Analytical Biochemistry 448 (2014) 14–22

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Analysis of plant nucleotide sugars by hydrophilic interaction liquid chromatography and tandem mass spectrometry Jun Ito a, Thomas Herter a,b, Edward E.K. Baidoo a, Jeemeng Lao a, Miguel E. Vega-Sánchez a, A. Michelle Smith-Moritz a, Paul D. Adams a,c, Jay D. Keasling a,c,d, Björn Usadel b,e,f, Christopher J. Petzold a, Joshua L. Heazlewood a,⇑ a

Joint BioEnergy Institute and Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA Max Planck Institute of Molecular Plant Physiology, Potsdam-Golm 14476, Germany Department of Bioengineering, University of California, Berkeley, CA 94720, USA d Department of Chemical and Biomolecular Engineering, University of California, Berkeley, CA 94720, USA e RWTH Aachen University, Institute for Biology I, Aachen 52056, Germany f Forschungszentrum Jülich, IBG-2: Plant Sciences, Jülich 52425, Germany b c

a r t i c l e

i n f o

Article history: Received 23 August 2013 Received in revised form 11 November 2013 Accepted 22 November 2013 Available online 1 December 2013 Keywords: Nucleotide sugars Plant cell walls Hydrophilic interaction liquid chromatography Arabidopsis Rice Selected reaction monitoring

a b s t r a c t Understanding the intricate metabolic processes involved in plant cell wall biosynthesis is limited by difficulties in performing sensitive quantification of many involved compounds. Hydrophilic interaction liquid chromatography is a useful technique for the analysis of hydrophilic metabolites from complex biological extracts and forms the basis of this method to quantify plant cell wall precursors. A zwitterionic silica-based stationary phase has been used to separate hydrophilic nucleotide sugars involved in cell wall biosynthesis from milligram amounts of leaf tissue. A tandem mass spectrometry operating in selected reaction monitoring mode was used to quantify nucleotide sugars. This method was highly repeatable and quantified 12 nucleotide sugars at low femtomole quantities, with linear responses up to four orders of magnitude to several 100 pmol. The method was also successfully applied to the analysis of purified leaf extracts from two model plant species with variations in their cell wall sugar compositions and indicated significant differences in the levels of 6 out of 12 nucleotide sugars. The plant nucleotide sugar extraction procedure was demonstrated to have good recovery rates with minimal matrix effects. The approach results in a significant improvement in sensitivity when applied to plant samples over currently employed techniques. Ó 2013 Elsevier Inc. All rights reserved.

Plant cell walls have been the focus of recent efforts to convert biomass into liquid transportation fuels with the intention of providing a sustainable alternative to fossil fuels [1]. The majority of nucleotide sugar substrates of plant cell wall polymers are synthesized through a series of nucleotide sugar interconverting enzymes in the cytosol and Golgi apparatus from UDP-a-D-glucose or GDP-a-D-mannose [2]. Cell wall UDP-sugar precursors include UDP-a-D-glucose (UDP-Glc), -a-D-galactose (UDP-Gal), -a-D-glucuronate (UDP-GlcA), -a-D-galacturonate (UDP-GalA), -a-D-xylose (UDP-Xyl), -a-D-apiose (UDP-Api), -b-L-arabinose (UDP-Ara) and -b-L-rhamnose (UDP-Rha) [3]. Cell wall GDP-sugar precursors include GDP-a-D-glucose (GDP-Glc), -a-D-mannose (GDP-Man), -b-L-galactose (GDP-Gal), and -b-L-fucose (GDP-Fuc) [3]. Other nucleotide sugar precursors include CMP-D-ketodeoxyoctonate ⇑ Corresponding author. Address: Joint BioEnergy Institute, Lawrence Berkeley National Laboratory, One Cyclotron Road MS978-4466, Berkeley, CA 94720, USA. Fax: +1 510 486 4253. E-mail address: [email protected] (J.L. Heazlewood). 0003-2697/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ab.2013.11.026

(CMP-Kdo), which is an activated acid sugar that is incorporated into pectic rhamnogalacturonan II fractions of primary cell walls of higher plants [4]. There are also numerous minor nucleotide sugars that also exist in plants, although evidence for their incorporation into plant cell walls is rare or unknown [5]. Glycosyltransferases utilize these activated nucleotide sugar donors as substrates to produce the major plant cell wall polysaccharides: cellulose, and matrix polysaccharides (e.g., hemicellulose and pectin) [6]. Reverse genetic studies of enzymes involved in cell wall biosynthesis using the model dicot plant Arabidopsis thaliana have shown that nucleotide sugars involved in cell wall biosynthesis are essential for normal plant growth and development. For example, the mur1 mutant is a nonfunctional cytosolic GDP-D-mannose 40 ,60 -dehydratase catalyzing the first step in converting GDP-Man into GDP-Fuc. In the pectic component rhamnogalacturonan-II of Arabidopsis mur1 mutants, GDP-Fuc is substituted with GDP-Gal, preventing the formation of borate-dependent dimers and mur1 plants display dwarf phenotypes [7]. In addition, the mur4 mutant is a Golgi-targeted UDP-xylose 40 -epimerase partially defective in the last step of UDP-Ara synthesis. Analyses of

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Analysis of plant nucleotide sugars by LC-MS/MS / J. Ito et al. / Anal. Biochem. 448 (2014) 14–22

leaf samples from Arabidopsis mur4 plants show a 50% decrease in Larabinose in its cell wall compared with leaves from wild-type plants [8,9]. Finally, knockout mutants of two nucleotide sugar mutases (RGP1 and RGP2) that interconvert UDP-L-arabinopyranose (UDP-Arap) to UDP-L-arabinofuranose (UDP-Araf) have markedly lower total L-arabinose content (12 to 31%) compared with wild-type plants [10]. Down regulation of their expression levels was detrimental to the development of affected plants, along with virtually no L-arabinose in their cell walls [10]. Measuring metabolic changes within the network of cell wall biosynthetic reactions is difficult because of the high number of metabolites involved. Metabolic analysis of plant cell wall biosynthesis requires a highly sensitive and robust method for detecting changes in levels of precursors such as nucleotide sugars. Recently a number of quantitative methods based on porous graphitic carbon (PGC)1 [11], anion-exchange [12,13], and ion-pair reversed-phase chromatography [14] coupled to mass spectrometry (MS) have been developed. Several of these approaches have been employed to directly measure nucleotide sugars from plant material, namely Arabidopsis thaliana cell cultures and rosette leaves [11,12]. An emerging chromatographic method has gained interest for its capacity to separate polar metabolites from complex biological mixtures. Hydrophilic interaction liquid chromatography (HILIC) consists of a polar chromatographic surface with the starting mobile phase containing low aqueous content in low-polarity solvent (i.e., acetonitrile) [15,16]. Polar compounds are retained in the water-rich hydrophilic stationary phase away from the solvent mobile phase. They are eluted in order of increasing polarity under higher aqueous conditions in the mobile phase [16]. Low overall aqueous content (typically 5 to 40%) and the limited amount of salts required in the mobile phase solutions allow HILIC to be highly compatible with electrospray ionization (ESI) mass spectrometry. A recent metabolomic study applied zwitterionic silica (ZIC)-based stationary phase in HILIC mode to separate and quantify over 200 hydrophilic intracellular metabolites, including several nucleotide sugars from extracts of b-lactam antibiotic fermentation broths, demonstrating the utility of the separation approach [17]. Here, we present a highly sensitive and robust LC–MS/MS method using ZIC-HILIC coupled with a triple quadrupole operating in multiple reaction monitoring mode to compare nucleotide sugar levels from leaves of two plants with different cell wall compositions: the model dicot plant species, Arabidopsis thaliana, and the model monocot plant species rice (Oryza sativa).

