Auxin Regulates the Initiation and Radial Position of Plant Lateral Organs

Inhibition of Polar Auxin Transport Blocks Leaf Initiation but Not Meristem Propagation

Vegetative tomato shoot apices were cultured on a synthetic medium containing the auxin transport inhibitor N-1-naphthylphthalamic acid (NPA). This treatment completely inhibited the formation of new leaf primordia, whereas the meristem continued to grow and form stem tissue, leading to a pinlike structure devoid of leaves (cf. Figures 1A and 1B with Figure 1C). Preexisting leaf primordia continued to develop, but leaflets were not initiated and the leaf blade was deformed (Figure 1A, P₁ and P₂). Note that at the beginning of the experiment, the preexisting primordia of the apex shown in Figure 1A (P₁ and P₂) were at the stages of P₁ and P₂ shown in Figure 1C. Axillary meristems grew out (Figure 1A), indicating that apical dominance was reduced. The structurally unrelated auxin transport inhibitors 2,3,5-triiodobenzoic acid (TIBA) and 9-hydroxyfluorene-9-carboxylic acid (HFCA) gave similar results, but the flavonoids quercetin and apigenin, which are putative endogenous regulators of auxin efflux (Jacobs and Rubery, 1988), had no effect. Although 10 μM NPA always blocked primordium formation, elongation of the pins was variable. Approximately 50% of the pins grew to only a few hundred micrometers long and then ceased to grow, whereas the remaining pins recovered and grew as much as several millimeters. For further studies, these vigorously growing pins were transferred weekly to new plates containing 10 μM NPA, at which time the base of the pins was trimmed to leave ∼200 μm of stem length.

The pinlike structures, further referred to as NPA pins, exhibited small, densely staining cells at the tip that were arranged in layers as in control meristems (cf. Figures 1D and 1H; Steeves and Sussex, 1989). The tip of the NPA pins expressed the ribosomal protein Rpl2, a general marker for biosynthetic activity (Figure 1E; Fleming et al., 1993). Similarly, LeT6, a gene encoding a homeobox protein that is expressed in meristems (Chen et al., 1997), was highly expressed in the same tissue (Figure 1F). Histone H4, which is a marker for cells during S phase (Fleming et al., 1993; Brandstätter et al., 1994), was expressed only in the most apical cells of the pins, whereas in control apices, histone H4 was also detected in the meristem flanks (Figures 1G and 1K). Because Rpl2, LeT6, and histone H4 are expressed in meristems (Figures 1I to 1K), their presence indicates that NPA pins contain meristematic tissues at the tip. Cortical stripes that stained with toluidine blue (Figures 1D and 1H) and express Rpl2 (Figures 1E and 1I) presumably represent provascular strands and therefore indicate that the basic stem anatomy in NPA pins was not affected. Thus, auxin transport inhibitors specifically inhibit primordium initiation but not meristem self-perpetuation or formation of stem tissues.

Local Treatment with Indole-3-Acetic Acid Induces Leaf Primordia on NPA Pins

Although auxin is thought to be produced in the shoot apex, whether it is produced in the meristem itself is not known (Davies, 1995). If auxin is produced in the meristem, then NPA would be expected to cause accumulation of auxin within the meristem, and any resulting inhibition of leaf initiation could be attributed to inhibitory concentrations of auxin. If auxin is not produced in the meristem but is transported there from subtending tissues by an NPA-sensitive transporter, then NPA treatment would deplete auxin in the meristem, and inhibition of organ formation would result from the decrease in auxin concentrations in the meristem. In the latter case, application of auxin to the meristem would be expected to rescue organogenesis.

We applied small amounts (estimated as <5% of the volume of the meristem) of lanolin paste containing 0.1 to 30 mM indole-3-acetic acid (IAA) to various positions on the NPA pins, particularly on the flank and tip of the meristem. By taking into account retention in lanolin and photooxidation and diffusion in the tissue, we can reasonably expect these applied concentrations to cover the physiologic concentration range of IAA (Uggla et al., 1996). Treatment with auxin at the flank of the meristems induced the formation of bulges, whereas lanolin alone had no effect (Figures 2A and 2B, and Table 1). These bulges, which always coincided with the radial position of IAA application, differentiated into leaf primordia (Figures 2C and 2D). In the longitudinal (apical–basal) dimension, the primordia always formed at a fixed position from the summit, that is, within a ring-shaped zone roughly equivalent to the peripheral zone in untreated meristems (Steeves and Sussex, 1989; Lyndon, 1990, 1998).

