Sequence analysis and functional characterization of the promoter of the Picea glauca Cinnamyl Alcohol Dehydrogenase gene in transgenic white spruce plants

GENETIC TRANSFORMATION AND HYBRIDIZATION

Sequence analysis and functional characterization of the promoterof the Picea glauca Cinnamyl Alcohol Dehydrogenase genein transgenic white spruce plants

Frank Bedon Æ Caroline Levasseur ÆJacqueline Grima-Pettenati Æ Armand Seguin ÆJohn MacKay

Received: 3 November 2008 / Revised: 16 February 2009 / Accepted: 17 February 2009 / Published online: 14 March 2009

� Springer-Verlag 2009

Abstract The enzyme Cinnamyl Alcohol Dehydrogenase

(CAD) catalyses the last step of lignin monomer synthesis,

and is considered as a molecular marker of cell wall lig-

nification in different plants species. Here, we report the

isolation and analysis of 50 flanking genomic DNA regions

upstream to the CAD gene, from two conifers, i.e. white

spruce (Picea glauca (Moench) Voss) and loblolly pine

(Pinus taeda L.). Sequence comparisons with available

CAD gene promoters from angiosperms highlighted the

conservation of cis-elements matching MYB, WRKY and

bHLH binding sites. Functional characterization of the

P. glauca CAD promoter used P. glauca seedlings stably

transformed with a DNA fragment of 1,163 base pairs

(PgCAD) fused to the b-glucuronidase (GUS) gene. His-

tochemical observations of different vegetative organs of

the transgenic trees showed that this sequence was suffi-

cient to drive GUS expression in lignifying tissues, and

more specifically in differentiating xylem cells. Quantita-

tive RT-PCR experiments also indicated that the native

CAD gene was preferentially expressed in differentiating

xylem both in stems and roots. In addition, GUS expression

driven by the PgCAD promoter was wound-inducible

which was consistent with the accumulation of CAD

mRNA in response to jasmonate application and mechan-

ical wounding. The spruce CAD promoter represents a

valuable tool for research and biotechnology applications

related to xylem and wood.

Keywords Conifer � Cinnamyl alcohol dehydrogenase

(CAD) � Lignin � Vascular tissues � Cis-regulatory

elements � Jasmonate � Wounding

Introduction

Wood formation in plants depends on the differentiation of

secondary xylem from the vascular cambium. This differ-

entiation entails several genetically controlled events and

produces xylem cells with unique morphological and

chemical properties which enable efficient solute transport

and mechanical support. The process can be divided into

four major steps: cell division, bi-directional cell expan-

sion, formation of a lignified secondary cell wall (SCW)

and programmed cell death (Plomion et al. 2001). In

trees, the major SCW constituents are cellulose (40–50%),

Communicated by L. Jouanin.

Electronic supplementary material The online version of thisarticle (doi:10.1007/s00299-009-0688-0) contains supplementarymaterial, which is available to authorized users.

F. Bedon � J. MacKay (&)

Centre d’Etude de la Foret, Universite Laval, Quebec,

QC G1V 0A6, Canada

e-mail: [email protected]

F. Bedon

e-mail: [email protected]

F. Bedon � J. Grima-Pettenati

UMR UPS/CNRS 5546, Pole de Biotechnologies Vegetales,

24 chemin de Borde Rouge, BP42617, Auzeville Tolosane,

31326 Castanet Tolosan, France

C. Levasseur � A. Seguin

Natural Resources Canada, Canadian Forest Service,

Laurentian Forestry Centre, Quebec, QC G1V 4C7, Canada

Present Address:F. Bedon

UMR Biodiversite Genes Communaute 1202, INRA,

Equipe de Genetique, 69 route d’Arcachon,

33612 Cestas cedex, France

123

Plant Cell Rep (2009) 28:787–800

DOI 10.1007/s00299-009-0688-0

hemicellulose (around 25%) and lignins (25–35%) (Plom-

ion et al. 2001). The cellulose and hemicellulose network

shape the SCW which is impregnated and reinforced by

lignins. The lignin polymers act as bounding agents,

responsible for making the cell wall more rigid and rela-

tively waterproof, as well as more resistant to biotic attack

or degradation (Lange et al. 1995).

Lignin precursor synthesis derives from the phenyl-

propanoid pathway which leads to several aromatic

derivatives like stilbenes, hydroxycoumarines and flavo-

noids, in addition to lignins and lignans (Boerjan et al.

2003). The lignin specific pathway involves two enzymes:

cinnamoyl coenzymeA reductase (CCR) and cinnamyl

alcohol dehydrogenase (CAD) which reduce cinnamoyl-

CoA to form p-hydroxycinnamyl alcohols, referred to as

monolignols. CAD has often been proposed as a marker for

lignification in conifers and angiosperm trees (Boerjan

et al. 2003; Boudet et al. 2004).

The CAD genes form a small gene family that have been

well characterized by genetic approaches and mutant

analysis in several angiosperms species including Euca-

lyptus, poplar and Arabidopsis (Grima-Pettenati et al.

1993; Baucher et al. 1996; Sibout et al. 2005). Specific

CAD gene sequences have unambiguously been linked to

lignin amount and composition, as well as SCW formation

and structure (for a review Boudet et al. 2004). Transcript

profiling has shown the vascular preferential expression of

CAD in different angiosperm species, and functional

analyses of cis-regulatory elements have identified poten-

tial mechanisms controlling this expression (Feuillet et al.

1995; Hawkins et al. 1997; Lauvergeat et al. 2002; Sibout

et al. 2003; Raes et al. 2003; Seguin et al. 1997; Baghdady

et al. 2006). Several experimental evidences link tran-

scriptional control of the Eucalyptus CAD gene (EgCAD2)

to the activity of two R2R3-MYB transcription factors,

EgMYB2 and EgMYB1 which are able to specifically bind

MBSIIG sites in the cis-region of the promoter required for

xylem expression (Lauvergeat et al. 2002; Goicoechea

et al. 2005; Legay et al. 2007). EgMYB2 activates tran-

scription while EgMYB1 represses it.

