Progress 09/01/07 to 08/31/10
Outputs
OUTPUTS: AIM1 - Function of vt1/spi1 in inflorescence development. Quantitative analysis showed that spi1 mutants have fewer branches, spikelets, florets and floral organs (Gallavotti et al 2008). SEM and histology showed that spi1 mutants do not initiate axillary meristems. Double mutant analysis with teosinte branched1 (tb1), showed that spi1 also plays a role in vegetative axillary meristem initiation Measurements of cell size showed that the inflorescences of spi1 mutants are short due defects in cell size (Barazesh et al 2009). AIM 2 -Cloning and molecular characterization of vt1/spi1. In collaboration with Bob Schmidts lab, we cloned the spi1 gene by positional cloning and showed that spi1 encodes a YUC-like gene involved in auxin biosynthesis (Gallavotti et al 2008). Sequencing of four alleles identified different mutations confirming the correct gene had been cloned. RT PCR showed that the spi1 was expressed in vegetative and reproductive tissues. RNA in situ hybridization showed that spi1 was expressed as axillary meristems and lateral primordia initiate (Gallavotti et al 2008). AIM3 - Interaction of vt1/spi1 with bif2 and ba1. By realtime RTPCR, bif2 and ba1 were expressed at lower levels in spi1 mutants and spi1 expression was reduced in spi1 and bif2 mutants (Barazesh, 2008). spi1;bif2 double mutants had a synergistic interaction producing semi-dwarf plants with fewer leaves indicating that both spi1 and bif2 function in vegetative development (Gallavotti et al 2008). spi1;ba1 double mutants had an additive interaction (Barazesh et al 2009). spi1;Bif1 double mutants resembled Bif1 mutants (Barazesh et al 2009). AIM4 - Interaction of vt1/spi1 with auxin. spi1 mutants had lower levels of PIN gene expression (Gallavotti et al 2008). Direct measurement of auxin levels by GC/MS (in collaboration with Jerry Cohens lab), showed that spi1 mutants had a reduction in auxin levels (Phillips et al 2011). AIM 5 -Mapping of vanishing tassel2 (vt2). We fine mapped vt2. A tryptophan (TRP) aminotransferase in the interval was a good candidate gene for vt2 due to its role in auxin biosynthesis. Sequencing revealed mutations in seven different vt2 alleles indicating that vt2 encodes a TRP aminotransferase (Phillips et al 2011). In addition, analysis of auxin levels in spi1;vt2 double mutants, showed that spi1 and vt2 function in the same pathway for auxin biosynthesis. Analysis of the phenotype of vt2 mutants showed that vt2 mutants made fewer leaves indicating that vt2 functions in leaf formation. vt2 mutants, like spi1 mutants, had fewer branches and spikelets due to defects in axillary meristem initiation and the inflorescences were short due to defects in cell size. vt2 was expressed in all tissues by RT PCR and within the inflorescence, RNA in situ hybridization showed that vt2 was expressed in the epidermis. We also analyzed the interaction of vt2 with tb1, bif2 and ba1 (Phillips et al 2011). DISSEMINATION - Results from this grant were disseminated in talks at nine conferences and seminars at 18 US universities. Posters were presented at three conferences. PARTICIPANTS: Paula McSteen, Project Director, supervision of research and dissemination of information. Solmaz Barazesh, Ph.D. awarded, Dec 2008, Cloning and characterization of spi1. Kim Phillips, M.Sc. awarded, Dec 2009, Cloning and characterization of vt2. Andrea Skirpan, Postdoctoral scholar/Research Associate, Expression analysis of spi1 and vt2. Cima Nowbakht, Research Technologist, Further characterization of the spi1 mutant. Chris Hudson, undergraduate student, fine mapping of vt2. Kyle Barnoff, undergraduate student, assistance with genotyping double mutants. Adam Stubert, undergraduate student, assistance with genotyping double mutants. Collaborators: Andrea Gallavotti and Robert Schmidt, UCSD, cloning of spi1. David Jackson, CSHL, PIN1:YFP marker line expression in spi1. Xing Liu and Jerry Cohen, University of Minnesota, measurement of auxin levels. Simon Malcomber, California State University-Long Beach, phylogenetic analysis. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: We changed the name of vanishing tassel1 (vt1) to sparse inflorescence1 (spi1). We originally proposed just to fine map vt2. Due to advances in maize genomics research, we succeeded in identifying the vt2 gene by positional cloning. In addition to performing all of the experiments proposed to characterize spi1, we performed the same characterization experiments on vt2. In essence, for this project, we produced twice as much as originally proposed.
