Ectopic expression of the Sesuvium portulacastrum genes SpHDZ39 and SpHDZ41 enhances cadmium tolerance in Arabidopsis

Ectopic expression of the Sesuvium portulacastrum genes SpHDZ39 and SpHDZ41 enhances cadmium tolerance in Arabidopsis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Ectopic expression of the Sesuvium portulacastrum genes SpHDZ39 and SpHDZ41 enhances cadmium tolerance in Arabidopsis Tongjing Cui, Yuxin Li, Xiaoyou Wu, Fengyan Fang, Yaoxiu Li, Yingyi Yu, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8285582/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 12 Feb, 2026 Read the published version in Functional & Integrative Genomics → Version 1 posted 13 You are reading this latest preprint version Abstract The homeodomain-leucine zippers ( HDZs ) function as key regulators to regulating plant growth, development, and environmental responses. Its functions have been extensively investigated in many plant species, while the HDZs family in Sesuvium portulacastrum has not been thoroughly explored. We carried out a genome wide analysis of S. portulacastrum to identify and characterize the SpHDZs . The functional of SpHDZ39 and SpHDZ41 were investigated through subcellular localization, transient expression, and yeast one hybrid assays. Their roles in the response to cadmium stress were further analyzed using quantitative real time polymerase chain reaction and transgenic approaches. We identified 46 SpHDZs in the S. portulacastrum genome and classified into four subgroups. The SpHDZs were unevenly distributed across 21 chromosomes. Their physicochemical features were examined, including isoelectric points, hydrophobicity, stability, and predicted secondary and tertiary structures. Gene duplication analysis revealed that all identified duplications resulted from whole-genome or segmental events, and most gene pairs exhibited signs of purifying selection. Promoter analysis indicated the presence of numerous cis -elements associated with stress signaling and development. Expression profiling of heavy metal and salt treatment identified a subset of SpHDZs that respond to these treatments, of which, SpHDZ39 and SpHDZ41 showed significant induction under cadmium treatment. Both SpHDZ39 and SpHDZ41 functioned as transcription factors, and transgenic experiments demonstrated that they enhanced cadmium tolerance by promoting root growth and increasing biomass. These results contribute to a deeper understanding of SpHDZs function in halophytic species and provide candidate genes for future applications in low cadmium crop breeding. Sesuvium portulacastrum HDZ gene family Cd tolerance SpHDZ39 SpHDZ41 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Sesuvium portulacastrum , a salt-tolerant herb that grows as either annual or perennial is widely found in coastal zones across tropics and subtropics, and exhibits remarkable resistance to drought stress and salinity (Sukhorukov et al., 2021 ). S. portulacastrum is well-adapted to saline environments, characterized by succulent, prostrate stems, adventitious roots arising from nodes enabling clonal propagation, and habitats such as sandy shores, limestone coastlines, salt flats, and marshes (Zhang et al., 2024 ). Its leaves are opposite and linear-lanceolate, equipped with epidermal wax coatings and water-storage tissues that collectively maintain cellular water balance under salt stress (Sukhorukov et al., 2018 ). S. portulacastrum can efficiently utilize both sodium and potassium ions. Seedling growth remains largely stable even when exposed to high concentration of sodium chloride (NaCl), with only slight curling observed in young leaves. The plant rapidly accumulates osmoprotective substances in salt stress, which helps sustain photosynthetic performance (Muchate et al., 2016 ; Kulkarni et al., 2024 ). NaCl also alleviates cadmium (Cd) toxicity by change water content, redox balance, while promoting the synthesis of chelating agents like glutathione and proline (Mariem et al., 2014 ; Wali et al., 2016 ; Peng et al., 2019 ). This species shows strong environmental adaptability, including tolerance to heavy metal stress (Ghnaya et al., 2005 ; Lokhande et al., 2020 ). After copper (Cu) is absorbed by the roots, the plant regulates metal ion homeostasis through transport proteins belonging to families such as ZIP, which control Cu uptake and internal distribution (Colangelo & Guerinot, 2006 ; Alagarasan et al., 2017 ). Under different salt conditions, S. portulacastrum is capable of retaining high levels of heavy metals in its root tissues (Bonaventure et al., 2025 ). Plant reduces oxidative damage caused by Cu through enhanced antioxidant enzyme activity, stimulation of MAPK signaling and ascorbate-glutathione cycle. Genes relate to metal binding and transport also contribute to Cu detoxification by directing it into vacuoles, where it is safely compartmentalized (Xiang et al., 2025 ). In addition to its physiological resilience, this halophyte is valued for its nutritional and medicinal properties. It is considered an edible marine plant, rich in minerals, vitamin C, vitamin K, and antioxidant substances (Mathieu & Meissa, 2007 ; Lokhande et al., 2009 ; Boxman et al., 2018 ;). High levels of phenolics and other bioactive compounds are responsible for its traditional use in treating diseases and suppressing microbial growth (Magwa et al., 2006 ; Chandrasekaran et al., 2011 ). From an ecological standpoint, S. portulacastrum contributes to environmental restoration. It is used in floating bed systems to absorb excess nitrogen and phosphorus from nutrient-rich waters and can also serve as feed in aquaculture settings (Liu et al., 2019 ; Senff et al., 2020 ). Its strong capacity for sodium absorption supports soil desalination, making degraded lands more suitable for the cultivation of salt-sensitive crops (Rabhi et al., 2010 ). The Homeodomain-Leucine Zippers ( HDZs ) encode transcription factors central to plant growth, development, and environmental responses (Henriksson et al., 2005 ). These proteins are defined by a conserved homeodomain (HD), responsible for sequence-specific DNA binding, and a leucine zipper (LZ) domain that mediates dimerization and DNA recognition (Moens & Selleri, 2006 ). HDZs are classified into four subfamilies based on their structural motifs and functional diversity (Ariel et al., 2007 ). HDZ I and HDZ II subfamilies contain only HD and LZ domains, while HDZ III and HDZ IV include additional motifs such as the START domain, and in HDZ III, the MEKHLA domain, which are associated with hormonal and developmental signaling pathways (Ariel et al., 2007 ; Zhu et al., 2018 ). HDZ I plays a key role in mediating abiotic stress responses (Gu et al., 2019 ; Li et al., 2022 ). HDZ II is primarily involved in regulating light signaling and morphogenesis (Qi et al., 2013 ), while HDZ III regulates apical meristem development, vascular patterning, and organ polarity (Turchi et al., 2015 ). The HDZ IV subgroup is implicated in epidermal differentiation, root hair formation, and cuticle development (Chew et al., 2013 ; Schrick et al., 2023 ). In Arabidopsis , AtHB7 and AtHB12 function antagonistically for aluminum stress (Liu et al., 2020 ). Specifically, AtHB7 promotes stomatal closure to reduce water loss, while AtHB12 facilitates stomatal opening and is beneficial for seed production (Olsson et al., 2004 ; Ré et al., 2014 ). Moreover, AtHB21 , AtHB40 , and AtHB53 show upregulated expression under abotic stress conditions (Skinner & Gasser, 2009 ). The transcripts of OsHOX22 and OsHOX24 are significantly upregulated in both rice cultivars, whereas OsHOX6 shows only a slight increase in the drought-sensitive rice under prolonged drought conditions (Agalou et al., 2008 ). OsHOX22 fine-tunes drought and salinity adaptation by regulating the ABA-signaling cascade. (Zhang et al., 2012 ). The expression of OsHOX19 is enhanced by drought stress application in both drought-sensitive and drought-resistant cultivars (Harris et al., 2011 ). Overexpression of OsTF1L has been shown to promote lignin biosynthesis and stomatal closure, improving drought tolerance (Bang et al., 2019 ). In this work, we identified that 46 SpHDZs in the S. portulacastrum genome. These genes were classified into four subgroups, comprising 25 members in subgroup I, 6 members in subgroup II, 6 members in subgroup III, and 9 members in subgroup IV. The identified SpHDZs were unevenly distributed across 21 chromosomes, and both intra- and inter-species collinearity relationships were observed. Protein sequence analysis revealed the presence of six conserved motifs and structural domains, reflecting evolutionary conservation and gene duplication events. The exons number was ranged from 2 to 19, while introns varied between 1 and 5. The encoded proteins had lengths between 215 and 865 amino acids, with predicted molecular weights ranging from 24,651.80 to 94,974.01 Da. The theoretical isoelectric points (pI) were found to vary from 4.56 to 9.07. In addition, physicochemical analyses demonstrated wide variation in instability indices, aliphatic indices, and GRAVY values, which ranged from 43.50 to 75.89, 54.65 to 86.69, and − 1.174 to -0.062, respectively. Cis -element on the promoters associated with light responsiveness, hormone signaling, stress responses, and developmental processes. Transcriptome profiling under Cd, Cu, and NaCl treatment indicated that the expression of SpHDZs were differentially regulated in response to these abotic stress. The expression pattern of SpHDZ39 and SpHDZ41 were further validated by quantitative real-time PCR. In addition, subcellular localization and yeast one-hybrid assays confirmed that SpHDZ39 and SpHDZ41 function as transcription factors. Their overexpression in A. thaliana enhanced Cd tolerance. Overall, our results suggest that SpHDZs play important roles in Cd stress and may provide genetic resources for improving Cd tolerance in crops through molecular breeding strategies. Materials And Methods Identification of SpHDZs in S. portulacastrum genome The reference genome of S. portulacastrum was obtained from the laboratory at Guizhou University. AtHDZs sequences from Arabidopsis thaliana , available in the TAIR database, were used as queries to blast in the S. portulacastrum proteome using TBtools software. Candidate gene sequences were validated through the identification of conserved Pfam domains, specifically PF00046 and PF02183. Phylogenetic and sequence analysis Protein sequences of the identified SpHDZs, together with AtHDZs from A. thaliana and OsHDZs from O. sativa , were aligned and constructed a phylogenetic tree in MEGA11as described previously (Wu et al., 2025 ). The tree was edited and graphically refined using Adobe Illustrator. Protein subcellular localization was predicted using the WoLF PSORT online tool. Physicochemical properties such as molecular weight and isoelectric point were analyzed through the ProtParam server. Motif structures were identified using the MEME suite under default settings. Promoter analysis of cis -acting elements Promoter sequences extending 2,000 bps upstream of the start codon of each SpHDZ were extracted and analyzed for cis -acting elements using the PLANTCARE database. The results were visualized using the Simple BioSequence Viewer module in TBtools. To assess the distribution of regulatory elements, a statistical summary was generated using PivotCharts in Microsoft Excel, and a heatmap representing the abundance and classification of elements was produced using the HeatMap module in TBtools. Synteny analysis of the SpHDZs Syntenic relationships among SpHDZs within the S. portulacastrum genome and between S. portulacastrum and A. thaliana were investigated using TBtools. Data required for MCScanX-based analysis were processed using the File Merger module. The synteny maps were visualized with the Circle Gene Viewer function. Chromosomal localization of SpHDZs Chromosomal distribution of SpHDZs was analyzed using TBtools. The genomic annotation file and filtered gene list were imported, and genes were positioned based on their physical coordinates along the chromosomes. The resulting map was adjusted for clarity and visualization quality. Structural and evolutionary characterization The secondary structure of each SpHDZ protein was predicted using SOPMA, and the tertiary structure was modeled with SWISS-MODEL. Gene duplication patterns were determined based on synteny analysis, and types of duplication events were identified accordingly. Non-synonymous (Ka) and synonymous (Ks) substitution rates were calculated in TBtools to evaluate evolutionary pressure among gene pairs. Expression pattern of SpHDZ39 and SpHDZ41 To examine the expression of SpHDZ39 and SpHDZ41 under cd teatment, S. portulacastrum seedlings were treated