Genome-Wide Identification, Characterization, and Expression Analysis of the HD-Zip Gene Family in Lagerstroemia for Regulating Plant Height

The Homeodomain leucine zipper (HD-Zip) family of transcription factors is crucial in helping plants adapt to environmental changes and promoting their growth and development. Despite research on the HD-Zip family in various plants, studies in Lagerstroemia (crape myrtle) have not been reported. This study aimed to address this gap by comprehensively analyzing the HD-Zip gene family in crape myrtle. This study identified 52 HD-Zip genes in the genome of Lagerstroemia indica, designated as LinHDZ1-LinHDZ52. These genes were distributed across 22 chromosomes and grouped into 4 clusters (HD-Zip I-IV) based on their phylogenetic relationships. Most gene structures and motifs within each cluster were conserved. Analysis of protein properties, gene structure, conserved motifs, and cis-acting regulatory elements revealed diverse roles of LinHDZs in various biological contexts. Examining the expression patterns of these 52 genes in 6 tissues (shoot apical meristem, tender shoot, and mature shoot) of non-dwarf and dwarf crape myrtles revealed that 2 LinHDZs (LinHDZ24 and LinHDZ14) and 2 LinHDZs (LinHDZ9 and LinHDZ35) were respectively upregulated in tender shoot of non-dwarf crape myrtles and tender and mature shoots of dwarf crape myrtles, which suggested the important roles of these genes in regulate the shoot development of Lagerstroemia. In addition, the expression levels of 2 LinHDZs (LinHDZ23 and LinHDZ34) were significantly upregulated in the shoot apical meristem of non-dwarf crape myrtle. These genes were identified as key candidates for regulating Lagerstroemia plant height. This study enhanced the understanding of the functions of HD-Zip family members in the growth and development processes of woody plants and provided a theoretical basis for further studies on the molecular mechanisms underlying Lagerstroemia plant height.


