Lecture 2: Stomatal action and metabolism

1
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

2

• Lecture 1 Erratum Cavitation in Beech (page 7 HG
  Lecture 1) second bullet point should have said
  MINIMUM conductivity occurs in spring.. of
  course.. youd 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

3

• 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

4
(No Transcript)
5

• 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

6

• 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

7

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

8

• 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

9

• 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

10

• 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?
11

• 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

12

• 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?

13

• 2.3 Ion fluxes and exchange
• Dont 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
14
(No Transcript)
15

• 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

16

• 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??

17
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-
• IKout and IKin suggesting that the cell can
  independently control rates of inward and outward
  exchange of K..

and ion flux matches observed accumulation when
E -120mv
18

• 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

19

• 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 IKout 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

20
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

21

• 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

22

• 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