Author
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LU, MEI-YEH - PLANT BIOLOGY UOFI URBANA |
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BUSH, DANIEL |
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Submitted to: Proceedings of the National Academy of Sciences (PNAS)
Publication Type: Peer Reviewed Journal Publication Acceptance Date: 7/1/1998 Publication Date: N/A Citation: N/A Interpretive Summary: Plants leaves capture light energy from the sun and transform that energy into a useful form in the process called photosynthesis. The primary product of photosynthesis is sucrose. Generally, 50 to 80 percent of the sucrose synthesized during photosynthesis is transported from the leaf to supply many of the edible parts of the plant such as fruits, grains, and tubers. Alterations in the transport process are known to significantly affect crop productivity. We have identified an essential amino acid residue in the sucrose transporter protein that is responsible for the long distance transport of sugar from the leaves to the harvested tissues of the plant. In addition, we have used site-directed mutagenesis to replace that residue with other amino acids and we showed that some of these improve transport activity by over 400 percent. In addition to providing fundamental information about the structure and function of this essential transporter, these results represent a vital first step for using biotechnology to modify crop productivity by improving sucrose transport. Technical Abstract: The proton-sucrose symporter that mediates phloem loading is a key component of assimilating partitioning in most higher plants. Previous biochemical investigations showed that a DEPC-sensitive histidine residue is at or near the substrate-binding site of the symporter. Among the proton-sucrose symporters clones to date, only the histidine residue at position 65 of AtSUC1 is conserved across species. To test whether His65 is involved in the transport reaction, we have used site-specific mutagenesis and functional expression in yeast to determine the significance of this residue in the reaction mechanism. Among ten different substitutions of His65, L65H and R65H transport sucrose at higher rates than the wild-type symporter (increased Vmax). Other substitutions exhibited a range of activities. For example, the C65H substitution resulted in the complete loss of transport capacity while Q65H was almost as active as wild-type. RNA gel blot and protein blot analyses showed that, with the exception of Q65H, the variation in transport activity is due to the change in the protein structure as opposed to alterations in steady-state levels of mRNA or symporter protein. Significantly, functional symporters with substitutions of His65 were no longer sensitive to DEPC inactivation, suggesting this is the inhibitor-sensitive histidine residue. Taken together with our previous results, these data show that His65 is involved in sucrose binding and increased rates of transport activity implicate this region of the protein in the transport reaction. |
