Progress 09/01/08 to 08/31/12
Outputs
OUTPUTS: Our research studied the genetic mechanism of bract suppression in the cereal grasses by characterization of the maize mutant tassel sheath1 (tsh1), its orthologs in rice and barley (nl1 and trd), and the maize tsh4 mutant. These mutants were cloned and characterized during the funding period. We performed phylogenetic analysis of NL1 Tsh1 and TRD (NTT)-like genes from a broad sampling of grasses and outgroups to time the duplication events that created the three NTT-like genes in grass genomes. An NTT-like gene from the grass outgroup Cyperus was analyzed by in situ hybridization. We compared the function of Tsh1 and its Arabidopsis ortholog HANABA TARANU. We performed a screen for genetic enhancers of the weak tsh1-2 allele in over 2000 EMS-mutagenized families. Further analysis of the TSH1 regulatory network via mRNAseq analysis is in progress. Finally, we expanded our analysis of maize bract suppression by fine mapping of tsh2 and Few branched1 (Fbr1) mutants. CW presented this research at the Maize Genetics Conference (2009, 2010, 2011), FASEB conference (2010), and seminars at BYU (2009) Utah Plant Genetics Conference (2009) BYU-Hawaii (2011) and the Botanical Society of America (2011). DJ also gave multiple research presentations including seminars at Guelph (2009), Neuchatel, Switzerland (2009), and the JIC (2009), Sogang University, , Korea (2010), CSHL Asia Conference in Plant Biotechnology (2010), and seminars at Harvard and Tokyo University (2011). CW discussed maize and transgenics issues on the radio program Thinking Aloud (http://www.classical89.org/thinkingaloud/archive/episode/id=3/28/20 12) Collaborations were initiated with George Chuck and Sarah Hake at the PGEC, Albany, CA on tsh4 characterization. Torbert Rocheford (Purdue) provided fields to screen for tsh1-2 enhancers/suppressors. CW took a position at Brigham Young University in 2009 and continues to collaborate on bract suppression there. CW spent one week at the National Tropical Botanical Gardens in Kauai, HI collecting Joinvillea and Flagellaria tissue in collaboration with David Lawrence. Cliff Weil (Purdue University) is contributing to characterization of Fbr1, focusing on naturally occurring modifiers. Erik Vollbrecht (ISU) recently identified a Ds transposon in Tsh1 and is perform saturation mutagenesis screens to make novel alleles for detailed structure and function information. Finally, CW and Mike Muszynski (ISU) are collaborating to characterize an interaction between Tsh1 and Hairy sheath frayed1. Training was provided for 10 students, one technician and two postdocs. Students trained on in molecular biology and maize field work include a high school student, Kay Chen, and undergraduates Steven Williams, Dinesh Adhikary, Alicia Garff, Nicholas Rigby, Sidney McDonald, Holly Waddell, Jouber Santos, Zachary Golden, and Rachel Thayer. Ulises Hernandez (CSHL) was trained in maize field work and phenotyping. Seth Bybee was trained to perform phylogenetic analysis of NTT genes. Dissemination: CW discussed maize and transgenics issues on the radio program Thinking Aloud (http://www.classical89.org/thinkingaloud/archive/episode/id=3/28/20 12). PARTICIPANTS: Training was provided for 10 students, one technician and two postdocs. Students trained on in molecular biology and maize field work include a high school student, Kay Chen, and undergraduates Steven Williams, Dinesh Adhikary, Alicia Garff, Nicholas Rigby, Sidney McDonald, Holly Waddell, Jouber Santos, Zachary Golden, and Rachel Thayer. Ulises Hernandez (CSHL) was trained in maize field work and phenotyping. Seth Bybee was trained to perform phylogenetic analysis of NTT genes. Collaborations were initiated with George Chuck and Sarah Hake at the PGEC, Albany, CA on tsh4 characterization. Torbert Rocheford (Purdue) provided fields to screen for tsh1-2 enhancers/suppressors. CW took a position at Brigham Young University in 2009 and continues to collaborate on bract suppression there. CW spent one week at the National Tropical Botanical Gardens in Kauai, HI collecting Joinvillea and Flagellaria tissue in collaboration with David Lawrence. Cliff Weil (Purdue University) is contributing to characterization of Fbr1, focusing on naturally occurring modifiers. Erik Vollbrecht (ISU) recently identified a Ds transposon in Tsh1 and is perform saturation mutagenesis screens to make novel alleles for detailed structure and function information. Finally, CW and Mike Muszynski (ISU) are collaborating to characterize an interaction between Tsh1 and Hairy sheath frayed1. TARGET AUDIENCES: In addition to the training described above, DJ organized a CSHL workshop in Cereal Genomics that trained international and US students in cereal crop informatics and quantitative genetics. He also organized a CSHL Asia conference in Plant Genomes and Biotechnology, that trained students in modern approaches to crop plant biology. