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.     

Serine/threonine protein phosphatases: Multi-purpose enzymes in control of defense mechanisms

Depending on the threat to a plant, different pattern recognition receptors, such as receptor-like kinases, identify the stress and trigger action by appropriate defense response development.^1,2 The plant immunity system primary response to these challenges is rapid accumulation of phytohormones, such as ethylene (ET), salicylic acid (SA), and jasmonic acid (JA) and its derivatives. These phytohormones induce further signal transduction and appropriate defenses against biotic threats.^3,4 Phytohormones play crucial roles not only in the initiation of diverse downstream signaling events in plant defense but also in the activation of effective defenses through an essential process called signaling pathway crosstalk, a mechanism involved in transduction signals between two or more distinct, “linear signal transduction pathways simultaneously activated in the same cell.”⁵     

Modulation of plant HMG-CoA reductase by protein phosphatase 2A: Positive and negative control at a key node of metabolism

The enzyme HMG-CoA reductase (HMGR) has a key regulatory role in the mevalonate pathway for isoprenoid biosynthesis, critical not only for normal plant development, but also for the adaptation to demanding environmental conditions. Consistent with this notion, plant HMGR is modulated by many diverse endogenous signals and external stimuli. Protein phosphatase 2A (PP2A) is involved in auxin, abscisic acid, ethylene and brassinosteroid signaling and now emerges as a positive and negative multilevel regulator of plant HMGR, both during normal growth and in response to a variety of stress conditions. The interaction with HMGR is mediated by B″ regulatory subunits of PP2A, which are also calcium binding proteins. The new discoveries uncover the potential of PP2A to integrate developmental and calcium-mediated environmental signals in the control of plant HMGR.Key words: HMG-CoA reductase, HMGR, mevalonate pathway, isoprenoid biosynthesis, protein phosphatase 2A, PP2A, salt stressPlant 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase or HMGR) was detected for the first time long ago¹ and is modulated by many diverse endogenous signals and environmental factors. However, no protein factor interacting with and controlling plant HMGR in vivo had been described until recently.² The finding that HMGR is under multilevel control by protein phosphatase 2A (PP2A)² raises interesting hypotheses concerning the modulation of HMGR and, therefore, isoprenoid biosynthesis via the signal transduction network. In the present review we discuss these hypotheses, in connection the current knowledge on plant HMGR and PP2A. Other interesting reviews are available for a more detailed background on plant HMGR^3,4 or PP2A.^5–8     

