Both action potentials and variation potentials induce proteinase inhibitor gene expression in tomato

Tomato plants (Lycopersicon esculentum) accumulate proteinase inhibitor 2 (pin2) mRNA in response to insect attack, crushing and flaming in leaves distant from those treated. Most earlier work suggests that the systemic wound signals are chemical; here we try to determine whether electrical or physical (hydraulic) signals can also evoke pin expression. We used a mild flame to evoke a systemic hydraulic signal and its local electrical aftermath, the variation potential (VP), and we used an electric stimulus to evoke a systemic electrical signal, the action potential (AP). We determined the kinetic parameters of both the VP and AP. Flame-wounded plants essentially always exhibited major electrical responses throughout the plant and a several-fold increase in pin2 mRNA within 1 h. Electrically stimulated plants that generated and transmitted a signal (AP) into the analyzed leaf exhibited similarly large, rapid increases in pin2 mRNA levels. Plants which generated no signal, or signals of just a few microvolts, had unchanged levels of pin2 mRNA. Since the AP and VP both arrived in the receiving leaf before accumulation of pin2 mRNA began, we conclude that, in addition to the previously shown chemical signals, both hydraulically induced VPs and electrically induced APs are capable of evoking pin2 gene expression.     

Action potentials and variation potentials in sunflower: An analysis of their relationships and distinguishing characteristics

Sunflower plants ( Helianthus annuus L.) were given an electrical stimulus to the stem or a heat (flame)‐wound to a single leaf or a cotyledon. The resulting electrical activity was monitored with extracellular electrodes. An electrical stimulus applied to the stem frequently evoked an action potential (AP), but never a variation potential (VP). In contrast, a heat‐wound applied to a leaf virtually always elicited a VP, which was often accompanied by one or more superimposed spikes (putative APs). The kinetic parameters of the AP and the VP were investigated. The AP appears to propagate without decrement in velocity or magnitude, whereas the VP parameters decrease significantly with distance. The heat stimulus triggered rapid alterations in stem elongation/contraction, which preceded changes in electrical potential, indicating the transmission of a hydraulic signal. Light‐off and light‐on stimuli evoked negative‐ and positive‐going changes in extracellular electrical potential, respectively, corresponding to de‐ and hyper‐polarization of the plasma membrane. Membrane depolarization (extracellularly manifested as a VP) evoked by both the light‐off and heat‐wounding stimuli was able to trigger one or more APs. We interpret these results to suggest that APs are "genuine" electrical signals involving voltage‐gated ion channels or pumps, which can be evoked directly by electrical stimulation or indirectly by changes in membrane potential occurring during the VP or after the light‐off stimulus. In contrast, VPs appear to be a local (non‐transmissible) electrical consequence of the passage of a rapidly transmitted hydraulic signal in the xylem, presumably acting on mechanosensitive ion channels or pumps in adjacent living cells.     

Remote stimulation by heat induces characteristic membrane-potential responses in the veins of wild-type and abscisic acid-deficient tomato plants

The systemic induction of proteinase inhibitor genes in tomato plants is either mediated by fast electrical signals or alternatively by chemical messengers. In the present study we analyzed the pathway of the electrical signal. The question of which cell types are involved in this pathway of long-distance signaling within plants is still controversial. To identify these we inserted microelectrodes into the veins of tomato leaves (Lycopersicon esculentum Mill. cv. Moneymaker). A newly developed computer program and microcomputer interface enabled us to position these microelectrodes inside the vein with an accuracy of 1 μm. Due to this precision in positioning we were able to demonstrate that the pathway of the electrical signal is not restricted to a specific tissue type, e.g. the phloem. In particular, the entire vein contributes to the propagation of the electrical wave along the plant. Therefore, an apoplastic contribution to the long-distance signal transduction mechanism appears most likely. To furthermore investigate the involvement of cis-abscisic acid (ABA) in this long-distance signal transduction pathway, ABA-deficient tomato mutants (Lycopersicon esculentum cv. Sitiens) were used in comparison to the wild type. Significant differences between the membrane-potential relaxation kinetics of the wild type and the mutants could be detected. Wild-type tomato plants exhibited six characteristic classes of membrane-potential relaxation kinetics following heat treatment. In contrast, the ABA-deficient mutants were more restricted in terms of their relaxation upon heat stimulation. The responses in the membrane potential of all cells within a vein consisted of only three categories. In conclusion, ABA did not affect all cells within the vein in a similar manner. Single cells exhibited different response patterns to systemic heat application in the presence of ABA. Moreover, ABA had a pronounced effect on the resting potentials of individual cells within the veins of tomato. Received: 1 July 1997 / Accepted: 16 January 1998     

