT NO.
CT 2nd
11. PERIOD OF PROPOSED PROJECT DATES
From:
6/1/98       Through: 12/31/98
12. DURATION
REQUESTED

7 months
13. TYPE OF REQUEST (Check only one)
  [X] New       [   ] Renewal       [   ] Supplement       [   ] Resubmission
  [   ] Continuing Increment      [   ] PI Transfer [PRIOR USDA Award No. ______________ ]
14. FUNDS REQUESTED
(From Form CSREES-55)
    $ 33,077
16. a. PI/PD #1 PHONE NUMBER (Area Code) (860)-465-5327
    b. FAX NUMBER: (860)-465-5213
    c. INTERNET ADDRESS: koning@ecsu.ctstateu.edu
15. PRINCIPAL INVESTIGATOR(S)/PROJECT DIRECTOR(S)
  a. PI/PD #1 Name (First,Middle,Last) SS #* (Correspondent PI)     Ross Edward Koning 367-48-8687
  b. PI/PD #2 Name (First,Middle,Last) SS #*
 
17. PI/PD #1 BUSINESS ADDRESS (Include Department/Zip Code)
      Biology Department - Goddard Hall
      Eastern Connecticut State University
      Willimantic, CT 06226
  c. PI/PD #3 Name (First,Middle,Last) SS #*
 
*Submission of the Social Security Number is voluntary and will not affect the organization's eligibility for an award.
However, it is an integral part of the CSREES information system and will assist in the processing of the proposal.
18. TYPE OF PERFORMING ORGANIZATION
(Check one only)
01 [   ] USDA/S&E Laboratory
02 [   ] Other Federal Research Laboratory
03 [   ] State Agricultural Experiment Station (SAES)
04 [   ] Land-Grant University 1862
05 [   ] Land-Grant University 1890 or Tuskegee University
06 [   ] Private University or College
07 [X] Public University or College (Non Land-Grant)
08 [   ] Private Profit-making
09 [   ] Private Non-profit
10 [   ] State or Local Government
11 [   ] Veterinary School or College
12 [   ] 1994 Institution
13 [   ] Individual
14 [   ] Other (Specify)
15 [   ] Hispanic-serving Institution
19. WILL THE WORK IN THIS PROJECT INVOLVE RECOMBINANT DNA?
[ ] No       [ X] Yes (If yes, complete Form CSREES-662)
20. WILL THE WORK IN THIS PROJECT INVOLVE LIVING VERTEBRATE ANIMALS?
[ X] No       [   ] Yes (If yes, complete Form CSREES-662)
21. WILL THE WORK IN THIS PROJECT INVOLVE HUMAN SUBJECTS?
[ X] No       [   ] Yes (If yes, complete Form CSREES-662)
22. WILL THIS PROJECT BE SENT OR HAS IT BEEN SENT TO OTHER FUNDING AGENCIES, INCLUDING OTHER USDA AGENCIES?
[ X] No       [   ] Yes (If yes, list Agency acronym(s) & program(s))
By signing and submitting this proposal, the applicant is providing the required certifications set forth in 7 CFR Part 3017, as amended, regarding Debarment and Suspension and Drug-Free Workplace; and 7 CFR Part 3018 regarding Lobbying. Submission of the individual forms is not required. (Please read the Certifications and Instructions included in this kit before signing this form.) In addition, the applicant certifies that the information contained herein is true and complete to the best of its knowledge and accepts as to any award, the obligation to comply with the terms and conditions of Cooperative State Research, Education, and Extension Service in effect at the time of the award.
SIGNATURE OF PRINCIPAL INVESTIGATOR(S)/PROJECT DIRECTOR(S)
(All PI's/PD's listed in block 15 must sign if they are to be included in award document.)

 

DATE
SIGNATURE OF AUTHORIZED ORGANIZATIONAL REPRESENTATIVE
(Same as Item 3)
TITLE
Vice President for
Academic Affairs
DATE
      Form CSREES-661 (6/95)



Expression of Leaf Development Genes
in Transgenic Nicotiana sylvestris

Table of Contents

Application for Funding (CSREES-661)previous
Table of Contents this
Project Summary (CSREES-1232)next
Project Description1-4
      Research Interests and Goals1
      Sabbatic Research Project1-4
            Rationale and Significance1
            Research Methods2
            Limitations3
            Timetable3
      Enhancing Research Capabilities3
      Future Research Goals3-4
References to Project Description5
Host Documentation6
Curriculum vitae-Ross Koning7
Curriculum vitae-Neil McHale8
Conflict of Interest List (CSREES-1233) - Ross Koning9
Conflict of Interest List (CSREES-1233) - Neil McHale10
Budget (CSREES-55)11-12
Current and Pending Support (CSREES-663)13
Applicant Assurances (CSREES-662)14
NEPA (CSREES-1234)15