Plant growth and sample harvest Arabidopsis thaliana (Col-0) plants were grown with a 16 h photoperiod at 22 °C with 90 lmol m2 s1 illumination intensity during the day period. Arabidopsis rosettes from 4-week-old plants (three separate individuals) were sampled simultaneously in the middle of the light period. These were immediately frozen in liquid nitrogen and stored at 80 °C until used for metabolic extraction. Rice (Oryza sativa, cultivar Nipponbare) plants were grown in chambers under the following conditions: 12 h daylight, 470 lmol m2 s1 illumination intensity, 80% relative humidity, 26 °C for 1 h at the beginning and end of the cycle, and 28 °C for the remaining 10 h; 12 h dark, 80% relative humidity, 26 °C. Leaf material from three individual plants (4–5 weeks old) was sampled in the middle of the day period. These were immediately frozen in liquid nitrogen and stored at 80 °C until used for metabolic extraction. Monosaccharide composition analysis of extracted cell wall material Leaf material from three individual plants of either rice (4– 5 weeks old) or Arabidopsis (4 weeks old) was harvested during the day period and dried in an oven at 40 °C for 3 days. Dried material was ground with a bead beater (Retsch, GmbH, Germany) to a fine powder at 30 Hz for 1 to 2 min. Preparation and hydrolysis of alcohol-insoluble residues from ground material were separately prepared from three independent biological replicates for both Arabidopsis and rice as previously outlined [18]. Monosaccharide composition was measured by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAECPAD, Dionex, Sunnvale CA) using a CarboPac PA20 column as previously outlined [18]. Nucleotide sugar extraction from plant material After grinding the frozen leaf material to a fine powder using a bead beater (Retsch GmbH, Germany), nucleotide sugars were extracted from 10 mg fresh weight (FW) as previously described [19]. The freeze-dried extract was dissolved in 1.2 mL of 10 mM ammonium bicarbonate before using an ENVI-Carb SPE column (Sigma– Aldrich, St. Louis, MO) using a previously established purification protocol for bacterial samples [20]. Purified extracts were dried in a CentriVap Vacuum Concentrator System (Labconco, Kansas City, MO) and immediately stored at 80 °C. Hydrophilic interaction liquid chromatography (LC–MS/MS)

Materials and methods Nucleotide sugar standards and reagents All chemicals were analytical grade or higher and were used as received without any further purification. Nucleotide sugar standards were obtained from the following sources: UDP-a-D-xylose (p), UDP-b-L-arabinose (p), UDP-a-D-galacturonic acid (p) (Carbosource Services, Complex Carbohydrate Research Center, Athens, GA); UDP-a-D-glucuronic acid (p), UDP-a-D-glucose (p), UDP-a-D-galactose (p), UDP-N-acetyl-a-D-glucosamine, UDP-Nacetyl-a-D-galactosamine, GDP-a-D-mannose (p), GDP-b-L-fucose, GDP-a-D-glucose (p) (Sigma–Aldrich, St. Louis, MO); UDP-b-L-arabinose (f) (Peptides International, Louisville, KY). 1 Abbreviations used: HILIC, hydrophilic interaction liquid chromatography; LOD, limit of detection; LOQ, limit of quantification; PGC, porous graphitic carbon; ZIC, zwitterionic silica.

Metabolite extracts were initially reconstituted in 10 lL of 10 mM ammonium acetate (pH 7) and then diluted 1:10 with a solution of 94% acetonitrile and 10 mM ammonium acetate (pH 7) to produce 85% acetonitrile. Thus, FW equivalents of 300 to 700 lg of extracts (10 lL) in 85% acetonitrile, 10 mM ammonium acetate (pH 7) were used for analysis by LC–MS/MS. Liquid chromatography was performed on an 1100 series capillary HPLC system (Agilent Technologies, Santa Clara, CA) with a 20 lL flow sensor, a 40 lL sample loop, and appropriate capillaries for the flow rate used. During chromatographic runs, the injection volume was 10 lL, plate cooler temperature was set to 10 °C, and column compartment was 50 °C. Nucleotide sugars were separated with a ZIC-HILIC stationary phase column (150 mm  1 mm, 3.5 lm, 200 Å) and a ZIC-HILIC guard column cartridge (5 mm  1 mm, 5 lm, 200 Å) (Merck SeQuant, Umeå Sweden). The flow rate was 20 lL/min with the mix rate set at 400 lL/min and coupled directly to the mass spectrometer for analysis. The mobile phase was 10 mM ammonium acetate (pH 7), in (A) 90% acetonitrile and (B) H2O. At the start of the run, (A) was set at 85% for 2 min. Gradient

