Sugarcane DIRIGENT and O-METHYLTRANSFERASE promoters confer stem-regulated gene expression in diverse monocots
Mona B. Damaj · Siva P. Kumpatla · Chandrakanth Emani · Phillip D. Beremand · Avutu S. Reddy · Keerti S. Rathore · Marco T. Buenrostro-Nava · Ian S. Curtis · Terry L. Thomas · T. Erik Mirkov
Abstract Transcription proWling analysis identiWedSaccharum hybrid DIRIGENT (SHDIR16) and �-METHYL-TRANSFERASE (SHOMT), putative defense and Wberbiosynthesis-related genes that are highly expressed in thestem of sugarcane, a major sucrose accumulator and bio-mass producer. Promoters (Pro) of these genes were isolatedand fused to the �-glucuronidase (GUS) reporter gene.Transient and stable transgene expression analyses showedthat both ProDIR16:GUS and ProOMT:GUS retain the expres-sion characteristics of their respective endogenous genes insugarcane and function in orthologous monocot species,
including rice, maize and sorghum. Furthermore, both pro-moters conferred stem-regulated expression, which was fur-ther enhanced in the stem and induced in the leaf and root bysalicylic acid, jasmonic acid and methyl jasmonate, key reg-ulators of biotic and abiotic stresses. ProDIR16 and ProOMT
will enable functional gene analysis in monocots, and willfacilitate engineering monocots for improved carbon metab-olism, enhanced stress tolerance and bioenergy production.
Genomic tools such as regulated promoters that allowspatial and temporal control of gene expression enhance
Electronic supplementary material The online version of this article (doi:10.1007/s00425-010-1138-5) contains supplementary material, which is available to authorized users.
M. B. Damaj · M. T. Buenrostro-Nava · I. S. Curtis · T. E. Mirkov (&)Department of Plant Pathology and Microbiology, Texas AgriLife Research, Texas A&M System, Weslaco, TX 78596, USAe-mail: [email protected]; [email protected]
S. P. KumpatlaDepartment of Trait Genetics and Technologies, DowAgroSciences LLC, 9330 Zionsville Road, Indianapolis, IN 46268, USA
C. Emani · K. S. RathoreLaboratory for Crop Transformation, Institute for Plant Genomics and Biotechnology, Norman E. Borlaug Center for Southern Crop Improvement, Texas A&M University, College Station, TX 77843-2123, USA
P. D. Beremand · T. L. Thomas (&)Laboratory for Functional Genomics, Department of Biology, Texas A&M University, College Station, TX 77843-3258, USAe-mail: [email protected]
A. S. ReddyDepartment of Molecular Biology and Traits, DowAgroSciences LLC, 9330 Zionsville Road, Indianapolis, IN 46268, USA
our understanding of gene function and facilitate thegenetic improvement of important crop species. Regulatedtransgene expression is essential to ensure plant productiv-ity, viability and fertility, especially when the constitutiveexpression of the transgene is likely to compromise metab-olism or important aspects of meristem or embryo function(Lee et al. 2003; Karim et al. 2007; Pino et al. 2007).Depending on the improvement strategy, expression of thetransgene can be restricted to a given tissue, organ, ordevelopmental stage, or induced for a speciWc time (Maizeland Weigel 2004). Tissue-regulated expression is particu-larly advantageous for maximizing metabolic energy intotransgene products at targeted sites, thereby reducing theimpact on non-target tissues.
Unlike the current situation in dicot systems, success inpromoter development and use in monocots has been limited.Monocot promoters currently used are mostly derived fromhighly expressed constitutive genes, such as ubiquitin andactin (McElroy et al. 1990; Christensen et al. 1992; McElroyand Wu 1997; Lu et al. 2008; He et al. 2009). In particular, thefew promoters isolated and characterized in sugarcane, amajor sucrose accumulator and biomass producer, are of aconstitutive nature. These include promoters from ubiquitin 4and 9 genes (Wei et al. 1999, 2003; Albert and Wei 2003), anelongation factor 1� gene, a proline-rich protein-encodinggene (Yang et al. 2003), and Sugarcane bacilliform virus(Tzafrir et al. 1998; Braithwaite et al. 2004). Tissue-speciWcmonocot promoters have been developed that target geneexpression in leaf (Nomura et al. 2000), root (Yu et al. 2007),epidermis (Altpeter et al. 2005) and anther tapetum (Luo et al.2006). To date, functional monocot stem-expressed promotersare not available. This is a crucial deWcit for crops such assugarcane, where the stem comprises signiWcant biomass andcommercial value. Stem-regulated promoters would be ofgreat value in terms of improving plant biomass characteris-tics through enhanced carbon metabolism for sugar accumula-tion and/or high Wber content for biofuel feedstock (Sticklen2006). Such promoters would be speciWcally useful in manip-ulating key metabolic enzymes that are important for carbonpartitioning between cell wall Wber and soluble sugar in thesugarcane stem (Groenewald and Botha 2008).
The successful use of stem- and other tissue-regulated pro-moters depends to a large extent on overcoming the ability ofhighly polyploid species such as sugarcane to silence trans-genes, speciWcally those driven by native promoters. Severalgroups have reported on the silencing of endogenous stem-expressed promoters in sugarcane, a phenomenon not depen-dent on transgene copy number or sites of integration butassociated with developmental changes in ploidy levels(Potier et al. 2008a; Mudge et al. 2009). Overcoming thisobstacle is a major future challenge in obtaining functionalregulated promoters for the commercialization of transgenicsugarcane (Potier et al. 2008b).
In this paper, we report the application of high-through-put array diVerential screening to identify Saccharumhybrid (sugarcane) DIRIGENT (SHDIR16) and �-METH-YLTRANSFERASE (SHOMT) genes that are highlyexpressed in the stem and potentially involved in ligniWca-tion and defense responses. Expression of both SHDIR16and SHOMT genes was characterized by transcription pro-Wling as well as by functional analysis of their isolated pro-moters (Pro) that were fused to the �-glucuronidase (GUS)reporter gene, resulting in ProDIR16:GUS and ProOMT:GUS.Transient and stable expression analyses demonstrated thatProDIR16:GUS and ProOMT:GUS retain the expression char-acteristics of their respective endogenous genes in sugar-cane and function in orthologous monocot species. It isfurther shown that ProDIR16:GUS and ProOMT:GUS exhibit astem-regulated expression that is further enhanced in thestem and induced in the leaf and root by salicylic acid (SA),jasmonic acid (JA) and methyl jasmonate (MeJA), key reg-ulators of the protective plant responses to biotic and abi-otic stresses (Glazebrook et al. 2003). The value of ProDIR16
and ProOMT in functional gene analysis and in engineeringmonocots for improved carbon metabolism, enhancedstress tolerance and bioenergy production, is discussed.
Materials and methods
RNA isolation
A cDNA library was constructed from RNA extracted fromtissue obtained from top, middle and bottom regions of sug-arcane (Saccharum spp. hybrid, cultivar CP72-1210) stems.RNA extraction and poly(A) RNA enrichment was carriedout using the RNeasy plant Mini kit (Qiagen, Valencia,CA) and Oligotex mRNA Mini kit (Qiagen), respectively.
RNA used for microarray, northern blot and other expres-sion analyses was isolated according to Damaj et al. (2009).BrieXy, liquid nitrogen-frozen tissue in a buVer containing10 mM hydroxymethyl aminomethane (Tris–HCl, pH 7.5),1 mM ethylenedinitrilo-tetracetic acid (EDTA, pH 8.0),0.1 M sodium chloride, 1% (w/v) sodium dodecyl sulfate, 2%(w/v) polyvinylpyrrolidone and 1% 2-mercaptoethanol wasextracted twice with phenol (pH 4.3), chloroform, and iso-amyl alcohol (1.0:0.8:0.2); RNA was ethanol precipitated inthe presence of 3 M sodium acetate (pH 5.2) and reprecipi-tated with 4 M lithium chloride. Prior to use, RNA was treatedwith RNase-free DNase I (Ambion, Austin, TX).
Stem cDNA library construction and diVerential hybridization screens
Poly(A) RNA (100 ng) from each stem region was pooledand used to prepare cDNA using the SMART PCR cDNA
library construction kit (Clontech, Mountain View, CA).Size selected cDNA [0.5–3 kilobase pairs (kb)] was clonedinto pCR2.1 (Invitrogen, Carlsbad, CA).