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Our characterization of tsh1, nl1, and trd mutants from maize, rice and barley respectively, has demonstrated that all of these mutants are caused by disruption of a GATA zinc finger coding gene orthologous to HANABA TARANU of Arabidopsis. The Nl1/Tsh1/Trd (NTT) genes of these cereals regulate bract growth in the inflorescence. Bract suppression was likely an important step in cereal crop evolution, since the reallocation of resources to the flowers that occurs when bract growth is suppressed can enhance seed yield. Infact, the tsh loss of function mutants generally make fewer spikelets and seed. In situ expression analysis shows that the TSH orthologs are all expressed in the cells of the cryptic (suppressed) bract. Together these results show that grasses have a common mechanism of bract suppression involving NTT orthologs. In addition we demonstrated that the maize tsh4 mutant is Squamosa promoter Binding Protein6 (SBP6) a target of the microRNA Corngrass1. Together, NTT and tsh4 constitute a novel pathway for bract suppression that evolved independently from Arabidopsis and the Brassicaceae. Our ectopic expression studies of Tsh1 and HAN in the petals and stamens of Arabidopsis using the AP3 promoter have shown that both the maize and Arabidopsis protein have similar phenotypic consequences, including the inhibition of lateral organ growth and promotion of indeterminate callus-like tissue. Phylogenetic analysis of NTT and NTT-like genes isolated from grasses and their closest outgroups suggests that successive duplications created the three paralogous gene lineages known from fully sequenced grass genomes. In order to assess the likely ancestral function of Tsh/Ntt genes, we examined the expression in Cyperus, which has a single Tsh/Ntt gene that diverged before these duplication events. Cyperus NTT has an expression pattern similar to its Arabidopsis ortholog HANABA TARANU. These results are consistent with our hypothesis that a expression neofunctionalization in the Tsh1 lineage rather than a change in protein function was important in the evolution of the novel bract suppression pathway seen in the grass family, and provide a concrete example of neofunctionalization contributing to the generation of morphological innovation, which has been frequently hypothesized but rarely demonstrated. Multiple genetic enhancers of tsh1 have now been isolated, and many of these have strong pleiotropic effects on plant development including plant height and leaf width suggesting that Tsh1 interacts with fundamental regulators of plant development, possibly hormonal pathways. This potential Tsh1 hormone connection is substantiated by the genetic interaction of Tsh1 with Hsf1, which regulates cytokinin signaling. Positional cloning of tsh2 and Fbr1 are nearing completion. We have localized tsh2 to a narrow region that contains tsh4, and it is likely that these mutants are allelic. Fbr1 has been localized to a 1.1 Mb region on chromosome 6 that contains ~50 genes and several good candidates. Isolation of these additional loci will provide additional candidate genes to test their potential effect on cereal crop productivity.
Publications
- Whipple, C. J., Hall, D. H., DeBlasio, S., Taguchi-Shiobara, F., Schmidt, R. J., and Jackson, D. P. (2010). A conserved mechanism of bract suppression in the grass family. The Plant Cell 22:565-578. (featured on the cover and in an In this issue editorial)
- Chuck, G., Whipple, C., Jackson, D., and Hake, S. (2010). The Maize SBP-box Transcription factor encoded by tasselsheath4 regulates bract development and the establishment of meristem boundaries. Development 137:1243-1250.
- Eveland, A.L. and Jackson, D. (2011) Sugars, signaling, and plant development. J. Exp. Bot. (2012) 63 (9): 3367-3377.
- Whipple, C.W., Kebrom, T., Weber, A.L., Yang, F., Hall, D.H., Meeley, R. B., Schmidt, R.J., Doebley, J., Brutnell, T.B. and Jackson, D.P. (2011). grassy tillers1 promotes apical dominance in maize and responds to shade signals in the grasses. Proc Natl Acad Sci Plus, 108 (33) 13375-13376.
- Brutnell, T.P., Wang, L., Swartwood, K., Goldschmidt, A., Jackson, D., Zhu, X.G., Kellogg, E., VanEck, J. (2010). Setaria viridis: A Model for C4 Photosynthesis. Plant Cell, 22: p. 2537-44.
- Michael Pautler, Wakana Tanaka, Hiro-Yuki Hirano, David Jackson (2012, pending). Grass Meristems I: Shoot apical meristem maintenance, axillary meristem determinacy, and the floral transition. Plant and Cell Physiology.
