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Arogenate Dehydratase Isoenzymes Profoundly and Differentially Modulate Carbon Flux into Lignins*

  • Oliver R.A. Corea
    Footnotes
    Affiliations
    Institute of Biological Chemistry, Washington State University, Pullman, Washington 99164-6340
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  • Chanyoung Ki
    Footnotes
    Affiliations
    Institute of Biological Chemistry, Washington State University, Pullman, Washington 99164-6340
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  • Claudia L. Cardenas
    Affiliations
    Institute of Biological Chemistry, Washington State University, Pullman, Washington 99164-6340
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  • Sung-Jin Kim
    Affiliations
    Institute of Biological Chemistry, Washington State University, Pullman, Washington 99164-6340
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  • Sarah E. Brewer
    Affiliations
    Institute of Biological Chemistry, Washington State University, Pullman, Washington 99164-6340
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  • Ann M. Patten
    Affiliations
    Institute of Biological Chemistry, Washington State University, Pullman, Washington 99164-6340
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  • Laurence B. Davin
    Affiliations
    Institute of Biological Chemistry, Washington State University, Pullman, Washington 99164-6340
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  • Norman G. Lewis
    Correspondence
    To whom correspondence should be addressed. Tel.: 509-335-2682; Fax: 509-335-8206
    Affiliations
    Institute of Biological Chemistry, Washington State University, Pullman, Washington 99164-6340
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  • Author Footnotes
    * This work was supported by a grant from the Chemical Sciences, Geosciences and Biosciences Division, Office of Basic Energy Sciences DE-FG-0397ER20259, which involved all of the lignin, laser microscope dissection, and pyrolysis GC/MS work. Complementation studies were supported by United States Department of Agriculture-National Institute of Food and Agriculture-supported Northwest Advanced Renewable Alliance Grant 2011-68005-30416. This work was also supported by United States Department of Agriculture Grant 683A757612, the G. Thomas and Anita Hargrove Center for Plant Genomic Research, and the BioEnergy Science Center, which is supported by the Office of Biological and Environmental Research in the Department of Energy Office of Science. The latter support enabled some of the mutant lines to be screened/selected for subsequent analyses.
    This article contains supplemental Figs. S1–S4 and Tables S1–S4.
    1 Both authors contributed equally to this work.
Open AccessPublished:February 06, 2012DOI:https://doi.org/10.1074/jbc.M111.322164
      How carbon flux differentially occurs in vascular plants following photosynthesis for protein formation, phenylpropanoid metabolism (i.e. lignins), and other metabolic processes is not well understood. Our previous discovery/deduction that a six-membered arogenate dehydratase (ADT1–6) gene family encodes the final step in Phe biosynthesis in Arabidopsis thaliana raised the fascinating question whether individual ADT isoenzymes (or combinations thereof) differentially modulated carbon flux to lignins, proteins, etc. If so, unlike all other lignin pathway manipulations that target cell wall/cytosolic processes, this would be the first example of a plastid (chloroplast)-associated metabolic process influencing cell wall formation. Homozygous T-DNA insertion lines were thus obtained for five of the six ADTs and used to generate double, triple, and quadruple knockouts (KOs) in different combinations. The various mutants so obtained gave phenotypes with profound but distinct reductions in lignin amounts, encompassing a range spanning from near wild type levels to reductions of up to ∼68%. In the various KOs, there were also marked changes in guaiacyl:syringyl ratios ranging from ∼3:1 to 1:1, respectively; these changes were attributed to differential carbon flux into vascular bundles versus that into fiber cells. Laser microscope dissection/pyrolysis GC/MS, histochemical staining/lignin analyses, and pADT::GUS localization indicated that ADT5 preferentially affects carbon flux into the vascular bundles, whereas the adt3456 knock-out additionally greatly reduced carbon flux into fiber cells. This plastid-localized metabolic step can thus profoundly differentially affect carbon flux into lignins in distinct anatomical regions and provides incisive new insight into different factors affecting guaiacyl:syringyl ratios and lignin primary structure.

