Multiple database searches were performed to collect all members of the rice (Oryza sativa) and Poplar (Populustirchocarpa) KNOX family. The amino acid sequence of the 9 KNOX genes from Arabidopsis (Arabidopsis thaliana) were used as query sequences, BLAST programs (TBLASTN and BLASTP) on the Phytozome (http://www.phytozome.net/), TAIR (http://www.arabidopsis.org/), Rice Genome Annotation Project (http://rice.plantbiology.msu.edu/), NCBI (http://www.ncbi.nlm.nih.gov/) and PlantDB (http://www.plantgdb.org/) genome database were performed and the sequences hitting with 1e−6 or less were treated as conserved genes. To increase the extent of the database search results, position-specific iterated BLAST [30] search against the rice and Poplar database on the NCBI web site were also performed. All the sequences with conserved domains of KNOX protein except for those annotated as miRNA, retrotransposons or transposable elements were considered as KNOX candidates.

To generate the phylogenetic tree of each gene family, a multiple alignment analysis was performed with ClustalW and the rooted phylogenetic tree with branch length was constructed by UPGMA (Unweighted Pair Group Method with Arithmetic mean) method with amino acid sequences. In UPGMA the unweighted term indicates that all distances contribute equally to each average that is computed and does not refer to the math by which it is achieved and the proportional averaging in UPGMA produces an unweighted result. The UPGMA algorithm constructs a rooted tree (dendrogram) that reflects the structure present in a pairwise similarity matrix (or a dissimilarity matrix). At each step, the nearest two clusters are combined into a higher-level cluster. The distance between any two clusters A and B, each of size |A| and |B| is taken to be the average of all distances d(x, y) between pairs of objects x in A and y in B, that is, the mean distance between elements of each cluster. The UPGMA algorithm produces rooted dendrograms and requires a constant-rate assumption that is, it assumes an ultrametric tree in which the distances from the root to every branch tip are equal.

Gene structure display server (GSDS) program (http://gsds.cbi.pku.edu.cn/) [31] was used to illustrate exon/intron organization for individual KNOX genes by aligning the coding sequences (CDS) with their corresponding genomic sequences from Phytozome (http://www.phytozome.net). The visualization of gene features such as composition and position of exons and introns for genes offers visual presentation to integrate annotation, and to produce high-quality figures.

The program MEME (v4.9.0) (http://meme.nbcr.net/meme) [32] and ClustalW multiple alignment analyses (http://www.genome.jp/tools/clustalw) were used for the elucidation of conserved motifs in all deduced rice and Poplar KNOX protein sequences. The following parameters were used: number of repetitions any, maximum number of motifs 20, and the optimum motif widths were constrained to between 6 and 60 residues. The HMM (Hidden Markov Models) logos were used for visualization of domain conservation. Structural motif annotation was performed using the SMART (http://smart.embl-heidelberg.de) and Pfam (http://pfam.sanger.ac.uk) databases.

To identify the number of domains in KNOX protein, domain search were executed by Conserved Domains Database (http://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml) [33] and pfam database (http://pfam.sanger.ac.uk/) [3] with both local and global search strategy and expectation cut off (E value) 1.0 was set as the threshold for significance. Only significant domain found in rice and Poplar KNOX protein sequences were considered as a valid domain.

The 3D (three-dimensional structure) protein structure prediction of KNOXs in Arabidopsis, rice and Poplar were performed by PHYRE2 (Protein Homology/ AnalogY Recognition Engine) Protein Fold Recognition Server (www.sbg.bio.ic.ac.uk/phyre2/) [34] . PHYRE2 is web-based services for protein structure prediction using the principles and techniques of homology modeling. It is able to regularly generate reliable protein models when other widely used methods such as PSI-BLAST cannot.

All targets identified in this study were subjected to agriGO toolkit analysis (http://bioinfo.cau.edu.cn/agriGO) to investigate gene ontology [35] .

