KNOX Lost the OX: The Arabidopsis KNATM Gene Defines a Novel Class of KNOX Transcriptional Regulators Missing the Homeodomain W

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1 The Plant Cell, Vol. 20: , April 2008, ª 2008 American Society of Plant Biologists KNOX Lost the OX: The Arabidopsis KNATM Gene Defines a Novel Class of KNOX Transcriptional Regulators Missing the Homeodomain W Enrico Magnani a,b,1 and Sarah Hake a,b a Department of Plant and Microbial Biology, University of California, Berkeley, California, b Plant Gene Expression Center, U.S. Department of Agriculture Agriculture Research Service, Albany, California, 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. INTRODUCTION Homeodomain proteins are characterized by a highly conserved DNA binding domain called the homeodomain (Gehring et al., 1994). This domain has been implicated in important protein protein interactions that are required for fundamental aspects of plant and animal development. Three amino acid loop extension (TALE) homeodomain proteins contain three extra amino acids between helix-1 and helix-2 of the homeodomain (Bertolino et al., 1995). Five TALE protein classes (PBC, Myeloid ecotropic viral integration site [MEIS], Iroquois, TG-interacting factor and Mohawk) have been identified in Metazoans based on the conservation of the homeodomain and associated domains, while only two classes exist in plants (KNOTTED1-like homeobox [KNOX] and BEL1- like [BELL]) (Hake et al., 2004; Mukherjee and Burglin, 2007). Plant KNOX and animal MEIS proteins carry a conserved domain upstream of the homeodomain, collectively called MEINOX. This conservation suggests that an ancestral MEINOX-TALE protein predated the divergence of plants from fungi and metazoans (Burglin, 1997, 1998; Mukherjee and Burglin, 2007). The plant MEINOX domain is made of two smaller domains, KNOX1 and KNOX2, separated by a poorly conserved linker sequence. Yeast two-hybrid assays showed that the MEINOX domain is 1 Address correspondence to emagnani@berkeley.edu. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors ( is: Enrico Magnani (emagnani@berkeley.edu). W Online version contains Web-only data. necessary and sufficient to interact with BELL proteins (Bellaoui et al., 2001; Muller et al., 2001; Smith et al., 2002). However, both the MEINOX domain and the homeodomain mediate KNOX- KNOX dimerization (Muller et al., 2001; Nagasaki et al., 2001). Similarly, MEIS-A and MEIS-B domains (also called HM1 and HM2) constitute the metazoan MEINOX domain and allow MEIS proteins to interact with PBC proteins (Knoepfler et al., 1997; Rieckhof et al., 1997; Berthelsen et al., 1998; Ryoo et al., 1999). A strong functional relationship exists between the homeodomain and the MEINOX domain, but some observations indicate that the MEINOX domain can also work in a homeodomainindependent fashion. Alternative splicing of the MEIS Drosophila homothorax (hth) and its mouse ortholog meis1 genes generates homeodomainless isoforms that still encode the MEINOX domain. Interestingly, homeodomainless Hth is sufficient to carry out most Hth functions (Noro et al., 2006). The Meis2-e splicing form of the human Meis2 gene encodes a MEINOX domain and a truncated homeodomain unable to bind DNA. Meis2-e works as a dominant-negative regulator by competing with homeodomaincontaining isoforms (Yang et al., 2000). Similarly, Arabidopsis thaliana plants overexpressing an artificial homeodomainless SHOOTMERISTEMLESS (STM) KNOX protein show dominantnegative phenotypes (Markel et al., 2002). The eight KNOX genes of Arabidopsis fall into two phylogenetic classes. STM, BREVIPEDICELLUS (BP)/KNAT1, KNAT2, and KNAT6 belong to class I and are characteristically expressed in the shoot apical meristem (SAM), while class II KNAT3, KNAT4, KNAT5, and KNAT7 are more broadly expressed (Hake et al., 2004). Two knox loss-of-function phenotypes have been characterized and reveal a function in meristem establishment and

2 876 The Plant Cell maintenance and internode growth. Severe stm mutants do not form a SAM and have cotyledons fused at the petiole base (Long et al., 1996). bp plants are reduced in size because of irregularly shortened internodes and their pedicels point down (Douglas et al., 2002; Venglat et al., 2002). Mutants in the KNAT2 and KNAT6 genes do not show any visible phenotype, but KNAT6 contributes with STM to the maintenance of the SAM (Belles-Boix et al., 2006). In addition, Knox dominant mutants, KNOX-overexpressing lines, and loss-of-function mutants of KNOX negative regulators drastically affect leaf proximal-distal patterning (Hake et al., 2004). The Arabidopsis genome contains 13 BELL genes (Smith et al., 2004), and the function of six of them has been characterized. BEL1, the first discovered BELL in Arabidopsis, isinvolvedinthe development of ovule integuments (Reiser et al., 1995). Mutations in PENNYWISE (PNY) (also known as BELLRINGER, REPLUM- LESS or VAAMANA) disturb the Arabidopsis spiral phyllotactic pattern, while double mutations in PNY and its close paralog POUND-FOOLISH (PNF) arrest the