Signalling in Rhizobacteria-Induced Systemic Resistance in Arabidopsis thaliana

Abstract: To protect themselves from disease, plants have evolved sophisticated defence mechanisms in which the signal molecules salicylic acid, jasmonic acid and ethylene often play crucial roles. Elucidation of signalling pathways controlling disease resistance is a major objective in research on plant-pathogen interactions. The capacity of a plant to develop a broad spectrum, systemic acquired resistance (SAR) after primary infection with a necrotizing pathogen is well-known and its signal transduction pathway extensively studied. Plants of which the roots have been colonized by specific strains of non-pathogenic fluorescent Pseudomonas spp. develop a phenotypically similar form of protection that is called rhizobacteria-mediated induced systemic resistance (ISR). In contrast to pathogen-induced SAR, which is regulated by salicylic acid, rhizobacteria-mediated ISR is controlled by a signalling pathway in which jasmonic acid and ethylene play key roles. In the past eight years, the model plant species Arabidopsis thaliana was explored to study the molecular basis of rhizobacteria-mediated ISR. Here we review current knowledge of the signal transduction steps involved in the ISR pathway that leads from recognition of the rhizobacteria in the roots to systemic expression of broad-spectrum disease resistance in aboveground foliar tissues.     

Hormone sensitivity and plant adaptations to flooding

Plant hormones play a key role as mediators between environmental signals and adaptive plant responses. Auxin, ethylene and gibberellins are involved in the initiation of adaptive plant responses such as the development of adventitious roots and stimulated shoot elongation upon flooded conditions. These adaptive plastic responses in plants are frequently linked to changes in the concentrations of the hormones involved, but only rarely to shifts in sensitivity. Examples from ecophysiological research performed with species from the genusRumex demonstrate the importance of the hormone sensitivity concept in plant adaptations to flooding: (a)Rumex species can be grouped into three response categories according to the ethylene sensitivity of the youngest petioles: positive, negative and indifferent; (b) Sub-ambient oxygen concentrations sensitize petioles of wetlandRumex species to ethylene; (c) Enhanced ethylene levels sensitize petioles of wetlandRumex species to gibberellin; (d) Auxin is the primary plant hormone responsible for the initiation of adventitious roots in wetlandRumex species. However, a factor related to waterlogging, possibly ethylene, is required to sensitize the root-shoot junction to endogenous auxin.     

Contrasting interactions between ethylene and abscisic acid in Rumex species differing in submergence tolerance

Complete submergence of flooding-tolerant Rumex palustris plants strongly stimulates petiole elongation. This escape response is initiated by the accumulation of ethylene inside the submerged tissue. In contrast, petioles of flooding-intolerant Rumex acetosa do not increase their elongation rate under water even though ethylene also accumulates when they are submerged. Abscisic acid (ABA) was found to be a negative regulator of enhanced petiole growth in both species. In R. palustris, accumulated ethylene stimulated elongation by inhibiting biosynthesis of ABA via a reduction of RpNCED expression and enhancing degradation of ABA to phaseic acid. Externally applied ABA inhibited petiole elongation and prevented the upregulation of gibberellin A(1) normally found in submerged R. palustris. In R. acetosa submergence did not stimulate petiole elongation nor did it depress levels of ABA. However, if ABA concentrations in R. acetosa were first artificially reduced, submergence (but not ethylene) was then able to enhance petiole elongation strongly. This result suggests that in Rumex a decrease in ABA is a prerequisite for ethylene and other stimuli to promote elongation.     

Sensitivity to ethylene: the key factor in submergence-induced shoot elongation of Rumex

Rosettes of flooding-resistant Rumex palustris plants show a submergence-induced stimulation of elongation, which is confined to the petioles of young leaves. This response increases the probability of survival. It is induced by ethylene that accumulates in submerged tissues. Flooding-intolerant Rumex acetosella plants do not show this response. We investigated whether differences in shoot elongation between the species, between old and young leaves and between the petiole and leaf blade of a R. palustris plant result from differences in internal ethylene concentration or in sensitivity to the gas. Concentrations of free and conjugated ACC in petioles and leaf blades of R. palustris indicated that ethylene is synthesized throughout the submerged shoot, although production rates varied locally. Nevertheless, no differences in ethylene concentration were found between submerged leaves of various ages. In contrast, dose-response curves showed that only elongation of young petioles of R. palustris was sensitive to ethylene. In R. acetosella, elongation of all leaves was insensitive to ethylene. We conclude that variation in ethylene sensitivity rather than content explains the differences in submergence-induced shoot elongation between the two Rumex species and between leaves of R. palustris.     

