Functions of CiHY5 in regulating the salt tolerance of Chrysanthemum revealed by transgenic Chrysanthemum indicum

Functions of CiHY5 in regulating the salt tolerance of Chrysanthemum revealed by transgenic Chrysanthemum indicum | 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 Functions of CiHY5 in regulating the salt tolerance of Chrysanthemum revealed by transgenic Chrysanthemum indicum Bin Xia, Ziwei Li, Xiaowei Liu, Yujia Yang, Shengyan Chen, Bin Chen, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4699886/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Cultivated chrysanthemums are susceptible to abiotic stress, and the intricate polyploidy complicates the discovery of resistance genes. Chrysanthemum indicum is a native diploid species with strong resistance, which makes it an important resource for investigating stress resistance genes and improving genetic traits in ornamental chrysanthemum plants. Results In this study, we cloned the CiHY5 gene and generated both overexpressing CiHY5 (OE- CiHY5 ) and suppressing CiHY5 (RNAi- CiHY5 ) transgenic chrysanthemum lines. After salt stress treatment, compared with the WT plants, the OE- CiHY5 plants exhibited a lower Malondialdehyde content and less leaf electrolyte leakage and significantly greater antioxidant enzyme activity. In contrast, the physiological parameters of the RNAi- CiHY5 plants exhibited opposite trends. Moreover, the Na + /K + ratio in both the leaves and roots of the OE- CiHY5 plants significantly decreased in contrast with that in the leaves and roots of the WT and RNAi- CiHY5 plants. The qRT‒PCR results showed that the expression levels of downstream stress response genes, such as CiRAB18 , CiERF1 , CiABF2 , CiABF4 , and CiDREB1D, were significantly greater in the OE- CiHY5 plants than in the WT plants. Additionally, a yeast one-hybrid assay revealed that CiHY5 could directly bind to the promoter of CiABF4 and activate CiABF4 expression. Transient overexpression of CiABF4 in C. indicum leaf discs also improved salt stress tolerance. Conclusions Overall, we concluded that overexpressing CiHY5 enhanced but RNAi- CiHY5 reduced salt tolerance in C. indicum , acting as a pivotal candidate stress resistance gene that participates in the salt stress response at least partially in an ABA-dependent manner. The above findings demonstrated the molecular mechanisms underlying the CiHY5-mediated salt stress response and laid the foundation for the molecular breeding of chrysanthemum plants to improve resistance. Chrysanthemum CiHY5 transgenic Salt-stress ABA Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Background Plants may encounter a variety of adverse environmental conditions, such as extreme temperatures, water deficiencies, drought and salinity stresses, affecting their growth and productivity [ 1 ]. To overcome abiotic stresses, plants have evolved a series of mechanisms involving the regulation of transcription factors (TFs) for adaptive physiological and developmental processes, such as those in the bHLH, WRKY, bZIP, and MYB TF families [ 2 ]. As the dominant energy source for photosynthesis, light is an indispensable environmental factor for plant growth and development and plays pivotal roles in the process of mitigating abiotic stresses. Previous research has shown that plant growth inhibition induced by salt stress is strongly affected by light [ 3 ]. The salt tolerance of Arabidopsis is positively correlated with light intensity, indicating that light signals broadly regulate intricate molecular networks involved in salt stress [ 4 ]. ELONGATED HYPOCOTYL 5 (HY5), a basic leucine zipper (bZIP) family TF, has been reported to participate mainly in genome-wide large-scale transcriptional reprogramming induced by light-dark environment transformation and to act as a master regulator that regulates various physiological and biological processes, as well as abiotic stress responses; this gene is regarded as a pivotal candidate gene for the breeding of Chrysanthemum for abiotic stress tolerance [ 5 , 6 ]. Recent research has shown that HY5 physically interacts with COP1, a pivotal negative regulator of photomorphogenesis, resulting in the ubiquitination and degradation of HY5 to maintain homeostasis in etiolated plants [ 7 ]. Additionally, HY5 has been identified as a positive regulator of seedling photomorphogenesis, lateral root development and the root gravitropism response in hy5 mutants [ 8 , 9 ]. HY5 is considered a high-level hierarchical regulator of transcriptional networks involved in multiple signal transduction and metabolic pathways, such as pigment accumulation, nutrient assimilation, hormone signaling, and abiotic stress responses [ 10 – 17 ]. Approximately one-third of genes in Arabidopsis are directly or indirectly affected by HY5, and ~ 3000 of these genes directly target HY5 binding sites, as determined by ChIP-chip experiments [ 18 , 19 ]. This may be a critical factor underlying the multifarious roles of HY5 in abundant functions. Recent studies have elucidated the involvement of HY5 in secondary metabolism regulation. For instance, HY5 directly binds to MYB12/PFG1 , and MYB75/PAP1 and PIF3 regulate flavonoid biosynthesis and anthocyanin gene expression, further promoting pigment accumulation [ 20 – 22 ]. In a recent review, HY5 was shown to participate in response mechanisms by targeting downstream genes, such as QH6 (monoterpene β-pinene synthase), RAP2.1 and LEA18 (cold-responsive genes), WRKY genes (involved in defense responses), and NIA2 and NIR1 (nitrogen signaling responses), to regulate terpenoid biosynthesis, cold acclimation, plant cell death and nutrient assimilation [ 13 , 23 – 25 ]. Recent research has extensively investigated the interplay of HY5 with physically interacting proteins integrating signaling pathways involved in the response to abiotic stresses in plants. The expression of HY5 in the nucleus is affected by the nuclear and cytoplasmic distribution of COP1 at low temperatures, leading to the regulation of downstream cold-induced genes and cold adaptation in Arabidopsis [ 26 ]. A convergent study showed that HY5 directly or indirectly integrates cold-responsive and ABA signaling pathways by binding the promoters of CBF and MYB15 , resulting in sophisticated adaptive regulation leading to cold resistance in tomato [ 16 ]. Similarly, in apple, MdHY5 upregulated the expression of MdMYB108L via a CBF-dependent pathway, leading to enhanced cold tolerance in apple [ 27 ]. Furthermore, the overexpression of HY5 in tobacco compensates for the phenotypes of hy5 mutants and enhances tolerance to salt stress by regulating reduced ROS accumulation in transgenic lines [ 28 ]. More recently, HY5 was shown to maintain the modification of histone H3K4me3 by binding to the promoter of the Δ 1 -pyrroline-5-carboxylate synthetase 1 ( P5CS1 ) gene and contributing to the memorability of proline under salt stress, thereby contributing to the memory of proline under salt stress and enhancing salt resistance in Arabidopsis, along with rapid accumulation and expression upon exposure to salinity [ 25 ]. Moreover, comparative transcriptome analysis revealed that HY5 plays a central role in the ABA signal transduction pathway, which negatively regulates drought stress under green photoinduction conditions [ 29 ]. Over the course of evolution, hormones have become crucial means by which plants integrate complex transcriptional regulatory mechanisms to mitigate adverse effects and gain the adaptive capacity to resist external stresses [ 30 ]. Notably, previous work on the integration of the ABA signaling pathway and HY5 has indicated that HY5 mediates multiple regulatory mechanisms to survive under saline conditions. During the period of seed germination and seedling development, the high-affinity binding of HY5 to the promoter of ABI5 enhances resistance to environmental stresses, along with the rapid adaptability of plants to the root system and photoautotrophy after germination in an ABA signaling manner [ 31 ]. Subsequent studies have shown that HY5 additionally binds to the promoters of ABA RESPONSIVE ELEMENT BINDING FACTORS (AREB/ABF) bZIP TFs in Arabidopsis , which are homologous to ABI5 and are regarded as subfamily members of ABI5, such as ABF1, ABF3 and ABF4 [ 18 , 32 ]. In Arabidopsis , HY5/HYH and RSM1 independently or in some way independently induce the expression of ABI5- and ABI5 -targeted genes, thereby regulating ABA and abiotic stress responses [ 17 ]. Furthermore, HY5 mediates root architecture by promoting primary root growth and inhibiting lateral root growth via the ABA signaling pathway, further facilitating seedling establishment and soil composition absorption under drought and salinity stresses [ 9 , 33 ]. Owing to the long-term accumulation of natural and anthropic factors, soil salinization is one of the main abiotic stress conditions faced by plants, ultimately causing quality degradation and economic losses [ 34 ]. Chrysanthemum is highly appreciated as a cutting-flower industry material and is vulnerable to high salinity conditions. Chrysanthemum indicum , a diploid species native to China, is renowned for its ornamental and medicinal properties and is well adapted to adverse environmental conditions [ 35 , 36 ]. Previously, only research on HY5 in chrysanthemum ( Chrysantemum morifolium ) showed that downregulated expression of CmHY5 in response to high temperature positively promoted anthocyanin synthesis according to transcriptome data [ 37 ]. However, little is known about the ectopic expression of HY5 and its involvement in signaling pathways and abiotic stresses in other species. To further elucidate the function of HY5 in chrysanthemum, CiHY5 was isolated from C. indicum and characterized. We verified that overexpression of CiHY5 contributed to adaptive phenotypes and enhanced resistance to salt stress by binding to the promoter of the CiABF4 gene in an ABA-dependent manner. To some extent, we demonstrated that CiHY5 is a promising candidate gene for the response to salt stress for genetic engineering and the development of the chrysanthemum breeding industry. Materials and methods 2.1. Plant materials, growth conditions and treatments Chrysanthemum indicum , a native diploid species, was obtained from Hubei, China, and cultivated in the nursery of Northeast Forestry University for test materials. Te voucher specimens, C. indicum (KUN 1445652) was identified by Xinxin Zhu and its sheet was deposited in the herbaria KUN ( https://www.cvh.ac.cn/spms/detail.php?id=21c961b0 ). Rooted cuttings were transplanted into pots of the same size containing a mixture of V (humus):V (vermiculite):V (perlite) = 2:1:1, pH = 6.65 cultivation substrate under natural conditions (temperature of 23 ± 1°C, relative humidity of 65%-75%, 16 h/8 h light/dark photoperiod) [ 38 ]. For the light environment and UV-B irradiation treatments, two-week-old acclimated seedlings with consistent growth status were subjected to darkness, white light (intensity = 180 µmol m − 2 s − 1 , source: Osram L18W/30 tubes, NE, USA) and UV-B irradiation (intensity = 400 µmol m − 2 s − 1 , source: T12 306 nm narrowband UV-B tubes, Huaqiang, Nanjing) for 0, 1, 6, 24, 48 h, respectively. The consistent stative seedlings were precultured with Hoagland’s nutrient solution for one week and then transferred to the same solution containing 200 mmol L − 1 NaCl or 100 µmol L − 1 ABA for salt stress and ABA treatment [ 39 ]. The control groups were concurrently treated with Hoagland’s nutrient solution under the same growth conditions, and three replicates were performed. Following these treatments, the leaves were sampled for 0, 1, 3, 6, 12 and 24 h and immediately stored at -80°C in preparation for subsequent experiments. The roots, stems, leaves, buds and ray florets were sampled for tissue-specific expression analysis. The expression levels of CiHY5 were validated by quantitative real-time PCR (qRT‒PCR) with specific primers (Table S1 ). 2.2. Gene cloning, structure and phylogenetic analysis Based on the transcriptome data of C. indicum in previous work, we isolated the CiHY5 gene. CiHY5 was further identified via analysis of the chrysanthemum genome database ( http://www.amwayabrc.com/zh-cn/index.html ), the MUM GARDEN ( http://mum-garden.kazusa.or.jp/ ) and the TAIR website ( https://www.A.thaliana.org/index.jsp ) [ 40 , 41 ]. For sequence alignments and phylogenetic analysis, we used amino acid sequences of CiHY5 homologs in Arabidopsis and other species obtained from the online database NCBI ( https://blast.ncbi.nlm.nih.gov/Blast.cgi ) for alignment and analysis via ClustalX1.83 and MEGA7 via the neighbor-joining (NJ) method [ 42 , 43 ]. Specific primers were designed based on the genomic sequence containing the complete open reading frame (ORF) sequence and RNA interference (RNAi) fragment of CiHY5 , and enzyme cut sites were added at the 5’-terminus of the upstream and 3’-terminus of the downstream primers, respectively. The target products were amplified with C. indicum cDNA template by RT‒PCR according to KOD DNA Polymerase instructions (TOYOBO, Japan). The expected CiHY5 and CiHY5- RNAi fragments were inserted into the pEASY ®-Blunt Zero vector for reproduction and then transformed into Agrobacterium tumefaciens EHA105 receptor cells (Weidi, China) for integration into C. indicum leaves. All of the primer sequences are listed in Supplementary Table S1 . 2.3. RNA extraction, RT‒PCR and qRT‒PCR Total RNA from C. indicum was extracted with an RNA extraction kit (OMEGA, USA) and reverse transcribed to the first strand of cDNA using a ReverTra Ace-α Kit (TOYOBO, Japan). According to the initial RNA concentration, cDNA was diluted as a template to clone the CiHY5 and CiHY5- RNAi fragments by RT‒PCR, and the corresponding primer sequences are listed in Table S1 . qRT‒PCR was performed with three biological replicates of UltraSYBR Mixture (CWBIO, China) on an ABI Prism 7500 system (Applied Biosystems, USA) as follows: 95°C for 10 min; 40 cycles of 95°C for 15 s and 60°C for 1 min; and melting at 95°C for 15 s, 60°C for 1 min, 95°C for 15 s and 60°C for 15 s. The relative expression levels of genes were calculated using the 2 −ΔΔCT method with the reference gene CiEF1α (GenBank Accession No. KF305681) from C. indicum (Table S1 ). 2.4. Subcellular localization of CiHY5 The full-length coding region (without the stop codon) of the CiHY5 integrated green fluorescent protein (GFP) tag was used to construct the recombinant vector pBI121-CaMV35S:CiHY5::GFP ( 35S:CiHY5::GFP ), and the pBI121-CaMV 35S:GFP ( 35S:GFP ) empty vector was used as a positive control. The prepared infiltration buffer (10 mmol L − 1 MES, 150 µmol L − 1 AS and 10 mmol L − 1 MgCl 2 ) was used to suspend the centrifuged Agrobacterium liquid to OD 600 = 1.0, which was subsequently transformed with the recombinant vectors described above. Agrobacterium suspensions of 35S:CiHY5::GFP and 35S:GFP were injected into the abaxial surface of tobacco ( Nicotiana benthamiana ) leaves. After injection, the tobacco plants were cultivated in darkness for 48–72 h. Then, a disk of approximately 0.25 cm 2 in the area from the injected leaves was cut to observe the fluorescence signal [ 44 ]. The GFP signal was detected by a Zeiss LSM710 confocal laser scanning microscope (Nikon, Japan) with the following settings: 488 nm excitation and 525 nm emission wavelengths. 2.5. Construction of plant expression vectors and identification of transgenic plants For overexpression- CiHY5 vector construction, the complete ORF sequence of CiHY5 with XbaI/XmaI sites was cloned and integrated into the pBI121 vector using T4 DNA ligase (NEB, USA), and the resulting construct was named pBI121 - CiHY5 (Fig. S1 ). For RNAi- CiHY5 vector construction, a 242 bp specific sequence of CiHY5 was amplified into sense and antisense fragments containing XhoI/KpnI and XbaI/HindIII sites, respectively, in the pHANNIBAL vector, which automatically formed a repeat hairpin-silencing RNA (ihpRNA) structure to degrade the targeted mRNA. The ihpRNA structure with the 35S promoter and Nos terminator was then recombined into the pART27 vector pART27-CiHY5 (Fig. S1 ). The terminal vectors pBI121-CiHY5 and pART27-CiHY5 were separately inserted into Agrobacterium receptor cells and stored at -80°C in 50% glycerol. Leaves from two-week-old tissue culture-generated C. indicum plants were used as explants for Agrobacterium -mediated transformation. Infected leaves were cultured with differentiation media supplemented with specific antibiotics (5 mg L − 1 kanamycin, Kana for pBI121-CiHY5 , 1 mg L − 1 spectinomycin, and Spec for pART27-CiHY5 ) until the resistant buds were differentiated from the callus tissues. Then, the resistant seedlings were transferred to rootling media. Two weeks later, transgenic plants were selected and identified by RT‒PCR and qRT‒PCR analysis. Three transgenic C. indicum lines in which CiHY5 was overexpressed or suppressed (OE- CiHY5 and RNAi- CiHY5 ) or the wild-type (WT) control was transplanted into the same size pots consistent with the cultivation conditions described above. Transgenic lines with different expression levels (OE-2, 4, 7 and Ri-1, 2, 4) and WT seedlings were selected for subsequent experiments. 