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Article

Genome-Wide Classification and Evolutionary Analysis of the KNOX Gene Family in Plants

by
Zhi-Meng Gan
1,
Xiao-Yan Ai
2,
Chun-Gen Hu
1,* and
Jin-Zhi Zhang
1,*
1
National Key Laboratory for Germplasm Innovation & Utilization of Horticultural Crops, College of Horticulture and Forestry Science, Huazhong Agricultural University, Wuhan 430070, China
2
Institute of Pomology and Tea, Hubei Academy of Agricultural Sciences, Wuhan 430070, China
*
Authors to whom correspondence should be addressed.
Horticulturae 2023, 9(11), 1174; https://doi.org/10.3390/horticulturae9111174
Submission received: 20 September 2023 / Revised: 23 October 2023 / Accepted: 25 October 2023 / Published: 27 October 2023
(This article belongs to the Special Issue A Decade of Research towards to Horticultural Crop from Omics to Biotechnology)
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Abstract

:
The Knotted1-like homeobox (KNOX) gene family plays a pivotal role in regulating meristem activity, organ differentiation, and cell meristematic identity. However, there has been a lack of large-scale, systematic, and comprehensive comparative analyses to explore their expression patterns and evolutionary mechanisms. In this study, a total of 1425 KNOX genes were identified across 118 plant species. The result showed that higher plants exhibited a significantly higher abundance of KNOX genes compared to lower plants. Phylogenetic analysis revealed that all KNOX genes can be divided into two classes (class I and II) and evolved independently after species differentiation. An analysis of gene duplication or loss showed that gene loss was more common than gene duplication in lower plants within the KNOX gene family. These findings suggest that gene loss in the KNOX gene family occurs after events such as whole-genome duplication (WGD) or whole-genome triplication (WGT). In addition, conserved motif analysis was also conducted to uncover the evolutionary trajectories of KNOX genes. We found that three motifs (M1, M2, and M4) were present in nearly all KNOX genes, while four novel motifs (M7–M10) were lost in lower plants but present in higher plants. Moreover, the loss of certain motifs in the KNOX genes was also observed in higher plants, indicating sequence divergence in KNOX genes throughout evolution. To understand the expression patterns of KNOX genes, a gene expression pattern analysis was performed in A. thaliana and O. sativa. The results showed that class I KNOX genes exhibit conserved high expression in stems, suggesting their potential similar biological roles across different plant species and the conservation of their functions during evolution. Additionally, we analyzed the KNOX genes in the Citrus genus and closely related species, and we found that the number of KNOX genes evolved at a slower rate in these species, indicating a relatively conservative nature. In conclusion, this study provides valuable resources for the investigation of KNOX gene family evolution and function in plants.

1. Introduction

The KNOX gene family is one of the plant-specific homeobox transcription factors, belonging to the TALE (three amino acid loop extension) subclass, and it plays a vital role in leaf morphogenesis and shoot apical meristem (SAM) development [1,2,3]. Previous reports have shown that KNOX genes are very conserved and mainly contain four conserved domains (KNAT1, KNAT2, ELK, and Homeobox KN domain) [4,5]. The members of the KNOX gene family are divided into two classes based on their similarity and expression pattern, class I or class II [6]. In Arabidopsis, class I KNOX genes such as SHOOT MERISTEMLESS (STM), BREVI-PEDICELLUS (BP or KNAT1), KNAT2, and KNAT6 exhibit specific expression in meristems. These genes play a significant role in regulating plant organ development and shaping plant morphology. Among the class Ⅱ KNOX genes are KNAT3, KNAT4, KNAT5, and KNAT7, which are expressed in the tissues of all plants. Currently there are few studies on these genes and their functions in plants are not yet clear.
The first KNOX gene (KN1 or ZmKNAT1) was cloned and characterized in Zea mays [7,8]. Until now, the KNOX gene has been characterized in most species, such as 8 members in A. thaliana [6], 23 members in Brassica juncea [9], 20 members in Brassica napus [10], 9 members in Brassica oleracea [11], 12 members in Brassica rapa [9], 8 members in Cardamine hirsute [12], 8 members in Citrus clementina [13], 11 members in Cucumis sativus [14], 8 members in Fragaria vesca [15], 28 members in Glycine max [16], 8 members in Lactuca sativa [17], 17 members in Malus domestica [18], 10 members in Medicago truncatula [19], 7 members in Prunus persica [20], 7 members in Solanum lycopersicum [21], 8 members in Solanum tuberosum [22], 9 members in Triticum aestivum [23], and 9 members in Vitis vinifera [24]. These studies will provide a rich resource for further analysis of the function and evolution of the KNOX family gene in plants.
Currently, the functions of KNOX genes in model plants have been widely reported, such as the maintenance of apical meristems [25], leaf morphogenesis [26], stem elongation [27,28], and floral organ development [29,30]. In Arabidopsis, STM is expressed in the shoot meristem and interacts with CLAVATA3 (CLV3) to jointly maintain stem cell homeostasis [27,28]. Additionally, STM can prevent cell differentiation of in the meristems by maintaining the expression of WUSCHEL (WUS) [31], indicating that STM is essential for stem cell maintenance in the SAM. In terms of leaf morphogenesis, the abnormal expression of KNOX genes causes maize leaves to shrink and become “knotted” [7]. In plants with compound leaves, such as tomatoes, the class I KNOX genes Tkn1 and Tkn2/LeT6 are expressed in leaf primordia. When tkn1 and tkn2/let6 are mutated, it results in accelerated leaf differentiation and reduced leaf complexity [12,32]. In both M. truncatula and Cardamine hirsuta, KNOXI proteins are also necessary and sufficient for the formation of leaflets [33]. The ectopic expression of class I KNOX members can similarly enhance leaf complexity, indicating that class I KNOX genes play a role in regulating leaf development. For instance, PagKNAT2/6b downregulates the expression of GA20ox1 in poplar, inhibiting cell elongation and expansion, leading to a dwarf phenotype [34]. Similarly, similar phenotypes have been observed in A. thaliana [28], Oryza sativa [26], and Z. mays [35], indicating that class I KNOX genes play an important role in regulating plant cell elongation and expansion. Class I KNOX family genes also play a crucial role in floral morphogenesis. The ectopic expression of STM in Arabidopsis promotes the transformation of homologous ovules into carpels, with a reduction in the number of petals and other floral organs in stm mutants [36,37]. Furthermore, STM may interact with APETALA1 (AP1) during the regulation of the development of floral organs [38]. KNAT1 inhibits KNAT2/KNAT6 to ensure the normal upward orientation of the pedicel, while the ectopic expression of KNAT2 and KNAT6 in knat1 null mutants results in an abnormal inflorescence structure [29].
So far, there have been few comparative and evolutionary analysis studies across multiple different species. There are related transcription factor databases, such as the Plant Transcription Factor Database (PlantTFDB, http://planttfdb.gao-lab.org/, accessed on 12 July 2023) [39], which can query and analyze KNOX family genes. However, limited genomes, low quality, and the inability to perform targeted updates affect the comprehensiveness of identification. With the development of sequencing technology, an increasing number of genomes have been updated or assembled with higher quality, making it possible to identify the whole genome of KNOX genes in more species. Here, KNOX genes from 118 plants were comprehensively identified. Subsequently, we further conducted comparative analysis, gene duplication and loss analysis, conserved motif analysis, collinearity analysis, replication type identification, expression pattern analysis, and pan-genome analysis on KNOX genes.

