Genome-wide identification of CUC gene family and functional analysis of HcCUC1 in kenaf

CUP-SHAPED COTYLEDON (CUC) transcription factors have a central regulatory function in plant growth and development. However, their involvement in kenaf (Hibiscus cannabinus L.) remains largely unexplored. In this study, we conducted a comprehensive analysis to identify six HcCUC genes in the kenaf genome. Through bioinformatic analysis, we found that the kenaf HcCUC genes share similar motifs and highly conserved gene structures. Phylogenetic analysis categorized the six HcCUC genes into two groups, that shared similarities with CUC2 or CUC3 genes from other species. Collinearity analysis revealed the formation of 6 syntenic gene pairs among the HcCUC genes, and 8 homologous gene pairs with three AtCUC genes from Arabidopsis. To investigate tissue-specific expression, we analyzed transcriptome data, that showed differential expression of HcCUC genes, particularly in leaves during the seedling stage, buds during the maturation stage, and anthers at the dual-core period. Functional characterization of HcCUC1 was achieved through its overexpression in Arabidopsis, resulting in elongated cotyledons, absent of petioles and increased number of rosette leaf and lateral branches. qRT-PCR analysis revealed that HcCUC1 potentially influences leaf and lateral branch development by up-regulating the expression of auxin-related genes (AtYUC2, AtYUC4, AtPIN1, AtPIN3, AtPIN4) and leaf shape-related genes (AtKNAT2, AtKNAT6). Notably, overexpression of HcCUC1 down-regulated the expression of flowering-related genes (AtFT, AtAP1, AtLFY, AtFUL), causing delayed flowering. Overall, our findings emphasize the pivotal role of HcCUC1 in regulating leaf and lateral branch growth, development, and flowering time, provide valuable insights into the function and genetic regulation of HcCUC genes. Six HcCUC genes were identified in the kenaf genome, with HcCUC1 playing a role in regulating leaf and lateral branch growth and development, as well as flowering time.


