© 2012 The Authors. The Journal © 2012Blackwell Ltd Publishing PlantJournal © 2012The Authors. The Article', doi:10.1111/tpj.12012 'Accepted articleasan citethis of Record.Please version andtheVersion this between differences through the copyediting, typesetting, pagin buthasnotbeen review undergone fullpeer and forpublication accepted This articlehasbeen ofoneMammillaria as in the Cactaceae, evolving still is composition lignin Seedcoat examined. thatwere Opuntioidae thesubfamily in six did all as seeds, their , Cumarinia polymer, resulting in benzodioxane units. However, the species examined within the genera growing the onto monomers alcohol of caffeyl –O–4-coupling endwise by formed homopolymer within the subfamily Cactoidae possess coat lignin of the novel C-type only, which we show is a genera of most but members type, (G/S) guaiacyl/syringyl normal ofthe is of cacti tissues vegetative have now determined the lignin types in the seed coats of 130 different species. Lignin in the ofonegenus, andin cacti orchid of vanilla seed coats the in (C-lignin) polymer lignin catechyl unsuspected ahitherto described recently We have SUMMARY 1 Chen, Fang implications fortheevolution oflignindiversity C the Novel seedcoatligninsin Article type : OriginalArticle 03-Sep-2012 : Date Accepted :30-Aug-2012 Date Revised :08-Jul-2012 Date Received USA; 73401, USA; *For correspondence (fax +1 580 224 6692; e-mail [email protected] or [email protected]) [email protected]) or [email protected] e-mail 6692; 224 (fax 580 +1 correspondence *For Initiative, USA. 1710 USA; University Avenue,1710 Madison,WI53726, Accepted Article BiologyDivision, SamuelPlant Roberts NobleFoundation,2510Sam Noble Ardmore,Parkway, OK 4 DOE Great Lakes Bioenergy Research 1,3* 2 Yuki Tobimatsu, Department of Biochemistry, University of , 2, Lisa Jackson, and Center, Madison, WI,andWisconsin Bioenergy 1 ation andproofreadingprocess whichmay leadto actaceae: structure, distribution and distribution actaceae: structure, JohnRalph, 3 DOE Bioenergy Sciences Center, Oak Ridge, TN, ()mostly had normal G/S lignin in (Chen et al., PNAS 109: 1772-1777, 2012). We Wisconsin-Madison, EnzymeInstitute, 2,4 2,4 and Richard A. Dixon A. Richard and species ( 1,3*

M . lasiacantha ) generate lignin precursors diffe © 2012 The Authors. The Plant Journal © 2012Blackwell Ltd Publishing PlantJournal © 2012The Authors. The lignins aregenerally composed of hydroxylationaromatic and etal. Zhou Boerjan andChapple, inrecent 2002; studied (Humphreys years pathways biosynthetic The been formation to the leading cell ligninhave of wall the extensively polymer INTRODUCTION . coat, seed spectroscopy, resonance magnetic nuclear composition, lignin alcohol, caffeyl Cactaceae, Keywords: types. lignin newly discovered ofthese biosynthesis the underlying mechanisms for the findings these of the implications We discuss all. at nolignin contain species ofsome andseeds , in naturally occurring apolymer ofsuch report first the units, of5-hydroxy-guaiacyl composed lignin unusual possess only C-lignin, three methylation reactions had been blocked (Marita et al. rare units these lignin found transgenicwere inthe of only plantsin thefirstwhich or second alcoholscaffeyl and 5-hydroxyconiferyl (Figure 1a)are in‘normal’ not found lignins. Untilrecently, Catechyl (C) (5H) units and 5-hydroxyguaiacyl that de may respectivelyalcohols, (Figure 1a); natural angiosperm lowlignins have levels (<~2%)only of H-units. primarymonolignols, three the polymerization of by biosynthesized Accepted plantkingdom, isitthe andhow made cacti has theligninfrom of described. to be yet ‘endwise’ radicalcoupling reactions that typify lignification (Chen planifolia (Chen planifolia with dominantpolymer G units (Wagner the ligninof transgenic gymnosperm (radiata pine)tracheary the case catechylof units,the report only conc Article etal. We recentlyWe unexpectedthe observation made that the seed coats some of monocots ( Two obvious questions arise when considering a is derived from caffeyl isderived alcohol linkedmonomers headto tail into benzodioxane chains via the (cacti ) anddicots thegenus of , 2010). The, a 2010). seriesmonolignols, alcohols, hydroxycinnamyl areof formed by asequence of , 2012). Nuclear magnetic resonance spectrosc O Escobaria -methylation reactions (as well assuccessive side-chain reductions) to ring intheiraromatic subst p -hydroxyphenyl (H), (G),-hydroxyphenyl guaiacyl (S) andsyringyl units,that are species ( in planta et al. et Melocactus , 2011). erned the appearanceof asmall proportion C-unitsof in E. dasyacantha E. ? To date, we have only found this polymer in polymer this found have date, only ? To we ) a contain largeamounts of catechyl homopolymer , 1999; Ralph novel lignin polymer. novelWhat is ligninpolymer. its in distribution rive from polymerization of thecorresponding opy revealed that the C-lignin polymer from itution patternsitution (Boerjan element cultures, as part probably a of co- , et al. E. lloydii E. et al. etal. p , 2003; Abramson 2003; , -coumaryl, -coumaryl, andsinapylconiferyl, , 2012) (Figure 1b).The structure , 2001; Wagner 2001; , and E. zilziana etal. et al. et al. ) contain an contain ) , 2003).Natural , 2010; 2010; , , 2011). In 2011). , O Vanilla - Vanilla V. © 2012 The Authors. The Plant Journal © 2012Blackwell Ltd Publishing PlantJournal © 2012The Authors. The species)number of within the Cactaceae (Cacto samples of 130 different species of cactus, representative theof two major sub-families (based on the familythroughout Cactaceae, we usedthioacidolysis to determine from seed thelignincomposition the genus the of recently presence coat have described the seed We of C-lignin inthe of Distribution of C-lignin within the family Cactaceae RESULTS species and members of the Cactaceae, although itis intermediate stage in the evolution species possess anovel natural of primarily units, 5H lignincomposed which could beviewed asan in theseed coat is atrait that isstill evolving the structures of the non-classical fo ligninpolymers cactus, covering the two sub-families representative of thelargest number knownof species, andreport Here, biosynthesis. we determine the distribution and collectors, andthis makes them an excellent subj bydriven the commercial availability of a largenumber of these attractive species for amateur growers (Butterworth However, there hasbeenbiochemistry. considerable interest inthetaxonomy of this family seed coat. C-lignin.For whatever reason,of lack of monolignol appearsmethylation to be better tolerated inthe vascular architecture (Do al. et methyltransferase) inArabidopsis Medicagoleads or to adwarf phenotype with seriously distorted Understanding itsdistribution may