2.6. Transgenic plant phenotype observation and salt stress treatment Transgenic and WT C. indicum plants were subjected to tissue culture. Once the plants rooted had 4–5 true leaves, the plants of the selected transgenic and WT lines were transplanted into plastic pots for vegetative growth. Then, transgenic and WT plants in neat and consistent growth states were cultivated in plastic pots of the same size and subjected to conventional maintenance management at an incubation temperature of 23 ± 1°C and a relative humidity of 65%-75%. Two-month-old independent wild-type (WT) and transgenic plants were used to observe leaf area, internode length and diameter phenotypes. For salt stress, 50 ml of 200 mmol/L NaCl solution was added to each pot once every two days, and the oozed salt solution was removed from the trays to prevent salt loss until it was fully absorbed into the soil. Leaf relative water content (RWC), total chlorophyll content, malondialdehyde (MDA) content, proline content and antioxidant enzyme activities (superoxide dismutase, SOD and catalase, CAT) were determined from the 4th-6th leaves beneath terminal buds after 10 days of salt stress treatment (Takahashi et al., 2007; Wang et al., 2003; Wang et al., 2015). We collected roots less than 2 mm in diameter and leaves as materials to measure the relative contents of Na + and K + after 0 d and 10 d of salt stress by ICP‒MS using inductively coupled plasma‒mass spectrometry (ICP‒MS) with three biological replicates. 2.7. Isolation and in silico analysis of the promoter Thermal asymmetric interlaced PCR (TAIL-PCR) was conducted to amplify the promoter region of CiABF4 ( proCiABF4 ) using a Genome Walking Kit (Takara, USA) according to the manufacturer’s instructions. Four arbitrary degenerate primers ( AP1 , AP2 , AP3 , and AP4 ) were used as sense primers, and three specific primers ( SP1 / SP2 / SP3 - proCiABF4 ) with higher annealing temperatures were designed according to the 5’-terminal coding sequence of CiABF4 (Table S1 ). Three rounds of TAIL-PCR were employed to obtain amplified fragments of different lengths. After splicing and sequencing, a 1401 bp fragment was designated proCiABF4 . The cis -acting elements of proCiABF4 were analyzed via the PlantCARE database ( https://bioinformatics.psb.ugent.be/webtools/plantcare/html/ ). The sequenced promoter was ligated into the plant expression vector by replacing the CaMV35S promoter region between the BamHI and HindIII restriction endonuclease sites, generating the ProCiABF4:Gus vector. Agrobacterium strain EHA105 harboring ProCiABF4:Gus and 35S:Gus was cultured in LB liquid media supplemented with 20 mg/L rifampicin (Rif) and 50 mg/L Kana at approximately 28°C for 16 h until the OD600 reached 0.6–0.8. The ProCiABF4:Gus and 35S:Gus Agrobacterium strains were collected and suspended in infiltration buffer (10 mmol/L MES, 200 µmol/L AS, and 10 mmol/L MgCl 2 ) to an OD600 of 0.2–0.3. The ProCiABF4:Gus and 35S:Gus suspension solutions were used to infiltrate young leaves of 6-week-old tobacco ( N. benthamiana ) plants with syringes. The infected plants were placed in Gus staining solution for one night at 25–37°C in darkness, and the promoter activity was then observed after decolorization by soaking in 70% ethanol. Agrobacterium from 35S:Gus -infected leaves was used as the positive control. The primers used for promoter cloning and vector construction are listed in Table S1 . 2.8 Transactivation activity analysis and yeast one-hybrid assay For transactivation activity analysis, the full-length coding region of CiHY5 was fused to the GAL4 DNA binding domain of the pGBKT7 vector pGBKT7-CiHY5 ( BD-CiHY5 ) (Fig. S2 ). pGBKT7-CiHY5 + pGADT7-T ( BD-CiHY5 + AD-T , experimental group), pGBKT7-53 + pGADT7-T ( BD-53 + AD-T , positive control) and pGBKT7-Lam + pGADT7-T ( BD-Lam + AD-T , negative control) vectors were transformed into yeast competent cells according to the instructions of Y2HGold Chemically Competent Cell (Weidi, China), and transcriptional activation activity was detected by screening the growth status on SD/-Trp/-Leu and SD/-Trp/-Leu/-His/-Ade/X-α-gal (20 mg ml − 1 ) media. For the yeast one-hybrid (Y1H) assay, the full-length coding region of CiHY5 was inserted into the pGADT7-Rec2 ( AD-Rec2 ) vector with the SmaI enzyme cutting site via homologous complementary technology to construct the prey vector pGADT7-Rec2-CiHY5 ( AD-CiHY5 ) (Fig. S2 ). Subsequently, the CiABF4 promoter fragment was amplified from C. indicum genomic DNA and inserted into the pHIS2 vector with EcoRI and SacI enzyme cutting sites to construct the bait vector pHIS2 - proCiABF4 (Fig. S2 ). The AD-CiHY5 + pHIS2-proCiABF4 (experimental group) and AD-Rec2 + pHIS2-proCiABF4 (control group) vectors were cotransformed into Y187 Gold yeast (Weidi, China) according to the manufacturer’s instructions. Yeast cells were subsequently placed on SD/-Trp/-Leu media to verify growth status, and SD/-Trp/-Leu/-His media supplemented with 50 mmol/L 3-aminotriazole (3-AT) were used to verify the interaction between CiHY5 and the promoter of CiABF4 . The yeast cells were incubated at 30°C for 3 days. The RT‒PCR primers used for vector construction are listed in Table S1 . 2.9. Transient transformation of CiABF4 and salt treatment Based on previous transcriptome data, the full-length sequence of CiABF4 was aligned, and the complete ORF fragment was subsequently cloned. The amplified PCR product was purified and inserted into pBI121 to construct the overexpression- CiABF4 (OE -CiABF4 ) vector pBI121-CiABF4 , as described above. Fresh leaf discs of C. indicum were cut for two days of preculture and then infected with the suspended pBI121-CiABF4 strain by means of Agrobacterium -mediated transient transformation, which utilized a vacuum negative pressure infection method to generate a negative pressure environment to promote the effective infiltration of Agrobacterium into plant tissue cells to accelerate transformation for two days of coculture in a 30°C incubator [ 45 , 46 ]. Immediately, we transferred the uninfected (WT) and OE -CiABF4 leaf discs to MS media for two days, transferred them to salt-stress media supplemented with 50 mmol/L NaCl for 4 days, and finally allowed the plants to recover from the normal media for 4 days to observe the phenotypes. 2.10. Statistical analysis Statistical analyses were performed using Excel. Three biological replicates are presented as the means ± SDs, and the significance levels were compared with Duncan's multiple range test with one-way ANOVA ( p < 0.01) using SPSS 23. Results 3.1. Cloning and sequence analysis of CiHY5 from C. indicum To explore the functions of pivotal regulators of the light-mediated pathway in response to abiotic stresses in chrysanthemum, we isolated a member of the bZIP family, CiHY5 , from C. indicum transcriptome data. Based on the cDNA library, the complete 477 bp open reading frame (ORF) sequence of CiHY5 (GenBank accession number No. OP589306), which encodes a 158 amino acid polypeptide, was cloned, and the predicted molecular weight of the CiHY5 protein was 17.51 kDa. Sequence alignment revealed that CiHY5 contains a highly flexible and disordered N-terminal structure and a conserved bZIP_HY5-like domain in the C-terminus from amino acids 90 to 141, which may directly bind to G-box elements in promoter regions to activate gene expression and regulate signaling networks (Fig. 1 A). Additionally, CiHY5 contains a basic region from amino acids 77 to 109 to a stable tertiary structure and a leucine zipper (LZ) domain from amino acids 110 to 141, which are responsible for DNA binding and dimerization, respectively (Fig. 1 A). In the phylogenetic trees composed of CiHY5 and 10 HY5s in the other 10 species (Fig. 1 B), CiHY5 was classified with CmHY5, AaHY5 and HaHY5 from C. morifolium , Artemisia annua and Helianthus annuus in the same branch, and it was also close to AtHY5 from Arabidopsis . The length of the evolutionary branch represented the genetic variability and evolutionary distance, which indicated that CiHY5 and AaHY5 had the highest sequence similarities with the shorter branch, implying that the divergence of the HY5 family occurred later in chrysanthemum and Artemisia annua. Additionally, CiHY5 was more closely related to dicotyledons than to monocotyledons, such as OsHY5 and ZmHY5 from Oryza sativa and Zea mays , indicating the consistency of phylogenetic relationships and evolutionary conservation among CiHY5 and HY5s in other species. 3.2. Nucleus subcellular localization of CiHY5 and tissue-specific expression Agrobacterium -mediated transient transformation of N. benthamiana leaves with the 35S:CiHY5::GFP fusion plasmid resulted in a GFP signal only in the nucleus (Fig. 2 A). As a positive control, a GFP signal was observed throughout the cell after transfection with the 35S:GFP plasmid. CiHY5 was localized to the nucleus. Tissue-specific expression analysis revealed that the transcript levels of CiHY5 in the stems and leaves were greater than those in the other tissues (Fig. 2 B). Relatively lower expression levels were detected in ray florets, and the lowest expression levels were detected in roots and buds. The expression level of CiHY5 in leaves was approximately 1.4, 64.6 and 37.6 times greater than that in ray florets, buds and roots, respectively. Taken together, the gene expression and subcellular localization data suggested the potential functions and regulation of the CiHY5 transcription factor. 3.3. CiHY5 expression pattern analysis CiHY5 has received a great deal of attention due to its pivotal regulatory capacity in response to external conditions via the light signaling pathway. The qRT‒PCR results revealed that CiHY5 expression was strongly correlated with light conditions (Fig. S3). Both white light and UVB radiation significantly stimulated CiHY5 expression, but the expression of CiHY5 did not significantly change in darkness, suggesting that CiHY5 is an important gene involved in the response to the light signaling pathway. Under salt stress, the expression of CiHY5 significantly decreased and reached its lowest level at 1 h, which was 0.54 times that at 0 h (Fig. 2 C). Then, the expression levels gradually increased and peaked at 12 h at 1.52 to 0 h. When plants are subjected to environmental stresses, the hormone metabolism balance is disturbed. Interestingly, the expression trends of CiHY5 under 100 µmol/L ABA were similar to those under salt stress. At 1 h, the expression of CiHY5 decreased to the lowest level (0.39 to 0 h) and then gradually increased, but there were no significant differences at 6 h, 12 h or 24 h compared to 0 h under ABA treatment (Fig. 2 D). These findings suggested that CiHY5 may act as a pivotal component involved in light, salt stress and ABA signaling responses in C. indicum. 3.4. Identification of transgenic C. indicum To investigate the biological functions of CiHY5 in response to salt stress, the complete ORF (477 bp) and specific RNA interference (RNAi) fragment (242 bp) sequences of CiHY5 were cloned and inserted into the overexpression and RNAi vectors pBI121 and pART27 , respectively, and subsequently transformed into host cells (Fig. S1 , S4). Through Agrobacterium -mediated leaf disc transformation, we screened stable transgenic lines (Fig. S5). RT‒PCR was used to amplify 675 bp, 1293 bp and 2051 bp products from the Kanamycin (Kana) resistance tag, universal primers from the pBI121 vector and 35S promoter region primers, respectively, to verify the OE -CiHY5 transgenic plants, and a 789 bp product from the Spectinomycin (Spec) resistance tag primers was used to verify the RNAi -CiHY5 transgenic plants (Table S1 , Fig. S5). There were no amplification results for the above primers in the WT plants. Eventually, we obtained seven independent OE -CiHY5 transgenic lines (OE-1 to OE-7) and four RNAi -CiHY5 lines (Ri-1 to Ri-4) for further exploration. Compared with those in the WT plants, the relative expression levels of CiHY5 were significantly greater in the OE- CiHY5 lines and lower in the RNAi -CiHY5 lines, indicating the successful generation of transgenic C. indicum plants (Fig. S5). OE-2, OE-4, and OE-7 and the Ri-1, Ri-2, and Ri-4 lines with high, medium, and low expression levels of CiHY5 were selected for further investigation. 3.5. CiHY5 alters the growth and development of C. indicum To provide an overview of the phenotypes of the transgenic plants, we observed the phenotypes of the C. indicum WT, OE- CiHY5 and RNAi- CiHY5 lines. The leaf area, internode length and diameter of 8-week-old plants with consistent growth from cuttings were calculated. As shown in Fig. 3 A and Fig. 3 B, compared with those of the WT plants, the leaves of the OE -CiHY5 plants were greater, and the OE-2 line had the greatest leaf area among all the transgenic lines. In terms of leaf number, the leaf area of the RNAi -CiHY5 plants did not differ from that of the WT plants, but the leaf area of the Ri-1, Ri-2 and Ri-4 plants significantly decreased compared with that of the WT plants (Fig. 3 C). Further exploration revealed that the OE -CiHY5 plants exhibited more and stronger phenotypes than did the WT plants. In contrast, the RNAi- CiHY5 plants presented shorter heights and greater lignification at the bottom of the stems than did the WT plants (Fig. 3 D). Compared with the WT and RNAi-CiHY5 lines, the OE-CiHY5 lines exhibited obviously greater increases in both the internode length and diameter and greater synchronous growth (Fig. 3 F, G, H). Furthermore, the RNAi-CiHY5 plants had relatively more roots and earlier flowering than did the WT and OE-CiHY5 plants, which was in agreement with the findings in Arabidopsis (Fig. 3 E, Fig. S6). 3.6. CiHY5 affects salt stress tolerance in C. indicum Given the multifaceted roles of HY5 in the regulation of plant growth and development in Arabidopsis , we investigated the salt stress resistance of WT and transgenic plant lines subjected to 200 mmol/L NaCl for 10 days. During the first 5 days, the growth of the plants in the OE -CiHY5 and RNAi -CiHY5 lines did not exhibit obvious differences, but during the following days, the plants in the RNAi -CiHY5 lines were more severely injured by salt stress than were those in the WT and OE -CiHY5 lines; for example, the plants were curled and dehydrated with a brown color on a large range of leaves. In contrast to those in the WT and RNAi- CiHY5 lines, slight or limited injury occurred at the edge of the OE -CiHY5 leaves with erect stems during growth (Fig. 4 A, B). After water recovery for 10 d, 100% of the OE -CiHY5 plants survived and continued to sprout new buds from the apex and lateral shoots, whereas the survival rates of the WT and RNAi -CiHY5 plants in the stagnant growth state were only 66.67% and 44.44%, respectively (Fig. 4 C). To further elucidate the molecular mechanism involved in the response to salt stress influenced by CiHY5 , we measured physiological parameters and indexes in WT and transgenic plants. The RWC of leaves did not obviously differ between OE -CiHY5 and RNAi -CiHY5 plants before and after 10 d of salt stress but slightly decreased in the RNAi -CiHY5 line (Fig. S7). Compared with those of the WT plants, the electrolyte leakage rates of the OE-7, Ri-1 and Ri-2 lines decreased by 27.32% and increased by 19.56% and 21.36%, respectively, confirming that the OE -CiHY5 plants could maintain the integrity of the cell membrane under salinity stress but that the RNAi -CiHY5 plants could not (Fig. 4 D). Chlorophyll degradation indicates that photosynthesis is seriously impaired in plants under salinity stress. Under normal growth conditions, the total chlorophyll content of the OE -CiHY5 and RNAi- CiHY5 lines was distinctly different from that of the WT, but there was no apparent difference. However, under salt stress, a significant decrease in the total chlorophyll content in the Ri-1 ( p < 0.05 ) and Ri-4 lines ( p < 0.01 ) is shown in Fig. 4 E, indicating severe chlorophyll degradation and sensitivity to salt stress in the RNAi -CiHY5 plants. In terms of the antioxidant defense system, the SOD activity in the OE -CiHY5 lines significantly increased by 35.78%, 44.34% and 78.60% compared with that in the WT, and the CAT activity in the OE-7 line significantly increased by 36.31% compared with that in the WT (Fig. 4 F, G). Free proline accumulation and MDA reduction are closely related to the extent of injury caused by external environmental stresses in plants. As shown in Fig. 4 H and I, compared with those of the WT plants, the MDA content of the OE-4 and OE-7 lines significantly decreased, and the free proline content of all the RNAi -CiHY5 lines significantly decreased, implying that the OE -CiHY5 plants had a greater capacity to withstand salt stress and that the RNAi- CiHY5 plants had the opposite effect. Taken together, these results revealed that compared with the WT plants, the CiHY5-overexpressing and RNAi- CiHY5 -overexpressing plants exhibited greater and lower salinity resistance, respectively. 3.7. CiHY5 affects the homeostasis of Na + and K + under salt stress The influx and efflux of Na + and K + can reflect the metabolic capacity of cells and further infer salt stress resistance in plants. Under salt stress, the Na + content in the leaves of the three RNAi-CiHY5 lines significantly decreased by 18.77% and 27.84% in the OE-2 and OE-7 lines, respectively, and increased by 41.24%, 32.57% and 25.52%, respectively, compared with that in the WT (Fig. 5 A). However, no significant difference was found in either the K + or Na + content in roots or the K + content in leaves between the WT and transgenic lines (Fig. 5 B, C, D). Intriguingly, the ratio of Na + to K + significantly increased in both the roots and leaves of the Ri-1 and Ri-4 lines but decreased in the roots of the OE-2 and OE-7 lines under saline conditions (Fig. 5 E, F), although there was no obvious difference in the Na + or K + concentration in the roots. For the above six indicators, the trend of absorbed Na + and reduced K + in the OE -CiHY5 lines was opposite to that in the RNAi- CiHY5 lines under salt stress, with a decrease in Na + /K + in the OE -CiHY5 plants. 