      Introduction

      The final step of Phe biosynthesis, catalyzed by arogenate dehydratase (ADT)
      The abbreviations used are: ADT
      arogenate dehydratase
      H
      p-hydroxyphenyl
      G
      guaiacyl
      S
      syringyl
      VB
      vascular bundle
      IF
      interfascicular fiber
      CWR
      cell wall residues
      X-gluc
      5-bromo-4-chloro-3-indolyl-β-d-glucuronic acid
      GUS
      β-glucuronidase.
      in planta (
      • Jung E.
      • Zamir L.O.
      • Jensen R.A.
      Chloroplasts of higher plants synthesize l-phenylalanine via l-arogenate.
      ,
      • Siehl D.L.
      • Conn E.E.
      Kinetic and regulatory properties of arogenate dehydratase in seedlings of Sorghum bicolor (L.) Moench.
      ,
      • Cho M.-H.
      • Corea O.R.A.
      • Yang H.
      • Bedgar D.L.
      • Laskar D.D.
      • Anterola A.M.
      • Moog-Anterola F.A.
      • Hood R.L.
      • Kohalmi S.E.
      • Bernards M.A.
      • Kang C.
      • Davin L.B.
      • Lewis N.G.
      Phenylalanine biosynthesis in Arabidopsis thaliana. Identification and characterization of arogenate dehydratases.
      ), is potentially a major regulatory point due to both its pivotal position at the branch point of Tyr and Phe biosynthesis (see Fig. 1) and as a linkage point between plastid/chloroplast-localized shikimate-chorismate and cytosolic/membrane/cell wall-associated phenylpropanoid metabolic networks. Together, these pathways comprise some of the most metabolically intensive networks in vascular plants. Indeed, depending upon the species, up to 50% of captured photosynthetic carbon can be in the form of Phe-derived phenylpropanoids (
      • Lewis N.G.
      • Yamamoto E.
      ,
      • van Heerden P.S.
      • Towers G.H.
      • Lewis N.G.
      Nitrogen metabolism in lignifying Pinus taeda cell cultures.
      ). Furthermore, downstream phenylpropanoid-derived products can have important but distinct physiological functions in planta, including fragrances/flavors, defense molecules, UV protectants, pigments, and cell wall structural biopolymers, e.g. allyl/propenyl phenols, lignans (
      • Vassão D.G.
      • Kim K.-W.
      • Davin L.B.
      • Lewis N.G.
      ), flavonoids (
      • Forkmann G.
      • Heller W.
      ), (proantho)cyanidins, stilbenes (
      • Schröder J.
      ), phytoalexins (e.g. isoflavones) (
      • Dixon R.A.
      ), lignins (
      • Davin L.B.
      • Jourdes M.
      • Patten A.M.
      • Kim K.W.
      • Vassão D.G.
      • Lewis N.G.
      Dissection of lignin macromolecular configuration and assembly: comparison to related biochemical processes in allyl/propenyl phenol and lignan biosynthesis.
      ), and suberins (
      • Bernards M.A.
      • Lewis N.G.
      The macromolecular aromatic domain in suberized tissue: a changing paradigm.
      ,
      • Franke R.
      • Schreiber L.
      Suberin—a biopolyester forming apoplastic plant interfaces.
      ). The broad physiological functions of phenylpropanoid-derived metabolites thus translate into a diverse and ever changing demand for the pathway intermediate Phe in different tissues and organs, i.e. in addition to Phe utilization for protein synthesis and other metabolic pathways.
      Figure thumbnail gr1
      FIGURE 1Proposed biosynthetic pathways to Phe, Tyr, and Trp in plants for which body of evidence supports arogenate pathways to Phe/Tyr.
      Curiously, the question of pivotal regulatory metabolic networks upstream of Phe and profoundly altering carbon flux/allocation into phenylpropanoid/lignin metabolism versus protein synthesis etc. had essentially not been addressed before. Instead, previous biotechnological manipulations targeted the presumed entry point to the phenylpropanoid pathway, phenylalanine ammonia-lyase, as well as various downstream monolignol pathway steps (see Anterola and Lewis (
      • Anterola A.M.
      • Lewis N.G.
      Trends in lignin modification: a comprehensive analysis of the effects of genetic manipulations/mutations on lignification and vascular integrity.
      ) and Davin et al. (
      • Davin L.B.
      • Jourdes M.
      • Patten A.M.
      • Kim K.W.
      • Vassão D.G.
      • Lewis N.G.
      Dissection of lignin macromolecular configuration and assembly: comparison to related biochemical processes in allyl/propenyl phenol and lignan biosynthesis.
      ) for a discussion). Most of these approaches, however, did not take into consideration the potentially seamless integration of related upstream but differentially localized metabolic networks associated with carbon flux into phenylpropanoids and transcriptional regulation thereof. This was relevant because, in previous metabolic flux studies leading to monolignols in loblolly pine (Pinus taeda), it was established that factors apparently affecting Phe availability helped control/modulate carbon flux into phenylpropanoid metabolism (