Library archiving, high-density Wlter preparation andscreening were as described (Connell et al. 1998). A totalof 13,824 cDNA clones were archived in 384-well plates.Macroarrays were prepared by spotting ampliWed cDNAsonto high-density Wlters (Hybond-N+, Amersham Biosci-ences, Piscataway, NJ). Filters were probed with cDNAsprepared from pooled stem RNAs labeled with the DECA-prime II random priming kit (Applied Biosystems, FosterCity, CA). Following autoradiography, Wlters were strippedand re-probed with labeled leaf and root cDNAs.
Microarray preparation and analysis
Stem-expressed cDNAs were sequenced and used to con-struct a cDNA microarray. The nucleotide sequences ofthese cDNAs are available at http://enterprise.bio.tamu.edu/index_sugar.html. cDNAs were ampliWed by PCR andprinted on PL-100C poly-L-lysine-coated glass slides (CELAssociates, Pearland, TX) as described (Bailey et al. 2003).
AmpliWed RNA (aRNA) was synthesized (Message-Amp; Ambion) from sugarcane total RNAs. Randomlyprimed Xuorescent probes were produced from aRNA sam-ples using the 3DNA Array 350RP expression array detec-tion kit (Genisphere, HatWeld, PA). Hybridizations andwashings followed Genisphere’s suggestions.
Labeled arrays were scanned with an AVymetrix 428array scanner (AVymetrix, Santa Clara, CA). Resultingimages were analyzed with GenePixPro (Axon Instruments,Union City, CA). Data Wles were further analyzed usingGeneSpring (Agilent Technologies, Palo Alto, CA) to facil-itate normalization, parameter assignment and Wltering.Experimental values were divided by the control values andfurther normalized relative to the positive control sugarcanegenes: glutathione-S-transferase, G protein-coupled recep-tor, histone deacetylase, ribulose epimerase, tubulin andubiquitin. DiVerentially regulated genes were deWned as thosewith a twofold or higher amplitude change in their normal-ized ratios and a t-test p value of 0.05 or less. Two biologi-cal samples were used for each tissue type or time point.Each biological sample was used for three hybridizations.Six microarray hybridizations were conducted per sample.The t-test values were calculated based on the average of allsix pooled hybridization data.
Northern blot and quantitative RT-PCR analyses
Total RNA (15 �g per lane), fractionated on formaldehydedenaturing gels, was blotted onto nylon membranes(Nytran® SuperCharge, Schleicher and Schuell, Inc., NH)in 10£ SSPE buVer (Sambrook and Russell 2001). RNA
blots were hybridized with probes ampliWed by PCR usingprimers derived from the full-length coding region of eachtarget gene. PCR products were labeled with [�-32P] dATPby random priming using Klenow Exo¡ DNA polymerase(New England BioLabs Inc., Ipswich, MA). Hybridizedblots were visualized and quantiWed with the BAS-5000scanning system (FujiWlm Medical Systems USA, Stam-ford, CT). RNA loading and transfer eYciency was normal-ized relative to the band intensity of the sugarcane ubiquitingene.
First-strand cDNAs for quantitative RT-PCR (qRT-PCR) were synthesized using total RNA (2 �g) and theTaqMan® reverse transcription kit (Applied Biosystems).qRT-PCR was performed on an ABI PRISM 7700 (AppliedBiosystems) with the SYBR® Green PCR master mix(Applied Biosystems). Primers were designed with thePrimer Express version 1.5 software (Applied Biosystems).qRT-PCR was performed twice in triplicate with two bio-logical repeats. Results were analyzed with SDS version1.7 software (Applied Biosystems) and recorded as CT
(threshold cycle) values. Each transcript was quantiWed rel-ative to the sugarcane ubiquitin gene. Primer pairs used inthe qRT-PCR analysis were as follows: OMT [5�-AGATTCGGCAAGCTCTTCGAC-3� (F) and 5�-TTGCCACGATGTCCATGATG-3� (R)], DIR16 [5�-CCTGGGCGCTTCTACCAAC-3� (F) and 5�-ACACTTGTCGATCAAGCGTCG-3� (R)], DIR11 [5�-CATTCGGCAATTCTACTGGCA-3� (F) and 5�-GCGTCCAAAGAAACCGATGA-3� (R)],and ubiquitin [5�-CCAAACCCCGACGATCC-3� (F) and5�-TCTCGTACTTGTGCCGGTCC-3� (R)].
SHOMT and SHDIR16 genomic clone and promoter isolation
A Saccharum hybrid DIRIGENT16 (SHDIR16) genomicclone was isolated by screening a sugarcane (SaccharumoYcinarum £ S. spontaneum hybrid, cultivar R570) bacte-rial artiWcial chromosome (BAC) library SHCRBa (Clem-son University Genomics Institute, Clemson, SC) (Tomkinset al. 1999) with SHDIR16 cDNA, according to Damajet al. (2010). The SHDIR16 promoter was isolated by PCR-based genome walking in the BAC clone 159E16 DNAusing the Genome Walker kit (Clontech, Mountain View,CA). A 2.6-kb ampliWed PCR fragment was cloned intopGEM-T (Promega, Madison, WI) yielding the plasmidpSHDIR16/pGEM-T. The nucleotide sequence of theSHDIR16 coding and 5�-UTR/promoter regions (Supple-mentary material S1) was deposited into GenBank underthe accession number GU062718.
A Saccharum hybrid O-METHYLTRANSFERASE(SHOMT) genomic clone was isolated by screening a sug-arcane genomic library, constructed in Lambda DASH IIusing standard methods (Yang et al. 2003; Mirkov et al. 2008),
with full-length SHOMT cDNA. Southern blot analysisidentiWed a 4.3-kb XbaI SHOMT fragment that was sub-cloned into pUC19 yielding SHOMT/pUC19. A 2.9-kb pro-moter fragment was released from SHOMT/pUC19 as anXbaI/NcoI restriction digest. The sequence of the full-length SHOMT coding and 5�-untranslated (UTR)/promoterregions (Supplementary material S2) was deposited intoGenBank under the accession number GU062719.
Promoter-GUS constructs
A 2.9-kb XbaI/NcoI promoter fragment from SHOMT/pUC19 was cloned as a transcriptional fusion with the GUSgene ligated into the XbaI/NcoI-digested vector, GUSNOS/pUC19, (Van der Geest and Hall 1996) to yieldProOMT:GUS-1 for transformation of sugarcane, sorghumand maize. ProOMT:GUS-2, designed for rice transforma-tion, was constructed by releasing the SHOMT promoterfrom SHOMT/pUC19 as an XbaI/NcoI fragment and cloningit as a transcriptional fusion with the GUS gene in binaryvector, pCAMBIA1301 (CAMBIA, Brisbane, Australia),replacing the CaMV35S promoter.
A 2.6-kb SHDIR16 promoter fragment was ampliWedfrom the pSHDIR16/pGEM-T described above using theprimers: 5�-TCTGAGATAATACGACTCACTATAGGGCACGC-3� (F), and 5�-TGTACTAGTTATGGCAGCTACCGTT-3� (R). The PCR product was digested with XbaIand NcoI and cloned as a transcriptional fusion with theGUS gene into the XbaI/NcoI-digested vector, GUSNOS/pUC19, yielding ProDIR16:GUS-1 for transformation ofsugarcane, sorghum and maize. For rice transformation,the XbaI/NcoI-digested SHDIR16 PCR fragment wascloned as a transcriptional fusion with the GUS gene intothe XbaI/NcoI-digested binary vector, pCAMBIA1301,replacing the CaMV35S promoter and resulting inProDIR16:GUS-2.
The maize ubiquitin 1 (UBI1) promoter:GUS-pUC19construct (ProUBI1:GUS-1), pAHC27 (Christensen andQuail 1996) was used as a positive control for sugarcane,sorghum and maize transformations. For rice transforma-tion, the UBI1 promoter was excised from pAHC20(UBI1:BAR-pUC8 construct) (Christensen and Quail 1996)with BamHI and HindIII, Wlled in at the BamHI site usingT4 DNA polymerase (New England BioLabs Inc.), andcloned as a transcriptional fusion with the GUS gene intothe NcoI/HindIII (NcoI site Wlled in) digested binary vector,pCAMBIA1301, replacing the CaMV35S promoter to yieldProUBI1:GUS-2.