3.8. CiHY5 modulates the expression of stress-responsive genes related to the ABA signaling pathway Exogenous ABA treatment significantly downregulated the expression of CiHY5 from 1 h to 3 h (Fig. 2 D). We attempted to confirm whether the expression levels of related ABA-dependent or ABA-independent pathway genes were altered in the transgenic plants. As shown in Fig. 6 , compared with those in the WT, the expression levels of the ABA-dependent pathway genes CiRAB18 and CiERD7 significantly increased in the OE -CiHY5 line but decreased in the RNAi -CiHY5 line. ABA-independent pathway genes, such as CiDREB1D and CiERF1 , were significantly upregulated in the OE -CiHY5 plants but relatively downregulated in the RNAi -CiHY5 lines. Moreover, the expression levels of genes encoding a series of protein kinases and protein phosphatases involved in ABA signaling, such as CiPP2C and CiSnRK2 , were significantly variable. In addition, the expression of CiABF4 in the OE -CiHY5 plants was significantly greater than that in the WT plants, and the expression of CiABF4 in the OE-CiHY5 lines increased by 3.48-, 3.59- and 3.39-fold, respectively (Fig. 6 ). It has been reported that ABF4 is involved in the ABA signaling pathway and specifically binds to ABA-responsive elements (ABREs) through SnRK2 in response to salt stress. Additionally, a genome-wide study of HY5 target genes in Arabidopsis suggested that HY5 binds to ABF1 , ABF3 and ABF4 but not to ABF2 (Fernando et al., 2018). These findings suggested that CiHY5 likely affects salt stress tolerance by modulating the accumulation of CiABF4 in an ABA-dependent manner. 3.9. CiHY5 directly combined with the promoter of CiABF4 Given that OE -CiHY5 transgenic plants significantly induced CiABF4 expression, we investigated whether CiHY5 directly bound to the promoter of CiABF4 . According to the instructions of the Genome Walking Kit, we utilized TAIL-PCR technology to amplify the proCiABF4 fragment . Ultimately, we obtained a 1404 bp aligned sequence from the spliced products after three rounds of amplification; this sequence was regarded as proCiABF4 in this study (Fig. S8). The activity of proCiABF4 was verified by evaluation of the Gus gene expression of proCiABF4:Gus following visible blue in tobacco leaves (Fig. S8). Analysis of cis -regulatory elements revealed that a conserved G-box (light-responsive element) was present in the − 1104 bp to -1110 bp region in proCiABF4 (Fig. 7 A). As previously described, HY5 predominantly acts as a pivotal transcription factor by binding G-box cis -acting elements downstream of target genes to regulate physiological and developmental processes in plants. In addition, the proCiABF4 region also contained MeJA-responsive elements (CGTCA motif and TGACG motif), abscisic acid responsiveness elements (ABREs), MYB and MYC recognition sites, an auxin-responsive element (TGA element) and other light responsiveness elements (AE box, ATCT motif, Box 4) (Fig. S8). Transactivation activity analysis revealed that after cotransformation into Y2HGold cells, all the fusion plasmids grew normally on SD-Leu/-Trp media, but only the positive control ( BD-53 + AD-T ) grew and produced blue spots on SD/-Trp/-Leu/-His/-Ade/X-α-gal media (Fig. S9). This finding indicated that there was no transcription of downstream reporter genes through the combination of the BD DNA binding domain and the upstream activation sequence UAS in GAL4. These results indicated that CiHY5 could be used for further verification because it has no transcriptional activation activity. Yeast one-hybrid (Y1H) assay analysis showed that both the AD-CiHY5 + pHIS2-proCiABF4 (experimental group) and AD-Rec2 + pHIS2-proCiABF4 (control group) yeast strains grew normally on SD-Leu/-Trp plates, indicating that the bait and prey vectors were successfully cotransformed into yeast cells (Fig. 7 B). However, the yeast strains transformed with AD-CiHY5 + pHIS2-proCiABF4 obviously grew on SD-Leu/-Trp/-His selection media supplemented with 50 mmol/L 3-AT, but at the same time, the AD-Rec2 + pHIS2-proCiABF4 yeast strains failed to grow (Fig. 7 C). This result suggested that the GAL4 transcriptional domain activated PminHIS3 to express the His reporter gene in the experimental group, but the leakage of the reporter gene was inhibited by 50 mmol/L 3-AT, with no expression in the control group. Taken together, these results suggested that CiHY5 directly binds to the promoter of CiABF4 to activate downstream gene expression. 3.10. CiABF4 enhances salt tolerance in C. indicum The complete ORF sequence of CiABF4 , 1248 bp in length (GenBank accession number no. OP589307), encodes a 415-amino-acid polypeptide (Fig. S10). The phylogenetic analysis revealed that CiABF4 clustered with AtABF4, which possesses a conserved bZIP domain in the C-terminal region (Fig. S10). To clarify whether CiABF4 plays a pivotal role in the salt stress response in chrysanthemum, we performed Agrobacterium -mediated instantaneous transformation with the pBI121-CiABF4 vector to observe the phenotypes of C. indicum leaf discs under salt stress. As shown in Fig. S11A, no obvious phenotypic variations were detected in the leaves of the WT and overexpressing- CiABF4 (OE -CiABF4 ) plants before salt stress treatment. When 50 mmol/L NaCl was exposed to media for 5 days, in contrast with the green and complete leaf plates of OE -CiABF4 , the leaves of the WT plants were tawny, injured and susceptible to bacterial infection (Fig. S11B). After 5 days of normal growth, the OE -CiABF4 leaf plates gradually recovered and continued to differentiate, but most of the WT leaf plates still remained wilted and even putrid (Fig. S11C). These findings preliminarily verified that overexpression of CiABF4 increased the resistance of C. indicum to salt stress . Discussion In recent years, secondary soil salinization caused by human activities has become increasingly serious, especially unsustainable tillage and irrigation, resulting in reduced plant water utilization efficiency, further loss of osmotic pressure regulation in roots, sodium ion toxicity reactions, and eventually physiological damage in plants [ 47 , 48 ]. Transcription factors (TFs), as pivotal switches in signaling networks, provide targets for researching the molecular mechanisms of the external stress response. HY5, a member of the bZIP TF family, is considered a major regulator of the light signaling pathway and is involved in multiple growth and metabolic processes, such as cell elongation and proliferation, hypocotyl growth, chloroplast and pigment accumulation, and shade avoidance in plants [ 49 – 52 ]. There are highly conserved key domains that bind to COP1 in the N-terminus and the coiled coil bZIP domain in the C-terminus, which combines with many gene promoters with ACGT elements (ACE) in the HY5 protein, suggesting that HY5 plays extensive regulatory roles downstream of genes in response to multiple signal transduction pathways [ 53 ]. In addition, HY5 can readily form protein complexes at the peak in the early developmental stage of Arabidopsis . There is intricate crosstalk between hormone signaling pathways and abiotic stress responses in plants. As previously reported, HY5 promotes photomorphogenesis through the coordinated regulation of various hormonal signaling pathways, such as those involving gibberellin (GA), ethylene, abscisic acid (ABA) and brassinolide (BR) [ 54 – 56 ]. The nuclear and cytoplasmic distributions of HY5 affected by salt stress are positive factors that promote the binding of HY5 to the ABI5 promoter, thereby closely influencing the salt stress response in an ABA-dependent manner [ 55 ]. Mounting evidence indicates that HY5 is involved in a broad range of abiotic stress processes, such as ABA, cold, drought and salt stress; however, the specific mechanism of HY5-mediated gene regulation remains unclear [ 17 , 24 ]. Considering the extensive research on the potential conserved roles of HY5 in salt stress tolerance in Arabidopsis , extending research on the functions of HY5 to ornamental chrysanthemum is highly important. In our study, we cloned and characterized the CiHY5 gene and established OE- CiHY5 and RNAi- CiHY5 transgenic lines to explore the resistance of C. indicum to salt stress (Figs. S4 and S5). The temporal expression patterns of CiHY5 in response to salt and ABA treatments and the various expression levels of ABA-responsive genes in the WT and transgenic lines deepened our understanding of the vital roles of CiHY5 in mediating the salt stress response in C. indicum (Figs. 2 and 6 ). Phenotypic evidence revealed that CiHY5 overexpression improved salt tolerance in CiHY5 transgenic plants. Compared with the WT and RNAi-CiHY5 plants, the OE-CiHY5 plants exhibited more robust growth conditions, such as a larger leaf area, longer internode length, stronger stalks, and longer nutritional reproduction with delayed flowering (Figs. 3 and S6), which was consistent with previous findings in Arabidopsis [ 14 ]. Under salt stress, the physiological parameters of the OE -CiHY5 lines were lower than those of the WT plants in terms of electrolyte leakage, free proline content and MDA content (Fig. 4 D, H and I), illustrating that the accumulated osmotic system protected the integrity of the cell membrane and decreased cell injury and lipid peroxidation [ 44 ]. The OE -CiHY5 and RNAi -CiHY5 lines exhibited opposite trends in SOD activity, CAT activity and chlorophyll content (Fig. 4 E, F and G), indicating that the accumulation of active oxygen was effectively eliminated to increase the resistance of the OE -CiHY5 plants to salt stress. Similarly, HY5 is an indispensable regulator required for the accumulation of chlorophyll through binding to a subset of chlorophyll biosynthesis gene promoters in a light-dependent manner [ 57 ]. The exclusion and absorption of internal Na + or Cl − are the main strategies used by plants to adapt to salinity stress [ 58 ]. Under salt stress, the decrease in Na + /K + in the OE -CiHY5 lines implies that the intake of essential mineral nutrients, such as K + , was sufficient rather than the accumulation of Na + , indicating that the generation of defense mechanisms contributed to enhancing salt tolerance in the OE -CiHY5 plants (Fig. 5 ). Moreover, the variation trends of these indices in the RNAi -CiHY5 plants were opposite to those in the OE- CiHY5 plants under salt stress, indicating that the RNAi -CiHY5 plants were sensitive to salt stress. Limited research on the role of HY5 in salt stress has shown that the interaction of the HY5-HDA9 protein complex is significantly induced by salt stress, inhibiting the expression of the HsfA2 gene and improving salt tolerance in Arabidopsis [ 59 ]. In addition, the hy5 mutant displayed opposite sensitivities to salt stress treatment during the seed germination and 7 d seedling growth stages in Arabidopsis , reinforcing the idea that HY5 directly regulates numerous downstream genes to activate intricate regulatory networks [ 17 ]. Plants have evolved sophisticated mechanisms to precisely control stress responses via hormonal pathways. ABA induces the expression of stress-responsive genes, enabling the maintenance of homeostasis of the internal environment in plant cells under external stresses [ 60 ]. HY5 is extensively involved in ABA signaling and salt response during Arabidopsis growth and development. Early studies revealed that the expression of ABI5 in response to ABA and salt stress depended on the direct binding of HY5 to the ABI 5 promoter. HY5/HYH and RSM1 aggregated on the ABI5 promoter independently or somehow dependently regulated ABI5 and downstream of gene expression, thereby improving the adaptation to salt stress [ 17 ]. To investigate the potential regulatory mechanisms involved in the response to salt stress, sequence alignments and homology analyses were carried out with transcriptome data from C. indicum . Surprisingly, we only identified members highly homologous to ABI5, the ARBE/ABF subfamily family, which can specifically recognize ABRE (ABA-responsive) elements and act as key components of ABA signaling (Fig. S10). According to qRT‒PCR, CiABF4 was significantly upregulated in the OE -CiHY5 lines but not in the RNAi -CiHY5 lines (Fig. 6 ). These results supported our hypothesis that the CiHY5-CiABF4 module mediates the salt stress response via an ABA-dependent pathway according to previous studies showing that HY5 targets ABF4 downstream genes to induce ABF4 expression in Arabidopsis via a ChIP-chip identification assay [ 32 ]. Subsequently, the Y1H assay showed that CiHY5 directly bound to the promoter of CiABF4 , which was consistent with the results of a previous ChIP-chip study (Fig. 7 ) [ 61 ]. Compared with those in WT plants, the abf3 and abf4 mutants in Arabidopsis are more sensitive to salinity stress, and the chlorophyll content decreases and the yellowing rate increases after NaCl treatment [ 32 , 62 ]. Extensive research has provided evidence that the overexpression of ABF4 enhances resistance to salt stress and increases the RWC and photosynthetic rate of plants compared with those of WT plants in many species, such as potato, Ipomoea batatas and Arabidopsis [ 63 , 64 ]. Further analysis revealed that the instantaneous transformation of C. indicum leaves to OE-CiABF4 positively contributed to salt stress tolerance (Fig. S11). Therefore, we inferred that the upregulation of CiABF4 induced the expression of downstream stress-responsive genes, including CiRAB18 , CiERD7 , CiDREB1D and CiERF1 (Fig. 6 ), which in turn regulated plant resistance to salt stress in accordance with the function of ABI5 [ 65 ]. The combined evidence from the above results in concert with those of previous studies indicated that the CiHY5-CiABF4 module is positively involved in enhancing salt stress tolerance in part in an ABA-dependent manner. Furthermore, further efforts are required to obtain a thorough understanding of the role of CiHY5 in the integration of multiple signaling networks to regulate plant growth by identifying the binding sites for CiHY5-targeted genes and CiHY5-interacting proteins. Conclusions In conclusion, CiHY5 is a bZIP TF family member involved in the salt stress response in C. indicum . Taken together, the phenotypic, physiological parameter and molecular mechanism data revealed enhanced salt resistance in the OE- CiHY5 transgenic plants and reduced salt resistance in the RNAi -CiHY5 transgenic plants. Although the complex regulatory mechanism of CiHY5 in response to abiotic stress is not clear, our results revealed a possible CiHY5-CiABF4 module for potential ABA-dependent regulation of stress responses. This study provides insight into the molecular mechanism of salt tolerance and lays a foundation for the creation of new germplasm materials that can adapt to adverse environments in chrysanthemum. Abbreviations CiHY5, Chrysanthemum indicum ELONGATED HYPOCOTYL 5; bZIP, basic leucine zipper; ABA, abscisic acid; WT, wild type; ABF4, ABRE BINDING FACTOR 4; RNAi, RNA interference; OE, overexpressing. Declarations Data availability All data relevant to the results and analysis in this study are included in this article and its supplementary materials. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Ethics approval and consent to participate This study obtained ethical approval and collection permits from the relevant authorities at the collection sites prior to gathering plant materials. All collection activities were conducted in compliance with local laws, regulations, and ethical standards. Consent for publication Not applicable. Funding This work was financially supported by the Fundamental Research Funds for the Central Universities (grant number 2572020AW06), the National Natural Science Foundation of China (grant number 31870687) and the National Key Research and Development Program of China (grant number 2019YFD1001500). Author Contribution M.H. and Y.Z. coordinated the project and designed the experiments. Z.L., B.X. and X.L. performed the experiments and data analysis; B.C., H.L. and S.C. conducted the bioinformatics work; and B.X, Z.L, Y.Y, J.H. and M.H were involved in the preparation of the manuscript. All the authors contributed to the manuscript. Acknowledgement We are grateful to our lab colleagues for their contributions to the lab operations. References Saibo NJ, Lourenco T, Oliveira MM. Transcription factors and regulation of photosynthetic and related metabolism under environmental stresses. Ann Bot. 2009;103(4):609–23. Song M, Wang H, Ma H, Zheng C. 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HY5-HDA9 Module Transcriptionally Regulates Plant Autophagy in Response to Light-to-Dark Conversion and Nitrogen Starvation. Mol Plant. 2020;13(3):515–31. Huang HE, Ho MH, Chang H, Chao HY, Ger MJ. Overexpression of plant ferredoxin-like protein promotes salinity tolerance in rice (Oryza sativa). Plant Physiol Biochem. 2020;155:136–46. Gangappa SN, Botto JF. The Multifaceted Roles of HY5 in Plant Growth and Development. Mol Plant. 