Introduction
CUP-SHAPED COTYLEDON (CUC) transcription factors, belonging to the NAC family (NAM/ATAF/CUC), are crucial regulators of plant growth and development (Aslam et al. 2022;Taoka et al. 2004).Initially, AtCUC1 and AtCUC2 mutations were found in Arabidopsis and resulted in defects in cotyledon separation, sepal and stamen formation, and shoot apical meristem development (Aida et al. 1997).Further studies revealed the regulatory role of miR164 in modulating CUC transcription levels (Laufs et al. 2004;Mallory et al. 2004).Later studies revealed additional biological functions of CUC in plant growth and development.During bud development, CUC promotes regeneration of adventitious bud from callus tissue (Daimon et al. 2003;Kareem et al. 2015).CUC2 and CUC3 bind directly to the promoter of the ubiquitin-dependent peptidase DA1 (with UBIQUITIN-SPECIFIC PROTEASE15, UBP15, as substrate), and activate its expression.This regulatory module, consisting of CUC2/CUC3-DA1-UBP15, controls the initiation of lateral branches in Arabidopsis, thereby influencing plant architecture (Li et al. 2020).Moreover, CUC has been implicated in leaf morphology.The miRNA164-CUC2 regulatory module controls the depth of serration at the leaf margin in Arabidopsis.Overexpression of miR164-resistant CUC2 leads to enhanced leaf serrations, while CUC2 inactivation eliminates serrations in both miR164a mutants and wild-type plants (Nikovics et al. 2006).Additionally, as demonstrated by Hasson et al. (Hasson et al. 2011), CUC3 contributes to leaf serration formation but exhibits distinct function from CUC2.CUC2 is essential in the early stages of serration development, whereas CUC3 sustains its growth, albeit to a lesser extent than CUC2.In floral organ development, miR164c regulates the number of petals by modulating the transcript levels of CUC1 and CUC2 (Baker et al. 2005).The interaction efforts of HWS (HAWAIIAN SKIRT), CUC1, and CUC2 also affects floral organ number (Gonzalez-Carranza et al. 2017).Beyond Arabidopsis, the involvement of CUC in plant growth and development has been observed in various species.In Betula pendula, miR164 affects internode growth and leaf shape by targeting BpCUC2 during internode development (Liu et al. 2019).In strawberry, the FvemiR164-FveCUC2 regulatory module regulates leaf morphology and floral organ development (Zheng et al. 2019).In rice, OsCUC1 interacts with the leaf-rolling protein CURLED LEAF AND DWARF1 (CLD1) to control leaf morphology (Wang et al. 2021).These findings highlight the pivotal role of CUC in regulating plant growth and development.
Kenaf (Hibiscus cannabinus L.), an annual dicotyledonous plant in the Malvaceae family, is extensively cultivated in China, India, Bangladesh, and Thailand (Ramesh 2016).Kenaf possesses several advantages, including rapid growth, high biomass production, and wide adaptability (Chen et al. 2021).It is widely used in industries such as textile, papermaking, construction, automotive, and as a soil remediation agent for saline-alkali and heavy metal-contaminated soils (Danalatos and Archontoulis 2010;Ramesh 2016;Wei et al. 2019;Yue et al. 2022).The HcCUC genes of kenaf remain to be identified.Identification and analysis of the HcCUC genes of kenaf and elucidation of their gene function are of great significance for understanding the regulation of growth and development of kenaf.
In this study, bioinformatics analysis methods were used to identify six HcCUC genes in kenaf.Transgenic plants overexpressing HcCUC1 in Arabidopsis were generated to analyze the phenotypes and expression levels of the related metabolic regulatory genes and to investigate their gene functions.The results demonstrated differential expression of HcCUC genes.Overexpression of HcCUC1 resulted in abnormal phenotypes in transgenic Arabidopsis, including elongated cotyledons, and absent petioles, significantly increased rosette leaves and lateral branches, and delayed flowering time.Furthermore, the underlying mechanism was explored by examining the expression of related genes using RT-qPCR analysis.

Plant materials and growth conditions
Kenaf cultivar FuHong952 was used in this study.Sterilized seeds were placed in plastic seedling trays covered with filter paper.After approximately 4 days of growth in the culture room, evenly grown seedlings were selected and transferred to seedling basins containing 0.5×Hoagland solution for hydroponic cultivation.The temperature in the culture room was maintained at 25 °C, with a light/dark cycle of 16 h of light and 8 h of dark.Leaves of approximately 3-week-old kenaf seedlings were used for RNA extraction.
Arabidopsis thaliana WT (Col-0) was used in this study.After disinfection, seeds were sown in 1/2 MS medium containing 0.7% (w/v) agar and 3% (w/v) sucrose.They were then vernalized for two days at 4 °C in the dark and then transferred to a growth chamber.After about 10 days, the seedlings were transferred to soil for further growth.Arabidopsis plants were grown in soil under controlled environmental conditions at 22 °C with a photoperiod of 16 h of light and 8 h of darkness to observe natural senescence.

Identification ofHcCUCgenes in kenaf
The kenaf genome and annotation files were downloaded from the National Genomics Data Center under accession number GWHACDB00000000.1 (Zhang et al. 2020).The protein sequences of the CUC genes of Arabidopsis and other species (Carica papaya, Daucus carota, Arabidopsis lyrata subsp.Lyrate, Brassica rapa, Aquilegia coerulea, Cardamine hirsute, Raphanus sativus and Oryza sativa Japonica Group) were obtained from the National Center for Biotechnology Information (NCBI) public database.The protein accession numbers of the other species are listed in Supplementary Table 1.
Using TBtools (Chen et al. 2020), the AtCUC protein sequence of Arabidopsis was used as a probe to search for homologous protein sequences in the kenaf genome.After removing redundant sequences, sequences with an E-value < e − 5 were selected as candidate sequences.The Pfam database (http://pfam.xfam.org/)and the NCBI CDD database (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi)were consulted to determine whether the candidate sequences possessed complete conserved domains.The ExPASy proteomics server database (https://www.expasy.org/) was employed to analyze related physical and chemical properties, such as molecular weight (MW), isoelectric point (pI), grand average of hydropathicity (GRAVY), and instability index.Subcellular localization of HcCUC were predicted by CELLO v.2.5 (Yu et al. 2004) (http://cello.life.nctu.edu.tw/).

