Phytochemistry 105 (2014) 147–157

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UHPLC–MS/MS based target profiling of stress-induced phytohormones Kristy´na Floková, Danuše Tarkowská, Otto Miersch, Miroslav Strnad, Claus Wasternack, Ondrˇej Novák ⇑ ´ University, Laboratory of Growth Regulators, Centre of the Region Haná for Biotechnological and Agricultural Research, Institute of Experimental Botany AS CR & Palacky ˚ 11, CZ-78371 Olomouc, Czech Republic Šlechtitelu

a r t i c l e

i n f o

Article history: Received 19 February 2014 Received in revised form 12 May 2014 Available online 17 June 2014 Keywords: Stress-induced phytohormones Jasmonates Abscisic acid Salicylic acid Indole-3-acetic acid Arabidopsis thaliana Solid-phase extraction (SPE) Ultra-high performance liquid chromatography (UHPLC) Tandem mass spectrometry (MS/MS)

a b s t r a c t Stress-induced changes in phytohormone metabolite profiles have rapid effects on plant metabolic activity and growth. The jasmonates (JAs) are a group of fatty acid-derived stress response regulators with roles in numerous developmental processes. To elucidate their dual regulatory effects, which overlap with those of other important defence-signalling plant hormones such as salicylic acid (SA), abscisic acid (ABA) and indole-3-acetic acid (IAA), we have developed a highly efficient single-step clean-up procedure for their enrichment from complex plant matrices that enables their sensitive quantitative analysis using hyphenated mass spectrometry technique. The rapid extraction of minute quantities of plant material (less than 20 mg fresh weight, FW) into cold 10% methanol followed by one-step reversed-phase polymer-based solid phase extraction significantly reduced matrix effects and increased the recovery of labile JA analytes. This extraction and purification protocol was paired with a highly sensitive and validated ultra-high performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) method and used to simultaneously profile sixteen stress-induced phytohormones in minute plant material samples, including endogenous JA, several of its biosynthetic precursors and derivatives, as well as SA, ABA and IAA. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction Plants have evolved a number of unique defense mechanisms to adapt to changing ambient conditions. This ability to alter their growth and development is regulated by signalling of several phytohormones. Although the individual roles of jasmonates (JAs), abscisic acid (ABA) and salicylic acid (SA) in plants’ responses to various biotic and abiotic stresses are quite well established, stress-related JAs signalling in the modulation of numerous developmental processes remains to be clarified (Li et al., 2001; Linkies and Leubner-Metzger, 2012). The jasmonates are shortchain alkylcyclopentenone and alkylcyclopentanone carboxylates that are formed via the lipoxygenase pathway. The de novo synthesis of jasmonic acid (JA) and its subsequent metabolism are both crucial in controlling the level of the bioactive hormone (Wasternack and Kombrink, 2010). JA accumulates very rapidly in both local and distal sites of wounded model plant Arabidopsis leaf tissues: its level increases noticeably within around two minutes after injury (Glauser et al., 2009). Simultaneously, its volatile methyl ester (MeJA) is generated to enable the rapid transmission of JA signalling after its demethylation to free JA in target tissues ⇑ Corresponding author. Tel.: +420 58563 4853; fax: +420 58563 4870. E-mail address: [email protected] (O. Novák). http://dx.doi.org/10.1016/j.phytochem.2014.05.015 0031-9422/Ó 2014 Elsevier Ltd. All rights reserved.

(Stitz et al., 2011). Crucial is the conjugation of primarily synthesized (+)-7-iso-JA, the initial product of JA biosynthesis. This compound is conjugated with the amino acid isoleucine (Ile) to form the most active JA compound and the ligand of the JA-receptor, (+)-7-iso-JA-Ile, which plays a key role in JA signalling (Fonseca et al., 2009; Sheard et al., 2010). Other JA conjugates with primarily non-polar amino acids including leucine (Leu), valine (Val), phenylalanine (Phe), tyrosine (Tyr), tryptophan (Trp) and methionine (Met) have also been detected in various plant species and may have roles that go beyond environmental stress responses (Knöfel and Sembdner, 1995; Tamogami and Kodama, 1997). For example, JA-Trp acts as a potential regulator of auxin homeostasis via an unknown mechanism during root growth (Staswick, 2009; Guttierrez et al., 2012). In addition, a wide range of JA metabolites with different physiological functions have been described, including 11-OH-JA, 12-OH-JA, and 12-glucosylated or sulfonylated JA derivatives (Wasternack and Hause, 2013). Finally, both the biosynthetic precursor of JA, cis-(+)-12-oxo-phytodienic acid (OPDA), and its 16-carbon homolog dinor-OPDA (dn-OPDA) have been also shown to accumulate in wounded leaves (Stintzi et al., 2001). Modern target profiling analyses involve the simultaneous measurement of several phytohormonal classes in order to determine their individual significance and control mechanisms. Two main conventional hyphenated techniques, liquid chromatography–mass