- Wakana Tanaka, Michael Pautler, David Jackson, Hiro-Yuki Hirano (2012, pending). Grass Meristems II: Flower development and meristem fate. Plant and Cell Physiology.
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Progress 09/01/10 to 08/31/11
Outputs
OUTPUTS: Phylogenetic analysis of the Tsh1/Ntt1/Ntt2 gene family in the grass family was continued by isolating orthologs from the grass outgroups Ecdeiocolea and Flagellaria. These sequences were used to more accurately place the timing of the duplication events creating this relatively small gene family in the grasses. We performed in situ hybridization with the Cyperus NTT gene, which diverged before the Tsh1/Ntt1/Ntt2 duplications. Joinvillea inflorescence tissue was collected at the National Tropical Botanical garden and fixed for in situ hybridization and SEM analysis to determine the role of Tsh1 in this bracteate outgroup to the grass family. Transgenic maize expressing an RNAi construct for targeted degradation of Ntt1 in maize was created. We have been working to optimize mRNAseq methods for small amounts of tissue/ RNA that are available from immature maize inflorescences. Our optimized protocol works well for as few as ten 1mm inflorescences, and is therefore ready to proceed for tsh mutant transcriptomics. We have also optimized maize mRNAseq analysis pipelines. We continued our screen for new tsh mutants as well as enhancer of tsh (ent) mutants by screening 500 EMS mutagenized tsh1-2 M2 families. This screen produced five new ent* mutants, bringing the total ent* alleles identified to date to nine. Three novel tsh mutants were also identified which may represent novel loci. Finally we generated large (>2000 individuals) F2 mapping populations for the tsh2 and Fbr1 mutants. DNA was isolated from these populations and is currently being genotyped with flanking markers to fine map these mutants pursuant to positional cloning. Events: CW presented a poster at the Maize Genetics Conference (March - St. Charles, IL) and a seminar on the genetics of bract suppression at Brigham Young University - Hawaii (Feb - Laie, HI). DJ presented this work at a CSHL Asia Conference in Plant Genomes and Biotechnology (October 2010), and invited seminar at Harvard University and Tokyo University (May 2011). Collaborations: CW spent one week at the National Tropical Botanical Gardens in Kauai, HI collecting Joinvillea and Flagellaria tissue in a collaboration with David Lawrence. In addition we established a collaboration with Cliff Weil of Purdue University to characterize the Fbr1 mutant, with Dr. Weil focusing on identifying naturally occurring modifiers of Fbr1. The collaboration with Torbert Rocheford (Purdue University) to grow out the tsh1-2 enhancer screen continued as in previous years. Training: Three new BYU undergraduates, Sidney McDonald, Holly Waddell, and Jouber Santos received training in molecular biology (PCR, cloning, DNA extraction and sequencing) as well as maize field work. PARTICIPANTS: In addition to previous listed participants, three new BYU undergraduates, Sidney McDonald, Holly Waddell, and Jouber Santos received training in molecular biology (PCR, cloning, DNA extraction and sequencing) as well as maize field work. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
New knowledge, techniques, development of the discipline: A more complete phylogenetic analysis of Tsh/Ntt genes isolated from grasses and their closest outgroups suggests that successive duplications created the three paralogous gene lineages known from fully sequenced grass genomes. The first duplication created the Tsh and Ntt lineages and appears to have occurred after the divergence of the Restionaceae, but before the divergence of the grass outgroups Joinvillea and Ecdeiocolea. The second duplication event generated the Ntt1 and Ntt2 lineages, and this appears to have occurred just prior to diversification of the grass family, likely in the proposed whole genome duplication event. In order to assess the likely ancestral function of Tsh/Ntt genes, we examined the expression in Cyperus, which has a single Tsh/Ntt gene that diverged before these duplication events. Cyperus Tsh/Ntt has an expression pattern similar to its Arabidopsis ortholog HANABA TARANU. This observation, in combination with our previous demonstration that HAN and TSH1 proteins have similar activity and are both capable of suppressing leaf outgrowth, suggest that gene Tsh1/Ntt duplication was followed by expression neofunctionalization in the Tsh1 lineage creating a novel bract suppression pathway that is now found throughout the grass family. These findings provide a concrete example of neofunctionalization contributing to the generation of morphological innovation, which has been frequently hypothesized but rarely demonstrated. We are currently confirming these results by examining expression of Ntt genes in the grasses, which we expect to maintain the ancestral expression pattern as seen in Cyperus and Arabidopsis. We are also examining maize Ntt1 RNAi transgenic lines to assess the function of the Ntt1 lineage in the grasses, which we predict to be similar to HAN.
