Progress 01/01/11 to 12/31/11
Outputs
OUTPUTS: We have been working on three related projects in the past year. The first project involves studying the interaction between auxin, and the Afila (AF) and UNI genes in controlling the development of the pea compound leaf. We collected data on ontogenetic variation of leaf form (heteroblasty) of af uni and af tl uni plants to see how AF and UNI interact to control leaf development in pea. We measured auxin levels in shoot tips of a range of mutants and wildtype, to determine if there are genotypic differences especially in af, af tl, uni-tac and uni. We crossed the DR5::GUS reporter into a range of leaf mutants, including uni-tac, uni and af to determine if there are differences in auxin perception during morphogenesis. And we used qRT-PCR to look at gene expression of UNI, and other auxin-regulated genes to compare expression levels in shoot tips of a range of mutants and wildtype plants with the results of auxin measurements and auxin perception experiments. The results have been submitted for publication. Secondly, we are studying the expression of UNI and the Arabidopsis homologous gene LEAFY (LFY) in both Arabidopsis and pea and we are studying the differential regulation of their promoters in both species. We are making, testing and propagating a number of transgenic plants to conduct the study. Unfortunately the first round of transgenic pea plants made with UNI::GUS (13 individual transformants) and LFY::GUS (3 individual transformants) failed to express GUS. We are redoing the UNI::GUS transformations and have 5 new transgenic plants. Finally, we have crossed the pea homolog of the phantastica mutation, called crispa (cri), into a range of leaf mutants and expanded the set of isogenic lines available in pea to include cri, cri tl, cri af, cri tac, cri st and cri af tl. We also crossed DR5::GUS into the cri line. We are characterizing the phenotypes, morphologically and anatomically, of all lines to understand the role that CRI plays in pea leaf development in comparison to other model plant species. PARTICIPANTS: A post-doctoral researcher, V.J. Chetty has made important contributions to the three studies and has done the majority of work on the Arabidopsis and pea transformations. The auxin study is a collaboration with Dr. Jerry Cohen at the University of Minnesota and a granduate student in his lab, Xing Liu did an important auxin transport experiment. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
The Fabaceae is one of the ten largest families of flowering plants and one of the three most economically important. Leaf form variation is exceptional in the family, ranging from bipinnate to palmately compound, trifoliate and simple. As the range of leaf forms in the pea mutants replicates some of this total variation, genes identified in pea are potentially important for understanding leaf form variation with the family as a whole. Results from our studies may provide the genetic tools to initiate a biotechnology approach to maximize photosynthetic and other agronomic potential and of both peas, as well as other economically important legume species, especially the cool season legumes (i.e. Medicago, Cicer, Lens, Lathyrus, Lotus).
Publications
- D.A. DeMason and V.J. Chetty. 2011. Interactions between GA, auxin and UNI expression controlling shoot ontogeny, leaf morphogenesis, and auxin response in Pisum sativum (Fabaceae): Or how the uni-tac mutant is rescued. Amer. J. Bot. 98: 1-17.