Plant transformation
Sorghum (Sorghum bicolor L., Moench genotypeRTx437) and maize (Zea mays L. inbred line DKC 69-7)
seedlings were grown on germination media [Murashigeand Skoog or MS media (PlantMedia, Dublin, OH) with100 mg L¡1 myo-inositol and 0.11 mg L¡1 1-napthalene-acetic acid and 2.5 mg L¡1 kinetin] at room temperaturewith a light intensity of 700 �mol photons m¡2 s¡1. Tissuesections (0.25 mm diameter) of 12- to 30-day-old seedlingswere bombarded with gold particles coated with DNA fromProOMT:GUS-1 or ProDIR16:GUS-1 using the Bio-Rad PDS-1000/He gene gun (12.5 �g DNA per 1 mg gold particles)as described (Emani et al. 2002). Control stem sectionswere bombarded with particles coated with ProUBI1:GUS-1DNA. Transient GUS reporter gene expression was evalu-ated histochemically (JeVerson et al. 1987) by counting thenumber of blue spots; results were expressed as the meanaverage of the number of spots per tissue section relative toProUBI1:GUS-1.
Transformation of sugarcane callus (Cultivar CP72-1210) by particle gun bombardment with ProOMT:GUS-1,ProDIR16:GUS-1, or ProUBI1:GUS-1 DNA, as well as regen-eration of shoots and roots were performed as described(Gallo-Meagher and Irvine 1996; Ingelbrecht et al. 1999).A total of 8, 12 and 4 independent sugarcane linestransgenic for ProOMT:GUS-1, ProDIR16:GUS-1, andProUBI1:GUS-1 were generated, respectively. Stem, leaf androot tissues from 4-month-old control and transformedplants were used for histochemical and biochemical GUSreporter gene analyses (JeVerson et al. 1987).
Embryo-derived rice calli (Oryza sativa subspeciesjaponica, cultivar Taipei 309) and Agrobacterium tumefac-iens strain EHA105 (Hood et al. 1993), harboring thepCAMBIA1301 binary vector carrying the sugarcaneProOMT:GUS-2 or ProDIR16:GUS-2, or the maizeProUBI1:GUS-2 were used for rice transformation (Hieiet al. 1994; Aldemita and Hodges 1996). Calli were pre-cultured on N6 medium (Chu et al. 1975) for 5 days prior totransformation. Co-cultivation of calli with bacterial sus-pension was performed for 3 days in darkness at room tem-perature on N6 medium supplemented with 55.5 mMglucose and 200 �M acetosyringone and subsequent incu-bation on hygromycin-free N6 medium for 1 week in dark-ness at room temperature. Transformed calli weretransferred to N6 medium with hygromycin selection(50 mg L¡1) for two subcultures of 3 weeks each. Regener-ated green shoots were selected on rooting medium (hor-mone-free MS) with hygromycin (30 mg L¡1) for 2 weeks.Surviving plants were transferred to potting mix (Redi-earth mix, Scotts, Hope, AR) and grown to maturity in thegreenhouse at 28°C. A total of 8, 13 and 12 independentrice lines transgenic for ProOMT:GUS-2, ProDIR16:GUS-2,and ProUBI1:GUS-2 were generated, respectively. Culm,leaf and root explants of 4-month-old control and trans-formed plants were analyzed histochemically and biochem-ically for GUS expression (JeVerson et al. 1987).
Sugarcane (Cultivar CP72-1210) was grown in potting mix(Redi-earth mix, Scotts, Hope, AR) in a controlled-environ-ment greenhouse (28°C day with 14 h light/10 h dark).Four-month-old plants were treated with SA, JA, or MeJA(Sigma–Aldrich, Saint Louis, MO). Four-month-old riceplants carrying ProOMT:GUS, ProDIR16:GUS and ProUbi1:GUSwere grown under the same conditions and treated in thesame way as wild-type sugarcane.
Treatments were conducted by spraying plants with a5 mM SA solution in water and 0.05% (v/v) Tween-20 or a25 �M JA solution in 0.1% (v/v) ethanol and 0.05% (v/v)Tween-20, and kept at high humidity in the greenhouse.MeJA treatments were carried out by placing a cotton swabcontaining 1 ml of 100 �M solution in 0.1% (v/v) ethanoland 0.05% (v/v) Tween-20 at the soil surface near the mainstem of plants kept in clear plastic bags. Control plantswere treated identically except without the addition of SA,JA, or MeJA. Stems, leaves and roots of treated anduntreated control plants were collected at 0, 24 and 48 h oftreatment. Three plants were used for each time point.
Analysis of �-glucuronidase activity
Histochemical localization of �-glucuronidase (GUS)activity was performed according to JeVerson et al. (1987),using GUS buVer with X-Gluc (5-bromo-4-chloro-3-indolyl-�-D-glucuronic acid) (Rose ScientiWc Ltd., Alberta,Canada). Stained plant tissues were photographed with azoom stereomicroscope (Olympus SZX7, Olympus, CenterValley, PA).
Quantitative GUS assays were carried out using 4-meth-ylumbelliferyl-�-D-glucuronide (Rose ScientiWc Ltd.) assubstrate (JeVerson et al. 1987). Fluorescence was measuredusing a VersaFluor Xuorometer (Bio-Rad Laboratories,Hercules, CA). Protein concentrations were determinedwith the Bio-Rad protein assay kit.
Results
SHDIR16 and SHOMT genes are highly expressed in the sugarcane stem
Sugarcane stems amass sucrose, cellulose and lignin, sig-niWcant feedstock for biofuel and other biomass applica-tions. To identify stem-expressed genes, DNA macroarrayscontaining 13,824 cDNA clones derived from a cDNAlibrary enriched for sugarcane stem mRNAs were hybrid-ized with stem, leaf and root cDNA probes. A total of 229candidate stem-expressed sugarcane genes were identiWed(http://enterprise.bio.tamu.edu/index_sugar.html).
Subsequently, a DNA microarray containing candidatestem-expressed cDNAs was used to assess expression oftheir respective mRNAs in stems, leaves and roots. Sixty-Wve cDNAs on the microarray were expressed in stem two-fold or more compared to leaf or root (data not shown)(sequences available at http://enterprise.bio.tamu.edu/Sugarcane_cDNA_Sequences_Planta.txt and Supplemen-tary material S3). About two-thirds (42) of these stem-expressed cDNAs were similar to a gene family encodingdirigent (DIR) proteins (Casu et al. 2004; Ralph et al.2007). Expression of the 42 cDNAs, named Saccharumhybrid DIRIGENT-like (SHDIRs), ranged from 2.7- to 7.8-fold in stems relative to leaves and from 2.2- to 7.8-fold instems relative to roots (data not shown). The sequence ofone cDNA, SHDIR16, was obtained (GU062718) andshown to encode a protein of 187 amino acids that shares96% identity with that of the S. oYcinarum DIR1 (SoDIR1,AY421731) (Casu et al. 2004) (Fig. 1).
Another stem-expressed cDNA identiWed in the micro-array was a gene coding for O-methyltransferase-like protein,named Saccharum hybrid O-METHYLTRANSFERASE-likegene (SHOMT). Sequence analysis of SHOMT cDNA(GU062719) (1.085 kb) revealed that it encodes a 361 aminoacid protein, which is 72% identical to Zea mays O-methyl-transferase ZRP4 (ZmZRP4-2, NM_001155649) (Alexan-drov et al. 2009) (Fig. 2).
Northern gel blot and qRT-PCR analyses validatedmicroarray expression patterns of SHDIRs and SHOMT(Fig. 3; Table 1). For instance, the qRT-PCR analysisshowed that representative SHDIRs like SHDIR11 andSHDIR16 were expressed in stems relative to leaves by asmuch as 12.4-fold and 11-fold, respectively (Table 1).SHOMT expression was also observed to be higher in stemsrelative to leaves by more than fourfold. SHDIR16 andSHOMT genes were selected for further analysis.