transition from vegetative to reproductive phase (Byrne et al., 2003; Roeder et al., 2003; Smith and Hake, 2003; Bhatt et al., 2004; Smith et al., 2004). ARABI- DOPSIS THALIANA HOMEOBOX1 also controls floral competency by transcriptionally activating the flower repressor gene FLOWERING LOCUS C (Proveniers et al., 2007). SAWTOOTH1 (SAW1) and SAW2 negatively regulate BP, and the saw1 saw2 double mutant has increased leaf serration (Kumar et al., 2007). In this study, we identify and characterize KNATM, a novel Arabidopsis KNOX gene that encodes a MEINOX domain but lacks the homeodomain. Alternative splicing generates at least three KNATM isoforms that define a new KNOX phylogenetic class. KNATM selectively interacts with BELL proteins through the MEINOX domain. In addition, an acidic coiled-coil domain, termed BP-interacting domain (BPID), mediates KNATM-BP dimerization. KNATM is expressed in a polar pattern in organ primordia and at the boundary of mature organs. Arabidopsis plants overexpressing KNATM show defects in petiole elongation, leaf serration, and flowering time. Yeast transcriptional activation assays and analyses of Arabidopsis plants expressing KNATM carrying a transcriptional repression or activation domain highlight the role of KNATM in transcriptional regulation in a homeodomain-independent fashion. 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.

3 KNATM: A Homeodomainless KNOX Protein 877 RESULTS A Novel Member of the Arabidopsis KNOX Family To find Arabidopsis KNOX-related proteins, we used all Arabidopsis KNOX full-length proteins and MEINOX domains as seeds in PSI-BLAST searches against the National Center for Biotechnology Information (NCBI, National Institutes of Health, Bethesda, MD) nonredundant (NR) Arabidopsis protein database. These searches revealed the Arabidopsis At1g14760 locus, which is predicted to code for a KNOX1 domain (Figure 1A). The 59 and 39 rapid amplification of cdna ends PCR analyses detected three splicing forms (Figures 1A and 1B). The Conserved Domain Database and the PFAM HMM library predicted the three protein isoforms to carry a KNOX1 domain, a full MEINOX domain, or a MEINOX domain missing the interdomain linker. In addition, the N-terminal region of all three isoforms contains a predicted coiledcoil acidic domain (Figure 1A). Based on sequence similarities to KNOX genes, we named these three splicing forms KNATM-A, KNATM-B, andknatm-c. RT-PCR analyses detected KNATM-A, KNATM-B, and KNATM-C transcripts in inflorescence and seedling at 40 cycles (Figure 1C) and in rosette leaf, root, stem, cauline leaf, and silique at 45 cycles (see Supplemental Figure 1 online). KNATM Phylogenetic Analysis We aligned the MEINOX domain of all Arabidopsis KNOX proteins, including KNATM-B, and built neighbor-joining, maximum parsimony, and UPGMA phylogenetic trees. The three algorithms group class I, class II, and KNATM MEINOX domains in three separate clades with bootstrap values >50. This result suggests that the KNATM-B protein does not fall into any existing KNOX classes and will be referred to as class M KNOX protein (Figure 1D). To find the homologs of KNATM in other plant species, the Arabidopsis KNATM proteins were used as seeds in BLAST and PSI-BLAST searches against the NCBI NR and the Joint Genome Institute Populus trichocarpa protein databases. Our analysis revealed KNATM homologs in Medicago truncatula, P. trichocarpa, and Solanum esculentum (see Supplemental Figure 2 online). Interestingly, we did not detect any clear homolog in monocotyledons. KNATM BELL Interaction KNATM-B is predicted to code for a full MEINOX domain and therefore to interact with BELL proteins. To test this hypothesis, we performed combinatorial yeast two-hybrid assays using KNATM-A, KNATM-B, and KNATM-C as baits and the BELL proteins PNY, PNF, SAW1, SAW2, and BEL1 as preys. We assessed the strength of the interactions using the URA3, lacz, and HIS3 reporter genes that show a decreasing level of stringency. To quantify the reporter gene expression levels, we used four well-characterized protein partners with a spectrum of interaction strength. KNATM-B was analyzed only with the most stringent lack-of-uracil selection because it selfactivates the HIS3 and lacz reporter genes. KNATM-B interacts with BEL1, SAW1, SAW2, and PNY, while KNATM-A and KNATM-C showed no interaction with BELL proteins (Figure 2). Interestingly, KNATM-B strongly interacts with PNY but not with its paralog PNF. KNATM-B interaction with PNY was confirmed in planta by bimolecular fluorescence complementation assays (Figures 4F and 4G). To test if the MEINOX domain is sufficient to mediate the interaction with BELL proteins, we created a deleted form of KNATM-B (DKNATM-B) that carries only the MEINOX domain. Interestingly, DKNATM-B does not self-activate and could be tested under more relaxed selections. DKNATM-B shows the same pattern of interaction of KNATM-B (Figure 2). In summary, our results show that KNATM-B selectively interacts with BELL proteins through the MEINOX domain. 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.

