Jasmonate biosynthesis enzyme allene oxide cyclase 2 mediates cold tolerance and pathogen resistance

Abstract

Allene oxide cyclase (AOC) is a key enzyme in the biosynthesis of jasmonic acid (JA), which is involved in plant growth and development as well as adaptation to environmental stresses. We identified the cold- and pathogen-responsive AOC2 gene from Medicago sativa subsp. falcata (MfAOC2) and its homolog MtAOC2 from Medicago truncatula. Heterologous expression of MfAOC2 in M. truncatula enhanced cold tolerance and resistance to the fungal pathogen Rhizoctonia solani, with greater accumulation of JA and higher transcript levels of JA downstream genes than in wild-type plants. In contrast, mutation of MtAOC2 reduced cold tolerance and pathogen resistance, with less accumulation of JA and lower transcript levels of JA downstream genes in the aoc2 mutant than in wild-type plants. The aoc2 phenotype and low levels of cold-responsive C-repeat-binding factor (CBF) transcripts could be rescued by expressing MfAOC2 in aoc2 plants or exogenous application of methyl jasmonate. Compared with wild-type plants, higher levels of CBF transcripts were observed in lines expressing MfAOC2 but lower levels of CBF transcripts were observed in the aoc2 mutant under cold conditions; superoxide dismutase, catalase, and ascorbate–peroxidase activities as well as proline concentrations were higher in MfAOC2-expressing lines but lower in the aoc2 mutant. These results suggest that expression of MfAOC2 or MtAOC2 promotes biosynthesis of JA, which positively regulates expression of CBF genes and antioxidant defense under cold conditions and expression of JA downstream genes after pathogen infection, leading to greater cold tolerance and pathogen resistance.

Introduction

Plants are often simultaneously subjected to a variety of biotic and abiotic stresses, which limit plant growth and development (Gong et al. 2020). Jasmonic acid (JA) is a phytohormone that regulates plant growth and development as well as biotic and abiotic stress responses. JA stimulates root development by directly suppressing the transcriptional function of ROOT HAIR DEFECTIVE 6 (RHD6) (Han et al. 2020). JA induces leaf senescence by activating expression of SENESCENCE-ASSOCIATED GENE 29 (SAG29) expression via myelocytomatosis protein 2 (MYC2) (Qi et al. 2015). Overexpression of allene oxide cyclase from Triticum aestivum (TaAOC1) in both bread wheat (T. aestivum) and Arabidopsis (Arabidopsis thaliana) enhances salt tolerance by regulating JA level (Zhao et al. 2014). JA is perceived by the receptor CORONATINE INSENSITIVE 1 (COI1), which interacts with JA zinc-finger inflorescence meristem (ZIM)-domain (JAZ) family proteins and induces the degradation of JAZs to derepress downstream transcription factors, resulting in rapid activation of JA-responsive genes (Thines et al. 2007). JA is also involved in regulation of cold tolerance, while blocking of JA biosynthesis or signaling results in hypersensitive response to freezing stress in Arabidopsis (Hu et al. 2013). Inducer of CBF Expression (ICE)1 and ICE2 activity is repressed through interacting with JAZ1 and JAZ4, which downregulates C-REPEAT BINDING FACTOR/DRE BINDING FACTOR1 (CBF/DREB1) pathway (Hu et al. 2013). MdJAZ1 and MdJAZ2 interact with Malus domestica B-box 37 protein (MdBBX37) to repress its transcriptional activity, while MdBBX37 binds to the promoters of MdCBF1 and MdCBF4 to activate their expression in apple (M. domestica) (An et al. 2021). HAN1 (“han” termed “chilling” in Chinese) catalyzes conversion of JA-Ile to the inactive form 12-hydroxy-JA-Ile (12OH-JA-Ile) and functions as a negative factor in regulation of JA-mediated chilling response in rice (Oryza sativa L.) (Mao et al. 2019). JA regulates cold tolerance in trifoliate orange (Poncirus trifoliata) through MYC2-BADH-like transcriptional regulatory module by modulating glycine betaine biosynthesis (Ming et al. 2021).

The roles of JA in plant immunity are well understood. JA-dependent defense responses are activated in response to herbivores or infection with necrotrophic pathogens which require host cell death to obtain nutrients (Glazebrook 2005). Perception of herbivore or necrotrophic pathogen attack promotes JA biosynthesis, while JA binds with the COI1-JAZ receptor to trigger degradation of JAZ repressor via the 26S proteasome, thereby derepressing MYC transcription factors for induced transcriptional reprogramming associated with plant defenses (Zhang et al. 2015). In addition, JA enhances transcription activity of ETHYLENE INSENSITIVE 3 (EIN3) and EIN3-LIKE 1 (EIL1) through the removal of JAZ proteins and further activates downstream defense genes, such as Ethylene Response Factor 1 (ERF1), OCTADECANOID-RESRESSIVE ARABIDOPSIS 59 (ORA59), and Plant Defensin 1.2 (PDF1.2), thereby protecting against necrotrophic pathogens (Zhu et al. 2011).

Jasmonate biosynthesis begins with peroxidization of α-linolenic acid (18:3) (α-LeA) to produce (13S)-hydroperoxyoctadecatrienoic acid (13-HPOT), catalyzed by 13-lipoxygenase (LOX). 13-HPOT is further converted to 12,13-(S)-epoxy-octadecatrienoic acid (12,13-EOT) and in turn (9S,13S)-12-oxo-phytodienoic acid (OPDA), catalyzed by allene oxide synthase (AOS) and allene oxide cyclase (AOC) sequentially. OPDA is subsequently reduced, catalyzed by OPDA reductase (OPR3), and converted to jasmonate via 3 cycles of β-oxidation (Hazman et al. 2015). Methyl jasmonate (MeJA) is the ester derivate of JA. AOC is the key enzyme in JA biosynthesis (Stenzel et al. 2012). Constitutive expression of the bread wheat AOC gene TaAOC1 lead to increased JA accumulation and enhanced salinity tolerance (Zhao et al. 2014). OsAOC transcript is induced by cold and drought (Du et al. 2013), while mutation of OsAOC leads to a phenotype susceptible to blast fungus Magnaporthe oryzae (Riemann et al. 2013). The AOC knockout mutants of rice are more susceptible to brown planthopper (BPH) attack compared with the wild-type plants, with the accumulation of attenuated defensive secondary metabolites (Xu et al. 2021). However, the strategy for improved cold and pathogen resistance in crops by JAs through modulation of AOC expression has not been reported.