UNITED STATES DEPARTMENT OF AGRICULTUREOMB Approved 0524-0033
COOPERATIVE STATE RESEARCH, EDUCATION, AND EXTENSION SERVICEExpires 5/00
NATIONAL RESEARCH INITIATIVE COMPETITIVE GRANTS PROGRAM



Principal Investigator(s):PROPOSAL TYPE
PI#1     Ross Edward Koning     Institution     Eastern CT State University     [ ] Standard Research Proposal
[ ] Conference
[ ] AREA Award
    [ ] Postdoctoral
    [ ] New Investigator
    Strengthening:
    [X] Career Enhancement
    [ ] Equipment
    [ ] Seed Grant
[ ] Standard Strengthening
PI#2Institution
PI#3Institution
PI#4Institution



Project Title: Expression of Leaf Development Genes in Transgenic Nicotiana sylvestris



Key Words: Transgenic, Leaf Development, Tobacco, Nicotiana, Agrobacterium



(Approximately 250 words)        PROJECT SUMMARY
The PI from a public undergraduate teaching university eligible for EPSCoR funds will add molecular genetic techniques to his research capabilities during a sabbatic leave at the Connecticut Agricultural Experiment Station. The sabbatical project will construct vectors containing the PHAN gene required for blade formation in Nicotiana tabacum. This vector will be transformed into lam-1 bladeless Nicotiana sylvestris. After selection, the regenerants will be analyzed for gene expression using antibodies against gene products and in situ hybridization of RNAs. This analysis will be repeated after additional transformation with vectors containing the 5'-antisense construct to silence expression, and vectors incorporating these genes with a copper-inducible transcription factor. This project should enable additional understanding of the allelic or epistatic relationship between these genes in various tissue layers during the development of a leaf primordium into a mature leaf. However this project is seminal in that the techniques learned by the PI here will be applied rather to the study of the development of flower parts to be subsequently undertaken at the home institution. The most likely initial project there would be to isolate the gene differentially expressed in long- and short-morph stamens in the flower of Torenia fournieri, and insert the 5'-antisense construct with the copper-inducible transcription system into these plants. Controlling of the timing of expression and silencing of the gene should result in flowers optimized for different pollenating insects by having either four short-morph or four long-morph stamens.

Form CSREES-1232 (4/94)



Expression of Leaf Development Genes
in Transgenic Nicotiana sylvestris

Project Description

Research Interests and Goals

My research career in plant growth and development began with study of the effect of surgical deletion of plant parts on internode elongation (Koning et al. 1977). I later moved on to study the growth and development of flower parts in Gaillardia. (Koning 1983a, 1983b, 1983c, 1984, 1986a). In these studies I elucidated the roles of traditional plant growth substances in standard and acid-induced growth mechanisms for stamens, corolla, and carpel.

In my first professional position, I extended this work primarily to Ipomoea as this plant has strict timing of the growth of its very large flower parts (Koning 1986b, 1986c; Koning and Raab 1987; Raab and Koning 1987a, 1987b, 1988; Kiss and Koning 1989, 1990; Kiss, Koning and Daie 1991) but also with brief attention to Fuchsia (Jones and Koning 1986) and other species. These efforts were clearly based upon traditional techniques of growth kinetics studies, microscopy, dose responses to applied growth substances, extraction, purification, and quantitation of endogenous plant growth substances including gibberellins, auxins, ethylene, and abscisic acid by gas chromatography, mass spectrometery, radio-immunoassay, and enzyme-linked immunoassay. Such techniques were the foundation of growth physiology in the 1970s and early 1980s.

Rapid advances in our understanding of hormone biology and flower development have come from cloning and molecular analysis of genes controlling basic steps in these pathways (Coen and Meyerowitz, 1991; Weigel and Meyerowitz, 1994). My research interests remain in studying floral development, but now with particular emphasis on patterns of genetic expression during the growth and expansion of flower parts. The development of a flower into a three-dimensional object is critical to pollination, fruit development, and seed-set. An understanding about how flowers achieve the correct form to position the anthers and stigma correctly to achieve pollination, and how the perianth develops to attract, support, and reward an animal pollinator can provide insight into how crop yields may be improved by optimizing flowers for increased pollination efficiency. The recent decline in North American honeybee populations due to parasites underscores the importance of knowing how to manipulate flower parts from a genetic point of view to optimize flower function in the face of the mix of available pollinators.

Sabbatic Research Project

Rationale and Significance. Early studies in comparative plant morphology suggested that flower parts were essentially modified leaves. This idea is now supported by genetic studies confirming that floral organs attain their identity by adding gene functions to a vegetative ground state (Bowman et al., 1991). The stamen of a flower can be interpreted as a reproductive leaf, a leaf without a blade. The filament under this interpretation is equivalent to a bladeless leaf. Indeed homeotic mutations have been known for decades to convert stamens into petaloids, hence "double" flowers. Such petaloids consist of the petiole-midrib axis (the filament) with marginal blade-like extensions. A first approach to study genetic expression in stamens might thus begin with study of bladeless leaf development.

