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Article

Genome-Wide Analysis of KNOX Genes: Identification, Evolution, Comparative Genomics, Expression Dynamics, and Sub-Cellular Localization in Brassica napus

1
College of Landscape Architecture and Art Design, Hunan Agricultural University, Changsha 410128, China
2
Orient Science & Technology College, Hunan Agricultural University, Changsha 410128, China
3
College of Bioscience and Biotechnology, Hunan Agricultural University, Changsha 410128, China
4
Key Laboratory of Crop Epigenetic Regulation and Development in Hunan Province, Changsha 410128, China
5
Yuelu Mountain Laboratory, Changsha 410128, China
*
Author to whom correspondence should be addressed.
Plants 2025, 14(14), 2167; https://doi.org/10.3390/plants14142167
Submission received: 15 June 2025 / Revised: 5 July 2025 / Accepted: 9 July 2025 / Published: 14 July 2025
(This article belongs to the Section Plant Genetics, Genomics and Biotechnology)

Abstract

KNOX genes play crucial roles in cell-fate determination and body plan specification during early embryogenesis. However, the specific gene structure and functional differentiation of KNOXs in Brassica napus is still unclear. We investigated KNOX genes in Brassica rapa (B. rapa), Brassica oleracea (B. oleracea), and Brassica napus (B. napus), which are polyploidy models with genome triplication after Arabidopsis-Brassiceae divergence. In total, 15, 14, and 32 KNOX genes were identified in B. rapa, B. oleracea, and B. napus, respectively. Phylogenetic analysis classified BnKNOXs (B. napus) into three classes with conserved domain organization. Synteny analysis indicated that BnKNOXs family expansion during allopolyploidization was mainly due to whole-gene and segmental duplications. Cis-element, gene structure, and expression pattern analyses showed high conservation within the same group. RNA-seq and qRT-PCR results divided BnKNOXs into three classes with distinct expression patterns: Class I exhibited moderate and specific expression in buds and inflorescence tips; Class III showed specific low expression in seeds and stamens; while the second class showed expression in most tissues. Sub-cellular localization results showed that the three candidate genes from the three classes exhibited distinct subcellular localizations, with BnSTM-C and BnKNAT3a-A predominantly in the nucleus and BnKNATM1-A in the cytoplasm indicating different expression patterns. Collectively, these findings provide a foundation for further functional studies of BnKNOX genes in B. napus.

1. Introduction

Homeotic genes encode proteins that include a conserved 60-amino-acid homeodomain, which is essential for regulating the expression levels of target genes and thus plays a pivotal role in the developmental processes of organisms [1,2]. These genes were first identified in fruit flies and found to be involved in segmental development as members of the Bithorax and Antennapedia complexes [3,4]. In the plant kingdom, maize Knotted-1 (Kn1) was the first identified gene that encodes a homeodomain-containing protein. Subsequently, KNOX genes have been cloned from a diverse array of plant species, revealing an expansion in the membership of the KNOX gene family in conjunction with the evolutionary development of multicellular diploid plants [5,6]. Unicellular green algae and red algae harbor a solitary KNOX gene, whereas in terrestrial plants, KNOX proteins are commonly encoded by a complement of genes within diverse gene families [7]. Plant homeotic genes are classified based on their sequence differentiation and the fusion with characteristic codomain sequences [8,9,10], resulting in their division into 14 distinct classes [11]. Knotted-like homeobox (KNOX) belongs to the three-amino acid loop extension super class, which is characterized by three additional residues between helixes 1 and 2, and it is regarded as one of the oldest homeobox gene classes [12,13,14].
Based on sequence similarity, structural characteristics, phylogenetic relationships, and expression patterns, KNOX genes are mainly divided into three classes [15,16,17], and they have been found in almost all plant species [6,18]. Class I and II KNOX proteins always contain KNOX1, KNOX2, ELK, and homeobox_KN domains [19]. The two KNOX domains, located at the N-terminus, play an important role in phenotypic change in transgenic plants [20,21]. The ELK domain encodes nuclear localization signals and facilitates interaction with other proteins as well as transcriptional inhibition [21]. The HD domain is located at the C-terminal and it is involved in DNA binding and homodimer formation [22]. KNOX subfamily in Class III lacks the ELK-HD domain. KNOX genes play a crucial role in regulating plant growth and development by controlling meristem and vascular cambium activity in the stem [23], axillary bud [24], leaf primordial [25], and gametophyte development [26,27].
In single-celled green algae (Chlamydomonas reinhardtii), the primary function of the KNOX protein is to regulate the sex of the gametophyte and the development of the fertilized egg [27]. In bryophytes (Physcomitrella patens), although the two KNOX genes are mainly expressed in sporophytes, the KNOX proteins encoded by them are significantly differentiated in function [28]. Class I KNOX is involved in promoting cell proliferation during the sporophyte development without inducing shoot formation of the moss Physcomitrella patens, while Class II KNOX protein inhibits gametophyte development [29]. KNOX in class I is essential for sporophyte meristem development in vascular plants but dispensable for gametophyte development in ferns [30]. In Arabidopsis thaliana, four Class I KNOX genes, including SHOOTMERISTEMLESS (STM, AT1G62360), KNAT1 (AT4G08150), KNAT2 (AT1G70510), and KNAT6 (AT1G23380), play crucial roles in the maintenance and function of the shoot apical meristem (SAM) [25,31]. Cytokinin can induce the expression levels of STM and KNAT1 in SAM. In addition, AtSTM was proved to be associated with carpel and inflorescence meristem development [22,32]. AtKNAT1 targets TCP15 to modulate filament elongation during stamen development [33]. KNAT6 and STM exhibit functional redundancy in the upkeep of the vegetative SAM, with KNAT6 being positioned at the regulatory boundaries within embryos through the STM/CUC2 (cup-shaped cotyledon 2) pathway [32]. Class II KNOX genes, including KNAT3 (AT5G25220), KNAT4 (AT5G11060), KNAT5 (AT4G32040), and KNAT7 (AT1G62990), exhibit diverse expression patterns [34,35,36]. Class II KNOX genes exhibit expression across various angiosperm organs, including roots, stems, leaves, and flowers, playing a pivotal role in the orchestration of plant organ differentiation. Furthermore, these genes are implicated in the negative regulation of secondary cell wall synthesis in the vascular bundle fibers of the inflorescence stem [37]. For instance, KNAT3 and KNAT7 play multiple roles in the development of secondary cell walls and redundantly regulate mucilage biosynthesis in Arabidopsis seeds [38]. Arabidopsis KNATM, which is defined as a novel KNOX transcriptional regulator, encodes a homeodomain protein expressed in both reproductive and vegetative meristems. It is also involved in leaf proximal-distal patterning [17], and its homologs belong to a new class [39]. Class I and Class II KNOX proteins, acting as transcription factors, share downstream targets but exert opposite effects: while Class I KNOX proteins activate target genes, Class II KNOX proteins negatively regulate these genes [7].
KNOX genes regulate plant development, with their role primarily elucidated in Arabidopsis [25,33]. Although extensive studies have identified KNOX genes in various species [30], limited knowledge exists regarding Brassica species. B. napus is an allotetraploid (AACC, 2n = 38) oilseed species providing almost 15% of vegetable oil worldwide with a complex genome, which was formed by natural hybridization between the two diploid species B. rapa (AA, 2n = 20) and B. oleracea (CC, 2n = 18). Genome-wide duplications and chromosomal rearrangements are pivotal in propelling the diversification and evolutionary processes of plant species. Consequently, the three Brassica species serve as an exemplary model for investigating the impact of extensive chromosomal modifications on genomic evolution following the divergence from Arabidopsis to the Brassicaceae family [40]. However, a comprehensive investigation of the KNOX gene family in Brassicaceae, especially in B. napus, remains unexplored. In this study, we identify and phylogenetically analyze the KNOX genes in Brassica species. Subsequently, we analyze the structures, cis-elements, and the expression patterns of the KNOX gene family, as well as sub-cellular localization in B. napus. This work provides new insights into the evolutionary significance of functional differentiation of these genes and lays a foundation for future research on KNOX genes in B. napus.

2. Results

2.1. Identification and Chromosome Map of KNOX Proteins and Genes from Brassica

A total of 15 KNOX proteins from B. rapa, 14 from B. oleracea, and 32 from B. napus were identified on the official website (BRAD (brassicadb.cn)), while an additional 89 members were screened from the genomes of several green lineage species (Phytozome (Phytozome), version 13.0) (Supplement File S1). KNOX proteins are exclusively found in higher plants, except one member with the KNOX2 domain identified in Ostreococcus lucimarinus; they cannot be detected in lower plants such as Chlamydomonas reinhardtii, Volvox carteri, and Phaeodactylum tricornutum, despite having HOX homologous (Supplement File S1).
The KNOX genes of B. rapa were mapped onto six chromosomes. Chromosome A09 showed the highest number of five KNOX genes, followed by chromosome A02/03 with three genes. Chromosomes A04, A07, and A10 did not contain any KNOX genes. Nine KNOX genes of B. oleracea were also mapped onto six chromosomes (C01-03, C05, C07, and C08), while BoSTM, BoKNAT4a, BoKNAT6a/6b, and BoKNAT7 could not be successfully mapped. In total, 29 KNOX genes of B. napus were mapped onto 14 chromosomes (six A-chromosomes and eight C-chromosomes). However, three C-genome specific genes (BnKNAT1b-C, BnKNAT4b-C and BnKNAT7a-C) could not be mapped (Figure 1).