In conifers, a single CAD gene has been linked to lignin

biosynthesis although different alleles may be detected in

some species (Galliano et al. 1993; MacKay et al. 1995;

Kirst et al. 2003). In loblolly pine the discovery and

detailed analysis of trees with a rare CAD mutant allele

(cad-n1) clearly established that CAD could modulate

lignin composition as a result of greatly reduced CAD

enzyme activity (MacKay et al. 1997). CAD transcripts

were found to be present in both lignifying tissues and non

lignifying tissues of loblolly pine and Norway spruce but

more abundant in the stem developing xylem (MacKay

et al. 1997; Koutaniemi et al. 2007). Moreover in white

spruce, CAD gene transcripts accumulated in compression

wood (Bedon et al. 2007), which is characterized by thicker

and more highly lignified secondary cell walls (Timell

1986). Functional promoter analysis of lignin/phenylprop-

anoids genes including CAD is still in its infancy in

conifers compared to angiosperm species, although a CAD

promoter from Pinus radiata has been histochemically

associated with stem lignifying tissues (Wagner and Walter

2004). Considering the abundance of Pinaceae family

conifer trees in several parts of the world and their wide-

spread use in the forest products industry, investigations

related to these genes are of interest because of potential

breeding and biotechnology applications.

In this report, we isolated and analysed the sequences

upstream of the CAD gene from two conifers (white spruce

and loblolly pine). These sequences were analysed along

with angiosperm CAD promoters (Eucalyptus and Ara-

bidopsis) to identify common cis-regulatory motifs and

compare the distribution patterns of these motifs. We

assessed spatial and temporal activity of the putative CAD

promoter from white spruce (Picea glauca, ProPgCAD)

fused to the b-glucuronidase (GUS) gene in stably trans-

formed spruce trees. The accumulation of GUS and native

CAD transcripts, and the localisation of GUS staining were

monitored in different plant tissues. Moreover, mechanical

wounding and jasmonate application were used to assess

the potential regulation of the CAD gene in the response to

stresses.

Materials and methods

Isolation of putative CAD promoter fragments

from white spruce and loblolly pine

The Picea glauca (white spruce) and Pinus taeda (loblolly

pine) cinnamyl alcohol dehydrohenase upstream flanking

sequences were isolated by a PCR-based genome walking

procedure using the GenomeWalker Universal Kit (BD

Biosciences, Palo Alto, CA, USA). For P. glauca, genomic

DNA was extracted from young needles using the Genomic-

Tip Kit (Qiagen, Mississauga, ON, Canada). Two non-

overlapping gene specific primers (ProPgCAD-GSP1:

50-TGTAAGTGTAAGGGGACAAATGGCCAC-30 and

ProPgCAD-GSP2: 50-AGTCCCGAGCTGCATATCCTGT

AACAG-30) were designed based on the CAD cDNA

sequences obtained from TIGR Picea gene index (TC21142)

which was most similar to CAD sequences shown to be

involved in lignification in loblolly pine (MacKay et al.

1995) and Norway spruce (Galliano et al. 1993). All PCR

reactions were performed using Advantage 2 Polymerase

mix (BD Biosciences, Palo Alto, CA, USA). Two rounds of

PCR amplification using the ProPgCAD-GSP1 primer with

an adapter-specific primer and the nested PCR amplification

788 Plant Cell Rep (2009) 28:787–800

123

with ProPgCAD-GSP2 and another adapter-specific primer

gave a fragment of 1,163 base pairs (bp) fragment. All PCR

products were ligated to pCR2.1 with the TA cloning Kit

(Invitrogen, Carlsbad, CA, USA) and sequenced. Based on

this sequence we used the high fidelity Taq DNA polymerase

(Invitrogen) to amplify the ProPgCAD from spruce genomic

DNA with forward and reverse primers containing restric-

tions sites (ProPgCAD-Forward (PstI): 50-CTGCAGTAC

TATAGGGCACGCGTGGTCGACGGC-30, ProPgCAD-

Reverse (EcoRI): 50-GAATTCTTTTTTAAAACCACTT

CACAGGATTCG-30). The PCR used a reaction volume of

25 uL with the following steps: pre-denaturation at 94�C for

15 min, followed by 34 cycles of 92�C for 1 min, 60�C for

1 min, 72�C for 1 min, and finishing with 72�C for 5 min.

Similar methods were employed to isolate the P. taeda

genomic flanking sequence with the following modifica-

tions. Genomic DNA was isolated from a single

megagametophyte obtained from the tree genotype 7-56.

DNA fragments amplified by genome walking PCR were

cloned into the pT7Blue vector (Stratagene). The final

genomic fragment giving the P. taeda CAD promoter

(729 bp) was amplified using the following primers

(without restriction sites): ProPtCAD–Forward: 50-CG

GAGGAGTTAGCCCAAAAAGTTAT-30, and ProPt-

CAD–Reverse: 50-TCAAGAGTGGCAGATTCCGCAGT

AAA-30.A CAD promoter construct was prepared to drive GUS

gene expression as follows. The 1,163 bp spruce promoter

fragment cloned into pCR2.1 (Invitrogen) was digested

with SmaI and EcoRI (New England Biolabs, Beverly,

MA, USA). The CaMV 35S promoter from pRT 106

(Topfer et al. 1993) was removed by digestion with HincII

and EcoRI, and replaced with the spruce promoter to create

pRT-CAD-polyA. The GUS reporter gene was then added at

the BamHI and SacII sites from the original pRT 106

polylinker. The GUS coding sequence was obtained from

pCAMBIA2301 (www.cambia.org) which was amplified

with primer containing the BamHI and SacII restriction

sites. The resulting vector was called pRT-CAD::GUS-

polyA. A control vector, that did not contain the putative

CAD promoter, was also produced. Finally the CAD::GUS-

polyA and control vector cassettes were obtained by

digestion with SbfI of pRT-CAD::GUS-polyA, and ligated

into pCAMBIA2300 digested with PstI. The resulting

ProPgCAD::GUS and control promoter-less vector con-

structs were transferred into the A. tumefaciens strain C58

pMP90.