Effect of Waterlogged Soil Conditions on the Production of Ethylene and on Water Relationships in Tomato Plants

The roots of tomato plants (Lycopersicon esculentum Mill., cv.Moneymaker) were exposed to low concentrations of oxygen bywaterlogging the soil or by growing the plants in nutrient solutionflushed with nitrogen gas. After 24 h, the rate of ethyleneproduction by the petioles, main stem, and shoot apex was increasedby 4–6-fold and the petioles developed epinastic curvatures.Removing the roots did not reproduce these responses. The amountsof ethylene produced by shoot tissues in response to physicalwounding was greatly increased by waterlogging the soil. The production of ethylene by roots was suppressed by the absenceof oxygen. When the roots were transferred back to an aerobicenvironment ethylene production quickly exceeded that observedin roots maintained continuously in aerobic conditions. The enhanced rate of ethylene production in the shoots occurredin the absence of increased water stress as measured with aleaf pressure chamber; leaf water potentials were increasedrather than decreased by waterlogging for 30 h or more. Thiswas associated with stomatal closure and reduced transpiration.Resistance to water flow through the plant increased as transpirationdecreased in response to waterlogging. However, at similar ratesof transpiration, resistance was normally lower in waterloggedplants than in controls.     

Ethylene-induced differential growth of petioles in Arabidopsis. Analyzing natural variation, response kinetics, and regulation

Plants can reorient their organs in response to changes in environmental conditions. In some species, ethylene can induce resource-directed growth by stimulating a more vertical orientation of the petioles (hyponasty) and enhanced elongation. In this study on Arabidopsis (Arabidopsis thaliana), we show significant natural variation in ethylene-induced petiole elongation and hyponastic growth. This hyponastic growth was rapidly induced and also reversible because the petioles returned to normal after ethylene withdrawal. To unravel the mechanisms behind the natural variation, two contrasting accessions in ethylene-induced hyponasty were studied in detail. Columbia-0 showed a strong hyponastic response to ethylene, whereas this response was almost absent in Landsberg erecta (Ler). To test whether Ler is capable of showing hyponastic growth at all, several signals were applied. From all the signals applied, only spectrally neutral shade (20 micromol m(-2) s(-1)) could induce a strong hyponastic response in Ler. Therefore, Ler has the capacity for hyponastic growth. Furthermore, the lack of ethylene-induced hyponastic growth in Ler is not the result of already-saturating ethylene production rates or insensitivity to ethylene, as an ethylene-responsive gene was up-regulated upon ethylene treatment in the petioles. Therefore, we conclude that Ler is missing an essential component between the primary ethylene signal transduction chain and a downstream part of the hyponastic growth signal transduction pathway.     

Ethylene Sensitivity and Response Sensor Expression in Petioles of Rumex Species at Low O2 and High CO2 Concentrations

Rumex palustris, a flooding-tolerant plant, elongates its petioles in response to complete submergence. This response can be partly mimicked by enhanced ethylene levels and low O2 concentrations. High levels of CO2 do not markedly affect petiole elongation in R. palustris. Experiments with ethylene synthesis and action inhibitors demonstrate that treatment with low O2 concentrations enhances petiole extension by shifting sensitivity to ethylene without changing the rate of ethylene production. The expression level of the R. palustris gene coding for the putative ethylene receptor (RP-ERS1) is up-regulated by 3% O2 and increases after 20 min of exposure to a low concentration of O2, thus preceding the first significant increase in elongation observable after 40 to 50 min. In the flooding-sensitive species Rumex acetosa, submergence results in a different response pattern: petiole growth of the submerged plants is the same as for control plants. Exposure of R. acetosa to enhanced ethylene levels strongly inhibits petiole growth. This inhibitory effect of ethylene on R. acetosa can be reduced by both low levels of O2 and/or high concentrations of CO2.     

De-submergence-induced ethylene production in Rumex palustris: regulation and ecophysiological significance

Rumex palustris responds to total submergence by increasing the elongation rate of young petioles. This favours survival by shortening the duration of submergence. Underwater elongation is stimulated by ethylene entrapped within the plant by surrounding water. However, abnormally fast extension rates were found to be maintained even when leaf tips emerged above the floodwater. This fast post-submergence growth was linked to a promotion of ethylene production that is presumed to compensate for losses brought about by ventilation. Three sources of ACC contributed to post-submergence ethylene production in R. palustris: (i) ACC that had accumulated in the roots during submergence and was transported in xylem sap to the shoot when stomata re-opened and transpiration resumed, (ii) ACC that had accumulated in the shoot during the preceding period of submergence and (iii) ACC produced de novo in the shoot following de-submergence. This new production of ethylene was associated with increased expression of an ACC synthase gene (RP-ACS1) and an ACC oxidase gene (RP-ACO1), increased ACC synthase activity and a doubling of ACC oxidase activity, measured in vitro. Out of seven species of Rumex examined, a de-submergence upsurge in ethylene production was seen only in shoots of those that had the ability to elongate fast when submerged.     

