15 NSFBIO SAUR regulation of stomatal aperture

Guard cells control photosynthesis and transpiration by regulating opening and closing of stomatal pores. We know much about how guard cells shrink in response to the drought stress hormone ABA, leading to stomatal closure. In contrast, we have sparse knowledge of the biochemical events by which guard cells swell to cause stomatal opening in response to light and diurnal rhythms. We have found that Arabidopsis SAUR (Small Auxin Up RNA) proteins can promote stomatal opening. This proposal will be to unravel under what conditions and by what mechanisms they do so.

SAUR genes are a large class in all land plants. Several Arabidopsis SAUR proteins localize to the plasma membrane and promote cell expansion. These proteins are normally short-lived, but can be stabilized in the plasma membrane. We know that closely related SAUR proteins promote growth by inhibiting membrane-associated protein phosphatases that otherwise dephosphorylate plasma membrane H+-ATPases at a key regulatory site and increase H+-ATPase proton pumping. The same SAURs also promote stomatal opening when expressed in guard cells and a subset of these are preferentially expressed in guard cells under conditions in which stomata are open, but are repressed by drought or ABA. By contrast, ABA activates other SAUR genes in guard cells which may have opposing effects on stomatal function. Thus, what we do not know is whether these opposing actions are unique, whether they overlap, or whether SAUR-mediated signalling affects other transport and homeostatic processes in guard cells.

Multiple transporters in both the plasma membrane and the tonoplast influence guard cell shrinking and swelling, and their activities often depend either directly or indirectly on the activation status of the others. Thus, to understand stomatal dynamics, it is essential to gain a global, systems view of guard cell transport and homeostasis. A major component of this project will exploit computational models that simulate these couplings using parameters based on experimental measurements. Starting points for current modelling will draw on past predictions that changes in H+-ATPase activity will have significant effects on stomatal aperture. For example, increased H+-ATPase activity in the model leads to slower stomatal closing at dusk and also to changed cytoplasmic concentrations of several other ions. Such knowledge will aid in experimental design and will also form a basis for subsequent validation.

The collaborating researchers have complementary expertise in guard cell physiology (Blatt), computational modeling (Blatt), biochemistry (Gray), genetics (Reed, Nagpal), and auxin response (Gray, Nagpal, Reed). The collaboration will pool resources and expertise developed in the three labs to make possible the multipronged approach.

This project is to address the roles of SAUR proteins in auxin-associated signalling of guard cells. The project is divided so that the Reed/Nagpal and Gray labs will characterize expression patterns and subcellular localization of candidate Arabidopsis SAUR and associated proteins, and determine how light and ABA may regulate their abundance or localization. They will also test whether these proteins interact, and assess the regulatory consequences of such interaction. Finally, they will generate mutant and transgenic plants with increased or decreased activity of these proteins.

The Blatt lab will vary selected parameters of guard cell systems models to predict which ion concentrations may be altered in the guard cells of the mutant and transgenic plants. The Blatt lab will also perform electrophysiological experiments to measure K+, Cl-, and H+ and other ion transport activities in guard cells to assess and refine the above models and to guide experimental analysis of key mutant lines. These analyses should allow fitting of the model to match the observed results, and make predictions about effects of additional genetic or physiological perturbations. Modelling will thus allow the workers to focus on meaningful experiments, and point to unanticipated emergent properties of the system. Cases where the experiments do not validate the predictions will also be interesting as they will suggest alternate mechanisms by which SAUR proteins may act, or needed adjustment of the parameters or couplings in the model.

Combined modeling, biochemical, genetic and electrophysiological approaches should yield important insight into stomatal opening mechanisms, and they may suggest synthetic biological approaches to manipulating stomatal kinetics for improved crop efficiencies.

This proposal is for fundamental research developing new concepts at the core of ideas emerging within the international cell biology community. The research should stimulate thinking about these topics and help facilitate a paradigm shift in approach. These studies will also extend recent developments by MRB and colleagues Reed/Nagpal and Gray in expanding our understanding of stomatal regulation by SAUR proteins and in its modelling. Thus, the research is expected to benefit fundamental researchers as well as industry through conceptual developments as well as the introduction of new technologies for the analysis of complex systems in vitro and in vivo. The research will feed into higher education programmes through research training at the postgraduate and postdoctoral levels. Finally it will help guide future efforts in applications to agricultural/industrial systems. MRB has established links with industrial/technology transfer partners (e.g. Agrisera, Plant Bioscience) and research institutes (JHI, NIH and JIC) to take advantage of these developments. Further details of these, and additional impacts will be found in the attached documentation.