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, 327-330Backround 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!! (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 matchesobserved accumulationwhen 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

• 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

Ion channel functions (from Schroeder et al 2001)

• 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