4 878 The Plant Cell KNATM KNOX Interaction A comprehensive yeast two-hybrid assay was conducted to study KNATM KNOX interaction. KNATM-A, KNATM-B, and KNATM-C were tested as baits against all Arabidopsis KNOX proteins. KNATM-B strongly dimerizes with BP. A mild interaction was also detected with KNAT3 and KNAT4. On the contrary, KNATM-A and KNATM-C do not show any interaction with KNOX proteins. In addition, KNATM isoforms do not dimerize with one other (Figure 2). DKNATM-B was also assayed against all KNOX proteins and showed no interaction (Figure 2). This finding demonstrates that the acidic coiled-coil domain is necessary to mediate the KNATM BP interaction, and we therefore named it the BPinteracting domain (BPID). Expression Analysis We conducted RNA in situ hybridization experiments to study KNATM expression in reproductive and vegetative meristems. We detected KNATM transcripts in the lateral domains of flower meristems but not in the inflorescence meristem (see Supplemental Figure 3 online). Our attempts to detect KNATM expression in vegetative apices did not produce clear results (data not shown). To confirm our in situ data, the KNATM promoter and genomic sequence was translationally fused to the uid-a reporter gene and stably transformed into Arabidopsis plants. b-glucuronidase activity was detected in the lateral domains of flower meristems from stage one to stage three, but not in the inflorescence meristem (Figures 3A and 3B). Similarly, KNATM was detected in leaf primordia, and its expression changes from lateral to proximal-lateral as organs develop, but it is excluded from the vegetative SAM (Figures 3C and 3D). In addition, KNATM expression demarcates leaf hydathodes, the adaxiallateral boundaries of mature lateral organs, and the boundaries of embryo cotyledons (Figures 3E to 3I). KNATM-B Subcellular Localization A KNATM-B translational fusion to the green fluorescent protein (GFP) was analyzed in transient assays using epidermal onion Figure 3. KNATM Expression as Observed in PKNATM-KNATM-uidA Arabidopsis Plants Stained to Detect b-glucuronidase Activity. (A) Serial transverse sections of an inflorescence. b-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. b-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.

