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O R I G I N A L P A P E R

Down-regulation of Leucaena leucocephala cinnamoyl CoAreductase (LlCCR) gene induces significant changes in phenotype,

soluble phenolic pools and lignin in transgenic tobacco

S. Prashant M. Srilakshmi Sunita S. Pramod Ranadheer K. Gupta

S. Anil Kumar S. Rao Karumanchi S. K. Rawal P. B. Kavi Kishor

Received: 3 May 2011 / Revised: 19 June 2011/ Accepted: 14 July 2011/ Published online: 17 August 2011 Springer-Verlag 2011

Abstract cDNA and genomic clones of cinnamoyl CoA

reductase measuring 1011 and 2992 bp were isolated froma leguminous pulpwood tree Leucaena leucocephala,

named as LlCCR. The cDNA exhibited 8085% homology

both at the nucleotide and amino acid levels with other

known sequences. The genomic sequence contained five

exons and four introns. Sense and antisense constructs of

LlCCR were introduced in tobacco plants to up and down-

regulate this key enzyme of lignification. The primary

transformants showed a good correlation between CCR

transcript levels and its activity. Most of the CCR down-

regulated lines displayed stunted growth and development,

wrinkled leaves and delayed senescence. These lines

accumulated unusual phenolics like ferulic and sinapic

acids in cell wall. Histochemical staining suggested

reduction in aldehyde units and increased syringyl over

guaiacyl (S/G) ratio of lignin. Anatomical studies showed

thin walled, elongated xylem fibres, collapsed vessels with

drastic reduction of secondary xylem. The transmission

electron microscopic studies revealed modification of

ultrastructure and topochemical distribution of wall poly-saccharides and lignin in the xylem fibres. CCR down-

regulated lines showed increased thickness of secondary

wall layers and poor lignification of S2 and S3 wall layers.

The severely down-regulated line AS17 exhibited 24.7%

reduction of Klason lignin with an increase of 15% holo-

cellulose content. Contrarily, the CCR up-regulated lines

exhibited robust growth, development and significant

increase in lignin content. The altered lignin profiles

observed in transgenic tobacco lines support a role for CCR

down-regulation in improving wood properties of L. leu-

cocephala exclusively used in the pulp and paper industry

of India.

Keywords Cinnamoyl CoA reductase Leucaena

leucocephala Down-regulation Cell wall ultrastructure

Phenolics Lignin

Introduction

Lignin, a heterogeneous phenolic polymer is present

mainly in the walls of secondary thickened cells of vascular

plants and represent 2030% of the dry weight of wood.

Lignin confers rigidity to the cell wall for structural support

and impermeability for transport of water and nutrients

over large distances. The intrinsic properties of the lignin

polymer have been essential for plants to adapt to a

terrestrial habitat, enabling them to grow upward, but are also

crucial in determining the value of plants as raw materials.

Lignin is a major concern for the pulp and paper industry as

it hinders the optimum utilization of the biomass and needs

to be extracted from the wood by harsh chemical condi-

tions to produce pure cellulose fibers (Peter et al. 2007).

Communicated by P. Kumar.

Electronic supplementary material The online version of thisarticle (doi:10.1007/s00299-011-1127-6) contains supplementarymaterial, which is available to authorized users.

S. Prashant M. Srilakshmi Sunita R. K. Gupta

S. Anil Kumar P. B. Kavi Kishor (&)

Department of Genetics, Osmania University,

Hyderabad 500 007, India

e-mail: pbkavi@yahoo.com

S. Pramod S. Rao Karumanchi

Department of Biosciences, Sardar Patel University,

Vallabh Vidyanagar, Anand 388120, India

S. K. Rawal

Ajeet Seeds Ltd, 233 Chitegaon, Aurangabad 431105, India

123

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DOI 10.1007/s00299-011-1127-6

http://dx.doi.org/10.1007/s00299-011-1127-6http://dx.doi.org/10.1007/s00299-011-1127-6

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Paper industry mainly uses bamboos, Eucalyptus, Acacia,

Populus, Casuarina, Picea and Pinus species as a source of

raw material for paper and pulp production. Though all

these species are important to the paper industry world-

wide, Leucaena species is exclusively used in India

because of its easy acclimatization and high rate of biomass

production. About 25% of raw material comes from this

genus. Lignin content is rather high in this species(2532%) and hence needs to be reduced.

Over the past decade, considerable attention has been

focused on understanding the lignin biosynthetic pathway

and on exploring the potential of genetic engineering to

tailor lignin content and composition for industrial appli-

cations (Baucher et al. 2003; Boudet et al. 2003). The

picture that emerges out of research on the individual roles

of the monolignol biosynthetic genes is that down-regula-

tion of PAL, C4H, 4CL, C3H, CCoAOMT, CCR and to a

lesser extent CAD have a prominent effect on lignin con-

tent (Vanholme et al. 2008). Lignin composition can be

altered as well. Cinnamoyl-CoA reductase (CCR) occupiesa key position between phenylpropanoid metabolism and

lignin specific branch. As a first step committed to the

lignin branch pathway (Lacombe et al. 1997), CCR may be

considered as a potential control point regulating the car-

bon flux towards lignins and therefore its down-regulation

could affect the lignin content. In the present study, we

isolated both cDNA and genomic clones encoding CCR

from L.leucocephala and evaluated the effect of up and

down-regulation of CCR gene in tobacco.

Materials and methods

Isolation ofCCR cDNA and genomic clones

Two sets of degenerate oligonucleotide primers for cDNA

amplification were designed based on the conserved amino

acid sequences identified by multiple sequence alignment

of orthologous sequences available in the NCBI database

using ClustalW program (Thompson et al. 1997). The

primers were: CCR F1: 50-CGCCTCCCCCGTGACNG

AYGAYCC-30; CCR R1: 50-GTCTTGGCGGAGCCGKY

NARRTAYTT-30; CCR F2: 50-CCGTGAGGGGCAAAG

YNMGNAAYCC-30; CCR R2: 50-CACCGTCTTGCCGT

AGCARTACCARTT-30. Total RNA was extracted from

xylem tissue of L. leucocephala using TRIZOL reagent

(Invitrogen, USA). Single stranded cDNAs were synthe-

sized with reverse transcriptase using poly T primer (MBI-

Fermentas, Germany), according to the manufacturers

instructions. Partial sequences were amplified by using

cDNA as a template for PCR with 10 pico moles each of

degenerate primer, 0.2 mM dNTP, 1.5 mM MgCl2, and 1

U Taq polymerase per 25 ll volume of reaction. PCR was

performed with a denaturation temperature of 94C for

5 min, then 30 cycles of 94C for 30 s, 54C for 45 s, and

72C for 1 min, with final extension at 72C for 5 min.

The partial amplicons were sequenced and the sequence

was used to design gene specific oligonucleotide primers

according to Frohman et al. (1988) for 50 and 30 RACE PCR.

Both the 50 and 30 RACE cDNAs were synthesized

according to manufacturers instructions (SMART RACEcDNA Amplification Kit, Clontech, CA, USA). Then 30

RACE PCR was performed using the gene specific forward

primer CCR RF 50-CACGGCTTCTCCAGTCACAGA-

CAAC-30 and universal primer mix. In the same way, 50

RACE was performed using gene specific reverse primer

CCR RR 50-CACTGCCTTCCCATAGCAATACCAG-3 0

and universal primer mix. The 30 and 50 RACE PCR prod-

ucts containing 30 UTR, 50 UTR respectively, were analyzed

on agarose gel and sub-cloned into pTZ-TA cloning vector

(MBI Fermentas, USA) and sequenced. The sequencing

results of RACE PCR products were used for character-

ization of translation start site, stop codon and also the 30

and 50 UTRs. After analyzing the 30 and 50 UTRs, CCR gene

specific primers CCR FF: 50-CATATGGGCAGCGTCG

AAGGAGA-30 and CCR FR: 50-GTCGACTCATTGATC

AAGTTTGCTGCCGG-3 0 were designed and used for the

amplification ofCCR full length cDNA. BLAST (Altschul

et al. 1990) search program was used for sequence com-

parisons in NCBI database. Analysis for conserved

sequences in CCR was performed using the ClustalW pro-

gram. PCR was performed with the primers designed for

CCR cDNA using genomic DNA of L. leucocephala as a

template to isolate the genomic clone ofCCR. The genomic

amplicon containing exons and introns were characterized

by Genewise software from EMBL tools.