The subcellular localization of KNOX proteins were predicted by SVM based server ESL pred (http://www.imtech.res.in/raghava/eslpred/submit.html), and ProtComp 9.0 server (http://linux1.softberry.com/berry.phtml?topic=protcomppl&group=help&subgroup=proloc). Further, species-specific localization prediction system was utilized for Arabidopsis (AtSubP, http://bioinfo3.noble.org/AtSubP) [36] .

For Poplar, the expression profile for each KNOX gene was obtained by evaluating its EST representation among 19 cDNA libraries derived from different tissues and/or developmental stages available at PopGenIE (http://www.popgenie.org/) [37] . The absolute mode was used.

For rice, expression support for each gene model was explored through gene expression evidence search page (http://rice.plantbiology.msu.edu/expression.shtml) available at MSU. Expression data were derived from NCBI Sequence Read Archive (SRA). Sequence reads were mapped to the version 7 pseudomolecules with Tophat. Expression abundances for RNAseq libraries were calculated with Cufflinks [38] [39] . The Sequence Read Archive (SRA) stores raw sequencing data from the next generation of sequencing platforms including Roche 454 GS System®, Illumina Genome Analyzer®, Applied Biosystems SOLiD® System, HelicosHeliscope®, Complete Genomics®, and Pacific Biosciences SMRT®.

By BLAST(BLASTp and BLASTn) search against multiple databases with query sequences of nine previously published KNOX genes from Arabidopsis, the candidate KNOX genes in rice and Poplar were further analyzed to confirm the presence of KNOX1, KNOX2, ELK and Homeobox_KN domain through Pfam program and Conserved Domain Search Service (CD Search) in NCBI. Excluding the redundant sequences, 13 rice (Oryza sativa. L) KNOX genes and 15 Poplar (populoustirchocarpa) KNOX genes were identified (Table 1).

According to the conserved amino acid sequences, all these KNOXs genes in

^a:The sequence information was from Phytozome database (http://www.phytozome.net/); The longest isoform was chosen if there were more than one alternative splicing isoform available.

Arabidopsis, rice and Poplar divided into three groups, class I, class II and class III, which agreed with representative genes of Arabidopsis (Figure 1). Among which, 22 KNOX genes belonged to class I, including 4 Arabidopsis KNOX genes, 9 Popolus KNOX genes and nine rice KNOX genes, 13 genes belonged to class II and 2 genes belonged to class III.

Class I was further divided into 3 subclasses, designated as IA, IB and IC. Class IC comprises only 4 rice genes, LOC_Os03g56140, LOC_Os03g56110, LOC_Os03g47022 and LOC_Os03g47042, showed ambiguous clustering from other phylogenetic classes (Figure 1).

Class II included IIA and IIB two subclasses. KNAT7, together with rice gene LOC_Os03go3164 and Poplar member POPTR_0001s08550 represented class IIA subclass. Besides Arabidopsis KNAT3, KNAT4 and KNAT5, 3 rice members, LOC_Os08g19650, LOC_Os02g08544 and LOC_Os06g43860 and 4 Poplar members, POPTR_0006s20440, POPTR_0018s12200, POPTR_0006s27560, and POPTR_0018s02210 were classified into class IIB clade (Figure 1).

Poplar KNOX gene POPTR_0012s04040 fell into class III together with Arabidopsis KNOX member KNATM, showed big divergences from the other clades and indicated that class III was new branch in the phylogeny (Figure 1).