Alfalfa (Medicago sativa L.) is a perennial high-quality leguminous forage with the large cultivation and the wide distribution in the world (Brummer 2004). Low temperature is the major abiotic stress to limit alfalfa production (Liu et al. 2022). For example, it leads to 33% yield loss due to cold temperature in Iran (Pourshirazi et al. 2022). The accumulated sugars or total nonstructure carbohydrate levels in roots during cold acclimation is associated with cold tolerance in alfalfa (Cunningham et al. 2001; Seppanen et al. 2018). Ca²⁺ signaling via calmodulin-like proteins (CMLs) is involved in regulation of cold tolerance in alfalfa and Medicago truncatula, a model leguminous plant (Sun et al. 2021; Yu et al. 2022). Soluble sugars such as sucrose, myo-inositol, galactinol, and raffinose are also associated with cold tolerance in M. sativa subsp. falcata (L.) (hereafter M. falcata) that is closely related to alfalfa with great cold tolerance (Tan et al. 2013; Zhuo et al. 2013). In addition, regulation of several cold-responsive (COR) genes from M. falcata, such as S-ADENOSYLMETHIONINE SYNTHETASE (MfSAMS1), ETHYLENE RESPONSE FACTOR (MfERF1), AUXIN INDUCED IN ROOT CULTURE 12 (MfAIR12), and COLD TOLERANCE LRR-RLK1 (CTRK1), on cold tolerance has been documented (Guo et al. 2014; Zhuo et al. 2018; Geng et al. 2021; Wang, Shi, et al. 2021). Apart from cold, alfalfa production and quality are largely reduced by diseases (Nutter et al. 2002; Zhang, Xia, et al. 2021). Rhizoctonia solani is 1 of the major pathogens associated with root rot in alfalfa (Anderson et al. 2013; Zhang, Yu, et al. 2021). The disease caused by the pathogen is hard to control due to its ability to overwinter as sclerotia or mycelia in the soil or in crop debris (Okubara et al. 2014; Ajayi-Oyetunde and Bradley 2018). The disease occurs when alfalfa is exposed to disastrous weather, such as abnormal coldness after spring comes, while the freezing injury causes easy invasion of pathogens for a serious disease. Given the regulation of JA in plant response to both biotic and abiotic stresses, it is hypothesized that cold tolerance and disease resistance in alfalfa should be improved by modulating AOC expression for promoted JA biosynthesis.

In our previous investigation, an upregulated AOC2 was harvested in a cDNA library of M. falcata responsive to cold (Pang et al. 2009), implying a potential role of MfAOC2 in cold tolerance of M. falcata. The objective of this study was to examine the role of JAs in regulation of cold tolerance and pathogen resistance through modulation of AOC expression. The expression of MfAOC2 from M. falcata and MtAOC2 from M. truncatula in response to cold and pathogen infection in combination with analysis of cold and pathogen resistance in overexpressing lines of MfAOC2 and the Tnt1 retrotransposon insertion mutant of MtAOC2 were investigated. The results validated that AOC2 confers cold tolerance and resistance to R. solani infection through JA signaling pathway.

Results

Freezing aggravates the infection of necrotrophic fungus R. solani to M. truncatula

The effect of freezing on infection of necrotrophic fungus R. solani was examined. Freezing treatment resulted in increased ion leakage and decreased survival rate in M. truncatula (Supplemental Fig. S1, A and B). Survival rate was further decreased after inoculation with R. solani, with lower level in the inoculated plants as compared with the noninoculated control (Supplemental Fig. S1, B and C). Likely, fresh weight of the survival plants was decreased in the freezing-treated plant, and it was further decreased by inoculation R. solani, with lower level in the inoculated plants than in the control plants (Supplemental Fig. S1D). The results indicated that freezing-treated plants were more sensitive to infection of pathogens compared with the control.

Molecular characterization and spatial expression of MfAOC2

To examine the function of AOC2, the coding sequence of MfAOC2 (OQ291228) was cloned from M. falcata. It has an open reading frame (ORF) of 759 bp and encodes a peptide of 253 amino acids with a molecular weight of 27.72 kDa and the isoelectric point (pI) at 9.72. Phylogenetic tree analysis showed that MfAOC2 was mostly close to MtAOC2 (MTR_5g053950) in M. truncatula among 6 MtAOC members (Supplemental Fig. S2A). An allene motif that is conserved in AOC family proteins was located at the C-terminal from 77 to 250 amino acids (Supplemental Fig. S2B).

The spatial expression analysis showed that MfAOC2 transcript could be detected in root, stem, leaf, and flower, with relative high level in root, stem, and leaf (Fig. 1A), while MtAOC2 transcript could be detected in root, stem, leaf, flower, and seed with higher level in root and stem (Fig. 1B). In addition, relative expression of MfAOC2 and MtAOC2 was higher than that of the other MtAOCs. Analysis of the other AOC genes in M. truncatula showed that MtAOC1, MtAOC3, MtAOC4, MtAOC5, and MtAOC6 transcripts were major expressed in root and seed (Supplemental Fig. S3, A to E).

Figure 1.

Relative spatiotemporal expression and transcript levels of MfAOC2 and MtAOC2 in response to cold treatment and R. solani inoculation. Total RNA was isolated from leaves, stems, roots, flowers, and seeds for examining the spatiotemporal expression of MfAOC2A) and MtAOC2B) using RT-qPCR, while transcript levels of MfAOC2C, E) and MtAOC2D, F) in leaves and hypocotyls in response to treatments with cold (5 °C) and inoculation of R. solani were analyzed, respectively. Means of 3 replicates and standard errors are presented; the same letter above the column indicates no significant difference at P < 0.05 using Duncan's test.

