Progress 12/15/09 to 06/14/13
Outputs
Target Audience: This project has trained 3 postdoctoral fellows, and 2 undergraduate students. The information learned from this project was also incorporated into 2 undergraduate courses at Duke. Some of the data had been published or will be published in peer reviewed journals. Changes/Problems: This project had a 6-month no-cost extension in order to complete our studies proposed in Specific Aim 1. The delay in completing one of the proposed goals in this aim was mainly because our first set of tomato transformation for one of the constructs did not yield any viable transformants. After changing to a different binary vector, our transformation was successful and multiple homozygous lines have been generated. The extension was needed to complete our study on the phenotypes of these lines. What opportunities for training and professional development has the project provided? Three postdoctoral researchers (Drs. R. Zentella, J. Hu and J. Dayan) were involved in this project. The PI met with the postdoctoral researchers individually biweekly to discuss experimental design and data analysis. They were encouraged to formulate new hypotheses, design experiments to test the hypotheses, and develop problem solving skills. There were also plenty opportunities for the postdocs to give oral presentations. The lab had weekly meetings throughout the year. Each lab member presented once every 1-2 months. Yearly, each postdoc also presented his data at the Developmental, Cell and Molecular Biology seminar series within the Biology Department at Duke, and at the NC Plant Molecular Biology Consortium annual retreat. In addition, each postdoc was encouraged to present at domestic and international conferences. To prepare for presentations outside the lab, the PI worked with each postdoc to improve the quality of the presentation. As for improving writing skills, the PI worked with the postdoc to write manuscripts, and to prepare figures. The postdoc also participated in grant writing. Three undergraduate students were also supported to conduct experiments. Each of these students was mentored by a postdoc in the lab. How have the results been disseminated to communities of interest? Publications in journals and presentations at conferences What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Specific Aim 1: Promotion of Parthenocarpy in Tomato by Altering GA Responses Phytohormone gibberellin (GA) is known to promote parthenocarpic fruit growth, but how its signaling pathway is involved in parthenocarpy is largely unclear. Here our goal is to study whether upstream GA signaling components are involved in parthenocarpy, using genetic mutants and transgenic lines. SlDELLA is a GA signaling repressor, whereas SlSLY1 is the likely ortholog of the Arabidopsis SLY1 that is a positive regulator of GA signaling. We showed that parthenocarpy is strongly enhanced in procera, which is a DELLA loss-of-function tomato mutant. We also generated transgenic tomato plants that overexpress SlSLY1 or a dominant allele At-sly1-d. Parthenocarpic growth was observed in an At-sly1-d overexpression line, although weaker when compared to procera. In summary, inhibition of SlDELLA function is more effective than inhibition of SLY1 to promote parthenocarpy in tomato. Specific Aim 2: Analysis of the SlDELLA Expression Pattern in Ovary and Fruit To monitor DELLA protein levels in developing fruits, we generated transgenic tomato lines (in the procera background) that carry a SlDELLA promoter:6xHis-3xFLAG-SlDELLA construct. This DELLA fusion protein is functional in planta because it rescued the procera phenotype. We found that SlDELLA is highest in the ovules, intermediate in the placenta and lowest in the ovule walls (pericarps) at -2 and 0 day (anthesis). To analyze SlDELLA expression pattern in more detail, we also generated transgenic tomato lines that carry either SlDELLA promoter::GUS or SlDELLA promoter::HA-YPet-SlDELLA constructs. In unfertilized ovary and young fruit, the SlDELLA::GUS signal is exclusively localized in placenta. In older fruit, however, both placenta and pericarp show SlDELLA expression. The YPet-SlDELLA construct is functional since it rescues the procera phenotypes partially, and the YPet-SlDELLA fusion protein is degraded in response to GA. With the application of PAC, an inhibitor of GA biosynthesis, YFP-SlDELLA signal in young fruit was also found to be localized to placenta. It is unclear why our reporter lines showed stronger SlDELLA signal in the placenta than in the ovules or pericarp. Nevertheless, our study indicates that SlDELLA is expressed in unfertilized ovary and in young fruit. Specific Aim 3: Identification and Characterization of DELLA Interacting Transcription Factors in Fruit Development Previous studies suggested that DELLA functions through interaction with other transcription factors. We performed yeast-two-hybrid (Y2H) screen to identify such novel factors during fruit development. Several SlDELLA-interacting factors were identified from a tomato cDNA library. We focused on one of them, SlARF2 (Auxin Response Factor 2) because identification of SlARF2 as a DELLA interactor presented an exciting potential mechanism for auxin-GA interaction during fruit development. Because SlARF7 has been shown previously to regulate parthenocarpy, we tested whether SlARF7 also interacts with DELLA. Y2H assays showed that both full-length SlARF2 and SlARF7 interact with DELLA. Interestingly, SlARF2 binds to DELLA via its C-terminal dimerization domain (CTD), suggesting that DELLA may regulate ARF activity by interfering with ARF-ARF or ARF-AUX/IAA dimerization. This potential mechanism would be distinct from previous results suggesting that DELLA antagonizes several classes of transcription factors (bHLH, EIN3 and BZR1) by binding to their DNA-binding domains. Our Y2H assays also showed that SlIAA9 interacts with SlARF7, but not with DELLA. These results support that IAA9-ARF7 heterodimer represses fruit initiation in tomato. ARF7 contains the glutamine-rich transcription activation sequence. We propose that in the absence of IAA9 or DELLA, ARF7 activates fruit initiation genes by forming homo- or hetero-dimers (with other ARFs).
