- Original article
- Open Access
Characterization of Jasmonoyl-Isoleucine (JA-Ile) Hormonal Catabolic Pathways in Rice upon Wounding and Salt Stress
- Mohamed Hazman1, 2,
- Martin Sühnel3,
- Sandra Schäfer3,
- Julie Zumsteg1,
- Agnès Lesot1,
- Fréderic Beltran4,
- Valentin Marquis1,
- Laurence Herrgott1,
- Laurence Miesch4,
- Michael Riemann†3Email author and
- Thierry Heitz†1Email authorView ORCID ID profile
© The Author(s). 2019
- Received: 15 March 2019
- Accepted: 5 June 2019
- Published: 25 June 2019
Jasmonate (JA) signaling and functions have been established in rice development and response to a range of biotic or abiotic stress conditions. However, information on the molecular actors and mechanisms underlying turnover of the bioactive jasmonoyl-isoleucine (JA-Ile) is very limited in this plant species.
Here we explored two gene families in rice in which some members were described previously in Arabidopsis to encode enzymes metabolizing JA-Ile hormone, namely cytochrome P450 of the CYP94 subfamily (CYP94, 20 members) and amidohydrolases (AH, 9 members). The CYP94D subclade, of unknown function, was most represented in the rice genome with about 10 genes. We used phylogeny and gene expression analysis to narrow the study to candidate members that could mediate JA-Ile catabolism upon leaf wounding used as mimic of insect chewing or seedling exposure to salt, two stresses triggering jasmonate metabolism and signaling. Both treatments induced specific transcriptional changes, along with accumulation of JA-Ile and a complex array of oxidized jasmonate catabolites, with some of these responses being abolished in the JASMONATE RESISTANT 1 (jar1) mutant. However, upon response to salt, a lower dependence on JAR1 was evidenced. Dynamics of CYP94B5, CYP94C2, CYP94C4 and AH7 transcripts matched best the accumulation of JA-Ile catabolites. To gain direct insight into JA-Ile metabolizing activities, recombinant expression of some selected genes was undertaken in yeast and bacteria. CYP94B5 was demonstrated to catalyze C12-hydroxylation of JA-Ile, whereas similarly to its Arabidopsis bi-functional homolog IAR3, AH8 performed cleavage of JA-Ile and auxin-alanine conjugates.
Our data shed light on two rice gene families encoding enzymes related to hormone homeostasis. Expression data along with JA profiling and functional analysis identifies likely actors of JA-Ile catabolism in rice seedlings. This knowledge will now enable to better understand the metabolic fate of JA-Ile and engineer optimized JA signaling under stress conditions.
- Jasmonate catabolism
- Salt stress
Jasmonate hormones (JAs) play major roles in plant development and their responses to environmental challenges. Essential functions of this hormonal pathway have been demonstrated in vegetative or reproductive processes, and adaptation to an array of adverse conditions, particularly in Arabidopsis and other dicotyledonous plants (Wasternack and Hause 2013). Through complex interplays with other phytohormones, JAs control a wide spectrum of induced responses, including the accumulation of defense proteins, or enzymes directing the formation of specialized metabolites, both increasing survival capacity against herbivorous insects or necrotrophic microbial pathogens (Campos et al. 2014). In addition, JA signaling also impacts tolerance to different types of abiotic stress (Kazan 2015), making this hormone a critical hub for translating environmental cues into relevant physiological adaptations.
The core jasmonate biosynthetic pathway is initiated when linolenic acid from plastidial membranes is converted by the successive action of lipoxygenase, allene oxide synthase and allene oxide cyclase into the precursor 12-oxo-phytodienoic acid (OPDA). This compound is then further reduced by OPDA-reductase 3 (OPR3) followed by three rounds of ß-oxidation to form the inactive pro-hormone jasmonic acid (JA) (Schaller and Stintzi 2009). Among many possible modification routes described for JA (Wasternack and Hause 2013), its conjugation to isoleucine by Jasmonate Resistant 1 (JAR1) constitutes the critical activation step, and resulting jasmonoyl-isoleucine (JA-Ile) acts as the master regulator of most JA responses in higher plants (Campos et al. 2014; Heitz et al. 2016). This occurs when JA-Ile promotes assembly of the F-box protein CORONATINE INSENSITIVE 1 (COI1) with one of numerous JASMONATE-ZIM-domain (JAZ) repressor proteins and disrupts their transcription-repressing function at promoter regions of target genes. Upon ligand-dependent co-receptor assembly, JAZ are tagged by ubiquitin E3 ligase complexes before proteolytic degradation, allowing transcription of JA-responsive genes to proceed and execute the numerous adaptation responses (Chini et al. 2007; De Geyter et al. 2012; Thines et al. 2007).
