Next Article in Journal
Lipidomics—Paving the Road towards Better Insight and Precision Medicine in Rare Metabolic Diseases
Previous Article in Journal
Multi-Step Extracellular Matrix Remodelling and Stiffening in the Development of Idiopathic Pulmonary Fibrosis
Previous Article in Special Issue
Genome-Wide Identification, Evolution, and Expression Pattern Analysis of the GATA Gene Family in Tartary Buckwheat (Fagopyrum tataricum)
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
IJMS
Volume 24
Issue 2
10.3390/ijms24021704
ijms-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Jasmonic Acid-Induced β-Cyclocitral Confers Resistance to Bacterial Blight and Negatively Affects Abscisic Acid Biosynthesis in Rice

by
Shiduku Taniguchi
,
Aya Takeda
,
Masaki Kiryu
and
Kenji Gomi
*
Faculty of Agriculture, Kagawa University, Miki 761-0795, Kagawa, Japan
*
Author to whom correspondence should be addressed.
Int. J. Mol. Sci. 2023, 24(2), 1704; https://doi.org/10.3390/ijms24021704
Submission received: 12 December 2022 / Revised: 10 January 2023 / Accepted: 12 January 2023 / Published: 15 January 2023
(This article belongs to the Special Issue Jasmonic Acid in Plants)
Browse Figures
Versions Notes

Abstract

:
Jasmonic acid (JA) regulates the production of several plant volatiles that are involved in plant defense mechanisms. In this study, we report that the JA-responsive volatile apocarotenoid, β-cyclocitral (β-cyc), negatively affects abscisic acid (ABA) biosynthesis and induces a defense response against Xanthomonas oryzae pv. oryzae (Xoo), which causes bacterial blight in rice (Oryza sativa L.). JA-induced accumulation of β-cyc was regulated by OsJAZ8, a repressor of JA signaling in rice. Treatment with β-cyc induced resistance against Xoo and upregulated the expression of defense-related genes in rice. Conversely, the expression of ABA-responsive genes, including ABA-biosynthesis genes, was downregulated by JA and β-cyc treatment, resulting in a decrease in ABA levels in rice. β-cyc did not inhibit the ABA-dependent interactions between OsPYL/RCAR5 and OsPP2C49 in yeast cells. Furthermore, we revealed that JA-responsive rice carotenoid cleavage dioxygenase 4b (OsCCD4b) was localized in the chloroplast and produced β-cyc both in vitro and in planta. These results suggest that β-cyc plays an important role in the JA-mediated resistance against Xoo in rice.

1. Introduction

Rice (Oryza sativa L.) is an important crop worldwide and has a precise regulatory network that responds to many environmental stresses, including pathogen attack. In rice plants, a few plant hormones induce the expression of numerous defense-related genes, under pathogen attack. The plant hormone, jasmonic acid (JA), plays an important role in the regulation of defense responses in rice [1]. JA induces disease resistance against the hemibiotrophic pathogen, Xanthomonas oryzae pv. oryzae (Xoo), which causes rice bacterial blight [2]. In contrast, the expression of JA-responsive genes is downregulated by virulent Xoo infection in rice [3]. A study revealed that treatment with cell-wall-degrading enzymes derived from Xoo activates JA signaling and induces Xoo resistance in rice [4]. JA upregulates the expression of defense-related genes by activating transcription factors (TFs), such as OsMYC2, OsbHLH034, and RICE EARLY RESPONSIVE TO JASMONATE1 (RERJ1) [5,6,7]. OsMYC2 regulates the expression of early JA-responsive defense-related genes in rice [5]. OsMYC2 and OsbHLH034 interact with several rice JA repressors, JASMONATE ZIM (JAZ) proteins, which forms a complex with NOVEL INTERACTOR OF JAZ (NINJA) [5,6,8]. The degradation of JAZ proteins triggers the release of TFs that regulate the expression of genes involved in JA signaling [9,10]. Transgenic rice plants overexpressing the C-terminal-deleted OsJAZ8ΔC exhibit a JA-insensitive phenotype and decreased JA-induced resistance against Xoo [10]. Moreover, overexpression of OsNINJA1 and its interactors, OsSRO1a and OsFHA1, results in the suppression of OsMYC2-dependent defense responses in rice [8,11,12]. Additionally, a WRKY-type TF, OsWRKY72, directly binds to the promoter of the rice JA biosynthesis gene, OsAOS1, and represses its transcription, resulting in the reduction of endogenous JA levels and an increase in Xoo susceptibility in rice [13]. In rice, lipase-like protein, OsEDS1, acts as a positive regulator in the JA-mediated defense response against Xoo, whereas in Arabidopsis, AtEDS1 regulates the salicylic acid (SA) signaling pathway in response to biotrophic pathogen infection [14].
Xoo is a vascular pathogen that colonizes the xylem vessels [15] and infects living cells through the pit membranes that separate the xylem lumen from xylem parenchyma cells. Furthermore, infection with an avirulent Xoo strain induces thickening of the xylem secondary walls and reduces pit diameter in rice, thus resulting in the reduction of Xoo access to xylem parenchyma cells [16]. Lignin is an essential component of the response leading to the thickening of the xylem secondary walls and its accumulation is an important event to protect Xoo infection in rice [16,17,18,19]. Xoo-derived lipopolysaccharides induce upregulation of lignin biosynthesis genes in rice [20]. OsbHLH034 acts as a positive regulator of lignin biosynthesis for the secondary-wall thickening of xylem vessels in rice [6]. Altogether, these results indicate that JA signaling plays an important role in Xoo resistance in rice.
In rice, JA also induces the accumulation of several plant volatiles that are involved in Xoo resistance [21,22,23,24]. The C7 volatile, (E,E)-2,4-heptadienal, has an antibacterial activity against Xoo [25]. Additionally, JA-induced volatile terpenoids, such as monoterpenes and sesquiterpenes, exhibit antibacterial activities against Xoo [22,23,24]. The monoterpene, γ-terpinene, suppresses Xoo growth by damaging the plasma membrane of the Xoo cells [22]. Furthermore, geraniol, a monoterpene, inhibits Xoo growth by suppressing the expression of cell-division-related genes of Xoo [24]. These results suggest that rice possesses several defense mechanisms to suppress Xoo infection, and that the JA-responsive volatiles exhibit antibacterial activities against Xoo in rice. Furthermore, plant volatiles function as signaling compounds to induce Xoo resistance in rice. The C6 volatile, (E)-2-hexenal, shows antibacterial activity against Xoo, and its vapor treatment induces the upregulation of many defense-related genes in rice [26]. Linalool, a JA-responsive monoterpene, also induces disease resistance against Xoo, although it has no antibacterial activity against Xoo in rice [21,25]. Linalool-accumulating transgenic rice plants, produced by overexpressing linalool synthase, exhibited enhanced disease resistance against Xoo. Microarray analysis revealed that linalool induces the expression of many defense-related genes in rice [21]. Furthermore, linalool biosynthesis is regulated by OsJAZ8 in rice [21]. RERJ1 interacts with OsMYC2 and positively regulates linalool production by upregulating linalool synthase [7]. These results strongly indicate that JA-responsive volatiles play both direct and indirect roles in JA-mediated defense responses in rice.
We previously identified a few JA-responsive volatiles with no antibacterial activity against Xoo [21,25]. However, except for linalool, the biological functions of these volatiles in the rice defense response against Xoo have not been investigated. In this study, we focused on the JA-responsive volatile, β-cyclocitral (β-cyc), and investigated its role in the JA-mediated defense response against Xoo in rice. We demonstrated that β-cyc negatively affects abscisic acid (ABA) biosynthesis. It has been revealed that ABA suppresses SA signaling and has a negative effect on Xoo resistance in rice [27]. Furthermore, we showed that in rice, β-cyc is synthesized from β-carotene by the enzymatic reaction of a JA-induced carotenoid cleavage dioxygenase, OsCCD4b.