Accepted Article monolignol the two of knock-down or knock-out double example, For stem and leaves. the of vascular tissue due tothefact that blocking ligninmethylation isproblematic for theplantifallowed to inoccur the methylated monolignols were blocked. The fact that beenC-lignin hadnot discovered earlier islikely Theoretically, the presumed caffeyl precursor alcohol beformed C-ligninwould ifallroutes of to the The Cactaceae represent a large butrelatively unexplored plantfamily as regards their O et al. -methyltransferases (caffeicacid , 2002; Nyffeler, 2002; Butterworth and Wallace, 2004; Edwards 2004; Wallace, and Butterworth 2002; Nyffeler, 2002; , Melocactus , 2007; Zhou 2007; , (Chen shed light themechanisms unde on of seedof co et al. et al. et within the Cactaceae. We also show thatsome cactus ats with C-lignin. O , 2012). To determine whether C-lignin isfound whether Todetermine , 2012). ideae and ), -methyltransferase andcaffeoyl CoA-3- , but, without2010) surprisingly, measurableformation of lignin typesof composition across species 130 of und. Theresults indicate that possession C-lignin of more thanlikely th ect for the study seedcoatof lignindiversity and rlying its rlying biosynthesis. V. planifolia at it occurs elsewhere. and including 47 different andincluding 47 etal. and some members O , 2005),largely - O - guaiacyl (5H) unitsin the lignin (see oflevels ligninasassessed thioacidolysis; the by Mammillaria evolving inthe genus evolving C-lignin(Figure only 2c). Thus, possession of C-ligninintheseed coat appears to beatrait that isstill levels) in 15 sequence analysis (Crozier, 2005). Interestingly, alt publishedphylogeny previously of analysis compositional theon of broad the taxonomy orders within the Cactaceae andon a detailed, predominantly G/S lignin in their seeds, and seeds of members of the genus Cactoideae, members of the related genera © 2012 The Authors. The Plant Journal © 2012Blackwell Ltd Publishing PlantJournal © 2012The Authors. The however, that thelignin in the interface,spine base,stem/root and roots of mature caffeylof alcohol-derived units (Figure2a). Analysis of cell wall preparations from the stems, spines, the lignininseed coat theof Brazilian cactus excess of 30 (Chen reactions that typify lignification (Figure1b), and with caffeyl alcohol linkedmonomers headto benzodioxanetail into chains via the ‘endwise’ radical coupling C-lignin intheseed coat of C-lignin from cactiis alinear benzodioxane polymer genera. Table S1presents the complete data set fo genera A (in the thesubfamily Opuntioideae presence within for its found evidence we no Cactoidae; subfamily subfamilies, tribes and genera. From this analysis, is it clear that C-ligninisprimarily found within the Table 1places thelignincomposition data for ea Accepted Articlethioacidolysis. In afewspecies,the ligninlevels the seed,whereas asmaller number contained G/S lignin traces areshown inFigure2. thioacidolysis species Most C-ligninas contained lignintype within the only includingoverall lignin thioacidolysis analyzed, and notesyields on seed Representativecolor. ustrocylindropuntia Mammillaria and

