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Heartwater, Ehrlichia ruminantium infection in cattle, sheep, goats, and some wild ruminants, is an economically important disease in Africa characterized by high mortality rates in susceptible populations. In South Africa, the current commercial heartwater vaccine is an infection and treatment type of immunization using virulent live E. ruminantium organisms generated from blood of infected sheep with subsequent treatment of the animals with antibiotics at specific times during the course of infection. This vaccine has several inherent problems preventing its wide use as the vaccine must be administered intravenously and it does not protect against all the South African field isolates. A vaccine based on inactivation of Zimbabwean E. ruminantium Mbizi strain organisms produced in endothelial cell cultures can be a sustainable option because it will not require antibiotic treatment and will be safe as there is no potential for reversion to virulence. Previous data generated in laboratory trials and under natural field setting provides support for this vaccine approach. Four inactivated vaccine formulations using the E. ruminantium Mbizi strain were tested for their efficacy in Merino sheep compared to an unvaccinated control group (11 sheep per group). Two vaccines were prepared by beta-propiolactone (BPL) inactivation, and two were inactivated with binary ethylenimine (BEI) while purification was done with both percoll and polyethylene glycol (PEG). The four vaccine preparations were formulated with Montanide ISA 50V2 adjuvant and administered twice subcutaneously (2 ml per dose) at an interval of 4 weeks. All groups were challenged with a virulent homologous cell-cultured E. ruminantium inoculated via the intra-venous route on day 56. The primary variable of efficacy was measured by the percentage survival rate or mortality between the Controls and Vaccine Groups. Three vaccine formulations (BEI/Percoll (Group 3), BEI/PEG (Group 4), BPL/Percoll, (Group 1) had a significantly higher percent of animal surviving challenge compared to the unvaccinated control (p-values 0.001, 0.035, 0.030, respectively). The highest number of survivors was obtained in Group 3 BEI/Percoll; 10/11 (91%). Groups 4 (BEI/PEG) and Group 1 (BPL/Percoll) produced similar percentage of survivals of 64%. In contrast, the lowest survival rate of 50% was observed in Group 2 (BPL/PEG) which was numerically different but not significantly different from the unvaccinated control which had an 18% survival rate (2/11). The inactivated vaccine using BEI or BPL as inactivating agents blended with ISA 50 adjuvant induced protective immunity against challenge. The BEI/Percoll (Group 3) vaccination regimen was most efficacious against a lethal heartwater challenge as it significantly protected sheep against mortality which is the most important aspect of heartwater infections. Future work should be directed towards improvement of this vaccine formulation especially from the down-stream processing point of view as the percoll method is not scalable for commercialization purposes.

Summary The potential of low viscosity silicone-based impression material for measuring stomatal aperture on intact leaves was investigated. It was found that the probability of successful replication of Commelina communis L. stomata depended on the width of the stomatal pores. The wider pores in a sample were more likely to be measured, so the resulting estimated mean was an overestimate of the true mean. This bias in estimates of stomatal aperture from impressions was studied and quantified. A transformation is described which was used to adjust apparent aperture measurements from impressions to give more accurate estimates of stomatal apertures on intact leaves of C. communis. Maximum absolute errors due to the bias usually occurred at apparent apertures on impressions of between 2 and 5 μm but were proportionally greatest below 3μm, where they often exceeded 40%. At a given aperture, the error depended upon the dispersion of the stomatal apertures about their mean and was greatest when this was large.

Variation in stomatal development and physiology of mature leaves from Alnus glutinosa plants grown under reference (current ambient, 360 μmol mol

This review examines contemporary views of the role of plant hormones in the control of physiological processes. Past and present difficulties with nomenclature encapsulate the problems inherent in using the 'classic' hormone concept in plants, with their distinctive multicellular organization. Chemical control may be a more relevant notion. However, control may also reside in the responding tissue via changes in sensitivity, or as combined control, where response is dictated by both sensitivity and concentration. Criteria for demonstrating these modes of action are reviewed, as well as frameworks for deciding whether hormone transport is involved. Problems of measuring relevant hormone concentrations are discussed. Methods for measuring and comparing tissue sensitivity to hormones are outlined and relative control is introduced as a means of assessing the importance of hormonal control against a background of other influences. While animals and plants appear to have coinherited homologueous intracellular signalling systems, at the whole organism level modes of hormone action may diverge. It is postulated that the synthesis-transport-action mechanism of action may be just one of several possible ways that phytohormones could control physiological processes. Twelve separate roles are discussed, and it is suggested that some of these could operate simultaneously to the plant's advantage. Contents Summary 375 I. Introduction 376 II. The history of the hormone concept in plant systems 376 III. Issues of nomenclature 380 IV. The need for sound conceptual frameworks in plant hormone research 382 V. Development of criteria for chemical control 384 VI. Identification and quantitative analysis of plant hormones 387 VII. Hormone transport in plants 389 VIII. Hormone sensitivity and its quantification 390 IX. Roles of receptors, second messengers and signal amplification in hormone sensitivity changes 393 X. Relative control as a pivotal concept 395 XI. Diversity of physiological roles for chemical influences in plants 397 XII. Concluding remarks 400 Acknowledgements 402 References 402.