Dr. Neil McHale at the Connecticut Agricultural Experiment Station has an ongoing project on genetic approaches to mechanisms controlling formation of the leaf blade. The leaf blade offers a unique experimental system for a genetic approach to basic events in organ formation because it is a dispensable structure. Mutations defining several steps in formation and growth of the blade primordium in Nicotiana sylvestris have been recovered as viable bladeless mutants. The lam1 mutant produces bladeless leaves throughout the life cycle which grow to full length but fail to expand in the lateral plane (McHale 1993). The mutation similarly blocks lateral expansion of the corolla tip and the lateral growth and fusion of the carpel primordia, indicating that LAM1 is also a requirement for developing floral organs. A bladeless mutant of Antirrhinum (phantastica) with very similar phenotype to lam1 was described recently (Waites and Hudson 1995). Histological analysis of phan mutants indicates that this gene is involved in establishing dorsal cell identity, a basic requirement for adaxial flattening and lateral growth of vegetative and floral organs. Four phan alleles were characterized where the cotyledons and first three pairs of leaves generate a blade, but thereafter leaves are reduced to needle-like structures lacking all dorsal cell types. One of the phan mutant alleles was generated by a transposon insertion allowing cloning and sequence analysis of the PHAN gene, which shows homology to the myb family of transcription factors. Thus it appears that genetic approaches are producing the tools necessary to investigate dorsoventrality at the molecular level, opening new avenues for understanding the spatial coordinates guiding development of leaves and flowers.

The striking phenotypic similarity between the lam1 and phan mutants of tobacco and Antirrhinum led to efforts to determine whether LAM and PHAN are homologous genes. The PHAN gene was obtained from Dr. Andrew Hudson and used to generate a hybridization probe based on a conserved region of the myb domain. A tobacco leaf cDNA library in ZAPII was screened and a full length cDNA (NTPHAN, 1.4 kb) with close homology to PHAN has been isolated.

Research Methods. lam1 complementation. Our first priority will be to determine whether LAM1 is a PHAN homologue. We will be testing this directly by cloning the PHAN homologue (NTPHAN) from a N. tabacum leaf cDNA library for a transgenic approach to complementation. We have a putative full length clone which upon confirmation will be introduced into the lam1 background by Agrobacterium mediated transformation to look for blade restoration. In the meantime, we will conduct a heterologous complementation experiment inserting the Arabidopsis homologue of PHAN (ATPHAN), into the lam1 background and looking for blade restoration.

Since lam1 mutant leaves regenerate shoots readily in tissue culture, we will introduce these constructs directly into the mutant and screen for blade formation in transgenic shoots. The constructs will also be introduced into LAM1/lam1 heterozygotes, allowing an opportunity to look for evidence of a hyperdorsalized phenotype associated with PHAN overexpression. These transgenics will then be grown to maturity and self-pollinated to test for lam1 complementation in the T1 progeny.

If ATPHAN or NTPHAN restores blade formation in lam1, we will pursue these avenues:

Sequence analysis. We will isolate LAM1 wild-type and lam1 mutant alleles from N. sylvestris genomic libraries for sequence analysis, with particular attention to characterization of the mutant allele.

Expression analysis. NTPHAN cDNA will be employed for a detailed in situ expression analysis in the wild-type meristem, leaf primordia and blade mesophyll. Expression of NTPHAN in the meristem would confirm that dorsalization begins at a very early stage in formation of the leaf primordium. Since dorsalizing information is required throughout blade expansion, it will be of particular interest to determine whether LAM1 protein localizes to dorsal cell layers during this stage. We will use NTPHAN cDNA to generate a fusion protein in E. coli and produce a LAM1 antibody for an in situ analysis of protein distribution.

Inducible LAM1 transgenics. If NTPHAN restores blade formation in lam1 mutants, this provides an experimental system for introducing this function into developing mutant primordia at different times in development using an inducible promoter. We have obtained the two element copper-controllable vector system based on the yeast metallo-regulatory transcription factor ACE1 (Mett et al., 1993). This system requires construction of a two transgenic lines, one (driver) with ACE1 driven by a constitutive promoter and a second transgenic (target) with a gene of interest behind a 46 bp TATA CaMV 35S sequence linked to four repeats of the ACE1 binding site. The strains are hybridized to obtain double transgenic progeny. Application of exogenous copper produces a conformational change in the ACE1 driver protein allowing binding to the target promoter, activating transcription of the gene (Mett et al., 1996). Our approach here will be to introduce a NTPHAN target transgene into the lam1 mutant and an ACE1 driver transgene into a LAM1/lam1 heterozygote to allow hybridization and recovery of F1 lam1/lam1 homozygotes carrying the driver and target transgenes. Copper-controlled induction of NTPHAN expression at different developmental stages would allow us to test directly the developmental window for restoration of dorsoventrality during leaf development.

