Pea compound leaf architecture is regulated by interactions among the genes UNIFOLIATA, cochleata, afila, and tendril-lessn

Abstract

The compound leaf primordium of pea represents a marginal blastozone that initiates organ primordia, in an acropetal manner, from its growing distal region. The UNIFOLIATA (UNI) gene is important in marginal blastozone maintenance because loss or reduction of its function results in uni mutant leaves of reduced complexity. In this study, we show that UNI is expressed in the leaf blastozone over the period in which organ primordia are initiated and is downregulated at the time of leaf primordium determination. Prolonged UNI expression was associated with increased blastozone activity in the complex leaves of afila (af), cochleata (coch), and afila tendril-less (af tl) mutant plants. Our analysis suggests that UNI expression is negatively regulated by COCH in stipule primordia, by AF in proximal leaflet primordia, and by AF and TL in distal and terminal tendril primordia. We propose that the control of UNI expression by AF, TL, and COCH is important in the regulation of blastozone activity and pattern formation in the compound leaf primordium of the pea.

Figure 1.

Morphology of Wild-Type and Mutant Pea Leaves. (A) A wild-type (JI 1194) compound leaf showing a basal pair of stipules, a petiole and a blade comprising a pair of proximal leaflets, two distal pairs of tendrils, and a terminal tendril, all borne on a rachis. (B) A uni (JI 2171) mutant leaf with a basal pair of stipules and the leaf blade reduced to a unifoliate form. (C) A tl (JI 1197) mutant leaf with a basal pair of stipules and leaflets at all positions on the leaf blade. (D) An af (JI 1195) mutant leaf with a basal pair of stipules and all positions on the leaf blade occupied by branching rachides bearing tendrils. (E) An af tl (JI 1199) double mutant leaf with a basal pair of stipules and all positions on the leaf blade occupied by branching rachides terminating in small leaflets. (F) A coch (JI 2165) mutant leaf showing that each structure occupying the position of a stipule mimicks the organization of the leaf blade with a pair of leaflets, pairs of tendrils, and a terminal tendril.

Figure 2.

SEM Showing the Effects of the uni Mutation on Leaf Primordium Initiation and Early Development. (A) and (B) Two-week-old wild-type (JI 2171) vegetative shoot apices. (C) and (D) Two-week-old sibling uni mutant (JI 2171) vegetative shoot apices. The white arrow in (C) indicates a central crease marking the beginning of lamina folding of a single, terminal leaflet. A, shoot apex; P1 to P4, plastochron 1 to plastochron 4 of leaf development; S1 to S3, stipule primordia present on P1 to P3 marginal blastozones; L2 to L4, proximal leaflet primordia present on P2 to P4 marginal blastozones; T3 and T4, tendril primordia present on P3 and P4 marginal blastozones. .

Figure 3.

SEM Showing the Effects of the tl, af, and af tl Mutations on Early Leaf Development. (A) and (B) Two-week-old wild-type (JI 1194) vegetative shoot apices. White arrowheads indicate leaflet primordia. A, vegetative shoot apex; P1 to P4, plastochron 1 to plastochron 4 of leaf development; S2 to S4, stipule primordia present on P2 to P4 marginal blastozones. (C) and (D) Two-week-old tl mutant (JI 1197) vegetative shoot apices. White arrowheads indicate leaflet primordia. A, vegetative shoot apex; P1 to P4, plastochron 1 to plastochron 4 of leaf development; S3 and S4, stipule primordia present on P3 and P4 marginal blastozones. (E) and (F) Two-week-old af mutant (JI 1195) vegetative shoot apices. White arrowhead in (E) indicates a secondary blastozone, initiating tertiary primordia acropetally. These tertiary primordia will develop into tendrils, indicated by a white arrowhead in (F). A, vegetative shoot apex; P3 to P6, plastochron 3 to plastochron 6 of leaf development; S4 and S5, stipule primordia present on P4 and P5 primary marginal blastozones. (G) and (H) Two-week-old af tl double mutant (JI 1199) vegetative shoot apices. White arrowhead in (G) indicates a secondary blastozone initiating tertiary blastozones acropetally. The distal tip of a secondary blastozone, marked by the P6 label in (H), has been removed. Quaternary leaflet primordia, derived from tertiary blastozones, are indicated with white arrowheads in (H). A, vegetative shoot apex; P2 to P6, plastochron 2 to plastochron 6 of leaf development; S3 and S5, stipule primordia present on P3 and P5 primary marginal blastozones. .

Figure 4.

Localization of UNI Transcripts in Wild-Type, tl, af, and af tl Mutant Samples by RNA in Situ Hybridization on Frontal Longitudinal Sections. Sections were taken from 2-week-old seedlings. (A) and (B) Wild type (JI 1194). (C) tl mutant (JI 1197). (D) af mutant (JI 1195). (E) to (H) af tl double mutant (JI 1199). Sections shown in (A) and (C) to (G) were hybridized with an antisense digoxigenin-labeled UNI probe, section shown in (B) was hybridized with a sense digoxigenin-labeled UNI probe, and section shown in (H) was hybridized with an antisense digoxigenin-labeled HH3 probe. The plastochronic ages indicated in (B) apply to sections (A) to (E) and (H), where (A) and (E) are in midplastochron, (C) and (D) are very early in a plastochron, and (B) and (H) are late in a plastochron. White arrowheads in (A) and (C) indicate the positions of lateral organs that emerged in front of the plane of section. The section in (D) is lateral to P1 on the shoot apex, reducing the view of the P2 and P3 blastozones. A, vegetative shoot apex; L, leaflet; P1 to P7, plastochronic age of leaf primordia; PR, primary marginal blastozone; S, secondary blastozone; T, tertiary blastozone.