Sequences analysis and accession numbers

Sequences analysis performed in this study used publicly

available 50 upstream sequences of cinnamyl-alcohol

dehydrogenase (CAD), including 50UTR, from Arabidopsis

thaliana, AtCAD-C (At3g19450, 1,696 bp) and AtCAD-D

(At4g34230, 1,917 bp), and Eucalyptus gunnii EgCAD2

(X75480, 2,615 bp), which have been functionally char-

acterized in planta by fusion with the GUS gene (uid A)

reporter (Sibout et al. 2003; Feuillet et al. 1995; Hawkins

et al. 1997; Lauvergeat et al. 2002; Baghdady et al. 2006).

Accession numbers of the 50 upstream sequence from white

spruce CAD is FJ428229 (1,163 bp) and FJ428228

(829 bp) for loblolly pine.

Putative cis-regulatory elements shown in this study and

their positions were identified by the use of the PLACE

software (Higo et al. 1999); the PlantCare software (Rom-

bauts et al. 1999) was used only for the Fig. 1. The graphic

representation of the cis-elements distribution along the

ProCAD sequences was produced with Microsoft� Excel

2002; the ‘‘radar’’ representation was used for the Fig. S1.

Determination of the CAD promoter transcription start

site by 50-RACE

The putative transcription start site for the loblolly pine

CAD sequence has been reported in MacKay et al. (1995).

The transcription start site of the spruce CAD promoter was

determined by 50-rapid amplification of cDNA ends (50-RACE). Total RNA was extracted from stem apex (new

flush of the year) following the procedure of Chang et al.

(1993) as described in Bedon et al. (2007). 50-RACE cDNA

synthesis and PCR was carried out using the SMART

RACE cDNA Amplification Kit (BD Biosciences Clon-

tech, CA, USA) with 1 lg of total RNA from stem apex.

Touchdown PCR for the nested 50-RACE reaction was

performed with the following parameters: five cycles of

two steps at 94�C for 30 s and 72�C for 3 min, five cycles

of tree steps at 94�C for 30 s, 70�C for 30 s and 72�C for

3 min and 25 cycles of tree steps at 94�C for 30 s, 68�C for

30 s and 72�C for 3 min. The universal primers provided

by the manufacturer were used with the CAD specific

reverse primer (PgCAD-R5: 50-CTTGAGTAGGGGTG

CCGTCATGGTT-30). The 50-RACE PCR products were

cloned into pCR2.1 (Invitrogen, Carlsbad, CA, USA) and

eight positive clones were sequenced and aligned with the

spruce CAD promoter sequence to determine the tran-

scription start site.

Transgenic plant production materials

The embryogenic spruce tissues line Pg653 (Picea glauca

[Moench] Voss) was transformed using co-cultivation with

the A. tumefaciens strain C58 pMP90 containing the

ProPgCAD::GUS or the empty vector constructs as

described in Klimaszewska et al. (2001, 2004). After

co-cultivation, explants were decontaminated from A. tum-

efaciens with cefotaxim and transferred onto fresh medium

Plant Cell Rep (2009) 28:787–800 789

123

containing cefotaxim alone. Kanamycin resistant embryo-

genic tissues were screened according to the procedure

described by Klimaszewska et al. (2004), and were further

confirmed positive by X-gluc tests. Three transgenic lines

containing the ProPgCAD::GUS construct, representing

independent transformation events and exhibiting a range of

X-gluc activity levels and one control line (empty vector),

were selected for somatic embryo maturation and seedling

production. Transgenic plantlets were transplanted into a

mix of moss, vermiculite and turface (ratio 4:2:1), and grown

in a mist environment for 15 days before being transferred to

the greenhouse with a 16 h day/8 h night photoperiod at

24�C day/20�C night. The plants were taken through three

vegetative growth cycles of 3 months each, separated by

8-week dormancy periods during which plants were placed

in the dark at 4�C (i.e. corresponding to 3-year-old trees).

Following the first dormancy the plants were transferred to

3-l pots. All plants including controls were fertilized weekly

during vegetative growth with 20 g/L of N–P–K. All

experiments used a randomized distribution of lines and

treatments in the greenhouse.

Histochemical staining and b-glucuronidase assays

Sample preparation for GUS histochemical staining of

transgenic (ProPgCAD::GUS) and control trees was

adapted from Hawkins et al. (1997). Briefly, samples of

roots, needles, apical stems (new flush or terminal leader),

and segments of the main stem were transversally cut to

5–50 mm in length depending the organ and pre-treated

30 min in cold acetone 90% to block wounding effects

and to facilitate substrate penetration (Hemerly et al.

Fig. 1 Nucleotide sequence of

a 1,163-bp fragment

immediately upstream of the

gene coding for cinnamyl

alcohol dehydrogenase (CAD)

in Picea glauca. Numbering

starts at the transcription start

site (?1) determined by 50-RACE; the 50 UTR is in italics.

Putative cis-regulatory elements

were identified by both

PlantCare (Rombauts et al.

1999) and PLACE software

(Higo et al. 1999), except for

MYBST1 and

BOXLCOREDCPAL, by

PLACE only (double linesboxes). TATA-boxes and

CAAT-boxes are in bold;

CCAAT-boxes are framed and

underlined when fused to

CAAT-boxes. Dotted line boxesare for putative transcription

factors binding sites: ERE,

‘‘Ethylene Responsive

Element’’; CCA1, ‘‘Myb-related

transcription factor CCA1’’;

WRKY; MRE, ‘‘Myb-

Response Elements’’. DOF

transcription factor binding sites

(AAAG) are underlined

790 Plant Cell Rep (2009) 28:787–800

123

1993). Then, samples were rinsed twice with 100 mM

potassium phosphate buffer (pH 8), and incubated in

5-bromo-4-chloro-3-indoyl-b-D-glucuronate at 37�C in

the dark under vacuum for 6–12 h until a blue stain was

clearly visible. Finally, samples were fixed under vacuum

overnight in PBS 0.05 M (pH 7) containing 2% of para-

formaldehyde and 2.5% of glutaraldehyde, then moved to

fresh fixation buffer and stored at 4�C until used for hand

sections or embedded in paraffin for thin transversal

sections.

