Antagonistic roles for KNOX1 and KNOX2 genes in patterning the land plant body plan following an ancient gene duplication

Abstract

Neofunctionalization following gene duplication is thought to be one of the key drivers in generating evolutionary novelty. A gene duplication in a common ancestor of land plants produced two classes of KNOTTED-like TALE homeobox genes, class I (KNOX1) and class II (KNOX2). KNOX1 genes are linked to tissue proliferation and maintenance of meristematic potentials of flowering plant and moss sporophytes, and modulation of KNOX1 activity is implicated in contributing to leaf shape diversity of flowering plants. While KNOX2 function has been shown to repress the gametophytic (haploid) developmental program during moss sporophyte (diploid) development, little is known about KNOX2 function in flowering plants, hindering syntheses regarding the relationship between two classes of KNOX genes in the context of land plant evolution. Arabidopsis plants harboring loss-of-function KNOX2 alleles exhibit impaired differentiation of all aerial organs and have highly complex leaves, phenocopying gain-of-function KNOX1 alleles. Conversely, gain-of-function KNOX2 alleles in conjunction with a presumptive heterodimeric BELL TALE homeobox partner suppressed SAM activity in Arabidopsis and reduced leaf complexity in the Arabidopsis relative Cardamine hirsuta, reminiscent of loss-of-function KNOX1 alleles. Little evidence was found indicative of epistasis or mutual repression between KNOX1 and KNOX2 genes. KNOX proteins heterodimerize with BELL TALE homeobox proteins to form functional complexes, and contrary to earlier reports based on in vitro and heterologous expression, we find high selectivity between KNOX and BELL partners in vivo. Thus, KNOX2 genes confer opposing activities rather than redundant roles with KNOX1 genes, and together they act to direct the development of all above-ground organs of the Arabidopsis sporophyte. We infer that following the KNOX1/KNOX2 gene duplication in an ancestor of land plants, neofunctionalization led to evolution of antagonistic biochemical activity thereby facilitating the evolution of more complex sporophyte transcriptional networks, providing plasticity for the morphological evolution of land plant body plans.

Fig 1. Phylogeny and expression patterns of KNOX and BELL genes.

(A) Phylogenetic relationships of KNOX and BELL gene families in the plant lineage. Available sequence information suggests the gene duplication producing the KNOX and BELL genes occurred before the divergence of red and green algae. A gene duplication in the lineage leading to land plants created KNOX1 and KNOX2 genes from an ancestral algal KNOX gene. Estimated dates for some nodes are listed in millions of years before present (Mya). (B) In Arabidopsis, KNOX1, KNOX2, and BELL proteins are encoded by 4, 4, and 13 genes, respectively. In addition, KNATM encodes for a KNOX-related protein lacking a homeodomain. Detailed phylogenetic analyses of KNOX genes are presented in S1 and S2 Figs. (C) Schematic depiction of expression patterns for Arabidopsis KNOX1, KNOX2, and BELL genes based on previous literature [75] and publicly available transcriptome data (for details, see S3 and S4 Figs.). KNOX1 and some BELL genes, e.g., PNF and PNY, are primarily expressed in meristematic tissues while KNOX2 and other BELL genes such as SAW1 and SAW2 are expressed in differentiating organs. KNOX2 genes are highlighted in red.

Fig 2. KNOX2 mutant phenotypes and KNAT5 expression patterns.

(A-C) 10-day-old seedlings of wild-type (WT, A), knat3 knat5 (designated as knat35, B), and knat345 (C). (D-E) Wild-type (D) and knat345 (E) plants after bolting; 5 weeks-old plants are shown. (F-G) Representative wild-type (F) and knat345 (G) rosette leaves. (H-L) First cauline (stem) leaves of wild-type (H), knat3 knat4 (designated as knat34, I), knat3 knat4 knat5/+ (designated as knat34, 5/+, J), knat3 knat5 (K), and knat345 (L) plants; progressive loss of the KNOX2 activity results in increasingly serrated leaves. (M-N) Wild-type (M) and knat345 (N) flowers. (O-R) Mature wild-type (O, Q) and knat345 (P, R) ovules and embryo sacs. An arrow marks ectopic formation of tracheary elements. (S-V) _pro KNAT5:KNAT5-GUS expression in developing embryos (S-T), vegetative shoot apex (U), leaf (V), and ovules (W). _pro KNAT5:KNAT5-GUS is not detected in the shoot apical meristem (marked by an arrowhead) and the youngest leaf primordium (marked with an arrow). An asterisk indicates a stipule. (X) _pro KNAT4:GUS expression in vegetative shoot apex. For additional expression data, see S10 Fig. Plants are all in the Columbia (Col) background. Scale bars, 1 cm.

Fig 3. Genetic interactions between BELL and KNOX2 genes.

(A-E) 10 days-old seedlings of _pro 35S:KNAT3 (A), _pro STM>>SAW2 (B), _pro 35S:KNAT3 _pro STM>>SAW2 (C), _pro STM>>KNAT5 _pro STM>>SAW2 (D), and stm-11 (E), showing resemblance between stm (E) and plants expressing both KNOX2 and SAW2 proteins in the meristem (C, D). Defective meristems are marked with arrows. In (B-D), expression of SAW2 or KNAT5 alone, or both genes together is transactivated by the STM regulatory sequence. (F) Introducing the _pro 35S:amiR ¹⁵⁹ -KNAT345–1 construct did not alter the seedling lethal phenotype of stm-11 plants. Shown is a 25 days-old plant. (G-J) Gynoecia from wild-type (G, emasculated and unpollinated), bel1–154 (H), knat345 (I), and bel1–154 blh1–114 (J) flowers. (I-J) Unfertilized gynoecia turn yellow. See S9 Fig. for more data. This phenotype was not observed in emasculated wild-type (G), bel1 (H), and blh1–114 plants. Plants in (B, D, E, F) are in the Landsberg erecta (Ler) background. A plant in (C) is in the Col/Ler mixed background. All other plants are in the Col background. Scale bars, 0.5 mm.

