Methyl Jasmonate Elicits Distinctive Hydrolyzable Tannin, Favonoid, And Phyto‑oxylipin Responses in Pomegranate (Punica Granatum L.) Leaves Part 2

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Lipids were converted to free fatty acids and phyto-oxylipins in MeJA-treated pomegranate leaves

Besides flavonoids and anthocyanins, changes in lipid molecules were also evident in pomegranate leaves at 72-h after MeJA induction(Fig. 5a). Of the 102 differentially accumulated metabolites, 23 were lipids, fatty acids, or phyto-oxylipins(Table S6).In contrast to monoacylglycerol (MAG)(18:4)isomer 1 that showed increased accumulation, seven glycerolipids, including MAG(18:1)isomer, MAG (18:3)isomer4,galactosyl monoacylglycerol (DGMG)(18:2)isomer,DGMG(18:2)isomer2,DGMG(18:2)iso-mer3, monogalactosyl monoacylglycerol(MGMG)(18:2)isomers, and MGMG(18:2)isomer2, were decreased by 3-5 folds when MeJA was applied to the leaves(Fig.5a;Table S6).The phospholipids LysoPC 18:0 and 18:1 exhibited an over two-fold increase in MeJA-treated leaves (Table S6).

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Exogenous MeJA application elevated the levels of JA and isoleucine (JA-Ile)by 4.3-fold and 2.8-fold, respectively, in pomegranate leaves(Table S6). Various non-JA Phyto-oxylipins, including fatty acid hydroperoxides, epoxy fatty acids, hydroxy fatty acids, keto fatty acids, and acetylenic fatty acids, notably increased in MeJA-treated leaves (Fig.5a; Table S6). Enhanced accumulation of punicic acid, a PUFA(18:3) that is abundant in pomegranate seed oil, was also observed in leaves treated with MeJA(Table S6).

Fig.3 Flavonoid and anthocyanin levels were altered upon methyl jasmonate (MeJA) application. Metabolites with increased, decreased, and non-significantly changed accumulation (two-fold threshold) in MeJA-treated pomegranate leaves compared to mock-treated controls, harvested at 72-h post-treatment, are colored in green, red, and blue, respectively. Pathway intermediates that were not detected in the liquid chromatography-electrospray ionization tan-dem mass spectrometry analysis are shown in black. Dashed arrows indicate multiple reaction steps.

Biochemical characterization of a candidate LOX for fatty acid modification in MeJA-treated pomegranate leaves

To explore metabolic genes that are involved in the alternation of fatty acid metabolism towards Phyto-oxylipins, transcriptomes of mock- and MeJA-treated pomegranate leaves were analyzed for DEGs that are annotated as lipases or LOXs.Two lipase genes Pgr004895 and Pgr001125 showed enhanced expression in the 24-h post-MeJA treatment transcriptomes and were confirmed for significantly increased expression by real-time qPCR analysis (Fig.5b). On the other hand, the lipase Pgr024441 showed reduced expression in the transcriptome analysis, which was not supported by the real-time qPCR result (Fig. 5b). Interestingly, elevated expression of Pgr025417, a putative LOX, was observed in transcriptomes of leaves collected at 2-h, 6-h,24-h, and 72-h after MeJA treatment (Fig. S1). Real-time qPCR analysis further indicated a continuously increased expression of Pgr025417, ranging from 4-to 20-fold, in leaves collected from 2 to 72-h after MeJA treatment (Fig.5c).

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To determine whether the putative LOX (Pgr025417)was involved in the increased Phyto-oxylipin production in MeJA-treated leaves, its sequence and activity were examined. When the amino acid sequence of Pgr025417 was ana-lyzed, His, Asn, and Ile residues that correspond to those coordinating the iron atom at the active site of soybean LOX L-1(Minor et al.1996)were found in Pgr025417(Fig.6a). In addition, PgrO25417 is predicted to be localized to the chloroplast (likelihood probability of 0.9096)with the signal peptide cleaved between 64 and 65 aa(Fig. 6a). Among the 11 putative LOXs (786 aa to 970 aa) identified in the annotated pomegranate genome,5 other LOXs: Pgr025413, Pgr025418, Pgr009839, Pgr016852, and Pgr013780 are predicted to be chloroplastic besides Pgr025417 and group with type II LOXs characterized in other plants (Fig.6b). Interestingly,while Pgr008562,Pgr025678,Pgr018982,and PgrO18980 are clustered with dicotyledonous type I LOXs. Pgr020032 is more distantly related to the other type ILOXs (Fig.6b).To determine the enzymatic activity of PgrO25417, the recombinant protein(906 aa,~102 kDa; Fig.6c)was assayed using linoleic acid as substrate. Oxidized products of linoleic acid were produced by Pgr025417 as demonstrated by a greater absorption at 598 nm when increased amounts of recombinant protein were present in the reaction mixture (Fig.6d).

