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A 13C isotope labeling method for the measurement of lignin metabolic flux in Arabidopsis stems
© The Author(s) 2018
Received: 8 February 2018
Accepted: 16 June 2018
Published: 23 June 2018
Metabolic fluxes represent the functional phenotypes of biochemical pathways and are essential to reveal the distribution of precursors among metabolic networks. Although analysis of metabolic fluxes, facilitated by stable isotope labeling and mass spectrometry detection, has been applied in the studies of plant metabolism, we lack experimental measurements for carbon flux towards lignin, one of the most abundant polymers in nature.
We developed a feeding strategy of excised Arabidopsis stems with 13C labeled phenylalanine (Phe) for the analysis of lignin biosynthetic flux. We optimized the feeding methods and found the stems continued to grow and lignify. Consistent with lignification profiles along the stems, higher levels of phenylpropanoids and activities of lignin biosynthetic enzymes were detected in the base of the stem. In the feeding experiments, 13C labeled Phe was quickly accumulated and used for the synthesis of phenylpropanoid intermediates and lignin. The intermediates displayed two different patterns of labeling kinetics during the feeding period. Analysis of lignin showed rapid incorporation of label into all three subunits in the polymers.
Our feeding results demonstrate the effectiveness of the stem feeding system and suggest a potential application for the investigations of other aspects in plant metabolism. The supply of exogenous Phe leading to a higher lignin deposition rate indicates the availability of Phe is a determining factor for lignification rates.
Metabolic fluxes quantify the activities of biochemical networks that functionally integrate metabolites, enzymes, and their interactions . Mathematical analysis of metabolic fluxes is becoming increasingly attractive to assess the responses of metabolic pathways in plants to genetic or environmental perturbations . Stable isotope labeling experiments coupled with mass spectrometry (MS) quantification have been used to measure the flux within metabolic networks in various plant systems and expand our knowledge of metabolism and the control of flux [3, 4]. For example, Szecowka et al.  supplied 13CO2 to intact Arabidopsis rosettes to estimate intracellular fluxes of photosynthesis and central carbon metabolism. Developing seeds of Arabidopsis, oilseed rape, and Brassica napus were fed with 13C labeled glucose or sucrose to elucidate fluxes in lipid synthesis and other primary metabolism [6–8]. Flux analysis has also been applied to unravel phenylpropanoid metabolism in plants. Matsuda et al. utilized deuterium labeled Phe to analyze metabolic flux towards defense-related products in potato tubers upon wounding stress or elicitor treatment [9–11]. Similarly, labeled Phe was provided to petunia flowers to address flux distribution through CoA-dependent and -independent pathways for benzenoid biosynthesis [3, 12]. A recent study with labeled glucose revealed that expression of the Arabidopsis transcription factor MYB12 induces primary and specialized metabolism to enhance flavonoid production in tomato fruits . In contrast, there have been no measurements of metabolic flux into lignin, the most abundant carbon sink derived from phenylpropanoid metabolism.
Lignin is a heterogeneous aromatic polymer that constitutes approximately 20–30% of carbon fixed by land plants . Deposited together with polysaccharides in the plant secondary cell wall, lignin provides mechanical strength and hydrophobicity for plants to stand upright and to transport water and nutrients through vascular structures . Although important for plants, lignin is a major recalcitrance factor for forage digestibility, efficient paper-pulping, and biofuel production . Alteration of lignin content and monomer composition in natural mutants and genetically engineered plants improves the efficiency of utilization of lignocellulosic biomass [17, 18], and has thereby motivated many studies of lignin biosynthesis and its regulation. Lignin in dicots is derived mainly from three monomers, p-coumaryl alcohol (H lignin), coniferyl alcohol (G lignin), and sinapyl alcohol (S lignin), all of which are synthesized from the phenylpropanoid pathway (Additional file 1: Figure S1) [15, 19, 20]. Phenylpropanoid metabolism starts with deamination of phenylalanine (Phe) by Phe ammonia lyase (PAL), the ring of which is subsequently hydroxylated by three cytochrome P450 enzymes  and O-methylated [22, 23], with some of these steps occurring at the level of CoA and shikimate esters of the hydroxycinnamic acids [24–26]. Ultimately, CoA thioesters are the substrates for reduction to their corresponding aldehydes and finally the alcohols commonly known as monolignols. Despite a comprehensive knowledge of the kinetics of most of the lignin biosynthetic enzymes and steady-state accumulation of metabolites involved, we lack systematic and quantitative measurements of the carbon flux into and within this branched metabolic pathway. Accurate quantification and prediction of lignin biosynthetic flux in planta will guide our understanding of controlling steps in the pathway and facilitate future genetic manipulations of lignification.