In this mechanism, the levels and dynamics of JA-Ile are of crucial importance to direct a timely orchestration of transcriptional cascades (Du et al. 2017; Hickman et al. 2017). Many biotic and abiotic stress conditions induce accumulation of JA/JA-Ile and presumed catabolic derivatives and only recently have new metabolic relationships been established within this hormone family (Heitz et al. 2016; Koo 2018). In particular, metabolic studies in Arabidopsis have revealed the existence of two enzymatic JA-Ile turnover routes that are induced along with biosynthetic and signaling pathways (Additional file 1: Figure S1). The first one consists in a two-step oxidation mediated by three cytochromes P450 of the 94 family (CYP94B1, B3 and C1) and the resulting 12OH-JA-Ile and 12COOH-JA-Ile products have strongly reduced capacity to promote COI1-JAZ co-receptor formation (Aubert et al. 2015; Heitz et al. 2012; Kitaoka et al. 2011; Koo et al. 2011, 2014). They are therefore considered as largely inactive derivatives. The second JA-Ile elimination pathway consists in conjugate cleavage by IAR3 and ILL6, two members of a multi-functional amido-hydrolase family, that release JA from JA-Ile, but also 12OH-JA from 12OH-JA-Ile (Widemann et al. 2013; Woldemariam et al. 2012; Zhang et al. 2016). Loss and mostly gain-of-function experiments have established that these enzymes deplete specifically JA-Ile hormone pools to attenuate JA signaling and contribute to the proper regulation and termination of JA-mediated processes. Despite of their importance, the nature and regulation of JA-Ile turnover pathways have not been investigated in crop species and may have significant impact on some agricultural traits with regards to stress tolerance. Rice is particularly suited for such a purpose as a model cereal and a major food crop. Recent research has identified candidate genes for most components of JA biosynthetic and signaling pathway and several features have been addressed functionally (Dhakarey et al. 2016; Liu et al. 2015; Riemann et al. 2015). Functions were described for example in photomorphogenesis (Svyatyna et al. 2014), spikelet development (Cai et al. 2014; Liu et al. 2017), control in the transition between juvenile and adult phases (Hibara et al. 2016), defense against insects (Lu et al. 2015; Ye et al. 2012) and pathogens (Riemann et al. 2013; Yamada et al. 2012). In addition, significant impacts of the JA pathway on abiotic stress tolerance were reported in this species (Dhakarey et al. 2016). The most documented case is probably salt stress resilience for which JA signaling seems to be detrimental, based on several reports. We showed previously that a JA-deficient rice mutant performs better upon salt exposure than wild-type (Hazman et al. 2015). Similarly, suppressing (OsJAZ9) or enhancing (OsJAZ8) salt-induction of JAZ repressors improves tolerance to this stress (Peethambaran et al. 2018; Wu et al. 2015). This data supports a previous report by Kurotani and co-workers (Kurotani et al. 2015a) showing that a rice line overexpressing CYP94C2b, a putative ortholog of the JA-Ile-oxidase CYP94C1 in Arabidopsis displays enhanced survival upon salt exposure. This notion was further extended by the finding of a positive correlation between expression levels of this gene and salt tolerance in an extensive collection of tolerant rice lines (Kurotani et al. 2015b), suggesting a possible link between JA-Ile catabolism and salt stress tolerance.
In the present work, we explored rice JA-Ile catabolism under two distinct stresses known to trigger JA biosynthesis and signaling, namely mechanical wounding used as a proxy for attack by chewing insects, and salt exposure as a relevant abiotic stress that increasingly threatens rice productivity under agricultural conditions (Shrivastava and Kumar 2015). We established detailed kinetic jasmonate profiles in seedlings, including catabolic derivatives, and examined the requirement of the conjugating enzyme JAR1 for each stress. We next mined the rice genome to explore the two known gene families, CYP94 and amido-hydrolase (AH), in which some Arabidopsis members encode JA-Ile-metabolizing activities as described above. We identified through phylogenic analysis and gene expression studies candidate rice members that could potentially encode such activities. By recombinant protein expression, we were able to demonstrate for one CYP94 and one AH JA-Ile oxidation or cleaving activities, respectively. The study sheds light onto new players of JA hormone turnover and increases our understanding of JA-Ile homeostasis in rice.
Occurrence and Diversity of CYP94 and Amidohydrolase (AH) Genes in the Rice Genome
Rice amidohydrolase (AH) gene family was similarly investigated and 9 predicted proteins were identified. Figure 1b shows their phylogenetic relationships with the 7 Arabidopsis AH protein sequences (Rampey et al. 2004). Five members (OsAH2 through OsAH6) cluster with AtILR1 that prefers auxin-amino acid conjugates as substrates (Zhang et al. 2016). Two other isoforms, OsAH1 and OsAH9 were more distantly related to previously characterized enzymes. The closest rice homolog of AtIAR3, the conserved bifunctional AH cleaving both JA-amino acid and indole acetic acid-amino acid conjugates (Widemann et al. 2013; Zhang et al. 2016) was OsAH8 (78/64% similar/identical). Finally, OsAH7 protein sequence was closely related (75/63% similar/identical) to JA-Ile-specific AtILL6.
Specific CYP94 and AH Gene Isoforms Display Dynamic Expression upon Wound Stress
To investigate stress responsiveness of members in both gene families, expression studies were conducted in leaves using kinetic responses to wounding. Specific primer pairs were designed for genes with unique 3’UTR sequences available from databases, comprising 14 OsCYP94 genes and all 9 OsAH genes (Fig. 1). This criterion was met for CYP94C2a, but not for the very similar (91%) and formerly reported CYP94C2b gene (Kurotani et al. 2015a) for which no specific primer pair could be designed. Most analyzed OsCYP94 genes exhibited wound-induced expression, either as a rapid and transient pulse peaking at 0.5 h post-wounding (hpw), as for CYP94B4, CYP94B5, CYP94C2a, CYP94C3, CYP94C4, or as a steady increase over the 6 h period studied, for CYP94D5a, CYP94D7, CYP94D9, CYP94E2 (Additional file 2: Figure S2). However, overall expression levels were extremely variable between genes, and ranged over 3 orders of magnitude. Despite of detectable wound-triggered changes, some isoforms displayed very low expression levels, like CYP4B4, CYP94C3, CYP94D5, CYP94D11, CYP94D12, CYP94D15, and CYP94E2, and were not further studied.
A similar approach was taken to visualize AH gene expression. Slow increase upon wounding was recorded for AH1, AH2, AH3, AH6, and AH8 transcripts, with maximal expression at later time points (Additional file 3: Figure S3). Because of rapid accumulation of JAs within the first hour after leaf wounding in Arabidopsis (Glauser et al. 2008; Heitz et al. 2012; Widemann et al. 2013), we speculated that genes whose expression was rapidly induced to high levels by wounding would be best candidates to participate in rice JA metabolism. AH7 combined a rapid and transient induction peaking at 0.5 hpw and relatively high expression, whereas AH8 displayed the highest basal expression and moderate induction by 3 hpw.
