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

2. Cellulose biosynthesis Tree Biotechnology 570 billion tons of carbon sequestered in nature 75% 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 have enormous impact on global economy that also has great ecological significance.

3. 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

4. Doblin et al. 2002

5. Sjostrom E. 1993 Primary wall Secondary wall

6. Cellulose heterogeneity in Trees Primary wall (P) Content: <20% DP: 500-2000 Low crystallinity 30% MF angle: 50-90 0 Expanding cell wall Secondary wall (S2) Content: ~50% DP: ~14,000 High crystallinity 50% MF angle: 10-20 0 Rigidity and strength Two different types of Cellulose synthases might be involved in biogenesis of primary and secondary cell walls!

7. 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

8. Sucrose + UDP SUSY UDPG microtubules CESA KOR.... microfibrils PM Glucan chain Joshi et al., 2004 New Phytologist 164: 53-61

9. 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

10. Arabidopsis genome sequencing and mutant studies enabled identification of at least ten distinct CesA genes. 0.1 AtCESA7 AtCESA2 AtCESA9 AtCESA5 AtCESA6 AtCESA1 AtCESA10 AtCESA4 AtCESA8 AtCESA3 (rsw1) (irx3) (prc1) (irx1) (ixr1) (irx5)

11. Clone namecDNA length%identity % similarity toArabidopsis CesA PtrCesA1 3232 bp 83 88AtCesA8 (irx1)* PtrCesA23277 bp 87 91AtCesA7 (irx3)* PtrCesA33401 bp 79 85AtCesA4 (irx5) PtrCesA43640 bp 87 91AtCesA1 (rsw1) PtrCesA53532 bp 89 91 AtCesA3 (ixr1)* PtrCesA63773 bp 74 81 AtCesA6 (prc1)* PtrCesA73809 bp 85 90AtCesA6 (prc1)* Isolation of cellulose biosynthesis-related cDNAs from aspen xylem cDNA library Using 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

12. PtrCesA3PtrCesA2PtrCesA1 abc def PtrCesA3PtrCesA2PtrCesA1 a d b e c f PtrCesA1 PtrCesA2 PtrCesA3 zn HVRI A CSR B A B C D E F PtrCesA1 PtrCesA2 PtrCesA3

13. 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

14. Summary of poplar 17 CesA genes grouped into 9 types AtCesA1 and AtCesA104A, 4B (VI, XVIII)P2: 2 AtCesA2, 5, 6, 97A, 7B, 8A, 8BP4:4 (V, VII) (II, V) AtCesA72A, 2B (VI, XVIII)S1:2 AtCesA81A, 1B (IV, XI)S1:2 AtCesA43A (II)S1:1 AtCesA35A, 5B, 9A, 9BPS1:4 (I, VI, IX, XVI) ?6A, 6BP (rice-like) (XVIII, SCAFFOLD 133) Arabidopsis PoplarWhere? Ratio expressed? A:P

15. I II III IV VIII 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 X X XX XXXX X