Structural diversity of KNOXs in rice and Poplar analyzed based on the alignment of cDNA and corresponding genomic sequences by GSDS revealed that KNOX members within the same subfamilies shared very similar gene structure in terms of either intron numbers or exon lengths except for class IC.KNOXs in class IA shared a structure with 5 exons and the third and fourth exons were separated by a long phase 1 intron. According to the sequences from Phytozome, class III member KNATM had only one intron, whether it had 5’ and 3’ UTR still need to be identified (Figure 1). Nonetheless, the gene structures in 4 rice members of subclass IC appeared to be more variable. LOC_Os03g56110 had a same structure as subclass IA, LOC_Os03g56140 had 4 exons and a long phase 1 intron interval between the third and fourth exon. Both of LOC_Os03g47042 and LOC_Os03g47022 had 3 exons separated by two phase 0 introns, the 5’ UTR and 3’UTR of gene LOC_Os03g47042 were not available and the gene LOC_Os03g47022 had 3’URT only, that showed similar features with KNATM in gene structure.

In addition, the intron phases were highly conserved in KNOX genes within each class. Class I and Class III always was in phase 0 and 1, while 50% genes in class II was in phases 2 and 0 (Figure 1), resulting in a significant excess of non-symmetrical exons. That suggested splicing phases were also highly conserved during the evolution and there was a strong correlation between the phylogeny and exon/intron structure of the KNOX gene family in rice and Poplar.

Although the intron phases with respect to codons were remarkably well-conserved within the same subfamilies, there were striking distinctions in the arrangement of introns and intron phases among subfamilies of KNOX IB (Figure 1). The conservation of intron phases within KNOX subfamilies and the striking dissimilarity between subfamilies may reciprocally lend support to the results from phylogenetic analysis and genome duplication, and provide further supports to plant KNOX subfamilies definition.

The domain conservation of 13 rice and 15 Poplar KNOX proteins were analyzed by performing a multiple sequence alignment with Arabidopsis KNOX amino acid sequences by ClustalW (Figure S1, Figure S2, Figure S3) and MEME program. No KNOX2 domain was found in rice gene LOC_Os03g56140. Both ELK domain and Homeobox_KN domain were not found in rice sequences LOC_Os03g47042 and LOC_Os03g47022 (Figure 2). These two genes showed similar characters as class III member, Arabidopsis KNATM and POPTR_0012s04040, which characterized as no ELK domain and Homeobox_KN domain (Figure 2).

For other class I genes and class II genes, KNOX1, KNOX2, ELK and Homeobox_KN domains were highly conserved (Figure 2). The KNOX1 domain was completely conserved in all of the 22 class I sequences and the E-Value was as high as 4.3e-309 (Figure S1).

The domain variation of three genes in class IC showed consistence with their divergence in phylogenic analysis. And the domain similarities among LOC_Os03g47042, LOC_Os03g47022, KNATM and POPTR_0012s04040 suggested that the gene cluster analysis sometimes cannot classify the high divergence of the motif, especially combined with its short length and the occurrence of many ancient paralogs when large number of sequences being examined, which can be complementary by domain analysis. Same results were also obtained through CD (conserved domain) search in NCBI and Pfam (Table S1).

The 3D protein structures of KNOXs in rice and Poplar predicated by PHYRE2 showed variances within different subclasses. All of the members in class IA, class IB and class II members had highly conserved 3D structures (Figure 3(a)) featured 3 helices, among which helices I and II were typically connected by a loop structure, helices II and helices III formed a helix-turn-helix motif. The structure of KNAT4 was a little different which included a set of 3 helices structure at N terminal and a set of 5 helices structure at C terminal (Figure 3(b)). A bipartite arrangement of these two set helix structure was separated by a linker region. This kind of structure configuration hinted the functional diversity of KNAT4 in Arabidopsis development.