      • Anterola A.M.
      • van Rensburg H.
      • van Heerden P.S.
      • Davin L.B.
      • Lewis N.G.
      Multi-site modulation of flux during monolignol formation in loblolly pine (Pinus taeda).
      ,
      • Anterola A.M.
      • Jeon J.-H.
      • Davin L.B.
      • Lewis N.G.
      Transcriptional control of monolignol biosynthesis in Pinus taeda: factors affecting monolignol ratios and carbon allocation in phenylpropanoid metabolism.
      ), i.e. rather than phenylalanine ammonia-lyase having a central rate-limiting role as had often been reported due to its entry point position to phenylpropanoids.
      The ADT family was thus considered a potentially promising candidate for involvement in regulating the previously documented changes in Phe availability in plants due to its branch point position in the shikimate-chorismate pathway and its sensitivity to feedback inhibition by Phe. Indeed, we had previously characterized all six ADT isoenzymes from Arabidopsis thaliana and provided molecular and biochemical evidence supporting the arogenate route as the major mode of Phe biosynthesis (Fig. 1) (
      • Cho M.-H.
      • Corea O.R.A.
      • Yang H.
      • Bedgar D.L.
      • Laskar D.D.
      • Anterola A.M.
      • Moog-Anterola F.A.
      • Hood R.L.
      • Kohalmi S.E.
      • Bernards M.A.
      • Kang C.
      • Davin L.B.
      • Lewis N.G.
      Phenylalanine biosynthesis in Arabidopsis thaliana. Identification and characterization of arogenate dehydratases.
      ). Specifically, three isoenzymes, ADT3, ADT4, and ADT5, demonstrated exclusive substrate preference for arogenate, whereas isoenzymes ADT1, ADT2, and ADT6 displayed instead a strong substrate preference for arogenate but also had a limited ability to utilize prephenate (
      • Cho M.-H.
      • Corea O.R.A.
      • Yang H.
      • Bedgar D.L.
      • Laskar D.D.
      • Anterola A.M.
      • Moog-Anterola F.A.
      • Hood R.L.
      • Kohalmi S.E.
      • Bernards M.A.
      • Kang C.
      • Davin L.B.
      • Lewis N.G.
      Phenylalanine biosynthesis in Arabidopsis thaliana. Identification and characterization of arogenate dehydratases.
      ). Further confirmatory observations of a strong substrate preference for arogenate were subsequently made for one rice ADT isoenzyme (
      • Yamada T.
      • Matsuda F.
      • Kasai K.
      • Fukuoka S.
      • Kitamura K.
      • Tozawa Y.
      • Miyagawa H.
      • Wakasa K.
      Mutation of a rice gene encoding a phenylalanine biosynthetic enzyme results in accumulation of phenylalanine and tryptophan.
      ) and three petunia ADT isoenzymes (
      • Maeda H.
      • Shasany A.K.
      • Schnepp J.
      • Orlova I.
      • Taguchi G.
      • Cooper B.R.
      • Rhodes D.
      • Pichersky E.
      • Dudareva N.
      RNAi suppression of arogenate dehydratase1 reveals that phenylalanine is synthesized predominantly via the arogenate pathway in petunia petals.
      ). Feedback inhibition of ADTs was also demonstrated to be an important factor influencing Phe biosynthesis, accumulation, and turnover as feedback-insensitive ADTs in both rice and Arabidopsis were found to accumulate approximately 55 and 160 times more Phe, respectively, compared with wild type (WT) (
      • Yamada T.
      • Matsuda F.
      • Kasai K.
      • Fukuoka S.
      • Kitamura K.
      • Tozawa Y.
      • Miyagawa H.
      • Wakasa K.
      Mutation of a rice gene encoding a phenylalanine biosynthetic enzyme results in accumulation of phenylalanine and tryptophan.
      ). Thus, given the important role of ADT and considering the vast range of uses for Phe in planta, it was instructive to determine whether different isoenzymes potentially participate in distinct metabolic networks involving Phe.
      To begin to delineate the potential individual physiological contributions of specific ADT isoenzymes, Arabidopsis lines containing knock-outs (KOs) of single and multiple ADT genes were generated and then initially analyzed for potential differential effects on phenylpropanoid metabolism (specifically lignification). It was thus established that lines with a combination of ADT4 and ADT5 KOs had profoundly altered lignin contents, including the various triple and quadruple KOs involving those isoenzymes, that led to even more pronounced effects. This is, therefore, the first demonstration that modulation of a network pathway step (ADT) upstream of phenylpropanoid metabolism, localized in plastids/chloroplasts, can differentially alter carbon allocation/flux into lignification (phenylpropanoid metabolism) versus formation of Phe for either protein synthesis or some other metabolic pathway.