The role of receptor interactions in regulating ethylene signal transduction

The phytohormone ethylene is perceived in Arabidopsis by a five-member receptor family. Earlier work has demonstrated that the basic functional unit for an ethylene receptor is a disulfide-linked homodimer. We recently reported in The Journal of Biological Chemistry that the ethylene-receptor ETR1 physically associates with other ethylene receptors through higher order interactions, suggesting the existence of receptor clusters. Here we consider the implications of such clusters upon the mechanism of ethylene signal transduction. In particular, we consider how such clustering provides a cooperative mechanism, akin to what has been found for the prokaryotic chemoreceptors, by which plant sensitivity to ethylene may be increased. In addition, we consider how the dominant ethylene insensitivity conferred by some receptor mutations, such as etr1-1, may also be propagated by interactions among members of the ethylene receptor family.Key words: ethylene, receptor, ETR1, cooperativity, ArabidopsisThe plant hormone ethylene regulates growth and development, and is perceived by a five-member family of receptors (ETR1, ERS1, ETR2, ERS2 and EIN4) in Arabidopsis.¹ Genetic analysis indicates that ethylene receptors are functionally redundant and negatively regulate ethylene responses through interactions with the Raf-like kinase CTR1.^2–5 The functional unit of an ethylene receptor in a disulfide-linked homodimer, with each homodimer capable of binding one ethylene molecule.^6,7 However, several observations suggest that propagation of the ethylene signal through the receptors is likely to involve more than just ethylene-induced changes within individual receptor homodimers. First, Arabidopsis is amazingly sensitive to ethylene and can respond to ethylene concentrations as low as 0.2 nl/L,⁸ 300-fold lower than the K_d of the receptors for ethylene, which suggests that some mechanism exists for amplifying the input signal.^7,9 Second, ethylene-insensitive mutations in the binding sites of the receptors exhibit greater dominance than would be predicted solely from a lesion within one member of the receptor family.¹⁰In our paper published in The Journal of Biological Chemistry,¹¹ we demonstrate that the Arabidopsis ethylene receptor ETR1 physically associates with other ethylene receptors through higher order interactions. Such physical interactions suggest that the receptors exist in plants as clusters, and that models for cooperative signaling previously applied to the histidine-kinaselinked chemoreceptors of bacteria may also be applicable to the evolutionarily related ethylene receptors of plants. In bacteria, the highly packed chemoreceptors are found in clusters at one or both poles of the cell.^12,13 Structural studies indicate that chemoreceptors can associate to form a ‘trimer of dimers’^14,15 and also support the possibility that domain swapping may occur to produce a large interconnected array of receptors. 16 Our studies indicate that ethylene receptors can interact through their cytosolic GAF domains, identifying one possible interface through which conformational changes could be propagated in an ethylene receptor cluster.A higher-order cooperative mechanism among the ethylene receptors may explain the high sensitivity of plants to ethylene. In this model, the ethylene receptors amplify ethylene signaling by lateral signal output. Binding of ethylene to one receptor induces the conformation change of the receptor from a tense state (T) to a relaxed state (R). This conformational change is then propagated to other empty receptors in the cluster due to their physical associations with the receptor in the R state. As a result empty receptors also adopt the relaxed state (R′), resulting in amplification of the initial signal. It should be noted here that mutational evidence supports the unbound state of the receptors (T state) as being the lower energy conformation of the receptors.¹⁷ Thus, according to this model, part of the energy from ligand binding would be used to transmit conformational changes to the neighboring receptors.An alternative model that may also explain the high sensitivity of ethylene responsiveness in plants, and one that is not necessarily incompatible with the previous model, is a conjugation model.¹⁸ Here it is hypothesized that, due to the physical proximity of the ethylene receptors, that ethylene released from one receptor then binds to another receptor rather than diffusing away. Through this conjugation mechanism, one ethylene molecule could amplify its signal by converting the conformations of multiple ethylene receptors from the ethylene-unbound state (T) to the ethylene-bound state (R). This model is based on several assumptions. One assumption is that a single ethylene molecule can bind ethylene receptors in the same cluster multiple times due to the dynamic binding of ethylene and ethylene receptor. A second assumption is that, after ethylene is released from one ethylene receptor, the recovery time for that receptor to resume the T state is longer than the time required for the released ethylene to bind to and convert another receptor from the T to the R state.Models for cooperativity need to also explain the dominant ethylene insensitivity of various mutant receptors such as etr1-1, in which a missense mutation results in a receptor incapable of binding ethylene. Several studies indicate that the etr1-1 mutant receptor acts cooperatively to affect the signal output from other wild-type receptors (i.e., the presence of the etr1-1 receptor in its T state increases the likelihood of other receptors adopting the T state).^10,11 This observation can be most readily explained if the dominant ethylene-insensitive mutations result in a receptor that requires more energy to undergo the T to R transition than do the wild-type receptors. For example, the etr1-1 mutation may increase the stability of the T form (a T′ state). There is evidence to support this possibility. The etr1-1 missense mutation results in a receptor unable to chelate a copper cofactor necessary for ethylene binding,¹⁹ but the effects of this mutation on signaling are different from wild-type receptors that lack their copper cofactor. The etr1-1 mutant receptor appears locked in its T state, whereas wild-type receptors lacking the copper cofactor appear to be in the R state.²⁰ Thus etr1-1 is truly a gain-of-function mutation that alters the conformation of the receptor in ways not necessarily predicted from just the loss of the copper cofactor.In conclusion, we have attempted here to provide models that can resolve an apparent contradiction in the cooperative signaling behavior exhibited by ethylene receptors. The high sensitivity of plants to ethylene suggest cooperative changes in which an R state can be propagated within a receptor cluster, but the dominance of the ethylene ethylene-insensitive mutant etr1-1 suggests that the T state can also be propagated within a receptor cluster. It should be born in mind, however, that ethylene signaling is mediated by multiple signaling components. The ethylene receptors regulate ethylene responses through interaction with and modulation of CTR1 kinase activity. Thus, the total kinase activity of CTR1 represents the signal output from the receptors. This situation is very similar to that of the bacterial chemoreceptors, which regulate the activity of an associated histidine kinase, and, as with the chemoreceptors, the stoichiometry of CTR1 interactions with the ethylene receptors and the means by which its kinase activity is regulated are important for the elucidation of the mechanism of ethylene signal transduction.     

