Physiological aspects of stomatal function

 

Changes in apoplastic pH and membrane potential in leaves in relation to stomatal responses to CO₂, malate, abscisic acid or interruption of water supply.

by Hedrich R., Neimanis S., Savchenko G., Felle H. H., Kaiser W. M., Heber U. (2001)

in Planta 213: 594–601 –

[PubMed]

Abstract

Low CO2 concentrations open CO2-sensitive stomata whereas elevated CO2 levels close them. This CO2 response is maintained in the dark. To elucidate mechanisms underlying the dark CO2 response we introduced pH- and potential-sensitive dyes into the apoplast of leaves. After mounting excised leaves in a gas-exchange chamber, changes in extracellular proton concentration and transmembrane potential differences as well as transpiration and respiration were simultaneously monitored.

Upon an increase in CO2 concentration transient changes in apoplastic pH (occasionally brief acidification, but always followed by alkalinization) and in membrane potential (brief hyperpolarization followed by depolarization) accompanied stomatal closure.

Alkalinization and depolarization were also observed when leaves were challenged with abscisic acid or when water flow was interrupted. During stomatal opening in response to CO2-free air the apoplastic pH increased while the membrane potential initially depolarized before it transiently hyperpolarized.

To examine whether changes in apoplastic malate concentrations represent a closing signal for stomata, malate was fed into the transpiration stream. Although malate caused apoplastic alkalinization and membrane depolarization reminiscent of the effects observed with CO2 and abscisic acid, this dicarboxylate closed the stomata only partially and less effectively than CO2.

Apoplastic alkalinization was also observed and stomata closed partially when KCl was fed to the leaves. Respiration increased on feeding of malate or KCl, or while abscisic acid closed the stomata.

From these results we conclude that CO2 signals modulate the activity of plasma-membrane ion channels and of plasmalemma H+-ATPases during changes in stomatal aperture. Responses to potassium malate and KCl are not restricted to guard cells and neighbouring cells.

 

CO2-triggered chloride release and kinetics of the onset of stomatal closure

Photo credit: NCBI

Effect of light off (L. off) on guard cell apoplastic Cl⁻ activity (pCl) after adaptation to different CO₂ levels (given in the bar on the top). The data basis is three experiments with different leaves.

CO₂-triggered chloride release from guard cells in intact fava bean leaves: kinetics of the onset of stomatal closure.

by Hanstein S. M., Felle H. H. (2002)

in Plant Physiol 130:940–950 – 10.1101/gad.1550707. –

CrossRef PubMed PubMedCentral – PubMed CentralView ArticlePubMed

Abstract

The influence of CO(2) on Cl(-) release from guard cells was investigated within the intact leaf by monitoring the Cl(-) activity in the apoplastic fluid of guard cells with a Cl(-)-sensitive microelectrode.

In illuminated leaves adapted to a CO(2) concentration within the cuvette of 350 microL L(-1), an increase of 250 microL L(-1) CO(2) triggered a transient rise in the apoplastic Cl(-) activity from 3 to 14 mM within 10 min. This Cl(-) response was similar to the Cl(-) efflux evoked by turning off the light, when the substomatal CO(2) was kept constant (CO(2) clamp).

Without CO(2) clamp, substomatal CO(2) increased by 120 microL L(-1) upon “light off.” The response to an increase in CO(2) within the cuvette from 250 to 500 microL L(-1) in dark-adapted leaves was equivalent to the response to an increase from 350 to 600 microL L(-1) in the light.

No Cl(-) efflux was triggered by 2-min CO(2) pulses (150-800 microL L(-1)). After a switch from 350 microL L(-1) to CO(2)-free cuvette air, the guard cells were less sensitive to a rise in CO(2) and to light off, but the sensitivity to both stimuli partially recovered.

Changes in CO(2) also caused changes of the guard cell apoplastic voltage, which were generally faster than the observed Cl(-) responses, and which also promptly occurred when CO(2) did not initiate Cl(-) efflux.

The comparatively slow activation of Cl(-) efflux by CO(2) indicates that an intermediate effector derived from CO(2) has to accumulate to fully activate plasma membrane anion channels of guard cells.

Ionic activities in the apoplast of the sub-stomatal cavity

Photo credit: Wiley

(b) Photographs of stomata representative of leaves with maximal and with minimal transpiration. The vitality of the closed guard cell was tested with neutral red. Bar = 25 µm.

Dynamics of ionic activities in the apoplast of the sub-stomatal cavity of intact Vicia faba leaves during stomatal closure evoked by ABA and darkness

by Felle H. H., Hanstein S., Steinmeyer R., Hedrich R.

(2000)

in The Plant Journal, 2000, 24, 3, 297 – DOI: 10.1046/j.1365-313x.2000.00878.x

Wiley Online Library – 

http://onlinelibrary.wiley.com/doi/10.1046/j.1365-313x.2000.00878.x/full 

Transpiration of Vicia faba leaves and corresponding stomata opening. (a) Light-and ABA-dependent changes in transpiration (mmol H2O m−2 sec−1) of Vicia faba leaves. Increase in water vapour loss (stomatal opening) was induced by light in a CO2-free atmosphere. At the time indicated, 100 µm ABA were added to the transpiration stream. The delay between ABA application to the petiole of the excised leaf and decrease in transpiration (stomatal closure) is mainly due to the time required for xylem transport of ABA. The stomatal apertures of fully transpiring leaves (plateau) and those treated with ABA were monitored on a CLSM. – http://onlinelibrary.wiley.com/store/10.1046/j.1365-313x.2000.00878.x/asset/image_n/TPJ_878_f1.gif?v=1&t=iojs63xw&s=0efeb91939c56a46e187dc830e52952061d101f3

Summary

Stomatal movement is accomplished by changes in the ionic content within guard cells as well as in the cell wall of the surrounding stomatal pore.

In this study, the sub-stomatal apoplastic activities of K⁺, Cl^–, Ca²⁺ and H⁺ were continuously monitored by inserting ion-selective micro-electrodes through the open stomata of intact Vicia faba leaves.

In light-adapted leaves, the mean activities were 2.59 mm (K⁺), 1.26 mm (Cl^–), 64 µm(Ca²⁺) and 89 µm (H⁺). Stomatal closure was investigated through exposure to abscisic acid (ABA), sudden darkness or both. Feeding the leaves with ABA through the cut petiole initially resulted in peaks after 9–10 min, in which Ca²⁺ and H⁺ activities transiently decreased, and Cl^– and K⁺ activities transiently increased. Thereafter, Ca²⁺, H⁺ and Cl^– activities completely recovered, while K⁺ activity approached an elevated level of around 10 mm within 20 min.

Similar responses were observed following sudden darkness, with the difference that Cl^– and Ca²⁺ activities recovered more slowly. Addition of ABA to dark-adapted leaves evoked responses of Cl^– and Ca²⁺ similar to those observed in the light.

K⁺ activity, starting from its elevated level, responded to ABA with a transient increase peaking around 16 mm, but then returned to its dark level. During stomatal closure, membrane potential changes in mesophyll cells showed no correlation with the K⁺ kinetics in the sub-stomatal cavity.

We thus conclude that the increase in K⁺ activity mainly resulted from K⁺ release by the guard cells, indicating apoplastic compartmentation. Based on the close correlation between Cl^– and Ca²⁺ changes, we suggest that anion channels are activated by a rise in cytosolic free Ca²⁺, a process which activates depolarization-activated K⁺ release channels.