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spectrometry (LC–MS) and gas chromatography–mass spectrometry (GC–MS), have been widely used to determine the levels of endogenous phytohormones and thereafter their involvement in plant stress signalling. The development of an effective extraction and sample enrichment protocols together with selective and sensitive single-run analytical methods has made it possible to quantify the levels of JA, ABA, SA, IAA, and their metabolites in diverse plant tissues (Durgbanshi et al., 2005; Forcat et al., 2008; Matsuura et al., 2009; Müller and Munné-Bosch, 2011; Balcke et al., 2012; Liu et al., 2012). When selecting extraction solvents for these signalling molecules, it is important to account for their weak acidity (pKa = 4–5) and structural diversity. JAs and ABA are readily extracted using water-miscible organic solvents such as methanol, ethanol or acetone (Creelman and Mullet, 1995; Yoshihara et al., 1996; Durgbanshi et al., 2005; Balcke et al., 2012; Liu et al., 2012; Glauser et al., 2014). Isopropanol and methyl tert-butyl ether (MTBE) are also widely used for extraction in both targeted and non-targeted metabolomic studies aiming to investigate changes in phytohormonal profiles (Glauser et al., 2008; Stumpe et al., 2010; Müller and Munné-Bosch, 2011; Ternes et al., 2011). While organic solvents offer better analyte solubility, sample contamination with pigments and other interfering compounds can be minimized by extraction into a buffer of neutral or acidic pH such as phosphate-sodium buffer (Prinsen et al., 2000; Novák et al., 2012). The processes used for further sample enrichment should be designed to suit the final analysis. The target profiling of many metabolites from crude plant extracts may be hindered by signal suppression, due to strong matrix effects. These can be mitigated by using solid-phase extraction (SPE), which has been used for decades in bioanalysis and nowadays is more employed than non-selective liquid–liquid partitioning and subsequent filtration (Dobrev et al., 2005; Fan et al., 2011; Glauser and Wolfender, 2013). Several multistep SPE methods combining silica-based reversed-phase (RP) sorbents with long alkyl chains (C18) or polymer-based RP materials with ion-exchange properties (mixed-mode sorbents) have proven to be effective for purifying acidic plant hormones (Baldwin et al., 1997; Dobrev et al., 2005; Balcke et al., 2012; Jikumaru et al., 2013). Because many plant hormones of interest are present only at trace levels in tissue samples and are non-volatile (with the exception of the volatile MeJA), RP-based separations using capillary-LC or ultra-high performance liquid chromatography (UHPLC) systems interfaced with electrospray tandem mass spectrometry (ESI–MS/MS) have become popular for their analysis (Wilbert et al., 1998; Segarra et al., 2006; Forcat et al., 2008; Müller and Munné-Bosch, 2011; Balcke et al., 2012). Non-destructive LC methods are also preferred due to minimal and straightforward sample preparation without the time-consuming analyte derivatisation procedures required by GC–MS approaches (Mueller et al., 1993, 2006; Engelberth et al., 2003). Many publications have described simultaneous quantitative analyses of stress-induced phytohormones such as JA, SA, and ABA together with the other phytohormone classes (cytokinins, auxins and gibberellins) using highly selective MS monitoring of precursor-to-product ion transitions - MRM mode (Tamogami and Kodama, 1997; Wilbert et al., 1998; Balcke et al., 2012; Kojima and Sakakibara, 2012). Here, we describe a novel sensitive and selective profiling method for analysing a broad range of jasmonates and other stress-induced phytohormones including SA, ABA, and IAA. The new methodology significantly reduces the impact of matrix effects by using optimized conditions for sample extraction and purification, and allows the determination of analytes with diverse physicochemical properties present in the plant tissue at very low levels. The combination of an effective SPE process using a polymeric reversed-phase sorbent with a sensitive UHPLC–ESI–MS/ MS method enabled the exact quantification of sixteen compounds

(13 JAs, SA, ABA, and IAA) in minute Arabidopsis leaf tissues. Our novel high-sensitive method provides detailed insights into the changes in phytohormone profiles that occur following wounding and will be applicable in studies of many stress-related responses and developmental processes involving these phytohormones. 2. Results and discussion The methodology reported herein was primarily designed to establish a sensitive MS-based approach for the simultaneous profiling of stress-induced phytohormones including most of the JA metabolites (Fig. 1). The protocol was developed to enable the rapid and effective extraction of target compounds from a minimal quantity of plant material, with efficient isolation and effective enrichment together with highly selective and extremely sensitive analysis. 2.1. Optimization of extraction and purification protocols In general, the main purpose of sample preparation procedures prior to an analyte detection using selected analytical method is to reduce sample complexity while maintaining high extraction efficiency of target compounds – in this case, low-abundance phytohormones. Depending on the chemical properties of the extraction solvent, crude plant extracts may contain large quantities of substances that will interfere with subsequent analyses (e.g., proteins, carbohydrates, pigments and lipids). To develop an improved extraction protocol, we optimized the method of sample processing (extraction and purification) and sample size in order to minimize analyte losses and reduce contamination with non-polar extractable substances. To begin with, the extraction efficiencies of four cold methanolic solvents (80% methanol, 50% methanol, 10% methanol, and 10% methanol acidified with 0.1% formic acid) were tested. Arabidopsis shoot extracts with a fresh weight of 20 mg each were prepared in quadruplicate for each extraction solution and spiked with a mixture of JAs standards of 20 pmol each with the exceptions of MeJA (50 pmol) and trans/cis-(+)-OPDA (100 pmol). After flow-through purification using an OasisÒ HLB (HLB) cartridge, the total yields for the JAs were 25 ± 14%, 27 ± 12%, and 56 ± 22% for cold 80%, 50% and 10% methanolic solutions, respectively. When the experiment was repeated using an acidified solution of aqueous methanol (10%), the overall JAs recovery for the extraction and one-step SPE protocol was 53 ± 28%. Interestingly, the yield of acidic plant hormones was not affected by acidifying the extraction solution. In accordance with previously published data (Urbanová et al., 2013), the plant pigment content of the extracts increased rapidly with the methanol content of the extraction solution, yielding samples of insufficient purity for UHPLC–MS/MS analysis. Therefore, the optimized amount of sample and type of organic extraction solvent would reduce ion suppression or enhancement caused by the sample matrix and interferences from metabolites as well as sample throughput by increasing the sample preparation time (Novák et al., 2012). Consequently, all subsequent experiments were performed using 20 mg samples extracted with unacidified 10% methanol. To maximize the sensitivity and selectivity of the final MS-based analysis, we sought to combine the efficient extraction protocol described above with a purification protocol that would afford high analyte recovery. We therefore decided to use a one-step purification protocol based on a HLB column washed with 10% methanol and eluted using 80% methanol (Fig. 2). The HLB sorbent is described as a macroporous copolymer [poly-(divinylbenzeneco-N-vinylpyrrolidone)] with both hydrophilic and lipophilic retention characteristics. This polymeric reversed-phase sorbent has been also preferred SPE material for one-step SPE of acidic plant

