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First published online July 1, 2009; 10.1104/pp.109.141481 Plant Physiology 151:1096-1113 (2009) © 2009 American Society of Plant Biologists OPEN ACCESS ARTICLE
Integrated Metabolite and Transcript Profiling Identify a Biosynthetic Mechanism for Hispidol in Medicago truncatula Cell Cultures1,[C],[W],[OA]Plant Biology Division, Samuel Roberts Noble Foundation, Ardmore, Oklahoma 73401 (M.A.F., B.E.D., M.N., R.A.D., L.W.S.); Pharmacognosy Department, Faculty of Pharmacy, Cairo University, Cairo, Egypt 11562 (M.A.F.); Department of Biology, Colorado State University, Fort Collins, Colorado 80523 (B.E.D.); and Departamento de Química, Instituto de Ciências Exatas, Universidade Federal de Minas Gerais, Campus Pampulha, Belo Horizonte, Minas Gerais, 31270–901, Brazil (Â.d.F.)
Metabolic profiling of elicited barrel medic (Medicago truncatula) cell cultures using high-performance liquid chromatography coupled to photodiode and mass spectrometry detection revealed the accumulation of the aurone hispidol (6-hydroxy-2-[(4-hydroxyphenyl)methylidene]-1-benzofuran-3-one) as a major response to yeast elicitor. Parallel, large-scale transcriptome profiling indicated that three peroxidases, MtPRX1, MtPRX2, and MtPRX3, were coordinately induced with the accumulation of hispidol. MtPRX1 and MtPRX2 exhibited aurone synthase activity based upon in vitro substrate specificity and product profiles of recombinant proteins expressed in Escherichia coli. Hispidol possessed significant antifungal activity relative to other M. truncatula phenylpropanoids tested but has not been reported in this species before and was not found in differentiated roots in which high levels of the peroxidase transcripts accumulated. We propose that hispidol is formed in cell cultures by metabolic spillover when the pool of its precursor, isoliquiritigenin, builds up as a result of an imbalance between the upstream and downstream segments of the phenylpropanoid pathway, reflecting the plasticity of plant secondary metabolism. The results illustrate that integration of metabolomics and transcriptomics in genetically reprogrammed plant cell cultures is a powerful approach for the discovery of novel bioactive secondary metabolites and the mechanisms underlying their generation.
Phenylpropanoid metabolism encompasses a complex network of branching biochemical pathways that collectively provide plants with thousands of compounds that have diverse functions in plants, most notably in defense, such as cell wall strengthening and repair (e.g. lignin and suberin), antimicrobial activity (e.g. furanocoumarin, pterocarpan, and isoflavonoid phytoalexins), and as signaling compounds such as luteolin (Peters et al., 1986  ) and apigenin (Peters and Long, 1988). In the core pathway, Phe is converted into p-coumaroyl-CoA, and the condensation of this molecule with three molecules of malonyl-CoA, via chalcone synthase activity, yields 4,2',4',6'-tetrahydroxychalcone (naringenin chalcone), which is the primary C15 flavanone skeleton from which a large number of flavonoid subclasses diverge (Fig. 1
). In legumes, the 6'-deoxy chalcone isoliquiritigenin is produced through the concerted activity of chalcone synthase and chalcone reductase (Ralston et al., 2005). This trihydroxychalcone can be further metabolized by chalcone isomerase to form liquiritigenin, a precursor for a range of 5-deoxy flavones, flavonols, and isoflavones (Fig. 1). Exceptions to this biosynthetic route are the aurones, a flavonoid subclass that can be directly synthesized from isoliquiritigenin (Strack, 1997). Aurones (Fig. 1) serve significant roles in flower pigmentation (Nakayama et al., 2001) and defense responses (Paré et al., 1991). In humans, aurones have recently drawn much attention for their therapeutic potential as anticancer, antidiabetic, antibacterial, antiparasitic, and antihormonal agents (Boumendjel, 2003). While flavone, flavonol, and isoflavone biosynthetic pathways have been extensively studied using different molecular and biochemical approaches, only a few studies on aurone biosynthesis have been reported.