Transcriptional response to exogenous MeJA application in pomegranate leaves

To understand whether the changes in HTs,flavonoids, lipids, fatty acids, and phyto-oxylipins are regulated transcription-ally, transcriptomes of MeJA-and mock-treated pomegranate leaves collected at2-h,6-h,24-h,and72-h were compared and identified 34 transcription factors(TFs) that showed differen-tial expression (Log,FCl>1,adjusted P<0.05)(Table 1);31 of these TFs also exhibited significantly changed expression by real-time qPCR analysis (Fig.7). Notably, a Zinc-finger TF Pgr009895 showed increased expression at three out of the four time points(2-h,24-h,and 72-h)(Fig.7a).The expression of Pgr002863(PCF5-like),Pgr002859(bZIP1)and Pgr006935(auxin response factor)was increased, and PgrO11269 (MYB) decreased upon MeJA elicitation at two time points(Fig. 7a, b).Both PgrO09366(anthocyanin regu-latory C1 protein/MYB)and PgrO03015(MYB)displayed increased expression at 6-h, but decreased expression at 72-h (Fig. 7b).The expression of 12 TFs was enhanced at only one time point,including Pgr009357(2-h),Pgr027831 (2-h),Pgr023581(6-h),Pgr009363(6-h),Pgr000147 (6-h), Pgr021507(6-h), Pgr021504(6-h),Pgr020147(24-h), Pgr025715(72-h),Pgr023629(72-h),Pgr015728(72-h),and Pgr004388(72-h)(Table 1;Fig.7b,c).On the other hand, the expression of 12 TFs was suppressed in MeJA-treated pomegranate leaves at one time point, including Pgr013499 (2-h),Pgr023409 (2-h),Pgr017106(6-h),Pgr010911(24-h),Pgr017568(72-h),Pgr024750(72-h),Pgr004878(72-h)Pgr002084(72-h),Pgr008889(72-h),Pgr004532(72-h), Pgr020131 (72-h),and Pgr002400(72-h)(Table1;Fig.7c).

To assess the transcriptional regulation of candidate genes that may function in modulating flavonoid and fatty acid/Phyto-oxylipin pathways upon MeJA induction, TF-binding sites in the promoter regions of the candidate genes were predicted using PlantRegMap (Tian et al.2020) with TFs bioinformatically identified from Eucalyptus Grandis, which is closely related to pomegranate in Myrtales(Table S7). For PgrO25417(putative LOX), 80 binding sites of 65 TFs were identified, including abundant binding sites for MYB (10 TFs.15 sites)and WRKY(11 TFs,18 sites), but without binding sites for bipolar zinc finger TFs(Table S7). Because CHS and CHI are positioned at the entry point of flavonoid and anthocyanin biosynthetic pathways and showed reduced gene expression in transcriptome and real-time qPCR analyses(Figs.S1 and 4), the promoters of putative CHS(Pgr005566)and CHI(Pgr025966) were also analyzed for putative TF-binding sites. For CHS, there are 104 binding sites of 76 TFs, with MYB(20 TFs,23 sites)and LH (13 TFs, 23 sites)being the most abundant TFs(Table S7). For CHI, there are 93 binding sites of 78 TFs, with bHLH (16 TFs,23 sites)and WRKY(13 TFs,13 sites) being the most abundant TFs (Table S7).

Discussion

Unique metabolic changes induced by exogenous MeJA application suggest functions of HTs, methylated flavones/flavonols, and Phyto-oxylipins in pomegranate response to MeJA.

