• Stomatal Biology

• Guard cells change their size and

shape in response to internal and
external stimuli
– Results in alteration of stomatal
aperture
– Pore size is regulated by uptake and
release of water to change turgor
pressure
• Aerial leaves typically have most
stomates interspersed on lower
(abaxial) surface, but often on
upper (adaxial) surface as well
• Floating leaves lack stomates on
abaxial side
• Figure 10.1 Light-stimulated
stomatal opening in detached
epidermis of Vicia faba
• Stomatal aperture follows
photosynthetically active
radiation at the leaf surface

• Light mediates stomatal aperture

• Photosynthetic component
– Can be opened partially by red light
– inhibited by DCMU’s competition
in the e- transport chain
– CO2-depletion effect can be
eliminated by using epidermal peels
• Specific blue light component
– Co-irradiation after saturating
photosynthetic response allows
separation of the two effects
 – Reversible
– Guard cell-specific irradiation
• Figure 10.3 Response of stomata
to blue light under a red-light
background
• Figure 10.4 Action spectrum for
blue light–stimulated stomatal
opening (under a red-light
background)

• Isolated guard cell protoplasts

swell in blue light, which allows
study of mechanism
– Water uptake driven by ion uptake
and the resulting lower Ψs
– Sensitive to poisoning of PM H+-
ATPase by orthovanadate
• Figure 10.5 Blue light–stimulated
swelling of guard cell protoplasts
• Figure 10.6 Acidification of a
suspension medium of guard cell
protoplasts of V. faba stimulated
by a 30-s pulse of blue light
• Figure 10.7 Activation of the H+-
ATPase at the plasma membrane
of guard cell protoplasts by
fusicoccin and blue light can be
measured as electric current in
patch clamp experiments
• At least three separable
osmoregulatory pathways in
guard cells
 – Proton pumping drives uptake of K+
and Cl- ions, and malate2- produced
from starch metabolites
– Sucrose accumulation from starch
metabolism
– Sucrose production from
photosynthetic carbon fixation
• Figure 10.8 Three distinct
osmoregulatory pathways in
guard cells (Part 1)
• Figure 10.8 Three distinct
osmoregulatory pathways in
guard cells (Part 2)
• Figure 10.8 Three distinct
osmoregulatory pathways in
guard cells (Part 3)
• Sucrose and K+ (and counterions
Cl-/malate2-) act together to
provide the more negative Ψs for
water uptake
– Circadian change in major
component:
– K+ is primarily responsible for
opening, while decrease in sucrose
is responsible for closing
• Figure 10.9 Daily course of
changes in stomatal aperture, and
in potassium and sucrose content,
of guard cells from intact leaves
of broad bean (V. faba)
• Multiple Blue-light receptors
• One of the receptors regulating Blue-
specific opening is the carotenoid
zeaxanthin
 – npq1 mutants lack low intensity
component of closing in some plants
– Reducing agent dithiothreitol (DTT)
enhances accumulation of zeaxanthin and
sensitivity to blue light
– phot1/2 mutants also have reduced
opening
• Action spectra consistent with both
carotenoid and flavin receptors
• Zeaxanthin accumulation tracks
sensitivity to blue light

• Figure 10.10 Blue-light

sensitivity of the zeaxanthinless
mutant npq1 and of the
phototropin-less double mutant
phot1/phot2
• Figure 10.11 Zeaxanthin content
of guard cells correlates with
 photosynthetically active
radiation and stomatal apertures
• Figure 10.12 Absorption
spectrum of zeaxanthin in ethanol
• Figure 10.13 Role of zeaxanthin
in blue-light sensing in guard
cells

• Blue light pulse-induced opening

is reversed/suppressed by a
subsequent green light pulse
• phot1/phot2 have a significant
effect on regulating the decreased
aperture response to green
• Figure 10.14 Blue/green
reversibility of stomatal
movements
• Figure 10.15 Green light
regulates stomatal apertures in the
intact leaf
• Figure 10.16 Action spectrum for
blue light–stimulated stomatal
opening and for its reversal by
green light

• Photobiology/Photophysiology
are powerful tools for
understanding light receptors,
signaling pathways, and responses
 – Action spectra, reversibility,
lag/escape times, fluence
responsiveness
– Genetic experiments (e.g. mutants,
transgenics)
– Pharmacological agents (DCMU,
DTT, CCCP, orthovanadate, CN-)

• However…
– Confounding factors (e.g. screening
pigments, alternative explanations)
– Pleiotropic effects of gene
mutations (zeaxanthins also act in
photoprotection and as antennae
pigments, and hormone ABA
derived from violaxanthin)
– Reagents often have multiple effects
(e.g. reducing agents like DTT;
Altering H+-ATPase will affect
many transport pathways, pH
responses, etc.)