The whole-cell patch-clamp method has been used to measure Ca2+ influx through otherwise K+-selective channels in the plasma membrane surrounding protoplasts from guard cells of Vicia faba. These channels are activated by membrane hyperpolarization. The resulting K+ influx contributes to the increase in guard cell turgor which causes stomatal opening during the regulation of leaf-air gas exchange. We find that after opening the K+ channels by hyperpolarization, depolarization of the membrane results in tail current at voltages where there is no electrochemical force to drive K+ inward through the channels. Tail current remains when the reversal potential for permeant ions other than Ca2+ is more negative than or equal to the K+ equilibrium potential (−47 mV), indicating that the current is due to Ca2+ influx through the K+ channels prior to their closure. Decreasing internal [Ca2+] (Cai) from 200 to 2 nm or increasing the external [Ca2+] (Cao) from 1 to 10 mm increases the amplitude of tail current and shifts the observed reversal potential to more positive values. Such increases in the electrochemical force driving Ca2+ influx also decrease the amplitude of time-activated current, indicating that Ca2+ permeation is slower than K+ permeation, and so causes a partial block. Increasing Caoalso (i) causes a positive shift in the voltage dependence of current, presumably by decreasing the membrane surface potential, and (ii) results in a U-shaped current-voltage relationship with peak inward current ca. −160 mV, indicating that the Ca2− block is voltage dependent and suggesting that the cation binding site is within the electric field of the membrane. K+ channels in Zea mays guard cells also appear to have a Cai-, and Cao-dependent ability to mediate Ca2+ influx. We suggest that the inwardly rectiying K+ channels are part of a regulatory mechanism for Cai. Changes in Caoand (associated) changes in Cairegulate a variety of intracellular processes and ion fluxes, including the K+ and anion fluxes associated with stomatal aperture change.
RECENT investigations suggest that cytoplasmic D-myo-inositol 1,4,5-trisphosphate (InsP3) functions as a second messenger in plants, as in animals, coupling environmental and other stimuli to intracellular Ca2+ release1,2. Cytoplasmic levels of InsP3 and the turnover of several probable precursors in plants are affected by physiological stimuli—including light, osmotic stress and the phytohormone indoleacetic acid3–5—and InsP3 activates Ca2+ channels6 and Ca2+ flux across plant vacuolar7 and microsomal membranes8. Complementary data also link changes in cytoplasmic free Ca2+ to several physiological responses, notably in guard cells which regulate gas exchange through the stomatal pores of higher plant leaves. Recent evidence indicates that guard cell K+ channels and, hence, K+ flux for stomatal movements9 may be controlled by cytoplasmic Ca2+ (ref. 10). So far, however, direct evidence of a role for InsP3 in signalling in plants has remained elusive. Here we report that InsP3 released from an inactive, photolabile precursor, the P5-l-(2-nitrophenyl)ethyl ester of InsP3 (caged InsP3)11 reversibly inactivates K+ channels thought to mediate K+ uptake by guard cells from Vicia faba L. while simultaneously activating an apparently time-independent, inward current to depolarize the membrane potential and promote K+ efflux through a second class of K+ channels12,13. The data are consistent with a transient rise in cytoplasmic free Ca2+ (ref. 9) and demonstrate that intact guard cells are competent to use InsP3 in signal cascades controlling ion flux through K+ channels.
We describe and compare inward and outward whole-cell K+ currents across the plasma membrane surrounding guard-cell protoplasts from the dicotyledon, Vicia faba, and the graminaceous monocotyledon, Zea mays. Macrosopic whole-cell current is considered in terms of microscopic single-channel activity, which involves discrete steps between conducting (open) and nonconducting (closed) states of the channel protein. Kinetic equations are used to model the number of open and closed states for channels conducting K+ influx (K(in)) and K+ efflux (K(out)) in the two species, and to calculate the rate at which open-closed transitions occur. The opening and closure of K(in) channels in both Vicia and Zea follow single-exponential timecourses, indicating that K(in)-channel proteins in each species simply fluctuate between one open and one closed state. In both species, opening of K(in) channels is voltage-independent, but closure of K(in) channels is faster at more positive membrane potentials. In response to identical voltage stimuli, K(in) channels in Zea open and close approximately three times as fast as in Vicia. In contrast to K(in), K(out) channels in Zea open and close more slowly than in Vicia. The closure of K(out) channels follows a single-exponential timecourse in each species, indicating one open state. The kinetics of K(out)-channel opening are more complicated and indicate the presence of at least two (Vicia) or three (Zea) closed states.
In this study, Roman nettle (Urtica pilulifera L.) seedlings grown singly in standard pots containing compost were exposed to two different levels of aluminum and cadmium (100 μM and 200 μM) and water stress (moderate and severe stress) treatments. Measurements of stomatal perimeters, diameters and areas from the epidermal sections in lower surfaces of young expanded leaves of main stem and first lateral branches were examined by image processing and analysis software. The data proved that all stomata were affected significantly, but with varying responses, in all treated plants compared to control plants. Excluding severe water stress (WS 2), the data from first lateral branch leaves showed slight sensitivity to all stress treatments. Nevertheless, there were no statistically significant differences between stomatal measurements from main stem and first lateral branch leaves. Particularly, reduction in stomatal diameters of both main stem and first lateral branches in severe water stressed plants, reducing by 26.45% and 48.09% respectively; suggest that this could be a response of U. pilulifera to drier environments.