2. Materials and Methods

2.1. Data Collection

In this study, a total of 118 genomes were selected, including 2 Rhodophyta, 8 Chlorophyta, 1 basal plant, and 107 land plants (2 basal angiosperms, 26 monocots, 74 eudicots and 5 non-angiosperms) (Table S1). It is noteworthy that 60 of these land plants are horticultural plants, including 23 fruit trees (Actinidia chinensis, Amborellatrichopoda, Ananas comosus, Arachis ipaensis, Citrullus lanatus, C. clementina, Citrus maxima, Citrus sinensis, Cucumis melo, Diospyros lotus, Ficus carica, F. vesca, M. domestica, Mangifera indica, Musa acuminata, Musa acuminata subsp, Pistacia vera, Prunus avium, Prunus dulcis, Prunus persica, Punica granatum, Vitis riparia, V. vinifera), 24 vegetables (Aquilegia coerulea, Arachis duranensis, Asparagus officinalis, Beta vulgaris, B. juncea, B. napus, B. oleracea, B. rapa, Capsicum annuum, Carica papaya, Cicer arietinum, Cucumis sativus, Daucus carota, G. max, Juglans regia, L. sativa, Manihot esculenta, Olea europaea, Phaseolus vulgaris, Pisum sativum, S. dulcamara, S. lycopersicum, Solanum pennellii, S. tuberosum), and 13 other horticultural plants (Elaeis guineensis, Eucalyptus grandis, Gossypium arboreum, Ipomoea triloba, Kalanchoe laxiflora, Lotus japonicus, M. truncatula, Nymphaea colorata, Picea abies, Prunus mume, Rosa chinensis, Salix purpurea, Salvia miltiorrhiza).
The genome data and annotation information for A. thaliana were downloaded from the Arabidopsis Information Resource (http://www.arabidopsis.org/, accessed on 5 August 2023), data for O. sativa were downloaded from the Rice Genome Annotation Project (http://rice.plantbiology.msu.edu/, accessed on 5 August 2023), data for 67 species were downloaded from the EnsemblPlants database (https://ftp.ebi.ac.uk/ensemblgenomes/pub/release-57/plants/, accessed on 5 August 2023), data for 30 species were downloaded from the NCBI Refseq database (https://ftp.ncbi.nlm.nih.gov/genomes/refseq/plant/, accessed on 5 August 2023), and the other 19 species were downloaded from the Phytozome database (https://phytozome-next.jgi.doe.gov/, accessed on 5 August 2023). The 17 pan-genome sequences of Citrinae species were downloaded from the CPBD database (http://citrus.hzau.edu.cn/, accessed on 10 August 2023) [40].
The transcriptome data of A. thaliana and O. sativa were downloaded from the Sequence Read Archive (SRA, https://www.ncbi.nlm.nih.gov/sra/, accessed on 12 August 2023) The dataset for A. thaliana consisted of 94 root samples, 24 stem samples, 315 leaf samples, 133 flower samples, 36 seed samples, 60 embryo samples, and 370 seedling samples. Furthermore, we integrated 281 samples that underwent hormone treatments, with 38 samples treated with IAA, 113 samples with ABA, 21 samples with GA, and 109 samples with JA. The dataset for O. sativa encompassed 487 samples obtained from various tissues of rice, along with 48 samples treated with different hormone conditions. Specifically, the rice dataset comprised 125 root samples, 7 stem samples, 242 leaf samples, 8 flower samples, 56 seed samples, 7 embryo samples, and 42 seedling samples.

2.2. Identification of KNOX Gene

The KNOX genes were identified by using the KNOX protein sequences of A. thaliana [6] and O. sativa [41] as the query (BLASTP, -perc_identit y > 50 and e-value < 1 × 10−10) and the hidden Markov model file of the KNOX1 domain (PF03790), KNOX2 domain (PF03791), ELK domain (PF03789), and Homeobox KN domain (PF05920) as probes (HMMER3.0). Furthermore, the CDD databases (https://www.ncbi.nlm.nih.gov/cdd/, accessed on 15 August 2023), Pfam databases (http://pfam.xfam.org/, accessed on 15 August 2023) and SMART (http://smart.embl.de/, accessed on 15 August 2023) databases were used to ensure the result accuracy.