      EXPERIMENTAL PROCEDURES

      All commercial kits were used according to the manufacturer's instructions with any minor deviations noted.

      Generation and Confirmation of Single, Double, Triple, and Quadruple ADT Knock-out Lines

      T-DNA insertion lines for all six ADT genes in Arabidopsis (supplemental Table S1) were obtained from either the Salk Institute Genomic Analysis Laboratory (
      • Alonso J.M.
      • Stepanova A.N.
      • Leisse T.J.
      • Kim C.J.
      • Chen H.
      • Shinn P.
      • Stevenson D.K.
      • Zimmerman J.
      • Barajas P.
      • Cheuk R.
      • Gadrinab C.
      • Heller C.
      • Jeske A.
      • Koesema E.
      • Meyers C.C.
      • Parker H.
      • Prednis L.
      • Ansari Y.
      • Choy N.
      • Deen H.
      • Geralt M.
      • Hazari N.
      • Hom E.
      • Karnes M.
      • Mulholland C.
      • Ndubaku R.
      • Schmidt I.
      • Guzman P.
      • Aguilar-Henonin L.
      • Schmid M.
      • Weigel D.
      • Carter D.E.
      • Marchand T.
      • Risseeuw E.
      • Brogden D.
      • Zeko A.
      • Crosby W.L.
      • Berry C.C.
      • Ecker J.R.
      Genome-wide insertional mutagenesis of Arabidopsis thaliana.
      ) or the Institut National de la Recherche Agronomique (
      • Samson F.
      • Brunaud V.
      • Balzergue S.
      • Dubreucq B.
      • Lepiniec L.
      • Pelletier G.
      • Caboche M.
      • Lecharny A.
      FLAGdb/FST: a database of mapped flanking insertion sites (FSTs) of Arabidopsis thaliana T-DNA transformants.
      ). For each T-DNA insertion line, DNA was extracted from leaves of individual plants using the RedExtract kit (Sigma), and these samples were then individually used as a template for two PCRs with different primer sets. For Salk Institute Genomic Analysis Laboratory lines, gene-specific left and right primers LP + RP, respectively, were used to amplify WT-specific PCR products, and left border primer site “c1” (LBc1) + RP were used to amplify T-DNA-specific PCR products (supplemental Table S1; LBc1, 5′-CACAATCCCACTATCCTTCGC-3′). For the Institut National de la Recherche Agronomique line, the T-DNA-specific primer FLAG-LB (5′-GACGTAACATAAGGGACTGACC-3′) was substituted for LBc1. Homozygous T-DNA insertion lines were identified as those having T-DNA-specific PCR products only, and these were sequenced to confirm the presence and the specific site of each T-DNA insertion. ADT KO lines were confirmed using RT-PCR with primers designed to the 3′-end of each ADT mRNA transcript. Confirmed single KO lines were then crossed together to generate double heterozygous ADT KOs in all combinations, and double homozygous lines were identified in the subsequent generation using the same PCR screening approach described above. The same strategy was used to create triple and quadruple KO lines using double and triple KO parental lines, respectively. Each double, triple, and quadruple KO line was independently confirmed using the PCR strategy described above.

      Complementation of adt5 Line

      Complementation was carried out by expressing ADT5 under the control of its native promoter in the adt5 KO line. First, a 1888-bp fragment upstream of ADT5 was amplified using promoter-specific primers (see supplemental Table S2). The ADT5 coding gene was then cloned into the pENTRTM/D-TOPO® vector to generate an entry construct. After full sequence verification, the entry vector construct was subcloned into a pK2GW7 binary vector (
      • Karimi M.
      • Inzé D.
      • Depicker A.
      GATEWAY vectors for Agrobacterium-mediated plant transformation.
      ). The cauliflower mosaic virus 35S promoter of the vector construct was next swapped for the ADT5 promoter following established procedures (
      • Kim S.J.
      • Kim K.W.
      • Cho M.H.
      • Franceschi V.R.
      • Davin L.B.
      • Lewis N.G.
      Expression of cinnamyl alcohol dehydrogenases and their putative homologues during Arabidopsis thaliana growth and development: lessons for database annotations?.
      ). The confirmed construct was transformed into Agrobacterium and used to transform the adt5 KO line using standard protocols (
      • Clough S.J.
      • Bent A.F.
      Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana.
      ).