2.2. Phylogenetic and Classification Analyses of KNOX Protein Family

  • To analyze the evolutionary patterns of KNOX, we constructed a comprehensive phylogenetic tree for 150 KNOX proteins, including the following: 32 from B. napus, 16 from Populus trichocarpa/Zea mays, 15 from B. rapa, 14 from B. oleracea, 13 from Oryza sativa, 11 from Brachypodium distachyon, 9 from Arabidopsis thaliana, 8 from Fragaria vesca, 7 from Medicago truncatula, 4 from Physcomitrella patens/Selaginella moellendorffii, and 1 from O. lucimarinus (Figure 2; Supplement File S1). Subsequently, excluding PpKNAT2a/2b and OlKNAT7 due to their inability to be classified into Class I or Class II despite sharing the same domain organization as others (similar results were obtained using different phylogenetic trees constructed by Neighbor-Joining or Maximum Likelihood methods), we successfully categorized the remaining set of 147 KNOX proteins into three distinct classes. Notably in Brassica species specifically, B. napus not only encompasses all KNOX proteins found in both B. rapa and B. oleracea but also exhibits an additional three unique members within its repertoire (Figure 2; Supplement File S1).

2.2.1. Phylogenetic and Domain Analyses of Class I

Arabidopsis Class I KNOX genes play a crucial role in shoot apical meristem (SAM) activity, carpel development, sporophyte development, and abscission zone development [41]. Class I specifically encompasses KNOX proteins found in vascular plants, including 2 members in S. moellendorffii, 3 in M. truncatula, 4 in A. thaliana, 5 in B. rapa/B. oleracea/F. vesca, 7 in B. distachyon, 9 in P. trichocarpa/O. sativa, 10 in B. napus, and 11 in Z. mays (Figure 3; Supplement File S1). Monocots exhibit a higher number of AtKNAT1 homologous proteins compared to dicots, while maintaining highly conserved domain organizations characterized by the major domains: KNOX1 (PF03790), KNOX2 (PF03791), ELK (PF03789), and Homeobox (PF00046). The phylogenetic relationships of the protein classes are closely linked to plant species evolution (Figure 3). Within Class I, KNOX genes can be categorized into three branches labeled as Groups I, II, and III. Group I represents the STM group with a conserved domain organization. Group II KNAT1 comprises the largest group. Most species including maize and rice possess more than two copies of KNAT1. Notably, the Up-frameshift suppressor 2 (Upf2) domain is identified within AtKNAT1 and FvKNAT1 [42]. The Upf2 domain is conserved in eukaryotes and is crucial for mRNA decay [43]. Epstein–Barr virus nuclear antigen 3 (EBNA-3, PF05009), which is an EBNA family member that responds to stimulated Epstein–Barr virus-specific T cells during adoptive immunotherapy is found in BoKNAT1 [44]. However, OsKNAT1f/1g and BdKNAT1d lack the ELK and Homeobox domains, respectively. Group III includes AtKNAT2/AtKNAT6 and its homologs. In addition to the major domains, FvKNAT6a contains an Integrator complex subunit 2 (INTS2, PF14750) domain, that is involved in snRNA transcription and processing. PtKANT6c contains an S-methyl trans domain (Homocysteine S-methyltransferase, pfam02574), and PtKANT6f contains a functionally unknown TMEM156 domain. Fern has similar domain organizations (Figure 3).
In Class I, both B. rapa and B. oleracea exhibit a total of five members, including two homologs of AtKNAT6 as well as one homolog each of AtSTM, AtKNAT1, and AtKNAT2 (Figure 3; Supplement File S2). The domain organizations remain highly conserved except for the duplicated BrKNAT1 members BrKNAT6a/BoKNAT6a which lack ELK and Homeobox domains [17] and are not detected among the Group III proteins that also play roles in KNOX transcriptional regulation and leaf proximal-distal patterning.
B. napus contains 11 homologous proteins (BoKNAT1 duplication) of B. rapa and B. olereace. The domain organizations of B. napus KNOX proteins exhibit a high degree of conservation with their respective donors. Notably, BnKNAT1a-A shows an increased EBV-NA3 domain compared to BrKNAT1, while BnKNAT6b-A lacks the ELK and Homeobox found in BrKNAT6b. Some sequence lengths also exhibit variability, such as BnSTM-C compared to BoSTM and BnKNAT6a-A relative to BrKNAT6, Figure 3). All Class I KNOX genes of the three Brassica species are syntenic to corresponding genes in Arabidopsis, except BnKNAT1b-C and BnKNAT6a-C (Supplement File S2). The syntenic genes suggest existence of the orthologs between B. napus and its parental species, namely, B. rapa and B. oleracea. In addition to inheriting most KNOX genes from its parents, new KNOX genes are also present in the genome of B. napus (BnKNAT1b-C, BnKNAT4b-C, BnKNAT6a-C, BnKNAT7a-A, BnKNAT7a-C, BnKNAT7c-C), while the BoKNAT6a ortholog is lost.

2.2.2. Phylogenetic and Domain Analyses of Class II

The functions of Class-II KNOX genes remain unclear, but they are potentially involved in the regulation of tissue differentiation, seed germination, root development and secondary wall formation [45,46]. Class-II may contain older KNOX proteins, and can be detected in all higher plants. A total of 2 members are present in P. patens/S. moellendorffii/F. vesca, 3 in M. truncatula, 4 in A. thaliana/B. distachyon/O. sativa, 6 in Z. mays/P. trichocarpa, 7 in B. oleracea, 8 in B. rapa and 17 in B. napus (three copies of BoKNAT7) (Figure 4; Supplement File S1). Similar to Class I KNOX genes, the major domains found within Class II include KNOX1, KNOX2, ELK and Homeobox domain. The phylogenetic tree reveals three distinct groups: Group I consists of KNAT3/KNAT4, Group II contains KNAT5 and Group III includes KNAT7. KNOX proteins of fern and moss are at the root of Group-I. Group-I contains multiple members and two or three homologous proteins from each organism except for the eight members from B. napus, four from P. trichocarpa and one from strawberry. The phylogenetic relationships among Group I proteins are related to plant species evolution (Figure 4). The domain organizations within Group-I are conserved except for BoKNAT4a and BnKNAT4a-C/4b-C. Group II exclusively comprises Cruciferae. AtKNAT5 possesses an additional enterotoxin motif in the heat-labile enterotoxin alpha chain. BnKNAT5a-A/5a-C have additional Virul-Fac motif. Group III encompasses AtKNAT7 and its homologs with conserved domain organizations except for ZmKNAT7a, which lacks the ELK domain, and BnKNA7a-C/7b-C which only possesses the KNOX1 domains.
All KNOX genes are duplicated in Brassica, with the exception of B. oleracea due to the presence of only one copy of KNAT7 in Class-II. Duplicated genes of Brassica encode proteins with conserved domain organizations. Based on the conserved relationships with Arabidopsis homologs, it is suggested that BrKNAT3a/3b and BoKNAT3a/3b may be involved in seed germination and early seedling development, while BrKNAT7a/7b and BoKNAT7 play roles in secondary wall formation.
B. napus has more than two of the sums of B. rapa and B. oleracea and the domain organizations are much conserved with their donors, except for BnKNAT4a-C lacking the ELK domain, BnKNAT7b-C/7c-C containing only the KNOX1 domain, and BnKNAT5a-A/5a-C carrying an additional Virul-Fac domain (pfam10139, Figure 4). Similar to Class I genes, all Class II genes of B. rapa and B. oleracea show synteny with Arabidopsis homologs except for BnKNAT4b-C/7a-A/7a-C/7c-C (Supplement File S2).

2.2.3. Phylogenetic and Domain Analyses of Class-III

The KNATM, a novel KNOX subfamily, is maintained by a homeodomain-independent mechanism [17,19]. A bioinformatic analysis shows that KNATM is found only in dicots and that it lacks the ELK and Homeobox domain (Figure 5; Supplement File S1). Class III contains only one member each in F. vesca, M. truncatula, A. thaliana and P. trichocarpa, B. rapa and B. oleracea contain duplicated KNATMs, whereas B. napus has quadruple KNATMs.
The relationships of Class III proteins are related to plant evolution but domain organizations are not well conserved (Figure 5). PtKNATM, FvKNATM and MtKNATM conserve KNOX1 and KNOX2 domain organizations. AtKNATM only possesses the KNOX1 domain, whereas BoKNATM2 and BnKNAT2-C contain the KNOX2 domain. BrKNATM2 has KNOX2 and a Fer4_NifH domain (PF00142), which is found in various proteins that share a common ATP-binding domain. Conversely, BnKNATM2-A displays typical KNOX1, KNOX2 and P-loop NTPase domains. In addition to the KNOX1 and KNOX2 domains, BrKNATM1 and BoKNATM1 have an additional Chlamydia polymorphic membrane protein middle (ChlamPMP_M) domain (PF07548). However, their homologs BnKNATM1-A/1-C in B. napus lack these domains (Figure 5). Similar to Class II genes, all genes in Class III from B. rapa and B. oleracea show synteny with Arabidopsis homologs (Supplement File S2).