Coryphantha et al. Mammillaria , 2012). Analysis by of composition ligninmonomer , 2012). thioacidolysis indicated that species, seeds the ofone species, V. planifolia , Grusonia ) (Butterworth and ) (Butterworth Wallace, low G/S contain either ligninor very 2004) vegetative tissues of the plantwas appeared low, and very itwashard toassigna composition by generamultiple within the . below). These data aresummarized , (Chen Maihueniopsis Coryphantha etal. ch genusstudiedinaphylogenet analyses suggested traces bothof Sand5-hydroxy- Melocactus salvadorensis hough we found only G/S lignin (or G/Shough wefound low only lignin(or lignin very r the analysis of the seed , 2012) is a linear polymer derived almost totally from plants of Melocactus by thioacidolysis indicated, averagea number degree polymerizationof in , Nopalea and (with noevidence for M. lasiacantha M. Cactoideae based onplastidDNA Mammillaria or comprised predominantly of GandS Opuntia). However, within the in Figure 3, which places the was likewise composed only appear tocontain , contained highlevels of lignins of the species Escobaria the presence of C-lignin). ic context based on (alsorelated to 4c). Lignin levelswere likewise extremely low to absent in the expected signalswerespectra, and seen for cell wall glucan,xylan and arabinanpolymers (Figure the lack of lignin in these seeds, although an unassigned peak was detected in the aromatic region of the micromeris Coryphantha © 2012 The Authors. The Plant Journal © 2012Blackwell Ltd Publishing PlantJournal © 2012The Authors. The the seeds of units, asis andThe hightypical 2010). for Landucci, most of level lignins (Ralph C-ligninin angiosperm as beingrichin-aryl ether units interrogated by whole cell wall NMR analysis (Figure 4b); theligninistypical G-rich ligninandisseen G/S the entire cell walls from roots. andin the stem the baseof at being found lignin, little most very contained the plant of body small units, with Hunits amounts of and no evide The structure theof G/S“classical” lignininseeds of Cactaceae the within types lignin ofother Structures biosynthesized purely fromcaffeyl alcohol, as we previously determined for the Vanilla seedcoat lignin. present. All these datasuggest that the are virtually nonexistent in this seed. indicated that the seed lignin isalmost exclusively composed Cunits; of typical G and Sligninaromatics for the observed previously acetylated the of dissolution/acetylation using the Acceptedresinol of amounts lignins units intypical from G/S coniferylderived and alcohols,sinapyl were absent, whereas small units-aryl Thenormaldimeric inthepolymer. acyclic ether units Article dominant for ligninunitsaccounting over were ofthe theidentifiable The98% benzodioxanes resolved. rings benzodioxane polysaccharides such asglucans, arabinansandxylans; the-, -, and-correlations from units benzodioxane the from The genus genus The To determine whether

and M. lasiacanthaM. withinthe Cactoideae (Cr E. greggii M. salvadorensis III VI units, whichalso can be generated by t , as well as lower-level contributions from failed torelease any ligninunits (Figure2d). Whole cell wall NMR confirmed M. salvadorensis M. V. planifolia (Figure2c) was confirmed bywhole cell wall NMR analysis (FigureS2a). VI Melocactus were clearly observed with along the signalsfrom typical cell wall isphylogenetically distinct from DMSO/NMI solvent system(Luand Ralph, 2003).The 2D HSQCspectra I , with more modest amounts of phenylcoumaran phenylcoumaran of amounts modest more with , seeds (Figure displayed4a) essentially identical signals tothose In the high-field aliphatic regions high-field aliphatic In the ozier, 2005) (Figure Melocactus C-lignin(Chenetal. seed were analyzed by solution-state NMR via complete C-ligninisstructurally similar to that found in nce the C presence for of main units S1).The (Figure seed ligninisessentially ahomopolymer Mammillaria densispina Mammillaria prolifera 3). Thioacidolysis of seeds of polymerization caffeyl alcohol,of are , 2012).The aromaticspectrum HSQC clearly Mammillaria cis I , whichare the linkage predominant -benzodioxane rings of theHSQC spec (Figure 2b) was , and Escobaria , aspecies that, theon II andresinol Epithelantha trum, the signals VI V. planifolia trans c , were - III

, cell wall NMR suggests that theligninfromseeds of whole 2f), (Figure S monomers G and/or amounts of comparable with along monomers 5H of amounts a similar way as those from caffeyl alcohol (Figure benzodioxane units The aliphatic regions of the spectra from those seeds clearly indicate the massive presence of 2003; Ralph 5-hydroxyconiferylincorporating alcohol isolated from transgenic plants (Marita non-etherified units 5H areresolved be andcould related revealed deepbluestaining purple around directly reflect the ligninthioacidolysis yield from the seed (Table S1). Stainingwith blue toluidine HSQC spectra of seed of in these The results suggest that the unusual monolignol, 5- products thioacidolyis couldand 5H beclearly identified G,Samounts unitsof (5-hydroxyguaiacyl) andunusual5H (Figure2f). resolved, Although poorly theS seedcoats from containing C-lignins.TheGC G/S or traces for these yields from examined contain highlevels lignintheir of G/S seeds (Table S1, Figure 2e). Incontrast, the thioacidolysis S units and minimal levels GunitsS unitsandminimal of (andnoCunits) (Figure 4e). units (Figure4d), andthat from © 2012 The Authors. The Plant Journal © 2012Blackwell Ltd Publishing PlantJournal © 2012The Authors. The Acceptedgreatest in (Figure 5). Lignin/phenolic autofluorescence was seen images inconfocal of allseed coats, and was and S ligninonly), species, morphological feature in the cactus seed coat, we examined the coats from imbibed seeds offour determine To whether possession of different lignintypes was associated with any specific compositions lignin different coats with seed of cactus Morphology ArticleMammillaria basis of chloroplast sequence,DNA is closelyvery related to Escobaria