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Figure 1.

Inhibition of Leaf Primordium Initiation by the Auxin Transport Inhibitor NPA and Expression of Diagnostic Genes in NPA-Treated Apices.

(A) Culture of tomato apices on NPA-containing medium results in a naked pin (arrowhead), outgrowth of axillary meristems (A), and altered development of preexisting primordia (P₁ and P₂, with P₁ being the youngest primordium at the beginning of the experiment).

(B) Close-up of an NPA pin visualized by low-vacuum scanning electron microscopy.

(C) Control tomato apex with the meristem (arrowhead) and the three youngest leaf primordia (P₁ to P₃, with P₁ being the youngest), as well as the leaf bases of older primordia (P₄ and P₆).

(D) to (G) Longitudinal sections of NPA pins.

(H) to (K) Longitudinal sections of control apices.

(D) and (H) show staining with toluidine blue. (E) to (G) and (I) to (K) are the result of in situ hybridizations. (E) and (I) show hybridization with a probe against Rpl2, which encodes a ribosomal protein. (F) and (J) show hybridization with a probe against the homeobox gene LeT6. (G) and (K) are the result of hybridization with a probe against histone H4. Arrowheads indicate the meristem; L, leaflet primordium; P, primordium. Bar in (A) = 2 mm; bars in (B) to (K) = 100 μm.

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Figure 2.

Induction of Leaves on NPA Pins by the Natural Auxin IAA.

(A) NPA pin 4 days after treatment with control paste (white arrowhead) at the flank of the meristem (black arrowhead).

(B) to (D) Induction of a leaf primordium (arrows in [B] to [D]; P in [D]) on an NPA pin treated with red lanolin paste containing 10 mM IAA (white arrowheads in [B] to [D]). Black arrowheads point to the meristem. The same pin was photographed after 1 day (B), 2 days (C), and 4 days (D).

(E) Leaf induced by IAA on an NPA pin.

(F) to (K) Leaf primordia induced on NPA pins 1 week after local IAA treatment (red paste) at concentrations of 0.1, 0.3, 1, 3, 10, and 30 mM, respectively.

M, meristem; P, primordium. Bars in (A) to (D) and (F) to (K) = 100 μm; bar in (E) = 0.5 cm.

When auxin was applied to the very tip of the meristems, bulges never initiated at the tip but rather emerged at the flank at unpredictable radial positions (data not shown). The primordia induced by IAA exhibited dorsoventrality, turned green, and formed the trichomes typical for leaves (Figures 2C to 2E). At later stages, lateral leaflets were formed in ∼50% of the cases; after 2 months, the leaves had reached a length of several centimeters and exhibited all of the structures found in normal leaves (Figure 2E). However, the proportions of the different parts deviated from those of normal leaves; in particular, the petiole was thicker than normal, and only one pair of leaflets was formed (Figure 2E). The size of induced leaf primordia depended on the concentration of auxin (Figures 2F to 2K). Whereas lower concentrations (0.1 and 0.3 mM) induced primordia of approximately normal size (Figures 2F and 2G), higher concentrations (1 to 30 mM) induced primordia that were wider than normal (Figures 2H to 2J) and, in some cases, encompassed the entire meristem (Figure 2K). The frequency of primordium initiation was also dependent on auxin concentration (Table 1).

These experiments show that the NPA pins contain functional meristems capable of leaf formation when supplied with auxin. The site of auxin treatment determined the site of primordium formation in the radial position, but in the longitudinal position, primordia always originated from a ring-shaped region at the flanks of the meristem, as in natural leaf formation. The concentration of auxin determined the number of cells recruited into primordia.

Various growth regulators that have been shown to control cell division and cell expansion in other systems are therefore candidates for a role in primordium formation. We applied gibberellin A₃, the kinetin 6-benzylaminopurine, and brassinolide to the flanks of the meristems of NPA pins. In addition, the effects of fusicoccin, a compound that mimics some aspects of IAA-induced growth by activating H⁺ extrusion into the cell wall (Marrè, 1979), were tested. In contrast to IAA, none of these substances was able to induce primordium formation (Table 2). Thus, induction of primordium formation by IAA on NPA pins is a specific effect.