2016;9(10):1353–65. Kim S, Kang JY, Cho DI, Park JH, Kim SY. ABF2, an ABRE-binding bZIP factor, is an essential component of glucose signaling and its overexpression affects multiple stress tolerance. Plant J. 2004;40(1):75–87. Muniz Garcia MN, Cortelezzi JI, Fumagalli M, Capiati DA. Expression of the Arabidopsis ABF4 gene in potato increases tuber yield, improves tuber quality and enhances salt and drought tolerance. Plant Mol Biol. 2018;98(1–2):137–52. Wang W, Qiu X, Yang Y, Kim HS, Jia X, Yu H, Kwak SS. Sweetpotato bZIP Transcription Factor IbABF4 Confers Tolerance to Multiple Abiotic Stresses. Front Plant Sci. 2019;10:630. Xu Y, Zhao X, Aiwaili P, Mu X, Zhao M, Zhao J, Cheng L, Ma C, Gao J, Hong B. A zinc finger protein BBX19 interacts with ABF3 to affect drought tolerance negatively in chrysanthemum. Plant J. 2020;103(5):1783–95. Additional Declarations No competing interests reported. Supplementary Files SupplementalInformation.docx Supplementalgeloriginalplot.pptx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4699886","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":342885903,"identity":"4a3ee33c-9af0-4cae-a7f4-6fec3900f243","order_by":0,"name":"Bin Xia","email":"","orcid":"","institution":"Northeast Forestry University","correspondingAuthor":false,"prefix":"","firstName":"Bin","middleName":"","lastName":"Xia","suffix":""},{"id":342885904,"identity":"496d5125-2498-4809-a6e1-cc311fba87af","order_by":1,"name":"Ziwei Li","email":"","orcid":"","institution":"Northeast Forestry University","correspondingAuthor":false,"prefix":"","firstName":"Ziwei","middleName":"","lastName":"Li","suffix":""},{"id":342885905,"identity":"0f81505d-e38e-4f61-b009-aa09659c57ef","order_by":2,"name":"Xiaowei Liu","email":"","orcid":"","institution":"Northeast Forestry University","correspondingAuthor":false,"prefix":"","firstName":"Xiaowei","middleName":"","lastName":"Liu","suffix":""},{"id":342885906,"identity":"bceeffcb-c754-452d-89f2-02711fd8462e","order_by":3,"name":"Yujia Yang","email":"","orcid":"","institution":"Northeast Forestry University","correspondingAuthor":false,"prefix":"","firstName":"Yujia","middleName":"","lastName":"Yang","suffix":""},{"id":342885907,"identity":"a5dc28ff-e916-4b3a-aa49-15cd25f6e45f","order_by":4,"name":"Shengyan Chen","email":"","orcid":"","institution":"Northeast Forestry University","correspondingAuthor":false,"prefix":"","firstName":"Shengyan","middleName":"","lastName":"Chen","suffix":""},{"id":342885908,"identity":"908bf4c3-4d68-44db-82f0-d23f94cb8317","order_by":5,"name":"Bin Chen","email":"","orcid":"","institution":"Northeast Forestry University","correspondingAuthor":false,"prefix":"","firstName":"Bin","middleName":"","lastName":"Chen","suffix":""},{"id":342885909,"identity":"ee5ecf91-d9bf-4db2-8d31-9585efc347a1","order_by":6,"name":"Hongyao Li","email":"","orcid":"","institution":"Northeast Forestry University","correspondingAuthor":false,"prefix":"","firstName":"Hongyao","middleName":"","lastName":"Li","suffix":""},{"id":342885910,"identity":"a6250aa5-2257-4c29-a3ad-19c2a3bebfcf","order_by":7,"name":"Jinxiu Han","email":"","orcid":"","institution":"Northeast Forestry University","correspondingAuthor":false,"prefix":"","firstName":"Jinxiu","middleName":"","lastName":"Han","suffix":""},{"id":342885911,"identity":"dcb18fae-22f7-45cb-9fbb-95727efb3e17","order_by":8,"name":"Yunwei Zhou","email":"","orcid":"","institution":"Jilin Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Yunwei","middleName":"","lastName":"Zhou","suffix":""},{"id":342885912,"identity":"14c87d67-1f85-486b-b4f8-4f282ea82235","order_by":9,"name":"Miao He","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0klEQVRIiWNgGAWjYBACAyA+wMBgw8DADKQY2IjXkgZUnUCCFiA4TIIWc/buxAM/Ks7n8bPxGDB8KDvMwD+7Ab8Wy56zGw72nLldLNnGY8A449xhBok7Bwg47EbuhgO8bbcTN9zvMWDmbTvMYCCRQEDL/bcbDv5tO5e4/xiPAfNforTc4N1wmLftQOIGoF+YGYnSciZ3w2GZM8mJM46xFRzsOZfOI3GDkJbjZzd/fFNhl9jfxrzxwY8yazn+GQS0oIADQMxDgvpRMApGwSgYBbgAAJEFSJDGH6FNAAAAAElFTkSuQmCC","orcid":"","institution":"Northeast Forestry University","correspondingAuthor":true,"prefix":"","firstName":"Miao","middleName":"","lastName":"He","suffix":""}],"badges":[],"createdAt":"2024-07-07 10:51:45","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4699886/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4699886/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":62953561,"identity":"fb8b6839-5698-4169-be25-40308eb13d7f","added_by":"auto","created_at":"2024-08-21 11:50:23","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":594266,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe deduced protein sequence and homologues proteins of CiHY5 in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eC. indicum\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and HY5s in other 10 plant species from NCBI database.\u003c/strong\u003e \u003cstrong\u003e(A)\u003c/strong\u003e Multiple sequence alignments of CiHY5 with other HY5s proteins. The Basic region, Leucine Zipper domain and bZIP_HY5-like domain were respectively underlined in black, blue and red.\u003cstrong\u003e (B) \u003c/strong\u003ePhylogenetic tree analysis of CiHY5 with other HY5s proteins using Neighbor-joining method. The numbers on the nodes indicated bootstrap values from 1000 replicates. The scale bar indicated the relative genetic distance. The GenBank accession numbers of HY5s were listed as follows: CiHY5, \u003cem\u003eChrysanthemum indicum\u003c/em\u003e(OP589306), AaHY5, \u003cem\u003eArtemisia annua\u003c/em\u003e(PWA48328.1), AtHY5, \u003cem\u003eArabidopsis thaliana\u003c/em\u003e(NP_001330553.1), HaHY5, \u003cem\u003eHelianthus annuus\u003c/em\u003e (XP_022023437.1), MdHY5, \u003cem\u003eMalus domestica\u003c/em\u003e (NP 001280752.1), NtHY5, \u003cem\u003eNicotiana tabacum\u003c/em\u003e (XP_016452266.1), OsHY5, \u003cem\u003eOryza sativa\u003c/em\u003e (XP_015617496.1), SmHY5, \u003cem\u003eSolanum melongena\u003c/em\u003e (KT895426.1), VvHY5, \u003cem\u003eVitis vinifera\u003c/em\u003e (AGX85877.1) and ZmHY5, \u003cem\u003eZea mays\u003c/em\u003e (XP_008655674.1).\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4699886/v1/ff7be15affabbd1e10db6bef.jpeg"},{"id":62954350,"identity":"dbec2484-04b6-40fb-ae6f-6b8f95d2b8f1","added_by":"auto","created_at":"2024-08-21 11:58:23","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":178064,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSubcellular localization and expression levels of CiHY5. (A)\u003c/strong\u003e 35S:CiHY5::GFP fusion protein was localized to the nucleus. 35S:CiHY5::GFP construct was injected into \u003cem\u003eNicotiana benthamiana\u003c/em\u003e leaves and observed by confocal laser scanning microscopy. 35S:GFP served as a positive control. GFP, green fluorescent protein. Images were dark field (left) showing green fluorescence, bright field (middle) showing the morphology of the cells, and merged (right) showing a combination. Scale bars: 20 µm. \u003cstrong\u003e(B) \u003c/strong\u003eThe transcript abundance of \u003cem\u003eCiHY5 \u003c/em\u003ein root, stem, leaf, bud and ray floret of \u003cem\u003eC. indicum\u003c/em\u003e.\u003cstrong\u003e (C) \u003c/strong\u003eand\u003cstrong\u003e (D) \u003c/strong\u003eExpression levels of \u003cem\u003eCiHY5\u003c/em\u003e in leaves upon 200 mmol L\u003csup\u003e-1\u003c/sup\u003e salt and 100 μmol L\u003csup\u003e-1\u003c/sup\u003e ABA treatments in different time points. Data were presented as means of three biological replicates. The error bars represented SD and asterisks (*) indicated significant differences (“*”, \u003cem\u003eP \u0026lt; 0.05\u003c/em\u003e; “**”, \u003cem\u003eP \u0026lt; 0.01\u003c/em\u003e) compared with WT by Student’s t-test analysis. The same as following figures.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4699886/v1/a672142510408b8d47a390f8.jpeg"},{"id":62953562,"identity":"fceee321-fa78-4586-94a5-830c7beff08b","added_by":"auto","created_at":"2024-08-21 11:50:23","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":356605,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhenotypes and morphological indexes of WT and transgenic plants. (A)\u003c/strong\u003e The leaves of two-month-old WT, \u003cem\u003eOE-CiHY5\u003c/em\u003e and \u003cem\u003eRNAi-CiHY5\u003c/em\u003e plants from top to bottom were observed for leaf phenotypes. The scales were below the first leaves. \u003cstrong\u003e(B)\u003c/strong\u003e The number of leaves in WT, \u003cem\u003eOE-CiHY5\u003c/em\u003e and \u003cem\u003eRNAi-CiHY5\u003c/em\u003e plants.\u003cstrong\u003e (C)\u003c/strong\u003e The area of leaves (cm\u003csup\u003e2\u003c/sup\u003e) in WT, \u003cem\u003eOE-CiHY5\u003c/em\u003e and \u003cem\u003eRNAi-CiHY5\u003c/em\u003e plants. \u003cstrong\u003e(D)\u003c/strong\u003e Two-month-old WT and transgenic plants which developed from the cuttings of the same size. \u003cstrong\u003e(E) \u003c/strong\u003eRoot growth of two-week-old WT and transgenic plants. \u003cstrong\u003e(F) \u003c/strong\u003eSixteen internodes of WT, \u003cem\u003eOE-CiHY5\u003c/em\u003e and \u003cem\u003eRNAi-CiHY5\u003c/em\u003e lines were sliced from top to bottom and arranged from left to right. Bar = 10 mm.\u003cstrong\u003e (G) \u003c/strong\u003eand\u003cstrong\u003e (H)\u003c/strong\u003e Internode length and diameter of WT, \u003cem\u003eOE-CiHY5\u003c/em\u003e and \u003cem\u003eRNAi-CiHY5\u003c/em\u003e lines. Data were presented as means of three biological replicates, and error bars represented SD. Asterisks (“*”, \u003cem\u003eP \u0026lt; 0.05\u003c/em\u003e; “**” \u003cem\u003eP \u0026lt; 0.01\u003c/em\u003e) indicated significant differences compared to WT by Student’s t-test.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4699886/v1/bee2ea7cca3c596dc72d8949.jpeg"},{"id":62954352,"identity":"e84380f5-ede2-41f2-9bd3-878ff3cbf1f6","added_by":"auto","created_at":"2024-08-21 11:58:24","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":414127,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSalt stress tolerance of WT and transgenic plants.\u003c/strong\u003e \u003cstrong\u003e(A)\u003c/strong\u003e WT, \u003cem\u003eOE-CiHY5\u003c/em\u003e and \u003cem\u003eRNAi-CiHY5\u003c/em\u003e plant lines before salt stress treatment.\u003cstrong\u003e (B) \u003c/strong\u003ePhenotypes of WT, \u003cem\u003eOE-CiHY5\u003c/em\u003e and \u003cem\u003eRNAi-CiHY5\u003c/em\u003e plants subjected to 200 mM L\u003csup\u003e-1\u003c/sup\u003e NaCl treatment for 10 days.\u003cstrong\u003e (C)\u003c/strong\u003e Recovery of WT, \u003cem\u003eOE-CiHY5\u003c/em\u003e and \u003cem\u003eRNAi-CiHY5\u003c/em\u003e plants for 10 days rehydration in normal conditions after 10 days salt stress. (D) Leaf electrolyte leak (%).\u003cstrong\u003e (E) \u003c/strong\u003eTotal chlorophyll content (mg·g\u003csup\u003e-1\u003c/sup\u003e).\u003cstrong\u003e (F) \u003c/strong\u003eSuperoxide dismutase (SOD) activity (U·g\u003csup\u003e-1\u003c/sup\u003e). \u003cstrong\u003e(G) \u003c/strong\u003eCatalase (CAT) activity (U·g\u003csup\u003e-1\u003c/sup\u003e·min\u003csup\u003e-1\u003c/sup\u003e). \u003cstrong\u003e(H)\u003c/strong\u003e Malondialdehyde (MDA) content (umol·g\u003csup\u003e-1\u003c/sup\u003e).\u003cstrong\u003e (I) \u003c/strong\u003eProfile content (ug·g\u003csup\u003e-1\u003c/sup\u003e). Data were presented as means of three biological replicates, and error bars represented SD. Asterisks (“*”, \u003cem\u003eP \u0026lt; 0.05\u003c/em\u003e; “**” \u003cem\u003eP \u0026lt; 0.01\u003c/em\u003e) indicated significant differences compared to WT by Student’s t-test.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4699886/v1/d19e4c1a0c5ea4b5202b6cd8.jpeg"},{"id":62953560,"identity":"bc08e0ff-88a9-4da7-88af-0355be089b48","added_by":"auto","created_at":"2024-08-21 11:50:23","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":898297,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNa\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e and K\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e relative contents of WT,\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e OE-CiHY5\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eRNAi-CiHY5\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e plants under normal and 200 mM NaCl treatment conditions.\u003c/strong\u003e \u003cstrong\u003e(A)\u003c/strong\u003e and \u003cstrong\u003e(B)\u003c/strong\u003e Leaf Na\u003csup\u003e+ \u003c/sup\u003eand K\u003csup\u003e+ \u003c/sup\u003erelative content. \u003cstrong\u003e(C) \u003c/strong\u003eand\u003cstrong\u003e (D)\u003c/strong\u003e Root Na\u003csup\u003e+ \u003c/sup\u003eand K\u003csup\u003e+ \u003c/sup\u003erelative content. \u003cstrong\u003e(E)\u003c/strong\u003e and\u003cstrong\u003e (F) \u003c/strong\u003eNa\u003csup\u003e+ \u003c/sup\u003e: K\u003csup\u003e+ \u003c/sup\u003eratio in leaf and root. Data were presented as means of three biological replicates, and error bars represented SD. Asterisks (“*”, \u003cem\u003eP \u0026lt; 0.05\u003c/em\u003e; “**” \u003cem\u003eP \u0026lt; 0.01\u003c/em\u003e) indicated significant differences compared to WT by Student’s t-test.\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4699886/v1/5739c24fd21b9def05593378.jpeg"},{"id":62953565,"identity":"684d8e72-2fcc-4c86-a713-1dc75f8e2e76","added_by":"auto","created_at":"2024-08-21 11:50:24","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":233980,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpressions of genes related to the abscisic acid (ABA)-dependent or -independent pathway in WT, \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eOE-CiHY5\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eRNAi-CiHY5\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e plants.\u003c/strong\u003e Quantitative real-time PCR was executed to evaluate the expression levels of genes by 2\u003csup\u003e−ΔΔCT\u003c/sup\u003e calculation method. \u003cem\u003eCiEF1α\u003c/em\u003e was used as reference gene. Data were presented as means of three biological replicates, and error bars represented SD. Asterisks (“*”, \u003cem\u003eP \u0026lt; 0.05\u003c/em\u003e; “**” \u003cem\u003eP \u0026lt; 0.01\u003c/em\u003e) indicated significant differences compared to WT by Student’s t-test.\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4699886/v1/587dba3758a0482d88c9b455.jpeg"},{"id":62955098,"identity":"73ab8a41-de82-4fbd-89dc-0be661f002a7","added_by":"auto","created_at":"2024-08-21 12:06:24","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":149442,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCis-response elements analysis of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eCiABF4\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e promoter and yeast one-hybrid assay.\u003c/strong\u003e \u003cstrong\u003e(A)\u003c/strong\u003e Schematic representation of the 1404 bp \u003cem\u003eCiABF4\u003c/em\u003e promoter. Rectangle corresponded to putative G-box (AACGTG, -1104 to -1110 bp) binding element motif with CiHY5. \u003cstrong\u003e(B)\u003c/strong\u003e and\u003cstrong\u003e (C) \u003c/strong\u003eAnalysis of CiHY5 binding to the \u003cem\u003eCiABF4\u003c/em\u003epromoter. The empty prey vector (\u003cem\u003eAD-Rec2\u003c/em\u003e) was used as negative control. Interactions between bait and prey were determined by cell growth on SD-Trp/-Leu medium plate, and containing 50mM 3-AT SD-Trp/-Leu/-His medium plate to inhibit the leaked HIS gene expression.\u003c/p\u003e","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4699886/v1/584f5bb7d33a3f301cb6628f.jpeg"},{"id":63312699,"identity":"48ea140c-5622-4cc0-90c2-6d5681e53497","added_by":"auto","created_at":"2024-08-26 20:44:09","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4025669,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4699886/v1/a53acb32-d2d3-4f38-a4fe-f459e4bb4e3a.pdf"},{"id":62953558,"identity":"263d694b-ff05-4b77-a1cb-d776eef2ac1a","added_by":"auto","created_at":"2024-08-21 11:50:23","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1281650,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-4699886/v1/8580587b1b6e47e411c7fba2.docx"},{"id":62953563,"identity":"f951843c-102b-455f-992e-612cbb4ad417","added_by":"auto","created_at":"2024-08-21 11:50:24","extension":"pptx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":33147371,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementalgeloriginalplot.pptx","url":"https://assets-eu.researchsquare.com/files/rs-4699886/v1/f704cf78a7695fa6eee0ef43.pptx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Functions of CiHY5 in regulating the salt tolerance of Chrysanthemum revealed by transgenic Chrysanthemum indicum","fulltext":[{"header":"Background","content":"\u003cp\u003ePlants may encounter a variety of adverse environmental conditions, such as extreme temperatures, water deficiencies, drought and salinity stresses, affecting their growth and productivity [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. To overcome abiotic stresses, plants have evolved a series of mechanisms involving the regulation of transcription factors (TFs) for adaptive physiological and developmental processes, such as those in the bHLH, WRKY, bZIP, and MYB TF families [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. As the dominant energy source for photosynthesis, light is an indispensable environmental factor for plant growth and development and plays pivotal roles in the process of mitigating abiotic stresses. Previous research has shown that plant growth inhibition induced by salt stress is strongly affected by light [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The salt tolerance of \u003cem\u003eArabidopsis\u003c/em\u003e is positively correlated with light intensity, indicating that light signals broadly regulate intricate molecular networks involved in salt stress [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. ELONGATED HYPOCOTYL 5 (HY5), a basic leucine zipper (bZIP) family TF, has been reported to participate mainly in genome-wide large-scale transcriptional reprogramming induced by light-dark environment transformation and to act as a master regulator that regulates various physiological and biological processes, as well as abiotic stress responses; this gene is regarded as a pivotal candidate gene for the breeding of Chrysanthemum for abiotic stress tolerance [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eRecent research has shown that HY5 physically interacts with COP1, a pivotal negative regulator of photomorphogenesis, resulting in the