If the complementation experiments indicate that PHAN and LAM1 are different genes, we will pursue these alternative avenues:

PHAN expression analysis. The NTPHAN cDNA and antibody will be extremely valuable molecular markers if they in fact localize to dorsal cell types in the emerging primordium and expanding leaf blade in wild-type plants. We will examine this by in situ hybridization, focusing first on the leaf mesophyll where dorsal cell types are arranged in distinct layers and have a readily recognizable morphology.

Antisense NTPHAN transgenics. Constructs overexpressing NTPHAN cDNA in the sense and antisense orientations will also be introduced into N. sylvestris wild-type to generate bladeless transgenics. These transgenics will provide experimental material for detailed histological studies and construction of double mutants with lam1 to determine the temporal and epistatic relationships of the NTPHAN and LAM1 genes in the blade pathway.

Limitations. Possible pitfalls in this line of research include the loss of genetic material after transformation (lack of genetic stability of the plasmid-insert or recombination site), the chance of somaclonal variation among the regenerants due to the culture techniques, insufficient time to raise antibodies specific enough to detect gene products, low level expression of one or the other genes in the transformants such that hybridization results are equivocal, folding back of the 5'-antisense gene mRNA would prevent its silencing the sense homolog, and transformation of only one of the two parts of the inducible yeast transcription-factor system or subsequent loss of one of the two parts after transformation. Of course this long list of potential problems is potentially frustrating, but the main point of this sabbatic project is to learn techniques. It is hoped that these techniques will prove successful in the project, and every effort will be made to avoid these problems through generating suitably large numbers of regenerants and rigorously screening abnormal individuals. On the other hand, even if the tobacco leaf project is doomed by a mine-field of happenstances, the techniques learned in the work will make the sabbatic leave a successful experience for me.

Timetable. My timetable for this project is to begin vector construction starting in late May 1998 and proceed through the proposed steps in the sequence mentioned above as far as is possible by the end of December 1998 given whatever delays and pitfalls are experienced in the laboratory.


Enhancing Research Capabilities

Whether or not the project completely clarifies the interaction of PHAN and lam1 in transgenic plants, I expect this work will achieve my most important goal--to gain experience in modern techniques in plant physiology. The prime objective of the project is to return from the sabbatic leave with the new skills mentioned above in manipulating genomes and observing genetic expression of manipulated plants. Upon return to ECSU, I plan to apply these new-learned techniques to answering questions about flower development at the molecular genetics level.

Future Research Goals

A few years ago I started a project using the traditional growth analysis and hormone analysis techniques I already possess in asking the question about the basis for the difference between the two pairs of stamens in the flower of Torenia fournieri. In this flower, one pair of stamens is exceedingly short, and the other pair is nearly ten times longer. I studied the differences morphologically which indicated more cell elongation and more cell division in the longer pair. Plant growth substances applied in vitro to filaments, excised early in development so that they were not yet different in size, indicated that hormones could only explain part of the difference. Long-morph filaments respond well to applied hormones and short-morph filaments are much less responsive. However, it was not possible to convert a short-morph into an apparent long-morph or vice versa. This result implies that there is a more fundamental difference between the two morphs that deserves further study at a genetic level. At that point I had to drop the project because I was at the end of my abilities to explore the situation any further.

With the experience gained in this sabbatical project, I hope to be able to return to my own laboratory and begin to dissect this problem a bit more. Both morphs of stamens in this flower presumably have a complete diploid genome, but one morph is expressing a gene or genes involved with filament elongation while the other morph is not. This problem is very similar to the sabbatic project--a transgenic tobaccco which has a PHAN gene and a lam1 gene (particularly if these are epistatic genes rather than complementary alleles). After I isolate a gene by screening a short-morph library with a long-morph probe or vice-versa, I can then follow along the lines of my sabbatic project to study the expression of that gene. I think an important step in this case will be to construct a 5'-antisense vector with an inducible transcription factor (combining two parts of the sabbatic project). This way I can perhaps silence the one gene at will during flower development to force the short-morph into growth as a long-morph stamen or vice versa.

The Torenia flower has two morphs of stamen that permit the flower to be pollinated by two sizes of vector. The long morph stamens hold the anther high off the perianth landing platform and require a large pollinator (bumblebee). The short morph stamens are held in the base of the flower and deposit pollen on smaller pollinators (honeybees, orchard mason bees, etc). If I can learn to silence or constitutively express the gene that differentiates the two morphs, then I should be able to force Torenia to have four long-morph stamens (and improve pollination efficiency with bumblebees) or to have four short-morph stamens (and improve pollination efficiency with orchard mason bees). If successful, this project would provide a practical demonstration of how crop yield might be improved for districts with pollinator availability problems of one sort or another.