2. Results

2.1. JA-Responsive β-Cyc Induces Resistance to Rice Bacterial Blight

Our previous study showed that transgenic rice plants overexpressing OsJAZ8 with a truncated Jas domain (OsJAZ8ΔC) were insensitive to JA [10]. Large-scale microarray analysis indicated that overexpression of OsJAZ8ΔC altered the expression of JA-responsive genes, including defense-related genes, in rice. Furthermore, OsJAZ8ΔC negatively regulates JA-induced resistance to Xoo in rice [10]. β-cyc accumulation was not observed in these plants after JA treatment (Figure 1A). This result suggests that the biosynthesis of beta-cyc in rice is induced by JA and is at least partly dependent on OsJAZ8. As shown in Figure 1A, β-cyc is a rice metabolite induced by JA and produced during Xoo infection [21]. However, previous research has reported that β-cyc has no antibacterial activity against Xoo [25]. Therefore, we decided to investigate the role of β-cyc in Xoo resistance in rice. To do this, we applied β-cyc to rice plants and evaluated its effect on disease resistance to Xoo. Rice leaf blades were inoculated with virulent Xoo 24 h after treatment with β-cyc, and the blight lesion lengths were measured after 2 weeks. As shown in Figure 1B, the blight lesions on the β-cyc-treated rice plants were significantly shorter than those on the mock-treated rice plants. This result indicates that β-cyc has bioactivity in inducing Xoo resistance in rice.

2.2. β-Cyc Negatively Affects the Expression of ABA-Responsive Genes and ABA Biosynthesis

The absence of antibacterial activity of β-cyc against Xoo suggests that β-cyc may play a regulatory role in rice defense mechanism(s). Thus, we performed a DNA microarray using the Agilent rice 44 K custom oligo DNA microarray to identify β-cyc-responsive genes in rice. Using the criterion of a two-fold increase or decrease in the average expression levels, we extracted differentially expressed spots in β-cyc-treated rice (Supplementary Table S1). Some of the upregulated genes were associated with the plant defense mechanism, suggesting that they are involved in the β-cyc-induced resistance against Xoo. The characteristic defense-related genes are presented in Table 1. To validate the results obtained from microarray analysis, we analyzed the expression of defense-related genes presented in Table 1 using RT–qPCR. The results showed almost the same tendency compared with the microarray results (Figure 2).
Analysis of the microarray data revealed that the expression of some ABA-responsive genes was downregulated by β-cyc treatment [28] (Table 1). Thus, we performed RT-qPCR analysis using the ABA-responsive genes and 9-cis carotenoid cleavage dioxygenases (NCEDs; OsNCED2, OsNCED3a, and OsNCED9), which are involved in ABA biosynthesis [29]. The expression of all the tested genes was confirmed to be upregulated by ABA treatment (Figure 3). Conversely, the expression of all the genes, except for the dehydrin family protein [Os02g44870 (20)], was downregulated by both JA and β-cyc treatments (Figure 3).
Subsequently, we measured the amounts of ABA in the leaf blades post treatments with JA and β-cyc. The levels of ABA in the JA- and β-cyc-treated rice plants were significantly lower than those in mock-treated rice plants (Figure 4A,B). In addition, the levels of β-cyc in the ABA-treated rice plants were significantly lower than those in mock-treated rice plants (Figure 4C). The levels of β-carotene, a precursor for ABA biosynthesis [30], in JA-treated rice plants were not significantly different from those in mock-treated rice plants (Figure 4D).

2.3. β-Cyc Negatively Affects ABA-Induced Susceptibility to Xoo

JA induces Xoo resistance in rice, while ABA enhances Xoo susceptibility [10,27]. When rice plants were inoculated with virulent Xoo after treatment with ABA for 24 h, the blight lesions were significantly longer than those on the mock-treated rice plants (Figure 5A). However, when the ABA-treated rice plants were subsequently treated with JA or β-cyc for 24 h, the blight lesions in leaf blades were significantly shorter than those of the ABA-treated rice plants (Figure 5A,B).

2.4. β-Cyc Has No Effect on ABA Perception on OsPYL/RCAR5

ABA binds to pyrabactin-resistant like/regulatory components of ABA receptors 5, OsPYL/RCAR5, which forms a complex with a protein phosphatase, OsPP2C49, to regulate ABA signaling in rice [31]. The ABA-dependent interaction between OsPYL/RCAR5 and OsPP2C49 was demonstrated using a yeast two-hybrid system (Y2H) [31]. We added β-cyc to the Y2H medium to investigate whether β-cyc interferes with the ABA-dependent OsPYL/RCAR5-OsPP2C49 interaction, and the result revealed that there was no effect on the ABA-dependent OsPYL/RCAR5-OsPP2C49 interaction at any concentration of β-cyc (Figure 6).

2.5. Identification of a Rice β-Cyc Biosynthesis Gene, OsCCD4b

Recently, it was demonstrated that in saffron (Crocus sativus), β-cyc is produced from β-carotene by carotenoid cleavage dioxygenase (CCD)4c, CsCCD4c [32]. The rice genome contains two CCD4 family genes, OsCCD4a and OsCCD4b [29]. Thus, we investigated the expression of CCD4 and other CCD family genes after JA, ABA, and β-cyc treatments [29]. The expression of OsCCD4b was significantly upregulated by JA treatment (Figure 7A). It has been reported that β-carotene are accumulated in chloroplasts [33]. To investigate localization of OsCCD4b, we constructed an OsCCD4b-GFP fusion gene and transiently expressed the fusion product in rice protoplast cells. This analysis showed that OsCCD4b was localized in the chloroplasts of transformed rice cells (Figure 7B).
To determine the products catalyzed by OsCCD4b, we prepared recombinant histidine-tagged-OsCCD4b (His-OsCCD4b) protein and tested its enzymatic activity using β-carotene as a substrate. The catalyzed products were analyzed using gas chromatography-mass spectrometry (GC-MS). Three major peaks were detected in the His-OsCCD4b reaction with β-carotene (Figure 7C), which were not detected when the total proteins from E. coli with an empty vector were analyzed (Figure 7C). The two products were identified as β-cyc and β-ionone by comparing their mass spectra and retention times with those of the authentic β-cyc and β-ionone, respectively. The other product was unknown (Figure 7C).
To assess the enzymatic activity of OsCCD4b in planta, we generated transgenic rice plants overexpressing OsCCD4b and confirmed the expression of the transgenes using RT-PCR (Figure 7D). Two independent lines, namely lines six and thirteen, were used for further experiments. The levels of β-cyc and β-ionone in OsCCD4b-overexpressing rice plants were significantly higher than those in the wildtype (WT) plants (Figure 7E,F).