2.3. Analysis of Cis-Acting Elements of BnKNOX Gene Promoters and Gene Structure in B. napus

The cis-acting elements of promoters specifically bind to transcription factors to form transcription initiation complexes, which initiate gene expression. Therefore, we identified 717 cis-elements belonging to 26 different types (Figure 6). These cis-elements could be divided into three groups, plant growth and development, phytohormone responses and abiotic stress responses. For instance, we detected 12 light-responsive cis-elements involved in growth and development with a cumulative occurrence of 388: ACE, AE-box, ATCT-motif, Box 4, GA-motif, GATA-motif, G-Box, GT1-motif, I-box, L-box, MRE and TCT-motif (Supplement Files S8). Among the cis-acting elements involved in hormone response, ABRE, GAREs (GARE-motif and P-box), O2-site, TCA-element, TGA-element and the MeJA-responsive (CGTCA-motif and TGACG-motif) were identified in the promoter elements regions of 59, 18, 14, 28, 18 and 98 occurrences respectively. Additionally, drought and low temperature-stress related cis-acting elements were also detected in the promoter regions of BnKNOX genes. These findings indicate that BnKNOXs may be pivotal in modulating the growth of plants development and may help elucidate precise functions of the proteins from the BnKNOX genes family.
In order to explore the structural diversity of BnKNOXs, a comparative analysis was conducted on the gene structures. Visual analysis revealed that while most family members exhibit similar counts of exons and introns, there is variation in their length. Furthermore, it was observed that the majority of these members possess 3–7 exons, with the exception of BnKNAT7a-C which contains 2 exons, and the BnKNAT7c-C gene which contains only 1 exon. The distribution pattern of exons and introns appears to be intricate, suggesting a potential correlation with phylogenetic subgrouping.

2.4. Gene Collinearity and Duplication of BnKNOXs in B. napus

The gene collinearity analysis facilitates the discovery of homologous sequences within species, which can be used as evidence of the whole genome duplication events. We detected 26 duplication events, 15 of which occurred between subgenome A and C (Figure 7). Notably, the BnKNOXs on chromosome A05 were not collinear with BnKNOX genes on other chromosomes (Figure 7, Supplement File S4). In chromosomes, gene family can expand by tandem, segmental replication or whole genome [47]. In BnKNOXs gene family, 24 pairs were amplified by fragment replication, and only 1 pair (BnKNAT7b-A/BnKNAT7a-A) were amplified by tandem replication (Supplement File S5). This result suggested that fragment replication events contributed most to the expansion of the BnKNOXs in B. napus. Previous studies show that duplication of genes can prevent the loss of function caused by genetic mutation [48,49].
To analyse the mechanism by which BnKNOX gene family evolved, the Ka/Ks ratio was calculated on the 26 pairs of genes with collinearity. The results showed that the Ka/Ks ratio of all duplicated BnKNOX gene pairs were <1 (Supplementary File S4), which indicates that this family was subject to purifying negative selection throughout evolution. Additionally, the duplication events date was estimated around 0.5–31 MYA (Million Years Ago).
Inter-chromosomal relationship of the BnKNOXs in B. napus genome. The red lines show the syntenic blocks, while the gray lines collinear blocks in the whole genome.

2.5. Expression Patterns of BnKNOXs in Different Tissues from BrassicaEDB

The expression patterns of a gene are closely related to its function. For a comprehensive understanding of BnKNOXs functions, we analyzed all members’ expression patterns during flowering and fruiting transition stage using available transcriptome data in different tissues including young leaves, roots, seeds, flowers, siliques (Figure 8A). The results showed that all BnKNOX genes were expressed in at least 1 tissue indicating they may play a vital role during the developmental stage. Remarkably, the expression pattern of BnKNOXs in different tissues was quite different among the three subgroups, while the members clustered in the same group showed a relatively similar expression pattern (Figure 8A). It means that BnKNOXs may affect diverse biological functions in different tissues. The transcripts of most genes from group II were relatively highly observed in most of the tissues, reflecting their ubiquitous roles in plant development. The four BnKNOXs (BnKNATM1-A, BnKNATM1-C, BnKNATM2-A and BnKNATM2-C) in group III were expressed at very low levels in all tissues except for higher expression in mature seed coat.
Similarly, BnKNOXs from group I were concentratedly expressed only in stem, root, and inflorescence tip. For example, BnSTM-A, BnSTM-C and BnKNAT1a-C were highly expressed in various stages of stem, suggesting their important roles in stem development.

2.6. BnKNOXs Expression Levels of in Reproductive Organs by qRT-PCR

Numerous studies have demonstrated the significant roles of KNOX genes in floral organs [50] and fruit development [51]. To confirm the temporal and spatial expression patterns of BnKNOX genes, we subsequently conducted qRT-PCR experiments in various tissues, including buds (bolting bud, 0.8 cm bud, 1.2 cm bud, 1.6 cm bud), floral organs (sepal, petal, anther and stamen), seeds, siliques (1 cm silique, 3 cm silique and 5 cm silique) and young leaves (Figure 8B). The expression level of BnSTM-A in young leaves was used as a reference with a value set at 1. Overall, the qRT-PCR results are consistent with the transcriptome data trends observed. For example, the expression profile showed that Class I and Class II KNOX genes were expressed broadly in the bud development compared with Class III group. Notably, BnKNAT3a-A and BnKNAT3a-C displayed high expression levels in bolting buds, suggesting their potential involvement in inflorescence formation. Furthermore, we found significantly elevated expressions of five BnKNOXs (BnKNAT7a-A, BnKNAT7a-C, BnKNAT7b-A, BnKNAT7b-C and BnKNAT7c-C) specifically in stigma indicating their putative roles in stigma development. Interestingly, BnKNOXs in Class III (BnKNATM1-A, BnKNATM1-C, BnKNATM2-A, BnKNATM2-C) were specifically highly expressed in seeds, implying their crucial functions during seed development processes. Additionally, BnKNAT3a-C and BnKNAT3b-A might play vital roles during silique development stage. Moreover, gene members belonging to the same group exhibited similar expression characteristics.

2.7. Subcellular Localization Analysis

The subcellular localization of the BnKNOX proteins was initially predicted to be nuclear using Cell-PLoc 2.0. However, the experimental results showed that BnSTM-C and BnKNAT3a-A were predominantly localized to the nucleus, while BnKNATM1-A was found in the cytoplasm. In contrast, GFP alone exhibited a diffuse pattern throughout the entire cell (Figure 9).

3. Discussion

3.1. Conservation and Evolution of KNOX Proteins in Plants

Genome evolutionary events and developmental biology reveal historical relationships, genome contractions and expansions, the evolution of functions and genome architecture [52]. KNOX genes represent a subfamily within the homeobox gene family, which is a key regulator of cell fate and body plan specification at the early stages of embryogenesis in higher organisms [53]. Screening and classification of KNOX proteins from lower to higher plants have been conducted on the basis of sequence conservation and domain organization. KNOX genes have been divided into Class I and Class II on the basis of sequences and expression patterns [8].
Based on the domain organization of KNOX1 and KNOX2, it is evident that KNOX genes are relatively recent additions to the plant kingdom, with their presence limited to higher plants (from moss to flowering plants). Even the homeobox gene family is ancient and conserved with their copies increasing during evolution (Figure 2; Supplement File S1). The classification of KNOX proteins into three classes (Figure 2). Class I and Class II are similar to the previous results [8]. AtKNATM, a novel KNOX member lacking a homeodomain domain [17] is classified as Class III (Class KNATM) and exclusively found in dicots. These findings are consistent with previous studies (Figure 2) [39]. Class II is highly conserved and present in all higher plants except Group II, which is restricted to Cruciferae. On the other hand, Class I and Class III are specific to vascular plants or dicots (Figure 2). Domain organization analysis shows that Class I/II carry conserved KNOX1, KNOX2, ELK and homeobox domains (Figure 3 and Figure 4), while Class III lacks the latter two domains (Figure 5). The differentiation of KNOX genes occurred during the evolutionary process of organisms. It is likely that Class I and Class II emerged after the divergence between higher/lower plants, whereas Class III appeared following the separation of dicots/monocots (Figure 2).