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Fig. 1. Structures of the analysed phytohormones and their abbreviations.

hormones in multiple recently developed methods (Izumi et al., 2009; Novák et al., 2012). By performing low-specific purification of 20 mg plant extract samples, we were able to avoid exceeding the sorbent’s capacity and to increase the process efficiency (PE) for all jasmonates as well as IAA, ABA and SA (Table 1). As written above, acidification of the extraction solvent weakly improved the yields of more polar analytes but did not affect the overall PE rate of target analytes, which was 53% for acidified 10% methanol and 56% for non-acidified 10% methanol. Most of the tested metabolites were strongly retained under our optimized loading, washing and elution conditions as shown in Fig. 2, giving more than 50% PE of the purification method for 20 mg FW samples (see Table 1). In particular, adequate PEs were achieved for the key compounds

but volatile MeJA and non-polar trans/cis-(+)-OPDA, whose PEs ranged from 13% to 38% for 20 mg FW samples. On the other hand, HLB cartridges contain 30 mg of highly retentive sorbent and should be able to accommodate up to 10 mg FW extract without any recovery interference. Our data clearly indicate that the 24-day-old Arabidopsis tissues contain higher amount of interfering compounds, which decrease the efficiency of purification process as well as the efficiency of MS ionization (see Section 2.2). To investigate this issue, we also examined the compound-specific two-step SPE protocol (combination of HLB/MAX columns), which afforded high yields of IAA and ABA, with overall mean PE of 70% and 80% for experiments in the presence and absence of a plant matrix, respectively (Supplemental Fig. S1). However, the recoveries for the more

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K. Floková et al. / Phytochemistry 105 (2014) 147–157 Table 1 Process efficiency (PE) of the low-specific one-step SPE protocol. All experiments were performed in quadruplicate using an aqueous solution of methanol (10% MeOH/H2O, v/v) and 20 mg FW Arabidopsis leaf tissue extracts spiked before extraction with known quantities of the target compounds: 20 pmol of all JAs and derivatives other than MeJA (50 pmol) and trans/cis-(+)-OPDA (100 pmol), 10 pmol of ABA and IAA, 100 pmol of SA. Spiked extraction solution (Blank) and tissue extracts (20 mg FW) were purified using one-step (OasisÒ HLB) SPE protocol, then analysed by UHPLC– ESI–MS/MS. Process efficiency expressed as the ratio of the mean peak area of an analyte spiked before extraction to the mean peak area of the same analyte standards multiplied by 100. Compound

cis-(+)-OPDA trans-OPDA OPC-4 OPC-6 ( )-JA MeJA 9,10-dh-JA P 11-OH-JA/12-OH-JA JA-Ile JA-Val JA-Phe JA-Trp SA ABA IAA a

Fig. 2. Sample preparation process for acidic phytohormone determination using the low-specific one-step SPE protocols. Plant material (10–25 mg FW) was extracted using an aqueous solution of methanol (10% MeOH/H2O, v/v) with stable isotope-labelled standards. The extracts were purified using the HLB sorbent. All obtained fractions containing neutral and acidic compounds were evaporated to dryness, dissolved in 25 ll of acetonitrile/10 mM HCOOH (15:85, v/v) and 10 ll was injected directly into the UHPLC–ESI–MS/MS system.

hydrophilic SA and most of the JAs varied more widely, from 14% to 53% for samples of 20 mg FW, and the highest recoveries for MeJA were only 4%. The oxylipin intermediates in JA biosynthesis, trans-OPDA and cis-(+)-OPDA, were present at levels below the limit of detection. Overall, these results show that the two-step SPE process with two evaporation steps can significantly reduce the effectiveness of SPE methods for determining diverse JA metabolites due to their high volatility, lowest chemical stability and/or irreversible retention on the sorbent. Therefore, the optimized one-step SPE conditions were duly incorporated into the newly developed extraction protocol and used in conjunction with a highly selective and sensitive UHPLC–ESI–MS/MS method.

2.2. Development of UHPLC–MS/MS method At present, acidic plant hormones are generally quantified by MS using the isotope dilution technique (Rittenberg and Foster,

PEa (%) Blank

20 mg FW

16 ± 0 18 ± 0 92 ± 9 50 ± 2 77 ± 4 13 ± 0 84 ± 3 96 ± 2 93 ± 6 83 ± 1 96 ± 7 70 ± 4 89 ± 3 90 ± 1 69 ± 1

38 ± 1 38 ± 1 89 ± 11 55 ± 7 43 ± 1 13 ± 0 73 ± 3 60 ± 5 84 ± 12 64 ± 4 95 ± 9 72 ± 7 75 ± 3 53 ± 1 94 ± 11

All quoted values are means ± SD (n = 4).

1940). GC–MS is useful for the quantitative analysis of JAs (Miersch and Wasternack, 2000; Engelberth et al., 2003). However, several recent reports have demonstrated that LC–MS can also be a very potent tool for quantifying JAs, ABA, IAA and SA (Wilbert et al., 1998; Segarra et al., 2006; Forcat et al., 2008; Müller and Munné-Bosch, 2011; Balcke et al., 2012). The rapid development of chromatographic techniques such as UHPLC using sub-2-micron particle columns has greatly improved the speed, separation, resolution and sensitivity of LC-based analyses relative to those achieved with conventional HPLC. LC-based separations of JA and its metabolites are typically performed with silica- or polymerbased columns (Forcat et al., 2008; Matsuura et al., 2009; Müller and Munné-Bosch, 2011; Balcke et al., 2012; Liu et al., 2012). Our UHPLC method uses an Acquity UPLCÒ CSH™ (Charged Surface Hybrid) column because it provided a better peak shape and peak-to-peak resolution than the Acquity UPLCÒ BEH column (Urbanová et al., 2013; Supplemental Fig. S2). Optimal separation was achieved by using 10 mM HCOOH and acetonitrile as solvents A and B (see Fig. 3). Twelve of the 16 acidic plant hormone metabolites listed in Table 2 were fully resolved under these reversedphase UHPLC conditions. The non-polar analytes cis-(+)-OPDA and trans-OPDA, were adequately separated under these conditions, with retention times of 19.51 and 19.75 min, respectively. However, the chromatographic resolution of the most polar analytes, 11-OH-JA and 12-OH-JA, and the JA epimers, ( )-JA and (+)-7-iso-JA, was insufficient to enable their separate quantification. Consequently, they were quantified together; the combined P measurements are denoted by the sum 11-OH-JA/12-OH-JA and total JA. Recently, Glauser and co-workers (2008) independently reported the RP-based separation of both hydroxyjasmonates on an Acquity UPLCÒ BEH column using isocratic gradient elution with aqueous formic acid/acetonitrile (92:8, v/v). Their chromatographic conditions can thus be used to separate 11-OH-JA and 12-OH-JA in cases where doing so might provide additional biological insights. Hyphenated MS techniques, particularly GC–MS or LC–MS, are suitable for high-throughput analysis, providing excellent separation and high sensitivity. However, complex multi-component

