Abscisic acid is essential for rewiring of jasmonic acid-dependent defenses during herbivory
Summary (4 min read)
Results
- ABA-and ET-dependency of JA-dependent defense gene expression upon P. rapae feeding Here, the authors investigated whether ABA and ET have a role in the differential expression of the MYC-and the ERF-branch during induction of JA-dependent defense signaling by P. rapae feeding.
- Expression of the MYC-branch marker gene VSP2 and the ERFbranch marker gene PDF1.2 was monitored in wild-type Col-0, MYC2-impaired mutant jin1-7 (hereafter called myc2), MYC2, MYC3, MYC4 triple mutant myc2,3,4, ABA biosynthesis mutant aba2-1 and ET response mutant ein2-1. First-instar P. rapae caterpillars were allowed to feed for 24 h on the different Arabidopsis genotypes, after which they were removed.
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- The copyright holder for this preprint (which was this version posted August 28, 2019.
- PDF1.2 transcript levels were very low in both Col-0 and ein2-1.
Hormone accumulation upon P. rapae feeding
- To study whether the mutants used in this study are affected in herbivore-induced levels of jasmonates (JAs; JA, the biologically highly active conjugate JA-Ile and the JA-precursor OPDA) and ABA the authors monitored their accumulation in response to P. rapae feeding.
- Subsequently, hormone levels were measured in caterpillar-damaged leaves at different time points after caterpillar removal.
- Figure 2 shows that P. rapae feeding induced the accumulation of JA, JA-Ile, OPDA and ABA in Col-0 wild-type plants, confirming previous findings (Vos et al., 2013b) .
- This indicates that the biosynthesis of JAs is not significantly affected by the myc2 mutation and only relatively late affected by the aba2-1 mutation.
- The positive control, infection with the necrotrophic fungus B. cinerea, showed strongly enhanced ET production , whereas P. rapae infestation did not lead to changes in ET production over a 72-h feeding period compared to non-treated control plants .
The role of ABA in regulation of MYC/ERF antagonism
- To further investigate the role of ABA in the regulation of the MYC/ERF antagonism upon feeding by P. rapae, the authors determined the effect of exogenously applied ABA on the P. rapae-induced expression levels of VSP2 and PDF1.2.
- On the other hand, ABA application diminished the high P. rapae-induced PDF1.2 transcript levels in myc2 and aba2-1 plants at 30 h. CC-BY-NC-ND 4.0 International license available under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
- The copyright holder for this preprint (which was this version posted August 28, 2019.
- This indicates that ABA antagonizes the activation of the ERF-branch independently of the MYC2, MYC3 and MYC4 transcription factors.
- To investigate whether the preference of P. rapae caterpillars for the ERFbranch-expressing myc2 and aba2-1 mutant plants coincides with increased performance of the caterpillars on these genotypes, the authors assessed their growth in nochoice assays with Col-0, myc2, myc2,3,4, aba2-1, ein2-1, and JA-nonresponsive coi1-1 plants.
Discussion
- The complex plant immune regulatory network that is activated upon recognition of attackers is largely controlled by plant hormones (Pieterse et al., 2012) .
- CC-BY-NC-ND 4.0 International license available under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
- ABA is required for P. rapae-induced activation of the MYC-branch and repression of the ERF-branch Also in maize and rice plants, an increased production of JAs and ABA has been demonstrated upon root herbivory (Erb et al., 2009; Lu et al., 2015) .
- The ABA treatment stimulated the herbivore-induced MYC-branch in Col-0 plants, while in myc2 and myc2,3,4 plants ABA treatment strongly inhibited the enhanced expression of the ERF-branch .
ABA antagonizes the ERF-branch downstream of ORA59 at the GCC-box
- Analysis of the 35S::ORA59 transgenic line showed that ABA is able to suppress PDF1.2 even when ectopic ORA59 expression levels are constitutively high .
- Previously, Van der Does et al. ( 2013) investigated the suppressive effect of SA on JA-induced PDF1.2 expression.
- They also found that SA could suppress activation of PDF1.2 in the 35S::ORA59 line.
- Moreover, they reported that the GCCbox, which is present in the promoter of PDF1.2, and required for the JA-responsive expression, is essential and sufficient for transcriptional suppression by SA.
- Together, these data point towards a similar mechanism for SA-dependent and ABA-dependent suppression of the expression levels of ORA59 and PDF1.2 at the level of transcriptional regulation at the GCC-box.
Strong activation of the ET pathway is necessary for suppression of the MYC-branch
- The production of JA-Ile, JA and especially ABA was enhanced in the ein2-1 plants compared with Col-0 upon P. rapae feeding , suggesting that in wild-type plants basal activity of the ET pathway can inhibit herbivory-induced production of JA and ABA, which tempers the activation of the MYC-branch.
- This ET treatment led to activation of the ERF-branch during P. rapae feeding, while the MYC-branch was suppressed .
- CC-BY-NC-ND 4.0 International license available under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
- The feeding preference of P. rapae caterpillars for aba2-1 and myc2 plants was not obviously correlated with enhanced performance (weight gain) on these mutants in no-choice assays , which corresponds with the observation that the ERF-branch activating B. cinerea infection or ACC pretreatment did not affect caterpillar performance .
- Plants were cultivated in a growth chamber with a 10-h day and 14-h night cycle at 70% relative humidity and 21°C.
Chemical treatments
- For gene expression analysis, plants were treated with MeJA (Serva, Brunschwig Chemie, Amsterdam, the Netherlands) or ABA (Sigma, Steinheim, Germany) by dipping the rosettes in a solution containing either 100 µM MeJA, 100 µM ABA or a combination of both chemicals and 0.015% (v/v) Silwet L77 (Van Meeuwen Chemicals BV, Weesp, the Netherlands) 24 h before caterpillar feeding.