3. Discussion

JA-induced accumulation of β-cyc was partly regulated by OsJAZ8, suggesting that the activation of the TF that is repressed by OsJAZ8 is essential for JA-dependent β-cyc biosynthesis. OsMYC2 is an OsJAZ8-interacting TF, but microarray analysis of OsMYC2-overexpressing rice plants revealed that it does not regulate OsCCD4b expression [5]. These results suggest the presence of other uncharacterized TF(s) that play an important role in JA-induced biosynthesis of β-cyc in rice. Although it has no antibacterial activity against Xoo in rice [25], treatment with β-cyc induced Xoo resistance, suggesting that β-cyc acts as a signaling compound to induce the JA-mediated defense response against Xoo. Indeed, in this study, we revealed that the expression of many defense-related genes, including peroxidase and Bowman-Birk type proteinase inhibitor, was upregulated by β-cyc, which are also upregulated by JA and linalool [10,21]. A β-cyc-responsive peroxidase, OsPrx115, is a secretory-type class III peroxidase involved in lignin biosynthesis [34,35]. Other rice class III peroxidases, OsPrx38 and OsPrx114, play important roles in Xoo resistance by producing lignin, which is essential for the thickening of secondary cell walls [6,16]. The expression of OsPrx38 is regulated by JA-inducible OsbHLH034 [6] and is secreted into the xylem vessels in rice [36]. OsPrx114 is strongly induced by inoculation with avirulent Xoo and is secreted into the xylem lumen and walls of xylem parenchyma cells in rice [16]. In addition, it has been reported that transgenic rice plants overexpressing a Bowman-Birk type proteinase inhibitor gene exhibited enhanced resistance against Xoo [37]. These results suggest that β-cyc-induced resistance is at least partly due to the coordinated expression of these defense-related genes. Further studies, using transgenic rice plants overexpressing OsPrx115 and β-cyc-responsive Bowman-Birk proteinase inhibitors are required to clarify their respective effects on β-cyc-induced Xoo resistance in rice.
It has been revealed that ABA has a negative effect on Xoo resistance in rice [27]. Rice ABA-deficient mutants exhibit resistance to Xoo, and it is thought that the ABA-regulated water potential is involved in Xoo resistance [38]. A NAC-type TF, ONAC066, positively regulates Xoo resistance by suppressing ABA signaling in rice [39]. Furthermore, ABA has been reported to interact antagonistically with JA and downregulate the expression of the JA biosynthesis gene, OsAOS2, and a JA-responsive TF, OsJAmyb, in rice [40]. Indeed, ABA treatment suppresses JA production in rice [41]. In this study, we found that exogenous application of β-cyc triggers downregulation of ABA-responsive genes, including ABA-biosynthesis genes, and suppresses ABA-induced susceptibility to Xoo in rice. We demonstrated that β-cyc has no effect on the ABA-dependent interaction between OsPYL/RCAR5-OsPP2C49 in yeast cells, suggesting that β-cyc does not inhibit ABA signaling by directly binding to the ABA receptor, OsPYL/RCAR5. The rice genome reportedly contains at least 13 PYL/RCAR and 10 PP2C family genes [31]. Therefore, further analysis of the inhibitory effect of β-cyc on the interactions between other combinations of PYL/RCARs and PP2Cs is required.
Recently, it was reported that β-cyc directly binds to Arabidopsis 1-deoxy-D-xylulose 5-phosphate synthase (AtDXS), a key enzyme in the plastid-localized 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway, and inhibits its enzymatic activity in vitro [42]. The MEP pathway supplies precursors for many metabolites, including carotenoids and their derivatives such as β-carotene and ABA [33]. This finding supports our results, which show a decrease in the ABA levels after JA/β-cyc treatment. However, our previous studies revealed that the production of monoterpenes, such as linalool, γ-terpinene, and geraniol, which are produced via the MEP pathway, is also induced by JA treatment in rice [21,22,24], suggesting that the recognition mechanism of β-cyc in rice is different from that in Arabidopsis. Therefore, identification of β-cyc receptors involved in the rice defense mechanism is needed to clarify the biological functions of β-cyc.
It has been reported that ABA also suppresses SA signaling by downregulating OsWRKY45 and OsNPR1 expressions [27]. OsWRKY45 was characterized as a key TF involved in SA-mediated disease resistance against Xoo [43]. Recently, it has been reported that OsWRKY45 is a JA-responsive gene and the OsWRKY45-dependent signaling pathway is activated by OsVQ13 in the JA-induced resistance against Xoo in rice [44]. OsNPR1 is a rice homologue of an Arabidopsis SA receptor, AtNPR1, and its expression is upregulated by JA [45]. OsNPR1 is degraded by an E3 ubiquitin ligase, OsCUL3a, and oscul3a mutant exhibits increased resistance to Xoo by activating both JA- and SA-signaling pathways [46]. These results indicate that both JA- and SA-signaling pathways interact coordinately in the rice defense response, termed “Common Defense System” (CDS) [47]. Analysis of the microarray data revealed that neither OsWRKY45 nor OsNPR1 were upregulated by β-cyc treatment, suggesting that β-cyc might have no effect on CDS-mediated signaling in rice. However, further studies are required on the involvement of the β-cyc in CDS-mediated defense responses to clarify JA-repressed ABA signaling in rice defense responses.
β-Cyc is a volatile apocarotenoid derived from the oxidative cleavage of β-carotene [48]. The enzymatic cleavage of carotenoids is catalyzed by CCD and NCED family proteins. NCEDs cleave the 11,12 (11′,12′) double bond of 9-cis-violaxanthin or 9-cis-neoxanthin and are key enzymes in ABA production [49]. CCDs in plants are categorized into four families: CCD1, CCD4, CCD7, and CCD8 [29], among which CCD7 and CCD8 are involved in the biosynthesis of strigolactones, which are plant hormones that regulate plant growth and development [50]. CCD1 has low substrate specificity in vitro and produces several types of apocarotenoids, including β-ionone, along with a wide range of carotenoids [30]. In Arabidopsis, β-cyc and β-ionone are thought to be produced by non-enzymatic oxidative cleavage of β-carotene, but not by CCD-enzymatic cleavage of β-carotene, because these volatiles were normally produced in quadruple mutant for AtCCD1, 4, 7, and 8 [51]. In contrast, it has been reported that saffron and citrus (Citrus clementina) CCD4 proteins, CsCCD4c and CcCCD4b1, respectively, can produce β-cyc in vitro [32,52]. Moreover, CsCCD4c can produce β-ionone [32]. In this study, we demonstrated that OsCCD4b can produce both β-cyc and β-ionone in vitro and in planta. We could not investigate the β-cyc-mediated defense responses in the OsCCD4b-ox rice plants, because β-ionone had also accumulated in the plants. We previously found that accumulation of β-ionone was induced by JA treatment in rice [21], suggesting that β-ionone may also be produced by OsCCD4b in JA-treated rice plants. β-Ionone has no antibacterial activity against Xoo [25], further studies on β-ionone may provide new insights on the defense responses in rice.

4. Materials and Methods

4.1. Plant Materials, Chemical Treatments, and Bacterial Inoculation

The growth conditions of the rice plants (Oryza sativa L. cv. Hinohikari and Nipponbare) and Xoo (strain 7174) were set as previously described by Onohata and Gomi [6]. Nipponbare was used for production of transgenic rice plants. Xoo strain is virulent to both Hinohikari and Nipponbare cultivars. Fully opened fifth-leaf blades of rice plants were inoculated using the clipping inoculation technique [53], and the lengths of the blight lesions were measured. OsJAZ8ΔC-overexpressing transgenic rice plants produced previously by Yamada et al. [10] were used to analyze the JAZ-dependent regulation of β-cyc. Treatment with 100 μM JA (Sigma, St. Louis, MO, USA), 50 μM ABA (Sigma), and 10 μM β-cyc (Wako, Osaka, Japan) for 24 h was performed as previously described by Taniguchi et al. [21].

4.2. Microarray Analysis

An Agilent Rice Oligo Microarray (44 K, custom-made; Agilent Technologies, Redwood City, CA, USA) was used for the microarray analysis. Seedlings of the five-leaf stage rice plants were treated with 10 μM β-cyc for 24 h and a microarray analysis was performed as described by Taniguchi et al. [21]. The microarray data files are stored in the Gene Expression Omnibus Database (accession number GSE152023).