Publications
- No publications reported this period
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Progress 09/01/09 to 08/31/10
Outputs
OUTPUTS: Activities: In order to understand the duplication events that led to the paralogous Tassel sheath1 (Tsh1) and Neckleaf1-Tsh1-Third outer glume-like1 and 2 (Ntt1/Ntt2) clades that are present in the grasses we have cloned and isolated Tsh1 and Ntt orthologs from early diverging grass lineages and outgroups to the grass family. To date we have isolated orthologs from the grasses Streptochaeta, Pharus, and Lithachne, as well as the non-grass outgroups Joinvillea, Elegia, Cyperus, and Chaemedoria. Arabidopsis lines expressing either HANABA TARANU (HAN the Arabidopsis Tsh1 ortholog) or Tsh1 under the control of an AP3 driver were created and characterized to test the ability of HAN and TSH1 to ectopically inhibit leaf growth. Approximately 400 more M2 families from mutagenesis of the weak tsh1-2 allele were screened for enhancers and suppressors. The recessive maize tsh2 mutant and the dominant Fbr mutant, both of which produce ectopic bracts were mapped by bulked segregant analysis, and flanking markers were identified to positionally clone tsh2. Three new putative alleles of tsh2 were isolated by screening an M1 non-complementation screen generated in the previous year. Mutator insertions in a maize specific duplicate of Tsh1 (Tsh1b) were characterized. Sasha Goldschmidt and Andrea Eveland at CSHL are working to optimize next-gen profiling methods for immature maize inflorescences that will be used to profile tsh mutants and further understand the genetic network for bract suppression. Events: CW presented work on Tsh1 and Tsh4 for the Plant and Wildlife Sciences departmental seminar as an invited speaker (BYU October 2009) and at the Utah Plant Genetics Meeting (University of Utah November 2009). DJ presented invited talks about this project at Sogang University, Seoul, Korea, and at a conference on Plant Biotechnology in Suzhou, China. Collaborations: CW attained a faculty position at Brigham Young University and continues to work on all the aims of this proposal through a subcontract to BYU. Torbert Rocheford (Purdue University) provided field space to screen for tsh1-2 enhancers/suppressors and novel tsh2 alleles. Training: Seth Bybee was employed as a postdoctoral associate (BYU) to amplify Tsh1/Ntt orthologs and perform a phylogenetic analysis. Several undergraduates (Steven Williams, Dinesh Adhikary, Alicia Garff, and Nicholas Rigby) were trained during the summer of 2010 at BYU on molecular biology (PCR) and genetic mapping in maize, as well as maize field genetics. Ulises Hernandez (CSHL) was trained in maize field work and phenotyping. PARTICIPANTS: Clint Whipple continues to be the main personnel on this project, working in close contact with project director Dave Jackson. Clint completed his post doctoral training at CSHL in August 2009 , and assumed an Assistant Professor position at Brigham Young University. His continued work on this project there is funded by a sub-contract from CSHL. Seth Bybee was employed as a postdoctoral associate (BYU) to amplify Tsh1/Ntt orthologs and perform a phylogenetic analysis. Several undergraduates (Steven Williams, Dinesh Adhikary, Alicia Garff, and Nicholas Rigby also contributed to the project. At CSHL, Sasha Goldschmidt (post doc) contributed to imaging of TSH1-YFP transgenic lines, and Ulises Hernandez (technician) helped with field work and genotyping. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
New knowledge, techniques, development of the discipline: Our phylogenetic analysis of Tsh1 and Ntt orthologs from grasses and close outgroups to the grass family show that the duplication that created the Tsh1 and Ntt lineages occurred before the most recent common ancestor of the grass family as both lineages are present in the non-grass Joinvillea. This result is surprising since Joinvillea fails to suppress bract growth, yet has the gene Tsh1 lineage, which clearly performs a bract suppression function throughout the grass family. Further work will be necessary to see if Tsh1 orthologs are expressed in bracts of species like Joinvillea that do not have bract suppression as seen in the grasses. Our ectopic expression studies of Tsh1 and HAN in the petals and stamens of Arabidopsis using the AP3 promoter have shown that both the maize and Arabidopsis protein have similar phenotypic consequences, including the inhibition of lateral organ growth and promotion of indeterminate callus-like tissue. These results are consistent with our hypothesis that a change of expression in the Tsh1 lineage rather than a change in protein function was important in the evolution of the novel bract suppression pathway seen in the grass family. Five new tsh1-2 enhancers were identified in our mutant screen, and these are similar to enhancers identified in the previous year in that the mutant phenotypes are highly pleiotropic, affecting more than just bract suppression. Further characterization of these mutants should provide insight into Tsh1 function. The maize tsh2 mutant was found to map to chromosome 7, and the Few branched1 mutant to chromosome 6. An exonic Mutator insertion was found in a maize specific duplicate of Tsh1 (Tsh1b) by reverse genetic screening. Plants with the insertion had a weak bract growth phenotype indicating that this duplicate may be partially redundant with Tsh1.
