Methyl Jasmonate Elicits Distinctive Hydrolyzable Tannin, Favonoid, And Phyto‑oxylipin Responses in Pomegranate (Punica Granatum L.) Leaves Part 1

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Abstract: Methyl jasmonate (MeJA) produced in plants can mediate their response to environmental stresses. Exogenous application of MeJA has also been shown to activate signaling pathways and induce phytoalexin accumulation in many plant species. To understand how pomegranate plants respond biochemically to environmental stresses, metabolite analysis was conducted in pomegranate leaves subjected to MeJA application and revealed unique changes in hydrolyzable tannins, flavonoids, and Phyto-oxylipins. Additionally, transcriptome and real-time qPCR analyses of mock- and MeJA-treated pomegranate leaves identified differentially expressed metabolic genes and transcription factors that are potentially involved in the control of hydrolyzable tannin, flavonoid, and Phyto-oxylipin pathways. Molecular, biochemical, and bioinformatic characterization of the only lipoxygenase with sustained, MeJA-induced expression showed that it is capable of oxidizing polyunsaturated fatty acids, though not located in the subcellular compartment where non-jasmonate (non-JA) Phyto-oxylipins were produced. These results collectively suggested that while the broad suppression of flavonoids and anthocyanins is at least partially controlled at the transcriptional level, the induced biosynthesis of non-JA Phyto-oxylipins is likely not regulated transcriptionally. Overall, a better understanding of how pomegranate leaves respond to environmental stresses will not only promote plant health and productivity but also have an impact on human health as fruits produced by pomegranate plants are a rich source of nutritional compounds.

Keywords: Methyl jasmonate. Flavonoid·Anthocyanin· Fatty acid.Phyto-oxylipin·Lipoxygenase

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Introduction

When wounded or attacked by herbivores and pathogens, plants produce and emit methyl jasmonate(MeJA), which is perceived by non-wounded plant tissues and neighboring plants to activate defense response(Cheong and Choi 2003). Additionally, exogenous application of MeJA to a plant has been shown to elicit signaling pathways as well as the production of pathogenesis-related proteins and defense chemicals known as phytoalexins. Phenolic phytoalexins, e.g. flavonoids and anthocyanins, have exhibited increased accumulation in response to MeJA treatment in different plants, such as Arabidopsis thaliana, grape(Vitis vinifera), banana(Musa acuminate), apple(Malus Domestica), and red raspberry(Rubus idaeus)(Pandey et al.2016; Portu et al.2015; De Geyteret al.2012; Shafiq et al.2011; Flo-res and Ruiz del Castillo 2014). The biosynthesis of flavonoids and anthocyanins begins with the formation of naringenin chalcone from coumaroyl CoA and three molecules of malonyl CoA catalyzed by chalcone synthase (CHS)and the subsequent isomerization of naringenin chalcone to naringenin by chalcone isomerase(CHI). Naringenin is then used to generate the core flavonoid and anthocyanin skeletons, which are further modified with glycosyl, methyl, hydroxyl, and prenyl functional groups to give rise to diverse structures and functions(Tian et al. 2008).

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In addition to phenolic phytoalexins, the production of Phyto-oxylipins upon MeJA induction has also been reported in a few plant species (Deboever et al.2020). Phyto-oxylipins are oxygenated fatty acids and derivatives that play a role in plant growth, development, stress response, and innate immunity(Wasternack and Feussner 2018). The initial step of Phyto-oxylipin biosynthesis involves oxidation of polyunsaturated fatty acids(PUFAs) to fatty acid hydroperoxides (HPOs)by lipoxygenases(LOXs)(Andreou and Feussner 2009). Plant LOXs are grouped into two subfamilies according to their protein sequences; type ILOXs are highly homologous(>75% similarity)and do not contain a signal peptide, whereas type II LOXs possess an overall low sequence similarity(<35%)but all contain a chloroplast target peptide (Feussner and Wasternack 2002). Plant LOXs can also be classified based on enzymatic activities;9-LOXs and 13-LOXs target the C-9 and C-13 position of the fatty acid substrate, respectively (Feussner and Wasternack 2002). HPOs generated by LOXs can be further transformed to various phyto-oxylipins, such as hydroxy fatty acids by reductases, keto fatty acids by LOXs, epoxy fatty acids by per-oxygenases(PXGs), and dihydroxy fatty acids by LOXs or α-dioxygenase α-DOXs). Notably,13-hydroperoxy-linolenic acid(an HPO)produced from linolenic acid by 13-LOX can initiate a series of reactions to form jasmonic acid(JA), MeJA, and the bioactive JA-isoleucine conjugate.