MfAOC2 and MtAOC2 regulated cold tolerance

The role of MfAOC2 and MtAOC2 in regulation of cold tolerance was investigated. Transcript levels of MfAOC2 and MtAOC2 in response to cold were measured. Both MfAOC2 and MtAOC2 transcripts were induced after 2 h of cold treatment, followed by decrease (Fig. 1, C and D). Transgenic M. truncatula plants overexpressing MfAOC2 were generated (Supplemental Fig. S4A), because transformation protocol in M. falcata was failed to be established in our laboratory. Three homozygous lines (OE1, OE6, and OE7) with 19 to 45 times increased MfAOC2 transcript level were selected for further investigations. Cold-acclimated (CA) plants had lower TEL₅₀ than nonacclimated (NA) ones, and more negative TEL₅₀ were observed in transgenic plants than in wild type (WT) under both NA and CA conditions (Fig. 2A). Similarly, survival rates were increased in CA plants compared with NA ones, and higher levels were observed in transgenic plants than in WT (Fig. 2, B and C). The results indicated that cold tolerance was increased by CA treatment, while overexpression of MfAOC2 resulted in increased cold tolerance in M. truncatula.

Figure 2.

Analysis of cold tolerance in transgenic M. truncatula overexpressing MfAOC2 in comparison with the wild type. Electrolyte leakage of excised leaves from transgenic plants was measured to calculate the temperature that resulted in 50% electrolyte leakage (TEL₅₀) A). Survival rates were measured after plants were treated by freezing at −5 °C (NA plants) or −7 °C (CA plants) for 6 h B). After 4 d of recovery at room temperature, the freeze-treated and control M. truncatula plants were photographed C). Means of 3 replicates and standard errors are presented; the same letter above the column indicates no significant difference at P < 0.05 using Duncan's test.

One homozygous mutant line of MtAOC2, aoc2 with Tnt1 insertion in the exon, was identified (Supplemental Fig. S4B). Compared to WT, MtAOC2 transcript was not detected in aoc2 mutant (Supplemental Fig. S4C). In addition, 3 complementing lines by expressing MfAOC2 in aoc2 (MfAOC2::aoc2) were generated (Supplemental Fig. S4D), and 1 line (MfAOC2::aoc2-1) was used for investigations. Compared to WT, aoc2 mutant had increased TEL₅₀ under both NA and CA conditions, while the complementing plants had similar TEL₅₀ with WT (Fig. 3A). Consistently, survival rate was decreased in aoc2 mutant compared with WT under both NA and CA conditions, which was restored by expressing MfAOC2 in complementing plants (Fig. 3, B and C). The results indicated that MtAOC2 mutation resulted in decreased cold tolerance, which could be rescued by expressing MfAOC2.

Figure 3.

Analysis of cold tolerance in aoc2 mutant and the complementing plants. Plants were exposed to 7 d of CA at 5 °C by placing in a growth chamber, while those growing at room temperature were used as NA control. The temperature resulting in 50% electrolyte leakage (TEL₅₀) A) and survival rate B) in aoc2 mutant and complementing plants (MfAOC2::aoc2) were measured after plants were subjected to freezing at −7 °C (CA plants) or −5 °C (NA plants) for 6 h. After 4 d of recovery at room temperature, the freezing-treated and control plants were photographed C). Means of 3 replicates and standard errors are presented; the same letter above the column indicates no significant difference at P < 0.05 using Duncan's test.

MfAOC2 mediated JA accumulation in response to cold stress

To understand the mechanisms involved in the altered cold tolerance in overexpressing lines and aoc2 mutant, JA concentrations were measured. Compared to WT, JAs including JA, MeJA, JA-ILE, JA-Val, and OPDA levels were lower in either overexpressing lines or the mutant under NA control condition, and the lowest level was observed in the mutant. JA, MeJA, JA-ILE, and JA-Val levels were increased in all genotypes of plants after cold treatment, with higher levels in overexpressing lines and lower levels in the mutant. In addition, JA level was higher than MeJA, JA-ILE, and JA-Val levels under both control and cold conditions (Fig. 4, A to D). The results indicated that MfAOC2 expression altered JA, MEJA, JA-ILE, and JA-Val accumulation.

Figure 4.

Analysis of JA concentrations in response to cold. JA A), MeJA B), JA-ILE C), and JA-Val D) in MfAOC2 overexpressing lines and aoc2 mutant were measured after plants were exposed for 7 d of CA at 5 °C or NA as control. Means of 3 replicates and standard errors are presented; the same letter above the column indicates no significant difference at P < 0.05 using Duncan's test.

COR CBF pathway was altered by AOC2 expression

Four CBF transcription factor genes and 2 JA-responsive genes in response to cold were examined. MtCBF1, MtCBF2, MtCBF2, MtCBF3, MtCBF4, MtJAZ, and MtMYC2 transcripts showed no substantial difference among genotypes under control condition. The transcripts of MtCBF1, MtCBF2, MtCBF3, MtCBF4, MtJAZ, and MtMYC2 were induced in WT and overexpressing lines after 2 h of cold treatment with higher levels in overexpressing lines than in WT, but the transcripts could not be induced in aoc2 mutant (Fig. 5, A to F). The low levels of MtCBF1, MtCBF2, MtCBF3, MtCBF4, MtJAZ, and MtMYC2 in aoc2 mutant could be rescued by expressing MfAOC2 that were shown in the complementing plants (Supplemental Fig. S5, A to F). The results indicated that MfAOC2 or MtAOC2 is essential for cold-induced transcripts of MtCBF1, MtCBF2, MtCBF3, MtCBF4, MtJAZ, and MtMYC2.

Figure 5.