Preparation of CCR sense and antisense constructs

The sense and antisense constructs contain the full length

cDNA (LlCCR) encoding cinnamoyl CoA reductase

enzyme ofL. leucocephala. The full length cDNA (LlCCR)

was obtained from cDNA library by PCR using the prim-

ersCCR FF: 50-CATATGGGCAGCGTCGAAGGAGA-

30 and CCR FR: 50-GTCGACTCATTGATCAAGTTTG

CTGCCGG-30. The LlCCR cDNA amplicon was then

stabilized in a pTZ TA cloning vector (MBI, Fermentas,

USA). Due to lack of compatible sites between the pTZ TA

vector and the plant expression vector pCAMBIA1301, the

full length cDNA was initially subcloned into an interme-

diate vector, pRT100 and subsequently into pCAM-

BIA1301. Plasmid pTZ TA, containing the LlCCR cDNA,

was digested with BamHI, SacI and XbaI, SmaI to release

the sense and antisense fragments respectively. These

fragments were subsequently cloned into linearized

pRT100 vectors digested with the same restriction

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endonucleases. The recombinant pRT100 vectors were

digested with the restriction enzyme HindIII to release the

sense and antisense LlCCR cDNA constructs along with

CaMV35S promoter and NOS polyA terminator. These

cassettes were cloned into pCAMBIA1301 vectors at the

HindIII sites respectively. The constructs (pCAMBIA-

sense and antisense LlCCR) were then transferred to

Agrobacterium tumefaciens strain LBA4404 by the freeze-thaw method (Holsters et al. 1978) for subsequent plant

transformation experiments.

Plant transformation and regeneration

Nicotiana tabacum variety Xanthi was transformed by

pCAMBIA1301-sense and antisense LlCCR constructs

using the leaf-disk method (Horsch et al. 1985). Untrans-

formed tobacco plants served as controls. Hygromycin

(50 mg/l) was used as a selective agent during in vitro

regeneration. Differentiation of shoots was achieved on MS

medium supplemented with 1 mg/l 6-benzylaminopurine(BA) and 0.1 mg/l napthaleneacetic acid (NAA). Rooting

was obtained on MS basal medium devoid of growth reg-

ulators. Cephotaxime was used at 250 mg/l during in vitro

regeneration to remove excess bacterial growth. Trans-

formed plants were grown in vitro for 6 weeks under a

light-dark regime of 16 h (2030 mE m-2 s-1, 27C)/8 h

(27C) and then transferred to soil and grown to maturity in

the green house. T0 transformants were allowed to self

pollinate to obtain homozygous lines. T1 seeds were har-

vested and subjected to selection on germination medium

containing hygromycin.

Molecular characterization of transformants

Genomic DNA was isolated according to Doyle and Doyle

(1990), purified and quantified by measuring absorbance at

260 and 280 nm. PCR was performed with DNA isolated

from transgenic and control plants using L. leucocephala

CCR gene specific primers. The reaction volume for all

PCR reactions was set to 25 ll. PCR amplifications were

carried out starting with an initial denaturation at 94C for

5 min, denaturation at 94C for 30 s, annealing at 58C for

45 s for CCR primers and extension at 72C for 90 min.

These steps were repeated for 35 cycles followed by a final

extension for 5 min at 72C. The reaction mixture without

a template was run as a negative control. Positive controls

for CCR were also included. Amplified DNA fragments

were separated by gel electrophoresis in 1.2% agarose gel.

Genomic DNA isolated from the leaves of transgenic

tobacco and control plants (15 lg each) was digested with

EcoRI, separated by agarose gel electrophoresis and blotted

onto nylon membranes (Hybond N?, Amersham Biosci-

ences, UK) using standard protocols (Sambrook and

Russell 2001). The labelling, hybridization and detection

methods were performed according to the manufacturers

instructions. pCAMBIA-sense and antisense LlCCR plas-

mids were digested with HindIII (1.7 kb each) and used as

probes after labelling with non-radioactive AlkPhos direct

system (Amersham Biosciences, UK). Both positive and

negative controls were included.

RT-PCR analysis

Total RNA from transformed and untransformed (control)

tissues were extracted using TRIZOL reagent according to

the manufacturers instructions (InVitrogen, USA). The

concentration and purity of RNA samples were checked

using UV-VIS spectrophotometer. 5 lg of total RNA was

taken for first strand cDNA synthesis using oligo-dT (20)

and M-MuLV reverse transcriptase (MBI Fermentas,

USA), following manufacturers instructions. After the first

strand cDNA synthesis, the reaction was terminated by heat

inactivation at 70C for 10 min. PCR was performed forthe amplification of CCR gene sequence using LlCCR

cDNA specific primers. A total volume of 25 ll of PCR

mix was prepared in a sterile 0.2 ml Eppendorf tube with

10 pmol/ll each of both forward and reverse primers,

0.22 ll of first strand cDNA as a template, 50 lM each

dNTP, 1.5 mM MgCl2 and 1 U of Taq polymerase. The

standard reaction conditions carried were initial denatur-

ation at 94C for 5 min, followed by 35 cycles of 94C for

30 s, 58C for 45 s, 72C for 90 min and a final extension

of 10 min at 72C. The reaction mixture without a template

was run as a negative control. Positive controls for CCR

gene were also included. Amplified DNA fragments were

separated by gel electrophoresis in 1.2% agarose gel.

Protein extraction and CCR enzyme assay

CCR enzyme assays were conducted on 34-months old

transgenic tobacco plants acclimatized in the green house.

The xylem tissue from the bottom of the stem (23 cm

high) was scrapped and ground in liquid nitrogen and

proteins were extracted at 4C in 0.1 M Tris-HCl pH 7.5,

2% (w/v) PEG 6000, 5 mM DTT, 2% (w/v) PVPP. The

crude extract was centrifuged for 10 min at 10,000 rpm at

4C twice and the supernatant was used for enzyme assay.

Activity of CCR enzyme (oxidation of coniferaldehyde,

reverse reaction) was determined by the increase in

absorbance at 366 nm (Luderitz and Grisebach 1981). At

366 nm wavelength, the change in the absorbance was the

result of the increase in the absorbance of feruloyl CoA

ester and of the decrease in absorbance of NADPH and

coniferaldehyde. The buffer soluble protein concentration

was measured by the method of Bradford (1976). The

incubation mixture contained 0.1 mM coniferaldehyde,

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0.35 mM coenzyme A, 0.25 mM NADP, 100 lg of total

protein extract and 0.2 M Tris HCl buffer pH 7.8 in a total

volume of 1 ml. The assay was performed for 10 min at

30C.

Histochemical staining, autofluorescence

and anatomical studies in CCR down-regulated plants

Cross sections of stems were prepared from fresh samples

of CCR up and down-regulated transgenic tobacco plants

using the sliding microtome (Reichert-Jung Nussloch,

Germany) and phloroglucinol-HCl (Weisner reaction;

Speer 1987), Maule (Iiyama and Pant 1988) reactions were

performed. Autofluorescence was detected on a Leitz

microscope equipped with a fluorescence device using

340380 nm excitation wavelength and 430 nm barrier

filters in conjunction with a Leitz 50 W HBO mercury

burner. For further validation of the down-regulation, a set

of anatomical studies as detailed below were carried out.