Figure 5.

Localization of UNI Transcripts in Wild-Type, tl, af, and af tl Mutant Samples by RNA in Situ Hybridization on Transverse Sections. Sections taken from 2-week-old seedlings hybridized with an antisense digoxigenin-labeled UNI probe. (A) Wild type (JI 1194). (B) tl mutant (JI 1197). (C) and (D) af mutant (JI 1195). (E) and (F) af tl double mutant (JI 1199). White arrowheads in (A) and (B) indicate lateral primordia on P3 and P4 marginal blastozones where transcripts were not detected. The black arrowheads in (C) to (E) indicate secondary lateral blastozones on P3, P4, and P5 primary blastozones where UNI transcripts were detected. White arrowhead in (D) indicates a tertiary primordium in which UNI expression was downregulated compared with the expression in an adjacent secondary blastozone (marked with a black arrowhead). Black arrowheads in (F) indicate tertiary blastozones where UNI transcripts were detected. (A) to (C) and (E) were taken ∼40 μm below the shoot apex; (D) and (F) were taken ∼70 μm above the shoot apex. A, vegetative shoot apex; P2 to P5, plastochronic age of leaf primordia; S3 to S6, stipule primordia present on P3 to P6 marginal blastozones.

Figure 6.

SEM Showing Effects of the coch Mutation on Early Stipule Development and Localization of UNI Transcripts by RNA in Situ Hybridization. (A) and (B) Two-week-old coch mutant (JI 2165) vegetative shoot apices. Black arrow in (B) indicates a compound stipule (S5) with two pairs of lateral organ primordia. (C) and (D) Transverse sections taken from a 2-week-old coch seedling hybridized with an antisense digoxigenin-labeled UNI probe. (C) was taken ∼40 μm below the shoot apex, and (D) was taken ∼100 μm below the shoot apex, at a slightly oblique angle. A, vegetative shoot apex; P1 to P6, plastochronic age of leaf primordia; S1 to S6, stipule primordia present on P1 to P6 marginal blastozones. .

Figure 7.

Morphology of a uni af tl Triple Mutant and Localization of PSKN1 Transcripts by RNA in Situ Hybridization. (A) Nodes 5 and above of a uni af tl plant before flowering. The white arrow indicates a leaf at node 12 with two pairs of leaflets, borne on a rachis (obscured from view). (B) Frontal longitudinal section of a 3-week-old uni af tl (XM 7175) shoot apex hybridized to an antisense PSKN1 probe. (C) Frontal longitudinal section of a 3-week-old uni (JI 2171) shoot apex hybridized to an antisense PSKN1 probe. (D) Frontal longitudinal section of a 3-week-old wild-type (JI 1194) shoot apex hybridized to an antisense PSKN1 probe. The black arrowhead indicates an inflorescence meristem developing in the axil of the P2 marginal blastozone. A, shoot apex; LS, lateral shoot; P1 to P7, plastochron 1 to plastochron 7 of leaf development; V, developing vasculature of the main stem.

Figure 8.

Summary Diagram Showing UNI Expression in Vegetative Shoot Apices of Wild-Type, tl, af, af tl, and coch Mutant Seedlings. Representations of transverse sections through the shoot apex are followed through the early stages of leaf development (arrows). The main shoot axis is shown as a large green circle, blastozones in which UNI expression was detected are shown as red ovals, and organs in which UNI expression was not detected are shown as blue ovals. Small black double-headed arrows represent the rachis and rachides. The plastochronic age of each marginal blastozone is shown at left (P1 to P6). During P1 and P2, the morphology and pattern of UNI expression in all the genotypes studied was the same (top center), but during P3, both the morphology and the expression patterns changed. In the far left-hand column, UNI expression was detected in the primary marginal blastozones of wild-type and tl mutant seedlings, up to and during P4. Lateral primordia that arose from these showed no signal and formed determinate organs (stipules, leaflets, and tendrils). In the second column from the left, UNI transcripts accumulated in primary blastozones and in the secondary blastozones that arose during P3 in both af and af tl mutant leaves. Both of these mutants initiated tertiary branches late in P3 and during P4 (second and third columns). In af mutant leaves, the tertiary branches were determinate tendril primordia showing no detectable UNI expression (second column), whereas af tl tertiary blastozones showed UNI signal, remained indeterminate, and exhibited quaternary branching during P5 (third column). In af tl mutant leaves, UNI transcripts were detected in the primary marginal blastozone up to P6. In the far right-hand column, UNI expression was detected in the primary marginal blastozones of coch mutant seedlings, up to and during P4, but not in the lateral leaflet or tendril organ primordia that arose from these. During P3 and P4, UNI expression was detected in stipule primordia that behaved like blastozones and initiated lateral leaflet and tendril organ primordia (these lateral primordia are not shown). After this time, no further signal was detected.

Figure 9.

Stipule Morphology in Mutant Backgrounds. (A) A coch uni double mutant. Stipules at five nodes are indicated with white arrows. Node 11 is at the bottom of the panel; node 15, with an axial flower, is at the top. (B) An st af tl (JI 1201) plant with a compound stipule opposite a simple stipule at node 18. The compound stipule is indicated with a white arrow.