Fig 4. Genetic interactions between KNOX1 and KNOX2 genes.

(A-B) Morphology of _pro 35S:amiR ¹⁵⁹ -KNAT345–1 plants in wild-type (A) and bp-9 knat2–5 knat6–1 mutant (B) backgrounds. 6 weeks-old plants are shown. (C-E) F1 plants expressing both _pro BLS:STM and _pro 35S:amiR ¹⁵⁹ -KNAT345–1 constructs (D) show a stronger serration phenotype than either parental line (C, E). All plants shown are hemizygous for the transgene(s) and 2 weeks old. (F-G) Morphology of knat3 knat5 (F) and knat2–5 knat3 knat5 knat6–1 (G) plants grown for 5 weeks. (H) A representative bp knat345 rosette leaf with deeply lobed margins, as seen in knat345. (I-J) The bp inflorescence phenotype (I) is observed in bp knat345 infloresences (J), indicating additive effects of mutations in these genes. (H-J) Plants were grown for 2 months. (K-P) KNOX1 reporter expression in wild-type (K-M) and knat3 knat5 (N-P) plants. Shown are _pro BP:GUS expression in 17 days-old plants (K, N), _pro KNAT2:GUS expression in 20 days-old plants (L, O), and _pro STM:GUS expression in 21 days-old plants (M, P). No ectopic expression of _pro BP:GUS was detected during stages of leaf development when lobes are forming in knat3 knat5 plants (K, N), but ectopic expression was observed at leaf serration tips after their development. _pro KNAT2:GUS and _pro STM:GUS expression patterns in knat3 knat5 plants are similar to those in wild-type plants (L-M, O-P). Occasionally, longer incubation detected _pro STM:GUS activity in the sinus of wild-type and mutant leaves (M). _pro KNAT2:GUS and _pro STM:GUS were analyzed in the mixed genetic background (refer to S1 Table for details), and other plants are in the Col background. Scale bars in I, J, 3 mm, K, L, N, 100 μm and in M, O, P, 200 μm.

Fig 5. KNOX1 and KNOX2 converge on CUC activity.

(A) One-month old cuc2 knat345 quadruple mutant. A mutation in the CUC2 gene largely suppresses the leaf serration phenotype of knat345 plants. Compare with a knat345 plant in Fig. 2E. See S13 Fig. for additional data. (B-C) Representative knat345 (B) and cuc2 knat345 (C) rosette leaves, demonstrating marginal leaf lobing is suppressed by a cuc2 mutation. Shown are the 10th leaves from 2 month-old plants of each genotype. Note that other mutant phenotypes, including leaf size and female sterility, are not suppressed by the cuc2 mutation. (D-F) _pro 35S:KNAT3 partially suppresses the leaf lobing phenotype of _pro BLS:STM plants. 12 days-old plants are shown. (G-H) Second leaves of wild-type (G) and _pro 35S:amiR ¹⁵⁹ -KNAT345–2 (H) Cardamine hirsuta plants grown for four weeks. (G) In wild-type plants, the first and second leaves always consist of a single, undivided, lamina. (H) Reducing KNOX2 activity by consitutively expressing an amiRNA that targets Cardamine hirsuta orthologues of KNAT3, KNAT4, and KNAT5 genes (_pro 35S:amiR ¹⁵⁹ -KNAT345–2) results in plants with an extra lateral leaflet (marked with an arrow) on the second leaf. (I-J) Third leaves removed from one month-old wild-type (I) and _pro 35S:KNAT3 (J) Cardamine hirsuta plants. (I) In wild-type plants, the third leaf typically consists of three leaflets. (J) Introduction of gain-of-function KNOX2 alleles (constitutive expression of the KNAT3 gene from Arabidopsis; _pro 35S:KNAT3) results in an undivided third leaf in strong lines. Plants in (A-F) are all in the Col background.

Fig 6. Proposed KNOX functions during land plant evolution.

Along the phylogeny of plants, the primary functions for BELL (depicted as B) and KNOX (K) proteins, as well as the gene copy number, are presented for Chlamydomonas (a unicellular Chlorophyte alga), Physcomitrella (a moss), and Arabidopsis (a flowering plant). The ancestral conditions at branches were deduced from our phylogenetic analyses (S1 and S17 Figs.). In each life cycle, a red arrow indicates meiosis, and haploid (grey) and diploid (green) stages are color-coded. In Chlamydomonas the plus gamete expresses a BELL (depicted as B) protein while the minus gamete expresses a KNOX (K) protein; upon gamete fusion the KNOX and BELL proteins heterodimerize and regulate zygotic gene expression. Prior to the origin of land plants, a gene duplication in an ancestral KNOX gene generated two subclasses, KNOX1 (K1) and KNOX2 (K2) genes. In Physcomitrella, KNOX1 activity maintains tissue proliferation during sporophyte (diploid) development while KNOX2 represses the haploid genetic program during the diploid generation. In Arabidopsis, KNOX1 activity promotes meristem maintenance, and our study demonstrates that KNOX2 activity promotes tissue differentiation, perhaps via repression of meristematic functions, in the diploid generation. We propose that (1) the gene duplication producing KNOX1 and KNOX2 paralogs and ensuing neofunctionalization was instrumental in the evolution of a complex multicellular diploid generations in land plants and (2) the diversification of KNOX/BELL modules during land plant evolution facilitated the evolution of ever more complex diploid sporophyte body plans.