The role of oxygen in submergence-induced petiole elongation in Rumex palustris: in situ measurements of oxygen in petioles of intact plants using micro-electrodes

In a study on the mechanism of stimulated petiole elongation in submerged plants, oxygen concentrations in petioles of the flood-tolerant plant Rumex palustris were measured with micro-electrodes. Short-term submergence lowered petiole partial oxygen pressure to c . 19 kPa whereas prolonged submergence under continuous illumination depressed oxygen levels to c . 8–12 kPa after 24 h. Oxygen levels in petioles depended on the presence of the lamina, even in submerged conditions, and on available light. In darkness, petiole oxygen levels in submerged plants dropped quickly to values as low as 0.5–4 kPa. It is hypothesized that prolonged submergence in the light is accompanied by a decrease in carbon dioxide in the petiole. Submergence-enhanced petiolar elongation rate was compared with emergent plants. Peak daily elongation rates occurred at the end of the dark period in emergent plants, but in the middle of the light period in submerged plants. We suggest that this shift in daily elongation pattern is induced by dependence of growth on photosynthetically derived oxygen in submerged plants. Implications of reduced oxygen for ethylene production are raised. Levels of 1- aminocyclopropane-1-carboxylic acid synthase and 1-aminocyclopropane-1-carboxylic acid oxidase and ethylene sensitivity are cited as potential factors in hypoxia-induced ethylene release.     

The roles of ethylene, auxin, abscisic acid, and gibberellin in the hyponastic growth of submerged Rumex palustris petioles

Rumex palustris responds to complete submergence with upward movement of the younger petioles. This so-called hyponastic response, in combination with stimulated petiole elongation, brings the leaf blade above the water surface and restores contact with the atmosphere. We made a detailed study of this differential growth process, encompassing the complete range of the known signal transduction pathway: from the cellular localization of differential growth, to the hormonal regulation, and the possible involvement of a cell wall loosening protein (expansin) as a downstream target. We show that hyponastic growth is caused by differential cell elongation across the petiole base, with cells on the abaxial (lower) surface elongating faster than cells on the adaxial (upper) surface. Pharmacological studies and endogenous hormone measurements revealed that ethylene, auxin, abscisic acid (ABA), and gibberellin regulate different and sometimes overlapping stages of hyponastic growth. Initiation of hyponastic growth and (maintenance of) the maximum petiole angle are regulated by ethylene, ABA, and auxin, whereas the speed of the response is influenced by ethylene, ABA, and gibberellin. We found that a submergence-induced differential redistribution of endogenous indole-3-acetic acid in the petiole base could play a role in maintenance of the response, but not in the onset of hyponastic growth. Since submergence does not induce a differential expression of expansins across the petiole base, it is unlikely that this cell wall loosening protein is the downstream target for the hormones that regulate the differential cell elongation leading to submergence-induced hyponastic growth in R. palustris.     

The vascular pathogen Verticillium longisporum requires a jasmonic acid-independent COI1 function in roots to elicit disease symptoms in Arabidopsis shoots

Verticillium longisporum is a soil-borne vascular pathogen that causes reduced shoot growth and early senescence in Arabidopsis (Arabidopsis thaliana). Here, we report that these disease symptoms are less pronounced in plants that lack the receptor of the plant defense hormone jasmonic acid (JA), CORONATINE INSENSITIVE1 (COI1). Initial colonization of the roots was comparable in wild-type and coi1 plants, and fungal DNA accumulated to almost similar levels in petioles of wild-type and coi1 plants at 10 d post infection. Completion of the fungal life cycle was impaired in coi1, as indicated by the reduced number of plants with microsclerotia, which are detected on dead plant material at late stages of the disease. Contrary to the expectation that the hormone receptor mutant coi1 should display the same phenotype as the corresponding hormone biosynthesis mutant delayed dehiscence2 (dde2), dde2 plants developed wild-type-like disease symptoms. Marker genes of the JA and the JA/ethylene defense pathway were induced in petioles of wild-type plants but not in petioles of dde2 plants, indicating that fungal compounds that would activate the known COI1-dependent signal transduction chain were absent. Grafting experiments revealed that the susceptibility-enhancing COI1 function acts in the roots. Moreover, we show that the coi1-mediated tolerance is not due to the hyperactivation of the salicylic acid pathway. Together, our results have unraveled a novel COI1 function in the roots that acts independently from JA-isoleucine or any JA-isoleucine mimic. This COI1 activity is required for a yet unknown root-to-shoot signaling process that enables V. longisporum to elicit disease symptoms in Arabidopsis.     