      Arabidopsis Growth and Harvest Conditions

      All confirmed homozygous KO, complementation, and WT lines were grown in soil with four plants per pot in Washington State University greenhouses (16-h days, 27–28 °C; 8-h nights, 24–26 °C; 200-ppm nitrogen-based fertilizer added 5 days a week). For lignin analyses, the main stems of at least 48 plants were harvested weekly from after initial stem emergence up to maturity (∼3.5–10 weeks). The weights and lengths of 20 inflorescence stems from each line were measured, and stems were then subsequently cut into 0.5–1-cm-long pieces, lyophilized, and stored at room temperature prior to lignin analyses. For histochemical staining, two main stems for each ADT KO and WT line were harvested at 7 weeks.

      Real Time RT-PCR Analysis of ADT KO Lines

      Stem tissues for WT and selected ADT KO lines were harvested 5 weeks after planting, flash frozen in liquid N2, and stored at −80 °C until use. Frozen tissue was ground using a mortar and pestle, and ∼90–110 mg was transferred to a 1.5-ml microcentrifuge tube. Total RNA was extracted using the SpectrumTM Plant Total RNA Extraction kit (Sigma-Aldrich). RNA quantity and quality were assessed using a Nanodrop 2000c spectrometer (Thermo Fisher Scientific Inc.), and mRNA (1 μg) was reverse transcribed to cDNA using Superscript III (Invitrogen).
      Gene-specific primers for each ADT isoform and housekeeping gene TIP41-like (AT4G34270; Ref.
      • Czechowski T.
      • Stitt M.
      • Altmann T.
      • Udvardi M.K.
      • Scheible W.R.
      Genome-wide identification and testing of superior reference genes for transcript normalization in Arabidopsis.
      ) were designed using Primer Premier 6.10 software (Premier Biosoft International) (see supplemental Table S2). The SYBR Green Real Time RT-PCR kit (Invitrogen) was used for real time RT-PCRs using 0.05 μg of cDNA and 62.5 pmol of primers for each reaction. Triplicate reactions were run on an Mx 3505P Real Time Thermocycler (Stratagene), and data were analyzed with Mx Pro QPCR software (Stratagene).

      Arogenate Dehydratase Assays

      The following method from Jung et al. (
      • Jung E.
      • Zamir L.O.
      • Jensen R.A.
      Chloroplasts of higher plants synthesize l-phenylalanine via l-arogenate.
      ), modified by Maeda et al. (
      • Maeda H.
      • Shasany A.K.
      • Schnepp J.
      • Orlova I.
      • Taguchi G.
      • Cooper B.R.
      • Rhodes D.
      • Pichersky E.
      • Dudareva N.
      RNAi suppression of arogenate dehydratase1 reveals that phenylalanine is synthesized predominantly via the arogenate pathway in petunia petals.
      ), was applied for assaying ADT activity in Arabidopsis stems. Approximately 20 g of stem tissue was harvested, immediately ground in liquid N2, and extracted with 30 ml of buffer (20 mm Tris-HCl, pH 8.0, 1 mm EDTA, 1 mm dithiothreitol, 10% glycerol, 1 mm phenylmethylsulfonyl fluoride, 35 mg of leupeptin, and 35 ml of plant cell and tissue extract protease inhibitor mixture (Sigma-Aldrich)). The crude lysate was then subjected to an ammonium sulfate precipitation, and both the 20–40 and 40–80% fractions were collected. Each fraction was desalted with a PD-10 column (GE Healthcare) and then concentrated to ∼500 μl using an Amicon Ultra-4 Centrifugal Filter (Millipore). An aliquot (5 μl) of each protein extract (containing 30 and 370 μg for the 20–40% and 40–80% fractions, respectively) was added to the total volume of the 12-μl reaction mixture containing 250 μm arogenate and 20 mm Tris-HCl, pH 8.0. After incubation at 37 °C for 15 min, the reaction was stopped by addition of 10 μl of MeOH with 2 μl of 10 mm alanine added as an internal standard. The assay mixtures were vortexed and centrifuged, half of the sample was derivatized with N-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide and analyzed by GC/MS, and the other half was derivatized using the Pico-tag system (Waters) and analyzed by HPLC as described previously (
      • Cho M.-H.
      • Corea O.R.A.
      • Yang H.
      • Bedgar D.L.
      • Laskar D.D.
      • Anterola A.M.
      • Moog-Anterola F.A.
      • Hood R.L.
      • Kohalmi S.E.
      • Bernards M.A.
      • Kang C.
      • Davin L.B.
      • Lewis N.G.
      Phenylalanine biosynthesis in Arabidopsis thaliana. Identification and characterization of arogenate dehydratases.
      ,
      • van Heerden P.S.
      • Towers G.H.
      • Lewis N.G.
      Nitrogen metabolism in lignifying Pinus taeda cell cultures.
      ). No enzymatic phenylalanine formation was detected under the conditions used.