Relative expression of cold- and JA-responsive genes in MfAOC2 overexpressing lines and aoc2 mutant. Relative expression levels of MtCBF1A), MtCBF2B), MtCBF3C), and MtCBF4D) were determined after 2 h of cold treatment, while those of MtJAZE) and MtMYC2F) were determined after 24 h of cold treatment. Means of 3 replicates and standard errors are presented; the same letter above the column indicates no significant difference at P < 0.05 using Duncan's test.

Exogenous application of MeJA rescued the defect of cold tolerance in aoc2 mutant

To examine whether the altered cold tolerance in aoc2 mutant is related to JA level, the TEL₅₀ in aoc2 and WT plants in response to exogenous application of MeJA was detected. The TEL₅₀ was higher in aoc2 than in WT under NA and CA conditions, but it was decreased after application of MeJA and reached to similar level with that in WT under NA and CA conditions (Supplemental Fig. S6, A and B), indicating that application of MeJA rescued the defect in cold tolerance in aoc2. The results indicated that MfAOC2 or MtAOC2 expression regulated cold tolerance through altering JA synthesis and accumulation. In addition, application of MeJA resulted in greatly increased MtCBF1 transcript in aoc2 mutant but not in WT under cold conditions, which led to the similar level of MtCBF1 between WT and aoc2 (Supplemental Fig. S7A). MeJA treatment resulted in greatly increased transcripts of MtCBF2, MtCBF3, MtCBF4, MtJAZ, and MtMYC2 in both WT and aoc2 mutant under cold stress, and the transcript levels of MtCBF2 and MtCBF4 in aoc2 reached to that in WT. Although the transcript levels of MtCBF3, MtJAZ, and MtMYC2 were lower in aoc2 than that in WT, the increase folds by MeJA in aoc2 were higher than that in WT (Supplemental Fig. S7, B to F). The results indicated that cold-induced transcripts of MtCBF1, MtCBF2, MtCBF3, MtCBF4, MtJAZ, and MtMYC2 were mediated by accumulation of JAs as a result of MfAOC2 or MtAOC2 expression.

Homeostasis of reactive oxygen species was affected by MfAOC2 expression

Activities of antioxidant enzymes including superoxide dismutase (SOD), catalase (CAT), and ascorbate-dependent peroxidase (APX) and proline concentration were measured in all genotypes in response to cold treatment. SOD, CAT, and APX activities and proline level showed no significant difference among genotypes under control condition. They were increased in all plants except CAT activity in aoc2 mutant after cold treatment, while higher levels were observed in overexpressing lines but lower levels were maintained in aoc2 mutant compared with WT (Fig. 6, A to D). H₂O₂ was not accumulated in all plants under control condition. Lower levels were accumulated in overexpressing lines, but higher level was in aoc2 mutant compared with WT after cold treatment (Fig. 6E).

Figure 6.

Antioxidant enzyme activities, proline concentration, and H₂O₂ level in response to cold. SOD A), CAT B), and APX C) activities, proline D), and H₂O₂E) level in leaves of MfAOC2 overexpressing lines and aoc2 mutant were measured after 7 d of CA at 5 °C. Bar, 1 mm. Means of 3 replicates and standard errors are presented; the same letter above the column indicates no significant difference at P < 0.05 using Duncan's test.

MfAOC2 and MtAOC2 expression regulated R. solani resistance in M. truncatula

To investigate the roles of MfAOC2 and MtAOC2 in disease resistance, the roots of M. falcata and M. truncatula were inoculated with fresh R. solani mycelia. Reverse transcription quantitative PCR (RT-qPCR) analysis showed that MfAOC2 transcript was significantly induced at 12-h postinfection (hpi) followed by decrease (Fig. 1E), while MtAOC2 transcript was induced at 36 hpi (Fig. 1F). To explain the above differential responses between 2 species, a 1,016 bp of promoter fragment of MfAOC2 was amplified using HiTail-PCR (OQ65629), while the MtAOC2 promoter sequence was searched on database. Based on analysis of 1,016 bp of the promoter sequence of MfAOC2 and MtAOC2, 6 EIRE cis-acting elements that respond to fungal infection were observed in the MfAOC2 promoter, while 3 EIRE and 2 W-box that respond to fungal infection were shown in the MtAOC2 promoter (Supplemental Fig. S8). The results indicated that difference in MfAOC2 and MtAOC2 transcripts in response to R. solani was associated with numbers and types of cis-acting elements in the promoter regions. Resistance to R. solani in MfAOC2 overexpressing lines and aoc2 mutant was evaluated after 14 d of infection. Compared to WT, higher survival rate and fresh weight of the surviving plants were observed in overexpressing lines, and lower levels were observed in aoc2 mutant (Fig. 7, A to C). In addition, the complementing plants had the similar survival rate and fresh weight with WT, which were higher than those in aoc2 mutant (Fig. 7, D to F). The results indicated that MfAOC2 and MtAOC2 positively regulated resistance to R. solani infection.

Figure 7.

Analysis of disease resistance in MfAOC2 overexpressing lines and aoc2 mutant as well as the complementing line (MfAOC2::aoc2-1) in comparison with the wild type. Survival rates were measured at 14 d after inoculation of R. solani in overexpressing lines and aoc2 mutant A) or in aoc2 mutant and the complementing line D) at the same time, followed by weighing the fresh weight of the survival plants B, C, E, F). Bar, 3 cm. Means of 3 replicates and standard errors are presented; the same letter above the column indicates no significant difference at P < 0.05 using Duncan's test.

Transcripts of the downstream genes in JA signaling associated with plant immunity were determined at 36 hpi. Compared to the noninoculated control, MtJAZ, MtMYC2, MtVSP2, and MtLOX2 transcripts were significantly upregulated after R. solani inoculation (Fig. 8). Higher transcript levels of MtMYC2, MtJAZ, MtVSP2, and MtLOX2 were observed in overexpressing lines as compared with WT, while lower levels were in aoc2 mutant (Fig. 8, A to D). In addition, the altered expression of above genes in aoc2 mutant was rescued in the complementing plants (Supplemental Fig. S9). The results indicated that MfAOC2 or MtAOC2 expression regulated expression of the downstream genes in JA signaling pathway after infection by R. solani.

