Rainer Stahlberg

The Propagation of Slow Wave Potentials in Pea Epicotyls

Slow wave potentials are considered to be electric long-distance signals specific for plants, although there are conflicting ideas about a chemical, electrical, or hydraulic mode of propagation. These ideas were tested by comparing the propagation of hydraulic and electric signals in epicotyls of pea (Pisum sativum L). A hydraulic signal in the form of a defined step increase in xylem pressure (Px) was applied to the root of intact seedlings and propagated nearly instantly through the epicotyl axis while its amplitude decreased with distance from the pressure chamber. This decremental propagation was caused by a leaky xylem and created an axial Px gradient in the epicotyl. Simultaneously along the epicotyl surface, depolarizations appeared with lag times that increased acropetally with distance from the pressure chamber from 5 s to 3 min. When measured at a constant distance, the lag times increased as the size of the applied pressure steps decreased. We conclude that the Px gradient in the epicotyl caused local depolarizations with acropetally increasing lag times, which have the appearance of an electric signal propagating with a rate of 20 to 30 mm min-1. This static description of the slow wave potentials challenges its traditional classification as a propagating electric signal.

Slow Wave Potentials — a Propagating Electrical Signal Unique to Higher Plants

Plants have at least three kinds of propagating electrical signals. In addition to a sustained wound potential (WP) that stops a few millimeters from dying cells, these signals are action potentials (APs) and slow wave potentials (SWPs). All three signals consist of a transient change in the membrane potential of plant cells (depolarization and subsequent repolarization), but only SWPs and APs make use of the vascular bundles to achieve a potentially systemic spread through the entire plant. The principal difference used to differentiate SWPs from APs is that SWPs show longer, delayed repolarizations. Unfortunately, SWP repolarizations also show a large range of variation that makes a distinction difficult. SWPs and APs do differ more clearly, however, in the causal factors stimulating their appearance, the ionic mechanisms of their depolarization and repolarization phases as well as the mechanisms and pathways of propagation. The depolarizations of a SWP arise with an increase in turgor pressure cells experience in the wake of a hydraulic pressure wave that spreads through the xylem conduits after rain, embolism, bending, local wounds, organ excision and local burning. The generation of APs occurs under different environmental and internal influences (e.g. touch, light changes, cold treatment, cell expansion) that — mediated through varying generator potentials — trigger a voltage-dependent depolarization spike in an all-or-nothing manner. While APs and WPs can be triggered in excised organs, SWPs depend on the pressure difference between the atmosphere and an intact plant interior. High humidity and prolonged darkness will also suppress SWP signaling. The ionic mechanism of the SWP is thought to involve a transient shutdown of a P-type H+-ATPase in the plasma membrane and differs from the mechanism underlying APs. Another defining characteristic of SWPs is the hydraulic mode of propagation that enables them — but not APs — to pass through killed or poisoned areas. Unlike APs they can easily communicate between leaf and stem. SWPs can move in both directions of the plant axis, while their amplitudes show a decrement of about 2.5% cm−1 and move with speeds that can be slower than APs in darkness and faster in bright light. The SWPs move with a rapid pressure increase that establishes an axial pressure gradient in the xylem. This gradient translates distance (perhaps via changing kinetics in the rise of turgor pressure) into increasing lag phases for the pressure-induced depolarizations in the epidermis cells. Haberlandt (1890), after studying propagating responses in Mimosa pudica, suggested the existence of hydraulically propagated electric potentials at a time when only APs were conceivable. It took a century to realize that such signals do exist and that they coincide with the characteristics of SWPs rather than those of APs. Moreover, we begin to understand that SWPs are not only ubiquitous among higher plants but represent a unique, defining characteristic without parallels in lower plants or animals.

The effect of light on membrane potential, apoplastic pH and cell expansion in leaves of Pisum sativum L. var. Argenteum.