with 200 µM Cd for 0, 7, 14, and 21 days. Total RNA was extracted from root tissues using a commercial RNA extraction kit. First-strand cDNA was synthesized using a reverse transcription kit. The SpGAPDH was used as an internal control. Quantitative real-time PCR was performed with three biological and three technical replicates per treatment group with gene special primers (Table S5 ). Relative gene expression levels were calculated using the 2⁻ΔΔCT method. Subcellular localization of SpHDZ39 and SpHDZ41 The coding sequences of SpHDZ39 and SpHDZ41 without stop codons were cloned into vectors, generating 35S::SpHDZ39-GFP and 35S::SpHDZ41-GFP constructs with gene special primers (Tab S5). These constructs were transformed into Agrobacterium tumefaciens and infiltrated into tobacco leaves. After dark incubation, fluorescence signals were observed at 72 hours post-inoculation using a Leica TCS SP8 confocal microscope (Leica, Wetzlar, Germany). The 35S::TksARF3-mCherry construct served as a nuclear marker for co-localization assessment (Wu et al., 2025 ). DNA-binding activity of SpHDZ39 and SpHDZ41 To assess DNA-binding activity, the coding regions of SpHDZ39 and SpHDZ41 were cloned into the pGADT7-rec2 vector with gene special primers (Table S5 ). Synthetic DNA sequences containing triple tandem repeat binding motifs, including MA0573, MA0573_mutant, MA0008, and MA0008_mutant, were inserted individually into the pHIS2 vector with gene special primers (Table S5 ). These constructs were co-transformed into Saccharomyces cerevisiae strain Y187 using a polyethylene glycol and lithium acetate protocol. Protein-DNA interaction was assessed by growing transformants on SD/-Leu/-Trp (DDO) and SD/-His/-Leu/-Trp (TDO) media. Plates supplemented with 50 mM 3-aminotriazole were used for further validation of DNA-binding specificity. Plant transformation The constructs 35S::SpHDZ39-GFP and 35S::SpHDZ41-GFP were introduced into Agrobacterium tumefaciens GV3101. Transformation of A. thaliana was performed using the floral dip method, and the resulting seeds were collected for subsequent selection and analysis. Results Genome-wide identification and classification of SpHDZs A comprehensive genome-wide analysis uncovered 46 HDZs in S. portulacastrum . Based on phylogenetic relationships with A. thaliana and O. sativa , these genes were categorized into HDZ I, HDZ II, HDZ III, and HDZ IV subgroups (Elhiti & Stasolla, 2009 ). Among them, 25 SpHDZs belonged to subgroup I, 6 to subgroup II, another 6 to subgroup III, and 9 to subgroup IV (Fig. 1 ). Based on chromosomal locations and gene annotations, the 46 SpHDZs were renamed as SpHDZ1 to SpHDZ46 , sequentially (Table S1 ). Among them, SpHDZ16 encodes the longest protein, consisting of 865 amino acids and exhibiting the highest molecular weight, whereas SpHDZ34 encodes the shortest protein, with 215 amino acids and the lowest molecular weight. The predicted isoelectric points (pI) of the SpHDZs ranged from 4.56 (SpHDZ14) to 9.07 (SpHDZ19). All SpHDZs displayed negative grand average of hydropathicity (GRAVY) values, ranging from − 1.174 (SpHDZ41) to -0.062 (SpHDZ4), suggesting that these proteins are generally hydrophilic. The aliphatic index values varied between 54.65 (SpHDZ39) and 86.69 (SpHDZ17), while all proteins had instability index values above 43.50(SpHDZ26) and below 75.89(SpHDZ7), indicating potential instability in vitro . Subcellular localization predictions indicated that 41 SpHDZs are primarily localized in the nucleus. Notable exceptions include SpHDZ17, SpHDZ35, and SpHDZ43, which are predicted to localized in the cytoplasm, as well as SpHDZ22 and SpHDZ33, which are likely localized in the chloroplast. Chromosomal distribution and synteny All 46 SpHDZs were successfully mapped across 21 chromosomes in S. portulacastrum (Fig. 2 ). The highest gene density was found on Chr7, Chr10, and Chr11, each containing four SpHDZs . Chr2, Chr3, and Chr5 each harbored three genes. Chr1, Chr8, Chr12, Chr13, Chr14, Chr18, Chr20, Chr21, Chr23, and Chr24 contained two SpHDZs , while Chr4, Chr15, Chr16, Chr17, and Chr19 were each associated with only one SpHDZ (Fig. 2 ). To explore the structural diversity and potential functional roles of SpHDZs, conserved domain analysis was conducted. Members within the same subgroup generally exhibited similar domain architectures, whereas marked variation was observed among different subfamilies. The HD and LZ domains were consistently present in most SpHDZ I proteins, aligning with findings in other species. An exception was SpHDZ34, which lacked the LZ domain despite being classified in the SpHDZ I group. All six HDZ II proteins retained the LZ domain; however, only SpHDZ1 possessed the HD domain, while the remaining members, SpHDZ13, SpHDZ19, SpHDZ25, SpHDZ30, and SpHDZ38, lacked it. Members of the HDZ III and IV subfamilies all contained both HD and START domains. Notably, HDZ III members also harbored a C-terminal MEKHLA domain, indicating a higher degree of structural complexity and possible functional divergence (Fig. 3 B). Motif analysis revealed that Motif1, Motif2, and Motif3 were highly conserved across all SpHDZs. Functional annotation showed that Motif1 and Motif2 together formed the canonical HD domain (Fig. 3 A). Gene structure analysis demonstrated that varying exon-intron organizations across subfamilies. Most SpHDZs contained 1 to 5 introns, but exon numbers ranged from 2 to 19. The HDZ III subgroup showed the most complex gene structure, with 18 to 19 exons. This was followed by HDZ IV, whose members had 8 to 11 exons. In contrast, HDZ I and II members generally exhibited simpler gene structures, typically containing only 2 to 4 exons (Fig. 3 C). Cis -acting regulatory element analysis of the SpHDZs Cis -acting regulatory elements are crucial for transcriptional regulation and directly impact gene function. To investigate potential regulatory mechanisms, the 2,000 base pairs upstream promoter regions of SpHDZs were analyzed for the presence of cis -acting regulatory elements (Fig. 4 A). Identified elements were grouped into light-responsive, hormone-responsive, biotic/abiotic stress-responsive, and plant growth and development-related elements (Fig. 4 B). Among these, hormone-responsive elements were the most prevalent, totaling 248, with jasmonate-responsive elements (JAREs) being the most dominant, present in over 59% of the genes. Light-responsive elements were the second most common, followed by stress-related elements, which included MYB-DRE (41.17%), ARE (35.88%), LTRE (11.76%), DSRE (7.06%), and ANX-Enh (4.12%). The plant development-related elements group was the least represented, with meristem-related motifs being the most enriched within this category. Collinearity analysis of the SpHDZs Collinearity analysis using MCScanX identified 39 collinear gene pairs within the SpHDZs , spanning all four subfamilies (Fig. 5 B). These duplication events, primarily resulting from whole-genome duplication (WGD) or segmental duplication (Table S2 ), likely contributed to genome expansion and evolutionary diversification. The polyploidization or local genomic rearrangements driven events may enhance genetic variability, promoting functional innovation and adaptive responses. To evaluate evolutionary pressures acting on these duplicated genes, nonsynonymous/synonymous substitution rate (Ka/Ks) ratios were calculated (Table S3 ). It showed that most gene pairs showed Ka/Ks values between 0.03 and 0.88 (Ka/Ks < 1), indicating that purifying selection has predominantly preserved protein function by eliminating deleterious mutations. Notably, only SpHDZ7 / SpHDZ8 pair exhibited a Ka/Ks ratio of 1.09, suggesting positive selection, which may have driven functional divergence through advantageous amino acid changes. Structural modeling of these collinear gene products revealed substantial tertiary structural conservation, reinforcing the presence of functional constraints. Furthermore, interspecies synteny analysis between S. portulacastrum and A. thaliana uncovered 57 orthologous gene pairs, while SpHDZ24 , SpHDZ30 , and SpHDZ45 were lacked identifiable syntenic counterparts (Fig. 5 A). This high level of conservation reflects shared evolutionary ancestry and underscores the structural and functional preservation of the SpHDZ family across plant species. Expression profiling of SpHDZs under different Abiotic stress conditions To assess the transcriptional responses of SpHDZs to salt stress, RNA-seq data from salt-treated S. portulacastrum leaves were analyzed under varying NaCl concentrations (Fig. 6 C). The results revealed diverse expression trends, with both upregulation and downregulation observed across different genes as salt levels increased. The SpHDZ1 , SpHDZ6 , SpHDZ7 , SpHDZ8 , SpHDZ10 , SpHDZ24 , SpHDZ26 , SpHDZ29 , SpHDZ30 , SpHDZ34 , SpHDZ40 , and SpHDZ41 were exhibited marked upregulation under salt concentrations. Notably, 10 of these genes belonged to the HDZ I subgroup, while SpHDZ1 and SpHDZ30 were classified under HDZ II. Conversely, SpHDZ5 , SpHDZ16 , SpHDZ20 , SpHDZ22 , SpHDZ23 , and SpHDZ32 were displayed consistent downregulation. Several other genes showed peak expression at intermediate salt concentrations. To explore the involvement of SpHDZs in heavy metal stress responses, transcriptomic data were analyzed from roots and leaves subjected to Cd and Cu treatment. The SpHDZ6 , SpHDZ13 , SpHDZ14 , SpHDZ29 , and SpHDZ38 were upregulated with prolonged Cd exposure compared with control conditions in roots (Fig. 6 A). However, most genes were also upregulated at 7 days of Cd exposure compared with control, while SpHDZ4 , SpHDZ5 , SpHDZ6 , SpHDZ14 , SpHDZ17 , and SpHDZ33 maintained elevated expression at either 7 days, 14 days, or 21 days, compared with control in leaves (Fig. 6 A). In contrast, SpHDZ10 , SpHDZ23 , SpHDZ25 , SpHDZ31 , SpHDZ32 , and SpHDZ45 showed downregulated with Cd treatment compared with control. Furthermore, the expression of SpHDZ39 and SpHDZ41 under Cd treatment was validated by qRT-PCR (Fig. 6 D). Data analysis revealed that the expression level of SpHDZ39 showed a gradual downregulation trend with the extension of Cd stress duration. In contrast, SpHDZ41 exhibited an upregulation at 7 days of treatment, followed by a downregulation relative to the control group as the stress duration prolonged. Similarly, SpHDZ5 , SpHDZ15 , SpHDZ16 , and SpHDZ17 showed upregulation at 50 µM Cu treatment compared with control, SpHDZ7 , SpHDZ8 , SpHDZ26 , SpHDZ29 , and SpHDZ41 showed upregulation at 100 µM Cu treatment, SpHDZ4 , SpHDZ6 , SpHDZ18 , SpHDZ24 , and SpHDZ46 were induced at 200 µM Cu treatment, compared with control in roots, respectively (Fig. 6 B). In leaves, exposure to 50 µM Cu elicited significant upregulation of SpHDZ12 , SpHDZ23 , SpHDZ32 , and SpHDZ34 compared with untreated controls. Treatment with 100 µM Cu induced the expression of SpHDZ10 , SpHDZ14 , and SpHDZ40 . Following 200 µM Cu exposure, SpHDZ6 , SpHDZ7, SpHDZ8, SpHDZ29 , and SpHDZ41 were markedly upregulated, whereas SpHDZ2 , SpHDZ11, SpHDZ15 , SpHDZ20 , SpHDZ22 , SpHDZ23 , SpHDZ39 , and SpHDZ45 exhibited significant downregulation relative to control levels (Fig. 6 B). Those results suggested that different SpHDZs may response to different abiotic stress. SpHDZ39 and SpHDZ41 function as transcription factors SpHDZ39 and SpHDZ41 were predicted to be localized in the nucleus based on WOLF PSORT analysis (Table S1 ), and this prediction was experimentally confirmed through transient expression assays in tobacco leaves (Fig. 7 A). Constructs expressing SpHDZ39-GFP or SpHDZ41-GFP were co-expressed with the nuclear marker TksARF3-mCherry in leaf epidermal cells. The observed overlap of fluorescence signals confirmed that both proteins were localized in the nucleus. To further examine their DNA-binding capacity, yeast one-hybrid assays were conducted. Yeast cells co-transformed with pHIS2-MA0573 or pHIS2-MA0008 and pGADT7-rec2-SpHDZ39 grew normally on TDO medium (Fig. 7 B). In contrast, yeast cells carrying the mutant versions of these sequences or the negative control failed to grow under the same conditions. Similar binding behavior was observed for SpHDZ41 (Fig. 7 C), indicating that both transcription factors specifically recognize and bind these DNA motifs. Taken together, those results showed that both SpHDZ39 and SpHDZ41 function as a transcription factor. Overexpression of SpHDZ39 and SpHDZ41 improved Cd tolerance in A. thaliana We generated A. thaliana plants transgene overexpressing SpHDZ39 ( SpHDZ39OE ) and SpHDZ41 ( SpHDZ41OE ) (Fig. 8 A). Segregating T 3 plants from two independent SpHDZ39OE and SpHDZ41OE plants were subjected to 75 µM Cd treatment. Both SpHDZ39OE and SpHDZ41OE plants exhibited stronger growth compared with wild-type plants (Fig. 8 