Alamethicin-induced electrical long distance signaling in plants

Systemic signals induced by wounding and/or pathogen or herbivore attack may be realized by either chemical or mechanical signals. In plants a variety of electrical phenomena have been described and may be considered as signal-transducing events; such as variation potentials (VPs) and action potentials (APs) which propagate over long distances and hence are able to carry information from organ to organ. In addition, we recently described a new type of electrical long-distance signal that propagates systemically, i.e., from leaf to leaf, the “system potential” (SP). This was possible only by establishing a non-invasive method with micro-electrodes positioned in substomatal cavities of open stomata and recording apoplastic responses. Using this technical approach, we investigated the function of the peptaibole alamethicin (ALA), a channel-forming peptide from Trichoderma viride, which is widely used as agent to induce various physiological and defence responses in eukaryotic cells including plants. Although the ability of ALA to initiate changes in membrane potentials in plants has always been postulated it has never been demonstrated. Here we show that both local and long-distance electrical signals, namely depolarization, can be induced by ALA treatment.Key words: alamethicin, long distance electrical signal, depolarization, non-invasive recordingPeptaibols are linear membrane active peptide antibiotics produced by various fungi.^1,2 They are characterized by the presence of an unusual amino acid, α-aminoisobutyric acid, and a C-terminal hydroxylated amino acid and are generated by non-ribosomal peptide synthetases.^2,3 Due to their amphiphilic nature they self-associate into oligomeric ion-channels that span the width of lipid bilayer membranes.³ Peptaibols exhibit antibiotic activity against bacteria and fungi, tissue damage in insect larvae, as well as cytolytic activity towards mammalian cells.² ALA is a voltage-gated ion channel-forming peptide mixture that consists of at least 12 compounds each containing 20 amino acid residues.^1,2 This peptaibole is often used to elicit typical systemic responses in plants, many of which are in the context of indirect defences, such as the induction and accumulation of secondary metabolites in general, volatile compounds, and the phytohormones jasmonic acid and salicylic acid but also tendril coiling is induced.^4–7 Moreover, ALA has been shown to permeabilize mitochondria and the plasma membrane of tobacco suspension culture cells but not the tonoplast for small molecules.⁸ Thus, ALA is a valuable tool in plant science but its primary biological activity in plant tissues has never been shown experimentally.Because of the channel-forming properties of ALA, effects on the membrane potential of cells are very likely. Therefore, to monitor ALA application-elicited electrical signals, we employed the non-invasive method with microelectrodes placed in the apoplasm of the sub-stomatal cavities of open stomata. Whereas electrical signals in plants such as action potentials and variation potentials have been well documented in literature,⁹ this particular approach has been successfully applied for the detection and characterization of the novel system potentials.^10,11 With an intracellular recording the depolarization of a membrane occurs when the cell interior becomes less negative; whereas for the apoplastic recording used here, the inverse argument holds true. To avoid confusion, we follow the convention and call an apoplastic hyperpolarization a depolarization.^10,11ALA (mixture of several isoforms purchased from Sigma-Aldrich, Germany) was tested on the dicotyledonous lima bean (Phaseolus lunatus L.) and the monocotyledonous barley (Hordeum vulgare L.). As shown in Figure 1, upon a 5 nM ALA stimulus a 40 mV depolarization was induced and detected at a distance of 5 cm on the same leaf in lima bean. Systemic electric propagation was tested and demonstrated in barley (Fig. 2). We used barley for these experiments because we learned from earlier studies that electrical signals are not propagated between the primary leaves of lima bean whereas in barley electrical signals pass nodes and internodia more easily. Application of 25 nM ALA on one leaf (S-leaf) resulted in a 45 mV depolarization response at a distant of 25 cm on a different leaf (T-leaf), indicating that ALA induced a systemic electrical response that moved from one to the other leaf. In both plants the velocity of the propagating depolarization signal was calculated to be 2.5 cm min⁻¹. Whereas these results basically demonstrate the ability of ALA to initiate electrical signals in plant tissues both locally and systemically, the molecular interconnections between these electrical phenomena, the induction and synthesis of phytohormones as well as secondary metabolites, and how the ALA signal is transduced mechanistically, remains to be elucidated.Open in a separate windowFigure 1Local response of Phaseolus lunatus to Alamethicin. (A) Experimental setup for the measurement of apoplastic voltage changes using microelectrodes positioned in the sub-stomatal cavities of P. lunatus.¹¹ Stimuli and measurements of apoplastic voltage changes were performed on the same leaf with a distance of approximately 5 cm. The tip of the leaf was submerged in 5 mM KCl with 0.1 mM CaCl₂, pH 5, and the solution was connected to earth with a reference electrode filled with 0.5 M KCl. (B) The voltage response to 5 nM Alamethicin (ALA) (Sigma-Aldrich, Germany) added to a cut injury of the P. lunatus leaf at the indicated time shows a hyperpolarization of the apoplast (negative shift of the apoplastic potential), suggesting depolarization of the symplast. A typical recording of one out of three independent experiments is presented.Open in a separate windowFigure 2Systemic response of Hordeum vulgare to Alamethicin. (A) Experimental setup for the measurement of apoplastic voltage changes using microelectrodes positioned in the sub-stomatal cavities of H. vulgare.¹¹ Stimuli were applied to one leaf (Stimulus leaf, S-leaf), and measurements of apoplastic voltage changes were performed on a second leaf (Target leaf, T-leaf) with a distance of approximately 25 cm. The tip of the T-leaf was submerged in 5 mM KCl with 0.1 mM CaCl₂, pH 5, and the solution was connected to earth with a reference electrode filled with 0.5 M KCl. (B) The voltage response to 25 nM Alamethicin (ALA) (Sigma-Aldrich, Germany) added to a cut injury of the H. vulgare S-leaf at the indicated time shows a hyperpolarization of the apoplast (negative shift of the apoplastic potential), suggesting depolarization of the symplast. A typical recording of one out of three independent experiments is presented.     