Impacts
The spi1 gene has been determined to encode a YUC-like flavin mononoxygenase involved in localized auxin biosynthesis. Phylogenetic analysis shows that the spi1 gene belongs to a monocot specific clade and there has been a radiation of this gene family in both monocots and eudicots. In Arabidopsis the gene family has much higher redundancy, in fact, knockout of four genes are required to see the same phenotype as the single knockout in maize. As genes in the spi1/YUC family are required for auxin biosynthesis, which plays a critical role in organ formation, an intriguing possibility is that the evolution of the spi1/YUC gene family may have played a role in the evolution of different plant forms. As the spi1 gene plays a critical role in tiller formation, ear formation, kernel number and tassel formation, spi1 plays a fundamental role in yield in corn which is an important crop in the US. The vt2 gene also plays a critical role in vegetative and reproductive development in corn. The vt2 gene has encodes a TRP aminotransferase involved in localized auxin biosynthesis. Based on work in Arabidopsis, the spi1 and vt2 genes were previously proposed to be involved in separate pathways for auxin biosynthesis. Due to the reduced redundancy of the two gene families in maize, we were able to test this hypothesis through construction of double mutants and analysis of auxin levels. A major impact of our results on the field of plant biology has been to show that, in fact, these two genes may be functioning in the same auxin biosynthetic pathway. The cloning of spi1 and vt2 provides the first indication of a role for these genes in auxin dependent development in corn or any other monocot plant species. Our work has led to a greater understanding of the role of auxin biosynthesis in reproductive development (production of the tassel and ear) in corn and hence has the potential to improve crop and biomass yield for food and fuel.
Publications
- A. Gallavotti, S. Barazesh, S. Malcomber, D. Hall, D. Jackson, R.J. Schmidt, P. McSteen (2008) sparse inflorescence1 encodes a monocot-specific YUCCA-like gene required for vegetative and reproductive development in maize. Proceedings of the National Academy of Sciences USA, 105:15196-15201.
- S. Barazesh and P. McSteen (2008) Hormonal control of grass inflorescence development. Trends in Plant Sciences, 13:656-662.
- S. Barazesh (2008) The function of barren inflorescence1 and sparse inflorescence1 in maize inflorescence development. Ph.D Thesis. The Pennslyvania State University.
- S. Barazesh, C. Nowbakht, and P. McSteen (2009) sparse inflorescence1, barren inflorescence1 and barren stalk1 promote cell elongation in maize inflorescence development. Genetics, 182: 403-406.
- P. McSteen (2009) Hormonal regulation of branching in grasses. Plant Physiology, 149: 46-55.
- K.A Phillips (2009) The roles of vanishing tassel2 and developmental disaster1 in maize vegetative and inflorescence development. M.Sc. Thesis. The Pennslyvania State University.
- P. McSteen (2010) Auxin and Monocot Development. In "Auxin Signaling: From Synthesis to Systems Biology". Edited by D. Weijers, K. Ljung, O. Leyser, M. Estelle. Cold Spring Harbor Perspectives in Biology, 2 (3) a001479.
- K.A. Phillips, A.L. Skirpan, X. Liu, A. Christensen, T.L. Slewinski, C. Hudson, S. Barazesh, J.D. Cohen, S. Malcomber and P. McSteen (2011) vanishing tassel2 encodes a grass-specific tryptophan aminotransferase required for vegetative and reproductive development in maize. Plant Cell, 23, 550-566.
