Mol. Cells 2018; 41(8): 724-732
Published online July 10, 2018
https://doi.org/10.14348/molcells.2018.0104
© The Korean Society for Molecular and Cellular Biology
Correspondence to : *Correspondence: cmryu@kribb.re.kr
Plant defence responses to various biotic stresses via systemic acquired resistance (SAR) are induced by avirulent pathogens and chemical compounds, including certain plant hormones in volatile form, such as methyl salicylate and methyl jasmonate. SAR refers to the observation that, when a local part of a plant is exposed to elicitors, the entire plant exhibits a resistance response. In the natural environment, plants are continuously exposed to avirulent pathogens that induce SAR and volatile emissions affecting neighbouring plants as well as the plant itself. However, the underlying mechanism has not been intensively studied. In this study, we evaluated whether plants “memorise” the previous activation of plant immunity when exposed repeatedly to plant defensive volatiles such as methyl salicylate and methyl jasmonate. We hypothesised that stronger SAR responses would occur in plants treated with repeated applications of the volatile plant defence compound MeSA than in those exposed to a single or no treatment.
Keywords methyl salicylate, plant immunity, plant memory, SAR, VOC
Plants respond to pathogen attack by activating both local and systemic defences aimed at inhibiting the growth and spreading of the pathogen (Fu and Dong, 2013; Shah and Zeier, 2013). The defence response, systemic acquired resistance (SAR), develops following localised foliar infection by diverse avirulent pathogens and is expressed systemically (Fu and Dong, 2013; Hammond-Kosack and Jones, 1996; Shah and Zeier, 2013). During this process, leaves become primed to activate more rapid and/or stronger defence responses following attack by pathogens or insects or in response to abiotic stress upon secondary infection (Conrath et al., 2015; Martinez-Medina et al., 2016). SAR provides the benefits of enhanced protection without the costs associated with constitutive expression of stress-related genes (Bruce et al., 2007; Heil and Baldwin, 2002; Jung et al., 2009). This plant defence priming mechanism can be elicited by the exogenous application of chemicals as well as exposure to stress cues (Lyon, 2007). This plant response was recently attributed to “plant memory” (Crisp et al., 2016), a theory that has also been referred to as “plant stress memory” and “defence priming” (Crisp et al., 2016). In the natural state, plants are continuously exposed to avirulent pathogens that cause SAR and volatile emissions that affect neighbouring plants as well as the plant itself. However, little is known about the underlying mechanism.
Leaves infected with SAR-inducing bacteria produce modified compounds that confer disease resistance to systemic tissues (previously unexposed plants) (Fu and Dong, 2013; Van Bel and Gaupels, 2004), which indicates that a mobile systemic signal(s) is involved in SAR (Gao et al., 2015; Park et al., 2007; Shah et al., 2014). Previously, salicylic acid (SA) was postulated to be this mobile signal because it induces defence responses when applied to plants, moves systemically, is found in phloem exudates of infected leaves and is required in systemic tissue for SAR (Jung et al., 2009; Park et al., 2007). However, later grafting studies showed that infected, SA-deficient rootstocks can trigger SAR in wild-type scions, implying that SA is not a mobile SAR signal (Vernooij et al., 1994). SA is synthesised via the shikimic acid pathway, which bifurcates into two branches after the biosynthesis of chorismic acid. Both branches contribute to SA biosynthesis and are required for SAR (Wildermuth et al., 2001). SA accumulation alone is insufficient to induce SAR (Cameron et al., 1999). For instance, exogenous application of the 3C sugar alcohol glycerol-3-phosphate (G3P) or dicarboxylic acid azelaic acid (AzA) to induce SAR in wild-type plants does not induce SA accumulation. However, G3P or AzA cannot confer SAR in
Since 2007, when MeSA was found to function as a mobile SAR signal, up to 13 compounds have been shown to move throughout the plant as SAR signals via the vascular system. Representative molecules include the abietane diterpenoid dehydroabietinal (DA), the lysine catabolite pipecolic acid (Pip), G3P and AzA (Chanda et al., 2011; Chaturvedi et al., 2012; Jung et al., 2009; Mandal et al., 2012; Návarová et al., 2012). These four signalling molecules are related to SA accumulation/signalling. Long-distance signalling by specific volatile organic compounds (VOCs) such as the SA-derivative MeSA has also been reported. Notably, green-leaf volatiles and herbivore-induced VOCs induce resistance from locally damaged leaves to the entire plant (Kost and Heil, 2006; Yi et al., 2009). These VOCs can also affect other plants by moving through the air. This so-called “plant–plant communication” can occur between taxonomically unrelated plants. For instance, lima bean (