New evidence for a multi-functional role of herbivore-induced plant volatiles in defense against herbivores

A diverse, often species-specific, array of herbivore-induced plant volatiles (HIPVs) are commonly emitted from plants after herbivore attack. Although research in the last 3 decades indicates a multi-functional role of these HIPVs, the evolutionary rationale underpinning HIPV emissions remains an open question. Many studies have documented that HIPVs can attract natural enemies, and some studies indicate that neighboring plants may eavesdrop their undamaged neighbors and induce or prime their own defenses prior to herbivore attack. Both of these ecological roles for HIPVs are risky strategies for the emitting plant. In a recent paper, we reported that most branches within a blueberry bush share limited vascular connectivity, which restricts the systemic movement of internal signals. Blueberry branches circumvent this limitation by responding to HIPVs emitted from neighboring branches of the same plant: exposure to HIPVs increases levels of defensive signaling hormones, changes their defensive status, and makes undamaged branches more resistant to herbivores. Similar findings have been reported recently for sagebrush, poplar and lima beans, where intra-plant communication played a role in activating or priming defenses against herbivores. Thus, there is increasing evidence that intra-plant communication occurs in a wide range of taxonomically unrelated plant species. While the degree to which this phenomenon increases a plant’s fitness remains to be determined in most cases, we here argue that withinplant signaling provides more adaptive benefit for HIPV emissions than does between-plant signaling or attraction of predators. That is, the emission of HIPVs might have evolved primarily to protect undamaged parts of the plant against potential enemies, and neighboring plants and predators of herbivores later co-opted such HIPV signals for their own benefit.Key words: intra-plant signaling, plantplant communication, eavesdropping, systemic wound signals, plant defense, tri-trophic interactionsPlants often emit a unique blend of volatiles in response to herbivore attack. The emission of these herbivore-induced plant volatiles (HIPVs) is an active response to herbivore feeding, producing a blend of volatiles that is distinct from those emitted following mechanical injury alone.¹ Their emission can be variable; while some compounds follow a diurnal pattern with increasing amounts during the time of high photosynthesis,^2,3 others are emitted primarily at night.⁴ In some cases, the HIPV blend produced also differs depending on the species of herbivore feeding on the plant.⁵ This specificity is thought to be due to chemicals in the herbivore’s regurgitant, such as the fatty-acid amino-acid conjugate volicitin, that activate the emission of volatiles in plants.^6,7 Furthermore, HIPVs are emitted not only from the site of damage, but also at times from systemically undamaged parts of the plant.⁸ This and other systemic responses are, however, restricted within a plant such that only parts of the plant that share vascular connections with the damaged tissue receive wound signals and have the potential to respond.^9,10The ecological role of HIPVs has been a subject of fascination and the evolutionary advantage gained for plants by emitting HIPVs remains an unresolved topic of discussion. While some HIPV compounds, and some of their precursors, have sufficient volatility that their release is essentially inevitable after synthesis,¹¹ most tend to be tightly regulated. Assuming that HIPV emissions evolved as a result of trophic interactions among plants, herbivores, and natural enemies, there are four general ecological roles that HIPVs may play: (1) a direct negative effect on the herbivore, (2) a signal to alert natural enemies of the herbivore, (3) a warning signal to nearby undamaged plants, and (4) a systemic warning signal within the damaged plant (Fig. 1). The first two potential roles involve the manipulation of animal behavior, while the last two may alter plant “behavior”.Open in a separate windowFigure 1Herbivore-induced plant volatiles (HIPVs) play multiple roles in interactions among plants, herbivores, and natural enemies (possible interactions are depicted by arrows). Some of them benefit the HIPV-emitting plant (Emitter); these positive interactions include repellent effects on herbivores, attraction of natural enemies of herbivores, activation or priming of defenses in unwounded parts within the emitting plant (within-plant signaling), and growth inhibitory effects on neighboring plants (Receiver) through allelopathy. On the other hand, HIPVs may negatively affect the emitting plant by attracting herbivores or natural enemies (e.g., certain parasitoids) that result in increased damage. Finally, neighboring plants may “eavesdrop” from the emitting plant by responding to HIPVs (between-plant signaling). This latter interaction may be negative to the emitter if it is outcompeted by neighbors who receive wound signals, but beneficial to the receiving plant. Drawing by Robert Holdcraft.Scents can have a demonstrable effect on animal behavior. With respect to plant-herbivore interactions, scents can provide information about the status of a plant to herbivores and their natural enemies. For example, HIPVs may repel adults moths searching for oviposition sites,³ which has been interpreted from the perspective of either a plant minimizing damage or, perhaps more realistically, an adult moth searching for an undamaged, high quality resource for her offspring. Conversely, HIPV-emitting plants may increase their chance of being injured if herbivores are attracted to these volatiles.