Expression of Leaf Development Genes
in Transgenic Nicotiana sylvestris

References to Project Description



Neil A. McHale
Department of Biochemistry & Genetics
The Connecticut Agricultural Experiment Station
P. O. Box 1106, New Haven, CT 06504
Phone: (203) 789-7218
FAX: (203) 789-7232
Email: nmchale@caes.state.ct.us

To Those Concerned:

As department head and host, I am writing in support of the grant application of Dr. Ross Koning for a 6 month sabbatical leave here at the Experiment Station. We are currently involved in research aimed at understanding the molecular mechanisms governing leaf development. We are cloning genes involved in this process from cDNA libraries, performing the DNA sequence analysis and making the appropriate vectors to insert these genes into model plant systems (Arabidopsis and Nicotiana) for further analysis. Dr. Koning feels that direct laboratory experience with these mainstay procedures for plant molecular biology would greatly enhance the academic opportunities he can offer to his students at Eastern Connecticut State University. I fully agree with his assessment and would be happy to provide the necessary space in our laboratories, growth rooms and greenhouses for his work.

One aspect of our work requires the production of transgenic plants with constitutive and inducible promoters controlling expression of inserted genes. These vectors are being constructed in the lab now and will be available to Dr. Koning when he arrives. To help get Dr. Koning up to speed as soon as possible, some assistance from one of the technicians in our molecular biology lab will be available to him. Dr. Koning will also be able to participate in our lab meetings and have continuous access to assistance and advice from Dr. Neil Schultes and myself as the work proceeds. I think the arrangement will be quite satisfactory and provide Ross with some very valuable molecular expertise to bring home to ECSU.

Neil A. McHale
Head, Biochemistry & Genetics
The Conn. Agric. Expt. Station
P.O. Box 1106
New Haven CT 06504



Expression of Leaf Development Genes
in Transgenic Nicotiana sylvestris

Curriculum Vitae - Sabbatical Applicant

Ross E. Koning       Full CV--http://plantphys.info/personal.shtml
Biology Department - Goddard Hall
Eastern Connecticut State University
Willimantic, CT 06226

Education:
BS 1975
MS 1976
PhD 1981
University of Michigan, Ann Arbor

Positions:
Professor:Eastern Connecticut State UniversityFall 1994 to present
Associate Professor:Eastern Connecticut State UniversityFall 1989-Fall 1994
Assistant Professor:Eastern Connecticut State UniversityFall 1987-Fall 1989
Assistant Professor:Rutgers UniversityFall 1981-Spring 1987
Visiting Instructor:Eastern Michigan UniversitySummers 1978 & 1979
InstructorUniversity of MichiganSpring 1978
Resident Fellow:University of MichiganFall 1980-Winter 1981
Teaching Assistant:University of MichiganFall 1975-Winter 1980
University of Michigan Distinguished Teaching Award1979

Memberships:
National Association of Biology Teachers
Botanical Society of America--Special Papers Editor (Amer. J. Bot.);
    Physiology Newsletter Editor; Physiology Program Director;
    Endowment Fund Administrator
American Society of Plant Physiologists
Sigma Xi

Publications:
62 Published Abstracts for Meetings Presentations (Career total)
23 Publications Prior to those listed below

Publications (1993-present), academic refereed journals/books
Santaniello, C. R. and R. E. Koning. 1996. Are radishes really allelopathic to lettuce? American Biology Teacher 58:102-103. Catherine was an undergraduate student in biology. Full text in color also at: http://plantphys.info/research/abt.58.102.3.html
Koning, R. E. 1996. Standards for effective presentations. Invited book chapter in: Units, symbols, and terminology for plant physiology: a reference for presentation of research results in the plant sciences. F. B. Salisbury, Editor. pp. 188-201. Oxford University Press; New York.

Grants received (1993-present)
1994-1996 Mashantucket Pequot Tribe. Lake-of-Isle Vegetation Survey. 1995: $52,583.36 1996: $69,926.26
    Co-authored with C. Cid and P. Elliott
1993-1997 AAUP-CSU Research Grant Awards (annual awards-two selected here)
    Inheritance of phytohormone mutations in Brassica rapa. 1995 $3500
    Linkage of phytohormone mutations in Brassica rapa. 1996 $2600



Expression of Leaf Development Genes
in Transgenic Nicotiana sylvestris

Curriculum Vitae - Host

Neil A. McHale
Department of Biochemistry and Genetics
Connecticut Agricultural Experiment Station
New Haven, CT 06504

Education:
B.A., Biology, Merrimack College, 1973
M.S., Genetics, University of New Hampshire, 1976
Ph.D., Horticulture/Genetics, University of Minnesota, 1980

Positions:
Connecticut Agricultural Experiment Station, New Haven, CT: Assistant Geneticist, 1983-1989; Associate Geneticist, 1989-1994, Department Head, 1994-present.
University of Connecticut, Storrs, CT: Adjunct Professor, Department Molecular and Cellular Biology, 1990-1993.
International Plant Research Institute, San Carlos, CA: Assistant Geneticist, 1980-1983