In this study, we evaluated whether plants memorise the previous activation of plant immunity when exposed repeatedly to plant defensive volatiles such as MeSA and methyl jasmonate (MeJA). To investigate volatile-induced plant memory, we designed a new experimental system and obtained phenotypic evidence for VOC-mediated defence priming in wild tobacco (
Seeds of Arabidopsis
To investigate whether MeSA and MeJA have a direct inhibitory effect against
Following inoculation with pathogen, leaf tissue was harvested 0, 12, 24, 36 and 48 h after inoculation with
Plant growth parameters such as shoot weight, chlorophyll content and leaf senescence were measured at 7 days after the second exposure in 6-week-old plants. Chlorophyll concentration was measured using a SPAD-502 meter (Konica-Minolta, Japan) (Ling et al., 2011). This meter is used to determine the amount of chlorophyll in a leaf as the SPAD (single-photon avalanche diode) value (value indicating chlorophyll content), which is used as a measure of plant health. Leaf tissues were harvested for these measurements using a circular punch cork borer, yielding 1 cm diameter leaf discs with an area of 0.785 cm2. SPAD values were recorded using exactly the same leaves from the same plants and 12 independent plants. The fresh weight, i.e., shoot weight per treatment per plant, was measured using 12 plants.
Analysis of variance for the experimental datasets was performed using JMP software version 5.0 (SAS Institute Inc., USA;
We utilised in vitro-grown
To examine whether SAR-related defence genes were more strongly upregulated in response to MeSA-MeSA than Water-MeSA treatment, we measured the transcript levels of SA signalling-related genes, i.e.,
Previous studies suggest that the induction of SAR results in the inhibition of plant growth via a mechanism referred to as “allocation fitness cost” (Heil and Baldwin, 2002). In the current study, we measured fresh shoot weight at 6 weeks in plants under the conditions shown in Fig. 1. There were no differences in fresh shoot weight among MeSA-MeSA, Water-MeSA and Water-Water (control) plants. However, MeSA-MeJA significantly reduced fresh shoot weight (Figs. 3A and 3B). The total shoot weight per plant was 65.4 mg for MeSA-MeSA, 50.9 mg for MeSA-MeJA, 67 mg for Water-MeSA and 68.3 mg for Water-Water treatment (Figs. 3A and 3B). However, under MeSA-MeSA treatment, the SPAD value decreased by 2 compared with the control. For MeSA-MeJA treatment, which inhibited plant growth, the SPAD value decreased by 8 compared with the control (Figs. 3A and 3C). In addition, the number of senescent leaves in the MeSA-MeSA treatment group was 1.5-fold higher than that in the Water-MeSA treatment group. Under MeSA-MeJA treatment, the number of senescent leaves was approximately 16-fold that of the control (Figs. 3A and 3D). These results indicate that MeSA-MeSA treatment did not affect plant vegetative growth, as reflected by shoot length, but it did increase leaf senescence.
To evaluate the effect of repeated applications of MeSA on SAR against the necrotrophic pathogen
Next, we used the Incu Tissue system to test the effects of repeated applications of MeSA on plant defence pathway signalling in the
Plant defence-related VOCs play an essential role in plant–plant communication (Heil and Bueno, 2007; Kost and Heil, 2006; Yi et al., 2009). Of the many studies investigating plant VOC emissions in response to herbivore damage and the occurrence of insect resistance in neighbouring plants, SAR against pathogenic microbes (such as viruses, bacteria and fungi) induced by plant VOCs has been intensively investigated only recently (Chen et al., 2008; Heil and Bueno, 2007; Karban et al., 2006). Furthermore, plants under natural conditions are continuously exposed to VOCs from neighbouring plants. In the current study, we developed a simplified system to test the concept of plant memory in which the repeated application of the plant defence VOC MeSA elicited higher plant protection capacity than the control and the expression of SAR marker genes
While the elicitation of SAR by extracellular application of plant defence-related VOCs (airborne signals) is well known (Heil and Adame-Álvarez, 2010; Yi et al., 2009), the increase in SAR in response to repeated applications of VOCs has not previously been demonstrated. The current study was designed to evaluate the plant memory effects of sequential releases of defensive airborne signals such as MeSA and MeJA on SAR. We previously reported that lima bean (
Plants exhibit resistance to specific pathogens using different resistance signalling pathways, such as the SA and JA pathways (Kunkel and Brooks, 2002). SA signalling is generally effective against biotrophs, while JA signalling is generally effective against necrotrophs. Notably, in the current study, plants exposed to MeSA twice showed greater resistance against
Another interesting phenomenon is the occurrence of VOC-mediated signalling crosstalk in plant memory. Subsequent treatment with MeJA instead of a second MeSA treatment suppressed SA-dependent plant defence responses, such as resistance to the biotrophic pathogen