Objective The aim of this study was to evaluate the predictive value of the dynamic morphological development process between cleavage-stage and blastocyst-stage embryos. Study design A retrospective study was executed between 2015 and 2017 at Ghent University Hospital. A total of 996 first fresh IVF/ICSI cycles resulting in a single embryo transfer on day 5 were included. Embryos were scored on day 3 and day 5 as excellent, good, moderate or poor based on Alpha/ESHRE guidelines and Gardner and Schoolcraft scoring-system. If embryos changed category between day 3 and 5, the number of steps (between excellent; good; moderate; poor) in positive and negative direction was expressed. Results On day 5, the ongoing pregnancy rate (OPR) of excellent embryos was 37.4 %. Univariate analyses showed that on day 5, both a higher cell stage, better inner cell mass and better trophectoderm were significantly associated with an ongoing pregnancy. In case of deterioration in quality of individual embryos between day 3 and day 5, the OPR was significantly lower. Conversely, improvement of embryo quality between day 3 and day 5 showed higher ongoing pregnancy rates (overall OPR of good day-3 embryos improving to excellent day-5 embryos: 30 %; moderate day 3 to excellent day 5: 50 %; poor day 3 to excellent day 5: 42 %; poor day 3 to good day 5: 20 %; poor day 3 to moderate day 5: 16 %). When embryos improved from poor on day 3 to excellent day 5 the OPR was significantly higher in comparison with embryos that did not change in quality scoring during development (steady embryos) (OR: 1.785, p Conclusion Our results suggest that it is more likely to achieve an ongoing pregnancy when transferring an embryo that has improved in quality between days 3 and 5 as opposed to one that has remained stable.

This paper introduces a new approach to the adaption of user interfaces by identifying input errors from a given user interface through modeled error automata. These error automata will also be used to generate reconfiguration rules for formal reconfiguration of a user interface on its logical layer paired with reconfiguration patterns derived from psychological guidelines. These guidelines seek to describe concepts, such as enhancing situation awareness through physical rearrangement in certain control situations, or by increasing or decreasing the level of automation. The whole process, starting with the generation of error automata and ending with reconfiguration of the user interface, is implemented based on a formal approach to user interface modeling and reconfiguration, as well as on formal description of deterministic finite automata.

If a current of electrons flows through a normal conductor (in contrast to a superconductor), it is impeded by local scattering at defects as well as phonon scattering. Both effects contribute to the voltage drop observed for a macroscopic complex system as described by Ohm's law. Although this concept is well established, it has not yet been measured around individual defects on the atomic scale. We have measured the voltage drop at a monatomic step in real space by restricting the current to a surface layer. For the Si(111)-( [see text]3 x [see text]3)-Ag surface a monotonous transition with a width below 1 nm was found. A numerical analysis of the data maps the current flow through the complex network and the interplay between defect-free terraces and monatomic steps.

If the sample and the tip of a scanning tunneling microscope are at different temperatures a ‘thermovoltage’ arises which is superposed on the external bias. Although it is small it can be measured with high accuracy applying scanning tunneling potentiometry. Since it depends on the details of the electronic states contributing to the tunneling process it provides useful information, e.g. on interference patterns of electronic surface states, on the distribution of different chemical elements on a surface etc. Results on homogeneous as well as on inhomogeneous metallic surfaces will be shown. The correlation between the thermovoltage and the electronic states will be discussed for the Si(111) 7×7 surface.

Variation in stomatal development and physiology of mature leaves from Alnus glutinosa plants grown under reference (current ambient, 360 μmol mol−1 CO2) and double ambient (720 μmol mol−1 CO2) carbon dioxide (CO2) mole fractions is assessed in terms of relative plant growth, stomatal characters (i.e. stomatal index and density) and leaf photosynthetic characters. This is the first study to consider the effects of elevated CO2 concentration on the distribution of stomata and epidermal cells across the whole leaf and to try to ascertain the cause of intraleaf variation. In general, a doubling of the atmospheric CO2 concentration enhanced plant growth and significantly increased stomatal index. However, there was no significant change in relative stomatal density. Under elevated CO2 concentration there was a significant decrease in stomatal conductance and an increase in assimilation rate. However, no significant differences were found for the maximum rate of carboxylation (Vcmax) and the light saturated rate of electron transport (Jmax) between the control and elevated CO2 treatment.