Aureusidin synthase, a polyphenol oxidase (PPO) homolog, has been identified in snapdragon (Antirrhinum majus) as a key enzyme involved in aurone biosynthesis (Nakayama et al., 2000 ). The original mechanism for aureusidin synthase proposed the 3-hydroxylation and oxidative cyclization, involving 2', -dehydrogenation, of naringenin chalcone to produce aureusidin (4,6,3',4'-tetrahydroxyaurone). It was subsequently shown that auresidin synthase could likewise form the corresponding aurone sulfuretin by similar 3-hydroxylation and oxidative cyclization of isoliquiritigenin (Fig. 1; Nakayama et al., 2001). An alternate mechanism proposed for aurone biosynthesis in soybean (Glycine max) seedlings involves a hydrogen peroxide (H2O2)-dependent peroxidase (PRX; Wong, 1967; Rathmell and Bendall, 1972) that catalyzes the oxidation of isoliquiritigenin to yield a 2-(-hydroxybenzyl)coumaranone derivative, a hydrated form of aurone, which is then spontaneously dehydrated to form the aurone hispidol (Fig. 1). However, no plant PRX involved in aurone biosynthesis has been functionally characterized in legumes to date (Strack, 1997; Nakayama et al., 2001).
Plant PRXs (EC 1.11.17) are ubiquitous, heme-containing glycoproteins that catalyze the oxidation of diverse organic and inorganic substances at the expense of H2O2. Higher plants possess a number of PRX isoenzymes that are usually classified as anionic, neutral, or cationic based upon their pI (Barz et al., 1990
Large-scale metabolite profiling (metabolomics) is a powerful tool for analyzing metabolism and gene function (Sumner et al., 2003
Barrel medic (Medicago truncatula) is a rapidly developing model for legume biology (Cook, 1999
Hispidol and Hispidol-4'-O-β-D-Glucoside Are Novel Phenylpropanoid Compounds Induced in the Response of M. truncatula to YE
A large-scale elicitation experiment was conducted using liquid suspension cell cultures and two elicitors (YE and MeJA) to generate an integrated global data set (transcriptome, proteome, and metabolome) and to facilitate gene discovery and novel insight into biotic and abiotic stress responses associated with natural product pathways in M. truncatula. Triplicate biological samples from control and elicited cell cultures were harvested at 21 different time points between 0 and 48 h postelicitation for each elicitor and each replicate from independent culture flasks (Broeckling et al., 2005
The effect on phenylpropanoid metabolite pools was most dramatic in cell cultures treated with YE. M. truncatula and alfalfa (Medicago sativa) cell cultures are known to respond to YE by transcriptional induction of isoflavonoid biosynthetic genes and the downstream accumulation of the isoflavonoid-derived pterocarpan phytoalexin medicarpin (Suzuki et al., 2005
The basic aurone nucleus has a Mr of 222, and the 32-D mass difference between the aurone nucleus and P2 suggests the presence of two additional hydroxyl groups on either the A or B ring of P2. Several authentic aurone standards having a Mr of 254, the same as P2, were analyzed using the same HPLC-MS conditions, of which only hispidol (6,4'-dihydroxyaurone) matched P2 in retention time and spectral characteristics. Thus, P2 was identified as hispidol, a compound previously detected in soybean seedlings (Wong, 1967 ). P1 was identified as hispidol-4'-O-β-D-glucoside through HPLC-PDA-MS and cocharacterization with an authentic standard. This is, to our knowledge, the first report of the presence of these aurones in M. truncatula. Higher intracellular levels of hispidol glucoside rather than hispidol were observed in response to YE (Fig. 2A). This might indicate a rapid in vivo conversion of hispidol to hispidol glucoside or a limiting availability of isoliquiritigenin. However, metabolite profiling of the extracellular medium from YE-treated cell cultures showed that hispidol accumulation was induced up to 50-fold (Supplemental Fig. S1), whereas, hispidol glucoside was below the detection limit of the HPLC-PDA-MS. The large accumulation of hispidol in the medium suggests that the lower intracellular levels of hispidol compared with hispidol glucoside are likely due to secretion of this compound into the medium.
The induction of hispidol and hispidol glucoside in response to YE suggested that these compounds might be defense-related or antimicrobial compounds. Thus, antifungal activity assays were conducted with the fungal pathogen Phoma medicaginis, which causes spring black stem and leaf spot disease in alfalfa, to assess potential biological activities of hispidol and its glucoside. Other endogenous Medicago metabolites induced in response to YE were also tested and included formononetin, ononin, afrormosin, irisolidone, and isoliquiritigenin. All phenolics were tested at a concentration of 100 µM. Coumestrol, a known antimicrobial compound in alfalfa, was included in the assay as a positive control and showed the strongest inhibition of fungal growth, followed by hispidol, afrormosin, ononin, and hispidol-4'-O-β-D-glucoside (Fig. 3 ). Thus, hispidol can be classified as a potential and relatively potent antifungal compound in M. truncatula cell cultures. The direct precursor of hispidol, isoliquiritigenin, showed very weak antifungal activity against P. medicaginis.