Conserved motif and gene structure analysis
The online tool MEME (Bailey et al. 2015) was used to analyze the conserved motifs of the kenaf HcCUC family proteins.The parameter for motif identification was set to 10, while other parameters were kept as default.TBtools was utilized to visualize the results of conserved motif prediction and gene structure.

Construction of phylogenetic tree and collinearity analysis
The phylogenetic tree of the kenaf HcCUC gene family was constructed using the maximum likelihood method with MEGA-X software (Kumar et al. 2018).The phylogenetic tree, depicting the relationship between kenaf HcCUC proteins and those of other species was constructed using the Neighbor-joining method.The bootstrap repeat number was set to 1000.Tbtools software was used to build the whole-genome protein database for kenaf and Arabidopsis.Subsequently, BLASTp comparison was performed, and collinearity analysis of homologous genes was conducted using MCScanX software (Wang et al. 2012).The resulting collinearity map was generated to illustrate the distribution of homologous genes on chromosomes.

Tissue-specific expression analysis
The tissue expression pattern of HcCUC genes was analyzed using raw RNA sequencing data obtained from the NCBI SRA database (https://www.ncbi.nlm.nih.gov/sra/) and presented in Supplementary Table 2. First, the RNAseq data were standardized to TPM (transcripts per million reads).And then, log 2 (TPM + 1) conversion was applied to visualize the data in the form of heat maps.Furthermore, RT-qPCR was performed to verify some of the results.

Gene cloning and transgenic plant generation
The coding sequence of HcCUC1 (Hc.01G004330.t1) was cloned from kenaf cDNA using primers HcCUC1-F1/HcCUC1-R1 (Supplementary Table 3).The amplified fragment was then ligated to the BamH I and Sac I sites of the pBI121-GUS vector through homologous recombination.The specific steps for this procedure can be found in the instructions provided with the ClonExpress II® One Step Cloning Kit (Vazyme, China).The constructed expression vector was confirmed by sequencing.The constructed pBI121-HcCUC1 expression vector was transformed into Arabidopsis using Agrobacterium tumefaciens GV3101 via the inflorescence dipping method (Clough and Bent 1998).Subsequent experiment was performed with pure and thirdgeneration (T 3 ) Arabidopsis plant.

Expression analysis by RT-qPCR
Total RNA was extracted using Total RNA Extraction Reagent (Vazyme, China) According to the manufacturer's instructions according to the manufacturer's instructions, cDNA was synthesized by reverse transcription using HiS-cript® II 1st Strand cDNA Synthesis Kit (+ gDNA wiper) (Vazyme, China).Quantitative PCR was performed using the F ChamQ Universal SYBR qPCR Master Mix premix (Vazyme, China).AtTIP41-like (AT4G34270.1) was employed as the internal reference gene in Arabidopsis, whereas HcActin (Hc.17G007860.t1) was used in kenaf.These reference genes were used to detect the expression of the genes of interest using the primers listed in Supplementary Table 3.The relative transcript levels of the respective genes in Arabidopsis were calculated using the 2^− ΔΔCT formula, while the transcript levels of the six HcCUC genes in various tissues of kenaf were calculated using the 2^-ΔCT formula.All treatments were performed using three independent biological replicates.Statistical analysis was conducted using IBM SPSS 25.0.