Time-Resolved Analysis of Signals Involved in Systemic Induction of Pin2 Gene Expression

The systemic induction of proteinase inhibitor genes in tomato plants is mediated either by electrical signals, hydraulic signals or chemical messengers. In the present study we analyzed the effects of mechanical wounding, heat treatment and electrical current application on wild-type tomato plants (Lycopersicon esculentum Mill, cv Moneymaker) and ABA-deficient mutants of tomato (sitiens). Kinetic studies revealed that systemic Pin2 gene expression could be slightly induced by the fast transient membrane potential change which left the damaged leaf within 30–60s after wounding. Moreover, a signal leaving the damaged tissue between 2 and 4 minutes after wounding was responsible for a significant amplification of Pin2 gene expression. This signal could either be a decrease in turgor pressure, which occurred 3–4min after treatment, or a slow electrical transient. In addition, mechanical wounding and electrical current seem to involve ABA to induce changes in membrane potential and to promote Pin2 gene expression. In contrast, heat triggers fast and slow electrical transients leading to an induction of Pin2 gene expression within the plant independently of ABA. Turgor pressure, in turn, is presumably adjusted in relation to ionic movements across the membrane, elucidated by membrane potential recordings. In conclusion, wound-induced changes in membrane potential seem to be dependent on the endogenous level of ABA. These shifts in membrane potentials, in turn, are involved in regulation of turgor pressure within the plant.     

Kinetics of wound-induced hydraulic signals and variation potentials in wheat seedlings

Displacement transducers were used to demonstrate that localised wounding causes a rapid and systemic increase in leaf thickness in seedlings of wheat (Triticum durum Desf. cv. Iva). These increases are interpreted as reflecting wound-induced hydraulic signals. The duration of the wound-induced increase was found to be about 1 h or more, and it was shown that repeated wounds could induce repeated responses. The increase occurred even when plants had no access to an external water supply. Change in leaf thickness was shown closely to reflect change in leaf water potential. The velocity and kinetics of the wound-induced hydraulic signal were measured using multiple transducers ranged along a single leaf. The front of the signal was shown to travel through the plant at rates of at least 10 cm · s^–1. Development of the increase in leaf thickness was found to be relatively faster furthest from the wound. Onset of the change in leaf thickness in leaves remote from the wound was shown to precede onset of changes in surface electrical potential (variation potential) which are also induced by wounding. In contrast to reports from other species, variation potentials in wheat were here shown to spread extremely rapidly, at rates similar to that of the hydraulic signal. These data support the view that wound-induced hydraulic signals are the trigger for variation potentials in wheat.Symbol: _w water potential Grateful thanks are due to Paul Springer of the HRI (Wellesbourne) mechanical workshop for building equipment, and to H.G. Jones for helpful discussion.     

