Sucrose-mediated transcriptional regulation of sucrose symporter activity in the phloem.

This project was based on our discovery that sucrose acts as a signaling molecule that regulates the activity of a proton-sucrose symporter in sugar beet leaf tissue. A major objective here was determining how sucrose transporter activity is being regulated. When sucrose accumulates in the phloem sucrose transport activity drops dramatically. Western blots of plasma membrane proteins isolated from sucrose treated leaves showed that the loss of sucrose transport activity was proportional to a decline in symporter abundance, demonstrating that sucrose transport is regulated by changes in the amount of BvSUT1 protein. BvSUT1 transcript levels decreased in parallel with the loss of sucrose transport activity. Nuclear run-on experiments demonstrated that BvSUT1 gene transcription was repressed significantly in nuclei from leaves fed 100 mM exogenous sucrose, showing that sucrose-dependent modulation of BvSUT1 mRNA levels is mediated by changes in transcription. To identify which secondary messenger systems might be involved in regulating symporter activity, we used a variety of pharmacological agents to probe for a role of calcium or protein phosphorylation in sucrose signaling. In a detailed analysis, only okadaic acid altered sucrose transport activity. These results suggest a protein phosphatase is involved. We hypothesized that protein kinase inhibitors would have a neutral affect or increase symporter transcription. Transpirational feeding of the protein kinase inhibitor staurosporine had no impact on sucrose transport while calphostin C, an inhibitor of protein kinase C, caused a 60% increase. These data provided good evidence that protein phosphorylation plays a central role in regulating sucrose symporter expression and sucrose transport activity. To determine whether protein phosphorylation is involved in sucrose regulation of proton-sucrose symporter activity, we pre-fed leaves with staurosporine for 4 h and then fed the treated leaves water or 100 mM sucrose for an additional 20 h. Sucrose transport activity was higher than the water control in both staurosporine/water- and staurosporine/sucrose-fed leaves. In contrast, sucrose transport activity was only 40% of the water control in sucrose-fed leaves. Taken together, these results showed that a phosphorylation-dependent signal transduction pathway is involved in sucrose-mediated regulation of BvSUT1 gene expression, sucrose transport activity, and ultimately phloem loading. Publications originating from this work: Vaughn MW, GN. Harrington, and DR Bush 2002. Sucrose-mediated transcriptional regulation of sucrose symporter activity in the phloem. Proc. Natl. Acad. Sci. USA 99:10876-10880 Ransom-Hodgkins W, MW Vaughn, and DR Bush 2003. Protein phosphorylation mediates a key step in sucrose-regulation of the expression and transport activity of a beet proton-sucrose symporter. Planta 217:483-489 Harrington GN and Bush DR 2003. The bifunctional role of hexokinase in metabolism and glucose signaling. Plant Cell 15: 2493-249