A Conserved Molecular Framework for Compound Leaf Development

Thomas Blein,¹ Amada Pulido,¹ Aurélie Vialette-Guiraud,^1* Krisztina Nikovics,¹ Halima Morin,^1,2 Angela Hay,³ Ida Elisabeth Johansen,⁴ Miltos Tsiantis,³ Patrick Laufs¹

^* Present address: Laboratoire de Reproduction et Développement des Plantes, UMR INRA-CNRS-Ecole Normale Supérieure de Lyon, Université de Lyon I, 46 allée d'Italie, 69364 Lyon Cedex 07, France.

Present address: Unité INSERM U552, Institut Universitaire d'Hématologie, Batiment HAYEM, Hôpital Saint Louis, 1, avenue Claude Vellefaux, 75475 Paris Cedex 10, France.

To whom correspondence should be addressed. E-mail: laufs{at}versailles.inra.fr

Leaves of seed plants can be simple, with a single leaf blade, or compound when divided into distinct leaflets (1, 2). Additionally, margins of both simple and compound leaves can elaborate less-pronounced incisions such as serrations or lobes. Regardless of the final shape, leaves are initiated as simple primordia from the shoot apical meristem. Primordia of compound leaves maintain an organogenic region at their margin from which leaflet primordia emerge (1, 2). Two different pathways have been recruited to promote this organogenic activity during the multiple independent origins of compound leaves in seed plants. One pathway involves expression in the primordia of compound leaves of class 1 homeodomain KNOTTED1–like (KNOXI) transcription factors that were initially identified for their role in maintenance of meristem identity (3–5). This pathway is active in a wide range of flowering seed plants, including Solanum lycopersicum and Cardamine hirsuta. A second pathway involving the UNIFOLIATA (UNI) gene is found in Pisum sativum, which does not express KNOXI genes in the leaf primordium. UNI encodes a member of the LEAFY (LFY) family of transcription factors, initially identified for its role in floral meristem identity (6, 7). Despite progress in understanding what promotes the organogenic potential of compound leaves, the mechanistic basis of leaflet formation and delimitation is less clear. The generation of activity maxima of auxin, a small indolic hormone, is one such mechanism that facilitates initiation and separation of both leaves at the shoot apical meristem and leaflets from the rachis (8–10). Other key regulators of organ initiation and delimitation are the NAM/CUC3 genes, which are members of a large evolutionarily conserved family of plant transcription factors that are subdivided into NAM (NO APICAL MERISTEM) and CUC3 (CUP-SHAPED COTYLEDON3) clades (11–14). They are expressed in the boundary of organ primordia, where they repress growth to allow organ separation (15). In addition, they are involved in meristem establishment via their activation of KNOXI expression (16).

Because previous work showed that AtCUC2 is required for Arabidopsis leaf serration (17), we hypothesized that NAM/CUC3 genes could have a broader role in leaf dissection. To test this hypothesis, we analyzed the function of NAM/CUC3 genes in a selection of five eudicots with compound leaves (Aquilegia caerulea, S. lycopersicum, S. tuberosum, C. hirsuta, and P. sativum) that show contrasting phylogenetic positions, genetic controls and patterning of leaflet development, dissection of leaflet margins, and leaflet specialization (Fig. 1, A, E, I, M, and R, and fig. S1) (18). We cloned 11 NAM/CUC3 genes from these species, and phylogenetic analysis showed that they group either into NAM (AcNAM, SlNAM, StNAM, PsNAM1, PsNAM2, ChCUC1, and ChCUC2) or CUC3 (AcCUC3, StCUC3, PsCUC3, and ChCUC3) clades (fig. S2). The NAM/CUC3 genes had a typical expression pattern in the boundary domain at the base of organ primordia, a pattern that is complementary to the cell proliferation marker HISTONE H4 (fig. S3). This suggested conserved roles in defining boundary and organ separation by local repression of cell proliferation.

