Biomass derived from transgenic tobacco expressing the Arabidopsis CESA3ixr1-2 gene exhibits...

Acta Biologica Hungarica 65(2), pp. 189–204 (2014)DOI: 10.1556/ABiol.65.2014.2.7

0236-5383/$ 20.00 © 2014 Akadémiai Kiadó, Budapest

Biomass derived from transgenic toBacco expressing tHe ArABIDOpsIs CEsA3Ixr1–2 gene

exHiBits improved saccHarificationDipak kumar Sahoo * and Indu B. MaItI

Ktrdc, college of agriculture, food and environment, University of Kentucky, Lexington, KY 40546-0236, Usa

(received: July 3, 2013; accepted: november 21, 2013)

studies in Arabidopsis thaliana and Nicotiana tabacum L. variety samsun nn demonstrated that expres-sion of the CEsA3 cellulose synthase gene that contains a point mutation, named ixr1–2, results in greater conversion of plant-derived cellulose to fermentable sugars. the present study was designed to examine the improved enzymatic saccharification efficiency of lignocellulosic biomass of tobacco plants express-ing AtCEsA3ixr1–2. three-month-old AtCEsA3ixr1–2 transgenic and wild-type tobacco plants (Nicotiana tabacum L. variety samsun nn) were grown in the presence and absence of isoxaben. Biomass obtained from leaf, stem, and root tissues were analyzed for enzymatic saccharification rates. During enzymatic saccharification, 45% and 25% more sugar was released from transgenic leaf and stem samples, respec-tively, when compared to the wild-type samples. This gain in saccharification efficiency was achieved without chemical or heat pretreatment. additionally, leaf and stem biomass from transgenic AtCEsA3ixr1–2 requires a reduced amount of enzyme for saccharification compared to biomass from wild-type plants. from a practical standpoint, a similar strategy could be employed to introduce the mutated cesa into energy crops like poplar and switchgrass to improve the efficiency of biomass conversion.

Keywords: Nicotiana tabacum – biofuel – biomass – AtCEsA3ixr1–2

introdUction

Biofuels have arisen as a potential alternative, renewable source of bio-energy main-ly because of increasing demand for energy and the environmental problems associ-ated with petroleum consumption. Lignocellulosic biomass derived from plant cell walls has an advantage as a biofuel feedstock due to its abundance and high rates of production. as part of the Us energy independence and security act (eisa) of 2007, the renewable fuel standard mandates that 36 billion gallons of biofuels are to be produced annually by 2022, of which 16 billion gallons are expected to come from cellulosic feed stocks (U.s. department of energy genomic science program; http://genomicscience.energy.gov). the greatest barrier to using lignocellulosic biomass as an industrial feedstock is its indigestibility due to the recalcitrant nature of plant cell walls [18, 27]. the heterogeneous nature of biomass particles, accessible surface

* Corresponding author; e-mail address: [email protected]; [email protected]

190 dIpak kuMar Sahoo and Indu B. MaItI

Acta Biologica Hungarica 65, 2014

area, crystallinity of the cellulose, its protection by lignin, and sheathing by hemicel-lulose all contribute to the recalcitrance of lignocellulosic biomass to hydrolysis [19]. cellulose, the most abundant biopolymer on earth, comprises roughly one-third of the total biomass of many plants [30]. In general, it represents 10–14% of the dry weight of primary cell walls, 40–60% of secondary cell walls, and up to 98% of the dry weight of specialized cells, such as cotton fibers [13].

