Mechanisms of derived unitegmy among Impatiens species

Abstract

Morphological transitions associated with ovule diversification provide unique opportunities for studies of developmental evolution. Here, we investigate the underlying mechanisms of one such transition, reduction in integument number, which has occurred several times among diverse angiosperms. In particular, reduction in integument number occurred early in the history of the asterids, a large clade comprising approximately one-third of all flowering plants. Unlike the vast majority of other eudicots, nearly all asterids have a single integument, with the only exceptions in the Ericales, a sister group to the other asterids. Impatiens, a genus of the Ericales, includes species with one integument, two integuments, or an apparently intermediate bifid integument. A comparison of the development of representative Impatiens species and analysis of the expression patterns of putative orthologs of the Arabidopsis thaliana ovule development gene INNER NO OUTER (INO) has enabled us to propose a mechanism responsible for morphological transitions between integument types in this group. We attribute transitions between each of the three integument morphologies to congenital fusion via a combination of variation in the location of subdermal growth beneath primordia and the merging of primordia. Evidence of multiple transitions in integument morphology among Impatiens species suggests that control of underlying developmental programs is relatively plastic and that changes in a small number of genes may have been responsible for the transitions. Our expression data also indicate that the role of INO in the outgrowth and abaxial-adaxial polarity of the outer integument has been conserved between two divergent angiosperms, the rosid Arabidopsis and the asterid Impatiens.

Figure 1.

Scanning Electron Microscopy of Developing Impatiens Ovules. In all panels, the gynoapical side of the ovules is toward the left. Bars = 50 μm. (A) to (C) I. hookeriana. (D) to (F) I. balsamina. (G) to (I) I. niamniamensis. (A) Ovule primordia are initiated basipetally in the gynoecium, with the inner integument (ii) and nucellus (n) becoming apparent in the more fully developed ovules. (B) The outer integument (oi) grows to partially cover the inner integument. (C) At maturity, only the inner integument fully encloses the nucellus and contributes to the micropyle (m). (D) Ovule primordia are fewer in number but otherwise are initiated similarly to I. hookeriana. (E) The lobe derived from the outer integument does not extend as far as the lobe derived from the inner integument. (F) Only the inner integument lobe encloses the nucellus and forms the micropyle. (G) Ovule primordia are initiated similarly to I. hookeriana and I. balsamina. (H) The single integument (i) grows by mostly anticlinal divisions to cover the nucellus. (I) The single integument forms the micropyle and completely envelops the embryo sac.

Figure 2.

Light Microscopy of Developing Impatiens Ovules. In all panels, the gynoapical side of the ovules is toward the top and the placenta is to the right. Bars = 50 μm. (A) to (C) I. hookeriana. (D) to (F) I. balsamina. (G) to (I) I. niamniamensis. (A) The inner integument (ii) is initiated by anticlinal divisions in the L1 at the chalaza (c) as the megaspore mother cell (mmc) differentiates in the nucellus (n). (B) The outer integument (oi) primordium arises by anticlinal divisions in the L1 on the flanks of the inner integument. (C) In the mature ovule, the inner integument protrudes beyond the outer integument, which is partly derived from periclinal divisions in the L2 of the chalaza. (D) The inner integument primordium arises by anticlinal and subsequent periclinal divisions of the L1. (E) The outer integument is initiated in the L1, and both inner and outer integument primordia grow out by periclinal divisions in the L2 of the chalaza. (F) In the mature ovule, the single integument has two lobes, which are derived from the inner and outer integument primordia. (G) The single integument (i) primordium arises in the same way as the inner integument of I. hookeriana and I. balsamina from the L1. (H) Early growth of the single integument occurs by anticlinal and periclinal divisions in the L1-derived initials of the single integument. (I) Divisions of L2 layers in the chalaza contribute to the single massive integument of the mature ovule. The inner layers of the integument differentiate into a densely staining endothelium.

Figure 3.

Alignment of Predicted Amino Acid Sequences for Arabidopsis YABBY Protein Family Members, Nymphaea Orthologs (Yamada et al., 2003), and Impatiens INO Orthologs. Amino acids unique to all INO orthologs are highlighted in black, and those common only to Arabidopsis and Impatiens are highlighted in gray. Sequences for Impatiens INO orthologs are incomplete. Nc, Nymphaea colorata; Na, Nymphaea alba; Is, Impatiens sodenii; Ib, Impatiens balsamina; In, Impatiens niamniamensis; Ih, Impatiens hookeriana; Iw, Impatiens walleriana; At, Arabidopsis thaliana.

Figure 4.

One of the Three Shortest Trees Showing Possible Relationships among Impatiens INO Proteins and Other Published YABBY Proteins. The phylogram was constructed using the amino acid sequence alignment shown in Figure 3. The bootstrap values are shown for clades with >50% bootstrap support (excepting the group comprising the I. niamniamensis, I. balsamina, and I. sodenii sequences, in which the 74% bootstrap support value did not fit on the figure beside the very short branches). Within the INO group, including INO from Nymphaea (N. alba and N. colorata) and Arabidopsis (At), the bitegmic Impatiens species studied, I. hookeriana (Ih), was sister to the clade including the unitegmic and intermediate species I. balsamina (Ib), I. sodenii (Is), I. walleriana (Iw), and I. niamniamensis (In).

Figure 5.

In Situ Hybridization with Antisense I. niamniamensis INO and Sense I. niamniamensis INO Probes on Developing Impatiens Ovules. (A) to (D) I. hookeriana. (E) to (H) I. balsamina. (I), (K), and (L) I. niamniamensis. (J) I. sodenii. (A) to (C) Expression of INO is detected only in the outer integument (oi) and decreases to an undetectable level late in development. (E ) to (G) Expression of INO is detected only toward the base of the single-lobed integument. (G) As the ovule nears maturity, expression of INO diminishes. (I) to (K) Expression of INO orthologs is detected only proximally and in the outermost layer of the single integument (i). (D), (H), and (L) No hybridization was detected in I. niamniamensis INO sense controls. ii, inner integument; n, nucellus. Arrowheads indicate locations of in situ hybridization signals that might be difficult to discern. Bars = 50 μm.

Figure 6.

Model for the Derivation of Integument Morphologies in Impatiens. Shading represents areas of subdermal growth. Green indicates the location of INO ortholog expression, and red indicates the location of endothelium. (A) Development of a bitegmic ovule with subdermal growth occurring beneath an outer integument primordium. (B) Development of an intermediate ovule with subdermal growth occurring beneath both inner and outer integument primordia. (C) Development of a unitegmic ovule with subdermal growth occurring beneath the single integument primordium that is derived from the fusion of two ancestral primordia.

Figure 7.

Distribution of Integument Morphologies among Impatiens and Relatives. A subset of the neighbor-joining tree of Yuan et al. (2004) showing those species for which integument morphology has been described. Numbers above nodes indicate the highest bootstrap values associated with the indicated clade in the full analysis of Yuan et al. (2004). Integument morphology is illustrated according to the included pattern key.