Spatiotemporal variation of leaf epidermal cell growth: a quantitative analysis of Arabidopsis thaliana wild-type and triple cyclinD3 mutant plants

Abstract

Background and aims: The epidermis of an expanding dicot leaf is a mosaic of cells differing in identity, size and differentiation stage. Here hypotheses are tested that in such a cell mosaic growth is heterogeneous and changes with time, and that this heterogeneity is not dependent on the cell cycle regulation per se.

Methods: Shape, size and growth of individual cells were followed with the aid of sequential replicas in expanding leaves of wild-type Arabidopsis thaliana and triple cyclinD3 mutant plants, and combined with ploidy estimation using epi-fluorescence microscopy.

Key results: Relative growth rates in area of individual epidermal cells or small cell groups differ several fold from those of adjacent cells, and change in time. This spatial and temporal variation is not related to the size of either the cell or the nucleus. Shape changes and growth within an individual cell are also heterogeneous: anticlinal wall waviness appears at different times in different wall portions; portions of the cell periphery in contact with different neighbours grow with different rates. This variation is not related to cell growth anisotropy. The heterogeneity is typical for both the wild type and cycD3.

Conclusions: Growth of leaf epidermis exhibits spatiotemporal variability.

Fig. 1.

A fragment of A. thaliana Col shoot with aerial rosettes growing in cauline leaf axils. Scale bar = 10 mm (A). Leaf portions used in cellular parameters and growth computation overlaid on an SEM micrograph of an exemplary Col leaf. Scale bar = 500 µm (B). Nucleus size and DNA content assessment (C–F). The surface area of cell nuclei measured in central optical sections is linearly dependent on the DAPI fluorescence intensity (in 10⁶) of the same nuclei (y = 1·1 × 10⁻⁵ x + 7·74; R² = 0·596) measured in image cytometry in arbitrary units (C). To obtain values of various cellular parameters for individual cells, epi-fluorescence micrographs are taken from a whole leaf preparation at a different focus so that the anticlinal cell wall outlines (D) and the nucleus outlines (E) are distinct. The same fragment of epidermis is recognized in the nail polish replica (F) in which other cellular parameters are assessed. Exemplary cells visible in all the images are labelled with asterisks; arrowheads point to their nuclei. Scale bars = 20 µm.

Fig. 2.

Pavement cell morphogenesis shown in sequences of SEM micrographs of the abaxial epidermis of expanding leaves. The time at which the replicas were taken is given on the top of each column of micrographs. Formation of wavy anticlinal cell walls is shown for proximal portions of Col (A, D) and cycD3 (B, C) leaves. Anticlinal wall segments of exemplary large cells, labelled by 1, exhibit different shapes at contacts with cells 2, 3 and 4 (A, B). Black arrows (C, D) point to cell wall segments on which secondary invaginations are formed; white arrows (C) label the wall that first bulges into the larger cell and afterwards attains a wavy shape; white arrowheads (D) point to the wall in which the wave amplitude increases in time without formation of secondary invaginations. Occurrence of divisions in jigsaw puzzle-shaped pavement cells (pointed by arrows) is exemplified by a proximal portion of Col leaf (E). Scale bars = 20 µm.

Fig. 3.

Variation of cellular parameters measured in nail polish replicas of expanding leaf epidermis (A–C) and test for their dependence (D–F). Three cellular parameters are examined: cell surface area (A); relative growth rate in cell area (B); and nucleus section area (C). In box-type plots (A–C) all measurements for large groups of approx. 100 cells, made in the first replicas of all the leaves in a given genotype, are taken together. The relative growth rates between the first and the second replicas only are considered (B). In (C) measurements are from all the leaves in a given genotype, separate for pavement cells and stomata guard cells (GC). A solid line within each box represents the median. The box delimits the first and third quantiles. Whiskers extend from each end of the box to the adjacent values in the data as long as the most extreme values are within 1·5 times the interquartile range from the ends of the box. Crosses represent outliers, i.e. data with values beyond the ends of the whiskers. (D) Cell surface area is linearly dependent on the nucleus surface area both in Col (y = 28·98 x – 142·69; R² = 0·651) and in cycD3 (y = 34·94 x – 462·76; R² = 0·628). (E) The relative growth rate in area during the time interval directly preceding material fixation for the nucleus size measurement is not linearly dependent on the nucleus surface area, both in Col (R² = 0·108) and in cycD3 (R² = 0·028). (F) The relative growth rate in area (in the time interval between taking the first and the second replica), plotted with respect to the initial cell surface area, also shows no linear dependence either in Col (R² = 0·09) or in cycD3 (R² = 0·122). Stomatal guard cells or guard mother cells are excluded from this analysis.

Fig. 4.

Local spatial variation in relative growth rate in cell area computed from nail polish replicas, for large groups of cells from the distal (A–C) and proximal (D–F) portion of an expanding Col leaf (initial leaf lamina length was 1·4 mm). The time at which the replica was taken is given in the lower right corner of each map. The orientation of all maps is such that the leaf midrib is vertical and the apical direction points upward. Maps representing the relative growth rates are plotted on the cell pattern as it appeared at the beginning of the given time interval (A, B; D, E). In the last maps of each row the cell outlines only are plotted. In these maps, pairs of stomata guard cells are labelled with ‘x’. Exemplary groups of cells that differ in growth rate from surrounding cells are labelled with arrows. Scale bar = 100 µm.

Fig. 5.

Local spatial variation in relative growth rate in cell area computed from nail polish replicas, for large groups of cells from the distal (A–C) and proximal (D–F) portion of a expanding cycD3 leaf (1·7 mm long). Labelling as in Fig. 4. Scale bar = 100 µm.

Fig. 6.

Scanning electron micrographs (A–C) and growth parameter plots (D–K) computed from the sequence of epoxy resin replicas taken from the proximal portion of an expanding Col leaf (initial lamina length was 2·8 mm). The time at which the replica was taken is given in the lower right corner of each micrograph. Only cells for which the parameters were computed are shown in the micrographs. Colour maps representing growth parameters are plotted on the cell pattern as it appeared at the beginning of the given time interval. Growth parameters visualized in maps are: relative growth rate in approximated cell area (D, E); relative growth rate in length of straight line segments between two vertices joined by the same anticlinal cell wall (F, G); coefficient of variation (CV) of the growth rates in length of segments belonging to individual cells (H, I); directions of maximal growth rate and average cell growth anisotropy (J, K). Growth rates in length are plotted as maps in which the colour code is assigned to the considered line segment. In anisotropy plots, short line segments representing the direction of maximal growth are plotted on colour maps representing average cell growth anisotropy. An exemplary small group of cells and a single cell outlined in black change their areal growth rates in consecutive time intervals. Dots mark cells with similar growth anisotropy but a different CV. Scale bar = 50 µm.

Fig. 7.

Scanning electron micrographs (A–C) and growth parameter plots (D–K) for the sequence of replicas taken from the proximal portion of an expanding cycD3 leaf (1·7 mm initial length). Labelling as in Fig. 6. Exemplary adjacent cells of similar size that differ a lot in growth rates in area are labelled with asterisks; arrows point to an exemplary pair of cells exhibiting different values of the coefficient of variation despite the similar size. Scale bar = 50 µm.

Fig. 8.

Scanning electron micrographs (A–C) and growth parameter plots (D–K) for the sequence of replicas taken from the proximal portion of an expanding cycD3 leaf. This epidermis fragment originates from the same sequence of replicas as that shown in Fig. 7 but was located farther away from the leaf lamina base. Labelling as in Figs 6 and 7. Scale bar = 50 µm.