Reliable estimation of phenylpropanoid flux towards lignin and further analysis of flux regulation depend on precise quantification of phenylpropanoids and their isotopologues from appropriate plant tissue fed with isotope-labeled precursor. Arabidopsis stems deposit lignin at substantial levels and thus make a good system in which to analyze lignin biosynthetic flux not only in wild-type plants, but also in genetically modified plants with perturbations in Phe synthesis or lignin biosynthesis itself [19, 27]. Plants transport water and nutrients via the transpiration stream, which continues to function after plant organs have been removed from the parent plant. This property permitted experiments in which isotope-labeled amino acids or NH4NO3 were fed to white lupin, wheat, poplar, and Arabidopsis to study nitrogen transport and metabolism [28–31]. Similarly, stable isotope feeding assays of excised Arabidopsis leaves or whole rosettes were developed for the investigation of carbon partitioning in sink and source leaves . In this study, we fed excised inflorescence stems of Arabidopsis with 13C6-ring labeled Phe ([13C6]-Phe) to trace phenylpropanoid metabolism. Our recently developed liquid chromatography coupled with tandem MS (LC/MS–MS) based phenylpropanoid metabolic profiling method provides fast and comprehensive analysis of pathway intermediates and end products . All six isotopically-labeled carbons (13C) in the Phe ring are maintained in all intermediates and in lignin, allowing downstream labeled compounds to be distinctly detected and accurately quantified by MS. Furthermore, incorporation of label into lignin can be measured to estimate flux to the major pathway end product. We have optimized the feeding conditions and analyzed metabolite pool sizes and isotopic abundances, selected enzyme activities and lignin deposition during the feeding process. Over a time course of only 360 min, [13C6]-Phe rapidly labeled soluble intermediates and lignin, demonstrating the efficiency of the feeding strategy. These data together can be used for mathematical modeling of lignin deposition and the exploration of the regulation of lignin biosynthesis in plants [3, 9–12].
Establishment of a stem feeding system using wild-type Arabidopsis
To perform stable isotope labeling experiments, we first established an experimental system using Arabidopsis stems that can be fed with exogenous [13C6]-Phe to synthesize labeled phenylpropanoids. We cut Arabidopsis stems under water and then transferred them into liquid Murashige and Skoog medium (MS medium) supplemented with [13C6]-Phe in 1.5 mL tubes (Additional file 1: Figure S2, see “Methods” section for details). Excising stems under water prevented cavitation of the xylem so that the transpiration stream would not be blocked.
Phenylpropanoid metabolism is not homogeneous along the stem
In addition to metabolite analysis, PAL and 4-coumarate: CoA ligase (4CL) enzyme activities were measured to assess the heterogeneity of lignin biosynthetic enzymes along the Arabidopsis inflorescence stem axis (Fig. 3b). PAL and 4CL both showed higher activities in the basal stems compared to those in the apical region. The enzyme activities together with the soluble metabolite pool sizes suggest that the stems have a larger phenylpropanoid flux in the base than the top, again consistent with the stem base being the best tissue for analysis. We thus chose to harvest this basal 2 cm fragment of stems for analysis in our feeding experiments.