ubiquitination and degradation of HY5 to maintain homeostasis in etiolated plants [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Additionally, HY5 has been identified as a positive regulator of seedling photomorphogenesis, lateral root development and the root gravitropism response in \u003cem\u003ehy5\u003c/em\u003e mutants [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. HY5 is considered a high-level hierarchical regulator of transcriptional networks involved in multiple signal transduction and metabolic pathways, such as pigment accumulation, nutrient assimilation, hormone signaling, and abiotic stress responses [\u003cspan additionalcitationids=\"CR11 CR12 CR13 CR14 CR15 CR16\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Approximately one-third of genes in \u003cem\u003eArabidopsis\u003c/em\u003e are directly or indirectly affected by HY5, and ~\u0026thinsp;3000 of these genes directly target HY5 binding sites, as determined by ChIP-chip experiments [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. This may be a critical factor underlying the multifarious roles of HY5 in abundant functions. Recent studies have elucidated the involvement of HY5 in secondary metabolism regulation. For instance, HY5 directly binds to \u003cem\u003eMYB12/PFG1\u003c/em\u003e, and \u003cem\u003eMYB75/PAP1\u003c/em\u003e and \u003cem\u003ePIF3\u003c/em\u003e regulate flavonoid biosynthesis and anthocyanin gene expression, further promoting pigment accumulation [\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. In a recent review, HY5 was shown to participate in response mechanisms by targeting downstream genes, such as \u003cem\u003eQH6\u003c/em\u003e (monoterpene β-pinene synthase), \u003cem\u003eRAP2.1\u003c/em\u003e and \u003cem\u003eLEA18\u003c/em\u003e (cold-responsive genes), \u003cem\u003eWRKY\u003c/em\u003e genes (involved in defense responses), and \u003cem\u003eNIA2\u003c/em\u003e and \u003cem\u003eNIR1\u003c/em\u003e (nitrogen signaling responses), to regulate terpenoid biosynthesis, cold acclimation, plant cell death and nutrient assimilation [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eRecent research has extensively investigated the interplay of HY5 with physically interacting proteins integrating signaling pathways involved in the response to abiotic stresses in plants. The expression of HY5 in the nucleus is affected by the nuclear and cytoplasmic distribution of COP1 at low temperatures, leading to the regulation of downstream cold-induced genes and cold adaptation in \u003cem\u003eArabidopsis\u003c/em\u003e [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. A convergent study showed that HY5 directly or indirectly integrates cold-responsive and ABA signaling pathways by binding the promoters of \u003cem\u003eCBF\u003c/em\u003e and \u003cem\u003eMYB15\u003c/em\u003e, resulting in sophisticated adaptive regulation leading to cold resistance in tomato [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Similarly, in apple, MdHY5 upregulated the expression of \u003cem\u003eMdMYB108L\u003c/em\u003e via a CBF-dependent pathway, leading to enhanced cold tolerance in apple [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFurthermore, the overexpression of \u003cem\u003eHY5\u003c/em\u003e in tobacco compensates for the phenotypes of \u003cem\u003ehy5\u003c/em\u003e mutants and enhances tolerance to salt stress by regulating reduced ROS accumulation in transgenic lines [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. More recently, HY5 was shown to maintain the modification of histone H3K4me3 by binding to the promoter of the \u003cem\u003eΔ\u003c/em\u003e\u003csup\u003e\u003cem\u003e1\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-pyrroline-5-carboxylate synthetase 1\u003c/em\u003e (\u003cem\u003eP5CS1\u003c/em\u003e) gene and contributing to the memorability of proline under salt stress, thereby contributing to the memory of proline under salt stress and enhancing salt resistance in Arabidopsis, along with rapid accumulation and expression upon exposure to salinity [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Moreover, comparative transcriptome analysis revealed that HY5 plays a central role in the ABA signal transduction pathway, which negatively regulates drought stress under green photoinduction conditions [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOver the course of evolution, hormones have become crucial means by which plants integrate complex transcriptional regulatory mechanisms to mitigate adverse effects and gain the adaptive capacity to resist external stresses [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Notably, previous work on the integration of the ABA signaling pathway and HY5 has indicated that HY5 mediates multiple regulatory mechanisms to survive under saline conditions. During the period of seed germination and seedling development, the high-affinity binding of HY5 to the promoter of \u003cem\u003eABI5\u003c/em\u003e enhances resistance to environmental stresses, along with the rapid adaptability of plants to the root system and photoautotrophy after germination in an ABA signaling manner [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Subsequent studies have shown that HY5 additionally binds to the promoters of ABA RESPONSIVE ELEMENT BINDING FACTORS (AREB/ABF) bZIP TFs in \u003cem\u003eArabidopsis\u003c/em\u003e, which are homologous to ABI5 and are regarded as subfamily members of ABI5, such as ABF1, ABF3 and ABF4 [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. In \u003cem\u003eArabidopsis\u003c/em\u003e, HY5/HYH and RSM1 independently or in some way independently induce the expression of \u003cem\u003eABI5-\u003c/em\u003e and \u003cem\u003eABI5\u003c/em\u003e-targeted genes, thereby regulating ABA and abiotic stress responses [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Furthermore, HY5 mediates root architecture by promoting primary root growth and inhibiting lateral root growth via the ABA signaling pathway, further facilitating seedling establishment and soil composition absorption under drought and salinity stresses [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOwing to the long-term accumulation of natural and anthropic factors, soil salinization is one of the main abiotic stress conditions faced by plants, ultimately causing quality degradation and economic losses [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Chrysanthemum is highly appreciated as a cutting-flower industry material and is vulnerable to high salinity conditions. \u003cem\u003eChrysanthemum indicum\u003c/em\u003e, a diploid species native to China, is renowned for its ornamental and medicinal properties and is well adapted to adverse environmental conditions [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Previously, only research on HY5 in chrysanthemum (\u003cem\u003eChrysantemum morifolium\u003c/em\u003e) showed that downregulated expression of \u003cem\u003eCmHY5\u003c/em\u003e in response to high temperature positively promoted anthocyanin synthesis according to transcriptome data [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. However, little is known about the ectopic expression of HY5 and its involvement in signaling pathways and abiotic stresses in other species. To further elucidate the function of \u003cem\u003eHY5\u003c/em\u003e in chrysanthemum, \u003cem\u003eCiHY5\u003c/em\u003e was isolated from \u003cem\u003eC. indicum\u003c/em\u003e and characterized. We verified that overexpression of \u003cem\u003eCiHY5\u003c/em\u003e contributed to adaptive phenotypes and enhanced resistance to salt stress by binding to the promoter of the \u003cem\u003eCiABF4\u003c/em\u003e gene in an ABA-dependent manner. To some extent, we demonstrated that \u003cem\u003eCiHY5\u003c/em\u003e is a promising candidate gene for the response to salt stress for genetic engineering and the development of the chrysanthemum breeding industry.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Plant materials, growth conditions and treatments\u003c/h2\u003e \u003cp\u003e \u003cem\u003eChrysanthemum indicum\u003c/em\u003e, a native diploid species, was obtained from Hubei, China, and cultivated in the nursery of Northeast Forestry University for test materials. Te voucher specimens, \u003cem\u003eC. indicum\u003c/em\u003e (KUN 1445652) was identified by Xinxin Zhu and its sheet was deposited in the herbaria KUN (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.cvh.ac.cn/spms/detail.php?id=21c961b0\u003c/span\u003e\u003cspan address=\"https://www.cvh.ac.cn/spms/detail.php?id=21c961b0\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Rooted cuttings were transplanted into pots of the same size containing a mixture of V (humus):V (vermiculite):V (perlite)\u0026thinsp;=\u0026thinsp;2:1:1, pH\u0026thinsp;=\u0026thinsp;6.65 cultivation substrate under natural conditions (temperature of 23\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C, relative humidity of 65%-75%, 16 h/8 h light/dark photoperiod) [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. For the light environment and UV-B irradiation treatments, two-week-old acclimated seedlings with consistent growth status were subjected to darkness, white light (intensity\u0026thinsp;=\u0026thinsp;180 \u0026micro;mol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, source: Osram L18W/30 tubes, NE, USA) and UV-B irradiation (intensity\u0026thinsp;=\u0026thinsp;400 \u0026micro;mol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, source: T12 306 nm narrowband UV-B tubes, Huaqiang, Nanjing) for 0, 1, 6, 24, 48 h, respectively. The consistent stative seedlings were precultured with Hoagland\u0026rsquo;s nutrient solution for one week and then transferred to the same solution containing 200 mmol L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e NaCl or 100 \u0026micro;mol L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e ABA for salt stress and ABA treatment [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. The control groups were concurrently treated with Hoagland\u0026rsquo;s nutrient solution under the same growth conditions, and three replicates were performed. Following these treatments, the leaves were sampled for 0, 1, 3, 6, 12 and 24 h and immediately stored at -80\u0026deg;C in preparation for subsequent experiments. The roots, stems, leaves, buds and ray florets were sampled for tissue-specific expression analysis. The expression levels of \u003cem\u003eCiHY5\u003c/em\u003e were validated by quantitative real-time PCR (qRT‒PCR) with specific primers (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Gene cloning, structure and phylogenetic analysis\u003c/h2\u003e \u003cp\u003eBased on the transcriptome data of \u003cem\u003eC. indicum\u003c/em\u003e in previous work, we isolated the \u003cem\u003eCiHY5\u003c/em\u003e gene. CiHY5 was further identified via analysis of the chrysanthemum genome database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.amwayabrc.com/zh-cn/index.html\u003c/span\u003e\u003cspan address=\"http://www.amwayabrc.com/zh-cn/index.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), the MUM GARDEN (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://mum-garden.kazusa.or.jp/\u003c/span\u003e\u003cspan address=\"http://mum-garden.kazusa.or.jp/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and the TAIR website (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.A.thaliana.org/index.jsp\u003c/span\u003e\u003cspan address=\"https://www.A.thaliana.org/index.jsp\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. For sequence alignments and phylogenetic analysis, we used amino acid sequences of CiHY5 homologs in \u003cem\u003eArabidopsis\u003c/em\u003e and other species obtained from the online database NCBI (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://blast.ncbi.nlm.nih.gov/Blast.cgi\u003c/span\u003e\u003cspan address=\"https://blast.ncbi.nlm.nih.gov/Blast.cgi\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) for alignment and analysis via ClustalX1.83 and MEGA7 via the neighbor-joining (NJ) method [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Specific primers were designed based on the genomic sequence containing the complete open reading frame (ORF) sequence and RNA interference (RNAi) fragment of \u003cem\u003eCiHY5\u003c/em\u003e, and enzyme cut sites were added at the 5\u0026rsquo;-terminus of the upstream and 3\u0026rsquo;-terminus of the downstream primers, respectively. The target products were amplified with \u003cem\u003eC. indicum\u003c/em\u003e cDNA template by RT‒PCR according to KOD DNA Polymerase instructions (TOYOBO, Japan). The expected \u003cem\u003eCiHY5\u003c/em\u003e and \u003cem\u003eCiHY5-\u003c/em\u003eRNAi fragments were inserted into the \u003cem\u003epEASY\u003c/em\u003e\u0026reg;-Blunt Zero vector for reproduction and then transformed into \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e EHA105 receptor cells (Weidi, China) for integration into \u003cem\u003eC. indicum\u003c/em\u003e leaves. All of the primer sequences are listed in Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. RNA extraction, RT‒PCR and qRT‒PCR\u003c/h2\u003e \u003cp\u003eTotal RNA from \u003cem\u003eC. indicum\u003c/em\u003e was extracted with an RNA extraction kit (OMEGA, USA) and reverse transcribed to the first strand of cDNA using a ReverTra Ace-α Kit (TOYOBO, Japan). According to the initial RNA concentration, cDNA was diluted as a template to clone the \u003cem\u003eCiHY5\u003c/em\u003e and \u003cem\u003eCiHY5-\u003c/em\u003eRNAi fragments by RT‒PCR, and the corresponding primer sequences are listed in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. qRT‒PCR was performed with three biological replicates of UltraSYBR Mixture (CWBIO, China) on an ABI Prism 7500 system (Applied Biosystems, USA) as follows: 95\u0026deg;C for 10 min; 40 cycles of 95\u0026deg;C for 15 s and 60\u0026deg;C for 1 min; and melting at 95\u0026deg;C for 15 s, 60\u0026deg;C for 1 min, 95\u0026deg;C for 15 s and 60\u0026deg;C for 15 s. The relative expression levels of genes were calculated using the 2\u003csup\u003e\u0026minus;ΔΔCT\u003c/sup\u003e method with the reference gene \u003cem\u003eCiEF1α\u003c/em\u003e (GenBank Accession No. KF305681) from \u003cem\u003eC. indicum\u003c/em\u003e (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Subcellular localization of CiHY5\u003c/h2\u003e \u003cp\u003eThe full-length coding region (without the stop codon) of the \u003cem\u003eCiHY5\u003c/em\u003e integrated green fluorescent protein (GFP) tag was used to construct the recombinant vector \u003cem\u003epBI121-CaMV35S:CiHY5::GFP\u003c/em\u003e (\u003cem\u003e35S:CiHY5::GFP\u003c/em\u003e), and the \u003cem\u003epBI121-CaMV 35S:GFP\u003c/em\u003e (\u003cem\u003e35S:GFP\u003c/em\u003e) empty vector was used as a positive control. The prepared infiltration buffer (10 mmol L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e MES, 150 \u0026micro;mol L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e AS and 10 mmol L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e MgCl\u003csub\u003e2\u003c/sub\u003e) was used to suspend the centrifuged \u003cem\u003eAgrobacterium\u003c/em\u003e liquid to OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;1.0, which was subsequently transformed with the recombinant vectors described above. \u003cem\u003eAgrobacterium\u003c/em\u003e suspensions of \u003cem\u003e35S:CiHY5::GFP\u003c/em\u003e and \u003cem\u003e35S:GFP\u003c/em\u003e were injected into the abaxial surface of tobacco (\u003cem\u003eNicotiana benthamiana\u003c/em\u003e) leaves. After injection, the tobacco plants were cultivated in darkness for 48\u0026ndash;72 h. Then, a disk of approximately 0.25 cm\u003csup\u003e2\u003c/sup\u003e in the area from the injected leaves was cut to observe the fluorescence signal [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. The GFP signal was detected by a Zeiss LSM710 confocal laser scanning microscope (Nikon, Japan) with the following settings: 488 nm excitation and 525 nm emission wavelengths.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Construction of plant expression vectors and identification of transgenic plants\u003c/h2\u003e \u003cp\u003eFor overexpression-\u003cem\u003eCiHY5\u003c/em\u003e vector construction, the complete ORF sequence of \u003cem\u003eCiHY5\u003c/em\u003e with XbaI/XmaI sites was cloned and integrated into the \u003cem\u003epBI121\u003c/em\u003e vector using T4 DNA ligase (NEB, USA), and the resulting construct was named \u003cem\u003epBI121\u003c/em\u003e-\u003cem\u003eCiHY5\u003c/em\u003e (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). For RNAi-\u003cem\u003eCiHY5\u003c/em\u003e vector construction, a 242 bp specific sequence of \u003cem\u003eCiHY5\u003c/em\u003e was amplified into sense and antisense fragments containing XhoI/KpnI and XbaI/HindIII sites, respectively, in the \u003cem\u003epHANNIBAL\u003c/em\u003e vector, which automatically formed a repeat hairpin-silencing RNA (ihpRNA) structure to degrade the targeted mRNA. The ihpRNA structure with the 35S promoter and Nos terminator was then recombined into the \u003cem\u003epART27\u003c/em\u003e vector \u003cem\u003epART27-CiHY5\u003c/em\u003e (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The terminal vectors \u003cem\u003epBI121-CiHY5\u003c/em\u003e and \u003cem\u003epART27-CiHY5\u003c/em\u003e were separately inserted into \u003cem\u003eAgrobacterium\u003c/em\u003e receptor cells and stored at -80\u0026deg;C in 50% glycerol.