Free hand sections of stem were stained in 0.05% ToluidineBlue O (Sigma, T-3260) prepared in benzoate buffer (pH

6.5) for 15 min and washed several times in water. Stem

samples from control, lignin up and down-regulated plants

were macerated to measure the length and width of fibres

and vessel elements. Small matchstick size stem pieces

were macerated by incubating in Jeffreys fluid (Berlyn and

Miksche 1976). After thorough washing in water, the

macerated elements were stained with safranin O (Sigma,

S-2255) before mounting in 50% glycerol. The length and

width of fibres and vessel elements were measured with an

ocular micrometer scale mounted in a research microscope.

For each parameter, 100 readings were taken from

randomly selected elements and they were statistically

analyzed to determine the mean values. Stained sections

were observed and photographed using a Carl Zeiss

microscope (KS 300) and Image Analyzer.

Transmission electron microscopy analysis

For ultrastructural studies, small slices (1 mm in thickness)

taken from the base of the stem by freehand sectioning with

a razor blade were fixed in 2.5% glutaraldehyde in phos-

phate buffer (pH 7.2) followed by 2% osmium tetroxide

for overnight. After dehydration through a graded ethanol

series up to 80% (v/v) and infiltration, the samples were

embedded in Spurrs resin (Spurr 1969). Ultrathin sections

on nickel and gold grids were subjected to potassium

permanganate (Donaldson 1992) and periodic acid- thio-

carbohydrazide-silver proteinate (PATAg) (Thiery 1967)

staining for localization of lignin and cell wall polysac-

charides respectively and observed under TEM (Philips,

Tecnai) at an acceleration voltage of 80 kV. Micrographs

were taken with CCD camera (Keenview, Olympus Soft

Imaging Solutions, USA). For measuring the fibre wall

thickness and proportion of secondary wall layers in fibres,

mean values from 30 random observations were taken from

ultrathin sections.

Metabolite analysis

To determine whether CCR gene up and down-regulation

had any impact on phenyl propanoid metabolism, reverse

phase HPLC was performed to observe UV absorbent low

molecular weight phenolics. Transgenic tobacco stem

tissue was ground under liquid nitrogen in a mortar and

pestle. Ground tissue (100 mg) was extracted with 1.5 ml

of methanol/water/HCl (48.5:48.5:1) for 4 h at 50C and

then centrifuged for 10 min at 150009g. The supernatant

was removed from the pellet; distilled water (1 ml) was

added followed by an equal volume of ethyl ether. The

sample was mixed thoroughly and left for phase separation.

The upper phase was removed and retained, while theextract was again extracted with a second volume of ethyl

ether, removed, and pooled. The ether phase was then dried

in vacuo, resuspended in methanol and analyzed by HPLC.

A dionex summit HPLC system fitted with a reverse-phase

2.0 mm 9 150-mm pursuit column (water 5-lm particle

size) autosampler and a photodiode array detector was used

for methanolic profiling. The methanolic extracts were

eluted from the column with a linear gradient of 100% A to

80% B over 60 min followed by a 10 min wash with 100%

B, and finally reclimated with 100% A for 10 min, where

eluant A is 5% acetic acid and eluant B is a 75:25 mix of

20% acetic acid and acetonitrile. The flow rate for analysiswas 0.2 ml/min, column temperature was 45C and

detection was at 320 nm.

Lignin and holocellulose analysis

Lignin content in the transgenic tobacco lines and controls

was estimated by the standard Klason lignin method

(Dence 1992). Whole mature stems of transgenic and

control tobacco plants were harvested, dried and ball mil-

led to fine powder. The powdered wood sample (0.91.1 g)

of each was sequentially extracted with cyclohexane:

ethanol (7:3 v/v) using a soxhlet apparatus for 67 h. The

resulting cell wall residue was treated with 72% sulphuric

acid for 2 h and subsequently boiled for 4 h under constant

volume conditions. The mix, after cooling was filtered

through a glass Gooch crucible, washed with warm

(*50C) deionized water to remove residual acids and

dried overnight at 105C. The dry crucibles were weighed

to determine Klason lignin (acid-insoluble lignin) gravi-

metrically. Lignin content was reported as percentage of

the original weight of cell wall residues.

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The powdered wood sample (0.91.1 g) was taken from

transgenic and control tobacco stems treated with 80 ml of

ethanol. With gentle swirling, 20 ml of concentrated HNO3was added. The flask was connected to water cooled

refluxing condenser and heated on a boiling water bath for

1 h. 1 g of filter aid was added and then the contents in the

flask were filtered through Gooch crucible. The crucible

was washed thrice with 15 ml of ethanol. The contentswere then transferred to the original flask and the above

steps were repeated twice. After third treatment, all the

contents from the flask were transferred to the crucible

using ethanol and a glass rod fitted with a policeman. The

crucible was washed thrice with 15 ml of distilled water

and then dried in an oven at 105 3C for 2 h. The cru-

cible was cooled for 1 h in a desiccator containing silica

gel and weighed. The crucible was then placed in a muffle

furnace heated to 500550C and allowed to stand for 1 h.

The crucible was removed, allowed to cool in a desiccator

containing silica gel and reweighed to determine holocel-

lulose content. Holocellulose content was reported as per-centage of the original weight of cell wall residues.

Results

Isolation ofCCR cDNA and genomic clones

The coding sequence of CCR cDNA (LlCCR) measured

1011 bp (AM263501) encoding a putative peptide of 336

amino acids with a predicted molecular weight of

36.52 kDa. The nucleotide sequence showed 84% homol-

ogy with Linum, 83% with Prunus and 80% with Fragaria

at the nucleotide level. The deduced amino acid sequence

showed high identity with the orthologs from Solanum

tuberosum (82%), Eucalyptus gunnii (76%) and Populus

trichocarpa (76%). PCR was performed with the primers

designed for CCR cDNA using genomic DNA of L. leu-

cocephala as a template to isolate the genomic clone of

CCR. The amplicon measured 2992 bp (AM262869) and

upon analysis using Genewise software revealed that CCR

gene has 5 exons and 4 introns.

Construction of CCR sense and antisense vectors

For up and down-regulating L.leucocephala CCR gene

expression in tobacco, sense and antisense constructs ofLlCCR cDNA were prepared in pCAMBIA 1301 under the

control of the CaMV 35S promoter. For antisense construct

preparation, the full length LlCCR cDNA was inserted in

reverse orientation under the control of CaMV 35S pro-

moter. The CCR sense-pCAMBIA1301 and antisense-

pCAMBIA1301 vectors (Fig. 1a, b) were mobilized into

the Agrobacterium tumefaciens strain LBA4404. These

agrobacterial strains were used to carry out genetic trans-

formation of tobacco.