4.3. Quantification of Plant Volatiles, ABA, and β-Carotene

The amounts of β-cyc and β-ionone in the leaf blades were measured using GC-MS, as previously described by Kiryu et al. [23]. GC-MS analysis was performed using a GCMS-QP2010SE (Shimadzu, Kyoto, Japan) fitted with a DB-WAX column (60 m × 0.25 mm, 0.25 μm film thickness; J and W Scientific, Folsom, CA, USA) according to the manufacturer’s instructions. The compounds were identified by comparing their mass spectra with those of a database (Wiley10) and their retention times with those of authentic β-cyc and β-ionone. The amount of ABA in the leaf blades was measured using the Phytodetek ABA Test Kit (Agdia, Elkhart, IN, USA), according to the manufacturer’s instructions. β-Carotene was extracted from the leaf blades, as previously described by Zhou et al. [54] and was measured using high-performance liquid chromatography (HPLC). HPLC analysis was performed using a Prominence (Shimadzu, Kyoto, Japan) fitted with an Xbridge C18 column (4.6 mm × 250 mm, 5 μm; Waters, Milford, MA, USA). The extracts were eluted with MeOH-MTBE-H2O [81:15:4 (v/v), solvent A] and MeOH-MTBE-H2O [10:90:4 (v/v), solvent B]. The linier elution gradients started with 100 % of solvent A for 14.5 min at a flow rate of 1 mL min−1and were followed by a gradual increase of solvent B, reaching 100% in 2 min, maintaining 100% solvent B for 13.5 min. The peak at 440 nm was identified by comparing the retention time with that of HPLC-grade β-carotene purchased from Sigma-Aldrich (St. Louis, MO, USA).

4.4. Reverse Transcription-Quantitative PCR (RT-qPCR)

After each treatment, total RNA was extracted from the rice leaf blades using TRIzol (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instructions. Fourth-leaf blades were used for RT-qPCR analysis, and four leaf blades were used per replicate. RT-qPCR was performed as described by Gomi et al. [26]. The sequences of the gene-specific primers used in RT-qPCR are shown in Supplementary Table S2.

4.5. Yeast Two-Hybrid (Y2H) System

Y2H analysis was performed as previously described by Kim et al. [31] using the MATCHMAKER Y2H system [Clontech (Takara Bio), Shiga, Japan]. The OsPYL/RCAR5 (Os05g12260) and OsPP2C49 (Os05g38290) cDNAs were ligated into pGBKT7 and pGADT7 vectors, respectively. The plasmids were then introduced into the AH109 yeast strain. The selection media were supplemented with 10 μM ABA and different concentrations of β-cyc. The plates were incubated at 30 °C for 5 days.

4.6. Transient Localization Assay

For the construction of OsCCD4b-GFP, the ORF of OsCCD4b (Os12g24800) without the stop codon was amplified by PCR and subcloned into the corresponding site on the pE7133-GFP vector [55], fusing GFP in-frame to the C-terminus of OsCCD4b. The OsCCD4b-GFP fusion protein was introduced into rice protoplast cells using polyethylene glycol 3350, according to Bart et al. [56]. Localization analysis of OsCCD4b was performed as previously described by Kiryu et al. [23]. GFP fluorescence was observed using a BIOREVO BZ-9000 fluorescent microscope (Keyence, Osaka, Japan) equipped with a GFP-specific filter unit.

4.7. Functional Expression of OsCCD4b in Escherichia coli

The ORF of OsCCD4b truncated the putative chloroplast transit sequence was ligated in-frame into the pCold II vector (Takara, Shiga, Japan) and the histidine-tagged proteins produced in Escherichia coli were purified using a HisTrapTM HP column (GE Healthcare, Buckinghamshire, UK) according to the manufacturer’s instructions. The purified fusion protein was dialyzed using an assay buffer [50 mM NaPO4 (pH 7.2) containing 300 mM NaCl, 5 μM FeSO4, and 5 mM ascorbic acid.]

4.8. Enzyme Assay and Analysis of Volatiles

Enzyme activity was assayed using approximately 40 μg of purified His-tagged OsCCD4b protein in a 15 mL sealed Spelco vial (Spelco, St. Louis, MO, USA) containing 1 mL of the assay buffer. The reaction was initiated by the addition of β-carotene (55 μM final concentration). After incubation at 30 °C for 4 h, the headspace above the sample was trapped for 10 min at 50 °C using a polydimethylsiloxane (PDMS)/divinylbenzene (DVB)-coated solid-phase microextraction (SPME) fiber (Supelco, Bellafonte, PA, USA) for GC-MS. The GC-MS conditions were set as described by Kiryu et al. [23].

4.9. Production of OsCCD4b-Overexpressing Rice Plants

The ORF of OsCCD4b was ligated into the pBI333-EN4 vector [57]. The binary vector was introduced into Agrobacterium tumefaciens EHA101 by electroporation [58]. Rice transformation was performed as described by Hiei et al. [59], and the transgenic plants were selected on a medium containing 50 mgL−1 of hygromycin. Second and third generation plants were used for the experiments. To verify the expression of the transgene, RT-PCR was performed using the OneStep RT-PCR kit (QIAGEN, Hilden, Germany) with the OsCCD4b-specific forward primer and Nos terminator-specific reverse primer. The sequences used for RT-PCR are as follows: OsCCD4bF, 5’- CAGAGCTAGACATTGTTGCAGAAG-3’ and tNOSR, 5’-GTATAATTGCGGGACTCTAATC-3’; actin, forward, 5’-CCTGGAATCCATGAGACCAC-3’ and reverse, 5’-ACACCAACAATCCCAAACAGAG-3’.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms24021704/s1.

Author Contributions

Conceptualization, K.G.; formal analysis, S.T., A.T. and M.K.; writing—original draft preparation, S.T. and K.G.; writing—review and editing, K.G. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported in part by the Funding Program for Next Generation World-Leading Researchers from Japan Society for Promotion of Science (No. GS022).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

All data generated or analyzed during this study are included in this published article and its supplementary information files. The microarray data files are stored in the Gene Expression Omnibus Database (accession number GSE152023).