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Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: We have been working on three related projects in the past year. The first, which involved using genetic, tissue culture and physiological methods, as well as DR5::GUS expression and qRT-PCR, to explore the interactions between the hormones gibberellic acid (GA) and auxin and Unifoliata (UNI) gene expression that control leaf morphogenesis in pea. We found that the rate of increase in leaf complexity during shoot ontogeny (i.e. heteroblasty) and adult leaf complexity are controlled by GA through the UNI gene. Leaves on greenhouse grown uni-tac mutants are rescued by weekly GA or auxin applications. Auxin responsiveness is reduced in uni-tac shoot and root tips and in wild type shoot tips treated with auxin transport inhibitors. GA and auxin increase UNI mRNA levels in uni-tac as well as that of other transcription factors. Therefore, GA and auxin positively promote leaf dissection during leaf morphogenesis in pea. GA-generated elaboration of leaf morphogenesis is in distinct contrast to that in other species, such as tomato. Instead, GA and auxin play common and supportive roles in pea leaf morphogenesis as they do in many other aspects of plant development. These data have been written up and are in press. The second project involves studying the interaction between auxin, and the Afila (AF) and UNI genes in controlling the development of the pea compound leaf. We collected data on ontogenetic variation of leaf form (heteroblasty) of af uni and af tl uni plants to see how AF and UNI interact to control leaf development in pea. We measured auxin levels in shoot tips of a range of mutants and wildtype, to determine if there are genotypic differences especially in af, uni-tac and uni. We crossed the DR5::GUS reporter into a range of leaf mutants, including uni-tac, uni and af to determine if there are differences in auxin perception. And finally, we used qRT-PCR to look at gene expression of UNI, and other auxin-regulated genes to compare expression levels in shoot tips of a range of mutants and wildtype plants with the results of auxin measurements and auxin perception experiments. We are finishing up these experiments for publication. Finally, we are studying the expression of UNI and the Arabidopsis homologous gene LEAFY (LFY) in both Arabidopsis and pea and we are studying the differential regulation of their promoters in both species. We are making, testing and propagating a number of transgenic plants to conduct the study. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
The Fabaceae is one of the ten largest families of flowering plants and one of the three most economically important. Leaf form variation is exceptional in the family, ranging from bipinnate to palmately compound, trifoliate and simple. As the range of leaf forms in the pea mutants replicates some of this total variation, genes identified in pea are potentially important for understanding leaf form variation with the family as a whole. Results from our studies may provide the genetic tools to initiate a biotechnology approach to maximize photosynthetic and other agronomic potential and of both peas, as well as other economically important legume species, especially the cool season legumes (i.e. Medicago, Cicer, Lens, Lathyrus, Lotus).
Publications
- DeMason, D.A. and V. J. Chetty. 2011. Interactions between GA, auxin and UNI expression controlling shoot ontogeny, leaf morphogenesis and auxin response in garden pea; or how the uni-tac mutant is rescued. American Journal of Botany (in press).
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Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: We are continuing the process of crossing the DR5::GUS transgene into the various leaf mutants of pea. We have completed the process for uni-tac, tendrilless, afila, stipuleless, crispa. We completed the process for uni but possibly because this allele is in a different background from our isogenic lines, it didn't express well and we are backcrossing. We are growing out F2 lines of insecatus, sinuate leaf and arthritic. Seeds are being accumulated for af tendrilless, nana, le Nana and LE Nana. Shoot ontogeny data are being collected for LE Nana, le Nana and nana to test the effect on GA levels on leaf development. Greenhouse failures both last winter and this past fall have prevented the collection of publishable data. Using MUG assays and GUS staining we have determined that DR5 is less responsive to auxin treatment in uni-tac than in WT. We have transformed Arabidopsis with UNI::GUS and we are accumulating seeds to study its expression in comparison to LFY::GUS. We are actively working on transforming pea with both constructs. Finally, expression of auxin-regulated genes is being compared in shoot tips of the different genotypes by qRT-PCR. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
The Fabaceae is one of the ten largest families of flowering plants and one of the three most economically important. Leaf form variation is exceptional in the family, ranging from bipinnate to palmately compound, trifoliate and simple. As the range of leaf forms in the pea mutants replicates some of this total variation, genes identified in pea are potentially important for understanding leaf form variation with the family as a whole. Results from our studies may provide the genetic tools to initiate a biotechnology approach to maximize photosynthetic and other agronomic potential and of both peas, as well as other economically important legume species, especially the cool season legumes (i.e. Medicago, Cicer, Lens, Lathyrus, Lotus).
Publications
- DeMason, D.A. and Chawla, R. 2009. Raising anti-PIN1 polyclonal antibodies for pea. Pisum Genetics 41: 15-20.
- Sundrish Sharma, Darleen A DeMason, Bahman Ehdaie and J Giles Waines. 2010. Dosage effect of the short arm of chromosome 1 of rye on root morphology and anatomy in bread wheat. J. Exp. Bot. (in press).