This paper describes spatio-temporal variation in gas exchange over the lower surface of primary leaves of glasshouse-grown Phaseolus vulgaris L. plants. Simultaneous measurements of assimilation and water vapour conductance were made with a small area cuvette attached to an infra-red gas analyser. The plants were kept in glass chambers so that the external gaseous environment could be controlled. Observations are reported from four half-hour periods during a day in which the ambient PPFD, while variable, was close to saturating for photosynthesis. 'Snapshot' measurements of gas exchange were made at 20 positions on the leaf surface using a stratified random out-to-in strategy, which avoided disturbance of yet-to-be-measured sites. Data were mapped using the 'Unimap' cartographic program. For any given measurement period, gas exchange varied greatly over the leaf surface : typically, net assimilation (A) varied by over 4-fold and leaf conductance (g I ) by over 3-fold. Estimated intracellular pressures of CO 2 and leaf temperatures showed less relative variation both in space and time. Comparing measurement periods, the spatial patterns of variation in A and g, were dissimilar. Moreover, at different sites on the leaf, the trends in a given variable could be in opposite directions, while external conditions were relatively constant. Although the correlation between A and g, was significant overall, there was a large degree of scatter in the data and zones of high g, often corresponded to areas of low A. Depending on the basis of calculation, A was as much as 63% lower than a value predicted on the basis of steady-state measurements for the appropriate mean irradiance. It was not possible to deduce from the relationships between pairs of variables which factors were most important in determining A and g I at any given time or space, but g I did not appear to be the only factor limiting A. It is hypothesized that the observed variation in gas exchange, the lack of close correspondence between g 1 and A and the reduction in photosynthesis compared with the apparent potential value are all phenomena that arise from differences in the induction times for these variables following changes in conditions, interacting with other factors associated with position on the leaf.

Investigations on sun and shade leaves allow students to compare plant growth patterns in different environments and to appreciate plants as slow-motion combatants for light as a resource Plants can respond developmental^ to many environmental stimuli. Sun and shade leaves are an example of an acclimation to light intensity. This paper describes how this phenomenon can be used as a stimulating vehicle for teaching plant ecophysiology to undergraduates. Detailed protocols for investigations are outlined, with an appendix relating to studies in schools. We also provide representative data and introduce topics for classroom or tutorial discussion.

The aim of this research was to investigate the nature of heterogeneity in stomatal conductance and, in particular, to determine whether the characteristic 'patchy' pattern of water infiltration is reflected in measurements on individual stomata. Silicone rubber replicas were made of primary leaves of glasshouse-grown Phaseolus vulgaris L. plants, and the leaves were then infiltrated with water at controlled subatmospheric gas pressures according to their estimated or measured stomatal conductance. Seven leaves examined in detail all showed patchy infiltration, and the mean size of infiltrated areas was negatively correlated with the prevailing stomatal conductance. In four of the leaves, a one millimetre wide transect across the leaf was selected for further detailed study. Measurements of mean peristomatal groove distance (PGD) and stomatal frequency were made along the transect and related to the state of infiltration. Analysis of variance indicated that, in all four cases, variation in PGD among patches was highly significant, but there was no significant difference between patches of different infiltration categories. Thus, local (patch-level) variation in stomatal aperture appeared to bear no relation to the infiltration status of the patches. The dominant source of stomatal variability was between individual pores in the same locality, which accounted for 82% or more of the total variability. Taking into account variation in stomatal frequency, correlations between predicted stomatal conductance and the extent of infiltration were significant in only one out of the seven leaves studied. Possible reasons for these results are discussed. It is suggested that the infiltration method misrepresents the underlying state of the stomata as being either open or closed, when there is little evidence for this from measurements of stomatal dimensions. For these unstressed plants under relatively stable conditions, it is concluded that the 'unit of variability' in stomatal heterogeneity may rest at the individual pore ('micro') scale, rather than at the areolar patch ('macro') scale, or above.

Procedures are described for estimating photosynthetic characteristics using a portable infra-red gas analysis (IRGA) system. Once the effects of stomatal limitation on CO2 assimilation have been established, up to ten parameters of photosynthesis can be estimated for a single leaf within 2 h, including: photosynthetic efficiency and capacity on both photon and CO2 bases; compensation irradiances and CO2 compensation concentrations; and light and dark respiration rates. These measurements can be made in the laboratory, glasshouse or field with relative ease. Methods for obtaining near instantaneous ("snapshot") measurements of leaf photosynthesis are also described, using carefully pre-set conditions within the leaf cuvette. Representative results are shown for Phaseolus vulgaris L. Important aspects of the procedure's experimental design, assumptions made in the analysis, and limitations of this approach are analysed.

