Compound leaf development and evolution in the legumes

Abstract

Across vascular plants, Class 1 KNOTTED1-like (KNOX1) genes appear to play a critical role in the development of compound leaves. An exception to this trend is found in the Fabaceae, where pea (Pisum sativum) uses UNIFOLIATA, an ortholog of the floral regulators FLORICAULA (FLO) and LEAFY (LFY), in place of KNOX1 genes to regulate compound leaf development. To assess the phylogenetic distribution of KNOX1-independent compound leaf development, a survey of KNOX1 protein expression across the Fabaceae was undertaken. The majority of compound-leafed Fabaceae have expression of KNOX1 proteins associated with developing compound leaves. However, in a large subclade of the Fabaceae, the inverted repeat-lacking clade (IRLC), of which pea is a member, KNOX1 expression is not associated with compound leaves. These data suggest that the FLO/LFY gene may function in place of KNOX1 genes in generating compound leaves throughout the IRLC. The contribution of FLO/LFY to leaf complexity in a member of the Fabaceae outside of the IRLC was examined by reducing expression of FLO/LFY orthologs in transgenic soybean (Glycine max). Transgenic plants with reduced FLO/LFY expression showed only slight reductions in leaflet number. Overexpression of a KNOX1 gene in alfalfa (Medicago sativa), a member of the IRLC, resulted in an increase in leaflet number. This implies that KNOX1 targets, which promote compound leaf development, are present in alfalfa and are still sensitive to KNOX1 regulation. These data suggest that KNOX1 genes and the FLO/LFY gene may have played partially overlapping roles in compound leaf development in ancestral Fabaceae but that the FLO/LFY gene took over this role in the IRLC.

Figure 1.

Simplified Representation of Phylogenetic Relationships in Fabaceae. Topology based on maximum parsimony and Bayesian analyses of plastid matK gene sequences, adapted from Figure 6 of Wojciechowski et al. (2004). Nested within the paraphyletic subfamily Caesalpinioideae, the position of monophyletic groups corresponding to the subfamilies Mimosoideae and Papilionoideae are indicated by black circles. The Millettioids, Hologalegina, and Robinioid clades are denoted with gray circles. The IRLC is shown with a white circle. Representative taxa from these clades are indicated. Numbers at selected nodes are bootstrap proportions (above) and Bayesian posterior probabilities (below). Taxa sampled for KNOX1 protein expression are shown in bold and marked by an asterisk; taxa showing no KNOX1 expression are underlined.

Figure 2.

KNOX1 Expression Is Downregulated in the Incipient Leaf Primodium in Members of the IRLC. Alfalfa (A) and Chinese wisteria (B) as well as members of the Millettioids clade soybean (C) and bean (D). Arrows denote incipient primordia. Bars = 10 μm.

Figure 3.

Mature Leaf Form and KNOX1 Immunolocalization Patterns in Select Members of the IRLC. Compound-leafed IRLC members pea ([A] and [E]), wisteria ([B] and [F]), alfalfa ([C] and [G]), and fava bean ([D] and [H]) have KNOX1 expression restricted to the SAM and no expression in developing leaves. Arrows point to developing leaves lacking KNOX1 expression. Bars = 30 μm.

Figure 4.

Mature Leaf Form and KNOX1 Immunolocalization Patterns in Select Members of Non-IRLC Fabaceae. Compound-leafed Fabaceae outside of the IRLC soybean ([A] and [F]), M. pudica ([B] and [G]), L. japonicus ([C] and [H]), A. hindsii ([D] and [I]), and bean ([E] and [J]) have KNOX1 expression in the SAM and in developing leaves. Insets of (F), (I), and (J) show details of leaves and meristems from Acacia, soybean, and bean, respectively. Dashed boxes depict regions magnified in insets. Arrows point to KNOX1 expression in leaves. Bars = 30 μm in all cases, except in insets of (F) and (J) and red boxed inset of (I), where bars = 10 μm.

Figure 5.

KNOX1 Expression Patterns Support the Fusion Hypothesis for the Unifoliolate Cercis Leaf. (A) Morphology of the unifoliolate Cercis leaf. (B) KNOX1 proteins are localized within the SAM and developing leaves of Cercis. Bar = 30 μm.

Figure 6.

Phenotypes of Soybean Gm LFYRNAi Lines. (A) Wild-type soybean flower. (B) Gm LFYRNAi line ST40-141 with altered floral development. (C) Wild-type soybean shoot with two simple, opposite leaves at the first node and one trifoliolate leaf at the second node. (D) Line ST40-99 with two simplified opposite leaves at the second node. (E) Line ST40-189 with two opposite leaves, one simple and the other trifoliolate, at the second node. (F) Line ST40-189 with two opposite leaves at the second node. Note that one leaf appears to have two fused leaflets. (G) Another individual from line ST40-189 with one simple leaf at the second node. (H) Line ST40-114 with two simple opposite leaves at the first node (the second leaf is highly reduced) and a simple leaf at the second node.

Figure 7.

Overexpression of a KNOX1 Gene in Alfalfa. (A) Wild-type alfalfa. Bar = 2 cm. (B) 35S:LeT6 line with reduced internodes, reduced petioles, and small leaves. Bar = 2 cm. (C) 35S:LeT6 line 59-015 produces leaves with extra leaflets (arrow). (D) 35S:LeT6 line 59-067 produces leaflets that are deeply lobed (asterisk) and serrated (arrowheads).

Figure 8.

Quantitative RT-PCR Analysis of LeT6 RNA Levels in Several Alfalfa Transgenic Lines and the Wild Type. LeT6 transcript levels were normalized to Ms GAPDH mRNA and are shown in relative units of expression. Bars represent se over four technical replicates.