NPA and IAA Interfere with Primordium Positioning at the Meristem

If local differences in auxin concentration determine leaf position, then it should be possible to disrupt phyllotaxis in a normal meristem by manipulating the auxin concentrations present with exogenous auxin as well as with auxin transport inhibitors. With this in mind, we applied auxin or auxin transport inhibitors to the I₁ and I² positions of normal meristems, that is, at the positions where the next two primordia would form (Figure 3A), or to the entire flank of the meristem. Application of IAA to I₁, the site of incipient primordium formation, increased the size of the primordium to be formed at this position (cf. Figures 3B and 3C; see Table 3). Specifically, the base of the primordium was enlarged, often to an extent that it was thicker than the next older primordium, suggesting that more cells were engaged in primordium initiation than normal. Treatment at I₂ (between P₁ and P₂) caused the initiation of ectopic primordia (Figure 3D and Table 3), which were fused to various degrees with P₁. When the entire flank of the meristem was treated with IAA, oversized fused primordia were induced (Figure 3E and Table 3). Note that in Figures 3D and 3E, tissues on different sides of P₁ were induced to form primordia. In Figure 3D, these tissues were in the space between P₁ and P₂, whereas in Figure 3E, they were in the space between P₁ and I₁. This indicates that on both sides of P₁, the flank is able to form primordia if supplied with auxin. However, in the proximity of P₂, the flank tissue did not respond to IAA; consequently, whereas ectopic primordia were always fused to some extent with P₁, they were never fused with P₂ (Figures 3D and 3E). Perhaps at stage P₂, primordia are surrounded by an inhibitory field (Lyndon, 1990).

To determine the effects of local inhibition of auxin transport within the meristem, apices were treated with NPA at I₁. Primordium formation was inhibited at the site of treatment (Figure 3F), whereas on the opposite side of the meristem, that is, at I₂, a primordium was initiated, resulting in a reversal of phyllotaxis. The auxin transport inhibitors 2,3,5-triiodobenzoic acid and 9-hydroxyfluorene-9-carboxylic acid had similar effects, whereas apigenin and quercetin had no effects (Table 3). Taken together, auxin and NPA both affected organ positioning but had opposite effects.

Phyllotaxis in Recovering NPA Pins Is Variable

Leaf positioning is known to be affected by preexisting leaf primordia (Snow and Snow, 1931, 1933; Callos and Medford, 1994). The NPA pins are devoid of leaves and therefore provide a simple system with which to test the role of preexisting leaves. After 5 weeks of culture on NPA and repeated removal of the basal stem portion, the pins lacked any trace of leaves. Three days after transfer to synthetic medium without NPA, pins spontaneously initiated new primordia (Figure 4A). These always originated from the flank of the meristem. Primordium formation continued and resulted in distichous (Figure 4B; 30%), spiral (Figure 4C; 18%), or whorled phyllotaxis (Figure 4D; 27%); the remaining pins either were fasciated or did not recover at all (n = 60). Interestingly, recovering apices returned to spiral phyllotaxis after production of a few whorled leaves (Figure 4E). In general, the apices initiated axillary meristems soon after primordium formation (Figures 4B and 4C). These results show that preexisting leaves are not required for primordium formation per se but for correct phyllotactic patterning. Both the occurrence of distichous phyllotaxis and the symmetry observed in whorled phyllotaxis (Figures 4B and 4D) suggest the involvement of lateral inhibition in the patterning of the plant apex (Lyndon, 1990; Meinhardt, 1994).

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Figure 3.

Effects of Treatments of Meristems with IAA and NPA.

(A) Schematic representation of a tomato apex with the youngest and the second youngest primordia (P₁ and P₂, respectively). The large circle delimits the apical meristem, and the small circle marks the central zone, which is not involved in organogenesis. The sites of incipient primordium formation (I₁) and the site of the following primordium (I₂) are indicated as yellow areas.

(B) Control treatment with lanolin (red paste) at the site of incipient primordium formation (I₁). The primordium that formed at the I₁ position has a normal size relative to P₁ (cf. with Figure 1C).

(C) Increased primordium size caused by IAA treatment (red paste) at I₁. P₁ denotes the preexisting primordium. Note that the base of I₁ is extended toward P₁.

(D) Induction of an ectopic primordium by IAA (red paste) at the I₂ position (between preexisting primordia P₁ and P₂). The ectopic primordium is fused to P₁.