Molecular characterization of transgenic tobacco lines

From the surviving explants, several GUS positive trans-

formants were recovered. 15 independent transformants

were regenerated with CCR sense construct while 27

independent transformants were regenerated with CCR

antisense construct. Of these, five independent transfor-

mants (3-months-old) from each CCR sense and antisense

constructs were selected along with untransformed controls

and analysed by PCR and Southern blotting. The LlCCR

gene specific primers were used for PCR confirmation of

transgenic tobacco plants with LlCCR sense and antisense

transgenes. All positive transformants (both sense and

antisense) showed a 1011 bp band (Fig. 2a, b) of CCR

gene. The same were not noticed in untransformed con-

trols. Genomic DNA from CCR sense and antisense T0transgenic lines were digested with HindIII that has single

restriction site within the T-DNA along with the DNA from

untransformed control plants. The plasmid DNA of anti-

sense-pCAMBIA1301 served as a positive control. DNA

Fig. 1 The LlCCR sense and antisense constructs. CaMV 35S

Cauliflower mosaic virus 35S RNA promoter, Sense LlCCR CCR

cDNA ofL. leucocephala in sense orientation, Antisense LlCCR CCR

cDNA of L.leucocephala in antisense orientation, Nos poly A

termination sequence of the nopaline synthase gene, T poly A

termination sequence of the CaMV 35S RNA, hpt II hygromycin

phosphotransferase gene, GUSb-glucuronidase gene

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samples that were digested with the above enzyme were

transferred onto nitrocellulose membrane after electro-

phoresis. The membrane was hybridized with CCR gene

specific probe. The autoradiogram generated after exposing

the blot to the film showed positive hybridization at the

1011 bp region for each putative sense and antisense CCR

transgenic plant samples (Figs. S1A and B) except in

untransformed control plants. The signals on the blot

indicated the gene integration in all transgenic plants.

RT-PCR analysis and CCR activity in transgenic

tobacco

RT-PCR analysis and CCR enzyme assay were performed

on 34-month-old, five independent lines each ofCCR sense

and antisense transgenics along with untransformed controls

acclimatized in the green house. RT-PCR analysis showed

less transcript levels in CCR antisense lines compared to

sense lines in the xylem tissue (Fig. S2). On the other hand,

untransformed control tobacco plants did not show any

amplification. This indicated the up and down-regulation ofCCR gene in transgenic lines. All the five antisense CCR

transgenic lines showed differing amounts of reduction in

CCRactivity as compared to control. However, the degree of

silencing varied in different individual lines, giving rise to

widely different levels of CCR enzyme activity in geneti-

cally identical plants. While antisense lines AS2 and AS3

showed 69.3 and 58.6% reduction in activity respectively,

line AS17 recorded 74.6% reduction. Lines AS4 and AS12

displayed a reduction of 28.2 and 48.4%, respectively. Of the

five sense transformants, S2 and S27 exhibited 62.6 and

41.2% increase in CCR activity respectively. Likewise, S6

and S9 also showed 25.3 and 30.6% increase in CCRactivity. However, line S5 exhibited the highest increase of

72.3% in CCR activity as compared to untransformed con-

trol. The results are summarized in Fig. 3.

Morphology of CCR sense and antisense transgenic

tobacco plants

When grown in vitro, there were no developmental dif-

ferences among the primary transformants. However, after

transfer to the greenhouse, the transgenic plants exhibited

differences in morphological features. The CCR down-

regulated plants displayed varied phenotypes especially

AS2 and AS17 lines. The general growth of the plants was

affected; the antisense plants grew to two-thirds the size of

the control plants (Fig. S3 A), while the CCR sense plants

grew to the height of untransformed plants Fig. S3 B. The

leaves were stunted in antisense plants, wrinkled and curled(Fig. 4a, b), clear venation and paler photosynthetic tissue

was observed compared to controls (Fig. 4c, d). Weak stem

with decreased internodal length (1 cm) was observed in

the CCR down-regulated plant compared to control

(1.5 cm) and up-regulated plant (2 cm) (Fig. 4e). More-

over, both flowering and senescence were delayed by

1520 days in contrast to control and CCR up-regulated

plants. But the line AS4 showed normal phenotype like that

of controls. On the other hand, CCR up-regulated plants

Fig. 2 PCR analysis of CCR sense and antisense transgenic tobacco plants with CCR gene primers. Putative CCR sense (a) and antisense

(b) transgenics showing CCR gene amplification

Fig. 3 Enzyme assay of CCR in antisense and sense transgenic

tobacco lines. The CCR activity of each sample was read in triplicate

and each bar represents the average values taken from six replicates

from two independent experiments. Error bars are standard devia-

tions. Significantly different values are represented by asterisk (*) and

the values are significantly different at P B 0.05. CCR antisense

transgenics showed very low specific activity as compared to controls

indicating the down-regulation of CCR enzyme. Sense transgenics

showed a high specific activity as compared to controls indicating theup-regulation of CCR enzyme. C Control, AS2 AS3, AS4, AS12 and

AS17 CCR antisense transgenic lines. S2, S5, S6, S27 and S9 CCR

sense transgenic lines

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exhibited robust growth and development compared to

controls. Transgenic line S5 grew to a height of 75 cm,

exhibited expanded leaves, strong stem with a diameter of

4.8 cm and increased internodal length of 2 cm. However,

approximately 5% of all transformants, either from sense or

antisense lines, were dwarf in nature. The lines AS9, AS11

and S4 survived for 23 months in green house but died

without flowering. Table 1 provides the data of morpho-

logical features of the sense and antisense CCR T0 trans-genic plants.

Orange-brown coloration of debarked stem ofCCR

transgenics

All the CCR down-regulated transformants after

34 months of growth in the greenhouse exhibited orange

brown coloration of debarked stems compared to untrans-

formed controls (Fig. 5). This phenotype was previously

observed in transgenic tobacco with severely depressed

CCR activity (Piquemal et al. 1998). However, the color

faded soon after peeling the bark. Furthermore, the color-

ation was generally more pronounced in the basal part of

the stem, whereas it was not noticed towards the apical end.

The intensity of the colour was corelatable with the degree

of reduction in CCR activity. AS17 line which showed

maximum reduction (74.6%) in CCR activity displayed

darker orange-brown colouration of the stem than the otherlines.

Histochemical and autofluorescence changes associated

with CCR up and down-regulation

To investigate whether reduced CCR activity in the trans-

genic tobacco was corelatable with modified lignin content

or composition, stem sections from severely down-regu-

lated lines AS2 and AS17, untransformed control and

Fig. 4 Morphology of leaf and

stem of CCR sense and

antisense transgenic lines.

a, b Comparison of leaf

morphology of sense and

antisense transgenic lines with

control. C Control, AS17 CCR

antisense line. c Photosynthetic

tissue in leaf of control plant.

d Paler photosynthetic tissue in

leaf of AS17 transgenic line.

Stem internodal lengths of sense

transgenic line

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up-regulated lines S5 and S2 were subjected to different

histochemical staining by Weisner and Maules reagents.

Phloroglucinol reacts with the hydroxycinnamaldehyde and

benzldehyde groups of lignin and displays a typical pink

color of the cell wall in the control. The Wiesner reaction

showed intermediate intensity of staining in control plants

(Fig. 6a), whereas the down-regulated plants showed fee-

ble staining due to less lignin on the cell walls (Fig. 6b).

The Wiesner reaction showed intense red colouration oncell walls in the secondary xylem of CCR up-regulated

plants (Fig. 6c). Moreover, lignin distribution on the walls

of vessels was uneven in down-regulated plants compared

to that of control and up-regulated plants. Protoxylem

elements also showed less lignin on the walls of down-

regulated plants compared to that of control plants. Maules

reaction revealed the S/G ratio in CCR modulated xylem

elements. Control plants showed vessels and fibres with

reddish brown colour indicating combination of S and G

units (Fig. 6d). The fibre walls of CCR down-regulated

plants showed intense reddish colour due to increase in S

units (Fig. 6e) whereas the up-regulated plants showed

intense brown colour on vessel walls and fibres with

brownish red colour indicating increase in G units of lignin

(Fig. 6f). Together, these data suggest that CCR down-

regulation reduces the level of hydroxycinnamaldehydes

and increases S-units of lignin. Autofluorescence of stem

sections, excited by long wave length UV, displayed blue

colour in the xylem areas. In CCR down-regulated plants,

autofluorescence was less pronounced as compared tountransformed controls due to down-regulation of lignin

(Fig. 6g), and the highest intensity in the vessel elements in

control and up-regulated transformants (Fig. 6h, i). These

results thus show a positive correlation between CCR

activity and overall lignin content and composition in the

transgenics.