\u003c/p\u003e \u003cp\u003eLeaves from two-week-old tissue culture-generated \u003cem\u003eC. indicum\u003c/em\u003e plants were used as explants for \u003cem\u003eAgrobacterium\u003c/em\u003e-mediated transformation. Infected leaves were cultured with differentiation media supplemented with specific antibiotics (5 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e kanamycin, Kana for \u003cem\u003epBI121-CiHY5\u003c/em\u003e, 1 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e spectinomycin, and Spec for \u003cem\u003epART27-CiHY5\u003c/em\u003e) until the resistant buds were differentiated from the callus tissues. Then, the resistant seedlings were transferred to rootling media. Two weeks later, transgenic plants were selected and identified by RT‒PCR and qRT‒PCR analysis. Three transgenic \u003cem\u003eC. indicum\u003c/em\u003e lines \u003cem\u003ein which CiHY5 was overexpressed or suppressed\u003c/em\u003e (OE-\u003cem\u003eCiHY5\u003c/em\u003e and RNAi-\u003cem\u003eCiHY5\u003c/em\u003e) or the wild-type (WT) control was transplanted into the same size pots consistent with the cultivation conditions described above. Transgenic lines with different expression levels (OE-2, 4, 7 and Ri-1, 2, 4) and WT seedlings were selected for subsequent experiments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Transgenic plant phenotype observation and salt stress treatment\u003c/h2\u003e \u003cp\u003eTransgenic and WT \u003cem\u003eC. indicum\u003c/em\u003e plants were subjected to tissue culture. Once the plants rooted had 4\u0026ndash;5 true leaves, the plants of the selected transgenic and WT lines were transplanted into plastic pots for vegetative growth. Then, transgenic and WT plants in neat and consistent growth states were cultivated in plastic pots of the same size and subjected to conventional maintenance management at an incubation temperature of 23\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C and a relative humidity of 65%-75%. Two-month-old independent wild-type (WT) and transgenic plants were used to observe leaf area, internode length and diameter phenotypes.\u003c/p\u003e \u003cp\u003eFor salt stress, 50 ml of 200 mmol/L NaCl solution was added to each pot once every two days, and the oozed salt solution was removed from the trays to prevent salt loss until it was fully absorbed into the soil. Leaf relative water content (RWC), total chlorophyll content, malondialdehyde (MDA) content, proline content and antioxidant enzyme activities (superoxide dismutase, SOD and catalase, CAT) were determined from the 4th-6th leaves beneath terminal buds after 10 days of salt stress treatment (Takahashi et al., 2007; Wang et al., 2003; Wang et al., 2015). We collected roots less than 2 mm in diameter and leaves as materials to measure the relative contents of Na\u003csup\u003e+\u003c/sup\u003e and K\u003csup\u003e+\u003c/sup\u003e after 0 d and 10 d of salt stress by ICP‒MS using inductively coupled plasma‒mass spectrometry (ICP‒MS) with three biological replicates.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Isolation and in silico analysis of the promoter\u003c/h2\u003e \u003cp\u003eThermal asymmetric interlaced PCR (TAIL-PCR) was conducted to amplify the promoter region of \u003cem\u003eCiABF4\u003c/em\u003e (\u003cem\u003eproCiABF4\u003c/em\u003e) using a Genome Walking Kit (Takara, USA) according to the manufacturer\u0026rsquo;s instructions. Four arbitrary degenerate primers (\u003cem\u003eAP1\u003c/em\u003e, \u003cem\u003eAP2\u003c/em\u003e, \u003cem\u003eAP3\u003c/em\u003e, and \u003cem\u003eAP4\u003c/em\u003e) were used as sense primers, and three specific primers (\u003cem\u003eSP1\u003c/em\u003e/\u003cem\u003eSP2\u003c/em\u003e/\u003cem\u003eSP3\u003c/em\u003e-\u003cem\u003eproCiABF4\u003c/em\u003e) with higher annealing temperatures were designed according to the 5\u0026rsquo;-terminal coding sequence of \u003cem\u003eCiABF4\u003c/em\u003e (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Three rounds of TAIL-PCR were employed to obtain amplified fragments of different lengths. After splicing and sequencing, a 1401 bp fragment was designated \u003cem\u003eproCiABF4\u003c/em\u003e. The \u003cem\u003ecis\u003c/em\u003e-acting elements of \u003cem\u003eproCiABF4\u003c/em\u003e were analyzed via the PlantCARE database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://bioinformatics.psb.ugent.be/webtools/plantcare/html/\u003c/span\u003e\u003cspan address=\"https://bioinformatics.psb.ugent.be/webtools/plantcare/html/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe sequenced promoter was ligated into the plant expression vector by replacing the CaMV35S promoter region between the BamHI and HindIII restriction endonuclease sites, generating the \u003cem\u003eProCiABF4:Gus\u003c/em\u003e vector. \u003cem\u003eAgrobacterium\u003c/em\u003e strain EHA105 harboring \u003cem\u003eProCiABF4:Gus\u003c/em\u003e and \u003cem\u003e35S:Gus\u003c/em\u003e was cultured in LB liquid media supplemented with 20 mg/L rifampicin (Rif) and 50 mg/L Kana at approximately 28\u0026deg;C for 16 h until the OD600 reached 0.6\u0026ndash;0.8. The \u003cem\u003eProCiABF4:Gus\u003c/em\u003e and \u003cem\u003e35S:Gus Agrobacterium\u003c/em\u003e strains were collected and suspended in infiltration buffer (10 mmol/L MES, 200 \u0026micro;mol/L AS, and 10 mmol/L MgCl\u003csub\u003e2\u003c/sub\u003e) to an OD600 of 0.2\u0026ndash;0.3. The \u003cem\u003eProCiABF4:Gus\u003c/em\u003e and \u003cem\u003e35S:Gus\u003c/em\u003e suspension solutions were used to infiltrate young leaves of 6-week-old tobacco (\u003cem\u003eN. benthamiana\u003c/em\u003e) plants with syringes. The infected plants were placed in Gus staining solution for one night at 25\u0026ndash;37\u0026deg;C in darkness, and the promoter activity was then observed after decolorization by soaking in 70% ethanol. \u003cem\u003eAgrobacterium\u003c/em\u003e from \u003cem\u003e35S:Gus\u003c/em\u003e-infected leaves was used as the positive control. The primers used for promoter cloning and vector construction are listed in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e2.8 \u003cem\u003eTransactivation activity analysis and yeast one-hybrid assay\u003c/em\u003e\u003c/p\u003e \u003cp\u003eFor transactivation activity analysis, the full-length coding region of \u003cem\u003eCiHY5\u003c/em\u003e was fused to the GAL4 DNA binding domain of the \u003cem\u003epGBKT7\u003c/em\u003e vector \u003cem\u003epGBKT7-CiHY5\u003c/em\u003e (\u003cem\u003eBD-CiHY5\u003c/em\u003e) (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). \u003cem\u003epGBKT7-CiHY5\u0026thinsp;+\u0026thinsp;pGADT7-T\u003c/em\u003e (\u003cem\u003eBD-CiHY5\u0026thinsp;+\u0026thinsp;AD-T\u003c/em\u003e, experimental group), \u003cem\u003epGBKT7-53\u0026thinsp;+\u0026thinsp;pGADT7-T\u003c/em\u003e (\u003cem\u003eBD-53\u0026thinsp;+\u0026thinsp;AD-T\u003c/em\u003e, positive control) and \u003cem\u003epGBKT7-Lam\u0026thinsp;+\u0026thinsp;pGADT7-T\u003c/em\u003e (\u003cem\u003eBD-Lam\u0026thinsp;+\u0026thinsp;AD-T\u003c/em\u003e, negative control) vectors were transformed into yeast competent cells according to the instructions of Y2HGold Chemically Competent Cell (Weidi, China), and transcriptional activation activity was detected by screening the growth status on SD/-Trp/-Leu and SD/-Trp/-Leu/-His/-Ade/X-α-gal (20 mg ml\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) media.\u003c/p\u003e \u003cp\u003eFor the yeast one-hybrid (Y1H) assay, the full-length coding region of \u003cem\u003eCiHY5\u003c/em\u003e was inserted into the \u003cem\u003epGADT7-Rec2\u003c/em\u003e (\u003cem\u003eAD-Rec2\u003c/em\u003e) vector with the SmaI enzyme cutting site via homologous complementary technology to construct the prey vector \u003cem\u003epGADT7-Rec2-CiHY5\u003c/em\u003e (\u003cem\u003eAD-CiHY5\u003c/em\u003e) (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). Subsequently, \u003cem\u003ethe CiABF4\u003c/em\u003e promoter fragment was amplified from \u003cem\u003eC. indicum\u003c/em\u003e genomic DNA and inserted into the \u003cem\u003epHIS2\u003c/em\u003e vector with EcoRI and SacI enzyme cutting sites to construct the bait vector \u003cem\u003epHIS2\u003c/em\u003e-\u003cem\u003eproCiABF4\u003c/em\u003e (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). The \u003cem\u003eAD-CiHY5\u003c/em\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003epHIS2-proCiABF4\u003c/em\u003e (experimental group) and \u003cem\u003eAD-Rec2\u003c/em\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003epHIS2-proCiABF4\u003c/em\u003e (control group) vectors were cotransformed into Y187 Gold yeast (Weidi, China) according to the manufacturer\u0026rsquo;s instructions. Yeast cells were subsequently placed on SD/-Trp/-Leu media to verify growth status, and SD/-Trp/-Leu/-His media supplemented with 50 mmol/L 3-aminotriazole (3-AT) were used to verify the interaction between CiHY5 and the promoter of \u003cem\u003eCiABF4\u003c/em\u003e. The yeast cells were incubated at 30\u0026deg;C for 3 days. The RT‒PCR primers used for vector construction are listed in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.9. Transient transformation of CiABF4 and salt treatment\u003c/h2\u003e \u003cp\u003eBased on previous transcriptome data, the full-length sequence of \u003cem\u003eCiABF4\u003c/em\u003e was aligned, and the complete ORF fragment was subsequently cloned. The amplified PCR product was purified and inserted into \u003cem\u003epBI121\u003c/em\u003e to construct the overexpression-\u003cem\u003eCiABF4\u003c/em\u003e (OE\u003cem\u003e-CiABF4\u003c/em\u003e) vector \u003cem\u003epBI121-CiABF4\u003c/em\u003e, as described above. Fresh leaf discs of \u003cem\u003eC. indicum\u003c/em\u003e were cut for two days of preculture and then infected with the suspended \u003cem\u003epBI121-CiABF4\u003c/em\u003e strain by means of \u003cem\u003eAgrobacterium\u003c/em\u003e-mediated transient transformation, which utilized a vacuum negative pressure infection method to generate a negative pressure environment to promote the effective infiltration of \u003cem\u003eAgrobacterium\u003c/em\u003e into plant tissue cells to accelerate transformation for two days of coculture in a 30\u0026deg;C incubator [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Immediately, we transferred the uninfected (WT) and OE\u003cem\u003e-CiABF4\u003c/em\u003e leaf discs to MS media for two days, transferred them to salt-stress media supplemented with 50 mmol/L NaCl for 4 days, and finally allowed the plants to recover from the normal media for 4 days to observe the phenotypes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.10. Statistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analyses were performed using Excel. Three biological replicates are presented as the means\u0026thinsp;\u0026plusmn;\u0026thinsp;SDs, and the significance levels were compared with Duncan's multiple range test with one-way ANOVA (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.01)\u003c/em\u003e using SPSS 23.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Cloning and sequence analysis of CiHY5 from C. indicum\u003c/h2\u003e \u003cp\u003eTo explore the functions of pivotal regulators of the light-mediated pathway in response to abiotic stresses in chrysanthemum, we isolated a member of the bZIP family, \u003cem\u003eCiHY5\u003c/em\u003e, from \u003cem\u003eC. indicum\u003c/em\u003e transcriptome data. Based on the cDNA library, the complete 477 bp open reading frame (ORF) sequence of \u003cem\u003eCiHY5\u003c/em\u003e (GenBank accession number No. OP589306), which encodes a 158 amino acid polypeptide, was cloned, and the predicted molecular weight of the CiHY5 protein was 17.51 kDa. Sequence alignment revealed that CiHY5 contains a highly flexible and disordered N-terminal structure and a conserved bZIP_HY5-like domain in the C-terminus from amino acids 90 to 141, which may directly bind to G-box elements in promoter regions to activate gene expression and regulate signaling networks (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Additionally, CiHY5 contains a basic region from amino acids 77 to 109 to a stable tertiary structure and a leucine zipper (LZ) domain from amino acids 110 to 141, which are responsible for DNA binding and dimerization, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003eIn the phylogenetic trees composed of CiHY5 and 10 HY5s in the other 10 species (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB), CiHY5 was classified with CmHY5, AaHY5 and HaHY5 from \u003cem\u003eC. morifolium\u003c/em\u003e, \u003cem\u003eArtemisia annua\u003c/em\u003e and \u003cem\u003eHelianthus annuus\u003c/em\u003e in the same branch, and it was also close to AtHY5 from \u003cem\u003eArabidopsis\u003c/em\u003e. The length of the evolutionary branch represented the genetic variability and evolutionary distance, which indicated that CiHY5 and AaHY5 had the highest sequence similarities with the shorter branch, implying that the divergence of the HY5 family occurred later in chrysanthemum and \u003cem\u003eArtemisia annua.\u003c/em\u003e Additionally, CiHY5 was more closely related to dicotyledons than to monocotyledons, such as OsHY5 and ZmHY5 from \u003cem\u003eOryza sativa\u003c/em\u003e and \u003cem\u003eZea mays\u003c/em\u003e, indicating the consistency of phylogenetic relationships and evolutionary conservation among CiHY5 and HY5s in other species.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Nucleus subcellular localization of CiHY5 and tissue-specific expression\u003c/h2\u003e \u003cp\u003e \u003cem\u003eAgrobacterium\u003c/em\u003e-mediated transient transformation of \u003cem\u003eN. benthamiana\u003c/em\u003e leaves with the \u003cem\u003e35S:CiHY5::GFP\u003c/em\u003e fusion plasmid resulted in a GFP signal only in the nucleus (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). As a positive control, a GFP signal was observed throughout the cell after transfection with the \u003cem\u003e35S:GFP\u003c/em\u003e plasmid. CiHY5 was localized to the nucleus.\u003c/p\u003e \u003cp\u003eTissue-specific expression analysis revealed that the transcript levels of \u003cem\u003eCiHY5\u003c/em\u003e in the stems and leaves were greater than those in the other tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Relatively lower expression levels were detected in ray florets, and the lowest expression levels were detected in roots and buds. The expression level of \u003cem\u003eCiHY5\u003c/em\u003e in leaves was approximately 1.4, 64.6 and 37.6 times greater than that in ray florets, buds and roots, respectively. Taken together, the gene expression and subcellular localization data suggested the potential functions and regulation of the CiHY5 transcription factor.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.3. CiHY5 expression pattern analysis\u003c/h2\u003e \u003cp\u003e \u003cem\u003eCiHY5\u003c/em\u003e has received a great deal of attention due to its pivotal regulatory capacity in response to external conditions via the light signaling pathway. The qRT‒PCR results revealed that \u003cem\u003eCiHY5\u003c/em\u003e expression was strongly correlated with light conditions (Fig. S3). Both white light and UVB radiation significantly stimulated \u003cem\u003eCiHY5\u003c/em\u003e expression, but the expression of \u003cem\u003eCiHY5\u003c/em\u003e did not significantly change in darkness, suggesting that \u003cem\u003eCiHY5\u003c/em\u003e is an important gene involved in the response to the light signaling pathway. Under salt stress, the expression of \u003cem\u003eCiHY5\u003c/em\u003e significantly decreased and reached its lowest level at 1 h, which was 0.54 times that at 0 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Then, the expression levels gradually increased and peaked at 12 h at 1.52 to 0 h. When plants are subjected to environmental stresses, the hormone metabolism balance is disturbed. Interestingly, the expression trends of \u003cem\u003eCiHY5\u003c/em\u003e under 100 \u0026micro;mol/L ABA were similar to those under salt stress. At 1 h, the expression of \u003cem\u003eCiHY5\u003c/em\u003e decreased to the lowest level (0.39 to 0 h) and then gradually increased, but there were no significant differences at 6 h, 12 h or 24 h compared to 0 h under ABA treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). These findings suggested that \u003cem\u003eCiHY5\u003c/em\u003e may act as a pivotal component involved in light, salt stress and ABA signaling responses in \u003cem\u003eC. indicum.\u003c/em\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Identification of transgenic C. indicum\u003c/h2\u003e \u003cp\u003eTo investigate the biological functions of \u003cem\u003eCiHY5\u003c/em\u003e in response to salt stress, the complete ORF (477 bp) and specific RNA interference (RNAi) fragment (242 bp) sequences of \u003cem\u003eCiHY5\u003c/em\u003e were cloned and inserted into the overexpression and RNAi vectors \u003cem\u003epBI121\u003c/em\u003e and \u003cem\u003epART27\u003c/em\u003e, respectively, and subsequently transformed into host cells (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e, S4). Through \u003cem\u003eAgrobacterium\u003c/em\u003e-mediated leaf disc transformation, we screened stable transgenic lines (Fig. S5). RT‒PCR was used to amplify 675 bp, 1293 bp and 2051 bp products from the Kanamycin (Kana) resistance tag, universal primers from the \u003cem\u003epBI121\u003c/em\u003e vector and 35S promoter region primers, respectively, to verify the OE\u003cem\u003e-CiHY5\u003c/em\u003e transgenic plants, and a 789 bp product from the Spectinomycin (Spec) resistance tag primers was used to verify \u003cem\u003ethe\u003c/em\u003e RNAi\u003cem\u003e-CiHY5\u003c/em\u003e transgenic plants (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e, Fig. S5). There were no amplification results for the above primers in the WT plants. Eventually, we obtained seven independent OE\u003cem\u003e-CiHY5\u003c/em\u003e transgenic lines (OE-1 to OE-7) and four RNAi\u003cem\u003e-CiHY5\u003c/em\u003e lines (Ri-1 to Ri-4) for further exploration. Compared with those in the WT plants, the relative expression levels of \u003cem\u003eCiHY5\u003c/em\u003e were significantly greater in the OE-\u003cem\u003eCiHY5\u003c/em\u003e lines and lower in the RNAi\u003cem\u003e-CiHY5\u003c/em\u003e lines, indicating the successful generation of transgenic \u003cem\u003eC. indicum\u003c/em\u003e plants (Fig. S5). OE-2, OE-4, and OE-7 and the Ri-1, Ri-2, and Ri-4 lines with high, medium, and low expression levels of \u003cem\u003eCiHY5\u003c/em\u003e were selected for further investigation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.5. CiHY5 alters the growth and development of C. indicum\u003c/h2\u003e \u003cp\u003eTo provide an overview of the phenotypes of the transgenic plants, we observed the phenotypes of the C. indicum WT, OE-\u003cem\u003eCiHY5\u003c/em\u003e and RNAi-\u003cem\u003eCiHY5\u003c/em\u003e lines. The leaf area, internode length and diameter of 8-week-old plants with consistent growth from cuttings were calculated. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, compared with those of the WT plants, the leaves of the OE\u003cem\u003e-CiHY5\u003c/em\u003e plants were greater, and the OE-2 line had the greatest leaf area among all the transgenic lines. In terms of leaf number, the leaf area of the RNAi\u003cem\u003e-CiHY5\u003c/em\u003e plants did not differ from that of the WT plants, but the leaf area of the Ri-1, Ri-2 and Ri-4 plants significantly decreased compared with that of the WT plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Further exploration revealed that the OE\u003cem\u003e-CiHY5\u003c/em\u003e plants exhibited more and stronger phenotypes than did the WT plants. In contrast, the RNAi-\u003cem\u003eCiHY5\u003c/em\u003e plants presented shorter heights and greater lignification at the bottom of the stems than did the WT plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Compared with the WT and RNAi-CiHY5 lines, the OE-CiHY5 lines exhibited obviously greater increases in both the internode length and diameter and greater synchronous growth (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF, G, H). Furthermore, the RNAi-CiHY5 plants had relatively more roots and earlier flowering than did the WT and OE-CiHY5 plants, which was in agreement with the findings in Arabidopsis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE, Fig. S6).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.6. CiHY5 affects salt stress tolerance in C. indicum\u003c/h2\u003e \u003cp\u003eGiven the multifaceted roles of HY5 in the regulation of plant growth and development in \u003cem\u003eArabidopsis\u003c/em\u003e, we investigated the salt stress resistance of WT and transgenic plant lines subjected to 200 mmol/L NaCl for 10 days. During the first 5 days, the growth of the plants in the OE\u003cem\u003e-CiHY5\u003c/em\u003e and RNAi\u003cem\u003e-CiHY5\u003c/em\u003e lines did not exhibit obvious differences, but during the following days, the plants in the RNAi\u003cem\u003e-CiHY5\u003c/em\u003e lines were more severely injured by salt stress than were those in the WT and OE\u003cem\u003e-CiHY5\u003c/em\u003e lines; for example, the plants were curled and dehydrated with a brown color on a large range of leaves. In contrast to those in the WT and RNAi-\u003cem\u003eCiHY5\u003c/em\u003e lines, slight or limited injury occurred at the edge of the OE\u003cem\u003e-CiHY5\u003c/em\u003e leaves with erect stems during growth (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, B). After water recovery for 10 d, 100% of the OE\u003cem\u003e-CiHY5\u003c/em\u003e plants survived and continued to sprout new buds from the apex and lateral shoots, whereas the survival rates of the WT and RNAi\u003cem\u003e-CiHY5\u003c/em\u003e plants in the stagnant growth state were only 66.67% and 44.44%, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eTo further elucidate the molecular mechanism involved in the response to salt stress influenced by \u003cem\u003eCiHY5\u003c/em\u003e, we measured physiological parameters and indexes in WT and transgenic plants. The RWC of leaves did not obviously differ between OE\u003cem\u003e-CiHY5\u003c/em\u003e and RNAi\u003cem\u003e-CiHY5\u003c/em\u003e plants before and after 10 d of salt stress but slightly decreased in the RNAi\u003cem\u003e-CiHY5\u003c/em\u003e line (Fig. S7). Compared with those of the WT plants, the electrolyte leakage rates of the OE-7, Ri-1 and Ri-2 lines decreased by 27.32% and increased by 19.56% and 21.36%, respectively, confirming that the OE\u003cem\u003e-CiHY5\u003c/em\u003e plants could maintain the integrity of the cell membrane under salinity stress but that the RNAi\u003cem\u003e-CiHY5\u003c/em\u003e plants could not (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). Chlorophyll degradation indicates that photosynthesis is seriously impaired in plants under salinity stress. Under normal growth conditions, the total chlorophyll content of the OE\u003cem\u003e-CiHY5\u003c/em\u003e and RNAi-\u003cem\u003eCiHY5\u003c/em\u003e lines was distinctly different from that of the WT, but there was no apparent difference. However, under salt stress, a significant decrease in the total chlorophyll content in the Ri-1 (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e) and Ri-4 lines (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003c/em\u003e) is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE, indicating severe chlorophyll degradation and sensitivity to salt stress in the RNAi\u003cem\u003e-CiHY5\u003c/em\u003e plants.\u003c/p\u003e \u003cp\u003eIn terms of the antioxidant defense system, the SOD activity in the OE\u003cem\u003e-CiHY5\u003c/em\u003e lines significantly increased by 35.78%, 44.34% and 78.60% compared with that in the WT, and the CAT activity in the OE-7 line significantly increased by 36.31% compared with that in the WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF, G). Free proline accumulation and MDA reduction are closely related to the extent of injury caused by external environmental stresses in plants. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH and I, compared with those of the WT plants, the MDA content of the OE-4 and OE-7 lines significantly decreased, and the free proline content of all the RNAi\u003cem\u003e-CiHY5\u003c/em\u003e lines significantly decreased, implying that the OE\u003cem\u003e-CiHY5\u003c/em\u003e plants had a greater capacity to withstand salt stress and that the RNAi-\u003cem\u003eCiHY5\u003c/em\u003e plants had the opposite effect. Taken together, these results \u003cem\u003erevealed that compared with the WT plants, the CiHY5-overexpressing\u003c/em\u003e and RNAi-\u003cem\u003eCiHY5\u003c/em\u003e-overexpressing plants exhibited greater and lower salinity resistance, respectively.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.7. CiHY5 affects the homeostasis of Na\u003csup\u003e+\u003c/sup\u003e and K\u003csup\u003e+\u003c/sup\u003e under salt stress\u003c/h2\u003e \u003cp\u003eThe influx and efflux of Na\u003csup\u003e+\u003c/sup\u003e and K\u003csup\u003e+\u003c/sup\u003e can reflect the metabolic capacity of cells and further infer salt stress resistance in plants. Under salt stress, the Na\u0026thinsp;+\u0026thinsp;content in the leaves of the three RNAi-CiHY5 lines significantly decreased by 18.77% and 27.84% in the OE-2 and OE-7 lines, respectively, and increased by 41.24%, 32.57% and 25.52%, respectively, compared with that in the WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). However, no significant difference was found in either the K\u003csup\u003e+\u003c/sup\u003e or Na\u003csup\u003e+\u003c/sup\u003e content in roots or the K\u003csup\u003e+\u003c/sup\u003e content in leaves between the WT and transgenic lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, C, D). Intriguingly, the ratio of Na\u003csup\u003e+\u003c/sup\u003e to K\u003csup\u003e+\u003c/sup\u003e significantly increased in both the roots and leaves of the Ri-1 and Ri-4 lines but decreased in the roots of the OE-2 and OE-7 lines under saline conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE, F), although there was no obvious difference in the Na\u003csup\u003e+\u003c/sup\u003e or K\u003csup\u003e+\u003c/sup\u003e concentration in the roots. For the above six indicators, the trend of absorbed Na\u003csup\u003e+\u003c/sup\u003e and reduced K\u003csup\u003e+\u003c/sup\u003e in the OE\u003cem\u003e-CiHY5\u003c/em\u003e lines was opposite to that in the RNAi-\u003cem\u003eCiHY5\u003c/em\u003e lines under salt stress, with a decrease in Na\u003csup\u003e+\u003c/sup\u003e/K\u003csup\u003e+\u003c/sup\u003e in the OE\u003cem\u003e-CiHY5\u003c/em\u003e plants.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.8. CiHY5 modulates the expression of stress-responsive genes related to the ABA signaling pathway\u003c/h2\u003e \u003cp\u003eExogenous ABA treatment significantly downregulated the expression of \u003cem\u003eCiHY5\u003c/em\u003e from 1 h to 3 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). We attempted to confirm whether the expression levels of related ABA-dependent or ABA-independent pathway genes were altered in the transgenic plants. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, compared with those in the WT, the expression levels of the ABA-dependent pathway genes \u003cem\u003eCiRAB18\u003c/em\u003e and \u003cem\u003eCiERD7\u003c/em\u003e significantly increased in the OE\u003cem\u003e-CiHY5\u003c/em\u003e line but decreased in the RNAi\u003cem\u003e-CiHY5\u003c/em\u003e line. ABA-independent pathway genes, such as \u003cem\u003eCiDREB1D\u003c/em\u003e and \u003cem\u003eCiERF1\u003c/em\u003e, were significantly upregulated in the OE\u003cem\u003e-CiHY5\u003c/em\u003e plants but relatively downregulated in the RNAi\u003cem\u003e-CiHY5\u003c/em\u003e lines. Moreover, the expression levels of genes encoding a series of protein kinases and protein phosphatases involved in ABA signaling, such as \u003cem\u003eCiPP2C\u003c/em\u003e and \u003cem\u003eCiSnRK2\u003c/em\u003e, were significantly variable. In addition, the expression of \u003cem\u003eCiABF4\u003c/em\u003e in the OE\u003cem\u003e-CiHY5\u003c/em\u003e plants was significantly greater than that in the WT plants, and the expression of CiABF4 in the OE-CiHY5 lines increased by 3.48-, 3.59- and 3.39-fold, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). It has been reported that \u003cem\u003eABF4\u003c/em\u003e is involved in the ABA signaling pathway and specifically binds to ABA-responsive elements (ABREs) through \u003cem\u003eSnRK2\u003c/em\u003e in response to salt stress. Additionally, a genome-wide study of HY5 target genes in \u003cem\u003eArabidopsis\u003c/em\u003e suggested that HY5 binds to \u003cem\u003eABF1\u003c/em\u003e, \u003cem\u003eABF3\u003c/em\u003e and \u003cem\u003eABF4\u003c/em\u003e but not to ABF2 (Fernando et al., 2018). These findings suggested that \u003cem\u003eCiHY5\u003c/em\u003e likely affects salt stress tolerance by modulating the accumulation of \u003cem\u003eCiABF4\u003c/em\u003e in an ABA-dependent manner.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.9. CiHY5 directly combined with the promoter of CiABF4\u003c/h2\u003e \u003cp\u003eGiven that OE\u003cem\u003e-CiHY5\u003c/em\u003e transgenic plants significantly \u003cem\u003einduced CiABF4\u003c/em\u003e expression, we investigated whether CiHY5 directly bound to the promoter of \u003cem\u003eCiABF4\u003c/em\u003e. According to the instructions of the Genome Walking Kit, we utilized TAIL-PCR technology to amplify the \u003cem\u003eproCiABF4 fragment\u003c/em\u003e. Ultimately, we obtained a 1404 bp aligned sequence from the spliced products after three rounds of amplification; this sequence was regarded as \u003cem\u003eproCiABF4\u003c/em\u003e in this study (Fig. S8). The activity of \u003cem\u003eproCiABF4\u003c/em\u003e was verified by evaluation of the \u003cem\u003eGus\u003c/em\u003e gene expression of \u003cem\u003eproCiABF4:Gus\u003c/em\u003e following visible blue in tobacco leaves (Fig. S8). Analysis of \u003cem\u003ecis\u003c/em\u003e-regulatory elements revealed that a conserved G-box (light-responsive element) was present in the \u0026minus;\u0026thinsp;1104 bp to -1110 bp region in \u003cem\u003eproCiABF4\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). As previously described, HY5 predominantly acts as a pivotal transcription factor by binding G-box \u003cem\u003ecis\u003c/em\u003e-acting elements downstream of target genes to regulate physiological and developmental processes in plants. In addition, the \u003cem\u003eproCiABF4\u003c/em\u003e region also contained MeJA-responsive elements (CGTCA motif and TGACG motif), abscisic acid responsiveness elements (ABREs), MYB and MYC recognition sites, an auxin-responsive element (TGA element) and other light responsiveness elements (AE box, ATCT motif, Box 4) (Fig. S8).\u003c/p\u003e \u003cp\u003eTransactivation activity analysis revealed that after cotransformation into Y2HGold cells, all the fusion plasmids grew normally on SD-Leu/-Trp media, but only the positive control (\u003cem\u003eBD-53\u0026thinsp;+\u0026thinsp;AD-T\u003c/em\u003e) grew and produced blue spots on SD/-Trp/-Leu/-His/-Ade/X-α-gal media (Fig. S9). This finding indicated that there was no transcription of downstream reporter genes through the combination of the BD DNA binding domain and the upstream activation sequence UAS in GAL4. These results indicated that CiHY5 could be used for further verification because it has no transcriptional activation activity. Yeast one-hybrid (Y1H) assay analysis showed that both \u003cem\u003ethe AD-CiHY5\u003c/em\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003epHIS2-proCiABF4\u003c/em\u003e (experimental group) and \u003cem\u003eAD-Rec2\u003c/em\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003epHIS2-proCiABF4\u003c/em\u003e (control group) yeast strains grew normally on SD-Leu/-Trp plates, indicating that the bait and prey vectors were successfully cotransformed into yeast cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). However, the yeast strains transformed with \u003cem\u003eAD-CiHY5\u003c/em\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003epHIS2-proCiABF4\u003c/em\u003e obviously grew on SD-Leu/-Trp/-His selection media supplemented with 50 mmol/L 3-AT, but at the same time, the \u003cem\u003eAD-Rec2\u003c/em\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003epHIS2-proCiABF4\u003c/em\u003e yeast strains failed to grow (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). This result suggested that the GAL4 transcriptional domain activated \u003cem\u003ePminHIS3\u003c/em\u003e to express the \u003cem\u003eHis\u003c/em\u003e reporter gene in the experimental group, but the leakage of the reporter gene was inhibited by 50 mmol/L 3-AT, with no expression in the control group. Taken together, these results suggested that CiHY5 directly binds to the promoter of \u003cem\u003eCiABF4\u003c/em\u003e to activate downstream gene expression.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.10. CiABF4 enhances salt tolerance in C. indicum\u003c/h2\u003e \u003cp\u003eThe complete ORF sequence of \u003cem\u003eCiABF4\u003c/em\u003e, 1248 bp in length (GenBank accession number no. OP589307), encodes a 415-amino-acid polypeptide (Fig. S10). The phylogenetic analysis revealed that CiABF4 clustered with AtABF4, which possesses a conserved bZIP domain in the C-terminal region (Fig. S10).