5 KNATM: A Homeodomainless KNOX Protein 879 cells as well as in stable Arabidopsis transformants. In both cases, GFP was detected in the cytoplasm and the nucleus (Figures 4A to 4E). Consistent with these results, KNATM-B does not carry any known localization signal and is expected to freely move through the nuclear pores because of its size (15.7 kd). To test if KNATM-B localization changes when it forms dimers, we performed bimolecular fluorescence complementation assays. A KNATM-B translational fusion to the N-terminal fragment of the yellow fluorescent protein (YFP) and a PNY translational fusion to the C-terminal fragment of YFP were transiently cotransformed into epidermal onion cells. YFP was preferentially detected in the cytoplasm (Figures 4F and 4G), suggesting that KNATM-BELL dimers are confined in the cytoplasm. Individual constructs were transformed as controls and did not generate fluorescence. KNATM Overexpression To gain insight into the function of KNATM, we undertook multiple approaches to knock out the gene and created overexpression lines. Because no T-DNA or TILLING lines affecting KNATM expression or function were available, we produced RNA interference and artificial microrna constructs that failed to significantly knock down KNATM (data not shown). We also generated Arabidopsis transgenic lines carrying KNATM-B under the control of the constitutive cauliflower mosaic virus 35S promoter (35S). KNATM-B expression in these lines was confirmed by RNA gel blot analyses (see Supplemental Figure 4 online). The overexpression lines show a series of leaf development defects. The petioles of cotyledons and rosette leaves are more elongated compared with the wild type (Figures 5A to 5D). The increased petiole length of cotyledons and the fifth, sixth, seventh, and eighth rosette leaves occurs at the expense of the lamina, which is significantly shorter. In addition, lamina expansion is impaired and results in narrower leaves (Figures 5A to 5D and 5H). Rosette leaves are curled down and more serrated (Figures 5C, 5D, and 5G). Increased serration is also observed in cauline leaves (Figures 5E and 5F). Figure 5G shows cauline leaves curled up and wrapped around the flowering stem during the first stages of stem elongation. A scanning electron microscope analysis of 35S-KNATM-B leaves showed no defects in petiole and adaxial lamina cell types (Figures 7L and 7M). On the contrary, abaxial lamina cells are more elongated than in the wild type (Figures 7P and 7Q). Furthermore, plants overexpressing KNATM-B bolt approximately 5 d later, producing four additional rosette leaves compared with the wild type (Figure 5H). Arabidopsis 35S-KNATM-A, 35S-KNATM-C, and 35S- DKNATM-B transgenic lines were also generated and do not show any visible phenotype (Figure 8B). These data point out the functional importance of BPID and MEINOX domains. KNATM SAW1 Genetic Interaction 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) A49,6-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. KNATM-B and SAW1 strong interaction and overlapping expression patterns in the hydathodes suggest a functional relationship. In addition, leaf serration observed in 35S-KNATM-B plants resembles the saw1 saw2 double mutant (Kumar et al., 2007). To investigate their genetic interaction, we generated transgenic lines overexpressing both SAW1 and KNATM-B. 35S-SAW1 plants show reduced size and male sterility (Figure 6B) (Kumar et al., 2007). The double overexpression of KNATM-B and SAW1 fully suppresses both single overexpression phenotypes, resulting in a wild-type-looking plant (Figure 6). Taken together, our data show an antagonistic effect of KNATM-B and SAW1. KNATM BP Genetic Interaction To better characterize the BP KNATM interaction, we generated Arabidopsis transgenic lines overexpressing both BP and KNATM-B. Double overexpression seedlings are smaller than single lines and show a synergistic phenotype on the first two rosette leaves (Figures 7A to 7D). While the first true leaf of 35S- KNATM-B and 35S-BP plants is overall correctly shaped, it is deeply lobed with reduced lamina surface in the double overexpression line (Figures 7E to 7K). In addition, some lamina tissue curves to form hollow protuberances similar to the knots observed in maize (Zea mays) Knotted1 plants (Vollbrecht et al., 1991) (Figures 7H to 7K). The analysis of leaf cell types revealed

6 880 The Plant Cell 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).

7 KNATM: A Homeodomainless KNOX Protein 881 levels similar to those seen with the KNATM-B-DB monomer, while PNY-DB, BEL1-DB, and KNATM-B-NLS do not activate on their own. On the contrary, KNATM-B-NLS loses its activation activity when coexpressed with BP-DB (Figure 8A). We further analyzed the role of KNATM-B in transcriptional regulation by generating Arabidopsis lines overexpressing KNATM-B or DKNATM-B fused to the GAL4 transcriptional activation domain (AD) or the SRDX transcriptional repression domain (RD) (Hiratsu et al., 2003; Rutherford et al., 2005). Arabidopsis lines overexpressing DKNATM-B-AD or DKNATM-B-RD resemble wild-type plants, confirming the importance of the BPID domain (Figure 8B). We then tested 33 35S-KNATM-B-RD independent T2 lines and selected the three with the strongest phenotype for further analyses. These transgenic lines have similar or higher KNATM-B expression levels than the 35S-KNATM-B high expresser (Figure 8E; see Supplemental Figure 4 online). Overall, the 35S-KNATM-B-RD leaves are similar to those of 35S-KNATM-B plants but do not curl down (Figures 8C to 8E). This finding suggests that RD suppresses KNATM-B transcriptional activation activity responsible for leaf downcurling, implying that KNATM-B is directly involved in transcriptional regulation. 