Figure 8.

Analysis of transcript levels of JA-responsive genes and pathogenesis-related protein genes in MfAOC2 overexpressing lines and aoc2 plants in response to inoculation of R. solani. Transcript levels of MtJAZA), MtMYC2B), MtVSP2C), and MtLOX2D) were determined at 36 hpi of R. solani. Means of 3 replicates and standard errors are presented; the same letter above the column indicates no significant difference at P < 0.05 using Duncan's test.

Discussion

Plants are often simultaneously subjected to biotic and abiotic stresses, which limit largely crop productivity and quality (Gong et al. 2020). It is important to identify the genes involved in regulation on biotic and abiotic stress responses for engineering crops with multiple resistances. CA in the fall increases survival of alfalfa plants against the freeze during overwintering. The sudden decrease in temperature in the spring results in damages to alfalfa plants, while the freezing injury–caused wound makes it more susceptible to be infected by pathogen invasions. R. solani is a soil-borne fungal pathogen that persists in soil and plant debris for long periods in the absence of plant hosts. Once attached to the host, R. solani secretes enzymes to disrupt cell walls, invades into plant cells, and kills them (Abdoulaye et al. 2019). R. solani is 1 of the major pathogens that caused root rot in alfalfa (Zhang, Yu, et al. 2021). JA is involved in regulation of plant cold tolerance and defense against herbivores and necrotrophic pathogens (Cao et al. 2009; Jin et al. 2009; Hu et al. 2013; Campos et al. 2014). The freezing aggravated infection of necrotrophic fungus R. solani to M. truncatula was observed in the present study, and the role of MfAOC2 and MtAOC2 in regulation on cold tolerance and R. solani resistance was examined.

AOC is a key enzyme catalyzing JA biosynthesis, and its overexpression results in JA accumulation in plants under stressed conditions (Lu et al. 2014; Ruan et al. 2019). There are 4 AOCs in the genomes of Arabidopsis (Stenzel et al. 2012), and 6 AOCs were found in M. truncatula in the present study. MfAOC2 from M. falcata and its homolog MtAOC2 in M. truncatula were identified. Relative expression of MfAOC2 and MtAOC2 was higher than that of the other MtAOCs in different tissues, indicating that MfAOC2 and MtAOC2 might be more important than other homologs in JA biosynthesis. JA levels were lower in aoc2 mutant than in WT, confirming the role of MtAOC2 in JA biosynthesis. Unexpectedly, levels of JA and its derivatives were not increased but decreased in MfAOC2 overexpressing lines under control condition. JA biosynthesis is a complex and dynamic process, which takes place in 3 cellular compartments including plastids, cytosol, and peroxisomes (Heitz et al. 2016). There may be some unknown factors like substrate availability or transport of intermediates across subcellular membranes that influence JA biosynthesis and metabolism in MfAOC2 overexpressing lines under control condition. This case is consistent with that JA showed no substantial difference in transgenic Arabidopsis and WT under control condition, but JA was accumulated more in wound leaves in overexpressing lines than in WT under salt stress (Zhao et al. 2014). Likely, overexpressing AOC did not alter the basal level of JA under normal condition in Nicotiana tabacum and A. thaliana plants (Laudert et al. 2000). JAs were increased in all plants after cold treatment with higher levels in MfAOC2 overexpressing lines but lower in aoc2 mutant compared with WT, which is associated with the induced MfAOC2 and MtAOC2 transcripts by cold treatment. The results suggest that MfAOC2 and MtAOC2 confer the increased JA accumulation under low temperature.

Overexpression of TaAOC increased salt tolerance in transgenic wheat and Arabidopsis (Zhao et al. 2014), but the osaoc mutants were less sensitive to salt stress in rice (Hazman et al. 2015), indicating diversified functions of AOC in different plant species in response to salt stress. Overexpressing AOC from Camptotheca acuminata improves tolerance against low temperature and salt stress in tobacco (N. tabacum) plants, although JA concentration and the underlying mechanisms were not investigated (Pi et al. 2009). We documented that overexpression of MfAOC2 resulted in increased cold tolerance, while the mutant aoc2 showed a decreased cold tolerance, which was rescued by expressing MfAOC2, suggesting that MfAOC2 and MtAOC2 regulate cold tolerance positively. Exogenous application of MeJA rescued the decreased cold tolerance in mtaoc2 mutant, indicating that the increased cold tolerance in MfAOC2 overexpressing lines or decreased cold tolerance in aoc2 mutant resulted from the altered JA concentrations. Exogenous MeJA treatment improves cold tolerance in Arabidopsis, rice, and other plant species (Cao et al. 2009; Jin et al. 2009; Hu et al. 2013; Zhao et al. 2013; An et al. 2021).

The CBF cold response pathway plays a key role in regulation of plant cold tolerance by activating 10% to 20% of the COR genes during CA (Jia et al. 2016; Zhao et al. 2016). Several investigations reveal that JA involves in cold responses in plants by regulating CBF pathway (Zhao et al. 2013; Hu et al. 2013, 2017; An et al. 2021). MYC2, the component in JA signaling pathway, interacts with ICE1 in Arabidopsis and banana for activating CBF pathway (Hu et al. 2013; Zhao et al. 2013). MtCBF1, MtCBF2, MtCBF3, MtCBF4, MtJAZ, and MtMYC2 transcript levels were maintained higher in MfAOC2 overexpressing lines but lower in aoc2 mutant, and the low levels in aoc2 mutant could be recovered by exogenous application of MeJA or expression of MfAOC2 in the complementing plants. The results suggest that MfAOC2 and MtAOC2 expression improved cold tolerance via JA signaling that in turn mediated the COR CBF pathway.