3.2. KNOX Orthologs Among B. napus, B. rapa and B. oleracea

The Brassica species, including B. napus, B. rapa, and B. oleracea, are economically important crops and serve as model plants for studying gene duplication events. These species are closely related to Arabidopsis and have been extensively sequenced in genomics research. Genomics has undergone large-scale gene silencing or elimination events after the divergence of the Arabidopsis and Brassica lineages. A chromosome duplication event might have occurred between the evolution of Arabidopsis and B. rapa. The diploid B. rapa genome has undergone genome triplication and lost genes [43,54,55]. The diploid B. oleracea genome has been actively rearranged since the divergence from Arabidopsis, potentially due to polyploidization [56,57]. The duplication of KNOX genes occurs after the separation of Brassica from Arabidopsis (Supplement Files S1 and S2). The number of KNOX genes in Brassica (15 in B. rapa and 14 in B. oleracea) is less than twice the number in Arabidopsis (9). Whole genomic duplication may have included KNOX gene duplications as most homologous genes in Brassica map to different chromosomes. They have also undergone large-scale gene silencing or elimination events after the divergence of the Arabidopsis and Brassica lineages. Synteny information and mutually best hit approach can be used to identify orthologous gene pairs, which tend to keep their original functions among different genomes [58]. The homologous KNOX genes of B. rapa and B. oleracea fall into the same clades in the phylogenetic tree and are all syntenic with corresponding Arabidopsis genes. Hence, these KNOX genes are orthologs (Figure 2, Figure 3, Figure 4 and Figure 5; Supplement File S2). Allopolyploid B. napus includes 32 KNOX genes, three more than the combined total of the KNOX genes in B. rapa and B. oleracea (Supplement Files S2 and S3). Compared with Arabidopsis, B. napus has six nonsyntenic genes, namely, BnKNAT7a-A/1b-C/4b-C and BnKNAT6a-C/7a-C/7c-C. The former exhibit mutual best hits with BrKNAT7a and BoKNAT1/4b along with their respective orthologous genes, while it is possible that BnKNAT6a-C/7a-C/7c-C represent paralogous duplicates generated after B. napus formation (Supplement Files S2 and S3).
The subgenomes of B. napus exhibit a lower frequency of homeologous exchange [59]. The homologs of B. rapa and B. napus can be Reciprocal Best BLASTp except BnKNAT3a-A. While all KNOX proteins from B. oleracea show significant hits against the homologs of B. rapa, five proteins from B. rapa (BrSTM, BrKNAT6a/6b/7a/7b) fail to return to corresponding targets of B. oleracea. Similar events are observed between B. oleracea and the C- genome of B. napus (Supplement File S3). These findings suggest that KNOX homeologous exchange events may occur more frequently in the C-genome than in the A-genome. Furthermore, we observe that BoKNAT1/3a/4b best hits the A-genome homologs of B. napus and that BrKNAT6b best hits the C- genome homologs BnKNAT6b-C (Supplement File S3). The function of a few Brassica KNOX orthologs might be high of conservation. The orthologs of the homeobox gene BREVIPEDICELLUS/KNAT1 in three Brassica species are conserved at the nucleotide and amino acid levels, and BnKNAT1 complements the Arabidopsis bp mutation [60].

3.3. KNOX Gene Duplication and Diversity in B. napus, B. rapa and B. oleracea

Gene duplications increase molecular diversity and serve as a foundation for organismal evolution. High-frequency polyploidy plays a crucial role in plant evolution. Although major chromosomal rearrangements have not been observed, homeologous recombination events between A- and C-genomes happen commonly [61]. B. rapa has duplicated BrKNAT3/4/5/6/7, whereas B. oleracea has duplicated BoKNAT3/4/5/6. The syntenic analysis of KNOX genes between Arabidopsis and Brassica species shows synteny between BrKNAT3a/3b/BoKNAT3a/3b and AtKNAT3, BrKNAT4a/4b/BoKNAT4a/4b and AtKNAT4, BrKNAT5a/5b/BoKNAT5a/5b and AtKNAT5, BrKNAT6a/6b/BoKNAT6a/6b and AtKNAT6, and BrKNAT7a/7b and AtKNAT7 (Supplement File S2). We speculate that the duplication of BrKNAT7a/7b may be due to segmental rearrangement since it is mapped to chromosome A09. Others may result from genomic duplication. The confirmation of four genes in B. oleracea remains uncertain due to the lack of clarity regarding their physical mapping on the chromosomes. Pseudogenization, conservation of gene function, subfunctionalization and neofunctionalization are recognized as the main evolutionary fates of duplicated genes [62]. Neofunctionalization and subfunctionalization have been proposed as important processes driving the retention of duplicated genes [63]. Many mechanisms exist to silence or eliminate the duplicated genes [64].
Shifts of domain organization and expression patterns suggest the differentiation of functions. We find that most of the KNOX proteins are conserved, but several homologous genes have or lose some domains relative to the donors. Both Brassica species have two conserved copies of KNAT3 and KNAT5, one conserved copy of KNAT6 and two differentiated copies of KNATM. In addition, B. rapa has two conserved copies of BrKNAT4 and BrKNAT7, while B. oleracea shows conservation for BoKNAT4 but divergence for BoKNAT6 on the contrary.
BnKNAT1a-A/BnKNAT5a-A/5a-C (Figure 3), and BnKNATM2-A (Figure 5) have EBV-NA3, Virul-Fac and KNOX1 domains in comparison to BrKNAT1a/BrKNAT5a/BoKNAT5a (Figure 3) and BrKNATM2 (Figure 5), respectively. BnKNAT4a-C (Figure 4) lacks ELK and Nit-Regul-home domains, while BnKNATM1-A/1-C (Figure 5) lacks ChlamPMP_M domain relative to BoKNAT4a (Figure 4) and BrKNATM1/BoKNATM1 (Figure 5), correspondingly. In addition, BnKNAT1a-C (Figure 3) loses the KNOX1 domain, but BnKNAT7b-C/7c-C (Figure 4) only carry the KNOX1 domain. The majority of duplicated genes in allopolyploids evolve independently, and their functional diversification is a prominent feature of the long-term evolution of polyploids [65]. Changes in domain organizations suggest that KNOX genes also evolve and that the functions of these homologous genes may be differentiated in B. napus. Several members undergo pseudogenization, whereas others experience neofunctionalization or subfunctionalization.

3.4. Potential Roles of BnKNOX Genes Related to Plant Growth and Development

During our research, the analysis of transcriptomic data for 32 BnKNOX genes has served as a crucial foundation for conducting functional analysis. The qRT-PCR results of this study complement and confirm the transcriptome data. According to the qRT-PCR results, significant variations in the expression profiles of 32 BnKNOX transcripts were observed across different reproductive tissues. These differences were found to be closely associated with specific cis-elements identified in the promoter region of BnKNOXs, including auxin-responsive elements, light-responsive elements, circadian control elements, and hormone response elements. Class I KNOX genes are reported mainly expressed in meristem and regulating the transcription [28]. BnSTM-A and BnSTM-C share resembling cis-elements, gene structures and expression patterns, suggesting that they may have similar functions in plant development. Similar to the expression pattern of Class I genes that AtSTM/AtKNAT1/AtKNAT2/KNAT6 were highly expressed in inflorescence tissues in Arabidopsis, the genes of Class I in B. napus were highly expressed in different development stages of Buds. Previous studies have shown that Class II KNOX genes AtKNAT3/AtKNAT7 play an essential role in mucilage production in the early development stage of Arabidopsis seeds. Interestingly, our findings indicate that the BnKNOX genes in Class III (BnKNATM1-A/1-C, BnKNATM2-A/2-C) exhibited pronounced expression patterns in seeds, suggesting species-specific and diverse gene functions. Moreover, the high expression levels of BnKNAT7a-A/a-C/7b-A/7b-C/7c-C in stigma suggest their potential involvement in regulating female gametophytes morphogenesis. Additionally, the expression patterns of duplicated gene pairs (Supplement File S5) might differ from that of the genes in their subgroups. For example, BnKNAT3a-A is mainly expressed in all floral tissues except stigmas and seeds while BnKNAT4a-A is expressed only in the development stage of buds. BnKNAT7a-A and BnKNAT7c-C showed high expression in anther and stamen. These findings indicate that gene duplication could potentially drive structural diversity and result in functional disproportionation or redundancy. The subcellular localization studies lend weight to the possibility that distinct gene subfamilies may have experienced functional divergence.
Overall, these findings provide valuable insights for further investigating the role of these genes in plant morphogenesis regulation and adaptation to environmental changes.

4. Materials and Methods

4.1. KNOX Protein Identification and Chromosome Map Construction

Sequences of Arabidopsis KNOX proteins, namely, AtSTM (AT1G62360), AtKNAT1 (AT4G08150), AtKNAT2 (AT1G70510), AtKNAT6 (AT1G23380), AtKNAT3 (AT5G25220), AtKNAT4 (AT5G11060), AtKNAT5 (AT4G32040), AtKNAT7 (AT1G62990) and AtKNATM (AT1G14760), were used as queries to search target proteins from the Brassica website (http://brassicadb.cn, Version 3.0) and the Phytozome (http://www.phytozome.net/, version 13.0). The recovered protein sequences were then used as queries in Blastp in TAIR (TAIR—BLAST (arabidopsis.org)), and the domains of sequences with hits to relevant KNOX proteins in Arabidopsis were analyzed. The gene locus information of B. rapa, B. oleracea and B. napus was used to generate chromosome maps by using the MapChart 2.2 program [66] (Voorrips, 2002). A syntenic analysis of KNOX genes in the Arabidopsis and Brassica species was conducted on the website (http://brassicadb.cn, Version 3.0).

4.2. Analysis of Protein Domain Organization

The domain organization of protein sequences was analyzed using NCBI-CD searches of the Pfam database (NCBI Conserved Domain Search) [67]. The low-complexity filter was turned off, the Expect Value was set at 10 to detect short domains or regions of minimal conservation and domains were verified and named according to the SMART database (SMART: Main page (embl-heidelberg.de)).

4.3. Phylogenetic Analysis

Multiple sequence alignments were performed using the Clustal W program [68]. The resulting file was subjected to a phylogenic analysis using the MEGA 11.0 program [69]. Trees were constructed on the basis of the protein sequences using Neighbor-Joining methods with parameters (Pairwise deletion option; p-distance substitution model; and 1000 Bootstrap test). The duplicated genes and homologs of Brassica were confirmed via the phylogenetic tree and syntenic analysis (BRAD (brassicadb.cn)).

4.4. Analysis of Cis-Acting Elements of BnKNOXs Promoters and Gene Structures in B. napus

For the investigation of cis-acting elements with BnKNOXs, the 2000 bp upstream gene sequences were extracted by TBtools [70], and then the cis-acting elements were identified by PlantCARE database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/). Cis-acting element numbers and annotation were visualized using TBtools (V.2.315). To further explore the characteristics of BnKNOXs, exon-intron structure was analyzed using the Gene Structure Display Server and gene structures were visualized by TBtools.