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Fig. 3. Chromatographic separation of JAs, IAA, SA and ABA standards by UHPLC–ESI–MS/MS. The figure shows the MRM sums for 16 phytohormones represented by 10 pmol of each compound per injection using an Acquity UPLCÒ CSH™ C18 2.1  100 mm, 1.7 lm column.

Table 2 Optimized UHPLC–MS/MS parameters for the quantification of JA, some of its biosynthetic precursors and derivatives, SA, ABA and IAA. Compound

Retention time (min)

11-OH-JA 12-OH-JA IAA SA ABA ( )-JA JA-Val 9,10-dh-JA OPC-4 JA-Ile JA-Trp JA-Phe MeJA OPC-6 cis-(+)-OPDA trans-OPDA

2.34 ± 0.02 2.34 ± 0.02 6.15 ± 0.02 8.14 ± 0.03 8.90 ± 0.01 10.88 ± 0.01 11.91 ± 0.01 12.59 ± 0.01 13.70 ± 0.01 13.78 ± 0.01 13.89 ± 0.01 14.20 ± 0.01 14.64 ± 0.01 16.97 ± 0.01 19.51 ± 0.02 19.75 ± 0.01

Scan mode

+

+

+ + + + + + +

MRM transition

Cone voltage (V)

Collision energy (eV)

LOD (fmol)

Linear range (pmol)

R2

225.2 > 58.2 225.2 > 58.2 176.3 > 130.2 137.1 > 92.8 263.2 > 153.1 209.2 > 58.8 310.3 > 151.3 211.2 > 58.8 237.2 > 58.8 324.3 > 151.2 397.3 > 351.3 358.8 > 151.2 225.3 > 151.2 267.3 > 135.2 293.3 > 275.3 293.3 > 275.3

30 30 30 25 23 30 30 30 30 30 30 30 30 30 30 30

23 23 20 13 12 23 23 23 23 23 17 23 15 20 15 20

50 50 10 50 0.5 5 0.5 10 50 0.5 10 0.05 10 50 10 10

0.1–100 0.1–100 0.05–100 0.1–100 0.01–100 0.05–250 0.01–500 0.05–250 0.1–500 0.01–100 0.05–100 0.005–500 0.05–250 0.1–250 0.1–250 0.05–500

0.997 0.997 0.998 0.994 0.997 0.998 0.999 0.999 0.996 0.991 0.992 0.997 0.996 0.995 0.999 0.998

plant matrices can limit the efficiency of MS ionization (Novák et al., 2012). Having established a highly sensitive method for acidic phytohormone metabolite analysis, we tested the extent to which the plant matrix from our samples suppressed the MS signals of interest. Purified Arabidopsis shoot extracts were spiked with 20 pmol of each JA metabolite other than MeJA (50 pmol) and trans/cis-(+)-OPDA (100 pmol). The signal intensity for each analyte in the presence of the plant matrix was compared to that observed for samples of unused extraction solution (10% methanol) spiked in the same way. A weak matrix effect was observed, causing a slight reduction in MS signal intensity - the mean reduction for all JA metabolites was 21 ± 2% (Supplemental Table S1). All of the acidic plant hormones and their metabolites were amenable to LC–MS analysis in both positive and negative ESI mode using polarity switching without derivatization (Table 2). In keeping with previously published results (Wilbert et al., 1998; Segarra et al., 2006; Forcat et al., 2008; Müller and Munné-Bosch, 2011; Balcke et al., 2012), all analytes generated strong signals corresponding to their protonated [M+H]+ and de-protonated [M H] molecular ions. The most abundant product ions for each precursor were selected for diagnostic MRM analysis (Table 2). Based on the

reproducibility of the retention times for each analyte (Table 2), the 29 min period required for the chromatographic separation of the target analytes was divided into 10 scan segments, with dwell times ranging from 0.1–1.2 s to provide 16 data points across a chromatographic peak (see Section 4.5.). It should be noted that UHPLC–ESI–MS/MS methods for the profiling of stress-induced phytohormones sometimes have relatively high limits of detection due to the complexity of plant matrices. The modest matrix effects seen for samples extracted and purified using our new protocol underline its likely usefulness in phytohormone profiling. 2.3. Method validation To determine the sensitivity parameters of the new method, 13-point calibration curves were constructed for each target analyte based on the response areas for triplicate injections of serially diluted authentic standard solutions. The analyte content of the dilutions ranged from 0.001–500 pmol and each dilution also contained a known amount of an appropriate deuterium-labelled internal standard (3 pmol of [2H5]-OPDA, 5 pmol of [2H6]-JA and [2H2]-JA-Ile, 10 pmol of [2H4]-SA, [2H6]-ABA, [2H5]-IAA). The

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Table 3 Method validation – method precision and accuracy for the low-specific SPE procedure. Extracts of 20 mg FW Arabidopsis leaves were spiked with 10 pmol (a) or 100 pmol (b) of authentic standards, purified by one-step SPE using the OasisÒ HLB sorbent, and analysed by UHPLC–MS/MS. Compound