Wounding Induces Strong, Partially JAR1-Dependent JA Accumulation and Catabolism in Rice
Responses of JA-Ile Catabolic Genes to Salt Stress
Salt Stress Triggers JA Accumulation and Catabolism
Functional Analysis of CYP94 and AH Proteins
AH7 and AH8 coding sequences were cloned in expression vector pHMGWA, and expression of double-tagged 6xHis-Maltose Binding Protein (MBP)-AH was induced in bacterial cells. Fusion proteins of the expected sizes (His-MBP-AH8: 86.4 kDa; His-MBP-AH7: 92.7 kDa) could be isolated. His-MBP-AH8 was produced abundantly in soluble form and was purified by dextrin affinity chromatography followed by gel-filtration (Additional file 5: Figure S5a). His-MBP-AH7 expressed at lower abundance and was captured in bacterial lysates by immobilized metal affinity chromatography (IMAC) (Additional file 5: Figure S5b). Both proteins were next incubated with hormone-amino acid conjugates, typically JA- or auxin-amino acid conjugates, that were previously described as in vivo substrates of this class of enzymes (Rampey et al. 2004; Widemann et al. 2013; Zhang et al. 2016). Recombinant OsAH7 and OsAH8 were incubated with either JA-Ile, or with its oxidized derivative 12OH-JA-Ile, or with the auxin conjugate IAA-Ala. Corresponding unconjugated hormonal compounds were searched for as evidence of cleaving activity. No activity could be recorded for OsAH7 with either substrate (not shown). In contrast, chromatograms shown in Fig. 6b illustrate that OsAH8, similarly to its Arabidopsis homolog AtIAR3, is able to generate free JA, 12OH-JA and IAA from their respective conjugated substrates.
Jasmonate signaling is governing major aspects of plant development and adaptation to environmental stress. Recent reports have revealed positive and negative contributions of JA signaling to biotic and abiotic threats (Hazman et al. 2015; Peethambaran et al. 2018; Riemann et al. 2013, 2015; Wu et al. 2015), calling for a better understanding of hormonal regulation. Catabolic processes affecting bioactive JA-Ile hormone homeostasis have been elucidated in Arabidopsis and shown to attenuate JA signaling (Heitz et al. 2012; Koo et al. 2011; Koo and Howe 2012; Widemann et al. 2013; Zhang et al. 2016). The precise actors and mechanisms of JA-Ile turnover were unknown in rice, but conservation of the oxidative pathway in this species was suggested by the correlation of OsCYP94C2b overexpression with higher in planta JA-Ile oxidation, leading to increased salt tolerance (Kurotani et al. 2015a, 2015b). Here we explored the rice CYP94 and AH gene families, in a search for orthologs of the Arabidopsis JA-Ile oxidation and deconjugation enzymes (Heitz et al. 2016; Koo and Howe 2012). Similarity search readily identified large gene families; strikingly, subclades B and C of CYP94 encoding suspected JA-Ile oxidases only represented a minor portion of the OsCYP94 gene family, in contrast to subclade D of unknown activity that displays up to 10 members. The additional occurrence of 3 subclade E genes illustrates the high diversification of CYP94 genes/proteins in rice and questions the existence of distinct enzymatic activities. In contrast, the AH gene family, with 9 members in rice had a comparable complexity to the 7-member Arabidopsis family.
To identify stress-responsive members, we examined transcriptional behavior of a subset of OsCYP94 and all OsAH genes, in response to leaf wounding or salt exposure, two stresses known to cause rapid/massive, or slower changes in JA metabolism, respectively. Only members of the CYP94B and C subclades were found to display a stereotypical expression peak at 0.5–1 hpw that parallels the transient accumulation of JA-Ile, their presumed substrate, upon wounding (Heitz et al. 2012; Wakuta et al. 2011). Despite of sharing this profile, CYP94B4 and CYP94C3 have very low expression and likely are not major players of JA-Ile oxidation in wounded tissues. In contrast, OsCYP94B5, C2a and C4 displayed much higher expression, and similarly to Arabidopsis characterized CYP94B1, B3 and C1 genes (Heitz et al. 2012), their induction was partially dependent on JA-Ile biosynthesis, as evidenced in the jar1–1 mutant. Quantitative, kinetic JA profiling was performed to reveal the characteristics of JA/JA-Ile catabolism in rice. Main features reported in Arabidopsis were conserved, with slower accumulation of its two oxidized derivatives. Therefore, OsCYP94B5, C2a and C4 are the prime candidates for wound-induced JA-Ile turn-over in rice. All 3 Ile-conjugates were nearly absent in jar1–1, but the minor conjugate JA-Phe was more abundant in this mutant, indicating it is produced by another conjugating enzyme, possibly boosted by the enhanced accumulation of JA precursor. JAR2, the closest JAR homolog in the GH3 family of rice, is unable to form JA-Phe in vitro (Svyatyna et al. 2014), and we established that its transcript levels decreased upon wounding in our experiments (Additional file 4: Figure S4B). Therefore, an unknown enzyme, possibly in the GH3 family may be involved in JA-Phe formation (Wakuta et al. 2011). In the AH family, OsAH7 and OsAH8 behaved also similarly to their respective Arabidopsis AtILL6 and AtIAR3 closest homologs (Widemann et al. 2013), with a stable but high expression for OsAH8, and a dynamic pulse with weaker expression for OsAH7. These features do not allow to draw firm conclusions as to which AH contributes most to JA-Ile cleavage.