SHDIR16 and SHOMT are related to genes involved in lignin formation and polymerization
Analysis of the amino acid sequence of SHDIR16 demon-strated that it contains the conserved motifs I, II, III, IV andV (Ralph et al. 2006, 2007) that are common to a number ofDIRs from graminaceous angiosperms, speciWcally thosefrom sugarcane, sorghum, maize, barley and wheat, and toseveral DIRs from the woody gymnosperm, Thuja plicata(western red cedar), and angiosperms Forsythia £ interme-dia (border Forsythia) and Podophyllum peltatum (mayap-ple) that were found to direct monolignol couplingreactions (Davin et al. 1997; Xia et al. 2000; Kim et al.2002a) (Fig. 1). SHDIR16 also shared these motifs with thedisease resistance-response (DRR) family proteins (Fristen-sky et al. 1988; Culley et al. 1995; Alexandrov et al. 2009)(Fig. 1).
Analysis of the amino acid sequence of SHOMTrevealed the presence of residues and motifs that are com-mon to a representative number of O-methyltransferasesthat are potentially involved in ligniWcation. SHOMT wasfound to contain residues and motifs (A, B, C, J, K and L)(Fig. 2a, blue box) that were previously identiWed as beinghighly conserved in O-methyltransferases that require
S-adenosyl-L-methionine (SAM) as a substrate (Joshi andChiang 1998; Selman-Housein et al. 1999). SHOMT alsocontained the active site residues involved in lignin mono-mer binding, namely Phe-170, Met-122, Met-174 and Met-319 (Fig. 2a, dark orange and yellow), that are common toMedicago truncatula hydroxyisoXavonone 4�-O-methyl-transferase (MtH4�OMT, AY942158) (Liu et al. 2006) and
Fig. 1 Amino acid sequence alignment of SHDIR16 and other relatedgenes. Sequences were aligned using the ClustalW multiple sequencealignment program (Version 1.4). Gene abbreviations and Genbankaccession numbers are as follows: Saccharum spp. hybrid (sugarcanehybrid) dirigent-like protein (SHDIR16, GU062718); S. oYcinarum(sugarcane) dirigents, SoDIR1 (AAR00251), SoDIR2 (CAF25234)and SoDIR3 (AAV50047); Zea mays (maize) dirigents: ZmDIR-A(NM_001158343), ZmDIR-B (NM_001156165), ZmDIR1 (AAF71261),ZmDIR3 (NM_001158356) and ZmDIR9 (NM_001157043); Z. maysdisease resistance-response (DRR) genes: ZmDRR1 (NM_001157553), ZmDRR2 (NM_001158590), ZmDRR3 (NM_001156097)and ZmDRR4 (NM_001158233); Sorghum bicolor (sorghum) dirigent(SbDIR1, AAM94289); Hordeum vulgare (barley) dirigents: HvDIR1(AAA87042), HvDIR2 (AAA87041) and HvDIR3 (AAB72098); Trit-icum aestivum (wheat) dirigents: TaDIR1 (AAC49284), TaDIR2(AAM46813), TaDIR3 (BAA32786) and TaDIR4 (AAR20919);Oryza sativa (rice) dirigents: OsDIR5 (AK108922), OsDIR11(AK106022) and OsDIR15 (AK108983); O. sativa DRRs: OsDRR1(AC115686/AAM74358), OsDRR3 (AP003749.3/BAC16397),OsDRR4 (AP003749.3/BAC16399), OsDRR5 (AP005292.5/
BAC45193), OsDRR6 (CM000132.1/EEC82520), OsDRR7(AP005292.5/BAC45199), OsDRR9 (CM000144/EAZ40847), Os-DRR10 (AP003765.5/BAC19943) and OsDRR11 (AP004342.5/BAC20739); Thuja plicata (western red cedar) dirigents: TpDIR1(AAF25359), TpDIR2 (AAF25360), TpDIR3 (AAF25361), TpDIR4(AAF25362), TpDIR5 (AAF25363), TpDIR6 (AAF25364), TpDIR7(AAF25365), TpDIR8 (AAF25366) and TpDIR9 (AAL92120); Tsugaheterophylla (western hemlock) dirigent (ThDIR1, AAF25367);Forsythia £ intermedia (border Forsythia shrub) dirigents: FiDIR1(AF210061) and FiDIR2 (AF210062); Pisum sativum (pea) DRR(PsDRR206-d, PSU11716); Populus trichocarpa (western balsampoplar) DRRs: PtDRR1 (XM_002297959), PtDRR2 (XM_002297960), PtDRR3 (XM_002297961), PtDRR5 (XM_002303581) andPtDRR6 (XM_002304498); Podophyllum peltatum (mayapple) diri-gent (PpDIR, AF352736); and Agrostis stolonifera (bentgrass) dirigent(AsDIR1, AAY41607). Numbering of amino acids is based on theSHDIR16 sequence. The conserved motifs are highlighted in black,and their sequences are indicated; a aliphatic, c charged, n negativelycharged, h hydrophobic, p polar, s small, u tiny
M. sativa caVeic acid/5-hydroxyferulic acid 3/5-O-methyl-transferase (MsCOMT, M63853) (Zubieta et al. 2002). Cat-alytic residues that are essential for MtH4�OMT andMsCOMT methyltransferase activity, such as His-264,were also found in SHOMT (Fig. 2a, dark orange and yel-low). Phylogenetic analysis indicated that SHOMT formeda large cluster with ZmZRP4-2, MtH4�OMT, MsCOMT,Populus tremuloides caVeic acid/5-hydroxyferulic acidO-methyltransferase (Ptrbi-OMT, U13171), Arabidopsis tha-liana O-methyltransferase 1 (AtOMT1, NM_124796), Zeamays O-methyltransferase (newly annotated as caVeic acid3-O-methyltransferase, ZmOMT/ZmCOMT, M73235), andS. oYcinarum caVeic acid 3-O-methyltransferase (SoCOMT,AJ231133) (Fig. 2b). Cinnamyl alcohol dehydrogenases(CADs) and cinnamoyl-CoA reductases (CCRs), alsoknown to be involved in lignin biosynthesis, were not partof this cluster, as these enzymes require NAD/NADP(H),and not SAM, as a cofactor for their activity (Pichon et al.1998; Sattler et al. 2009) (Fig. 2b).
ProDIR16 and ProOMT drive GUS expression in a tissue-speciWc manner
Nineteen SHDIR16 clones were isolated from the screeningof a sugarcane BAC genomic library with SHDIR16 cDNA.Genomic walking in SHDIR16 BAC (159E16) DNA gener-ated a 2.6-kb SHDIR16 putative promoter fragment(ProDIR16) (GU062718; Supplementary material S1).
Nine SHOMT clones were isolated from the screening ofa sugarcane � genomic library with full-length SHOMTcDNA. The nucleotide sequence of a 4.3-kb fragment fromone SHOMT genomic clone was determined, and a 2.9-kbSHOMT putative promoter fragment (ProOMT) was isolatedfrom this clone (GU062719; Supplementary material S2).
The reporter gene, �-glucuronidase (GUS), was fused tothe putative stem-regulated promoters, SHDIR16 (2.6 kb)and SHOMT (2.9 kb) yielding ProDIR16:GUS andProOMT:GUS, respectively. These constructs were used tostably transform sugarcane and rice, and to transientlytransform maize and sorghum. GUS gene presence andcopy number in stably transformed plants was veriWed bygenomic Southern blot analysis (data not shown).
ProDIR16 and ProOMT drive GUS expression in sugarcane stem and rice culm
SigniWcant �-glucuronidase (GUS) expression was histo-chemically detected in stems, especially in nodes andvascular bundles of transgenic sugarcane carryingProDIR16:GUS (Fig. 4b) or ProOMT:GUS (Fig. 4c). GUSexpression in leaves (Fig. 4e, f) and roots (Fig. 4h, i) wasundetectable. Nontransformed sugarcane tissues exhibitedno signiWcant GUS expression (Fig. 4a, d, g). Quantitative
analysis also indicated that GUS activity levels ofProDIR16:GUS and ProOMT:GUS sugarcane plants were sig-niWcantly higher in stems than in leaves and roots (Table 2).Increases in GUS activity of ProDIR16:GUS sugarcane stemswere 4.6- to 39.1-fold compared to leaves and 4.5- to 27.1-fold compared to roots. Stems from ProOMT:GUS sugarcaneplants exhibited 2.8- to 9.8-fold more GUS activity thanleaves and 2.1- to 8.5-fold more than roots. Stem GUSactivity levels were higher for ProDIR16:GUS than forProOMT:GUS sugarcane plants. ProUBI1:GUS sugarcaneplants displayed higher GUS activity levels in leaves androots than in stems.
