Genome-Wide Analysis of KNOX Genes in Brassicaceae: Evolution, Comparative Genomics, and Expression Dynamics in B. napus Floral and Silique Development

doi:10.21203/rs.3.rs-5297629/v1

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Genome-Wide Analysis of KNOX Genes in Brassicaceae: Evolution, Comparative Genomics, and Expression Dynamics in B. napus Floral and Silique Development

https://doi.org/10.21203/rs.3.rs-5297629/v1

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Background

Knotted-like homeobox (KNOX) genes, belonging to a subfamily of the homeobox gene family, play crucial roles in cell fate determination and body plan specification during early embryogenesis in higher organisms. Brassica rapa (B. rapa, AA), Brassica oleracea (B. oleracea, CC) and their natural hybridization named Brassica napus (B. napus, AACC) are excellent models for the study of polyploidy genes because they undego genome triplication events after Arabidopsis-Brassiceae divergence. Moreover, the specific gene structure and functional differentiation of KNOXs in B. napus is still unclear.

Methods and results

KNOX homologs from the three Brassica species, namely, B. rapa, B. oleracea and B. napus were downloaded from the Brassica database. Their evolutionary conservation and classification were analyzed with bioinformatics tools. This study identified 32, 15 and 14 KNOX genes in the genome of B. napus, B. rapa and B. oleracea, respectively. Phylogenetic analysis revealed that KNOXs can be classified into three classes based on their structural characteristics. The KNOX homologue proteins across the three Brassica species consistently share a highly conserved domain organization. Synteny analysis indicated that the KNOX gene family of B. napus expanded during allopolyploidization, with whole-gene duplication and segmental duplication being the primary contributors to the majority of KNOX gene duplications. Further analyses of the cis-elements, gene structures and expression patterns of KNOX genes in B. napus showed high conservation among members within the same group. RNA-seq data clearly divided BnKNOXs into three classes: 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 shows expression in most tissues. qRT-PCR results indicated widespread involvement of KNOX genes in reproductive organ development.

Conclusions

The evolutionary conservation and diversification of KNOX proteins are significant in plant evolution and species formation, providing a robust foundation following the Arabidopsis-Brassiceae divergence. The gene structure, cis-elements, and tissue-specific expression patterns underscore the sequence and functional differences among BnKNOX genes. The distinct roles of BnKNOX genes in reproductive development are highlighted. These findings lay the groundwork for further functional studies of BnKNOX genes in B. napus.

KNOX

Phylogenetic analysis

Domain organization

Brassicaceae

Brassica napus

1. 15, 14 and 32 KNOX genes were identified in , and , respectively.

2. The evolutionary characteristics, -elements, gene structures of in plant evolution and species formation and establishes a robust foundation for further functional analyses in .

3. Transcriptional expression profiles emphasized the roles of genes in floral and silique development.

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 (Morata et al., 2022; Jia et al., 2023). Homeotic genes were first detected in fruit flies and found to be involved in segmental development as members of the Bithorax and Antennapedia complexes (McGinnis et al., 1984; Irish, 2003). 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 (Li et al. 2023; Dai et al., 2023). 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 (Tsuda and Hake, 2015). Plant homeotic genes have been classified on the basis of sequence differentiation and fusion with characteristic codomain sequences (Bharathan et al., 1997; Chan et al., 1998; Wen et al., 2023), resulting in their division into 14 distinct classes (Pick, 2016). Knotted-like homeobox (KNOX) belongs to the three amino acid loop extension superclass, which is characterized by three additional residues between helixes 1 and 2, and it is regarded as one of the oldest homeobox gene classes (Hii EPW et al., 2023; Bertolino et al., 1995; Bürglin, 1997).

Based on sequence similarity, structural characteristics, phylogenetic relationships and expression patterns, KNOX genes are mainly divided into three classes (Mukherjee et al., 2009; Kerstetter et al., 1994; Magnani and Hake 2008), and they have been found in almost all plant species (Dai et al., 2023; Wang et al., 2023a). Class I and II KNOX proteins always contain KNOX1, KNOX2, ELK, and homeobox_KN domains (Gao et al., 2015). The two KNOX domains, located at the N-terminus, play an important role in phenotypic change of transgenic plants (Bellaoui et al., 2001; Jia et al., 2023). The ELK domain encodes nuclear localization signals and facilitates interaction with other proteins as well as transcriptional inhibition (Nagasaki et al. 2001). The HD domain is located at the C-terminal and it is involved in DNA binding and homodimer formation (Scofield et al., 2006). 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 (Rüscher et al., 2021), axillary bud (Yang et al., 2023), leaf primordial (Hay and Tsiantis, 2009), and gametophyte development (Ruiz-Estévez et al., 2017; Hisanaga et al., 2021).