Two species of Melocactus salvadorensis Escobaria E. vivpara arizonica E. dasyacantha et al. et species we observed to possess C-lignininseeds(Figure 3). and , 2001). There are several aromatic correlations (gray) that are currently not assigned. seeds. cell Whole wall NMR strongly supports thisfinding.Inthe aromatic region of arizonica Escobaria vivipara IVthat begeneratedcould from alcoholthe in of polymerization 5-hydroxyconiferyl Mammillaria ( Escobaria E. dasyacanthus , lloydii and E. zilziana and albicolumnaria and species, whereas the seed coat of the M. lasiacantha lasiacantha M. zilziana Mammillaria lasiacantha and isalso predominantly composed 5Hof units,with lowlevels of structures (corresponding to pits) inthe seed coats theof zilziana (G/S lignin by only), fluorescence and lightmicroscopy were very lowand the GC traces were atypical of those and assigned tentatively usingthe data lignins NMR from 1b). Whereas thioacidol (Figure 5c,d). The level autofluoresenceof didnot hydroxyconiferyl alcohol, participates inlignification hydroxyconiferyl E. dasyacantha (Figure 4d,e), clear sign vivipara by selectedby ion inset).monitoring (Figure2f, M. lasiacanthaM. [subspecies (C-ligninonly), is composed iscomposed totallyalmost of 5H Escobaria Melocactus ysis released only small arizonica (Crozier, 2005), the only the 2005), (Crozier, als from etherified and seeds displayed small Escobaria lloydii etal. and species showed , 1999,2001, neomexicana (5H and (5H ]) been selected for on the basis of environment. America ( evolving in neither C-nor G-signatures inthe other four species. Thus, itis clear that seed lignin composition is still showing analyzed G/S hadhighlevels andanunexpected inthreespecies of polymer species lignin; levelslow G/S lignin,andof one, sixteen species of

Mammillaria the genera within the subfamily G/Snormal type typical angiosperms,of and dicotyledonous that this toC-lignininmost of hasevolved © 2012 The Authors. The Plant Journal © 2012Blackwell Ltd Publishing PlantJournal © 2012The Authors. The analysisclearlyNMR indicated that theligninfrom Identification of a novel, naturally occurring 5H lignin ( their seeds. These species cover (Figure 3), andwe assume that it will contain “classical” G/S lignininseeds. Pereskia is regardedas being basaltoall familiesother withinthe Cactaceae (Edwards analyze ligninwithin the subfamilies Maihuenioideae (comprising only one species) or Pereskioideae; found (or atleastnot is not within common) the subfamily Opuntioideae. ancestral more We didnot Our dataindicate that C-lignin iswidelydistributed wi Distribution of C-lignin within the Cactaceae Discussion weakermuch stainingandlack of isrelatedstructure/staining andisprobably to taxonomy littleaffected by lignin composition. followed by internal trapping of the quinone by this apparently internal the explains methide trapping thequinone intermediate); very followed of 5Hof units. AsinC-lignin,these are characterized by chains benzodioxane of units –O–4-coupling (from AcceptedAcanthocalycium Article Our dataindicate that the s Within the Cactoideae, all members of fiftysix Pediocactus and Escobaria , where several species (possibly all inthe case of C ) through the tropics ( Mammillaria ). Thus, thepossession not of C-lignin does appear to beatrait thathas obviously Mammillaria examined, hadhigh four of levels G/S ligninintheseeds,eleven hadvery Cactoideae, with an enormous geographical and climatic range, from Argentina M. lasiacantha M. thepurplepit-likestructures. eed lignininthe ancestral cl . Melocactus , had high levels of C-lignin only. All sevenEscobaria the exception of Escobaria dasyacantha , thin the subfamily thin thesubfamily Cactoideae, butis apparently genera contained analyzed C-ligninonly within

ades within the Cactaceae is of the oryphantha ) to cold, regions mountainous North of These data indicate that the seed coat Coryphantha seeds consists almost entirely ) retain the G/S type. Of the the Of type. G/S the retain ) , Escobaria et al. and , 2005) 2005) , vegetative tissues (Do al. et Rather, plant growthisseriously disrupted, and very biosynthesis monolignol (Ye methyltransferase (CCoAOMT,pr theenzyme et al. mutant mutant contains C-lignin(Do the thecoat seed of whether investigate is not possible to it does not seeds, set mutant knock-out © 2012 The Authors. The Plant Journal © 2012Blackwell Ltd Publishing PlantJournal © 2012The Authors. The COMT canresult inrelatively normalplants with very high levels of 5H lignins in vascular tissue (Marita position, itis likely seed-specific innature. Inthisre work) suggests that ifagenetic mutation has occurred to block monolignol methylation at either possess C-ligninintheseeds appearsto be G/S ofthenormal type ((Chen substitution on the hydroxyl atposition 3. The fact that the lignin in the vegetative tissues of plants that position 5 of the aromatic ring G/S type. lignincommon 5H differsfrom Slignininthe lack of asingle group on methyl atthe hydroxyl hypothesis regardingthe evolution lignintypes of within theCactaceae. The ancestral form islikely the Based the on currently accepted pathw biochemical Biochemical mechanisms underlying seed lignin types within the Cactaceae created up-regulation by inCOMT-deficientof F5H backgrounds (Vanholme 2003; Ralph mutationthrough or RNAsilencing, invascular tissues severalof plantspecies (Marita artificially though knock-out of units been benzodioxane have created alcohol-derived containing 5-hydroxyconiferyl polymers Lignin lignins represent, to thebestof residues makes them resistant to release by this approach(Marita low lignincontent of these samples separate andS/(G) 5H polymers, be remains to dete Gunits(minimal) present. Whether the ligninin zilziana butalignincomposed2010) entirelyof units 5H hasnever beenreported. The lignin inseeds of of