2.3. Multiple Sequence Alignment and Phylogenetic Analyses

The phylogenetic tree was made according to the relationship between the species according NCBI taxonomy and plotted by using the R package, ggtree [42]. The phylogenetic tree was constructed by using Mafft (v7.471) [43] for the protein sequences and using IQ-tree (v2.1.11) [44] (the LG and JTT model was used, and bootstrap replications were set at 1000) for the maximum likelihood tree construction and plotted using the R package, ggtree.

2.4. Gene Duplication and Loss Inference

Gene duplication and loss were identified using the phylogenetic species of trees and genes using the java software, Notung (v2.9) [45]. All of this information on the phylogenetic trees was illustrated using the R package, ggtree.

2.5. Collinearity Analysis and Replication Type Identification of KNOX Gene

The collinearity of the KNOX gene among seven representative plants was detected using MCScanX software [46]. Firstly, the protein sequences of these species were aligned using the blastp program with an e-value of 10−5. Then, the collinear blocks were detected using MCScanX with the default parameters. Finally, the gene duplication types (WGD/segmental, tandem, singleton, dispersed, proximal duplications, and singleton) were identified using a duplicate gene classifier program from the MCScanX software. A significance analysis of the duplication type for the KNOX genes compared with the whole-genome genes was conducted using the χ2 test (p <  0.01).

2.6. Expression Pattern Analysis of the KNOX Gene

We conducted a search in databases such as the Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo/, accessed on 11 August 2023), the Sequence Read Archive (https://www.ncbi.nlm.nih.gov/sra/, accessed on 11 August 2023), the European Nucleotide Archive (https://www.ebi.ac.uk, accessed on 11 August 2023), and the DNA Data Bank (https://www.ddbj.nig.ac.jp/ddbj/, accessed on 11 August 2023) of Japan to retrieve samples of different tissues and hormone treatments in Arabidopsis and rice. Subsequently, we downloaded the raw data from all libraries and performed the analysis using a standardized pipeline (fastp–STAR–featureCounts). Ultimately, we obtained normalized FPKM (fragments per kilobase of transcript per million mapped reads) values in Araport11 for each library. Afterwards, the data were further processed and analyzed using Python. Grouping and statistical analysis were performed to gain insights into the data. Additionally, visualizations such as box plots and heatmaps were generated.

2.7. Nonsynonymous (Ka) and Synonymous (Ks) Substitution Rate Calculation Analysis

The calculation of non-synonymous (Ka) and synonymous (Ks) substitution rates for the KNOX gene pairs was performed using a model averaging approach and the Ka/Ks Calculator software [47]. This software enabled the estimation of Ka and Ks substitution rates, which were subsequently used to calculate the Ka/Ks ratio. Python was then utilized for grouping the data and generating visualizations.

3. Results

3.1. Identifying the KNOX Gene in 118 Plants

A total of 1425 KNOX genes were identified in 118 plants (Figure 1; Table S1). Among these species there are 2 Rhodophyta, 8 Chlorophyta, 1 basal plant and 107 land plants, which are further divided into 26 monocots, 74 eudicots, 2 basal angiosperms, and 5 non-angiosperms (Amborella trichopoda, Nymphaea colorata, Marchantia polymorpha, Physcomitrium patens, Picea abies, Selaginella moellendorffii, Sphagnum fallax). Among these land plants, 60 are horticultural plants, 23 are fruit trees, 24 are vegetables, and 13 are other horticultural plants (Figure 1 and Table S1).
Statistical analysis of the identified KNOX genes found that the average number of KNOX genes was 13.3 and most species (81, 68.64%) had >8 KNOX genes compared to A. thaliana (8) (Figure 1 and Figure S1a). Subsequently, we further calculated the length ratio of the KNOX gene (average length of KNOX genes/average length of all genes); the results showed that the length ratio of the KNOX gene was 55 (55.61%), between 0.8 and 1.2, which is greater than >1.2 (51, 46.22%) (Figure 1 and Figure S1b).

3.2. Comparative Analysis of the KNOX Gene in 118 Plants

Investigating the evolution of plant gene families is of great significance for exploring the process of plant evolution and morphological development [48,49]. The statistical analysis found that the number of KNOX genes in higher plants is significantly higher than in lower plants (Figure 1 and Figure 2a). Among the top 10 species with a higher proportion of KNOX genes, all species belong to higher plants, including seven eudicots and three monocots (Figure 2b and Table S1). Monocot species include P. dactylifera (date palm), M. acuminata (banana), and B. distachyon (brachypodium). Interestingly, the three species with the highest proportion of KNOX genes belong to the Malvaceae family and include G. raimondii, G. hirsutum, and G. arboretum (Figure 2b and Table S1). These results indicate that the leaf morphology and development of the Malvaceae family may exhibit higher diversity and complexity.
Most of the lower plants did not have any detected KNOX genes, and only one KNOX gene was detected in two Ostreococcus species, including O. lucimarinus and O. tauri (Table S1). Among the eight other species with a lower proportion of KNOX genes, were four eudicots, one monocot, and three other higher plants (Figure 2b). The only monocotyledon was T. dicoccoides (emmer), an allopolyploid plant with a total of 295,286 genes (Table S1). This result implies that the KNOX gene did not increase linearly with genome doubling, it is speculated that there may be some negative regulatory mechanism in T. dicoccoides that inhibits the replication of the KNOX gene or promotes the deletion of gene family members, which inhibits the expansion of the KNOX gene family. Additionally, among the three other higher plants, P. abies (Norway spruce), S. fallax (Peat moss), and P. patens (a moss) have the lowest proportion of KNOX gene family members. In P. patens, only three KNOX genes were detected out of the total 86,669 genes in the whole genome, accounting for only 0.0035% of all genes (Figure 1 and Table S1). These results suggest that these ancient plant species may utilize alternative approaches to regulate the leaf morphology and development instead of relying on KNOX genes.
To investigate the evolution of the KNOX gene in plants, we constructed an interspecies phylogenetic tree using 1425 KNOX proteins from 118 plant species (Figure 2c). The results show that all KNOX proteins were grouped into two clades (class I and class II), and, corresponding to previous reports [5], the number of class I KNOX proteins was significantly higher than the number in class II. This shows that the evolution process of KNOX genes is relatively conservative. Moreover, most branches contain KNOX genes from different plant classifications. This result indicates that the KNOX proteins of each branch evolved independently after species divergence. Also, KNOX genes are clearly separated in some clades into monocot and eudicot species. These results suggest that the KNOX genes may generate sequence variation during evolution.