Introduction
Transcription factors (TFs) play crucial roles in various aspects of plant growth [1], development [2], cell cycle and cell metabolism [3], and stress response [4].One important TF family is the HD-Zip transcription factor family, which contains two highly conserved domains: the homeobox domain (HD) and the leucine zipper (LZ).This TF family is plantspecific [5].The HD domain is a conserved motif comprising approximately 60 amino acid sequences, which fold to form a triple-helix DNA structure responsible for DNA binding [6].The LZ domain mediates protein dimerization [3].Based on the presence of conserved domains, functional characteristics, and motifs, the HD-Zip family can be classified into four subfamilies: HD-Zip I, II, III, and IV [7].
Genes 2024, 15 The protein structure of the HD-Zip I subfamily is relatively simple, comprising only the HD and LZ domains.Besides these domains, the HD-Zip II subfamily also possesses a conserved Cys, Pro, Ser, Cys, and Glu (CPSCE) motif, located adjacent to the LZ in the C-terminal direction [8].Both the HD-Zip III and IV subfamilies contain START and SAD domains [9].However, compared with the HD-Zip IV subfamily, proteins in the HD-Zip III subfamily have an additional methionine-glutamic acid-lysine-histidine-leucine-alanine (MEKHLA) domain at the C-terminus [10].
The HD-Zip gene family plays a crucial role in plant growth and development, with members of the same subfamily generally exhibiting similar gene functions and involvement in biological processes.HD-Zip I members are mainly associated with responses to abiotic stresses [11].For instance, it has been reported that the expression of LlHOX6 and LlHB16, members of the HD-Zip I subfamily, influences plant heat tolerance [12,13].Additionally, Tang et al. revealed that the ectopic expression of JcHDZ21, a member of HD-Zip I, reduced the tolerance to salt stress in Arabidopsis [14].Conversely, the expression of MdHB7 enhanced the salt tolerance in apple plants [15].HD-Zip II proteins mainly played essential roles in the auxin signal transduction pathway [16], organogenesis, and photosynthetic processes [17,18].The overexpression of HD-Zip II gene ATHB2 reduced auxin response and affected leaf development in Arabidopsis [19].Sasake et al. reported that EcHB1 belonging to the HD-Zip II subfamily increased photosynthesis and drought tolerance [20].HD-Zip III was involved in various development processes, including shoot apical meristem development, vascular development, and regulation of plant stems and leaf polarity [21,22].PtrHB4, a member of the HD-Zip III subfamily, affected the development of vascular cambium by regulating auxin signaling in poplar [23].The HD-Zip III activator ZIC2 promoted Arabidopsis shoot regeneration by limiting auxin transport [24].HD-Zip IV genes were reported to play essential roles in regulating multiple physiological processes in plants, such as stomata, trichome, epidermis, and root hair development [19], as well as anthocyanin metabolism, trichome modeling, synthesis and transport of lipids, and protection of plants against biotic and abiotic stresses [25].For instance, the HD-Zip IV gene Roc8 was reported to regulate rice bulliform cell size and lignin content [26].OCL4 expression inhibited the development of maize epidermal hair and also affected cell division and differentiation to varying degrees [27].
Crape myrtle is a valuable ornamental plant with diverse plant types, abundant flowers, and a long blooming period [28].The various plant types of crape myrtles cater to different landscaping needs.However, the molecular regulatory mechanisms governing plant types in L. indica remain unclear.Previous studies have identified internode length as a key factor affecting the Lagerstroemia plant height.Internode length is determined by cell division in the shoot apical meristem (SAM) and cell elongation in shoot segments.Studies have revealed that auxin and gibberellin are the primary hormones regulating internode length in crape myrtle [29][30][31].Although the crucial roles of the HD-Zip family in auxin signal transduction and SAM development are increasingly recognized [12,[32][33][34], their specific roles in Lagerstroemia have not yet been investigated.
This study involved an analysis of the HD-Zip gene family in Lagerstroemia, resulting in the identification of 52 HD-Zip genes within the genome of L. indica.Various aspects of these genes were investigated, including their chromosome location, protein physicochemical properties, gene structure, conserved domains, phylogenetic relationships, and the steadystate element of the HD-Zip gene family of Lagerstroemia.Additionally, the expression patterns of the 52 LinHDZs were examined across six tissues of non-dwarf and dwarf crape myrtles using transcriptome analysis (data not yet published).Further, a subset of 12 genes was selected for validation based on transcriptome data.These findings provide a theoretical basis for further exploration of the regulatory mechanisms of the HD-Zip family in controlling the plant height of crape myrtles.

Analysis of Physicochemical Properties of HD-Zip Proteins in L. indica
The physical and chemical properties, such as relative molecular mass, theoretical isoelectric point, and number of amino acids of HD-Zip proteins, were predicted using TBtools software (version 2.052) [36].The subcellular localization of the L. indica HD-Zip proteins was determined using the online tool Cello (http://cello.life.nctu.edu.tw/,access date: 7 December 2023).

Chromosome Localization Analysis of LinHDZ Genes
The chromosome distribution of the selected LinHDZ genes was predicted with Gene Location Visualize from GTF/GFF function of TBtools software.

Phylogenetic Tree Analysis of HD-Zip Genes in L. indica
A. thaliana, O. sativa, and P. trichocarpa were chosen as outgroup species, and the selected LinHDZ genes were used as the predicted population.Multiple sequence alignment analysis was performed using the ClustalW function of MEGA 6.0 software.The phylogenetic tree was constructed using the maximum likelihood method with the Jones-Taylor-Thornton (JTT) amino acid substitution model, and the bootstrap value was set to 1000 iterations [37].The resulting phylogenetic tree was visually enhanced using iTOL (https://itol.embl.de,access date: 16 December 2023).The HD-Zip proteins used are presented in Table 1.The structures of the LinHDZ genes were predicted using GSDS (http://gsds.gao-lab.org, access date: 9 December 2023).Conserved motifs were analyzed using the MEME online tool with default parameters (https://meme-suite.org/meme/tools/fimo, access date: 9 December 2023) [38].The 2000 bp upstream of the 5 ′ untranslated region of the LinHDZ genes was identified using the PlantCARE tool (http://bioinformatics.psb.ugent.be/webtools/plantcare/html,access date: 10 December 2023) to predict the cisacting regulatory elements.The results were visualized using the visualization function of TBtools software.