Fig. 1. Leaf shape and expression of the NAM/CUC3 genes during leaf development in eudicots: A. caerulea [(A) to (D)], S. lycopersicum [(E) to (H)], S. tuberosum [(I) to (L)], C. hirsuta [(M) to (Q)], and P. sativum [(R) to (U)]. (A) A. caerulea leaf formed by three leaflets subdivided into three majors lobes (arrow), each of which is dissected (arrowhead). (B and C) In a young leaf primordium, AcNAM and AcCUC3 are expressed in relation to the formation of the leaflet primordia (arrows). AcNAM expression is restricted to the distal side of the primordium marked in (B) by an asterisk. (D) AcNAM expression is coincident with further leaflet primordia (ltp) dissection (arrowhead). (E) S. lycopersicum leaf formed by primary (I), secondary (II), and intercalary (Int) leaflets that have dissected margins. (F and G) SlNAM expression precedes leaflet outgrowth [arrow in (F)] and marks the distal boundary of young or older leaflet primordia (asterisks). (H) SlNAM is expressed in relation with the serration of older leaflet (lt) margins (arrowheads). (I) S. tuberosum leaf formed by primary leaflets with entire leaf margins. (J to L) StNAM and StCUC3 are expressed during early stages of leaflet initiation (J) and are still detected at later stages [(K) and (L)]. Asterisk in (K) indicates a young leaflet primordium showing StNAM expression only on its distal part. (M) A rosette leaf of C. hirsuta formed by several leaflets that show mild incision of their margins. (N to Q) ChCUC1, ChCUC2, and ChCUC3 are expressed during leaflet initiation and at later stages. ChCUC expression is limited to the distal part of young [asterisk in (N)] and older [asterisks in (Q)] primordia. (R) P. sativum leaf formed by several pairs of proximal leaflets (lt) and distal tendrils (tdl). Leafletlike stipules (st) subtend the leaf. (S to U) The PsNAM1/2 and PsCUC3 genes are expressed during leaflet and tendril primordia development. PsNAM1/2 is expressed in the distal boundary of young [asterisk in (S)] and older [asterisks in (T)] leaflet primordia. Scale bars indicate 1 cm [(A), (E), (I), (M), and (R)] or 0.1 mm [(B) to (D), (F) to (H), (J) to (L), (N) to (Q), and (S) to (U)]. [View Larger Version of this Image (96K GIF file)]

To determine whether the NAM/CUC3 genes have a role in defining compound leaf morphology, we examined their expression during leaf development. A similar expression pattern was observed in all species examined (Fig. 1 and fig. S4). NAM/CUC3 genes were expressed in a narrow strip of cells at the distal boundary of leaflet primordia, whereas no expression was observed in the proximal region (e.g., Fig. 1, B, G, K, N, and U, and fig. S3). NAM/CUC3 expression preceded the actual outgrowth of leaflet primordia (e.g., Fig. 1, F and J, and fig. S3). Given the diversity of the species analyzed here, this conserved NAM/CUC3 gene expression pattern is likely to reflect a fundamental mechanism of leaflet formation. NAM genes were also expressed later in association with A. caerulea and S. lycopersicum leaflet margin dissection (Fig. 1, D and H), as shown for the simple Arabidopsis thaliana leaf (17).

Next, we undertook a series of functional analyses in A. caerulea, S. lycopersicum, P. sativum, and C. hirsuta combining three different methods. Down-regulation of NAM and/or CUC3 expression in A. caerulea, P. sativum, and S. lycopersicum by transient virus-induced gene silencing (VIGS) (fig. S5, A, C, and D) led to three specific leaf developmental defects that were never observed in control VIGS experiments (Fig. 2, fig. S6, and tables S1 to S3). First, the extent of serration or lobing at the leaf margin was reduced. Silencing of SlNAM was sufficient to produce smooth leaflet margins in S. lycopersicum (Fig. 2F and fig. S6), whereas full smoothing of the A. caerulea leaf margin required the simultaneous silencing of AcNAM and AcCUC3 (Fig. 2A and fig. S6). Second, all lines silenced for NAM/CUC3 except A. caerulea showed fusions of the leaflets with the rachis or between leaflets, and P. sativum showed tendril fusions (Fig. 2, D and F, and fig. S6). This indicated that NAM/CUC3 expression at the base of outgrowing leaflets is required for their separation, a function resembling that of organ primordia separation in the meristem. Third, the number of leaflets was reduced. A. caerulea silenced for AcNAM or AcNAM/AcCUC3 formed a single leaf blade, and fewer leaflets and tendrils were formed in P. sativum after PsNAM1/2 and PsNAM1/2/PsCUC3 silencing (Fig. 2, A, C, and D, and fig. S6). The number of primary leaflets was slightly reduced in SlNAM-silenced plants, whereas secondary and intercalary leaflet numbers were highly reduced (Fig. 2F and fig. S6).