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 ¼ 1 cm. abnormally shaped trichomes with supernumerary branches. The adaxial epidermal cells develop correctly, while petiole-like elongated cells invade the abaxial surface (Figures 7I to 7S). These findings suggest a synergistic interaction between BP and KNATM-B. KNATM-B Transcriptional Activation Activity The KNATM-B transcriptional activation activity, first observed in the yeast two-hybrid experiments, was tested with a quantitative assay. KNATM-B fused to the GAL4 DNA binding domain (DB) activates lacz to around 0.15 b-gal units. On the contrary, KNATM-A-DB and DKNATM-B-DB do not show any activation, revealing the importance of BPID and KNOX2 domains to drive activation (Figure 8A). To test whether KNATM-B activates transcription as a dimer with KNOX or BELL proteins, we coexpressed PNY, BEL1, or BP fused to GAL4-DB with KNATM-B fused to a nuclear localization signal (NLS). KNATM-B-NLS/BELL-DB dimers activate lacz to DISCUSSION TALE homeodomain proteins are conserved in animal and plants and play a key role in developmental programs. Plant KNOX and animal MEIS TALE proteins share a MEINOX domain in addition to the homeodomain. The MEINOX domain has always been found associated to a homeodomain and mediates MEIS-PBC and KNOX-BELL interactions. Arabidopsis KNOX transcription factors are involved in the establishment and maintenance of the SAM and in the control of phyllotaxy (Hake et al., 2004). The KNOX MEINOX domain is necessary and sufficient to interact with BELL proteins (Bellaoui et al., 2001; Muller et al., 2001; Smith et al., 2002). On the contrary, both the MEINOX domain and the homeodomain mediate KNOX KNOX interactions (Muller et al., 2001; Nagasaki et al., 2001). An in silico analysis of the Arabidopsis proteome allowed us to identify KNATM, a new KNOX protein lacking the homeodomain. Alternative splicing of KNATM produces three isoforms (KNATM-A, KNATM-B, and KNATM-C) coding for a partial or a complete MEINOX domain. We demonstrate that KNATM-B selectively interacts with BELL and KNOX proteins. The KNATM-B expression pattern and overexpression phenotypes suggest a function in leaf proximal/distal patterning. We speculate that KNATM-B regulates transcription by sequestering BELL transcription factors and being part of a transcriptional complex. Figure 5. (continued). (F) 35S-KNATM-B cauline leaves (10-week-old plant). Bars ¼ 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 6 SE (n ¼ 10). Asterisks indicate statistically significant differences (Student s t test, P < 0.05) of transgenic lines compared with the wild type.

8 882 The Plant Cell 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, and35s-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 ¼ 1cmin(A) to (D), 1mmin(E) to (K), and 100 mm in(l) to (S). All plants are 3 weeks old.

9 KNATM: A Homeodomainless KNOX Protein 883 KNOX Phylogeny Conservation of the MEINOX domain between plant KNOX and animal MEIS TALE proteins indicates that an ancestral MEINOX-homeodomain protein predated plant animal divergence (Burglin, 1997, 1998; Mukherjee and Burglin, 2007). Nevertheless, nothing is known about the evolution of the MEINOX domain. The finding of KNATM suggests two hypotheses: (1) the MEINOX domain and the homeodomain coevolved and KNATM lost the homeodomain; (2) the MEINOX domain evolved independently of the homeodomain. In favor of the first hypothesis, KNATM is the only protein of this kind described so far. On the other hand, MEIS homeodomainless splicing isoforms (Yang et al., 2000; Noro et al., 2006) might suggest an ancient shuffling of a HOMEOBOX gene with a MEINOX gene, in agreement with the second hypothesis. The discovery of the KNATM homolog in poplar suggests that KNATM originated early in the evolution of dicotyledons and argues against the hypothesis of KNATM being a pseudogene. On the contrary, we did not detect any homolog in monocotyledons. One hypothesis is that the KNATM homolog in monocotyledons is a canonic KNOX protein with the homeodomain being redundant. Alternatively, KNATM originated in dicotyledons, was lost in monocotyledons, or has still to be found. Our phylogenetic analysis of the KNOX family indicates that KNATM has diverged considerably from the other KNOX proteins. Therefore, we propose to introduce a new KNOX phylogenetic class that we named class M. KNATM Interactions The MEINOX domain is necessary and sufficient to mediate KNOX BELL interaction (Bellaoui et al., 2001; Muller et al., 2001; Smith et al., 2002). In agreement with these data, KNATM-B bears a full MEINOX domain and dimerizes with BELL proteins. Furthermore, KNATM-A and KNATM-C, which lack a complete Figure 8. KNATM Transcriptional Activation Activity. (A) Quantitative b-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 6 SE (n ¼ 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 ¼ 1cmin(C) and (D).