Age-dependent changes of photosynthetic responses induced by electrical signals in wheat seedlings

Electrical signals in plants, namely, the action potential (AP) and variation potential (VP) alter the activity of many processes, including photosynthesis. The functional responses induced by electrical signals vary in direction and amplitude, which might be determined by variable conditions of plants prior to stimulation, by the development stage in particular. In this work, the parameters of VP-induced photosynthetic responses were analyzed at various stages of wheat seedling development. Local wounding of the second leaf in wheat plants induced the propagation of VP and altered the activity of photosynthesis at a distance from the wound location. The amplitude of VP was enlarged when the seedling age increased from 11 to 18 days. The VP-induced photosynthetic response changed with age both qualitatively and quantitatively. The amplitude of VP-induced changes in CO₂ assimilation and nonphotochemical quenching (NPQ) increased with age, which might be due to the increase in VP amplitude and associated changes in Ca²⁺ and H⁺ concentrations. The quantum yield of photosystem II photoreaction was subject to age-dependent changes: the photochemical quantum yield (γ(PSII)) was found to increase after VP in young leaves, whereas the decline in γ(PSII) was observed after the VP propagation in mature leaves. The results may explain the diversity of photosynthetic responses caused by the electrical signals.     

Characterization of the Variation Potential in Sunflower

A major candidate for intercellular signaling in higher plants is the stimulus-induced systemic change in membrane potential known as variation potential (VP). We investigated the mechanism of occurrence and long-distance propagation of VP in sunflower (Helianthus annuus L.) plants. Here we present evidence of the relationship among injury-induced changes in xylem tension, turgor pressure, and electrical potential. Although locally applied wounding did trigger a change in membrane potential, it evoked even faster changes in tissue deformation, apparently resulting from pressure surges rapidly transmitted through the xylem and experienced throughout the plant. Externally applied pressure mimicked flame wounding by triggering an electrical response resembling VP. Our findings suggest that VP in sunflower is not a propagating change in electrical potential and not the consequence of chemicals transmitted via the xylem, affecting ligand-modulated ion channels. Instead, VP appears to result from the surge in pressure in the xylem causing a change in activity of mechanosensitive, stretch-responsive ion channels or pumps in adjacent, living cells. The ensuing ion flux evokes local plasma membrane depolarization, which is monitored extracellularly as VP.     

Proton cellular influx as a probable mechanism of variation potential influence on photosynthesis in pea

Electrical signals (action potential and variation potential, VP) caused by environmental stimuli are known to induce various physiological responses in plants, including changes in photosynthesis; however, their functional mechanisms remain unclear. In this study, the influence of VP on photosynthesis in pea (Pisum sativum L.) was investigated and the proton participation in this process analysed. VP, induced by local heating, inactivated photosynthesis and activated respiration, with the initiation of the photosynthetic response connected with inactivation of the photosynthetic dark stage; however, direct VP influence on the light stage was also probable. VP generation was accompanied with pH increases in apoplasts (0.17–0.30 pH unit) and decreases in cytoplasm (0.18–0.60 pH unit), which probably reflected H⁺‐ATPase inactivation and H⁺ influx during this electrical event. Imitation of H⁺ influx using the protonophore carbonyl cyanide m‐chlorophenylhydrazone (CCCP) induced a photosynthetic response that was similar with a VP‐induced response. Experiments on chloroplast suspensions showed that decreased external pH also induced an analogous response and that its magnitude depended on the magnitude of pH change. Thus, the present results showed that proton cellular influx was the probable mechanism of VP's influence on photosynthesis in pea. Potential means of action for this influence are discussed.     

Electrical and chemical signals involved in short-term systemic photosynthetic responses of tobacco plants to local burning