Pomegranate (Punica granatum L.)is a specialty horticultural crop valued for the abundant phenolic compounds in its fruit, such as flavonoids, anthocyanins, and hydrolyzable tannins (HTs)that are derived from an intermediate of the shikimate pathway (Ono et al.2016). Many studies have thus far focused on the role of pomegranate phenolics in alleviating stresses and illness in humans(Wu and Tian 2017).In contrast, little is known about the function of phenolics and other phytochemicals in defending pomegranate against abiotic and biotic factors(e.g. wounding, pathogens, MeJA induction) in leaves and fruits. Two recent reports evaluated pre-harvest MeJA treatment on the postharvest quality of pomegranate fruits(Koushesh Saba and Zarei 2019; Garcia-Pastor et al.2020). Total anthocyanins, flavonoids, and phenolics of MeJA-treated fruits, but not leaves, were analyzed collectively using a spectrophotometer. One of the studies also analyzed individual anthocyanins using mass spectrometry(Garcia-Pastor et al.2020). However, it remains unclear how pomegranate plant tissues, either leaves or fruits, respond to environmental stresses prior to the fruit set.

To begin dissecting interactions between pomegranate plants and environmental factors, we investigated the metabolic response of pomegranate leaves to exogenous MeJA application using high-performance liquid chromatography(HPLC)and liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). Unique changes in HTs, flavonoids, and anthocyanins as well as alterations in lipids, fatty acids, and Phyto-oxylipins were observed in pomegranate leaves treated with MeJA. Comparative transcriptome analysis, validated by real-time qPCR analysis, revealed that structural and/or regulatory genes involved in HT, flavonoid, anthocyanin, and Phyto-oxylipin metabolism were differentially expressed in mock- and MeJA-treated pomegranate leaves. The only LOX gene that exhibited a sustained upregulated expression in MeJA-treated leaves was subjected to further molecular, biochemical, and phylogenetic characterization.

Materials and methods

Chemicals

The β-glucogallin and pentagalloylglucose standards were purchased from Shanghai Yuanye Bio-Technology Co. Ltd (Shanghai, China). Chemicals used in the LOX assay were obtained from the following vendors: 3-(dimethylamino)benzoic acid (DMAB)(Adamas Reagent, Co., Ltd., Shanghai, China), linoleic acid(Sigma-Aldrich, St.Louis, MO, USA),3-methyl-2-benzothiazolinone(MBTH) and hemoglobin (Sangon Biotech Co., Ltd., Shanghai, China).

Plant materials

Pomegranate fruits and seeds(cv. Wonderful) were generously provided by the Panzhihua Academy of Agricultural and Forest Sciences and identified by Dr. Binjie Ge at Shanghai Chenshan Botanical Garden. A voucher specimen (No. CSHO173966) was deposited at the herbarium of Shanghai Chenshan Botanical Garden, Shanghai, China. Pomegranate seedlings were grown in a temperature-controlled growth room for6weeks at 25℃℃ and 16 h light/8h dark. The MeJA concentration for spray application to plant tissues reportedly ranges from 100 to 250 μM（Ku et al.2014; Hickman et al.2017). Different concentrations of MeJA were initially applied to pomegranate leaves, of which 200μM MeJA led to a discernable metabolic response in the preliminary analysis and was used for the metabolite and gene expression analyses described in this study. Prior to the MeJA treatment, half of the pomegranate plants were moved to another growth room with similar conditions. While plants in one growth room were sprayed with 200 μM MeJA， those in the other growth room were sprayed with water(ie. mock control).At 2-h,3-h,6-h,12-h,24-h, 30-h,36-h,48-h,and72-h after the treatment,leaves from3 to 5 mock- or MeJA-treated plants were pooled,which constitute one biological replicate. Three biological replicates were collected for the mock- and MeJA-treatment experiments; each biological replicate was divided into aliquots for metabolite profiling and gene expression analyses.

