KNOX lost the OX: the Arabidopsis KNATM gene defines a novel class of KNOX transcriptional regulators missing the homeodomain

Abstract

Three amino acid loop extension (TALE) homeodomain transcriptional regulators play a central role in plant and animal developmental programs. Plant KNOTTED1-like homeobox (KNOX) and animal Myeloid ecotropic viral integration site (MEIS) proteins share a TALE homeodomain and a MEINOX (MEIS-KNOX) domain, suggesting that an ancestral MEINOX-TALE protein predates the divergence of plants from fungi and animals. In this study, we identify and characterize the Arabidopsis thaliana KNATM gene, which encodes a MEINOX domain but not a homeodomain. Phylogenetic analysis of the KNOX family places KNATM in a new class and shows conservation in dicotyledons. We demonstrate that KNATM selectively interacts with Arabidopsis BELL TALE proteins through the MEINOX domain. The homeodomain is known to be necessary for KNOX-KNOX interaction. On the contrary, KNATM specifically dimerizes with the KNOX protein BREVIPEDICELLUS through an acidic coiled-coil domain. KNATM is expressed in proximal-lateral domains of organ primordia and at the boundary of mature organs; in accordance, genetic analyses identify a function for KNATM in leaf proximal-distal patterning. In vivo domain analyses highlighted KNATM functional regions and revealed a role as transcriptional regulator. Taken together, our data reveal a homeodomain-independent mechanism of KNOX dimerization and transcriptional regulation.

Figure 1.

Comparison of KNATM to Arabidopsis KNOX Proteins. (A) Alignment of the KNATM proteins with the MEINOX domain of all Arabidopsis KNOX proteins. The BPID domain is highlighted in green, the KNOX1 domain is in orange, and the KNOX2 domain is in red. The numbers above the alignment indicate the probability that each residue of KNATM-B is part of a coiled-coil structure measured in a 14-, 21-, or 28-residue window. (B) Scheme of the KNATM splicing forms. Rectangles and lines represent exons and noncoding sequences, respectively. (C) RT-PCR analyses (40 cycles) conducted with KNATM primers. ACTIN2 (ACT2) primers were used as positive control. The Control lane contains no input RNA. (D) Neighbor-joining tree built on the multiple sequence alignment of all Arabidopsis KNOX MEINOX domains. Numbers at the nodes represent bootstrap values obtained with neighbor-joining (black), UPGMA (green), and maximum parsimony (red) algorithms.

Figure 2.

KNATM Interaction with BELL and KNOX Proteins. Results of the yeast two-hybrid assay. Baits are highlighted in green and preys in blue. Interactions were assessed using combined readouts from the URA3, lacZ, and HIS3 reporter genes. Strong, medium, mild, and no interactions are indicated by three plus signs (red), two plus signs (orange), one plus sign (yellow), and a minus sign (white), respectively.

Figure 3.

KNATM Expression as Observed in PKNATM-KNATM-uidA Arabidopsis Plants Stained to Detect β-Glucuronidase Activity. (A) Serial transverse sections of an inflorescence. β-Glucuronidase activity is detected in the lateral domains of flower meristems. (B) Serial longitudinal sections of an inflorescence. (C) Serial transverse sections of a vegetative apex. β-Glucuronidase activity is detected in the lateral domains of leaf primordia. (D) Serial longitudinal sections of a vegetative apex. (E) Inflorescence. (F) Longitudinal section of an inflorescence stem. (G) Transversal section of an inflorescence node. (H) Seedling. (I) Mature embryo and close-up of a cotyledon hydathode.

Figure 4.

KNATM-B Subcellular Localization. (A) Phase contrast image of epidermal onion cells transiently expressing KNATM-B fused to GFP. The arrowhead indicates the nucleus. (B) GFP fluorescence image of epidermal onion cells transiently expressing KNATM-B fused to GFP. The arrowhead indicates the nucleus. (C) GFP fluorescence image of Arabidopsis petal cells stably expressing KNATM-B fused to GFP. (D) A 4′,6-diamidino-2-phenylindole (DAPI) fluorescence image of Arabidopsis petal cells stably expressing KNATM-B fused to GFP. (E) Superimposed GFP and DAPI fluorescence images of Arabidopsis petal cells stably expressing KNATM-B fused to GFP. (F) Phase contrast image of epidermal onion cells transiently expressing KNATM-B fused to the N-terminal fragment of YFP and PNY fused to the C-terminal fragment of YFP. The arrowhead indicates the nucleus. (G) YFP fluorescence image of epidermal onion cells transiently expressing KNATM-B fused to the N-terminal fragment of YFP and PNY fused to the C-terminal fragment of YFP. The arrowhead indicates the nucleus.