Transcriptional responses to salt exposure were contrasted. Only OsCYP94C2a was induced, similarly to OsJAZ11, and accompanied the increase in JA-Ile and its catabolites, whereas OsCYP94B5 and OsCYP94C4 were essentially down-regulated, as well as OsCYP94D7 and D9. Therefore, OsCYP94C2a is the most likely actor of JA-Ile oxidation upon salt-stress, lending support to earlier data by Kurotani et al. (2015a, 2015b), who identified in a genetic screen the related OsCYP94C2b as a major single-gene contributor to salt tolerance. OsCYP94D7 and D9 decrease could underlie the need to shutdown a distinct enzyme activity upon salt response. OsAH7 and OsAH8 transcripts only fluctuated marginally. As upon wounding, JA was hyperaccumulated in jar1–1 in salt-reacting leaves, indicating reduced JA metabolization, but Ile-conjugates were less reduced in this latter material, suggesting the contribution of another conjugating enzyme than JAR1. JAR2, the closest candidate to fulfill this function, is able to form JA-Ile in vitro (Svyatyna et al. 2014), but its contribution to salt-induced JA-Ile accumulation is not supported by the down-regulation of its transcripts. Currently, we are lacking information on alternative enzyme activities in the GH3 families, hence, more efforts are needed to clarify JAR1-independent JA conjugation. The fact that oxidized derivatives are less affected by jar1–1 mutation than JA-Ile may indicate that they undergo less deconjugation in this background and are therefore more stable.
Lastly, we set out to characterize directly enzyme activities of recombinant proteins. Absence of activity in OsCYP94C2a and C4 in yeast microsomes may be attributed to inactive conformation and/or insufficient expression. When carbon monoxide spectra of microsomes obtained from yeast transformed with OsCYP94C2a and C4 were recorded, a peak at about 420 nm rather than 450 nm was observed, suggesting the presence of misfolded heme-containing proteins (Luthra et al. 2011).
Our failure to assay recombinant OsCYP94C2a enzyme activity, combined with the absence of information on stress-induced expression of the closely related OsCYP94C2b gene (Kurotani et al. 2015a) limits the knowledge of the relative contribution of these two isoforms to JA-Ile turnover. Affinity-purified OsAH7 also proved inactive, possibly due to mis-folding or hindrance by the MBP fusion. This is reminiscent of AtILL6, its Arabidopsis counterpart that is also notoriously difficult to express/assay (Widemann et al. 2013). However, for two enzymes, we could demonstrate JA-Ile-metabolizing activity. OsCYP94B5 displays JA-Ile oxidase activity in vitro, consistent with its Arabidopsis CYP94B orthologs (Heitz et al. 2012; Kitaoka et al. 2011; Koo et al. 2014). Rendering CYP94B5 expression salt-responsive for enhanced JA-Ile oxidative inactivation could be an attractive strategy to engineer salt-tolerance in rice. OsAH8 could be obtained in the active form, and its ability to cleave JA-aa and IAA-aa conjugates was shared with the bi-functional Arabidopsis IAR3 (Widemann et al. 2013; Zhang et al. 2016).
Our work sheds light on rice CYP94 and AH gene families, in a context of hormone conjugate metabolism. CYP94 is so far limited to jasmonate substrates while some AH also readily catalyze cleavage of auxin conjugates (Zhang et al. 2016). The present evidence suggests that the JA-Ile catabolic network architecture is conserved in rice, both in terms of phylogeny and catalytic capacities: the closest homologous proteins display similar activities to enzymes characterized in Arabidopsis, but some unexpected regulations were also recorded. These findings and tools pave the way for stress-specific manipulation of JA-Ile catabolism for optimized output of the JA signaling pathway under various stresses.
Dr. D. Nelson (University of Tennesse, USA) kindly provided the names of CYP94 isoforms. The full length amino acid of rice cytochrome P450 (CYP94) and amidohydrolase (AH) sequences were extracted from rice databases MSU (http://rice.plantbiology.msu.edu) and RAP (http://rapdb.dna.affrc.go.jp) via blasting CYP94 and AH Arabidopsis protein sequences. CYP94 sequences were matched with sequences provided by D. Nelson. All sequences were then manually curated and loci identified in rice genome databases. A total of deduced 26 CYP94 and 16 AH full-length amino acid sequences were analyzed using the phylogeny suite (Dereeper et al. 2008) and DENDROSCOPE v3 (Huson and Scornavacca 2012).
Plant Materials, Growth and Stress Conditions
In this study, Oryza sativa L. ssp. japonica cv. Nipponbare was used as the wild type, and jar1–1 (Riemann et al. 2008) was used as mutant. The caryopses were dehusked and surface sterilized according to (Hazman et al. 2015) and sowed on 0.4% phytoagar containing 5% MS salts (Duchefa, Haarlem, The Netherlands) and incubated for 7 or 10 days in a culture room under continuous light at 25 °C. For wounding stress, 10-day old seedlings were subjected to manual wounding using either metal forceps (Additional file 2: Figure S2, Additional file 3: Figure S3) or scissors (Fig. 2 and Fig. 3) to produce consistent wounds along the second leaf (5 and 7 wounds/ leaf, respectively). Wounded leaves were harvested kinetically (0, 0.5, 1, 3 and 6 h after wounding) in 3 replicates and then stored at − 80 °C for quantitative gene expression analysis and jasmonate profiling. For salt stress, plant cultivation and treatments were accomplished according to Hazman et al. (2015). Briefly, surface sterilized caryopses of both WT and jar1–1 mutants were pre-germinated on 0.4% phytoagar for 7 days under continuous light of 120 μmol m− 2 s− 1 at 25 °C. The seedlings were transferred to microtubes with cut ends that were inserted on floating racks placed to a cylindric glass container containing 5% MS medium as nutrient solution for extra 3 days. Subsequently, the solution was replaced by the same solution (control time course) or the same solution containing NaCl (100 mM). Leaves of both control and stressed plants were harvested kinetically (0, 6, 24 and 72 h after transferring into final solution) in multiple replicates and then stored at − 80 °C for profiling of gene expression or jasmonate content.