Selection of Putative Hispidol Synthases
Two primary mechanisms for aurone biosynthesis have been proposed based upon PPO (Nakayama et al., 2000
Potential involvement of PPOs in hispidol biosynthesis was investigated. BLASTn queries of the aureusidin synthase described previously (Nakayama et al., 2000 Putative PRXs were then evaluated as potential hispidol synthases. The custom M. truncatula oligonucleotide microarray chip included 88 putative PRX TC sequences. Ten of the PRXs showed altered expression in response to YE (Fig. 4A ) and seven in response to MeJA (Fig. 4B; P < 0.05). Of the YE-modulated PRX TCs, six (TC106558, TC106564, TC106484, TC110836, TC102451, and TC107670) were up-regulated and the others (TC102226, TC107346, TC102451, and TC101009) were down-regulated. The down-regulated TCs were excluded as candidates (Fig. 4, A and B). Two of the YE-responding TCs (TC106564 and TC102451) were also induced in response to MeJA treatment (Fig. 4, A and B) and, therefore, excluded, since metabolite profile data showed no induction of hispidol or hispidol glucoside in response to MeJA (Fig. 4E). The induction of TC107670 was observed only at single time point (i.e. after 2 h of YE treatment), which was inconsistent with the metabolite data. Therefore, this TC was also excluded, leaving three putative candidate TCs (TC110836, TC106484, and TC106558). Replicated Affymetrix microarray analyses (Fig. 4C) were also performed at critical time points (2 and 24 h postelicitation). The induction of all candidate TCs was verified in the replicated Affymetrix microarray data and further confirmed by more rigorous quantitative reverse transcription (qRT)-PCR analysis of total RNA from MeJA- and YE-treated cells (Fig. 4, D and F, respectively). In general, the qRT-PCR and Affymetrix microarray expression levels were consistent with the 16K custom oligonucleotide microarray results. For example, the data showed that YE elicitor induced a significant transient increase from 0.5 to 4 h in TC106558, whereas TC106484 and TC110836 showed a sustained increase from 2 to 24 h (Fig. 4). At the metabolite level, both hispidol and hispidol-4'-O-β-D-glucoside showed similar induction kinetics, with a sustained increase from 8 to 36 h postelicitation (Fig. 4G). Both qRT-PCR and microarray (Fig. 4) analyses showed no change or a mild decrease in the same three TCs in response to MeJA, which correlated with no change or moderate declines in both hispidol and hispidol glucoside levels (Fig. 4E). In summary, the transcript profiles and qRT-PCR data correlated well with those of hispidol and hispidol glucoside accumulation, supporting the hypothesis that TC110836, TC106484, and TC106558 might be involved in hispidol formation.
Nucleotide Sequence Analysis of PRX Genes in M. truncatula
TC110836, TC106484, and TC106558 (designated MtPRX1, MtPRX2, and MtPRX3, respectively) were fully sequenced from both strands. MtPRX1, MtPRX2, and MtPRX3 encoded proteins containing 323, 355, and 357 amino acids with calculated pI values of 8.9, 7.5, and 8.7, respectively. Strong amino acid similarity (79% identity at the amino acid level) was observed between MtPRX2 and MtPRX3, whereas MtPRX1 shared only 54% identity with the other two PRXs. Deduced amino acid sequences showed all structural motifs required for functional class III PRX proteins. Highly conserved residues characteristic of heme-containing PRXs (Passardi et al., 2004
A C-terminal propeptide that targets PRXs for vacuolar import (Welinder et al., 2002 ) was present in MtPRX2 and MtPRX3 but not in MtPRX1 (Fig. 5A). However, the possibility still exists that MtPRX1 is targeted to the vacuole due to its strong cationic nature (pI 8.9), as most cationic PRXs are vacuole localized due to the acidic nature of the vacuole (Ros Barcelo et al., 2003). In addition, many vacuole-targeted proteins contain still undefined sorting signals (Carter et al., 2004).