Determined spiked content (pmol)a

Method precision (% RSD)a

Method accuracy (% bias)a

Determined spiked content (pmol)b

Method precision (% RSD)b

Method accuracy (% bias)b

cis-(+)-OPDA trans-OPDA OPC-4 OPC-6 ( )-JA MeJA 9,10-dh-JA P 11-OH-JA/12-OH-JA JA-Ile JA-Val JA-Phe JA-Trp SA ABA IAA

NC 11.0 ± 0.54 10.4 ± 0.6 11.3 ± 0.9 10.1 ± 0.7 11.1 ± 1.7 10.5 ± 0.4 NC 10.4 ± 0.1 11.3 ± 0.2 11.9 ± 0.3 10.9 ± 0.6 11.6 ± 0.9 9.3 ± 0.2 9.3 ± 0.3

NC 4.9 5.3 7.9 7.2 15.0 3.4 NC 1.1 1.9 2.0 5.8 7.5 2.0 3.5

NC 10.2 3.8 13.4 1.2 11.3 5.3 NC 3.9 12.9 19.0 8.5 16.3 7.0 7.3

104.2 ± 9.2 110.0 ± 3.1 102.8 ± 1.9 112.7 ± 12.9 94.2 ± 3.1 47.8 ± 3.6 113.8 ± 2.2 NC 104.3 ± 0.8 109.4 ± 1.0 113.9 ± 2.9 98.2 ± 2.3 109.1 ± 4.1 87.6 ± 2.2 98.6 ± 1.4

8.8 2.8 1.8 11.4 3.3 7.5 1.9 NC 0.7 0.9 2.6 2.3 3.7 2.5 1.5

4.2 10.0 2.8 12.7 5.8 52.3 13.8 NC 4.3 9.4 14.0 1.8 9.1 12.4 1.4

All values are means ± SD (n = 4), NC denotes non-calculated values.

resulting data were transformed using the logarithm function and plotted. These plots had linear regions extending over at least seven points of the calibration curves for most of target compounds. Most of the curves had a linear region extending from 5( 50) fmol to 100 pmol with R2 values of P0.991 (Table 2). Limits of detection (LOD) for each compound were determined as the amount of compound that would be expected to yield a signalto-noise ratio (S/N) of P3. Different metabolite groups had different LODs, reflecting their physicochemical properties and ionization behaviour. The highest S/N ratio was achieved for the amide conjugates JA-Phe and JA-Val/JA-Trp, for which the limits of detection were close to 50 and 500 amol, respectively (Table 2). The minimum detectable amounts of the other target metabolites were all 50 fmol or less. Importantly, the method’s LOD and linearity were comparable to or better than those of other published methods for the determination of JAs and acidic plant hormones (Balcke et al., 2012; Liu et al., 2012). Overall, the method’s high sensitivity enables the facile analysis and determination of stress-induced hormones in extracts derived from 20 mg FW Arabidopsis leaf samples. The effectiveness of the new method was demonstrated by spiking aliquots of a blank plant matrix (20 mg FW) with a standard mixture containing 10 or 100 pmol of authentic standards for all of the studied analytes and 20 pmol of the corresponding internal standards ([2H6]-JA, [2H2]-JA-Ile, [2H5]-OPDA, [2H4]-SA, [2H6]-ABA, [2H5]-IAA). After analyte extraction with 10% MeOH and isolation by one-step SPE, UHPLC–ESI–MS/MS analysis was performed as described above. The amount of each added compound present after extraction and analysis was determined by the standard isotope dilution method using appropriate stable isotope-labelled standards. The endogenous levels of each target analyte were determined by analysing non-spiked Arabidopsis leaf extracts and subtracted from the amount of added standard. In addition, deuterium-labelled JA was successfully used to quantify all of the other JA metabolites of interest (the measured analyte contents of the spiked samples are listed in Table 3). The method’s precision was determined by analysing four replicate samples spiked at 10 pmol per analyte and another four spiked at 100 pmol per analyte, and determining the relative standard deviation for each spiking level (RSD%). In addition, the method’s accuracy was assessed in terms of its percentage bias (Table 3). Because the hydroxylated JA derivatives were not well separated (Fig. 3), it was impossible to validate the measured results for 11-OH-JA and 12-OH-JA individually. Moreover, the relatively high endogenous levels of cis-(+)-OPDA in Arabidopsis tissues made it

Fig. 4. Experimental design. Half of the fully developed rosette leaves of 24 day-old A. thaliana plants were wounded using forceps on the one side of the central vein to induce mechanical stress (black cross). Both local (wounded) and distal (unwounded) leaves were harvested and screened to determine their stressinduced phytohormone metabolite profiles.

impossible to detect small variations in it abundance at the low picomolar level (10 pmol). Precision and accuracy estimates for this compound were therefore based on data for blank samples and matrix samples spiked with 100 pmol of the appropriate standard (Table 3). The mean precision obtained in spiking experiments with Arabidopsis extracts was 4.4%, with a range of 0.7–15%, and the mean accuracy for compounds other than MeJA was 3.6% with a range of 12.4–19.0%. The accuracy for MeJA was 52.2% based on samples spiked with 100 pmol of standard. The very low recovery obtained for highly volatile MeJA was most likely due to analyte loss during sample evaporation after SPE. Matrix-free control experiments using spiked extraction solution samples (Supplemental Table S2) yielded similar precision and accuracy results, with overall means of 3.6 ± 1.1% and 1.1 ± 13.0%, respectively. This demonstrates the power of stable isotope dilution assays to compensate for losses or inefficiencies in the sample preparation process and for ion suppression effects during MS analysis. These validation experiments and the calculated accuracy and precision of the new method demonstrate its reliability and usefulness for the routine determination of acidic phytohormone levels (including those of most jasmonate metabolites) in minute samples of plant material.