3.3. Gene Duplication and Loss Analysis of the KNOX Gene

Gene duplication or loss is prevalent in plant evolution and plays an important role in WGD (whole-genome duplication) or WGT (whole-genome triploidy) events [50]. To elucidate the evolution of the KNOX gene, we selected 17 representative plants for gene duplication and loss analysis, including two Chlorophyta (O. lucimarinus, O. tauri), two Bryophyta (S. fallax, P. patens), one Lycopodiophyta (S. moellendorffii), one Gymnospermae (P. abies), one basal Angiospermae (A. trichopoda), five eudicots (A. thaliana, C. clementina, F. vesca, M. domestica, and V. vinifera), and five monocots (M. acuminata, O. sativa, P. dactylifera, Z. mays, and Z. marina) (Figure 3a). To obtain the gene duplication and loss of KNOX genes during the evolutionary process, we used the Notung software to reconstruct the species tree and gene tree of these 17 representative plants and 20 typical dicotyledonous plants.
In the most recent common ancestor (MRCA) of these 17 representative plants, 29 genes underwent duplication without any gene loss (Figure 3a). However, in the common ancestors of two green algae (O. lucimarinus and O. tauri) and two bryophyte phyla (S. fallax and P. patens), gene loss occurred, but no gene duplication was found, and they lost 24 and 25 genes, respectively. In the common ancestors of mosses and other higher plants, 28 genes were duplicated and only one gene was lost. Although most WGD and WGT events occur in most higher plants, the gene loss in the KNOX gene family was more prevalent than duplications in S. moellendorffii (Lycopodiophyta), P. abies (Gymnospermae) and A. trichopoda (basal Angiospermae) (Figure 3a). These results indicated that the loss of the KNOX gene family may be involved in the processes of WGD or WGT during the plant evolution process.
In addition, we further analyzed the duplication and loss of KNOX genes in 20 typical dicotyledonous plants (Figure 3b). In the MRCA of these 20 plants, 141 genes underwent duplication without any gene loss, and in 16 species, the losses of the KNOX genes were more prevalent than duplications. This result further suggests that KNOX gene loss occurs after WGD or WGT events in most eudicots.

3.4. Conserved Motifs Analysis of the KNOX Gene

Conserved motif analysis can reveal the conserved patterns of a gene family [51]. Here, we explored the conserved motif of the KNOX gene in seven representative plants, ranging from O. tauri (lower plants) to the higher plants, including the Bryophyta species (P. patens), the Lycopodiophyta species (S. moellendorffii), the Gymnospermae species (P. abies), the basal angiosperm species (A. trichopoda), the monocot species (Z. mays and O. sativa), and the eudicot species (C. sinensis and A. thaliana) (Figure 4a and Table S1).
In total, 66 KNOX genes were identified from the whole genomes of nine species. The largest number of KNOX genes was detected in Z. mays (13), followed by S. moellendorffii (12), and O. sativa (11) (Figure 4a and Table S1). However, there was only one KNOX gene in the lower plant (O. tauri). To explore the phylogenetic relationship and classification of KNOX genes, a maximum likelihood tree was constructed using protein sequences. The results revealed that all of the KNOX genes could be divided into two groups, which we defined as class I and class II (Figure 4a).
Furthermore, we detected the motifs (motifs (M) 1–10) of the KNOX genes using the MEME program (Figure 4a and Table S2). A total of 479 motifs were detected with an average of 7.3 motifs per gene, and most genes (58, 85.29%) had >4 motifs. M4 was the longest, followed by M1 and M2. Three motifs (M1, M2, and M4) were present in almost all KNOX genes. However, some of these motifs were also absent. Furthermore, the Pfam analysis showed that the M1 motif was also partial in S. moellendorffii and A. trichopoda (Figure 4a). In addition, we also found that the M5 motif was present in almost all class I genes, while it was absent in almost all of the class II group. Similarly, M6 and M10 were only detected in the KNOX genes of class II and class I. These motifs might be associated with the functional specificity of a different class of KNOX genes. These findings suggest that the motifs within the same class exhibited a high degree of similarity, aligning with the phylogenetic relationship among these genes.
Moreover, the motif evolutionary trajectories of the KNOX gene were obtained to investigate the evolutionary trajectories of the domains of the KNOX genes (Figure 4b and Table S2). In the lower plant (O. tauri), only six motifs (M1, M2, M3, M4, M5, and M6) were detected, while four motifs (M7, M8, M9, and M10) were completely lost. In the bryophyte (P. patens), all 10 motifs were present, but M3, M5, M6, M7, M8, M9, and M10 were partially lost in some KNOX proteins. M5, M7, M9, and M10 were completely lost in Lycopodiophyta (S. moellendorffii) and M6, M8, M9 and M10 were completely lost in Gymnospermae (P.abies), while some motifs were partially lost in some genes. In two Monocots and two eudicots, M1, M2, and M4 were completely retained in all KNOX proteins. Further WebLogo analysis of all of the motif sequence revealed amino acid variability (Figure 4c). This result indicated that the occurrence of sequence divergence in the KNOX gene throughout the evolutionary course of the species.