Anatomical studies of stem sections ofCCR transgenics

The amount of secondary xylem was reduced significantly

in CCR down-regulated plants like AS17 compared to that

of up-regulated (S5) and untransformed plants (Fig. 7). Thecambial zone of CCR down-regulated plants was dormant

with 23 cell layers, in contrast to 46 cell layers in con-

trols and CCR up-regulated plants (Fig. 8ac). The sec-

ondary xylem of CCR down-regulated plants showed

collapsed vessels in contrast to round to angular shaped

vessels, mostly solitary to radial multiples of 23 cells

found in controls and CCR up-regulated plants. Vessel

walls bulged into lumen side in all down-regulated plants.

Fibres were thin walled, ray parenchyma were broader

Table 1 Morphological features of CCR sense and antisense transgenic tobacco lines

Plant Age

(months)

Mature leaf

length (cm)

Mature leaf

breadth (cm)

Stem diameter

(cm)

Plant height

(cm)

Morphological features

Control 4 29.5 0.50 11.0 0.34 4.0 0.10 73.0 0.50 Normal phenotype

AS 2 4.5 19.6 0.20* 6.5 0.50* 3.2 0.20* 57.5 0.50* Stunted phenotype, wrinkled leaves

AS 3 4.5 25.3 0.30* 9.3 0.30* 3.5 0.50 68.3 0.30* Stunted phenotype, wrinkled leaves

AS 4 4.5 32.0 0.50 11.8 0.30 4.5 0.50 75.0 0.50*

Normal phenotypeAS 9 2.5 17.3 0.30* 7.5 0.50* 2.5 0.50* 37.0 0.50* Dwarfed phenotype, wrinkled foliage

AS 12 4.5 30.0 0.30 11.5 0.50 4.0 0.20 65.5 0.50*

Normal, partially wrinkled foliage

AS 11 2.5 15.5 0.50* 6.5 0.50* 2.5 0.50* 35.5 0.50* Dwarfed phenotype, wrinkled foliage

AS 17 4 18.5 0.50* 6.0 0.20* 3.0 0.30* 52.0 0.50* Stunted phenotype, wrinkled leaves

S 2 4 30.0 0.40 11.0 0.60 4.8 0.80* 74.0 0.50* Normal phenotype, straight stem

S 4 2 15.5 0.50* 5.6 0.20* 2.5 0.50* 28.5 0.50* Dwarfed phenotype, wrinkled foliage

S 5 4 36.5 0.50* 16.0 0.30* 5.0 0.30* 72.5 0.50 Robust phenotype, expanded foliage

S 6 4 33.0 0.50 12.6 0.60* 3.7 0.40 78.0 0.50* Normal phenotype, straight stem

S 9 4 37.5 0.50* 13.2 0.20* 4.5 0.60 72.5 0.50 Normal phenotype, straight stem

S 27 4 34.5 0.40 13.5 0.50* 4.2 0.20 70.0 0.50* Normal phenotype, straight stem

Significantly different values are represented by asterisk (*) and the mean values are significantly different at P B 0.05

Fig. 5 Pattern of xylem colouration in CCR down-regulated trans-

genic line AS17 in comparison with control

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compared to that of control plants. Phloem fibres were thin

walled and appeared as single to group of 23 cells unlike

the controls and up-regulated plants in which fibres were

thick walled and appeared as group of 35 cells. Consid-

erable reduction in the frequency of vessels in xylem was

seen in CCR down-regulated plants, compared to controls.

Interestingly, wider vessel elements with slight decreased

density were noticed in CCR up-regulated plants. Thechange in vessel dimensions can be correlated with the

lignin. If the lignin content is decreased, vessels can

expand and elongate much because of more cellulose.

Length and width of the vessel elements are also inversely

correlated. These changes in the vessel dimensions clearly

indicate the decrease in lignin content (Table 2). Lignin

modulation resulted in a reduction of secondary xylem

production. The mean vessel density changed significantly

in CCR up-regulated and down-regulated plants in

comparison with untransformed controls (14 3.2,

22 3.4 and 29 3.4 per 5 mm2 area, respectively). In

both untransformed and CCR up-regulated plants, vessel

distribution was prominent from xylem that is nearer to

cambium till pith region whereas in CCR down-regulated

plants, few vessels were found adjacent to cambium but

most of the vessels were observed in close proximity to

pith. The vessel element dimensions were measured frommacerated xylem tissues. The length of the vessel elements

increased in CCR down-regulated plants. The mean lengths

of vessel element in the control, CCR up-regulated and

down-regulated plants were 344 53, 472 92and

433 96 lm, respectively. The width of vessel elements

was reduced in CCR up and down-regulated plants com-

pared to that of untransformed control plants. The radial

extent of secondary xylem, which is the source of active

lignification, was significantly reduced in the CCR

Fig. 6 Histochemical and autofluorescence analysis of lignin in CCR

sense and antisense transgenic tobacco plants. Phloroglucinol staining

of transverse stem section of control (a), CCR down-regulated line

AS17 (b) and CCR up-regulated line S5 (c). Maules staining of

transverse stem section of control (d), CCR down-regulated line AS17

(e) and CCR up-regulated line S5 (f). Autofluorescence analysis of

transverse stem section of control (g), CCR down-regulated line AS17

(h) and CCR up-regulated line S5 (i)

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down-regulated plants in contrast to that of control and

CCR up-regulated plants. The anatomical changes associ-

ated with lignin modulation are shown in Table 2. The fibre

wall thickness showed variation among lignin-modulated

plants compared to that of untransformed controls. CCR

down-regulated plants exhibited thin walled xylem fibres

compared to untransformed and CCR up-regulated plants.

The vessels were mostly round to angular in shape in bothcontrol and CCR up-regulated plants, whereas deformed

vessels were observed in CCR down-regulated plants

(Fig. 8df). CCR down-regulated plants showed elongated

and wider fibres compared to that of up-regulated plants.

Unlike that of untransformed controls, the fibre length

increased in CCR up-regulated plants but the fibre width

showed a corresponding reduction also. The mean values

of length and width of fibres in untransformed control,

CCR up-regulated and down-regulated plants were

819 88, 907 87, 998 114 lm and 23 3.4,

24 4.7 and 24 3.0 lm, respectively (Table 2).

Transmission electron microscopy studies of stem

sections of CCR transgenics

General contrasting of polysaccharides in different mor-

phological regions of fibre cell walls was carried out by

periodate oxidation-thiocarbohydrazidesilver proteinate

method (PATAg) (Thiery 1969). The three sub layers of

secondary wall (S1, S2 and S3) in fibres were visualized

clearly by enhanced reactivity to PATAg staining. The

middle lamellae and S2 layers of fibres were highly reac-

tive to peroxidate oxidation as shown by their electron

dense staining compared to less reactive S1 and S3 layers

(Fig. S4 A). The CCR down-regulated plants were char-

acterized by an increase in proportion of secondary wall

layers particularly S2 layer of the secondary wall (Fig. S4

B). On the other hand, CCR up-regulated plants showed

decrease in proportion of secondary wall layers compared

to that of control plants. The topochemical distribution of

lignin in different morphological regions of fibre cell wall

was carried out using KMnO4 method (Donaldson 1992).

