Spatial and temporal analyses of expansion and cell cycle in sunflower leaves. A common pattern of development for all zones of a leaf and different leaves of a plant

Abstract

We have investigated the spatial distributions of expansion and cell cycle in sunflower (Helianthus annuus L.) leaves located at two positions on the stem, from leaf initiation to the end of expansion. Relative expansion rate (RER) was analyzed by following the deformation of a grid drawn on the lamina; relative division rate (RDR) and flow-cytometry data were obtained in four zones perpendicular to the midrib. Calculations for determining in situ durations of the cell cycle and of S-G2-M in the epidermis are proposed. Area and cell number of a given leaf zone increased exponentially during the first two-thirds of the development duration. RER and RDR were constant and similar in all zones of a leaf and in all studied leaves during this period. Reduction in RER occurred afterward with a tip-to-base gradient and lagged behind that of RDR by 4 to 5 d in all zones. After a long period of constancy, cell-cycle duration increased rapidly and simultaneously within a leaf zone, with cells blocked in the G0-G1 phase of the cycle. Cells that began their cycle after the end of the period with exponential increase in cell number could not finish it, suggesting that they abruptly lost their competence to cross a critical step of the cycle. Differences in area and in cell number among zones of a leaf and among leaves of a plant essentially depended on the timing of two events, cessation of exponential expansion and of exponential division.

Figure 1

Spatial and temporal changes in morphology and in expansion of leaf 8. A to D, Photographs at the time of initiation on the apex (A, bar = 0.1 mm), on d 7 (B, bar = 1 mm), and 13 (C, bar = 5 mm) after initiation, and at the end of expansion (D, bar = 10 mm). The deformation of the grid of points drawn on the lamina can be seen by comparing C and D. E, Spatial distribution of RER calculated in 120 triangles on d 14 (left) and 18 (right). F, Mean cell area as a function of the distance to the leaf tip (expressed as the percentage of total leaf length) on eight dates after initiation: d 9, ▪; d 11, ♦; d 13, ▴; d 15, •; d 17, □; d 19, ⋄; d 21, ▵; and d 23 ○.

Figure 2

Spatial analyses of RER and RDR in the leaf. A, Grid of 70 points was drawn on the lamina, defining 120 triangles for calculation of local RERs. Coordinates of triangle vertices were defined in a system with the origin at the point of petiole insertion, the y axis along the midrib, and the x axis perpendicular to it. RDR was analyzed in four zones perpendicular to the midrib, T, MT, MB, and B, making up a series of triangles as indicated. B, Calculation of the area corresponding to transect i, for calculation of mean cell area per leaf. It was calculated as the area of the trapezoid defined by transects i − 1 and i + 1, and the edges of the leaf. W_i, Leaf width at the y coordinate of transect i.

Figure 4

Change with time in epidermal cell number in the whole leaf and in zones drawn on the lamina of leaves 8 (A) and 16 (B). Corresponding changes with time in RDR in the whole leaf and in the leaf zones are shown in C and D. Insets, Logarithmic representation of change with time in leaf cell number for leaves 8 (A) and 16 (B). For better legibility, leaf RDR is shown only during the period while cell division is exponential. Symbols are as in Figure 3.

Figure 3

Change with time in the area of the whole leaf and of zones drawn on the lamina of leaves 8 (A) and 16 (B). Corresponding changes with time in RER in the whole leaf and in the leaf zones are shown in C and D. Insets, Logarithmic representations of changes with time in leaf area for leaves 8 (A) and 16 (B). Symbols represent either the whole leaf (○) or one of the four zones, B (▪), MB (♦), MT (□), and T(▵), as shown in Figure 2. For better legibility, intervals of confidence at 0.95 are presented every 2nd d for the whole leaf or at the end of expansion for each zone. Whole-leaf RER is shown only during the period while leaf expansion is exponential. Dotted lines link RER of the whole leaf at the end of this period to the first measured RER in B.

Figure 5

Change with time in epidermal cell area in zones of leaves 8 (A) and 16 (B). Symbols are as in Figure 3. Intervals of confidence at 0.95 are presented every 2nd d for better legibility.

Figure 6

Frequency distribution of epidermal cell area in leaf 8 on d 9 or after the period with exponential increase in cell number (d 15 and 19 after initiation). A to C present the frequency distribution analyzed over the whole leaf. D to F present that analyzed in zone B only. Note that scales of x axes differ among the panels.

Figure 7

In situ analysis of cell cycle. A and B show changes with time in the duration of the cell cycle, as calculated in Equation 9, in leaves 8 (left) and 16 (right) and in zones drawn on the lamina of both leaves. C and D show changes with time in the percentage of nuclei in the S-G2-M phase analyzed by flow cytometry. E and F show changes with time in the duration of the S-G2-M phase as calculated in Equation 13. Symbols are as in Figure 3. The oblique dotted lines in A and B represent the times that remain available before completion of division in the base of the leaf. When duration of the cell cycle exceeds this limit, a mean cell in zone B will not have time to complete its cycle. Intervals of confidence at 0.95 are presented for flow-cytometry data.