10 884 The Plant Cell MEINOX domain, do not interact with any BELL protein tested. DKNATM-B, which contains only the MEINOX domain, shows a pattern of interactions identical to KNATM-B and demonstrates that the MEINOX domain is sufficient to dimerize with BELL proteins. These findings validate our bioinformatics prediction by confirming the nature of the KNATM MEINOX domain. In addition, KNATM-B selectivity for specific BELL proteins highlights the importance of protein protein interaction in regulating KNOX-BELL activity. A phylogenetic analysis of BELL proteins suggests that PNY and PNF are paralogs originated from the most recent Arabidopsis genome duplication (Smith et al., 2004). In agreement with this hypothesis, they share a high level of protein sequence similarity, and they are expressed in the same domains. Nevertheless, they show different loss-of-function phenotypes (Smith and Hake, 2003; Smith et al., 2004). Intriguingly, KNATM-B is coexpressed in flower meristems with both PNY and PNF but selectively interacts with PNY, providing a possible molecular explanation for their different phenotypes. KNOX KNOX interaction is mediated by the MEINOX domain and the homeodomain (Muller et al., 2001; Nagasaki et al., 2001). KNATM-B interaction with BP is the first example of a homeodomain-independent KNOX-KNOX dimerization. KNATM-A and KNATM-C do not interact with BP and confirm the importance of the MEINOX domain for the dimerization. The BPID domain compensates for the absence of a homeodomain, as shown by DKNATM-B lack of interaction. KNATM-B does not dimerize with the other KNATM isoforms, suggesting that it relies on homeodomain partners to perform its function. Conversely, KNATM-A and KNATM-C might work in different protein networks. KNATM Developmental Function The inability to significantly knock down KNATM-B transcripts through RNA interference and artificial microrna as well as the absence of a loss of function allele might suggest that KNATM-B is essential for embryo or gametophyte development. In agreement with this hypothesis, KNATM-B is expressed in embryos, in tissues that give rise to the gametophytes, and in the gametophytes themselves (Figure 3I; see Supplemental Figure 5 online). Although the final answer on KNATM-B developmental function should come from a loss-of-function allele, KNATM-B expression pattern combined with overexpression analyses give some functional hints. 35S-KNATM-B plants offer an advantage compared with many overexpression lines that generate phenotypes caused by spatial misexpression of the transgene. KNATM-B does not carry a DNA binding domain and therefore may affect only cells where its partners are expressed. Furthermore, KNATM selectivity for BELL and KNOX proteins further diminish the risk of disturbing any cell expressing BELL or KNOX proteins. In agreement with these observations, most phenotypic defects caused by KNATM-B overexpression occur in tissues where KNATM-B is normally expressed. 35S-KNATM-B elongated petioles and wild-type KNATM-B proximal expression in leaf primordia suggest a role in proximaldistal patterning. Elongated abaxial lamina cells might be interpreted as a sign of proximalization, indicating that KNATM-B specifies petiole cell fate. The leaf proximal-distal axis is even more disturbed when both BP and KNATM-B are overexpressed, 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. suggesting that they might control polarity establishment at the leaf boundary of wild-type plants where they colocalize. The overexpressor serrated leaf phenotype and the wild-type KNATM-B expression in hydathodes might indicate that KNATM-B is involved in leaf medial-lateral patterning. SAW1 and SAW2 BELL genes are expressed in hydathodes, and the double mutant has increased leaf serration (Kumar et al., 2007). These observations suggest that KNATM-B might sequester SAW1 and SAW2 and shape wild-type leaf lamina by controlling free SAW1 SAW2 dosage at the hydathodes. In agreement with this hypothesis, KNATM-B and SAW1 double overexpression plants clearly show a mutually antagonistic effect. If our rationale is correct, 35S-SAW1 plants may reveal KNATM-B loss-offunction phenotypes. Reduced petiole elongation in plants overexpressing SAW1 may be due to KNATM-B titration. Finally, KNATM-B might be involved in perceiving or generating flowering signals, as suggested by the 35S-KNATM-B late flowering phenotype and its wild-type expression in leaf primordia. KNATM-B interaction with PNY might be functionally important in regulating flowering time since pny pnf double mutant does not undergo flowering transition (Smith et al., 2004). KNATM Molecular Function Since KNATM-B lacks a homeodomain and can interact with KNOX and BELL proteins, we suggest two not mutually exclusive hypotheses: (1) KNATM-B might sequester BELL and/or KNOX proteins; (2) KNATM-B might work as a transcriptional cofactor with a DNA binding protein. In the first scenario, KNATM-B overexpression should result in a loss-of-function phenotype of its partners. 