natural enzymatic digestion of plant matter is a slow and complex process, which must be simplified for it to be viable for commercial biofuel production. The rela-tively poor accessibility of cellulose to the enzyme cellulase is due to the recalcitrant nature of the cell wall as a result of strong associations between plant cell wall com-ponents. cellulose can be hydrolytically broken down into glucose either enzymati-cally by cellulase or chemically by sulfuric or other acids. the processing of lignocel-lulosic biomass to ethanol consists of four major steps: pretreatment, hydrolysis, fer-mentation, and product separation/purification. Pretreatment is required to break the lignin seal, disrupt the crystalline structure of cellulose, and to alter the structure of cellulosic biomass, making cellulose more accessible to the hydrolyzing enzymes. pretreatment is considered as one of the most expensive processing steps in cellulosic biomass-to-fermentable sugars conversion, with costs as high as 30¢/gallon ethanol produced [19]. increasing overall sugar production and decreasing processing expens-es can be accomplished by (a) improving pretreatment [19, 25, 26, 34], (b) enhancing cellulase performance [12, 18, 35], (c) recycling cellulase as well as decreasing enzyme production costs by producing cellulase enzymes within the crop biomass [14, 17, 31, 32, 36], (d) improving the surface area accessible to cellulase for achieving a high sugar yield [22], and (e) producing less recalcitrant bioenergy plants by regulating the lignin content through manipulation of different lignin biosynthetic pathway genes [6, 20]. this can also be done by increasing cellulose and decreasing lignin content by expressing an Arabidopsis transcription factor, sHine [1], or by modifying cellulose crystallinity by expressing a mutated cellulose synthase gene [10, 11].

it has been well documented in Arabidopsis thaliana that genetic modification of cellulose structure can be achieved by mutation of cellulose synthase (cesa) cata-lytic component like AtCEsA1 and AtCEsA3 [9, 11]. it has also been demonstrated in Arabidopsis that a point mutation in the CEsA3 cellulose synthase gene, called ixr1–2, results in genetic modification of cellulose microfibril structure, which leads to greater conversion of plant biomass to fermentable sugars [9, 11]. furthermore, it has been established that overexpressing GFp fused to the Arabidopsis CEsA3ixr1–2 gene (AtCEsA3ixr1–2) in transgenic tobacco makes the plants resistant to the herbicide isoxaben (100 nm). cellulose extracts from the leaf and stem of these transgenic tobacco plants exhibited a 50–60% increase in hydrolytic efficiency [24]. Because transgenic plants expressing GFp-AtCEsA3ixr1–2 exhibit a higher lignin content and also have irregular lignin deposition patterns [24], the crude biomass from these plants was tested to evaluate its suitability for biofuel production. Here we report that leaf and stem biomass from transgenic tobacco plants expressing GFp-AtCEsA3ixr1–2 during saccharification requires reduced amounts of enzymes, and this apparent gain in saccharification efficiency can be achieved without chemical or heat pretreatment.

atcesa3ixr1–2 tobacco biomass saccharification 191


materiaLs and metHods

Chemicals and enzymes

all chemicals and reagents used were of analytical grade or higher and were obtained from Sigma-Aldrich, FMC-BioPolymer, Fisher Scientific, Riedel den Haan, and BdH as applicable. restriction enzymes and dna modifying enzymes were pur-chased from invitrogen, Life technologies (Usa). nitrocellulose membranes for western blot analysis were obtained from schleicher & schuell (Keene, nH, Usa).

plant growth conditions

twelve independent transgenic plant lines expressing the GFp-AtCEsA3ixr1–2 gene fusion were analyzed. transgenic and wild-type tobacco plants were grown in the presence and absence of isoxaben for cellulose content estimation and saccharifica-tion studies as described earlier [24]. Briefly, transgenic GFp-AtCEsA3ixr1–2 seedlings (3 weeks old, homozygous lines) selected in the presence of kanamycin (250 µg/ml) and wild-type tobacco seedlings (Nicotiana tabacum cv. samsun nn) were exposed to 50 nm isoxaben for 1 week followed by 4 weeks in 20 nm isoxaben [24]. Both wild-type and transgenic plants were then transferred to the greenhouse and grown under standard conditions for four weeks without any isoxaben treatment.

GFp visualization by confocal laser scanning microscopy

fluorescence images of leaf cells of transgenic tobacco plants expressing GFp-AtCEsA3ixr1–2 and wild-type tobacco plants were captured with a confocal laser scan-ning microscope (tcs sp5; Leica microsystems cms gmbH, d-68165 mannheim, germany) using Las af (Leica application suite advanced fluorescence) 1.8.1 build 1390 software and the pL fLUotar objective (10.0x/n.a.0.3 drY) with confocal pinhole set at airy 1 and 1× zoom factor for improved resolution with eight bits as described earlier [3, 16]. An argon laser (30%) with AOTF for 488 nm (at 40%) was used for excitation of GFP [15, 23], and the fluorescence emissions were collected between 501 and 580 nm with the photomultiplier tube (pmt) detector gain set at 1150 v.