3.5. Collinearity Analysis and Replication Type Identification of KNOX Gene

Gene duplication patterns may reflect gene family expansion mechanisms [52]. MCScanX was employed to analyze the entire genome as well as the KNOX genes, identifying five types of gene duplication, including singleton, dispersed, proximal, tandem, and WGD/segmental duplications (Figure 5 and Table S3). In all selected species, there were no KNOX genes classified under the proximal and tandem duplication types. As for singletons, only O. sativa and Z. mays possessed one singleton, while the remaining species did not have any. The majority of the KNOX genes belonged to the dispersed duplication type.
In A. thaliana, C. clementina, O. sativa, S. moellendorffii, and Z. mays, several KNOX genes were identified as belonging to the WGD/segmental duplication type (Figure 5c and Table S3). In C. clementina, the proportion of KNOX genes belonging to the WGD/segmental duplication type was 50.00% (Table S3). This proportion was significantly higher than the average proportion of all genome genes belonging to the WGD/segmental duplication type, which was 30.28% (p < 0.01). Therefore, the WGD/segmental duplication type may play a crucial role in the expansion of the KNOX gene family in C. clementina.

3.6. Expression Pattern Analysis of the KNOX Gene in Model Plants

To investigate the expression pattern of the KNOX genes, a comprehensive analysis was conducted using a dataset that comprised 1032 samples obtained from diverse tissues of A. thaliana, as well as samples treated with various hormones (Tables S4 and S6). Through a visual expression analysis of the FPKM values, it was found that all class I KNOX genes (AtSTM, AtKNAT1, AtKNAT2, and AtKNAT6) are highly expressed in the stem while class II KNOX genes are not, but they have a small amount of expression in different tissues, This indicates that class I KNOX genes may be maintaining meristem activity, and play a key role in the process of organ differentiation (Figure 6a–d and Figure S2). In addition, a further cluster analysis of its response to hormones also found that the expression of AtSTM, AtBP1, AtKNAT2, and AtKNAT6 belonging to the class I KNOX gene in SAM under IAA treatment conditions was significantly higher than that in other tissues (Figure 6e and Table S6), further indicating that class I KNOX genes may cooperate with IAA to regulate the development of SAM.
To assess the differences between monocotyledonous and dicotyledonous plants, we also collected transcriptomic data for O. sativa as a representative monocot plant (Tables S5 and S7). The results revealed that the class I KNOX gene exhibited not only high expression levels in the stems but also prominent expression in the root, seed, and embryo tissues (Figure 6f–i). In contrast, among the class II KNOX genes, except for OSH43 which showed significant up-regulation in seeds, the remaining genes demonstrated low levels of expression across different tissues (Figure S3). Therefore, these findings suggest the conservation of the developmental and functional roles of class I KNOX genes in these tissues across monocotyledonous and dicotyledonous plants. These results imply that these genes may play similar biological roles in different plant types, and their functions have been conserved during evolution. This conservation may be related to the importance and functionality of these tissues.

3.7. Pan-Genome Analysis of the KNOX Gene Family in 17 Citrinae

To understand the evolutionary relationships of the KNOX gene among different species in one genus, we performed KNOX family analysis in the Citrus genus and its closely related species. We selected a total of 17 species, including Murraya paniculata, Atalantia buxfoliata, Citrus trifoliata, Citrus mangshanensis, Citrus ichangensis, Citrus linwuensis, Fortunella hindsii, Citrus media, Citrus australasica, Citrus hongheensis, Citrus grandis (L.) Osbeck.cv. “Zipiyou”, Citrus grandis (L.) Osbeck. cv. “Majiayou”, Citrus sinensis, Citrus reticulata, Citrus clementina, and Citrus reticulata Blanco cv. “Ponkan”. We performed gene family identification analysis and identified a total of 164 KNOX genes (Figure 7a and Table S8).
Among them, M. paniculata, a close relative of the citrus genus, has nine KNOX family genes, which is the same as A. thaliana, F. hindsii, A. buxfoliata, C. mangshanensis, C. media, and C. ichangensis, which are primitive or wild species of the Citrus genus with little difference from modern cultivated species. All of them have 10 KNOX genes, more than M. paniculata, indicating gene duplication events in the KNOX gene family. Only C. linwuensis has 12 KNOX genes, which may be related to its larger genome size. C. trifoliata and C. grandis have eight and seven KNOX genes, respectively, suggesting slower evolution in these species (Figure 7a and Table S8).
In order to further explore the evolutionary relationship of KNOX genes in the Rutaceae species, we performed multiple sequence alignment between the 164 identified KNOX proteins and 9 A. thaliana KNOX proteins and used FastTree to construct a phylogenetic tree (Figure 7b). All KNOX proteins were also divided into two categories, class I and class II. Among them, the class II genes of most citrus species are relatively conservative, with only one or missing orthologous genes, and class I genes basically have gene duplication, indicating that class I KNOX genes have evolved more actively in citrus species.
To explore the origin of duplicated KNOX genes, the MCScanX analysis package was used to examine the duplication types of each KNOX gene, including WGD/segmented duplications, tandem duplications, proximal duplications, and dispersed duplications (Table S9). Among them, citrus mainly includes two types, WGD/segmented duplication and dispersed duplication, accounting for 22.22–80% and 20–77% of the total KNOX genes in the species, respectively, indicating that these two repeat types played a key role in the expansion of the KNOX gene family. Collinearity analysis also yielded the same results (Figure 5a and Figure 7c).
The Ka/Ks ratio reflects the selection pressure on duplicated genes. To explore the selective pressure of KNOX family genes in duplications, we calculated the Ka/Ks ratio of WGD/segmented duplication in the KNOX genes and plotted the Ks value and Ka/Ks ratio scatter plot of all repeated KNOX gene pairs (Figure 7d and Table S10). Among the 17 citrus species, the Ka/Ks ratios of all gene pairs ranged from 0.045 to 0.878, indicating that all repetitive genes in citrus were subject to purification selection, indicating that citrus KNOX genes are relatively conserved. In summary, the KNOX genes show a relatively conservative nature in Rutaceae plants and have undergone limited expansion.