In silico expression analysis was performed for MtPRX1, MtPRX2, and MtPRX3 to evaluate the spatial expression of these PRXs (Fig. 6
). Relative expression level data were extracted from an expression atlas study where Affymetrix microarray data were collected for a series of M. truncatula tissues, including leaf, petiole, stem, vegetative bud, flower, pods, and unnodulated root, as well as nodulated root developmental time course and a seed developmental time course (Benedito et al., 2008
Biochemical Characterization of Recombinant PRX Enzymes To determine the biochemical activities of each of the three candidate PRXs, their cDNAs were subcloned into a pET-28a expression vector and expressed in Escherichia coli BL21 (DE3) cells. Western-blot analysis of the total protein extracts from isopropyl β-D-1-thiogalactopyranoside (IPTG)-induced cells harboring MtPRX1, MtPRX2, and MtPRX3 clones showed the presence of a single protein band for each MtPRX between 34 and 39 kD and corresponding to the predicted size of the recombinant proteins (Fig. 7 ). No protein bands were detected in cultures harboring an empty pET-28a vector (Fig. 7). Recombinant PRX proteins in cell lysates were tested for enzymatic activity against guaicol, a phenolic substrate commonly utilized in PRX enzymatic assays with equal amounts of total lysate protein used in each enzymatic reaction (see "Materials and Methods"). PRX enzymatic activity was detected with all recombinant proteins: MtPRX1, 111 ± 15 nmol mg–1 min–1; MtPRX2, 105 ± 21 nmol mg–1 min–1; and MtPRX3, 220 ± 44 nmol mg–1 min–1. It should be noted that variation in PRX enzymatic activity in cell lysates for MtPRX1, -2, and -3 could be attributed to minor differences in expression levels of the recombinant proteins, as purified recombinant proteins could not be isolated. No PRX activity was detected in the cell lysate containing the empty vector. To determine whether these PRXs could be involved in hispidol biosynthesis, isoliquiritigenin was tested as a substrate and the products were analyzed using HPLC-PDA-MS. In vitro assays showed the formation of hispidol from isoliquiritigenin using cell lysates expressing each of the three different PRXs (Fig. 8, B–D ). No aurone products were observed in the in vitro assays of control cells expressing the empty vector (Fig. 8A). Although MtPRX2 and MtPRX3 protein sequences are 79% identical (Fig. 5A), their product profiles were quite different with isoliquiritigenin as a substrate. MtPRX2 yielded mainly hispidol (Fig. 8C), whereas MtPRX3 formed predominantly a quinol vinyl ether (QVE) and hispidol in lower amounts (Figs. 8D and 11). Furthermore, QVE was an observed product of horseradish peroxidase and not detected in elicited M. truncatula cell cultures.
Isoliquiritigenin-4-O-glucoside (Zhu et al., 2003 ) was also converted to hispidol glucoside by the recombinant PRXs (Fig. 11; Supplemental Fig. S2). A considerable proportion of the isoliquiritigenin glucoside was isomerized in all in vitro assays to liquiritigenin glucoside, as flavanones are the thermodynamically more stable isomers of chalcones (Boland and Wong, 1975). In the case of MtPRX3, a QVE glucoside was the major product (Fig. 11).
To further assess MtPRX1 to -3 as putative vacuolar PRXs, enzymatic assays were performed that monitored the formation of hispidol from isoliquiritigenin by MtPRX1 to -3 as a function of pH over the range of pH 3 to 9. Vacuolar enzymes tend to have lower optimum pH, consistent with the acidic environment of the vacuole, which is typically pH 5.5 (Taiz, 1992
The ability of the recombinant MtPRXs to synthesize aurones from a variety of chalcone substrates was evaluated to determine the specificity of these enzymatic reactions and the underlying mechanism(s) (Fig. 11). Chalcones tested either lacked one of the three (4, 2', or 4') hydroxyl groups present in isoliquiritigenin or contained an additional hydroxyl group on either the A or B ring. Chalcones lacking the 2' or 4-hydroxyl functions (i.e. compounds 6, 7, and 20) generated no aurones, whereas the absence of a 4' hydroxyl group (3) did not impede enzymatic activity and the respective aurone was produced as monitored by HPLC-PDA-MS. Interestingly, chalcone analogs having a Glc at position 4 (2) were also acceptable substrates for aurone production, whereas those with a methoxy group at this position (8) were not, highlighting the crucial role of ring substituents at this position on enzymatic activity. Interestingly, naringenin chalcone that serves as a substrate for aureusidin formation by PPO in snapdragon (Nakayama et al., 2000
Integrated Metabolite and Transcript Profiling Identify Hispidol and Putative PRXs
PRX multigene families have been found in all species thus far; however, the assignment of individual PRXs to a specific in vivo function still remains difficult (Quiroga et al., 2000
All cloned MtPRXs catalyzed the oxidation of isoliquiritigenin and its glucoside to the corresponding aurone as a single product or one of multiple products. However, only MtPRX1 and MtPRX2 are likely to be functional hispidol synthases. Although MtPRX3 catalyzes hispidol biosynthesis, it also catalyzes a greater quantitative production of a QVE that is believed to originate from the rearrangement of the 2-( The specificity of MtPRX1 and MtPRX2 as hispidol synthases was supported by the coordinated accumulation of their transcripts in response to YE using the 16K M. truncatula oligonucleotide microarry data (Fig. 4) with hispidol and hispidol glucoside. This observation was confirmed in replicated Affymetrix microarray analyses at 2 and 24 h postelicitation and by qRT-PCR. Other candidate M. truncatula PRXs were discounted based upon the nonstatistically significant accumulation of their transcripts. Examples include TC101009, TC107670, TC107346, TC106564, and TC102226.