Accepted Articlereported elsewhere.lignins willbe 5H Escobaria , 2001, 2003; Morreel 2003; 2001, , wasalso composed primarily of 5Hunits, although inthiscase there were alsoS unitsand et al. et , 2001). Arabidopsis transgenics with highlevels particularly benzodioxanes of were et al. et , 2007; Zhou 2007; , etal. et al. (Figure1a), whereas C-lignin G differsfrom lignininthelack of methyl 5-hydroxyconiferaldehyde 5- our our knowledge, thefirst examples naturallyof occurring lignins. all-5H , 2004), knock out of both COMT and caffeyl Coenzyme-A 3- , 1994)) does, 1994)) not result C-lignin inplantsproducing in vascular tissues. , 2007). In only one report to date have C-units been reported in the reported C-units date have report to been In one only , 2007). whendetermined by thioacidolysis, as the linkagepattern of 5H et al. , 2010). Since the Arabidopsis COMT/CCoAOMT double Arabidopsis COMT/CCoAOMT double Since the , 2010). imarily responsible for the 3- E. zilziana spect, it isinteresting th ay to wesuggestthefollowing monolignols, rmined. Isolation andmore low levels normalof G-ligninsareproduced inthe seeds is of mixed type, oris composed of O -methyltransferase (COMT), either et al et ., 2003). The 2003). ., et al. et O at, although knock out of -methylation reaction in et al , 2012) and the present detailed characterization detailed ., 2010; Weng Escobaria et al. , 1999, 2001, O - seed coat et al. E. ,

silencing (or avoidance) possibly of monolignol any fitness advantages to cacti, andthe molecular genetic mechanisms thatresult intissue-specific possible thatpossible C-ligninformation in detrimen vascular tissue is 2011). Even so, this ligninisstillpredominantly theof G type (Wagner Mammillaria the evolution theof family, andappearsto be still evolving inatleast two related genera ( iscomposition areliable taxonomic marker, itis interesting thatthis trait does in general correlate with lignin propose not that we do and Wallace, Crozier, Although Butterworth 2005). 2004; 2002; © 2012 The Authors. The Plant Journal © 2012Blackwell Ltd Publishing PlantJournal © 2012The Authors. The Accepted recently acquired more butcanalsowere be easily lost. rule out thepossibility that Cand5H seed coat lignins are ancestral, and that the methylation functions across limited understanding seedcoatour of lignincomposition theplant makes kingdom itdifficult to the result of loss function aprocessof of species that contains both lignins in C andG/S coat,the seed consistent with C-lignin being formation’s Cin theevolution of lignin.Itisimportant to note th 2010). In this latter case, the 5Hligninseen insome also able to catalyze the 3- require loss function of COMTof both and CCoAOMT, is knownabout seed-specific regulation of thepathwa Article Zhong (Zhong andYe, 2007; biosynthesis recently beenmajor advances in C1 of thatenzyme metabolism produces groups lignification. specific methyl have Althoughfor there to possible function account for theloss C-lignin lossof through or formation of expression of an CCoAOMT or aof regulatory gene controlling CCoAOMT expression in the seed. Alternatively, itis species of controlling COMT expression inthe seed coat) will result inaccumulation of the insome5H ligninsfound COMT (either through mutation a of seed-specific form theof chemical reactivity of the caffeyl alcohol monomer), butissomehow tolerated in the seed coat. tissues, non-seed lignin of in liquidculturesof Although we Although favor the above models, which are by supported data our within the Cactaceae, The taxonomy theof Cactaceae is complex andconstantly underrevision (Butterworth In thelight of these considerations, we prop Escobaria ). include questions forMajor futurestudy wh . Similarly, formation require of C-lignins would mutation of a seed-specificform of O -methylation of precursorsmonolignol (Parvathi our understanding our of thetranscriptional control of monolignol et al. that isalso required ligninbiosynthesis. for G/S Pinus radiataPinus , 2008; Demura and Ye, 2010; Zhao and Dixon, 2011), little little 2011), and Zhao andYe, Dixon, 2010; Demura , 2008; O -methylation. deposition Lignin in plantseed is coats a Escobaria at we have, to date, seenno example a of cactus ose that seed-coat-specific loss of expression of y. Itis y. also possible that C-lignin might formation because recent evidence because recent ether seed lignincompositional traits provide down-regulated inCCoAOMT (Wagner tal to plantgrowth (p species could be viewed as intermediate COMT gene of geneor a regulatory et al. , 2011). Itis therefore etal. has implicated COMT as , 2001; Zhou ossibly because of the and Escobaria et al. etal. et al. et , , , and water (3times each), then freeze-dried before lignin analysis. with ahammer. All samples were extracted with chloroform/methanol (2:1, v/v), 100% methanol, Metuchen, NJ)SamplePrep, unde dissected afrom juvenileplant of numberscatalog arelisted in TableS1. and depressus levitestatus neomontanus Melocactus obtusipetalusMelocactus ascencionis silicicola with adiamond knife a on Leica EM UC7ultramicrotome (Leica Mikrosysteme GmbH, Vienna,Austria), Ltd., Reading, Berkshire, UK). The resin was polymerized at 55°C for 3days. Serial 1μ tetroxide, dehydrated in a graded buffered saline(PBS, Samples pH7.2). were then washed with postPBS, fixed with 1% (v/v) osmium Sciences, Hatfield, and (v/v)PA) 4% paraformald glutaraldehyde fixed in (v/v) (upto (Electron were Microscopygerminated they 1 2.5% month), © 2012 The Authors. The Plant Journal © 2012Blackwell Ltd Publishing PlantJournal © 2012The Authors. The Acceptedrefrigerator for 3 days for before vernalization exposure to constant lightat 24 (MS)Skoog salts and1%sucrose. After planti Seeds wereplanted Arabidopsis growth on (0.5%medium phyta-agar)consisting 0.5 of Murashige-X Light Microscopy Article (Sulphur, Oklahoma): Seeds the speciesof following were obtained from author’sthe corresponding (RAD) privatecollection Plant Materials EXPERIMENTAL PROCEDURES ligninbiosynthesis,controlling withimplications for thefieldadvancing bio-based products.of unexploredrelatively area, but clearlyone with Wigginsia erinacea Plant vegetative tissue samplesPlant groundvegetative were to usinga afine freezer millpowder (SPEX