¹² The more commonly accepted role of HIPVs in manipulating animal behavior is to attract natural enemies of the herbivores. This tri-trophic “cry for help”¹³ has a potential evolutionary benefit for both the plant emitting the volatiles and the natural enemies responding to this emission.^14–16 Although this idea makes sense in an evolutionary perspective, only a few studies have documented the occurrence of this phenomenon in natural systems.¹⁷ Indeed, the effectiveness of a cry for help depends on the presence of a helper and, equally importantly, the ability of the helper to increase plant fitness. In the case of predator attraction, the herbivore may be removed from the plant and consumed, thereby reducing damage for the emitting plant.¹⁸ However, insect herbivores infected by parasitoids, which also use HIPV cues to locate hosts,¹⁹ may also consume less plant material²⁰ but may also in some cases consume more plant material than unparasitized insect herbivores.²¹ Since there is currently no evidence that plants can modify HIPV blends to attract selectively predators versus parasitoids, an answered cry for help may not reliably decrease the total amount of damage to an emitting plant. Thus, the fact that natural enemies respond to HIPVs does not imply that these volatiles evolved for this purpose or that there is an adaptive advantage for a plant to use HIPVs to attract natural enemies. Rather, natural enemies of insect herbivores may have learned to co-opt the HIPV signal emitted by plants and, by doing so, increased their fitness irrespective of the ultimate fitness outcome to the plant.Though more controversial, scents can also have an effect on plant behavior.²² Early work suggested that HIPVs from wounded willows,²³ poplars²⁴ and sugar maples²⁴ could trigger defense responses from other neighboring conspecifics. More recent studies have shown that this signaling can occur between different species of plants.²⁵ While these results are intriguing, they appear to have little adaptive function from the perspective of an emitting plant, which could be facilitating the fitness of potential resource competitors. Further, unless the individual within the same plant species shared some degree of kinship,²⁶ an emitting plant would also be at a disadvantage by providing an HIPV wound signal to a conspecific that, in theory, occupies the same competitive niche space. On the other hand, unwounded conspecific should benefit from being able to ‘eavesdrop’ by detecting HIPVs from wounded plants as they share the same herbivore complex and thus are vulnerable to attack. Moreover, from a heterospecific receiver’s perspective, the benefits of eavesdropping can be confounded by the potential of mounting defenses against a signal generated by incompatible herbivores feeding on a different plant species.²⁷ So, eavesdropping may be adaptive for a receiving plant if it realizes increased fitness relative to a conspecific that did not receive the signal. The emitting plant derives no apparent adaptive benefit of using HIPVs to warn neighboring plants. However, the emitting plant may benefit if their HIPVs have inhibitory allelopathic activity on neighboring plants.²⁸Our recent work¹ highlighted another scenario by which an HIPV-emitting plant would derive a direct benefit from the emissions: when HIPVs act as systemic wound signals within damaged plants. We showed that branches of blueberry shrubs lack effective vascular connections and thus cannot transmit wound signals among branches via the vasculature. To compensate, HIPVs can be transmitted among branches and, in so doing, overcome the vascular constraints of the branching life history strategy. Exposure to HIPVs increased levels of defensive signaling hormones in undamaged branches, changed their defensive chemical status, and made them more resistant to herbivores.¹ This idea that HIPVs may function in intra-plant communication to activate or prime defenses in other parts of the emitting plant against future attack was first suggested separately by Farmer²⁹ and Orians.⁹ The hypothesis was first tested with mechanically clipped wild sagebrush,³⁰ and it was further tested with insect herbivores of wild lima bean³¹ and hybrid poplar.³² Under this scenario, the emitting plant derives a direct benefit from the HIPVs, providing an unambiguous fitness advantage.So, what is the most beneficial factor to a plant for emitting volatiles in response to herbivore feeding? In terms of maximizing the potential benefit and minimizing the potential risk to the emitting plant, the function of HIPVs in mediating systemic wound signaling clearly provides the greatest potential adaptive advantage. Thus, we propose that the primary adaptive benefit for the evolution of HIPVs is to signal and protect unwounded parts of the attacked plant with high risk of infestation against herbivores. Later, these volatiles provided cues that led to adaptive fitness advantages for neighboring plants and natural enemies of herbivores, which may or may not benefit the HIPV-emitting plant. Indeed, ecologically adaptive advantages have emerged and contribute to a diverse, multi-functional chemical ecology mediated by HIPVs.     