Cellulose content measurement

crude cell walls were prepared from leaf, stem, and root samples from both trans-genic and wild-type tobacco plants as described earlier by Reiter et al. [21]. Briefly, the leaf, stem and root tissue samples were homogenized using a scientific grinder (arthur H. thomas co., philadelphia, pa, Usa) equipped with a 1-mm sieve.



Twenty-five milligrams of ground plant material was incubated in 1 ml of 70% etha-nol overnight at 65 °C, and washed twice with 1 ml 70% ethanol for 1 h and once with 1 ml acetone for 5 min. the samples were dried under vacuum after removing the final wash solution. Cellulose content was determined according to the methods described by Updegraff [33]. in brief, 5 mg samples of the dried leaf, stem, and root extracts from either wild-type or transgenic plants were weighed out in five replicates and boiled in aceticnitric reagent (acetic acid:nitric acid:water, 8:1:2) for 30 min to remove lignin and hemicellulose. the samples were allowed to cool to room tem-perature and the reagents were carefully removed. the plant cell wall material was washed twice, first with 8 ml MQ-water and then with 4 ml acetone, and dried under vacuum. The cellulose samples were then hydrolyzed in 67% sulfuric acid for 1 h and the glucose content of the samples was determined by the anthrone method [29]. a 25 µl aliquot of each sulfuric acid hydrolyzed sample was mixed with 475 µl water and 1 ml 0.3% anthrone in concentrated sulfuric acid on ice. The samples were boiled for 5 min then immediately returned to ice. the absorbance of the samples was measured using a Uv-visible spectrophotometer (shimadzu, Usa) at 620 nm and compared with a standard curve obtained from known (10–50 mm) concentrations of glucose (a new standard curve was made for each set of reactions). the cellulose content was calculated by multiplying the measured glucose concentration of each sample by the total volume of the assay and then by the hydration correction factor (0.9) to correct for the water molecule added upon hydrolytic release of each glucose residue from the cellulose polymer.

Microscale enzymatic saccharification

Enzymatic saccharification of lignocellulosic material was carried out in accordance with the laboratory analytical procedure #009 (Lap-009) of the national renewable Energy Laboratory (NREL) with little modification [11]. Celluclast 1.5 L (cellulase from Trichoderma reesei) having an activity of 486 endoglucanase units/ml for cel-lulase [45.6 filter paper units (FPU) – as standardized in LAP-009] and Novozyme 188 (cellobiase from Aspergillus niger) with an activity of 134 cellobiase units (cBU) per ml were purchased from sigma-aldrich (st Louis, mo, Usa). the enzyme cock-tail was obtained by mixing equal volumes of the two enzymes. the cellulose content of transgenic and wild-type tobacco plants was measured spectrophotometrically (thermofischer Biomate3, madison, Wi, Usa) on homogeneous, ground samples of leaf, stem, and root tissue obtained from ten tobacco plants; the sugar release values are a percentage of this total. the total reaction volume of 2 ml contained dry biomass samples equivalent to 0.02 g cellulose with a range of enzyme concentrations of 60, 30, 15 and 7.5 fpU. the samples were positioned horizontally in an innova 4300 incubator/shaker (New Brunswick Scientific, New Brunswick, NJ, USA) at 50 °C while shaking at 100 rpm using a 1-inch orbit. Enzyme blanks and Whatman #1 filter paper were digested alongside the samples. the progress of the reaction was meas-



ured by taking representative 100 µl aliquots at 0, 3, 6, 12, 24, 48, 72 and 120 h and determining the glucose concentration using a Yellow spring instruments (Ysi)-glucose analyzer standardized for glucose determination using Ysi buffer and mem-branes purchased from YSI (Yellow Springs, OH, USA) [9]. In order to confirm that cellulose was being converted, the fermentable sugars released by enzymatic hydrol-ysis were analyzed by HPLC [11], which indicated that >90% of the sugar was glu-cose. Another similar microscale enzymatic saccharification experiment was con-ducted by taking dry biomass samples equivalent to 0.02 g cellulose from leaf, stems and root of wild-type and transgenic plants grown in the presence and absence of isoxaben with enzyme concentrations of 60 fpU.