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Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: The DR5::GUS transgene is being crossed into various leaf form mutants of pea. Crossing is complete for uni-tac, and tendrilless. Homozygous expression is being obtained for crispa, and reduced stipule. F2 generations are being grown out for uni, and arthritic. F1 plants are being grown out for nana and crosses will be done this year for sinuate leaf. Nine genotypes differing in internal gibberellic acid content (high, low and very low) have been constructed for normal peas, uni-tac and afila. Shoot ontogeny and leaf complexity data have been accumulated for uni-tac and afila and are being obtained this year for normal. Auxin and gibberellic acid applications have been done on uni-tac plants grown in the greenhouse. MUG assays and GUS staining are being done on root and shoot tips of the genotypes as they are available. DR5 expression under different conditions and hormone treatments are being evaluated. Anti-GUS assays have been attempted with disappointing results. Oxidative activity assays have been attempted with inconsistent results. All double and triple mutant combinations of cri have been made and seeds are being propagated for experiments. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
The Fabaceae is one of the ten largest families of flowering plants and one of the three most economically important. Leaf form variation is exceptional in this family, ranging from bipinnate to palmately compound, trifoliate and simple. As the range of leaf forms in the pea mutants replicates some of this total variation, genes identified in pea are potentially important for understanding leaf form variation within the family as a whole. Results from our studies may provide the genetic tools to initiate a biotechnology approach to maximize photosynthetic and other agronomic potentials and of both peas, as well as other economically important legume species, especially the cool season legumes (i.e. Medicago, Cicer, Lens, Lathyrus, Lotus).
Publications
- DeMason, D.A. and P. L. Polowick. 2009. Patterns of DR5::GUS expression in organs of pea (Pisum sativum). Int. J. Plant Sci. 170:1-11.
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Progress 01/01/07 to 12/31/07
Outputs
OUTPUTS: Objective 1. Demonstration of auxin gradient. All tissue culture experiments are complete. Transformation of the WT line with DR5::GUS is complete and initial experiments show that GUS is expressed in all plant parts expected. A manuscript is in preparation. Crosses have been made to observe expression in afila, tendrilless, crispa, stipuleless and uni-tac backgrounds as well. Seed is being propagated for experimentation. Experimentation to determine if an oxidizing state, often associated with high auxin response is associated with DR5::GUS expression is in progress. Objective 2. PIN characterization and expression. Expression studies of PsPIN1 are complete and published. Antibodies raised against the central, gene-specific region have been tested using western blotting and immunolocalization. These are adequate for publication. Experiments are in progress. Expression studies of PsPK2, thought to regulate PIN1 are continuing. Transgenic studies in Arabidopsis are complete
and published. PsPK2::GUS transgenic pea plants have been made and seed is being propagated for experiments. Objective 3. Characterization of Uni expression. Previous attempts to construct a reporter for transformation of pea and Arabidopsis have been unsuccessful. We are starting to work with the crispa mutant by crossing this allele into our isogenic lines to study morphology, anatomy of stipules and leaflets as well as gene expression. Seed is being propagated for experiments.
PARTICIPANTS: Darleen DeMason, Project Director and Fang Bai, graduate student worked directly on the project during the past year. Collaborators, Dr. Karl Niklas and Edward Cobb collaborated on growth analyses. These colleagues worked at their own expense at Cornell University. This work is in press with the Amer. J. Bot. One undergraudate student (Janessa Semke) received training during the past year and an intern (Gideon Von Ende) from Holland also received training.
TARGET AUDIENCES: Target audiences include the research community, both basic and applied, as well as USDA research personnel interested in improvement of pea and other cool season legumes as commerical crops.
PROJECT MODIFICATIONS: No major changes were made.
Impacts
The Fabaceae is one of the ten largest families of flowering plants and one of the three most economically important. Leaf form variation is exceptional in this family, ranging from bipinnate to palmately compound, trifoliate and simple. As the range of leaf forms in the pea mutants replicates some of this total variation, genes identified in pea are potentially important for understanding leaf form variation within the family as a whole. Results from our studies may provide the genetic tools to initiate a biotechnology approach to maximize photosynthetic and other agronomic potentials and of both peas, as well as other economically important legume species, especially the cool season legumes (i.e. Medicago, Cicer, Lens, Lathyrus, Lotus).