B). The average root length of SpHDZ39OE plants ranged from 2.2 ± 0.2 cm to 2.4 ± 0.3 cm, while wild-type plants showed an average of 1.8 ± 0.2 cm (Fig. 8 C). SpHDZ41OE plants displayed longer roots ranging from 4.2 ± 0.3 cm to 4.9 ± 0.2 cm, compared with 4.0 ± 0.1 cm in wild type (Fig. 8 C). Fresh weight measurements also indicated enhanced biomass. SpHDZ39OE plants reached 1.4 ± 0.1 mg/plant to 1.6 ± 0.2 mg/plant, whereas wild-type plants averaged 1.2 ± 0.2 mg/plant (Fig. 8 D). SpHDZ41OE plants showed values between 1.7 ± 0.2 mg/plant to 1.9 ± 0.2 mg/plant compared with 1.5 ± 0.1 mg/plant in wild type (Fig. 8 D). Taken together, those results suggest that SpHDZ39 and SpHDZ41 enhance Cd tolerance in A. thaliana by promoting root growth and biomass accumulation. Discussion HDZ is an evolutionarily conserved transcription factor that have been identified in Arabidopsis, rice, maize, soybean, wheat, and tomato (Henriksson et al., 2005 ; Kim et al., 2008 ; Chen et al., 2014 ; Zhang et al., 2014 ; Yue et al., 2018 ). In this study, we present the first comprehensive identification and characterization of 46 SpHDZs in the S. portulacastrum genome through systematic bioinformatics analysis. The gene number in S. portulacastrum is equal to that in Arabidopsis , suggesting possible evolutionary and functional parallels between the two species. From an evolutionary perspective, the identical number of HDZs may reflect conserved gene retention, with minimal gene loss or expansion across lineages. This pattern supports the notion that the HDZs has undergone strong evolutionary constraints, preserving its core structure across species. Functionally, the conserved gene number implies that similar molecular mechanisms may operate in both species to regulate development and environmental adaptation. This finding lays the groundwork for future investigations into the molecular mechanisms underlying stress tolerance in S. portulacastrum . Previous studies have indicated that the abundance of HDZs are not directly related to genome size but is shaped by tandem and segmental duplication events (Zhao et al., 2011 ). Phylogenetic and domain structure analyses classified the 46 identified genes into four subfamilies, with HDZ I containing the largest number of members. This classification is consistent with results in other plants, supporting the evolutionary conservation of the HDZs . Gene structure and motif composition analyses revealed that members of the HDZ I subgroup contain highly conserved HD and LZ domains. In contrast, HDZ III and IV subfamilies include START domains, and HDZ III genes additionally contain MEKHLA domains at their C-terminus, consistent with patterns reported in other species. The structural features vary significantly among subfamilies, suggesting potential functional divergence. Subcellular localization prediction showed that most SpHDZs are localized in the nucleus, while a few are present in the cytoplasm or chloroplast. Transient expression assays confirmed that SpHDZ39 and SpHDZ41 are nuclear proteins, with DNA-binding activities supported by yeast one-hybrid assays. These results indicate that these genes likely function as transcriptional regulators. Collinearity analysis revealed 57 syntenic gene pairs between S. portulacastrum and Arabidopsis , including 38 pairs involving HDZs . This indicates limited gene family expansion and suggests a relatively stable evolutionary trajectory for the HDZ family in S. portulacastrum . Cis -element analysis of promoter regions showed that SpHDZs are associated with a wide range of regulatory functions, including hormone response, abiotic and biotic stress responses, developmental control, and light signaling (Zhou et al., 2025 ). Notably, drought-related elements were more frequently enriched in the HDZ III and IV subfamilies. This contrasts with patterns reported in other plants, where HDZ I genes typically play central roles in drought response (Li et al., 2022 ). For example, in Arabidopsis , AtHB7 and AtHB12, which belong to HDZ I, are involved in ABA-mediated regulation of stomatal movement and osmotic balance under drought conditions. This divergence suggests that, in S. portulacastrum , HDZ III and IV genes may have evolved to compensate for or replace some of the functions typically attributed to HDZ I genes. Given that S. portulacastrum naturally inhabits saline environments, this may represent a unique adaptation shaped by long-term exposure to salt stress. In Zea mays , overexpression of ZmHDZ9 enhances drought resistance in transgenic Arabidopsis (Qiu et al., 2022 ), further supporting the idea of functional plasticity within HDZ subfamilies. These findings raise the possibility that HDZ III and IV genes in S. portulacastrum may have been selectively co-opted for stress regulation. Further functional studies are needed to confirm this hypothesis. Transcriptome data under NaCl treatment revealed 13 SpHDZs responsive to salt stress. Among these, SpHDZ1 and SpHDZ30 belonged to the HDZ II subgroup, while the remaining 11 genes were classified under HDZ I. This pattern aligns with findings in soybean, where most salt-induced HDZs also belong to subgroup I and II (Chen et al., 2014 ). It has been reported that sodium ions stimulate V-ATPase activity, promoting the accumulation of soluble sugars in vacuoles and chloroplasts to maintain osmotic balance (Yi et al., 2014 ). These processes enhance salt tolerance in S. portulacastrum . While both sodium ions and chloride ions are toxic to most plants (Adams & Shin, 2014 ; Lutts & Lefèvre, 2015 ), in this halophyte, chloride ions appear to be the main contributor to toxicity, while sodium ions have been shown to promote shoot development and leaf succulence (Wang et al., 2012 ). Under NaCl, Cd, and Cu treatment, multiple SpHDZs exhibited altered expression. Notably, SpHDZ6 from subgroup I was upregulated in response to all three treatments, suggesting a central role in multistress tolerance, consistent with previous findings regarding HDZ I function in salt adaptation (Li et al., 2022 ). qRT-PCR results showed that SpHDZ39 expression declined progressively under Cd stress, while SpHDZ41 initially increased at day 7, followed by a decrease over time. A similar regulatory pattern has been observed for Oshox22 , which influences ABA biosynthesis and mediates stress responses in rice (Zhang et al., 2012 ). Conversely, overexpression of JcHDZ21 has been shown to increase salt sensitivity in Arabidopsis (Tang et al., 2023 ), indicating that HDZ function can vary significantly depending on genetic context. Additional genes such as SpHDZ4 , SpHDZ14 , SpHDZ24 , and SpHDZ29 also responded to multiple stress treatments, although their precise functions remain unclear. Further experimental validation is required to explore their roles in regulating plant responses to environmental challenges. These genes may offer valuable targets for enhancing crop resistance to Cd stress through genetic improvement strategies. Conclusions We identified 46 SpHDZs in S. portulacastrum , distributed unevenly across 21 chromosomes. Based on gene structures and conserved motifs, these SpHDZs were classified into four subgroups, each associated with distinct domain features. All duplication events identified were attributed to whole-genome or segmental duplication, and 39 duplicated gene pairs were detected. Among these, nearly all gene pairs had Ka/Ks ratios below one, indicating that purifying selection has maintained their sequence conservation. Promoter analysis revealed the presence of 703 cis -regulatory elements, which were categorized into four functional types related to light response, hormone signaling, environmental stress, and developmental regulation. These elements were distributed unevenly among the gene subgroups, suggesting potential functional divergence. Expression profiling identified twelve SpHDZs involved in salt stress responses and twelve others responsive to heavy metal treatment. Further verification using qRT-PCR confirmed that SpHDZ39 and SpHDZ41 showed altered expression patterns under Cd treatment. Subcellular localization predictions showed that most SpHDZs are targeted to the nucleus, with a few located in the chloroplast or cytoplasm. Experimental validation further confirmed that SpHDZ39 and SpHDZ41 act as nuclear transcription factors, as demonstrated by localization and yeast one-hybrid assays. Overexpression of these genes in A. thaliana enhanced Cd tolerance, supporting their role in stress regulation. These findings contribute to a deeper understanding of the molecular mechanisms underlying abiotic stress adaptation in S. portulacastrum . The identified genes offer promising targets for enhancing Cd tolerance in crops through genetic engineering and molecular breeding strategies. Declarations Acknowledgments The authors express their sincere gratitude to Dr. Xuchu Wang in Guizhou University for his critical reading and polishing of this manuscript. Author Contributions S. H. and T. C. initiated and designed the project, S. H., T. C., Y. L., X. W., F. F., Y. L., Y. Y., G. Z., Z. N., and Y. W. reviewing the referenced articles and preparing the Figures and tables, S. H. and T. C. writing and revising the manuscript. All authors approved the final version of the text. Funding This work was supported by the National Natural Science Foundation of Guizhou University (Guida Tegang Hezi [2025] No. 02), the Guizhou Provincial Basic Research Program (Natural Science) General Program (No. [2025] 660), the Innovation Center for Academicians Team of Hainan Province and the specific research fund of The Innovation Platform for Academicians of Hainan Province (No. YSPTZX202309). Data Availability Statement Data will be made available on request. Ethical approval and consent to participate Not applicable. Consent for publication All authors have read and approved the final version of the manuscript and consent to its submission for publica tion. The authors affirm that this work is original and has not been published previously, nor is it currently under consideration for publication elsewhere. Competing interests The authors declare no competing interests. References Adams E, Shin R (2014). 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The phylogenetic tree was constructed \u003cem\u003evia\u003c/em\u003e the Neighbor Joining method with 1000 bootstrap replicates by MEGA7. The number on the node indicates the supported percentage. Subclades are indicated by different colors.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8285582/v1/2c1e97c058ee60d8bfe8dc0d.png"},{"id":97986163,"identity":"6cf2963a-da85-4355-90d9-ffffc2d65cbf","added_by":"auto","created_at":"2025-12-11 13:48:42","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":847497,"visible":true,"origin":"","legend":"\u003cp\u003eChromosome distribution of \u003cem\u003eSpHDZ\u003c/em\u003e genes in \u003cem\u003eS. portulacastrum\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8285582/v1/25458de112dcb57ecab37214.png"},{"id":97986164,"identity":"334188e1-33da-42aa-8bd2-2247ba919f2e","added_by":"auto","created_at":"2025-12-11 13:48:42","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1394141,"visible":true,"origin":"","legend":"\u003cp\u003eGene and protein structure of 46 SpHDZs.\u003cstrong\u003e \u003c/strong\u003eA. Motifs in the detected 46 SpHDZs. B. Conserved structural domains in the 46 SpHDZs. C. Gene structure analysis of the 46 \u003cem\u003eSpHDZs\u003c/em\u003e. The horizontal coordinate represents the length of the gene/amino acid sequence.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8285582/v1/3cbe3dd955e93123ef88b89f.png"},{"id":97986168,"identity":"ff8c5368-3abf-40d0-b2a5-a9ce72cda525","added_by":"auto","created_at":"2025-12-11 13:48:42","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":3632622,"visible":true,"origin":"","legend":"\u003cp\u003ePredicted \u003cem\u003ecis\u003c/em\u003e-elements in the promoter regions of \u003cem\u003eSpHDZs\u003c/em\u003e. A. Category of \u003cem\u003ecis\u003c/em\u003e-elements in \u003cem\u003eSpHDZs\u003c/em\u003epromoter. B. Number of \u003cem\u003ecis\u003c/em\u003e-elements in \u003cem\u003eSpHDZs\u003c/em\u003e promoter.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8285582/v1/7bcd6bf3fcfdd260a8184ece.png"},{"id":98423329,"identity":"a6db0150-7c3f-490c-85e0-5fe5170ed473","added_by":"auto","created_at":"2025-12-17 16:32:06","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2211597,"visible":true,"origin":"","legend":"\u003cp\u003eCollinearity analysis of \u003cem\u003eHDZs\u003c/em\u003e in \u003cem\u003eS. portulacastrum\u003c/em\u003e and \u003cem\u003eA. thaliana\u003c/em\u003e.