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Table 4 Maximal endogenous levels of analysed compounds in wounded and unwounded Arabidopsis leaves. Plant material (20 mg FW) was extracted, purified using the low-specific SPE procedure and analysed by UHPLC–MS/MS. Compound

cis-(+)-OPDA trans-OPDA OPC-6 OPC-4 JA MeJA 9,10-dh-JA P 11-OH-JA/12-OH-JA JA-Val JA-Ile JA-Trp JA-Phe SA ABA IAA

Phytohormonal content (pmol g

1

FW)

Unwounded (control) leaves [time 0 h]

Wounded (local) leaves [time]

Unwounded (distal) leaves [time]

4246.3 ± 394.3 191.6 ± 11.2 43.0 ± 0.8 22.2 ± 1.2 67.6 ± 7.9 123.4 ± 9.9 179.0 ± 12.1 67.2 ± 3.0 0.3 ± 0.0 1.1 ± 0.1 ND ND 202.1 ± 17.5 2.6 ± 0.1 20.4 ± 0.5

11041.6 ± 31.5 [0.5 h] 1464.4 ± 199.[24 h] 74.1 ± 1.0 [1.0 h] 304.3 ± 43.6 [1.0 h] 2186.5 ± 73.9 [0.5 h] 348.9 ± 9.6 [0.5 h] 347.5 ± 9.3 [1.5 h] 922.5 ± 35.4 [3.0 h] 1.2 ± 0.0 [3.0 h] 6.1 ± 0.2 [1.0 h] ND ND 1084.5 ± 49.1 [12 h] 15.3 ± 1.0 [1.0 h] 50.6 ± 5.4 [1.0 h]

8262.3 ± 910.2 [1.0 h] 515.8 ± 18.4.[1.0 h] 60.7 ± 2.5.[3.0 h] 158.4 ± 1.3.[1.0 h] 642.6 ± 56.4.[1.0 h] ND 193.4 ± 2.6.[0.5 h] 473.7 ± 43.6.[6.0 h] 0.4 ± 0.0.[1.0 h] 1.7 ± 0.1.[1.0 h] ND ND 855.6 ± 9.7 [12 h] 6.5 ± 0.3 [1.5 h] 38.8 ± 0.8 [1.0 h]

All values are means ± SD (n = 4); time after wounding (h); ND – not detected.

Fig. 5. Wound-induced accumulation of selected JAs – cis-(+)-OPDA (a), ( )-JA (b), JA-Ile (c) – and the other stress-induced phytohormones – SA (d), ABA (e), IAA (f) – in 24days-old Arabidopsis thaliana Col-0. Local, wounded (light bars) and distal unwounded (dark bars) leaves were monitored. Plant material (20 mg FW) was extracted and purified by one-step SPE using the HLB sorbent, followed by UHPLC–MS/MS analysis. All quoted values are means ± SD (n = 4).

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2.4. Quantification of selected phytohormones after mechanical stress Having established an effective one-step SPE purification and MS-based profiling protocol, it was used to investigate the temporal and spatial dynamics of phytohormone levels in 20 mg FW plant tissue samples. To reduce the impact of biological variation, sets of 20–25 Arabidopsis plants were used for metabolite profiling. Every second fully developed rosette leaf on each plant was mechanically wounded and samples were collected at 8 time-points over a 24-h collection period (Fig. 4). In total, 14 compounds were determined using the new UHPLC–ESI–MS/MS method in both local (wounded) and distal (unwounded) tissue samples (Table 4). Only two of the target metabolites, JA-Trp and JA-Phe, were present at levels below the limit of detection. There were no significant changes in the levels of any of the target metabolites in unwounded (control) Arabidopsis leaves over the 24 h sampling period (Supplemental Table S3). This demonstrates the stable conditions of the wounding experiments and the accuracy of stress-induced phytohormone determination using the new method. Some compounds were detected in minute quantities (>50 pmol g 1 FW), including ABA, IAA, JA biosynthetic precursors 3-oxo-2-(2-(Z)-pentenyl)cyclopentane-1-hexanoic acid (OPC-6) and 3-Oxo-2-(2-(Z)-pentenyl)cyclopentane-1-butyric acid (OPC-4) as well as JA amino acid conjugates JA-Val and JA-Ile. P Trans-OPDA, JA, MeJA, 9,10-dh-JA, 11-OH-JA/12-OH-JA. However, moderate quantities of SA were found (50–500 pmol g 1 FW) and cis-(+)-OPDA was detected at very high levels (>500 pmol g 1 FW) – see Supplemental Table S3. This distribution of metabolites and the trends in their expression over time following wounding are in good agreement with previous reports (Glauser et al., 2009; Stintzi et al., 2001).

Local wounding of leaf tissues caused a substantial accumulation of JA (Fig. 5b) that peaked 30 min after wounding at a level 33 times higher than in control leaves. JA levels also increased ninefold in distal unwounded leaves, reflecting the systemic response to wounding. In addition, there was a pronounced but less rapid increase in the abundance of the physiologically active compound JA-Ile, whose abundance peaked 1 h after wounding and then again 12 h after wounding (Fig. 5c). This increase was accompanied by the accumulation of cis-(+)-OPDA (Fig. 5a), which is the first cyclic compound in the JA biosynthetic pathway (Wasternack and Kombrink, 2010; Wasternack and Hause, 2013). A similar OPDA accumulation profile with two maxima at the same time points has been observed in wounded tomato leaves (Balcke et al., 2012). Our experimental conditions enabled the quantification of direct JA precursors based on the chromatographic separation of trans-OPDA from its enzymatically formed cis-(+) isomer (Fig. 6). It should be noted that the hexadecanoid pathway product dn-OPDA is also a direct precursor of JA in Arabidopsis (Wasternack and Kombrink, 2010) but was not analysed in this work. However, since our new method enables the efficient recovery and determination of OPDA, it should also be applicable to the determination of dn-OPDA in cases where this would provide valuable biological information. In contrast, the new method was not capable of separating the polar hydroxylated JA derivatives. These two compounds were therefore quantified together. Glauser and co-workers (2008) have observed stress-induced changes in the abundance of 11-OH-JA in Arabidopsis plants and shown that its profile is similar to that for 12-OH-JA. In keeping with previous results (Glauser et al., 2008; Glauser et al., 2009; Balcke et al., 2012), most of the JA biosynthetic precursors and derivatives behaved in a way that mirrored that of endogenous JA and JA-Ile.

