Progress 02/01/11 to 01/31/12
Outputs
OUTPUTS: All plant growth and development is dependent upon the proper control of carbohydrate partitioning; however, we do not know how this process is regulated at the genetic and molecular levels. Using a multidisciplinary approach spanning molecular genetics, physiology, and cell biology we are addressing this gap in our knowledge. We identified genes, termed the Tie-dyed (Tdy) loci, that when mutated result in the hyperaccumulation of carbohydrates within discrete regions of maize leaves. From previous characterizations, we proposed that these genes function as sugar or osmotic stress sensors to promote the export of sucrose from leaves. In addition to understanding the molecular functions of the Tdy genes, this project defined the molecular functions of the Sucrose transporter1 (SUT1) gene of maize. Aim 1 further characterized Tdy1 expression and function in carbon partitioning. We performed RNA in situ hybridizations and determined that Tdy1 was specifically expressed in developing phloem cells. We also transiently expressed a TDY1 fusion protein to the green fluorescent protein (GFP) and localized the protein to the endoplasmic reticulum. To confirm the expression in vivo, we constructed stable maize transgenic plants expressing the Tdy1::GFP translational fusion under the native promoter. This construct was transformed into maize, stable transformants expressing the gene recovered, and the expression pattern recapitulated our data from transient expression assays. Aim 2 proposed to characterize the contributions of SUT1 in maize to phloem sucrose loading. Through expression, morphometric, physiological, and biochemical investigations, we demonstrated that SUT1 has a critical function in importing sucrose into the phloem in maize leaves. Our results were the first demonstration of the importance of SUTs in phloem loading in grasses. Aim 3 proposed to characterize and clone Tdy2 and to map Tdy3. We map-based cloned the Tdy2 gene to a narrow interval containing approximately 35 genes. Through analyzing multiple alleles, we determined that Tdy2 encodes a callose synthase. Radiotracer and fluorescent dye loading assays determined that Tdy2 functions in phloem loading, but not in long-distance phloem transport. Ultrastructural studies pinpointed the blockage in tdy2 mutants at the plasmodesmata between the companion cells and sieve elements. We successfully mapped and performed phenotypic characterizations of Tdy3 (renamed psychedelic). PARTICIPANTS: Tom Slewinski was a graduate student who worked on the project. He received broad training in molecular biology, genetics, physiology, cell biology, and biochemistry. He completed his PhD and is currently a postdoctoral researcher at Cornell University. Adam Stubert was an undergraduate student who worked on the project. He received training in genetics and cell biology. He earned his BS degree and is currently enrolled in medical school. R. Frank Baker was a postdoctoral researcher supported by the grant. He received training in transmission electron microscopy, RNA in situ hybridizations, genetics, and biochemistry. Kristen Leach was a postdoctoral researcher supported by the grant. She received training in molecular biology and genetics. Michael Swyers is an undergraduate student supported by the grant. He received training in genetics and cell biology. TARGET AUDIENCES: Results of supported research were presented in the form of talks and posters to maize geneticists, plant biologists, and faculty at several universities and scientific conferences. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Our studies on the Tdy and Sut1 genes provide fundamental information on genes controlling carbohydrate partitioning in maize. Understanding how these genes regulate carbohydrate partitioning will have profound impacts on future efforts to modify the delivery of fixed carbon to roots, flowers and developing seeds.
Publications
- Slewinski, T.L., Baker, R.F., Stubert, A. and Braun, D.M., 2012, Tie-dyed2 encodes a callose synthase that functions in carbohydrate partitioning and vascular development in maize leaves. To be submitted to Plant Physiology.
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