Acknowledgments

We are grateful to Kazuya Akimitsu (Kagawa University) for his continuous support and helpful suggestions. We thank Yoko Nishizawa (National Institute of Agrobiological Sciences, NIAS) and Hisatoshi Kaku (NIAS) for providing the binary vector and Xoo strain, respectively. We also thank Ikuo Kataoka (Kagawa University), Masaru Satoh (National Agricultural Research Center for Kyushu Okinawa Region, NARO), and Hiroo Kanno (NARO) for laying the foundation for a part of this study.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Trang Nguyen, H.; Thi Mai To, H.; Lebrun, M.; Bellafiore, S.; Champion, A. Jasmonates-the master regulator of rice development, adaptation, and defense. Plants 2019, 8, 339. [Google Scholar] [CrossRef] [Green Version]
  2. Vleesschauwer, D.D.; Gheysen, G.; Höfte, M. Hormone defense networking in rice: Tales from a different world. Trends Plant Sci. 2013, 18, 555–565. [Google Scholar] [CrossRef] [PubMed]
  3. Tariq, R.; Ji, Z.; Wang, C.; Tang, Y.; Zou, L.; Sun, H.; Chen, G.; Zhao, K. RNA-Seq analysis of gene expression changes triggered by Xanthomonas oryzae pv. oryzae in a susceptible rice genotype. Rice 2019, 12, 44. [Google Scholar] [CrossRef] [Green Version]
  4. Ranjan, A.; Vadassery, J.; Patel, H.K.; Pandey, A.; Palaparthi, R.; Mithöfer, A.; Sonti, R.V. Upregulation of jasmonate biosynthesis and jasmonate-responsive genes in rice leaves in response to a bacterial pathogen mimic. Funct. Integr. Genomics 2015, 15, 363–373. [Google Scholar] [CrossRef]
  5. Uji, Y.; Taniguchi, S.; Tamaoki, D.; Shishido, H.; Akimitsu, K.; Gomi, K. Overexpression of OsMYC2 results in the up-regulation of early JA-responsive genes and bacterial blight resistance in rice. Plant Cell Physiol. 2016, 57, 1814–1827. [Google Scholar] [CrossRef]
  6. Onohata, T.; Gomi, K. Overexpression of jasmonate-responsive OsbHLH034 in rice results in the induction of bacterial blight-resistance via an increase in lignin biosynthesis. Plant Cell Rep. 2020, 39, 1175–1184. [Google Scholar] [CrossRef] [PubMed]
  7. Valea, I.; Motegi, A.; Kawamura, N.; Kawamoto, K.; Miyao, A.; Ozawa, R.; Takabayashi, J.; Gomi, K.; Nemoto, K.; Nozawa, A.; et al. The rice wound-inducible transcription factor RERJ1 sharing same signal transduction pathway with OsMYC2 is necessary for defense response to herbivory and bacterial blight. Plant Mol. Biol. 2022, 109, 651–666. [Google Scholar] [CrossRef] [PubMed]
  8. Kashihara, K.; Onohata, T.; Okamoto, Y.; Uji, Y.; Mochizuki, S.; Akimitsu, K.; Gomi, K. Overexpression of OsNINJA1 negatively affects a part of OsMYC2-mediated abiotic and biotic responses in rice. J. Plant Physiol. 2019, 232, 180–187. [Google Scholar] [CrossRef]
  9. Pauwels, L.; Goossens, A. The JAZ proteins: A crucial interface in the jasmonate signaling cascade. Plant Cell 2011, 23, 3089–3100. [Google Scholar] [CrossRef] [Green Version]
  10. Yamada, S.; Kano, A.; Tamaoki, D.; Miyamoto, A.; Shishido, H.; Miyoshi, S.; Taniguchi, S.; Akimitsu, K.; Gomi, K. Involvement of OsJAZ8 in jasmonate-induced resistance to bacterial blight in rice. Plant Cell Physiol. 2012, 53, 2060–2072. [Google Scholar] [CrossRef]
  11. Kashihara, K.; Onohata, T.; Yariuchi, R.; Tanaka, S.; Akimitsu, K.; Gomi, K. The overexpression of OsSRO1a, which encodes an OsNINJA1- and OsMYC2-interacting protein, negatively affects OsMYC2-mediated jasmonate signaling in rice. Plant Cell Rep. 2020, 39, 489–500. [Google Scholar] [CrossRef]
  12. Kashihara, K.; Ojiri, T.; Gomi, K. Overexpression of OsNINJA1-interacting protein OsFHA1 affects jasmonate-mediated OsMYC2 signaling but not disease resistance in rice. J. Gen. Plant Pathol. 2022, 88, 225–231. [Google Scholar] [CrossRef]
  13. Hou, Y.; Wang, Y.; Tang, L.; Tong, X.; Wang, L.; Liu, L.; Huang, S.; Zhang, J. SAPK10-mediated phosphorylation on WRKY72 releases its suppression on jasmonic acid biosynthesis and bacterial blight resistance. iScience 2019, 16, 499–510. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  14. Ke, Y.; Kang, Y.; Wu, M.; Liu, H.; Hui, S.; Zhang, Q.; Li, X.; Xiao, J.; Wang, S. Jasmonic acid-involved OsEDS1 signaling in rice-bacteria interactions. Rice 2019, 12, 25. [Google Scholar] [CrossRef] [Green Version]
  15. Tabei, H. Anatomical studies of rice plant affected with bacterial leaf blight, with special reference to stomatal infection at the coleoptile and the foliage leaf sheath of rice seedling. Ann. Phytopath. Sci. Jpn. 1967, 33, 12–16. [Google Scholar] [CrossRef]
  16. Hilaire, E.; Young, S.A.; Willard, L.H.; McGee, J.D.; Sweat, T.; Chittoor, J.M.; Guikema, J.A.; Leach, J.E. Vascular defense responses in rice: Peroxidase accumulation in xylem parenchyma cells and xylem wall thickening. Mol. Plant-Microbe Interact. 2001, 14, 1411–1419. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  17. Bart, R.S.; Chern, M.; Vega-Sánchez, M.E.; Canlas, P.; Ronald, P.C. Rice Snl6, a cinnamoyl-CoA reductase-like gene family member, is required for NH1-mediated immunity to Xanthomonas oryzae pv. oryzae. PLoS Genet. 2010, 6, e1001123. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  18. Song, A.; Xue, G.; Cui, P.; Fan, F.; Liu, H.; Yin, C.; Sun, W.; Liang, Y. The role of silicon in enhancing resistance to bacterial blight of hydroponic- and soil-cultured rice. Sci. Rep. 2016, 6, 24640. [Google Scholar] [CrossRef] [Green Version]
  19. Suzuki, G.; Fukuda, M.; Lucob-Agustin, N.; Inukai, Y.; Gomi, K. The Mutation of Rice MEDIATOR25, OsMED25, Induces Rice Bacterial Blight Resistance through Altering Jasmonate- and Auxin-Signaling. Plants 2022, 11, 1601. [Google Scholar] [CrossRef] [PubMed]
  20. Girija, A.M.; Kinathi, B.K.; Madhavi, M.B.; Ramesh, P.; Vungarala, S.; Patel, H.K.; Sonti, R.V. Rice leaf transcriptional profiling suggests a functional interplay between Xanthomonas oryzae pv. oryzae lipopolysaccharide and extracellular polysaccharide in modulation of defense responses during infection. Mol. Plant-Microbe Interact. 2017, 30, 16–27. [Google Scholar] [CrossRef]
  21. Taniguchi, S.; Hosokawa-Shinonaga, Y.; Tamaoki, D.; Yamada, S.; Akimitsu, K.; Gomi, K. Jasmonate induction of the monoterpene linalool confers resistance to rice bacterial blight and its biosynthesis is regulated by JAZ protein in rice. Plant Cell Environ. 2014, 37, 451–461. [Google Scholar] [CrossRef]
  22. Yoshitomi, K.; Taniguchi, S.; Tanaka, K.; Uji, Y.; Akimitsu, K.; Gomi, K. Rice terpene synthase 24 (OsTPS24) encodes a jasmonate-responsive monoterpene synthase that produces an antibacterial γ-terpinene against rice pathogen. J. Plant Physiol. 2016, 191, 120–126. [Google Scholar] [CrossRef] [PubMed]
  23. Kiryu, M.; Hamanaka, M.; Yoshitomi, K.; Mochizuki, S.; Akimitsu, K.; Gomi, K. Rice terpene synthase 18 (OsTPS18) encodes a sesquiterpene synthase that produces an antibacterial (E)-nerolidol against a bacterial pathogen of rice. J. Gen. Plant Pathol. 2018, 84, 221–229. [Google Scholar] [CrossRef]