However, the 3D structures of 4 rice gene members in class IC showed high variances. LOC_Os03g56110 and LOC_Os03g56140 still had typical 3 helices structure like other KNOX genes, while rice gene LOC_Os03g47042 had very short helices II, no typical loop structure between helices I and helices II and no helix-turn-helix motif formed by helices II and helices III, and LOC_Os03g47022 showed merged long helices, no loop, turn and other two helices (Figure 3(c)). The typical 3D protein structures against the 3-helix-structureagreed with the protein structure prediction of class III KNOXs (Figure 3(d)). Arabidopsis gene KNATM (AT1G14760) had a structure with two longer helices connected by a short linker, populous gene PORT_0012s04040 had a structure with unlinked helices, the first helices was disconnected with the rest two. The conservation and variances of 3D protein structure in class I was highly in accordance with phylogenic analysis and conserved domains analysis, which further supported they represented highly diverged lineage-specific KNOX sequences. These a typical 3D protein structures of LOC_Os03g47022, LOC_Os03g47042 and PORT_0012s04040 implied their special biological function and phylogenic divergence.

To understand the temporal and spatial expression patterns of rice and Poplar KNOX genes, we compared their expression patterns during development with the expression data derived from RNA-seq available at MUS and from PopGenIE respectively.

The diversity expression patterns of rice KNOX genes in 11 different tissues showed that except unavailable expression data for LOC_Os03g47042, most of the class I genes mainly expressed in pre-emergence inflorescence, post-emergence inflorescence, and pistil, and 25 DAP (days after pollination) embryo with different expression dynamics (Table 2), gene LOC_Os05g03884had highest expression in pistil and gene LOC_Os01g19694in 25 DAP embryo. LOC_Os07g03770 also had a significant expression in anther besides the highest expression in pre-emergence inflorescence with expression value as high as 52.75 (Table 2). Comparing to other class I genes, the gene LOC_Os03g51710 had expression in all of 11 tissues and its highest expression was in pre-emergence inflorescence, the value reached 5.86 (Table 2).

All of the 4 class II genes almost expressed in all of the 11 tissues which were analyzed and had the same expression pattern with strong expression in shoot, 4-leaf stage seedling and 20 days leaf, and the gene LOC_02g08544 showed a ubiquity expression (Table 2).

For Poplar, the 5 class I KNOX genes (POPTR_0008s19300, POPTR_0013s01000, POPTR_0005s01720, POPTR_0004s00650, and POPTR_0002s11400) had strong expression in root, mature leaf, internodes, young leaf and nodes, while no expression of gene POPTR_0010s05340, POPTR_0015s09030, POPTR_0012s08910, POPRT_0011s01600 and Class III gene POPTR_0002s11400 were available in these five tissues. The gene POPTR_0008s19300 had a stronger expression in

^a: expression support for each gene model was explored through gene expression evidence search page (http://rice.plantbiology.msu.edu/expression.shtml) available at MSU. Expression data were derived from NCBI Sequence Read Archive (SRA). ^b: T1: Seed-5 DAP; T2: Seed-10 DAP; T3: Shoots; T4: Seedling four-leaf stage; T5: Leave 20 days; T6: Post-emergence inflorescence; T7: Pre-emergence inflorescence; T8: Anther; T9: Pistil; T10: Embryo-25 DAP; T11: Endosperm-25 DAP.

nodes than in other four tissues. The gene POPTR_0013s01000 had stronger expression in both of internodes and nodes with the expression value of 9.37 and 9.47 respectively. The expression of gene POPTR_0005s01720, gene POPTR_0004s00650 and gene POPTR_0002s11400 in root, internodes and nodes were higher than in other tissues (Table 3). The class II KNOX genes showed evenly expression in root, mature leaf, internodes, young leaf and nodes with highest expression in mature leaf (Table 3).

Protein subcellular localization is crucial for protein function prediction. The subcellular location of KNOX members in rice (Table 2) and Poplar (Table 3) showed that except for three rice class IB members were still unknown, all others were located in nucleus, which implied their transcription factor functions.