Metabolite profiling analysis

Pomegranate leaves were lyophilized, weighed, and ground into a fine powder using zirconia beads in a bead beater(Mixer Mill MM 400, Retsch GmbH, Haan, Germany)for the 90s at 30 Hz. For HPLC analysis, the leaf sample was extracted in 70% methanol for 60 min under sonication and centrifuged at 13,000 rpm for 10 min. The supernatant was transferred to an HPLC vial， of which 30μL was injected onto a reverse phase HPLC(Agilent 1200, Agilent Technologies, Santa Clara, CA, USA) and analyzed as previously described(Wil-son et al.2019). Metabolites were detected by UV absorption at 254 nm,280 nm,320 nm, and 360 nm. Standard calibration curves of β-glucogallin and pentagalloylglucose were constructed; they were used for converting the areas of peaks that match the retention times and absorption spectra of β-glucogallin and pentagalloylglucose to the respective concentrations.

For LC-ESI-MS/MS analysis, the homogenized leaf sample(100 mg) was extracted in 1 mL of 70% methanol at 4℃Covernight.On the following day, the methanolic extract was centrifuged at 10,000×g for 10 min and the superna-tant was passed through a CNWBOND Carbon-GCB SPE cartridge（ANPEL， Shanghai， China） and a 0.22-μm syringe filter(ANPEL) prior to metabolite analysis.

The extract （2 μL） was analyzed using LC-ESI-MS/MS (Shim-pack UFLC, Shimadzu, Kyoto, Japan; QTRAP6500, Applied Biosystems, Foster City, CA, USA)and a reverse phase C1s column(ACQUITY UPLC HSS T3,1.8 βm, 2.1 mm×100 mm, Waters, Milford, MA, USA).Metabolites were eluted using solvents(A)water containing 0.04%acetic acid,and(B)acetonitrile containing0.04% acetic acid at a gradient of 0-11 min,95-5%A;11-12 min,5% A;12-12.1 min,5-95%A;12.1-15min,95%A.The flow rate was maintained at 0.4 mL min-1. Linear ion trap (LIT) and triple quadrupole(QQQ)MS scans were acquired in positive-and negative-ion modes. The turbo spray ion source was operated at 500℃C with an ionization voltage of 5500 V. The ion source gas I, gas I, and curtain gas were set at 55 psi,60 psi, and 25 psi, respectively. The collision gas (nitro-gen) was set at 5 psi. For the MRM analysis, declustering potential (DP)and collision energy(CE) were optimized for each precursor-product ion transition.

For metabolite identification, the LC-ESI-MS/MS data were compared with an MS2T library of commercial standards and previously identified compounds published in mass spectral databases (when commercial standards are not available)(Chen et al.2013). Pomegranate metabolites were annotated based on the retention times, accumulate m/z values and fragmentation patterns that match the MS2T library entries(Chen et al.2013). Metabolite quantification was performed using the MRM method as described by Dresen et al. (Dresen et al.2010). The biological replicates of each treatment (mock or MeJA)were averaged for comparative metabolite analysis. The Variable Importance in Projection(VIP) value was obtained from the Orthogonal Partial Least Squares Discriminant Analysis(OPLS-DA)model. Metabolites with ILog, FCl>1, and VIP>=1 were considered significantly changed.