  24. Kiyama, H.; Matsunaga, A.; Suzuki, G.; Gomi, K. Monoterpene geraniol produced by Rice terpene synthase 21 suppresses the expression of cell-division related genes in the rice bacterial pathogen, Xanthomonas oryzae pv. oryzae. Physiol. Mol. Plant Pathol. 2021, 115, 101673. [Google Scholar] [CrossRef]
  25. Tanaka, K.; Taniguchi, S.; Tamaoki, D.; Yoshitomi, K.; Akimitsu, K.; Gomi, K. Multiple roles of plant volatiles in jasmonate-induced defense response in rice. Plant Signal. Behav. 2014, 9, e29247. [Google Scholar] [CrossRef] [Green Version]
  26. Gomi, K.; Satoh, M.; Ozawa, R.; Shinonaga, Y.; Sanada, S.; Sasaki, K.; Matsumura, M.; Ohashi, Y.; Kanno, H.; Akimitsu, K.; et al. Role of hydroperoxide lyase in white-backed planthopper (Sogatella furcifera Horváth)-induced resistance to bacterial blight in rice, Oryza sativa L. Plant J. 2010, 61, 46–57. [Google Scholar] [CrossRef] [PubMed]
  27. Xu, J.; Audenaert, K.; Hofte, M.; Vlesschauwer, D.D. Abscisic Acid Promotes Susceptibility to the Rice Leaf Blight Pathogen Xanthomonas oryzae pv oryzae by Suppressing Salicylic Acid-Mediated Defenses. PLoS ONE 2013, 8, e67413. [Google Scholar] [CrossRef] [Green Version]
  28. Rabbani, M.A.; Maruyama, K.; Abe, H.; Khan, M.A.; Katsura, K.; Ito, Y.; Yoshiwara, K.; Seki, M.; Shinozaki, K.; Yamaguchi-Shinozaki, K. Monitoring expression profiles of rice genes under cold, drought, and high-salinity stresses and abscisic acid application using cDNA microarray and RNA gel-blot analyses. Plant Physiol. 2003, 133, 1755–1767. [Google Scholar] [CrossRef] [Green Version]
  29. Vallabhaneni, R.; Wurtzel, E.T. From epoxycarotenoids to ABA: The role of ABA 8′-hydroxylases in drought-stressed maize roots. Arch. Biochem. Biophys. 2010, 504, 112–117. [Google Scholar] [CrossRef] [Green Version]
  30. Auldridge, M.E.; McCarty, D.R.; Klee, H.J. Plant carotenoid cleavage oxygenases and their apocarotenoid products. Curr. Opin. Plant Biol. 2006, 9, 315–321. [Google Scholar] [CrossRef]
  31. Kim, H.; Hwang, H.; Hong, J.W.; Lee, Y.N.; Ahn, I.P.; Yoon, I.S.; Yoo, S.D.; Lee, S.; Lee, S.C.; Kim, B.G. A rice orthologue of the ABA receptor, OsPYL/RCAR5, is a positive regulator of the ABA signal transduction pathway in seed germination and early seedling growth. J. Exp. Bot. 2012, 63, 1013–1024. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  32. Rubio-Moraga, A.; Rambla, J.L.; Fernández-de-Carmen, A.; Trapero-Mozos, A.; Ahrazem, O.; Orzáez, D.; Granell, A.; Gómez-Gómez, L. New target carotenoids for CCD4 enzymes are revealed with the characterization of a novel stress-induced carotenoid cleavage dioxygenase gene from Crocus sativus. Plant Mol. Biol. 2014, 86, 555–569. [Google Scholar] [CrossRef] [PubMed]
  33. Sun, T.; Yuan, H.; Cao, H.; Yazdani, M.; Tadmor, Y.; Li, L. Carotenoid Metabolism in Plants: The Role of Plastids. Mol. Plant. 2018, 11, 58–74. [Google Scholar] [CrossRef] [Green Version]
  34. Passardi, F.; Longet, D.; Penel, C.; Dunand, C. The class III peroxidase multigenic family in rice and its evolution in land plants. Phytochemistry 2004, 65, 1879–1893. [Google Scholar] [CrossRef] [PubMed]
  35. Liu, Q.; Luo, L.; Zheng, L. Lignins: Biosynthesis and biological functions in plants. Int. J. Mol. Sci. 2018, 19, 335. [Google Scholar] [CrossRef] [Green Version]
  36. Aki, T.; Shigyo, M.; Nakano, R.; Yoneyama, T.; Yanagisawa, S. Nano scale proteomics revealed the presence of regulatory proteins including three FT-Like proteins in phloem and xylem saps from rice. Plant Cell Physiol. 2008, 49, 767–790. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  37. Pang, Z.; Zhou, Z.; Yin, D.; Lv, Q.; Wang, L.; Xu, X.; Wang, J.; Li, X.; Zhao, X.; Jiang, G.; et al. Transgenic rice plants overexpressing BBTI4 confer partial but broad-spectrum bacterial blight resistance. J. Plant Biol. 2013, 56, 383–390. [Google Scholar] [CrossRef]
  38. Zhang, D.; Tian, C.; Yin, K.; Wang, W.; Qiu, J.L. Postinvasive Bacterial Resistance Conferred by Open Stomata in Rice. Mol. Plant-Micribe Interact. 2019, 32, 255–266. [Google Scholar] [CrossRef] [Green Version]
  39. Liu, Q.; Yan, S.; Huang, W.; Yang, J.; Dong, J.; Zhang, S.; Zhao, J.; Yang, T.; Mao, X.; Zhu, X.; et al. NAC transcription factor ONAC066 positively regulates disease resistance by suppressing the ABA signaling pathway in rice. Plant Mol. Biol. 2018, 98, 289–302. [Google Scholar] [CrossRef]
  40. Nahar, K.; Kyndt, T.; Nzogela, Y.B.; Gheysen, G. Abscisic acid interacts antagonistically with classical defense pathways in rice–migratory nematode interaction. New Phytol. 2012, 196, 901–913. [Google Scholar] [CrossRef]
  41. Xie, K.; Li, L.; Zhang, H.; Wang, R.; Tan, X.; He, Y.; Hong, G.; Li, J.; Ming, F.; Yao, X.; et al. Abscisic acid negatively modulates plant defence against rice black-streaked dwarf virus infection by suppressing the jasmonate pathway and regulating reactive oxygen species levels in rice. Plant Cell Environ. 2018, 41, 2504–2514. [Google Scholar] [CrossRef] [PubMed]
  42. Mitra, S.; Estrada-Tejedor, R.; Volke, D.C.; Phillips, M.A.; Gershenzon, J.; Wright, L.P. Negative regulation of plastidial isoprenoid pathway by herbivore-induced β-cyclocitral in Arabidopsis thaliana. Proc. Natl. Acad. Sci. USA 2021, 118, e2008747118. [Google Scholar] [CrossRef] [PubMed]
  43. Shimono, M.; Sugano, S.; Nakayama, A.; Jiang, C.J.; Ono, K.; Toki, S.; Takatsuji, H. Rice WRKY45 Plays a Crucial Role in Benzothiadiazole-Inducible Blast Resistance. Plant Cell 2007, 19, 2064–2076. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  44. Uji, Y.; Kashihara, K.; Kiyama, H.; Mochizuki, S.; Akimitsu, K.; Gomi, K. Jasmonic acid-induced VQ-motif-containing protein OsVQ13 influences the OsWRKY45 signaling pathway and grain size by associating with OsMPK6 in rice. Int. J. Mol. Sci. 2019, 20, 2917. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  45. Yuan, Y.; Zhong, S.; Li, Q.; Zhu, Z.; Lou, Y.; Wang, L.; Wang, J.; Wang, M.; Li, Q.; Yang, D.; et al. Functional analysis of rice NPR1-like genes reveals that OsNPR1/NH1 is the rice orthologue conferring disease resistance with enhanced herbivore susceptibility. Plant Biotechnol. J. 2007, 5, 313–324. [Google Scholar] [CrossRef]
  46. Liu, Q.; Ning, Y.; Zhang, Y.; Yu, N.; Zhao, C.; Zhan, X.; Wu, W.; Chen, D.; Wei, X.; Wang, G.L.; et al. OsCUL3a Negatively Regulates Cell Death and Immunity by Degrading OsNPR1 in Rice. Plant Cell 2017, 29, 345–359. [Google Scholar] [CrossRef] [Green Version]
  47. Tamaoki, D.; Seo, S.; Yamada, S.; Kano, A.; Miyamoto, A.; Shishido, H.; Miyoshi, S.; Taniguchi, S.; Akimitsu, K.; Gomi, K. Jasmonic acid and salicylic acid activate a common defense system in rice. Plant Signaling Behav. 2013, 8, e24260. [Google Scholar] [CrossRef]
  48. Havaux, M. β-Cyclocitral and derivatives: Emerging molecular signals serving multiple biological functions. Plant Physiol. Biochem. 2020, 155, 35–41. [Google Scholar] [CrossRef]
  49. Nambara, E.; Marion-Poll, A. Abscisic acid biosynthesis and catabolism. Annu. Rev. Plant Biol. 2005, 56, 165–185. [Google Scholar] [CrossRef] [Green Version]