Publications
- Bai, F. and D.A. DeMason. 2008. Hormone interactions and regulation of PsPK2::GUS compared to DR5::GUS and PID::GUS in Arabidopsis thaliana. Amer. J. Bot. 95: 133-145.
- Niklas, K.L., DeMason, D.A., and E. D. Cobb. 2008. Genetic effects on the biomass partition and growth of Pisum and Lycopersicon. Amer. J. Bot. (in press).
- Bai, F. 2007. A functional study of the PsPK2 gene, a PINOID-like gene from pea (Pisum sativum). Ph.D. dissertation, University of California, Riverside.
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Progress 01/01/06 to 12/31/06
Outputs
Objective 1. Demonstration of auxin gradient. All tissue culture experiments are complete. Transformation of the WT line with DR5::GUS is complete and initial experiments show that GUS is being expressed in all plant parts expected. Descriptions of expression are underway. Crosses have been made to observe expression in afila, tendrilless, crispa, stipuleless and uni-tac backgrounds as well. Objective 2. PIN characterization and expression. Expression studies of PsPIN1 are complete. Antibodies raised against the central, gene-specific region have been tested using western blotting and immunolocalization. These are adequate for publication. Experiments are in progress. Expression studies of PsPK2, thought to regulate PIN1 are continuing. We are using transgenic techniques for these studies. Objective 3. Characterization of Uni expression. We are working on making a reporter gene construct for transformation of pea and Arabidopsis. We are starting to work with the
crispa mutant by crossing this allele into our isogenic lines to study morphology, anatomy of stipules and leaflets as well as gene expression.
Impacts
The Fabaceae is one of the ten largest families of flowering plants and one of the three most economically important. Leaf form variation is exceptional in this family, ranging from bipinnate to palmately compound, trifoliate and simple. As the range of leaf forms in the pea mutants replicates some of this total variation, genes identified in pea are potentially important for understanding leaf form variation within the family as a whole. Results from our studies may provide the genetic tools to initiate a biotechnology approach to maximize photosynthetic and other agronomic potentials of peas, as well as other economically important legume species, especially the cool season legumes (i.e. Medicago, Cicer, Lens, Lathyrus, Lotus).
Publications
- Bai, F. and D.A. DeMason. 2006. Hormone interactions and regulation of Unifoliata, PsPK2, PsPIN1, and LE gene expression in pea (Pisum sativum) shoot tops. Plant Cell Physiol. 47: 935-948.
- DeMason, D.A. and N. Weeden. 2006. Two Argonaute1 genes from pea. Pisum Genet. 38: 3-9.
- Bai, F., J.C. Watson, J. Walling, N. Weeden, A.A. Santner, and D.A. DeMason. 2005. Molecular characterization and expression of PsPK2, a PINOID-like gene from pea (Pisum sativum). Plant Sci. 168: 1281-1291.
- DeMason, D.A. 2006. Auxin/gibberellin interactions in pea leaf morphogenesis. Bot. J. Linn. Soc. 150: 45-59.
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Progress 01/01/05 to 12/31/05
Outputs
Objective 1. Demonstration of auxin gradient. All tissue culture experiments are complete. Auxin rescue of the uni-tac mutant was successful. GA inhibition via paclobutrazol produced results similar to those of the auxin transport inhibitors. GA rescued the uni-tac mutant as well. Careful observations of a severe GA mutant of pea (nana1) have been completed and these plants resemble uni mutants in several morphological features. This work has been published. na1 has been crossed with le uni-tac to make a set of three lines that differ in GA content. Transformation of the WT line with DR5::GUS is complete and initial experiments show that GUS is being expressed in leaf primordium tips. Propagation of these lines is in progress and crosses have been started to observe expression in afila and uni-tac backgrounds as well. Objective 2. PIN characterization and expression. Expression studies of PsPIN1 are complete have continued. Antibodies raised against the central,
gene-specific region have been tested using western blotting and immunolocalization. These are adequate for publication. Expression studies of PIN1 and PsPK2, thought to regulate PIN1, are continuing. These genes are differentially expressed in the pea leaf mutants and are regulated by GA and auxin. Objective 3. Characterization of Uni expression. The Uni promoter has been isolated and sequenced. Potential auxin and GA regulatory domains are present. Short term induction experiments to determine regulatory characteristics of Uni are complete. Characterization of the sil mutants are still in progress.