In silico expression analyses were also performed for MtPRX1, MtPRX2, and MtPRX3 to evaluate the spatial expression of these PRXs (Fig. 6). MtPRX1 was most highly expressed in flower, pods, and roots. MtPRX2 expression levels were approximately 200-fold higher in roots and nodules than any other tissues. These data illustrate the high tissue specificity of MtPRX2, which would translate to the present cell cultures that were initiated from roots (Broeckling et al., 2005 To better assess the substrate specificities of each of the PRXs by kinetic analysis, multiple approaches were exhaustively pursued to express and purify the recombinant MtPRX proteins. The proteins were heterologously expressed in E. coli, yeast, and insect cell systems. In all systems, only low levels of the PRXs were observed to accumulate, suggesting a potent cell toxicity of these enzymes in prokaryotic and eukaryotic systems. Enrichment attempts using immobilized metal affinity chromatography to purify the His-tagged proteins were also unsuccessful, and it is assumed that the His tag was nonaccessible for affinity binding and purification. Thus, quantitative kinetic data for these PRXs could not be obtained.
The mechanism for the PRX-mediated conversion of isoliquiritigenin to hispidol proposed here is based upon that originally described by Wong (1967)
The substrate specificity of the MtPRXs differed from that of the snapdragon flower PPO. Interestingly, naringenin chalcone, which serves as a substrate for aureusidin formation by PPO in snapdragon (Nakayama et al., 2000 ), generated no aurone when incubated with expressed MtPRX1 to -3 (Fig. 11). However, naringenin chalcone does not accumulate in YE-elicited M. truncatula cell cultures (Farag et al., 2007). Thus, naringenin chalcone is less likely to be a primary in vivo substrate of the MtPRXs and/or is rapidly converted to the flavone via chalcone isomerase. In contrast, significantly elevated levels of isoliquiritigenin and its glucoside were observed and are more logical in vivo substrates for the MtPRXs.
Snapdragon aureusidin synthase also utilizes naringenin chalcone glucoside rather than naringenin chalcone as a substrate (Ono et al., 2006
Substitution of the 4-hydroxyl with a methoxy group (8) prevented conversion to aurone by any of the three MtPRXs, whereas Glc attachment at this position (2) did not. Furthermore, the 4-methoxy-2',4'-dihydroxychalcone substrate only yielded a flavanone dimer (16) with MtPRX2 and MtPRX3 (Fig. 11); trace levels were detected with MtPRX1. Protection of the 4-hydroxyl group with a methoxy group inhibits the formation of a B ring radical species. Instead, a phenoxy radical species is initiated in the A ring at the 2'-hydroxyl, which undergoes further cyclization, furnishing the pyrone radical (Fig. 10
, compound B) and dimerization to form compound C in Figure 10. Under our experimental conditions, the chalcones 6 and 7 (Fig. 11
), which both contain 2'- and 4'-hydroxyl groups, also produced the corresponding dimers. This suggests that the absence of a hydroxyl group at C4 or its protection with a methyl group provides the mechanistic diversion toward dimer formation. In contrast, the presence of the sugar hydroxyl groups in isoliquiritigenin-4-O-glucoside may contribute to aurone formation. Homolytic cleavage of the O-H bonds of the sugar may also result in the formation of radical species (Fig. 10, compound E) that then follow a similar reaction mechanism, as proposed in Figure 9 and the previous literature (Wong, 1967