  50. Zwanenburg, B.; Blanco-Ania, D. Strigolactones: New plant hormones in the spotlight. J. Exp. Bot. 2018, 69, 2205–2218. [Google Scholar] [CrossRef]
  51. Ramel, F.; Mialoundama, A.S.; Havaux, M. Nonenzymic carotenoid oxidation and photooxidative stress signalling in plants. J. Exp. Bot. 2013, 64, 799–805. [Google Scholar] [CrossRef] [PubMed]
  52. Rodrigo, M.J.; Alquézar, B.; Alós, E.; Medina, V.; Carmona, L.; Bruno, M.; Al-Babili, S.; Zacarías, L. A novel carotenoid cleavage activity involved in the biosynthesis of Citrus fruit-specific apocarotenoid pigments. J. Exp. Bot. 2013, 64, 4461–4478. [Google Scholar] [CrossRef] [PubMed]
  53. Kauffman, H.E.; Reddy, A.P.K.; Hsieh, S.P.Y.; Merca, S.D. An improved technique for evaluating resistance of rice varieties to Xanthomonas oryzae. Plant Dis. Rep. 1973, 57, 537–541. [Google Scholar]
  54. Zhou, K.; Ren, Y.; Lv, J.; Wang, Y.; Liu, F.; Zhou, F.; Zhao, S.; Chen, S.; Peng, C.; Zhang, X.; et al. Young Leaf Chlorosis 1, a chloroplast-localized gene required for chlorophyll and lutein accumulation during early leaf development in rice. Planta 2013, 237, 279–292. [Google Scholar] [CrossRef]
  55. Mitsuhara, I.; Ugaki, M.; Hirochika, H.; Ohshima, M.; Murakami, T.; Gotoh, Y.; Katayose, Y.; Nakamura, S.; Honkura, R.; Nishimiya, S.; et al. Efficient promoter cassettes for enhanced expression of foreign genes in dicotyledonous and monocotyledonous plants. Plant Cell Physiol. 1996, 37, 49–59. [Google Scholar] [CrossRef] [Green Version]
  56. Bart, R.; Chern, M.; Park, C.J.; Bartley, L.; Ronald, P.C. A novel system for gene silencing using siRNAs in rice leaf and stem-derived protoplasts. Plant Methods 2006, 2, 13. [Google Scholar] [CrossRef] [Green Version]
  57. Nishizawa, Y.; Nishio, Z.; Nakazono, K.; Soma, M.; Nakajima, E.; Ugaki, M.; Hibi, T. Enhanced resistance to blast (Magnaporthe grisea) in transgenic Japonica rice by constitutive expression of rice chitinase. Theor. Appl. Genet. 1999, 99, 383–390. [Google Scholar] [CrossRef] [PubMed]
  58. Wen-jun, S.; Forde, B.G. Efficient transformation of Agrobacterium spp. by high voltage electroporation. Nucleic Acids Res. 1989, 17, 8385. [Google Scholar] [CrossRef]
  59. Hiei, Y.; Ohta, S.; Komari, T.; Kumashiro, T. Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. Plant J. 1994, 6, 271–282. [Google Scholar] [CrossRef]
Ijms 24 01704 g001 550
Figure 1. The biosynthesis of JA-inducible beta-cyc is partly dependent on OsJAZ8, and exogenous application of β-cyc increases disease resistance against Xanthomonas oryzae pv. oryzae (Xoo). (A) The levels of β-cyc in wildtype (WT) and OsJAZ8ΔC-overexpressing plants 24 h after jasmonic acid (JA) treatment (JA). Data were analyzed using the Tukey–Kramer test [n = 4 for WT Mock (M); n = 6 for WT JA (JA); n = 3 for line 14 and line 17 Mock (M); n = 4 for line 14 JA; and n = 3 for line 17 JA]. Values are expressed as mean ± SE. Means accompanied by different letters are significantly different at p < 0.05. (B) The length of lesions on β-cyc-treated leaf blades 2 weeks after inoculation with Xoo. Values are expressed as means ± SE (n = 12). An asterisk represents statistically significant difference from the mock-treated control at p < 0.05 (Student’s t-test).
Figure 1. The biosynthesis of JA-inducible beta-cyc is partly dependent on OsJAZ8, and exogenous application of β-cyc increases disease resistance against Xanthomonas oryzae pv. oryzae (Xoo). (A) The levels of β-cyc in wildtype (WT) and OsJAZ8ΔC-overexpressing plants 24 h after jasmonic acid (JA) treatment (JA). Data were analyzed using the Tukey–Kramer test [n = 4 for WT Mock (M); n = 6 for WT JA (JA); n = 3 for line 14 and line 17 Mock (M); n = 4 for line 14 JA; and n = 3 for line 17 JA]. Values are expressed as mean ± SE. Means accompanied by different letters are significantly different at p < 0.05. (B) The length of lesions on β-cyc-treated leaf blades 2 weeks after inoculation with Xoo. Values are expressed as means ± SE (n = 12). An asterisk represents statistically significant difference from the mock-treated control at p < 0.05 (Student’s t-test).
Ijms 24 01704 g001
Ijms 24 01704 g002 550
Figure 2. Validation of the microarray results with RT-qPCR. Expression of the selected defense-related genes after β-cyc treatment was analyzed using RT-qPCR. The numbers in parentheses are the same as those in Table 1. Values are expressed as means ± SE (n = 4). Asterisks represent statistically significant difference from the mock-treated control at p < 0.05 (Student’s t-test).
Figure 2. Validation of the microarray results with RT-qPCR. Expression of the selected defense-related genes after β-cyc treatment was analyzed using RT-qPCR. The numbers in parentheses are the same as those in Table 1. Values are expressed as means ± SE (n = 4). Asterisks represent statistically significant difference from the mock-treated control at p < 0.05 (Student’s t-test).
Ijms 24 01704 g002
Ijms 24 01704 g003 550
Figure 3. RT-qPCR analysis of abscisic acid (ABA)-responsive genes after ABA, JA, and β-cyc treatment. The numbers in parentheses are the same as those in Table 1. Values are expressed as means ± SE (n = 3). Asterisks represent statistically significant difference from the mock-treated control at p < 0.05 (Student’s t-test).
Figure 3. RT-qPCR analysis of abscisic acid (ABA)-responsive genes after ABA, JA, and β-cyc treatment. The numbers in parentheses are the same as those in Table 1. Values are expressed as means ± SE (n = 3). Asterisks represent statistically significant difference from the mock-treated control at p < 0.05 (Student’s t-test).
Ijms 24 01704 g003
Ijms 24 01704 g004 550
Figure 4. JA and β-cyc negatively affect ABA biosynthesis. (A) ABA levels after 24 h of JA treatment. Values are expressed as means ± SE (n = 6). (B) ABA levels after 24 h of β-cyc treatment. Values are expressed as means ± SE (n = 7). (C) β-Cyc levels after 24 h of ABA treatment. Values are expressed as means ± SE (n = 6). (D) β-Carotene levels after 24 h of JA treatment. Values are expressed as means ± SE (n = 4). Asterisks represent statistically significant difference from the mock-treated control at p < 0.05 (Student’s t-test).
Figure 4. JA and β-cyc negatively affect ABA biosynthesis. (A) ABA levels after 24 h of JA treatment. Values are expressed as means ± SE (n = 6). (B) ABA levels after 24 h of β-cyc treatment. Values are expressed as means ± SE (n = 7). (C) β-Cyc levels after 24 h of ABA treatment. Values are expressed as means ± SE (n = 6). (D) β-Carotene levels after 24 h of JA treatment. Values are expressed as means ± SE (n = 4). Asterisks represent statistically significant difference from the mock-treated control at p < 0.05 (Student’s t-test).
Ijms 24 01704 g004
Ijms 24 01704 g005 550