It is important for plants to maintain reactive oxygen species (ROS) homeostasis under stress conditions as a result of upregulation of antioxidant defense system to scavenge the accumulated ROS (Foyer and Noctor 2013). SOD, CAT, and APX are the major antioxidant enzymes for scavenging of O₂⁻ and H₂O₂; the activities are usually increased in Medicago plants in response to cold (Guo et al. 2014; Zhang et al. 2019; Geng et al. 2021). Proline is the most common compatible osmolyte for osmotic regulation and ROS scavenging in plants (Szabados and Savouré 2010). SOD, CAT, and APX activities and proline level were increased after cold treatment, with higher levels in MfAOC2 overexpressing lines but lower levels in aoc2 mutant compared with WT. The altered antioxidant enzyme activity and proline level showed consistence with less accumulated H₂O₂ in overexpressing lines and more accumulated H₂O₂ in aoc2 mutant after cold treatment. The genes encoding antioxidant enzymes and P5CSs were not included in the altered downstream genes in cbf1 cbf2 cbf3 triple mutants under low temperature (Jia et al. 2016). The triple mutant data reveal that cold-induced antioxidant enzyme expression and proline accumulation are not regulated directly by CBF pathway (Jia et al. 2016; Zhao et al. 2016). Our results suggest that the altered antioxidant defense and proline accumulation are associated with the JA regulated cold tolerance as a result of MfACO2 and MtAOC2 expression.

It has been well known that JA signaling mediates plant resistance to pathogen infections (Campos et al. 2014; Ingle et al. 2015; Zhang et al. 2017). Disease resistance to necrotrophic fungus R. solani was increased in MfAOC2 overexpressing lines but decreased in aoc2 mutant. The defect in disease resistance in aoc2 mutant could be rescued by expressing MfAOC2. The results suggest that MfAOC2 and MtAOC2 regulated disease resistance positively. Likely, osaoc mutant with decreased JA levels is more susceptible to the blast fungus M. oryzae (Riemann et al. 2013). Transcript levels of MtJAZ, MtMYC2, MtVSP2, and MtLOX2, the downstream genes associated with disease resistance in JA signaling, were induced by inoculation with R. solani, with higher levels in MfAOC2 overexpressing lines but lower level in aoc2 mutant. Nevertheless, the altered disease resistance to R. solani infection by MfAOC2 and MtAOC2 expression was associated with JA signaling and the downstream genes.

Although it is well known that JA is involved in regulating plant tolerance against abiotic and biotic stresses (Cao et al. 2009; Jin et al. 2009; Hu et al. 2013; Campos et al. 2014), this investigation provided evidence that cold and disease resistance to R. solani infection could be improved simultaneously by modification of JA biosynthesis through overexpressing MfAOC2 or MtAOC2 in M. truncatula, indicating that MfAOC2 and MtAOC2 are probably valuable in crop improvements. Likely, overexpression of AOC results in enhanced salt tolerance in wheat and N. tabacum (Pi et al. 2009; Zhao et al. 2014). In addition, JAs were not accumulated in MfAOC2 overexpressing lines and several plant species under control condition, but accumulated under stressed conditions in M. truncatula and several plant species (Laudert et al. 2000; Zhao et al. 2014; Lu et al. 2014; Ruan et al. 2019). The above results are important for the application of MfAOC2 in crop improvements to avoid excessive accumulation of JA under control condition, because excessive accumulation of JA causes overactivation of defense response and leads to reduced plant growth (Yang et al. 2012; Li et al. 2022). It is suggested that MfAOC2 and MtAOC2 are valuable candidate genes for plant resistance breeding.

In summary, regulation of the cold- and pathogen-responsive MfAOC2 and its homolog MtAOC2 on cold tolerance and R. solani resistance was validated in the present study. MfAOC2 and MtAOC2 confer cold tolerance and pathogen resistance through promoted biosynthesis of JAs and JA signaling that regulates CBF expression and antioxidant defense. The results suggest that MfAOC2 and MtAOC2 are valuable candidate genes to be applied for improvements on crop cold tolerance and pathogen resistance in the future.

Materials and methods

Plant growth and treatments

The aoc2 mutant with Tnt1 retrotransposon insertion (NF7708) was obtained from Noble Research Institute (https://medicago-mutant.dasnr.okstate.edu/mutant/index.php). The Tnt1 insertion site was confirmed by transposon display PCR (Tadege et al. 2008; Cheng et al. 2014), and the homozygous line was harvested for investigations. M. sativa subsp. falcata (L.), M. truncatula cv. R108, aoc2 M. truncatula mutant, and the complementing plants were grown in 15-cm-diameter plastic pots containing a mixture of peat and perlite (3:1, v/v) in greenhouse with temperature about 25 °C under natural light. Six-week-old plants were placed in a growth chamber at 5 °C with 16-h light for cold treatment with a relative humidity of 75%, while those in growth chamber at room temperature were used as NA control. Total RNA from the third compound leaf was isolated at the time points as indicated in the figures, while the third compound leaf (0.1 g) from the top of each plant was sampled for physiological measurements after 7 d of cold treatment (CA). For exogenous application with MeJA, aoc2 and the wild-type (R108) were sprayed with 50 μM MeJA solution containing Tween-20 (0.01% v/v) or water as control. Total RNA was isolated from leaves of the plants treated at 5 °C for 2 h. Cold tolerance was measured 7 d after spraying MeJA.

Fungal culture conditions and inoculation

R. solani previously isolated from root rot alfalfa in the field was stored in the laboratory at 10 °C. R. solani cultures were inoculated onto potato dextrose agar (PDA) and allowed to grow at 25 °C for 5 d. The medium with mycelium was chopped and mixed with sterilized vermiculite with 1 dish of medium in each pot. The germinated seeds were placed in pots containing the above vermiculite, and the seedlings were grown in a growth chamber at 25 °C with 60% humidity and a photoperiod of 16-h/8-h light/dark. Ten seedlings were maintained in each pot with 3 replicates. The surviving plants were counted, and fresh weight of seedlings was weighed after 15 d of cultivation.

