Lecture 2: Stomatal action and metabolism Teaching aims: to introduce the structure, function and metabolic regulation of stomatal guard cells Learning outcomes: to understand the complex interplay between turgor of guard cells and epidermal cells, driven by the active accumulation of ions and solutes, which occurs across both plasmalemma and tonoplast

Lecture 1 Erratum: Cavitation in Beech (page 7 HG Lecture 1): second bullet point should have said “MINIMUM” conductivity occurs in spring.. of course.. you’d noticed that already!! 2.1 Regulation of transpiration 2.2 Stomata: structure and function 2.3 Sensing the environment 2.3 Ion fluxes and exchange 2.4 Ion channels and patch clamping 2.5 Signalling and control at plasmalemma and tonoplast Key references: Assmann SM and Wang X-Q (2001) Guard cells and environmental responses Current opinion in Plant Biology 4, 421-428 Shroeder JI et al 2001 Guard cell abscisic acid signalling and engineering drought hardiness in plants Nature 410, 327-330 Backround texts: Taiz and Zeiger

2.1 Regulation of transpiration Water loss is driven by the leaf to air vapour pressure difference Absolute water vapour concentration highly temperature dependent so we need to know leaf temperature precisely Evaporation will lead to leaf cooling

2.1 Regulation of transpiration Boundary layer and stomatal resistances control water loss from leaf Figure shows that in moving air, transpiration increases linearly with stomatal aperture; in still air, stomata only exert control when closing- but there are many adaptations to reduce Rair Resistance analogue: cuticle and stomatal resistances are in parallel, boundary layer in series; in diagram shown, cuticle and stomatal resistance is (1 x 70)/ (70 + 1) = 0.99 s cm-1; total R = 0.35 + 0.99 = 1.34 s cm-1 (units equivalent to time for one molecule of water to diffuse 1 cm)- but closed stomata approximate to an infinite resistance Conductances: calculated simply as (cm s-1) equivalent to distance one molecule diffuses in one second, are finite and easier to quantitate in practise

Ecologically, we can make some generalisations about maximal leaf conductance Largely, this will tie in with the need to restrict cavitation and capacity for the plant to recharge water status overnight Porometer: express conductance as a molar flux per m2 of leaf surface

2.2 Stomata: structure and function Antagonism between guard cell and epidermal turgor Ultrastructural modifications

2.3 Sensing the environment Feedback from internal CO2 and leaf water content (sensed partly by carbohydrate supply; hydropassive feedback due to direct effects on water supply); Abscisic acid a key signal from roots and mesophyll. Feedforward : guard cells have chloroplasts (sense light) and water is evaporated directly around the guard cell complex to alter GC turgor

Evidence that guard cells respond to vapour pressure independent of leaf water status Shoot water potential is constant, but stomatal conductance declines in drying air

Stomatal patchiness (Mott and Buckley 2000 TIPS 5, 258-262) Stomatal aperture is not randomly distributed across a leaf Chlorophyll fluorescence can be used to track spatial patterns of photosynthesis Overall leaf conductance shows a decline under high VPD….. ..but stomatal apertures are patchy!! How is this linking brought about?

Hydraulic coupling between adjacent guard cells LHS stomate increases aperture, decreasing adjoining epidermal cell turgor Relaxation of RHS stomate allows transpiration to occur and increases loss of epidermal turgor Effect is propagated through stomata until a vein is reached Other feedback / feedforward loops will eventually constrain opening

2.3 Ion fluxes and exchange Using a pressure probe, guard cell turgor can be measured directly Aperture and turgor are (virtually) linearly related What ionic fluxes lead to the generation of turgor? How are these processes energised?

2.3 Ion fluxes and exchange Don’t mention the starch -sugar hypothesis, I used to counsel Just remember the primary active H+ transport coupled to secondary ion transport processes of K+ and Cl- Add in a twist of malate2-, synthesised via PEP carboxylase So starch degradation in the light is not used osmotically to increase turgor…….(I said)…. …. and see the fantastic profiles of ions which exchange across guard cell, companion cell and epidermis:

So there is a role for sucrose after all So there is a role for sucrose after all!! (see Taiz and Zeiger; Zeiger and Zhu, J exp Bot, 1998, 433- 442) In Vicia faba (broad bean), potassium accumulation drives early morning opening, to be replaced by sucrose accumulation later in the day Sucrose comes from starch hydrolysis, CO2 fixation in the GC chloroplast and apoplastic import from the mesophyll

2.4 Ion channels and patch clamping Patch clamping allows the current carried by individual K+ channels to be distinguished in cell attached configuration If cell is depolarised to –120 mv, see three channels open successively Now if you had voltage clamped to –60 mv, and 11 mM K+ outside and 105 mM K+ inside, what flux would you expect??

Whole-cell configuration Remember the Nernst equation- K+ is in passive equilibrium, so there is NO net flux (and NO current flowing) Patch clamping can be used to resolve two types of channel- IK+out and IK+in – suggesting that the cell can independently control rates of inward and outward exchange of K+….. …and ion flux matches observed accumulation when E = -120mv

Ion channels Of course, there are two membranes to consider And driving forces will differ, with the elegant work of Enid MacRobbie first to show how the two are co-ordinated using tracer efflux experiments

the plasmamembrane is hyperpolarised by the H+ pump, driving the influx of other transporters There are up to 4 inward K+ channels Sucrose co-transport and Cl- channels osmotic accumulation Outward K+ channels and anion channels allow passive ion efflux, provided that some process has initially depolarised cells by activating anion efflux Responses to the environment (water deficit, cold, oxidative stress) mediated by calcium ABA is detected by an (as yet) unidentified receptor which induces an increase in intracellular Ca2+, which is either imported or released from intracellular stores; Slow and /or fast responding anion channels open, depolarising cell and activating IK+out channels; 90% of ions must first leave the vacuole, and Ca2+ stimulates VK channels and release of K+, although FV channels can mediate K+ release in response to cytosolic pH changes

Ion channel functions (from Schroeder et al 2001) ABA also inhibits ion uptake, and elevated Ca2+ inhibits the ATPase and K uptake channels; Calcium is the key to various signalling pathways which control ion fluxes and trugor generation and loss

Conclusions: Water loss is effectively controlled entirely by stomata, with boundary layers important at low windspeeds. While we can best define the entire pathway via a resistance analogue, in practise we translate water losses into finite conductances, which can be used to characterise vegetation types Guard cell ultrastructure and epidermal cell antagonism are the key to controlling the aperture between two guard cells Guard cells can “sense” vapour pressure and light intensity directly (feedforward responses), and response to internal CO2 concentration and leaf and soil water status Guard cell turgor is generated by accumulating K, Cl , malate and sucrose, energised by chloroplast and/or mitochondria and a blue light photoreceptor Stomata do not respond homogeneously (though we generally ignore patchiness when measuring leaf-level as exchange

Patch clamping allows the operation of individual channels to be distinguished The membrane potential can be seen to control ion fluxes, demonstrating the Nernst potential (no net flux) and that a hyperpolarised E or 120 mv can account for the observed rates of K accumulation Ion Accumulation and export are controlled by a range of anion and cation channels in tonoplast and plasmamembrane ABA is a key inhibitor of stomatal opening, and elicits a range of signalling responses controlled by intracellular Calcium