In untransformed plants, lignin distribution was high in the

cell corner middle lamellae, S1 and S3 layers of secondary

wall while S2 layer showed moderate staining intensity(Fig. S4 C). Though CCR down-regulated plants showed an

increase in secondary wall layer thickness, lignin distri-

bution was limited to middle lamellae and S1 layer,

whereas, S2 and S3 layer showed electron translucent areas

indicating extensive decrease in lignin content in these wall

layers (Fig. S4 D) On the other hand, fibres in the CCR

up-regulated plants showed a similar pattern of lignin

distribution in middle lamella, S1 and S2 layers while S3

layer showed intense staining indicating more lignin con-

tent in this layer (Fig. S4 E) compared to that of controls.

HPLC analyses of soluble phenolics

The CCR down-regulated lines showed enhanced accu-

mulation of ferulic acid as compared to controls and up-

regulated lines indicating a redirection of the carbon flux

through the phenylpropanoid pathway towards ferulic acid.

Such an increased levels of cell wall-linked ferulic acid

have earlier been reported in CCR-deficient Arabidopsis

thaliana (Goujon et al. 2003; Mir Derikvand et al. 2008),

tobacco (Piquemal et al. 1998; Chabannes et al. 2001b) and

poplar (Leple et al. 2007). In contrast to these CCR-defi-

cient dicots CCR1 maize mutant Zmccr1- did not release

higher amounts of ferulic acid when subjected to alkaline

hydrolysis (Tamasloukht et al. 2011). Syringic, p-coumaric

and sinapic acids were considerably higher in down-regu-

lated transgenics with a decrease in vanillin content. The

Fig. 7 Pattern of secondary

xylem in CCR sense and

antisense transgenic tobacco

plants. a Control, b AS17 and

c S5. The amount of secondary

xylem was reduced in CCR

down-regulated plants as

compared to control and

up-regulated plants.

Magnification 94. Scale bar 50

micron

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severely down-regulated line AS17 showed 74-folds

increase in ferulic acid, 1.5-folds increase in sinapic acid

and 12.9-folds increase in syringic acid accumulation

compared to the controls. But CCR up-regulated plants

recorded marginal increase in syringic, ferulic, p-coumaric

acids and vanillin as compared to controls. However, S5

line showed nine fold increased accumulation of sinapic

acid (Table 3).

Altered lignin and holocellulose contents inCCR down-regulated transgenics

To analyze whether CCR down-regulation reduces lignin

content, as indicated by phloroglucinolHCl and Maule

staining, stem tissues of CCR sense, antisense transgenic

and untransformed control tobacco plants were subjected to

Klason lignin analysis. The lignin content was measured

from cell wall residue (CWR) obtained by solvent extrac-

tion of the powdered xylem tissues. In the control, acid-

insoluble lignin constituted approximately 22% of total cell

wall residues, while the reduction in lignin content in

severely down-regulated line AS17 was 24.7% (Table 4),

whereas it was 22.5 and 17.8% in AS2 and AS3 lines

respectively compared to that of control. In contrast, the

line AS4 with normal phenotype showed 6.71% decrease in

lignin content compared to control. The lignin content of

over-expressed CCR transgenics was similar to that of

controls except in S5 and S2 lines which displayed a

maximum increase of 15.6 and 7.55%, respectively, dem-onstrating a significant increase in lignin content in these

transgenics (Table 4). These results thus show a positive

correlation between CCR activity and overall lignin con-

tent in the plants. The CCR antisense lignin deficient

transgenic lines exhibited 515% increase in holocellulose

content. The increase in holocellulose content in AS17 line

was 15.53% (Table 4), while the CCR sense transgenic

plants showed marginal increase of 1.52.5% in holocel-

lulose content in comparison with that of controls.

Fig. 8 Anatomical changes associated with CCR up and down-regulation in transgenic tobacco plants. Toulidine O Blue staining of transverse

stem sections of control (a, d) CCR antisense (b, e) and sense (c, f) transgenic tobacco plants. Magnification 910 and 940. Scale bar50 micron

Table 2 Anatomical

observations of CCR sense and

antisense transgenic lines

Significantly different values

are represented by asterisk (*)

and the mean values are

significantly different at

P B 0.05

Anatomical observation Control Antisense line Sense line

Vessel element length (lm) 344 50 433 96 472 92

Vessel element width (lm) 85 14 77 9.0 76 22

Vessel element density per 5 mm2 area 29 3.4 22 3.4* 14 3.2*

Fibre length (lm) 819 88 998 114 907 87

Fibre width (lm) 23 3.4 24 3.0 24 4.7

Fibre wall thickness (lm) 4.9 0.45 3.9 0.3* 5 0.5

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Discussion

High homology of both nucleotide and amino acid

sequences of Leucaena CCR with other known sequences

indicate that the sequences are highly conserved among

different families as also suggested by OMalley et al.(1992). The homology is higher with dicots compared to

monocots. Leucaena CCR amino acid sequence showed

high homology with other pulpwood yielding tree species

like Eucalyptus gunnii and Populus trichocarpa.

LlCCR down-regulation affected plant growth

and morphology

LlCCR down-regulation affected general growth and

development of the transgenic tobacco plants. The antisense

lines grew to two-thirds the size of the control plants. These

plants exhibited stunted, wrinkled, curled leaves with clearvenation and paler photosynthetic tissue compared to that of

control plants. Weak stem with decreased thickness and

internodal length was observed. These phenotypic modifi-

cations observed are apparently a direct consequence of the

significant lignin depletion in the cell walls as was also

pointed by Piquemal et al. (1998). Cell wall strength is

altered as shown by a decrease in stem mechanical strength.

On the other hand, the possibility that some of the pheno-

typic alterations observed in severe CCR down-regulated

lines may be due to a decrease in low molecular weight

phenolics derived from monolignols such as dehydroco-

niferyl glucosides cannot be excluded. Such glucosides

have been shown to be involved in signal transduction of

cytokinin-mediated cell division (Teutonico et al. 1991).

However, some of the CCR down-regulated lines like AS4showed normal phenotype like that of untransformed plants.

This might be due to the differential expression of antisense

genes which depend on the position of transgene insertion in

the genome (Van der Krol et al. 1988). The efficiency of the

antisense effect may also depend on the transcriptional

regulation of the endogenous gene (Atanassova et al. 1995).

Moreover, some of the CCR down-regulated plants showed

delayed flowering and biomass reduction while in other

cases there was no such difference in phenotype. Now, it is

not known if the altered flowering is linked to the reduction

in lignin content or to other induced series of metabolic

changes including phenolic compounds that occur in theseplants. Most of the CCR up-regulated plants exhibited

normal growth and phenotype like that of untransformed

controls, while line S5 showed robust growth with expan-

ded leaves, strong stem with increased internodal length and

high biomass. In the context of growth effects associated

with the suppression of CCR gene, wall-bound phenolics

present at elevated levels in the transgenic lines reflect

changes in the metabolic flow of hydroxycinnamic

acids. The hydroxycinnamic acids were found to exhibit

Table 3 HPLC analysis of low molecular weight phenolics in CCR sense and antisense tobacco lines

Sample Syringic acid Vanillin p-Coumaric acid Ferulic acid Sinapic acid

Control 0.164 0.13 0.115 0.06 0.185 0.17 0.102 0.02 0.665 0.08

Antisense (AS 17) 2.180 0.09* 0.050 0.03* 1.012 0.06* 7.447 0.32* 1.193 0.33*

Sense (S 5) 0.1935 0.02 0.177 0.09 0.401 0.02* 0.385 0.01 5.676 0.254*

The concentration of phenolic compounds is expressed in lg/mg

Significantly different values are represented by asterisk (*) and the mean values are significantly different at P B 0.05