MeJA has a demonstrated role in eliciting stress responses in plants(Cheong and Choi 2003). The accumulation of two HT pathway intermediates β-glucogallin and Penta-galloylglucose in leaves was induced at 30-h after MeJA application (Fig.2b), suggesting that they act in response to abiotic and biotic stresses in the environment. This result also corroborates the function of HTs in protecting pomegranate from abiotic stresses in the tissue of fruit peels (Schwartz et al.2009; Habashi et al.2019). Correspond-ing to the rise in HT metabolite accumulation, shikimate, and HT biosynthetic pathway genes also showed increased expression (Fig.1). Previous biochemical characterization of four grapevines (Vitis vinifera) SDH isoforms indicated that only VvSDH3 and VySDH4 could produce gallic acid from 3-dehydroshikimate(Bontpart et al.2016). According to our transcriptome and real-time qPCR analyses, PgSDH3_1 and PgSDH3_2, homologs of WvSDH3,but not PgSDH4,a homolog of VSDH4, exhibited induced expression by MeJA (Figs. S1 and 1). This observation suggests that PgSDH3_1 and PgSDH3_2 are likely involved in the synthesis of HTs in response to MeJA treatment and environmental stresses. Additionally, enhanced expression of PgUGT8423 and PgUGT84A24, which encode enzymes catalyzing the committed step of HT biosynthesis, by MeJA application further supports the role of HTs in stress response.

At 72-h after MeJA induction, an overall suppression of flavonoids and anthocyanins were found in pomegranate leaves (Fig.3; Table S6), which is contrary to the increased accumulation of these compounds observed in many other plants (Pandey et al.2016; De Geyter et al.2012; Shafiq et al.2011; Flores and Ruiz del Castillo 2014; Portu et al. 2015). However, three methylated flavones and flavanols: di-O-methyl quercetin, selling 5-O-hexoside, and chrysoe-riol O-hexosyl-O-hexoside accumulated at a greater level despite the reduction of non-methylated biosynthetic precursors(Fig.3; Table S6). Mono-and di-O-methylated quercetin is secreted from trichomes of solanaceous species and has been proposed to act in plant defense, possibly in a species-specific manner（Wollenweber and Dörr 1995; Roda et al.2003). It was also shown that glycosides of chrysoberyl, luteolin, and apigenin deterred feeding of aquatic herbivores on Potamogeton lucens(pondweed) leaves in vitro assays (Erhard et al.2007). The induced accumulation of methylated flavones/flavonols in pomegranate leaves suggests that they may play a role in MeJA-elicited stress response, and have implications for defense against wounding, pathogens, and herbivores in this tree species.

Besides HTs, flavonoids, and anthocyanins, additional notable metabolic changes included the mobilization of free fatty acids from lipids and the biosynthesis of Phyto-oxylipins at 72-h after MeJA application(Fig.5a). The threefold increase in punicic acid(18:3, cis-9,trans-11, cis-13) upon MeJA induction suggests a potential role of this PUFA with a three conjugated double bond system in stress signaling or direct chemical defense of pomegranate plants (Table S6). The concurrent reduction of MAG, MGMG, and DGMG also suggest that punicic acid could be usu-ally conjugated to these glycerolipids in pomegranate leaves (Fig. 5a; Table S6). Of the phyto-oxylipins, the rise in JA and JA-Ile in MeJA-treated pomegranate leaves could be due to demethylation of the exogenously applied MeJA to form JA, which is subsequently converted to JA-Ile(Table S6)(Stuhlfelder et al.2004). It may also suggest that MeJA can directly regulate the biosynthesis of JA and its derivatives in pomegranate. Volatile compounds derived from Phyto-oxylipins are reportedly involved in signaling response to wounding and pathogen attacks (Lim et al.2017). Recent studies have also revealed direct antimicrobial roles of non-JA Phyto-oxylipins, though the mechanistic basis for such functions is still unclear (Deboever et al. 2020). The inducible production of Phyto-oxylipins in pomegranate corroborates the previous observations in other plants. It remains to be determined whether these phyto-oxylipins are biocidal or function to stimulate an innate immune response in pomegranate.