Total RNA Extraction and Quantitative Real-Time PCR
For data shown in Additional file 2: Figure S2, Additional file 3: Figure S3, total RNA was isolated from the shoots of control, wounded and salt treated plants using Trizol Reagent (Molecular Research Center, Cincinatti, USA) according to the manufacturer’s instructions. The cDNA synthesis was performed with cDNA synthesis kit (Invitrogen, Carlsbad, USA) using 1 μg total RNA as a template. RT-qPCR (Reverse Transcriptase – quantitative PCR) was performed on 10 ng cDNA with a SYBR green dye protocol using LightCycler 480 II instrument (Roche Applied Science, Penzberg, Germany) as follows: 95 °C for 3 min, and 40 cycles (95 °C for 15 s, annealing at 66 °C for 30 s and extension at 72 °C for 30 s). The gene expression levels in three biological replicates were calculated using the ∆∆Ct method.
For data shown in Fig. 2 and Fig. 4, RNA-extraction was performed using the innuPREP Plant RNA KIT (Analytik Jena AG, Jena, Germany) according to instructions provided by the manufacturer. The RNA was transcribed via a first strand cDNA using MuLV reverse transcriptase (New England Biolabs, Frankfurt, Germany) and oligo dT-primers in a two-step reaction. Quantitative analysis was performed on a CFX Touch real-time PCR system (Bio-Rad, Munich, Germany) according to the protocol of Svyatyna et al. (2014). The primer sequences for the genes of interest and reference genes are listed in Additional file 6: Table S1.
Recombinant Heterologous Expression of OsCYP94B5 in Yeast and OsAH8 in Bacteria
Coding sequence of OsCYP94B5, OsCYP94B4 and OsCYP94C2a was generated by PCR amplification from rice genomic DNA cultivar Nipponbare. The forward and reverse primer sequences are given in Additional file 6: Table S1. The amplified PCR product was initially cloned in pGEM-T easy vector according to manufacturer instructions (Promega) and then sequenced to verify error-free insert that was cloned in the BamH1 and EcoR1 sites of the yeast expression vector pYeDP60. OsCYP94B5, OsCYP94B4 and OsCYP94C2a proteins were produced in yeast optimized heterologous system as described in Heitz et al. (2012). P450 expression and quality control was performed by differential spectrophotometry as described in Gavira et al. (2013).
For heterologous expression of OsAH7 and OsAH8, open reading frame sequence deleted of the 69 (AH7) or 78 (AH8) N-terminal signal peptide-encoding nucleotides was amplified using a mixture (8:1) of hotstart Taq Polymerase and Phusion Taq Polymerase (Thermo Scientific, Illkirch-Graffenstaden, France) prior to the cloning in the pDONOR-Zeo vector (Thermo Scientific) in the DH5α Escherichia coli strain. Inserts with error-free sequences were recombined into the expression vector pHMGWA. Plasmid was further transformed into the E. coli Rosetta 2 (DE3) strain (Merck, Darmstadt, Germany). Ice-cold bacterial pellets from isopropyl ß-D-1-thiogalactopyranoside-induced (0.5 mM) cultures were collected and kept frozen until use. For AH7 purification, pellet was thawed on ice for 30 min while resuspending in lysis buffer (50 mM Tris–HCl pH 7.5, 300 mM NaCl, 20 mM imidazole, 3 mg/ml lysozyme) to an OD600 of 20 units. Bacteria were lysed by pulsed-sonication on ice for 4 min. Lysate was clarified by centrifugation, supernatant was filtered and diluted with 1 vol buffer (Tris-HCl 50 mM pH 8, NaCl 300 mM, glycerol 5%) before loading on a His-Trap FF (1 ml capacity) column mounted on an Äkta purifier system (GE Healthcare, Velizy, France) and equilibrated with the same buffer. His-tagged protein was eluted with equilibration buffer containing 500 mM imidazole. For AH8 purification, bacterial pellet was resuspended in 1 x Phosphate Buffered Saline (PBS) pH 7.3 buffer to an OD600 of 20 units before lysis by pulsed sonication on ice for 4 min. Clarified lysate was loaded on a MBP-Trap (1 ml capacity) column pre-equilibrated with PBS solution and protein was eluted with equilibration buffer complemented with 10 mM maltose. After analysis by SDS–PAGE, selected fractions were concentrated to 500 μL and loaded on a Superdex 200 10/300 column equilibrated in 1x PBS pH 7.3. Concentration of proteins of interest was estimated via quantification with Bradford reagent using a bovine serum albumin calibration series.
Enzyme Assays and Analysis
For CYP94 assay, microsome incubations were performed as described in Widemann et al. (2015) with the following modifications: JA-Ile substrate concentration was 50 μM and the reaction was stopped with 150 μL methanol containing 0.2% acetic acid. The assays were centrifuged at 10000 g and the supernatant was used for LC-MS/MS analysis.
Amidohydrolase assay was performed in 200 μL as described in Widemann et al. (2013) using 10 μg affinity-purified protein and 30 μM substrates (JA-Ile, 12OH-JA-Ile or IAA). Both types of enzyme activities were analyzed by LC-MS as described in Widemann et al. (2015) on a Waters Quattro Premier XE (Waters, Mildorf, MA USA) instrument, using the following detection parameters: in negative mode: JA 209 > 59; 12OH-JA 225 > 59; 12OH-JA-Ile 338 > 130; 12COOH-JA-Ile 352 > 130; in positive mode: JA-Ile 324 > 151; IAA-Ala 247 > 130; IAA 176 > 130.