Fig. 2. Reducing NAM/CUC3 activity leads to a simplification of compound leaves. (A) Successive leaves formed on an A. caerulea plant silenced for the AcPDS, AcNAM, and AcCUC3 genes. Note the progressive smoothening of the leaflet margins from leaf 1 to 3 (arrowheads). At the final stage (leaf 4), corresponding to early silencing, a simple leaf with an entire margin is formed. (B) Control leaf of P. sativum silenced for PsPDS with three pairs of leaflets and three pairs of tendrils subtended by a pair of stipules (st). (C) P. sativum leaf silenced for PsPDS, PsNAM1/2, and PsCUC3 formed by one pair of leaflets and one pair of tendrils separated by a long, organless rachis (arrow). (D) P. sativum leaf silenced for PsPDS, PsNAM1/2, and PsCUC3 showing fusions between leaflets (left arrow) and between a leaflet and the rachis (right arrow). (E) Control leaf of S. lycopersicum silenced for SlPDS showing primary (I), secondary (II), and intercalary (Int) leaflets. (F) S. lycopersicum leaf silenced for SlPDS and SlNAM showing smoothed leaf margins (arrowhead), fusions between leaflets (arrow), and fewer leaflets. (G) Leaf of gob3 that harbors a mutation in the SlNAM gene and is similar to a SlNAM-silenced leaf. (H) Rosette leaf number 8 of wild-type (WT) C. hirsuta and of a line with reduced CUC expression (2x35S:MIR164b ChCUC3 RNAi). A reduced number of leaflets leads to a long, leafletless petiole and the fusion of the leaflets (arrows). (I) First cauline leaf of WT C. hirsuta and of a plant silenced for ChCUC3 showing fewer and smoothed leaflets (arrowhead). Scale bars, 1 cm. [View Larger Version of this Image (46K GIF file)]

The S. lycopersicum goblet mutant confirmed the role of the SlNAM gene during compound leaf ontogeny. The goblet mutant displays developmental defects reminiscent of nam/cuc3 mutants in other plant species (19), and sequencing of three goblet mutants revealed that each harbored a mutation in the SlNAM gene (fig. S7). Leaves regenerated from ectopic meristems of these mutants showed the same morphological defects as SlNAM-silenced plants (Fig. 2G).

In C. hirsuta, we stably reduced the expression of all three NAM/CUC3 genes by overexpressing MIR164b, which gives rise to mature microRNA164 that directs ChCUC1 and ChCUC2 repression, and silencing ChCUC3 through hairpin-mediated RNA interference (RNAi) (fig. S5B). Individually, MIR164b overexpression and ChCUC3 RNAi led to leaflet fusions and reduced leaflet lobing and number. The severity of these defects was increased in double transgenics (Fig. 2, H and I, and fig. S8).

Altogether, our data revealed a conserved requirement for NAM/CUC3 genes during leaflet formation, leaflet separation, and margin dissection. In addition, we showed the existence of a morphological continuity from leaf margin to leaflet dissection, which is facilitated at the molecular level by a common underlying mechanism involving NAM/CUC3 genes.

Next, we investigated NAM/CUC3 gene expression in response to modifications of other regulators of compound leaf development. First, we observed no NAM/CUC3 expression in the simple leaves of S. lycopersicum Lanceolate (La) mutants containing a gain-of-function TEOSINTE BRANCHED1/CYCLOIDEA/PCF (TCP) TCP gene (20) and in P. sativum uni mutants (fig. S9), consistent with NAM/CUC3 expression being required for leaflet formation. Second, we analyzed C. hirsuta transgenics bearing a KNOTTED1-GR fusion (3). A 2-day-long activation of the KNOTTED1-GR fusion led to increased ChCUC1-3 expression (Fig. 3A). ChCUC activity was required for the induction of ectopic leaflets by KNOTTED1-GR (3) because they did not form when the ChCUC genes were silenced (Fig. 3B). Altogether, these analyses showed that LA, UNI, and KNOXI genes influence NAM/CUC3 expression, which in turn regulate leaflet formation.