Long-distance transport of signals during symbiosis: Are nodule formation and mycorrhization autoregulated in a similar way?

Legumes enter nodule symbioses with nitrogen-fixing bacteria (rhizobia), whereas most flowering plants establish symbiotic associations with arbuscular mycorrhizal (AM) fungi. Once first steps of symbiosis are initiated, nodule formation and mycorrhization in legumes is negatively controlled by a shoot-derived inhibitor (SDI), a phenomenon termed autoregulation. According to current views, autoregulation of nodulation and mycorrhization in legumes is regulated in a similar way. CLE peptides induced in response to rhizobial nodulation signals (Nod factors) have been proposed to represent the ascending long-distance signals to the shoot. Although not proven yet, these CLE peptides are likely perceived by leucine-rich repeat (LRR) autoregulation receptor kinases in the shoot. Autoregulation of mycorrhization in non-legumes is reminiscent to the phenomenon of “systemic acquired resistance” in plant-pathogen interactions.Key words: arbuscular mycorrhiza, autoregulation, CLE peptides, mutant, nodulation, split-root systemUnder natural conditions, growth of plants is often limited by the availability of nutrients such as nitrogen and phosphorous. Plants have therefore developed strategies to acquire nutrients with the help of soil microorganisms. Most land plants enter mutualistic root symbioses with arbuscular mycorrhizal (AM) fungi, whereas legumes form special root nodules containing nitrogen-fixing bacteria, so-called rhizobia.^1–4 Establishment and maintenance of symbiosis requires plant resources, such as photosynthetically assimilated carbon. To minimize these costs, host plants are able to control the degree of their symbiotic interactions. Above a critical threshold level further establishment of symbiosis is restricted—a feedback phenomenon termed autoregulation of symbiosis. Autoregulation can be experimentally demonstrated in split-root systems. When legume roots are already infected by rhizobia on one side of a split-root, further nodule development is “systemically” inhibited on the other side. Similarly, prior colonization of split-roots by AM fungi on one half suppresses later fungal root colonization on the other half. Hence, important elements of the symbiotic autoregulation circuit are not only localized in roots, but also in aerial parts of the plant, implicating transport of signals in vascular bundles (Fig. 1). Whereas autoregulation of nodulation in legumes has been studied for many decades,^5–9 the first publications clearly stating a shoot-controlled autoregulation of mycorrhization in split-root systems appeared in 2000 for the non-legume barley (Hordeum vulgare) and thereafter for alfalfa (Medicago sativa) and soybean (Glycine max).^10–13 The data from these split-root experiments are supported by the findings that supernodulating (or hypernodulating) loss-of-autoregulation mutants displayed either an increased degree of AM colonization and/or a higher abundance of arbuscules.^14–16Open in a separate windowFigure 1Proposed model of shoot-controlled autoregulation of symbiosis in a split-root system. Prior infection of root A by rhizobia or AM fungi systemically suppresses later establishment of symbiosis in root B. Expression of specific CLE peptides (and/or other peptide hormones) is induced in response to rhizobial nodulation signals (Nod factors) and perhaps also in response to colonization by AM fungi (stage 1). The CLE peptides (and/or other signals) are then presumed to be transported in the xylem to the shoot, where they are perceived by leucine-rich repeat (LRR) autoregulation receptor kinases (stage 2). As a result of autoregulation signaling in the shoot, an unknown shoot-derived inhibitor (SDI) is produced (stage 3) and transported as a phloem-mobile signal to the root. Perception and action of the SDI signal in roots would then inhibit nodulation and root colonization by AM fungi (stage 4).     

AtAGP18, a lysine-rich arabinogalactan protein in Arabidopsis thaliana,functions in plant growth and development as a putative co-receptor for signal transduction

