T. A. Mansfield

Changes in stomatal behavior and guard cell cytosolic free calcium in response to oxidative stress

We have investigated the cellular basis for the effects of oxidative stress on stomatal behavior using stomatal bioassay and ratio photometric techniques. Two oxidative treatments were employed in this study: (a) methyl viologen, which generates superoxide radicals, and (b) H2O2. Both methyl viologen and H2O2 inhibited stomatal opening and promoted stomatal closure. At concentrations [less than or equal to]10–5 M, the effects of methyl viologen and H2O2 on stomatal behavior were reversible and were abolished by 2 mM EGTA or 10 [mu]M verapamil. In addition, at 10–5 M, i.e. the maximum concentration at which the effects of the treatments were prevented by EGTA or verapamil, methyl viologen and H2O2 caused an increase in guard cell cytosolic free Ca2+ ([Ca2+]i), which was abolished in the presence of EGTA. Therefore, at low concentrations of methyl viologen and H2O2, removal of extracellular Ca2+ prevented both the oxidative stress-induced changes in stomatal aperture and the associated increases in [Ca2+]i. This suggests that in this concentration range the effects of the treatments are Ca2+-dependent and are mediated by changes in [Ca2+]i. In contrast, at concentrations of methyl viologan and H2O2 > 10–5 M, EGTA and verapamil had no effect. However, in this concentration range the effects of the treatments were irreversible and correlated with a marked reduction in membrane integrity and guard cell viability. This suggests that at high concentrations the effects of methyl viologen and H2O2 may be due to changes in membrane integrity. The implications of oxidative stress-induced increases in [Ca2+]i and the possible disruption of guard-cell Ca2+ homeostasis are discussed in relation to the processes of Ca2+-based signal transduction in stomatal guard cells and the control of stomatal aperture.

Effects of abscisic acid on potassium uptake and starch content of stomatal guard cells

SummaryAbscisic acid (ABA) at a concentration of 100 μm reduced the mean stomatal aperture on isolated epidermis of Commelina communis from 9.5 to 3.1 μm. This closure resulted from a fall in osmotic pressure of the guard cells from 14.1 to 9.8 bars; the osmotic pressure of the subsidiary cells did not change significantly. Histochemical tests showed that the potassium concentration in guard cells was reduced by ABA-treatment, while the starch content of the chloroplasts increased. ABA was found to exert a significant effect on Rb86 uptake into leaf discs, but this was relatively small in magnitude. It is concluded that ABA has a greater effect on ion uptake into guard cells than into the leaf tissues as a whole.Recent hypotheses of the stomatal mechanism are discussed in relation to these new observations, and the rejection by some writers of any major role for starch hydrolysis is challenged. Evidence from several sources suggests that starch disappearance occurs simultaneously with K+ entry into guard cells. Breakdown of starch may lead to formation of organic anions, with which K+ uptake may be associated. In this case starch breakdown would contribute as much to the increased osmotic pressure as does K+ uptake.

Hormones as regulators of water balance.

The development of strategies which enable growth to continue without excessive consumption of limited water resources has played a vital part in the evolution of plants which can survive in terrestrial environments. Research over the last two decades has established a clear role for plant hormones in governing the water economy of plants. By influencing stomatal behavior they can control the expenditure of water, and by regulating the growth and activities of roots, they can exert some control over the uptake of water. Our knowledge of the role of hormones in relation to stomatal functioning is now progressing rapidly and it is appropriate to devote most of this chapter to this topic. Studies of roots have not progressed so rapidly, but nevertheless we can also recognise an important participation of plant hormones in governing activities in roots, which may often complement effects on stomata to provide an integrated strategy for improving water balance.

Effects of Light Quality on Apical Dominance in Xanthium strumarium and the Associated Changes in Endogenous Levels of Abscisic Acid and Cytokinins

SummaryApical dominance in Xanthium strumarium was influenced by the quality of illumination received at the end of the photoperiod. The involvement of the red/far-red regions of the spectrum was apparent. The persistence of the effects was partially dependent on the age of the individual buds concerned. Plants receiving 30 minutes of illumination from tungsten lamps after a 16-hour photoperiod from fluorescent tubes failed to branch, whereas plants given an identical photoperiod, both in terms of day-length and photosynthetically available light energy, but lacking the far-red from tungsten lamps, branched profusely.The influence of the spectral distribution of illumination on the levels of cytokinins and abseisic acid in the plant, and the correlation with the degree of branching, is presented and discussed. The cytokinin content was much higher in inhibited than released buds. The cytokinins present were probably not able to particinate in bud growth because of an accumulation of inhibitors resembling abscisic acid. The concentration of the inhibitors in inhibited buds was 50 to 250 times that occurring in all other plant parts examined.

A simple bioassay for detecting “antitranspirant” activity of naturally occurring compounds such as abscisic acid

SummaryIsolated epidermal strips of Commelina communis L. showed progressively smaller stomatal openings when incubated in abscisic acid solutions ranging in concentration from 10-8 to 10-4 M. The effects were reproducible and did not appear to be affected by the presence of auxin, gibberellic acid or kinetin. This specificity suggests that this method may prove valuable as a quick, sensitive bioassay for abscisic acid and other related compounds which might be used as antitranspirants on field crops. The fungal toxin fusicoccin, previously reported to cause increased stomatal opening on intact leaves, partially reversed the closure induced by abscisic acid.