Memberships:
American Society of Plant Physiologists Sigma Xi

Publications since 1990:
McHale, NA 1990. Chloroplast mutations from protoplast-derived cultures of tobacco. In: Perspectives in Biochemical and Genetic Regulation of Photosynthesis. Zelitch I, ed, Wiley-Liss, New York, pp 103-117 McHale, NA, EE Kawata, and AY Cheung 1990. Plastid disruption in a thiamine-requiring mutant of Nicotiana sylvestris blocks accumulation of specific nuclear and plastid mRNAs. Mol Gen Genet 221:203-209 Salvucci, ME, RR Drake, KP Broadbent, BE Haley, KR Hanson, and NA McHale 1990. Identification of the 64 kilodalton chloroplast stromal phosphoprotein as phosphoglucomutase. Plant Physiol 93:1212-1218 Havir, EA and NA McHale 1990. Purification and characterization of an isozyme of catalase with enhanced peroxidatic activity from leaves of Nicotiana sylvestris. Arch Biochem Biophys 283:491-495 Hanson, KR and NA McHale 1991. Concerning the influence of photoperiod and daylength on the Nicotiana sylvestris "starchless" mutant. (Letter to the Editor) Flowering Newsletter, 11,30 Zelitch, I, EA Havir, B McGonigle, NA McHale, and T Nelson 1991. Leaf catalase mRNA and catalase-protein levels in a high-catalase tobacco mutant with O2-resistant photosynthesis. Plant Physiol 97:1592-1595 McHale, NA 1992 A nuclear mutation blocking initiation of the lamina in leaves of Nicotiana sylvestris. Planta 186: 355-360 McHale, NA 1993 The LAM-1 and FAT genes control development of the leaf blade in Nicotiana. Plant Cell 5: 1029-1038 Carland, F.M. and McHale, N.A. 1996. LOP1: a gene involved in auxin transport and vascular patterning in Arabidopsis. Development 122: 1811-1819.



UNITED STATES DEPARTMENT OF AGRICULTUREOMB Approved 0524-0033
COOPERATIVE STATE RESEARCH, EDUCATION, AND EXTENSION SERVICEExpires 5/00
NATIONAL RESEARCH INITIATIVE COMPETITIVE GRANTS PROGRAM
Conflict of Interest List

Name:     Ross Edward Koning    

For each investigator (and other personnel as described in the application kit or program description), list ALPHABETICALLY the full names of only the individuals in the following categories. Additional pages may be used as necessary. A conflict of interest list for each investigator must be submitted before a proposal is considered complete. Inclusion of a C.V. or publication list in the proposal is not sufficient.

All co-authors on publications within the past four years, including pending publications and submissions

All collaborators on research projects within the past four years, including current and planned collaborations

All thesis or postdoctoral advisees/advisors within the past four years

All persons in your field with whom you have had a consulting/financial arrangement/other conflict-of-interest within the past four years

Other investigators working in the applicant's specific research area are not in conflict of interest with the applicant unless those investigators fall within one of the listed categories.

CO-AUTHORS CO-AUTHORS COLLABORATORS ADVISEES/ADVISORS
Catherine R. Santaniello
Frank B. Salisbury
  Steven Lamoureux
Patricia Tellekamp
Carmen R. Cid
Phillip F. Elliott
 
OTHER - SPECIFY
NATURE
 
Form CSREES-1233 (11/96)



UNITED STATES DEPARTMENT OF AGRICULTUREOMB Approved 0524-0033
COOPERATIVE STATE RESEARCH, EDUCATION, AND EXTENSION SERVICEExpires 5/00
NATIONAL RESEARCH INITIATIVE COMPETITIVE GRANTS PROGRAM
Conflict of Interest List

Name:     Neil A. McHale    

For each investigator (and other personnel as described in the application kit or program description), list ALPHABETICALLY the full names of only the individuals in the following categories. Additional pages may be used as necessary. A conflict of interest list for each investigator must be submitted before a proposal is considered complete. Inclusion of a C.V. or publication list in the proposal is not sufficient.

All co-authors on publications within the past four years, including pending publications and submissions

All collaborators on research projects within the past four years, including current and planned collaborations

All thesis or postdoctoral advisees/advisors within the past four years

All persons in your field with whom you have had a consulting/financial arrangement/other conflict-of-interest within the past four years

Other investigators working in the applicant's specific research area are not in conflict of interest with the applicant unless those investigators fall within one of the listed categories.