Arabinogalactan-proteins (AGPs) are a class of hyperglycosylated, hydroxyproline-rich glycoproteins that are widely distributed in the plant kingdom. AtAGP17, 18 and 19 are homologous genes encoding three classical lysine-rich AGPs in Arabidopsis. We observed subcellular localization of AtAGP18 at the plasma membrane by expressing a translational fusion gene construction of AtAGP18 attached to a green fluorescent protein (GFP) tag in Arabidopsis plants. We also overexpressed AtAGP18 without the GFP tag in Arabidopsis plants, and the resulting transgenic plants had a short, bushy phenotype. Here we discuss putative roles of AtAGP18 as a glycosylphosphatidylinositol (GPI)-anchored protein involved in a signal transduction pathway regulating plant growth and development.Key words: Arabidopsis thaliana, arabinogalactan-proteins, co-receptor, glycosylphosphatidylinositol, lipid rafts, overexpressionArabinogalactan-proteins (AGPs) are plant cell surface glycoproteins or proteoglycans which are thought to play important roles in various aspects of plant growth and development, such as somatic embryogenesis, cell proliferation and elongation, pattern formation and hormone signaling.¹ The lysine-rich classical AGP subfamily in Arabidopsis contains three members: AtAGP17, 18 and 19. The subcellular localization of AtAGP17 and AtAGP18 was previously studied in our laboratory by expressing GFP-AtAGP17/18 fusion proteins in tobacco cell cultures.^2,3 In a recent report, we used Arabidopsis plants to overexpress GFP-AtAGP17/18/19 fusion proteins to observe subcellular localization of the lysine-rich AGPs in planta, in contrast to our previous plant cell culture work.⁴ Moreover, the lysine-rich AGPs alone (i.e., AtAGP17/18/19 without the GFP tag) were overexpressed in Arabidopsis plants, and only AtAGP18 overexpressors had a distinctive phenotype. This phenotype included shorter stems, more branches and less seeds, indicating a role for AtAGP18 in plant growth and development.⁴ In this addendum, we further discuss the putative biological role of AtAGP18 on a molecular level and its possible mode of action in cellular signaling.Classical AGPs are frequently predicted to have a glycosylphosphatidylinositol (GPI) anchor, which would allow for the localization of such AGPs to the outer surface of the plasma membrane. Biochemical analyses were carried out to support this hypothesis in tobacco, pear,⁵ rose⁶ and Arabidopsis.⁷ The lysine-rich classical AGPs, AtAGP17 and 18, were predicted to have a GPI anchor.⁸ To test this idea, tobacco cell cultures expressing GFP-AtAGP17/18 fusion proteins were plasmolyzed and GFP fluorescence was observed on the plasma membrane.^2,3 To corroborate this finding in planta, GFP-AtAGP17/18 were expressed in Arabidopsis plants and leaf trichome cells were plasmolyzed. Enhanced GFP fluorescence was observed at the plasma membrane of these transgenic trichome cells, indicating the presence of GFP-AtAGP17/18 at the plasma membrane.⁴ The localization of these lysine-rich classical AGPs at the plasma membrane suggests possible biological roles in sensing extracellular signals. They are likely associated with lipid rafts involved in cell signaling for the following reasons. In plants as well as animals, there are sterol-enriched, detergent-resistant plasma membrane microdomains called lipid rafts. Lipid rafts are known to be involved in signal transduction and are enriched in transmembrane receptors and GPI-anchored proteins, including AGPs.^9–11 The accumulation of these proteins in such microdomains may allow for interactions between these proteins in sensing extracellular signals which lead to various intracellular events. Interestingly, a recent study shows that lipid rafts from hybrid aspen cells contain callose synthase and cellulose synthase, and these enzymes are active since in vitro polysaccharide synthesis by the isolated detergent-resistant membranes was observed. These results demonstrate that lipid rafts are involved in cell wall polysaccharide biosynthesis.¹² In addition, an Arabidopsis pnt mutant study shows GPI-anchored proteins are required in cell wall synthesis and morphogenesis.¹³ These observations, coupled with previous observations that cellulose synthases as well as AGPs interact with microtubules, suggest that AGPs in lipid rafts may have a role in signal events, including those regulating cellulose and/or callose biosynthesis or deposition.^14,15To examine the role of LeAGP-1, a lysine-rich AGP in tomato, transgenic tomato plants were produced which expressed GFP-LeAGP-1 under the control of the cauliflower mosaic virus 35S promoter.¹⁶ The tomato LeAGP-1 overexpressors and Arabidopsis AtAGP18 overexpressors both have a bushy phenotype similar to transgenic tobacco plants overproducing cytokinins.^4,16,17 Cytokinins are an important class of plant hormones involved in many plant growth and development processes, such as cell growth and division, differentiation and other physiological processes.¹⁸ Therefore, Sun et al. proposed that LeAGP-1 might function in concert with the cytokinin signal transduction pathway.¹⁶ Since the overexpression phenotypes of AtAGP18 are similar to those of LeAGP-1, AtAGP18 is also likely associated with the cytokinin signal transduction pathway. The prevailing model for cytokinin signaling in Arabidopsis is similar to the two-component system in bacteria and yeast. In this model, the cytokinin receptor contains an extracellular domain, a kinase domain and a receiver domain. When the cytokinin receptor senses cytokinin signals, it auto-phosphorylates at a His residue in the kinase domain. The phosphoryl group is then transferred to an Asp residue in the receiver domain. Subsequently, the phosphoryl group is transferred to a His residue in the histidine phosphotransfer protein (Hpt) and the Hpt translocates to the nucleus and transfers the phosphoryl group to an Asp residue in a downstream response regulator to activate it.¹⁹ This model is consistent with our hypothesis since the cytokinin receptor in this model is a receptor kinase located in the plasma membrane with an extra-cellular domain that can potentially interact with AtAGP18. AtAGP18 may function as a co-receptor that first binds to cytokinins, then either directly interacts with cytokinin receptors or brings the cytokinins to cytokinin receptors in the plasma membrane. The first scenario is analogous to the interaction of contactin and contactin-associated protein (Caspr) in neurons. In this model, contactin is a GPI-anchored protein on the cell surface that binds to signal molecules and interacts with the transmembrane receptor Caspr to transmit signals to the cell interior.²⁰ The second scenario is analogous to fibroblast growth factor (FGF) signal activation in which heparan sulfate proteoglycans bind to FGF molecules and bring them to the FGF receptor.²¹Based on all the above observations and findings, a hypothetical model for AtAGP18 function is proposed in Figure 1. The model shows AtAGP18 located on the outer surface of the plasma membrane in lipid rafts where it could act as a co-receptor to sense extracellular signals (such as cytokinin) and interact with transmembrane proteins, possibly receptor kinases or ion channels, in the lipid rafts to initiate signaling by triggering various intracellular events. Interestingly, receptor tyrosine kinases and ion channels are known to be present in lipid rafts.^9,22 Moreover, AGPs are likely associated with ion channels since addition of the AGP-binding reagent Yariv phenylglycoside resulted in elevated cytoplasmic calcium concentrations in tobacco cells and lily pollen tubes.^15,23,24 Clearly, additional work will be required to verify such a model, and to better understand how AtAGP18 might sense extracellular signals and interact with the transmembrane proteins in the lipid rafts.Open in a separate windowFigure 1Model for atAGP18 functioning in cellular signaling to control plant growth and development. In this model, lipid rafts are enriched in glycosphingolipids, sterols, transmembrane proteins (such as receptors, receptor kinases and ion channel proteins) and GPI-anchored proteins including AtAGP18. (a) AtAGP18 acts as a co-receptor by binding to signaling molecules and directly interacting with transmembrane proteins in the lipid rafts. (B) AtAGP18 acts as a co-receptor by binding to signaling molecules and bringing the signaling molecules to transmembrane proteins in the lipid rafts. Upon activation by the extracellular signals, the transmembrane proteins initiate signaling and lead to various intracellular events (e.g., phosphorylation similar to the two-component signaling system, influx of calcium ions). The different components of the AtAGP18 molecule and the various lipid components of lipid rafts and plasma membrane are shown in the boxed inset. Hpt, histidine phosphotransfer protein.     