Transcriptome analysis

Total RNA was extracted from pomegranate leaves using TRIzol reagent (Invitrogen, Carlsbad, CA, USA)and quantified using Nanodrop2000(ThermoFisher Scientific, Waltham, MA, USA). The integrity of the RNA samples was verified through separation on an agarose gel(no visible degradation)and determination of the O.D.20/28o ratio (between 1.8 and 2.2)using Nanodrop2000. Enrichment of mRNA from total RNA was carried out using the oligo (dT) magnetic beads(Invitrogen).mRNA-Seq libraries were constructed using the Truseq RNA library preparation kit (Illumina, San Diego, CA, USA). Transcriptome analysis was conducted on Illumina HiSeq4000 and 55-60 million of 150-bp paired-end reads(PE150) were obtained for each sample library.

The raw sequence data were processed by removing the adaptor sequences as well as short(<50 bp), low quality (Q<30), and poly(>10%)reads using SeqPrep (https://github.com/jstjohn/seqprep)and Sickle (HTTPS:GitHub. com/Joshi/sickle).Over 95% of the cleaned reads were uniquely mapped to the reference pomegranate genome for each sample library using HISAT2(Kim et al.2015) and the mapped reads were assembled using StringTie(Pertea et al.2015). The assembled transcriptome sequences were annotated using NCBI NR(ftp://ftp.ncbi.nlm.nih.gov/blast/db/). Transcript abundance was determined by the RNA-Seq by Expectation-Maximization(RSEM) method and expressed as Transcripts Per Kilobase Million(TPM)(Li and Dewey 2011). Differential gene expression analysis was performed using DESeq2 (Love et al.2014), with a thresh-old of the log, FCl>1, and adjusted P value<0.05.

Real-time qPCR analysis

Total RNA was extracted from mock- and MeJA-treated pomegranate leaves using the RNAprep Pure Plant Kit (Tiangen Biotech Co., Ltd., Beijing, China). Reverse transcription (RT) was performed using total RNA and the PrimeScriptTM RT Reagent Kit(Takara Bio Inc., Kusatsu, Japan).Quantitative PCR(qPCR) was carried out using the TB GreenTM Premix Ex Taq TM(Tli RNaseH Plus)kit (Takara) and a StepOnePlus Real-Time PCR System(Ther-moFisher Scientific). Melting curve analysis was conducted and showed a single amplification product for each primer pair. For the RT-qPCR analysis, three biological replicates and each with three technical replicates were examined for mock- and MeJA-treated samples. Gene expression was analyzed using the comparative C,(△AC) method (Livak and Schmittgen 2001), and significance levels were determined using a two-tailed Student's t-test. The primer sequences for the real-time qPCR analysis and the amplification efficiencies of the primer pairs are shown in Table S1.

Expression and purification of recombinant proteins and enzyme assays

The open reading frame of Pgr025417 (encoding a putative LOX) was synthesized for optimal codon usage in E. coli(Genewiz, Suzhou, China)and cloned in pET28a.The recombinant plasmid was transformed into E. coli BL21 (DE3) cells. A 5-mL Luria Bertani (LB)culture with 50μg mL-Ikanamycin was started from a single colony and incubated overnight with shaking at 37C. The overnight culture was used to inoculate a 100-mL LB medium with 50ug mL-'kanamycin and allowed to grow to an O.D.600 of 0.5. Isopropyl-β-D-thiogalactoside(IPTG) was then added to a final concentration of 0.1 mM for induction of protein expression. After incubation with shaking at 16℃℃ for 18 h, the cells were harvested by centrifugation. The cell pellets were resuspended in the lysis buffer(50 mM NaH, PO, pH 7.4,300 mM NaCl,10 mM imidazole) and homogenized using a cell disruptor(Constant Systems Ltd, Northants, UK).His-tagged proteins were purified using Ni-NTA beads (ThermoFisher Scientific)with the wash buffer(50 mM NaH,PO, pH 7.4,300 mM NaCl,25 mM imidazole)and the elution buffer(50 mM NaH,PO,pH 7.4,300 mM NaCl, 500 mM imidazole).The purified proteins were separated on a 10% SDS-PAGE gel for visualization of protein purity. The concentration of the purified proteins was determined using the Bradford assay (Bradford 1976).