The expression levels of defence genes in tobacco and
Previous investigations of chemical triggers eliciting plant immunity have involved the direct drenching of plants with compounds that induce resistance in a dose-dependent manner. This method has been successfully applied to pepper roots and cucumber seeds, leading to defence priming under field conditions for 4 consecutive years (Choi et al., 2014; Song and Ryu, 2013). In addition, 4-week-old pepper plants were dip-treated with 1 mM 3-pentanol solution before being transplanted into the field. This process elicited induced resistance in 2 year field trials without affecting fruit yield. Drench application of the volatiles 3-pentanol and 2-butanone upregulated the defence-related gene
These findings demonstrate that, even when applied by drenching, water-soluble VOCs can help recruit a natural enemy of aphids due to the odours that they spread and may ultimately prevent plant disease and insect damage by eliciting induced resistance, even under open field conditions (Song et al., 2013). Successful cases of the use of VOCs for disease prevention in the field were recently reported (Choi et al., 2014). Nonetheless, the primary challenge to field application of VOCs is developing adequate chemical treatment methods. The application of volatiles has other drawbacks as well, including the high rate of volatile diffusion after application in the open field, inconsistent levels of effectiveness and negative effects on plant growth. However, with the increasing use of indoor cultivation systems, such as greenhouses and glasshouses, the chances for successfully applying VOCs to growing crops have increased (Kim et al., 2016). Eliciting induced resistance in plants through the use of VOCs without affecting plant growth may lead to the development of new biocontrol methods for future use in agriculture. Therefore, if we take advantage of the plant memory concept observed in this study, the agricultural application of VOCs to induce plant defence responses should be more feasible.
Mol. Cells 2018; 41(8): 724-732
Published online August 31, 2018 https://doi.org/10.14348/molcells.2018.0104
Copyright © The Korean Society for Molecular and Cellular Biology.
Geun Cheol Song1, and Choong-Min Ryu1,2,*
1Molecular Phytobacteriology Laboratory, KRIBB, Daejeon 34141, Korea, 2Biosystems and Bioengineering Program, University of Science and Technology (UST), Daejeon 34113, Korea
Correspondence to:*Correspondence: cmryu@kribb.re.kr
Plant defence responses to various biotic stresses via systemic acquired resistance (SAR) are induced by avirulent pathogens and chemical compounds, including certain plant hormones in volatile form, such as methyl salicylate and methyl jasmonate. SAR refers to the observation that, when a local part of a plant is exposed to elicitors, the entire plant exhibits a resistance response. In the natural environment, plants are continuously exposed to avirulent pathogens that induce SAR and volatile emissions affecting neighbouring plants as well as the plant itself. However, the underlying mechanism has not been intensively studied. In this study, we evaluated whether plants “memorise” the previous activation of plant immunity when exposed repeatedly to plant defensive volatiles such as methyl salicylate and methyl jasmonate. We hypothesised that stronger SAR responses would occur in plants treated with repeated applications of the volatile plant defence compound MeSA than in those exposed to a single or no treatment.
Keywords: methyl salicylate, plant immunity, plant memory, SAR, VOC
Plants respond to pathogen attack by activating both local and systemic defences aimed at inhibiting the growth and spreading of the pathogen (Fu and Dong, 2013; Shah and Zeier, 2013). The defence response, systemic acquired resistance (SAR), develops following localised foliar infection by diverse avirulent pathogens and is expressed systemically (Fu and Dong, 2013; Hammond-Kosack and Jones, 1996; Shah and Zeier, 2013). During this process, leaves become primed to activate more rapid and/or stronger defence responses following attack by pathogens or insects or in response to abiotic stress upon secondary infection (Conrath et al., 2015; Martinez-Medina et al., 2016). SAR provides the benefits of enhanced protection without the costs associated with constitutive expression of stress-related genes (Bruce et al., 2007; Heil and Baldwin, 2002; Jung et al., 2009). This plant defence priming mechanism can be elicited by the exogenous application of chemicals as well as exposure to stress cues (Lyon, 2007). This plant response was recently attributed to “plant memory” (Crisp et al., 2016), a theory that has also been referred to as “plant stress memory” and “defence priming” (Crisp et al., 2016). In the natural state, plants are continuously exposed to avirulent pathogens that cause SAR and volatile emissions that affect neighbouring plants as well as the plant itself. However, little is known about the underlying mechanism.