Identification and basic physicochemical properties analysis of kenafHcCUCgenes
We identified six HcCUC genes in kenaf by Blastp alignment, CDD, and Pfam database analysis.To facilitate distinction, we renamed them based on their chromosome positions (Table 1).The predicted HcCUC proteins ranged

Conserved motif and gene structure analysis
We constructed a phylogenetic tree based on the predicted full-length protein sequences of HcCUC.The HcCUC proteins were divided into two groups (I and II) (Fig. 1a), and within each group, HcCUC had very similar conserved motifs and gene structures (Fig. 1b, c).Motifs 1-4 were present in all HcCUC proteins, with motifs 1-3 corresponding to the conserved NAM domain of HcCUC proteins.Motifs 5-9 were unique to group I HcCUC proteins, while motif 10 was specific to group II HcCUC proteins (Fig. 1b).Most HcCUC genes had a similar number and arrangement of coding regions, with no 3' untranslated regions.HcCUC1/3/4/5/6 had three coding regions and no 5' in length from 327 to 386 amino acid residues and in molecular masse from 36,300.8Da to 42,780.41Da.The values of the isoelectric point (pI) ranged from 5.81 to 8.48, with three members having pI value of 7. The aliphatic index ranged from 58.5 to 69.51.The proteins exhibited negative hydrophilicity, with the average total hydrophilicity (GRAVY) ranging from − 0.388 to -0.554.The instability coefficient ranged from 24.96 to 52.75.HcCUC2 and HcCUC5 were identified as stable proteins with an instability coefficient below 40, while the remaining members were classified as unstable proteins with an instability coefficient above 40.The subcellular localization of the six HcCUC genes was predicted using CELLO v.2.5, and the results are shown in Table 1.All six HcCUC genes were localized in the nucleus.  of the HcCUC gene family.Additionally, collinearity analysis identified eight homologous gene pairs (HcCUC1/ AtCUC1, HcCUC1/AtCUC2, HcCUC2/AtCUC3, HcCUC3/ AtCUC1, HcCUC4/AtCUC1, HcCUC5/AtCUC3, HcCUC6/ AtCUC1, HcCUC6/AtCUC2) formed between six HcCUCs and three AtCUCs (Fig. 2b).These homologous gene pairs likely evolved from a common ancestor and have a close evolutionary relationship.

Tissue-specific expression analysis of theHcCUCgene
We analyzed the expression patterns of the HcCUC genes using RNA-seq data obtained from various organs of kenaf.
The results, depicted in Fig. 3a, reveal distinct expression patterns among the different gene members.For instance, the expression of HcCUC6 is difficult to detect in different tissues and stages.Conversely, HcCUC4 and HcCUC5 showed different levels of expression in most tissues.In paticular, HcCUC genes were predominantly expressed in leaves during the seedling stage, in buds during the mature stage, and in anthers during the dual-core period.All members exhibited detectable expression in anthers during the dual-core period.
Additionally, with the exception of HcCUC6, all members displayed expression in leaves during the seedling stage and in buds at the 2 cm stage of maturity.These findings suggest that HcCUC genes may play pivotal roles in the development of these specific tissues.Finally, we validated some of the results through quantitative real-time PCR (qPCR) and untranslated region.In contrast, HcCUC2 differed in structure from the other HcCUC genes, with four coding regions and one 5' untranslated region.The structural differentiation of HcCUC2 may contribute to its functional divergence.