Jasmonate profiling of rice leaves was performed as follows: about 50–100 mg frozen plant material was extracted with 8 volumes of ice-cold extraction solution (MeOH:water:acetic acid 70:29:0.5) containing 9,10-dihydro-JA and 9,10-dihydro-JA-Ile as internal standards for workup recovery. Grinding was performed with a glass-bead Precellys tissue homogenizer (Bertin Instruments, Montigny-le-Bretonneux, France) in 2 mL screw-capped tubes. After 30 min incubation at 4 °C on a rotating wheel, homogenates were cleared before concentration under a stream of N2 and overnight conservation at − 20 °C. After a second centrifuge step, extracts were submitted to LC-MS/MS analysis on an EvoQ Elite LC-TQ (Bruker, Palaiseau, France). Column and chromatographic conditions were as described in Smirnova et al. (2017). Absolute quantifications were achieved by comparison of sample signals with dose–response curves established with pure compounds. Compound specific detections were performed in Multiple Reaction Monitoring Mode (MRM) using transitions described in Additional file 7: Table S2.
All statistical analysis were performed using InfoStat 2015d (http:// www.infostat.com.ar). Comparisons of sample means were per- formed by one-way analysis of variance (P < 0.05 or P < 0.01) and Tukey’s post-hoc multiple comparisons tests (P < 0.05 or P < 0.01), and significant differences of means were determined.
We thank David Nelson (University of Tennessee, USA) for providing CYP94 sequences used for phylogeny. We are grateful to Pauline Delcros for help in OsAH7 expression and analysis.
MH was supported by a joint post-doctoral fellowship from the Institut Français d’Egypte (IFE, Cairo, Egypt) and the Science and Technology Development Fund (STDF, Egypt). MS was supported by the Erasmus exchange program of the European Union. VM and FB were recipients of a predoctoral fellowship from the Université de Strasbourg and the Ministère de l’Enseignement Supérieur et de la Recherche. Exchanges between KIT and IBMP were supported by Campus France Procope and German Academic Exchange Service (DAAD) funds. Publication of this article has been funded through the Open Access Publishing Fund of Karlsruhe Institute of Technology.
MH, MS and SS performed plant treatment and analysis. LH purified recombinant protein, AL and MH produced yeast microsomes. MH, MS and VM performed enzyme assays. JZ performed LC-MS analysis. FB and LM performed chemical synthesis of JAs. TH and MR designed research and wrote the paper. All authors read and approved the final manuscript.
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The authors declare that they have no competing interests.
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- Aubert Y, Widemann E, Miesch L, Pinot F, Heitz T (2015) CYP94-mediated jasmonoyl-isoleucine hormone oxidation shapes jasmonate profiles and attenuates defence responses to Botrytis cinerea infection. J Exp Bot 66:3879–3892View ArticleGoogle Scholar
- Cai Q, Yuan Z, Chen M, Yin C, Luo Z, Zhao X, Liang W, Hu J, Zhang D (2014) Jasmonic acid regulates spikelet development in rice. Nat Commun 5:3476View ArticleGoogle Scholar
- Campos ML, Kang JH, Howe GA (2014) Jasmonate-triggered plant immunity. J Chem Ecol 40:657–675View ArticleGoogle Scholar
- Chini A, Fonseca S, Fernandez G, Adie B, Chico JM, Lorenzo O, Garcia-Casado G, Lopez-Vidriero I, Lozano FM, Ponce MR, Micol JL, Solano R (2007) The JAZ family of repressors is the missing link in jasmonate signalling. Nature 448:666–671View ArticleGoogle Scholar
- De Geyter N, Gholami A, Goormachtig S, Goossens A (2012) Transcriptional machineries in jasmonate-elicited plant secondary metabolism. Trends Plant Sci 17:349–359View ArticleGoogle Scholar
- Dereeper A, Guignon V, Blanc G, Audic S, Buffet S, Chevenet F, Dufayard JF, Guindon S, Lefort V, Lescot M, Claverie JM, Gascuel O (2008) Phylogeny.fr: robust phylogenetic analysis for the non-specialist. Nucleic Acids Res 36:W465–W469View ArticleGoogle Scholar
- Dhakarey R, Kodackattumannil Peethambaran P, Riemann M (2016) Functional analysis of Jasmonates in Rice through mutant approaches. Plants (Basel) 5(1) https://doi.org/10.3390/plants5010015 View ArticleGoogle Scholar
- Du M, Zhao J, Tzeng DTW, Liu Y, Deng L, Yang T, Zhai Q, Wu F, Huang Z, Zhou M, Wang Q, Chen Q, Zhong S, Li CB, Li C (2017) MYC2 orchestrates a hierarchical transcriptional cascade that regulates jasmonate-mediated plant immunity in tomato. Plant Cell 29:1883–1906View ArticleGoogle Scholar
- Gavira C, Hofer R, Lesot A, Lambert F, Zucca J, Werck-Reichhart D (2013) Challenges and pitfalls of P450-dependent (+)-valencene bioconversion by Saccharomyces cerevisiae. Metab Eng 18:25–35View ArticleGoogle Scholar