Expression Profile Analysis of L. indica Using RNA-Seq Datasets
Transcriptome sequencing was conducted on six tissues-shoot apical meristem (SAM), tender shoot (TS), and mature shoot (MS) of both non-dwarf crape myrtle (S) and dwarf crape myrtle (D)-to investigate the regulation of HD-Zip genes on plant height in Lagerstroemia.A total of 3 µg of RNA for each sample was prepared for sequencing.Libraries were generated using an Illumina Novaseq platform.The gene expression levels were estimated using FPKM values.Differential expression analyses of S_TS vs. S_SAM, S_MS vs. S_SAM, D_TS vs. D_SAM, D_MS vs. D_SAM, and D_SAM vs. S_SAM were conducted using the DESeq2 R package (version 1.20.0), with genes having a p-value ≤ 0.05 and |log2 (fold change)| ≥ 1 considered as differentially expressed genes (Tables S2-S6).Three biological replicates were set for each tissue site.Tissue-specific expression patterns of LinHDZ genes were then analyzed.

Quantitative Real-Time-Polymerase Chain Reaction
The total RNA were extracted from different tissues of dwarf and non-dwarf crape myrtle using a total RNA extraction kit (Tiangen, Beijing, China) for qRT-PCR analysis, which was also used for the RNA-seq analysis.A total of 500 ng of RNA was reversetranscribed into cDNA using the Prime Script RT reagent kit (TaKaRa, Dalian, China).The quantitative real-time polymerase chain reaction (qRT-PCR) was performed using the ACEX96 real-time PCR detection instrument (Bio-Rad, Hercules, CA, USA).EF-1α (Gen Bank ID: MG704141) was selected as the internal reference gene.The PCR system and procedures were conducted as previously described [39].The expression levels were calculated using the 2 −∆∆Ct method, and the expression of each tissue was repeated three times biologically.All primer sequences are provided in Table S1.

Genome-Wide Identification of HD-Zip Genes in L. indica
The genome of L. indica was analyzed using the BLAST tool in the PFAM database to identify potential HD-Zip genes.Then, the SMART and CD-search tools were used to confirm the existence of conserved HD and LZ domains, resulting in the identification of 52 HD-Zip genes designated as LinHDZ1-52 (Table 1).The analysis revealed that the encoded proteins ranged in length from 222 (LinHDZ16 and LinHDZ52) to 916 (LinHDZ35) amino acids, with an average length of 450 amino acids.The molecular weights of these LinHDZs ranged from 24.617 kDa (LinHDZ52) to 100.398 kDa (LinHDZ35), with an average value of 50.072 (Table 1).The isoelectric points of these proteins ranged from 4.63 (Lin-HDZ33) to 9.66 (LinHDZ40), with an average of 6.67.Subcellular localization analysis indicated that most LinHDZs were localized at the nucleus, whereas 10 were found in the cell membrane.Further details about LinHDZs are provided in Table 1.

Phylogenetic Analysis of HD-Zip in L. indica
To elucidate the evolutionary relationship of HD-Zip genes between L. indica and other species, a phylogenetic tree was constructed using 48 A. thaliana HD-Zip, 40 O. sativa HD-Zip, and 63 P. trichocarpa HD-Zip proteins (Figure 2).The results revealed that the crape myrtle HD-Zip family could be categorized into four subfamilies (HD-Zip I-IV).The phylogenetic tree of HD-Zip genes from these four species demonstrated that the HD-Zip I subfamily had the largest number of representatives, followed by the HD-Zip II and IV subfamilies.Conversely, the HD-Zip III subfamily had the least number of representatives, with five in A. thaliana, five in O. sativa, eight in P. trichocarpa, and seven in L. indica (Figure 2).The phylogenetic tree with bootstrap values is shown in Figure S1.Generally, genes with close evolutionary relationships might have similar structures or biological functions.