\u003cstrong\u003e \u003c/strong\u003eA. Collinearity analysis of \u003cem\u003eHDZs\u003c/em\u003ebetween \u003cem\u003eS. portulacastrum \u003c/em\u003eand \u003cem\u003eA. thaliana\u003c/em\u003e. B. Synteny analysis of \u003cem\u003eSpHDZs\u003c/em\u003e in \u003cem\u003eS. portulacastrum\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8285582/v1/a8cbb6402a449f312d05d3eb.png"},{"id":98423325,"identity":"690684c4-8920-422e-8844-51b03fbfc1e5","added_by":"auto","created_at":"2025-12-17 16:32:06","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":3478133,"visible":true,"origin":"","legend":"\u003cp\u003eExpression patterns of \u003cem\u003eSpHDZs\u003c/em\u003e in \u003cem\u003eS. portulacastrum\u003c/em\u003e under various\u003cem\u003e \u003c/em\u003eabiotic. A. Fold change of \u003cem\u003eSpHDZs\u003c/em\u003eunder Cd stress. B. Fold change of \u003cem\u003eSpHDZs\u003c/em\u003e under Cu stress. C. Fold change of \u003cem\u003eSpHDZs\u003c/em\u003e under salt stress. D. Relative expression patterns of \u003cem\u003eSpHDZ39\u003c/em\u003eand \u003cem\u003eSpHDZ41 \u003c/em\u003eunder Cd treatment analyzed by qRT-PCR. Asterisks indicate a significant difference between day 0 and day 7, 14, 21 Cd treatment as determined by 2-tailed Student’s \u003cem\u003et\u003c/em\u003e test at **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 or *\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8285582/v1/093da94e09d7c190c9d38a31.png"},{"id":98424165,"identity":"2cb5bc76-dfc3-436a-bc99-f28b961f88f9","added_by":"auto","created_at":"2025-12-17 16:33:01","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":2281600,"visible":true,"origin":"","legend":"\u003cp\u003eTranscriptional activation assay of SpHDZ39 and SpHDZ41.\u003cstrong\u003e \u003c/strong\u003eA. Subcellular localization of SpHDZ39 and SpHDZ41. \u003cem\u003eTaraxacum kok-saghyz \u003c/em\u003eARF3 protein fused to mCherry was used as a nucleus marker. Scale bars correspond to 20 μm. B. Schematic diagram of MA0573 and MA0008 of SpHDZ39 and SpHDZ41 binding site. C. Analysis of the transcriptional activity of SpHDZ39 and SpHDZ41.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-8285582/v1/dad00f1fb5c2d4ca53b9facd.png"},{"id":98424187,"identity":"4fb61f89-9773-46cf-95bf-6c3a06a7bfb6","added_by":"auto","created_at":"2025-12-17 16:33:03","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":615883,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOverexpression of the \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eSpHDZ39\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eand \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eSpHDZ41\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e improving Cd tolerance in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eA. thaliana\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. \u003c/strong\u003eA. Expression level of \u003cem\u003eSpHDZ39\u003c/em\u003eand \u003cem\u003eSpHDZ41 \u003c/em\u003ein transgene-plants.\u003cem\u003e \u003c/em\u003eB. Root morphology of \u003cem\u003eSpHDZ39OE\u003c/em\u003eand \u003cem\u003eSpHDZ41OE \u003c/em\u003eunder\u003cem\u003e \u003c/em\u003eCd treatment. C. Root length of \u003cem\u003eSpHDZ39OE\u003c/em\u003eand \u003cem\u003eSpHDZ41OE \u003c/em\u003eunder\u003cem\u003e \u003c/em\u003eCd treatment. D. Fresh weight of \u003cem\u003eSpHDZ39OE\u003c/em\u003eand \u003cem\u003eSpHDZ41OE \u003c/em\u003eunder\u003cem\u003e \u003c/em\u003eCd treatment. Asterisks indicate significant difference determined by one-tail Student’s \u003cem\u003et\u003c/em\u003e-test (**\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 or *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-8285582/v1/28ed7bfa85348f6ecde0d8ca.png"},{"id":102785399,"identity":"a6167220-9f14-4917-9000-6bdbdd607ce2","added_by":"auto","created_at":"2026-02-16 16:06:20","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":16008144,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8285582/v1/bea3896d-2269-4f45-95cf-b1d6870361f5.pdf"},{"id":98424257,"identity":"07d18ab8-3733-4252-9703-26446bc7b24e","added_by":"auto","created_at":"2025-12-17 16:33:07","extension":"pptx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":886989,"visible":true,"origin":"","legend":"","description":"","filename":"CuiSupplementFigures.pptx","url":"https://assets-eu.researchsquare.com/files/rs-8285582/v1/195417744665b74e55ff5bbf.pptx"},{"id":97986174,"identity":"59108fee-5a16-4dd3-99ac-9c25214d6665","added_by":"auto","created_at":"2025-12-11 13:48:43","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":233219,"visible":true,"origin":"","legend":"","description":"","filename":"CuiSupplementTables.docx","url":"https://assets-eu.researchsquare.com/files/rs-8285582/v1/2028c175323f34630a7ae3d7.docx"},{"id":98425138,"identity":"48180acd-f85c-4f42-b7d9-80654f198e13","added_by":"auto","created_at":"2025-12-17 16:34:23","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":17492,"visible":true,"origin":"","legend":"","description":"","filename":"TableS6.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8285582/v1/541b2403b83074a80b7bc080.xlsx"},{"id":97986178,"identity":"d7943396-35c7-4435-8751-8897f82680fa","added_by":"auto","created_at":"2025-12-11 13:48:43","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":16377,"visible":true,"origin":"","legend":"","description":"","filename":"TableS7.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8285582/v1/c139181b25f5b0fb0553c438.xlsx"},{"id":97986173,"identity":"c6be30f4-4f2e-493d-8111-139d969a9c25","added_by":"auto","created_at":"2025-12-11 13:48:42","extension":"xlsx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":14857,"visible":true,"origin":"","legend":"","description":"","filename":"TableS8.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8285582/v1/03477b2bdb1fe08b72afb373.xlsx"},{"id":98424314,"identity":"1f500516-f60c-4aa0-b44d-b302379785a4","added_by":"auto","created_at":"2025-12-17 16:33:10","extension":"docx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":14641,"visible":true,"origin":"","legend":"","description":"","filename":"Supportinginformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-8285582/v1/e1e4203461ac39f4cadae0d6.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Ectopic expression of the Sesuvium portulacastrum genes SpHDZ39 and SpHDZ41 enhances cadmium tolerance in Arabidopsis","fulltext":[{"header":"Introduction","content":"\u003cp\u003e\u003cem\u003eSesuvium portulacastrum\u003c/em\u003e, a salt-tolerant herb that grows as either annual or perennial is widely found in coastal zones across tropics and subtropics, and exhibits remarkable resistance to drought stress and salinity (Sukhorukov et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). \u003cem\u003eS. portulacastrum\u003c/em\u003e is well-adapted to saline environments, characterized by succulent, prostrate stems, adventitious roots arising from nodes enabling clonal propagation, and habitats such as sandy shores, limestone coastlines, salt flats, and marshes (Zhang et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Its leaves are opposite and linear-lanceolate, equipped with epidermal wax coatings and water-storage tissues that collectively maintain cellular water balance under salt stress (Sukhorukov et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cem\u003eS. portulacastrum\u003c/em\u003e can efficiently utilize both sodium and potassium ions. Seedling growth remains largely stable even when exposed to high concentration of sodium chloride (NaCl), with only slight curling observed in young leaves. The plant rapidly accumulates osmoprotective substances in salt stress, which helps sustain photosynthetic performance (Muchate et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Kulkarni et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). NaCl also alleviates cadmium (Cd) toxicity by change water content, redox balance, while promoting the synthesis of chelating agents like glutathione and proline (Mariem et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Wali et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Peng et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). This species shows strong environmental adaptability, including tolerance to heavy metal stress (Ghnaya et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Lokhande et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). After copper (Cu) is absorbed by the roots, the plant regulates metal ion homeostasis through transport proteins belonging to families such as ZIP, which control Cu uptake and internal distribution (Colangelo \u0026amp; Guerinot, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Alagarasan et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Under different salt conditions, \u003cem\u003eS. portulacastrum\u003c/em\u003e is capable of retaining high levels of heavy metals in its root tissues (Bonaventure et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Plant reduces oxidative damage caused by Cu through enhanced antioxidant enzyme activity, stimulation of MAPK signaling and ascorbate-glutathione cycle. Genes relate to metal binding and transport also contribute to Cu detoxification by directing it into vacuoles, where it is safely compartmentalized (Xiang et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). In addition to its physiological resilience, this halophyte is valued for its nutritional and medicinal properties. It is considered an edible marine plant, rich in minerals, vitamin C, vitamin K, and antioxidant substances (Mathieu \u0026amp; Meissa, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Lokhande et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Boxman et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2018\u003c/span\u003e;). High levels of phenolics and other bioactive compounds are responsible for its traditional use in treating diseases and suppressing microbial growth (Magwa et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Chandrasekaran et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). From an ecological standpoint, \u003cem\u003eS. portulacastrum\u003c/em\u003e contributes to environmental restoration. It is used in floating bed systems to absorb excess nitrogen and phosphorus from nutrient-rich waters and can also serve as feed in aquaculture settings (Liu et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Senff et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Its strong capacity for sodium absorption supports soil desalination, making degraded lands more suitable for the cultivation of salt-sensitive crops (Rabhi et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe \u003cem\u003eHomeodomain-Leucine Zippers\u003c/em\u003e (\u003cem\u003eHDZs\u003c/em\u003e) encode transcription factors central to plant growth, development, and environmental responses (Henriksson et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). These proteins are defined by a conserved homeodomain (HD), responsible for sequence-specific DNA binding, and a leucine zipper (LZ) domain that mediates dimerization and DNA recognition (Moens \u0026amp; Selleri, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). HDZs are classified into four subfamilies based on their structural motifs and functional diversity (Ariel et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). HDZ I and HDZ II subfamilies contain only HD and LZ domains, while HDZ III and HDZ IV include additional motifs such as the START domain, and in HDZ III, the MEKHLA domain, which are associated with hormonal and developmental signaling pathways (Ariel et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Zhu et al., \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cem\u003eHDZ I\u003c/em\u003e plays a key role in mediating abiotic stress responses (Gu et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Li et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). \u003cem\u003eHDZ II\u003c/em\u003e is primarily involved in regulating light signaling and morphogenesis (Qi et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), while \u003cem\u003eHDZ III\u003c/em\u003e regulates apical meristem development, vascular patterning, and organ polarity (Turchi et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The \u003cem\u003eHDZ IV\u003c/em\u003e subgroup is implicated in epidermal differentiation, root hair formation, and cuticle development (Chew et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Schrick et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In \u003cem\u003eArabidopsis\u003c/em\u003e, \u003cem\u003eAtHB7\u003c/em\u003e and \u003cem\u003eAtHB12\u003c/em\u003e function antagonistically for aluminum stress (Liu et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Specifically, \u003cem\u003eAtHB7\u003c/em\u003e promotes stomatal closure to reduce water loss, while \u003cem\u003eAtHB12\u003c/em\u003e facilitates