The Ways and Means of Boosting Cellulose Production in Transgenic Trees Chandrashekhar P. Joshi Michigan Technological University Houghton, MI, USA
Jan 09, 2016
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