A number of studies have highlighted differenc- es in the density of stomata between Vitis species, but few have examined differences between varieties of V. vinifera. The density and size of the stomata in the lower epidermis of leaves belonging to 12 grapevine varieties, a direct producer hybrid (DPH) involving a V. vinifera and a non-vinifera parent, and the non-vini- fera rootstocks ‘SO4’ and ‘110-Richter’, were therefore examined. Transparent nail polish peel prints of the area between the main and right lateral veins were pro- duced for 10 leaves per variety. These prints were then examined under a light microscope and the number of stomata in a unit area of 0.196 mm2 counted. Image analysis software was then used to measure the length and width of all those counted. Rootstock ‘SO4’, ‘Chas- selas Dorée’, ‘Albariño’ and ‘Cabernet Sauvignon’ had the highest stomatal densities (all > 34 stomata per unit area), while ‘Castañal’, ‘Torrontés’ and ‘Caiño Blan- co’ and ‘Jacquez’ (DPH), had the smallest (all < 26.50 stomata per unit area). ‘Treixadura’ and ‘Caiño Blan- co’ had significantly longer and wider stomata than all the other varieties examined; the DPH ‘Jacquez’ had among the shortest and narrowest. No relationship was seen, however, between mean varietal leaf size and the stomatal density or stomatal size; nor was any seen be- tween the variables examined and the condition of be- longing to V. vinifera or not.
Two-year-old beech and Norway spruce seedlings were exposed to a combination of ozone and acid mist treatments in open-top chambers in Scotland during the months of July through to September 1988. Replicate pairs of chambers received charcoal-filtered air (control), ozone-enriched air (140 nl ozone litre−1) or 140 nl ozone litre−1 plus a synthetic acid mist (pH 2·5) composed of ammonium nitrate and sulphuric acid. Field measurements of assimilation and stomatal conductance were made during August. In addition, measurements of assimilation and conductance were made during September in the laboratory. Light response curves of assimilation and conductance were determined using a GENSTAT nonrectangular hyperbolic model. During February 1988/9 the Norway spruce were subject to a four day warming period at 12°C and the light response of assimilation determined. The same plants were then subject to a 3-h night-time frost of −10°C. The following day the time-course of the recovery of assimilation was determined.
It was found that ozone fumigation did not influence the light response of assimilation of beech trees in the field, although stomatal conductance was reduced in the ozone-fumigated trees. The rate of light-saturated assimilation of Norway spruce was increased by ozone fumigation when measured in the field. Measurements of assimilation of Norway spruce made during the winter showed that prior to rewarming there was no difference in the rate of light-saturated assimilation for control and ozone-fumigated trees. However, the ozone plus acid mist-treated trees exhibited a significantly higher rate. The 4-day period of warming to 12°C increased the rate of light-saturated assimilation in all treatments but only the ozone plus acid mist-treated trees showed a significant increase. Following a 3-h frost to −10°C the control trees exhibited a reduction in the rate of light-saturated assimilation (Amax) to 80% of the pre-frost value. In comparison, following the frost, the ozone-fumigated trees showed an Amax of 74% of the pre-frost value. The ozone plus acid mist-treated trees showed an Amax of 64% of the pre-frost trees. The time taken for Amax to attain 50% of the pre-frost value increased from 30 min (control) to 85 min for ozone-fumigated trees to 190 min (ozone plus acid mist). These results are discussed in relation to the impact of mild, short-term frosts, which are known to occur with greater frequency than extreme, more catastrophic frost events. A simple conceptual framework is proposed to explain the variable results obtained in the literature with respect to the impact of ozone upon tree physiology.
Leaves of in vitro-grown plantlets and greenhouse-grow plants of ‘Totem’ strawberry (Fragaria .times. ananassa Duch.) were compared using scanning and light microscopy. Each apex and marginal serration of in vitro- and greenhouse-grow leaves had a terminal hydathode region. The leaf teeth were composed of an acuminate-mucronate tip, obscured in greenhouse-grown plants by an abaxial cluster of thick-walled unicellular trichomes, and a subterminal, adaxial group of sunken water pores. Water pores and stomata of plantlet leaves were open, whereas greenhouse-grown plant leaves had closed water pores and stomata or comparatively small apertures. Internally, the hydathodes of greenhouse-grown plants and cultured plantlets were delimited by a bundle sheath thart extended below the vascular tissues, approaching the adaxial epidermis on each side of the zone of water pores. Between the epidermis and the vascular tissues were loosely arranged epithem cells. The hydathodes of plantlet leaves were smaller than greenhouse-grown plants, with fewer water pores and reduced epithem.