Short-term (up to 1 h) systemic responses of tobacco (Nicotiana tabacum cv. Samsun) plants to local burning of an upper leaf were studied by measuring the following variables in a distant leaf: extracellular electrical potentials (EEPs); gas exchange parameters; fast chlorophyll fluorescence induction; and endogenous concentrations of three putative chemical signaling compounds—abscisic (ABA), jasmonic (JA), and salicylic (SA) acids. The first detected response to local burning in the distant leaves was in EEP, which started to decline within 10–20 s of the beginning of the treatment, fell sharply for ca. 1–3 min, and then tended to recover within the following hour. The measured gasometric parameters (stomatal conductance and the rates of transpiration and CO₂ assimilation) started to decrease 5–7 min after local burning, suggesting that the electrical signals may induce stomatal closure. These changes were accompanied by systemic increases in the endogenous ABA concentration followed by huge systemic rises in endogenous JA levels started after ca. 15 min, providing the first evidence of short-term systemic accumulation of these plant hormones in responses to local burning. Furthermore, JA appears to have an inhibitory effect on CO₂ assimilation. The correlations between the kinetics of the systemic EEP, stomatal, photosynthetic, ABA, and JA responses suggest that (1) electrical signals (probably induced by a propagating hydraulic signal) may trigger chemical defense-related signaling pathways in tobacco plants; (2) both electrical and chemical signals are interactively involved in the induction of short-term systemic stomatal closure and subsequent reductions in the rate of transpiration and CO₂ assimilation after local burning events.     

Development of transgenic pearl millet (Pennisetum glaucum (L.) R. Br.) plants resistant to downy mildew

Transgenic pearl millet lines expressing pin gene—exhibiting high resistance to downy mildew pathogen, Sclerospora graminicola—were produced using particle-inflow-gun (PIG) method. Shoot-tip-derived embryogenic calli were co-bombarded with plasmids containing pin and bar genes driven by CaMV 35S promoter. Bombarded calli were cultured on MS medium with phosphinothricin as a selection agent. Primary transformants 1T₀, 2T₀, and 3T₀ showed the presence of both bar and pin coding sequences as evidenced by PCR and Southern blot analysis, respectively. T₁ progenies of three primary transformants, when evaluated for downy mildew resistance, segregated into resistant and susceptible phenotypes. T₁ plants resistant to downy mildew invariably exhibited tolerance to Basta suggesting co-segregation of pin and bar genes. Further, the downy mildew resistant 1T₁ plants were found positive for pin gene in Southern and Northern analyses thereby confirming stable integration, expression, and transmission of pin gene. 1T₂ progenies of 1T₀ conformed to dihybrid segregation of 15 resistant:1 susceptible plants.     

The relationship between wound-induced proteinase inhibitors and hydraulic signals in tomato seedlings

Localized wounding is known to induce systemic proteinase inhibitors (PI) in seedlings of tomato (Lycopersicon esculentum L.). Inhibitors of elastase (EC 3.4.21.36) were shown here to be among those systemically induced by wounding, and a simple rapid assay for PI based on elastase was developed. Using this assay, the nature of the systemic signalling system (‘PIIF’) was investigated. Hydraulic signals were shown to be induced in tomato by localized wounds. These signals travelled throughout the plant well within the lag time before appearance of systemic wound-induced PI. A number of correlations were drawn between the occurrence of the hydraulic signals and induction of systemic PI, suggesting that hydraulic signals might be the PIIF, or a component of it. It was shown that systemic hydraulic signals could be triggered, without significant wounding, by excision of a single leaflet through the submerged petiole. These hydraulic signals were similar in both kinetics and magnitude to those induced by localized wounding. However, they did not induce systemic PI. In addition, it was shown that systemic events almost as rapid as wound-induced hydraulic signals could be induced without wounding, under certain environmental conditions. This indicates that rapid hydraulic signals do not provide a specific signal of wounding. These findings demonstrate that hydraulic signals per se are not the PIIF.     

Enhancement of tolerance to soft rot disease in the transgenic Chinese cabbage (<Emphasis Type="Italic">Brassica rapa</Emphasis> L. ssp. <Emphasis Type="Italic">pekinensis</Emphasis>) inbred line,Kenshin