Phylogenetic Analysis of HD-Zip in L. indica
To elucidate the evolutionary relationship of HD-Zip genes between L. indica and other species, a phylogenetic tree was constructed using 48 A. thaliana HD-Zip, 40 O. sativa HD-Zip, and 63 P. trichocarpa HD-Zip proteins (Figure 2).The results revealed that the crape myrtle HD-Zip family could be categorized into four subfamilies (HD-Zip I-IV).The phylogenetic tree of HD-Zip genes from these four species demonstrated that the HD-Zip I subfamily had the largest number of representatives, followed by the HD-Zip II and IV subfamilies.Conversely, the HD-Zip III subfamily had the least number of representatives, with five in A. thaliana, five in O. sativa, eight in P. trichocarpa, and seven in L. indica (Figure 2).The phylogenetic tree with bootstrap values is shown in Figure S1.Generally, genes with close evolutionary relationships might have similar structures or biological functions.

Analysis of Conserved Motifs and Gene Structure of LinHDZs
This study examined conserved motifs and gene structures to gain further insight into the evolutionary relationships and validate the classification accuracy of HD-Zip proteins in L. indica.Using the MEME online tool, the present study predicted the composition of conserved motifs in LinHDZ proteins, identifying 10 motifs.The motif composition within the same subfamily was largely consistent, indicating functional similarity among LinHDZs within the same subfamily due to the shared domain distribution.Conserved motif analysis revealed that all 52 LinHDZs contained motifs 1-3 corresponding to the HD and LZ domains, underscoring the importance of the two domains in LinHDZ expression.The high conservation of motifs 1-3 in LinHDZs aligned with the characteristic structural properties of HD-Zip proteins.Furthermore, HD-Zip I and II exclusively contained HD

Analysis of Conserved Motifs and Gene Structure of LinHDZs
This study examined conserved motifs and gene structures to gain further insight into the evolutionary relationships and validate the classification accuracy of HD-Zip proteins in L. indica.Using the MEME online tool, the present study predicted the composition of conserved motifs in LinHDZ proteins, identifying 10 motifs.The motif composition within the same subfamily was largely consistent, indicating functional similarity among LinHDZs within the same subfamily due to the shared domain distribution.Conserved motif analysis revealed that all 52 LinHDZs contained motifs 1-3 corresponding to the HD and LZ domains, underscoring the importance of the two domains in LinHDZ expression.The high conservation of motifs 1-3 in LinHDZs aligned with the characteristic structural properties of HD-Zip proteins.Furthermore, HD-Zip I and II exclusively contained HD and LZ domains, whereas HD-Zip III and IV also included a START domain constituted by motifs 4, 5, and 8.In addition, the HD-Zip III subfamily featured a special MEKHLA domain, denoted by motif 6 (Figure 3a).and LZ domains, whereas HD-Zip III and IV also included a START domain constituted by motifs 4, 5, and 8.In addition, the HD-Zip III subfamily featured a special MEKHLA domain, denoted by motif 6 (Figure 3a).
The analysis of gene structure revealed significant differences among the four subfamilies of HD-Zip genes, with members within the same subfamily exhibiting similar numbers of exons and introns.The exon-intron structure of HD-Zip I and II subfamilies appeared simpler compared with that of HD-Zip III and IV subfamilies.Specifically, HD-Zip I and II subfamilies predominantly had 3 to 4 exons, whereas HD-Zip III and IV subfamilies exceeded 10 exons (Figure 3b).These findings suggested that the HD-Zip gene family might have undergone exon supplementation or deletion during evolution.

Analysis of cis-Regulatory Element in LinHDZs
As a regulatory factor controlling gene transcription and expression, cis-elements are indispensable in uncovering gene function [40].The cis-regulatory elements of the LinHDZs (the 2 kb upstream of promoter region) were predicted to investigate the transcriptional characteristics and gene function using PlantCARE (Figure 4).Twenty-two cisacting elements were detected in LinHDZs.Hormone-related elements, such as ABA, GA, IAA, SA, and MeJA response elements, were mainly distributed in HD-Zip I and II subfamilies.Among these, the cis-acting element involved in abscisic acid responsiveness was the most abundant (187), distributed among 52 LinHDZ genes.Stress response-related elements were mainly distributed in the HD-Zip I subfamily, with the cis-acting element involved in low-temperature responsiveness being the most prevalent (55), distributed The analysis of gene structure revealed significant differences among the four subfamilies of HD-Zip genes, with members within the same subfamily exhibiting similar numbers of exons and introns.The exon-intron structure of HD-Zip I and II subfamilies appeared simpler compared with that of HD-Zip III and IV subfamilies.Specifically, HD-Zip I and II subfamilies predominantly had 3 to 4 exons, whereas HD-Zip III and IV subfamilies exceeded 10 exons (Figure 3b).These findings suggested that the HD-Zip gene family might have undergone exon supplementation or deletion during evolution.