Phylogenetic and collinearity analysis
To gain further insight into the phylogeny of HcCUCs, we constructed a phylogenetic tree using 22 CUC protein sequences from other species, along with the six HcCUC protein sequences (Fig. 2a).The six HcCUC proteins were categorized into two distinct groups, group I and group V. Within group I, HcCUC1, HcCUC3, HcCUC4, and HcCUC6 showed close relationships with CpCUC2.In group V, HcCUC2 and HcCUC5 were most closely related to CpCUC3 and DcCUC3.Among the three AtCUC proteins found in the model plant Arabidopsis, AtCUC2 and AtCUC3 were placed in group I and group V, respectively, along with HcCUC proteins.The relationship between the two AtCUC proteins and the HcCUC proteins is closer, suggesting that CUC proteins within the same cluster may have similar biological functions.Gene duplication events are crucial for gene family evolution and often lead to gene amplification and the emergence of new functional genes.Collinearity analysis revealed that six fragment repeat gene pairs (HcCUC1/3, HcCUC1/4, HcCUC1/6, HcCUC2/5, HcCUC3/4, HcCUC4/6) formed among the six HcCUC genes (Fig. 2b).This finding highlights the importance of fragment repeats in the amplification of rosette leaves, on average about 20, compared to about 8 rosette leaves in wild-type (Fig. 4b, d).During bolting, the wild-type plants grew preferentially on the main stem, while the transgenic plants showed simultaneous growth on the main stem and multiple lateral branches, with a significantly higher number of lateral branches compared to the wild-type (Figs.4e and 5b-c).These results indicate that heterologous expression of HcCUC1 affects leaf and lateral branch growth and development in Arabidopsis, resulting in altered cotyledon morphology and increased number of rosette leaf and lateral branch.

Overexpression ofHcCUC1delayed the flowering time ofArabidopsis
In addition to the observed variations in leaves and lateral branches, our investigation unveiled significant differences in flowering time between wild-type and transgenic Arabidopsis plants.In the HcCUC1 overexpressing lines, both bolting and flowering were delayed by approximately four days compared to the wild type (Fig. 5a, b, d, e).Notably, the transgenic lines had lower plant height during the early growth stage due to delayed bolting, but no significant difference in overall plant height was observed between the transgenic lines and the wild-type plants throughout the growth period (Fig. 5c, f).These findings suggest that found that the validation results were overall consistent with the transcriptomic data.(Fig. 3b)

Overexpression ofHcCUC1regulated the growth and development ofArabidopsisleaves and lateral branches
To further investigate the biological functions of HcCUC genes, we cloned HcCUC1, which exhibited high expression in leaves at the seedling stage, buds at the mature stage, and anthers at dual-core period of kenaf, and showed a collinearity relationship with AtCUC2 based on phylogenetic analysis.
The study revealed significant differences between the wild-type and the overexpression lines in terms of cotyledon morphology and rosette leaf number.Compared to the wild-type, the overexpression lines had narrower and elongated cotyledon blades that lacked petioles (Fig. 4a).As for the rosette leaves, the overexpression lines showed clustered growth with smaller leaf size and a higher number regulate leaf and lateral branch growth and development by upregulating the transcript levels of auxin and leaf morphology-related genes.However, when the transcript level of flower-related genes was examined, it was observed that transgenic Arabidopsis exhibited a significant downregulation of the transcription level of flowering control genes AtFT, AtAP1, AtLFY, and AtFUL compared with the wild type plants (Fig. 6c).This indicates that the heterologous expression of HcCUC1 can delay flowering in Arabidopsis by downregulating the transcript levels of flowering-related genes.

Discussion
The plant-specific transcription factor CUC is integral to various aspects of plant growth and development, including the establishment of organ boundaries, shoot apical meristem formation, and development of leaves, lateral branches, and floral organs (Aida et al. 1997;Aslam et al. 2022;Gonzalez-Carranza et al. 2017;Hasson et al. 2011).Although this transcription factor has been identified in heterologous expression of HcCUC1 effectively delays the flowering time in Arabidopsis without affecting the final plant height.