Is the Diageotropic Tomato Ethylene Deficient?

The production of ethylene by a mutant form of tomato (Lycopersicon esculentum Mill. cv. Ailsa Craig) with diageotropic shoot growth was compared with that from non-mutant, upright plants. No difference in the rate of production by segments of petiole or stem apex was observed. The amounts of ethylene produced by excised segments of petiole from diageotropic and upright plants in response to wounding were also comparable. When the roots of either kind of plant were exposed to anaerobic conditions, the production of ethylene increased in the petioles; in ordinary plants this was associated with epinastic curvature, while in diageotropic plants the direction of shoot extension became reorientated from the horizontal to a more upright position. Exogenous ethylene gas had similar effects. These results support the view that the mutant has a modified response mechanism to gravity and to ethylene rather than an abnormally slow rate of ethylene production in the shoot. Since applying inhibitors of ethylene action to non-mutant, upright plants did not induce diageotropism, endogenous ethylene seems unlikely to play a significant role in maintaining their upright orientation. The roots of both kinds of plant produced large amounts of ethylene, although the rate for diageotropic roots was about 37% less than that of roots from normal plants. Application of indol-3-ylacetic acid increased the production of ethylene by all roots but those from mutant plants were less responsive.     

Auxin and Ethylene Regulate Elongation Responses to Neighbor Proximity Signals Independent of Gibberellin and DELLA Proteins in Arabidopsis

Plants modify growth in response to the proximity of neighbors. Among these growth adjustments are shade avoidance responses, such as enhanced elongation of stems and petioles, that help plants to reach the light and outgrow their competitors. Neighbor detection occurs through photoreceptor-mediated detection of light spectral changes (i.e. reduced red:far-red ratio [R:FR] and reduced blue light intensity). We recently showed that physiological regulation of these responses occurs through light-mediated degradation of nuclear, growth-inhibiting DELLA proteins, but this appeared to be only part of the full mechanism. Here, we present how two hormones, auxin and ethylene, coregulate DELLAs but regulate shade avoidance responses through DELLA-independent mechanisms in Arabidopsis (Arabidopsis thaliana). Auxin appears to be required for both seedling and mature plant shoot elongation responses to low blue light and low R:FR, respectively. Auxin action is increased upon exposure to low R:FR and low blue light, and auxin inhibition abolishes the elongation responses to these light cues. Ethylene action is increased during the mature plant response to low R:FR, and this growth response is abolished by ethylene insensitivity. However, ethylene is also a direct volatile neighbor detection signal that induces strong elongation in seedlings, possibly in an auxin-dependent manner. We propose that this novel ethylene and auxin control of shade avoidance interacts with DELLA abundance but also controls independent targets to regulate adaptive growth responses to surrounding vegetation.     

Brassinosteroid-induced epinasty in tomato plants

The effects of root treatments of brassinosteroid (BR) on the growth and development of hydroponically grown tomato plants (Lycopersicon esculentum Mill cv Heinz 1350) were evaluated. There was a dramatic increase in petiole bending when the plants were treated with 0.5 to 1.0 micromolar BR. The leaf angle of the treated plants was almost three times that of untreated controls. BR-induced epinasty appeared to be due to stimulation of ethylene production. Excised petioles from BR-treated plants produced more than twice as much ethylene as did untreated controls. As ethylene production increased, the degree of petiole bending also increased, and inhibition of ethylene production by AOA or CoCl₂ also inhibited epinasty. BR-treated plants had increased levels of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) in the leaf tissue. ACC appeared to accumulate primarily in the petioles with the greatest amount of ACC accumulating in the youngest petioles. Time course evaluations revealed that BR treatment stimulated ACC production. As ACC accumulated, ethylene increased, resulting in epinasty. Little or no ACC was found in the xylem sap, indicating that there was a signal transported from the roots which stimulated ACC synthesis in the leaf tissue.     