Table 4 Lignin and

holocellulose analysis in mature

stems of CCR sense and

antisense transgenic lines

Significantly different values

are represented by asterisk (*)

and the mean values are

significantly different at

P B 0.05

Sample Klason lignin (%) % Increase or

decrease in lignin

Holocellulose (%) % Increase in

holocellulose

Control 22.02 0.32 61.62 0.69

AS 2 17.69 0.19* 22.5 69.31 0.49* 12.47

AS 3 18.10 0.15* 17.8 67.61 0.32 9.75

AS 4 20.56 0.16 6.71 64.42 0.28* 4.54

AS 12 19.07 0.12 13.4 67.31 0.22 9.23

AS 17 16.58 0.23* 24.7 71.19 0.23* 15.53

S 2 23.71 0.25 ?7.55 63.23 0.18 2.56S 5 25.54 0.13* ?15.6 62.98 0.45 2.15

S 6 22.31 0.19 ?1.22 64.42 0.32* 4.54

S 27 23.13 0.22 ?4.95 62.82 0.22*

1.94

S 9 22.32 0.15 ?1.27 62.61 0.49* 1.60

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growth-stimulating (Lee and Skoog 1965) and auxin-pro-

tecting activities (Nitsch and Nitsch 1962; Zenk and Muller

1963). Further, CCR may play a role in modulating the

phenylpropanoid metabolism leading to the synthesis of

flavonoids (Hahlbrock and Grisebach 1979) which through

their role as potential auxin transport regulators (Shirley

1996) may affect growth (Ruegger et al. 1997). During

growth and development of woody plants, compensatoryregulation of lignin and cellulose was suggested to be

associated with radial auxin gradient. The lignin biosyn-

thesis, carbohydrate levels and altered phenylpropanoid flux

may be involved in the growth enhancement as observed in

CCR up-regulated plants. The spatial and temporal control

of lignification is critical in plant support, water transport

and disease resistance. In the present study, it was observed

that the CCR down-regulated plants were more prone to

pest attack especially to lepidopteron larval forms in com-

parison with control and CCR up-regulated plants. This

could be because plants with compromised abilities to

synthesise normal quantities of lignin lose their capacity tosupport the plant body (Zhong et al. 1997; Jones et al. 2001)

and to defend themselves against pathogens (Franke et al.

2002a). The impact of lignin pathway disruption can have

undesirable phenotypes such as reduced yield, sterility,

greater drought susceptibility, lodging, and reduced resis-

tance to insect pests and microbial pathogens (Pedersen

et al. 2005). Reports of these consequences in CCR deficient

plants are scarce. However, increased susceptibility of

transgenic plants deficient for lignin biosynthetic pathway

genes to microbial pathogens has been reported. Suscepti-

bility toward the fungal pathogen Cercospora nicotianae

was reported for tobacco repressed in PAL expression

(Maher et al. 1994). Sorghum bmr-6CAD mutants showed

altered colonization by Fusarium ssp (Funnel et al. 2006).

CCR down-regulated plants displayed orange-brown

colouration of the xylem with collapsed vessels and thin

walled fibres

In transgenic tobacco, reduction in CCR activity resulted in

orange-brown colouration of the xylem. This indicates

major changes in cell wall composition. The presence of

unusual phenolics (such as ferulic and sinapic acids) in the

cell wall may account for this color, because semi-in vivo

incorporation of these two hydroxycinnamic acids into

stem sections resulted in a comparable phenotype (Pique-

mal et al. 1998; Leple et al. 2007). The coloration observed

in CCR down-regulated plants is reported to be different

from that observed in CAD or O-methyl transferase (OMT)

suppressed plants which were due to incorporation of

conjugated cinnamaldehydes into lignin polymer (Van

Doorsselaere et al. 1995). Stunted growth and collapsed

vasculature were noticed in 4CLdown-regulated transgenic

poplar (Kitin et al. 2010; Voelker et al. 2010). Such a

phenomenon was recorded in the present study also. The

reduced vessel density, presence of contorted and collapsed

vessels in CCR down-regulated plants is probably due to

insufficient lignification to withstand the tension generated

during transpiration. Reduced fibre wall thickness in CCR

down-regulated plants may be primarily due to reduction in

lignin deposition on the secondary wall of fibres. The pri-marily selective decrease in lignification of fibre wall could

be a compensatory mechanism to maintain the structural

integrity of vessel. This in turn may affect plant growth and

development by decreasing water and solute transport

efficiency (Piquemal et al. 1998). A delay in lignification

and development has been reported for the CCR Arabid-

opsis mutants (Laskar et al. 2006; Mir Derikvand et al.

2008). These observations suggest that a substantial

decrease in lignin content (almost half of the normal con-

centration) is incompatible with normal development.

Voelker et al. (2011) reported a 2040% reduction in lignin

content in 4CL down-regulated transgenic poplar wasassociated with increased vulnerability to embolism, shoot

dieback and mortality. Transgenic event AS17 also dis-

played xylem vulnerability to embolism and reduced

growth (biomass as well as leaf area). Vessels were also

irregular in this event. In contrast to collapsed xylem

phenotype observed in CCR down-regulated angiosperm

species, xylem vessels of maize CCR 1 mutant Zmccr1-

stained red with phloroglucinol and showed correctly

formed xylem vessels (Tamasloukht et al. 2011). The

above results underscore the need for adequate lignification

for mechanical support of the stem, water transport, growth

and survival of the plant.

CCR down-regulation modified ultrastructure

and topochemical distribution of polysaccharides

and lignin of cell wall

Structural and histochemical studies are important in lignin

genetic engineering experiments to clarify the spatial effect

of structural modification of lignin following transforma-

tion (Chabannes et al. 2001). The present study reveals that

the ultrastructure and topochemical distribution of poly-

saccharides and lignin in the fibre walls have been exten-

sively modified following up and down-regulation ofCCR

gene. The proportion of S2 layer of secondary wall in CCR

down-regulated plants have been increased and shows high

reactivity to PATAg and low reactivity to KMnO4 staining

indicating an increase and decrease in polysaccharides and

lignin, respectively. The increase in total cellulose content

may be a result of such modification in the fibre wall.

Important alternations in the fibre cell walls ofCCR down-

regulated plants have already been reported. In Arabidopsis

and tobacco, down-regulation ofCCR resulted in loosening

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of cellulose microfibrils leading to the reduced cell wall

cohesion (Goujon et al. 2003; Pincon et al. 2001).

Chabannes et al. (2001a) showed that the reduction of

lignin deposition in tobacco was not uniform in the cell

wall but that the S2 and S3 layers of the fibres and vessels

were mainly influenced. Similarly, lignin deposition was

mainly affected in the inner S2 layer of fibres in CCR

down-regulated Arabidopsis plants (Goujon et al. 2003).Our study also showed a drastic decrease in lignin distri-

bution in S2 and S3 layer of fibre secondary wall. While,

the CCR up-regulated plants showed an increase in lignin

distribution in secondary wall layers particularly in S3

layer. Being the innermost layer, S3 layer forms the part of

wood cell wall which is first to come in contact with wood

protecting and modifying chemicals such as preservatives,

pulping agents and microbial enzymes (Singh et al. 2002).

Therefore, S3 layer is an important part of the wood cell

walls from the point of wood processing and utilization.

Hence, the observed increase in proportion of secondary

wall layers rich in polysaccharides and reduction in lignindistribution in inner wall layers in the CCR down-regulated

plants are important structural and topochemical modifi-

cations in the fibre walls for the wood technologist from the

point of its utility for paper and pulp industry.