Determinations of GUS enzyme activity were per-

formed as described in Cote and Rutledge (2003). DNA

concentrations were determined in reference to a standard

curve obtained from a solutions series of standard DNA

(Lambda DNA, Roche, Indianapolis, IN, USA). GUS

activities were measured twice on two samples per tree in

the third vegetative growth cycle and for two different

trees per line.

Isolation of RNA from transgenic spruce and RT-qPCR

analysis

All samples were immediately frozen in liquid nitrogen

upon collection and stored at -80�C. Different tissues were

collected from the three transgenic lines and the control,

and were analysed separately. Stem sections were taken

from the base of the main stem (three trees per line, ana-

lysed separately), and included all stem tissues from pith to

xylem, phloem and bark. Young needles were collected

from the apical region of the stem. Samples were ground

and total RNA extracted as described in Bedon et al.

(2007). Tissues were also collected from two 33-year-old

trees, as described (Bedon et al. 2007). RNA quality was

verified with a bioAnalyser instrument (model 2100, RNA

6000 Nano Assay kit, Agilent Technologies, Santa Clara,

USA). RNA and DNA concentrations were determined

optically with a Multiskan Spectrum spectrophotometer

(Thermo Scientific, Waltham, USA). One microgram of

total RNA was treated with amplification grade DNAse I

(Invitrogen, Carlsbad, USA), checked for integrity with a

bioAnalyser, and reversed transcribed with SuperScript II

RT (Invitrogen, Carlsbad, USA). cDNAs were diluted

fivefold, and used as template in a PCR reaction mixture

(15 lL) of 7.5 lL of 2X Quantitect SYBR Green I mixture

(Qiagen, Germantown, USA), 0.9 lL of primers (0.3 lM

forward and 0.3 lM reverse), 2 lL of cDNA, and 4.6 lL

of RNase-free water. Reactions were assembled in

LightCycler� 480 Multiwell plate 384 (Roche, Basel,

Switzerland) using a pipetting robot (EpMotion 5075,

Eppendorf, Hamburg, Germany). The thermal cycling used

the LightCycler� 480 (Roche), with a 15-min activation

period at 95�C followed by 40 cycles (95�C for 10 s; 55�C

for 60 s; 72�C for 30 s) with a fluorescence reading taken

at the end of each cycle. Melting curve analysis at the end

of cycling was used to verify that there was single ampli-

fication. Crossing point (Cp) values were determined with

the LC480 software, and standard curves were used to

transform Cp values into numbers of transcript molecules.

Standard curves were based on dilution series covering five

orders of magnitude (10-1–10-6 ng/lL) prepared for each

plasmid containing cDNA (spruce EST clones and vector

constructs) linearized with EcoRI or BamHI, purified on

Qiaquick columns (Qiagen) and verified on a bioAnalyser

(model 2100, DNA 1000 LabChip kit., Agilent Technolo-

gies). Transcript levels were normalized against the

transcript level of elongation factor 1-alpha (EF1-alpha)

or cell division cycle 2 (CDC2). Primer pairs were designed

to target 30-UTR of the genes with Primer 3 software to

ensure gene specificity (Rozen and Skaletsky 2000). The

forward and reverse primer pairs are as follows (amplicon

length and spruce EST clone ID indicated in brackets):

CDC2 (96 bp, GQ0197_L17), 50-GTGCAGAGAAAAA

GTCGAAC-30 and 50-CCACACCATATGTTCCTTCT-30;35S-terminator used for b-glucuronidase (GUS) gene

expression (101 bp, no EST), 50-CAAAAATCACCAGT

CTCTCTCT-30 and 50-ACCCTATAAGAACCCTAATT

CC-30; Defensin (169 pb, GQ0131_B10), 50-CTACTAC

TGTGATCTTCTCTGGTTT-30 and 50-GTAAGTAAAA

GATAACCTGAACCAC-30. Primer pairs of CAD (spruce

EST clone ID: GQ00410_D06) and EF1-a (spruce EST

clone ID: GQ0041-B09) are given in Bedon et al. (2007).

Jasmonate induction and mechanical wounding of wild

type white spruce

Two-year-old white spruce seedlings (Picea glauca clone

653) were used to analyse gene transcript levels in response

to jasmonate application (JA) and mechanical wounding

(W). The seedlings were grown in greenhouse as described

for the transgenic trees. Jasmonate (Sigma-Aldrich, Buchs,

Switzerland) was dissolved in methanol to a concentration

of 800 mM and diluted in water (0.05% Tween) to 800 lM

just before being sprayed onto four seedlings (12 ml/

seedling). Four other seedlings were sprayed with the

methanol (0.1%) only as control. Mechanical wounding

was performed by twisting the entire stem of the seedlings

(from the stem apex to the base) about ten times with

forceps; four other seedlings were not wounded (control).

Exactly 24 h after the treatments were applied, tissues were

sampled from control and treated plants (JA and W) by first

removing the apical stem including needles and then

immediately harvesting of the main stem without branches

or needles.

Plant Cell Rep (2009) 28:787–800 791

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Results

Sequence analysis of CAD promoters from two conifers

Genomic DNA fragments upstream of the cinnamyl alco-

hol deshydrogenase (CAD) gene were isolated by using a

PCR based genome walking procedure from Picea glauca

(1,163 bp) and Pinus taeda (829 bp). The cinnamyl alcohol

dehydrogenase aminoacid coding sequences between lob-

lolly pine and white spruce were both 357 amino acids in

length and shared 95% identity, strongly suggesting that

these two genes represent orthologues. The transcription

start site (?1) of these conifer CAD genes were located 76

nucleotides upstream of the presumed ATG initiation

codon, by 50-RACE analysis in P. glauca (Fig. 1) and in P.

taeda (MacKay et al. 1995), indicating that the first tran-

scribed nucleotide was an adenine in both cases. Pair-wise

alignment of the 76 nucleotides identified as 50-UTR in

pine and spruce gave 88% identity; however, sequences

upstream of the two 50-UTR regions were less conserved

(data not shown).

The structure of the P. glauca genomic fragment is

shown in detail in Fig. 1. Its most proximal putative

TATA-box was located between nucleotides -30 and -22

and an alternative TATA-box was between –504 and –498.