35S-KNATM-B plants show leaf serration as in the saw1 saw2 double mutant. Furthermore, KNATM-B and SAW1 show an antagonistic role in double overexpression lines. KNATM-B may prevent SAW1 and SAW2 from taking part in

11 KNATM: A Homeodomainless KNOX Protein 885 transcriptional complexes by titrating them in an inactive dimer and/or in the cytoplasm. In favor of the second hypothesis, KNATM-B transcriptional activation activity observed in yeast suggests a direct involvement in transcriptional regulation. The SRDX transcriptional repression domain partially suppresses KNATM-B overexpression phenotype and suggests that KNATM-B takes an active part in a transcriptional initiation complex. KNATM-A, KNATM-C, DKNATM-B, DKNATM-B-AD, or DKNATM-B-RD overexpression lines do not show any visible phenotype and highlight the functional importance of MEINOX and BPID domains. Taken together, our data suggest that KNATM-B modulates SAW1 and SAW2 activity by titration. KNATM-B might also regulate transcription by working in a transcriptional complex with a DNA binding protein (Figure 9). METHODS Bioinformatics The NR database of protein sequences (NCBI, National Institutes of Health, Bethesda, MD) was searched using BLAST or PSI-BLAST with E-value cutoffs of 10 and 1e-03, respectively. The Joint Genome Initiative Populus trichocarpa protein database ( Poptr1_1/Poptr1_1.home.html) was searched using BLAST. Sequences were submitted to the Conserved Domain Database (Marchler-Bauer et al., 2003) and PFAM (Bateman et al., 2004) protein structure prediction HMM library for domain analysis. Phylogenetic Analysis Muscle 3.2 (Edgar, 2004) was used for multiple sequence alignments. The phylogenetic trees were constructed using neighbor-joining, maximum parsimony, and UPGMA algorithms implemented in the MEGA software suite ( 10,000 bootstrap replicates were performed. COILS was used to predict coiled-coils (Lupas et al., 1991). Molecular Biology RNA gel blotting, PCR, and RT-PCR analyses were conducted as previously described (Sambrook and Russell, 2001). The Invitrogen GeneRacer Core kit was used to perform 59 and 39 rapid amplification of cdna ends PCR according to the manufacturer s instructions. RNA in situ hybridization analyses were conducted as previously described (Jackson, 1991) using the full-length KNATM-B coding sequence as the probe. b-glucuronidase activity was tested as previously described (Jefferson, 1989). Cloning KNATM-A, KNATM-B, and KNATM-C coding sequences were PCR amplified with or without (NOSTOP) the stop codon and cloned into pentr/d-topo (Invitrogen) according to the manufacturer s instructions. The KNATM-B coding sequence was PCR amplified from nucleotide 94 (DKNATM-B) fused to a start codon with or without (NOSTOP) the stop codon and cloned into pentr/d-topo according to the manufacturer s instructions. KNOX and BELL coding sequences were PCR amplified and cloned into pentr/sd/d-topo (Invitrogen) according to the manufacturer s instructions. For yeast two-hybrid and transcriptional activation assays, KNOX, BELL, KNATM-A, KNATM-B, DKNATM-B, and KNATM-C coding sequences were mobilized from pentr/sd/d-topo or pentr/d-topo into pdest22 (Invitrogen) and pdest32 (Invitrogen) according to the manufacturer s instructions. pexp-ad502 (Invitrogen) was digested with SalI and XhoI restriction enzymes and religated to create pexp-nls502 that lacks the sequence encoding the GAL4 transcriptional AD. A Gateway cassette (Invitrogen) was cloned into the pexp-nls502 SmaI site. The KNATM-B coding sequence was mobilized from pentr/d-topo into pexp-nls502 according to the manufacturer s instructions and used in yeast transcriptional activation assays. The sequence encoding the SRDX transcriptional