The modified flavonoid and anthocyanin, but not phyto-oxylipin, metabolism upon MeJA induction is at least partially regulated at the transcriptional level

Consistent with the generally decreased accumulation of flavonoids and anthocyanins, genes encoding CHS and CHI, two enzymes positioned at the entry point of flavonoid and anthocyanin biosynthesis, showed reduced expression in MeJA-treated leaves(Fig.4). Intriguingly, a pomegranate homolog(PgrO09366)of the maize MYB TF anthocyanin regulatory protein C1 was initially upregulated in MeJA-treated leaves at 6-h but then downregulated in MeJA-treated leaves at 72-h(Table 1; Fig. 7b). A pomegranate homolog (PgrO10911)of the maize MYB TF P was down-regulated in MeJA-treated leaves at 24-h (Table 1; Fig.7c). As demonstrated in studies in maize, Cl activates several genes in flavonoid and anthocyanin biosynthesis, such as CHS(C2), dihydro flavonol reductase(A1), anthocyanidin 3-O-glucosyltransferase(BZ1), and leucoanthocyani-din dioxygenase(A2)(Lesnick and Chandler 1998; Sainz et al.1997; Quattrocchio et al.1993). P, on the other hand, controls the expression of A1, but not BZ1(Grotewold et al.1994).In maize, C1 (MYB TF)works in concert with R(bHLH TF)to regulate the expression of anthocyanin biosynthetic genes(Mol et al.1998; Goff et al.1992). Pgr024750, a bHLH TF, was also downregulated in MeJA-treated leaves at 72-h and could be a potential partner of the pomegranate C1 homolog (Table 1; Fig.7c). Furthermore, binding sites for MYB and bHLH TFs were identified in the promoter regions of putative pomegranate CHS and CHI through bioinformatics analysis(Table S7). Taken together, these results suggest that the overall decrease in flavonoids and anthocyanins is at least partially attained through transcriptional control of the early-step biosynthetic genes.

A reduction of several glycerolipids was observed in MeJA-treated leaves(Fig.5). However, pomegranate homologs(GenBank accession numbers: XP_031374952 and XP_031374953)of the Arabidopsis Wrinkled1 (ArWRI1), an APETALA2(AP2) family TF that is considered a"master" regulator for plant lipid biosynthesis(Cernac and Benning 2004), expressed similarly in mock- and MeJA-treated leaves (data not shown). Functional characterization of the TFs responsive to MeJA induction (Table 1; Fig. 7)could potentially reveal a regulatory role for lipid metabolism in pomegranate leaves.

The pomegranate LOX(Pgr025417)with continuously increased expression after exogenous MeJA application, catalyzed fatty acid oxidation and was predicted to localize to the chloroplast (Figs.5c and 6). Therefore, Pgr025417 is likely involved in JA biosynthesis that takes place in this subcellular compartment(Fig.6). Of the 11 putative LOXs in pomegranate,5 are cytosolic (type I) and 6 are chloro-plastic(type II) (Fig.6b). Since non-JA Phyto-oxylipins are synthesized in the cytosol (Ponce de Le6n et al.2015), one or more of the type ILOXs may be responsible for convert-ing PUFA to non-JA Phyto-oxylipins upon MeJA induction. Considering that the LOX(Pgr025417) with a sustained increase in gene expression in MeJA-treated leaves is located in a subcellular compartment different from non-JA Phyto-oxylipins, the enzymes responsible for producing MeJA-induced non-JA Phyto-oxylipins are likely not modulated transcriptionally.

Exogenous MeJA application induced transcriptional responses in pomegranate leaves

A recent study in Arabidopsis revealed that a complex regulatory network enriched with MYC(bHLH), ethylene response factor (ERF), and MYB family TFs was involved in the early MeJA-induced transcriptional response (Hickman et al.2017).Pomegranate homologs of ERF TFs, including Pgr013499(2-h),PgrO00147(6-h),and PgrO21504 (6-h),and homologs of MYB TFs, including PgrO09357 (2-h),Pgr009366(6-h),Pgr003015(6-h),and Pgr011269 (6-h), were among the TFs with varied expression in MeJA-treated leaves collected at early time points(Table 1;Fig.7). As discussed above, pomegranate homologs of the maize flavonoid and anthocyanin regulatory proteins C1 and P (MYB TFs), and a bHLH TF showed decreased expression and could potentially control the overall reduction of flavonoids and anthocyanins. In addition, the promoter region of LOX(PgrO25417)contains binding sites of WRKY, MYB, and bZIP TFs(Table S7), which suggests that they could be regulated by the differentially expressed TFs belonging to these TF families in response to the MeJA application.