4. Discussion

With the rapid development and advancements in sequencing technology, an increasing number of plant genomes have been completely sequenced. This provides valuable resources for studying the functional aspects of plant gene families and has become a prominent area of research in understanding the evolution of plant gene families [35,53]. For instance, Cenci et al. (2022) utilized nine flowering plant genomes to unravel the important roles and evolutionary history of the GELP (GDSL-Type Esterase/Lipase) gene family in plant development [54]. Additionally, Qiao et al. (2019) developed a workflow called DupGen_finder, which identified different types of gene duplication events in 141 sequenced plant genomes, shedding light on the impact of gene and genome duplications on biodiversity evolution [55]. Furthermore, the evolutionary history of the same gene family across all species within a particular plant family can also be effectively analyzed [56]. In this study, we utilized a total of 118 plant genomes, all of which underwent comprehensive whole genome sequencing. Notably, some of these genomes were recently updated or represented higher-quality assemblies, such as the T2T genomes of A. thaliana [57], O. sativa [26], and Z. mays [58]. Among the selected species, there were 2 Rhodophyta, 8 Chlorophyta, 1 basal plant, and 107 land plants. The land plants were further classified into 26 monocots, 74 eudicots, 2 basal angiosperms, and 5 non-angiosperms. Compared to the PlantTFDB [39], the selection of species in this study offered greater comprehensiveness and representativeness. Additionally, the genomes utilized in this study exhibited higher quality. These factors collectively provide essential references for obtaining a thorough understanding of the evolution and functional changes within the KNOX gene family.
Our analysis found that the number of KNOX genes in higher plants is significantly higher than that in lower plants. This may be due to the fact that many WGD or WGT events have occurred in plants during the evolutionary process [59]. Especially in higher plants, which adapt to different environments, leaf morphogenesis occurs with higher diversity and complexity [49,60]. This finding is consistent with the discovery by Yu et al. (2022) that the proportion of heat shock transcription factors (Hsf) family genes is higher in the monocotyledonous plant banana (M. acuminata). This may be attributed to the fact that banana is a tropical fruit and requires a higher proportion of heat-tolerant genes to adapt to high-temperature environments. These results indicate a synergistic relationship between changes in the number of gene families and environmental adaptations [61]. Interestingly, three species with a higher proportion of KNOX genes belong to the Malvaceae family, further demonstrating that WGD or WGT events can affect the number of KNOX genes present, which it is consistent with previous reports that WGD and WGT events affect the number of Hs family genes in Brassicaceae species [61,62,63,64]. In gene duplication and loss analysis, the number of KNOX gene losses in most lycophytes, gymnosperms and angiosperms is greater than the number of gene duplications, indicating that frequent KNOX gene loss events occurring in higher plants occurred after WGD or WGT events.
Conserved motif analysis can reveal conserved patterns in gene families [43]. We found that three motifs (M1, M2, and M4) were present in nearly all KNOX genes and showed that the KNOX genes are structurally conserved. When we performed replication-type identification on seven plants, we found that the dispersed and WGD/segmental duplication type is the main type in the KNOX gene family. This is consistent with previous reports. In eudicots, segmental duplication is caused by whole genome duplication events [65,66]. Similar situations have been reported in other gene families, such as the MADS-box family gene [67], NAC transcription factors [68] and TCP transcription factors [69].
A. thaliana and O. sativa are typical model plants for dicotyledonous and monocotyledonous plants, respectively, and are often used for comparative analysis to reveal the conservation or differences in gene function and evolution [70]. In this study, we found that all class I KNOX genes are highly expressed in the stem, while class II KNOX genes are not, but have a small amount of expression in different tissues in A. thaliana, Consistent with extensive previous reports, class I KNOX members are mainly expressed in meristems and play a role in transcriptional regulation [3]. Among them, STM is highly expressed in the SAM, which maintains embryonic development [71]. KNAT1 is recorded throughout the stem and inflorescence, but not in the leaves [72]. KNAT2 is highly expressed in the SAM base and inflorescence tissue, but is low in the leaves, affecting leaf and flower development [73]. KNAT6 is expressed on the lateral root edge and SAM and plays a role in lateral root initiation, meristem activity and organ formation [74,75]. There are few studies on class II KNOX genes, as their expression patterns are inconsistent with class I KNOX genes, and some of them are functionally redundant [76]. In O. sativa, similar results were also shown, indicating that the expression patterns of KNOX genes are conserved. When treated with hormones, it was found that the class I KNOX gene in SAM under IAA treatment conditions was significantly higher than that in other tissues, indicating that class I KNOX genes may cooperate with IAA to regulate the development of SAM. Related findings have also been reported in barley [77], tomato [78], and maize [79].
Citrus is the largest fruit tree family in the world, ranking first in terms of production and planting area among fruit crops [80]. There are abundant types of leaf morphology, indicating that there may be differences in the development of leaf morphogenesis [81]. Previous studies have shown that CiKN1 and CiKN6 are differentially transcribed in the initial stages of leaf formation in trifoliate orange and lemon, and both of them can be directly involved in leaf development regulation through the miR164a-CUC2 pathway, indicating that KNOX family genes are related to leaf morphogenesis [13]. Recently, the pan-genomes of 23 citrus accessions were reported [40], and KNOX gene family analysis was performed on 17 of them. The study found that there are differences in the number of KNOX genes between different species, reflecting the expansion of the KNOX gene in citrus, mainly including WGD/segmented duplication and dispersed duplication.