Conserved CAAT-box sequences were located near the

TATA-box, at -6, -30 and -63, as well as in the 50-UTR.

Potential regulatory elements were identified upstream of

the CAD gene (PLACE software, Higo et al. 1999; Plant-

Care software, Rombauts et al. 1999); they included

consensus sequences for the binding of transcription factor

proteins like WRKY (four elements), Myb-Response-Ele-

ment (MRE, four elements including one in the 50-UTR)

and a Ethylene-Responsive-Element (Fig. 1). They also

included the R1-MYB binding site (MYBST1) and the

R2R3-MYB binding site CADa found in the Eucalyptus

gunnii CAD2 gene (EgCAD2), designated as ‘‘BOXL-

COREDCPAL’’ in PLACE software (ACCWWCC). This

sequence element was required for the activation of Eg-

CAD2 (Goicoechea et al. 2005).

Comparative analysis of CAD promoter’s architecture

between different angiosperm and gymnosperm species

Searches to identify putative cis-regulatory elements

common among angiosperms and gymnosperms were

performed using the 50 upstream flanking sequences of

spruce (ProPgCAD) Pinus taeda, (ProPtCAD), Arabidop-

sis thaliana (ProAtCAD-C and D; Sibout et al. 2003) and

Eucalyptus gunnii, (ProEgCAD2; Feuillet et al. 1995;

Lauvergeat et al. 2002). Comparative analysis with the

PLACE software (Higo et al. 1999) detected a total of 29

different cis-elements between CAD promoters from

angiosperm and gymnosperm origin (Table S1). Moreover,

as some of these cis-elements have different names but are

identical or very similar sequences, we were able to iden-

tify at least 21 potential cis-regulatory elements

corresponding to putative binding sites for proteins like

MYB, bHLH, WRKY and DOF (Table S1). The number of

cis-elements detected depended upon the sequence length,

ranging from 122 for ProPtCAD (829 bp) to 377 for

ProEgCAD (2,615 bp) (Table S1) whereas the relative

frequency of specific types of cis-elements was not

dependent on sequence length; for example, the bHLH

binding sequence for ‘‘MYCCONSENSUSAT’’ (CAN-

NTG) was clearly over-represented in ProPgCAD (Fig. 2;

Table S1).

We investigated more specially seven of these 21 cis-

regulatory elements whose binding sequences have been

experimentally associated with R2R3-MYB (CCWACC),

R1-MYB (GATA), bHLH (CANNTG), CCT domain-

containing proteins (CCAAT), WRKY (TGAC), nuclear

response regulators (NGATT) and DOF (AAAG) proteins

(Table S1; Fig. 2). Their distribution was used to compare

the set of promoter sequences in more detail (Fig. 2;

Table S2), and was suggestive of particular signatures

among the CAD promoter sequences (Fig. 1; Table S2;

Fig. S1). For example, the pine and spruce sequences

appeared very similar overall and most clearly with

regard to their ‘‘ARR1AT and DOFCOREZM’’ motifs

occurrence profiles (Fig. 2). The three angiosperm

sequences also had distinguishing features; for example,

the distribution of the ‘‘ARR1AT’’ motif grouped them

together in a distinct set from the conifer sequences

(Fig. 2).

Interestingly, the Arabidopsis ProAtCAD-C and -D

appeared quite distinct from one another for the occurrence

profile of two motifs; ‘‘DOFCOREZM’’ and ‘‘GATA-

BOX’’ (Fig. 2; Fig. S1). Some specific elements showed a

unique variation in a single sequence; for example, the

bHLH binding site ‘‘MYCCONSENSUSAT’’ (CANNTG)

appeared to be over-represented in P. glauca sequence

compared to the other sequences including P. taeda. The

‘‘GATABOX’’ sites are more abundant in the first 1,200 bp

of upstream sequence in ProAtCAD-C than the others; but

more ‘‘GATABOX’’ sites are present after 1,200 bp in

ProAtCAD-D and ProEgCAD2 (Fig. S1). The putative

R2R3-MYB binding sites ‘‘MYBPZM’’ (CCWACC) were

generally less frequent and located in the proximal regions,

with two exceptions in E.gunnii (Fig. 2), although a few

closely related elements including ‘‘BOXLCOREDCPAL’’

(ACCWWCC) were also detected (Fig. 1; Table S1). Some

regions in the sequences also appeared to lack particular

cis-elements; for example, no ‘‘ARR1AT’’ site in a 175-bp

window of the angiosperms (-925 to -1,100, Fig. 2),

which is populated by other conserved sequence elements.

792 Plant Cell Rep (2009) 28:787–800

123

Molecular analysis of stably transformed

ProPgCAD::GUS spruce trees

Transgenic P. glauca seedlings were produced harbouring

a construct with the presumed CAD promoter sequence

(ProPgCAD) fused to the GUS gene. Plants were obtained

from independent transformation events (lines) that were

kanamycin resistants (see ‘‘Materials and methods’’), and

were grown in the greenhouse for three growth cycles

(i.e. corresponding to 3-year-old trees). The growth and

development of the three lines of transgenic trees

ProPgCAD::GUS (L1, L6 and L7) were not in anyway

distinguishable from that of the control line (empty vector

construct) (Fig. 3a). Enzymatic GUS protein assays (MUG

assays) conducted in needles provide an indication of

construct integrity, and showed that there was very little

variation in expression between these three lines ProPg-

CAD::GUS (Fig. 3b). Transcript accumulation was

determined for the ProPgCAD::GUS construct and com-

pared to the endogenous CAD gene, in the different

transgenic lines and one control line in the middle of the

third vegetative growth cycle (Fig. 3c). The transcript

levels for CAD were consistent across the different lines,

both in small stem segments taken at the base of the plant

(including xylem, phloem and bark) and needles. By

comparison, GUS transcripts were more abundant and

somewhat more variable across the different transgenic

lines (Fig. 3c) as might be expected due to positional

effects or transgene copy number variation. Furthermore,

the relative transcript abundance in the different lines was

not entirely consistent with the GUS activity (MUG

assays), especially in L1.