resULts

GFp expression in transgenic plants

independent transgenic plant lines generated with the GFp-AtCEsA3ixr1–2 gene [24] were analyzed. examination of the GFp-AtCEsA3ixr1–2 protein load in transgenic plants was extrapolated via Western blot assay against the n-terminal-fused gfp, and for AtCEsA3ixr1–2 by using primary anti-GFp or anti-AtCEsA3ixr1–2 antibodies as reported earlier [24] (data not shown). in the present study, transgenic lines express-ing the GFp-AtCEsA3ixr1–2 fusion protein were further evaluated by visualizing gfp fluorescence; confocal microscopy showed the presence of GFP at the leaf cell boundaries (fig. 1).

Saccharification rates of biomass derived from transgenic tobacco plants expressing atcesa3ixr1–2 grown in the absence of isoxaben

Based on the cellulose content determined in leaf, stem, and root tissues for the wild-type and AtCEsA3ixr1–2 transgenic plants, identical cellulose loadings were analyzed for recalcitrance to saccharification after 3, 6, 24, 48, and 72 h of enzymatic digestion using a commercial cellulase and cellobiase cocktail (enzymes from Trichoderma reesei and Aspergillus niger, respectively). the results for each tissue of the AtCEsA3ixr1–2 transgenic and wild-type plants grown in the absence of isoxaben were compared based on the percentage of cellulose converted into fermentable sugars. About 93% of the sugar was released from transgenic leaf samples, while 48% was released from wild-type leaf biomass samples during enzymatic saccharification using an excess amount of enzyme (60 FPU) (Fig. 2A). For the stem samples, 68% of the sugar was released from transgenic and 42% was released from wild-type bio-mass samples during enzymatic saccharification with excess enzyme (60 FPU). This gain of 45% (in case of leaf biomass) 26% (in case of stem biomass) in saccharifica-tion efficiency was achieved without chemical or heat pretreatment (Fig. 2A and 2B).



Fig.

1. c

onfo

cal l

aser

scan

ning

mic

rosc

opy

of g

fp in

tran

sgen

ic to

bacc

o pl

ants

exp

ress

ing

GFp

-AtC

EsA3

irx1

–2. a

ccum

ulat

ion

of G

Fp-A

tCEs

A3ix

r1–2

at th

e ce

ll bo

unda

ries

was

vis

ualiz

ed b

y co

nfoc

al la

ser s

cann

ing

mic

rosc

ope

in le

af c

ells

of t

rans

geni

c pl

ants

(T);

no fl

uore

scen

ce w

as d

etec

ted

in w

ild-ty

pe (W

) pl

ants



Fig.

2.

Sacc

harifi

catio

n an

alys

is o

f bi

omas

s de

rived

fro

m t

rans

geni

c to

bacc

o pl

ants

exp

ress

ing

AtC

EsA3

ixr1

–2 g

row

n in

abs

ence

of

isox

aben

. Sa

ccha

rifica

tion

effic

ienc

y re

porte

d as

% o

f cel

lulo

se c

onve

rted

to g

luco

se a

t 3, 6

, 24,

48,

and

72

h fr

om le

af (A

), st

em (B

) and

root

(C) b

iom

ass o

f wild

-ty

pe (W

) and

tran

sgen

ic (t

) tob

acco

pla

nts

grow

n in

abs

ence

of i

soxa

ben

(-ix

o). d

ata

are

expr

esse

d as

mea

n ±

s.d

. of

5 ob

serv

atio

ns a

nd w

ere

sub-

ject

ed to

the

unpa

ired

stud

ent’s

t-te

st. S

tatis

tical

sig

nific

ance

was

acc

epte

d at

p <

0.05

. Sac

char

ifica

tion

anal

ysis

of l

eaf a

nd s

tem

bio

mas

s be

twee

n w

ild

(W) a

nd tr

ansg

enic

(T) p

lant

s w

ere

foun

d to

diff

er s

igni

fican

tly a

t p <

0.05

as

indi

cate

d by

ast

eris

ks. a

t lea

st s

even

inde

pend

ent t

rans

geni

c pl

ant l

ines

w

ere

gene

rate

d fo

r the

GFp

-AtC

EsA3

ixr1

–2 g

ene,

and

five

pla

nts

per l

ine

wer

e an

alyz

ed; d

ata

from

one

repr

esen

tativ

e lin

e is

sho

wn

in th

e fig

ure



approximately 1.5–2 times more sugar was released from stem biomass of trans-genic plants than from wild-type plants after only 3 h of incubation. for root biomass, no significant changes in saccharification rates were detected between transgenic and wild-type plants (fig. 2c).

Saccharification analysis of biomass derived from transgenic tobacco plants expressing atcesa3ixr1–2 grown in the presence of isoxaben

When both transgenic and wild-type plants were grown in the presence of isoxaben, 73% of the sugar was released from transgenic leaf samples, while 41% sugar was released from wild-type leaf biomass during enzymatic saccharification with excess enzyme (60 FPU) (Fig. 3A). Thus, ~32% more sugar was released from leaf biomass of transgenic plants than from wild-type plants. moreover, 3–3.5 times more sugar was released from transgenic leaf samples than wild-type samples within 24 h of enzymatic hydrolysis. In stem samples, 55% of the sugar was released from the trans-genic plants and 38% was released from wild-type plants during enzymatic sacchari-fication with an excess of enzyme (60 FPU) (Fig. 3B). This gain of 17% in sacchari-fication efficiency was achieved without chemical or heat pretreatment. Approximately 2.5 times more sugar was released from stem biomass of transgenic plants than from wild-type plants after only 3 hours of incubation. For root biomass, no significant changes in saccharification rates were detected between transgenic and wild-type plants (fig. 3c).

Saccharification analysis of leaf and stem biomass from transgenic tobacco plants expressing atcesa3ixr1–2 using different

enzyme concentrations

To gain a better understanding of the increased saccharification efficiency in the transgenic plants expressing AtCEsA3ixr1–2, a more detailed saccharification experi-ment was conducted to determine the kinetics of the reaction by measuring six time points from 3 to 72 h at four different enzyme concentrations ranging from 7.5 to 60 fpU.

the results indicated that at 48 h under conditions of excessive enzyme (60 fpU), the fermentable sugar released from leaf samples was >90% for the AtCEsA3ixr1–2 transgenic and ~48% for the wild-type plants (Fig. 4A). In addition, the AtCEsA3ixr1–2 transgenic leaf biomass released a greater percentage of sugar at each time point up to 48 hours during the reaction (fig. 4a), and more sugar was released by AtCEsA3ixr1–2 using the lowest enzyme concentration (7.5 fpU) than from the wild-type using a 4-fold higher enzyme concentration (30 fpU) (compare figs 4B and 4d). also, in the presence of excess enzyme (60 fpU), wild-type leaf biomass released ~48% of the fermentable sugar, while one-fourth this amount of enzyme



Fig.

3.

Sacc

harifi

catio

n an

alys

is o

f bi

omas

s de

rived

fro

m t

rans

geni

c to

bacc

o pl

ants

exp

ress

ing

AtC

EsA3

ixr1

–2 g

row

n in

pre

senc

e of

iso

xabe

n.

Sacc

harifi

catio

n ef

ficie

ncy

repo

rted

as %

of c

ellu

lose

con

verte

d to

glu

cose

at 3

, 6, 2

4, 4

8, a

nd 7

2 h

from

leaf

(A),

stem

(B) a

nd ro

ot (C

) bio

mas

s of

w

ild-ty

pe (W

) and

tran

sgen

ic (t

) tob

acco

pla

nts

grow

n in

pre

senc

e of

isox

aben

(+ix

o). d

ata

are

expr

esse

d as

mea

n ± s

.d. o

f 5 o

bser

vatio

ns a

nd w

ere

subj

ecte

d to

the

unpa

ired

stud

ent’s

t-te

st. S

tatis

tical

sig

nific

ance

was

acc

epte

d at

p <

0.05

. Sac

char

ifica

tion

anal

ysis

of

leaf

, ste

m, a

nd r

oot b

iom

ass

betw

een

wild

(W) a

nd tr

ansg

enic

(T) p

lant

s w

ere

foun

d to

diff

er s

igni

fican

tly a

t p <

0.05

as

indi

cate

d by

ast

eris

ks. a

t lea

st s

even

inde

pend

ent t

rans

-ge

nic

plan

t lin

es w

ere

gene

rate

d fo

r the

GFp

-AtC

EsA3

ixr1

–2 g

ene,

and

five

pla

nts p

er li

ne w

ere

anal

yzed

; dat

a fr

om o

ne re

pres

enta

tive

line

is sh

own

in

the

figur

e



Fig.

4. S

acch

arifi

catio

n ra

tes o

f bio

mas

s der

ived

from

leaf

of t

rans

geni

c to

bacc

o pl

ants

exp

ress

ing

AtC

EsA3

ixr1

–2 a

s a fu

nctio

n of

diff

eren

t enz

yme

conc

en-

tratio

ns. S

acch

arifi

catio

n ef

ficie

ncy

expr

esse

d as

% o

f cel

lulo

se c

onve

rted

to g

luco

se a

t 3, 6

, 12,

24,

48,

and

72

h fr

om le

af ti

ssue

s of

wild

-type

(W) a

nd

trans

geni

c (t

) pla

nts u

sing

60

(a),

30 (B

), 15

(c),

and

7.5

(d) f

pU o

f enz

yme.

dat

a ar

e ex

pres

sed

as m

ean ±

s.d

. of 5

obs

erva

tions

and

wer

e su

bjec

ted

to

the

unpa

ired

stud

ent’s

t-te

st. S

tatis

tical

sig

nific

ance

was

acc

epte

d at

p <

0.05

. Sac

char

ifica

tion

rate

s of

wild

(W

) an

d tra

nsge

nic

(T)

plan

ts w

ere

sign

ifi-

cant

ly d

iffer

ent a

t p <

0.05

as i

ndic

ated

by

aste

risks

. at l

east

seve

n in

depe

nden

t tra

nsge

nic

plan

t lin

es w

ere

gene

rate

d fo

r the

GFp

-AtC

EsA3

ixr1

–2 g

ene,

and

fiv

e pl

ants

per

line

wer

e an

alyz

ed; d

ata

from

one

repr

esen

tativ

e lin

e is

sho

wn

in th

e fig

ure



Fig.

5. S

acch

arifi

catio

n ra

tes

of s

tem

bio

mas

s of

tran

sgen

ic to

bacc

o pl

ants

exp

ress

ing

AtC

EsA3

ixr1

–2 a

s a

func

tion

of d

iffer

ent e

nzym

e co

ncen

tratio

ns.

Sacc

harifi

catio

n ef

ficie

ncy

repo

rted

as %

of c

ellu

lose

con

verte

d to

glu

cose

at 3

, 6, 1

2, 2

4, 4

8, a

nd 7

2 h

from

leaf

tiss

ues

of w

ild-ty

pe (W

) and

tran

sgen

ic

(t) p

lant

s usi

ng 6

0 (a

), 30

(B),

15 (c

), an

d 7.

5 (d

) fpU

of e

nzym

e. d

ata

are

expr

esse

d as

mea

n ± s

.d. o

f 5 o

bser

vatio

ns a

nd w

ere

subj

ecte

d to

the

unpa

ired

stud

ent’s

t-te

st. S

tatis

tical

sig

nific

ance

was

acc

epte

d at

p <

0.05

. Sac

char

ifica

tion

rate

s of

wild

(W) a

nd tr

ansg

enic

(T) p

lant

s w

ere

foun

d to

diff

er s

igni

fi-ca

ntly

at p

< 0.

05 a

s ind

icat

ed b

y as

teris

ks. a

t lea

st se

ven

inde

pend

ent t

rans

geni

c pl

ant l

ines

wer

e ge

nera

ted

for t

he G

Fp-A

tCEs

A3ix

r1–2

gene

, and

five

pla

nts

per l

ine

wer

e an

alyz

ed; d

ata

from

one

repr

esen

tativ

e lin

e is

sho

wn

in th

e fig

ure



(15 fpU) was required to release the same amount of sugar from the transgenic leaf biomass samples (fig. 4c).

these results indicated that at 72 h under conditions of excessive enzyme (60 FPU), the fermentable sugar released from stem samples was >65% for the AtCEsA3ixr1–2 transgenic and ~43% for the wild-type plants (Fig. 5A). In the presence of 30 FPU of the enzyme, wild-type stem biomass released ~17% of the sugar, whereas the same saccharification rate could be achieved in transgenic stem samples with only 25% as much enzyme (7.5 FPU) (Fig. 5B–D).

discUssion

it is possible to reduce the recalcitrance of plant cell walls to enzymatic hydrolysis by modifying the cellulose to make it more susceptible without any prior chemical or heat treatments. in Arabidopsis thaliana, the CEsA3ixr1–2 mutation results in greater conversion of plant biomass to fermentable sugar than in either wild-type or CEsA6ixr2,1 mutant plants [10, 11]. moreover, over-expression of the Arabidopsis CEsA3ixr1–2 gene (AtCEsA3ixr1–2) in tobacco (Nicotiana tabacum L. variety samsun nn) renders the transgenic plants resistant to the herbicide isoxaben [24]. enzymatic saccharification of the leaf- and stem-derived cellulose from the transgenic plants expressing GFp-AtCEsA3ixr1–2 is about 50% more efficient than in the untransformed wild-type plants [24]. on the other hand, over-expression of GFp-AtCEsA3ixr1–2 in tobacco causes aberrant spatial distribution of lignified secondary cell wall tissue, accompanied by a dramatic increase in acid-insoluble lignin content [24]. Hence, the saccharification of crude biomass needs to be studied for evaluation of its suitability for biofuel production. the GFp-AtCEsA3ixr1–2 transgenic plants expressed resistance to the cellulose biosynthesis inhibitory herbicide isoxaben, and exhibited a distinct prostrate to decumbent growth form as reported earlier [24]. furthermore, in the transgenic tobacco plants, the presence of the GFp-AtCEsA3ixr1–2 fusion protein was confirmed by the presence of GFP fluorescence in leaf cells (Fig. 1). Our result cor-roborated the earlier findings of Desprez et al. [8], who confirmed the presence of the gfp-cesa3 fusion protein in the cellulose synthase complex by transient expression studies in Nicotiana benthamiana. due to exposure to isoxaben, the growth of wild-type plants was inhibited, and they exhibited more root radial swelling compared to transgenic plants, as previously reported [24].

During enzymatic saccharification, approximately 45% and 25% more sugar was released from transgenic leaf and stem biomass samples, respectively, when com-pared to the wild-type samples. At a practical level, this gain in saccharification effi-ciency alone represents a remarkable improvement in bioconversion without addi-tional costly chemical or heat pretreatments [31]. although pretreatment of plant biomass with heat, physical force, and/or chemical catalysts renders the cellulose more available to enzymatic hydrolysis, it comes at an additional financial cost [4]. Furthermore, byproducts of the harsh pretreatments necessary to facilitate saccharifi-cation can inhibit the growth of microorganisms used to produce biofuels. the pro-



cess of saccharification of leaf and stem biomass from transgenic plants grown in the presence of isoxaben released 40% and 13% less sugars, respectively, compared to the transgenic plants grown in the absence of isoxaben (figs 2 and 3). this might be due to the increased lignin content in isoxaben-exposed plants [24]. several factors are known to alter the digestibility of cellulose, including lignification [5] and crystal-linity [9, 11]. regardless of the abnormal lignin accumulation observed in transgenic plants that was reported earlier [24], biomass from these plants had a higher level of saccharification than did biomass from wild-type plants (compare Fig. 2 to Fig. 3).

The increased rate of saccharification in tissues from the transgenic plants clearly indicates altered cellulose structure and crystallinity [9, 11]. the higher rate of sac-charification found in this study also corroborates prior results in the Arabidopsis CEsA3ixr1–2 mutant. It is evident from previous studies that cellulose microfibrils from the Arabidopsis CEsA3ixr1–2 mutant displayed inherent structural changes, hav-ing a more amorphous and mobile cellulose-like structure. moreover, the CEsA3ixr1–2 mutation displayed higher relative cesa velocity and reduced crystallinity [9, 11]. Hence, the expression of AtCEsA3ixr1–2 in tobacco results in the formation of aberrant cellulose with reduced crystallinity due to integration of the mutant AtCEsA3ixr1–2 subunit into the tobacco cellulose synthase complex (csc). it was also clearly evi-dent from our previous study that the catalytic efficiency (Kcat) value showed a 54–66% and 40–51% increase in hydrolytic efficiency in the AtCEsA3ixr1–2 leaf and stem cellulose extracts, respectively, demonstrating that the cellulose derived from leaf and stem tissues of tobacco plants over-expressing AtCEsA3ixr1-2 is 40 to 60% more amenable to enzymatic saccharification [24]. Hence, the crude leaf and stem biomass also exhibited an increased saccharification rate compared to wild-type plant biomass as demonstrated in the present study, in spite of the higher lignin content. moreover, transgenic leaf and stem biomass required about one fourth the amount of hydrolytic enzymes to release equal amount of soluble sugars during saccharification than did wild-type tissues. This further confirms that leaf and stem biomass derived from the transgenic tobacco plants is a preferred substrate for enzymatic saccharifica-tion than is wild-type plant-derived biomass.

genes such as the Corngrass1 microrna gene from maize, or the Arabidopsis transcription factor-like sHINE have been shown to improve digestibility, or reduce lignin and increase cellulose contents, when over-expressed in several other plant species including switchgrass and rice [1, 7]. Here we show that constitutive expres-sion of AtCEsA3ixr1–2 in tobacco is capable of modifying cellulose, making it more accessible to enzymatic digestion. in the search for alternative biofuel plant resourc-es, tobacco has been largely unnoticed as it is generally considered to be an expensive crop that is primarily grown for smoking [2]. However, like hardwood trees, tobacco can be coppiced to stimulate re-sprouting from the stump after cutting; thus, multiple harvests of green biomass are possible in a single growing season. moreover, closer examination identifies tobacco, which is cultivated in more than 100 countries world-wide, as an outstanding industrial biomass crop, which can generate up to 170 t/ha of green tissues when close-grown for biomass production rather than for smoking [28]. On, average stalk biomass produced in burley, dark air-cured, and dark fire-cured



tobacco is 75%, 30%, and 30%, respectively, compared to leaf biomass. Our findings suggest a potential new use for tobacco stalks as a substrate for biofuel. When grown for energy production instead for smoking, tobacco can generate a large amount of inexpensive biomass more efficiently than almost any other agricultural crop. From a practical point of view, a similar strategy could be employed to translate mutated cesa catalytic peptides involved in primary and secondary cell wall formation into energy crops like poplar, corn and switchgrass to improve the efficiency of biomass conversion to fermentable sugar.

acKnoWLedgements

We are very much grateful to Kentucky tobacco research and development center (Ktrdc) for facili-ties and support. this work was supported by the KY state Ktrdc grant to iBm. the authors would like to thank dr. david Zaitlin of the University of Kentucky for critical reading and necessary corrections of the manuscript. also, we would like to thank ms. Bonnie Kinney for her excellent care of the experimen-tal tobacco plants, dr. darby Harris for technical guidance, and dr. seth deBolt for important sugges-tions. the anonymous reviewers and the editor are also acknowledged for suggestions that greatly improved the manuscript.

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