Abstract. The connection between three light responses of green leaf cells-membrane potential (Vm), H+ net efflux and growth, was analyzed. Illumination of mesophyll cells in leaves from Argenteum peas caused two rapid responses: (i) a de- and repolarization of Vm and (ii) an alkalinization of the apoplast. The rapid responses were completely eliminated by the photosynthetic inhibitor 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea (DCMU) but not affected by ortho-vanadate, an inhibitor of the plasma membrane (PM) H+-ATPase. The rapid changes were followed by a set of delayed responses: (i) a slow, gradual hyperpolarization of Vm, (ii) a gradual acidification of the mesophyll apoplast and (iii) an increased rate of elongation. These three light responses persisted under DCMU but were completely eliminated by vanadate. The data show that the delayed (in contrast to the rapid) responses were due to a stimulation of PM H+ pumps which occurred independently of non-cyclic photosynthetic electron transport and the “dark” processes depending on it. When the rapid responses were blocked by DCMU, light-induced acidification, hyperpolarization of the membrane potential and growth proceeded simultaneously. A shared (4-min) lag phase indicated slower signal processing in mesophyll than in epidermal cells where light stimulation of PM H+ pumps was rapid.

Rapid alterations in growth rate and electrical potentials upon stem excision in pea seedlings

Excision of the epicotyl base of pea (Pisum sativum L.) seedlings in air results in a fast drop in the growth rate and rapid transient membrane depolarization of the surface cells near the cut. Subsequent immersion of the cut end into solution leads to a rapid, transient rise in the epicotyl growth rate and an acropetally propagating depolarization with an amplitude of about 35 mV and a speed of approx. 1 mm · s−1. The same result can be achieved directly by excision of the pea epicotyl under water. Shape, amplitude and velocity of the depolarization characterize it as a “slow-wave potential”. These results indicate that the propagating depolarization is caused by a surge in water uptake. Neither a second surge in water uptake (measured as a rapid increase in growth rate when the cut end was placed in air and then back into solution) nor another cut can produce the depolarization a second time. Cyanide suppresses the electrical signal at the treated position without inhibiting its transmission through this area and its development in untreated parts of the epicotyl. The large depolarization and repolarization which occur in the epidermal and subepidermal cells are not associated with changes in cell input resistance. Both results indicate that it is a transient shut-down of the plasma-membrane proton pump rather than large ion fluxes which is causing the depolarization. We conclude that the slow wave potential is spread in the stem via a hydraulic surge occurring upon relief of the negative xylem pressure after the hydraulic resistance of the root has been removed by excision.

Induction and ionic basis of slow wave potentials in seedlings of Pisum sativum L.

Slow wave potentials (SWPs) are transient depolarizations which propagate substantial distances from their point of origin. They were induced in the epidermal cells of pea epicotyls by injurious methods such as root excision and heat treatment, as well as by externally applied, defined steps in xylem pressure (Px)at in the absence of wounding. The common principle of induction was a rapid increase in Px. Such a stimulus appeared under natural conditions after (i) bending of the epicotyl, (ii) wounding of the epidermis, (iii) rewatering of dehydrated roots, and (iv) embolism. The induced depolarization was not associated with a change in cell input resistance. This result and the ineffectiveness of ion channel blockers point to H+-pumps rather than ion channels as the ionic basis of the SWP. Stimuli such as excision, heat treatment and pressure steps, which generate SWPs, caused a transient increase in the fluorescence intensity of epicotyls loaded with the pH-indicator DM-NERF, a 2′, 7′-dimethyl derivative of rhodol, but not of those loaded with the pH indicator 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF). Matching kinetics of depolarization and pH response identify a transient inactivation of proton pumps in the plasma membrane as the causal mechanism of the SWP. Feeding pump inhibitors to the cut surface of excised epicotyls failed to chemically simulate a SWP; cyanide, azide and 2,4-dinitrophenol caused sustained, local depolarizations which did not propagate. Of all tested substances, only sodium cholate caused a transient and propagating depolarization whose arrival in the growing region of the epicotyl coincided with a transient growth rate reduction.

Long-term inhibition by auxin of leaf blade expansion in bean and Arabidopsis.