Figure 5.

KNATM-B Overexpression Arabidopsis Plants. (A) Wild-type seedling (3 weeks old). (B) 35S-KNATM-B seedling (3 weeks old). (C) Wild-type cotyledons and rosette leaves aligned in order of appearance (6-week-old plant). (D) 35S-KNATM-B cotyledons and rosette leaves aligned in order of appearance (6-week-old plant). (E) Wild-type cauline leaves (10-week-old plant). (F) 35S-KNATM-B cauline leaves (10-week-old plant). Bars = 1 cm in (A) to (F). (G) 35S-KNATM-B plant (7 weeks old). (H) Measurements of petiole length, lamina length, and lamina width of wild-type (purple), 35S-KNATM-B low expression (orange), 35S-KNATM-B high expression (red) cotyledons (cot), and rosette leaves (1 to 8) (six-week-old plants). Measurements of the total number of rosette leaves and days to bolting of wild-type (purple), 35S-KNATM-B low expression (orange), and 35S-KNATM-B high expression (red) plants. Values are reported as mean ± se (n = 10). Asterisks indicate statistically significant differences (Student's t test, P < 0.05) of transgenic lines compared with the wild type.

Figure 6.

KNATM-B/SAW1 Genetic Interaction. (A) Arabidopsis 35S-KNATM-B plant (6 weeks old). (B) Arabidopsis 35S-SAW1 plant (6 weeks old). (C) Arabidopsis 35S-KNATM-B/35S-SAW1 plant (6 weeks old). Bars = 1 cm.

Figure 7.

KNATM-B/BP Genetic Interaction. (A) Arabidopsis wild-type seedling. (B) Arabidopsis 35S-KNATM-B seedling. (C) Arabidopsis 35S-BP seedling. (D) Arabidopsis 35S-KNATM-B/35S-BP seedling. (E) Scanning electron microscopy image of an Arabidopsis wild-type first rosette leaf. (F) Scanning electron microscopy image of an Arabidopsis 35S-KNATM-B first rosette leaf. (G) Scanning electron microscopy image of an Arabidopsis 35S-BP first rosette leaf. (H) to (K) Scanning electron microscopy images of an Arabidopsis 35S-KNATM-B/35S-BP first rosette leaf. (L) to (O) Scanning electron microscopy images of the adaxial surface of an Arabidopsis wild-type, 35S-KNATM-B, 35S-BP, and 35S-KNATM-B/35S-BP first rosette leaf. (P) to (S) Scanning electron microscopy images of the abaxial surface of an Arabidopsis wild-type, 35S-KNATM-B, 35S-BP, and 35S-KNATM-B/35S-BP first rosette leaf, respectively. Bars = 1 cm in (A) to (D), 1 mm in (E) to (K), and 100 μm in (L) to (S). All plants are 3 weeks old.

Figure 8.

KNATM Transcriptional Activation Activity. (A) Quantitative β-galactosidase assays in yeast. DB, GAL4 DNA binding domain. (B) Scheme of the KNATM protein variants overexpressed in Arabidopsis and the phenotypes associated. A plus sign indicates the 35S-KNATM-B phenotype, and a minus sign indicates no visible phenotype. The BPID domain is highlighted in green, the KNOX1 domain in orange, the KNOX2 domain in red, the GAL4 transcriptional AD in violet, the SRDX transcriptional RD in light blue, and unknown domains in pink. (C) Arabidopsis 35S-KNATM-B seedling and first rosette leaf (3 weeks old). (D) Arabidopsis 35S-KNATM-B-RD seedling and first rosette leaf (3 weeks old). (E) Measurements of petiole length, lamina length, and lamina width of 35S-KNATM-B (red) and three independent lines of 35S-KNATM-B-RD (blue tones) Arabidopsis cotyledons (cot) and first and fourth rosette leaves (1 and 4) (6-week-old plants). Measurements of days to bolting of 35S-KNATM-B (red) and three independent lines of 35S-KNATM–B-RD (blue tones) Arabidopsis plants. Values are reported as mean ± se (n = 10). RNA gel blot analysis of KNATM-B expression in 35S-KNATM-B and three independent lines of 35S-KNATM-B-RD Arabidopsis plants. Bars = 1 cm in (C) and (D).

Figure 9.

Model of KNATM Molecular Function. We suggest that KNATM-B titrates SAW1 and SAW2 proteins in an inactive dimer and/or in the cytoplasm, inhibiting their transcriptional activity. KNATM-B might also take part in a transcriptional complex with a DNA binding protein.