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Progress 09/01/08 to 08/31/09
Outputs
OUTPUTS: AIM 1 - In year two we made progress on Aim 1c. We previously noted that spi1 mutants had a defect in tassel inflorescence length (Gallavotti et al 2008). In year two, using cell impressions and measurements of cell size, we discovered that the inflorescences of spi1 mutants were short due to the fact that cells in the inflorescence were short (Barazesh et al 2009). AIM 2 - In year two, we made a construct to test the expression of the spi1 gene in plants by fusing 3 copies of the yellow fluorescent protein (YFP) to 3kb of the spi1 promoter. The construct was transformed into maize by the Iowa State University Plant Transformation facility. We showed that the construct is expressed in an auxin inducible manner in callus tissue and are currently waiting on seed to be harvested from the transgenic plants to test the expression in the inflorescence. AIM 3 - In year two, we made progress on Aim 3c. We tested the genetic interaction of spi1 with ba1 (Barazesh et al 2009). spi1;ba1 double mutants had no ears like ba1 and no bract primordia in the tassel like spi1 but the double mutant was more severe than either single mutant with respect to the number of spikelets. We concluded that spi1 and ba1 function in separate pathways (Barazesh et al 2009). In an additional experiment, not originally proposed in the grant, we tested the genetic interaction of spi1 with Bif1, as Bif1 had similar defects to spi1 as well as defects in cell elongation in the inflorescence (Barazesh et al 2008, Barazesh et al 2009). We found that the spi1;Bif1 double mutants appeared to be similar to Bif1 mutants, indicating that the spi1 and bif1 genes may function in the same pathway (Barazesh et al 2009). AIM 5 - Last year, we reported that we had mapped vt2 between two markers idp98 (1.36cM) and umc1974 (0.12cM) on chromosome 8. In year two, we were able to further narrow the region to three BACs by developing single nucleotide polymorphism (SNP) markers from genes in the interval. A TRP aninotransferase gene in the interval was considered to be a good candidate gene for vt2 due to its role in auxin biosynthesis. Sequencing of this candidate gene revealed mutations in seven different vt2 alleles indicating that vt2 encodes a TRP aminotransferase (Phillips et al, in preparation). Homologous TRP aminotransferases have been shown in Arabidopsis to convert TRP to indole-3 -pyruvic acid (IPA) in the first step of one of the TRP dependent pathways for auxin biosynthesis. In addition, we were able to show by constructing and analyzing spi1;vt2 double mutants that the spi1 and vt2 pathways for TRP dependent auxin biosynthesis function independently (Phillips et al, in preparation). DISSEMINATION: In year two, results from this grant were disseminated in talks given by the PD at three conferences (Plant and Animal Genome, San Diego, CA in Jan 2009, SFB 572, Cologne, Germany in Jun 2009 and Auxin 2008, Marrakech, Morocco in Oct 2008) as well as seminars at nine universities. Two posters depicting results were presented at the 51st Maize Genetics Conference, St. Charles, IL in March 2009. PARTICIPANTS: Paula McSteen, Project Director, supervision of research and dissemination of information. Kim Phillips, M.Sc. student, Cloning and characterization of vt2. Solmaz Barazesh, Ph.D. awarded, Dec 2008. Cloning and characterization of vt1/spi1. Andrea Skirpan, Postdoctoral scholar/Research Associate, Further characterization of the spi1 and vt2 genes. Cima Nowbakht, Research Technologist. Further characterization of the vt1/spi1 mutant. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: We originally proposed just to fine map vt2 to provide the foundation for future work to clone the corresponding gene. Due to advances in maize genomics research we have in fact succeeded in identifying the vt2 gene by positional cloning.
Impacts
In the first year of the grant, we cloned vt1/spi1 and showed that vt1/spi1 functioned in the YUC pathway for TRP dependent auxin biosynthesis. Cloning of vt2 in the second year of the grant showed that vt2 functioned in the IPA pathway for TRP dependent auxin biosynthesis. Both of these results were the first indication of a role for these two pathways in auxin dependent development in maize or any other monocot plant species. Furthermore, our analysis of the interaction between vt1 and spi1 is the first confirmation in any plant species of the independence of these two auxin biosynthetic pathways. As both the spi1 and vt2 mutants have severe defects in reproductive development, the results indicate the importance of these two pathways for auxin biosynthesis in maize. As the development of the tassel and ear is critical for yield, these findings show the importance of auxin in controlling yield in maize, one of the most important crop plants to US agriculture.
Publications
- (1) S. Barazesh (2008) The function of barren inflorescence1 and sparse inflorescence1 in maize inflorescence development. Ph.D Thesis. The Pennslyvania State University. (2) S. Barazesh, C. Nowbakht,and P. McSteen (2009) sparse inflorescence1, barren inflorescence1 and barren stalk1 promote cell elongation in maize inflorescence development. Genetics,182:403-406. (3) P. McSteen (2009) Hormonal regulation of branching in grasses. Plant Physiology,149:46-55.