The variation in stomatal characters in leaves from one Alnus glutinosa (L.) Gaertn. tree is analysed. Measurements were taken from over 70 sites on the abaxial surfaces of representative ‘sun’ and ‘shade’ leaves having the same insertion point. The mean values of stomatal density and index in the shade leaf were significantly lower (71 and 93%, respectively) than those for the sun leaf. Within leaves, up to 2.5-fold differences in stomatal density values were observed. Contour maps derived from the data reveal non-random trends over the leaf surface. Correlations between stomatal density, epidermal cell density and stomatal index indicate that the variation in stomatal density within leaves arose primarily from local differences in stomatal differentiation, rather than from local differences in leaf expansion. This research demonstrates that a high level of variation in stomatal characters occurs both within and between leaves. We conclude that a well-defined sampling strategy should be used when estimating stomatal characters for (tree) leaves. Furthermore, the leaf's insertion point and situation within the tree crown should be taken into account. We discuss the implications of these findings for palaeoclimatic interpretations and emphasize the need for great caution when drawing conclusions based solely on stomatal characters.

We highlight the need to combine information about hormone concentrations and tissue sensitivity when assessing control of physiological phenomena by plant hormones. A quantitative approach to this problem is suggested and the method illustrated using data for stomatal closure induced by abscisic acid. The technique requires accurate estimates of hormone concentration at the putative site of action and information about the kinetics of responses in the form of dose-response curves obtained under relevant conditions. Extending this approach, hormonal control is seen as relative, in that it must be considered against a background of other controlling influences. Possible means of evaluating this 'control potential' from dose-response curves are discussed. Finally, we draw together the above in an analytical framework for assessing hormonal control. This involves (1) matching observed and predicted responses; (2) studying effects of manipulating hormone concentration or sensitivity ; and (3) assessing the relative control potential of the compound of interest.

Short- and medium-term stresses (1 and 24 h, respectively) were applied to detached leaves of Commelina communis L., resulting in both cases in a final leaf cell water potential (ψ w ) of about -1.0 MPa and relative water content (RWC) of about 74%. A long-term stress (14 d), applied to intact plants, resulted in a final leaf cell Ψ w of -0.57 MPa and RWC of 90%. All the stresses resulted in lower leaf conductances (g i ) compared to controls, although temporary increases in g i were observed early in the short- and medium term stresses. When epidermal strips were incubated in vitro, the stomatal opening potential was reduced by the stresses, possibly due to lower initial apertures caused by the stress

Fibres from plant sources have been used by man for many generations and there is increasing interest in the potential use of such fibres from non-tree sources for various applications. This review brings together most of the relevant literature on the botany, chemistry and processing. The different cell types are explained and those which produce fibres are highlighted in conjunction with the actual plants involved. The chemistry of the plant cell wall is addressed with special consideration to the components of fibre cell walls and the biosynthesis, where known, is explained. Finally, the various methods for processing fibres into valuable industrial raw materials are detailed along with some prospective new technologies.

Potato plants (Solanum tuberosum L. cv. Bintje) were grown in open-top chambers (OTCs) under three CO(2) levels (ambient and 24 h d(-1) seasonal mean concentrations of 550 and 680 micromol mol(-1)) and two O(3) levels (ambient and a seasonal mean 8 h d(-1) concentration of 50 nmol mol(-1)). The objectives were to determine the effects of season-long exposure to these key climate change gases on gas exchange, leaf thickness and epidermal characteristics. The experimental design also provided an ideal opportunity to examine within-leaf variation in epidermal characteristics at the whole-leaf level. Stomatal and epidermal cell density and stomatal index were measured at specific locations on the youngest fully expanded leaf (centre of lamina, mid-way between tip and base) and representative whole leaves from each treatment. Effects on leaf conductance, assimilation rate and instantaneous transpiration efficiency were determined by infrared gas analysis, while anatomical characteristics were examined using a combination of leaf impressions and thin sections. Exposure to elevated CO(2) or O(3) generally increased leaf thickness, leaf area, stomatal density, and assimilation rate, but reduced leaf conductance. The irregular stomatal distribution within leaves resulted from a combination of uneven differentiation and expansion of the epidermal cells. The results are discussed with reference to sampling protocols and the need to account for within-leaf variation when examining the impact of climate change or other environmental factors on epidermal characteristics.