Fig. 6. Representative MRM chromatograms of endogenous stress-induced compounds (JA, JA-Ile, trans/cis-(+)-OPDA, SA, ABA and IAA) in 20 mg FW of locally stressed Arabidopsis leaves 30 min after wounding.

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P However, 11-OH-JA/12-OH-JA responded more slowly: their levels peaked around 3 h after wounding. It is also noteworthy that the levels of another compound with a late accumulation pattern, JA-Val, correlated strongly with those of OH-JA (see Supplemental Table S4). The levels of other important defence-signalling plant hormones such as SA, ABA and IAA were also determined (Fig. 5d–f). Wounding caused increases in the levels of all of these compounds at both local and distal sites: the levels of ABA/IAA peaked after one hour while those of SA peaked after around 12 h. Overall, these results confirm that our new method for the MS-based target profiling of stress-induced phytohormones is a powerful tool with high levels of specificity and sensitivity. 3. Conclusions A highly sensitive MS-based method has been developed for the extraction and quantification of 16 stress-induced phytohormones including JA, its biosynthetic precursors and amino acid conjugates, SA, ABA and IAA. In addition, sample extraction and purification conditions were optimized to enable the efficient recovery of highly unstable analytes and to minimize the impact of matrix effects. To our knowledge, this is the first simple, sensitive and precise method that can be used to determine most jasmonate metabolites in minute plant tissue samples of less than 20 mg FW. The method has clear advantages over alternative techniques for the screening of stress-induced phytohormone levels due to its ability to accommodate very small samples and highly selective detection. Validation experiments were performed, demonstrating the method’s high selectivity and sensitivity. 4. Experimental 4.1. Chemicals and materials Authentic and stable isotopically labelled standards (Fig. 1) – ( )-JA, (±)-11-OH-JA, (±)-12-OH-JA, (±)-JA-Me, (±)-9,10-dh-JA, (±)-OPC-4, (±)-OPC-6, trans-OPDA, cis-(+)-OPDA, ( )-JA-L-Ile, ( )JA-L-Val, ( )-JA-L-Phe, ( )-JA-L-Trp, IAA, SA, (+)-cis,trans-ABA, [2H6]-(±)-JA, [2H2]-( )-JA-Ile, [2H5]-IAA, [2H4]-SA, [2H6]-(+)-cis,trans-ABA – were provided by Olchemim Ltd (Olomouc, Czech Republic). Cis-(+)-OPDA and [2H5]-OPDA were synthesized enzymatically from a-linolenic acid (Sigma Aldrich, St. Louis, MO, USA), and (17,17,17,18,18-[2H5])-linolenic acid ethyl ester (Larodan Fine Chemicals, Malmö, Sweden) using flaxseed extracts according to a modification of a procedure described elsewhere (Zimmerman and Feng, 1978). The resulting synthetic products were dissolved in 100% methanol (gradient grade, Merck, Darmstadt, Germany) and subsequently purified by semi-preparative HPLC system (Waters 1525 Binary HPLC Pump; JetStream 2 Plus column thermostat; Waters, Milford, MA, USA). The standard fraction was separated using an RP column (LiChrospherÒ 100 RP 18; 4  250 mm; 5 lm; Phenomenex, Torrance, California, USA) with a flow rate of 0.6 ml/min under isocratic conditions using 2% acetic acid/water (80% A, v/v) and methanol (20% B, v/v) as the mobile phase constituents. Detection was achieved using a Waters 2487 Dual k Absorbance Detector (Waters, Milford, MA, USA) at 223 nm. Acetonitrile with formic acid as an eluent additive (both of LC–MS grade) from Sigma–Aldrich (St. Louis, MO, USA) was used in the UHPLC–ESI–MS/MS analysis. All aqueous solutions were prepared using deionized (Milli-Q) water from a Simplicity 185 System (Millipore, Bedford, MA, USA). SPE sample enrichment and purification was performed using OasisÒ HLB (1 cc/30 mg) and OasisÒ MAX (1 cc/30 mg) columns purchased from Waters Co. (Milford, MA, USA).