For inoculation experiment, the seeds were sterilized for 30 min using 6.25% (v/v) NaClO solution, followed 3 washes of sterilized deionized water, and placed on 1/2 MS medium. Seedlings were grown for 10 d in a growth room at 25 °C with a photoperiod of 16-h/8-h light/dark. Taking a bit of R. solani culture to PDA plates through an inoculating needle, and grown on PDA at 25 °C for 5 d. The medium fulled with mycelium was then used for the inoculation. Hypocotyls of seedlings were inoculated on 1.5-cm-diameter medium blocks containing 5-d-old pathogen mycelia. The inoculation site was wrapped in absorbent paper, which was dampened with water, and 5 inoculated seedlings were wrapped together as a replicate. The inoculated seedlings were maintained in a greenhouse at 25 °C under dark condition. Total RNA was isolated from hypocotyls of seedlings surrounded by hyphae for 12, 24, and 36 h.

Cloning of MfAOC2 and its promoter

Total RNA was extracted from leaves using the RNAprep pure Plant Kit (Tiangen, Beijing, China) according to the manufacturer's instruction. One microgram of total RNA was used to synthesize cDNA, using the Prime Script RT reagent Kit with gDNA Eraser (Takara, Japan), and cDNA concentration was measured using NanoDrop 1000 (Thermo Fisher Scientific Inc., Waltham, MA, USA). The cDNA was diluted and used as template for cloning the ORF of MfAOC2 in a 100 μL of reaction solution using the primers Y1400 and Y1401. The primers were designed based on assembly of EST sequences of AOC2 from GenBank using SeqMan (DNASTAR Inc., Madison, WI, USA). For cloning of MfAOC2 promote, genomic DNA was isolated from 1-g leaves of M. falcata using hexadecyltrimethylammonium bromide (CTAB). The diluted DNA was used as template to amply the promoter region of MfAOC2 using the method of HiTail-PCR (Liu and Chen 2007). Three rounds of PCR were conducted using the universal primers LAD1-4 and AC1 in combination with 3 HiTail-PCR reverse primers R1, R2, and R3 that were designed at the 5′ end of ORF of MfAOC2. A 1,016 bp of MfAOC2 promoter fragment was collected. The primers used for RT-PCR and HiTail-PCR are listed in Supplemental Table S1.

Analysis of MfAOC2 and MfAOC2 promoter sequence

The phylogenetic relationship of MfAOC2 with AOCs in Arabidopsis and M. truncatula was analyzed using MEGA 7.0 software (https://www.megasoftware.net/) and the neighbor-joining method with 1,000 bootstrap replicates. Analysis of MfAOC2 sequence was performed using DNAMAN software. The cis-acting elements in 1,016 bp of promoter region MfAOC2 (OQ656292) and MtAOC2 such as W-box (TGAC), EIRE (GTCG), G-box (CACGTT), and ABRE (ACGTG) were predicted by online analysis tool (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/).

Identification of aoc2 mutant

One Tnt1 retrotransposon insertion mutant line (NF7708) was ordered from the Noble Research Institute (https://medicago-mutant.dasnr.okstate.edu/mutant/index.php). The Tnt1 insertion sites were confirmed by retrotransposon display PCR (Tadege et al. 2008), using primers Y010 (5′-CTTGGTGAAAAATAAAAATGTC-3′) and Y1355 (5′-GGTAAAGAGGCATGAGGCTCAGT-3′) for NF7708 as the forward and reverse primer of Tnt1. MtAOC2 expression in homozygous lines was determined using RT-PCR that was conducted in a 20 μL reaction mixture containing the first-strand cDNA as the template, deoxyribonucleotide triphosphate (dNTP), Taq DNA polymerase, and 2 primers Y1576 and Y1577. Amplification of ACTIN was used as an internal control using primers Y403 and Y404 (Supplemental Table S1).

RT-qPCR

For RT-qPCR, the diluted cDNA was used as a template in a 10 μL PCR mixture, containing 15 ng of cDNA, 200 nM each of forward and reverse primers, and 5 μL SYBR Premix Ex Taq (Takara Bio Inc.). RT-qPCR was performed using a SYBR Green PCR Master Mix kit (Takara, Kusatsu, Japan). A negative control without cDNA template was always included, and parallel reaction to amplify ACTIN was used to normalize the amount of template. The primers used for RT-qPCR were designed using the software tool BEACON DESIGNER (Premier Biosoft International, Palo Alto, CA) and listed in Supplemental Table S2. The primer specificity was validated by melting profiles, showing a single product-specific melting temperature. All PCR efficiencies were above 95%. The relative expression was calculated by 2^−ΔΔCt.

Generation of transgenic plants

The coding sequence of MfAOC2 was cloned to the pCAMBIA3301 binary expression vector under the control of CaMV 35S promoter (35S::MfAOC2). The CaMV 35S promoter in the above vector was replaced by a 1,016-bp promoter of MfAOC2 (P_MfAOC2) to construct the complementing vector (P_MfAOC2::MfAOC2). M. truncatula cv. R108 and the mutant aoc2 were transformed using Agrobacterium tumefaciens strain EHA105 harboring 35S::MfAOC2 and P_MfAOC2::MfAOC2, respectively, to generate transgenic plants overexpressing MfAOC2 (35S::MfAOC2) and complementing plants (P_MfAOC2::MfAOC2) as previously described (Sun et al. 2021). Homozygous lines were harvested after selection of the T₁ and T₂ plants by resistance to Basta (10 mg L⁻¹) and used for measurements.