- Glauser G, Grata E, Dubugnon L, Rudaz S, Farmer EE, Wolfender JL (2008) Spatial and temporal dynamics of jasmonate synthesis and accumulation in Arabidopsis in response to wounding. J Biol Chem 283:16400–16407View ArticleGoogle Scholar
- Hazman M, Hause B, Eiche E, Nick P, Riemann M (2015) Increased tolerance to salt stress in OPDA-deficient rice ALLENE OXIDE CYCLASE mutants is linked to an increased ROS-scavenging activity. J Exp Bot 66:3339–3352View ArticleGoogle Scholar
- Heitz T, Smirnova E, Widemann E, Aubert Y, Pinot F, Menard R (2016) The rise and fall of jasmonate biological activities. Subcell Biochem 86:405–426View ArticleGoogle Scholar
- Heitz T, Widemann E, Lugan R, Miesch L, Ullmann P, Desaubry L, Holder E, Grausem B, Kandel S, Miesch M, Werck-Reichhart D, Pinot F (2012) Cytochromes P450 CYP94C1 and CYP94B3 catalyze two successive oxidation steps of plant hormone Jasmonoyl-isoleucine for catabolic turnover. J Biol Chem 287:6296–6306View ArticleGoogle Scholar
- Hibara K, Isono M, Mimura M, Sentoku N, Kojima M, Sakakibara H, Kitomi Y, Yoshikawa T, Itoh J, Nagato Y (2016) Jasmonate regulates juvenile-to-adult phase transition in rice. Development 143:3407–3416View ArticleGoogle Scholar
- Hickman R, Van Verk MC, Van Dijken AJH, Mendes MP, Vroegop-Vos IA, Caarls L, Steenbergen M, Van der Nagel I, Wesselink GJ, Jironkin A, Talbot A, Rhodes J, De Vries M, Schuurink RC, Denby K, Pieterse CMJ, Van Wees SCM (2017) Architecture and dynamics of the jasmonic acid gene regulatory network. Plant Cell 29:2086–2105View ArticleGoogle Scholar
- Huson DH, Scornavacca C (2012) Dendroscope 3: an interactive tool for rooted phylogenetic trees and networks. Syst Biol 61:1061–1067View ArticleGoogle Scholar
- Kazan K (2015) Diverse roles of jasmonates and ethylene in abiotic stress tolerance. Trends Plant Sci 20:219–229View ArticleGoogle Scholar
- Kitaoka N, Kawaide H, Amano N, Matsubara T, Nabeta K, Takahashi K, Matsuura H (2014) CYP94B3 activity against jasmonic acid amino acid conjugates and the elucidation of 12-O-beta-glucopyranosyl-jasmonoyl-L-isoleucine as an additional metabolite. Phytochemistry 99:6–13View ArticleGoogle Scholar
- Kitaoka N, Matsubara T, Sato M, Takahashi K, Wakuta S, Kawaide H, Matsui H, Nabeta K, Matsuura H (2011) Arabidopsis CYP94B3 encodes jasmonyl-L-isoleucine 12-hydroxylase, a key enzyme in the oxidative catabolism of jasmonate. Plant Cell Physiol 52:1757–1765View ArticleGoogle Scholar
- Koo AJ (2018) Metabolism of the plant hormone jasmonate: a sentinel for tissue damage and master regulator of stress response. Phytochem Rev 17:51 https://doi.org/10.1007/s11101-017-9510-8:1-30 View ArticleGoogle Scholar
- Koo AJ, Cooke TF, Howe GA (2011) Cytochrome P450 CYP94B3 mediates catabolism and inactivation of the plant hormone jasmonoyl-L-isoleucine. Proc Natl Acad Sci U S A 108:9298–9303View ArticleGoogle Scholar
- Koo AJ, Howe GA (2012) Catabolism and deactivation of the lipid-derived hormone jasmonoyl-isoleucine. Front Plant Sci 3:19View ArticleGoogle Scholar
- Koo AJ, Thireault C, Zemelis S, Poudel AN, Zhang T, Kitaoka N, Brandizzi F, Matsuura H, Howe GA (2014) Endoplasmic reticulum-associated inactivation of the hormone jasmonoyl-L-isoleucine by multiple members of the cytochrome P450 94 family in Arabidopsis. J Biol Chem 289:29728–29738View ArticleGoogle Scholar
- Kurotani K, Hayashi K, Hatanaka S, Toda Y, Ogawa D, Ichikawa H, Ishimaru Y, Tashita R, Suzuki T, Ueda M, Hattori T, Takeda S (2015a) Elevated levels of CYP94 family gene expression alleviate the jasmonate response and enhance salt tolerance in rice. Plant Cell Physiol 56:779–789View ArticleGoogle Scholar
- Kurotani K, Yamanaka K, Toda Y, Ogawa D, Tanaka M, Kozawa H, Nakamura H, Hakata M, Ichikawa H, Hattori T, Takeda S (2015b) Stress tolerance profiling of a collection of extant salt-tolerant rice varieties and transgenic plants overexpressing abiotic stress tolerance genes. Plant Cell Physiol 56:1867–1876View ArticleGoogle Scholar
- Liu L, Zou Z, Qian K, Xia C, He Y, Zeng H, Zhou X, Riemann M, Yin C (2017) Jasmonic acid deficiency leads to scattered floret opening time in cytoplasmic male sterile rice Zhenshan 97A. J Exp Bot 68:4613–4625View ArticleGoogle Scholar
- Liu Z, Zhang S, Sun N, Liu H, Zhao Y, Liang Y, Zhang L, Han Y (2015) Functional diversity of jasmonates in rice. Rice (N Y) 8:42Google Scholar
- Lu J, Robert CA, Riemann M, Cosme M, Mene-Saffrane L, Massana J, Stout MJ, Lou Y, Gershenzon J, Erb M (2015) Induced jasmonate signaling leads to contrasting effects on root damage and herbivore performance. Plant Physiol 167:1100–1116View ArticleGoogle Scholar
- Luthra A, Denisov IG, Sligar SG (2011) Spectroscopic features of cytochrome P450 reaction intermediates. Arch Biochem Biophys 507:26–35View ArticleGoogle Scholar