GUS expression was conWned to culm tissues in trans-genic rice harboring ProDIR16:GUS (Fig. 5b) or ProOMT:GUS(Fig. 5c). Nontransformed rice tissues showed no signiW-cant GUS expression (Fig. 5a). Quantitative analysis alsorevealed higher GUS levels in culms than in leaves androots of ProDIR16:GUS and ProOMT:GUS rice plants(Table 2). GUS activity in ProOMT:GUS rice culms was19.1- to 36.1-fold higher compared to leaves and 25.7- to84.3-fold higher compared to roots. Culms fromProDIR16:GUS rice plants exhibited 5.8- to 11.4-fold moreGUS activity than leaves and 1.6- to 10.3-fold more thanroots. Culm GUS activity levels were higher forProOMT:GUS than for ProDIR16:GUS rice plants.ProUBI1:GUS rice plants showed higher GUS activity levelsin leaves and roots than in culms.
ProDIR16 and ProOMT confer vascular GUS expression in sugarcane stem and rice culm
Histochemical analysis of ProDIR16- and ProOMT-drivenGUS expression in sugarcane stem and rice culm revealedthat both promoters conferred vascular GUS expression. Insugarcane, GUS expression was associated with the bundlesheath cells of the sclerenchymatous tissue and cells sur-rounding the protoxylem and xylem for ProDIR16:GUS andProOMT:GUS plants (Fig. 6a, b). Phloem companion cellswere also stained for GUS in ProOMT:GUS (Fig. 6b) but notin ProDIR16:GUS sugarcane plants (Fig. 6a). In rice, ProDIR16
and ProOMT directed a diVerent pattern of GUS expressionin the culm vascular system, with signiWcant GUS expres-sion in the protoxylem region (Fig. 7a, b).
ProDIR16 and ProOMT confer stem expression in maize and sorghum
Tissue bombardment experiments demonstrated thatProDIR16:GUS and ProOMT:GUS conferred transient GUSexpression in maize (Supplementary material S4b, c) andsorghum stems (Supplementary material S5b,c ) but not inleaves (Supplementary material S4e, f and S5e, f) or roots(Supplementary material S4 h, i and S5 h, i). In contrast,
ProUBI1:GUS directed GUS expression in all tissues exam-ined in maize (Supplementary material S4a, d, g) and sor-ghum (Supplementary material S5a, d, g).
ProDIR16 and ProOMT sequences are enriched with tissue-speciWc regulatory elements
In silico analysis of the ProDIR16 and ProOMT sequences withPlantCARE Motif Sampler (Lescot et al. 2002) and PLACESignal Scan (Higo et al. 1999) predicted the presence of
several potential cis-acting DNA elements involved in theregulation of gene expression in vascular tissues. Motifs pre-viously associated with vascular tissue-speciWc expression,such as the ASL-box (CTTTA repeat) (Saha et al. 2007; Yinet al. 1997), Box P (CACCAAAC or AACCAAAC) (daCosta e Silva et al. 1993; Feuillet et al. 1995; Ito et al. 2000)and NTBBF1 (ACTTTA) (Baumann et al. 1999; Liu et al.2003), were identiWed in both ProDIR16 and ProOMT (Table 3).Other vascular tissue-speciWc elements such as BS1(AGCGGG) (Lacombe et al. 2000) and AC (ACI: ACC-TACC and ACII: ACCAACC) (PatzlaV et al. 2003; Fornaléet al. 2006) were only observed in ProOMT (Table 3).
ProDIR16 and ProOMT respond to stress regulators
Endogenous SHDIR and SHOMT genes are induced by stress regulators
Salicylic acid (SA), jasmonic acid (JA) and methyl jasmonate(MeJA) mediate plant responses to biotic and abiotic stresses(Glazebrook et al. 2003). Microarray analysis indicated thatSHOMT, SHDIR11 and SHDIR16 expression was regulatedby SA, JA and MeJA (data not shown). qRT-PCR demon-strated that expression of the SHDIRs was induced about20-fold (SHDIR16) and Wvefold (SHDIR11) by SA at 48 h(Table 4). Maximal induction of SHDIR11 and SHDIR16expression occurred at 24 h in response to JA and MeJA.SHOMT expression was maximally induced by SA (3.5-fold)at 48 h, and by JA and MeJA (2.3- to 2.5-fold) at 24 h.
ProDIR16 and ProOMT respond to SA, JA and MeJA in rice
ProDIR16:GUS and ProOMT:GUS rice lines were exposed to SA(5 mM), JA (25 �M) and MeJA (100 �M) for 0, 24 and 48 h.GUS activity in culms, leaves and roots was determined ateach time point. GUS activity levels were signiWcantlyenhanced in culms and induced in leaves and roots by thesetreatments (Table 5). GUS activity in nontransformed plantswas undetectable (not shown). SA maximally increased GUSexpression in culms of rice transgenic for ProDIR16:GUS orProOMT:GUS at 48 h. JA maximally enhanced GUS expressionin rice culms containing ProDIR16:GUS at 24 h andProOMT:GUS at 48 h. MeJA had the most signiWcant eVect onincreasing ProDIR16:GUS and ProOMT:GUS expressions. MeJAenhanced GUS expression in ProDIR16:GUS rice culms approx-imately 22-fold at 24 h, and in ProOMT:GUS rice culms by14.5-fold at 48 h. In leaves and roots of rice transgenic forProDIR16:GUS or ProOMT:GUS, SA induced GUS expression tomaximum levels at 48 h, with signiWcant induction observedfor ProDIR16:GUS. JA and MeJA induced GUS expression tomaximum levels in ProDIR16:GUS and ProOMT:GUS rice leavesand roots at 24 h, with a more pronounced induction inProDIR16:GUS roots.
Fig. 2 Phylogenetic relationships of SHOMT and representative genesencoding O-methyltransferases, cinnamyl alcohol dehydrogenases andcinnamoyl-CoA reductases that are potentially involved in ligniWca-tion. a Amino acid sequences were aligned using the ClustalW multi-ple sequence alignment program (Version 1.4). Gene abbreviationsand Genbank accession numbers are as follows: Saccharum spp. hy-brid (sugarcane hybrid) O-methyltransferase-like protein (SHOMT,GU062719), Medicago truncatula (barrel clover) hydroxyisoXavanone4�-O-methyltransferase (MtHI4�OMT, AY942158), Zea mays (maize)O-methyltransferase ZRP4 (ZmZRP4-2, NM_001155649), Z. maysO-methyltransferase (newly annotated as caVeic acid-3-O-methyl-transferase, ZmOMT or ZmCOMT, M73235), Arabidopsis thaliana(Arabidopsis) O-methyltransferase 1 (AtOMT1, NM_124796), Popu-lus tremuloides (quaking aspen) caVeic acid/5-hydroxyferulic acidO-methyltransferase (Ptrbi-OMT, U13171), S. oYcinarum (sugar-cane) caVeic acid 3-O-methyltransferase (SoCOMT, AJ231133),M. sativa (alfalfa) caVeic acid/5-hydroxyferulic acid 3/5-O-methyl-transferase (MsCOMT, M63853), cinnamyl alcohol dehydrogenasesfrom S. oYcinarum (SoCAD, AJ231135) and A. thaliana (AtCAD4,NM_112832 and AtCAD5, NM_001036711), and cinnamoyl-coen-zyme A reductases from S. oYcinarum (SoCCR, AJ231135), Z. mays(ZmCCR, X98083) and Lolium perenne (perennial ryegrass) (LpCCR,AY061888). Conserved residues and motifs are highlighted by diVer-ent colors. Motifs A, B, C, J, K and L that correspond to the S-adenosyl-L-methionine binding domains are boxed in blue, and their sequencesare indicated. b Phylogenetic tree was built using the UPGMA algo-rithm. The values on the branches are bootstrap proportions, with thelengths of branches being proportional to evolutionary distances be-tween species
Fig. 3 Stem-regulated expression of SHDIR16 and SHOMT. Relativeabundance of SHDIR and SHOMT steady-state transcript levels wasdetermined by northern gel blot analysis in sugarcane stem, leaf androot
ProDIR16 and ProOMT sequences are enriched with stress-responsive regulatory elements
In silico analysis of the ProDIR16 and ProOMT sequencesrevealed that they contain cis-elements conferring respon-
siveness to SA, the jasmonates (JA and MeJA) and otherabiotic and biotic stresses. These include the ASF1 motif(TGACG) (Rouster et al. 1997; Hwang et al. 2008) and theW-box (TTGAC) (Hiroyuki and Terauchi 2008; Hwanget al. 2008) found in both promoters, the GCC-box
Table 1 Expression of SHDIRs and SHOMT in sugarcane vegetative tissues
Relative abundance of mRNA transcripts of a representative set of SHDIRs (SHDIR11, SHDIR16) and SHOMT cDNAs was determined in sugar-cane stem, leaf and root by the indicated analysis. Expression is represented as the fold change in stem relative to leaf or root tissue and is reportedwith the standard error. Data represents two biological samples and three technical repetitions
Fig. 4 ProDIR16 and ProOMT di-rect GUS gene expression in the sugarcane stem. GUS activity was analyzed histochemically in transgenic sugarcane carrying ProOMT:GUS or ProDIR16:GUS in stems, leaves and roots. At least six plants (4-month-old) per line were examined for GUS stain-ing, and typical results are pre-sented. a–c transverse stem sections, d–f leaves, and g–i roots. Scale bar 2 mm
(GCCGCC) (Brown et al. 2003; Yang et al. 2005; Zhanget al. 2007) present in ProDIR16, and the T/G box(AACGTG) (Yamamoto et al. 2004; Wu et al. 2009) identi-Wed in ProOMT (Table 3).