stomatal opening and is beneficial for seed production (Olsson et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; R\u0026eacute; et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Moreover, \u003cem\u003eAtHB21\u003c/em\u003e, \u003cem\u003eAtHB40\u003c/em\u003e, and \u003cem\u003eAtHB53\u003c/em\u003e show upregulated expression under abotic stress conditions (Skinner \u0026amp; Gasser, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). The transcripts of \u003cem\u003eOsHOX22\u003c/em\u003e and \u003cem\u003eOsHOX24\u003c/em\u003e are significantly upregulated in both rice cultivars, whereas \u003cem\u003eOsHOX6\u003c/em\u003e shows only a slight increase in the drought-sensitive rice under prolonged drought conditions (Agalou et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). \u003cem\u003eOsHOX22\u003c/em\u003e fine-tunes drought and salinity adaptation by regulating the ABA-signaling cascade. (Zhang et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). The expression of \u003cem\u003eOsHOX19\u003c/em\u003e is enhanced by drought stress application in both drought-sensitive and drought-resistant cultivars (Harris et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Overexpression of \u003cem\u003eOsTF1L\u003c/em\u003e has been shown to promote lignin biosynthesis and stomatal closure, improving drought tolerance (Bang et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn this work, we identified that 46 \u003cem\u003eSpHDZs\u003c/em\u003e in the \u003cem\u003eS. portulacastrum\u003c/em\u003e genome. These genes were classified into four subgroups, comprising 25 members in subgroup I, 6 members in subgroup II, 6 members in subgroup III, and 9 members in subgroup IV. The identified \u003cem\u003eSpHDZs\u003c/em\u003e were unevenly distributed across 21 chromosomes, and both intra- and inter-species collinearity relationships were observed. Protein sequence analysis revealed the presence of six conserved motifs and structural domains, reflecting evolutionary conservation and gene duplication events. The exons number was ranged from 2 to 19, while introns varied between 1 and 5. The encoded proteins had lengths between 215 and 865 amino acids, with predicted molecular weights ranging from 24,651.80 to 94,974.01 Da. The theoretical isoelectric points (pI) were found to vary from 4.56 to 9.07. In addition, physicochemical analyses demonstrated wide variation in instability indices, aliphatic indices, and GRAVY values, which ranged from 43.50 to 75.89, 54.65 to 86.69, and \u0026minus;\u0026thinsp;1.174 to -0.062, respectively. \u003cem\u003eCis\u003c/em\u003e-element on the promoters associated with light responsiveness, hormone signaling, stress responses, and developmental processes. Transcriptome profiling under Cd, Cu, and NaCl treatment indicated that the expression of \u003cem\u003eSpHDZs\u003c/em\u003e were differentially regulated in response to these abotic stress. The expression pattern of \u003cem\u003eSpHDZ39\u003c/em\u003e and \u003cem\u003eSpHDZ41\u003c/em\u003e were further validated by quantitative real-time PCR. In addition, subcellular localization and yeast one-hybrid assays confirmed that SpHDZ39 and SpHDZ41 function as transcription factors. Their overexpression in \u003cem\u003eA. thaliana\u003c/em\u003e enhanced Cd tolerance. Overall, our results suggest that \u003cem\u003eSpHDZs\u003c/em\u003e play important roles in Cd stress and may provide genetic resources for improving Cd tolerance in crops through molecular breeding strategies.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cp\u003e\u003cb\u003eIdentification of\u003c/b\u003e \u003cb\u003eSpHDZs\u003c/b\u003e \u003cb\u003ein\u003c/b\u003e \u003cb\u003eS. portulacastrum\u003c/b\u003e \u003cb\u003egenome\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe reference genome of \u003cem\u003eS. portulacastrum\u003c/em\u003e was obtained from the laboratory at Guizhou University. AtHDZs sequences from \u003cem\u003eArabidopsis thaliana\u003c/em\u003e, available in the TAIR database, were used as queries to blast in the \u003cem\u003eS. portulacastrum\u003c/em\u003e proteome using TBtools software. Candidate gene sequences were validated through the identification of conserved Pfam domains, specifically PF00046 and PF02183.\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003ePhylogenetic and sequence analysis\u003c/h2\u003e\u003cp\u003eProtein sequences of the identified SpHDZs, together with AtHDZs from \u003cem\u003eA. thaliana\u003c/em\u003e and \u003cem\u003eOsHDZs\u003c/em\u003e from \u003cem\u003eO. sativa\u003c/em\u003e, were aligned and constructed a phylogenetic tree in MEGA11as described previously (Wu et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). The tree was edited and graphically refined using Adobe Illustrator. Protein subcellular localization was predicted using the WoLF PSORT online tool. Physicochemical properties such as molecular weight and isoelectric point were analyzed through the ProtParam server. Motif structures were identified using the MEME suite under default settings.\u003c/p\u003e\u003cp\u003e\u003cb\u003ePromoter analysis of\u003c/b\u003e \u003cb\u003ecis\u003c/b\u003e\u003cb\u003e-acting elements\u003c/b\u003e\u003c/p\u003e\u003cp\u003ePromoter sequences extending 2,000 bps upstream of the start codon of each \u003cem\u003eSpHDZ\u003c/em\u003e were extracted and analyzed for \u003cem\u003ecis\u003c/em\u003e-acting elements using the PLANTCARE database. The results were visualized using the Simple BioSequence Viewer module in TBtools. To assess the distribution of regulatory elements, a statistical summary was generated using PivotCharts in Microsoft Excel, and a heatmap representing the abundance and classification of elements was produced using the HeatMap module in TBtools.\u003c/p\u003e\u003cp\u003e\u003cb\u003eSynteny analysis of the\u003c/b\u003e \u003cb\u003eSpHDZs\u003c/b\u003e\u003c/p\u003e\u003cp\u003eSyntenic relationships among \u003cem\u003eSpHDZs\u003c/em\u003e within the \u003cem\u003eS. portulacastrum\u003c/em\u003e genome and between \u003cem\u003eS. portulacastrum\u003c/em\u003e and \u003cem\u003eA. thaliana\u003c/em\u003e were investigated using TBtools. Data required for MCScanX-based analysis were processed using the File Merger module. The synteny maps were visualized with the Circle Gene Viewer function.\u003c/p\u003e\u003cp\u003e\u003cb\u003eChromosomal localization of\u003c/b\u003e \u003cb\u003eSpHDZs\u003c/b\u003e\u003c/p\u003e\u003cp\u003eChromosomal distribution of \u003cem\u003eSpHDZs\u003c/em\u003e was analyzed using TBtools. The genomic annotation file and filtered gene list were imported, and genes were positioned based on their physical coordinates along the chromosomes. The resulting map was adjusted for clarity and visualization quality.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eStructural and evolutionary characterization\u003c/h3\u003e\n\u003cp\u003eThe secondary structure of each SpHDZ protein was predicted using SOPMA, and the tertiary structure was modeled with SWISS-MODEL. Gene duplication patterns were determined based on synteny analysis, and types of duplication events were identified accordingly. Non-synonymous (Ka) and synonymous (Ks) substitution rates were calculated in TBtools to evaluate evolutionary pressure among gene pairs.\u003c/p\u003e\u003cp\u003e\u003cb\u003eExpression pattern of\u003c/b\u003e \u003cb\u003eSpHDZ39\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003eSpHDZ41\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo examine the expression of \u003cem\u003eSpHDZ39\u003c/em\u003e and \u003cem\u003eSpHDZ41\u003c/em\u003e under cd teatment, \u003cem\u003eS. portulacastrum\u003c/em\u003e seedlings were treated with 200 \u0026micro;M Cd for 0, 7, 14, and 21 days. Total RNA was extracted from root tissues using a commercial RNA extraction kit. First-strand cDNA was synthesized using a reverse transcription kit. The \u003cem\u003eSpGAPDH\u003c/em\u003e was used as an internal control. Quantitative real-time PCR was performed with three biological and three technical replicates per treatment group with gene special primers (Table \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003e). Relative gene expression levels were calculated using the 2⁻ΔΔCT method.\u003c/p\u003e\n\u003ch3\u003eSubcellular localization of SpHDZ39 and SpHDZ41\u003c/h3\u003e\n\u003cp\u003eThe coding sequences of \u003cem\u003eSpHDZ39\u003c/em\u003e and \u003cem\u003eSpHDZ41\u003c/em\u003e without stop codons were cloned into vectors, generating 35S::SpHDZ39-GFP and 35S::SpHDZ41-GFP constructs with gene special primers (Tab S5). These constructs were transformed into \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e and infiltrated into tobacco leaves. After dark incubation, fluorescence signals were observed at 72 hours post-inoculation using a Leica TCS SP8 confocal microscope (Leica, Wetzlar, Germany). The 35S::TksARF3-mCherry construct served as a nuclear marker for co-localization assessment (Wu et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eDNA-binding activity of SpHDZ39 and SpHDZ41\u003c/h3\u003e\n\u003cp\u003eTo assess DNA-binding activity, the coding regions of \u003cem\u003eSpHDZ39\u003c/em\u003e and \u003cem\u003eSpHDZ41\u003c/em\u003e were cloned into the pGADT7-rec2 vector with gene special primers (Table \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003e). Synthetic DNA sequences containing triple tandem repeat binding motifs, including MA0573, MA0573_mutant, MA0008, and MA0008_mutant, were inserted individually into the pHIS2 vector with gene special primers (Table \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003e). These constructs were co-transformed into \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e strain Y187 using a polyethylene glycol and lithium acetate protocol. Protein-DNA interaction was assessed by growing transformants on SD/-Leu/-Trp (DDO) and SD/-His/-Leu/-Trp (TDO) media. Plates supplemented with 50 mM 3-aminotriazole were used for further validation of DNA-binding specificity.\u003c/p\u003e\n\u003ch3\u003ePlant transformation\u003c/h3\u003e\n\u003cp\u003eThe constructs 35S::SpHDZ39-GFP and 35S::SpHDZ41-GFP were introduced into \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e GV3101. Transformation of \u003cem\u003eA. thaliana\u003c/em\u003e was performed using the floral dip method, and the resulting seeds were collected for subsequent selection and analysis.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eGenome-wide identification and classification of\u003c/b\u003e \u003cb\u003eSpHDZs\u003c/b\u003e\u003c/p\u003e\u003cp\u003eA comprehensive genome-wide analysis uncovered 46 \u003cem\u003eHDZs\u003c/em\u003e in \u003cem\u003eS. portulacastrum\u003c/em\u003e. Based on phylogenetic relationships with \u003cem\u003eA. thaliana\u003c/em\u003e and \u003cem\u003eO. sativa\u003c/em\u003e, these genes were categorized into HDZ I, HDZ II, HDZ III, and HDZ IV subgroups (Elhiti \u0026amp; Stasolla, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Among them, 25 \u003cem\u003eSpHDZs\u003c/em\u003e belonged to subgroup I, 6 to subgroup II, another 6 to subgroup III, and 9 to subgroup IV (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eBased on chromosomal locations and gene annotations, the 46 \u003cem\u003eSpHDZs\u003c/em\u003e were renamed as \u003cem\u003eSpHDZ1\u003c/em\u003e to \u003cem\u003eSpHDZ46\u003c/em\u003e, sequentially (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Among them, \u003cem\u003eSpHDZ16\u003c/em\u003e encodes the longest protein, consisting of 865 amino acids and exhibiting the highest molecular weight, whereas \u003cem\u003eSpHDZ34\u003c/em\u003e encodes the shortest protein, with 215 amino acids and the lowest molecular weight. The predicted isoelectric points (pI) of the SpHDZs ranged from 4.56 (SpHDZ14) to 9.07 (SpHDZ19). All SpHDZs displayed negative grand average of hydropathicity (GRAVY) values, ranging from \u0026minus;\u0026thinsp;1.174 (SpHDZ41) to -0.062 (SpHDZ4), suggesting that these proteins are generally hydrophilic. The aliphatic index values varied between 54.65 (SpHDZ39) and 86.69 (SpHDZ17), while all proteins had instability index values above 43.50(SpHDZ26) and below 75.89(SpHDZ7), indicating potential instability in \u003cem\u003evitro\u003c/em\u003e. Subcellular localization predictions indicated that 41 SpHDZs are primarily localized in the nucleus. Notable exceptions include SpHDZ17, SpHDZ35, and SpHDZ43, which are predicted to localized in the cytoplasm, as well as SpHDZ22 and SpHDZ33, which are likely localized in the chloroplast.