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 (Hisanaga et al., 2021). 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 (Frangedakis et al. 2017). Class I KNOX is involved in promoting the cell proliferation during the sporophyte development without inducing shoot formation of the moss Physcomitrella patens, while Class II KNOX protein inhibits gametophyte development (Sakakibara et al., 2008). KNOX in class I is essential for sporophyte meristem development in vascular plant but dispensable for gametophyte development in ferns (Zhang et al. 2022a). 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) (Hay and Tsiantis, 2009; Scofield et al. 2013). Cytokinin can induce the expression levels of STM and KNAT1 in SAM. Besides, AtSTM was proved to be associated with carpel and inflorescence meristem development (Scofield et al. 2007; Nidhi et al. 2021). AtKNAT1 targets TCP15 to modulate filament elongation during stamen development (Gastaldi et al. 2023). 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 (Nidhi et al. 2021). Class II KNOX genes, including KNAT3 (AT5G25220), KNAT4 (AT5G11060), KNAT5 (AT4G32040) and KNAT7 (AT1G62990), exhibit diverse expression patterns (Reiser et al., 2000; Wang et al. 2020a; Wang et al. 2020b). 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 (Wang et al. 2021). For instance, KNAT3 and KNAT7 play multiple roles in the development of secondary cell walls and redundantly regulate mucilage biosynthesis in Arabidopsis seeds (Zhang et al. 2022b). 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 (Magnani et al., 2008), and its homologs belongs to a new class (Xiong et al., 2018). 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 (Tsuda and Hake, 2015).

KNOX genes regulate plant development, with their role primarily elucidated in Arabidopsis (Hay and Tsiantis, 2009; Gastaldi et al. 2023). Although extensive studies have identified KNOX genes in various species (Zhang et al. 2022a; Sun et al. 2023), limited knowledge exists regarding Brassica species. Brassica napus (B. napus) is an allotetraloid (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 Brassica rapa (AA, 2n = 20) and Brassica 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 (Wang et al. 2023b). 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 as well as the expression patterns of the KNOX gene family in B. napus. The work provides new insights into the evolutionary significance of functional differentiation of these genes and lay a foundation for future research on KNOX genes in B. napus.

2.1 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 (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).

2.2 Analysis of protein domain organization

The domain organization of protein sequences was analyzed using NCBI-CD searches of the Pfam database (http://ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) (Wang et al. 2023c). 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)).

2.3 Phylogenetic analysis

Multiple sequence alignments were performed using the Clustal W program (Thompson et al., 1994). The resulting file was subjected to a phylogenic analysis using the MEGA 11.0 program (Tamura et al., 2013). Trees were constructed on 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) ).

2.4 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 (Chen et al. 2023), 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. The further explore the characteristics of BnKNOXs, exon-intron structure was analyzed using the Gene Structure Display Server and gene structures were visualized by TBtools.

2.5 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-synonumous substitution rate (Ka) and synonymous substitution rate (Ks) value of the duplicated gene pairs were calculated by TBtools. The raito of Ka/Ks was to estimate the mode of selection and considered positive, negative or neutral selection when Ka/Ks ratio was > 1, <1 or = 1, respectively. Divergence time (million years ago, Mya) was estimated using the T = Ks/(2*1.5*10^− 8)*10^− 6 (Pi et al. 2021) .

2.6 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 (https://brassica.biodb.org/index). 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.

2.7 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 templated into synthesizing cDNA for qRT-PCR using the Hiscript III RT SuperMix. The qRT-PCR was completed using three technical and biological replicates. The relative expression levels were calculated by the 2^−ΔΔCt method. The relative expression data were analyzed and showed using TBtools.

3.1 Identification and chromosome map of KNOX proteins and genes from Brassica

A total 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 (doe.gov), version 13.0) (Supplement File 1). 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 genes (Supplement File 1).

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 (Fig. 1).

3.2 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: 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 (Fig. 2; Supplement File 1). 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 (Fig. 2; Supplement File 1).

3.3 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 (Zhao et al. 2020). 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 (Fig. 3; Supplement File 1). 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 (Fig. 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 (Wang et al., 2006). The Upf2 domain is conserved in eukaryotes and is crucial for mRNA decay (Yi et al. 2021). 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 (Wang et al., 2014). 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 (Fig. 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 (Fig. 3; Supplement File 2). The domain organizations remain highly conserved except for the duplicated BrKNAT1 members BrKNAT6a/BoKNAT6a which lack ELK and Homeobox domains (Magnani and Hake, 2008) 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 exhibiting a high degree of conservation with their respective donors. Notably, BnKNAT1-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, Fig. 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 2). 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.

