Auxin acts as a local morphogenetic trigger to specify lateral root founder cells

Abstract

Plants exhibit an exceptional adaptability to different environmental conditions. To a large extent, this adaptability depends on their ability to initiate and form new organs throughout their entire postembryonic life. Plant shoot and root systems unceasingly branch and form axillary shoots or lateral roots, respectively. The first event in the formation of a new organ is specification of founder cells. Several plant hormones, prominent among them auxin, have been implicated in the acquisition of founder cell identity by differentiated cells, but the mechanisms underlying this process are largely elusive. Here, we show that auxin and its local accumulation in root pericycle cells is a necessary and sufficient signal to respecify these cells into lateral root founder cells. Analysis of the alf4-1 mutant suggests that specification of founder cells and the subsequent activation of cell division leading to primordium formation represent two genetically separable events. Time-lapse experiments show that the activation of an auxin response is the earliest detectable event in founder cell specification. Accordingly, local activation of auxin response correlates absolutely with the acquisition of founder cell identity and precedes the actual formation of a lateral root primordium through patterned cell division. Local production and subsequent accumulation of auxin in single pericycle cells induced by Cre-Lox-based activation of auxin synthesis converts them into founder cells. Thus, auxin is the local instructive signal that is sufficient for acquisition of founder cell identity and can be considered a morphogenetic trigger in postembryonic plant organogenesis.

Fig. 1.

Auxin response-marked specification of lateral root founder cells precedes cell cycle activation in the pericycle. (A and B) DR5 activity throughout the lateral root formation starting from a presumptive founder cell (0); roman numbers are developmental stages in accordance with ref. . (A) Arabidopsis roots of the DR5rev::GFP line (n = 58). (B) Tomato roots of the DR5::GUS line (n = 46). Red arrowheads denote end walls of pericycle founder cells; black arrowheads denote periclinal cell walls. (A) CLSM images. (B) Nomarski optics. 0, merged Nomarski and CLSM; 0 and III, live unstained roots; I and II, live roots stained with neutral red; IV–VII, fixed roots. (C and D) Time-lapse analysis of live roots showing that pericycle founder cells are always accompanied by increased DR5 auxin response. Longitudinal unicellular (C) and bicellular (D) types of lateral root initiation. (Left) Pericycle cells at the beginning of the experiment. (Right) Images taken at the same focal plane 15 h later, showing primordia formed. Red arrowheads indicate end walls of founder cells. Yellow arrowheads indicate new cell walls formed. At the beginning of the experiments plants were 6 days old (C) and 7 days old (D). (E) DR5 activation in presumptive founder cell in 10-day homozygous alf4–1 mutant plant (n = 10); p, pericycle; px, protoxylem. (F) In Arabidopsis, DR5 can be activated in all pericycle cells by auxin treatment (10 μM NAA, 6 h); live roots were stained with neutral red. (Scale bars: A, B, and F, 20 μm; C–E, 25 μm.)

Fig. 2.

Heat shock-induced Cre/Lox based random iaaM expression. (A) Sectors of GUS-marked iaaM-expressing cells in root meristem induced by heat shock. (B) No sectors of GUS-marked iaaM expression were observed without the heat shock treatment. (C) LRP initiation was not affected by 30- or 45-min heat shock durations (Student's t test 2, P = 0.407), nor by 10- or 50-μM Trp treatments (Student's t test 1, P = 0.173). Simultaneous iaaM activation by 45-min heat shock and 50-μM Trp treatment led to a significant increase of LRP initiation over the heat shock alone (Student's t test 4, P < 0.00004) or Trp alone (Student's t test 3, P < 0.0003). The frequency of LRP initiation was scored 62 h after the heat shock in 10–15 seedlings per treatment (mean ± SE).

Fig. 3.

Local auxin production in pericycle cells triggers primordium formation. (A–E) Examples of sectors of GUS-marked iaaM-expressing cells induced by heat shock. (A) Sector of a single xylem-adjacent pericycle cell is shown. (B) After cell division two longitudinally abutted cells are formed; arrowhead indicates the new cell wall formed. (C) Such activation leads to formation of a fully stained primordium. (D and E) Activation of iaaM expression in only one of two longitudinally abutted cells can recruit the neighboring cell as the second founder cell and leads to staining of the progeny of only one founder cell (D) and development of a half-stained primordium (E). Arrowhead in D shows the position of end walls of a heat shock-activated and an independently specified pericycle founder cells. (F) Seedlings with random sectors of iaaM expression initiate 48% more primordia when auxin synthesis is induced by Trp treatment (black bar) as compared with untreated controls (white bar) (Student's t test; *, P < 0.0008). Accordingly, the proportion of GUS-positive primordia (indicating auxin-producing sectors) is 40% higher in Trp-treated seedlings (dotted black bar) as compared with untreated controls (dotted white bar). (G) Scoring of stained (iaaM expressing) and unstained primordia reveals that Trp treatment does not affect the iaaM-independent initiation of primordia, as the number of unstained primordia is not changed compared with the untreated controls (white bars) (Student's t test; *, P = 0.6,). The significant increase in the number of stained primordia (black bars) upon Trp treatment (Student's t test; **, P < 0.005) suggests that the local increase in the auxin level as a consequence of iaaM activity in pericycle sectors stimulates primordia initiation. The number of primordia was scored in 35–40 seedlings from two independent experiments (mean ± SE). (Scale bars: A–C, 25 μm; D and E, 40 μm.)

Fig. 4.

Random activation of IaaM leads to increased frequency of defects in LRP positioning. (A) In non-Trp-treated control, IaaM activated pericycle cell in close proximity of other LRP at the same xylem pole without LRP iniation. (B) In Trp-treated seedlings coincidental activation of IaaM in pericycle cell in close proximity of other LRP at the same xylem pole results in primordia initiation. Arrowheads indicate pericycle end walls. A total of 30–35 seedlings were analyzed 48 h after heat shock incubation. (Scale bar: 55 μm.)