      Free Amino Acid Analysis

      Total free amino acid pools were extracted from 5-week-old WT and ADT KO stems using methanol:chloroform:water (12:5:3) as described previously (
      • Corea O.R.A.
      • Ki C.
      • Cardenas C.L.
      • Davin L.B.
      • Lewis N.G.
      ). Amino acids were derivatized using the AccQ·TagTM Ultra Derivatization kit (Waters) and analyzed by ultraperformance liquid chromatography (Waters). Phe and Tyr levels in WT were ∼18 and ∼12 pmol/mg dry weight, respectively, whereas those of the ADT KO lines ranged from 12 to 21 and 5 to 11 pmol/mg dry weight, respectively, suggesting that no massive changes occurred in Phe/Tyr levels between WT and ADT KO lines.

      Histochemical Staining

      Histochemical staining and imaging of fresh hand-cut sections taken near the base of 7-week-old stems (Stage 3) (
      • Altamura M.M.
      • Possenti M.
      • Matteucci A.
      • Baima S.
      • Ruberti I.
      • Morelli G.
      Development of the vascular system in the inflorescence stem of Arabidopsis.
      ) of both mutants and WT lines were carried out as described previously by Patten et al. (
      • Patten A.M.
      • Jourdes M.
      • Cardenas C.L.
      • Laskar D.D.
      • Nakazawa Y.
      • Chung B.Y.
      • Franceschi V.R.
      • Davin L.B.
      • Lewis N.G.
      Probing native lignin macromolecular configuration in Arabidopsis thaliana in specific cell wall types: further insights into limited substrate degeneracy and assembly of the lignins of ref8, fah 1–2 and C4H::F5H lines.
      ). For detection of presumed guaiacyl (G) lignin, stem cross-sections were placed in a phloroglucinol-HCl (0.1%, w/v) solution for 30 min (
      • Ruzin S.E.
      ,
      • Wiesner J.
      Note über das Verhalten des Phloroglucins unde einiger verwandter Körper zur verholtzen Zellmembran.
      ), transferred to a glass slide, and observed under the differential interference contrast setting using an Olympus System Microscope, Model BHT (Olympus Optical Co., Ltd., Tokyo, Japan). The Mäule reaction was used for presumed syringyl (S) lignin component detection. Hand-cut cross-sections of stems were treated for 10 min with KMnO4 (0.5%, w/v; filtered through a 0.45-μm MillexHV filter) and rinsed with distilled H2O (
      • Mäule C.
      Das Verhalten verholzter Membranen gegen Kalium-permanganat, eine Holzreaktion neuer Art.
      ). Samples were then treated with HCl (0.1%, v/v) for 5 min, rinsed, mounted in concentrated NH4OH, and observed as described above.

      Laser Microscope Dissection of Vascular Bundles and Interfascicular Regions

      Laser microscope dissection was used to separate the vascular bundle (VB) and interfascicular fiber (IF) regions from 7-week-old stems (Stage 3) for WT and adt1/4/5 lines as described previously (
      • Patten A.M.
      • Jourdes M.
      • Cardenas C.L.
      • Laskar D.D.
      • Nakazawa Y.
      • Chung B.Y.
      • Franceschi V.R.
      • Davin L.B.
      • Lewis N.G.
      Probing native lignin macromolecular configuration in Arabidopsis thaliana in specific cell wall types: further insights into limited substrate degeneracy and assembly of the lignins of ref8, fah 1–2 and C4H::F5H lines.
      ). Briefly, sections were made using a Cryocut 1800 microtome (Leica Microsystems) and laser-microdissected using a P.A.L.M. Microbeam System (P.A.L.M. Microlaser Technologies) to isolate the VB and IF regions. Approximately 5000 samples from each region were individually collected for adt1/4/5 and WT, respectively. Each dissected sample was combined, then individually transferred to glass vials and extracted twice for 12 h with H2O:acetone (3:7, v/v) at RT, and dried in vacuo in preparation for pyrolysis GC/MS analyses.