For the LOX assay, linoleic acid was used as substrate in a two-step, colorimetric method with slight modifications （Anthon and Barrett 2008）. The 500-μL reaction mixture，including 50 mM Na-phosphate, pH 6, 10 mM DMAB, 0.5 mM linoleic acid,and various amounts of purified recombinant proteins(1.5 mg mL-), was incubated at 25 ℃ for 10 min.A second solution （500 μL）containing 0.2 mM MBTH and 0.1 mg mL-'hemoglobin was added to the reaction mixture, which was incubated for an additional5min. The reaction was terminated by adding 500μL 1%（w/v）sodium lauryl sulfate. Light absorption at 598 nm was determined.

Subcellular localization and phylogenetic analyses

A search of the annotated pomegranate genome(Qin et al. 2017)identified 1l putative full-length LOXs(786 aa to 970 aa),including Pgr025413,Pgr020032,Pgr025418,Pgr025417, PgrO18982, PgrO18980, PgrO16852(full-length sequence in GenBank XP_031395793),Pgr009839, Pgr008562, Pgr025678, and PgrO13780. Subcellular localization and cleavage sites of signal peptides for the pomegranate LOXs were predicted using TargetP 2.0(http://www.cbs.dtu.dk/servi ces/TargetP/)(Almagro Armenteros et al.2019).

TF-binding site analysis

To predict the binding sites of TFs, 1000 bp upstream of the ATG start codon of the target genes were obtained from GenBank and searched against the Eucalyptus Grandis TFs in PlantRegMap (version 5)(Tian et al.2020).The threshold value for binding site identification was set at P≤le-4.

Statistical analysis

Statistical analysis for the metabolite quantification, transcriptome, and real-time qPCR data is described in the respective sections.

Results

MeJA modulates cell signaling and metabolic pathways in pomegranate leaves

To understand the genome-wide transcriptional response of pomegranate to MeJA elicitation, transcriptomes of pomegranate leaves at 2-h,6-h,24-h, and 72-h after MeJA or mock treatment (each with three biological replicates)were analyzed (Fig. S1). Approximately 55 million raw sequence reads(2×150 bp paired-end) were obtained for each transcriptome with GC-content around 52% and Q30 values ranging from 91.6 to 95%(Table S2). For all transcriptomes, more than 96% of the cleaned sequence reads were mapped to the reference pomegranate genome (Qin et al.2017)(Table S3).A majority of the assembled transcripts were less than 1000 bp(34.3%),1000bp—2000 bp (32.9%),or 2000 bp—3000 bp(18.1%)(Table S4).

To identify pathways that are significantly enriched with differentially expressed genes (DEGs)at the above-mentioned time points, genes that show significantly different expression(log, FCl>1, adjusted P<0.05)between MeJA- and mock-treated leaves were compared to the Kyoto Encyclopedia of Genes and Genomes(KEGG)database (Fig.S1). Application of MeJA modulated the expression of genes in plant hormone and mitogen-activated protein kinase (MAPK)signaling pathways as well as fatty acid metabolism at all time points, with the only exception of those in plant hormone pathways at 24-h (Fig. S1). While changes in aromatic amino acid metabolic (including the shikimate pathway)genes became evident at 6-h after MeJA treatment (Fig.S1b),a surge of modified expression of flavonoid metabolic genes was observed for the 24-h and 72-h post-MeJA treatment leaves (Figs. Slc and S1d).

Shikimate and HT pathway genes and HT metabolites were induced in MeJA-treated pomegranate leave As revealed in the transcriptome and KEGG pathway enrichment analysis, three shikimate biosynthetic path-way genes showed upregulated expression in MeJA-treated leaves relative to mock controls at 6-h, including 3-deoxy-D-arabinose-heptulosonate-7-phosphate synthase (DAHPS), 3-dehydrogenate synthase(DHS), and the bifunctional 3-dehydrogenate dehydratase/shikimate dehydrogenase (DHQ/SDH; abbreviated as SDH)(Figs.S1b and la).In particular, three isoforms of pomegranate SDHs were identified and showed differential expression in the transcriptome analysis, Pgr020271, Pgr019030, and Pgr019029,