5. Conclusions

To gain a deeper understanding of the expression patterns and evolutionary mechanisms of KNOX family genes, we conducted an extensive, systematic, and comprehensive comparative analysis of KNOX family genes across 118 plant species. In this study, a total of 1425 KNOX genes were identified from these 118 plant species. Phylogenetic analysis classified them into two classes (class I and class II), and it was observed that they underwent independent evolution following species differentiation. Furthermore, it was noted that in non-angiosperm plants, gene loss was more prevalent than gene duplication within the KNOX gene family. Additionally, a conserved motif analysis revealed that three motifs (M1, M2, and M4) were present in nearly all KNOX genes, while four novel motifs (M7–M10) were lost in non-angiosperm plants, suggesting sequence divergence in KNOX genes during their evolution. Gene expression pattern analysis in Arabidopsis and rice showed that class I KNOX genes exhibited consistently high expression in stems, implying their potentially similar biological roles across different plant species and the conservation of their functions during evolution. Notably, this study marked the first pan-genome analysis of KNOX family genes in 17 citrus species. This analysis revealed that the number of KNOX family genes evolved at a slower rate in these citrus species, indicating a relatively conservative nature. These findings may offer new insights in the understanding of the evolutionary trajectory of KNOX genes and their contributions to leaf morphogenesis.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/horticulturae9111174/s1, Supporting information Figures S1–S3: Figure S1. (a) The statistics of KNOX gene number and percentage for three categories in plants (0, 1–8, ≥8). (b) The statistics of KNOX gene length ratio (average length of KNOX genes/average length of all genes), and their percentage for four categories (length ratio: 0, ≤0.8, 0.8–1.0, ≥1.2) in plants. Figure S2. (a–d) Box plots of expression patterns of AtKNAT3, AtKNAT4, AtKNAT5, and AtKNAT7 in seven different tissue species of A. thaliana. The Y-axis represents the FPKM of the genes. Rt: root; Sm: stem; Lf: leaf; Fr: flower; Sd: seed; Sg: seeding; Em: embryo. Figure S3. (a–g) Box plots of expression patterns of other class II KNOX genes in seven different tissue species of O. sativa. The Y-axis represents the FPKM of the gene. Rt: root; Sm: stem; Lf: leaf; Sd: seed; Sg: seeding; Em: embryo. Supporting information Tables S1–S10: Table S1. Information of identified KNOX gene in 118 plants. Table S2. Conserved motifs and domains analysis of identified KNOX gene in seven representative plants. Table S3. Percentage of each duplication type for KNOX gene and all genes in seven representative species. Table S4. Fpkm value of KNOX gene from diverse tissues in A. thaliana. Table S5. Fpkm value of KNOX gene from diverse tissues in O.sativa. Table S6. Fpkm value of KNOX gene underwent hormone treatments in A. thaliana. Table S7. Fpkm value of KNOX gene underwent hormone treatments in O.sativa. Table S8. Identifying the KNOX gene in 17 Citrinae. Table S9. Percentage of each duplication type for KNOX gene in 17 Citrinae. Table S10. Percentage of each duplication type for KNOX gene and all genes in seven representative species.

Author Contributions

Z.-M.G. collected and analyzed the data. C.-G.H. and J.-Z.Z. designed the study and the experiments. Z.-M.G. wrote the manuscript. X.-Y.A., C.-G.H. and J.-Z.Z. reviewed the manuscript. All authors discussed the data obtained. All authors have read and agreed to the published version of the manuscript.

Funding

This research was financially supported by the National Major Research and Development Plan (No. 2022YFD1200503) and the National Natural Science Foundation of China (Nos. 319702356, 32072521, and 31872045).

Conflicts of Interest

The authors have no conflict of interest to declare.