\u003c/p\u003e\n\u003ch3\u003eChromosomal distribution and synteny\u003c/h3\u003e\n\u003cp\u003eAll 46 \u003cem\u003eSpHDZs\u003c/em\u003e were successfully mapped across 21 chromosomes in \u003cem\u003eS. portulacastrum\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The highest gene density was found on Chr7, Chr10, and Chr11, each containing four \u003cem\u003eSpHDZs\u003c/em\u003e. Chr2, Chr3, and Chr5 each harbored three genes. Chr1, Chr8, Chr12, Chr13, Chr14, Chr18, Chr20, Chr21, Chr23, and Chr24 contained two \u003cem\u003eSpHDZs\u003c/em\u003e, while Chr4, Chr15, Chr16, Chr17, and Chr19 were each associated with only one \u003cem\u003eSpHDZ\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eTo explore the structural diversity and potential functional roles of SpHDZs, conserved domain analysis was conducted. Members within the same subgroup generally exhibited similar domain architectures, whereas marked variation was observed among different subfamilies. The HD and LZ domains were consistently present in most SpHDZ I proteins, aligning with findings in other species. An exception was SpHDZ34, which lacked the LZ domain despite being classified in the SpHDZ I group. All six HDZ II proteins retained the LZ domain; however, only SpHDZ1 possessed the HD domain, while the remaining members, SpHDZ13, SpHDZ19, SpHDZ25, SpHDZ30, and SpHDZ38, lacked it. Members of the HDZ III and IV subfamilies all contained both HD and START domains. Notably, HDZ III members also harbored a C-terminal MEKHLA domain, indicating a higher degree of structural complexity and possible functional divergence (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003eMotif analysis revealed that Motif1, Motif2, and Motif3 were highly conserved across all SpHDZs. Functional annotation showed that Motif1 and Motif2 together formed the canonical HD domain (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eA).\u003c/p\u003e\u003cp\u003eGene structure analysis demonstrated that varying exon-intron organizations across subfamilies. Most \u003cem\u003eSpHDZs\u003c/em\u003e contained 1 to 5 introns, but exon numbers ranged from 2 to 19. The HDZ III subgroup showed the most complex gene structure, with 18 to 19 exons. This was followed by HDZ IV, whose members had 8 to 11 exons. In contrast, HDZ I and II members generally exhibited simpler gene structures, typically containing only 2 to 4 exons (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003e\u003cb\u003eCis\u003c/b\u003e\u003cb\u003e-acting regulatory element analysis of the\u003c/b\u003e \u003cb\u003eSpHDZs\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eCis\u003c/em\u003e-acting regulatory elements are crucial for transcriptional regulation and directly impact gene function. To investigate potential regulatory mechanisms, the 2,000 base pairs upstream promoter regions of \u003cem\u003eSpHDZs\u003c/em\u003e were analyzed for the presence of \u003cem\u003ecis\u003c/em\u003e-acting regulatory elements (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Identified elements were grouped into light-responsive, hormone-responsive, biotic/abiotic stress-responsive, and plant growth and development-related elements (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003eAmong these, hormone-responsive elements were the most prevalent, totaling 248, with jasmonate-responsive elements (JAREs) being the most dominant, present in over 59% of the genes. Light-responsive elements were the second most common, followed by stress-related elements, which included MYB-DRE (41.17%), ARE (35.88%), LTRE (11.76%), DSRE (7.06%), and ANX-Enh (4.12%). The plant development-related elements group was the least represented, with meristem-related motifs being the most enriched within this category.\u003c/p\u003e\u003cp\u003e\u003cb\u003eCollinearity analysis of the\u003c/b\u003e \u003cb\u003eSpHDZs\u003c/b\u003e\u003c/p\u003e\u003cp\u003eCollinearity analysis using MCScanX identified 39 collinear gene pairs within the \u003cem\u003eSpHDZs\u003c/em\u003e, spanning all four subfamilies (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). These duplication events, primarily resulting from whole-genome duplication (WGD) or segmental duplication (Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e), likely contributed to genome expansion and evolutionary diversification. The polyploidization or local genomic rearrangements driven events may enhance genetic variability, promoting functional innovation and adaptive responses.\u003c/p\u003e\u003cp\u003eTo evaluate evolutionary pressures acting on these duplicated genes, nonsynonymous/synonymous substitution rate (Ka/Ks) ratios were calculated (Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e). It showed that most gene pairs showed Ka/Ks values between 0.03 and 0.88 (Ka/Ks\u0026thinsp;\u0026lt;\u0026thinsp;1), indicating that purifying selection has predominantly preserved protein function by eliminating deleterious mutations. Notably, only \u003cem\u003eSpHDZ7\u003c/em\u003e/\u003cem\u003eSpHDZ8\u003c/em\u003e pair exhibited a Ka/Ks ratio of 1.09, suggesting positive selection, which may have driven functional divergence through advantageous amino acid changes. Structural modeling of these collinear gene products revealed substantial tertiary structural conservation, reinforcing the presence of functional constraints. Furthermore, interspecies synteny analysis between \u003cem\u003eS. portulacastrum\u003c/em\u003e and \u003cem\u003eA. thaliana\u003c/em\u003e uncovered 57 orthologous gene pairs, while \u003cem\u003eSpHDZ24\u003c/em\u003e, \u003cem\u003eSpHDZ30\u003c/em\u003e, and \u003cem\u003eSpHDZ45\u003c/em\u003e were lacked identifiable syntenic counterparts (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). This high level of conservation reflects shared evolutionary ancestry and underscores the structural and functional preservation of the \u003cem\u003eSpHDZ\u003c/em\u003e family across plant species.\u003c/p\u003e\u003cp\u003e\u003cb\u003eExpression profiling of\u003c/b\u003e \u003cb\u003eSpHDZs\u003c/b\u003e \u003cb\u003eunder different Abiotic stress conditions\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo assess the transcriptional responses of \u003cem\u003eSpHDZs\u003c/em\u003e to salt stress, RNA-seq data from salt-treated \u003cem\u003eS. portulacastrum\u003c/em\u003e leaves were analyzed under varying NaCl concentrations (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). The results revealed diverse expression trends, with both upregulation and downregulation observed across different genes as salt levels increased. The \u003cem\u003eSpHDZ1\u003c/em\u003e, \u003cem\u003eSpHDZ6\u003c/em\u003e, \u003cem\u003eSpHDZ7\u003c/em\u003e, \u003cem\u003eSpHDZ8\u003c/em\u003e, \u003cem\u003eSpHDZ10\u003c/em\u003e, \u003cem\u003eSpHDZ24\u003c/em\u003e, \u003cem\u003eSpHDZ26\u003c/em\u003e, \u003cem\u003eSpHDZ29\u003c/em\u003e, \u003cem\u003eSpHDZ30\u003c/em\u003e, \u003cem\u003eSpHDZ34\u003c/em\u003e, \u003cem\u003eSpHDZ40\u003c/em\u003e, and \u003cem\u003eSpHDZ41\u003c/em\u003e were exhibited marked upregulation under salt concentrations. Notably, 10 of these genes belonged to the HDZ I subgroup, while \u003cem\u003eSpHDZ1\u003c/em\u003e and \u003cem\u003eSpHDZ30\u003c/em\u003e were classified under HDZ II. Conversely, \u003cem\u003eSpHDZ5\u003c/em\u003e, \u003cem\u003eSpHDZ16\u003c/em\u003e, \u003cem\u003eSpHDZ20\u003c/em\u003e, \u003cem\u003eSpHDZ22\u003c/em\u003e, \u003cem\u003eSpHDZ23\u003c/em\u003e, and \u003cem\u003eSpHDZ32\u003c/em\u003e were displayed consistent downregulation. Several other genes showed peak expression at intermediate salt concentrations.\u003c/p\u003e\u003cp\u003eTo explore the involvement of \u003cem\u003eSpHDZs\u003c/em\u003e in heavy metal stress responses, transcriptomic data were analyzed from roots and leaves subjected to Cd and Cu treatment. The \u003cem\u003eSpHDZ6\u003c/em\u003e, \u003cem\u003eSpHDZ13\u003c/em\u003e, \u003cem\u003eSpHDZ14\u003c/em\u003e, \u003cem\u003eSpHDZ29\u003c/em\u003e, and \u003cem\u003eSpHDZ38\u003c/em\u003e were upregulated with prolonged Cd exposure compared with control conditions in roots (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). However, most genes were also upregulated at 7 days of Cd exposure compared with control, while \u003cem\u003eSpHDZ4\u003c/em\u003e, \u003cem\u003eSpHDZ5\u003c/em\u003e, \u003cem\u003eSpHDZ6\u003c/em\u003e, \u003cem\u003eSpHDZ14\u003c/em\u003e, \u003cem\u003eSpHDZ17\u003c/em\u003e, and \u003cem\u003eSpHDZ33\u003c/em\u003e maintained elevated expression at either 7 days, 14 days, or 21 days, compared with control in leaves (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). In contrast, \u003cem\u003eSpHDZ10\u003c/em\u003e, \u003cem\u003eSpHDZ23\u003c/em\u003e, \u003cem\u003eSpHDZ25\u003c/em\u003e, \u003cem\u003eSpHDZ31\u003c/em\u003e, \u003cem\u003eSpHDZ32\u003c/em\u003e, and \u003cem\u003eSpHDZ45\u003c/em\u003e showed downregulated with Cd treatment compared with control. Furthermore, the expression of \u003cem\u003eSpHDZ39\u003c/em\u003e and \u003cem\u003eSpHDZ41\u003c/em\u003e under Cd treatment was validated by qRT-PCR (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). Data analysis revealed that the expression level of \u003cem\u003eSpHDZ39\u003c/em\u003e showed a gradual downregulation trend with the extension of Cd stress duration. In contrast, SpHDZ41 exhibited an upregulation at 7 days of treatment, followed by a downregulation relative to the control group as the stress duration prolonged. Similarly, \u003cem\u003eSpHDZ5\u003c/em\u003e, \u003cem\u003eSpHDZ15\u003c/em\u003e, \u003cem\u003eSpHDZ16\u003c/em\u003e, and \u003cem\u003eSpHDZ17\u003c/em\u003e showed upregulation at 50 \u0026micro;M Cu treatment compared with control, \u003cem\u003eSpHDZ7\u003c/em\u003e, \u003cem\u003eSpHDZ8\u003c/em\u003e, \u003cem\u003eSpHDZ26\u003c/em\u003e, \u003cem\u003eSpHDZ29\u003c/em\u003e, and \u003cem\u003eSpHDZ41\u003c/em\u003e showed upregulation at 100 \u0026micro;M Cu treatment, \u003cem\u003eSpHDZ4\u003c/em\u003e, \u003cem\u003eSpHDZ6\u003c/em\u003e, \u003cem\u003eSpHDZ18\u003c/em\u003e, \u003cem\u003eSpHDZ24\u003c/em\u003e, and \u003cem\u003eSpHDZ46\u003c/em\u003e were induced at 200 \u0026micro;M Cu treatment, compared with control in roots, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). In leaves, exposure to 50 \u0026micro;M Cu elicited significant upregulation of \u003cem\u003eSpHDZ12\u003c/em\u003e, \u003cem\u003eSpHDZ23\u003c/em\u003e, \u003cem\u003eSpHDZ32\u003c/em\u003e, and \u003cem\u003eSpHDZ34\u003c/em\u003e compared with untreated controls. Treatment with 100 \u0026micro;M Cu induced the expression of \u003cem\u003eSpHDZ10\u003c/em\u003e, \u003cem\u003eSpHDZ14\u003c/em\u003e, and \u003cem\u003eSpHDZ40\u003c/em\u003e. Following 200 \u0026micro;M Cu exposure, \u003cem\u003eSpHDZ6\u003c/em\u003e, \u003cem\u003eSpHDZ7, SpHDZ8, SpHDZ29\u003c/em\u003e, and \u003cem\u003eSpHDZ41\u003c/em\u003e were markedly upregulated, whereas \u003cem\u003eSpHDZ2\u003c/em\u003e, \u003cem\u003eSpHDZ11, SpHDZ15\u003c/em\u003e, \u003cem\u003eSpHDZ20\u003c/em\u003e, \u003cem\u003eSpHDZ22\u003c/em\u003e, \u003cem\u003eSpHDZ23\u003c/em\u003e, \u003cem\u003eSpHDZ39\u003c/em\u003e, and \u003cem\u003eSpHDZ45\u003c/em\u003e exhibited significant downregulation relative to control levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Those results suggested that different \u003cem\u003eSpHDZs\u003c/em\u003e may response to different abiotic stress.\u003c/p\u003e\n\u003ch3\u003eSpHDZ39 and SpHDZ41 function as transcription factors\u003c/h3\u003e\n\u003cp\u003eSpHDZ39 and SpHDZ41 were predicted to be localized in the nucleus based on WOLF PSORT analysis (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), and this prediction was experimentally confirmed through transient expression assays in tobacco leaves (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). Constructs expressing SpHDZ39-GFP or SpHDZ41-GFP were co-expressed with the nuclear marker TksARF3-mCherry in leaf epidermal cells. The observed overlap of fluorescence signals confirmed that both proteins were localized in the nucleus.\u003c/p\u003e\u003cp\u003eTo further examine their DNA-binding capacity, yeast one-hybrid assays were conducted. Yeast cells co-transformed with pHIS2-MA0573 or pHIS2-MA0008 and pGADT7-rec2-SpHDZ39 grew normally on TDO medium (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). In contrast, yeast cells carrying the mutant versions of these sequences or the negative control failed to grow under the same conditions. Similar binding behavior was observed for SpHDZ41 (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e7\u003c/span\u003eC), indicating that both transcription factors specifically recognize and bind these DNA motifs. Taken together, those results showed that both SpHDZ39 and SpHDZ41 function as a transcription factor.