Individual Cu-phthalocyanine molecules have been investigated by scanning tunnel microscopy on a closed packed film of C-60 at various temperatures. The molecules are found to bind asymmetrically to one C-60. While they remain in one position at low temperature, they can hop between six equivalent positions at higher temperatures performing a hindered eccentric rotation around the binding C-60 molecule. At room temperature only the time average over different positions is observed.

The ancestral angiosperm flower probably had many separate elements in each floral whorl (sepals, petals, stamens and carpels). Derived character states include ‘fusion' of elements within a whorl (cohesion) and fusion between whorls (adhesion), as well as epigyny and the emergence of the other floral elements from the apex of the fused carpels. This article considers the roles of epidermal fusion and intercalary growth in the phylogeny and ontogeny of fused floral elements, and the importance of fusion for angiosperm evolution.

Publisher Summary Stomata are variable in many attributes is evident to any scientist who has researched their structure or physiology. Heterogeneity in stomatal characteristics is found at many levels, from the size, frequency, and behavior of individual guard cells to the gas exchange of whole plants or stands of plants. This chapter outlines the major forms of heterogeneity to be found in characteristics associated with the development and function of stomata. A variety of methods has been used to study these phenomena, each having its advantages and disadvantages. It is possible to speculate on the causes of this heterogeneity, but much more research is required before it will be possible to make firm conclusions. At the whole–leaf scale there is a challenge to incorporate such complex influences as venation patterns, surface topography, hairs, leaf shape, wind direction, and turbulence. For physiological processes, the focus is shifting from consideration of spatial variability as a constant phenomenon towards considering it as a dynamic process Perhaps the most important rationale for working on this topic arises because it is fundamentally related to ones understanding of the stornatal mechanism itself. At the canopy level and beyond there is the need to consider further details such as the placement of the individual plant within a community and the influence of the community's activities on the individual.

In recent years, the subject of plant hormone sensitivity has given rise to a great deal of controversy. The efforts of A.J. Trewavas to stimulate a reappraisal of the role of sensitivity as a controller of response (Trewavas, 1981, 1982, 1983; Trewavas and Jones, 1981) at first met with extreme resistance, but it cannot be denied that he achieved his aim (Trewavas, 1991) and the concept of sensitivity is now widely discussed in texts and reviews. While it may be true that many past authors had mentioned the possibility of sensitivity changes (Cleland, 1983), and that many since have shown evidence for sensitivity changes (Trewavas, 1991), few have addressed the subject in a systematic manner For progress to be made, a sound conceptual framework is required so that relevant hypotheses may be formulated and tested (Firn, 1986; Weyerset al.1987). In a series of papers, we have explored methods of quantifying sensitivity using stomatal responses to abscisic acid (ABA) as a test system (Weyerset al.1987; Patersonet al.1988; 1990).

The local thermoelectric properties of a SeCl4-graphite intercalate compound (SeCl4-GIC) are studied by means of scanning-tunneling microscopy (STM). The analysis by micro-Raman spectroscopy suggested two domains, a “carbonlike” and a “SeCl4-GIC-like.” By using the thermovoltage that arises if a temperature difference between the tunneling tip and the sample of a STM is applied, areas with a difference in the local density of states can be distinguished with high lateral resolution. The differences in the local thermopower may be as large as several hundred μV/K. The aim was to estimate the contribution of the different domains to the thermopower of SeCl4-GIC.

Recently the ballistic transport through organic molecules could be analyzed with submolecular resolution by an extension of ballistic electron emission microscopy. In this work we compare the results of ballistic transport of electrons and holes through C₆₀ molecules deposited onto a Bismuth/Silicon Schottky diode. The study of hole transmission also exhibits molecular a resolved pattern in the transmission images showing the molecular periodicity of the C₆₀ layer. © 2008 IOP Publishing Ltd. CA Bobisch