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Figure 1. Comparative analysis of KNOX genes in 118 plants. (a) The abbreviated Latin name of each species. (b) The base classification of each species. (c) The Log10 (number of all genes) in each species. (d) The Log2 (number of KNOX genes) in each species. (e) The Log10 (number ratio of KNOX genes) in each species. (f) The length ratio of KNOX genes in each species.
Figure 1. Comparative analysis of KNOX genes in 118 plants. (a) The abbreviated Latin name of each species. (b) The base classification of each species. (c) The Log10 (number of all genes) in each species. (d) The Log2 (number of KNOX genes) in each species. (e) The Log10 (number ratio of KNOX genes) in each species. (f) The length ratio of KNOX genes in each species.
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Figure 2. Phylogenetic analysis of KNOX genes in plants. (a) Boxplot of KNOX genes for four categories of plants (Rhodophyta and Chlorophyta, Eudicots, Monocots, and other plants). (b) Number ratio of KNOX genes in representative plants. The gray gradient indicates the 10 plants with the highest ratio of KNOX genes, and the orange gradient indicates the 10 plants with the lowest ratio of KNOX genes. The Y-axis represents the -log10 ratio of the KNOX gene. (c) Phylogenetic tree using 1425 KNOX proteins from 118 plants. Cyan and magenta represent class I and class II KNOX genes, respectively.
Figure 2. Phylogenetic analysis of KNOX genes in plants. (a) Boxplot of KNOX genes for four categories of plants (Rhodophyta and Chlorophyta, Eudicots, Monocots, and other plants). (b) Number ratio of KNOX genes in representative plants. The gray gradient indicates the 10 plants with the highest ratio of KNOX genes, and the orange gradient indicates the 10 plants with the lowest ratio of KNOX genes. The Y-axis represents the -log10 ratio of the KNOX gene. (c) Phylogenetic tree using 1425 KNOX proteins from 118 plants. Cyan and magenta represent class I and class II KNOX genes, respectively.
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Figure 3. Gene duplication and loss analysis of KNOX gene. (a). Gene duplication and loss analysis of the KNOX gene in 17 representative plants. (b). Gene duplication and loss analysis of the KNOX gene in 20 represent eudicots. “+” and “−”, respectively represent gene duplication and loss, respectively, while the numerical values represent the number of genes.
Figure 3. Gene duplication and loss analysis of KNOX gene. (a). Gene duplication and loss analysis of the KNOX gene in 17 representative plants. (b). Gene duplication and loss analysis of the KNOX gene in 20 represent eudicots. “+” and “−”, respectively represent gene duplication and loss, respectively, while the numerical values represent the number of genes.
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Figure 4. Conserved motif analysis of the KNOX gene. (a) Conserved motif analysis and duplication-type analysis of the KNOX genes in nine representative plants. (b) The motif evolutionary trajectories of the KNOX genes in nine representative plants. Blank and dashed boxes indicate complete loss or nonexistence of the motif. White with an X indicates loss of the motif in a certain gene. Black indicates the motif is present in all genes. (c). WebLogo analysis of the four domains (KNOX1, KNOX2, ELK, and Homeobox domains).
Figure 4. Conserved motif analysis of the KNOX gene. (a) Conserved motif analysis and duplication-type analysis of the KNOX genes in nine representative plants. (b) The motif evolutionary trajectories of the KNOX genes in nine representative plants. Blank and dashed boxes indicate complete loss or nonexistence of the motif. White with an X indicates loss of the motif in a certain gene. Black indicates the motif is present in all genes. (c). WebLogo analysis of the four domains (KNOX1, KNOX2, ELK, and Homeobox domains).
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Figure 5. Number of KNOX genes and duplication types for each representative species. (a) The phylogenetic tree of the seven species. (b) The number of KNOX genes in the seven species. (c) The proportions of different replication types and genes belonging to different replication types in all KNOX genes in seven species. The number of blue quadrilaterals represents the occurrences of whole-genome duplication (WGD) events during the evolutionary process of the species, while the number of purple-red stars represents the occurrences of whole genome triplication (WGT) events during the evolutionary process of the species.
Figure 5. Number of KNOX genes and duplication types for each representative species. (a) The phylogenetic tree of the seven species. (b) The number of KNOX genes in the seven species. (c) The proportions of different replication types and genes belonging to different replication types in all KNOX genes in seven species. The number of blue quadrilaterals represents the occurrences of whole-genome duplication (WGD) events during the evolutionary process of the species, while the number of purple-red stars represents the occurrences of whole genome triplication (WGT) events during the evolutionary process of the species.
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Figure 6. Analysis of expression patterns of KNOX genes in different tissues and under different hormone treatments in A. thaliana and O. sativa. (a–d) Box plots of expression patterns of class I KNOX gene in seven different tissue species of A. thaliana. The Y-axis represents the FPKM of the genes. Rt: root; Sm: stem; Lf: leaf; Fr: flower; Sd: seed; Sg: seeding; Em: embryo. (e) Heatmap of expression pattern of KNOX gene in different tissues in A. thaliana under different hormone treatments. (f–i) Box plots of expression patterns of class I KNOX gene in seven different tissue species of O. sativa. The Y-axis represents the FPKM of the gene. Rt: root; Sm: stem; Lf: leaf; Sd: seed; Sg: seeding; Em: embryo. (j) Heat map of expression patterns of KNOX gene in different tissues in O. sativa under different hormone treatments.
Figure 6. Analysis of expression patterns of KNOX genes in different tissues and under different hormone treatments in A. thaliana and O. sativa. (a–d) Box plots of expression patterns of class I KNOX gene in seven different tissue species of A. thaliana. The Y-axis represents the FPKM of the genes. Rt: root; Sm: stem; Lf: leaf; Fr: flower; Sd: seed; Sg: seeding; Em: embryo. (e) Heatmap of expression pattern of KNOX gene in different tissues in A. thaliana under different hormone treatments. (f–i) Box plots of expression patterns of class I KNOX gene in seven different tissue species of O. sativa. The Y-axis represents the FPKM of the gene. Rt: root; Sm: stem; Lf: leaf; Sd: seed; Sg: seeding; Em: embryo. (j) Heat map of expression patterns of KNOX gene in different tissues in O. sativa under different hormone treatments.
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Figure 7. Pan-genome analysis of the KNOX genes in 17 Citrinae. (a) The phylogenetic tree and number of KNOX genes in the Citrus genus and its closely related species. (b) The phylogenetic tree of all of the KNOX proteins in the Citrus genus and its closely related species. (c) Collinear analysis of KNOX genes in C. clementine, C. grandis (L.) Osbeck cv. “Zipiyou”, and C. linwuensis. (d) Ka/Ks analysis scatter plot of different citrus KNOX family duplicate gene pairs, the X-axis is the Ks value, and the Y-axis is the corresponding Ka/Ks ratio.
Figure 7. Pan-genome analysis of the KNOX genes in 17 Citrinae. (a) The phylogenetic tree and number of KNOX genes in the Citrus genus and its closely related species. (b) The phylogenetic tree of all of the KNOX proteins in the Citrus genus and its closely related species. (c) Collinear analysis of KNOX genes in C. clementine, C. grandis (L.) Osbeck cv. “Zipiyou”, and C. linwuensis. (d) Ka/Ks analysis scatter plot of different citrus KNOX family duplicate gene pairs, the X-axis is the Ks value, and the Y-axis is the corresponding Ka/Ks ratio.
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Gan, Z.-M.; Ai, X.-Y.; Hu, C.-G.; Zhang, J.-Z. Genome-Wide Classification and Evolutionary Analysis of the KNOX Gene Family in Plants. Horticulturae 2023, 9, 1174. https://doi.org/10.3390/horticulturae9111174

AMA Style

Gan Z-M, Ai X-Y, Hu C-G, Zhang J-Z. Genome-Wide Classification and Evolutionary Analysis of the KNOX Gene Family in Plants. Horticulturae. 2023; 9(11):1174. https://doi.org/10.3390/horticulturae9111174

Chicago/Turabian Style

Gan, Zhi-Meng, Xiao-Yan Ai, Chun-Gen Hu, and Jin-Zhi Zhang. 2023. "Genome-Wide Classification and Evolutionary Analysis of the KNOX Gene Family in Plants" Horticulturae 9, no. 11: 1174. https://doi.org/10.3390/horticulturae9111174

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