To determine whether changes in the amount of shikimate and HT biosynthetic gene transcripts may affect the level of metabolites derived from these pathways, phenolic metabolites were extracted from leaves harvested at 24-h, 30-h. 36-h,48-h, and 72-h after MeJA or mock application and analyzed by HPLC(Fig.2). It should be noted that these time points were chosen to account for the time needed for protein synthesis and metabolite production and accumulation after the observed expression changes of shikimate and HT biosynthetic genes at 6-h. The retention times and absorption spectra of two metabolites eluted at 4.57 min (peak 1)and 24.98 min (peak 2) matched to those of the HT pathway intermediates β-glucogallin and Penta alloys-glucose, respectively(Figs. la and 2a). Both peaks showed significant changes in integrated peak areas at multiple time points (Fig.2b). Specifically, peak 1 increased in MeJA-treated leaves relative to mock controls at 30-h, 36-h, and 48-h(Fig.2b). Interestingly, peak 2in MeJA-treated leaves initially decreased at 24-h, but subsequently increased at 30-h and 36-h before returning to a level similar to mock controls at 48-h and 72-h (Fig.2b).

Reduction of most flavonoids and anthocyanins, as well as increased methylated flavones and flavonols, were apparent in MeJA-treated pomegranate leaves

To investigate whether exogenous application of MeJA may trigger broad-scale metabolic changes in pomegranate, metabolite profiling analysis was conducted on leaves collected at 72-h after mock- or MeJA treatment using LC-ESI-MS/MS. Metabolite annotation and quantification were performed using an MS/MS spectral tag (MS2T)library and multiple reaction monitoring(MRM), respectively. Of the 658 metabolites that were detected,29 showed increased and 73 exhibited decreased accumulation in MeJA-treated leaves compared to the mock controls (ILog, FCl>1; Tables S5 and S6). For the differentially accumulated metabolites, there was an overall enrichment of metabolites involved in plant secondary/specialized metabolism(67 of 102), particularly phenolic compounds (63 of 102) (Table S6).

Among phenolics, a concerted reduction in a wide range of flavonoids and anthocyanins(42 of the 73 decreased compounds)was apparent in MeJA-treated leaves(Fig. 3; Table S6). Intriguingly, three mono- or di-O-methylated flavones and flavonols, including di-O-methyl quercetin, chrysoberyl O-hexosyl-O-hexoside, and selling 5-O-hexoside, were increased in MeJA-treated leaves(Fig. 3; Table S6). Several intermediates of the flavonoid and anthocyanin pathways, including luteolin, chrysoberyl, dihydrokaempferol, dihydroquercetin, dihydromyricetin, epicatechin, delphinidin, and pelargonidin, were detectable but did not show significant changes in MeJA-treated leaves(Fig.3; Table S5). Hydroxycin-namoyl derivatives, isoflavones, and coumarins were among other phenolics that showed reduced accumulation upon MeJA induction (Table S6). By contrast, two phenolic acids, 2,3-dihydroxybenzoic acid and protocatechuic acid (3,4-dihydroxybenzoic acid), and a coumarin, 6-methyl coumarin, were increased in MeJA-treated leaves (Table S6).

Consistent with the largely reduced flavonoids and anthocyanins in MeJA-treated pomegranate leaves, the transcripts of two key enzymes for flavonoid and anthocyanin biosynthesis. CHS(Pgr005566)and CHI (Pgr025966), were significantly decreased at 6-h and 24-h after MeJA application according to the transcriptome analysis(Fig. 4). Real-time qPCR analysis was carried out to examine CHS and CHI expression with additional time points, including 2-h, 3-h, 6-h,12-h,24-h,48-h, and 72-h (Fig.4).CHS transcripts dropped in MeJA-treated leaves at 3-h and remained to be significantly lower than those in mock controls until 72-h, with the largest decrease at 12-h. In contrast, reduction in CHI expression was only significant at 24-h, 48-h, and 72-h post-MeJA treatment (Fig. 4).

This article is extracted from Planta (2021) 254:89 https://doi.org/10.1007/s00425-021-03735-9