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Analysis of plant nucleotide sugars by LC-MS/MS / J. Ito et al. / Anal. Biochem. 448 (2014) 14–22

elution was performed starting with 85% (A) to 45% (A) over a period of 15 min, then back to starting conditions (45 to 85% A) in 15 min, followed by a reequilibration period (85% A) of 10 min (total run time 42 min). An extended gradient (15 min) was used for reequilibration as recommended by the ZIC-HILIC column supplier (Merck SeQuant, Umeå Sweden). Reverse-phase ion-pair chromatography (LC–MS/MS) Liquid chromatography was performed on an 1100 series HPLC system (Agilent Technologies, Santa Clara, CA). The injection volume was 10 lL for three dilutions (1:2, 1:5, and 1:10) of plant metabolite extracts and the nucleotide sugars were separated using a reverse-phase Synergi 4u Hydro 80A-RP 150  1 mm column (Phenomenex, Torrance, CA) with a Micro-Guard 14  1 mm cartridge (Alltech Associates, Deerfield, IL) at a temperature of 24 °C. Separation of UDP-Glc from UDP-Gal was performed with an equilibration step of 10 min, followed by 35 min isocratic flow of 20 mM buffered triethylamine/acetic acid (TEAA) (pH 6) at a flow rate of 50 lL/min into the mass spectrometer. Electrospray ionization tandem mass spectrometry For detection of nucleotide sugars separated by ZIC-HILIC, a 5500 QTRAP LC/MS/MS system (AB Sciex, Foster City, CA) equipped with a TurboIonSpray ion source was used. Specific compounddependent MS parameters for each nucleotide sugar were determined by direct infusion at the MS interface of individual standards dissolved in 50% acetonitrile (concentration of 1 pmol lL1) at a flow rate of 20 lL min1. Declustering potential (DP), entrance potential (EP), and collision energy (CE) were adjusted for Q1/Q3 transitions. The specific precursor mass [MH], product ions, and collision energies applied are compiled in Table 1. The 5500 QTRAP system was operated in negative ion mode using the multiple reaction monitoring (MRM) scan type. The ion spray voltage was set at 4200 V, source temperature (TEM) at 400 °C, and IonSource gases 1 (GS1) and 2 (GS2) were both 20. MS/MS spectra were collected for 2.04 s with Q1 resolution set to low and Q3 resolution set to unit. All data were collected using Analyst 1.5.2 (AB Sciex, Foster City, CA). For the detection of nucleotide sugars separated by reverse-phase ion-pair chromatography, an API 2000 LC/MS/MS system (AB Sciex, Foster City, CA) equipped with a TurboIonSpray ion source was used. The system was operated in negative ion mode with Q1 scanning only. The ion spray voltage was set at 4200 V, TEM at 350 °C, GS1 was 30, and GS2 was 6. MS spectra were collected for 2.3 s with Q1 set to unit. Q1

Table 1 Specific settings used for the analysis of nucleotide sugar standards by selected reaction monitoring (SRM). Compound

Precursor ion [MH]

Product ion [MH]

Collision energy (eV)

UDP-Xyl UDP-Arap UDP-Araf UDP-Glc UDP-Gal UDP-GlcA UDP-GalA GDP-Fuc GDP-Glc GDP-Man UDP-GlcNAc UDP-GalNAc

535 535 535 565 565 579 579 588 604 604 606 606

323 323 323 323 323 403 403 442 362 442 385 385

30 30 30 33 33 30 30 30 33 36 33 33

Values were determined empirically through infusion of standards into the mass spectrometer.

scanning data were collected and analyzed with Analyst 1.4.2 (AB Sciex, Foster City, CA). Data analysis Nucleotide sugars from plant extracts were quantified by taking the integrated signal peak area using the Analyst 1.5.2 and MultiQuant 2.1 (build 2.1.1296.02.1) software packages (AB Sciex, Foster City, CA). These values were used to calculate the amount of substrate using linear regression from a calibration curve of known nucleotide sugar standards run at the start and end of the analysis period. A mixture of nucleotide sugar standards was used to create the standard curve comprising 2.5, 5, 10, 25, 50, 100, 250, 500 fmol, and 1, 2.5, 5, 10, 25, 100, 250 pmol. Calculations were undertaken using Microsoft Excel (Microsoft Corporation, WA). Linearity, repeatability, limit of detection, limit of quantification, and recovery To determine linearity, standard mixtures of nucleotide sugars ranging from 2.5 fmol to 250 pmol were analyzed to obtain an external calibration curve for each compound. Repeatability was determined by analyzing three different amounts of each standard compound (n = 3). The coefficient of variation (CV%) is 100  standard deviation/mean for the retention times of three different concentration levels of standards. Limit of detection (LOD) and limit of quantification (LOQ) were calculated for signal/noise ratios of 3:1 and 10:1, respectively, that had been determined during the measurements of linearity. Recovery was determined by the addition of each standard to plant extracts both before metabolite extraction and after metabolite extraction. This was undertaken both using a physiological amount of each nucleotide sugar (based on a plant extracts) and using three times this amount to test the effect of ion suppression. Recovery was determined with n = 4 biological samples for both before and after extraction.

Results and discussion Separation and detection of nucleotide sugar standards by ZIC-HILIC and tandem mass spectrometry The biosynthetic pathways of the principle nucleotide sugars incorporated into plant cell walls are largely known (Fig. 1) [2,3,5]. A major challenge in quantifying plant nucleotide sugars involved in cell wall biosynthesis by LC–MS/MS is to separate structural isomers such as UDP-Xyl/UDP-Ara, UDP-Glc/UDP-Gal, UDP-GlcA/UDP-GalA, and GDP-Glc/GDP-Man [12]. Other than GDP-Glc/GDP-Man, the aforementioned nucleotide sugar isomers undergo identical fragmentation, which necessitate their separation with optimized chromatographic conditions. Further complicating this issue, in plants UDP-Arap is converted by UDP-Ara mutases into UDP-Araf prior to its incorporation into matrix polysaccharides and deposition into the cell wall [10]. Prior work investigating a variety of hydrophilic metabolite standards had demonstrated the efficacy of employing HILIC for the separation of nucleotide sugars [17,21]. Consequently we sought to optimize the ZIC-HILIC LC–MS/MS approach for the separation and quantification of mixtures of nucleotide sugars with a focus on those commonly found in plant tissues. A total of 8 of the 12 nucleotide sugar standards were successfully separated by mass and/or retention time (Fig. 2). The nucleotide sugars standards UDP-Rha, UDP-Api, CMP-Kdo, and GDP-Gal were excluded from this analysis as they were not commercially available. In the case of CMP-Kdo and UDP-Api, both have been reported to be highly labile compounds

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Analysis of plant nucleotide sugars by LC-MS/MS / J. Ito et al. / Anal. Biochem. 448 (2014) 14–22

D-Man-1-P

GDP-Man

D-Man-6-P

GDP-Fuc

D-Fru-6-P

D-GlcN-6-P

D-GlcNAc-1-P

PPP

D-Glc-6-P

UDP-Gal

UDP-GlcNAc

GDP-Glc

D-Glc-1-P

UDP-Glc

UDP-GalNAc

CMP-Kdo

GDP-Gal

Kdo Kdo-8-P

UDP-Rha

SUCROSE UDP-GlcA

UDP-Api

UDP-GalA UDP-Xyl

UDP-Arap

UDP-Araf

Fig.1. Nucleotide sugar biosynthetic pathways in plants. Schematic pathways for common nucleotide sugar substrates incorporated into plant cell walls. Nucleotide sugars in black boxes were analyzed in this study. Abbreviations not defined in text: D-Glc-1-P, D-glucose-1-phosphate; D-Glc-6-P, D-glucose-6-phosphate; D-Fru-6P, D-fructose-6-phosphate; D-Man-6-P, D-mannose-6-phosphate; D-Man-1-P, D-mannose-1-phosphate; D-GlcN-6-P, D-glucosamine-6-phosphate; D-GlcNAc-1-P, N-acetyl-D-glucosamine-1-phosphate; PPP, pentose phosphate pathway; Kdo-8-P, ketodeoxyoctonate-8-phosphate.

with short half-lives of 34 min at pH 7.5 and 25 °C [22] and 97.2 min at pH 8.0 and 25 °C [23], respectively.