Analysis of cis-Regulatory Element in LinHDZs
As a regulatory factor controlling gene transcription and expression, cis-elements are indispensable in uncovering gene function [40].The cis-regulatory elements of the LinHDZs (the 2 kb upstream of promoter region) were predicted to investigate the transcriptional characteristics and gene function using PlantCARE (Figure 4).Twenty-two cis-acting elements were detected in LinHDZs.Hormone-related elements, such as ABA, GA, IAA, SA, and MeJA response elements, were mainly distributed in HD-Zip I and II subfamilies.Among these, the cis-acting element involved in abscisic acid responsiveness was the most abundant (187), distributed among 52 LinHDZ genes.Stress response-related elements were mainly distributed in the HD-Zip I subfamily, with the cis-acting element involved in lowtemperature responsiveness being the most prevalent (55), distributed among 29 LinHDZ genes.Functional elements related to growth and development were mainly distributed in HD-Zip II and III subfamilies, with the cis-acting regulatory element related to meristem expression having the largest number (33), distributed among 24 LinHDZ genes.The light-response element was present throughout the entire family.These results suggested that LinHDZ genes played crucial roles in affecting Lagerstroemia plant height and resisting external stress.
FOR PEER REVIEW 9 of 15 among 29 LinHDZ genes.Functional elements related to growth and development were mainly distributed in HD-Zip II and III subfamilies, with the cis-acting regulatory element related to meristem expression having the largest number (33), distributed among 24 LinHDZ genes.The light-response element was present throughout the entire family.These results suggested that LinHDZ genes played crucial roles in affecting Lagerstroemia plant height and resisting external stress.

Analysis of Tissue-Specific Expression Patterns of LinHDZs in Non-Dwarf and Dwarf Crape Myrtles
The expression levels of 52 LinHDZs in SAM, TS, and MS of non-dwarf and dwarf crape myrtles were analyzed using RNA-seq to investigate the mechanism of LinHDZ genes in regulating the plant height of L. indica.The expression patterns of LinHDZs across six tissues showed significant differences (Figure 5).The HD-Zip I subfamily exhibited expression in different tissues.In contrast, the HD-Zip II subfamily was mainly expressed in the shoot apical meristem of dwarf crape myrtle (D_SAM) and the TS and MS of nondwarf crape myrtle (S_TS and S_MS).HD-Zip III and IV subfamilies showed tissue-specific expression patterns, with the HD-Zip III subfamily primarily expressed in TS and MS.The expression levels of LinHDZ24, and LinHDZ14 in S_TS were significantly upregulated compared with those in S_SAM.The expression of LinHDZ9 and LinHDZ35 in both TS and MS of dwarf crape myrtles were about two times higher than those in D_SAM, while showed no significant differences in non-dwarf crape myrtles (Tables S2-S5).Additionally, the expression levels of LinHDZ23 and LinHDZ34 in the D_SAM were nearly two times lower than those in non-dwarf crape myrtle (Table S6).These results suggested that

Analysis of Tissue-Specific Expression Patterns of LinHDZs in Non-Dwarf and Dwarf Crape Myrtles
The expression levels of 52 LinHDZs in SAM, TS, and MS of non-dwarf and dwarf crape myrtles were analyzed using RNA-seq to investigate the mechanism of LinHDZ genes in regulating the plant height of L. indica.The expression patterns of LinHDZs across six tissues showed significant differences (Figure 5).The HD-Zip I subfamily exhibited expression in different tissues.In contrast, the HD-Zip II subfamily was mainly expressed in the shoot apical meristem of dwarf crape myrtle (D_SAM) and the TS and MS of non-dwarf crape myrtle (S_TS and S_MS).HD-Zip III and IV subfamilies showed tissue-specific expression patterns, with the HD-Zip III subfamily primarily expressed in TS and MS.The expression levels of LinHDZ24, and LinHDZ14 in S_TS were significantly upregulated compared with those in S_SAM.The expression of LinHDZ9 and LinHDZ35 in both TS and MS of dwarf crape myrtles were about two times higher than those in D_SAM, while showed no significant differences in non-dwarf crape myrtles (Tables S2-S5).Additionally, the expression levels of LinHDZ23 and LinHDZ34 in the D_SAM were nearly two times lower than those in non-dwarf crape myrtle (Table S6).These results suggested that the effects of HD-Zip genes on the growth and development of L. indica varied among subfamilies, with HD-Zip III and IV subfamilies suggested to play essential roles in regulating Lagerstroemia plant height.