To identify biological processes these KNOXs might participate in and whether they were differential in rice and Poplar, 15 Poplar KNOX genes and 13 rice KNOX genes were subjected to AgriGO toolkit analysis to investigate gene ontology. 13 rice genes were all involved in cellular process, regulation of biological process, biological regulation, metabolic process, transcription regulator activity and binding activity (Figure 4(a)). All 15 Poplar KNOXs genes were involved in DNA binding activity and more than 70% of the Poplar genes were involved in biological regulation and transcription regulator activity (Figure 4(b)). The

^a: the expression profile were obtained by evaluating its EST representation among 19 cDNA libraries derived from different tissues and/or developmental stages available at PopGenIE (http://www.popgenie.org/).

enrichment of rice and Poplar KNOXs involved in DNA binding was consistent with the subcellular predication that most of them were located in nucleus.

According to GO analysis, the functional group for transcription regulator activity, transcription factor activity and sequence specific DNA binding was highly enriched by KNOX target genes in rice and Poplar nucleus (Figure 5), but the enrichment of functional group for DNA binding and transcription factor activity was not as high as that of sequence-specific binding in Poplar. That showed all of rice KNOXs and most of Poplar KNOXs had roles in DNA binding and transcription regulator activity when considered their expression and subcellular analysis together.

Except for transcription factor activity and DNA binding, KNOXs target genes in rice were highly functionally related to RNA metabolic process, regulated RNA metabolic process and RNA biosynthesis process (Figure 5(a) and Figure 5(b)). It confirmed that rice KNOXs were involved in plant development as transcriptional factors. Poplar KNOXs target genes were weaker in regulation of RNA metabolic process and RNA biosynthesis process (Figure 5(c) and Figure 5(d)).

The KNOX genes in Arabidopsis are divided into class I, class II and class M

depending on their sequences and expression patterns [7] . According to sequences search and blast in this study, KNOXs homologous are scattered across rice and Poplar chromosome and can also be grouped into 3 classes based on sequences and gene structure (Figure 1). That indicated KNOXs were an ancient gene family and conserved highly across species. However, class IC consisted of only four rice genes, was divergent clustering between other phylogenetic classes. They most likely represented highly diverged lineage-specific KNOX sequences or the phylogenetic analysis could not resolve their evolutionary relationships.

Two rice genes, LOC_Os03g47016 and LOC_Os03g47036, were classified into KNOX gene family by Jain et al. (2008), while no KNOX1 and KNOX2 domains were found by us, so they were excluded from KNOX family in this study. However, two new KNOX genes, LOC_Os03g47042, LOC_Os03g47022 were firstly identified in this study and grouped into class IC according to the amino acid sequences, but when considered the gene structure, conserved domain and 3D protein structure all together, they showed more similar to Arabidopsis class M members and might be the KNATM orthologs in rice. As deep nodes and determining interclade relationships commonly showed low statistical support and varied between different phylogenetic methods which typically observed in protein phylogenies [40] , the ambiguous classifications indicated the gene cluster analysis sometimes cannot classify the high divergence of the motif, especially combined with its short length and the occurrence of many ancient paralogs when large number of sequences being examined, which can be complementary by domain analysis.

KNATM homolog was found only in dicots and placed in a new class in KNOX family [7] . The KNATM homolog in Poplar suggested that KNATM originated early in the evolution of dicotyledons. What’s more, two possible KNATM homolog genes were found in rice (Figure 1), it was the first time to identify MEINOX protein in monocotyledon and the discovery argued against the hypothesis that the KNATM homolog in monocotyledons is a canonic KNOX protein with the homeodomain being redundant or KNATM originated in dicotyledons was lost in monocotyledons. All of these argue against the hypothesis of KNATM being a pseudogene.

As key effectors involved in transcriptional regulation and hormonal signaling, the function diversity is performed by the KNOX members in different classes. Class IA, IB and Class II KNOXs in rice and Poplar all had 4 highly conserved domains and were located in nucleus, highly expressed in undifferentiated tissue (Table 2 and Table 3), functioned in transcriptional factor activity (Figure 5), that suggested them might participate in plant cell differentiation and plant morphogenesis, with similar functions as that in Arabidopsis.