- MeJA and ABA solutions were diluted from a 1000-fold concentrated stock in 96% ethanol.
- CC-BY-NC-ND 4.0 International license available under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
- Five-week-old plants were placed separately in the cuvettes and remained there for the duration of the experiment.
- For northern blot analysis, 15 µg of RNA was denatured using glyoxal and dimethyl sulfoxide (Sambrook et al., 1989) , electrophoretically separated on 1.5% agarose gel, and blotted onto Hybond-N + membranes (Amersham, 's-Hertogenbosch, the Netherlands) by capillary transfer.
Jasmonates and ABA analysis
- For JA, JA-Ile, OPDA and ABA concentration analysis, 50-100 mg of P. rapaeinfested damaged leaves as well as undamaged leaves from non-infested control plants were grinded.
- At the start of the extraction 1 ml of cold ethylacetate containing D6-SA (25 ng/ml) and D5-JA (25 ng/ml) was added to the samples as an internal standard in order to calculate the recovery of the hormones measured.
- Multiple reaction monitoring was performed for parent-ions and selected daughter-ions after negative ionization: JA 209/59 (fragmented under 12V collision energy), JA-Ile 322/130 (fragmented under 19V collision energy), OPDA 291/165 (fragmented under 18V collision energy) and ABA 263/153 (fragmented under 9V collision energy).
- Analytes were quantified using standard curves made for each individual compound.
Ethylene measurements
- ET production was measured in a laser-driven photoacoustic detection system (ETD-300, Sensor Sense, Nijmegen, the Netherlands) connected to a 6-channel valve control box in line with a flow-through system (Voesenek et al., 1990) .
- Five-week-old plants were placed in 2-l air-tight cuvettes (four plants per cuvette), which were incubated under growth chamber conditions.
- After an acclimation time of 2 h, the cuvettes were continuously flushed with air (flow rate: 0.9 l/h), directing the flowthrough air from the cuvettes into a photoacoustic cell for ET measurements.
- ET levels were measured over consecutive 0.5 h time intervals, after which the machine switched to the next cuvette (n=6).
GUS assays
- . CC-BY-NC-ND 4.0 International license available under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
- Shown; two-way ANOVA (treatment x time point), LSD test for multiple comparisons; RT-qPCR analysis of VSP2 and PDF1.2 gene expression at 30 h in leaves of Col-0, myc2, myc2,3,4, aba2-1 and ein2-1 plants that were treated with a mock solution or with 100 µM ABA 24 h prior to infestation with P. rapae.
- Indications above the brackets specify whether there is an overall statistically significant difference between myc2 and Col-0 (two-way ANOVA (treatment x genotype), LSD test for multiple comparisons; *** = P<0.001).
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"Abscisic acid is essential for rewi..." refers background in this paper
...…and oral secretion of the insects and respond with the production of nutritive value-diminishing enzymes, toxic compounds, or predator-attracting volatiles (Kessler and Baldwin, 2002; Lawrence and Koundal, 2002; Wittstock et al., 2004; Chen et al., 2005; Mithöfer and Boland, 2012; Dicke, 2016)....
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1,061 citations
"Abscisic acid is essential for rewi..." refers background in this paper
...…cultivation Seeds of Arabidopsis thaliana accession Col-0 and mutants jin1-7 (myc2), myc2,3,4, aba2-1, ein2-1 and coi1-1 (Koornneef et al., 1982; Feys et al., 1994; Alonso et al., 1999; Lorenzo et al., 2004; Fernández-Calvo et al., 2011) and the transgenic lines 35S::ORA59 and GCC::GUS (Pré et…...
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1,058 citations
"Abscisic acid is essential for rewi..." refers background in this paper
...2 induction after exogenous application of JA (Anderson et al., 2004)....
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...Furthermore, previous studies have indicated that ABA plays a co-regulating role in the activation of the MYC-branch (Anderson et al., 2004; Bodenhausen and Reymond, 2007; Sánchez-Vallet et al., 2012; Vos et al., 2013b)....
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...Several studies indicated that ABA co-regulates the JA-induced activation of the MYC-branch, while ET co-regulates activation of the ERF-branch (Penninckx et al., 1998; Lorenzo et al., 2003; Anderson et al., 2004; Lorenzo et al., 2004; Pré et al., 2008; Vos et al., 2013b)....
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...ABA-deficient mutants have been reported to be more susceptible to herbivory (Thaler and Bostock, 2004; Bodenhausen and Reymond, 2007; Dinh et al., 2013) and more resistant to necrotrophic pathogens (Anderson et al., 2004; Sánchez-Vallet et al., 2012)....
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...…controlled by the basic helix-loop-helix leucine zipper transcription factors MYC2, MYC3 and MYC4 leading to transcription of hundreds of JA-responsive MYC-branch regulated genes, including VSP1 and VSP2 (Anderson et al., 2004; Lorenzo et al., 2004; Fernández-Calvo et al., 2011; Niu et al., 2011)....
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1,031 citations
"Abscisic acid is essential for rewi..." refers background in this paper
...2 is impaired in both JA- and ethylene (ET)-insensitive mutants, indicating that joint activation of the JA and ET pathways is necessary for full expression of the ERF-branch (Penninckx et al., 1998; Lorenzo et al., 2003; Pré et al., 2008; Broekgaarden et al., 2015)....
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...Several studies indicated that ABA co-regulates the JA-induced activation of the MYC-branch, while ET co-regulates activation of the ERF-branch (Penninckx et al., 1998; Lorenzo et al., 2003; Anderson et al., 2004; Lorenzo et al., 2004; Pré et al., 2008; Vos et al., 2013b)....
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...2 expression (Penninckx et al., 1998)....
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