Soluble phenylpropanoids are rapidly labeled in stems fed with [13C6]-Phe
During the labeling period of 360 min, Phe and intermediates in the early part of the pathway rapidly approached isotopic steady state, whereas downstream metabolites showed a slower increase in labeling enrichment (Fig. 5). To achieve a quantitative overview of the labeling kinetics, we used hierarchical clustering to analyze the isotope enrichment profiles of the compounds measured. The hierarchical clustering based on squared Euclidian distance grouped the labeling percentage of phenylpropanoids into two patterns (Additional file 1: Figure S4). The first group contained Phe, p-coumarate, caffeate, ferulate, p-coumaraldehyde, p-coumaryl alcohol, and p-coumaroyl shikimate each of which exhibited rapid isotope enrichment in the first 80 min and a high final labeling of approximately 90%, except for p-coumaryl alcohol, which was 74%. In contrast, the second group including caffeoyl shikimate, coniferaldehyde, sinapaldehyde, coniferyl alcohol, sinapyl alcohol, and sinapate all showed slower increases in labeling, reaching less than 60% final labeling except for caffeoyl shikimate, which was slightly higher. These results are consistent with precursor-product relationships , where compounds closer to the fed substrate [13C6]-Phe are labeled faster and to a higher percentage than downstream product or than products with large endogenous pools. Furthermore, the efficient labeling of p-coumaryl alcohol provides an alternative or additional explanation for the high labeling of H-lignin that we previously observed (Fig. 2).
Isotopic label is incorporated into lignin in stems fed with [13C6]-Phe
We developed a stable isotope feeding system to measure phenylpropanoid flux in intact Arabidopsis stems. In comparison to previous tracer experiments for lignin biosynthesis using cell suspension cultures [39, 40], the whole stems in our system continue their normal growth and development (Fig. 1), and conduct in situ phenylpropanoid metabolism and lignification (Fig. 2). The excised wild-type stems, resembling the plants growing in soil, consume supplied [13C6]-Phe that rapidly and efficiently labels the soluble phenylpropanoid intermediates and lignin over a time course of 360 min. The measurement of metabolites together with enzyme activities for lignin biosynthesis will provide valuable in planta input for analysis of flux through the phenylpropanoid pathway.
Application of isotope labeling has a long history in investigation of phenylpropanoid metabolism. Half a century ago, 14C labeled precursors were fed to various plants and cell suspension cultures to solve the structure of the pathway [39, 40]. Recently, the widespread use of MS technology has made it feasible to employ stable isotope labeling for the systematic estimation of metabolic fluxes in biochemical networks . To analyze the flux through the branched lignin biosynthetic pathway in growing plants, an optimal feeding system needs to maintain physiologically normal conditions and active lignification. To this end, we chose intact Arabidopsis stems, where lignin is primarily deposited, excised from plants grown in soil. Previously, xylem sap extracted from plants was used as liquid medium to feed isotope labeled compounds to shoots of various plants [30, 31]. Because xylem sap contains amino acids including Phe [31, 41], we utilized a defined MS medium that avoids interference with endogenous unlabeled Phe and is practical to prepare in large volumes. The medium is adequate for stems to grow (Fig. 1) and even to develop fertile seeds when fresh medium was provided to the stems every day (data not shown). As the cauline leaves were all left intact, their transpiration promoted the rapid absorption of liquid MS medium together with the [13C6]-Phe at a rate of 1.4 µL min−1 (Additional file 1: Figure S3). When fed with 1 mM [13C6]-Phe, each stem on average took up approximately 500 nmol labeled Phe after 360 min, equivalent to 10% of the carbon fixed by photosynthesis in an Arabidopsis rosette of 200 mg in the same amount of time . At the end of the feeding experiment, the stem base (approximately 30 mg) deposited over 3.5 µmol g FW−1 labeled lignin (Fig. 8), accounting for more than 20% of the labeled Phe absorbed. This measurement of the end products gives an estimation of total flux entering the pathway.
The Arabidopsis stem is a complex organ that comprises different tissues and cells, each of which differs in the level of phenylpropanoids they synthesize and the nature of the end products they accumulate. To find a suitable tissue for analysis, we assessed phenylpropanoid metabolite content and enzyme activities along the inflorescence stems and throughout the feeding process. In these experiments we found that the base of the stems contained the highest levels of labeled precursor and activities of PAL and 4CL (Fig. 3), demonstrating a high phenylpropanoid flux, consistent with the lignin deposition pattern observed in Arabidopsis .