Validation of LinHDZ Expression
Twelve differentially expressed LinHDZ genes identified based on RNA-seq analysis (Figure 6) were subjected to qRT-PCR verification in SAM, TS, and MS tissues of both nondwarf and dwarf crape myrtles.The results revealed significant upregulation of LinHDZ24, LinHDZ14, whereas LinHDZ35 and LinHDZ9 were markedly downregulated

Validation of LinHDZ Expression
Twelve differentially expressed LinHDZ genes identified based on RNA-seq analysis (Figure 6) were subjected to qRT-PCR verification in SAM, TS, and MS tissues of both non-dwarf and dwarf crape myrtles.The results revealed significant upregulation of LinHDZ24, LinHDZ14, whereas LinHDZ35 and LinHDZ9 were markedly downregulated in S_TS.Additionally, LinHDZ23 and LinHDZ34 were significantly upregulated in S_SAM.The qRT-PCR results corroborated those obtained from RNA-seq analysis, indicating that these genes served as key candidates for regulating Lagerstroemia plant height.This study contributed to a deeper understanding of the molecular mechanisms underlying plant height regulation.indicating that these genes served as key candidates for regulating Lagerstroemia plant height.This study contributed to a deeper understanding of the molecular mechanisms underlying plant height regulation.

Discussion
Crape myrtle, being one of the most significant flowering plants in summer, has been widely used in gardens due to its diverse plant types.In recent years, dwarf crape myrtles have been increasingly favored by the garden market.However, the molecular mechanism behind the dwarf plant type of Lagerstroemia remains unclear.Identifying genes regulating plant height and exploring the regulatory mechanism of plant height can offer a crucial theoretical basis for enhancing plant types.
HD-Zip TFs play a crucial role in affecting plant growth, development, and resilience to environmental stress [41].To date, HD-Zip family genes have been systematically identified in several species, such as Chinese cabbage, chrysanthemum, oil palm, and watermelon [42][43][44][45].Various studies have demonstrated that HD-Zip III and IV genes mainly contributed to shoot and root meristem development, as well as cell proliferation regulation.However, the characteristics and functions of this gene family in crape myrtle have not yet been investigated.
This study identified 52 LinHDZ genes in the genome of L. indica and involved the