Freezing tolerance assay

Freezing tolerance was evaluated by survival rate and the temperature resulted in 50% ion leakage (TEL₅₀) as described previously (Guo et al. 2014; Yu et al. 2022). Five-week-old Medicago plants were placed in a growth chamber at 5 °C for 7 d of CA or at room temperature as NA control. The plants were subsequently subjected to freezing treatment by decreasing temperature at a rate of 1 °C h⁻¹ from 5 to −5 °C (for NA plants) and −7 °C (for CA plants) and were maintained for 1 h at the final temperature. The plants were then moved to a freezer and maintained overnight at 4 °C followed by placing in a greenhouse at room temperature for 4 d of recovery. Surviving plants were counted for calculating survival rate. For measurement of TEL₅₀, leaflets were placed in glass tubes preincubated on ice and maintained for 1 h. After ice was added on the top leaflets, the tubes were placed on a programmable freezer (model: Polystat cc1 & k6, Huber Unit, Offenburg, Germany) and were allowed to equilibrate for an additional hour at 0 °C. The temperature was decreased at a rate of 2 °C h⁻¹ to various freezing temperatures with holding for 1 h at each temperature. Tubes were then moved to a freezer and recovered overnight, followed by addition of 15 mL of deionized water in each tube. After shaking for 4 h, ion leakage was determined and calculated as previously described. TEL₅₀ was calculated using a fitted mode plot based on the above ion leakage data. At least 3 independent experiments were done and each experiment was performed with 3 technical replicates.

Determination of jasmonate concentrations

Fifty-day-old plants were placed in a growth chamber at 4 °C for 24 h or at room temperature as control. JA concentrations were measured using ultra-high-performance liquid chromatography linked with high-resolution mass spectrometry (LC–MS/MS), as described by Šimura et al. (2018). Briefly, 50 mg of fresh leaves was homogenized in liquid nitrogen using a TissueLyser II (Thermo Fisher Scientific), followed by extraction in 1 mL of methanol/water/formic acid (15:4:1, v/v/v) (1 mL) and purification on a nonselective reversed-phase solid-phase extraction (RP-SPE) using an Oasis HLB cartridge (Waters). JAs were eluted using 1 mL of 30% (v/v) acetonitrile and dried in vacuum concentrator, followed by dissolving in 100 μL of 30% (v/v) acetonitrile and transferring to insert-equipped vials. The purified extracts were analyzed using an UPLC–ESI–MS/MS system (UPLC, ExionLC AD, https://sciex.com.cn/; MS, Applied Biosystems 6500 Triple Quadrupole, https://sciex.com.cn/). The mobile UPLC phase consisted of binary gradients of acetonitrile containing 0.01% (v/v) formic acid and 0.01% (v/v) aqueous formic acid, with a flow rate of 0.5 mL min⁻¹.

Measurements of antioxidant enzyme activity and proline concentration

Fresh leaves (0.1 g) were homogenized in 1 mL of 50 mM phosphate buffer (pH 7.8) for extraction of SOD and CAT or 50 mM phosphate buffer (pH 7.0) containing 1 mM ascorbic acid and 1 mM EDTA for extraction of APX. After centrifugation at 12,000 × g for 15 min at 4 °C, the supernatants were collected for measurements of enzyme activity and protein concentration using Coomassie Brilliant Blue G-250 as described previously (Zhuo et al. 2018). One unit of SOD activity was defined as the amount of enzyme required for 50% inhibition of photochemical reduction of ρ-nitro blue tetrazolium chloride (NBT), while 1 unit of CAT or APX was defined as the amount of enzyme required for catalyzing the conversion of 1 μmol H₂O₂ (extinction coefficient 39.4 mM ⁻¹ cm⁻¹) or ascorbic acid (extinction coefficient 2.8 mM ⁻¹ 1 cm⁻¹) within 1 min. For determination of proline concentration, fresh leaves (0.1 g) were ground with pestle in 5 mL of 3% (v/v) sulfosalicylic acid. The homogenates were centrifuged at 15,000 × g for 10 min, and the supernatants were recovered for determination of proline as described previously using acid ninhydrin (Zhuo et al. 2018). Absorbance at 520 nm was measured, and proline concentration was calculated by comparing with a standard curve.

Statistical analysis

The physiological measurements were repeated 3 times from different plant samples. All data were subjected to analysis of variances according to the model for completely randomized design using an SPSS program (SPSS Inc., Chicago, IL). Differences among means of treatments and plant lines were evaluated using Duncan's test at 0.05 probability level.

Accession numbers

Sequence data from this article can be found in the GenBank/EMBL data libraries under accession number OQ291228.

Author contributions

L.Y., Q.S., B.G., J.S., Y.S., and Q.Y. performed the experiments; L.Y., Q.S., and H.Z. analyzed the experimental results; L.Y., Q.S., and B.Y. wrote the manuscript; Z.G. conceived the research; Z.G. and B.Y. designed the experiments.

Supplemental data

The following materials are available in the online version of this article.

Supplemental Figure S1. Analysis of disease resistance of M. truncatula in response to freezing treatment.

Supplemental Figure S2. Phylogenetic tree of MfAOC2 with 6 MtAOCs and multiple sequence alignment of MfAOC2 and MtAOC2.

Supplemental Figure S3. Analysis of relative spatiotemporal expression of MtAOC transcripts.

Supplemental Figure S4. Analysis of transgenic M. truncatula lines heterologously expressing MfAOC2, the aoc2 mutant, and complementing lines.

Supplemental Figure S5. Relative expression of cold- and jasmonate-responsive genes in the aoc2 mutant and the complementing plants in response to cold.

Supplemental Figure S6. Effect of exogenous application of MeJA on cold tolerance in the aoc2 mutant.

Supplemental Figure S7. Effect of exogenous application of MeJA on transcript levels of cold- and JA-responsive genes in the aoc2 mutant in response to cold.

Supplemental Figure S8. Elements in the promoters of MtAOC2 and MfAOC2 predicted using an online tool (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/).

Supplemental Figure S9. Relative expression of jasmonate-responsive genes in the aoc2 mutant and complemented plants in response to R. solani inoculation.

Funding

This work was supported by the National Natural Science Foundation of China (grant number 31971766).

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Author notes

Lei Yang and Qiguo Sun contributed equally.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (https://academic.oup.com/plphys/pages/General-Instructions) is Zhenfei Guo.

Conflict of interest statement. The authors declare that they have no conflict of interest.