Regulation of growth anisotropy in well-watered and water-stressed maize roots. II. Role Of cortical microtubules and cellulose microfibrils

Abstract

We tested the hypothesis that the degree of anisotropic expansion of plant tissues is controlled by the degree of alignment of cortical microtubules or cellulose microfibrils. Previously, for the primary root of maize (Zea mays L.), we quantified spatial profiles of expansion rate in length, radius, and circumference and the degree of growth anisotropy separately for the stele and cortex, as roots became thinner with time from germination or in response to low water potential (B.M. Liang, A.M. Dennings, R.E. Sharp, T.I. Baskin [1997] Plant Physiol 115:101-111). Here, for the same material, we quantified microtubule alignment with indirect immunofluorescence microscopy and microfibril alignment throughout the cell wall with polarized-light microscopy and from the innermost cell wall layer with electron microscopy. Throughout much of the growth zone, mean orientations of microtubules and microfibrils were transverse, consistent with their parallel alignment specifying the direction of maximal expansion rate (i.e. elongation). However, where microtubule alignment became helical, microfibrils often made helices of opposite handedness, showing that parallelism between these elements was not required for helical orientations. Finally, contrary to the hypothesis, the degree of growth anisotropy was not correlated with the degree of alignment of either microtubules or microfibrils. The mechanisms plants use to specify radial and tangential expansion rates remain uncharacterized.

Figure 7

Birefringent retardation as a function of distance from the apex of well-watered (ww) and water-stressed (ws) roots. Cortical cell walls were measured in approximately median-longitudinal sections in 2-μm semithin methacrylate sections. Data are means ± se of five roots, with two cell walls sampled at each position per section and three sections measured per root.

Figure 1

Longitudinal and radial strain rates as a function of distance from the apex for well-watered (ww, 24 and 48 h after transplanting) and water-stressed (ws, 48 h after transplanting) roots. The data are from Liang et al. (1997). A, Longitudinal strain rate. Arrows indicate positions where microtubule orientation changed from transverse to oblique (Fig. 3). B, Radial strain rate for the cortex. C, Growth anisotropy, calculated as the ratio of longitudinal to radial strain rates. Note that when radial strain rates are near 0 growth anisotropy tends toward infinity; these values are not plotted.

Figure 2

Micrographs of cortical microtubules in median-longitudinal sections showing the similar appearance of transverse, oblique, and longitudinal orientations in cells of the cortex of well-watered and water-stressed roots. A to D, Well-watered roots; E to H, water-stressed roots. Examples of transverse (A and E), oblique (B and F; right-handed helical), longitudinal (C and G) , and oblique (D and H; left-handed helical) microtubule orientations. Micrographs were obtained from peels, as described in Methods. Bar = 20 μm.

Figure 3

Microtubule orientation in cortical cells as a function of distance from the apex of well-watered (ww) and water-stressed (ws) roots. A, Mean microtubule angle measured for cells localized in median-longitudinal sections. B, The sds of the above distributions. At each position, 20 microtubules were sampled from two cells, and the data presented were pooled from measurements of 5 to 10 roots (100–200 microtubules measured per position).

Figure 4

Tangential (radial) strain rate, growth anisotropy, and microtubule orientation as a function of distance from the apex for the stele of well-watered (ww) and water-stressed (ws) roots. A, Tangential strain rate. B, Growth anisotropy, calculated as the ratio of longitudinal to tangential strain rates. Note that the longitudinal strain rate profile shown in Figure 1A is the same for all tissues. C, Mean microtubule angle measured is stelar parenchyma. D, The sds of the above distributions. Data in A and B are from Liang et al. (1997) and in C and D are averages of at least 100 microtubules measured per position from 5 to 10 roots.

Figure 5

Mean microtubule angle as a function of time from peak longitudinal strain rate. Mean microtubule angle for the cortex of well-watered (24 h) and water-stressed (48 h) roots and for the stele of the water-stressed roots were re-plotted versus time instead of position, taking advantage of the steady-state elongation kinetics and using the transformation method described by Silk et al. (1984).

Figure 6

Micrographs showing that exposure of the seedlings to 0°C for 6 min depolymerized cortical microtubules to the same extent for transverse, oblique, and longitudinal orientations in well-watered and water-stressed roots. A to D, Well-watered roots; E to H, water-stressed roots. Examples of transverse (A and E), oblique (B and F; right-handed helical), longitudinal (C and G), and oblique (D and H; left-handed helical) microtubule orientations. Bar = 20 μm.

Figure 8

Electron micrograph showing the appearance of microfibrils on the innermost layer of a longitudinal-radial cell wall of a cortical cell from a well-watered root. Image shows a cell with a net transverse orientation of microfibrils, approximately 5 mm from the apex. The longitudinal axis of the root is parallel to the side of the figure. Vibratome sections were extracted with carbonate and a metal-carbon replica was made as described in Methods. Bar = 400 nm.

Figure 9

Microfibril orientation as a function of distance from the apex of well-watered (ww) and water-stressed (ws) roots. A, Mean microfibril angle measured for cortical cells in longitudinal sections. B, The sds of the above distributions. Data are averages of 250 to 1800 microfibrils measured per position from three experiments with five roots each.

Figure 10

In water-stressed roots the handedness of helical orientations of microfibrils is in many cells opposite to that of the microtubules. Percentage of cortical cells with various classes of orientations of microtubules (MT, white bars) and microfibrils (MF, black bars) at 6 mm (A), 7 mm (B), 10.5 mm (C), and 11.5 mm (D) from the apex are shown. Percentages of cells with undefined (Un), transverse (Tr), right-handed helical (Rt), longitudinal (Long), and left-handed helical (Lft) orientations are also shown. Orientations of each element were measured in the same sections. Data for microtubules are means of 100 to 140 cells, and for microfibrils of 270 to 560 cells, from single median-longitudinal sections from five to seven roots.