Auxin regulates the initiation and radial position of plant lateral organs

Abstract

Leaves originate from the shoot apical meristem, a small mound of undifferentiated tissue at the tip of the stem. Leaf formation begins with the selection of a group of founder cells in the so-called peripheral zone at the flank of the meristem, followed by the initiation of local growth and finally morphogenesis of the resulting bulge into a differentiated leaf. Whereas the mechanisms controlling the switch between meristem propagation and leaf initiation are being identified by genetic and molecular analyses, the radial positioning of leaves, known as phyllotaxis, remains poorly understood. Hormones, especially auxin and gibberellin, are known to influence phyllotaxis, but their specific role in the determination of organ position is not clear. We show that inhibition of polar auxin transport blocks leaf formation at the vegetative tomato meristem, resulting in pinlike naked stems with an intact meristem at the tip. Microapplication of the natural auxin indole-3-acetic acid (IAA) to the apex of such pins restores leaf formation. Similarly, exogenous IAA induces flower formation on Arabidopsis pin-formed1-1 inflorescence apices, which are blocked in flower formation because of a mutation in a putative auxin transport protein. Our results show that auxin is required for and sufficient to induce organogenesis both in the vegetative tomato meristem and in the Arabidopsis inflorescence meristem. In this study, organogenesis always strictly coincided with the site of IAA application in the radial dimension, whereas in the apical-basal dimension, organ formation always occurred at a fixed distance from the summit of the meristem. We propose that auxin determines the radial position and the size of lateral organs but not the apical-basal position or the identity of the induced structures.

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. ; .

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. ; .

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. .

Figure 4.

Phyllotaxis in Pins Recovering from NPA. (A) Primordium initiation on an NPA pin 3 days after transfer to NPA-free medium. (B) to (E) Phyllotaxis on apices 7 days after transfer to NPA-free medium (n = 60). Shown are distichous phyllotaxis (B), spiral phyllotaxis (C), whorled phyllotaxis (D), and reversion from whorled to spiral phyllotaxis (E). A, axillary meristem; M, apical meristem; P, primordia initiated concomitantly; P₁, P₂, P₃, successive primordia, with P₁ being the youngest. .

Figure 5.

Leaf and Inflorescence Phenotype of the Arabidopsis pin1-1 Mutant and Induction of Flowers by IAA. (A) Wild-type Arabidopsis seedling with two leaf primordia. (B) pin1-1 seedling with one fused leaf primordium. (C) pin1-1 seedling with a cup-shaped leaf primordium. (D) Inflorescence apex of a pin1-1 mutant plant devoid of flowers. The arrowhead indicates the meristem. (E) to (H) Induction of flowers by treatment with 1 mM IAA (red paste) at the flank. Apices were analyzed 38 hr (arrow points to local bulge) (E), 4 days (F), 7 days (G), and 11 days (H) after treatment. Arrowheads in (E) to (H) denote the meristem. (I) Induction of a circular bulge (arrow) around the flank by treatment for 2 days with 1 mM IAA at the top of the meristem (arrowhead). (J) Circular flower induced after 6 days by treatment as given in (I). Note that the circular flower does not correspond to a single normal flower but rather is a ring of several flowers that are laterally fused; consequently, petaloid organs are found on the adaxial side of the circular carpel. In the center, the indeterminate inflorescence meristem is maintained (not visible). (K) Flowers (arrows) induced after 4 days by treatment as given in (I); in this case, the initial ring bulge broke into several individual flowers. Two of these are partially fused (on the lower left side). C, carpel; Co, cotyledon; F, flower primordium; P, petal; Pa, papilla; Pr, leaf primordium. ; ; .