The spruce CAD promoter confers a vascular

expression pattern

Histochemical localization of GUS activity driven by the

putative CAD promoter was performed on the same lines as

used for the molecular analysis, and revealed intense blue

staining in differentiating secondary vascular tissues

including xylem, cambial zone and phloem, at different

positions along the stem and at different stages of the

vegetative growth (i.e. beginning and middle of the vege-

tative growth cycle) (Fig. 4a, b). Histochemical GUS

staining was consistently observed in the differentiating

secondary xylem close to the cambium zone, in the three

lines investigated. Staining was strong both in the single

ring of secondary xylem formed in completely elongated

terminal leaders (data not shown), and along the second

growth ring further down the terminal stem (Fig. 4a, b).

The staining localized to differentiating tracheids near the

cambial zone as well as to ray parenchyma cells (Fig. 4b).

No evidence of GUS staining was observed in mature

tracheids localized further away from the cambial zone.

Histological observations on thin transversal sections from

the base of the main stem afforded a higher resolution of

the GUS staining in different cellular types of differenti-

ating xylem and phloem, cambium and parenchyma ray

cells in the compression wood (Fig. 4c, d). Similar local-

izations of the GUS staining were observed in roots,

needles and at the site of initiation of young branches

(Fig. 4e–h). These data indicate that the 1,163 bp fragment

CAD flanking sequence from spruce indeed drives sec-

ondary xylem preferential expression, in agreement with

Fig. 2 Multi-species distribution of the seven common types of cis-

regulatory elements detected in the proximal 1,200 base pairs of the

conifer and angiosperm CAD promoter sequences. The cis-regulatory

elements (nucleotide sequences) identified by the PLACE software

(Higo et al. 1999) are represented by diamonds (Table S1). The

promoters are represented by the horizontal black lines (?1 to

-1,200 bp). Pg: Picea glauca (ProPgCAD, 1,163 bp); Pt: Pinus taeda(ProPtCAD, 829 bp); Eg: Eucalyptus gunnii; At: Arabidopsisthaliana

Plant Cell Rep (2009) 28:787–800 793

123

the expression patterns of the endogenous CAD transcripts

which accumulated preferentially in differentiating xylem

from stems and roots compared to needles, bark, stem

differentiating phloem and root periderm (Fig. 5).

Induction by mechanical wounding and jasmonate

application

We also examined the accumulation of CAD transcripts

after mechanical wounding or the application of jasmonate,

because of the involvement of phenylpropanoid metabo-

lism in some defence reactions. Significant accumulation of

the CAD gene transcripts was observed upon wounding and

JA application in 2-year-old seedlings, both in the terminal

leader (including needles) and in the main stem tissues

(Fig. 6). The largest induction was in the main stem of

plants sprayed with JA, which is a known mediator of the

wound response (Wasternack 2007). A defensin gene

(Nicole et al. 2006) was used as a positive control for this

experiment since it also accumulates following mechanical

wounding or jasmonate application.

These observations also led us to examine whether GUS

activity would be enhanced upon wounding the cambial

zone in the transgenic trees. Despite the higher blue

staining in phloem compared to xylem tissues probably due

to the presence of more metabolically active cells in

phloem, we noted that insertion of a pin through the bark

near the base of the main stem for 3 days lead to intensified

GUS staining quite specifically in secondary xylem cells

(Fig. 7a), compared to unwounded trees (Fig. 7b). This

observation, although somewhat preliminary, is consistent

with CAD transcript accumulation data, and suggested that

this putative promoter sequence contained wound response

elements.

Discussion

In this paper, we report the sequences of the putative

promoter regions of two conifer CAD genes as well a

functional evaluation of a 1,163-bp genomic DNA frag-

ment from P. glauca. Several types of cis-regulatory

Fig. 3 Production and

characterization of transgenic

spruce expressing the

ProPgCAD::GUS construct.

a Phenotypes of three transgenic

lines L-1, L-6 and L-7harbouring the

ProPgCAD::GUS construct, and

the control line (C) having an

empty vector construct. b Mean

GUS enzyme activity (MUG

assay) in needles, with standard

errors (n = 4). c Mean

transcript levels determined by

RT-qPCR for the GUS and CADgenes, with standard errors

(n = 3 for stem section, n = 2

for needles); transcript levels for

the Elongation Factor 1-a gene

(EF1-a) are shown. Stem

sections included all stem

tissues from pith to xylem,

phloem and bark

794 Plant Cell Rep (2009) 28:787–800

123

elements were identified and many were found to be shared

with other CAD gene promoter sequences from an angio-

sperm tree Eucalyptus gunnii (ProEgCAD2, Lauvergeat

et al. 2002) and the herbaceous model plant Arabidopsis

thaliana (ProAtCAD-C and ProAtCAD-D, Sibout et al.

2003). Histochemical observations on 3-year-old trans-

genic spruce trees indicate that the ProPgCAD sequence is

able to drive the GUS expression in vascular tissues and is

wound-inducible. These findings were consistent with

transcripts abundance data for the native CAD gene which

is preferential to xylem tissues and is increased by

mechanical wounding and jasmonate application.

