Haiying Liang1, Nicole Brown2, John Carlson2, Ming Tien2 1 Clemson University, Clemson, SC 29634

2The Pennsylvania State University, University Park, PA 16802

A Novel Approach to Facilitate Accessibility of Cellulose and Hemicellulose by Introducing a Tyrosine Rich Peptide Gene In

Poplar Cell Walls

Energy Crisis

Don Tate III AMERICAN-STATESMAN ILLUSTRATION, from http://www.statesman.com/business/content/business/other/gas.html

Haiying Liang1, Nicole Brown2, John Carlson2, Ming Tien2 1 Clemson University, Clemson, SC 29634

2The Pennsylvania State University, University Park, PA 16802

A Novel Approach to Facilitate Accessibility of Cellulose and Hemicellulose by Introducing a Tyrosine Rich Peptide Gene In

Poplar Cell Walls

About Lignin

A major component of wood -raw material for pulp and paper production -a good renewable energy source

A component of lignocellulosic material -feedstock for livestock

2nd most abundant terrestrial biopolymer -approx. 30% of the organic carbon in the biosphere

Lignin Degradation is of Central Importance in Biomass Utilization

Undesired material in pulp and paper industries and

biomass utilization

Mosier et al. 2005

Difficult to degrade , no nutritional value

Approaches Being Taken

Schematic from Mosier et al. (2005) showing goals of pretreatment of lignocellulosic material

Expensive and environment-unfriendly

lignin network crystallinity of cellulose

1. Decrease lignin content2. Modify lignin monomer composition: G (guaiacyl) S (syringyl)

Biotechnology

PAL

C4H

CCR

CAD

4CL

COMT

monolignol synthesis

Essential Role of Lignin in Cell Wall

Essential component of cell wall:

- imparts rigidity to plants

- conducts water & solutes to different parts of plants

- provides physical barrier to invading pests

HypothesisFree radical coupling between lignol subunits and TYR will result in a lignin structure that can be partially hydrolyzed with proteases.

CHOHHC

HOCH2

H3COO

O

HCCH

CH2OH

HCHC

HOH2C

OCH3

OHOH3CO

OCH3

O

CHOHCH

CH2OH

OCH3

OCHOH

HCHOH2C

OCH3

CHOHCH

CH2OH

H3CO

HO

OCH3

O

N NO

O

R

RN N

O

O

R

RN N

O

O RN N

O

O

R

N NO

O

R

N NO

O R

OHOH

OCHOH

HC

HOH2C

OCH3

CHOHCH

CH2OH

H3CO

HO

OCH3

O

R

O

OCHOH

HC

HOH2C

OCH3R

Representative structure of peptide-cross-linked lignin via phenolic tyrosines.

1. Design TYR-rich peptide genes differing in length and sequence

2. Express transgene in lignifying tissue in poplar

3. Characterize transgenic plants:-Plant fitness -Lignin structure and lignin-tyrosine bonding in plants-Small scale pulping and ethanol production tests

Strategies

Strategy 1: Gene Design Binary Vector (Modified from pBI101)

Tyr rich gene (13%)

PAL promoter--CBG-leader

For secretion into the cell wall during lignification (Pinus contorta coniferin-

specific β-glucosidase)

Expression of PAL2-GUS gene fusion in poplar in the actively lignifying

phloem and xylem cells(PAL: Phenylalanine Ammonia-Lyase )

Transformation in Poplar

Ogy (P. deltoides Marsh. × P. nigra L.)

Transgenic lines1 Kb

Plasmid DNAWild type DNA

Example of PCR screening with transgene-specific primers

2W 2ZZ 2-II 2X M 4-2 Wt

1Kb

2Kb

Example of genomic southern hybridization

Transgenic Line

wt 2x 29 2-I 4c 4.2 2zz 10.1 2ee 2w 2-II

Ty

ros

ine

-ric

h t

ran

sg

en

e

ex

pre

ss

ion

(re

lati

ve

to

r1

8S

)

0.0

0.1

0.2

0.3

0.4

0.5

0.6

Strategy 2: Gene Expression Real-Time PCR

2-I 2X 2YY 2Z 2P

Strategy 3: CharacterizationHistochemical Staining of Lignin

Wt 2-5V 4-4 2EE

Phloroglucinol

Potassium permanganate

Phloroglucinol

Potassium permanganate

Strategy 3: CharacterizationKlason Lignin Content Analysis

wt

2yy

2-5

v

2I

2-5

2P

2II

2E

E

2A

2W

Lig

nin

Co

nte

nt

(% S

tem

Dry

We

igh

t)

05

101520253035

Further StudiesLocalization of TYR-rich peptide with antibodyPathogen susceptibility analysisTensile strength, lignin structure, etc.

Tyr

TyrLys

Glu

Lys

Glu

Lys

Glu

Tyr

Tyr

Tyr

Tyr

Tyr

Peptide showing salt bridging

Strategy 3: Characterizing Lignin-Protein Structure and Interactions

Isolated Lignin

MWL isolated from stemsHSQC and TOCSY (Hu et al.)

Compare to 13C-Tyrosine treated MWL fractions (from control plants)

Strategy 3: Characterizing Lignin-Protein Structure and Interactions

HSQC of MWL (Hu et al., Nature Biotechnol, 1999)

Solid samples

Method—CP/MAS NMR 15N Tyrosine tissues Plants supplied K15NO3

(Englesberger et al. 2006)

Cross polarization studies Basis: < 10 Å proximity Different 15N chemical shifts

should be detected for the various amino acids

Strategy 3: Characterizing Lignin-Protein Interactions at the Nanoscale

Morais et al. 1999, J. Braz. Chem. Soc.  

Lignin-Protein Interactions

Solid state NMR (CP/MAS)

Variable Contact Time Cross Polarization Proton spin-lattice relaxation time in the rotating frame

(HT1)

Common HT1indicates nanoscale homogeneity, while different HT1 indicates nanoscale phase separation

Strategy 3: Characterizing Lignin-Protein Interactions at the Nanoscale

Strategy 3: Thermal Characterization

DMA/DSCLignin Glass Transition Region

Before and after plasticization (DMA) Control and modified plants Extracted MWL

Strategy 3: Bioenergy Studies

•Pulping efficiency and ethanol production from modified tissues

•Does protease pre-treatment of tyrosine-rich transgenic poplar plants impact these processes?

0

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08

0.09

0.1

mg of weight loss /mg wood

chps

Wt T15 T17 T19 T28

Preliminary digestibility assay using protease K

Acknowledgments

• Alan Benesi, Penn State NMR Director• DOE & Huck Institutes for the Life Sciences at• Penn State for $$$