Purpose: To compare 2D (BAT) and 3D (IBeam) ultrasounds with seed markers (SM) for prostate localization, and to assess intrafractional prostate motion during external beam radiotherapy(EBRT).Method and Materials: Prior to daily prostate localization with SM, patients underwent localization with two ultrasound systems for one week and with one ultrasound system for remaining treatments. Ultrasound shifts were prospectively recorded and patients were shifted as per SM alignment. The differences in lateral (LR), craniocaudal (CC) and anterior‐posterior (AP) directions were compared assuming SM revealed the true position of the prostate. Intrafraction motion of the prostate was assessed by repeating the SM after EBRT treatment on four fractions. Results: From 20 patients, 455 I‐Beam and 343 BAT ultrasound scans were compared to SM localizations. I‐Beam had a mean error ± sd of 0.6±3.3mm in the LR direction, 2.2±4.8mm (CC) and 0.2±3.8mm (AP). BAT had a mean error ± sd of 0.0±3.8mm (LR), 0.8±4.5mm (CC), and −1.5±4.3mm (AP). Deviations >5mm occurred in 11%, 32% and 18% in the LR, CC and AP directions respectively for I‐Beam, and 11%, 24% and 23% respectively for BAT. Deviations >1cm occurred in 7% in the CC direction for I‐Beam. Intrafraction prostate motion is minimal with a mean displacement ⩽1.5mm. The greatest movement is in the AP direction with 4% of patients having >5mm displacement. Conclusion: Target localization with two US systems provide different results than SM localization. Our data suggest that PTV margin guidelines for BAT and I‐Beam should be greater than those for SM. The US margin requirements may also need to be greater than if no daily localization is performed. In addition, intrafractional prostate motion is minimal with large displacements occurring in

We outline a theoretical framework for comparing the relative effectiveness of hormonal and other factors in the control of physiological responses. This involves determining a 'baseline' set of conditions, then adjusting each factor in turn with the others held constant. The initial rate of response to each change in condition is estimated and compared among factors to arrive at a quantitative indication of control potential under the specified baseline conditions - the control coefficient. The suitability of the stomatal response as a model system for such studies is discussed. A portable infra-red gas analyser was used to investigate the control potential of abscisic acid (ABA), CO2 and light - measured as photosynthetic photon flux density (PPFD) - over responses of Phaseolus vulgaris L. stomata at baseline conditions appropriate to an unstressed plant under moderate light conditions. The ranking of control coefficients observed under these conditions was light>CO2>ABA. The control coefficients of CO2 and ABA were 0.28 and 0.11 times that of light, respectively. These results are discussed in relation to the sensitivity of the plant material and the experimental conditions. Implications for a hormonal role for ABA in the control of stomatal movements are considered. We conclude that this method can provide valuable information concerning the relative control potential of hormonal and other influences in the context of differences in baseline conditions and in relation to changes in sensitivity of plant material.

Weyers, J. D. B. and Paterson, N. W. 1987. Responses of Commelina communis stomata in vitro. J. exp. Bot. 38: 631-641. Analysis of the kinetics of movements of Commelina communis L. stomata in vitro revealed a sequence of opening and closing phases dependent on the incubation medium used and the physiological state of the plant material. In buffer containing 50 mol m "3 KC1 the sequence of aperture changes appeared to be influenced by equilibration of cell water potentials with that of the medium and by solute fluxes (dependent and independent on metabolic activity). The results indicate that the stomatal aperture after several hours of incubation may not always provide a reliable quantitative estimate of the ability of the stomata to operate. As a consequence, modifications are suggested to the ways in which experiments using epidermal strips are carried out and reported.

The effects of K+ concentration, light intensity and CO2 levels on the volume of Commelina communis L. guard cell protoplasts were studied. Two degrees of swelling response were observed, both dependent on an external supply of K+, but not necessarily on the supply of a permeant anion. The presence of K+ itself, independent of light or CO2 level, stimulated swelling at a relatively slow rate. When K+, light and low CO2 conditions were supplied together, the swelling was relatively rapid and of high magnitude. The rapid swelling was specific for K+ and Rb+ giving a half maximal effect after 2 h at a KCl concentration of about 18 mmol m−3. The addition of CaCl2 at 1 mol m−3 inhibited K+-dependent swelling under all conditions tested. The response to light and low CO2 levels by Commelina guard cell protoplasts is thought to reflect a high degree of physiological integrity.

Stomata of Commelina leaves pre-opened by incubation in moist air were found to close within 30 min when supplied with abscisic acid (ABA) via the transpiration stream. Radioactive ABA had similar effects, but allowed the distribution of the compound within the leaf to be measured and correlated with stomatal movements to give estimates of the sensitivity of Commelina stomata. On a whole-leaf basis, less than 163 fmol ABA per mm(2) leaf area were present at the time of complete stomatal closure. This was close to other published estimates. By taking epidermal (14)C measurements, however, it was possible to increase the accuracy of the estimate on the assumption that only ABA present in the epidermis was physiologically active. Thus, less than 235 amol ABA for stomatal complex were present at complete closure, and statistically significant narrowing of the stomatal aperture had occurred when between 12.6 and 45.4 amol per complex were present. The distribution of ABA within the epidermal tissue after transpiration-stream application was studied using microautoradiography, and the compound appeared to have accumulated within the stomatal complex.