Discussion
Promoters capable of driving desired spatial and/or tempo-ral transgene expression are critical to plant biotechnologyand in the elucidation of gene function. While there aremany promoter choices in dicots, this is not the case inmonocots. We have expanded the repertoire of promotersavailable for use in monocots by developing two stem-expressed promoters that are also inducible by SA, JA andMeJA, key regulators of biotic and abiotic stress signaling.The promoters were developed from the Saccharum hybridgenes, DIRIGENT (SHDIR16) and O-METHYLTRANS-FERASE (SHOMT); both are possibly involved in ligniWca-tion and defense pathways. These genes were identiWed bya combination of high-throughput macro- and micro-arrayscreening, and qRT-PCR analysis. Transient and stableexpression analyses using SHOMT- and SHDIR16-pro-moter-�-glucuronidase (GUS) fusions (ProDIR16:GUS andProOMT:GUS) showed that these promoters are functional insugarcane and in orthologous monocot species, such as sor-ghum, maize and rice, reproducing the expression patternsof endogenous SHDIR16 and SHOMT genes in sugarcane.
Phylogenetic relationships of SHDIR16 and SHOMT with other related genes
The function of several O-methyltransferases (OMTs) hasbeen discussed in relation to the biosynthesis of Xavonoids
and lignins (Kuroda 1983; Inoue et al. 1998). Transgenicplants altered in OMT expression have provided evidencefor the potential involvement of OMTs in ligniWcation andshed more light into the biosynthesis of monolignols (VanDoorsselaere et al. 1995; Tsai et al. 1998; Guo et al. 2001;Aldwin and Lewis 2002). The most striking eVect of thedown-regulation of caVeic acid O-methyltransferases(COMTs) is the reduction in syringyl (S)-type phenylpro-panoid/monolignol units and the incorporation of 5-hydroxyconiferyl alcohol into the lignin polymer (Lapierreet al. 1988; Jouanin et al. 2000; Boerjan et al. 2003). Forinstance, COMT-deWcient poplars show a 4.5-fold reduc-tion in the abundance of their sinapaldehyde monolignolbiosynthetic precursor, and consequently S lignin, and theaccumulation of high amounts of novel benzodioxanestructures derived from incorporation of the 5-hydroxyco-niferyl alcohol (Morreel et al. 2004). The incorporation ofthese uncommon monomers is in accordance with the com-binatorial random model that predicts that lignin forms viaradical coupling of monolignol units into the growing lig-nin polymer under chemical control in a near-random fash-ion (Adler 1977; Morreel et al. 2004). This model has beenrecently challenged by the discovery of specialized pro-teins, called dirigents (DIRs), that have been proposed toact as protein arrays in xylem and other ligniWed tissueswhere they control the monolignol coupling reactions thatlead to the formation of lignin (Davin et al. 1997; Davinand Lewis 2000). The plasticity to form lignin through ran-dom coupling would be an advantage in terms of defenseagainst pathogen invasion (Denton 1998). What needs to beaddressed, however, is how diVerent tissues within theplant are able to achieve variation in lignin composition(HatWeld and Vermerris 2001). Nevertheless, this variationcould be the result of controlled diVerential expression of
Table 2 SHDIR16 and SHOMT promoters drive GUS expression in the sugarcane stem and rice culm
Average GUS activity was measured in stems/culms, leaves and roots of 4-month-old sugarcane or rice lines (T1) transgenic for ProOMT:GUS andProDIR16:GUS. ProUBI1:GUS lines were included as a positive control. The number of independent ProDIR16:GUS, ProOMT:GUS and ProUBI1:GUStransgenic lines tested were 12, 8 and 4, respectively, for sugarcane, and 13, 8 and 12, respectively, for rice. GUS activity represents three biologicalsamples and three technical repetitions and is reported with the standard error. The range of each set of experiments is indicated in parentheses
Construct GUS activity [pmoles of 4-methylumbelliferone (MU)/min per �g protein]
lignin biosynthetic genes (Ralph et al. 2004). Hence, theDIR protein model certainly has merits but requires furtherexperimental evidence to enforce its strict involvement inligniWcation (Boerjan et al. 2003).
DIRs were the most abundant transcripts identiWed byour microarray analysis of the sugarcane stem. Besidestheir potential role in mediating the free radical coupling ofmonolignol phenols to yield lignans and lignins, DIRs havebeen implicated in disease resistance responses (Burlatet al. 2001; Wang and Fristensky 2001; Zhu et al. 2007),and share a dirigent-conserved domain with the diseaseresistance-response (DRR) family proteins (Fristenskyet al. 1988; Culley et al. 1995; Alexandrov et al. 2009).Comparison of the amino acid sequence of SHDIR16,which was selected for further analysis, with that of otherrelated genes showed that it shared the motifs I, II, III, IV andV that are conserved in DIR and DRR proteins (Ralph et al.2006, 2007), speciWcally those from woody gymnosperms
such as Thuja plicata (Kim et al. 2002a, b) and angio-sperms like Forsythia £ intermedia (Gang et al. 1999) andPodophyllum peltatum (Xia et al. 2000) (Fig. 1) that havebeen identiWed biochemically to direct the stereospeciWccoupling of E-coniferyl alcohol to produce the lignan pre-cursor (+)-pinoresinol. The sequence homology ofSHDIR16 to DIRs and DRRs reinforces its possible role inlignin-related defense responses.
