The mesoscale packing and crystal structure of cellulose microfibrils as well as temporal changes in cell wall composition and hydration during the development of cotton fibers from two species, Gossypium hirsutum and G. barbadense were studied using vibrational sum frequency generation (SFG), attenuated total refection infrared (ATR-IR), Fourier transform Raman (FT-Raman) spectroscopy and X-ray diffraction (XRD). The developmental stages analyzed (13-60 days post anthesis) included primary wall synthesis, transitional cell wall remodeling, secondary wall thickening via synthesis of nearly pure cellulose, and fiber maturation. ATR-IR and FT-Raman combined with principle component analysis revealed that fibers of both species undergo abrupt changes in the cellulose and matrix polymer contents during the transition to secondary cell wall synthesis. XRD revealed that cellulose crystal size and crystallinity increase similarly over time in both species. SFG analysis of fibers from un-opened bolls, which were stored in water then air dried, showed subtle differences between two species in the mesoscale ordering of cellulose microfibrils in the maturing secondary walls. In the samples of mature fibers dried on the plant after the boll split opened naturally, the difference in SFG spectra between species was negligible. Collectively, the results show that (a) SFG can uniquely reveal differences in cellulose fibril ordering in maturing cotton fibers before boll opening; and (b) illustrate the comparative usefulness of other commonly used spectroscopic analytical methods for cotton fiber analysis.
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