Analysis of membrane traffic in adaptive stress tolerance in plants

Stress-mediated adaptation is a key factor in the ability of plants to cope under long-term or repeated environmental challenge, and is especially important for stomata to compensate for gas exchange during extended periods of water deficit. Stomata in the epidermis of plant leaves play a vital role in regulating CO2 exchange for photosynthesis while minimising transpirational water loss between the inner leaf air space and the atmosphere. Guard cells surrounding the stomata take up inorganic ions and water, increasing in volume to open the stomatal pore when CO2 in the leaf is depleted; and they lose ions, other solutes and water, decreasing in volume to close the stomatal pore and conserve leaf water under stress, in the dark and when CO2 is high. Arguably, stomata are at the centre of a crisis in water availability that is expected to unfold over the next 20-30 years. Globally, water usage has increased 6-fold in the past 100 years, twice as fast as the human population, and is expected to double again before 2040, driven mainly by irrigation and agriculture. Clearly, a full understanding of guard cells and stomatal regulation will have significant implications for future efforts to improve the water use efficiency of agricultural crops and, consequently, for society to face these future challenges. In fact, we know a great deal about the cellular and molecular physiology of guard cells, especially in relation to abscisic acid (ABA), salinity and drought that triggrer stomata to close, and the depth and breadth of this information has helped to elevate the guard cell system to that of one of the best understood plant cell models. However, virtually all of this knowledge centres on the short-term responsive behaviour of guard cells. By contrast, our knowledge is remarkably poor of the mechanisms by which guard cells adjust stomatal movements to adapt to longer-term environmental stress. Recently, this laboratory observed that ABA triggers the traffic to an internal compartment of one of the plasma membrane K+ channels mediating ion fluxes in guard cells. This response shows all the hallmarks essential for a function in adaptive modulation of the channel population. It complements a second discovery that the Arabidopsis mutant Atsyp121, which is defective in a major vesicle trafficking protein at the plasma membrane, selectively impairs the ability of stomata to 'remember' the recent history of stress and remain closed (so-called 'progammed' stomatal closure). These discoveries firmly tie membrane vesicle trafficking to [Ca2+]i signalling and adaption in guard cells, and suggest that K+ channel traffic between the plasma membrane and the internal compartment is an important mechanism for adaptation in these cells. I propose to address this idea directly using cellular and molecular tools now available in the laboratory. The outcome will greatly extend our understanding of ABA signalling and adaptation in plants, and should add fundamentally to our knowledge of the dynamics of membrane traffic and ion channel control in eukaryotic cells generally.

Guard cells are able to adapt stomatal response to the past history of stress, as evinced by a number of well-defined behaviours such as so-called 'programmed' stomatal closure which reflects an ability to 'remember' the recent history of stress. Yet our knowledge is remarkably poor of the mechanisms that underlie this, and similar processes in plants and other eukaryotes. We recently discovered (i) that the water-stress hormone ABA triggers the traffic to an endosomal compartment of one of the plasma membrane K+ channels mediating ion fluxes in guard cells, and (ii) that the Arabidopsis mutant Atsyp121, which is defective in a major trafficking protein at the plasma membrane, impairs progammed stomatal closure. These discoveries firmly tie membrane traffic to [Ca2+]i signalling and adaption in guard cells. They lead to the postulate that K+ channel traffic is important to adapt the capacity for solute flux across the guard cell membrane to the recent history of stress. I propose to follow this line of enquiry to address three key questions underlying this postulate: (i) Is ABA-evoked channel traffic integrated with known ABA signalling pathways? (ii) Do the K+ channels recycle to the plasma membrane and is internalisation and/or recycling affected by the Atsyp121 mutant? Finally, (iii) Does the programmed closure stimulus and/or ABA evoke a traffic of other ion channel proteins associated with ABA signalling? The work will make use of a combination of confocal imaging and electrophysiological techniques, taking advantage of a toolchest of marker constructs and mutants now on hand in the laboratory, and will be supported by biochemical and cell fractionation studies. Various strategies will be used, including a combination of 'pulse-chase', localisation and kinetic analyses as well as in vivo epitope labelling methods.