In Arabidopsis, class I KNOX genes were mainly expressed in the meristematic tissues and regulate hormonal pathways to maintain meristematic cells in an undifferentiated state [5] . Our expression analysis found class I gene either in rice or Poplar mainly expressed in pre-emergence inflorescence, post-emergence inflorescence, pistil and 25 DAP embryo (Table 2), showed similar expression patterns as in Arabidopsis.

STM (AT1G62360) was classified into class IB in this study and expressed during early embryogenesis, its expression marked the entire SAM (Long et al. 1996) and was proposed to be essential for SAM formation and maintenance [41] [42] because STM inhibited the cellular differentiation normally associated with organogenesis and permitted the WUS-CLAVATA feedback loop to maintain the central stem cells [41] [43] . As two Poplar genes, POPTR_0004s00650 and POPRT_0011s01600 exactly fell into STM clade, they had similar characters as STM in phylogenic, protein structure and expression patterns, they probably functioned as the same as STM in Arabidopsis and it was worth to give a further investigation for their roles in cellular differentiation and interactions with other genes.

Two of three rice class IB KNOX genes, LOC_Os03g51690 and LOC_Os07g03770 had highest expression in pre-emergence inflorescence (Table 2) and fell into the same clade to KNAT1. Expression of Oskn3 marked the boundaries of different embryonic organs following SAM formation (Postma-Haarsma et al. 1999). The expression of KNAT1 covered the embryonic SAM, post-embryonic development SAM and the boundary of the inflorescence SAM (Venglat et al. 2002).LOC_Os03g51690 might also take part in the inflorescence architecture just like LOC_Os07g03770 and KNAT1.

KNAT2 was expressed during embryogenesis and marked the base of the SAM [15] [16] . Its’ promoter had been reported to be active in root tissue [17] . KNAT6 was expressed in the embryonic SAM, the SAM boundaries [15] and the phloem tissue of roots [18] . The gene POPTR_0008s19300 had strongest expression in nodes and might function in phloem tissue just like KNAT6 as they both fell into the same clade and had the similar expression profiles.

The 4 conserved domains of KNOX protein showed different molecular functions. KNOX1 played a role in suppressing target gene expression,KNOX2 was thought to be necessary for homo-dimerisation, ELK domain was required for the nuclear localization of these proteins, and Homeobox KN domain was a homeobox transcription factor conserved from fungi to human and plants [3] [4] [5] . Poplar gene POPTR_0012s04040, rice genesLOC_Os03g47042 and LOC_Os03g47022 had only the MEINOX domain as the same as KNATM (Figure 2), which indicated the functions owing to ELK and Homeobox-KN4 might be lost in these 3 genes. However, all of them were found to be expressed in nucleus (Table 2 and Table 3) and showed transcription activities by GO analysis (Figure 4 and Figure 5). Though strong functional relationship existed between the homeodomain (HD) and the MEINOX domain, some observations indicated that the MEINOX domain can also work in a homeodomain-independent fashion [44] . Class III members KNATM was found to be participated in transcriptional regulation in a homeodomain-independent fashion [7] . That indicated the KNATM homologues in rice and Poplar might function also independent of Homeoboxdomain. However, the BP-interacting domain reported by Magnani and Kake [7] was not conserved in rice homologues (Figure 3). The mechanisms of KNATM?BP interaction in rice KNATM homologues need further investigations.

Recent evidence suggested that auxin may play a major role in down-regulating KNOX genes during organ emergence [45] . And several clues pointed to a hormone-dependent down-regulation of KNOX genes in the incipient primordium [46] [47] , but the identity of the genetic factors responsible for this control is still unknown.

All of these hinted the known and new KNOXs identified in this study may regulate expression of their target genes to control cell differentiation and development in rice and Poplar. Though further experimental studies still need to be conducted to unravel their biological roles, the genome-wide analysis of KNOXs in rice and Poplar will help to discovery new KNOXs gene and provide a valuable resource for further functional analysis.