The feeding of [13C6]-Phe to Arabidopsis stems showed rapid incorporation of label into downstream phenylpropanoid metabolism. Like previous studies done in various plant systems [43–46], we observed quick and high accumulation of labeled hydroxycinnamic acids (Fig. 6). The labeling kinetics of p-coumarate and ferulate followed that of Phe, indicative of rapid isotope incorporation. Interestingly, the labeling percentage profiles of all phenylpropanoids measured showed two different patterns apparently based on whether or not their synthesis was dependent on the enzyme p-coumaroylshikimate 3′-hydroxylase (C3′H). The compounds that do not require C3′H for their synthesis displayed rapid increases in labeling enrichment and reach isotopic steady state within 80 min of feeding. In contrast, C3′H-dependent compounds were mostly labeled more slowly (Fig. 5). One possible explanation is that the relatively large pools of endogenous unlabeled coniferyl alcohol, sinapyl alcohol, and sinapate in the stems render the labeling slow, but it cannot explain the results seen with coniferaldehyde and sinapaldehyde which had much less accumulation (Fig. 6). Another possibility is that a slow (often rate-limiting) enzymatic step provokes high labeling of upstream intermediates and causes slow and low labeling in downstream products [4, 47]. C3′H is a cytochrome P450-dependent monooxygenase, a class of enzymes generally known to have low turnover [48, 49]. In the feeding experiments, the substantial accumulation of p-coumaroylshikimate indicates that C3′H might be saturated, becoming a limiting step for lignification. Caffeate and ferulate are two exceptions in the first group as both are 3′-hydroxylated but exhibited a labeling pattern like Phe and p-coumarate. The labeling percentage of ferulate is higher than its shikimate ester precursors, which seems to suggest p-coumarate is a close upstream precursor for caffeate and ferulate and that its conversion to these two downstream intermediates bypasses the HCT/C3′H/HCT/CSE steps. Supporting this model, it was shown that C4H and C3′H from poplar form a protein complex and that the recombinant proteins can catalyze the hydroxylation of p-coumarate in vitro .
Lignification in plants is tightly regulated transcriptionally and post-translationally to control carbon allocation into this major metabolic sink. Recent studies have shown that the expression of PAL genes is regulated by Mediator and transcriptional factors [51, 52] and that Kelch repeat F-box proteins mediate the degradation of PAL enzymes via the proteasome complex . PAL has often been considered as the rate-limiting step in lignin biosynthesis  and this may well be the case when PAL is down-regulated. In contrast, our study shows that in wild-type Arabidopsis plants, PAL is present in excess and the availability of Phe instead determines lignin deposition, a factor that has been undervalued in the past. Before feeding, stems had developed for 6 days in soil and deposited approximately 12 µmol g FW−1 of lignin. When fed with 1.0 mM [13C6]-Phe for only 360 min, stems synthesized over 3 µmol g FW−1 additional, labeled, lignin, accounting for 25% of the previous total, showing a higher lignification rate than would occur under normal, unfed, conditions. Given that PAL activity remains unaltered during the feeding period (Fig. 7), these data indicate that an enhanced supply of Phe increases lignin biosynthesis in plants, and that PAL is not saturated with endogenous Phe in vivo under normal conditions. Consistent with these observations, it has been reported that the Arabidopsis pal1 mutant has a 40% reduction in PAL activity but deposits wild-type levels of lignin [55, 56], supporting the idea that PAL in wild-type plants does not perform at maximal velocity and is not the rate-limiting enzyme.