4.5. Collinearity Analysis

The collinearity relationship of the BnKNOXs was detected by TBtools. The results were visualized by Advanced Circos. Based on the full-length-CDS sequence covering and identity of amino acid detected by Blastn/Blastp in NCBI, the non-synonymous substitution rate (Ka) and synonymous substitution rate (Ks) value of the duplicated gene pairs were calculated by TBtools. The ratio of Ka/Ks was used to estimate the mode of selection and considered positive, negative or neutral selection when Ka/Ks ratio was >1, <1 or =1, respectively [71,72]. Divergence time (million years ago, Mya) was estimated by using a Simple Ka/ Ks Calculator tool on TBtools software [73].

4.6. Expression Levels of BnKNOXs in Different Tissues

The expression files of BnKNOXs in various developmental stages (Bolting, Initial flowering, Podding and maturation) from the cultivar XiangYou15 (XY15) of B. napus were obtained through BrassicaEDB (BrassicaEDB - A Gene Expression Database for Brassica Crops). The log2FPKM (Fragments Per Kilobase of exon model per Million mapped fragments) values were used to evaluate gene expression in different tissues and were showed via TBtools.

4.7. Validation of Expression Levels of BnKNOXs by qRT-PCR

To determine the expression levels of BnKNOXs, total RNA was isolated from various tissues following the protocol of RNA isolater Total RNA Extraction Reagent. The obtained RNA (1 μg) was used as the template in synthesizing cDNA for qRT-PCR using the Hiscript III RT SuperMix (Vazyme, Nanjing, China). The qRT-PCR was completed using three technical and biological replicates. The relative expression levels were calculated by the 2−ΔΔCt method. In this study, to ensure the accuracy and reliability of gene expression analysis, two reference genes, PPR (Postsynaptic protein-related) and GDI1 (Guanosine nucleotide diphosphate dissociation inhibitor 1), were selected [74]. The geomean value of these reference genes was used to recalculate the ΔΔCt. The relative expression data were analyzed and shown using TBtools.

4.8. Subcellular Localization of the BnKNOX::GFP Fusion Protein

To determine the specific subcellular localization of the proteins encoded by the genes of interest, we selected one representative candidate gene from each of the three classes: BnSTM-C (Class I), BnKNATM1-A (Class II), and BnKNAT3a-A (Class III). The corresponding genes were amplified and inserted into the pART27 vector in-frame with GFP through homologous recombination. The constructs were introduced into Agrobacterium tumefaciens GV3101, which was then infiltrated into tobacco epidermal cells for transient expression in tobacco (Nicotiana tabacum L.) leaves. pART27 alone was used as control. After 3 days of infiltration, GFP fluorescence was visualized using scanning confocal laser microscopy. In this study, mCherry served as the nuclear localization marker (Red fluorescence).

5. Conclusions

Plant KNOX proteins can be divided into three classes. Class I and Class III are exclusively found in vascular plants or dicots, respectively, whereas Class II is the most conserved and found in all higher plants. B. napus and its parental species B. rapa and B. oleracea possess 15, 14 and 32 KNOX genes, respectively. During allotetraploid formation, B. napus shares most highly conserved KNOX genes with their A- or C- genome contributors. B. napus eliminates some KNOX orthologs from its parents, and forms new homologs due to gene duplication. The divergence of KNOX gene functions occurs due to shifts observed in a few duplicated genes or orthologous proteins between B. napus and its parental species B. rapa and B. olerecea during allotetraploid formation process. The KNOX gene evolution and function diversity during allotetraploid formation are crucial issues. The evolutionary relationship, gene structure, cis-acting elements, expression patterns and subcellular localization further indicate that members of the BnKNOX gene family exhibit both conservative characteristics as well as diversity. The results from sub-cellular localization studies strengthen the hypothesis that different subfamilies of genes have undergone functional differentiation. These findings provide fundamental insights into understanding the role of KNOXs in growth and development processes specific to Brassica napus.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/plants14142167/s1. Supplement File S1. KNOX homologs of plant species. Supplement File S2. Syntenic analysis of KNOX homologs from Arabidopsis and three Brassica species. Supplement File S3. Reciprocal Blastp analysis of KNOX homologs from three Brassica species. Supplement File S4. KNOX protein sequences in Class I. Supplement File S5. KNOX protein sequences in Class II. Supplement File S6. KNOX protein sequences in Class III. Supplement File S7. Information of cis-acting elements on the promoters of KNOX genes in B. napus. Supplement File S8. Collinear gene pairs in B. napus. Supplement File S9. Duplication genes of KNOX in B. napus. Supplement File S10. Detailed dates of RNA-seq and qRT-PCR. Supplement File S11. Primers list of KNOX genes in B. napus.

Author Contributions

Conceptualization, C.T.; methodology, software and validation, X.H.; formal analysis, R.Z.; investigation, Y.C.; resources, Y.C.; data curation, R.Z.; writing—original draft preparation, X.H.; writing—review and editing, visualization, supervision, and project administration and funding acquisition, C.T. All authors have read and agreed to the published version of the manuscript.

Funding

The research was funded by the Scientific Project of Educational Department of Hunan (22C0393, 23B0224) and the Hunan Provincial Natural Science Foundation (2023JJ30288).

Data Availability Statement

The original contributions presented in this study are included in the article/Supplementary Materials. Further inquiries can be directed to the corresponding author.

Acknowledgments

During the preparation of this manuscript/study, the authors used [TBtools, V.2.315] for the purposes of [bioinformation analysis]. The authors have reviewed and edited the output and take full responsibility for the content of this publication.