, Frailea heliosa Frailea , Notocacus roseoluteus L1186, , Melocactus guaricensis Melocactus , Melocactus delessertianus Melocactus Mammillaria prolifera capricorne . Seeds of theremaining species were , Mammillaria pilcayensis , Melocactus erythranthusMelocactus r liquid nitrogen. Seedsr liquidnitrogen. werebrokendown powderto hand by , Melocactus salvadorensis Melocactus ethanol series, and embedded inLR Opuntia ficus-indica , Melocactus salvadorensis Melocactus

Root, stem, stem base, spineand spine base tissues were var texensis , Melocactus curvispinus Melocactus ng, petri dishes containing seeds were placed in the , Astrophytum myriostigma potential for uncovering potential newregulatory for mechanisms ehyde (Electron ehyde (Electron Sciences) inphosphate Microscopy , Mammillaria spinossissima, , Melocactus ammotrophus Melocactus , Melocactus , Uebelmannia pectinifera . purchased from Me , Melocactus loboguerrori Melocactus , Melocactus cesiusMelocactus Whiteresin ResinCo. (London var var columnare , o Mammallaria glassii Mammallaria , C. Afterseeds were , Melocactus guitartii Melocactus Melocactus varpseudopectinifera sa Garden, Belen, NM; , sections were cut Melocactus Melocactus , , Discocactus Melocactus Melocactus var , © 2012 The Authors. The Plant Journal © 2012Blackwell Ltd Publishing PlantJournal © 2012The Authors. The withZrO rpm were ball-milled using cycle) (3×20 min, cooling 10-min aRetschPM100 ball-mill at600 vibrating Mansfield andRalph,2003; (Lu previously using thedimethylsulfoxide (DMSO)/ Selected seed samples were analyzed by solution-state NMR via complete dissolution/acetylation NMR Analysis 1986).approximately Briefly, 10freeze-dried mg of samples werereacted 0.2BF with 3mlof M Analytical thioacidolysis was according to the method (Lapierre described previously Thioacidolysis gainsettingsphotomultiplier were kept constant between specimens. a with nm diode laser blue 405 detected andemission nm. and intensity, Laser at460 pinhole laser scanning microscope (Leica Microsystems Heidelberg GmbH, Mannheim, Germany) illuminated immersed inCitifluor (Electron Microscopy Science) Instruments Inc.,Tokyo, Japan). For of visualization total ligninautofluorescence, μ 1 and stainedwith 1%(w/v) toluidine blueO andobserve mode (70 eV). Scanning range wasfrom 50-650 m/z. 1; 60 m × 0.25 mm; 0.25 m film thickness), a Packard 5890 series IIgas chromatograph with a 5971 series mass-selective detector (column: HP- GC/MSquantified by astheir trimethylsilyl deriva spectrometricchromatography/mass (GC/MS) analysis. Ligninthioacidolysis monomers were BSTFA + 1% TMCS (Pierce Biotechnology Inc., Rockford, IL), the samples were subjected to gas layers combinedorganic were anddriedunderastream waterml of was added, and the extractedmixture at 100 mixture dioxane/ethanethiol inan 8.75:1 etherate AcceptedBruker Biospin AVANCE 500 MHz spectrometer fitted with acryogenically cooled 5-mm TCI d inchloroform- dissolved then lyophilized to yield acetylated seed tissues (yield typically at 115-130%), which were totally l).Theresultant(1 precipitate wasrecovered filtr by added, andthen stirred for 2h atroom temperature. The mixture was poured into distilled water in dissolved DMSO/NMI (2:1, 3 vol/vol, ml) atroom temperature, acetic anhydride (1 ml) was Article 2 vesselscontaining ZrO (600 l) NMR for experiments. TheNMR spectra were acquired aon 2 ball bearings. The ball-milled seed Theball-milled tissue ballbearings. (~40 mg) was N -methylimidazole (NMI) solvent system asdescribed et al nd mass spectra were recorded in electron impact tives. was GC/MS performed on a Hewlett– ., 2012). Briefly, pre-extracted seeds (~100 mg) three times with 4 ml and observedunder a Leica TCS AOBSconfocal SP2 ation, washed withultrapure water l) (1 and d withan Nikon Optophot-2 (Nikon microscope of nitrogen. Afterof derivatization with ° C for 4 h. At the end of the reaction, 4 ofdichloromethane. The et al. sections were , 1985, 1985, , 3 -