Discussion
Crape myrtle, being one of the most significant flowering plants in summer, has been widely used in gardens due to its diverse plant types.In recent years, dwarf crape myrtles have been increasingly favored by the garden market.However, the molecular mechanism behind the dwarf plant type of Lagerstroemia remains unclear.Identifying genes regulating plant height and exploring the regulatory mechanism of plant height can offer a crucial theoretical basis for enhancing plant types.
HD-Zip TFs play a crucial role in affecting plant growth, development, and resilience to environmental stress [41].To date, HD-Zip family genes have been systematically identified in several species, such as Chinese cabbage, chrysanthemum, oil palm, and watermelon [42][43][44][45].Various studies have demonstrated that HD-Zip III and IV genes mainly contributed to shoot and root meristem development, as well as cell proliferation regulation.However, the characteristics and functions of this gene family in crape myrtle have not yet been investigated.This study identified 52 LinHDZ genes in the genome of L. indica and involved the genome-wide analyses of LinHDZs.Phylogenetic tree analysis revealed that HD-Zip proteins were classified into four HD-Zip I-IV subfamilies, consistent with the findings in other species such as A. thaliana [46], O. sativa [47], and P. trichocarpa [48].Most of these 52 LinHDZ genes belonged to HD-Zip I and II subfamilies.The HD-Zip III subfamily had the fewest LinHDZ genes (7/52), consistent with the proportions observed in other species such as Prunus mume (4/32) [49], A. thaliana (5/48) [46], and tomato (6/49) [50].Analyses of gene-conserved motifs and gene structures revealed similar numbers of conserved motifs and exons and introns among members of the same subfamily, further supporting the reliability of the phylogenetic relationship of LinHDZ genes.Additionally, members of the same subfamily exhibited similar expression patterns across six tissues of non-dwarf and dwarf crape myrtles.HD-Zip I genes showed diverse expression across different tissues, whereas most HD-Zip II genes were expressed in S_TS and S_MS.HD-Zip III and IV subfamilies exhibited tissue-specific expression, with a preference for shoots and SAM, respectively.This might be because the members of HD-Zip III and IV subfamilies are known to be highly conserved lineages [45], with regulatory effects on SAM development, vascular development, leaf and shoot polarity regulation, and auxin transport [21][22][23][24]33].These findings suggest that the four subfamilies of LinHDZ genes had different effects on the growth and development of L. indica.Previous studies have reported that HD-Zip gene members affect plant organ morphology.By combining tissue-specific expression patterns with previous findings, several members of HD-Zip III and IV subfamilies were implicated in regulating Lagerstroemia plant height.For instance, two LinHDZs (LinHDZ24, and LinHDZ14) from the HD-Zip III subfamily were significantly upregulated in S_TS, with LinHDZ14 being homologous to ATHB8, reported to be regulated by AUX/IAA involved in auxin signaling [51].Studies on Lagerstroemia plant architecture highlighted the significance of IAA and GA hormones in regulating plant height [31].Additionally, in O. sativa, the overexpression of OsHox32, a homologous gene of LinHDZ35, resulted in a semi-dwarf phenotype [52].In this study, LinHDZ35 was upregulated in D_TS and D_MS, suggesting its positive regulatory role in Lagerstroemia dwarfism.Previous studies have shown a complex regulatory relationship between the ATML1 of HD-Zip IV and GA signal transduction, with negative feedback regulation between ATML1/PDF2 and DELLA [53].In the present study, LinHDZ23 and LinHDZ34 from the HD-Zip IV subfamily were homologous to ATHDG11 and ATHDG 12, respectively.The latter were reported to play significant roles in regulating root and shoot meristems [54], suggesting their involvement in the cell proliferation of SAMs.
In summary, HD-Zip genes play a crucial role in the growth and development of crape myrtle.The findings of this study provide valuable insights into the role of HD-Zip genes in woody plants and might have significant implications for the breeding of L. indica, offering valuable references for future studies and applications in this area.

Conclusions
This study involved a comprehensive genome analysis of the HD-Zip family in L. indica.Fifty-two HD-Zip genes were identified and classified into four subfamilies: I, II, III, and IV.Gene structure and motif analysis revealed that the members within the same subfamily shared similar motifs and likely performed similar functions.Cis-acting element analysis indicated the presence of numerous hormone-related and stress-responsive cis-acting elements, suggesting their crucial role in regulating Lagerstroemia plant height and stress response.Tissue-specific expression profiling highlighted the significant impact of HD-Zip III and IV subfamilies on Lagerstroemia plant development.Moreover, six LinHDZs were identified as key candidates regulating the Lagerstroemia plant height, with LinHDZ24 and LinHDZ14 implicated in the positive regulation of branch elongation, LinHDZ9 and LinHDZ35 as negative regulators of shoot development, and LinHDZ23 and LinHDZ34 showing significant upregulation in S_SAM and playing roles in cell division.Overall, these findings not only enhance the understanding of the molecular mechanisms underlying HD-Zip family function in the growth and development of Lagerstroemia, but also provide insights for the molecular breeding of crape myrtle and other woody ornamental plants, as well as for further studies on these significant TFs.

15 Figure 1 .
Figure 1.Localization of HD-Zip genes on the chromosomes of Lagerstroemia indica.Each chromosome is represented by a green strip, with the approximate distribution of each LinHDZ gene marked on the strip in black font.