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. Morata, G.; Lawrence, P. An exciting period of Drosophila developmental biology: Of imaginal discs, clones, compartments, parasegments and homeotic genes. Dev. Biol. 2022, 484, 12–21. [Google Scholar] [CrossRef]
  2. Jia, P.; Wang, Y.; Sharif, R.; Dong, Q.L.; Liu, Y.; Luan, H.A.; Zhang, X.M.; Guo, S.P.; Qi, G.H. KNOTTED1-like homeobox (KNOX) transcription factors—Hubs in a plethora of networks: A review. Int. J. Biol. Macromol. 2023, 253, 126878. [Google Scholar] [CrossRef]
  3. McGinnis, W.; Levine, M.S.; Hafen, E.; Kuroiwa, A.; Gehring, W.J. A conserved DNA sequence in homoerotic genes of the Drosophila Antennapedia and bithorax complexes. Nature 1984, 308, 428–433. [Google Scholar] [CrossRef] [PubMed]
  4. Irish, V.F. The evolution of floral homeotic gene function. BioEssays 2003, 25, 637–646. [Google Scholar] [CrossRef]
  5. Li, G.; Manzoor, M.A.; Wang, G.; Chen, C.; Song, C. Comparative analysis of KNOX genes and their expression patterns under various treatments in Dendrobium huoshanense. Front. Plant Sci. 2023, 14, 1258533. [Google Scholar] [CrossRef] [PubMed]
  6. Dai, H.; Zheng, S.; Zhang, C.; Huang, R.; Yuan, L.; Tong, H. Identification and expression analysis of the KNOX genes during organogenesis and stress responseness in Camellia sinensis (L.) O. Kuntze. Mol. Genet. Genom. 2023, 298, 1559–1578. [Google Scholar] [CrossRef]
  7. Tsuda, K.; Hake, S. Diverse functions of knox transcription factors in the diploid body plan of plants. Curr. Opin. Plant Biol. 2015, 27, 91–96. [Google Scholar] [CrossRef] [PubMed]
  8. Bharathan, G.; Janssen, B.J.; Kellogg, E.A.; Sinha, N. Did homeodomain proteins duplicate before the origin of angiosperms, fungi, and metazoa? Proc. Natl. Acad. Sci. USA 1997, 94, 13749–13753. [Google Scholar] [CrossRef]
  9. Chan, R.L.; Gago, G.M.; Palena, C.M.; Gonzalez, D.H. Homeoboxes in plant development. Biochim. Biophys. Acta 1998, 1442, 1–19. [Google Scholar] [CrossRef]
  10. Wen, J.; Deng, M.; Zhao, K.; Zhou, H.; Wu, R.; Li, M.; Cheng, H.; Li, P.; Zhang, R.; Lv, J. Characterization of Plant Homeodomain Transcription Factor Genes Involved in Flower Development and Multiple Abiotic Stress Response in Pepper. Genes 2023, 14, 1737. [Google Scholar] [CrossRef]
  11. Pick, L. Hox genes, evo-devo, and the case of the ftz gene. Chromosoma 2016, 125, 535–551. [Google Scholar] [CrossRef] [PubMed]
  12. Hii, E.P.W.; Ramanathan, A.; Pandarathodiyil, A.K.; Wong, G.R.; Sekhar, E.V.S.; Binti Talib, R.; Zaini, Z.M.; Zain, R.B. Homeobox Genes in Odontogenic Lesions: A Scoping Review. Head. Neck Pathol. 2023, 17, 218–232. [Google Scholar] [CrossRef] [PubMed]
  13. Bertolino, E.; Reimund, B.; Wildt-Perinic, D.; Clerc, R.G. A novel homeobox protein which recognizes a TGT core and functionally interferes with a retinoid-responsive motif. J. Biol. Chem. 1995, 270, 3178–3188. [Google Scholar] [CrossRef]
  14. Bürglin, T.R. Analysis of TALE superclass homeobox genes (MEIS, PBC, KNOX, Iroquois, TGIF) reveals a novel domain conserved between plants and animals. Nucleic Acids Res. 1997, 25, 4173–4180. [Google Scholar] [CrossRef]
  15. Mukherjee, K.; Brocchieri, L.; Bürglin, T.R. A comprehensive classification and evolutionary analysis of plant homeobox genes. Mol. Biol. Evol. 2009, 26, 2775–2794. [Google Scholar] [CrossRef]
  16. Kerstetter, R.; Vollbrecht, E.; Lowe, B.; Veit, B.; Yamaguchi, J.; Hake, S. Sequence analysis and expression patterns divide the maize knotted1-like homeobox genes into two classes. Plant Cell 1994, 6, 1877–1887. [Google Scholar] [CrossRef]
  17. Magnani, E.; Hake, S. KNOX lost the OX: The Arabidopsis KNATM gene defines a novel class of KNOX transcriptional regulators missing the homeodomain. Plant Cell 2008, 20, 875–887. [Google Scholar] [CrossRef] [PubMed]
  18. Wang, X.; Zhang, J.; Chai, M.; Han, L.; Cao, X.; Zhang, J.; Kong, Y.; Fu, C.; Wang, Z.Y.; Mysore, K.S.; et al. The role of Class II KNOX family in controlling compound leaf patterning in Medicago truncatula. J. Integr. Plant Biol. 2023, 65, 2279–2291. [Google Scholar] [CrossRef]
  19. Gao, J.; Yang, X.; Zhao, W.; Lang, T.; Samuelsson, T. Evolution, diversification, and expression of KNOX proteins in plants. Front. Plant Sci. 2015, 6, 882. [Google Scholar] [CrossRef]
  20. Bellaoui, M.; Pidkowich, M.S.; Samach, A.; Kushalappa, K.; Kohalmi, S.E.; Modrusan, Z.; Crosby, W.L.; Haughn, G.W. The Arabidopsis BELL1 and KNOX TALE homeodomain proteins interact through a domain conserved between plants and animals. Plant Cell 2001, 13, 2455–2470. [Google Scholar] [CrossRef]
  21. Nagasaki, H.; Sakamoto, T.; Sato, Y.; Matsuoka, M. Functional analysis of the conserved domains of a rice KNOX homeodomain protein, OSH15. Plant Cell 2001, 13, 2085–2098. [Google Scholar] [CrossRef]
  22. Scofield, S.; Murray, J.A. KNOX gene function in plant stem cell niches. Plant Mol. Biol. 2006, 60, 929–946. [Google Scholar] [CrossRef]
  23. Rüscher, D.; Corra, l.J.M.; Carluccio, A.V.; Klemens, P.A.W.; Gisel, A.; Stavolone, L.; Neuhaus, H.E.; Ludewig, F.; Sonnewald, U.; Zierer, W. Auxin signaling and vascular cambium formation enable storage metabolism in cassava tuberous roots. J. Exp. Bot. 2021, 72, 3688–3703. [Google Scholar] [CrossRef]
  24. Yang, Q.; Cong, T.; Yao, Y.; Cheng, T.; Yuan, C.; Zhang, Q. KNOX Genes Were Involved in Regulating Axillary Bud Formation of Chrysanthemum × morifolium. Int. J. Mol. Sci. 2023, 24, 7081. [Google Scholar] [CrossRef]
  25. Hay, A.; Tsiantis, M. A KNOX family TALE. Curr. Opin. Plant Biol. 2009, 12, 593–598. [Google Scholar] [CrossRef]
  26. Ruiz-Estévez, M.; Bakkali, M.; Martín-Blázquez, R.; Garrido-Ramos, M. Identification and characterization of TALE Homeobox Genes in the endangered fern Vandenboschia speciosa. Genes 2017, 8, E275. [Google Scholar] [CrossRef]
  27. Hisanaga, T.; Fujimoto, S.; Cui, Y.; Sato, K.; Sano, R.; Yamaoka, S.; Kohchi, T.; Berger, F.; Nakajima, K. Deep evolutionary origin of gamete-directed zygote activation by KNOX/BELL transcription factors in green plants. eLife 2021, 10, e57090. [Google Scholar] [CrossRef]
  28. Frangedakis, E.; Saint-Marcoux, D.; Moody, L.A.; Rabbinowitsch, E.; Langdale, J.A. Nonreciprocal complementation of KNOX gene function in land plants. New Phytol. 2017, 216, 591–604. [Google Scholar] [CrossRef]
  29. Sakakibara, K.; Nishiyama, T.; Deguchi, H.; Hasebe, M. Class 1 KNOX genes are not involved in shoot development in the moss Physcomitrella patens but do function in sporophyte development. Evol. Dev. 2008, 10, 555–566. [Google Scholar] [CrossRef] [PubMed]
  30. Zhang, D.; Lan, S.; Yin, W.L.; Liu, Z.J. Genome-Wide Identification and Expression Pattern Analysis of KNOX Gene Family in Orchidaceae. Front. Plant Sci. 2022, 13, 901089. [Google Scholar] [CrossRef] [PubMed]
  31. Scofield, S.; Dewitte, W.; Nieuwland, J.; Murray, J.A. The Arabidopsis homeobox gene SHOOT MERISTEMLESS has cellular and meristem-organisational roles with differential requirements for cytokinin and CYCD3 activity. Plant J. 2013, 75, 53–66. [Google Scholar] [CrossRef]
  32. Nidhi, S.; Preciado, J.; Tie, L. Knox homologs shoot meristemless (STM) and KNAT6 are epistatic to CLAVATA3 (CLV3) during shoot meristem development in Arabidopsis thaliana. Mol. Biol. Rep. 2021, 48, 6291–6302. [Google Scholar] [CrossRef]
  33. Gastaldi, V.; Alem, A.L.; Mansilla, N.; Ariel, F.D.; Viola, I.L.; Lucero, L.E.; Gonzalez, D.H. BREVIPEDICELLUS/KNAT1 targets TCP15 to modulate filament elongation during Arabidopsis late stamen development. Plant Physiol. 2023, 191, 29–34. [Google Scholar] [CrossRef]
  34. Reiser, L.; Sánchez-Baracaldo, P.; Hake, S. Knots in the family tree: Evolutionary relationships and functions of knox homeobox genes. Plant Mol. Evol. 2000, 42, 151–166. [Google Scholar] [CrossRef]
  35. Wang, S.; Yamaguchi, M.; Grienenberger, E.; Martone, P.T.; Samuels, A.L.; Mansfield, S.D. The Class II KNOX genes KNAT3 and KNAT7 work cooperatively to influence deposition of secondary cell walls that provide mechanical support to Arabidopsis stems. Plant J. 2020, 101, 293–309. [Google Scholar] [CrossRef]
  36. Wang, Y.; Xu, Y.; Pei, S.; Lu, M.; Kong, Y.; Zhou, G.; Hu, R. KNAT7 regulates xylan biosynthesis in Arabidopsis seed-coat mucilage. J. Exp. Bot. 2020, 71, 4125–4139. [Google Scholar] [CrossRef]
  37. Wang, L.; Yang, X.; Gao, Y.; Yang, S. Genome-Wide Identification and Characterization of TALE Superfamily Genes in Soybean (Glycine max L.). Int. J. Mol. Sci. 2021, 22, 4117. [Google Scholar] [CrossRef]
  38. Zhang, Y.; Yin, Q.; Qin, W.; Gao, H.; Du, J.; Chen, J.; Li, H.; Zhou, G.; Wu, H.; Wu, A.M. The Class II KNOX family members KNAT3 and KNAT7 redundantly participate in Arabidopsis seed coat mucilage biosynthesis. J. Exp. Bot. 2022, 73, 3477–3495. [Google Scholar] [CrossRef]