spectra beingintegrated. Foranesti volume-integrated, andthe Sintegralswere logically halved; linearprediction usedfor wasnot and C, andthecarbon-2 units 5H were fromcarbon-6 correlations andnon-etherified etherified the carbon-2 correlation G, from thecarbon-2/6 correlation from S,thecarbon-2 correlation from of linear prediction (32 coefficients) inF1. For quantification of aromatic distributions (Figure2), level andsquaredcosine-bell GB= andone (LB=0.5, matched inF2 0.001), Gaussianapodization usingtheparameters describedcarried out previousl ( © 2012 The Authors. The Plant Journal © 2012Blackwell Ltd Publishing PlantJournal © 2012The Authors. The Fellowship supportPostdoctoral from theJapanSociety for the of Promotion Science (JSPS). Science (BER DE-AC05-00OR22725 and DE-FC02-07ER64494, respectively). YT acknowledges Centers, bysupported the Office of Biological andEnvironmental Research in the DOE Office of andthe US Department Foundation, for criticalZhao reading of the manuscript.This work was supported bytheSamuel Noble Roberts We thank JinNakashimaDr for assistance with light microscopy, andDrs Xiaoqiang WangandQiao ACKNOWLEDGEMENTS taxonomy used inthis article isbased Cactaceaeof the has under phylogeny and Eggli, The constantThe been (Nyffeler 2010). overall revision Phylogenetic Analysis reference ( used Bruker’sTopspin 3.1 software. (Mac) The cent gradient probe with inverse coils geometry(proton closest to thesample) andspectral processing side chainC composition composition within the genera plastidbased on DNA sequence analysis (Crozier, 20 (http://en.wikipedia.org/wiki/Classification_of_the_ the International fo Organization IV

Accepted Article ) (Fig. 2) were integrated; no correction factors were used(Mansfield

–H C / H contours ( contours : 77.0/7.26 ppm). Adiabatic 2D-HSQC (“hsqcet ppm). Adiabatic2D-HSQC : 77.0/7.26 I , II Mammillaria , r Study III mation of thevarious interuni ) or, because ofspectral overlap of the on that of the International Cact of Energy’sof Bioenergy Sciences andGreat LakesBioenergy , Coryphantha 05) wasused 05) to infer aspectsevolutionary of lignin Cactaceae), anda higher y (Chen et al., 2012). Processing usedtypical (Chenetal.,2012). y ral chloroform peaksral chloroform were usedasinternal and Escobaria t linkage types, thewell-resolved gpsisp2.2”) experiments weregpsisp2.2”) aceae Systematics Group (ICSG) of . ., 2012). 2012). et al., IV resolution phylogeny resolution phylogeny contours, the C the contours, –H

Do, C.-T., Pollet, B., Thevenin, J., Sibout, R., Denoue, D., Barriere, Y., Lapierre, C. and Jouanin, L. ( Demura,andYe, T Z.H. Regulation plantbiomass production. (2010) of ( B.S. Crozier, Chen, F., Tobimatsu, Y., Havkin-Frenkel, D., Dixon, R.A. and Ralph, J. J. ( andRalph, R.A. Dixon, D., Havkin-Frenkel, Y., Tobimatsu, F., Chen, Butterworth, C.A. and Wallace, R.S. ( Butterworth, C.A., Cota-Sanchez, J.H. and Wallace, R.S. ( Boerjan, W., Ralph, J. and Baucher, M. M. ( J.andBaucher, W., Ralph, Boerjan, Mammillaria lasiacantha © 2012 The Authors. The Plant Journal © 2012Blackwell Ltd Publishing PlantJournal © 2012The Authors. The Z. andShani, O. Shoseyov, M., Abramson, REFERENCES S2. Figure thioacidolysis. S1. Figure information foundof inthe version this article. supporting be online Additional may SUPPORTING INFORMATION Accepted Article study. present Table S1. Both caffeoylBoth Coenzyme A 3- emphasis emphasis on the genus Mammillaria Haw., PhD Thesis, University of Texas, Austin. alcohol inplantseeds.alcohol (Cactaceae: Cactoideae): based A phylogeny J. Bot. chloroplast sequencefrom andhypothesisvariation testing usingtheparametric bootstrap. 27, 257-270. lignocellulosic production and Sci. production Plant lignocellulosic processability. Details of origin, seed color andlignincontent/composition allspeciesof analyzed inthe Short-range and different content aLignin composition of organs by of Melocactus plant,asdetermined 2005)

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Zhou, R., Nakashima, J., Jackson, L., Shadle, G., Temple, S., Chen, F. and Dixon, R.A. ( Zhong, R. and Ye, Z.H. ( Zhong, R., Lee, C., Zhou, J., McCarthy, R.L. and Ye, Z.-H. ( ( R.A. andDixon, Q. Zhao, Ye, Z.H., Kneusel, R.E., Matern, U. and Varner, J.E. Analternative (1994) pathwaymethylation in lignin Weng, J.-K., Mo, H. and Chapple, C. ( C. andChapple, H. Mo, J.-K., Weng, Wagner A., Tobimatsu, Y., Phillips, L., Flint, H., Torr, K., Donaldson, L., Pears, L,.. and Ralph, J. ( J. and Ralph, L,.. Pears, L., Donaldson, K., H., Torr, Flint, L., Y., Phillips, Tobimatsu, A., Wagner © 2012 The Authors. The Plant Journal © 2012Blackwell Ltd Publishing PlantJournal © 2012The Authors. The Acceptedhydroxyconiferyl alcohol, respectively. and alcohol from caffeyl 5- the ligninchain.(b)Assembly ligninpolymers of C-ligninand5-H reactions biosynthesis, in monolignol andthe struct 1. Figure andTable toFigures Legends ArticleVanholme, R., Ralph, J., Akiyama, T., Lu, F., Rencoret Pazo, J., Christensen, J., Rohde, A., Morreel, K., Ralph, J., Lapierre, C., Lu, F.C., Marita, .JM., Pilate, G., VanDoorsselaere, J., Boerjan, W. and Jouanin, L. Parvathi, K., Chen, F., Guo, D., Blount, J.W. and Dixon, R.A. ( Natl. Acad. Sci. USA Natl. Acad.Sci. CoA reductases involved inparallelcinnamoyl routes to ligninin 2782. intheinvolved regulation of secondary cell wall biosynthesis in Arabidopsis. Cell Plant thought? biosynthesis in to to enrichment anunusual ofof pollen ligninanddisruption formation. wall CCoAOMT CCoAOMT suppression in modifies lignincomposition Plant J. Plant ligninsby out concomitant of up-regulation W. ( and B Boerjan, Van H., Reusel, Kim, R., DeRycke, Chem. hydroxyconiferyl alcohol into lignins of ( 25, 193-202. methyltransferases inalfalfa suggestnew pathways for 3- Lignin biosynthesis. Lignin (a)Simplified scheme 2001) NMR evidence2001) for benzodioxane structures resulting incorporation from of 5-