HcCUC1affects the expression of genes related to auxin, leaf development and flowering regulation
Some studies have shown that auxin plays a crucial role in leaf and lateral branch development (Xiong and Jiao 2019), while the flowering of plants is regulated by numerous genes (Fornara et al. 2010).By examining the transcript levels of selected genes in wild-type and transgenic Arabidopsis, it was found that transgenic Arabidopsis exhibited a general trend toward up regulation of transcript levels of the auxin biosynthesis-related genes AtYUC2 and AtYUC4 and auxin transport-related genes AtPIN1, AtPIN3, and AtPIN4 compared to the wild-type plants (Fig. 6a).Additionally, transcript levels of two leaf morphology-related genes, AtKNAT2 and AtKNAT6, were significantly higher in the transgenic plants compared to the wild-type (Fig. 6b).These results suggest that overexpression of HcCUC1 may speculation is also supported by our collinearity analysis (Fig. 2b), in which HcCUC2/5 and AtCUC3 form homologous gene pairs, whereas HcCUC1/6 and AtCUC1/2, and HcCUC3/4 and AtCUC1, also form homologous gene pairs.Such highly homologous genes are closely related in evolution and likely exhibit greater functional conservation.
Tissue expression analysis revealed that HcCUC genes were expressed mainly in leaves during the seedling stage, in buds during the mature stage, and in anthers during the dual-core period, with particular high expression in buds during the mature stage.This finding aligns with the highest expression level of BpCUC2 in buds (Liu et al. 2018) and the significant expression of LcCUC2L in leaf buds (Wen et al. 2022).Functional redundancy often exists among members of gene families.In Arabidopsis, single mutations in cuc1 and cuc2 result in defects in cotyledon, sepal, and stamen abscission and in the formation of shoot apical meristem.However, these defects are more pronounced in double mutants (Aida et al. 1997).The three AtCUC genes contribute significantly to the formation of shoot apical meristem and cotyledon boundary, and the functional redundancy between AtCUC1 and AtCUC2 appears to be greater than that between AtCUC3 and AtCUC1 or AtCUC3 and AtCUC2.Moreover, AtCUC1 and AtCUC2 are numerous species, there have been no studies on kenaf.Therefore, the aim of our study was to identify and characterize six kenaf HcCUC genes (HcCUC1-HcCUC6) from the kenaf genome.Gene structure alterations play an important role in the evolution of gene families (Guo et al. 2013).In kenaf, five of the six HcCUC genes exhibit remarkable structural similarity, indicating a high degree of conservation in their structural evolution.However, HcCUC2 showed some differences compared to the other five genes, suggesting that its structural divergence may also involve functional differences.(Fig. 1c).The arrangement of motifs can provide information about the similarities and differences among proteins within a gene family (Su et al. 2020).By combining phylogenetic analysis with the identification of conserved motifs in HcCUC genes, we observed that HcCUC2/5 formed one evolutionary branch, while HcCUC1/3/4/6 clustered together, closely resembling the cluster formed by AtCUC2 from Arabidopsis (Figs. 1b  and 2c).Furthermore, HcCUC1/3/4/6 exhibited a large number of motifs compared to HcCUC2/5 (Figs. 1b and  2c), aligning with the findings of Aslam et al. (Aslam et al. 2022) and Hasson et al. (Hasson et al. 2011).These results suggest that HcCUC1/3/4/6 may have functions similar to AtCUC2 in Arabidopsis but distinct from HcCUC2/5.This In Arabidopsis, the DA1 mutation has been shown to reduce the formation of lateral branches, while its direct substrate UBP15 inhibits axillary meristem initiation.Additionally, AtCUC2 and AtCUC3 have been found to directly bind to the DA1 promoter and activate its expression, thereby influencing the number of lateral branches.(Li et al. 2020).In our study, overexpression of HcCUC1 significantly increased the number of rosette leaf lateral branches in transgenic Arabidopsis, without affecting the final plant height (Figs.4e and 5b, c and f).This finding suggests that the regulation of CUC in lateral branch development is comparable between kenaf and Arabidopsis.Moreover, after overexpression of LcCUC2L in Arabidopsis, Wen et al. (Wen et al. 2022) observed abnormal cotyledon phenotype and increased number of rosette leaves in Arabidopsis, which similar to our study.In contrast to the effect of the OE-gh-pre164 line in Arabidopsis, overexpression of GhCUC2m, following mutation of the miR164 target, resulted in significantly fewer branches and shortened branch length in transgenic Arabidopsis.These results illustrate both conserved and divergent regulatory mechanisms of CUC in leaf and lateral branch development across different species.Although no significant difference in final plant height was observed, the plant height of HcCUC1 transgenic Arabidopsis remained persistently shorter than that of wild-type plants during the involved in primordium development, while only AtCUC2 is involved in leaf margin development (Hibara et al. 2006).HcCUC2 and HcCUC5 are homologous to AtCUC3, with HcCUC5 exhibiting significantly higher expression than HcCUC2.HcCUC1, HcCUC3, HcCUC4, and HcCUC6 are more closely related to AtCUC2.Although HcCUC1 and HcCUC6 are homologous to AtCUC2, HcCUC6 was undetectable in all tissues.Alternatively, HcCUC1 displayed relatively high expression levels in leaves during the seedling stage, in buds during the mature period, and in anthers during the dual-core period.Hence, it is speculated that HcCUC1, which belong to the same group as AtCUC2, and HcCUC5, which belong to the same group as AtCUC3, may play central role in the growth and development of kenaf.However, further studies are required to test this hypothesis.
