Guard Cells

Why are plant membranes and ion transport important?

Plant biomass is inextricably tied to mineral nutrients and water: increases in crop production depend fundamentally on increased nutrient and water use. On a global scale, the physiological consequences of stress, when water or mineral nutrients are limited, or when soils become saline, remain among the most important factors that influence vegetative plant growth and yield.

Indeed, following the Rome summit in 2004, the FAO identified crop mineral nutrition as vital to world food security (see this report in PDF).

Why study guard cells?

The primary defence of the plant against water loss and dehydration is to reduce evaporation from the leaves. Plants control water loss primarily by closing stomatal pores in the leaf surface. Closing these pores also reduces CO₂ entry to the leaf and so affects photosynthesis and vegetative yield. Stomatal regulation is linked to changes in plant water usage and increased water runoff that is associated with ongoing climate changes [see Gedney, et al. (2006) Nature 439,835]. Clearly, understanding how stomata function is of huge importance for agriculture, and the use of water on arable and irrigated lands.

The guard cells that surround stomatal pores are a focus of attention in fundamental research as well. These cells integrate both environmental and internal signals to control the size of the stomatal pore, and their unique situation within the leaf tissue has provided a wealth of experimental access points to signal cascades that regulate membrane ion transport. The past 20 years of research has raised the status of the guard cell to that of the undisputed cell "model", and some of the most exciting findings in eukaryotic cell signalling in recent years have come from work with guard cells, taking advantage of the "stomatal interface" between molecular genetics, biophysics, and cell biology.

(Image courtesy of Jim Haselhof, Cambridge.)

Guard cells open and close stomata by taking up and releasing solutes (mainly KCl and other K⁺ salts) to drive water movement across the plasma membrane. Both K⁺ and Cl^- channels participate and are coordinately regulated. Under conditions of water stress, for example, abscisic acid (ABA) activates the Cl^- channels and the subset of so-called outward-rectifying K⁺ channels to promote net KCl loss; ABA also suppresses the activity of a second subset of (inward-rectifying) K⁺ channels that normally mediate K⁺ uptake. Many aspects of how ABA, and other environmental stimuli, coordinate these changes in membrane transport have been well-characterised, but others remain a mystery.

At least two parallel signalling pathways operate in the guard cells, one mediated through cytosolic-free [Ca²⁺] ([Ca²⁺]_i) and the other through cytosolic pH (pH_i). Protein phosphorylation and reactive oxygen species (ROS) are also players in ion channel control of guard cells. Both the [Ca²⁺]_i and pH_i pathways contribute to K⁺ and Cl^- channel control during stomatal closure. A rise (and oscillations) in [Ca²⁺]_i has long been associated with control of the inward-rectifying K⁺channel. This Laboratory first showed that these [Ca²⁺]_i transients are coupled to oscillations in membrane voltage [Grabov and Blatt (1998) PNAS 95,4778]. The transients are triggered when Ca²⁺ enters across the plasma membrane through inward-rectifying Ca²⁺ channels which, in turn, are modulated by ABA [Hamilton, et al. (2000) PNAS 97,4967] and protein phosphorylation [Köhler and Blatt (2002) Plant J.32,185; Sokolovski, et al. Plant J (2005) 43,520]. All indications are that ABA targets a site closely associated with the Ca²⁺ channels at the plasma membrane.

Work from the Laboratory was first also to identify a role for pH_i in ABA-mediated control of K⁺ channels. Guard cell pH_i rises by as much as 0.3-0.5 units over periods of 5-10 min in ABA and promotes K⁺ efflux through the K⁺ channels [Blatt and Armstrong (1993) Planta 191,330]. Under some conditions, pH_i can rise by more than 0.5 units in 20-30 seconds [Thiel, et al.  (1993) PNAS 90,11493], a rise at least as dramatic as that of [Ca²⁺]_i! The pH_i signal is important for K⁺ channel regulation mediated by the ABI1 protein phosphatase [Armstrong, et al. (1995) PNAS 92,9520; Leube, et al. (1998) FEBS Lett 424,100]. How these changes in pH come about is still one of the big puzzles of guard cell biology.

One of the biggest questions still to be addressed is how these different transporters and signals work together to modulate solute flux and regulate stomatal aperture. To date the reductionist approach has proven less useful in understanding the mechanisms by which guard cells adjust stomata dynamically and how stomata integrate different signal inputs to achieve a range of pore size without a threshold of response. In short, the complexity of the guard cell system is beyond intuitive grasp; it requires a framework linking the kinetic behaviour of the individual components of the guard cell system to macroscopic stomatal dynamics. We are now developing a mathematical model of guard cell membrane transport and homeostasis with an open structure that permits its expansion through systematic comparison between predicted and observed behaviours.

We are preparing to release a quantitative systems dynamic model of the guard cell that will enable users to explore the interactions between components of the guard cell. The model is built around the HoTSig library that offers a flexible platform for development of homeostatic models of virtually any cell type. So far the model and OnGuard software has proven highly robust in accounting for complex behaviours observed in vivo and in interpreting the underlying physiology of guard cell homeostasis and stomatal dynamics.