Figure 5. JA and β-cyc negatively affect ABA-induced Xoo susceptibility. (A) The length of lesions on ABA or ABA/JA-treated leaf blades 8 days after inoculation with Xoo. Values are expressed as means ± SE. Data were analyzed using the Tukey–Kramer test [n = 12 for Mock (−); n = 11 for ABA and ABA/JA (+)]. (B) The length of lesions on ABA or ABA/β-cyc-treated leaf blades 8 days after inoculation with Xoo. Data were analyzed using the Tukey–Kramer test (n = 12 for all treatments). Values are expressed as means ± SE. Means accompanied by different letters are significantly different at p < 0.05.
Figure 5. JA and β-cyc negatively affect ABA-induced Xoo susceptibility. (A) The length of lesions on ABA or ABA/JA-treated leaf blades 8 days after inoculation with Xoo. Values are expressed as means ± SE. Data were analyzed using the Tukey–Kramer test [n = 12 for Mock (−); n = 11 for ABA and ABA/JA (+)]. (B) The length of lesions on ABA or ABA/β-cyc-treated leaf blades 8 days after inoculation with Xoo. Data were analyzed using the Tukey–Kramer test (n = 12 for all treatments). Values are expressed as means ± SE. Means accompanied by different letters are significantly different at p < 0.05.
Ijms 24 01704 g005
Ijms 24 01704 g006 550
Figure 6. β-cyc has no effect on the ABA-dependent interaction between OsPYL/RCAR5 and OsPPC2C49. The selection media (SD−4) were supplemented with 10 μM ABA and different concentrations of β-cyc.
Figure 6. β-cyc has no effect on the ABA-dependent interaction between OsPYL/RCAR5 and OsPPC2C49. The selection media (SD−4) were supplemented with 10 μM ABA and different concentrations of β-cyc.
Ijms 24 01704 g006
Ijms 24 01704 g007 550
Figure 7. Identification of β-cyc biosynthesis gene, OsCCD4b. (A) RT-qPCR analysis of rice CCD family genes after treatments with JA, ABA, and β-cyc. Values are expressed as means ± SE (n = 4). Asterisks represent statistically significant difference from the mock-treated control at p < 0.05 (Student’s t-test). (B) Subcellular localization of OsCCD4b in rice cells. OsCCD4b-GFP and GFP-only vectors were transferred into rice protoplast cells. Left panels (GFP) show a protoplast expressing the GFP only vector and right panels (OsCCD4b-GFP) show a protoplast expressing the OsCCD4b-GFP vector. Upper panels show fluorescence microscope images and lower panels show light microscope images. Green fluorescence corresponding to the localization of the proteins was detected using fluorescence microscopy. Bars = 10 μm. (C) GC profiles of the products catalyzed by His-OsCCD4b protein (upper panel) and by E. coli protein (lower panel) using β-carotene as the substrate. Uk: unknown. (D) RT-PCR analysis of OsCCD4b and actin expression in the WT and OsCCD4b-overexpressing (OsCCD4b-ox) rice plants. (E,F) β-cyc and β-ionone levels in OsCCD4b-overexpressing rice plants. Values are expressed as means ± SE. Data were analyzed using the Tukey–Kramer test (n = 7 for WT and line 6; n = 8 for line 13). Values are the mean ± SE. Means accompanied by different letters were significantly different at p < 0.05.
Figure 7. Identification of β-cyc biosynthesis gene, OsCCD4b. (A) RT-qPCR analysis of rice CCD family genes after treatments with JA, ABA, and β-cyc. Values are expressed as means ± SE (n = 4). Asterisks represent statistically significant difference from the mock-treated control at p < 0.05 (Student’s t-test). (B) Subcellular localization of OsCCD4b in rice cells. OsCCD4b-GFP and GFP-only vectors were transferred into rice protoplast cells. Left panels (GFP) show a protoplast expressing the GFP only vector and right panels (OsCCD4b-GFP) show a protoplast expressing the OsCCD4b-GFP vector. Upper panels show fluorescence microscope images and lower panels show light microscope images. Green fluorescence corresponding to the localization of the proteins was detected using fluorescence microscopy. Bars = 10 μm. (C) GC profiles of the products catalyzed by His-OsCCD4b protein (upper panel) and by E. coli protein (lower panel) using β-carotene as the substrate. Uk: unknown. (D) RT-PCR analysis of OsCCD4b and actin expression in the WT and OsCCD4b-overexpressing (OsCCD4b-ox) rice plants. (E,F) β-cyc and β-ionone levels in OsCCD4b-overexpressing rice plants. Values are expressed as means ± SE. Data were analyzed using the Tukey–Kramer test (n = 7 for WT and line 6; n = 8 for line 13). Values are the mean ± SE. Means accompanied by different letters were significantly different at p < 0.05.
Ijms 24 01704 g007
Table
Table 1. List of β-cyclocitral-responsive genes.
Table 1. List of β-cyclocitral-responsive genes.
WT + β-Cyc/WT
Accession NumberGene NameFold Change *q-ValueReference
Defense-related gene
(1) Os01g03360Bowman-Birk type bran trypsin inhibitor8.75<0.05This study
(2) Os09g36580Thaumatin-like protein 13.70<0.05This study
(3) Os12g25090Subtilisin/chymotrypsin inhibitor3.50<0.05This study
(4) Os01g71830Beta-1,3-glucanase3.44<0.05This study
(5) Os07g48060Peroxidase (OsPrx115)2.90<0.05This study
(6) Os01g03330Proteinase inhibitor I122.72<0.05This study
(7) Os05g31140Beta-glucanase2.55<0.05This study
(8) Os01g03340Bowman Birk trypsin inhibitor2.51<0.05This study
(9) Os12g38150Thaumatin2.25<0.05This study
(10) Os03g58940Lipid transfer protein2.01<0.05This study
ABA-responsive gene
(11) Os06g48300Protein phosphatase 2C family protein0.20<0.05[28]
(12) Os05g46480LEA-like protein0.22>0.05[28]
(13) Os05g04700Similar to ICT protein0.48>0.05[28]
(14) Os08g36440Abscisic acid and stress inducible (A22) gene0.50>0.05This study
(15) Os01g64000Similar to ABA response element binding factor0.54>0.05This study
(16) Os11g26760Dehydrin RAB 16C0.60>0.05[28]
(17) Os12g29400GRAM domain containing protein0.71>0.05[28]
(18) Os06g10880Similar to ABA-responsive element binding protein 10.71>0.05This study
(19) Os11g26750Dehydrin RAB 16D0.81>0.05[28]
(20) Os02g44870Dehydrin family protein0.97>0.05[28]
Fold changes (relative to WT plants) and false discovery rate (q-values) of defense-related and ABA-responsive genes on β-cyclocitral-treated rice plants from microarray analyses. * Values of fold change are means of 4 independent biological replications. The numbers in parentheses are the same as those in Figure 2 and Figure 3.
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Taniguchi, S.; Takeda, A.; Kiryu, M.; Gomi, K. Jasmonic Acid-Induced β-Cyclocitral Confers Resistance to Bacterial Blight and Negatively Affects Abscisic Acid Biosynthesis in Rice. Int. J. Mol. Sci. 2023, 24, 1704. https://doi.org/10.3390/ijms24021704

AMA Style

Taniguchi S, Takeda A, Kiryu M, Gomi K. Jasmonic Acid-Induced β-Cyclocitral Confers Resistance to Bacterial Blight and Negatively Affects Abscisic Acid Biosynthesis in Rice. International Journal of Molecular Sciences. 2023; 24(2):1704. https://doi.org/10.3390/ijms24021704

Chicago/Turabian Style

Taniguchi, Shiduku, Aya Takeda, Masaki Kiryu, and Kenji Gomi. 2023. "Jasmonic Acid-Induced β-Cyclocitral Confers Resistance to Bacterial Blight and Negatively Affects Abscisic Acid Biosynthesis in Rice" International Journal of Molecular Sciences 24, no. 2: 1704. https://doi.org/10.3390/ijms24021704

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop