The Ways and Means of Boosting Cellulose Production in Transgenic

Trees

Chandrashekhar P. JoshiMichigan Technological University

Houghton, MI, USA

Cellulose biosynthesisTree Biotechnology

570 billion tons of carbon sequestered in nature75% in plants: 427 billion tons

180 billion ton: cellulose

Forest products worth of over $200 billion are sold every year in the US alone! (AFPA) +Agricultural products

Genetic improvement of cellulose production in specific organs, tissues and cells of trees and crop plants will haveenormous impact on global economy that also has greatecological significance.

Cellulose is a deceptively simple molecule (Delmer, 1999)

• We do not know how every alternate glucose is flipped by 180 0

• We do not know how cellulose chains elongate and terminate?• Why microfibrils differ in number, crystallinity, and orientation?

• Enzyme activity determination is still problematical.

• Upon isolation, rosettes lose integrity and activity

• In vitro reconstruction of cellulose biosynthetic apparatus is still impossible

Plasma membrane bound rosette-like structures synthesize cellulose!

The first cellulose synthase (CesA) gene was reported only in 1996

Doblin et al. 2002

Sjostrom E. 1993

Primary wall

Secondary wall

Cellulose heterogeneity in Trees

• Primary wall (P)• Content: <20%• DP: 500-2000• Low crystallinity 30%• MF angle: 50-900

• Expanding cell wall

• Secondary wall (S2)• Content: ~50%• DP: ~14,000• High crystallinity 50%• MF angle: 10-200

• Rigidity and strength

Two different types of Cellulose synthases might be involved in biogenesis of primary and secondary cell walls!

Goal

Understanding the mechanism of cellulose biosynthesis in trees

may provide a direct means of boosting cellulose production in cell walls in terms of cellulose

quantity and quality

Sucrose

+ UDP

SUSYUDPG

microtubulesCESA CESA

KOR ..

microfibrils

PM

Glucan chain

Joshi et al., 2004 New Phytologist 164: 53-61

Aspen PtrCesA1 cDNA Isolated

• Full length clone: 3232 bp long

• Protein of 978 amino acids, 110 kDa

• Eight transmembrane domain anchor

• UDP-Glucose binding domain conserved

• Xylem-specific and tension stress responsive expression, a major player

Wu, Joshi, Chiang (2000) Plant Journal 22: 495-502

Zn HVRI A HVRII B

Arabidopsis genome sequencing and mutant studies enabled identification of at least ten distinct CesA genes.

0.1

AtCESA7

AtCESA2

AtCESA9AtCESA5AtCESA6

AtCESA1

AtCESA10

AtCESA4

AtCESA8

AtCESA3

(rsw1)

(irx3)

(prc1)

(irx1)

(ixr1)(irx5)

Clone name cDNA length %identity % similarity to Arabidopsis CesA

PtrCesA1 3232 bp 83 88 AtCesA8 (irx1)*PtrCesA2 3277 bp 87 91 AtCesA7 (irx3)*PtrCesA3 3401 bp 79 85 AtCesA4 (irx5)

PtrCesA4 3640 bp 87 91 AtCesA1 (rsw1)PtrCesA5 3532 bp 89 91 AtCesA3 (ixr1)*PtrCesA6 3773 bp 74 81 AtCesA6 (prc1)*PtrCesA7 3809 bp 85 90 AtCesA6 (prc1)*

Isolation of cellulose biosynthesis-related cDNAs from aspen xylem cDNA libraryUsing aspen CSR regions and other available CesA probes:

23-46% >90% 36-60 % >90%

HVRI A CSR B

•Published in Wu et al 2000, Samuga and Joshi, 2002, Kalluri and Joshi, 2003, Samuga and Joshi, 2004,and Kalluri and Joshi 2004

PtrCesA3PtrCesA2PtrCesA1

a b c

d e f

PtrCesA3PtrCesA2PtrCesA1

a

d

b

e

c

f PtrCesA1 PtrCesA2 PtrCesA3

PtrCesA1 PtrCesA2 PtrCesA3

zn HVRI A CSR B

A

B

C

D

E

F

0

200

400

600

800

1000

1200

1

A1 xylem

A1 tension

A1 comp

A1 leaf

A2 xylem

A2 ten

A2 comp

A2leaf

A3xylem

A3 ten

A3 comp

A3leaf

PtrCesA1 PtrCesA2 PtrCesA3

PtrCesA1, PtrCesA2 and PtrCesA3 are coordinately expressed in the developing

xylem and phloem fibers during stem development of aspen trees.

PtrCesA1, PtrCesA2 and PtrCesA3 are coordinately expressed in the tension

responsive manner during tension stress conditions.

The quantities of PtrCesA1, PtrCesA2 and PtrCesA3 are unequal

Summary of poplar 17 CesA genes grouped into 9 types

• AtCesA1 and AtCesA10 4A, 4B (VI, XVIII) P 2: 2

• AtCesA2, 5, 6, 9 7A, 7B, 8A, 8B P 4:4

(V, VII) (II, V)

• AtCesA7 2A, 2B (VI, XVIII) S 1:2

• AtCesA8 1A, 1B (IV, XI) S 1:2

• AtCesA4 3A (II) S 1:1

• AtCesA3 5A, 5B, 9A, 9B PS 1:4

(I, VI, IX, XVI)

• ? 6A, 6B P (rice-like)

(XVIII, SCAFFOLD 133)

Arabidopsis Poplar Where? Ratio expressed? A:P

III

III

IVVIII

VI

? Scaffold

VII

1 2 3 4 5 6 7 8 9 10

11 12 13 14 15 16 17 18 19

V

IX

Poplar CesA Gene Shuffling and duplication

XX X X

XX XX X