Plant OMTs, in contrast to mammalian OMTs, exhibitnarrow substrate speciWcities so that the sequence homol-ogy comparison, derived using programs such as BLAST,does not provide suYcient information on the enzyme func-tion or its substrate preference. The predicted three-dimen-sional structure of the SHOMT identiWed in our microarrayanalysis (Supplementary material S6) was thereforeobtained by homology-based modeling using M. truncatulahydroxyisoXavonone 4�-O-methyltransferase (MtHI4�OMT,AY942158) (Liu et al. 2006) as a template. SHOMT exhibited
Fig. 5 ProDIR16 and ProOMT direct GUS gene expression in the rice culm. GUS activity was analyzed histochemically in transgenic rice carrying ProOMT:GUS and ProDIR16:GUS in culms, leaves and roots. At least six plants (4-month-old) per line were examined for GUS staining, and typical results are shown. a–c transverse culm sec-tions, d–f leaves, and g–i roots. Scale bar 1.3 mm
structural similarities to MtHI4�OMT and M. sativa caVeicacid/5-hydroxyferulic acid 3/5-O-methyltransferase(MsCOMT, M63853), known to be implicated in ligninbiosynthesis (Zubieta et al. 2002), in terms of the presenceof the SAM substrate binding motifs (A, B, C, J, K and L)that are highly conserved in all SAM-dependent methyl-transferases (Joshi and Chiang 1998), the catalytic sitesessential for methyltransferase activity (mainly His-264)and the active site residues required for binding of mono-meric lignin precursors (namely M122, M174, M319 andF170) (Fig. 2a; Supplementary material S6). Amino acidsequence comparison revealed that SHOMT also sharedthese characteristic motifs and residues with a number ofgraminaceous OMTs and COMTs, namely S. oYcinarumcaVeic acid-O-methyltransferase (SoCOMT, AJ231133)(Selman-Housein et al. 1999), and maize OMT ZRP4(ZmZRP4-2, NM_001155649) (Alexandrov et al. 2009)and COMT (ZmCOMT/ZmOMT, M73235) (Collazo et al.1992) (Fig. 2a). This structural characterization predicts apossible functional role for SHOMT in ligniWcation. Fur-thermore, phylogenetic analysis showed that SHOMT clus-tered, not only with ZmZRP4-2, MtHI4�OMT, SoCOMT
and MsCOMT, but also with Arabidopsis thaliana O-meth-yltransferase 1 (AtOMT1), ZmCOMT/ZmOMT andP. tremuloides caVeic acid/5-hydroxyferulic acid O-meth-yltransferase (Ptrbi-OMT) that were implicated in ligninbiosynthesis. For instance, an Arabidopsis knockout mutantfor AtOMT1 was found to be defective in the methylation oflignin precursors derived from S monolignols (Goujonet al. 2003), while in maize, an 85% reduction in ZmCOMTactivity at the Xowering stage was suYcient to cause atwofold decrease in S unit content (Piquemal et al. 2002).Similarly, in tobacco transgenic for antisense Ptrbi-OMT,an average decrease of 25% in S unit content was alsoobserved together with an increase in 5-hydroxy-guaiacylunits (Dwivedi et al. 1994; Ni et al. 1994; Atanassova et al.1995).
Stem-regulated gene expression
SHDIR16 and SHOMT endogenous genes were preferen-tially expressed in the sugarcane stem. This is in agreementwith Casu et al. (2004) who reported on the abundance oftranscripts for one OMT (maize homolog, P47917, Held
Fig. 6 ProDIR16 and ProOMT direct GUS gene expression in sugarcanestem vasculature. GUS activity was analyzed histochemically in trans-verse sections of transgenic sugarcane stems carrying ProDIR16:GUS
(a) and ProOMT:GUS (b) and in nontransformed plants (c). x xylem, pxprotoxylem, mx metaxylem, p phloem, s sclerenchyma, pa storageparenchyma. Scale bar 50 �m
et al. 1993) and several variants of one SoDIR1 (S. oYcina-rum DIR1, AY421731) in maturing sugarcane stem inter-nodes. Expression of the sugarcane SoCOMT (AJ231133)(Selman-Housein et al. 1999) and Arabidopsis AtOMT1(NM_124796) (Goujon et al. 2003) was also restricted to thestem. In spruce, transcripts for six DIR genes were found tobe abundant in outer stem tissues (Ralph et al. 2006).
Histochemical localization of GUS expression directedby ProDIR16 and ProOMT in situ, in sugarcane and rice, pro-vides evidence for their activity in the stem, preferentiallyin the vascular bundle, internode and rind tissues thatparticipate in the developmentally regulated ligniWcationprocess. Both ProDIR16 and ProOMT drove GUS gene
expression in the bundle sheath cells of sclerenchymatoustissues and in cells surrounding the phloem, xylem andprotoxylem in the sugarcane stem. In this case, theSHOMT and SHDIR16 genes might be involved in thedevelopment of xylem, especially the protoxylem elementsthat are the Wrst to mature before the surrounding organshave elongated, possibly through activation of secondarycell wall production and ligniWcation. In addition, ProOMT
directed gene expression in companion cells and surround-ing cells of the phloem. Thus, SHOMT-driven expressiontakes place in a more widespread vascular tissue-speciWcmanner than SHDIR16-driven expression. The functionalsigniWcance of the expression of OMT as a structural gene
Fig. 7 ProDIR16 and ProOMT di-rect GUS gene expression in rice culm vasculature. GUS activity was analyzed histochemically in transverse sections of transgenic rice culms carrying ProOMT:GUS (a) and ProDIR16:GUS (b) and in nontransformed plants (c). x xy-lem, px protoxylem, mx metaxy-lem, p phloem, s sclerenchyma, pa storage parenchyma. Scale bar 50 �m
of the phenylpropanoid/lignin pathway in the phloemregion lies in its participation in phloem cellular processes.Incorporation of additional phloem-derived cells ensuresproper transport of organic nutrients to those cellsinvolved in the reinforcement of the plant axis to counter-act the increased weight of the growing plant (Torneroet al. 1996). Furthermore, phloem-regulated gene expres-sion can be beneWcial by imposing a decreased metabolicload on the plant.
In the rice culm, both ProDIR16 and ProOMT displayed aunique pattern of GUS expression in the protoxylem of the
culm vascular system, reXecting regulatory diVerencesbetween plant species. Even though the pattern ofexpression in transgenic rice is diVerent from that observedin sugarcane, it closely follows the distribution of cellsundergoing ligniWcation.
GUS expression directed by ProOMT in transgenic sugar-cane stem follows the same pattern of immunocytochemi-cal localization of the maize lignin biosynthetic enzymeZmCOMT/ZmOMT (M73235), speciWcally in the cell layersurrounding xylem vessels and in sclerenchyma cells ofsugarcane stems (Ruelland et al. 2003). More evidence is
Table 3 Putative regulatory motifs enriched in the SHDIR16 and SHOMT promoters
Motifs were identiWed by PLACE signal scan (http://www.dna.affrc.go.jp/PLACE/signalscan.html) and PlantCARE motif sampler (http://bioin-formatics.psb.ugent.be/webtools/plantcare/html)a The motif position is given by the number corresponding to the 5� nucleotide in the motif from the presumed translational start codon (promotersequences are provided in Supplementary materials S1 and S2)
Name and sequence of motif Function Occurrence and positiona of motif in promoter
Table 4 Kinetics of SHDIR and SHOMT expression in sugarcane stem induced by salicylic acid, jasmonic acid and methyl jasmonate
Relative abundance of mRNA transcripts of SHDIR11, SHDIR16 and SHOMT cDNAs was determined by qRT-PCR analysis in stems of sugarcaneplants (4-month-old) at 0, 24 and 48 h treatment with salicylic acid (5 mM), jasmonic acid (25 �M) or methyl jasmonate (100 �M). Expression isrepresented as the fold change at 24 h and 48 h relative to 0 h treatment and is given with the standard error. Data represents two biological samplesand three technical repetitions
provided from the mRNA accumulation of ZmCOMT/ZmOMT and GUS expression driven by its promoter in thexylematic parenchyma and lignifying cells of the xylem ofmaize roots (Collazo et al. 1992; Capellades et al. 1996).ProOMT expression proWle also matches that of the AtOMT1promoter in the xylem, mature phloem and diVerentiatingWbers of Arabidopsis stems (Goujon et al. 2003).
The GUS expression pattern of ProDIR16 is similar to theone displayed by three western red cedar DIR promoters inthe diVerentiated xylem bundle region of Arabidopsis stems(Kim et al. 2002a). It is also analogous to the localization ofDIR in Forsythia £ intermedia xylem ray cells, Wbers andvessels (Gang et al. 1999).