\u003c/p\u003e\u003cp\u003e\u003cb\u003eOverexpression of\u003c/b\u003e \u003cb\u003eSpHDZ39\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003eSpHDZ41\u003c/b\u003e \u003cb\u003eimproved Cd tolerance in\u003c/b\u003e \u003cb\u003eA. thaliana\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWe generated \u003cem\u003eA. thaliana\u003c/em\u003e plants transgene overexpressing \u003cem\u003eSpHDZ39\u003c/em\u003e (\u003cem\u003eSpHDZ39OE\u003c/em\u003e) and \u003cem\u003eSpHDZ41\u003c/em\u003e (\u003cem\u003eSpHDZ41OE\u003c/em\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e8\u003c/span\u003eA). Segregating T\u003csub\u003e3\u003c/sub\u003e plants from two independent \u003cem\u003eSpHDZ39OE\u003c/em\u003e and \u003cem\u003eSpHDZ41OE\u003c/em\u003e plants were subjected to 75 \u0026micro;M Cd treatment. Both \u003cem\u003eSpHDZ39OE\u003c/em\u003e and \u003cem\u003eSpHDZ41OE\u003c/em\u003e plants exhibited stronger growth compared with wild-type plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e8\u003c/span\u003eB). The average root length of \u003cem\u003eSpHDZ39OE\u003c/em\u003e plants ranged from 2.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 cm to 2.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3 cm, while wild-type plants showed an average of 1.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 cm (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e8\u003c/span\u003eC). \u003cem\u003eSpHDZ41OE\u003c/em\u003e plants displayed longer roots ranging from 4.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3 cm to 4.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 cm, compared with 4.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 cm in wild type (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e8\u003c/span\u003eC). Fresh weight measurements also indicated enhanced biomass. \u003cem\u003eSpHDZ39OE\u003c/em\u003e plants reached 1.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 mg/plant to 1.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 mg/plant, whereas wild-type plants averaged 1.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 mg/plant (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e8\u003c/span\u003eD). \u003cem\u003eSpHDZ41OE\u003c/em\u003e plants showed values between 1.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 mg/plant to 1.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 mg/plant compared with 1.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 mg/plant in wild type (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e8\u003c/span\u003eD). Taken together, those results suggest that \u003cem\u003eSpHDZ39\u003c/em\u003e and \u003cem\u003eSpHDZ41\u003c/em\u003e enhance Cd tolerance in \u003cem\u003eA. thaliana\u003c/em\u003e by promoting root growth and biomass accumulation.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eHDZ is an evolutionarily conserved transcription factor that have been identified in Arabidopsis, rice, maize, soybean, wheat, and tomato (Henriksson et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Kim et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Chen et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Zhang et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Yue et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). In this study, we present the first comprehensive identification and characterization of 46 \u003cem\u003eSpHDZs\u003c/em\u003e in the \u003cem\u003eS. portulacastrum\u003c/em\u003e genome through systematic bioinformatics analysis.\u003c/p\u003e\u003cp\u003eThe gene number in \u003cem\u003eS. portulacastrum\u003c/em\u003e is equal to that in \u003cem\u003eArabidopsis\u003c/em\u003e, suggesting possible evolutionary and functional parallels between the two species. From an evolutionary perspective, the identical number of \u003cem\u003eHDZs\u003c/em\u003e may reflect conserved gene retention, with minimal gene loss or expansion across lineages. This pattern supports the notion that the \u003cem\u003eHDZs\u003c/em\u003e has undergone strong evolutionary constraints, preserving its core structure across species. Functionally, the conserved gene number implies that similar molecular mechanisms may operate in both species to regulate development and environmental adaptation. This finding lays the groundwork for future investigations into the molecular mechanisms underlying stress tolerance in \u003cem\u003eS. portulacastrum\u003c/em\u003e. Previous studies have indicated that the abundance of \u003cem\u003eHDZs\u003c/em\u003e are not directly related to genome size but is shaped by tandem and segmental duplication events (Zhao et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Phylogenetic and domain structure analyses classified the 46 identified genes into four subfamilies, with HDZ I containing the largest number of members. This classification is consistent with results in other plants, supporting the evolutionary conservation of the \u003cem\u003eHDZs\u003c/em\u003e.\u003c/p\u003e\u003cp\u003eGene structure and motif composition analyses revealed that members of the HDZ I subgroup contain highly conserved HD and LZ domains. In contrast, HDZ III and IV subfamilies include START domains, and HDZ III genes additionally contain MEKHLA domains at their C-terminus, consistent with patterns reported in other species. The structural features vary significantly among subfamilies, suggesting potential functional divergence. Subcellular localization prediction showed that most SpHDZs are localized in the nucleus, while a few are present in the cytoplasm or chloroplast. Transient expression assays confirmed that SpHDZ39 and SpHDZ41 are nuclear proteins, with DNA-binding activities supported by yeast one-hybrid assays. These results indicate that these genes likely function as transcriptional regulators.\u003c/p\u003e\u003cp\u003eCollinearity analysis revealed 57 syntenic gene pairs between \u003cem\u003eS. portulacastrum\u003c/em\u003e and \u003cem\u003eArabidopsis\u003c/em\u003e, including 38 pairs involving \u003cem\u003eHDZs\u003c/em\u003e. This indicates limited gene family expansion and suggests a relatively stable evolutionary trajectory for the HDZ family in \u003cem\u003eS. portulacastrum\u003c/em\u003e.\u003c/p\u003e\u003cp\u003e\u003cem\u003eCis\u003c/em\u003e-element analysis of promoter regions showed that \u003cem\u003eSpHDZs\u003c/em\u003e are associated with a wide range of regulatory functions, including hormone response, abiotic and biotic stress responses, developmental control, and light signaling (Zhou et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Notably, drought-related elements were more frequently enriched in the HDZ III and IV subfamilies. This contrasts with patterns reported in other plants, where HDZ I genes typically play central roles in drought response (Li et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). For example, in \u003cem\u003eArabidopsis\u003c/em\u003e, AtHB7 and AtHB12, which belong to HDZ I, are involved in ABA-mediated regulation of stomatal movement and osmotic balance under drought conditions. This divergence suggests that, in \u003cem\u003eS. portulacastrum\u003c/em\u003e, HDZ III and IV genes may have evolved to compensate for or replace some of the functions typically attributed to HDZ I genes. Given that \u003cem\u003eS. portulacastrum\u003c/em\u003e naturally inhabits saline environments, this may represent a unique adaptation shaped by long-term exposure to salt stress. In \u003cem\u003eZea mays\u003c/em\u003e, overexpression of \u003cem\u003eZmHDZ9\u003c/em\u003e enhances drought resistance in transgenic \u003cem\u003eArabidopsis\u003c/em\u003e (Qiu et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), further supporting the idea of functional plasticity within HDZ subfamilies. These findings raise the possibility that HDZ III and IV genes in \u003cem\u003eS. portulacastrum\u003c/em\u003e may have been selectively co-opted for stress regulation. Further functional studies are needed to confirm this hypothesis.\u003c/p\u003e\u003cp\u003eTranscriptome data under NaCl treatment revealed 13 \u003cem\u003eSpHDZs\u003c/em\u003e responsive to salt stress. Among these, \u003cem\u003eSpHDZ1\u003c/em\u003e and \u003cem\u003eSpHDZ30\u003c/em\u003e belonged to the HDZ II subgroup, while the remaining 11 genes were classified under HDZ I. This pattern aligns with findings in soybean, where most salt-induced \u003cem\u003eHDZs\u003c/em\u003e also belong to subgroup I and II (Chen et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). It has been reported that sodium ions stimulate V-ATPase activity, promoting the accumulation of soluble sugars in vacuoles and chloroplasts to maintain osmotic balance (Yi et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). These processes enhance salt tolerance in \u003cem\u003eS. portulacastrum\u003c/em\u003e. While both sodium ions and chloride ions are toxic to most plants (Adams \u0026amp; Shin, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Lutts \u0026amp; Lef\u0026egrave;vre, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), in this halophyte, chloride ions appear to be the main contributor to toxicity, while sodium ions have been shown to promote shoot development and leaf succulence (Wang et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Under NaCl, Cd, and Cu treatment, multiple \u003cem\u003eSpHDZs\u003c/em\u003e exhibited altered expression. Notably, \u003cem\u003eSpHDZ6\u003c/em\u003e from subgroup I was upregulated in response to all three treatments, suggesting a central role in multistress tolerance, consistent with previous findings regarding HDZ I function in salt adaptation (Li et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). qRT-PCR results showed that \u003cem\u003eSpHDZ39\u003c/em\u003e expression declined progressively under Cd stress, while \u003cem\u003eSpHDZ41\u003c/em\u003e initially increased at day 7, followed by a decrease over time. A similar regulatory pattern has been observed for \u003cem\u003eOshox22\u003c/em\u003e, which influences ABA biosynthesis and mediates stress responses in rice (Zhang et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Conversely, overexpression of \u003cem\u003eJcHDZ21\u003c/em\u003e has been shown to increase salt sensitivity in \u003cem\u003eArabidopsis\u003c/em\u003e (Tang et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), indicating that HDZ function can vary significantly depending on genetic context. Additional genes such as \u003cem\u003eSpHDZ4\u003c/em\u003e, \u003cem\u003eSpHDZ14\u003c/em\u003e, \u003cem\u003eSpHDZ24\u003c/em\u003e, and \u003cem\u003eSpHDZ29\u003c/em\u003e also responded to multiple stress treatments, although their precise functions remain unclear. Further experimental validation is required to explore their roles in regulating plant responses to environmental challenges. These genes may offer valuable targets for enhancing crop resistance to Cd stress through genetic improvement strategies.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eWe identified 46 \u003cem\u003eSpHDZs\u003c/em\u003e in \u003cem\u003eS. portulacastrum\u003c/em\u003e, distributed unevenly across 21 chromosomes. Based on gene structures and conserved motifs, these \u003cem\u003eSpHDZs\u003c/em\u003e were classified into four subgroups, each associated with distinct domain features. All duplication events identified were attributed to whole-genome or segmental duplication, and 39 duplicated gene pairs were detected. Among these, nearly all gene pairs had Ka/Ks ratios below one, indicating that purifying selection has maintained their sequence conservation. Promoter analysis revealed the presence of 703 \u003cem\u003ecis\u003c/em\u003e-regulatory elements, which were categorized into four functional types related to light response, hormone signaling, environmental stress, and developmental regulation. These elements were distributed unevenly among the gene subgroups, suggesting potential functional divergence. Expression profiling identified twelve \u003cem\u003eSpHDZs\u003c/em\u003e involved in salt stress responses and twelve others responsive to heavy metal treatment. Further verification using qRT-PCR confirmed that \u003cem\u003eSpHDZ39\u003c/em\u003e and \u003cem\u003eSpHDZ41\u003c/em\u003e showed altered expression patterns under Cd treatment. Subcellular localization predictions showed that most SpHDZs are targeted to the nucleus, with a few located in the chloroplast or cytoplasm. Experimental validation further confirmed that SpHDZ39 and SpHDZ41 act as nuclear transcription factors, as demonstrated by localization and yeast one-hybrid assays. Overexpression of these genes in \u003cem\u003eA. thaliana\u003c/em\u003e enhanced Cd tolerance, supporting their role in stress regulation. These findings contribute to a deeper understanding of the molecular mechanisms underlying abiotic stress adaptation in \u003cem\u003eS. portulacastrum\u003c/em\u003e. The identified genes offer promising targets for enhancing Cd tolerance in crops through genetic engineering and molecular breeding strategies.