3.4 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 (Li et al., 2011; Furumizu et al., 2015). 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) (Fig. 4; Supplement File 1). 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 root of the 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 (Fig. 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 donators, 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, Fig. 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 2).

3.5 Phylogenetic and domain analyses of Class-III

The KNATM, a novel KNOX subfamily, is maintained by a homeodomain-independent mechanism (Magnani and Hake, 2008; Gao et al. 2015). A bioinformatic analysis shows that KNATM is found only in dicots and that it lacks the ELK and Homeobox domain (Fig. 5; Supplement File 1). 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 (Fig. 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 (Fig. 5). Similar to Class II genes, all genes in Class III from B. rapa and B. oleracea show synteny with Arabidopsis homologs (Supplement File 2).

3.6 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 (Fig. 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 (Table S4). 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 BnaKNOXs, 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.

3.7 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 (Fig. 7). Notably, the BnKNOXs on chromosome A05 were not collinear with BnKNOX genes on other chromosomes (Fig. 7, Supplement File 4). In chromosomes, gene family can expand by tandem, segmental replication or whole genome (Soylev et al. 2019). 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 5).This result suggested that fragment replication events contributes most to the expansion of the BnKNOXs in B. napus. Previous study shows that duplication of genes can prevent the loss of function caused by genetic mutation (Abdullah et al. 2022; Yadav et al. 2023).

To analysis 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 4), 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).

3.8 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 (Fig. 8A). The results showed that all BnKNOX genes were expressed at least 1 tissue indicating they may play a vital role during the developmental stage. Remarkably, the expression pattern of BnKNOXs in different tissue was quite different among the three subgroups, while the members clustered in the same group showed a relatively similar expression pattern (Fig. 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, 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.

3.9 BnKNOXs Expression levels of in reproductive organs by qRT-PCR

Numerous studies have demonstrated the significant roles of KNOX genes in floral organs (Box et al. 2012) and fruit development (Keren-Keiserman et al. 2022). 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 (Fig. 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, 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.

4.1 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 (Ferreira de Carvalho et al. 2021). 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 (Jain et al., 2008). Screening and classification of KNOX proteins from lower to higher plants have been conducted on the basis of the sequence conservation and domain organization. KNOX genes have been divided into Class I and Class II on the basis of sequences and expression patterns (Bharathan et al., 1999).

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 (Fig. 2; Supplement File 1). The classification of KNOX proteins into three classes (Fig. 2). Class I and Class II are similar to the previous results (Bharathan et al., 1999). AtKNATM, a novel KNOX member lacking a homeodomain domain (Magnani and Hake, 2008) is classified as Class III (Class KNATM) and exclusively found in dicots. These findings are consistent with previous studies (Fig. 2; Xiong et al., 2018). 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 (Fig. 2). Domain organization analysis shows that Class I/II carry conserved KNOX1, KNOX2, ELK and homeobox domains (Figs. 3, 4), while Class III lacks the latter two domains (Fig. 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 (Fig. 2).

4.2 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 (Mun et al., 2009; Wang et al., 2011; Singh et al. 2021). The diploid B. oleracea genome has been actively rearranged since the divergence from Arabidopsis, potentially due to polyploidization (Lukens et al., 2003; Zeng et al. 2023). The duplication of KNOX genes occurs after the separation of Brassica from Arabidopsis (Supplement Files 1, 2). 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 (Glover et al. 2021). 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 (Figs. 2–5; Supplement File 2). 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 2, 3). 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 File 2, 3).

The subgenomes of B. napus exhibit a lower frequency of homeologous exchange (Bird et al. 2021). 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 3). 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 hit the A- genome homologs of B. napus and that BrKNAT6b best hits the C- genome homologs BnKNAT6b-C (Supplement File 3). 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 (Dumonceaux et al., 2009).

4.3 KNOX gene duplication and diversities 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 observed, homeologous recombination events between A- and C- genomes happen commonly (Parkin et al., 2005). 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 2). We speculate that the duplication of BrKNAT7a/7b may 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 (Zhang, 2003). Neofunctionalization and subfunctionalization have been proposed as important processes driving the retention of duplicated genes (Roth et al., 2007). Many mechanisms exist to silence or eliminate the duplicated genes (Otto and Yong, 2002).