Many studies have reported that lignin monomer composition is altered in transgenic plants or mutants when flux into or within the phenylpropanoid pathway is reduced. In addition to decreased lignin content, an increased S/G ratio was seen in mutants with reduced levels of PAL, C4H, 4CL, or arogenate dehydratase, an enzyme in Phe biosynthesis [55, 57–59]. It has been suggested that these changes occur when enzymes with markedly different kinetic characteristics that act at pathway branch points compete for a common intermediate, thus leading to altered flux partitioning when the two enzymes’ substrate concentration is decreased . In contrast, experimental evidence to show the opposite change, increased total influx leading to a decreased S/G ratio, has not previously been reported. As this model predicts, we found that enhanced supply of Phe resulted in increased H lignin deposition and comparatively reduced S lignin synthesis (Fig. 8). In plants, cinnamoyl CoA reductase (CCR) competes with hydroxycinnamoyl CoA: shikimate hydroxycinnamoyl transferase (HCT) for p-coumaroyl CoA to drive flux towards H lignin (Additional file 1: Figure S1). It has been suggested that HCT associates with the complex of C4H and C3′H on the endoplasmic reticulum membrane, forming a metabolon that efficiently directs carbon towards G and S lignin in plants under normal conditions . This is suggested to occur even though the reported KM of Arabidopsis CCR1 (2.27 µM towards p-coumaroyl CoA, ) is smaller than the KM of HCT from tobacco (600 µM, 23]) and the maximal activity of CCR (200 pkat mg−1 protein) in Arabidopsis stem extracts is higher than that of HCT (35 pkat mg−1 protein) using p-coumaroyl CoA as substrate (unpublished data). When p-coumaroyl CoA accumulates and C3′H in the metabolon becomes saturated, CCR likely outcompetes HCT and generates higher levels of p-coumaraldehyde, which ultimately results in more H lignin. Similarly, ferulate 5-hydroxylase, the last cytochrome P450-dependent monooxygenase in the pathway, typically competes effectively with cinnamyl alcohol dehydrogenase and the yet-to-be identified transporter of coniferyl alcohol to synthesize S lignin (Additional file 1: Figure S1) . When high concentrations of coniferaldehyde and coniferyl alcohol are present, proportionally more flux escapes 5-hydroxylation leading to a decreased S/G ratio (Fig. 8). The precise control of flux by the enzymes at each branch points will be better understood by a more sophisticated and mechanistic mathematic model that includes the kinetics of the enzymes involved.
In summary, we established an experimental feeding system to supply intact Arabidopsis stems with [13C6]-Phe to investigate the metabolic flux towards lignin. Our analysis revealed that the availability of Phe determines lignin deposition rate and can alter distribution of flux towards three monolignols. The soluble phenylpropanoid metabolite and lignin measurements from dynamic isotope labeling experiments can be input for mathematical modeling of metabolic fluxes to quantitatively unravel the control of flux and to explore its regulation. In addition to the measurement of lignin biosynthetic flux in wild-type Arabidopsis, we envision a wider application of this stem feeding system. For example, mutants with defects in lignification can be fed with labeled precursor in parallel with the wild type to explore how genetic perturbation affects flux distribution within the phenylpropanoid pathway. Furthermore, given the abundance of Arabidopsis mutants in myriad biochemical pathways, isotope labeled substrates could be administered with this system such that their flux distribution could be described but in this case, reoptimization of the selected tissue would be warranted.
Plant material and growth conditions
Arabidopsis thaliana (Columbia-0) were grown in soil at 23 °C under light intensity of 150 µE m−2 s−1 in a photoperiod of 16-h light and 8-h dark in a growth chamber. Primary stems of 4-week-old plants were used for feeding experiments and enzyme activity assays.
Arabidopsis stems were fed with liquid MS medium containing [13C6]-Phe in 1.5 mL tubes in the same growth chamber as they were grown into maintain the same environmental conditions. [13C6]-Phe was filter-sterilized before being added into autoclaved MS medium. A steel nail was heated and used to form a hole in the lid of tubes so that the stems could be inserted through it. Plants were next removed from soil, and then the inflorescence stem was cut immediately above the rosette with a double-edged razor blade under water. The excised stem was immediately transferred into incubation solution and placed on a rack in the growth chamber (Additional file 1: Figure S2). Tubes were preloaded with 0.5 mL medium and refilled with fresh medium when necessary. After the specified incubation period, stem fragments were rinsed three times with water to remove [13C6]-Phe on the surface and frozen in liquid nitrogen. Ten stem fragments were harvested as one sample, and three biological replicates were generated for each time point. Depending on the biological questions asked and quantification methods employed, more biological replicates or stem tissue per sample may be optimized to achieve accurate measurements of metabolites. Samples were stored at − 70 °C until extraction.
Stem growth measurement
Stems of 4-week-old plants were cut under water as described above and used for growth analysis. Stems were photographed alongside a ruler immediately and after 3, 6, 9, 12, 24, 27, 30, 33, 36, and 48 h. Measurements of stem length from the base to the apical meristem were done using ImageJ in comparison to the ruler included in the photograph.