CCR down-regulation alters the phenolic profile

Changes in one branch of the phenolic metabolism path-

way can affect the metabolic flux in other pathways and it

is known that the phenyl propanoid pathway generates a

variety of plant compounds that are important for the

adaptation and survival of plants. The HPLC analysis of

soluble phenolics of CCR down-regulated plants revealed

the accumulation ferulic, sinapic, p-coumaric and syringic

acids as compared to up-regulated and untransformed

plants. According to Ralph et al. (2008), the lignins of CCR

deficient poplar, tobacco and Arabidopsis contained higher

amounts of G-CHR-CHR2 (R = SEt), a compound that

originates from the increased incorporation of ferulic acid

by bis-b-O-4 ethers. These results are in agreement with

earlier reports of Piquemal et al. (1998), Chabannes et al.

(2001), Leple et al. (2007) and Mir Derikvand et al. (2008).

These changes might, in certain cases, have an impact on

developmental programs since phenolic compounds for a

long time are known to interfere with the metabolism and

function of plant growth substances and secondary plant

products also (Chabannes et al. 2001b). Feruloyl-CoA, the

substrate for CCR, might be hydrolyzed to ferulic acid

resulting in its accumulation in CCR down-regulated

poplar plants as opined by Leple et al. (2007). Increased

incorporation of ferulic acid in the lignin polymer as

suggested by the higher recovery of the thioacidolysis

monomer G-CHR-CHR2 (R = SEt), signature of ferulic

acid-incorporation in lignin was reported in Arabidopsis

mutants deficient for cinnamoyl CoA reductase 1 (Mir

Derikvand et al. 2008). This finding suggests that CCR1

absence favours an increased transfer of ferulic acid to cell

wall polysaccharides. Subsequently, ferulic acid would be

converted to sinapic acid (Meyermans et al. 2000).

Reduced CCR activity resulted in reduced levels of co-

niferaldehyde and 5-hydroxyconiferaldehyde which mightfacilitate the conversion of ferulic acid to sinapic acid. Our

data indicate that down-regulation of CCR results in a

decreased flux of feruloyl-CoA to lignin and an increased

flux towards ferulic acid, which could be either detoxified

by glucosylation or alternatively exported to the cell wall

where it is cross-coupled with lignin (Leple et al. 2007).

Further, the evidence presented by Ralph et al. (2008)

suggest that ferulic acid is a previously unrecognized

monomer in lignification, being incorporated at low levels

in various types of normal plants and at elevated levels in

various CCR deficient transgenics. Significantly, ferulic

acid incorporation also provides a new mechanism bywhich branch points can occur in the lignin polymer. Thus,

the down-regulation ofCCR gene significantly affected the

quantity of soluble phenolics pointing the redirection of the

carbon flux through the phenylpropanoid pathway towards

these phenolics. Furthermore, specific suppression ofCCR

induced a new carbon partitioning between lignins and

other phenolic carbon sinks and could be exploited in the

future for metabolic engineering experiments aimed at

optimizing soluble phenolic profiles of plants for applied

purposes (Dixon et al. 1996).

CCR down-regulation alters quality and

quantity of lignin

Histochemical analysis by Wiesner reaction, Maules

staining and autofluorescence revealed altered lignification

in CCR down-regulated lines. Wiesner reaction (phloro-

glucinol-HCl), which specifically stains the cinnamalde-

hyde end groups (Adler et al. 1948), exhibited feeble

staining in the down-regulated plants and intense red

colouration in CCR up-regulated plants indicating low and

high levels of lignin content in the corresponding cell

walls. In contrast, xylem vessels of Zmccr1- were not

affected by the CCR mutation (Tamasloukht et al. 2011).

The reduced levels of cinnamaldehyde in CCR down-reg-

ulated plants could have contributed to feeble staining of

the cell wall compared to untransformed plants and CCR

up-regulated plants. Down-regulation of CCR resulted in

very low lignin autofluorescence induced by long wave-

length UV excitation, compared to control and CCR

up-regulated lines indicating reduction of overall lignin

content. Maules staining which is considered to stain spe-

cifically syringyl (S) units in lignin revealed high proportion

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of S units and reduced G units. Together, these data suggest

that CCR down-regulation reduce the level of hydroxyl

cinnamaldehydes but increase S units in lignin. These

results are in agreement with earlier reports on thioacidol-

ysis analysis in transgenic tobacco (Piquemal et al. 1998)

and poplar (Leple et al. 2007) which revealed an increase in

S/G ratio. Such an increased S/G ratio in the wood will

increase lignin extractability during Kraft pulping. Thispoints out that the CCR cDNA used in the present study may

code for an isomer which specifically controls the synthesis

of guaiacyl (G) units of lignin in L. leucocephala.

In the present study, one of the severely down-regulated

CCR transgenic lines AS17 which recorded approximately

74.6% reduction of CCR activity, also exhibited a signifi-

cant decrease of 24.7% in Klason lignin content compared

to that of untransformed controls. Likewise, other CCR

down-regulated lines also exhibited reduction in lignin

content. These results are in agreement with earlier reports

which revealed significant reduction of Klason lignin in

tobacco (Piquemal et al. 1998; Ralph et al. 1998; OCon-nell et al. 2002) and Arabidopsis (Goujon et al. 2003),

while 20% reduction in poplar (Leple et al. 2007). On the

other hand, lignin content of over-expressed CCR trans-

genics S5 and S2 displayed significant increase in lignin

quantity due to overexpression of the CCR gene. Taken

together, the results obtained by altering the expression of

CCR gene point out that this gene controls the quantity of

lignin produced in L. leucocephala. Thus, a positive

correlation between reduction in CCR activity and overall

lignin content in the transgenic tobacco plants can be

drawn corroborating with histochemical analysis and

autofluorescence studies.

Transgenic modifications that reduce lignin content

should result in greater carbon availability for primary

metabolism and growth. Proportional increase in cellulose

content associated with reduced lignin has been found in

transgenic tobacco (Chabannes et al. 2001a) and transgenic

poplar (Voelker et al.2010). We noticed such a phenomenon

in the present study also. The CCR down-regulated trans-

genic lines exhibited a 515% increase in holocellulose

content. Though there is a decrease in over all biomass of the

plants, increase in cellulose may be compensating the

reduction of lignin indicating the compensatory regulation

of deposition of these two structural cell wall components as

pointed byHu etal.(1999). On the other hand, the CCR sense

transgenic plants showed marginal increase of 1.52.5% in

holocellulose content in comparison with that of control.

Attempts of modifying the lignin content in the plant by

over- or under-expression of lignin biosynthetic pathway

genes like CCR under CaMV 35S, a constitutive promoter

are likely to cause significant changes in the overall plant

growth and development due to pleiotropic effect. This

phenomenon could have biased the results obtained in the

present study. However, usage of xylem specific promoter

likely will help in overcoming the risks and challenges

associated with up or down-regulating lignin content across

the whole plant.

CCR down-regulated transgenic lines with moderate

reduction in lignin and considerable increase in holocel-

lulose contents displaying normal phenotype could prove

beneficial for the paper industry because of high proportionof S units in these lines, which facilitates easier delignifi-

cation in Kraft pulping (Chiang et al. 1988; OConnell

et al. 2002). While CCR up-regulated plants with increased

lignin content and overall biomass could find potential

application in bioenergy industry. This study suggests a

potential way to increase or reduce lignin content in

L. leucocephala through genetic manipulation by up or

down-regulation of CCR gene, a potential control point in

lignin biosynthetic pathway.

Acknowledgments This work was supported by a grant from the

Council of Scientific and Industrial Research (CSIR-NMITLI), NewDelhi, India and we gratefully acknowledge the financial assistance.

We are also thankful to the University Grants Commission, New

Delhi for financial assistance in the form of funds for Centre for

Advanced Studies to the Department of Genetics, Osmania

University.

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