Relationship between Leaf Water Status and Endogenous Ethylene in Detached Leaves

The pattern of changes in the internal concentration of ethylene in response to water stress was investigated in species with leaves that do abscise and leaves that do not abscise. When leaves which abscise were detached and exposed to dry air for up to 6 hours, a continuous increase of internal ethylene was observed. In water-stressed leaves which do not abscise only a transient rise in ethylene occurred. The peak, which was attained after 30 to 120 minutes, depending on the species studied, was followed by a sharp decline to the initial level. The principal site of ethylene production in response to a short period of water stress was in the blades rather than the petioles in both types of leaves. The internal ethylene level in leaves was reduced by pretreatment with the ethoxy analog of rhizobitoxine (an inhibitor of ethylene biosynthesis) or by maintaining the leaves under subatmospheric pressure. The results obtained by these methods showed that ethylene was not involved in the mechanism of stomatal movement in either turgid or in stressed leaves. Also, the increase in leaf abscisic acid content and the depletion of gibberellins induced by water stress were not related to the internal concentration of ethylene in the detached leaf. The different patterns of drought-induced ethylene production observed in the blades of leaves which exhibit abscission compared with those which do not exhibit abscission may indicate the involvement of ethylene in a primary event in the process of leaf abscission induced by water stress.     

Physiology and Chemistry of Substances Accelerating Abscission in Senescent Petioles and Fruit Stalks

Senescent petioles of Coleus rehneltianus Berger. Phaseolus vulgaris L. cv. Saxa. Acer pseudoplatanus L., and senescent fruit stalks of Malus domestica Borkh. cv. Golden Delicious contain at least three abscission accelerating substances, which were isolated by extraction with methanol or with water and by diffusion into agar. They were purified by thin-layer chromatography and bioassayed in a special abscission test using Coleus explants. Two of these abscission accelerators could be conclusively identified by thin-layer chromatography and by gas chromatography as abscisic acid and xanthoxin. The third substance, which has acidic properties and is less polar than abscisic acid, could not be identified. The concentration and the absolute amount of abscisic acid in Coleus petioles were found to decrease during their development, young petioles having the highest concentration. No evidence was found that the three abscission accelerators or synthetic abscisic acid and xanthoxin affect the production of ethylene in Caleus explatns. The results obtained do not support the hypothesis that senescent petioles contain a specific “senescence factor”, which stimulates abscission via ethylene production.     

The ABA-mediated switch between submersed and emersed life-styles in aquatic macrophytes

Hydrophytes comprise aquatic macrophytes from various taxa that are able to sustain and to complete their lifecycle in a flooded environment. Their ancestors, however, underwent adaptive processes to withstand drought on land and became partially or completely independent of water for sexual reproduction. Interestingly, the step backwards into the high-density aquatic medium happened independently several times in numerous plant taxa. For flowering plants, this submersed life-style is especially difficult as they need to erect their floral organs above the water surface to be pollinated. Moreover, fresh-water plants evolved the adaptive mechanism of heterophylly, which enabled them to switch between a submersed and an emersed leaf morphology. The plant hormone abscisic acid (ABA) is a key factor of heterophylly induction in aquatic plants and is a major switch between a submersed and emersed life. The mechanisms of ABA signal perception and transduction appear to be conserved throughout the evolution of basal plants to angiosperms and from terrestrial to aquatic plants. This review summarizes the interplay of environmental factors that act through ABA to orchestrate adaptation of plants to their aquatic environment.     

Differences in mechanical and structural properties of surface and aerial petioles of the aquatic plant Nymphaea odorata subsp. tuberosa (Nymphaeaceae)

Lily pads (Nymphaea odorata) exhibit heterophylly where a single plant may have leaves that are submerged, floating, or above (aerial) the surface of the water. Lily pads are placed in a unique situation because each leaf form is exposed to a distinctly different set of mechanical demands. While surface petioles may be loaded in tension under conditions of wind or waves, aerial petioles are loaded in compression because they must support the weight of the lamina. Using standard techniques, we compared the mechanical and morphological properties of both surface and aerial leaf petioles. Structural stiffness (EI) and the second moment of area (I) were higher in aerial petioles, although we detected no differences in other mechanical values (elastic modulus [E], extension ratio, and breaking strength). Morphologically, aerial petioles had a thicker rind, with increased collenchyma tissue and sclereid cell frequency. Aerial petioles also had a larger cross-sectional area and were more elliptical. Thus, subtle changes in the distribution of materials, rather than differences in their makeup, differentiate petiole forms. We suggest that the growth of aerial petioles may be an adaptive response to shading, allowing aerial leaves to rise above a crowded water surface.