  39. Xiong, H.; Shi, A.; Wu, D.; Weng, Y.; Qin, J.; Ravelombola, W.S.; Shu, X.; Zhou, W. Genome-wide identification, classification and evolutionary expansion of KNOX gene family in Rice (Oryza sativa) and Populus (Populus trichocarpa). Am. J. Plant Sci. 2018, 9, 1071. [Google Scholar] [CrossRef]
  40. Wang, T.; van Dijk, A.D.J.; Bucher, J.; Liang, J.; Wu, J.; Bonnema, G.; Wang, X. Interploidy Introgression Shaped Adaptation during the Origin and Domestication History of Brassica napus. Mol. Biol. Evol. 2023, 40, msad199. [Google Scholar] [CrossRef]
  41. Zhao, Y.; Song, X.; Zhou, H.; Wei, K.; Jiang, C.; Wang, J.; Cao, Y.; Tang, F.; Zhao, S.; Lu, M.Z. KNAT2/6b, a class I KNOX gene, impedes xylem differentiation by regulating NAC domain transcription factors in poplar. New Phytol. 2020, 225, 1531–1544. [Google Scholar] [CrossRef]
  42. Wang, X.Q.; Xu, W.H.; Ma, L.G.; Fu, Z.M.; Deng, X.W.; Li, J.Y.; Wang, Y.H. Requirement of KNAT1/BP for the development of Abscission Zones in Arabidopsis thaliana. J. Integr. Plant Biol. 2006, 48, 15–26. [Google Scholar] [CrossRef]
  43. Yi, Z.; Sanjeev, M.; Singh, G. The Branched Nature of the Nonsense-Mediated mRNA Decay Pathway. Trends Genet. 2021, 37, 143–159. [Google Scholar] [CrossRef]
  44. Wang, Y.; Aïssi-Rothe, L.; Virion, J.M.; De Carvalho Bittencourt, M.; Ulas, N.; Audonnet, S.; Salmon, A.; Clement, L.; Venard, V.; Jeulin, H.; et al. Combination of Epstein-Barr virus nuclear antigen 1, 3 and lytic antigen BZLF1 peptide pools allows fast and efficient stimulation of Epstein-Barr virus-specific T cells for adoptive immunotherapy. Cytotherapy 2014, 16, 122–134. [Google Scholar] [CrossRef]
  45. Li, E.; Bhargava, A.; Qiang, W.; Friedmann, M.C.; Forneris, N.; Savidge, R.A.; Johnson, L.A.; Mansfield, S.D.; Ellis, B.E.; Douglas, C.J. The Class II KNOX gene KNAT7 negatively regulates secondary wall formation in Arabidopsis and is functionally conserved in Populus. New Phytol. 2012, 194, 102–115. [Google Scholar] [CrossRef]
  46. Furumizu, C.; Alvarez, J.P.; Sakakibara, K.; Bowman, J.L. Antagonistic roles for KNOX1 and KNOX2 genes in patterning the land plant body plan following an ancient gene duplication. PLoS Genet. 2015, 11, e1004980. [Google Scholar] [CrossRef]
  47. Soylev, A.; Le, T.M.; Amini, H.; Alkan, C.; Hormozdiari, F. Discovery of tandem and interspersed segmental duplications using high-throughput sequencing. Bioinformatics 2019, 35, 3923–3930. [Google Scholar] [CrossRef]
  48. Abdullah, M.; Sabir, I.A.; Shah, I.H.; Sajid, M.; Liu, X.; Jiu, S.; Manzoor, M.A.; Zhang, C. The role of gene duplication in the divergence of the sweet cherry. Plant Gene 2022, 32, 100379. [Google Scholar] [CrossRef]
  49. Yadav, S.; Chahar, N.; Lal, M.; Das, S. Phylogenetic and comparative genomics establishes origin of paralogy between homologs of AtMYB42 and AtMYB85 in last common ancestor of Brassicaceae via segmental duplication. Plant Gene 2023, 35, 100424. [Google Scholar] [CrossRef]
  50. Box, M.S.; Dodsworth, S.; Rudall, P.J.; Bateman, R.M.; Glover, B.J. Flower-specific KNOX phenotype in the orchid Dactylorhiza fuchsia. J. Exp. Bot. 2012, 63, 4811–4819. [Google Scholar] [CrossRef] [PubMed]
  51. Keren-Keiserman, A.; Shtern, A.; Levy, M.; Chalupowicz, D.; Furumizu, C.; Alvarez, J.P.; Amsalem, Z.; Arazi, T.; Alkalai-Tuvia, S.; Efroni, I.; et al. CLASS-II KNOX genes coordinate spatial and temporal ripening in tomato. Plant Physiol. 2022, 190, 657–668. [Google Scholar] [CrossRef] [PubMed]
  52. Ferreira de Carvalho, J.; Stoeckel, S.; Eber, F.; Lodé-Taburel, M.; Gilet, M.M.; Trotoux, G.; Morice, J.; Falentin, C.; Chèvre, A.M.; Rousseau-Gueutin, M. Untangling structural factors driving genome stabilization in nascent Brassica napus allopolyploids. New Phytol. 2021, 230, 2072–2084. [Google Scholar] [CrossRef] [PubMed]
  53. Jain, M.; Tyagi, A.K.; Khurana, J.P. Genome-wide identification, classification, evolutionary expansion and expression analyses of homeobox genes in rice. FEBS J. 2008, 275, 2845–2861. [Google Scholar] [CrossRef] [PubMed]
  54. Mun, J.H.; Kwon, S.J.; Yang, T.J.; Seol, Y.J.; Jin, M.; Kim, J.A.; Lim, M.H.; Kim, J.S.; Baek, S.; Choi, B.S.; et al. Genome-wide comparative analysis of the Brassica rapa gene space reveals genome shrinkage and differential loss of duplicated genes after whole genome triplication. Genome Biol. 2009, 10, R111. [Google Scholar] [CrossRef]
  55. Wang, X.; Wang, H.; Wang, J.; Sun, R.; Wu, J.; Liu, S.; Bai, Y.; Mun, J.H.; Bancroft, I.; Cheng, F.; et al. The genome of the mesopolyploid crop species Brassica rapa. Nat. Genet. 2011, 43, 1035–1039. [Google Scholar] [CrossRef]
  56. Lukens, L.; Zou, F.; Lydiate, D.; Parkin, I.; Osborn, T. Comparison of a Brassica oleracea genetic map with the genome of Arabidopsis thaliana. Genetics 2003, 164, 359–372. [Google Scholar] [CrossRef]
  57. Zeng, P.; Ge, X.; Li, Z. Transcriptional Interactions of Single B-Subgenome Chromosome with C-Subgenome in B. oleracea-nigra Additional Lines. Plants 2023, 12, 2029. [Google Scholar] [CrossRef]
  58. Glover, N.; Sheppard, S.; Dessimoz, C. Homoeolog Inference Methods Requiring Bidirectional Best Hits or Synteny Miss Many Pairs. Genome Biol. Evol. 2021, 13, evab077. [Google Scholar] [CrossRef]
  59. Bird, K.A.; Niederhuth, C.E.; Ou, S.; Gehan, M.; Pires, J.C.; Xiong, Z.; VanBuren, R.; Edger, P.P. Replaying the evolutionary tape to investigate subgenome dominance in allopolyploid Brassica napus. New Phytol. 2021, 230, 354–371. [Google Scholar] [CrossRef]
  60. Dumonceaux, T.; Venglat, S.P.; Kushalappa, K.; Selvaraj, G.; Datla, R. Molecular and functional characterization of Brassica BREVIPEDICELLUS orthologs involved in inflorescence architecture. Botany 2009, 87, 604–615. [Google Scholar] [CrossRef]
  61. Parkin, I.A.; Gulden, S.M.; Sharpe, A.G.; Lukens, L.; Trick, M.; Osborn, T.C.; Lydiate, D.J. Segmental structure of the Brassica napus genome based on comparative analysis with Arabidopsis thaliana. Genetics 2005, 171, 2765–2781. [Google Scholar] [CrossRef] [PubMed]
  62. Zhang, J. Evolution by gene duplication: An update. Trends Ecol. Evol. 2003, 18, 292–298. [Google Scholar] [CrossRef]
  63. Roth, C.; Rastogi, S.; Arvestad, L.; Dittmar, K.; Light, S.; Ekman, D.; Liberles, D.A. Evolution after gene duplication: Models, mechanisms, sequences, systems, and organisms. JEZ-B Mol. Dev. Evol. 2007, 308, 58–73. [Google Scholar] [CrossRef]
  64. Otto, S.P.; Yong, P. The evolution of gene duplicates. Adv. Genet. 2002, 46, 451–483. [Google Scholar] [CrossRef]
  65. Blanc, G.; Wolfe, K.H. Functional divergence of duplicated genes formed by polyploidy during Arabidopsis evolution. Plant Cell 2004, 16, 1679–1691. [Google Scholar] [CrossRef]
  66. Voorrips, R.E. MapChart: Software for the graphical presentation of linkage maps and QTLs. J. Hered. 2002, 93, 77–78. [Google Scholar] [CrossRef]
  67. Wang, J.; Chitsaz, F.; Derbyshire, M.K.; Gonzales, N.R.; Gwadz, M.; Lu, S.; Marchler, G.H.; Song, J.S.; Thanki, N.; Yamashita, R.A.; et al. The conserved domain database in 2023. Nucleic Acids Res. 2023, 51, 384–388. [Google Scholar] [CrossRef]
  68. Thompson, J.D.; Higgins, D.G.; Gibson, T.J. CLUSTAL W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 1994, 22, 4673–4680. [Google Scholar] [CrossRef] [PubMed]
  69. Tamura, K.; Stecher, G.; Peterso, F.A.; Kumar, S. MEGA6: Molecular evolutionary genetics analysis version 6.0. Mol. Biol. Evol. 2013, 30, 2725–2729. [Google Scholar] [CrossRef]
  70. Chen, C.; Wu, Y.; Li, J.; Wang, X.; Zeng, Z.; Xu, J.; Liu, Y.; Feng, J.; Chen, H.; He, Y.; et al. TBtools-II: A “one for all, all for one” bioinformatics platform for biological big-data mining. Mol. Plant 2023, 16, 1733–1742. [Google Scholar] [CrossRef]
  71. Pi, B.; Pan, J.; Xiao, M.; Hu, X.; Zhang, L.; Chen, M.; Liu, B.; Ruan, Y.; Huang, Y. Systematic analysis of CCCH zinc finger family in Brassica napus showed that BnRR-TZFs are involved in stress resistance. BMC Plant Biol. 2021, 21, 555. [Google Scholar] [CrossRef] [PubMed]
  72. Hurst, L.D. The Ka/Ks ratio: Diagnosing the form of sequence evolution. Trends Genet. 2002, 18, 486–487. [Google Scholar] [CrossRef] [PubMed]
  73. Chen, C.; Chen, H.; Zhang, Y.; Thomas, H.R.; Frank, M.H.; He, Y.; Xia, R. TBtools: An integrative toolkit developed for interactive analyses of big biological data. Mol. Plant 2020, 13, 1194–1202. [Google Scholar] [CrossRef] [PubMed]
  74. Wang, Z.; Yu, F.; Shi, D.; Wang, Y.; Xu, F.; Zeng, S. Selection and validation of reference genes for RT-qPCR analysis in Desmodium styracifolium Merr. 3 Biotech 2021, 11, 403. [Google Scholar] [CrossRef]
Figure 1. Chromosomal distribution of KNOX genes from Brassica. (A): A-genome of B. rapa and B. napus. A01–A06, A08, and A09 are the chromosomes from the A-genome of B. rapa; chrA01–A06, chrA08 and chr are the chromosomes from the A-genome of B. napus; (B): C-genome of B. rapa and B. napus. C01–C03, C05, C07, and C08 are the chromosomes from the C-genome of B. oleracea; ChrC01–C06 and C07–C09 are the chromosomes from the C-genome of B. napus. The marks on the left represent the chromosome sizes. The red and blue lines show the syntenic gene pair. The syntenic genes of BoSTM, BoKNAT4a, BoKNAT6a/6b, and BoKNAT7 are not shown due to their uncertain locus on chromosome. The red and blue lines show the syntenic gene pairs between B. napus and B. rapa or B. oleracea.