The silicone rubber impression technique was used to measure stomatal apertures in 9 mm2 sampling areas covering the entire lower surface of leaves of Commelina communis L. The data were analysed using a computer program which produced ‘iso-aperture' contours illustrating local differences in mean stomatal aperture. Little consistency was seen in the iso-aperture patterns among sampling times, although the stomata were always relatively closed at the leaf tip and base. When stomata in the middle of the lamina were open, those near to the leaf margin tended to be relatively closed. In places, gradients of mean stomatal aperture were as high as 1 μm mm−1. Measurements along a transect across the lower epidermis revealed no correlation of stomatal aperture with the presence of major veins in the mesophyll tissue. Variation in guard-cell size and stomatal frequency on the lower leaf surface was also analysed. The guard cells were smallest and the stomata more frequent near to the leaf margins. The significance of the results is discussed in relation to measurements of leaf conductance and models of stomatal function.

Summary Seeds of flacca , the wilty tomato mutant deficient in abscisic acid (ABA), had a shorter mean germination time than those of the control cultivar, Rheinlands Ruhm. Applied ABA delayed germination of both control and mutant seeds, the latter appearing to be more sensitive to the hormone. Transfer from ABA to water allowed germination levels to reach those of controls within two or three days. It is speculated that low endogenous ABA levels in flacca seeds might result in the observed precocious germination. Viviparous germination was observed within ripe flacca fruit which may also have resulted from the hormone deficiency. Flacca seedlings exhibited normal gravitropic behaviour. This finding is discussed in regard to current theories of hormonal control of gravitropism.

The non-osmotic volume (NOV) of Connnelina communis L. guard cells was estimated by observing the volumes of guard cell protoplasts incubated in mannitol solutions of different solute potential, and applying the Boyle-van't Hoff relation to the results. NOV values of between 517 and 1782 μm3 were obtained for different batches of protoplasts. There was a negative correlation between NOV and apparent protoplast solute contents, and the NOV and solute content were observed to alter when pretreatments affecting stomatal aperture were given. H is hypothesized that changes in guard cell chloroplast starch levels could account for variation in NOV and solute content. For closed stomata, it is calculated that the NOV could reduce the proportion of the total guard cell volume which is osmotically active by over 40%. Serious inaccuracy may thus result if the NOV is not taken into account in the estimation of guard cell solute potential or solute concentration from measurements of solute levels per cell. The error is maximal at low stomatal apertures.

Guard-cell protoplasts were isolated by enzymic digestion of the epidermis peeled from the abaxial surface of leaves from Commelina communis L. The protoplasts were separated from mesophyll-cell protoplasts and other contaminants by density-gradient centrifugation, and the purity of the preparations carefully and quantitatively assessed by light microscopy. The preparations of guard-cell protoplasts were then compared with mesophyll-cell protoplasts in terms of the activity of photosystem II as assessed by a) the light-induced evolution of oxygen under both steady-state and flashing light and b) the characteristics of photosystem-II chlorophyll fluorescence. In all experiments, clear photosystem-II activity was found in guard-cell protoplasts, although some subtle distinctions between guard-cell and mesophyll-cell protoplasts were found. The contribution of any contaimination by mesophyll-cell chlorophyll to guard-cell-protoplast signals was estimated to be less than 3% in all cases. The results indicate that photosystem II is present and active in guard cells of Commelina.

Commelina communis L. guard cell protoplasts were induced to swell under low CO2 conditions in the light while incubated in media containing KCl. Precise measurements of changes in the volume of the protoplasts were made including estimates of protoplast non-osmotic volume by Boyle-van't Hoff analysis. The amount of K+ which accumulated during the treatment was measured. The observed changes in osmotic volume could be accounted for by the uptake of K+ which appeared to be balanced by an anion or anions with an effective mean charge of – 1.63. The K+ flux rates occurring in guard cell protoplasts were sufficient to explain guard cell turgor regulation in vivo.

Closure of stomata of Commelina communis L. leaf epidermis caused by abscisic acid (ABA) was inhibited by sodium azide, potassium cyanide and hypoxic conditions. Azide was more effective than cyanide at low concentrations, but the cyanide effect could be enhanced by addition of salicylhydroxamic acid, providing indirect evidence for cyanide-resistant respiration in epidermal tissue. Azide also inhibited ABA-induced closure of 'isolated' stomata and shrinkage of guard cell protoplasts. The results indicate that metabolic energy is required for ABA action involving solute loss from the guard cells. Possible mechanisms of action are discussed.