Hispidol biosynthesis in M. truncatula is unlikely to occur via a PPO mechanism. This conclusion is based upon the nondetectable levels of all three putative PPOs present on the M. truncatula microarray and based upon our current mechanistic understanding. According to the reported PPO-catalyzed mechanism for aurone synthesis (Nakayama et al., 2001 ), the substrate must contain hydroxyl groups at the 2' and 4 positions, such that all products will then possess a 3,4-dihydroxy-substituted B ring (Fig. 1). Hispidol contains a 4-monohydroxy B ring and lacks the 3-hydroxyl B ring group (Fig. 1); thus, the production of hispidol via a PPO-catalyzed mechanism is unlikely. This argument is further supported by the fact that the predicted substrate required for the PPO production of hispidol would contain an unsubstituted B ring (i.e. 2',4'-dihydroxy chalcone or a 4-dehydroxy isoliquiritigenin), which has not been identified in legumes to date. Nakayama et al. (2000, 2001) also suggest that hispidol biosynthesis occurs via a PRX-mediated as opposed to a PPO-mediated mechanism. Furthermore, alfalfa leaves and stems have been reported to have relatively little if any PPO activity (Sullivan et al., 2004; Sullivan and Hatfield, 2006). The above evidence supports the role of PRXs and discounts the role of PPO-mediated hispidol biosynthesis in legumes.
Production of hispidol and hispidol glucoside is likely associated with the oxidative burst and production of H2O2, which are key components of plant disease resistance (Lamb and Dixon, 1997
On the basis of hispidol's relative antifungal activity and its inducible synthesis from distant precursors, hispidol meets the criteria for classification as a phytoalexin (VanEtten et al., 1994
Cell Cultures and Elicitation
Cell cultures derived from barrel medic (Medicago truncatula ‘Jemalong’ line A17) roots were initiated, subcultured, elicited with YE and MeJA, and harvested at 21 time points as described previously (Broeckling et al., 2005
Hispidol, coumestrol, afrormosin, and isoliquiritigenin-4-O-glucoside were purchased from Apin Chemicals. Hispidol 4'-O-β-D-glucoside and irisolidone were kindly provided by Dr. Tom Mabry (University of Texas, Austin). Guaicol was purchased from Sigma-Aldrich. Other tested compounds were purchased from Indofine. Solvents used were of HPLC-grade purity. Naringenin chalcone was synthesized from naringenin according to the method reported by Shimokoriyama (1957)
Phenolic compounds were extracted from lyophilized M. truncatula cells, 20 ± 0.06 mg, with 1.8 mL of 80% methanol containing 2 µg of umbelliferone (as an internal standard) for 10 h at room temperature using an orbital shaker in the dark. Extract aliquots (1.4 mL) were centrifuged at 3,000g for 60 min, and the supernatant was evaporated under a nitrogen stream until dry. The residue was resuspended in 300 µL of 45% methanol, and samples were analyzed by HPLC-PDA-MS. For analysis of phenolic compounds in the culture medium, the medium was vacuum filtered from cell cultures and a 20-mL aliquot was extracted three times with 25 mL of ethyl acetate spiked with 2 µg of umbelliferone (internal standard). The extracts were pooled, evaporated to dryness using nitrogen, dissolved in 300 µL of methanol, and analyzed by HPLC-PDA-MS.
