1. Structure and Composition of Developing Cotton Fibers N. Abidi 1, E. Hequet 1,2, L. Cabrales 1, J. Gannaway 2, T. Wilkins 3, L. Wells 2 1 Texas Tech University, ITC & PSS 2 Texas A&M – Texas Agricultural Experiment Stations 3 Texas Tech University, PSS Funded by USDA/ICRC and TDA/FFRP

2. Objective Investigate the structural changes and gain novel insight into cell wall structure and composition in developing cotton fibers using Fourier Transform Infrared and Thermogravimetric Analysis.

3. Materials Two cotton cultivars (Gossypium hirsutum L. cv. TX19 and TX55) were planted in a greenhouse in triplicate. On the day of flowering (0 day post anthesis= 0 dpa) individual flowers were tagged. Bolls were harvested at 10, 14, 17, 20, 36, 50, and 61 dpa and stored in liquid nitrogen. Samples were dehydrated.

4. Methods: Sample dehydration Sample dehydration procedure consists of washing the sample with acidified 2,2-dimethoxypropane (DMP) followed by 5 exchanges for 5 min each in 100% acetone. FTIR, TGA, SEM, and fiber cross-sections were performed.

5. Fourier Transform Infrared Spectroscopy

6. Methods: FTIR analysis Spectrum One equipped with Universal Attenuated Total Reflectance (UATR) was used. Thirty spectra per sample were acquired for each developmental stage (30 spectra x 3 replications). FTIR spectra were collected with spectrum resolution of 4 cm -1, with 32 co-added scans over the range 4000- 650 cm -1. FTIR spectra were baseline corrected, normalized and subjected to Principal Component Analysis (PCA).

7. 2918, 2850 Assy. CH 2, stretch Wax 1737 C=O stretch xyloglucans 1633 H-O-H Adsorbed water 1537 NH 2 deformation Proteins, amino-acids 1439 CH 2 scissoring Wax 1429 CH 2 sym. deformation Belongs to glucose units 1204 COH in-plane bending SCW formation 900  -linkage Glucose—O—glucose

8. Principal Components Analysis of FTIR spectra 10 to 20dpa 36 to 61dpa TX19 TX55 90 spectra per sample

9. Results: FTIR analysis FTIR results show that vibration bands assigned to non-cellulosic compounds in the FTIR spectra of fibers at 10, 14, 17, and 20 dpa are not detectable at 36 dpa and above. The results are consistent with the onset of the secondary cell wall synthesis around 21 dpa.

10. Results: FTIR analysis The advantages of using the UATR FTIR are:  No extraction is needed  No sample preparation is needed  Ease of analysis  Non-destructive testing allowing other tests to be performed on the sample

11. Thermogravimetric Analysis

12. Methods: TGA analysis Pyris 1 TGA was used. Thermograms were recorded between 37 and 600 o C with heating rate of 10 o C/min in a flow of N 2 at 20 mL/min. Percent weight losses were calculated between 37- 150 o C and 150-425 o C. First derivatives of the thermograms were calculated to determine the inflection points.

13. % weight loss of developing cotton fibers

14. % weight loss between 37 and 150 o C vs. days post anthesis Secondary cell wall >99% cellulose Primary cell wall ~30% cellulose

15. % weight loss between 37 and 150 o C vs. days post anthesis The decrease of the amount of adsorbed water from 10% to 5.5% when the fiber reaches 36 dpa could be due to a decrease in the specific surface area per unit mass and to an increase in the structural organization of the cellulose macromolecules in the secondary cell wall (increased crystallinity).

16. % weight loss between 150 and 450 o C vs. days post anthesis Secondary cell wall >99% cellulose Primary cell wall ~30% cellulose

17. The increase of the weight loss between 150 o C and 425 o C is attributed to the increase in the cellulose deposition starting at/or around 20 dpa. In addition to the decomposition of noncellulosic compounds, pyrolysis reactions of cellulose macromolecules also take place. % weight loss between 150 and 450oC vs. days post anthesis

18. First derivative thermogravimetry

19. Peak temperature vs. days post anthesis Secondary cell wall DP>14,000, %cryst. >60* Primary cell wall DP<3,000, %cryst. <30 * Hsieh et al, TRJ, 1997, 67, 529

20. The temperature of decomposition of cellulose increases with increasing developmental stage of the fibers. As the secondary cell wall becomes thicker (fibers older than 20 dpa), cellulose becomes dominant (DP>14,000) and organized (Cryst.> 60%), leading to an increase in the decomposition temperature. Peak temperature (cellulose decomposition) vs. days post anthesis

21. Scanning Electron Microscopy and Fiber cross-sections

22. Scanning Electron Microscope 10 dpa17 dpa At 10, 14, and 17 dpa, the fiber walls are extremely thin and the fibers are stuck together.

23. Scanning Electron Microscope At 20 dpa, the fiber can be individualized. They are composed essentially of primary cell wall. Beginning at 36 dpa, the appearance of twist indicates the presence of thicker secondary cell wall. 20 dpa36 dpa

24. Fiber cross-sections 36 dpa Cross-sections of fibers at 20 dpa, show mostly the thin primary cell wall while the secondary cell wall is not yet detectable. At 36 dpa, secondary cell wall thickening is clearly visible, demonstrating that SCW synthesis is well underway at this stage. 20 dpa

25. 61 dpa Fiber cross-sections By 61 dpa, the fiber cross-section shows thick secondary cell wall indicating that the fibers have reached full maturity.

26. Wall thickness vs. days post anthesis

27. % contribution of the primary wall to the total weight of the fiber vs. wall thickness WTWT WTWT Young fiberMature fiber Calculation made for fiber diameter=14  m ; density of cellulose=1.52g.cm -3 ; density of PCW=1.14 g.cm -3 ; thickness of the PCW = 0.4  m.

28. Conclusions Results showed that changes in the structure and composition occur during cotton fiber development. FTIR showed that vibration bands assigned to non- cellulosic compounds in the spectra of fibers at 10, 14, 17, 20 dpa are not detectable at 36 dpa. The onset of secondary cell wall is around 20 dpa. TGA results supported FTIR findings.