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2010
Citation:
Sun T-p. 2010. Gibberellin-GID1-DELLA: a pivotal regulatory module for plant growth and development. Plant Physiol 154: 567-70
- Type:
Journal Articles
Status:
Published
Year Published:
2011
Citation:
Sun T-p. 2011. The molecular mechanism and evolution of the GA-GID1-DELLA signaling module in plants. Curr Biol 21: R338-45
- Type:
Journal Articles
Status:
Published
Year Published:
2012
Citation:
Dayan J, Voronin N, Gong F, Sun T-p, Hedden P, Fromm H, Aloni R. 2012. Leaf-induced gibberellin signaling is essential for internode elongation, cambial activity and fiber differentiation along the stem. Plant Cell 24: 66-79
|
Progress 12/15/10 to 12/14/11
Outputs
OUTPUTS: The plant hormone gibberellin (GA) plays an important role in regulating seedless fruit (parthenocarpy) development. We plan to induce parthenocarpy by altering expression of known GA signaling components by overexpression and RNAi approaches in transgenic tomatos. We have generated multiple transgenic tomato lines with altered expression of GA signaling components. We are monitoring the effects of these transgenes on tomato fruit development. We also generated a tomato cDNA library using mRNAs from developing fruits, and are screening components that are interacting with a GA signaling repressor by yeast two-hybrid screens. This project has helped to train three postdocs and two undergraduate students. PARTICIPANTS: Jianhong Hu, a postdoc, has been working on generating and characterizing the transgenic tomato plants. Rodolfo Zentella, a postdoc, has constructed the plasmids for tomato transformation. Jonathan Dayan, a postdoc, has been characterizing GA-related phenotypes of tomato mutants. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
The plant hormone gibberellin (GA) plays a pivotal role in regulating fruit development in flowering plants. Elevated GA pathway activity promotes seedless fruit growth, which is a desirable trait for many fruit and vegetable crops. Tomato is an ideal system to study the role of GA in flashy fruit development. This project will use combinatorial approaches (including molecular biology, biochemistry and proteomics) to identify the key regulatory components in GA-mediate fruit development in tomato. The knowledge gained from this study will contribute to ways to promote seedless fruit production in crops.
Publications
- Dayan J, Voronin N, Gong F, Sun T-p, Hedden P, Fromm, H, Aloni, R (2012). Leaf-induced gibberellin signaling is essential for internode elongation, cambial activity and fiber differentiation along the stem. Plant Cell accepted
- Sun T-p. 2011. The molecular mechanism and evolution of the GA-GID1-DELLA signaling module in plants. Curr Biol 21: R338-45
|
Progress 12/15/09 to 12/14/10
Outputs
OUTPUTS: The plant hormone gibberellin (GA) plays an important role in regulating seedless fruit (parthenocarpy) development. We plan to induce parthenocarpy by altering expression of known GA signaling components by overexpression and RNAi approaches in transgenic tomatos. We are in the process of generating multiple transgenic tomato lines with altered expression of GA signaling components. We will monitor the effects of these transgenes on tomato fruit development. This project has helped to train two postdocs and two undergraduate students. PARTICIPANTS: Jianhong Hu, a postdoc, has been working on generating and characterizing the transgenic tomato plants. Rodolfo Zentella, a postdoc, has constructed the plasmids for tomato transformation. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
The plant hormone gibberellin (GA) plays a pivotal role in regulating fruit development in flowering plants. Elevated GA pathway activity promotes seedless fruit growth, which is a desirable trait for many fruit and vegetable crops. Tomato is an ideal system to study the role of GA in flashy fruit development. This project will use combinatorial approaches (including molecular biology, biochemistry and proteomics) to identify the key regulatory components in GA-mediate fruit development in tomato. The knowledge gained from this study will contribute to ways to promote seedless fruit production in crops.
Publications
- Sun T-p. 2010. Gibberellin-GID1-DELLA: a pivotal regulatory module for plant growth and development. Plant Physiol 154: 567-70
- Sun T-p. 2011. The molecular mechanism and evolution of the GA-GID1-DELLA signaling module in plants. Curr Biol 21: R338-45
- Dayan J, Voronin N, Gong F, Sun T-p, Hedden P, Fromm, H, Aloni, R (2012). Leaf-induced gibberellin signaling is essential for internode elongation, cambial activity and fiber differentiation along the stem. Plant Cell accepted
|
|