The role of KNOX genes in the evolution of morphological novelty in Streptocarpus

Abstract

The genus Streptocarpus comprises species with diverse body plans. Caulescent species produce leaves from a conventional shoot apical meristem (SAM), whereas acaulescent species lack a conventional SAM and produce only a single leaf (the unifoliate form) or clusters of leaves from the base of more mature leaves (the rosulate form). These distinct morphologies reflect fundamental differences in the role of the SAM and the process of leaf specification. A subfamily of KNOTTED-like homeobox (KNOX) genes are known to be important in regulating meristem function and leaf development in model species with conventional morphologies. To test the involvement of KNOX genes in Streptocarpus evolution, two parologous KNOX genes (SSTM1 and SSTM2) were isolated from species with different growth forms. Their phylogenetic analysis suggested a gene duplication before the subgeneric split of Streptocarpus and resolved species relationships, supporting multiple evolutionary origins of the rosulate and unifoliate morphologies. In S. saxorum, a caulescent species with a conventional SAM, KNOX proteins were expressed in the SAM and transiently downregulated in incipient leaf primordia. The ability of acaulescent species to initiate leaves from existing leaves was found to correlate with SSTM1 expression and KNOX protein accumulation in leaves and to reflect genetic differences at two loci. Neither locus corresponded to SSTM1, suggesting that cis-acting differences in SSTM1 regulation were not responsible for evolution of the rosulate and unifoliate forms. However, the involvement of KNOX proteins in leaf formation in rosulate species suggests that they have played an indirect role in the development of morphological diversity in Streptocarpus.

Figure 1.

Streptocarpus Growth Forms. (A) Unifoliate S. dunnii. (B) Caulescent S. saxorum. (C) Rosulate S. rexii. (D) S. dunnii (left) and S. rexii (right). The S. dunnii × S. rexii F1 hybrid (center) has a rosulate arrangement of multiple, large phyllomorphs.

Figure 2.

Structure and Phylogeny of SSTM1 Genes. (A) Alignment of inferred SSTM1 protein sequences from S. dunnii (SdSTM1), S. rexii (SrSTM1), and S. saxorum (SsSTM1) to Arabidopsis STM, showing shared conserved domains and intron positions (arrowheads). Black boxes highlight invariant amino acids, dark gray boxes amino acids conserved between Streptocarpus proteins, and light gray boxes conservative substitutions. Sequences were aligned using ClustalW. (B) Strict consensus of the four most parsimonious KNOX trees using homeobox cDNA sequences. Dicot STM-like genes are highlighted in black and Streptocarpus genes with arrowheads. Aaknox1, mkn1, and Crknox3 were included as outgroups and trees rooted on the algal sequence, Aaknox1. Bootstrap values are given above branches.

Figure 3.

DNA Gel Blot Analysis of Streptocarpus KNOX Genes. DNA gel blots of genomic DNA were probed at both high and low stringency with an SSTM1 fragment that includes the homeobox (left) or at high stringency with the poorly conserved 5′ region of SSTM1 or its 3′ untranslated region (right).

Figure 4.

SSTM Phylogenies for Streptocarpus. (A) Relationships between SSTM exon sequences flanking intron 3 derived from a midpoint-rooted majority rule consensus of 2480 most parsimonious trees. Exon sequences for SSTM1 genes with repetitive introns are included. Percentage of the majority is given above branches with bootstrap support below. Branches that collapse in the strict consensus are indicated with an asterisk. Taxa represented more than once are highlighted in red and blue. (B) Phylogeny of SSTM third intron shown as a midpoint rooted majority rule consensus of 9002 most parsimonious trees. For each paralogue (SSTM1 or SSTM2) Clade I (I) and Clade II (II) species are grouped. Clades of Madagascan species are indicated by black bars across their bases. Morphological types are indicated at the right (illustrations redrawn from Hilliard and Burtt [1971] and Jong [1978]).

Figure 5.

SSTM1 Expression Patterns. (A) to (C) The expression of SSTM1 in inflorescence apices, proximal and distal midvein, and leaf lamina was compared by RT-PCR with a ubiquitously expressed 40S ribosomal subunit gene as a control. (A) Caulescent S. saxorum. (B) Unifoliate S. dunnii. (C) Rosulate S. rexii. (D) SSTM1 expression was detected under the same conditions in the organogenic petiolode region of rosulate S. rexii but not in the petiolode of unifoliate S. dunnii.

Figure 6.

KNOX Immunolocalization in S. saxorum. (A) A vegetative apex of S. saxorum showing leaves in whorls of three. New leaves are initiated directly opposite a leaf in the preceding whorl (bar = 1 mm). (B) Transverse section of apex. Nuclear-localized KNOX proteins can be seen in the central, triangular SAM and in leaf veins (bar = 50 μm). (C) and (D) Longitudinal sections of different S. saxorum apices, showing staining in the central SAMs, SAMs in the axils of leaves (star), and in developing veins. An unstained region was detected at the site of leaf initiation from the SAM (arrow in [D]). Bar = 100 μm. (E) to (J) Transverse sections through leaves at increasing stages of maturity. Arrows indicate vascular localization of KNOX proteins. Bars = 25 μm in (E), 50 μm in (F) and (G), and 100 μm in (H) to (J).

Figure 7.

KNOX Immunolocalization in Acaulescent Species. The inflorescence meristems of S. rexii and S. dunnii are formed in an acropetal sequence from the groove meristem at the proximal midrib. Each inflorescence meristem (im) gives rise to lateral bracts (b), a terminal older flower (T1) proximally, and a younger distal flower (V1) or repetitions of that unit (nomenclature according to Weber [1982]). se, sepal; p, petal; a, anther; g, gynoecium; np, new phyllomorph; Infl, inflorescence. Bars = 0.5 mm. (A) to (D) S. dunnii. (A) Two floral meristems in longitudinal section. (B) A pair of floral meristems at an earlier stage in development, subtended by a bract. (C) Longitudinal section through a juvenile phyllomorph in which we were unable to detect KNOX expression. (D) Longitudinal section through a juvenile phyllomorph showing localized KNOX expression at its base. (E) to (H) S. rexii. New phyllomorphs arise in two ranks from the more proximal petiolode. (E) KNOX localization in a transverse section of an inflorescence, corresponding to plane 1 in (H), and new inflorescence meristem using antibodies raised against Arabidopsis STM. (F) An adjacent section to (E) probed only with secondary antibody (bar = 0.1 mm). (G) A longitudinal section through a flowering phyllomorph corresponding to plane 2 in (H). (I) The inset region of (G) at higher magnification showing KNOX expression in a mound of cells at the prospective site of phyllomorph initiation.

Figure 8.

Inheritance of SSTM1 Alleles and Expression in Different Growth Forms. (A) and (B) SSTM1 alleles were amplified from the F1 (S. rexii × unifoliate) parent of the backcross, from the unifoliate parent, and from a selection of their progeny and distinguished by restriction site polymorphisms after digestion. No correlation is apparent between SSTM1 genotype and morphology. (C) SSTM1 expression correlates with plant form in backcross plants.