Fig. 3. Interplay between the NAM/CUC3 genes and other regulators of compound leaf development. (A) After 2 days of induction of the KN1:GR fusion, the expression of ChCUC1, ChCUC2, and ChCUC3 was increased compared with that of WT C. hirsuta control plants. ChEF1 was used as an internal control. (B) Upon dexamethasone induction, ectopic leaflets were formed on the primary leaflets in C. hirsuta expressing a KN1-GR fusion (arrowheads). No ectopic leaflets were formed when the same construct is induced in a background with reduced ChCUC activity (2x35S:MIR164B ChCUC3 RNAi). (C) A reduction of SlNAM expression after SlPDS SlNAM VIGS was correlated with reduced expression of SlLFY and of the two KNOXI genes TKn1 and T6/TKn2. SlGAPDH was used as an internal control. (D) PsNAM1/2 and PsCUC3 expression was reduced in plants silenced for PsPDS PsNAM1/2 or PsPDS PsCUC3, respectively, and correlated with lower UNI expression. PsEF1 was used as an internal control. (E) The ChSTM:GUS reporter was expressed in the petiole and rachis of a WT C. hirsuta leaf, and its expression was strongly reduced in a background with reduced ChCUC activity (2x35S:MIR164B ChCUC3 RNAi). Scale bars, 1 mm in (D) and 1 cm in (E). [View Larger Version of this Image (62K GIF file)]

Conversely, we tested whether NAM/CUC3 genes had an effect on the expression of KNOXI and LFY-like genes. Accumulation of KNOXI (Tkn1 and Tkn2 in S. lycopersicum) and LFY-like (SlLFY in S. lycopersicum and UNI in P.sativum) transcripts was reduced in lines silenced for the NAM/CUC3 genes (Fig. 3, C and D). In line with these results, KNOXI reporter (ChSTM:GUS) expression was reduced in the developing leaf of a C. hirsuta line with reduced ChCUC activity (Fig. 3E). This indicated that NAM/CUC3 genes are required for proper expression of KNOXI/LFY-like genes during compound leaf development. Together, these findings advocate the existence of a feed-forward regulatory loop between NAM/CUC3 and KNOXI/LFY-like genes and indicate that this coordinately regulated expression controls leaflet formation.

We reveal a dual evolutionarily conserved role for NAM/CUC3 genes during eudicot leaf development (fig. S10). First, NAM/CUC3 are required to dissect compound leaves into leaflets and leaflet margins into serrations or lobes. This is a local, probably cell-autonomous function of the NAM/CUC3 genes because they are expressed in the boundary domain. Second, NAM/CUC3 genes are required for leaflet formation. This is likely to be a non–cell-autonomous effect of NAM/CUC3 genes. Differences between formation of a leaflet, a lobe, or a serration could depend on different capacities of cells to respond to NAM/CUC3 expression. For example, factors such as TCP proteins (20–22) may limit growth and prevent leaflet formation.

In contrast to the KNOX and LFY-like pathways, whose contributions vary between the compound-leafed eudicots, the requirement for NAM/CUC3 activity during leaflet formation is conserved in all species tested here and is likely to be extensively conserved within eudicots. Our results suggest that species-specific activity of either the KNOXI or the LFY pathway induces expression of NAM/CUC3 genes, which are responsible for leaflet formation and maintenance of KNOXI/LFY expression through a positive feedback loop.

The dual role of the NAM/CUC genes revealed here during leaf development could also exist in the plant apex, where the topology of NAM/CUC3 expression is similar to that observed during leaflet formation [i.e., they are expressed at the boundary between the meristem and the primordium (23)]. It will therefore be interesting to determine whether NAM/CUC3 proteins, in addition to their well-established role in organ separation at the apex, also contribute to the outgrowth of the leaf primordium and whether NAM/CUC3 action in leaves is mediated by auxin maxima (8–10). This evolutionarily conserved deployment of both NAM/CUC3 genes and auxin in both leaf and leaflet formation may reflect the common evolutionary origin of leaves from branched shoots (10).

Our results highlight an unexpected role for the interleaflet boundary domain patterned by NAM/CUC3 genes in directing novel axes of growth that give rise to leaflets. This role is conceptually similar to that of boundary domains acting during animal development (24, 25) and hence provides an example of a common developmental logic operating to sculpt organ form in evolutionary lineages where multicellularity evolved independently.

References and Notes

ADVERTISEMENT