KNAT1 and ERECTA regulate inflorescence architecture in Arabidopsis

Abstract

Plant architecture is dictated by morphogenetic factors that specify the number and symmetry of lateral organs as well as their positions relative to the primary axis. Mutants defective in the patterning of leaves and floral organs have provided new insights on the signaling pathways involved, but there is comparatively little information regarding aspects of the patterning of stems, which play a dominant role in architecture. To this end, we have characterized five alleles of the brevipedicellus mutant of Arabidopsis, which exhibits reduced internode and pedicel lengths, bends at nodes, and downward-oriented flowers and siliques. Bends in stems correlate with a loss of chlorenchyma tissue at the node adjacent to lateral organs and in the abaxial regions of pedicels. A stripe of achlorophyllous tissue extends basipetally from each node and is positioned over the vasculature that services the corresponding lateral organ. Map-based cloning and complementation studies revealed that a null mutation in the KNAT1 homeobox gene is responsible for these pleiotropic phenotypes. Our observation that wild-type Arabidopsis plants also downregulate chlorenchyma development adjacent to lateral organs leads us to propose that KNAT1 and ERECTA are required to restrict the action of an asymmetrically localized, vasculature-associated chlorenchyma repressor at the nodes. Our data indicate that it is feasible to alter the architecture of ornamental and crop plants by manipulating these genetically defined pathways.

Figure 1.

Inflorescence Phenotypes of Ler and bp-2 Plants. (A) Wild-type Ler inflorescence exhibiting upright flowers and a long pedicel. (B) bp-2 inflorescence exhibiting downward-pointing flowers and siliques and very short pedicels. (C) and (D) bp-2 plants exhibiting bends at nodes (arrowhead) and stripes of achlorophyllous tissue (arrows).

Figure 2.

Anatomy of Lan and Ler Stems. (A) Toluidine blue–stained cross-section of the proximal end of a Lan pedicel. The abaxial side is oriented toward the bottom. (B) Hand section through a Lan floral node showing radial asymmetry. Arrowheads point to regions in which chlorenchyma development is repressed. (C) Hand section above a Lan vegetative node illustrating a gap in chlorenchyma tissue directly above the lateral organ. AS, axillary stem; CL, cauline leaf. (D) Toluidine blue–stained cross-section of a Ler internode. Note that cortical cells contain numerous chloroplasts and that supporting sclerenchyma tissue is abundant. (E) Toluidine blue–stained cross-section at the proximal end of a Ler pedicel with the abaxial side oriented toward the bottom. (F) Hand section through the base of a Ler vegetative node showing that the region of achlorophyllous tissue includes stem regions at the base of lateral organs. The leaf base is indicated by the arrow. T, trichome.

Figure 3.

Morphological Analysis of bp-2. (A) Hand section above a vegetative node. Note that the region of achlorophyllous tissue is expanded relative to Lan (cf. with Figure 2C). (B) Hand section through an internode. Arrows indicate reductions in cortical chlorenchyma over two vascular bundles. (C) Hand section through a pedicel with the abaxial region at the bottom. (D) Toluidine blue–stained cross-section of an internode. The stripe region is defined by the arrows (cf. with Figure 2D). The epidermal cells are narrower, and chloroplast density is reduced in all cortical cell layers. Note that the stripe occurs over a vascular bundle. (E) Toluidine blue–stained pedicel cross-section. The abaxial region is located at the bottom, and chloroplasts and intercellular spaces are scarce (cf. with Figure 2E). (F) Safranin-stained stem epidermis showing a stripe that is devoid of stomata surrounded by nonstripe regions containing stomata. Black horizontal lines delimit the region of the stripe. (G) Subepidermal (L2) layer of (F). Note the large block-like cells underlying the stripe and the lack of intercellular space. In contrast, the shadows of stomata can be seen in the nonstripe areas, cells in this area are smaller and rounder, and considerable intercellular space exists. (H) Scanning electron micrograph of a bp stem illustrating the files of cells that constitute the stripe (arrow). (I) Scanning electron micrograph of a bp pedicel/stem junction showing files of cells on the pedicel abaxial surface. (J) Pedicel/stem junction of a Ler plant showing the abaxial surface of the pedicel. (K) bp/ER plant showing that a wild-type ER gene suppresses the severity of the pedicel angle. (L) Hand section through a bp/ER pedicel showing continuous distribution of mature chlorenchyma (cf. with Figure 3C) tissue. The abaxial side is oriented toward the bottom. (M) Toluidine blue–stained cross-section of a bp/ER pedicel. Bar in (H) = 400 μm; bars in (I) and (J) = 100 μm.

Figure 4.

Stripes Are Associated with Specific Underlying Vascular Bundles. (A) Left, diagram of a stem showing a node with its associated lateral organ. Right, hand sections of a bp stem corresponding to regions shown in the diagram. The top section is oriented with the stripe-associated lateral organ at the top. Subsequent sections are oriented similarly, which can be confirmed by observing the arrangement of vascular bundles. The position of a second stripe, initiated from a superior node, is marked by arrowheads. (B) Hand section through an internode of a Ler plant illustrating a continuous ring of chlorenchyma tissue.

Figure 5.

Positional Cloning of the BP Gene and Molecular Analysis of Five Mutant Alleles. (A) Summary of mapping data and chromosomal location of the BP locus. A scheme of chromosome 4 is shown at the top. Simple sequence length polymorphism (nga8, nga12) and restriction fragment length polymorphism (mi167) markers and their respective map positions are shown along with the bacterial artificial chromosome (BAC) tiling path and the number of recombinants determined by cleaved-amplified polymorphic sequence (CAPS) analysis (see Methods). Polymerase chain reaction and DNA gel blot analysis of Ler and mutant DNAs enabled us to localize the deletions to an area containing three genes (KNAT1, a putative xylan endohydrolyase, and a putative inositol 1,3,4-triphosphate 5/6 kinase-like gene). (B) DNA gel blot analysis of genomic DNA from wild-type Columbia (Col) and Landsberg erecta (Ler) and five bp mutants probed with a KNAT1 gene probe. The KNAT1 gene is deleted in bp-1, bp-2, bp-3, and bp-5 and has suffered a point mutation in bp-4 (Table 2). The fainter signal presumably is the result of cross-hybridization with other conserved homeodomain genes and provides a useful loading control.

Figure 6.

Expression of BP during Development and in Mutant Backgrounds. BP expression was examined in wild-type plants by both in situ hybridization ([A], [B], [D], [H], and [I]) and GUS staining of plants transformed with a BP::GUS transgene ([C], [E], [F], and [G]). (A) Late globular-stage embryo illustrating expression in cells destined to become the hypocotyl. (B) Late heart-stage embryo. BP expression also is observed below the shoot apical meristem. (C) GUS staining of a late torpedo-stage embryo showing intense hypocotyl staining. (D) Longitudinal section of an early torpedo embryo revealing expression at the base of leaf primordia. (E) GUS staining of an inflorescence apex. BP is expressed in pedicels, and faint staining can be observed in style cells of the gynoecium. (F) GUS staining of cortical tissue in an internode cross-section. (G) Cross-section through a gynoecium showing GUS staining of marginal tissue. (H) In situ hybridization of BP to a cross-section from an ap2-9 floral bud. In ap2-9, BP is expressed ectopically in first-whorl carpel-like sepals (arrow). (I) In situ hybridization with an ap2-2 plant revealing ectopic staining of sepals (arrow). (J) Floral phenotype of a bp-2 ap2-5 double mutant. Arrows point to ovules arising on the margins of the sepals. (K) Unaltered as2 leaf morphology in a bp-2 as2-1 double mutant.