Figure 1.
Three developmental stages selected for internode characterization in Brachypodium distachyon.
(A, C, E) Intact and (B, D, F) dissected plants when (A-B) the first internode was elongating, (C-D) the first inflorescence was emerging, and (E-F) the first internode was senesced. FI, first internode; S, spike inflorescence. Scale bars (in red) = 5 cm. Distal internodes are marked with an asterisk.
Figure 2.
Analysis of Brachypodium distachyon stem internode development.
(A) Stem and vascular bundle count. (B) Stem height (black) and stem fresh weight (gray). (C) Internode length (black) and stem area (gray). (D) Inner (black circle) and outer (black square) vascular bundle area, and interfascicular region area (gray diamond). (E) Cell wall thickness of xylem vessel and adjacent bundle fibers of inner (black circle) and outer (black square) vascular bundles, sclerenchyma nearest (gray diamond) and second nearest (gray triangle) to the bundle sheath. Growth stages correspond to elongation (E), inflorescence emergence (F), and senescence (S). Data are means ± standard deviation. Points annotated with the same letter are not significantly different at P < 0.05.
Figure 3.
Brachypodium distachyon internal stem internode anatomy with emphasis on vasculature.
(A) Cross section of whole stem and (B) higher magnification of the first stem internode. Red, inner vascular bundles; pink, outer vascular bundles; cyan, interfascicular region compromised mostly of sclerenchyma fibers; gray, pith; lime green, chlorenchyma and sclerenchyma cells comprise the cortex; brown, epidermis. (C) Vascular bundle illustration at high magnification. Green, bundle sheath (BS); purple, phloem (P); vermilion, companion cells; tan, xylem vessels (XV); red, xylem tracheids (XT); white, lacuna (Lc); orange, xylem parenchyma cells (XP); gray, parenchyma cells (Py); blue, sclerenchyma fibers (SF). (A-B) Bar = 0.1 mm, (C) bar = 0.01 mm.
Figure 4.
Cell wall thickness and lignin detection increases following stem internode elongation.
Whole stem (A, C, E, G, I, K) and higher magnification (B, D, F, H, J, L) of Brachypodium distachyon cross-sections stained with toluidine-blue (A-F) or the Wiesner reagent (G-L). (A-B, G-H) Elongating, (C-D, I-J) inflorescence emergence, and (E-F, K-L) senesced stem internode transverse cross-sections. Images were taken using brightfield microscopy. Scale bars = 0.1 mm.
Figure 5.
Florescent detection of lignin and indirect immunodetection of crystalline cellulose and xylan in stem internode.
Whole stem (A, C, E, G, I, K, M, O, Q) and higher magnification (B, D, F, H, J, L, N, P, R) of Brachypodium distachyon cross sections observing lignin autofluorescence (A-F) and immunolabeled CBM3a probe (G-L) and LM10 antibody (M-R). (A, B, G, H, M, N) Elongating, (C, D, I, J, O, P) inflorescence emergence, and (E, F, K, L, Q, R) senesced stem internode transverse cross-sections. Images were taken using wide field epifluorescence microscopy. Scale bars = 0.1 mm.
Figure 6.
Quantification of florescence detection of lignin and indirect immunodetection of crystalline cellulose and xylan in stem internode.
Corrected (A) total autofluorescence, (B) CBM3a indirect immunofluorescence, and (C) LM10 immunofluorescence of whole stem (back circle), inner (gray diamond) and outer (black square) vascular bundles, and interfascicular region (gray triangle). Data are means ± standard deviation. Points annotated with the same letter are not significantly different at P < 0.05.
Figure 7.
Characterization of wall composition changes associated with growth using FTIR spectroscopy.
Average line spectra of stem tissue sampled from elongating (green), flowering (orange), and senesced (blue) stages of development. Wavenumbers corresponding to absorbance peaks associated with cellulose, hemicellulose, and lignin are noted. Letters indicate significant differences at P < 0.05.