Based on our aforementioned analysis, we selected HcCUC1 for further functional investigation.Examination of Arabidopsis lines overexpressing HcCUC1 revealed notable effects on leaf development.Overexpression of HcCUC1 in transgenic Arabidopsis resulted in abnormal cotyledon development, petiole deletion, and the formation of slender cotyledons.In addition, a significant increase in the number of rosette leaves was observed in transgenic Arabidopsis (Fig. 4a, b, d).In contrast, Arabidopsis seedlings overexpressing AtCUC1 showed symmetrical sinuses on both sides of their cotyledons, rather than slender cotyledons (Hibara et al. 2003), while overexpression of AtCUC2 resulted in leaf wrinkles (Laufs et al. 2004).Furthermore, Hasson et al. (Hasson et al. 2011) found that overexpression Fig. 6 HcCUC1 affects the expression of genes related to auxin, leaf shape development and flowering regulation.a: Transcription levels of auxin-related genes in wild-type and transgenic Arabidopsis; b: Transcription level of leaf development-related genes in wild-type and transgenic Arabidopsis; c: Transcription levels of genes involved in flowering regulation in wild-type and transgenic Arabidopsis.Data showed mean ± SD (n = 3), different letters represent significant differences (one-way ANOVA, Duncan's test, P < 0.05) motifs and exhibit tissue-specific expression.Overexpression of HcCUC1 in transgenic Arabidopsis resulted in elongated cotyledons, absence of petioles, a significant increase in the number of rosette leaves and lateral branches, and a delayed flowering time.HcCUC1 may regulate leaf and lateral branch development by up-regulating the expression of auxin-related genes (AtYUC2, AtYUC4, AtPIN1, AtPIN3, AtPIN4) and leaf shape-related genes (AtKNAT2, AtKNAT6).Additionally, HcCUC1 may control bolting and flowering time by down-regulating the expression of flowering-related genes (AtFT, AtAP1, AtLFY, AtFUL).
early growth stage (Fig. 5b, c, f).A similar growth retardation was observed in transgenic Arabidopsis seedlings overexpressing CUC1 in the study conducted by Hibara et al. (Hibara et al. 2003).Therefore, we speculate that the overexpression of HcCUC1 causes delayed growth in transgenic Arabidopsis, resulting in an initial height advantage over wild-type plants.This delay in growth could potentially contribute to the delayed bolting and flowering time observed in transgenic Arabidopsis (Fig. 5a, b, d, e).
Previous studies have demonstrated that PIN-FORMED1 (PIN1), an auxin efflux carrier, regulates the expression of AtCUC2 (Aida et al. 2002).Notably, AtCUC2 was found to be highly expressed on an auxin-rich callus induction medium (Gordon et al. 2007).This highlights the regulatory network encompassing auxin, AtPIN1, and AtCUC2, all of which play a role in leaf development (Bilsborough et al. 2011).Additionally, auxin has also been link to branch formation (Brewer et al. 2015;Muller and Leyser 2011).We observed up-regulation of auxin synthesis-related genes (AtYUC2, AtYUC4), auxin transport-related genes (AtPIN1, AtPIN3, AtPIN4), and leaf development-related genes (AtKNAT2, AtKNAT6) in the transgenic Arabidopsis by examining transcription levels of auxin-related and leaf development-related genes in wild-type and transgenic Arabidopsis (Fig. 6a, b).These findings are consistent with the results reported by Wen et al. (Wen et al. 2022).Thus, it is plausible that HcCUC1 regulates leaf and lateral branch development by up-regulating the transcription levels of auxin-related and leaf shape-related genes.Numerous factors regulate flowering in plants, and multiple genes are involved in the regulation of floral induction.For instance, FLOWERING LOCUS T (FT) integrates signals from diverse pathways and transmits floral induction signals to downstream floral meristem recognition genes such as LEAFY (LFY), APETALA1 (AP1), and FRUITFULL (FUL) (Amasino 2010;Fornara et al. 2010).Notably, the transcription levels of flowering-related genes (AtFT, AtAP1, AtLFY, AtFUL) were significantly lower in transgenic Arabidopsis compared to wild-type Arabidopsis (Fig. 6c).This suggests that HcCUC1 may delay flowering in Arabidopsis by down-regulating the transcription levels of floweringrelated genes.However, the specific regulatory mechanism of HcCUC1 in Arabidopsis remains unclear, and it is worth noting that the regulatory mechanism in kenaf may differ from that of Arabidopsis.