Chromatographic separation of structural isomers using ZIC-HILIC Despite testing various LC conditions which included varying pH between 3 and 8, ammonium acetate concentrations (10 or 20 mM), column temperatures, gradient slopes, solvent concentrations, and flow rates 20 to 50 lL min1, we were unable to separate the structural isomers UDP-Glc/UDP-Gal or the N-acetylated amino-sugars UDP-N-acetyl-D-galactosamine (UDP-GalNAc)/UDPN-acetyl-D-glucosamine (UDP-GlcNAc) using ZIC-HILIC chromatography (Fig. 2). A previous chromatographic separation technique applied to the analysis of nucleotide sugars which employed porous graphitic carbon (LC–MS) was also unable to separate the UDP-GlcNAc/GalNAc isomers [11]. Only the separation procedure employing highperformance anion-exchange chromatography (LC–MS/MS) [12] has been successful in separating these isomers, although this technique was not successful in separating GDP-Man/Glc. Both UDP-GlcNAc and UDP-GalNAc are major substrates for protein glycosylation in eukaryotic systems but are not incorporated into polysaccharide polymers of the cell wall. Although they were indistinguishable when applying the ZIC-HILIC separation approach, plants do not undertake GalNAc O-glycosylation of proteins unless engineered with both a GlcNAc C4-epimerase and a UDP-GalNAc polypeptide N-acetylgalactosaminyltransferases [24,25]. As a

consequence, the UDP-GlcNAc/GalNAc signal measured from plant samples can be assumed to essentially constitute UDP-GlcNAc. The chromatographic separation of the structural isomers UDPGlc/Gal by ZIC-HILIC was also not successful under conditions tested. In contrast, both recent approaches employing porous graphitic carbon (LC–MS) [11] or high-performance anion-exchange chromatography (LC–MS/MS) [12] have successfully distinguished these isomers. Nonetheless, we could demonstrate using standards that the calibration curves for UDP-Glc and UDP-Gal are almost identical when using ZIC-HILIC and LC–MS/MS (Fig. S1). Consequently, if the abundance and presence of UDP-Glc and UDP-Gal are required, samples can be analyzed using a complimentary technique, such as a reverse-phase LC–MS/MS method with an ion-pairing agent [26] to determine their respective intensity ratios. This value can then be used to estimate their abundances in samples analyzed by ZIC-HILIC as demonstrated below. Performance of ZIC-HILIC coupled to mass spectrometry Different concentrations of standards were used to obtain an external calibration curve for each available nucleotide sugar. Coupling the ZIC-HILIC separation technique to mass spectrometry provided good sensitivity of nucleotide sugar standards at low femtomole levels. For most nucleotide sugars, their calibration curves demonstrated linearity across a wide range of concentrations from low femtomole to upper levels of 100 to 250 pmol resulting in R2 > 0.98 for all standards (Table 2). The exceptions were UDP-Arap and UDP-Araf which had narrower linear ranges that extended to 25 pmol and UDP-Glc and UDP-Gal which extended to 50 pmol. The limits of detection varied from 2.5 and 5 fmol and the limits of quantification were between 5 and 20 fmol (Table 2). This was a considerable improvement on the high-performance anion-exchange chromatography separation technique (LC–MS/MS) examining plant nucleotide sugars which report LODs of 0.3 to 34 pmol and LOQs of 1 to 111 pmol [12]. LOD measurements with the porous graphitic carbon LC–MS method [11] were only performed on four nucleotide sugars: UDP-GlcNAc, UDP-Glc, GDPFuc, and UDP-GlcA using 25 fmol in each case. This was because the authors focused on the separation of nucleotides and nucleotide sugars on PGC columns, and not on the conditions for their detection [11]. These significant differences in LOD and LOQ for ZIC-HILIC highlight the unique compatibility of this separation technique when coupled to mass spectrometry. The repeatability of the ZIC-HILIC method was examined by running triplicates for three different concentrations of standard mixtures (Table 2). The coefficients of variation (CV%) of retention times for nucleotide sugars were calculated to be less than 8.34, 3.48, and 3.90%, for 100 fmol, 1 pmol, and 10 pmol of standard mixtures, respectively. Overall, these numbers indicated repeatability of the LC–MS/MS method with the nucleotide sugar standard mixtures. An assessment of sample recovery and matrix effects using ZICHILIC was explored using standard additions of nucleotide sugars to plant leaf extracts. The amount of standard applied was initially determined from the physiological values measured in plant samples (Table 3). Sample recovery for the majority of nucleotide sugars was within an acceptable range with recovery rates ranging from ca. 84 to 110% (standard before extraction, Table 3). Only the standard addition of 3-fold physiological amounts of UDP-GlcNAc/GalNAc (120 pmol) and UDP-Glc/Gal (2800 pmol) gave less than adequate recovery rates with values of 78.9 and 75.4%, respectively. Overall, these data also indicate minimal matrix effects during the ZIC-HILIC technique. This was further explored through standard additions after extraction (Table 3). Generally, the matrix had a minor impact on detection and quantification even with very high amounts of standard (3-fold) which was also used to assess ion

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Analysis of plant nucleotide sugars by LC-MS/MS / J. Ito et al. / Anal. Biochem. 448 (2014) 14–22 100

TIC 0 100

UDP-Glc/Gal

Transition: 565/323

0 100

UDP-GlcA

Relative Intensities (%)

UDP-GalA

Transition: 579/403

0 100

UDP-Xyl

UDP-Arap

Transition: 535/323

UDP-Araf 0

100

UDP-GlcNAc/GalNAc

Transition: 606/385

0 100

GDP-Man Transition: 604/424

0 100

GDP-Glc

0 100

Transition: 604/362

GDP-Fuc Transition: 588/442

0

0

42

Time (min)