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors express their sincere gratitude to Dr. Xuchu Wang in Guizhou University for his critical reading and polishing of this manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eS. H. and T. C. initiated and designed the project, S. H., T. C., Y. L., X. W., F. F., Y. L., Y. Y., G. Z., Z. N., and Y. W. reviewing the referenced articles and preparing the Figures and tables, S. H. and T. C. writing and revising the manuscript. All authors approved the final version of the text.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of Guizhou University (Guida Tegang Hezi [2025] No. 02), the Guizhou Provincial Basic Research Program (Natural Science) General Program (No. [2025] 660), the Innovation Center for Academicians Team of Hainan Province and the specific research fund of The Innovation Platform for Academicians of Hainan Province (No. YSPTZX202309).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData will be made available on request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval and consent to participate\u003c/strong\u003e Not applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e All authors have read and approved the final version of the manuscript and consent to its submission for publica tion. The authors affirm that this work is original and has not been published previously, nor is it currently under consideration for publication elsewhere.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e The authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003cp\u003eAdams E, Shin R (2014). Transport, signaling, and homeostasis of potassium and sodium in plants. J Integr Plant Biol. 56:231-249. https://doi.org/ 10.1111/jipb.12159\u003c/p\u003e\n\u003cp\u003eAgalou A, Purwantomo S, Overnäs E, Johannesson H, Zhu X, Estiati A, de Kam RJ, Engström P, Slamet-Loedin IH, Zhu Z, Wang M, Xiong L, Meijer AH, Ouwerkerk PB (2008). A genome-wide survey of HD-Zip genes in rice and analysis of drought-responsive family members. Plant Mol Biol. 66:87-103. https://doi.org/ 10.1007/s11103-007-9255-7\u003c/p\u003e\n\u003cp\u003eAlagarasan G, Dubey M, Aswathy KS, Chandel G (2017). 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Expression-based discovery of candidate ovule development regulators through transcriptional profiling of ovule mutants. BMC Plant Biol. 9:29. https://doi.org/ 10.1186/1471-2229-9-29\u003c/p\u003e\n\u003cp\u003eSukhorukov AP, Nilova MV, Erst AS, Kushunina M, Baider C, Verloove F, Salas-Pascual M, Belyaeva IV, Krinitsina AA, Bruyns PV, Klak C (2018). Diagnostics, taxonomy, nomenclature and distribution of perennial \u003cem\u003eSesuvium\u003c/em\u003e (Aizoaceae) in Africa. PhytoKeys. 92:45-88. https://doi.org/ 10.3897/phytokeys.92.22205\u003c/p\u003e\n\u003cp\u003eSukhorukov AP, Sennikov AN, Nilova MV, Kushunina M, Belyaeva IV, Zaika MA, Hanáček MA (2021). A new endemic species of \u003cem\u003eSesuvium\u003c/em\u003e (Aizoaceae: Sesuvioideae) from the Caribbean Basin, with further notes on the genus composition in the West Indies. Kew Bull. 76,:651-674. https://doi.org/10.1007/s12225-021-09985-w\u003c/p\u003e\n\u003cp\u003eTang Y, Peng J, Lin J, Zhang M, Tian Y, Shang Y, Chen S, Bao X, Wang Q (2023). A HD-Zip I transcription factor from physic nut, \u003cem\u003eJcHDZ21\u003c/em\u003e, confers sensitive to salinity in transgenic Arabidopsis. Front Plant Sci. 14:1097265. https://doi.org/ 10.3389/fpls.2023.1097265\u003c/p\u003e\n\u003cp\u003eTurchi L, Baima S, Morelli G, Ruberti I (2015). Interplay of HD-Zip II and III transcription factors in auxin-regulated plant development. J Exp Bot. 66:5043-5053. https://doi.org/ 10.1093/jxb/erv174 \u003c/p\u003e\n\u003cp\u003eWali M, Gunsè B, Llugany M, Corrales I, Abdelly C, Poschenrieder C, Ghnaya T (2016). High salinity helps the halophyte \u003cem\u003eSesuvium portulacastrum\u003c/em\u003e in defense against Cd toxicity by maintaining redox balance and photosynthesis. Planta. 244:333-346. https://doi.org/ 10.1007/s00425-016-2515-5\u003c/p\u003e\n\u003cp\u003eWang D, Wang H, Han B, Wang B, Guo A, Zheng D, Liu C, Chang L, Peng M, Wang X (2012). Sodium instead of potassium and chloride is an important macronutrient to improve leaf succulence and shoot development for halophyte \u003cem\u003eSesuvium portulacastrum\u003c/em\u003e. Plant Physiol Biochem. 51:53-62. https://doi.org/ 10.1016/j.plaphy.2011.10.009 \u003c/p\u003e\n\u003cp\u003eWu X, Zhang C, Yao M, Fang F, Wang B, Jin F, Chen F, Ji M, Liu H, Yu Y, Wang J, Hui S, Wang X (2025). Auxin response factor 3 positively affects natural rubber biosynthesis by targeting farnesyl diphosphate synthase 1 in \u003cem\u003eTaraxacum kok-saghyz\u003c/em\u003e. Ind Crop Prod. 233:121485. https://doi.org/10.1016/j.indcrop.2025.121485 \u003c/p\u003e\n\u003cp\u003eXiang Z, Yuan B, Yang Y, Fang F, He M, Li Y, Hui S, Wang X (2025). 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Genes. 9:70. https://doi.org/ 10.3390/genes9020070\u003c/p\u003e\n\u003cp\u003eZhang S, Haider I, Kohlen W, Jiang L, Bouwmeester H, Meijer AH, Schluepmann H, Liu CM, Ouwerkerk PB (2012). Function of the HD-Zip I gene \u003cem\u003eOshox22\u003c/em\u003e in ABA-mediated drought and salt tolerances in rice. Plant Mol Biol. 80:571-585. https://doi.org/ 10.1007/s11103-012-9967-1.\u003c/p\u003e\n\u003cp\u003eZhang W, Wang D, Cao D, Chen J, Wei X (2024). Exploring the potentials of\u003cem\u003e Sesuvium portulacastrum\u003c/em\u003e L. for edibility and bioremediation of saline soils. Front Plant Sci. 15:1387102. https://doi.org/ 10.3389/fpls.2024.1387102\u003c/p\u003e\n\u003cp\u003eZhang Z, Chen X, Guan X, Liu Y, Chen H, Wang T, Mouekouba LD, Li J, Wang A (2014). A genome-wide survey of homeodomain-leucine zipper genes and analysis of cold-responsive HD-Zip I members' expression in tomato. Biosci Biotechnol Biochem. 78:1337-1349. https://doi.org/ 10.1080/09168451.2014.923292\u003c/p\u003e\n\u003cp\u003eZhao Y, Zhou Y, Jiang H, Li X, Gan D, Peng X, Zhu S, Cheng B (2011). Systematic analysis of sequences and expression patterns of drought-responsive members of the \u003cem\u003eHD-Zip\u003c/em\u003e gene family in maize. PLoS One. 6:e28488. https://doi.org/ 10.1371/journal.pone.0028488\u003c/p\u003e\n\u003cp\u003eZhou H, Li Y, Yuan B, Nie Q, Xiang Z, He L, Wang Y, Yang Z, Wang J, Hui S, Wang X (2025). Genome-wide analysis of ascorbate peroxidase and functional characterization of \u003cem\u003eSpAPX249b\u003c/em\u003e and \u003cem\u003eSpAPX285c\u003c/em\u003e for salt tolerance in \u003cem\u003eSesuvium portulacastrum\u003c/em\u003e L. Plant Cell Rep. 44:83. https://doi.org/ 10.1007/s00299-025-03466-1\u003c/p\u003e\n\u003cp\u003eZhu Y, Song D, Xu P, Sun J, Li L (2018). A \u003cem\u003eHD-ZIP III\u003c/em\u003e gene, \u003cem\u003ePtrHB4\u003c/em\u003e, is required for interfascicular cambium development in \u003cem\u003ePopulus\u003c/em\u003e. Plant Biotechnol J. 16:808-817. https://doi.org/ 10.1111/pbi.12830\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"functional-and-integrative-genomics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"fige","sideBox":"Learn more about [Functional \u0026 Integrative Genomics](http://link.springer.com/journal/10142)","snPcode":"10142","submissionUrl":"https://submission.nature.com/new-submission/10142/3","title":"Functional \u0026 Integrative Genomics","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Sesuvium portulacastrum, HDZ gene family, Cd tolerance, SpHDZ39, SpHDZ41","lastPublishedDoi":"10.21203/rs.3.rs-8285582/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8285582/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe \u003cem\u003ehomeodomain-leucine zippers\u003c/em\u003e (\u003cem\u003eHDZs\u003c/em\u003e) function as key regulators to regulating plant growth, development, and environmental responses. Its functions have been extensively investigated in many plant species, while the \u003cem\u003eHDZs\u003c/em\u003e family in \u003cem\u003eSesuvium portulacastrum\u003c/em\u003e has not been thoroughly explored. We carried out a genome wide analysis of \u003cem\u003eS. portulacastrum\u003c/em\u003e to identify and characterize the \u003cem\u003eSpHDZs\u003c/em\u003e. The functional of SpHDZ39 and SpHDZ41 were investigated through subcellular localization, transient expression, and yeast one hybrid assays. Their roles in the response to cadmium stress were further analyzed using quantitative real time polymerase chain reaction and transgenic approaches. We identified 46 \u003cem\u003eSpHDZs\u003c/em\u003e in the \u003cem\u003eS. portulacastrum\u003c/em\u003e genome and classified into four subgroups. The \u003cem\u003eSpHDZs\u003c/em\u003e were unevenly distributed across 21 chromosomes. Their physicochemical features were examined, including isoelectric points, hydrophobicity, stability, and predicted secondary and tertiary structures. Gene duplication analysis revealed that all identified duplications resulted from whole-genome or segmental events, and most gene pairs exhibited signs of purifying selection. Promoter analysis indicated the presence of numerous \u003cem\u003ecis\u003c/em\u003e-elements associated with stress signaling and development. Expression profiling of heavy metal and salt treatment identified a subset of \u003cem\u003eSpHDZs\u003c/em\u003e that respond to these treatments, of which, \u003cem\u003eSpHDZ39\u003c/em\u003e and \u003cem\u003eSpHDZ41\u003c/em\u003e showed significant induction under cadmium treatment. Both SpHDZ39 and SpHDZ41 functioned as transcription factors, and transgenic experiments demonstrated that they enhanced cadmium tolerance by promoting root growth and increasing biomass. These results contribute to a deeper understanding of \u003cem\u003eSpHDZs\u003c/em\u003e function in halophytic species and provide candidate genes for future applications in low cadmium crop breeding.\u003c/p\u003e","manuscriptTitle":"Ectopic expression of the Sesuvium portulacastrum genes SpHDZ39 and SpHDZ41 enhances cadmium tolerance in Arabidopsis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-11 13:48:38","doi":"10.21203/rs.3.rs-8285582/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-01-15T23:51:04+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-15T20:28:11+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-07T20:50:45+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-06T06:34:50+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"242322965835121915423033787012190488469","date":"2025-12-12T00:35:43+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"87931886210608153920482984096010852150","date":"2025-12-11T14:58:42+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"128837016680488226474872416109048654601","date":"2025-12-11T12:42:33+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"302210791085429452129409207026513724268","date":"2025-12-10T21:22:40+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"184085226413587717019983750118182430135","date":"2025-12-09T08:53:20+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-12-08T21:19:55+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-08T15:35:40+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-12-08T04:40:28+00:00","index":"","fulltext":""},{"type":"submitted","content":"Functional \u0026 Integrative Genomics","date":"2025-12-05T08:31:47+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"functional-and-integrative-genomics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"fige","sideBox":"Learn more about [Functional \u0026 Integrative Genomics](http://link.springer.com/journal/10142)","snPcode":"10142","submissionUrl":"https://submission.nature.com/new-submission/10142/3","title":"Functional \u0026 Integrative Genomics","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"3ca2b482-b1ef-4637-b939-00dae05ed7cd","owner":[],"postedDate":"December 11th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-02-16T16:03:22+00:00","versionOfRecord":{"articleIdentity":"rs-8285582","link":"https://doi.org/10.1007/s10142-026-01836-w","journal":{"identity":"functional-and-integrative-genomics","isVorOnly":false,"title":"Functional \u0026 Integrative Genomics"},"publishedOn":"2026-02-12 15:58:52","publishedOnDateReadable":"February 12th, 2026"},"versionCreatedAt":"2025-12-11 13:48:38","video":"","vorDoi":"10.1007/s10142-026-01836-w","vorDoiUrl":"https://doi.org/10.1007/s10142-026-01836-w","workflowStages":[]},"version":"v1","identity":"rs-8285582","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8285582","identity":"rs-8285582","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}