      Estimations of Lignin Contents and Compositions

      Extractive-free stem cell wall residues (CWR) were obtained through extraction with EtOH:toluene (1:1, v/v), EtOH, and distilled H2O as described previously (
      • Patten A.M.
      • Jourdes M.
      • Cardenas C.L.
      • Laskar D.D.
      • Nakazawa Y.
      • Chung B.Y.
      • Franceschi V.R.
      • Davin L.B.
      • Lewis N.G.
      Probing native lignin macromolecular configuration in Arabidopsis thaliana in specific cell wall types: further insights into limited substrate degeneracy and assembly of the lignins of ref8, fah 1–2 and C4H::F5H lines.
      ,
      • Laskar D.D.
      • Jourdes M.
      • Patten A.M.
      • Helms G.L.
      • Davin L.B.
      • Lewis N.G.
      The Arabidopsis cinnamoyl CoA reductase irx4 mutant has a delayed but coherent (normal) program of lignification.
      ,
      • Patten A.M.
      • Cardenas C.L.
      • Cochrane F.C.
      • Laskar D.D.
      • Bedgar D.L.
      • Davin L.B.
      • Lewis N.G.
      Reassessment of effects on lignification and vascular development in the irx4 Arabidopsis mutant.
      ,
      • Jourdes M.
      • Cardenas C.L.
      • Laskar D.D.
      • Moinuddin S.G.
      • Davin L.B.
      • Lewis N.G.
      Plant cell walls are enfeebled when attempting to preserve native lignin configuration with poly-p-hydroxycinnamaldehydes: evolutionary implications.
      ). Estimations of lignin contents were made using the acetyl bromide (AcBr) method (
      • Iiyama K.
      • Wallis A.F.
      Determination of lignin in herbaceous plants by an improved acetyl bromide procedure.
      ) as described in Blee et al. (
      • Blee K.
      • Choi J.W.
      • O'Connell A.P.
      • Jupe S.C.
      • Schuch W.
      • Lewis N.G.
      • Bolwell G.P.
      Antisense and sense expression of cDNA coding for CYP73A15, a class II cinnamate 4-hydroxylase, leads to a delayed and reduced production of lignin in tobacco.
      ) and adjusted for G:S monomeric ratios using estimated AcBr extinction coefficients (280 nm) of 15.31, 18.61, and 14.61 l g−1 cm−1 for p-hydroxyphenyl (H), G, and S units, respectively (
      • Jourdes M.
      • Cardenas C.L.
      • Laskar D.D.
      • Moinuddin S.G.
      • Davin L.B.
      • Lewis N.G.
      Plant cell walls are enfeebled when attempting to preserve native lignin configuration with poly-p-hydroxycinnamaldehydes: evolutionary implications.
      ). Cleavable monomeric (G/S) compositions and contents were also estimated by thioacidolysis using the general procedures of Lapierre et al. (
      • Lapierre C.
      • Monties B.
      • Rolando C.
      Thioacidolysis of poplar lignins: identification of monomeric syringyl products and characterization of guaiacyl-syringyl lignin fractions.
      ) and Rolando et al. (
      • Rolando C.
      • Monties B.
      • Lapierre C.
      ) as described by Blee et al. (
      • Blee K.
      • Choi J.W.
      • O'Connell A.P.
      • Jupe S.C.
      • Schuch W.
      • Lewis N.G.
      • Bolwell G.P.
      Antisense and sense expression of cDNA coding for CYP73A15, a class II cinnamate 4-hydroxylase, leads to a delayed and reduced production of lignin in tobacco.
      ) and quantified by comparison using standard curves with authentic standards. Pyrolysis GC/MS was carried out as described previously (
      • Patten A.M.
      • Jourdes M.
      • Cardenas C.L.
      • Laskar D.D.
      • Nakazawa Y.
      • Chung B.Y.
      • Franceschi V.R.
      • Davin L.B.
      • Lewis N.G.
      Probing native lignin macromolecular configuration in Arabidopsis thaliana in specific cell wall types: further insights into limited substrate degeneracy and assembly of the lignins of ref8, fah 1–2 and C4H::F5H lines.
      ) using a Shimadzu PYR-4A pyrolyzer interfaced with a Shimadzu GC-17A gas chromatograph and a Shimadzu QP-5000 mass spectrometer (Shimadzu Scientific Instruments Inc., Columbia, MD) with the following modifications. Vapor phase products were separated on a SPB-5 poly (5% diphenyl and 95% dimethyl siloxane) nonpolar column (30 m × 0.25 μm) (Sigma-Aldrich) using a 5 °C min−1 gradient beginning from 50 to 270 °C. Pyrolysis products were identified by comparison with the following authentic standards (see supplemental Fig. S1 for structures): phenol (1), 4-methylphenol (2), 3-methylphenol (3), guaiacol (4), 4-ethylphenol (5), 4-methylguaiacol (6), catechol (7), 4-vinylphenol (8), 4-allylphenol (9), 4-ethylguaiacol (10), 4-vinylguaiacol (11), trans-propenylphenol (12), eugenol (14), vanillin (15), trans-isoeugenol (17), acetovanillone (18), guaiacylacetone (19), 4-allyl-2,6-dimethoxyphenol (21), syringaldehyde (22), trans-coniferyl alcohol (24), acetosyringone (25), trans-sinapyl aldehyde (27), trans-sinapyl alcohol (28), and trans-coniferyl aldehyde (29) as well as with the literature mass spectroscopic fragmentation data of 2,6-dimethoxyphenol (13), 2,6-dimethoxy-4-methylphenol (16), 2,6-dimethoxy-4-vinylphenol (20), trans-2,6-dimethoxy-4-propenylphenol (23), and syringylacetone (26) (
      • Patten A.M.
      • Jourdes M.
      • Cardenas C.L.
      • Laskar D.D.
      • Nakazawa Y.
      • Chung B.Y.
      • Franceschi V.R.
      • Davin L.B.
      • Lewis N.G.
      Probing native lignin macromolecular configuration in Arabidopsis thaliana in specific cell wall types: further insights into limited substrate degeneracy and assembly of the lignins of ref8, fah 1–2 and C4H::F5H lines.
      ,
      • Faix O.
      • Meier D.
      • Fortmann I.
      Thermal degradation products of wood: a collection of electron-impact (EI) mass spectra of monomeric lignin-derived products.
      ,
      • Faix O.
      • Meier D.
      • Fortmann I.
      Thermal degradation products of wood. Gas chromatographic separation and mass spectrometric charcterization of monomeric lignin-derived products.
      ).