Comparative gene expression and metabolite analysis of MeJA-treated leaves facilitates the interrogation of diverse phenolic pathways in fruits

Understanding metabolic and transcriptional responses of pomegranate leaves to MeJA application is clearly pertinent to improving plant health and productivity(i.e. fruit production). In addition, the comparative gene expression and metabolite analysis of MeJA-treated leaves also provide an opportunity to elucidate the regulation of HT, flavonoid, and anthocyanin pathways that are present in both leaf and fruit tissues(Bar-Ya'akov et al.2019). The phenolic compounds HTs, flavonoids, and anthocyanins contribute greatly to the human health-beneficial activities of pomegranate fruits, yet the control of their production and accumulation has not been extensively explored in pomegranate (for flavonoids and anthocyanins)or any plant species (for HTs). For example, previous studies on MeJA-applied pomegranate fruits determined the levels of anthocyanins and flavonoids in fruits after harvest, without investigating the expression of structural or regulatory genes related to these metabolites (Koushesh Saba and Zarei 2019; Garcia-Pastor et al.2020). On the other hand, the current study uncovered TFs (e.g. MYB TFs) that showed differential expression in MeJA-and mock-treated leaves with expression patterns similar to those of HT, flavonoid, and anthocyanin biosynthetic genes (Fig.7; Table 1). These TFs are potentially involved in the regulation of HT, flavonoid, and anthocyanin pathways thus warranting further investigation in the future.

It is worth noting that our study revealed a specific increase in methylated flavonoids and general decreases in other flavonoids in pomegranate leaves treated with MeJA, suggesting a potential role of methylated flavonoids in defending pomegranate leaves against pathogens (Fig. 3; Table S6). The previous report on pomegranate fruits treated with MeJA showed enhanced flavonoid accumulation, though only the total flavonoid content was determined without quantifying individual flavonoid molecules(Koushesh Saba and Zarei 2019). As such, a more in-depth analysis of pomegranate fruits treated with MeJA is needed to allow for a side-by-side comparison of flavonoid changes in leaf and fruit tissues. Contrasting to increased anthocyanins in pomegranate fruits after MeJA applications(Garcia-Pastor et al.2020), the level of anthocyanins was reduced in MeJA-treated leaves(Fig.3; Table S6). This disparity could be due to the distinct roles that anthocyanins play in leaf and fruit tissues upon pathogen attacks. In fact, the differential accumulation of anthocyanins in leaves and fruits underscores the need for examining MeJA responses in different pomegranate tissues.

Concluding remarks

Our study revealed unique metabolic changes in HTs, flavonoids, anthocyanins, and Phyto-oxylipins in pomegranate leaves triggered by exogenous MeJA application. Transcriptome and biochemical analyses suggested that, while suppression of multiple flavonoids and anthocyanins occurs at least partially at the transcriptional level, increased bio-synthesis of non-JA Phyto-oxylipins is likely not controlled transcriptionally. This work instigates further investigations on the regulatory architecture of HT, flavonoid, and anthocyanin metabolism, the orchestration of metabolic responses in pomegranate leaves to MeJA application, as well as the role that metabolites with induced accumulation play in signaling and/or direct chemical defense.

Author contributions statement LC and LT conceived the study.LC and WS performed experiments. LC, WS, and LT analyzed data.LC, WS, and LT wrote the manuscript. All authors read and approved the final manuscript.

Supplementary Information The online version contains supplementary material available at https://doi.org/10.1007/s00425-021-03735-9.

Acknowledgments We thank colleagues at the Panzhihua Academy of Agricultural and Forestry Sciences for providing us with the pomegranate seeds used in this study. We also thank Dr. Binjie Ge at Shanghai Chenshan Botanical Garden for assistance in the identification and recording of the voucher specimen.

Funding This work was supported by the Science and Technology Commission of Shanghai Municipality under grant 14DZ2260400 and the Special Fund for Scientific Research of Shanghai Landscaping and City Appearance Administrative Bureau under grants G172403 and G182403.

Availability of data and materials The transcriptome datasets generated and analyzed during the current study are available in the Sequence Read Archive (SRA)at NCBI under the accession number PRJNA600139., The metabolite profiling datasets generated and analyzed during the current study are included in this published article and its supplementary files.

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