Fig.2. Ion chromatograms of nucleotide sugar standards analyzed by ZIC-HILIC and LC–MS/MS. A total of 100 fmol of a nucleotide sugar standard mix was separated and quantified using ZIC-HILIC LC–MS/MS. Total ion chromatogram (TIC) of the mixture of nucleotide sugar standards is presented in the top panel. The precursor/product ion MS/ MS transition for each nucleotide sugar is indicated in the top right corner of each panel. The LC conditions employed are outlined in the methods section. Table 2 Linearity, repeatability, and detection limits of nucleotide sugar standards analyzed by ZIC-HILIC and LC–MS/MS. Compound

UDP-Glc/Gal UDP-Arap UDP-Araf UDP-Xyl UDP-GlcA UDP-GalA UDP-GlcNAc/GalNAc GDP-Man GDP-Fuc GDP-Glc

Retention time (min)

18.00 18.00 15.40 17.20 21.00 20.50 15.70 20.90 19.85 20.40

Linear range (pmol)

0.005–50 0.005–25 0.005–25 0.005–100 0.02–250 0.05–250 0.01–250 0.01–250 0.005–250 0.005–100

Correlation coefficient (R2)

0.9814 0.9910 0.9980 0.9908 0.9927 0.9977 0.9885 0.9852 0.9957 0.9883

suppression. Values for the majority of nucleotide sugar standards were in the range of ca. 82 to 109% with the exception of UDP-GlcNAc/GalNAc (40 pmol and 120 pmol) with values 68.4 and 79.6% and UDP-Xyl (35 pmol) with a value of 77.8%. Taken together, high amounts of UDP-GlcNAc/GalNAc in a sample are likely to be affected by the sample matrix or result in ion suppression when using the ZIC-HILIC approach. Overall, these results demonstrate both the reliability of the nucleotide sugar extraction procedure on plant material and the minimal effects of the plant matrix on the separation and quantification of nucleotide sugars. Nucleotide sugar analysis of leaf extracts with differing cell wall compositions While cell walls of the model dicot plant, Arabidopsis, and model monocot, rice (Oryza sativa), are composed mainly of the b-1,

Repeatability (CV%)

Detection limit

100 fmol

1 pmol

10 pmol

LOD (fmol)

LOQ (fmol)

1.92 1.12 2.39 8.34 0.55 1.02 2.31 0.28 0.58 0.85

2.25 1.39 3.40 2.53 0.55 0.74 3.48 0.48 1.26 0.28

1.92 1.39 3.90 2.97 0.48 0.74 3.80 0.73 0.77 0.49

2.5 2.5 5 2.5 5 5 2.5 5 2.5 2.5

5 10 20 10 20 20 10 20 10 5

4-glucan polymer cellulose they contain different matrix polysaccharide structures [27–29]. Indeed, comparing the monosaccharide compositions of Arabidopsis and rice leaves showed significantly higher levels of fucose (Fuc), rhamnose (Rha), galactose (Gal), mannose (Man), and galacturonic acid (GalA) in Arabidopsis leaf extracts and a significantly higher level of glucose (Glc) and xylose (Xyl) in rice leaf samples (Fig. 3). These monosaccharide composition differences reflect the high amount of pectin in Arabidopsis leaves [30] and the presence of arabinoxylan and mixed linkage glucan in rice leaves [31]. Since nucleotide sugars are the precursors for these cell wall polysaccharides, we applied the highly sensitive ZIC-HILIC-based LC–MS/MS method to compare levels of nucleotide sugar precursors between leaves of Arabidopsis and rice (Fig. 4). Only small amounts of plant sample (500 lg FW) were needed in our analysis of nucleotide sugars, as higher amounts were outside

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Analysis of plant nucleotide sugars by LC-MS/MS / J. Ito et al. / Anal. Biochem. 448 (2014) 14–22

Table 3 Recovery of nucleotide sugars analyzed by ZIC-HILIC and LC–MS/MS. Compound

Amount relative to plant samples

Standard addition (pmol)

Standard before extraction % ± SD

Standard after extraction % ± SD

UDP-Glc/Gal (4:1)

1 3 1 3 – – 1 3 1 3 1 3 1 3 1 3 1 3 1 3

927 2800 54 162 – – 35 105 33 100 27 81 40 120 14 42 1.9 5.7 10 30

110.9 78.9 89.9 80.3 ND ND 99.7 108.3 104.6 110.9 105.0 119.4 101.0 75.4 85.3 85.6 99.7 101.1 84.0 98.1

89.2 82.3 89.2 82.3 ND ND 77.8 91.9 98.9 101.1 98.8 98.8 68.4 79.6 84.3 92.2 97.9 108.5 113.5 100.7

UDP-Arap UDP-Araf UDP-Xyl UDP-GlcA UDP-GalA UDP-GlcNAc/GalNAc (1:1) GDP-Man GDP-Fuc GDP-Glc

± ± ± ±

5.0 0.9 5.4 2.9

± ± ± ± ± ± ± ± ± ± ± ± ± ±

9.2 12.0 6.1 7.9 6.2 9.3 28.0 4.11 0.9 7.7 9.1 4.0 10.7 6.4

± ± ± ±

0.7 3.6 0.7 3.6

± ± ± ± ± ± ± ± ± ± ± ± ± ±

2.4 7.6 3.0 11.4 4.5 14.4 1.2 5.2 4.9 10.8 6.7 13.9 6.5 15.5

Recovery was not determined (ND) for UDP-Araf due to limited availability of the standard. For UDP-Glc/Gal and UDP-GlcNAc/GalNAc which could not be resolved using ZICHILIC, standard additions were applied using ratios of nucleotide sugars.

the linear range (of the calibration curve) necessary for quantification. This can be especially beneficial when plant samples of interest are only available in low quantities. A solid phase extraction (SPE) technique employing microporous amorphous carbon (ENVI-Carb) has previously been shown to extract nucleotide sugars from bacterial lysates [20]. We have now shown its suitability for plant extracts using standard additions and demonstrated excellent recovery rates (Table 3). The inclusion of this clean-up step is essential for the reliable analysis of these compounds from plant material using the ZIC-HILIC approach. Consequently, we used this SPE method to enrich nucleotide sugars from plant leaf extracts prior to their analysis by mass spectrometry. The total quantities of nucleotide sugars per milligram fresh weight for Arabidopsis and rice leaf samples are outlined in Table 4 and Fig. S2. Levels of UDP-Glc and UDP-Gal were calculated from peak area ratios of their separations in three different dilutions of Arabidopsis and rice samples by reverse-phase ion-pair chromatography LC–MS (Fig. S3). Significant differences between Arabidopsis and rice leaves were identified with UDP-Glc (1.19-fold higher in Arabidopsis), UDP-Gal (1.16-fold higher in Arabidopsis), UDP-GalA (3.46-fold higher in Arabidopsis), UDP–Araf (5.09-fold higher in rice), GDP-Fuc (5.64-fold higher in rice), and GDP-Glc (2.1-fold higher in rice) varying between the two species. Previous