Conclusions
The six HcCUC genes in kenaf, which homologous to CUC2 or CUC3 in other species can be classified into two categories.They are relatively conserved in terms of structure and

Fig. 1
Fig. 1 Phylogenetic tree, conserved motif and gene structure of HcCUC.a: Phylogenetic tree of HcCUC; b: Conserved motifs of HcCUC; c: Gene structure of HcCUC

Fig. 2
Fig. 2 Phylogenetic and collinear analysis.a: Neighbor-joining phylogenetic tree analysis of CUC protein sequences between kenaf and other species; b: Collinearity of HcCUCs and collinearity of HcCUCs-

Fig. 3
Fig. 3 Expression of HcCUC genes in different tissues.a: Heatmap of HcCUC genes expression in different tissues based on RNA-seq.Color scale represents transcripts per million reads (TPM) normalized log 2 (TPM + 1).b: RT-qPCR was used to quantify the expression levels of 6 HcCUC in some tissues of kenaf.Data are presented as mean ± SD (n = 3), different letters represent significant differences (one-way ANOVA, Duncan's test, P < 0.05).SS: Seedling stage; EGP: Exuberant growth period; MP: Mature period; DCP: Dual-core period

Fig. 4
Fig. 4 Overexpression of HcCUC1 regulates the growth and development of Arabidopsis leaves and lateral branches.a: Arabidopsis at 5 days old; b: Arabidopsis at 21 days old; c: The expression level of HcCUC1 in wild-type and transgenic plants, data are presented as mean ± SD (n = 3), the asterisks indicate significant differences (Student's t test, P < 0.01); d: Number of rosette leaves of wild type and transgenic plants at 21 days of age; e: Number of lateral rosette branches of wild type and transgenic plants at 31 days of age.Data in d and e showed mean ± SD (n = 10), different letters represent significant differences (one-way ANOVA, Duncan's test, P < 0.05)

Fig. 5
Fig. 5 Overexpression of HcCUC1 delayed the flowering time of Arabidopsis.a: Arabidopsis at 23 days old; b: Arabidopsis at 31 days old; c: Arabidopsis at 56 days old; d: Bolting time of wild type and transgenic plants; e: Flowering time of wild type and transgenic plants; f: Height of wild type and transgenic plants at 56 days of age.Data in d-f showed mean ± SD (n = 10), different letters represent significant differences (one-way ANOVA, Duncan's test, P < 0.05)

Table 1
Basic information of CUC genes in kenaf Gene name