EFB530 Plant Physiology

Stomatal physiology

Regulation of stomatal aperture is the major mechanism the plant has under metabolic control to change the resistance to water loss.  By closing stomata, the main path for the efflux of water vapor is largely shut off, but at the same time, so is the main path for the influx of CO₂ for photosynthesis.  Stomata are also excellent cells for the study of membrane transport, because stomatal opening and closing involves dramatic ion flux.

opening is triggered by:

• red light - perceived by chlorophyll
• blue light - perceived by a xanthophyll acting as a blue light photoreceptor
• low internal CO₂ concentration (a measure of high photosynthetic activity in the leaf)
• circadian rhythm

closure is stimulated by:

• dark
• ABA (abscisic acid)=stress hormone
• high internal CO₂ concentration (a measure of low photosynthetic activity)
• circadian rhythm

Mechanism of stomatal opening

plasma membrane H⁺-ATPase is activated, it pumps H⁺ out of the cell

• generates a stronger membrane potential (gets more negative, originally -100 mV, it goes to -150 or -180 mV)
• we say that the membrane is hyperpolarized

triggers inward-specific K⁺ channels to open

• these channels are gated, they can open and close in response to membrane potential
• K⁺ diffuses in down its electrochemical gradient
• K⁺ concentration can increase from ~100 mM to 400 - 800 mM

Cl^- also diffuses in to balance the positive charge of the K⁺

• through the day, guard cells also make sucrose as osmoticum and malate^2- to balance the K⁺ and lower the pH

the accumulation of ions makes the water potential of the guard cells (Y_w) more negative

• water enters the cells, moving down the water potential gradient, increasing turgor pressure and causing the guard cells to swell

stomatal opening occurs due to changes in cell volume in the guard cells

• the radial arrangement of cellulose microfibrils in the guard cell walls forces the pore to open when guard cells swell

opening can also be triggered by a fungal toxin, fusicoccin, which hyperactivates the H⁺-ATPase

Stomatal closure

closure is initiated by

• shutting down the H⁺-ATPase
• opening anion (like Cl^-) channels allowing anions to flow out
• this dissipates much of the membrane potential (the charge difference becomes less negative, going back to the original membrane potential of ~-100 mV)

inward-specific K⁺ channels close, outward-specific K⁺ channels open (these were gated shut before)

• K⁺ diffuses out of the cell, again down its electrochemical potential
• the water potential in the guard cells becomes less negative
• water flows out of the cells, reducing turgor pressure, & they shrink

reduced volume of the guard cells causes the stomatal pore to collapse shut

closure is also induced (artificially) by vanadate (a H⁺-ATPase inhibitor) and CCCP (dissipates a H⁺ gradient)

Guard cells have been used to study ion transport

we know a great deal about ion channels and pumps by using a technique called patch clamping

First: generate protoplasts

• harvest epidermal peels
• degrade the cell walls using enzymes that breakdown cellulose
• guard cell walls are thicker than the other epidermal cells, they last longer in the enzyme solution
• cell wall breakdown releases protoplasts into the medium, which must have the proper osmoticum so that the protoplasts don't burst

Second: capture a protoplast onto a micropipette, apply a slight suction to hold it there

• this pipette has a salt solution in the pipette & an electrode
• the protoplast is sitting in a bath solution w/ the other electrode

Third: can use two modes

• pull off a "patch" of membrane = patch mode
• suck in a portion of the membrane, so that the pipette is accessing the cytoplasm = whole cell mode

Fourth: can "clamp" the voltage difference across the membrane using the electrodes

• can measure current each time an ion crosses the membrane

Leaf movements caused by pulvini also involves K⁺ flux and cell volume changes

the pulvinus is a region with special cells, called motor cells, at the base of leaf petioles

• the pulvini cause the leaf to droop, hinging at the base of the petiole

volumes of cells on the opposing sides of the leaf or leaflet change

• one side swells, other side shrinks
• this causes the leaf to move

the pulvini use a mechanism similar to guard cells

• the H⁺-ATPase generates a membrane potential
• K⁺ diffuses in down an electrochemical gradient
• water flows in down a water potential gradient

the cells on the other side carry out the reverse process to shrink

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