Leaves infected with SAR-inducing bacteria produce modified compounds that confer disease resistance to systemic tissues (previously unexposed plants) (Fu and Dong, 2013; Van Bel and Gaupels, 2004), which indicates that a mobile systemic signal(s) is involved in SAR (Gao et al., 2015; Park et al., 2007; Shah et al., 2014). Previously, salicylic acid (SA) was postulated to be this mobile signal because it induces defence responses when applied to plants, moves systemically, is found in phloem exudates of infected leaves and is required in systemic tissue for SAR (Jung et al., 2009; Park et al., 2007). However, later grafting studies showed that infected, SA-deficient rootstocks can trigger SAR in wild-type scions, implying that SA is not a mobile SAR signal (Vernooij et al., 1994). SA is synthesised via the shikimic acid pathway, which bifurcates into two branches after the biosynthesis of chorismic acid. Both branches contribute to SA biosynthesis and are required for SAR (Wildermuth et al., 2001). SA accumulation alone is insufficient to induce SAR (Cameron et al., 1999). For instance, exogenous application of the 3C sugar alcohol glycerol-3-phosphate (G3P) or dicarboxylic acid azelaic acid (AzA) to induce SAR in wild-type plants does not induce SA accumulation. However, G3P or AzA cannot confer SAR in
Since 2007, when MeSA was found to function as a mobile SAR signal, up to 13 compounds have been shown to move throughout the plant as SAR signals via the vascular system. Representative molecules include the abietane diterpenoid dehydroabietinal (DA), the lysine catabolite pipecolic acid (Pip), G3P and AzA (Chanda et al., 2011; Chaturvedi et al., 2012; Jung et al., 2009; Mandal et al., 2012; Návarová et al., 2012). These four signalling molecules are related to SA accumulation/signalling. Long-distance signalling by specific volatile organic compounds (VOCs) such as the SA-derivative MeSA has also been reported. Notably, green-leaf volatiles and herbivore-induced VOCs induce resistance from locally damaged leaves to the entire plant (Kost and Heil, 2006; Yi et al., 2009). These VOCs can also affect other plants by moving through the air. This so-called “plant–plant communication” can occur between taxonomically unrelated plants. For instance, lima bean (
In this study, we evaluated whether plants memorise the previous activation of plant immunity when exposed repeatedly to plant defensive volatiles such as MeSA and methyl jasmonate (MeJA). To investigate volatile-induced plant memory, we designed a new experimental system and obtained phenotypic evidence for VOC-mediated defence priming in wild tobacco (
Seeds of Arabidopsis
To investigate whether MeSA and MeJA have a direct inhibitory effect against
Following inoculation with pathogen, leaf tissue was harvested 0, 12, 24, 36 and 48 h after inoculation with
Plant growth parameters such as shoot weight, chlorophyll content and leaf senescence were measured at 7 days after the second exposure in 6-week-old plants. Chlorophyll concentration was measured using a SPAD-502 meter (Konica-Minolta, Japan) (Ling et al., 2011). This meter is used to determine the amount of chlorophyll in a leaf as the SPAD (single-photon avalanche diode) value (value indicating chlorophyll content), which is used as a measure of plant health. Leaf tissues were harvested for these measurements using a circular punch cork borer, yielding 1 cm diameter leaf discs with an area of 0.785 cm2. SPAD values were recorded using exactly the same leaves from the same plants and 12 independent plants. The fresh weight, i.e., shoot weight per treatment per plant, was measured using 12 plants.