Studies of the mechanism of action of fusicoccin, the fungal toxin that induces wilting, and its interaction with abscisic acid.

SummaryEffects of fusicoccin alone and together with abscisic acid were observed on the stomatal complex of Commelina communis. The experimental material consisted of isolated epidermal strips incubated in a medium containing the ions required for stomatal opening. Fusicoccin stimulated opening and this was accompanied by potassium entry into the guard cells, and hydrolysis of the starch in their chloroplasts. Abscisic acid alone inhibited potassium entry and starch hydrolysis, but these effects could be almost entirely overcome by fusicoccin.Attempts were made to measure the solute potential of the guard cells under the various treatments. Abscisic acid clearly increased their solute potential, but no absolute measurements could be made in the presence of fusicoccin owing to a failure of plasmolysis even with mannitol solutions of solute potential as low as —35 bars. Experiments using isotopically labelled mannitol indicated a massive uptake into the epidermis in the presence of fusicoccin.The mechanism of stimulation of stomatal opening by fusicoccin probably depends in part on a stimulation of the normal processes associated with opening in the guard cells, but may also involve release of pressure due to destruction of the surrounding cells. The effectiveness of this toxin under natural conditions may depend on its ability to counteract effects of abscisic acid, the stress hormone that induces stomatal closure.

Detection and preliminary identification of endogenous antitranspirants in water-stressed Sorghum plants.

SummarySorghum plants that had been subjected to different degrees of water stress were examined for the occurrence of endogenous compounds capable of inducing stomatal closure, i.e. “antitranspirants”. Acidic extracts contained increased amounts of abscisic acid (ABA) as the amount of stress increased, but another highly active compound easily distinguished from ABA also accumulated. This compound, also found in neutral extracts, was probably all trans-farnesol, an isoprenoid alcohol which, like ABA, is a sesquiterpenoid. Highly dilute solutions of “commercial” farnesol induced stomatal closure when applied to isolated epidermis of Commelina.

Effects of atmospheric sulphur dioxide on microbial activity in decomposing forest litter

Abstract At extremely high concentrations, relative to those occurring in the atmosphere, sulphur dioxide (SO 2 ) has known antimicrobial properties. There is also circumstantial evidence, based on field surveys, to indicate that the occurrence and activities of a number of phylloplane fungi and soil microorganisms are correlated with atmospheric concentrations of SO 2 occurring in parts of Europe and North America. The results of these studies need to be corroborated by controlled fumigation experiments applying realistic concentrations of SO 2 . Unfortunately such experiments have been rare. The suggestion that SO 2 may be affecting soil microorganisms merits serious consideration because of the fundamental role of these organisms in maintaining soil fertility, especially in forests. Events in the forest litter layer are considered to be particularly important because it forms an interface between the atmosphere and the soil system. The research described in this paper involved exposing leaf litter (from a Pinus sylvestris L. stand and a mixed deciduous woodland) to arithmetic mean concentrations of SO 2 of ⩽0.050 μ l 1 −1 in controlled field-based experiments lasting up to 215 days. Fungal cultures, isolated from the pine litter, were also fumigated with ⩽0.053 μ l 1 −1 SO 2 in laboratory-based studies. Results showed that arithmetic mean concentrations of SO 2 as low as 0.015 μl 1 −1 significantly reduced microbial activity (respiration) in both pine and deciduous litter in the open-air fumigation experiment. Results should also be interpreted in relation to the peak SO 2 concentrations (often considerably higher than arithmetic means) to which the litter was exposed. Pure cultures of Cladosporium cladosporioides (Fres.) de Vries and Coniothyrium olivaceum Bonord, isolated from the litter, were shown to be sensitive to SO 2 concentrations of ⩽0.053 μ l 1 t -1 in laboratory-based fumigations. It is concluded that the dry deposition of SO 2 to forest soils may have important implications for nutrient cycling processes and therefore forest productivity and community structure.

All-trans-farnesol: a naturally occurring antitranspirant?

SummaryLipid extracts from Sorghum sudanese Stapf (cv Piper) that had been subjected to different degrees of water stress contained increasing amounts of a sesquiterpenoid, other than abscisic acid, as the amount of stress increased and subsequent bioassay showed that this substance was capable of inducing stomatal closure. Infra-red, nuclear magnetic resonance and mass spectral analysis after final purification by preparative gas-liquid chromatography showed conclusively that this substance was identical to all-trans-farnesol. Various derivatives, homologues and isoprenologues of all-trans-farnesol were tested for their ability to close stomata. Only trans-nerolidol approached all-trans-farnesol in activity when applied to isolated epidermis of Commelina communis L. Stomatal closure was induced slightly faster and more completely in the presence of all-trans-farnesol than in the presence of ABA at a similar molar concentration.

The activity of abscisic acid analogues as inhibitors of stomatal opening.

SummaryTen analogues of abscisic acid (ABA) have been tested for their activity in assays based on the induction of stomatal closing on isolated epidermis of Commelina communis L., and the inhibition of opening on intact leaves of Xanthium strumarium L. Three of the analogues showed activity comparable to that of ABA on the stomata of Commelina. The same analogues also showed similar activity and persistence as ABA when applied to the leaf surfaces of Xanthium. Molecular requirements for activity on stomata agree closely with those for activity in the Avena coleoptile assay. The possible value of the analogues as antitranspirants is considered.