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Progress 09/01/07 to 08/31/08
Outputs
OUTPUTS: We changed the name of the mutant from vanishing tassel1 (vt1) to sparse inflorescence1 (spi1). For publication purposes we thought that this name better reflected the phenotype of the mutant. Aim1 - Function of vt1/spi1 in inflorescence development. 1a) Effect of the vt1/spi1 mutation on the mature tassel and ear phenotype. By quantitative analysis of the mature spi tassel phenotype, we showed that spi1- ref mutants have fewer branches, spikelets, florets and floral organs (Gallavotti et al 2008). 1b) Determine the role of vt1/spi1 in axillary meristem initiation. We showed using SEM and histology of developing tassels that spi1 mutants do not initiate axillary meristems (Gallavotti et al 2008). Using double mutant analysis with teosinte branched1, we showed that spi1 does play a role in vegetative axillary meristem initiation (Gallavotti et al 2008). Aim 2 - Cloning and molecular characterization of vt1/spi1. 2a) Identification of the vt1/spi1 coding region. 2b) Confirmation of cloning: Characterization of other alleles. In collaboration with Bob Schmidt's lab, UCSD, the spi1 gene was identified by map based cloning (Gallavotti et al 2008). The spi region was delineated to a region containing four BACs. A YUC gene family member was identified that was considered a good candidate due to its role in auxin biosynthesis. Sequencing of this candidate gene in different alleles identified multiple types of mutations confirming that the correct gene has been cloned. spi1-01-008-16 has an EMS induced change in a conserved domain, spi1-E914 and spi1-ref have in frame deletions in conserved domains, spi1-125 and spi-126 have the same Mutator transposon insertion in the 5'UTR (Gallavotti et al 2008). 2c) Expression analysis of vt1/spi1. RT PCR analysis showed that the spi1 gene is widely expressed in vegetative and reproductive tissues except roots (Gallavotti et al 2008). RNA insitu hybridization showed that the spi1 is expressed in a transient manner as axillary meristems and lateral primordia initiate (Gallavotti et al 2008). Aim 3 - Interaction of vt1/spi1 with bif2 and ba1. 3c) Genetic interaction of vt1/spi1 with other bif mutants. In an unexpected development spi1;bif2 double mutants had a synergistic interaction during vegetative development producing semi-dwarf plants with fewer leaves (Gallavotti et al 2008). Aim 4 - Interaction of vt1/spi1 with auxin. 4a) Auxin distribution in the vt1/spi1 mutant. We showed that spi1 mutants have lower levels of PIN gene expression using PIN1a:YFP line (Gallavotti et al 2008). We showed that spi1 mutants did not have a statistically significant difference in auxin levels by measuring auxin levels by GC/MS in collaboration with Jerry Cohen's lab, University of Minneosta (unpublished). Aim 5 - Mapping of vanishing tassel2 (vt2). We fine mapped vt2 to a region on chromosome 8 between markers idp98 (1.36cM) and umc1974 (0.23cm). Additional markers are being developed to further fine map. The results have been disseminated at talks at the Keystone conference (Feb), the Maize Genetics Conference (March), the FASEB meeting (August) and at multiple invited talks at universities. PARTICIPANTS: Paula McSteen, principal investigator, supervision of research and dissemination of information through talks and publications. Solmaz Barazesh, PhD student, characterization of vt1/spi1. Andrea Skirpan, postdoctoral scholar, further characterization of vt1/spi1. Kim Phillips, PhD student, fine mapping vt2. Chris Hudson, part time undergraduate student, Spring 2008, fine mapping vt2. Kyle Barnoff, part time undergraduate student, Summer 2008, field help. Collaborators: Robert Schmidt, UCSD. Andrea Gallavotti, UCSD. Dave Jackson, CSHL. Simon Malcomber, CSULB. Jerry Cohen, U Minnesota. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The spi1 gene has been determined to encode a YUC-like flavin mononoxygenase involved in localized auxin biosynthesis. Phylogenetic analysis shows that the spi1 gene belongs to a monocot specific clade and there has been a radiation of this gene family in both monocots and eudicots. Interesting the spi1 gene differs in expression pattern and function from its ortholog in rice indicating that there has been diversification of this gene family even in grasses. In Arabidopsis the gene family has much higher redundancy, in fact, knockout of four genes are required to see the same phenotype as the single knockout in maize. As genes in the spi1/YUC family are required for auxin biosynthesis, which plays a critical role in organ formation, an intriguing possibility is that the evolution of the spi1/YUC gene family may have played a role in the evolution of different plant forms. As the spi1 gene plays a critical role in tiller formation, ear formation, kernel number and tassel formation, spi1 plays a fundamental role in yield in corn which is an important crop in the US. Our work has led to a greater understanding of the role of auxin biosynthesis in reproductive development (production of the tassel and ear) in corn and hence has the potential to improve crop and biomass yield for food and fuel.
Publications
- Gallavotti, A., Barazesh, S., Malcomber, S., Hall, D., Jackson, D., Schmidt, R.J., and McSteen, P. (2008). sparse inflorescence1 encodes a monocot-specific YUCCA-like gene required for vegetative and reproductive development in maize. Proceedings of the National Academy of Sciences USA, 105:15196-15201.
- Barazesh, S., and McSteen, P. (2008). Hormonal control of grass inflorescence development. Trends in Plant Sciences, 13:656-662.
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