The role of auxin in controlling leaf expansion remains unclear. Experimental increases to normal auxin levels in expanding leaves have shown conflicting results, with both increases and decreases in leaf growth having been measured. Therefore, the effects of both auxin application and adjustment of endogenous leaf auxin levels on midrib elongation and final leaf size (fresh weight and area) were examined in attached primary monofoliate leaves of the common bean (Phaseolus vulgaris) and in early Arabidopsis rosette leaves. Aqueous auxin application inhibited long-term leaf blade elongation. Bean leaves, initially 40 to 50 mm in length, treated once with α-naphthalene acetic acid (1.0 mm), were, after 6 d, approximately 80% the length and weight of controls. When applied at 1.0 and 0.1 mm, α-naphthalene acetic acid significantly inhibited long-term leaf growth. The weak auxin, β-naphthalene acetic acid, was effective at 1.0 mm; and a weak acid control, benzoic acid, was ineffective. Indole-3-acetic acid (1 μm, 10 μm, 0.1 mm, and 1 mm) required daily application to be effective at any concentration. Application of the auxin transport inhibitor, 1-N-naphthylphthalamic acid (1% [w/w] in lanolin), to petioles also inhibited long-term leaf growth. This treatment also was found to lead to a sustained elevation of leaf free indole-3-acetic acid content relative to untreated control leaves. Auxin-induced inhibition of leaf growth appeared not to be mediated by auxin-induced ethylene synthesis because growth inhibition was not rescued by inhibition of ethylene synthesis. Also, petiole treatment of Arabidopsis with 1-N-naphthylphthalamic acid similarly inhibited leaf growth of both wild-type plants and ethylene-insensitive ein4 mutants.

Shade-Induced Action Potentials in Helianthus annuus L. Originate Primarily from the Epicotyl.

Repeated observations that shading (a drastic reduction in illumination rate) increased the generation of spikes (rapidly reversed depolarizations) in leaves and stems of many cucumber and sunflower plants suggests a phenomenon widespread among plant organs and species. Although shaded leaves occasionally generate spikes and have been suggested to trigger systemic action potentials (APs) in sunflower stems, we never found leaf-generated spikes to propagate out of the leaf and into the stem. On the contrary, our data consistently implicate the epicotyl as the location where most spikes and APs (propagating spikes) originate. Microelectrode studies of light and shading responses in mesophyll cells of leaf strips and in epidermis/cortex cells of epicotyl segments confirm this conclusion and show that spike induction is not confined to intact plants. 90% of the epicotyl-generated APs undergo basipetal propagation to the lower epicotyl, hypocotyl and root. They propagate with an average rate of 2 ± 0.3 mm s−1 and always undergo a large decrement from the hypocotyl to the root. The few epicotyl-derived APs that can be tracked to leaf blades (< 10%) undergo either a large decrement or fail to be transmitted at all. Occasionally (5% of the observations) spikes were be generated in hypocotyl and lower epicotyl that moved towards the upper epicotyl unaltered, decremented, or amplified. This study confirms that plant APs arise to natural, nontraumatic changes. In simultaneous recordings with epicotyl growth, AP generation was found to parallel the acceleration of stem growth under shade. The possible relatedness of both processes must be further investigated.

Chlorophyll is not the primary photoreceptor for the stimulation of P-type H+ pump and growth in variegated leaves of Coleus x hybridus.

Abstract. There has been persisting controversy over the role of photosynthesis in the stimulation of the plasma membrane H+-ATPase and growth of dicotyledonous leaves by light. To investigate this, we compared the effects of light on growth, H+ net efflux and membrane potential (Vm) of strips which contained either only chlorophyll-free (white) mesophyll cells or chlorophyll-containing (green) cells cut from variegated Coleus leaves. White mesophyll cells responded to white, blue and red light with a hyperpolarization of Vm, an acidification of the apoplast and a promotion of growth, all of which began after a lag of 2–7 min. In contrast, green mesophyll cells showed a biphasic light response in which the hyperpolarization and the acidification were preceded by a rapid depolarization of Vm and an alkalinization of the apoplast. Nevertheless, green and white tissues showed comparable growth promotions in response to light. The light response of the leaf mesophyll is a composite of two separate photosystems. The initial depolarization and alkalinization are mediated by photosynthesis and blocked by 3-(3,4-dichlorophenyl)-1,1-dimethylurea. The slower hyperpolarization, acidification and growth response, on the other hand, are clearly in response to light absorption by pigments other than chlorophyll.

Sensors and actuators inherent in biological species

This paper addresses examples of sensing and active mechanisms inherent in some biological species where both plants and animals cases are discussed: mechanosensors and actuators in Venus Fly Trap and cucumber tendrils, chemosensors in insects, two cases of interactions between different kingdoms, (i) cotton plant smart defense system and (ii) bird-of-paradise flower and hamming bird interaction. All these cases lead us to recognize how energy-efficient and flexible the biological sensors and actuators are. This review reveals the importance of integration of sensing and actuation functions into an autonomous system if we make biomimetic design of a set of new autonomous systems which can sense and actuate under a number of different stimuli and threats.

Historical Introduction to Plant Electrophysiology

Plant Electrophysiology – Theory & Methods (ed. by Volkov)