Characterization of sugar transporter for nectar secretion and development of fluorescent-based membrane tension sensors [electronic resource]

The transport of sugars is critical for plant growth and development since sugars provide the source of energy. Sugars must be transported from within the cells in which they are synthesized to their destination. Cellular membranes are selectively permeable to sugars, because they contain specific membrane proteins that transport sugars in and out of the cell, either actively against the concentration gradient through the expenditure of energy or passively down the concentration gradient. Here, Arabidopsis nectaries were used as a model system to investigate how sugars, the major components of nectar, are secreted. The molecular mechanisms driving nectar secretion had not been determined. I identified and characterized a novel nectary-specific sugar transporter, SWEET9. I showed that Arabidopsis SWEET9 has all the hallmarks of a transporter responsible for nectar secretion by confirming its expression in nectaries, sucrose transport activity, plasma membrane localization, and its function as a sucrose bi-directional transporter. Importantly, sweet9 knockout mutants lost their ability to secrete nectar, while SWEET9 overexpression led to increased nectar secretion. Based on additional experiments, I propose a model, in which starch-derived hexoses are re-synthesized to produce high concentrations of sucrose in the cytosol of the nectary parenchyma. Sucrose is subsequently secreted into the extracellular space via SWEET9, where it is hydrolyzed by an apoplasmic invertase, potentially creating a large enough osmotic gradient to sustain water efflux into the extracellular space and generate nectar containing a mixture of sucrose, glucose and fructose. Plants carrying mutations in SWEET9 can now be used to study, for example, why Arabidopsis, a predominantly self-fertilizing plant, retains nectar production, or to generate mutants with varying sugar levels in nectar to study plant-pollinator interactions. Our findings led to new questions, regarding the actual sugar gradients, and whether the gradients are sufficient to explain osmotically driven nectar secretion, or whether alternative factors are required. Osmotic gradients play critical roles in many other processes, e.g. the root response to moisture changes in soil, cell expansion and regulation of stomatal aperture. To develop tools that allow us to monitor osmotic gradients, I took advantage of mechanosensitive channels (MS channels) to develop fluorescence-based "membrane tension sensors" that may be able to detect changes in membrane tension that are caused by either osmolality changes or mechanostimulation in intact cells with high spatial and temporal resolution. In response to hypoosmotic stress, MS channels change their conformation to trigger channel opening to adjust turgor. By fusing MS channels to a donor and an acceptor fluorophore, I created Förster Resonance Energy Transfer (FRET) sensors that report conformational rearrangements occurring during changes in membrane tension. The optical readout of these sensors is a change in ratio of acceptor over donor fluorophore intensities. When expressed in yeast, the Oztrac sensors report a shift from hypo- to hyperosmotic conditions, which decrease turgor pressure, and reduce membrane tension. When expressed in Arabidopsis, the sensor showed a ratio change in response to mechanically stimulation in individual root cells as well as to compression forces when roots encountered a barrier, or when roots were exposed to mechanical forces during root growth or mechanical stress treatments. Future goals include the implementation of FRET sugar sensors in nectaries to measure the intra- and extracellular sugar concentration of nectary cells, and the analysis of membrane tension during nectar secretion. The membrane tension sensors will require extensive characterization and optimization, but are expected to have wide applications for studying mechanical forces during plant growth and development. Membrane tension sensors can also be applied to create a temporally resolved membrane tension maps during cellular growth and development and to study cytoskeleton/cell wall-membrane interactions.

Keywords: Biology.
Advisor: Frommer Wolf B.
Barton Kathryn
Bergmann Dominique advisor. Thesis advisor