RD was synthesized in vitro flanked by MluI restriction sites and cloned into pentr/d-topo- KNATM-B-NOSTOP and pentr/d-topo-dknatm-b-nostop AscI restriction sites (Hiratsu et al., 2003). KNATM-B and DKNATM-B fused to AD were PCR amplified from pdest22-knatm-b and pdest22- DKNATM-B, respectively, and cloned into pentr/d-topo according to the manufacturer s instructions. BP, KNATM-A, KNATM-B, DKNATM-B, KNATM-B-AD, DKNATM-B-AD, KNATM-B-RD, and DKNATM-B-RD coding sequences were mobilized from pentr/sd/d-topo or pentr/ D-TOPO into pmdc32 (Curtis and Grossniklaus, 2003) according to the manufacturer s instructions and used in overexpression experiments. KNATM-B-NOSTOP was mobilized from pentr/d-topo into pmdc83 (Curtis and Grossniklaus, 2003) according to the manufacturer s instructions for subcellular localization experiments. A Gateway cassette (Invitrogen) was cloned into pspyne-35s and pspyce-35s (Walter et al., 2004) SmaI sites. KNATM-B-NOSTOP and PNY-NOSTOP were mobilized from pentr/d-topo into pspyne-35s and pspyce-35s, respectively, according to the manufacturer s instructions for protein protein interaction and subcellular localization experiments. Genomic sequence (2334 bp) upstream of the KNATM-B stop codon was PCR amplified, cloned into pentr/d-topo, and mobilized into pmdc163 (Curtis and Grossniklaus, 2003) according to the manufacturer s instructions and used in expression analysis. Yeast Two-Hybrid and Transcriptional Activation Assays The Invitrogen ProQuest Two-Hybrid system with Gateway Technology was used in yeast two-hybrid and transcriptional activation assays according to the manufacturer s instructions. Plant and Genetic Materials Seeds of Arabidopsis thaliana, ecotype Columbia-0, were used for all experiments. The bp-9 allele and the 35S-SAW1 transgenic line were previously described (Mele et al., 2003; Kumar et al., 2007). Plants were grown in the greenhouse under long-day conditions for flowering time measurements and in vitro under constant fluorescent illumination for all other analyses. Transgenic Plants For stable transformation, Agrobacterium tumefaciens strain GV3101 was used to transform Arabidopsis plants by the floral dip method (Clough and Bent, 1998). For transient transformation, onion epidermal cells were transformed by particle bombardment using a Biolistic PDS-1000/He unit (Bio-Rad) as previously described (Sanford et al., 1993). Microscopy GFP and YFP visualizations in onion epidermal peels were performed 24 to 36 h after bombardment using a Zeiss Axiophot microscope.

12 886 The Plant Cell DAPI and GFP fluorescence were visualized in Arabidopsis petal cells using a Zeiss LSM510 confocal laser scanning microscope, with the fluorescein isothiocyanate channel and the UV channel, respectively. Arabidopsis tissue for scanning electron microscopy was fixed in FAA (50% ethanol, 5% acetic acid, and 3.7% formaldehyde) for 1 h at 48Cand dehydrated through an ethanol series to 100% ethanol. The samples were critical point dried, sputter coated with gold palladium for 45 s, and viewed on a Hitachi S-4700 scanning electron microscope at an accelerating voltage of 5 kv. Accession Numbers The Arabidopsis Genome Initiative locus identifier of the KNATM gene is At1g The GenBank accession numbers of KNATM homolog genes are AC (Medicago truncatula) and AF (Solanum esculentum). The Joint Genome Initiative accession number of the KNATM homolog in Populus trichocarpa is fgenesh4_pg.c_lg_xii Supplemental Data The following materials are available in the online version of this article. Supplemental Figure 1. KNATM-B Expression. Supplemental Figure 2. KNATM Homologs. Supplemental Figure 3. KNATM Expression as Detected by RNA in Situ Hybridization. Supplemental Figure 4. KNATM-B Expression in 35S-KNATM-B Arabidopsis Plants. Supplemental Figure 5. KNATM Expression in the Gametophytes. Supplemental Data Set 1. Alignment of the KNATM Proteins with the MEINOX Domain of All Arabidopsis KNOX Proteins in FASTA Format. Supplemental Data Set 2. Alignment of the Arabidopsis KNATM-B with Its Homologs in Medicago truncatula, Solanum esculentum, and Populus trichocarpa in FASTA Format. ACKNOWLEDGMENTS We thank the members of Sarah Hake s and Jennifer Fletcher s labs and Robert Blanvillain and Lisa Harper for valuable discussions and technical advice. We also thank George W. 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