Lateral root initiation in Arabidopsis: developmental window, spatial patterning, density and predictability

Abstract

Background and aims: The basic regulatory mechanisms that control lateral root (LR) initiation are still poorly understood. An attempt is made to characterize the pattern and timing of LR initiation, to define a developmental window in which LR initiation takes place and to address the question of whether LR initiation is predictable.

Methods: The spatial patterning of LRs and LR primordia (LRPs) on cleared root preparations were characterized. New measures of LR and LRP densities (number of LRs and/or LRPs divided by the length of the root portions where they are present) were introduced and illustrate the shortcomings of the more customarily used measure through a comparative analysis of the mutant aux1-7. The enhancer trap line J0121 was used to monitor LR initiation in time-lapse experiments and a plasmolysis-based method was developed to determine the number of pericycle cells between successive LRPs.

Key results: LRP initiation occurred strictly acropetally and no de novo initiation events were found between already developed LRs or LRPs. However, LRPs did not become LRs in a similar pattern. The longitudinal spacing of lateral organs was variable and the distance between lateral organs was proportional to the number of cells and the time between initiations of successive LRPs. There was a strong tendency towards alternation in LR initiation between the two pericycle cell files adjacent to the protoxylem poles. LR density increased with time due to the emergence of slowly developing LRPs and appears to be unique for individual Arabidopsis accessions.

Conclusions: In Arabidopsis there is a narrow developmental window for LR initiation, and no specific cell-count or distance-measuring mechanisms have been found that determine the site of successive initiation events. Nevertheless, the branching density and lateral organ density (density of LRs and LRPs) are accession-specific, and based on the latter density the average distance between successive LRs can be predicted.

Fig. 1.

Expression pattern of green fluorescent protein (GFP) in the enhancer trap line J0121 observed on roots orientated in the protoxylem plane. (A) Root apical meristem. (B) Portion of the elongation zone; note, in the upper root portion expression is detected in the pericycle (4th cell layer). (C–I) Root portions in the differentiation zone. (D–F) Stages I, II and III of lateral root primordium development, respectively. (G) Stage IV of primordium development. (H) Recently emerged lateral root. (I) Junction between primary and lateral root. Before observations, roots of 8 d (A, B, D–H), 16 d (C) and 30 d (I) after germination were divided into 5–10-mm portions and then stained with propidium iodide. Green, GFP fluorescence; red, propidium iodide. Scale bars: A, I = 50 µm; B–H = 20 µm.

Fig. 2.

Summary of the experimental method for the mapping of lateral root formation. (A) Diagram illustrating the experimental method allowing for analysis of the same root portion (blue rectangles) at day 8 and later at day 15. (B) At day 8, live roots (right) were examined and lateral roots and lateral root primordia were visualized using a combination of epifluorescence and Nomarski optics (green insets). Schematic representations (left) were rendered of each root including inter-lateral organ distance (mm) and zonation. Red dot indicates the presence of a stage I primordium. (C) At day 15, live roots were examined again and primordium initiation and lateral root formation were analysed. Scale bars = 200 µm (for root portion) and 50 µm (for all green insets).

Fig. 3.

Roots of J0121 subjected to plasmolysis with 1 m sorbitol. Root in protoxylem (A) and protophloem (B) plane. (A) Merged image of green fluorescent protein (GFP) fluorescence and Nomarski bright-field; note, plasmolysed cells in epidermis (ep) and cortex (c). Arrowheads indicate approximate position of pericycle end-walls. Scale bars = 50 µm.

Fig. 4.

Correlation analysis of interactions between inter-lateral organ distance and time required for initiation of successive lateral organs, between inter-lateral organ distance and the rate of root growth at time of initiation (C24, Col-0 and J0121), and between inter-lateral organ distance and the number of pericycle cells (J0121). All data are shown and regression lines are drawn. Equations and correlation coefficients, and number of measurements (n) are given.

Fig. 5.

Histogram of the distribution of the relative length of the branching root zone (%) in 8-d Col-0 plants (n = 101). The relative length of this zone was calculated for each plant as a percentage of the total root length.

Fig. 6.

Changes in the density of lateral organs in J0121, C24, Col-0 and aux1-7 plants 8 and 16 d after germination. Customary density was calculated as the number of emerged lateral roots divided by the total root length. The lateral organ density (LOR) was calculated as the number of all lateral organs divided by the root portion where they are present. The branching density (LR-Z1) was calculated as the number of emerged lateral roots divided by the length of the root portion where LRs are emerged (zone one). The density of primordia within this zone (LRP-Z1) was calculated as the number of primordia within the zone divided by the length of the zone. The density of primordia within zone two (LRP-Z2) was calculated as the number of primordia within the zone (see Fig. 2B) divided by the length of the zone. Combined data of at least two independent experiments; n is indicated in each case.