LC/MS–MS analysis of soluble metabolites
DFRC/GC/FID and DFRC/GC/MS
Stems were analyzed for lignin content following the method established by Lu and Ralph 1997 . Briefly samples were ground in liquid nitrogen washed five times with 10 mL 70% ethanol (v/v) at 80 °C and once with acetone. The dried cell wall residue was weighed and dissolved in 2.5 mL of acetyl bromide/glacial acetic acid (20:80 v/v) mixture containing 0.2 mg internal standard (4 4′-ethyldenebisphenol) overnight at room temperature. The mixture was dried under nitrogen gas then dissolved in 2 mL of dioxane/glacial acetic acid/H2O (50:40:10 v/v/v). 50 mg zinc dust was added to the mixture vortexed stirred for 25 min and applied to a solid phase extraction column (Discovery® DSC-18 SPE tube) pre-conditioned with 95% ethanol (v/v) and H2O. The column was washed with 5 mL 25% ethanol (v/v) and DFRC reactions products were eluted with 2.5 mL 96% ethanol (v/v) then dried under nitrogen gas. The sample was acetylated with 0.5 mL acetic anhydride/pyridine (60/40 v/v) overnight and dried again under nitrogen gas. The sample was then dissolved in 200 µL dichloromethane and 1 µL of the final product analyzed by GC/FID or GC/MS.
Acetyl bromide lignin analysis
Total lignin content in the stem tissue was quantified using acetyl bromide lignin analysis method described in 64] with minor revisions. Briefly the basal 0–2 cm fragments of stems of 4-week-old Arabidopsis were harvested and 15 biological replicates were included for analysis. The stem tissue was ground in liquid nitrogen and washed in 0.1 M sodium phosphate buffer (pH 7.2) at 50 °C for 30 min followed by five washes with 10 mL 70% ethanol (v/v) at 80 °C and once with 2 mL acetone. The dried cell wall residue was weighed and dissolved in 2.5 mL of acetyl bromide/glacial acetic acid (25:75 v/v) overnight at room temperature. A control without cell wall residue was included. The dissolved samples were completely transferred into 50 mL volumetric flasks containing 2.5 mL 2 M NaOH and 12 mL acetic acid. 0.5 mL freshly prepared 7.5 M hydroxylamine hydrochloride was added into each sample followed by 35 mL acetic acid. The sample was mixed and allowed to settle before the volume was brought to 50 mL with acetic acid. Absorbance at 280 nm was measured on spectrophotometer using the control as blank. Extinction coefficient 23.20 g−1 L cm−1 was used to calculate total lignin content.
PAL and 4CL enzyme assays
Stem tissue was harvested and frozen in liquid nitrogen. Crude protein was extracted from ground tissue with Tris–HCl buffer at pH of 7.8 and desalted on a gel filtration column (Sephadex™ G-50 fine GE Healthcare). PAL and 4CL assays were conducted following the method in Klempien 2010 65]. Each PAL assay contained 100 mM Tris–HCl buffer pH 7.5 5 mM Phe and 5 µL protein extract in a final volume of 50 µL. The reactions were incubated at 23 °C for 120 min. The 4CL assay contained 100 mM Tris–HCl buffer pH 7.5 5 mM MgCl2 5 mM ATP 1 mM p-coumarate 0.3 mM CoA and 2 µL protein extract in a final volume of 40 µL. Each reaction was incubated at 23 °C for 20 min. Assay products were quantified on HPLC with cinnamate and synthesized p-coumaroyl CoA as standards respectively. Protein concentrations were measured with Bradford assay using bovine serum albumin as standard.
PW, LG, and CC designed the research. PW, LG, RJ, AK, and RM performed the experiments and analyzed the data. PW and CC wrote the manuscript. All authors read and approved the final manuscript.
We thank Dr. John Ralph (Department of Biochemistry and Department of Biological Systems Engineering, Great Lakes Bioenergy Research Center, University of Wisconsin) for providing standards for analysis of lignin monomers in DFRC method.
Availability of data and materials
All data generated or analyzed during this study are included in this published article and its supplementary information files.
The authors declare that they have no competing interests.
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This work was supported by the U.S. Department of Energy, Office of Science, Office of Biological and Environmental Research, Genomic Science program, under Award Number DE-SC0008628.
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