CO-AUTHORS CO-AUTHORS COLLABORATORS ADVISEES/ADVISORS
Timothy Nelson
Steve Dellaporta
Francine Carland
Michael Marcotrigiano
Robert Bernatzky
     
OTHER - SPECIFY
NATURE
 
Form CSREES-1233 (11/96)



UNITED STATES DEPARTMENT OF AGRICULTUREOMB Approved 0524-0022
COOPERATIVE STATE RESEARCH, EDUCATION, AND EXTENSION SERVICEExpires 5/31/98
BUDGET
ORGANIZATION AND ADDRESS
Eastern Connectict State University
83 Windham Street, Willimantic, CT 06226
USDA AWARD NO.
PRINCIPAL INVESTIGATOR(S)/PROJECT DIRECTOR(S)
Ross E. Koning
Duration Proposed
Months:    
7    
Duration Awarded
Months:            
FUNDS REQUESTED BY PROPOSER FUNDS APPROVED BY CSREES (If Different)
A. Salaries and WagesCSREES FUNDED WORK MONTHS
    1. No. of Senior PersonnelCalendarAcademicSummer
        a.     1     (Co)-PI(s)/PD(s)  3$ 19,365 
        b.           Senior Associates     
    2. No. of Other Personnel (Non-Faculty)
        a.___Research Associates-Postdoctorate
     
        b.___Other Professionals     
        c. Graduate Students  
        d. Prebaccalaureate Students  
        e. Secretarial-Clerical  
        f. Technical, Shop and Other  
Total Salaries and Wages....................19,365 
B. Fringe Benefits (If charged as Direct Costs) 18.85% of line A3,650 
C.Total Salaries, Wages, and Fringe Benefits (A plus B)......23,015 
D. Nonexpendable Equipment (Attach supporting data. List items and dollar amounts for each item.)  
E. Materials and Supplies6,000 
F. Travel
    1. Domestic (Including Canada)
    2. Foreign (List destination and amount for each trip.)
  
G. Publication Costs/Page Charges  
H. Computer (ADPE) Costs  
I. All Other Direct Costs (Attach supporting data. List items and dollar amounts. Details of subcontracts, including work statements and budget, should be explained in full in proposal.)  
J. Total Direct Costs (C through I) ................................29,015 
K. Indirect Costs If Applicable (Specify rate(s) and base(s) for on/off campus activity. Where both are involved, identify itemized costs included in on/off campus bases.)
last-negotiated off-base 53.09%..we accept limit of 14% of total line J
4,062 
L. Total Direct and Indirect Costs (J plus K).......................33,077 
M. Other  
N. Total Amount of This Request ..................................$ 33,077 
O. Cost Sharing (If Required Provide Details)$
NOTE: Signatures required only for Revised BudgetThis is Revision No. 0
NAME AND TITLE (Type or print)SIGNATUREDATE
Principal Investigator/Project Director

 

  
Authorized Organizational Representative

 

  
      Form CSREES-55 (6/95)



Expression of Leaf Development Genes
in Transgenic Nicotiana sylvestris

Budget Comments

Home Institution Support for the Sabbatical Project

Eastern Connecticut State University has granted me the sabbatical leave with full pay for the mid-August through December period of this application, so the investigator support requested in this appplication is only for the three months mid-May to mid-August. Much of this amount will be used to cover my commute from Willimantic to New Haven over the entire seven months.

Facilities and Equipment Available at Connecticut Agricultural Exeriment Station

Facilities include a biochemistry lab equipped with a Superspeed (Sorvall RC-5B) and ultracentrifuge (Beckman L5-65), Gilford-250 spectrophotometer, Eppendorf microcentrifuge, liquid scintillation counter, Mettler balances, Savant Speed Vac concentrator, Perkin-Elmer HPLC and radioactive flow detector with Hewlett-Packard integrators. Electrophoresis equipment is available for vertical slabgels (Hoefer SE 600) and western blotting (TE 50 Transphor), electroelution (BioRad 422), slot-blotting (American Bionetics), horizontal RNA and DNA fractionation and DNA sequencing (Fisher FB-SEQ-3545, FB 600 power supply). PCR work is done with a Perkin Elmer thermal cycler. There is also a vacuum drier, a Fisher (FB 9W) densitometer, vacuum and convection ovens, incubators (Fisher 630D), water baths and a Revco (-80íC) freezer. Nucleic acid hybridizations are carried out in a G24 New Brunswick incubator-shaker, and in a Labnet midi-oven. The tissue culture lab is equipped with two sterile hoods, incubators, and an autoclave. The histological lab is equipped with a fume hood, Zeiss microtome, and a Zeiss Axiophot light microscope. The station is also equipped for ultrastructural work in a lab with a fume hood, binocular microscope, ultramicrotome and a Zeiss EM 10 electron microscope. The station also has access to a fully-equipped scanning electron microscopy lab at nearby Yale University.

Budget Justification (supplies for 7-month sabbatical)

Histology and ultrastructure. Slides, cover glass, stains, butanol, paraffin, plastic resin, mounting resin, film, developer, microtome blades. Usage expected at $1,000.
Plastic disposables and glassware. Usage expected at $1,000.
Greenhouse supplies. Usage expected at $500.
Enzymes and biologicals. Restriction enzymes, labelling kits, polymerases, standards. Usage expected at $2000.
Chemicals and reagents. Agarose and acrylamide, buffers, salts, resins, CsCl, nucleotides, detergents, antibiotics. Usage expected at $1000.
Film and developers. X-ray film and developer, polaroid film. Usage expected at $500.