analyses using the porous graphitic carbon LC–MS method had not successfully detected GDP-Glc in Arabidopsis leaves [11].While the high-performance anion-exchange chromatography (LC–MS/MS) technique was unable to separate GDP-Glc from GDP-Man [12], here the ZIC-HILIC method was sensitive enough to quantify this low-abundant nucleotide sugar in both Arabidopsis and rice leaves (Table 4). Although we observed some cross-talk with GDP-Man (Fig. 4), GDP-Glc could be differentiated and quantified from plant extracts. The ZIC-HILIC technique was also capable of consistently detecting a transition for UDP-Rha (549/323 m/z) as a significant peak at 16.9 min in both Arabidopsis and rice leaf samples (Fig. 4). However, this transition still needs to be verified once the standard becomes commercially available. This analysis and quantification of cell wall substrates from Arabidopsis and rice leaves and the corresponding composition of their cell walls enable a cursory examination of their relationship. The most dramatic differences in cell wall composition are those of GalA in Arabidopsis leaves and Xyl in rice leaves (Fig. 3). No significant differences were observed for UDP-Xyl between Arabidopsis and rice, while UDP-GalA were ca. 3-fold higher in Arabidopsis leaves. Biosynthesis of these two nucleotide sugars diverges at UDP-GlcA (Fig. 1); the similar UDP-Xyl levels may indicate a default route, with production of UDP-GalA more tightly regulated.

70

Relative content (mol%)

60

Arabidopsis

50

Rice

40

30 20 10 0

*Fuc

*Rha

Ara

*Gal *Glc *Xyl Monosaccharides

*Man

*GalA

GlcA

Fig.3. Monosaccharide composition of Arabidopsis and rice leaf material. Cell wall monosaccharide compositions of rosette leaves from Arabidopsis plants (dark gray bars, mean ± SD, n = 3 biological replicates) were compared to leaves of rice plants (light gray bars, mean ± SD, n = 3 biological replicates). Sugars marked with an asterisk (⁄) showed significant differences (P < 0.05) between Arabidopsis and rice leaf samples, calculated by two-tailed Student’s t test.

20

Analysis of plant nucleotide sugars by LC-MS/MS / J. Ito et al. / Anal. Biochem. 448 (2014) 14–22

A

TIC

B

TIC

UDP-Glc/Gal

UDP-Glc/Gal

UDP-GalA

UDP-GalA UDP-GlcA

Relative Intensities

UDP-Xyl

UDP-Arap

UDP-Xyl

UDP-GlcA

UDP-GlcNAc/GalNAc

Transition 535/323 UDP-GlcNAc/GalNAc

GDP-Man

GDP-Man

GDP-Fuc

*

GDP-Fuc

*

Transition 606/385

Transition 604/424

Transition 604/362

GDP-Glc

GDP-Glc

Transition 579/403

UDP-Arap

UDP-Araf

UDP-Araf

Transition 565/323

Transition 588/442

Transition 549/323

Time (min) Fig.4. Ion chromatograms of nucleotide sugars from the two different plant samples. Enriched nucleotide sugars from (A) Arabidopsis and (B) rice leaves were separated and quantified by ZIC-HILIC LC–MS/MS. Total ion chromatograms (TIC) of (A) Arabidopsis and (B) rice leaves are presented in the top two panels. The precursor/product ion MS/ MS transitions for each nucleotide sugar are indicated on the right of the chromatograms. The bottom panels both show a transition (549/323) at 16.9 min marked by an asterisk (⁄) representing UDP-Rha. This could not be verified because a UDP-Rha standard is not commercially available.

Table 4 Nucleotide sugar concentrations in leaves from Arabidopsis and rice plants. Nucleotide sugar

Arabidopsis (pmol mg FW1)

Rice (pmol mg FW1)

P value

UDP-Glc UDP-Gal UDP-GalA UDP-GlcA UDP-Araf UDP-Arap UDP-Xyl UDP-GlcNAc/ GalNAc GDP-Man GDP-Fuc GDP-Glc

41.74 ± 0.82a 11.82 ± 0.24a 1.21 ± 0.30 0.47 ± 0.20 0.05 ± 0.02 6.40 ± 0.46 3.84 ± 0.36 3.81 ± 0.68

35.14 ± 2.23a 10.17 ± 0.64a 0.35 ± 0.02 0.17 ± 0.03 0.27 ± 0.03 7.80 ± 1.47 3.49 ± 0.72 2.59 ± 0.37

0.009⁄ 0.014⁄ 0.008⁄ 0.060 0.001⁄ 0.189 0.493 0.051

0.55 ± 0.20 0.11 ± 0.02 0.015 ± 0.006b

0.71 ± 0.07 0.62 ± 0.14 0.032 ± 0.003

0.256 0.003⁄ 0.010⁄

Values are given as the mean ± standard deviation of three biological replicates. a Values were calculated using ratios outlined in Fig. S3. b Amount detected during analysis (1.5 fmol) was below limit of quantitation (LOQ) as outlined in Table 2. As calculated by two-tailed Student’s t test, P values marked with an asterisk were significantly different (P < 0.05) between plant species.

Although a recent analysis of metabolic flux of nucleotide sugars in Arabidopsis cell cultures did not indicate differential carbon flow at this point in the pathway [13]. There were also a number of opposite relationships observed, including GDP-Fuc/Fuc content and

UDP-Glc/Glc content. In Arabidopsis leaves, Fuc content was significantly higher, but GDP-Fuc levels were lower while in rice leaves Glc comprised a higher proportion of the cell wall but the UDP-Glc levels were lower. These examples may reflect demand on these metabolic pools during cell wall biosynthesis. Since GDP-Fuc is synthesized via GDP-Man (Fig. 1), which has similar levels in both species, again it is possible that these data indicate a high degree of regulation at this point in the pathway.