Evolutionarily conserved photoperiod mechanisms in plants: When did plant photoperiodic signaling appear?

Day-length and the circadian clock control critical aspects of plant development such as the onset of reproduction by the photoperiodic pathway.¹ CONSTANS (CO) regulates the expression of a florigenic mobile signal from leaves to the apical meristem and thus is central to the regulation of photoperiodic flowering.² This regulatory control is present in all higher plants,³ but the time in evolution when it arose was unknown. We have shown that the genomes of green microalgae encode members of the CONSTANS-like (COL) protein family. One of these genes, the Chlamydomonas reinhardtii CO homolog (CrCO), can complement the co mutation in Arabidopsis.⁴ CrCO expression is controlled by the clock and photoperiod in Chlamydomonas and at the same time is involved in the correct timing of several circadian output processes such as the accumulation of starch or the coordination of cell growth and division. We have proposed that, since very early in the evolutionary lineage that gave rise to higher plants, CO homologs have been involved in the photoperiod control of important developmental processes, and that the recruitment of COL proteins in other roles may have been crucial for their evolutionary success.Key words: photoperiod, flowering, constans, signaling, Arabidopsis, ChlamydomomasPlants have adapted several physiological mechanisms to finely respond to external signals and coordinate the correct timing of important developmental processes.⁵ This way, different diurnal and seasonal potentially deterring or optimal situations can be predicted and an adequate response prepared in advance. In the case of the control of reproduction, predicting the best time of the year to flower confers a fitness improvement directly related to seed productivity and thus is naturally selected as an important trait in plant evolution.⁶ Plants and algae posses sophisticated mechanisms to measure time, such as a circadian clock or a photoperiod control to detect day-length and probably temperature.^7,8 In photoperiodic flowering, the protein CONSTANS plays a central role because it activates in the leaves the expression of FLOWERING LOCUS T (FT). The small FT protein is able to move through the vascular bundles from the leaves to the apical meristem and trigger the program that transforms it into a reproductive meristem.² Eventually, these changes induce the production of the flower.We have recently described⁴ that in green microalgae the mechanism detecting photoperiod signals involves a CO homolog (CrCO) with a crucial role. CrCO expression is controlled by photoperiod and at the same time regulates several output processes of the clock such as starch accumulation or the onset of cell division.