- Peethambaran PK, Glenz R, Honinger S, Shahinul Islam SM, Hummel S, Harter K, Kolukisaoglu U, Meynard D, Guiderdoni E, Nick P, Riemann M (2018) Salt-inducible expression of OsJAZ8 improves resilience against salt-stress. BMC Plant Biol 18:311View ArticleGoogle Scholar
- Rampey RA, LeClere S, Kowalczyk M, Ljung K, Sandberg G, Bartel B (2004) A family of auxin-conjugate hydrolases that contributes to free indole-3-acetic acid levels during Arabidopsis germination. Plant Physiol 135:978–988View ArticleGoogle Scholar
- Riemann M, Dhakarey R, Hazman M, Miro B, Kohli A, Nick P (2015) Exploring jasmonates in the hormonal network of drought and salinity responses. Front Plant Sci 6:1077View ArticleGoogle Scholar
- Riemann M, Haga K, Shimizu T, Okada K, Ando S, Mochizuki S, Nishizawa Y, Yamanouchi U, Nick P, Yano M, Minami E, Takano M, Yamane H, Iino M (2013) Identification of rice Allene oxide cyclase mutants and the function of jasmonate for defence against Magnaporthe oryzae. Plant J 74:226–238View ArticleGoogle Scholar
- Riemann M, Riemann M, Takano M (2008) Rice JASMONATE RESISTANT 1 is involved in phytochrome and jasmonate signalling. Plant Cell Environ 31:783–792View ArticleGoogle Scholar
- Schaller A, Stintzi A (2009) Enzymes in jasmonate biosynthesis - structure, function, regulation. Phytochemistry 70:1532–1538View ArticleGoogle Scholar
- Shrivastava P, Kumar R (2015) Soil salinity: a serious environmental issue and plant growth promoting bacteria as one of the tools for its alleviation. Saudi J Biol Sci 22:123–131View ArticleGoogle Scholar
- Smirnova E, Marquis V, Poirier L, Aubert Y, Zumsteg J, Menard R, Miesch L, Heitz T (2017) Jasmonic acid oxidase 2 hydroxylates jasmonic acid and represses basal defense and resistance responses against Botrytis cinerea infection. Mol Plant 10:1159–1173View ArticleGoogle Scholar
- Svyatyna K, Jikumaru Y, Brendel R, Reichelt M, Mithofer A, Takano M, Kamiya Y, Nick P, Riemann M (2014) Light induces jasmonate-isoleucine conjugation via OsJAR1-dependent and -independent pathways in rice. Plant Cell Environ 37:827–839View ArticleGoogle Scholar
- Thines B, Katsir L, Melotto M, Niu Y, Mandaokar A, Liu G, Nomura K, He SY, Howe GA, Browse J (2007) JAZ repressor proteins are targets of the SCF(COI1) complex during jasmonate signalling. Nature 448:661–665View ArticleGoogle Scholar
- Wakuta S, Suzuki E, Saburi W, Matsuura H, Nabeta K, Imai R, Matsui H (2011) OsJAR1 and OsJAR2 are jasmonyl-L-isoleucine synthases involved in wound- and pathogen-induced jasmonic acid signalling. Biochem Biophys Res Commun 409:634–639View ArticleGoogle Scholar
- Wasternack C, Hause B (2013) Jasmonates: biosynthesis, perception, signal transduction and action in plant stress response, growth and development. An update to the 2007 review in annals of botany. Ann Bot 111:1021–1058View ArticleGoogle Scholar
- Widemann E, Grausem B, Renault H, Pineau E, Heinrich C, Lugan R, Ullmann P, Miesch L, Aubert Y, Miesch M, Heitz T, Pinot F (2015) Sequential oxidation of Jasmonoyl-phenylalanine and Jasmonoyl-isoleucine by multiple cytochrome P450 of the CYP94 family through newly identified aldehyde intermediates. Phytochemistry 117:388–399View ArticleGoogle Scholar
- Widemann E, Miesch L, Lugan R, Holder E, Heinrich C, Aubert Y, Miesch M, Pinot F, Heitz T (2013) The amidohydrolases IAR3 and ILL6 contribute to jasmonoyl-isoleucine hormone turnover and generate 12-hydroxyjasmonic acid upon wounding in Arabidopsis leaves. J Biol Chem 288:31701–31714View ArticleGoogle Scholar
- Woldemariam MG, Onkokesung N, Baldwin IT, Galis I (2012) Jasmonoyl-L-isoleucine hydrolase 1 (JIH1) regulates jasmonoyl-L-isoleucine levels and attenuates plant defenses against herbivores. Plant J 72:758–767View ArticleGoogle Scholar
- Wu H, Ye H, Yao R, Zhang T, Xiong L (2015) OsJAZ9 acts as a transcriptional regulator in jasmonate signaling and modulates salt stress tolerance in rice. Plant Sci 232:1–12View ArticleGoogle Scholar
- Yamada S, Kano A, Tamaoki D, Miyamoto A, Shishido H, Miyoshi S, Taniguchi S, Akimitsu K, Gomi K (2012) Involvement of OsJAZ8 in jasmonate-induced resistance to bacterial blight in rice. Plant Cell Physiol 53:2060–2072View ArticleGoogle Scholar
- Ye M, Luo SM, Xie JF, Li YF, Xu T, Liu Y, Song YY, Zhu-Salzman K, Zeng RS (2012) Silencing COI1 in rice increases susceptibility to chewing insects and impairs inducible defense. PLoS One 7:e36214View ArticleGoogle Scholar
- Zhang T, Poudel AN, Jewell JB, Kitaoka N, Staswick P, Matsuura H, Koo AJ (2016) Hormone crosstalk in wound stress response: wound-inducible amidohydrolases can simultaneously regulate jasmonate and auxin homeostasis in Arabidopsis thaliana. J Exp Bot 67:2107–2120View ArticleGoogle Scholar