Conservation of cis-regulatory elements among CAD

gene promoters

Comparative analysis integrating gene expression and

promoter function is a powerful approach to detect

Fig. 4 Histochemical

localization of GUS activity

driven by the CAD gene

promoter. Tissues were sampled

during the third vegetative

growth cycle. Histochemical

assays for GUS activity were

performed as described by

Jefferson (1987). Representative

GUS staining patterns from

among three transgenic lines

(L-1, L-6 and L-7) transformed

with the ProPgCAD::GUSconstruct. a, b, f, h Hand cut

section, c–e transversal sections

of 8 lm, a beginning of the

vegetative growth cycle,

b middle of the vegetative

growth cycle, a and b are the

second growth ring formation

from the pith (409),

c compression wood showing

parenchyma ray cells and

thicker xylem cell wall after

30 days of tree bending to 45�(1009), d differentiating xylem

tissue (1009), e vascular trace

leading to a branch (1009),

f transversal section of a needle

(1009), g roots, h transversal

section of young secondary root

(1009). CZ cambial zone,

Ph. phloem, Xy. xylem,

R parenchyma cell ray,

Pa parenchyma tissue,

CW compression wood,

CP cortical parenchyma tissue,

En endodermis, Ex exodermis

Plant Cell Rep (2009) 28:787–800 795

123

cis-regulatory elements between co-regulated genes in a

given condition, whether it involves different promoters

within the same species or among distant species (Ma and

Bohnert 2007; Haberer et al. 2006). In woody plants like

trees, this approach may be useful to identify key cis-reg-

ulatory elements for the genes, whose transcripts are

associated with the secondary cell wall during wood for-

mation. Nevertheless, very few reports on promoter

sequences analysis are based on trees species and even less

on gymnosperm species. Investigation of orthologous

genes to identify common regulatory elements is based on

the assumption that important transcription factor binding

sites are conserved through evolution. Here, the nucleotide

sequence flanking the spruce CAD gene contains several

motifs, which are similar to cis-elements previously iden-

tified and characterized as binding sequences for

transcription factors like MYB, WRKY and bHLH. Over-

all, as many as 21 types of cis-regulatory sequences were

found to be in common among the five putatively orthol-

ogous CAD promoters suggesting that regulatory networks

are maintained between angiosperms and gymnosperms.

The number and distribution of each type of cis-regulatory

elements varied between the promoter sequences. As

expected the shorter putative consensus sequences were

more abundant than the longer ones. Interestingly, four

times more potential bHLH cis-elements (CANNTG) were

detected in the spruce CAD promoter than in the other CAD

promoters studied. bHLH proteins are known to interact

with MYB and WD40 proteins to form a complex able to

trans-activate gene expression of a subset of phenylprop-

anoid genes and to initiate multiple cellular differentiation

pathways in a range of plants (Lepiniec et al. 2006; Hart-

mann et al. 2005). The PgCAD promoter also contains the

CADa R2R3-MYB binding site motif (ACCWWCC)

described in the Eucalyptus CAD2 and shown to be

involved in the transcriptional regulation (Goicoechea et al.

2005). The same cis-element was found one time in the

proximal promoter (450–40 nucleotides upstream the

transcription start site) of each of the sequences investi-

gated here, suggesting its role may be conserved for

transcriptional control of the CAD gene by MYB factors in

distantly related species.

By comparing the distribution of frequent cis-elements

along CAD gene promoter sequences, we are able to point to

potential structural conservation. For example, one of the

most similar patterns (i.e. number and distribution)

involved the DNA-binding-with-one-finger (i.e. DOF)

binding sites found in ProPgCAD and in ProAtCAD-C, both

of which are believed to be key for lignin accumulation,

Fig. 5 CAD transcript profile in spruce organs and tissues. Transcript

levels were determined by RT-qPCR in tissues from two different 33-

year-old trees and normalized with elongation factor (EF1-a). Nneedles; stem tissues: P periderm; Ph differentiating phloem; Xdifferentiating xylem; Root tissues: PPh root periderm with differ-

entiating phloem; X root differentiating xylem. Data are based on

three technical repetitions per tree, i.e. six measurements per data

point. Vertical bars are standard errors. Significance of differential

transcript accumulation between organs and tissues was evaluated

with t-test (LSD, SAS software version 9), at P B 0.05; letters A and

B indicate significant differences

Fig. 6 Transcript accumulation of CAD and Defensin after mechan-

ical wounding or application of jasmonic acid. Transcript levels were

determined by RT-qPCR in clonal replicates of 2-year-old P. glauca.

Tissues from four separate replicates per treatment were sampled 24 h

after mechanical wounding (W) or jasmonic acid spraying (JA) (see

‘‘Materials and methods’’). Fold change is the number of transcript

molecules (normalised with the housekeeping gene) in each treated

plant (W or JA) divided by that of the appropriate control plant

(unwounded or JA solvent sprayed) in the same replicate. A mean fold

change and standard error were computed from the fourfold change

values. AS ? Needles: apical stem (terminal leader) with needles,

Stem: whole main stem including bark and xylem together. Verticalbars are standard errors (n = 4). Significance of differential transcript

accumulation between JA and W was evaluated with Student’s t-test

at P B 0.05 (*) and P B 0.01 (**); ns not significant

796 Plant Cell Rep (2009) 28:787–800

123

indicating potential inter-species conservation of this tran-

scriptional element. Indeed, Skirycz et al. (2007) provided

evidence that Arabidopsis thaliana DOF4;2 influences

phenylpropanoid metabolism in an environmental- and

tissue-specific manner. Moreover, MYB and DOF tran-

scription factors may interact (Diaz et al. 2002; Isabel-

LaMoneda et al. 2003). The two conifer sequences also

appeared to have conserved profiles for ‘‘DOFCOREZM’’

and ‘‘ARR1AT’’ (binding sites for nuclear response regu-

lators) despite the different lengths of sequence analysed.

Cis-elements that are conserved among species could rep-

resent sequences which are maintained by natural selection

pressure to preserve function. Hence, the patterns observed

in CAD promoter regions could serve to generate hypoth-

eses regarding the possible role of specific cis-regulatory

elements to be tested through functional analysis, such as

promoter deletions and gel shift assays.

Vascular expression driven by the spruce CAD gene

promoter

The reported vascular GUS staining determined by the

spruce CAD promoter is in agreement with other studies in

different angiosperms tree species (Feuillet et al. 1995;

Hawkins et al. 1997; Lauvergeat et al. 2002). These

observations are consistent with the CAD gene mRNA

abundance data indicating xylem preferential accumulation

in mature wild type white spruce (this report) and in young

Norway spruce trees (Koutaniemi et al. 2007). In the E.

gunnii CAD2 promoter, the proximal region from 457 to

241 bp upstream the start codon was shown to be essential

for vascular cambium/xylem-specific expression (Lau-

vergeat et al. 2002), which was later linked to the R2R3-

MYB binding site CADa (ACCWWCC) (Goicoechea et al.

2005). Our analysis of the PgCAD promoter identified

R2R3-MYB consensus binding sequence site in a similar

position (402 bp upstream the start codon). Further inves-

tigation such as promoter deletions would indicate whether

this conserved sequence is also involved in xylem-prefer-

ential expression in spruce.