Closure of stomata by abscisic acid (ABA) was studied by floating leaf epidermal strips of Commelina communis L. in PIPES buffer (pH 6.8) containing a range of KCl concentrations. Control apertures were greatest at high concentrations of the salt, and the effects of ABA, in terms of closure, were most pronounced below 100 mol m(-3) KCl. Stomata opened on strips floated on buffer plus 50 mol m(-3) KCl and closed within 10 min when transferred to the same medium plus 0.1 mol m(-3) ABA. [2-(14)C]ABA was used to study uptake and distribution of the hormone by the epidermal strips. It was calculated that no more than 6 fmol ABA were present per stomatal complex at the time of closure, although uptake continued thereafter. Microautoradiography indicated that radioactivity from [2-(14)C]ABA accumulated in the stomatal complex at or near the guard cells within 20 min. TLC was used to examine the state of the label after 1 h incubation. Efflux of label from preincubated tissue appeared to occur in three phases (t1/2=7.2 s, 4.0 min, 35.2 min). Efflux was correlated with stomatal re-opening. The results confirm that ABA can accumulate in the epidermis of C. communis.

A definition of plant hormone sensitivity is proposed which is based on the initial rates of responses at different hormone concentrations. By analogy with enzyme kinetics, it is concluded that simple concentration-response curves can be described by a function containing three 'sensitivity parameters'. Objective methods for determining the values of these parameters and for comparing curves are described. These methods are used to describe the responses of Commelina communis L. stomata to abscisic acid. If certain assumptions are valid, the sensitivity parameters can be assigned physicochemical meanings. To validate the assumptions, certain experimental criteria must be fulfilled and these are discussed.

The degree of success achieved in obtaining protoplasts in consistent yields and quality can vary enormously. This can depend on the plant species or tissue used and its age (e.g. Watts et al. 1974) or on the nature of the digestion medium (e.g. Zeiger and Hepler 1976). In parallel with the increase in popularity of protoplasts as experimental tools, more commercial cellulases and pectinases have become available and the use of various non-enzyme components in isolation media has become more common. Unfortunately, our knowledge of the properties of enzyme mixtures in use or of other “additives” has not kept pace, and there is a need for quantitative data on the effects of particular products on protoplast yield and physiology. In this chapter we present the results of a study on the properties of Cellulysin, a commercial product in general use, and describe some effects of various additives to the isolation medium.

Guard cell protoplasts (GCP’s) were first studied by Zeiger and Hepler (1976), although Davey et al. (1974) had earlier experimented with isolated epidermal cell protoplasts from tobacco leaves. The microchamber method pioneered by Zeiger and Hepler allowed the observation of small numbers of GCP’s adhering to the remnants of strips of leaf epidermis, but did not allow a full range of microbiological techniques to be employed (Zeiger 1981). The full potential of the protoplast technique could only be realised when methods were developed for obtaining high yields of GCP’s and for their purification (Schnabl et al. 1978, Outlaw et al. 1981, Fitzsimons and Weyers 1983).

Photosynthesis of Phaseolus vulgaris L. leaves was measured after treatment with microcystin-LR (MC-LR), a potent cyanobacterial toxin and inhibitor of protein phosphatases 1 and 2A. The net photosynthetic rate fell by over 50 % within 8 h of dipping leaves in MC-LR solution. This inhibitory effect was observed when leaves were treated once with concentrations above 10 -2 mol m -3 . At 10 -2 mol m -3 , the inhibition caused by MC-LR was transient, and net photosynthesis rates essentially recovered after 5d, whereas at 10 -1 mol m -3 , the net photosynthesis rate in treated leaves was still 42% of controls after 8 d. Necrosis was observed at the higher concentration, but not usually below 10 -2 mol m -3 . Analysis of net photosynthetic rate as a function of internal CO 2 concentration and photosynthetically active photon flux density indicated that both the CO 2 -saturated rate of photosynthesis and the carboxylation efficiency were lowered when MC-LR-treated leaves were exposed to photosynthetically-saturating light. When the leaves were exposed to 2.0x10 -5 mol m -3 MC-LR solution repeatedly, the photosynthetic rate was significantly reduced after 7 d, suggesting that intermittent exposure of P. vulgaris leaves to low concentrations of MC-LR brings about an effect on photosynthesis more inhibitory than that of a single exposure to high concentrations of MC-LR. These results indicate that relatively low concentrations of MC-LR cause damage to the photosynthetic apparatus of P. vulgaris in situ. We discuss the significance of these findings in relation to current spray irrigation practice for crop plants involving the use of water containing cyanobacterial blooms and microcystins.

cost of equipment (Pearcy et al., 1989; Weyers and Meidner, 1990; Willmer and Fricker, 1996). An important It is proposed that a measurement of the peristomatal criterion for choosing a technique is scale of measurement: groove distance (PGD) of guard cells on surface depending on the purpose of the study, researchers may