      Generation of pADT::GUS Expression Lines

      Regions upstream of ADT4 and ADT5 (∼1800 bp each) were amplified using the primer sets shown in supplemental Table S2. Addition of PstI and PciI restriction sites to forward and reverse primers, respectively, facilitated directional cloning into the pCAMBIA 1305.2 vector. Confirmed pADT::GUS-pCAMBIA constructs were transformed into Agrobacterium for stable transformation of WT Arabidopsis (
      • Clough S.J.
      • Bent A.F.
      Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana.
      ). Second generation transformed (T2) plants were treated with X-gluc for visualization of GUS expression as described previously (
      • Kim S.J.
      • Kim K.W.
      • Cho M.H.
      • Franceschi V.R.
      • Davin L.B.
      • Lewis N.G.
      Expression of cinnamyl alcohol dehydrogenases and their putative homologues during Arabidopsis thaliana growth and development: lessons for database annotations?.
      ,
      • Kim K.-W.
      • Franceschi V.R.
      • Davin L.B.
      • Lewis N.G.
      ). Stems were hand-sectioned, stained with 0.25 mm X-gluc solution for 45 min, and observed under a stereomicroscope (Leica, Model MZ8) with fiber optics (NCL 150). Images were taken using a Leica camera (Model DFC 425C) and Leica Application Suite 35.

      Acknowledgments

      We express gratitude to Julianna Gothard for growing Arabidopsis wild type and mutant plants; Ericka Duncan, Victor Barona, Andres Lozano Garcia, and Luis Oleas Chavez for laser microscope dissection of vascular bundles and interfascicular fibers; and Diana Bedgar for arogenate dehydratase assays and amino acid analyses. We also thank Dr. Michael Knoblauch and Dr. Valerie Lynch-Holm of the Franceschi Microscopy and Imaging Center for use of the microscopy facility.

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