In contrast to angiosperms, few phenylpropanoid gene

promoter sequences and GUS histochemical localization

have been reported for conifer trees. Nevertheless, pro-

moter analyses of a caffeoyl-CoA O-methyltransferase

(CCoAOMT) from Pinus taeda, a multi-functional

O-methyltransferase (AEOMT) and a cinnamyl alcohol

dehydrogenase (CAD) from Pinus radiata have been

associated within lignifying tissues (Li et al. 1999; Moyle

et al. 2002; Wagner and Walter 2004). The GUS gene

driven by the spruce CAD promoter was expressed in the

vascular cambial zone and in differentiating xylem cells of

stems and roots as well as in parenchyma ray cells or in

needles. Our observations indicate that spruce CAD

expression overlaps with primary or secondary developing

xylem tissue formation in different organs. The GUS

staining associated with the expression of the spruce CAD

promoter localized most strongly to secondary xylem in the

elongating terminal leader, as well as in second and third

year growth rings further down the main stem, but was also

linked to primary xylem in needles. The GUS expression

was also localized to developing xylem tissues in other

parts of the trees, including primary and secondary roots. In

angiosperms, similar spatial and temporal patterns of

expression were previously shown for phenylpropanoid

genes including CAD and CCR from Eucalyptus (Feuillet

et al. 1995; Hawkins et al. 1997; Lacombe et al. 2000;

Baghdady et al. 2006) as well as COMT from B. pendula,

Tiimonen et al. (2007).

CAD gene expression in other biological processes

Lignification can occur during various stresses including

bending, drought and pathogen attack involving CAD in

other biological processes than lignification for structural

needs (Vance et al. 1980; Hose et al. 2001; Timell 1986).

Consequently GUS staining was expected to be induced

Fig. 7 Histochemical

localisation of GUS activity

after wounding. Transversal

hand-cut sections at the base of

3-year-old spruce trees (1009)

transformed with the

ProPgCAD::GUS construct.

Triangle (dotted lines) shows

the site of wounding: a pin was

inserted through the bark and

left for 3 days (b), control (a).CZ cambial zone, Ph. phloem,

Xy. xylem

Plant Cell Rep (2009) 28:787–800 797

123

upon different types of stresses and to localize to other

tissues than xylem.

CAD gene transcripts have been shown to accumulate

during compression wood formation (Bedon et al. 2007)

which is characterized by thicker xylem cell walls among

other features compared to control trees and to opposite

wood (Timell 1986). We observed that bending transgenic

trees led to stronger GUS staining in the ray parenchyma

cells, as well as CAD gene transcript accumulation. The

enhanced GUS staining in ray cells could be explained by

the fact that both xylem cells and parenchyma ray cells are

able to produce monolignols in a ‘‘cell-cooperation’’ sys-

tem (Feuillet et al. 1995; Hawkins et al. 1997; Baghdady

et al. 2006). Moreover, CAD gene transcripts accumulated

within 24 h following wounding and jasmonate applica-

tion. The spruce CAD gene promoter (ProPgCAD::GUS)

was also activated in bark and xylem tissues after

wounding of the cambial zone. These observations are

consistent with findings from other conifer species. For

example, several phenylpropanoid gene transcripts were

upregulated upon infection or wounding in Pinus sylvestris

seedling roots, and in Norway spruce trees with increased

lignin content in the bark (Koutaniemi et al. 2007; Adomas

et al. 2007). Putative cis-elements such as WRKY binding

sequences and the GT1-motif detected in the spruce pro-

moter may play roles in pathogen and wound induced gene

expression (Park et al. 2004, Eulgem and Somssich 2007).

Further analysis of theses sequence motifs may provide

clues as to the potential mechanism for regulating stress

related expression of the CAD gene in different tissues or

organs.

Data presented here also indicate that the spruce CAD

gene promoter drove GUS expression in roots tips where

there is no lignin deposition. Similar results have been

reported for phenylpropanoid gene promoters in other

plants, e.g. Eucalyptus CCR promoter (Lacombe et al.

2000), CAD from Eucalyptus and Arabidopsis (Lauvergeat

et al. 2002; Sibout et al. 2003). These findings could be

explained by the fact that derivatives of the phenylpropa-

noid-monolignol pathways, such as lignans and

dihydroconiferylglucosides (DCGs) were shown to be

components of the signal transduction in cytokinin-medi-

ated cell division (Teutonico et al. 1991; Lynn and Chang

1990). Indeed, cytokinins represent a major signal for root

development and are also known to be essential regulators

for vascular cell differentiation (Aloni et al. 2006). Inter-

estingly, one of the most abundant motifs detected in CAD

gene promoters matches the binding site for authentic

response regulator transcription factor (ARR), involved in

cytokinin-mediated regulation for root protoxylem differ-

entiation (Sakai et al. 2001; Yokoyama et al. 2007).

In conclusion, this study described a 1,163-bp fragment

of genomic DNA upstream of the spruce CAD gene which

drove GUS expression preferentially in vascular tissues and

was wound inducible. Comparative analyses of angiosperm

and conifer sequences highlighted the conservation of

putative cis-elements in the spruce CAD promoter which

remain to be tested but are potentially involved in vascular

and stress-related expression. Moreover, the spruce CAD

promoter fragment represents a useful molecular tool to

investigate the transcriptional regulation in vascular devel-

opment and to drive the tissue preferential expression of

other genes in secondary xylem cells of conifer trees or

gymnosperms more generally. Zhao et al. (2005) followed a

similar approach by using the cinnamate 4-hydroxylase

gene promoter from Populus tomentosa to reduce lignin

content in transgenic tobacco by downregulating another

phenylpropanoid gene (CCoAOMT) specifically in tissues

which lignify most intensely. White spruce is an economi-

cally important tree for the wood products industry;

therefore applied outcomes of such lignin gene character-

ization may include uses in tree breeding and biotechnology.

Acknowledgments We are grateful to Francoise Pelletier and

Laurence Tremblay for excellent assistance in tissue culture and

growth of the plants in greenhouse. We acknowledge Denis Lachance

for helpful advice for the jasmonate application methods. This

research was supported by funding from Genome Canada and Gen-

ome Quebec to JM and AS for the ARBOREA project.

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