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, 2763-

(a) and(e), staining. (a-d),autofluorescence ligninandphenolic (e-h), compounds. of staining withtoluidine blue O. 5. Figure dasyacantha Succulent Plant Study. X = lignindetectable.no suggested bytheInternational Cact aceae Systematics (ICSG) Group oftheInternational Organization for Table 1. (g), © 2012 The Authors. The Plant Journal © 2012Blackwell Ltd Publishing PlantJournal © 2012The Authors. The 4. Figure fulldetailsfor speciesof analyzed. species in to addition those listed within aspecific region of the detailed phylogenetic tree. See Table S1 indicated. The two numbered boxes with nooutline indicate the lignincomposition of andnumber with black show outlines the number speciesanalyzed of with individual thelignin composition indicated by red,C-lignin;color: blue,G/S gree lignin; with(2005), additional speciesnotanalyzed heredeleted the Cactoideae (this only represen based phylogeny, chloroplaston sequenceDNA data, shown for certain closelyrelated species within orders currently recognized the (from basal Pereskioidaeae to the Cactoideae), with a detailedmore 3. Figure (SIM) (S chromatogram (5H and 299 monitoring 357 of monomer) m/z monomer). m/z species indicated. IS,docosane internalstandard. The inserts in 2f show the partial selective-ion- 2. Figure Accepted salvadorensis Melocactus Article E. vivipara arizonica Seed lignincompositions generawithin 47 of cacti, according to thetaxonomy organized Cactus seed coat morphology revealed by fluorescence microscopy and blue toluidine O Short-range Phylogenetic relationships andseed lignincomposition of cacti. The scheme shows the five Gas traceschromatogram of the thioacidolysis products seeds of ligninfrom thecactusof Melocactus salvadorensis Melocactus (d),and Escobaria zilziana 13 C– (G and Slignin).(d)(h), (a), Mammillaria densispina (a), 1 H correlation (HSQC) spectra ofacetylated whole seed cellwalls from ts a small fraction of the order). (C ligninonly). (b) and(f), (e). Mammillaria lasiacantha n, 5H lignin; gray, lignin; nodetectablen, 5H lignin. Theboxes (b), forsimplification. is Lignin composition Escobaria lloydii Escobaria The latter istaken from Crozier (C lignin only). (5H andSlignin).(c) and (c), Escobaria Escobaria © 2012 The Authors. The Plant Journal © 2012Blackwell Ltd Publishing PlantJournal © 2012The Authors. The Accepted Article Cacteae Cactoideae Browningiae Browningia Subfamily Tribe Genus 1 C # of species Seed lignin type Pachycereeae Pachycereeae Notocactaceae Hylocereeae Calymmanthae Cereus 1 C 0 Copiapoa 2 C (0) Carnegia 1 C Ariocarpus Ariocarpus 3 C roye 3 C Echinocereus 3 Parodia 3 Frailea C 4 Eulychnia C Eriosyce 1 1 C C Uebelmannia Pilosocereus Melocactus 1 Discocactus C 1 C 15 C 1 C tobccu 1 C Turbinicarpus 1 Thelocactus Strombocactus Stenocactus 4 C 3 X C Sclerocactus Pediocactus 2 C Mammillaria 5 2 Ferocactus C 1 C Escobaria 16 Epithelantha G/S, 2 C Echinomastus 7 G/S, Echinocactus X, Coryphantha 5H C Astrophytum 1 C 1 C 11 G/S 3 C © 2012 The Authors. The Plant Journal © 2012Blackwell Ltd Publishing PlantJournal © 2012The Authors. The

Accepted Study. X Plant = nolignindetectable. (ICSG) of theInternational for SucculentOrganization theInternationalby Cactaceae Systematics Group cacti, according to theorganized taxonomy suggested Table 1. Article Pereskioideae Austrocylindropuntieae Opuntioideae Maihuenioideae Seed lignincompositions within 47 genera of Trichocereeae Rhipsalideae Tephrocacteae Pterocacteae Opuntieae Cylindropuntieae Opuntia 2 G/S 0 0 Maihueniopsis 1 G/S cnhclcu C 1 Grusonia 1 G/S eui C C C 4 1 3 C C Mila C 2 1 1 1 C 2 3 C C C 4 C 1 Stenocereus Polaskia 1 Pachycereus 1 Escontria C C 1 C utoyidouta 1 G/S 1 Austrocylindropuntia 0 0

© 2012 The Authors. The Plant Journal © 2012Blackwell Ltd Publishing PlantJournal © 2012The Authors. The Accepted Article

© 2012 The Authors. The Plant Journal © 2012Blackwell Ltd Publishing PlantJournal © 2012The Authors. The Accepted Article

© 2012 The Authors. The Plant Journal © 2012Blackwell Ltd Publishing PlantJournal © 2012The Authors. The Accepted Article

© 2012 The Authors. The Plant Journal © 2012Blackwell Ltd Publishing PlantJournal © 2012The Authors. The Accepted Article