We developed a transgenic Chinese cabbage (Brassica rapa L. ssp. pekinensis) inbred line, Kenshin, with high tolerance to soft rot disease. Tolerance was conferred by expression of N-acyl-homoserine lactonase (AHL-lactonase) in Chinese cabbage through an efficient Agrobacterium-mediated transformation method. To synthesize and express the AHL-lactonase in Chinese cabbage, the plant was transformed with the aii gene (AHL-lactonase gene from Bacillus sp. GH02) fused to the PinII signal peptide (protease inhibitor II from potato). Five transgenic lines were selected by growth on hygromycin-containing medium (3.7% transformation efficiency). Southern blot analysis showed that the transgene was stably integrated into the genome. Among these five transgenic lines, single copy number integrations were observed in four lines and a double copy number integration was observed in one transgenic line. Northern blot analysis confirmed that pinIISP-aii fusion gene was expressed in all the transgenic lines. Soft rot disease tolerance was evaluated at tissue and seedling stage. Transgenic plants showed a significantly enhanced tolerance (2–3-fold) to soft rot disease compared to wild-type plants. Thus, expression of the fusion gene pinIISP-aii reduces susceptibility to soft rot disease in Chinese cabbage. We conclude that the recombinant AHL-lactonase, encoded by aii, can effectively quench bacterial quorum-sensing and prevent bacterial population density-dependent infections. To the best of our knowledge, the present study is the first to demonstrate the transformation of Chinese cabbage inbred line Kenshin, and the first to describe the effect of the fusion gene pinIISP-aii on enhancement of soft rot disease tolerance.     

Electrical signals in higher plants: Mechanisms of generation and propagation

Local stimulation induces generation and propagation of electric signals in higher plants. Noninvasive stimulus induces an action potential and damaging influences lead to the variation potential. The mechanism of the generation of an action potential is rather complex in nature and is associated with both activation of ion channels (Ca²⁺, Cl^–, and K⁺) and transient change in the activity of the plasma membrane H⁺-ATPase. Generation of the variation potential, the duration of which is considerably longer than that of the action potential, is based on transient inactivation of the electrogenic pump; however, passive ion fluxes also contribute to such process, which causes qualitative similarity of the mechanisms of action potential and variation potential generation. Propagation of electrical signals mainly occurs in conducting bundles; thus, transfer of an action potential is associated with vascular parenchyma and sieve elements, while the variation potential is connected to the xylem vessels. The mechanism of the distribution the action potential is similar to nerve impulse transmission, while generation of the variation potential is induced by transfer of a chemical substance, whose propagation is accelerated by a hydraulic wave.     

Tobacco plants epigenetically suppressed in phenylalanine ammonia-lyase expression do not develop systemic acquired resistance in response to infection by tobacco mosaic virus

The response of tobacco (Nicotiana tabacum L. cv. Xanthinc) plants, epigenetically suppressed for phenylalanine ammonia-lyase (PAL) activity, was studied following infection by tobacco mosaic virus (TMV). These plants contain a bean PAL2 transgene in the sense orientation, and have reduced endogenous tobacco PAL mRNA and suppressed production of phenylpropanoid products. Lesions induced by TMV infection of PAL-suppressed plants are markedly different in appearance from those induced on control plants that have lost the bean transgene through segregation, with a reduced deposition of phenofics. However, they develop at the same rate as on control tobacco, and pathogenesis-related (PR) proteins are induced normally upon primary infection. The levels of free salicylic acid (SA) produced in primary inoculated leaves of PAL-suppressed plants are approximately fourfold lower than in control plants after 84 h, and a similar reduction is observed in systemic leaves. PR proteins are not induced in systemic leaves of PAL-suppressed plants, and secondary infection with TMV does not result in the restriction of lesion size and number seen in control plants undergoing systemic acquired resistance (SAR). In grafting experiments between wild-type and PAL-suppressed tobacco, the SAR response can be transmitted from a PAL-suppressed root-stock, but SAR is not observed if the scion is PAL-suppressed. This indicates that, even if SA is the systemic signal for establishment of SAR, the amount of pre-existing phenylpropanoid compounds in systemic leaves, or the ability to synthesize further phenylpropanoids in response to the systemic signal, may be important for the establishment of SAR. Treatment of PAL-suppressed plants with dichloro-isonicotinic acid (INA) induces PR protein expression and SAR against subsequent TMV infection. However, treatment with SA, while inducing PR proteins, only partially restores SAR, further suggesting that de novo synthesis of SA, and/or the presence or synthesis of other phenylpropanoids, is required for expression of resistance in systemic leaves.     