An Agilent 1100 Series II HPLC system (Agilent Technologies) equipped with a PDA detector was coupled to a Bruker Esquire ion-trap mass spectrometer equipped with an electrospray ionization source. UV spectra were obtained by scanning from 200 to 600 nm. A reverse-phase, C18, 5-µm, 4.6- x 250-mm column (J.T. Baker) was used for separations. Separations were achieved using a linear gradient of 5% to 90% (v/v) B in 70 min. The mobile phase consisted of eluent A (0.1% [v/v] acetic acid in water) and eluent B (100% acetonitrile). The flow rate was 0.8 mL min–1, and the temperature of the column was kept at 28°C. Both positive and negative ion mass spectra were acquired. Positive ion electrospray ionization was performed using an ion source voltage of 4.0 kV and a capillary offset voltage of 86.0 V. Nebulization was aided with a coaxial nitrogen sheath gas provided at a pressure of 60 p.s.i. Desolvation was aided using a counter current nitrogen flow set at a pressure of 12 p.s.i. and a capillary temperature of 300°C. Mass spectra were recorded over the range of 50 to 2,200 m/z. The Bruker ion-trap mass spectrometer was operated under an ion current control of approximately 10,000 with a maximum acquire time of 100 ms. Chromatographic peak areas of cell culture extracts were normalized based on the areas of recovered internal standard peaks. Relative metabolite abundances were calculated using a custom PEARL script to extract peak areas of individual ions characteristic of each component (Broeckling et al., 2006
Collected P1 fractions were evaporated, resuspended in 200 µL of water, and subjected to β-glucosidase hydrolysis for 8 h at 37°C (Mabry et al., 1970
RNA Isolation, cDNA Labeling, and Hybridization
Custom M. truncatula Oligonucleotide Microarray Analysis Each cDNA sample was hybridized against a reference cDNA sample that was prepared from a common pool of reference cDNA, and a reference design of the micorarray experiment was performed. Reference RNA was purified from separate M. truncatula ‘Jemalong’ (line A17) cell suspension cultures (maintained for approximately 3 years in subculture) and pooled for use. Cy3 dye (Amersham Biosciences) was used for labeling reference RNA, and Cy5 was used for experimental RNA. The oligonucleotide microarray data are publicly available in a custom database for "omics" data (DOME; http://medicago.vbi.vt.edu/data.html).
Affymetrix M. truncatula Microarray Analysis
Microarray Data Processing and Analysis
Microarray results were confirmed by qRT-PCR using TC-specific primers for tissue collected at 0 min, 30 min, 2 h, 4 h, 12 h, and 24 h postelicitation. RNA was isolated from triplicate control and elicited cells. Ten micrograms of purified total RNA was treated with Turbo DNA-free DNase I (Ambion) according to the manufacturer's protocol, purified by RNeasy MinElute columns (Qiagen), and checked for genomic DNA contamination by PCR using the M. truncatula-specific actin primers MtActin-for (5'-TCAATGTGCCTGCCATGTATGT-3') and MtActin-rev (5'-ACTCACACCGTCACCAGAATCC-3'). RNA integrity was evaluated with an Agilent 2100 Bioanalyzer using RNA nanochips. RT reactions were performed using SuperScript III reverse transcriptase (Invitrogen) according to the manufacturer's protocol. PCRs were performed on an optical 384-well plate with an ABI PRISM 7900 HT sequence detection system (Applied Biosystems) using SYBR Green to monitor double-stranded DNA synthesis. PCRs (10 µL final volume) contained 5 µL of SYBR Green Master Mix reagent (Applied Biosystems), 1 µL of cDNA, and 200 nM of each gene-specific primer: MtPrx1for (5'-GTATGCCCCGGAGTTGTATC-3'), MtPrx1rev (5'-CCGTTTTAGCATCTGTTCTTCC-3'), MtPrx2for (5'-GTGCTTACTGGAAAGAAAGGAGA-3'), MtPrx2rev (5'-GAGGTCACACTAGCAAGATCCA-3'), MtPrx3for (5'-GAAACAAGGAGAGATTAGAAAACA-3'), and MtPrx3rev (5'-CATTGAGCTAACCATACCCTCATC-3'). PCRs were performed as described elsewhere (Czechowski et al., 2005
BLAST searches were performed using BLASTp version 2.2.2 (http://www.ncbi.nih.gov/BLAST). The amino acid sequences were aligned using ClustalW, and the similarities were calculated using Megalign (DNASTAR). Publicly available programs (http://www.expacy.ch/tools/pi_tools.html/ and http://www.scripps.edu/
M. truncatula EST clones NF041G08NR (i.e. MtPRX1 corresponding to TIGR release 8.0 TC110836), NF092A02EC (i.e. MtPRX2 corresponding to TIGR release 8.0 TC106484), and NF089H06PL (i.e. MtPRX3 corresponding to TIGR release 8.0 TC106558) were sequenced from both ends using M13 primers and found to contain full-length sequences. cDNA sequences were PCR amplified from pBluescript II SK+ (Stratagene) using primers with EcoRI and XhoI (MtPRX1 and MtPRX3) or SacI and HindIII (MtPRX2) sites (underlined): MtPRX1F, 5'-AGGATTGAATTCATGGCTCCTTTAAATTGT-3'; MtPRX1R, 5'-TTAACCTCGAGATTAGTTGGTTTTTCTACA-3'; MtPRX2F, 5'-CACATAGAGCTCAAAATGAATTCCCTTAGT-3'; MtPRX2R, 5'-ACCCACAAGCTTTTAGATTGAACTAACCAT-3'; MtPRX3F, 5'-TCACAAGAATTCATGAACTCCCTTCGTGCT-3'; and MrPRX3R, 5'-CAATCACTCGAGTTACATTGAGCTA-3'. Purified PCR products were digested with appropriate restriction enzymes (Novagen) for 2 h and purified from an agarose gel using the QIAquick gel extraction kit (Qiagen). The excised PCR products were cloned into appropriately digested pET-28a (+) vector (Novagen). The resulting plasmids were sequenced to confirm identity.