Shifts of domain organization and expression patterns suggest the differentiation of functions (Duarte et al., 2006). 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 (Fig. 3), and BnKNATM2-A (Fig. 5) have EBV-NA3, Virul-Fac and KNOX1 domains in comparison to BrKNAT1a/BrKNAT5a/BoKNAT5a (Fig. 3) and BrKNATM2 (Fig. 5), respectively. BnKNAT4a-C (Fig. 4) lacks ELK and Nit-Regul-home domains, while BnKNATM1-A/1-C (Fig. 5) lacks ChlamPMP_M domain relative to BoKNAT4a (Fig. 4) and BrKNATM1/BoKNATM1 (Fig. 5), correspongdingly. In addition, BnKNAT1a-C (Fig. 3) loses the KNOX1 domain, but BnKNAT7b-C/7c-C (Fig. 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 (Blanc et al., 2004). 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.

4.4 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 confirmed 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 (Frangedakis et al. 2017). 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 shown that Class II KNOX gene 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 5) might differ from that of the genes in their subgroups. For example, BnKNAT3a-A mainly expressed in all floral tissues except stigmas and seeds while BnKNAT4a-A expressed only in the development stage of buds. BnKNAT7a-A and BnKNAT7c-C showed high expression special in anther and stamen. These results highlight how gene duplication can lead to structural diversity and functional disproportionation or redundancy. Overall, these findings provide valuable insights for further investigating the role of these genes in plant morphogenesis regulation and adaptation to environmental changes.

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 (AACC) and its parental species B. rapa (AA) and B. oleracea (CC) 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,and expression patterns further indicate that members of the BnKNOX gene family exhibit both conservative characteristics as well as diversity. These findings provide fundamental insights into understanding the role of KNOX in growth and development processes specific to Brassica napus.

Authors’ contributions

Xiaoli He conducted all data research and analyses. Chengfang Tan conceived the project and review the manuscript.

Supplement Files:

Supplement File 1 KNOX homologs of plant species

Supplement File 2 Syntenic analysis of KNOX homologs from Arabidopsis and three Brassica species

Supplement File 3 Reciprocal Blastp analysis of KNOX homologs from three Brassica species

Supplement File 4 Collinear gene pairs in B. napus

Supplement File 5 Duplication genes of KNOX in B. napus.

Data availability

Data will be made available on request.

Acknowledgements

The research was funded by the Scientific Project of Educational Department of Hunan (23B0224).

Competing Interests: Authors declare don't have competing interests related to the work submitted for publication.

Ethical approval: This declaration does not apply to our study.

Consent to participate: This declaration does not apply to our study.

Consent fot publication: This declaration does not apply to our study

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No competing interests reported.

SupplementTable1KNOXProteinslistusedinthispaper.docx
Supplement File 1 KNOX homologs of plant species
SupplementTable2Syntenicanalysis.docx
Supplement File 2 Syntenic analysis of KNOX homologs from Arabidopsis and three Brassica species
SupplementTable3ReciprocalBlastpanalysis.docx
Supplement File 3 Reciprocal Blastp analysis of KNOX homologs from three Brassica species
Supplementfile4DuplicationgenesofKNOXinB.napus.xlsx
Supplement File 4 Collinear gene pairs in B. napus
Supplementfile5CollineargenepairsinB.napus.xlsx
Supplement File 5 Duplication genes of KNOX in B. napus.

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Genome-Wide Analysis of KNOX Genes in Brassicaceae: Evolution, Comparative Genomics, and Expression Dynamics in B. napus Floral and Silique Development

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Abstract

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Highlights

1. INTRODUCTION

2. MATERIALS AND METHODS

2.1 KNOX protein identification and chromosome map construction

2.2 Analysis of protein domain organization

2.3 Phylogenetic analysis

2.4 Analysis of cis-acting elements of BnKNOXs promoters and gene structures in B. napus

2.5 Collinearity analysis

2.6 Expression levels of BnKNOXs in different tissues

2.7 Validation of expression levels of BnKNOXs by qRT-PCR

3. RESULTS

3.1 Identification and chromosome map of KNOX proteins and genes from Brassica

3.2 Phylogenetic and classification analyses of KNOX protein family

3.3 Phylogenetic and domain analyses of Class I

3.4 Phylogenetic and domain analyses of Class II

3.5 Phylogenetic and domain analyses of Class-III

3.6 Analysis of cis-acting elements of BnKNOX gene promoters and gene structure in B. napus

3.7 Gene collinearity and duplication of BnKNOXs in B. napus

3.8 Expression patterns of BnKNOXs in different tissues from BrassicaEDB

3.9 BnKNOXs Expression levels of in reproductive organs by qRT-PCR

4. DISCUSSION

4.1 Conservation and evolution of KNOX proteins in plants

4.2 KNOX orthologs among B. napus, B. rapa and B. oleracea

4.3 KNOX gene duplication and diversities in B. napus, B. rapa and B. oleracea

4.4 Potential roles of BnKNOX genes related to plant growth and development

5. CONCLUSIONS

Declarations

References

Additional Declarations

Supplementary Files

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