Chemical Signal as a Rapid Long-Distance Information Messenger After Local Wounding of a Plant?

A series of works have described an important role of chemical signaling compounds in generation of the stress response of plants in both the wounded and distant undamaged plant tissues. However, pure chemical signals are often not considered in the fast (minutes) long-distance signaling (systemic response) because of their slow propagation speed. Physical signals (electrical and hydraulic) or a combination of the physical and chemical signals (hydraulic dispersal of solutes) have been proposed as possible linkers of the local wound and the rapid systemic response. We have recently demonstrated an evidence for involvement of chemical compounds (jasmonic and abscisic acids) in the rapid (within 1 hour) inhibition of photosynthetic rate and stomata conductance in distant undamaged tobacco leaves after local burning. The aim of this addendum is to discuss plausible mechanisms of a rapid long-distance chemical signaling and the putative interactions between the physical and chemical signals leading to the fast systemic response.Key Words: tobacco, local burning, systemic response, hydraulic surge, electrical signal, abscisic acid, jasmonic acidPlants have evolved an amazingly complex system of defence-related strategies to protect themselves upon local wounding.^1–7 Important characteristics of self-defence responses of plants are their velocity and ubiquity. Indeed, fast (minutes to hours) responses to injurious factors have been detected in the site of injury and in distant regions (systemic response) in various plants.^8–11 These findings suggest that a signal generated by an attack to one leaf is transmitted through a whole plant. Several kinds of chemical^3,6 and physical¹² signals induced by local wounding and even their combination¹³ have been implicated. However, a little is known about the interactions of these signals and about the mechanisms of initiation of the short-term systemic responses.We have used a model system—tobacco plants exposed to the local burning—to study the signals involved in rapid wound responses of photosynthetic apparatus.¹⁴ Local burning of an upper leaf of a tobacco plant induced rapid changes in surface electrical potential (within seconds) and a pronounced fast decline in the stomatal conductance, CO₂ assimilation and transpiration (within minutes) in the basipetal direction (Fig. 1). Moreover, we have detected a fast (within minutes) transient increase in levels of endogenous abscisic acid (ABA) followed by a huge rapid rise in endogenous jasmonic acid (JA) in the leaf below the burned one. ABA and jasmonates are known to be involved in signaling pathways leading to stomatal closure and downregulation of photosynthesis.^15,16 Increases in ABA and/or JA levels have only previously been detected in remote untreated tissues several hours after local wounding^8,9 suggesting that chemical signals are too slow to induce rapid systemic response. Previous works have reported that fast physical (electrical) signals play an essential role in short-term systemic photosynthetic responses.^11,17 However, a several-minutes delayed stomata closing response after the initiation of electrical potential changes has been reported in Mimosa¹⁸ and in our case in tobacco¹⁴ plants. Therefore, the guard cell deflation is most likely triggered not only by the electrical signal, but also by indirect factors. Based on close correlations, our results now provide a new evidence for the idea that chemical signals (ABA and mainly JA) participate in mediating the short-term systemic photosynthetic responses to local burning in tobacco plants.Open in a separate windowFigure 1The model of putative signalling pathways leading to the rapid systemic responses of tobacco plants to local burning. Hypothetical (dashed lines) local responses, generation of signals and transport processes and detected (full line) systemic responses are demonstrated. For details see the text.The question is how do the physical (electrical and/or hydraulic) and chemical signals act? They may independently induce specific elements of systemic responses. However, they are more likely to act in a coordinated, interactive fashion. In this scenario (see Fig. 1), within first minutes after the local burning, hydraulic surge transmitted basipetally and acropetally through the xylem would transport chemicals released at the wound site (hydraulic dispersal¹⁹) and evoke changes in the ion fluxes in surrounding living cells leading to the local electrical activity.^12,13 The hypothesis of hydraulic dispersal is supported by our preliminary experiments with the fluorescent dye Rhodamine B applied on cut petiole of the upper leaf of tobacco plants showing that solutes can be rapidly transported (within minutes) basipetally following wounding.The rapid kinetics and transient character of ABA accumulation¹⁴ suggest that the main transport mechanism is the hydraulic dispersal in xylem. The participation of ABA in the generation of systemic electrical activity and/or vice versa cannot be ruled out.^8,20A rapid hydraulically driven transport of chemicals in the xylem of wounded plant in a reversed (basipetal) direction^19,21 to transpiration stream is not generally accepted. Exposing of leaves of undamaged plants to radioactive labelled molecules to determine the speed of chemical signal transport could be misrepresent, because hydraulic signal is not generated in undamaged plants and then the detected transport speed is too slow. Moreover, previous work²² demonstrated that neither the mass flow itself, nor the associated pressure changes induce the systemic response (the proteinase inhibitor activity). Thus, the efficacy of chemical agents in rapid systemic signaling seems to depend on transport by the mass flows associated with hydraulic signals.¹⁹However, hydraulic dispersal acts only for minutes, until all water released at the wound site is exhausted.²¹ A requirement for hydraulic dispersal of any solute is its presence in the wounded tissue at the time of wounding.¹⁹ Detected slower kinetics of JA accumulation than in the case of ABA and the huge rise of JA levels¹⁴ indicate a systemic accumulation of JA also by some additional processes.Does additional JA accumulation result from de-novo synthesis in undamaged leaves as a response to physical signal or does it result from a JA transport from the wounded leaves? In the longer time-frame the phloem transport²³ should also be considered. Experiments with tomato plants have shown that de novo JA synthesis in distant leaves is not required for the systemic response and that biosynthesis of JA at the wound site is necessary for the generation of a systemic signal.⁷ Indeed, a short-term increase in endogenous concentrations of JA has been detected in wounded tissue in Nicotiana sylvestris⁹ and rice.¹⁰However, a rapid burst in the systemic JA accumulation found in our experiments¹⁴ would implicate an ultra-rapid and extreme JA accumulation at the wound site before its transport. The systemic JA accumulation (within 1 hour¹⁴) preceded the generation of enzymes involved in the JA biosynthesis in the wounded leaf.Thus, several processes are suggested to play a role in the ultra-rapid and huge JA accumulation:

1. initiation of JA accumulation by preexisting enzymes,²⁴
2. fast release of free JA from its storage pools in cells (e.g., JA-conjugates²⁵),
3. direct uptake of elicitors (JA) by the phloem of the wounded leaf and exchange between the xylem and phloem as a consequence of severe wounds,²⁶
4. the mass flow (containing remaining JA) driven mainly acropetally in the xylem by transpiration after damping the hydraulic surge,²¹
5. JA accumulation evoked by the fast transmitting physical (electrical or hydraulic) signal that leads to imbalances in ion fluxes,^8,12,27
6. JA accumulation (and subsequent transport) directly in the phloem, where JA biosynthetic enzymes are located (at least in tomato²⁴),
7. volatile chemical compounds (methylester of JA) spreading in the surrounding air of wounded leaf could serve as signaling molecules and sources of JA.^25,28

The relevance of the above mentioned mechanisms should be checked by further research. Complex quantitative and kinetic analysis of JA and ABA content, levels of its biosynthetic derivatives (also volatiles in the surrounding air) and simultaneous physical signal detection in wounded and distant unwounded tissues would fill the remaining void about their role and interactions in the wound signal transduction networks. In addition, a suppression of other signaling pathways with similar transport kinetics (e.g., volatile compounds transmission, systemin and oligosaccharides generation and/or transport, using mutant plants) would be useful.Substantial similarity between the rapid physical (electrical) signaling in animal nervous system compared with the physical (electrical) signaling in plants has already been reported.^29,30 Interaction of chemical and electrical signals is the process well documented for post-synaptic events in animals. Our data now strengthen the role of chemical signals next to the role of physical signals in plants in the rapid systemic wound response; such a role of chemicals in plants was often underestimated up to now.     