E. coli BL21 (DE3) cells harboring the pET-28a or MtPRX expression constructs were grown to an optical density of 600 nm of 0.8, and expression was initiated by addition of IPTG to a final concentration of 0.5 mM, with further incubation while shaking overnight at 20°C. Cultures were centrifuged, and pellets were resuspended in a lysis buffer (50 mM Tris, pH 8.0, 500 mM NaCl, 20 mM imidazole, pH 8.0, 10% glycerol, 1% Tween 20, and 1 mM phenylmethylsulfonyl fluoride). Cells were lysed by sonication (Biologics) and centrifuged, and the resulting supernatant was removed and assayed for PRX activity against guaicol. For PRX activity assays, cell lysates (50 µg of protein equivalent) were added to 2 mL of 0.1% guaiacol and 0.03% H2O2 in 50 mM potassium acetate, pH 6.0, and the increase in A470 over 3 min was measured using a Beckman Coulter DU800 spectrophotometer. For all PRX enzymatic reactions, equal amounts of protein were used as quantified using the Bradford (1976) Product profiling of chalcone analogs incubated with recombinant PRXs was assayed in 500 µL of 10 mM potassium phosphate buffer, pH 6.7, containing 50 µg of protein equivalent of cell lysate, 8 µL of 5 mM substrate dissolved in dimethyl sulfoxide, and 1 µL of 100 mM H2O2. Reactions were carried out at 37°C for 2 h and stopped by adding 3 volumes of ethyl acetate. After vortexing, the ethyl acetate phase was removed and evaporated to dryness under nitrogen, and the residue was resuspended in methanol prior to HPLC-PDA-MS analysis using the same instrumental conditions mentioned previously. All enzyme assays were performed in duplicate.
E. coli cell lysates were separated on a 12% Tris-HCl gel (30 µg of protein loaded per lane) and electroblotted onto a nitrocellulose membrane (Transblot; Bio-Rad) according to the procedure of Sambrook and Russell (2001)
Antifungal assays were conducted essentially as described (Blount et al., 1992 Sequence data from this article can be found in the GenBank/EMBL data libraries under the following accession numbers: MtPRX1, EF456703; MtPRX2, EF456704; MtPRX3, EF456705.
The following materials are available in the online version of this article.
We thank Prof. T.J. Mabry for his contribution of authentic hispidol glucoside and related compounds. We thank Drs. Michael Udvardi, Vagner Benedito, Yuhong Tang, and Ivone Torres-Jerez for supplying us with the M. truncatula microarray atlas data used to extract in silico expression data for MtPRX1, MtPRX2, and MtPRX3. We also thank all those involved in the large-scale culturing, elicitation, and rapid harvesting of M. truncatula suspension cultures: Lahoucine Achnine, Courtney Allen, Stacy Allen, Victor Asirvatham, Naveed Aziz, Jack W. Blount, Fang Chen, John Cooper, Anthony Duran, Patrick Fennell, Xian Zhi He, Lisa Jackson, Parvathi Kota, Changjun Liu, Srinu Reddy, Gail Shadle, Shashi Sharma, Hideyuki Suzuki, Ivone Torres-Jerez, Bonnie Watson, and Deyu Xie. Received May 18, 2009; accepted June 29, 2009; published July 1, 2009.
1 This work was supported by the National Science Foundation Plant Genome Research Program (grant no. DBI–0109732).  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 (www.plantphysiol.org) is: Lloyd W. Sumner (lwsumner{at}noble.org).
[C] Some figures in this article are displayed in color online but in black and white in the print edition.
[W] The online version of this article contains Web-only data.
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.109.141481 * Corresponding author; e-mail lwsumner{at}noble.org.
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max at 390 nm, implying the presence of a highly conjugated chromophore typical of aurones. Aurones absorb near the visible region due to an extended conjugated double bond/pi system found in chalcones, flavones, or isoflavones (Mabry et al., 1970
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