Impacts
The Fabaceae is one of the ten largest families of flowering plants and one of the three most economically important. Leaf form variation is exceptional in this family, ranging from bipinnate to palmately compound, trifoliate and simple. As the range of leaf forms in the pea mutants replicates some of this total variation, genes identified in pea are potentially important for understanding leaf form variation within the family as a whole. Results from our studies may provide the genetic tools to maximize photosynthetic potentials of both peas themselves, as well as other economically important legume species, especially the cool season legumes (i.e. Medicago, Cicer, Lens, Lathyrus, Lotus).
Publications
- DeMason, D.A. and R. Chawla. 2004. Roles of auxin and Uni in leaf morphogenesis of the afila genotype of pea (Pisum sativum). Int. J. Plant Sci. 165: 707-722.
- Soto-Estrada, A., H. Forster, D.A. DeMason, and J.E. Adaskaveg. 2005. Initial infection and colonization of leaves and stems of cling peach by Tranzschelia discolor. Phytopathology 95: 942-950.
- DeMason, D.A. 2005. Auxin-cytokinin and auxin-gibberellin interactions during morphogenesis of the compound leaves of pea (Pisum sativum). Planta 222:151-166.
- DeMason, D.A. 2005. Extending Marxs isogenic lines in search of Uni function. Pisum Genetics 37: 10-14.
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Progress 01/01/04 to 12/31/04
Outputs
Objective 1 - Demonstration of auxin gradient. All tissue culture experiments are complete. Auxin rescue of the uni-tac mutant was successful. GA inhibition via paclobutrazol produced results similar to those of the auxin transport inhibitors. GA rescued the uni-tac mutant as well. Careful observations of a severe GA mutant of pea (nana1) have been completed and these plants resemble uni mutants in three ways: they produce more leaves during shoot ontogeny, they flower late and they have reduced numbers of pinna pairs on their leaves. A manuscript has been submitted describing these results. Transformation of the WT line with DR5::GUS is complete and initial experiments show that GUS is being expressed in leaf primordium tips. Objective 2 - PIN characterization and expression. Expression studies of PsPIN1 are complete and published. The central, gene-specific region was cloned in an expression vector and expressed in bacteria for peptide purification. This was
successful and antigen was sent to a commercial lab for polyclonal antibody preparation. This same region was cloned into a vector for making RNA probes for in situ localization. In addition, characterization of a kinase (PsPK2), thought to regulate PIN1, was cloned and characterized. This manuscript is in press. Objective 3 - Characterization of Uni expression. The Uni promoter has been isolated and sequenced. Potential auxin and GA regulatory domains are present. Short term induction experiments to determine regulatory characteristics of Uni are in progress. Characterization of the sil mutants are also in progress.
Impacts
Results of these studies are expected to provide the genetic tools to maximize photosynthetic and agronomic potential of peas and other economically important legume species in the future.
Publications
- Chawla, R. and D.A. DeMason. 2004. Molecular expression of PsPIN1, a putative auxin efflux carrier gene from pea (Pisum sativum). Plant Growth Reg. 44:1-14.
- DeMason, D.A. and R. Chawla. 2004. Roles of auxin and Uni in leaf morphogenesis of the afila genotye of pea (Pisum sativum). Int. J. Plant Sci. 165:707-722.
- Bai, F., J.C. Watson, J. Walling, N. Weeden, A.A. Santner, and D.A. DeMason. 2005. Molecular characterization and expression of PsPK2, a PINOID-like gene from pea (Pisum sativum). Plant Sci. (in press).
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