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4.2. Plant growth and wounding treatment Arabidopsis thaliana (ecotype Col-0) was used in all experiments. Seedlings were grown in a growth chamber under long day conditions at a light intensity of 100 lE m 2 s 1 (16 h light, 24 °C/8 h dark, 18 °C) for 24 days. To control for biological variation, about 200 Arabidopsis seedlings were grown and four biological replicates involving 20–25 plants each were used in the quantification experiment. After 24 days, forceps were used to cause mechanical damage covering 20% of the leaf area on half of the fully developed rosette leaves of each plant in order to induce stress (Fig. 4). Wounded and unwounded systemic leaves were harvested separately at 8 time points (0; 0.5; 1; 1.5; 3; 6; 12; and 24 h) and immediately frozen in liquid nitrogen. Control plants were harvested before and after the experiment. Samples were stored at 80 °C until extraction and analysis. 4.3. Sample extraction For the quantification experiments, the amount of plant material of 20–25 mg (fresh weight, FW) were ground into a fine powder using a mortar and pestle under liquid nitrogen and individually transferred into 2 ml plastic microtubes (Eppendorf, Germany), containing 2-mm ceria-stabilized zirconium oxide beads (Retsch GmbH & Co. KG, Haan, Germany). Stable isotopically labelled internal standards ([2H6]-(±)-JA, [2H2]-( )-JA-Ile, [2H5]OPDA, [2H5]-IAA, [2H4]-SA, [2H6]-(+)-cis,trans-ABA) were added (20 pmol to each sample) in order to determine analyte recovery during purification procedure and to validate the determination of their corresponding counterparts. The frozen leaf material was homogenized in 1 ml of ice cold 10% MeOH/H2O (v/v) extraction solution using an MM 301 vibration mill with (Retsch GmbH & Co. KG, Haan, Germany) at a frequency of 27 Hz for 3 min. Samples were sonicated for 3 min at 4 °C using a laboratory ultrasonicator with an ice block-filled bathtub (Transsonic T310, Elma GmbH & Co KG, Singen, Germany) and subsequently extracted using a benchtop laboratory rotator Stuart SB3 (BibbyScientific Ltd., Staffordshire, UK) for 20 min at 4 °C. After centrifugation (3 min, 20 000 rpm, 4 °C; BeckmanAvanti™ 30) the supernatants were transferred into clean plastic microtubes and the pelet was reextracted with 1 ml of ice cold 10% MeOH/H2O (v/v) by quick short spin stirring (vortex, Velp Scintifica, Usmate, Italy) and separated by centrifugation (3 min, 20,000 rpm, 4 °C). The combined supernatants were further purified according to the scheme shown in Fig. 2. 4.4. Solid-phase extraction In the first purification step, all samples were pre-concentrated by RP polymer-based solid phase extraction (OasisÒ HLB columns). The SPE sorbent was activated by 1 ml of 100% MeOH and equilibrated with 0.1% HCOOH/H2O (v/v). After loading the sample, the column was washed with 1 ml of extraction solution before the analytes were eluted with 3 ml of 80% MeOH/H2O (v/v). The samples intended for analysis after single-step SPE were then evaporated to dryness under gentle stream of nitrogen using a TurboVapÒ LV evaporation system (Caliper Life Sciences, Hopkinton, MA, USA) and stored in a freezer at 20 °C until analysis. For UHPLC–MS/MS analysis, the samples were reconstructed in 25 ll of the mobile phase (15% acetonitrile: 85% 10 mM HCOOH, v/v). For evaluation of the matrix effect and assay validation, three sets of samples were prepared and then analysed by UHPLC– ESI–MS/MS system. Simultaneously, non-spiked Arabidopsis leaf extracts were analysed and the endogenous levels of selected

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compounds were subtracted from the amount of added standard. In the first set, standards of the analytes present in the pure extraction solvent (10% methanol) were analysed directly at investigated concentrations as follows: 20 pmol of all JAs and derivatives other than MeJA (50 pmol) and trans/cis-(+)-OPDA (100 pmol), 10 pmol of ABA and IAA, 100 pmol of SA and 20 pmol of stable isotopelabelled standards. In the second set, 20 mg FW of plant tissues were first extracted and spiked before purification using one-step SPE using the HLB sorbent with 10, 20, 50, or 100 pmol of authentic and internal standards (depending on the natural abundance of the analyte in the plant tissue). In set 3, analytes were similarly spiked in the range 20–100 pmol after sample processing (extraction and purification) into plant extract samples (20 mg FW). The matrix effects (Supplemental Table S2) were calculated as the ratio of the mean peak area of an analyte spiked postextraction (set 3) to the mean peak area of the same analyte standards (set 1) multiplied by 100. The percent process efficiencies (Table 1; Supplemental Fig. S1) were determined as the mean peak area of the added standards before sample preparation (set 2) divided by the know mean peak area of standard solutions (set 1). For assessment of the method validation (Table 3), the concentration of each analyte was calculated using the standard isotope dilution method for each plant extract spiked before extraction and compared with the concentration of appropriate standard solution. Each experiment was performed in quadruplicate. 4.5. UHPLC–MS/MS conditions JA and its metabolites, ABA, SA, and IAA were analysed by an Acquity UPLCÒ System (Waters, Milford, MA, USA) coupled to a triple quadrupole mass spectrometer Xevo™ TQ MS (Waters MS Technologies, Manchester, UK), and 10 ll of each sample was injected onto a RP column (Acquity UPLCÒ CSH™ C18; 2.1  100 mm; 1.7 lm; Waters, Ireland) at a flow rate of 0.4 ml min 1. The column was maintained at 36 °C. The compounds of interest were separated by a gradient elution using 10 mM HCOOH (A) and acetonitrile (B) over 35 min. as follows: 0–5 min isocratic elution (15% A; v/v); 5–15 min linear gradient to 45% A; 15–28 min, logarithmic gradient to 48.6% A; 28–29 min linear gradient to 100% A. Finally, the column was washed with 100% acetonitrile and then equilibrated to the initial conditions (15% A, v/v) for 5 min. The eluate was introduced into the electrospray ion source of a tandem MS analyser and analysed using the following MS/MS conditions: source/desolvation temperature, 120/550 °C; cone/ desolvation gas flow, 70/650 L h 1; capillary voltage, 3 kV; cone voltage, 23–30 V; collision energy, 12–23 eV; collision gas flow (argon), 0.21 mL min 1. The analysed compounds and appropriate internal standards were quantified in multiple ion monitoring mode (MRM) using optimized MS conditions and continuous polarity-switching data measurements (Table 2). MRM transitions were recorded over each chromatographic run in ten targeted scan windows to obtain the greatest possible MS signal intensity for each compound: 1.5–3.0 min, 5.3–6.7 min, 7.0–9.0 min, 8.5–9.8 min, 10.3–11.5 min, 11.5–12.3 min, 12.1–14.5 min, 13.2–15.3 min, 16.0–18.0 min, and 17.5–20.7 min. The MassLynx™ software package (version 4.1, Waters, Milford, MA, USA) was used to control the instrument and to acquire and process all of the MS data. Acknowledgments We thank Karel Dolezˇal and René Lenobel for critical reading of the manuscript and Sees-editing Ltd. for careful revision of the manuscript. The Internal Grant Agency of Palacky´ University (IGA_PrF_2014006) is kindly acknowledged for financial support. This work was further funded by the Czech Science Foundation (Nr. 14-34792S) and by the Ministry of Education, Youth and

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MS based target profiling of stress-induced phytohormones.

Stress-induced changes in phytohormone metabolite profiles have rapid effects on plant metabolic activity and growth. The jasmonates (JAs) are a group...
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