(E) Induction of a large fused leaf primordium caused by IAA treatment (red paste) on the entire flank of the meristem. The fused leaf includes P₁ (right side) and I₁ (left side). P₂ (which would have been toward the viewer) was removed before scanning electron microscopic analysis.

(F) Inhibition of primordium initiation by NPA treatment at I₁ (red paste) and formation of an ectopic primordium at I₂ (arrow, between preexisting primordia P₁ and P₂).

Apices shown in (B) to (D) were examined 3 days after treatment; apices shown in (E) and (F) were analyzed 6 days after treatment. Bars in (B) to (F) = 100 μm.

Local Treatment with IAA Induces Flower Primordia on Inflorescence Apices of the Arabidopsis Mutant pin1-1

The effects of NPA on tomato apices resemble the phenotype of the Arabidopsis pin1-1 mutant (Okada et al., 1991). This mutant is affected in auxin transport in the inflorescence stem, the genetic defect for which was recently traced to a putative auxin efflux carrier (Gälweiler et al., 1998). The pin1-1 mutation affects leaf formation and development of the inflorescence. Two aspects of the mutant phenotype are particularly striking in the context of our experiments with tomato apices. In pin1-1 plants, leaves are often fused or cup shaped and are oversized from the initial stages of development (cf. Figure 5A with Figures 5B and 5C), suggesting that the number of cells recruited is larger than normal. This leaf phenotype resembles the leaves triggered on NPA pins by IAA at higher concentrations (Figures 2I to 2K). Second, mutant inflorescences are greatly reduced to a pinlike shoot that resembles the pins produced by tomato meristems in the presence of NPA (Okada et al., 1991; cf. Figures 1B and 5D).

In light of our results, the phenotype of the pin1-1 mutant might be caused by abnormally high amounts or mislocalization of auxin in vegetative meristems, leading to induction of broad leaf primordia, and by the lack of auxin in the apical inflorescence meristem, which prevents flower formation. To test this hypothesis, we applied auxin in lanolin paste to the flanks of inflorescence meristems of pin1-1 mutants. This treatment induced the formation of flower primordia at the site of treatment (85%; n = 80). In control treatments with lanolin, primordium formation was observed in only two cases (n = 57). A low frequency of spontaneous organ formation may be expected because pin1-1 mutant plants occasionally form aberrant flowers after the pins have reached a length of several centimeters (Okada et al., 1991). Therefore, for all further analyses of auxin-induced flower formation, we used young pins <1 cm long just after their emergence from the rosette. In such young pins, spontaneous organ formation was never observed.

Treatment with 1 mM IAA at the flank of pin1-1 apices induced bulges at the site of treatment after 38 hr (Figure 5E and Table 4), and the bulges later developed into flower primordia (Figure 5F). These flowers initiated two to four whorls of organs; after a week, they had reached a length of ∼0.5 mm (Figure 5G). Although the flowers reached normal sizes, their architecture was aberrant. Each had a large carpel with papillae at the tip surrounded by a variable number of petaloid organs (Figure 5H). Similar defects have been described for the flowers of Arabidopsis wild-type plants that had been treated with auxin transport inhibitors and in the escape flowers in pin1-1 mutants (Okada et al., 1991).

Treatments with 0.1 mM IAA at the flank had similar effects, but the frequency was less and the induced flowers were smaller than those developing after treatment with 1 mM IAA (Table 4). Application of IAA at 10 μM had no effect (Table 4). Treatment with 1 mM IAA at a position ∼100 μm below the flank induced only small bulges at a reduced frequency, and treatments 200 μm below the flank had no effect (Table 4), indicating that the maximal range of auxin diffusion in the apical tissues is between 100 and 200 μm. When Arabidopsis inflorescence pins were treated with 1 mM IAA on the summit of the meristem, ring-shaped bulges were induced around the flank of the entire meristem (Figure 5I and Table 4); however, the center of the meristem never responded to IAA treatment. At later times, the ring-shaped bulges developed into circular flowers that consisted of a large, ring-shaped carpel with petaloid organs both inside and outside the carpel (Figure 5J), or the ring broke into discrete flowers arranged in a circle (Figure 5K). Treatments with 0.1 mM IAA on the top resulted in the induction of incomplete ring-shaped bulges (Table 4); application on the top of IAA at 10 μM had no effect (Table 4).