Reported nucleotide sugar levels in Arabidopsis Concentrations of nucleotide sugars measured in Arabidopsis samples using the ZIC-HILIC technique resulted in some differences to previous analysis techniques. Although the prior application of the high-performance anion-exchange chromatography technique was applied to a different cell type, namely cell cultures, there was a considerable level of agreement between results [12]. Although data were presented as dry weight, a comparison of nucleotide sugar ratios (based on UDP-Glc) between our study indicates that only the level of UDP-Xyl was significantly different (an order of magnitude lower) in the cell cultures when compared to Arabidopsis leaf tissue (this study). In contrast, nucleotide sugar levels isolated from similar material (Arabidopsis leaf tissue) analyzed by porous graphitic carbon LC–MS were all generally an order of magnitude lower (ranging from 2.4 fmol mg FW1 for

21

Analysis of plant nucleotide sugars by LC-MS/MS / J. Ito et al. / Anal. Biochem. 448 (2014) 14–22

UDP-Araf to 280 fmol mg FW1 for UDP-Glc) [11]. When compared to measurements outlined in this study, values were generally in the pmol mg FW1 range for leaf material. However, the main objective of the porous graphitic carbon LC–MS study was to establish optimal chromatographic conditions for nucleotide and nucleotide sugar separations, with quantitative analysis of biological samples by MS a secondary concern [11]. Separation and identification of nucleotide sugar structural isomers in plant samples The analyses of plant samples in this study using the ZIC-HILIC LC–MS/MS procedure was specifically focused on the analysis and quantitation of major nucleotide sugars involved in cell wall biosynthesis. With the exception of UDP-Rha, which is both a major metabolite and a significant component of plant cell walls, we only targeted nucleotide sugars with available metabolic standards. Nonetheless, there are a number of structural isomers with potentially identical transition states that need to be considered when analyzing these data from plant samples. The separation and identification of the structural isomers UDPXyl, UDP-Araf and UDP-Arap could be successfully accomplished using ZIC-HILIC and LC–MS/MS. The branched-chain five carbon sugar apiose is generally a minor component of the pectin polymer rhamnogalacturonan II in the cell walls of plants and is likely present as UDP-Api in its activated form [32]. Although UDP-Api is an isomer of UDP-Xyl, UDP-Araf, and UDP-Arap, no evidence for this compound was present in our analysis of cell wall material from Arabidopsis or rice (Fig. 4). While this could indicate that the technique could not resolve this compound, it should be noted that the half-life of UDP-Api is less than 2 h at room temperature [23]. The identification and separation of UDP-Api has not been reported in plant samples by any of the recent mass spectrometry-based analysis procedures [11,12] and may further indicate the labile nature of this compound. The nucleotide sugars GDP-Gal and GDP-L-Gulose (GDP-Gul) are both synthesized from GDP-Man and are confirmed (GDP-Gal) or proposed (GDP-Gul) as intermediates in the biosynthesis of ascorbic acid [33]. Together with GDP-Glc, these four nucleotide sugars are structural isomers and consequently may complicate the analyses of complex metabolic samples. No evidence for either GDP-Gal or GDP-Gul could be observed from plant samples of Arabidopsis or rice employing either the 604/424 or the 604/362 transitions (Fig. 4). Thus, it is possible that under our conditions, ZIC-HILIC chromatography is not able to distinguish these nucleotide sugars. It has been reported that the application of porous graphitic carbon LC–MS was able to separate and identify GDPMan, GDP-Gul, and GDP-Gal in plant extracts from Arabidopsis and tobacco while no GDP-Glc was observed [11]. Although without standards for GDP-Gul, GDP-Gal, or GDP-Glc, it is unclear how the authors could distinguish between these isomers and thus as noted in their study, GDP-Gul and GDP-Glc are tentatively assigned. The high-performance anion-exchange chromatography (LC–MS/MS) technique did not report the presence of GDP-Gal or GDP-Gul [12]. Given the importance of ascorbic acid biosynthesis in plant development [34], it is possible that the reported GDPGlc or GDP-Man peaks identified in these studies (and our own analysis) may also contain GDP-Gal. Conclusion We have implemented a method based on ZIC-HILIC and selected reaction monitoring mass spectrometry to separate and quantify nucleotide sugars. The approach is sensitive and could be deployed on most biological material provided nucleotide sugar

standards are available for reliable quantitation. Using different quantities of nucleotide sugar standard mixtures, we demonstrated high sensitivity, linear range, and repeatability of the method. The extraction procedure and enrichment step showed reproducible recovery rates and resulted in a minimal impact from the sample matrix. Comparative quantitative analyses of Arabidopsis and rice leaf extracts validated its applicability to plant samples by revealing significant differences in concentrations of 6 out of 12 nucleotide sugars measured. This approach will provide a complementary analytical tool with other recently developed LC–MS/MS methods to study the dynamics of the complex metabolic processes involved in plant cell wall biosynthesis. Acknowledgments This work conducted by the Joint BioEnergy Institute was supported by the Office of Science, Office of Biological and Environmental Research, of the U.S. Department of Energy under Contract No. DE-AC02-05CH11231. The substrates UDP-xylose, UDP-arabinopyranose, and UDP-galacturonic acid were obtained from Carbosource Services (Athens, GA) which is supported in part by NSF-RCN Grant 0090281. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ab.2013.11.026. References [1] M. Pauly, K. Keegstra, Plant cell wall polymers as precursors for biofuels, Curr. Opin. Plant Biol. 13 (2008) 305–312. [2] G.J. Seifert, Nucleotide sugar interconversions and cell wall biosynthesis: how to bring the inside to the outside, Curr. Opin. Plant Biol. 7 (2004) 277–284. [3] W.D. Reiter, Biochemical genetics of nucleotide sugar interconversion reactions, Curr. Opin. Plant Biol. 11 (2008) 236–243. [4] W.S. York, A.G. Darvill, M. McNeil, P. Albersheim, 3-Deoxy-D-manno-2octulosonic acid (KDO) is a component of rhamnogalacturonan II, a pectic polysaccharide in the primary cell walls of plants, Carbohydr. Res. 138 (1985) 109–126. [5] M. Bar-Peled, M.A. O’Neill, Plant nucleotide sugar formation, interconversion, and salvage by sugar recycling, Annu. Rev. Plant Biol. 62 (2011) 127–155. [6] W.R. Scheible, M. Pauly, Glycosyltransferases and cell wall biosynthesis: novel players and insights, Curr. Opin. Plant Biol. 7 (2004) 285–295. [7] M.A. O’Neill, S. Eberhard, P. Albersheim, A.G. Darvill, Requirement of borate cross-linking of cell wall rhamnogalacturonan II for Arabidopsis growth, Science 294 (2001) 846–849. [8] E.G. Burget, W.D. Reiter, The mur4 mutant of arabidopsis is partially defective in the de novo synthesis of uridine diphospho L-arabinose, Plant Physiol. 121 (1999) 383–389. [9] E.G. Burget, R. Verma, M. Molhoj, W.D. Reiter, The biosynthesis of L-arabinose in plants: molecular cloning and characterization of a Golgi-localized UDP-Dxylose 4-epimerase encoded by the MUR4 gene of Arabidopsis, Plant Cell 15 (2003) 523–531. [10] C. Rautengarten, B. Ebert, T. Herter, C.J. Petzold, T. Ishii, A. Mukhopadhyay, B. Usadel, H.V. Scheller, The interconversion of UDP-arabinopyranose and UDParabinofuranose is indispensable for plant development in Arabidopsis, Plant Cell 23 (2011) 1373–1390. [11] M. Pabst, J. Grass, R. Fischl, R. Leonard, C. Jin, G. Hinterkorner, N. Borth, F. Altmann, Nucleotide and nucleotide sugar analysis by liquid chromatographyelectrospray ionization-mass spectrometry on surface-conditioned porous graphitic carbon, Anal. Chem. 82 (2010) 9782–9788. [12] A.P. Alonso, R.J. Piasecki, Y. Wang, R.W. LaClair, Y. Shachar-Hill, Quantifying the labeling and the levels of plant cell wall precursors using ion chromatography tandem mass spectrometry, Plant Physiol. 153 (2010) 915–924. [13] X. Chen, A.P. Alonso, Y. Shachar-Hill, Dynamic metabolic flux analysis of plant cell wall synthesis, Metab. Eng. 18 (2013) 78–85. [14] K. Nakajima, S. Kitazume, T. Angata, R. Fujinawa, K. Ohtsubo, E. Miyoshi, N. Taniguchi, Simultaneous determination of nucleotide sugars with ion-pair reversed-phase HPLC, Glycobiology 20 (2010) 865–871. [15] A.J. Alpert, Hydrophilic-interaction chromatography for the separation of peptides, nucleic acids and other polar compounds, J. Chromatogr. 499 (1990) 177–196. [16] P. Hemstrom, K. Irgum, Hydrophilic interaction chromatography, J. Sep. Sci. 29 (2006) 1784–1821. [17] B. Preinerstorfer, S. Schiesel, M. Lammerhofer, W. Lindner, Metabolic profiling of intracellular metabolites in fermentation broths from beta-lactam