\u003c/p\u003e \u003cp\u003eTo clarify whether \u003cem\u003eCiABF4\u003c/em\u003e plays a pivotal role in the salt stress response in chrysanthemum, we performed \u003cem\u003eAgrobacterium\u003c/em\u003e-mediated instantaneous transformation with the \u003cem\u003epBI121-CiABF4\u003c/em\u003e vector to observe the phenotypes of \u003cem\u003eC. indicum\u003c/em\u003e leaf discs under salt stress. As shown in Fig. S11A, no obvious phenotypic variations were detected in the leaves of the WT and overexpressing-\u003cem\u003eCiABF4\u003c/em\u003e (OE\u003cem\u003e-CiABF4\u003c/em\u003e) plants before salt stress treatment. When 50 mmol/L NaCl was exposed to media for 5 days, in contrast with the green and complete leaf plates of OE\u003cem\u003e-CiABF4\u003c/em\u003e, the leaves of the WT plants were tawny, injured and susceptible to bacterial infection (Fig. S11B). After 5 days of normal growth, the OE\u003cem\u003e-CiABF4\u003c/em\u003e leaf plates gradually recovered and continued to differentiate, but most of the WT leaf plates still remained wilted and even putrid (Fig. S11C). These findings preliminarily verified that overexpression of \u003cem\u003eCiABF4\u003c/em\u003e increased the resistance of \u003cem\u003eC. indicum to salt stress\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn recent years, secondary soil salinization caused by human activities has become increasingly serious, especially unsustainable tillage and irrigation, resulting in reduced plant water utilization efficiency, further loss of osmotic pressure regulation in roots, sodium ion toxicity reactions, and eventually physiological damage in plants [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Transcription factors (TFs), as pivotal switches in signaling networks, provide targets for researching the molecular mechanisms of the external stress response. HY5, a member of the bZIP TF family, is considered a major regulator of the light signaling pathway and is involved in multiple growth and metabolic processes, such as cell elongation and proliferation, hypocotyl growth, chloroplast and pigment accumulation, and shade avoidance in plants [\u003cspan additionalcitationids=\"CR50 CR51\" citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. There are highly conserved key domains that bind to COP1 in the N-terminus and the coiled coil bZIP domain in the C-terminus, which combines with many gene promoters with ACGT elements (ACE) in the HY5 protein, suggesting that HY5 plays extensive regulatory roles downstream of genes in response to multiple signal transduction pathways [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. In addition, HY5 can readily form protein complexes at the peak in the early developmental stage of \u003cem\u003eArabidopsis\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eThere is intricate crosstalk between hormone signaling pathways and abiotic stress responses in plants. As previously reported, HY5 promotes photomorphogenesis through the coordinated regulation of various hormonal signaling pathways, such as those involving gibberellin (GA), ethylene, abscisic acid (ABA) and brassinolide (BR) [\u003cspan additionalcitationids=\"CR55\" citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. The nuclear and cytoplasmic distributions of HY5 affected by salt stress are positive factors that promote the binding of HY5 to the \u003cem\u003eABI5\u003c/em\u003e promoter, thereby closely influencing the salt stress response in an ABA-dependent manner [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. Mounting evidence indicates that HY5 is involved in a broad range of abiotic stress processes, such as ABA, cold, drought and salt stress; however, the specific mechanism of HY5-mediated gene regulation remains unclear [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Considering the extensive research on the potential conserved roles of HY5 in salt stress tolerance in \u003cem\u003eArabidopsis\u003c/em\u003e, extending research on the functions of HY5 to ornamental chrysanthemum is highly important. In our study, we cloned and characterized the \u003cem\u003eCiHY5\u003c/em\u003e gene and established OE-\u003cem\u003eCiHY5\u003c/em\u003e and RNAi-\u003cem\u003eCiHY5\u003c/em\u003e transgenic lines to explore the resistance of \u003cem\u003eC. indicum\u003c/em\u003e to salt stress (Figs. S4 and S5). The temporal expression patterns of \u003cem\u003eCiHY5\u003c/em\u003e in response to salt and ABA treatments and the various expression levels of ABA-responsive genes in the WT and transgenic lines deepened our understanding of the vital roles of \u003cem\u003eCiHY5\u003c/em\u003e in mediating the salt stress response in \u003cem\u003eC. indicum\u003c/em\u003e (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ePhenotypic evidence revealed that \u003cem\u003eCiHY5\u003c/em\u003e overexpression improved salt tolerance in CiHY5 transgenic plants. Compared with the WT and RNAi-CiHY5 plants, the OE-CiHY5 plants exhibited more robust growth conditions, such as a larger leaf area, longer internode length, stronger stalks, and longer nutritional reproduction with delayed flowering (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and S6), which was consistent with previous findings in \u003cem\u003eArabidopsis\u003c/em\u003e [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eUnder salt stress, the physiological parameters of the OE\u003cem\u003e-CiHY5\u003c/em\u003e lines were lower than those of the WT plants in terms of electrolyte leakage, free proline content and MDA content (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD, H and I), illustrating that the accumulated osmotic system protected the integrity of the cell membrane and decreased cell injury and lipid peroxidation [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. The OE\u003cem\u003e-CiHY5\u003c/em\u003e and RNAi\u003cem\u003e-CiHY5\u003c/em\u003e lines exhibited opposite trends in SOD activity, CAT activity and chlorophyll content (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE, F and G), indicating that the accumulation of active oxygen was effectively eliminated to increase the resistance of the OE\u003cem\u003e-CiHY5\u003c/em\u003e plants to salt stress. Similarly, HY5 is an indispensable regulator required for the accumulation of chlorophyll through binding to a subset of chlorophyll biosynthesis gene promoters in a light-dependent manner [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. The exclusion and absorption of internal Na\u003csup\u003e+\u003c/sup\u003e or Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e are the main strategies used by plants to adapt to salinity stress [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. Under salt stress, the decrease in Na\u003csup\u003e+\u003c/sup\u003e/K\u003csup\u003e+\u003c/sup\u003e in the OE\u003cem\u003e-CiHY5\u003c/em\u003e lines implies that the intake of essential mineral nutrients, such as K\u003csup\u003e+\u003c/sup\u003e, was sufficient rather than the accumulation of Na\u003csup\u003e+\u003c/sup\u003e, indicating that the generation of defense mechanisms contributed to enhancing salt tolerance in the OE\u003cem\u003e-CiHY5\u003c/em\u003e plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Moreover, the variation trends of these indices in the RNAi\u003cem\u003e-CiHY5 plants\u003c/em\u003e were opposite to those in the OE-\u003cem\u003eCiHY5\u003c/em\u003e plants under salt stress, indicating that the RNAi\u003cem\u003e-CiHY5\u003c/em\u003e plants were sensitive to salt stress. Limited research on the role of HY5 in salt stress has shown that the interaction of the HY5-HDA9 protein complex is significantly induced by salt stress, inhibiting the expression of the \u003cem\u003eHsfA2\u003c/em\u003e gene and improving salt tolerance in \u003cem\u003eArabidopsis\u003c/em\u003e [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. In addition, the \u003cem\u003ehy5\u003c/em\u003e mutant displayed opposite sensitivities to salt stress treatment during the seed germination and 7 d seedling growth stages in \u003cem\u003eArabidopsis\u003c/em\u003e, reinforcing the idea that HY5 directly regulates numerous downstream genes to activate intricate regulatory networks [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePlants have evolved sophisticated mechanisms to precisely control stress responses via hormonal pathways. ABA induces the expression of stress-responsive genes, enabling the maintenance of homeostasis of the internal environment in plant cells under external stresses [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. HY5 is extensively involved in ABA signaling and salt response during \u003cem\u003eArabidopsis\u003c/em\u003e growth and development. Early studies revealed that the expression of \u003cem\u003eABI5\u003c/em\u003e in response to ABA and salt stress depended on the direct binding of HY5 to the \u003cem\u003eABI\u003c/em\u003e5 promoter. HY5/HYH and RSM1 aggregated on the \u003cem\u003eABI5\u003c/em\u003e promoter independently or somehow dependently regulated \u003cem\u003eABI5\u003c/em\u003e and downstream of gene expression, thereby improving the adaptation to salt stress [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. To investigate the potential regulatory mechanisms involved in the response to salt stress, sequence alignments and homology analyses were carried out with transcriptome data from \u003cem\u003eC. indicum\u003c/em\u003e. Surprisingly, we only identified members highly homologous to ABI5, the ARBE/ABF subfamily family, which can specifically recognize ABRE (ABA-responsive) elements and act as key components of ABA signaling (Fig. S10). According to qRT‒PCR, \u003cem\u003eCiABF4\u003c/em\u003e was significantly upregulated in the OE\u003cem\u003e-CiHY5\u003c/em\u003e lines but not in the RNAi\u003cem\u003e-CiHY5\u003c/em\u003e lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). These results supported our hypothesis that the CiHY5-CiABF4 module mediates the salt stress response via an ABA-dependent pathway according to previous studies showing that HY5 targets \u003cem\u003eABF4\u003c/em\u003e downstream genes to induce \u003cem\u003eABF4\u003c/em\u003e expression in \u003cem\u003eArabidopsis\u003c/em\u003e via a ChIP-chip identification assay [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Subsequently, the Y1H assay showed that CiHY5 directly bound to the promoter of \u003cem\u003eCiABF4\u003c/em\u003e, which was consistent with the results of a previous ChIP-chip study (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e) [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. Compared with those in WT plants, the \u003cem\u003eabf3\u003c/em\u003e and \u003cem\u003eabf4\u003c/em\u003e mutants in Arabidopsis are more sensitive to salinity stress, and the chlorophyll content decreases and the yellowing rate increases after NaCl treatment [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]. Extensive research has provided evidence that the overexpression of \u003cem\u003eABF4\u003c/em\u003e enhances resistance to salt stress and increases the RWC and photosynthetic rate of plants compared with those of WT plants in many species, such as potato, \u003cem\u003eIpomoea batatas\u003c/em\u003e and \u003cem\u003eArabidopsis\u003c/em\u003e [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e, \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFurther analysis revealed that the instantaneous transformation of \u003cem\u003eC. indicum\u003c/em\u003e leaves to OE-CiABF4 positively contributed to salt stress tolerance (Fig. S11). Therefore, we inferred that the upregulation of \u003cem\u003eCiABF4\u003c/em\u003e induced the expression of downstream stress-responsive genes, including \u003cem\u003eCiRAB18\u003c/em\u003e, \u003cem\u003eCiERD7\u003c/em\u003e, \u003cem\u003eCiDREB1D\u003c/em\u003e and \u003cem\u003eCiERF1\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e), which in turn regulated plant resistance to salt stress in accordance with the function of \u003cem\u003eABI5\u003c/em\u003e [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]. The combined evidence from the above results in concert with those of previous studies indicated that the CiHY5-CiABF4 module \u003cem\u003eis\u003c/em\u003e positively involved in enhancing salt stress tolerance in part in an ABA-dependent manner. Furthermore, further efforts are required to obtain a thorough understanding of the role of CiHY5 in the integration of multiple signaling networks to regulate plant growth by identifying the binding sites for CiHY5-targeted genes and CiHY5-interacting proteins.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn conclusion, CiHY5 is a bZIP TF family member involved in the salt stress response in \u003cem\u003eC. indicum\u003c/em\u003e. Taken together, the phenotypic, physiological parameter and molecular mechanism data revealed enhanced salt resistance in the OE-\u003cem\u003eCiHY5\u003c/em\u003e transgenic plants and reduced salt resistance in the RNAi\u003cem\u003e-CiHY5\u003c/em\u003e transgenic plants. Although the complex regulatory mechanism of \u003cem\u003eCiHY5\u003c/em\u003e in response to abiotic stress is not clear, our results revealed a possible CiHY5-CiABF4 module for potential ABA-dependent regulation of stress responses. This study provides insight into the molecular mechanism of salt tolerance and lays a foundation for the creation of new germplasm materials that can adapt to adverse environments in chrysanthemum.\u003c/p\u003e "},{"header":"Abbreviations","content":"\u003cp\u003eCiHY5, Chrysanthemum indicum ELONGATED HYPOCOTYL 5; bZIP, basic leucine zipper; ABA, abscisic acid; WT, wild type; ABF4, ABRE BINDING FACTOR 4; RNAi, RNA interference; OE, overexpressing.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003eData availability\u003c/h2\u003e \u003cp\u003eAll data relevant to the results and analysis in this study are included in this article and its supplementary materials.\u003c/p\u003e \u003c/div\u003e \u003ch2\u003eDeclaration of competing interest\u003c/h2\u003e \u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e \u003cp\u003e This study obtained ethical approval and collection permits from the relevant authorities at the collection sites prior to gathering plant materials. All collection activities were conducted in compliance with local laws, regulations, and ethical standards.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent for publication\u003c/strong\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was financially supported by the Fundamental Research Funds for the Central Universities (grant number 2572020AW06), the National Natural Science Foundation of China (grant number 31870687) and the National Key Research and Development Program of China (grant number 2019YFD1001500).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eM.H. and Y.Z. coordinated the project and designed the experiments. Z.L., B.X. and X.L. performed the experiments and data analysis; B.C., H.L. and S.C. conducted the bioinformatics work; and B.X, Z.L, Y.Y, J.H. and M.H were involved in the preparation of the manuscript. All the authors contributed to the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe are grateful to our lab colleagues for their contributions to the lab operations.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSaibo NJ, Lourenco T, Oliveira MM. Transcription factors and regulation of photosynthetic and related metabolism under environmental stresses. Ann Bot. 2009;103(4):609\u0026ndash;23.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSong M, Wang H, Ma H, Zheng C. Genome-wide analysis of JAZ family genes expression patterns during fig (Ficus carica L.) fruit development and in response to hormone treatment. BMC Genomics. 2022;23(1):170.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang C, Shen W, Yang L, Sun Y, Li X, Lai M, Wei J, Wang C, Xu Y, Li F, et al. 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Plant J. 2020;103(5):1783\u0026ndash;95.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Chrysanthemum, CiHY5, transgenic, Salt-stress, ABA","lastPublishedDoi":"10.21203/rs.3.rs-4699886/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4699886/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eCultivated chrysanthemums are susceptible to abiotic stress, and the intricate polyploidy complicates the discovery of resistance genes. \u003cem\u003eChrysanthemum indicum\u003c/em\u003e is a native diploid species with strong resistance, which makes it an important resource for investigating stress resistance genes and improving genetic traits in ornamental chrysanthemum plants.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eIn this study, we cloned the \u003cem\u003eCiHY5\u003c/em\u003e gene and generated both overexpressing \u003cem\u003eCiHY5\u003c/em\u003e (OE-\u003cem\u003eCiHY5\u003c/em\u003e) and suppressing \u003cem\u003eCiHY5\u003c/em\u003e (RNAi-\u003cem\u003eCiHY5\u003c/em\u003e) transgenic chrysanthemum lines. After salt stress treatment, compared with the WT plants, the OE-\u003cem\u003eCiHY5\u003c/em\u003e plants exhibited a lower Malondialdehyde content and less leaf electrolyte leakage and significantly greater antioxidant enzyme activity. In contrast, the physiological parameters of the RNAi-\u003cem\u003eCiHY5\u003c/em\u003e plants exhibited opposite trends. Moreover, the Na\u003csup\u003e+\u003c/sup\u003e/K\u003csup\u003e+\u003c/sup\u003e ratio in both the leaves and roots of the OE-\u003cem\u003eCiHY5\u003c/em\u003e plants significantly decreased in contrast with that in the leaves and roots of the WT and RNAi-\u003cem\u003eCiHY5\u003c/em\u003e plants. The qRT‒PCR results showed that the expression levels of downstream stress response genes, such as \u003cem\u003eCiRAB18\u003c/em\u003e, \u003cem\u003eCiERF1\u003c/em\u003e, \u003cem\u003eCiABF2\u003c/em\u003e, \u003cem\u003eCiABF4\u003c/em\u003e, and CiDREB1D, were significantly greater in the OE-\u003cem\u003eCiHY5\u003c/em\u003e plants than in the WT plants. Additionally, a yeast one-hybrid assay revealed that CiHY5 could directly bind to the promoter of \u003cem\u003eCiABF4\u003c/em\u003e and activate \u003cem\u003eCiABF4\u003c/em\u003e expression. Transient overexpression of CiABF4 in \u003cem\u003eC. indicum\u003c/em\u003e leaf discs also improved salt stress tolerance.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eOverall, we concluded that overexpressing \u003cem\u003eCiHY5\u003c/em\u003e enhanced but RNAi-\u003cem\u003eCiHY5\u003c/em\u003e reduced salt tolerance in \u003cem\u003eC. indicum\u003c/em\u003e, acting as a pivotal candidate stress resistance gene that participates in the salt stress response at least partially in an ABA-dependent manner. The above findings demonstrated the molecular mechanisms underlying the CiHY5-mediated salt stress response and laid the foundation for the molecular breeding of chrysanthemum plants to improve resistance.\u003ca class=\"FNLink\" href=\"#Fn1\" id=\"#FNLinkFn1\"\u003e\u003c/a\u003e\u003c/p\u003e","manuscriptTitle":"Functions of CiHY5 in regulating the salt tolerance of Chrysanthemum revealed by transgenic Chrysanthemum indicum","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-21 11:50:17","doi":"10.21203/rs.3.rs-4699886/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c7f20ec9-2f65-44ab-a406-e2a4cab6d5ba","owner":[],"postedDate":"August 21st, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-08-26T20:36:02+00:00","versionOfRecord":[],"versionCreatedAt":"2024-08-21 11:50:17","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4699886","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4699886","identity":"rs-4699886","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}