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The Ways and Means of Boosting Cellulose Production in Transgenic Trees

The Ways and Means of Boosting Cellulose Production in Transgenic Trees. Chandrashekhar P. Joshi Michigan Technological University Houghton, MI, USA. Cellulose biosynthesis. Tree Biotechnology 570 billion tons of carbon sequestered in nature 75% in plants : 427 billion tons

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The Ways and Means of Boosting Cellulose Production in Transgenic Trees

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  1. The Ways and Means of Boosting Cellulose Production in Transgenic Trees ChandrashekharP.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? Plasma membrane bound rosette-like structures synthesize cellulose! • Enzyme activity determination is still problematical. • Upon isolation, rosettes lose integrity and activity • In vitro reconstruction of cellulose biosynthetic apparatus is still impossible The first cellulose synthase (CesA) gene was reported only in 1996

  4. Doblin et al. 2002

  5. Secondary wall Primary wall Sjostrom E. 1993

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

  9. Aspen PtrCesA1 cDNA Isolated Zn HVRI A HVRII B • 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

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

  11. Isolation of cellulose biosynthesis-related cDNAs from aspen xylem cDNA library Using aspen CSR regions and other available CesA probes: 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)* • Published in Wu et al 2000, Samuga and Joshi, 2002, Kalluri and Joshi, 2003, Samuga and Joshi, 2004, • and Kalluri and Joshi 2004 HVRI A CSR B 23-46% >90% 36-60 % >90%

  12. A D zn HVRI A CSR B B PtrCesA1 PtrCesA2 PtrCesA3 PtrCesA1 PtrCesA2 PtrCesA3 E a b c PtrCesA1 PtrCesA2 PtrCesA3 d e f F PtrCesA1 PtrCesA2 PtrCesA3 C 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 Arabidopsis Poplar Where? Ratio expressed? A:P • 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)

  15. V VII X X X X III IX 1 2 3 4 5 6 7 8 9 10 II IV I VIII X X X X X 11 12 13 14 15 16 17 18 19 VI ? Scaffold Poplar CesA Gene Shuffling and duplication

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