22

[18]

[19]

[20]

[21]

[22] [23]

[24]

Analysis of plant nucleotide sugars by LC-MS/MS / J. Ito et al. / Anal. Biochem. 448 (2014) 14–22 antibiotics production by liquid chromatography–tandem mass spectrometry methods, J. Chromatogr. A 1217 (2010) 312–328. A.M. Smith-Moritz, M. Chern, J. Lao, W.H. Sze-To, J.L. Heazlewood, P.C. Ronald, M.E. Vega-Sanchez, Combining multivariate analysis and monosaccharide composition modeling to identify plant cell wall variations by Fourier transform near infrared spectroscopy, Plant Methods 7 (2011) 26. S. Arrivault, M. Guenther, A. Ivakov, R. Feil, D. Vosloh, J.T. van Dongen, R. Sulpice, M. Stitt, Use of reverse-phase liquid chromatography, linked to tandem mass spectrometry, to profile the Calvin cycle and other metabolic intermediates in Arabidopsis rosettes at different carbon dioxide concentrations, Plant J. 59 (2009) 826–839. J. Rabina, M. Maki, E.M. Savilahti, N. Jarvinen, L. Penttila, R. Renkonen, Analysis of nucleotide sugars from cell lysates by ion-pair solid-phase extraction and reversed-phase high-performance liquid chromatography, Glycoconj. J. 18 (2001) 799–805. S.U. Bajad, W. Lu, E.H. Kimball, J. Yuan, C. Peterson, J.D. Rabinowitz, Separation and quantitation of water soluble cellular metabolites by hydrophilic interaction chromatography–tandem mass spectrometry, J. Chromatogr. A 1125 (2006) 76–88. C.H. Lin, B.W. Murray, I.R. Ollmann, C.H. Wong, Why is CMPketodeoxyoctonate highly unstable?, Biochemistry 36 (1997) 780–785 P.K. Kindel, R.R. Watson, Synthesis, characterization and properties of uridine 50 -(-D-apio-D-furanosyl pyrophosphate), Biochem. J. 133 (1973) 227–241. E.P. Bennett, U. Mandel, H. Clausen, T.A. Gerken, T.A. Fritz, L.A. Tabak, Control of mucin-type O-glycosylation: a classification of the polypeptide GalNActransferase gene family, Glycobiology 22 (2012) 736–756.

[25] Z. Yang, E.P. Bennett, B. Jorgensen, D.P. Drew, E. Arigi, U. Mandel, P. Ulvskov, S.B. Levery, H. Clausen, B.L. Petersen, Toward stable genetic engineering of human O-glycosylation in plants, Plant Physiol. 160 (2012) 450–463. [26] D.C. Turnock, M.A.J. Ferguson, Sugar nucleotide pools of Trypanosoma brucei, Trypanosoma cruzi, and Leishmania major, Eukaryot. Cell 6 (2007) 1450–1463. [27] R.A. Burton, M.J. Gidley, G.B. Fincher, Heterogeneity in the chemistry, structure and function of plant cell walls, Nat. Chem. Biol. 6 (2010) 724–732. [28] H.V. Scheller, P. Ulvskov, Hemicelluloses, Annu. Rev. Plant Biol. 61 (2010) 263– 289. [29] J. Vogel, Unique aspects of the grass cell wall, Curr. Opin. Plant Biol. 11 (2008) 301–307. [30] E. Zablackis, J. Huang, B. Muller, A.G. Darvill, P. Albersheim, Characterization of the cell-wall polysaccharides of Arabidopsis thaliana leaves, Plant Physiol. 107 (1995) 1129–1138. [31] M.E. Vega-Sanchez, Y. Verhertbruggen, U. Christensen, X. Chen, V. Sharma, P. Varanasi, S.A. Jobling, M. Talbot, R.G. White, M. Joo, S. Singh, M. Auer, H.V. Scheller, P.C. Ronald, Loss of cellulose synthase-like F6 function affects mixedlinkage glucan deposition, cell wall mechanical properties, and defense responses in vegetative tissues of rice, Plant Physiol. 159 (2012) 56–69. [32] M. Molhoj, R. Verma, W.D. Reiter, The biosynthesis of the branched-chain sugar D-apiose in plants: functional cloning and characterization of a UDP-Dapiose/UDP-D-xylose synthase from Arabidopsis, Plant J. 35 (2003) 693– 703. [33] C.L. Linster, S.G. Clarke, L-Ascorbate biosynthesis in higher plants: the role of VTC2, Trends Plant Sci. 13 (2008) 567–573. [34] S.D. Veljovic-Jovanovic, C. Pignocchi, G. Noctor, C.H. Foyer, Low ascorbic acid in the vtc-1 mutant of Arabidopsis is associated with decreased growth and intracellular redistribution of the antioxidant system, Plant Physiol. 127 (2001) 426–435.