Figure 1. Chromosomal distribution of KNOX genes from Brassica. (A): A-genome of B. rapa and B. napus. A01–A06, A08, and A09 are the chromosomes from the A-genome of B. rapa; chrA01–A06, chrA08 and chr are the chromosomes from the A-genome of B. napus; (B): C-genome of B. rapa and B. napus. C01–C03, C05, C07, and C08 are the chromosomes from the C-genome of B. oleracea; ChrC01–C06 and C07–C09 are the chromosomes from the C-genome of B. napus. The marks on the left represent the chromosome sizes. The red and blue lines show the syntenic gene pair. The syntenic genes of BoSTM, BoKNAT4a, BoKNAT6a/6b, and BoKNAT7 are not shown due to their uncertain locus on chromosome. The red and blue lines show the syntenic gene pairs between B. napus and B. rapa or B. oleracea.
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Figure 2. Phylogenetic tree of plant KNOX proteins. This tree includes 150 KNOX proteins from Brassica napus (32), Populus trichocarpa (16), Zea mays (16), Brassica rapa (15), Brassica oleracea (14), Oryza sativa (13), Brachypodium distachyon (11), Arabidopsis thaliana (9), Fragaria vesca (8), Medicago truncatula (7), Physcomitrella patens (4), Selaginella moellendorffii (4), and Ostreococcus lucimarinus (1) genomes. Chlamydomonas reinhardtii, Volvox carteri, and Phaeodactylum tricornutum are not analyzed because of lacking KNOX domain proteins. The proteins were selected for the phylogenetic tree based on their homology to Arabidopsis. The KNOX proteins can be grouped into three classes, Class I-III, based on the phylogenetic tree and domain organization. PpKNAT2a/2b and OlKNAT6 are not included in any class and will not be discussed in this paper.
Figure 2. Phylogenetic tree of plant KNOX proteins. This tree includes 150 KNOX proteins from Brassica napus (32), Populus trichocarpa (16), Zea mays (16), Brassica rapa (15), Brassica oleracea (14), Oryza sativa (13), Brachypodium distachyon (11), Arabidopsis thaliana (9), Fragaria vesca (8), Medicago truncatula (7), Physcomitrella patens (4), Selaginella moellendorffii (4), and Ostreococcus lucimarinus (1) genomes. Chlamydomonas reinhardtii, Volvox carteri, and Phaeodactylum tricornutum are not analyzed because of lacking KNOX domain proteins. The proteins were selected for the phylogenetic tree based on their homology to Arabidopsis. The KNOX proteins can be grouped into three classes, Class I-III, based on the phylogenetic tree and domain organization. PpKNAT2a/2b and OlKNAT6 are not included in any class and will not be discussed in this paper.
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Figure 3. Phylogenetic and domain analyses of Class I. Monocotyledons have more copies of KNAT1, but no STM members. B. rapa and B. oleracea have homologous proteins to those in Arabidopsis Class I, and two homologs of AtKNAT6. B. napus includes both KNOX proteins of B. rapa and B. oleracea, but two copies of BoKNAT7. Domain organizations of homologous proteins are conserved.
Figure 3. Phylogenetic and domain analyses of Class I. Monocotyledons have more copies of KNAT1, but no STM members. B. rapa and B. oleracea have homologous proteins to those in Arabidopsis Class I, and two homologs of AtKNAT6. B. napus includes both KNOX proteins of B. rapa and B. oleracea, but two copies of BoKNAT7. Domain organizations of homologous proteins are conserved.
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Figure 4. Phylogenetic and domain analyses of Class II. Dicots have more members than monocots. B. rapa and B. oleracea contain duplicated conserved copies of almost all KNOX proteins. B. napus includes both KNOX proteins of B. rapa and B. oleracea, but three copies of BoKNAT7, and domain organization are similar to their donors.
Figure 4. Phylogenetic and domain analyses of Class II. Dicots have more members than monocots. B. rapa and B. oleracea contain duplicated conserved copies of almost all KNOX proteins. B. napus includes both KNOX proteins of B. rapa and B. oleracea, but three copies of BoKNAT7, and domain organization are similar to their donors.
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Figure 5. Phylogenetic and domain analyses of Class III. This class is only found in dicots. B. rapa and B. oleracea contain two copies of AtKNOX. B. napus includes both KNOX proteins of B. rapa and B. oleracea. Domain organizations of homologous KNOX of three Brassica species are diverse.
Figure 5. Phylogenetic and domain analyses of Class III. This class is only found in dicots. B. rapa and B. oleracea contain two copies of AtKNOX. B. napus includes both KNOX proteins of B. rapa and B. oleracea. Domain organizations of homologous KNOX of three Brassica species are diverse.
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Figure 6. Cis-elements and gene structure analysis of BnKNOXs family in B. napus. (A): Different cis-elements of BnKNOXs. The elements existed in the 2 kb upstream region of BnKNOXs, displayed in differently colored boxes. The core cis-acting elements CAAT and TATA boxes were identified in all promoters, not shown in the figure. (B): The exon-intron structure of BnKNOX genes. Exons, introns, and UTRs are indicated by yellow boxes, black lines and green boxes, respectively. The length of genes can be represented using the scale at the bottom.
Figure 6. Cis-elements and gene structure analysis of BnKNOXs family in B. napus. (A): Different cis-elements of BnKNOXs. The elements existed in the 2 kb upstream region of BnKNOXs, displayed in differently colored boxes. The core cis-acting elements CAAT and TATA boxes were identified in all promoters, not shown in the figure. (B): The exon-intron structure of BnKNOX genes. Exons, introns, and UTRs are indicated by yellow boxes, black lines and green boxes, respectively. The length of genes can be represented using the scale at the bottom.
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Figure 7. Gene collinearity and duplication of BnKNOXs in B. napus.
Figure 7. Gene collinearity and duplication of BnKNOXs in B. napus.
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Figure 8. Tissue-specific expression pattern of BnKNOXs genes. (A): The heat map of relative expression was generated using TBtools software. The data was collected from BrassicaEDB and normalized by log2FPKM transformed. (B): BnKNOXs Expression levels in reproductive organs by qRT-PCR.
Figure 8. Tissue-specific expression pattern of BnKNOXs genes. (A): The heat map of relative expression was generated using TBtools software. The data was collected from BrassicaEDB and normalized by log2FPKM transformed. (B): BnKNOXs Expression levels in reproductive organs by qRT-PCR.
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Figure 9. Sub-cellular localization of empty vector and three KNOX-GFP proteins. The empty vector and pART27-KNOX vectors were transformed into tobacco leaves, respectively, by using Agrobacterium tumefaciens mediated method. Three days later, the GFP (green color) and RFP (red color) fluorescence signals were observed by confocal microscopy.
Figure 9. Sub-cellular localization of empty vector and three KNOX-GFP proteins. The empty vector and pART27-KNOX vectors were transformed into tobacco leaves, respectively, by using Agrobacterium tumefaciens mediated method. Three days later, the GFP (green color) and RFP (red color) fluorescence signals were observed by confocal microscopy.
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He, X.; Zheng, R.; Chen, Y.; Tan, C. Genome-Wide Analysis of KNOX Genes: Identification, Evolution, Comparative Genomics, Expression Dynamics, and Sub-Cellular Localization in Brassica napus. Plants 2025, 14, 2167. https://doi.org/10.3390/plants14142167

AMA Style

He X, Zheng R, Chen Y, Tan C. Genome-Wide Analysis of KNOX Genes: Identification, Evolution, Comparative Genomics, Expression Dynamics, and Sub-Cellular Localization in Brassica napus. Plants. 2025; 14(14):2167. https://doi.org/10.3390/plants14142167

Chicago/Turabian Style

He, Xiaoli, Ruiyi Zheng, Yan Chen, and Chengfang Tan. 2025. "Genome-Wide Analysis of KNOX Genes: Identification, Evolution, Comparative Genomics, Expression Dynamics, and Sub-Cellular Localization in Brassica napus" Plants 14, no. 14: 2167. https://doi.org/10.3390/plants14142167

APA Style

He, X., Zheng, R., Chen, Y., & Tan, C. (2025). Genome-Wide Analysis of KNOX Genes: Identification, Evolution, Comparative Genomics, Expression Dynamics, and Sub-Cellular Localization in Brassica napus. Plants, 14(14), 2167. https://doi.org/10.3390/plants14142167

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