The tissue-speciWc activity of ProDIR16 and ProOMT corre-lates with the presence of vascular tissue-speciWc regula-tory motifs in their sequences. Box P-like motifs(MACCWAMC), common to ProDIR16 (CACCAAAC) andProOMT (AACCAAAC), were identiWed in several promot-ers of genes involved in the biosynthesis of lignin, lignansand phenylpropanoids (da Costa e Silva et al. 1993; Feuilletet al. 1995); they are speciWcally responsible for xylem-reg-ulated expression of parsley 4-coumarate:CoA ligase(HauVe et al. 1993) and rice peroxidase (Ito et al. 2000).ProOMT and ProDIR16 shared two additional regulatorymotifs, namely the NTBBF1 motif (ACTTTA) that confersvascular tissue-speciWc gene expression in rice and tobacco(Baumann et al. 1999; Liu et al. 2003), and the ASL-box(CTTTA repeat) present in phloem-speciWc promoters (Yinet al. 1997; Saha et al. 2007). Motifs that are unique toProOMT include the BS1 element, identiWed as essential forthe vascular expression of the lignin biosynthetic gene, cin-namoyl-CoA reductase, in Eucalyptus gunnii stem(Lacombe et al. 2000), and the AC-rich-elements, ACI(ACCTACC) and ACII (ACCAACC), shown to be particu-
larly important in the regulation of xylem/phloem-localizedactivity for genes involved in phenylpropanoid metabolism(PatzlaV et al. 2003) and lignin biosynthesis (Fornalé et al.2006). The fact that ProDIR16 and ProOMT are rich with vas-cular-speciWc regulatory motifs and confer gene expressionin vascular lignifying cells, as shown by histochemicalGUS localization, suggests a functional role for SHDIR16and SHOMT genes in ligniWcation.
Inducible gene expression
Another advantage of ProDIR16 and ProOMT is that transgeneexpression can be enhanced in the stem and induced in theleaf and root with one or more defense signaling regulatorssuch as SA, JA and MeJA. The kinetics of promoter activa-tion are consistent with the patterns of SA- and jasmonate-induction of the endogenous SHDIR16 and SHOMT mRNAexpression observed in the transcript proWling analysis.These results corroborate earlier studies on the inducibilityof several DIR and OMT homologs. Expression of 19unique DIRs in spruce stems (Ralph et al. 2006), 6 DIRs inthe bark of the conifer P. peltatum (Ralph et al. 2007), andfour DIRs, diVerent than SHDIR16, in sugarcane roots(Bower et al. 2005) was found to be up-regulated by MeJAtreatment. Similarly, transcripts for barley OMT (Lee et al.1997) and tobacco caVeic acid O-methyltransferase II(COMT II) (Toquin et al. 2003) were induced by exogenousapplications of jasmonates, mainly MeJA.
The kinetics of induction of ProDIR16 and ProOMT by SAand the jasmonates in transgenic rice follow a clear pattern ofinverse coordination. At 24-h of treatment, there is an increasein GUS transgene expression in response to the jasmonates,coinciding temporally with a decreased expression inresponse to SA. At 48-h of treatment, GUS expression is
Table 5 Response of ProDIR16:GUS and ProOMT:GUS to salicylic acid, jasmonic acid and methyl jasmonate in rice
GUS activity was measured in culms, leaves and roots of two highly expressing T1 rice independent lines (4-month-old) for each of ProDIR16:GUSand ProOMT:GUS at 0, 24 and 48 h treatment with salicylic acid (5 mM), jasmonic acid (25 �M) or methyl jasmonate (100 �M). GUS activity isrepresented as the fold change at 24 and 48 h relative to 0 h treatment and is reported with the standard error. Values represent three biologicalsamples and three technical repetitions
Transgenic GUS activity [pmoles of 4-methylumbelliferone (MU)/min per �g protein]
up-regulated by SA and down-regulated by the jasmonates.These results correlate with the inverse coordination in endog-enous levels of SA and JA seen during early wound responsein rice, which may be related mechanistically to cross talkbetween the SA and JA signaling pathways (Lee et al. 2004).Additionally, this inverse correlation was abolished in ourexpression systems by the simultaneous application of SA andMeJA, where transgene expression levels were constantlyinduced at 24 h and 48 h (data not shown), indicating thatexpression is strictly modulated by these hormones.
The GUS expression proWle in rice transgenic forProDIR16:GUS and ProOMT:GUS has been modiWed followingtreatment with each of SA, JA and MeJA. GUS localizationhas shifted from a stem-speciWc to a constitutive pattern,i.e. in stems, leaves and roots, through SA, JA and MeJA-inducible mechanisms. Expression can be rapidly turned onand oV by SA and the jasmonates in leaves and roots, mak-ing these promoter systems potentially useful for a broaderrange of applications.
The SA- and jasmonate-inducible activity of ProDIR16 andProOMT correlates with the occurence of stress-responsiveregulatory motifs in their sequences. The GCC-box(GCCGCC) is the jasmonate-responsive motif found inProDIR16 and that is common to the promoters of pathogene-sis-related (PR) genes encoding �-1,3-glucanase and chiti-nase (Yang et al. 2005; Zhang et al. 2007), and PLANTDEFENSIN1.2 (Brown et al. 2003). In ProOMT, six T/Gboxes (AACGTG) were identiWed; these are hybrid boxescomposed of T-box and G-box half sites, and responsible forthe jasmonate-responsive regulation of the promoters of chi-tinase genes such as Brassica juncea BjCH1 and tobaccoCHN48 (Yamamoto et al. 2004; Wu et al. 2009). The ASF1motif (TGACG), which prevails in both ProDIR16 and ProOMT,is known to mediate the MeJA and SA responses of promot-ers of PR genes such as the rice OsPR10a and of JA biosyn-thetic genes like the barley lipoxygenase 1 (Rouster et al.1997; Hwang et al. 2008). The W-box, a TTGACC elementalso abundant in both ProOMT and ProDIR16, was demonstratedto regulate the SA-, jasmonate- and pathogen-responsivenessof the promoters of phenylpropanoid genes such as tobaccoCOMT II (Toquin et al. 2003), and of PR genes like ricethaumatin-like and OsPR10a (Hiroyuki and Terauchi 2008;Hwang et al. 2008). The presence of SA- and jasmonate-responsive regulatory elements in ProDIR16 and ProOMT, aswell as the induction of these promoters and their endoge-nous gene expression by these signal defense molecules sup-ports the possible involvement of SHDIR16 and SHOMT inthe SA- and jasmonate-induced self-defense responses.
Potential applications
The speciWcity in regulating gene expression in the stem,especially in the vascular bundles, as well as the enhanced
response to induction by stress regulators of the newly iso-lated ProDIR16 and ProOMT make them useful tools formonocot crop improvement. Stem-regulated expressionwould be of great value for metabolic engineering toimprove plant biomass characteristics through enhancedcarbon metabolism for sugar accumulation or increasedWber content for biofuel feedstock, as well as for engineer-ing resistance to biotic and abiotic stresses. ProDIR16 andProOMT would be important in regulating the level of keymetabolic enzymes for carbon partitioning between sucroseaccumulation and cell wall Wber content. Constitutivedown-regulation of a key glycolytic enzyme, pyrophos-phate: fructose 6-phosphate 1-phosphotransferase (PFP)has been shown to increase sucrose levels in immatureinternodes and Wber contents in both immature and matureinternodes of sugarcane (Groenewald and Botha 2008). Theavailability of a stem-regulated promoter would allow PFPto be down-regulated in a tissue-speciWc manner, to maxi-mize sucrose production and biomass accumulation in bothdevelopmental stages of internodes relative to other tissues.
The fact that ProDIR16 and ProOMT share similarities inconferring gene expression patterns with promoters ofgenes involved in lignin formation and polymerizationdemonstrates their potential suitability for targeted trans-gene expression to modify lignin synthesis for improvingplant biomass.
Stem expression may also be exploited to develop virus-resistant lines by fusing antiviral constructs to ProDIR16 orProOMT, because many monocot viruses multiply and trans-locate in the vascular tissue (Yin et al. 1997; Opalka et al.1998). Additionally, enhancement of ProDIR16- and ProOMT-directed transgene expression with stress regulators couldprotect against important pests and opportunistic fungalpathogens through reinforcement of cell walls of internodesand vascular tissues.
Acknowledgments We are grateful to members of the Thomas Lab-oratory at Texas A&M University for discussions of results, data anal-ysis and support. We speciWcally acknowledge Dr. Andrew Tag andRick Hammer for bioinformatics support. We also thank Dr. Madhu-rababu Kunta (Citrus Center, Texas A&M University-Kingsville) forassistance in photography, and Brennick Langston (Texas AgriLifeResearch, Texas A&M System) for sequence submission to NCBI anddetermination of the three-dimensional structure of sugarcane O-meth-yltransferase. This work was supported by grants from the Texas Grainand Grass Gene Initiative Program (No. 06-0001), the Rio GrandeValley Sugar Growers Cooperative and Texas AgriLife Research,Texas A&M System.
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