Lecture 2: Stomatal action and metabolism
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Transcript of Lecture 2: Stomatal action and metabolism
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