Analysis of variance for the experimental datasets was performed using JMP software version 5.0 (SAS Institute Inc., USA;
We utilised in vitro-grown
To examine whether SAR-related defence genes were more strongly upregulated in response to MeSA-MeSA than Water-MeSA treatment, we measured the transcript levels of SA signalling-related genes, i.e.,
Previous studies suggest that the induction of SAR results in the inhibition of plant growth via a mechanism referred to as “allocation fitness cost” (Heil and Baldwin, 2002). In the current study, we measured fresh shoot weight at 6 weeks in plants under the conditions shown in Fig. 1. There were no differences in fresh shoot weight among MeSA-MeSA, Water-MeSA and Water-Water (control) plants. However, MeSA-MeJA significantly reduced fresh shoot weight (Figs. 3A and 3B). The total shoot weight per plant was 65.4 mg for MeSA-MeSA, 50.9 mg for MeSA-MeJA, 67 mg for Water-MeSA and 68.3 mg for Water-Water treatment (Figs. 3A and 3B). However, under MeSA-MeSA treatment, the SPAD value decreased by 2 compared with the control. For MeSA-MeJA treatment, which inhibited plant growth, the SPAD value decreased by 8 compared with the control (Figs. 3A and 3C). In addition, the number of senescent leaves in the MeSA-MeSA treatment group was 1.5-fold higher than that in the Water-MeSA treatment group. Under MeSA-MeJA treatment, the number of senescent leaves was approximately 16-fold that of the control (Figs. 3A and 3D). These results indicate that MeSA-MeSA treatment did not affect plant vegetative growth, as reflected by shoot length, but it did increase leaf senescence.
To evaluate the effect of repeated applications of MeSA on SAR against the necrotrophic pathogen
Next, we used the Incu Tissue system to test the effects of repeated applications of MeSA on plant defence pathway signalling in the
Plant defence-related VOCs play an essential role in plant–plant communication (Heil and Bueno, 2007; Kost and Heil, 2006; Yi et al., 2009). Of the many studies investigating plant VOC emissions in response to herbivore damage and the occurrence of insect resistance in neighbouring plants, SAR against pathogenic microbes (such as viruses, bacteria and fungi) induced by plant VOCs has been intensively investigated only recently (Chen et al., 2008; Heil and Bueno, 2007; Karban et al., 2006). Furthermore, plants under natural conditions are continuously exposed to VOCs from neighbouring plants. In the current study, we developed a simplified system to test the concept of plant memory in which the repeated application of the plant defence VOC MeSA elicited higher plant protection capacity than the control and the expression of SAR marker genes
While the elicitation of SAR by extracellular application of plant defence-related VOCs (airborne signals) is well known (Heil and Adame-Álvarez, 2010; Yi et al., 2009), the increase in SAR in response to repeated applications of VOCs has not previously been demonstrated. The current study was designed to evaluate the plant memory effects of sequential releases of defensive airborne signals such as MeSA and MeJA on SAR. We previously reported that lima bean (
Plants exhibit resistance to specific pathogens using different resistance signalling pathways, such as the SA and JA pathways (Kunkel and Brooks, 2002). SA signalling is generally effective against biotrophs, while JA signalling is generally effective against necrotrophs. Notably, in the current study, plants exposed to MeSA twice showed greater resistance against
Another interesting phenomenon is the occurrence of VOC-mediated signalling crosstalk in plant memory. Subsequent treatment with MeJA instead of a second MeSA treatment suppressed SA-dependent plant defence responses, such as resistance to the biotrophic pathogen
The expression levels of defence genes in tobacco and
Previous investigations of chemical triggers eliciting plant immunity have involved the direct drenching of plants with compounds that induce resistance in a dose-dependent manner. This method has been successfully applied to pepper roots and cucumber seeds, leading to defence priming under field conditions for 4 consecutive years (Choi et al., 2014; Song and Ryu, 2013). In addition, 4-week-old pepper plants were dip-treated with 1 mM 3-pentanol solution before being transplanted into the field. This process elicited induced resistance in 2 year field trials without affecting fruit yield. Drench application of the volatiles 3-pentanol and 2-butanone upregulated the defence-related gene
These findings demonstrate that, even when applied by drenching, water-soluble VOCs can help recruit a natural enemy of aphids due to the odours that they spread and may ultimately prevent plant disease and insect damage by eliciting induced resistance, even under open field conditions (Song et al., 2013). Successful cases of the use of VOCs for disease prevention in the field were recently reported (Choi et al., 2014). Nonetheless, the primary challenge to field application of VOCs is developing adequate chemical treatment methods. The application of volatiles has other drawbacks as well, including the high rate of volatile diffusion after application in the open field, inconsistent levels of effectiveness and negative effects on plant growth. However, with the increasing use of indoor cultivation systems, such as greenhouses and glasshouses, the chances for successfully applying VOCs to growing crops have increased (Kim et al., 2016). Eliciting induced resistance in plants through the use of VOCs without affecting plant growth may lead to the development of new biocontrol methods for future use in agriculture. Therefore, if we take advantage of the plant memory concept observed in this study, the agricultural application of VOCs to induce plant defence responses should be more feasible.
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