Pending Support

A grant proposal to the AAUP-CSU Research Grant Committee, a feature of the ECSU-AAUP Collective Bargaining Agreement, for $4000 has been submitted for additional funding to assist me in the first stages of technology transfer from the Connecticut Agricultural Experiment Station to Eastern Connecticut State University.



UNITED STATES DEPARTMENT OF AGRICULTUREOMB Approved 0524-0022
COOPERATIVE STATE RESEARCH, EDUCATION, AND EXTENSION SERVICEExpires 5/31/98
CURRENT AND PENDING SUPPORT

Instructions:
1. Record information for active and pending projects. (Concurrent submission of a proposal to other organizations will not prejudice its review by CSREES)
2. All current research to which principal investigator(s) and other senior personnel have committed a portion of their time must be listed, whether or not salary for the person involved is included in the budgets of the various projects.
3. Provide analogous information for all proposed research which is being considered by, or which will be submitted in the near future to, other possible sponsors including other USDA programs.
NAME
(List PI #1 first)
SUPPORTING AGENCY
AND AGENCY NUMBER
TOTAL $
AMOUNT
EFFECTIVE AND
EXPIRATION DATES
% OF TIME COMMITTED TITLE OF PROJECT
 Current

 

 

    
 
Ross Koning
Pending
CSU-AAUP Research Grant

 

 

 
$4,000
 
May 1998-
December 1998
(same)
 
same
 
Construction of Transgenic Plants and Comparison of the Expression of Three Leaf Development Genes in Nicotiana sylvestris.
      Form CSREES-663 (6/95)



UNITED STATES DEPARTMENT OF AGRICULTUREOMB Approved 0524-0033
COOPERATIVE STATE RESEARCH, EDUCATION, AND EXTENSION SERVICEExpires 5/00
National Environmental Policy Act Exclusions Form
Principal Investigator/Project Director Name
    Ross E. Koning
Institution
    Eastern Connecticut State University
Address
    Biology Department--Goddard Hall
    Willimantic, CT 06226

Under 7 CFR Part 3407 (CSREES's implementing regulations of the National Environmental Policy Act of 1969 (NEPA)), environmental data or documentation is required in order to assist CSREES in carrying out its responsibilities under NEPA, which includes determining whether proposed research requires the preparation of an environmental assessment or an environmental impact statement, or whether such research can be excluded from this requirement on the basis of several categories. Therefore, it is necessary for the applicant to advise CSREES whether the proposed research falls into one of the following Department of Agriculture or CSREES categorical exclusions, or whether the research does not fall into one of these exclusions (in which case the preparation of an environmental assessment or an environmental impact statement may be required). Even though the applicant considers that a proposed project may or may not fall within a categorical exclusion, CSREES may determine that an environmental assessment or an environmental impact statement is necessary for a proposed project should substantial controversy on environmental grounds exist or if other extraordinary conditions or circumstances are present that may cause such activity to have a significant environmental effect.

Please Read All of the Following and Check All Which Apply

[X] The proposed research falls under the categorical exclusion(s) indicated below:
Department of Agriculture Categorical Exclusions
(found at 7 CFR 1b.3 and restated at 7CFR 3407.6 (a)(1)(i) through (vii))
[ ] (i) Policy development, planning and implementation which are related to routine activities such as personnel, organizational changes, or similar administrative functions
[ ] (ii) Activities that deal solely with the functions of programs, such as program budget proposals, disbursements, and transfer or reprogramming of funds
[X] (iii) Inventories, research activities, and studies such as resource inventories and routine data collection when such actions are clearly limited in context and intensity
[ ] (iv) Educational and informational programs and activities
[ ] (v) Civil and criminal law enforcement and investigative activities
[ ] (vi) Activities that are advisory and consultative to other agencies and public and private entities, such as legal counseling and representation
[ ] (vii) Activities related to trade representation and market development activities abroad
CSREES Categorical Exclusions
(found at 7 CFR 3407.6(a)(2)(i) through (ii))

The following categories of CSREES actions are excluded because they have been found to have limited scope and intensity and to have no significant individual or cumulative impacts on the quality of the human environment:

(i) The following categories of research programs or projects of limited size and magnitude or with only short-term effects on the environment:
    [X] (A) Research conducted within any laboratory, greenhouse, or other contained facility where research practices and safeguards prevent environmental impacts
    [ ] (B) Surveys, inventories, and similar studies that have limited context and minimal intensity in terms of changes in the environment
    [ ] (C) Testing outside of the laboratory, such as in small isolated field plots, which involves the routine use of familiar chemicals or biological materials
[ ] (ii) Routine renovation, rehabilitation, or revitalization of physical facilities, including the acquisition and installation of equipment, where such activity is limited in scope and intensity

OR
[ ] Proposed research does not fall into one of the above categorical exclusions
(NOTE: If checked, please attach an explanation of the potential environmental impacts of the proposed research. May require completion of an environmental assessment or an environmental impact statement.)
    Form CSREES-1234 (4/94)


 
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