Figure 1 .
Figure 1.Localization of HD-Zip genes on the chromosomes of Lagerstroemia indica.Each chromosome is represented by a green strip, with the approximate distribution of each LinHDZ gene marked on the strip in black font.

Figure 2 .
Figure 2. Phylogenetic tree analysis of HD-Zip sequences of Lagerstroemia indica and other plants.All LinHDZ genes in Lagerstroemia indica were marked in red font.

Figure 2 .
Figure 2. Phylogenetic tree analysis of HD-Zip sequences of Lagerstroemia indica and other plants.All LinHDZ genes in Lagerstroemia indica were marked in red font.

Figure 3 .
Figure 3. Gene structure and conserved motif analysis of Lagerstroemia indica HD-Zip family.(a) Distribution of conserved motifs in LinHDZ proteins; (b) Exon-intron structure of LinHDZ genes.

Figure 3 .
Figure 3. Gene structure and conserved motif analysis of Lagerstroemia indica HD-Zip family.(a) Distribution of conserved motifs in LinHDZ proteins; (b) Exon-intron structure of LinHDZ genes.

Figure 4 .
Figure 4.The distribution of 22 cis-acting elements in LinHDZs of Lagerstroemia indica.Segments of 200 bp are used as a ruler.Different color boxes represent different types of cis-acting elements.

Figure 4 .
Figure 4.The distribution of 22 cis-acting elements in LinHDZs of Lagerstroemia indica.Segments of 200 bp are used as a ruler.Different color boxes represent different types of cis-acting elements.

Genes 2024 ,
15, x FOR PEER REVIEW 10 of 15subfamilies, with HD-Zip III and IV subfamilies suggested to play essential roles in regulating Lagerstroemia plant height.

Figure 5 .
Figure 5. Cluster analysis of LinHDZ expression profiles in Lagerstroemia indica.The heat map was generated based on the log2-transformed relative expression values of LinHDZs in the shoot apical meristem (S_SAM), tender stem (S_TS), and mature stem (S_MS) of non-dwarf crape myrtle, and the shoot apical meristem (D_SAM), tender stem (D_TS), and mature stem (D_MS) of dwarf crape myrtle.Expression levels are depicted using a color gradient ranging from blue (downregulated) to red (upregulated).

Figure 5 .
Figure 5. Cluster analysis of LinHDZ expression profiles in Lagerstroemia indica.The heat map was generated based on the log2-transformed relative expression values of LinHDZs in the shoot apical meristem (S_SAM), tender stem (S_TS), and mature stem (S_MS) of non-dwarf crape myrtle, and the shoot apical meristem (D_SAM), tender stem (D_TS), and mature stem (D_MS) of dwarf crape myrtle.Expression levels are depicted using a color gradient ranging from blue (downregulated) to red (upregulated).

Figure 6 .
Figure 6.Expression pattern analysis of HD-Zip genes in the shoot apical meristem (S_SAM), tender stem (S_TS), and mature stem (S_MS) of non-dwarf crape myrtle, and the shoot apical meristem (D_SAM), tender stem (D_TS), and mature stem (D_MS) of dwarf crape myrtle.The error bar shows the standard error between three biological replicates (n=3).EF-1α (Gen Bank ID: MG704141) was selected as the internal reference gene and the expression levels were calculated by the 2 −ΔΔCt method to normalize qRT-PCR.

Figure 6 .
Figure 6.Expression pattern analysis of HD-Zip genes in the shoot apical meristem (S_SAM), tender stem (S_TS), and mature stem (S_MS) of non-dwarf crape myrtle, and the shoot apical meristem (D_SAM), tender stem (D_TS), and mature stem (D_MS) of dwarf crape myrtle.The error bar shows the standard error between three biological replicates (n=3).EF-1α (Gen Bank ID: MG704141) was selected as the internal reference gene and the expression levels were calculated by the 2 −∆∆Ct method to normalize qRT-PCR.

Table 1 .
Characteristics of HD-Zip gene family members in Lagerstroemia indica.
Nuclear 2.5.Prediction of Gene Structure, Conserved Motif, and Cis-Acting Regulatory Elements