Publications
- Whipple, C. J., Hall, D. H., DeBlasio, S., Taguchi-Shiobara, F., Schmidt, R. J., and Jackson, D. P. (2010). A conserved mechanism of bract suppression in the grass family. The Plant Cell 22:565-578. (*featured on the cover and in an In this issue editorial)
- Chuck, G., Whipple, C., Jackson, D., and Hake, S. (2010). The Maize SBP-box Transcription factor encoded by tasselsheath4 regulates bract development and the establishment of meristem boundaries. Development 137:1243-1250.
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Progress 09/01/08 to 08/31/09
Outputs
OUTPUTS: Activities: Our research activities were centered on characterization of the maize mutant tassel sheath1 (tsh1), its orthologs in rice and barley (nl1 and trd), and the maize tsh4 mtutant. The genetic lesions were characterized for three independent tsh1 alleles, two nl1 alleles, two trd alleles, and two tsh4 alleles. The expression pattern was determined for Tsh1, Nl1, and Trd by RNA in situ hybridization. The protein localization was characterized for TSH1 using a TSH1-YFP fusion construct in transgenic maize. Approximately 600 M2 families were screened for enhancers and suppressors of the weak tsh1-2 allele. Events: CW presented an invited seminar on bract suppression in the grass family at the 25th symposium on Plant Biology "Evolution of Plant Development" Riverside, CA Jan 2009. In addition CW presented a poster on Tsh work at the 2009 Maize Genetics Conference (St. Charles, IL March 2009) and at the FASEB summer research conference "Mechanisms in Plant Development" (Saxtons River, VT August 2010). DJ presented results of this project in seminars at the University of Guelph, Neuchatel, Switzerland, and the John Innes Institute. Collaborations: George Chuck and Sarah Hake at the Plant Gene Expression Center in Albany, CA on tsh4 characterization. Torbert Rocheford (Purdue University) provided field space to screen for tsh1-2 enhancers/suppressors. Training: A high school student (Kay Chen) was trained on PCR, SSR-marker based mapping in maize and RT-PCR. A technician, Ulises Hernandez, was trained in maize field work, phenotyping and molecular genotyping. PARTICIPANTS: Clint Whipple is the postdoctoral researcher working on this project,and was responsible for the majority of the achievements, under the guidance of project director Dave Jackson. Clint received training in Arabidopsis and maize molecular genetics, imaging and informatics. In addition, a high school student, Kay Chen, received training in maize genetics and genetic mapping. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
New knowledge, techniques, development of the discipline: Our characterization of tsh1, nl1, and trd alleles from maize, rice and barley respectively, has demonstrated that all of these mutants are caused by disruption of a GATA zinc finger coding gene orthologous to HANABA TARANU of Arabidopsis. All of the mutants have a similar phenotype, namely de-repression of bract growth in the inflorescence. In situ expression analysis shows that the orthologs are all expressed in the cells of the cryptic (suppressed) bract. Together these results show that grasses have a common mechanism of bract suppression involving Tsh1/Nl1/Trd orthologs. Characterization of a TSH1-YFP construct in transgenic maize shows that the protein expression domain is similar to the RNA expression pattern. Phylogenetic analysis of Tsh1 orthologs and paralogs in the grass family indicate that a series of duplications created the Tsh1 lineage and a pair of closely related Nl1/Tsh1/Trd-like (Ntt1 and 2) genes. Since the closest gene in Arabidopsis has no role in bract suppression, and bract since suppression evolved independently in the grass and mustard families, or results suggest that the convergent evolution of bract suppression in grasses and mustards involves distinct genetic pathways. A likely explanation for the evolution of bract suppression in the grasses is that a gene duplication event created the novel Nl1/Tsh1/Trd lineage which evolved a new function in bract suppression, while the Ntt lineage maintains the ancestral function as described for HAN in Arabidopsis. We have begun an enhancer suppressor screen to identify novel factors in the maize bract suppression pathway, and have so far identified four putative novel enhancers of a weak tsh1-2 allele.
Publications
- No publications reported this period
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