Xylem cavitation and hydraulic control of stomatal conductance in Laurel (Laurus nobilis L.)

The possible link between stomatal conductance (g_L), leaf water potential ( Ψ _L) and xylem cavitation was studied in leaves and shoots of detached branches as well as of whole plants of Laurus nobilis L. (Laurel). Shoot cavitation induced complete stomatal closure in air‐dehydrated detached branches in less than 10 min. By contrast, a fine regulation of g_L in whole plants was the consequence of Ψ _L reaching the cavitation threshold ( Ψ _CAV) for shoots. A pulse of xylem cavitation in the shoots was paralleled by a decrease in g_L of about 50%, while Ψ _L stabilized at values preventing further xylem cavitation. In these experiments, no root signals were likely to be sent to the leaves from the roots in response to soil dryness because branches were either detached or whole plants were growing in constantly wet soil. The stomatal response to increasing evaporative demand appeared therefore to be the result of hydraulic signals generated during shoot cavitation. A negative feedback link is proposed between g_L and Ψ _CAV rather than with Ψ _L itself.     

Expression of a maize proteinase inhibitor gene is induced in response to wounding and fungal infection: systemic wound-response of a monocot gene

The isolation and characterization of cDNA and genomic clones encoding a proteinase inhibitor protein (MPI) in maize is reported. Accumulation of the MPI mRNA is induced in response to fungal infection in germinating maize embryos. The expression pattern of the MPI gene, in healthy and fungal infected maize tissues, was examined and compared with the expression pattern of a gene that codes for a pathogenesis-related protein (the PRms protein) from maize. These two genes are induced by fungal infection, however different signals trigger their activation. Accumulation of the proteinase inhibitor mRNA is more a consequence of the wound produced by the penetration and colonization of the host tissues by the pathogen, than the result of a direct molecular recognition of the pathogen by the plant, as is the case for the induction of the PRms gene. Wounding, or treatment with abscisic acid or methyl jasmonate, stimulate MPI mRNA accumulation, but not PRms mRNA accumulation. Local and systemic induction of the MPI gene expression in response to wounding occurs in maize plants. To the authors' knowledge, this is the first example of a gene from a monocotyledonous species that clearly shows a systemic wound response. The possible functional implications for the existence of different signal transduction pathways that simultaneously activate a battery of defense mechanisms against potential pathogens are discussed.     

Midday stomatal conductance is more related to stem rather than leaf water status in subtropical deciduous and evergreen broadleaf trees

Midday depressions in stomatal conductance (g_s) and photosynthesis are common in plants. The aim of this study was to understand the hydraulic determinants of midday g_s, the coordination between leaf and stem hydraulics and whether regulation of midday g_s differed between deciduous and evergreen broadleaf tree species in a subtropical cloud forest of Southwest (SW) China. We investigated leaf and stem hydraulics, midday leaf and stem water potentials, as well as midday g_s of co‐occurring deciduous and evergreen tree species. Midday g_s was correlated positively with midday stem water potential across both groups of species, but not with midday leaf water potential. Species with higher stem hydraulic conductivity and greater daily reliance on stem hydraulic capacitance were able to maintain higher stem water potential and higher g_s at midday. Deciduous species exhibited significantly higher stem hydraulic conductivity, greater reliance on stem capacitance, higher stem water potential and g_s at midday than evergreen species. Our results suggest that midday g_s is more associated with midday stem than with leaf water status, and that the functional significance of stomatal regulation in these broadleaf tree species is probably for preventing stem xylem dysfunction.