Genome-wide analysis of C-repeat binding factor (CBF) family members in Pennisetum glaucum and the function of PgCBF21 under cold stress

doi:10.21203/rs.3.rs-4916646/v1

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Research Article

Genome-wide analysis of C-repeat binding factor (CBF) family members in Pennisetum glaucum and the function of PgCBF21 under cold stress

https://doi.org/10.21203/rs.3.rs-4916646/v1

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Background

Pennisetum hybrid (Pennisetum glaucum x Pennisetum purpureum), a perennial forage grass of the Poaceae family, it has a well-developed root system, strong tillering ability, high biomass yield, and rich nutritional content, making it a premium forage grass. However, Pennisetum genera grasses are sensitive to low-temperature stress and lacks the ability to adapt to cold environments, severely limiting its cultivation in temperate and high-altitude regions.

Results

We identified 23 PgCBF genes, unevenly distributed across chromosomes 1 to 7. Physicochemical property analysis revealed that the amino acid count of PgCBF family members ranged from 187 to 324, with molecular weights between 19688.36 and 34271.70 Da and theoretical isoelectric points between 4.57 and 9.82. Promoter region analysis of PgCBF genes indicated the presence of various cis-acting elements involved in hormone response, tissue-specific expression, and stress response. Additionally, qRT-PCR results showed significant upregulation of PgCBF11 expression in stems following cold treatment, with increasing over 40-fold after 32 hours of cold treatment and about 20-fold after 8 hours of cold treatment. PgCBF21 exhibited a strong response in leaves after 8 hours of cold treatment, with expression levels rising more than 37-fold. Furthermore, through the overexpression of PgCBF21, we ascertained its capacity to enhance cold tolerance in yeast.

Conclusion

This study analyzed the molecular characteristics of the PgCBF family members in Pennisetum glaucum and identified several candidate genes responsive to cold stress, providing a theoretical foundation for future research on the innovative utilization of cold-resistant resources.

Pennisetum glaucum

PgCBF family members

Cold stress

PgCBF21

Temperature is one of the key factors affecting plant growth. Low-temperature stress, as a major environmental factor limiting plant growth and development [1]. C-repeat binding factor (CBF) transcription factors are critical transcription factors in plants' response to cold stress [2]. The CBF family belongs to the AP2/ERF (APETALA2/ethylene-responsive element-binding factor) type transcription activators, which contain a highly conserved AP2 domain with a 60-amino acid DNA-binding domain [3]. This domain specifically recognizes and binds to CRT/DRE (C-repeat/drought-responsive elements) cis-acting elements in promoters, promoting the expression of downstream cold-regulated (COR) genes and enhancing plant cold tolerance [4]. A distinguishing feature of CBF proteins from other AP2/ERF proteins is the presence of "signature sequences," namely PKKP/RAGRxKFxETRHP and DSAWR, located upstream and downstream of the AP2/ERF DNA-binding domain, respectively [5]. These signature sequences are highly conserved in CBF proteins from different plant species, indicating their important functional roles.

Plant cold signaling pathways can be divided into two types, namely CBF-dependent signaling pathways and CBF-independent signaling pathways. The CBF signaling pathway is a complex cold signal regulatory network, primarily regulating plant cold tolerance through the ICE1-CBF-COR cascade [6]. ICE1 (Inducer of CBF expression1) is considered a positive regulator of CBF genes [7]. ICE proteins become active through phosphorylation or interaction with related proteins upon receiving upstream cold signals, binding to the ICE box in the CBF gene promoter region to initiate CBF gene expression [8]. In Arabidopsis, ICE1 is inactive at normal temperatures but becomes activated under cold conditions, binding to the ICE1-box and inducing CBF gene expression [9]. Overexpression of OsICE1 and OsICE2 in Arabidopsis significantly enhanced seedling cold tolerance, indicating that these two OsICEs play positive roles in the activation of cold-responsive genes and the regulation of cold tolerance [10]. Besides ICE1, other transcription factors also bind to CBF promoters, participating in the transcriptional regulation of CBF under cold stress. Sheepgrass LcMYB4, as a positive regulator, improve the chilling and freezing tolerance in transgenic Arabidopsis [11]. PIF3 (Phytochrome-interacting factors) is a key transcription factor that inhibits photomorphogenesis. In Arabidopsis, PIF3 negatively regulates cold tolerance by directly binding to the CBF gene promoter and downregulating its expression [12]. OsbZIP52 gene can bind to the G-box motif in the CBF gene promoter, forming homodimers that negatively regulate cold tolerance in rice [13]. There is also mutual regulation among CBF family members. CBF2/DREB1 negatively regulates CBF1/DREB1B and CBF3/DREB1A, and the downstream gene COR can be normally expressed in Arabidopsis atcbf2/dreb1 mutant [14].

Under low-temperature condition, the free water content within plant cells decreases, indirectly causing mechanical damage, drought, and high osmotic stress, which are all forms of adversity stress [15]. Therefore, cold resistance is closely related to the ability to withstand mechanical damage, drought, and high osmotic stress. Among the six Arabidopsis AtCBF genes, AtCBF1, AtCBF2, and AtCBF3 are cold-responsive transcription factors, while AtCBF4 is associated with drought response [16]. 14 CBF/DREB1 transcription factors were identified in soybean, most of which were strongly induced by various abiotic stresses, including cold, drought, high salinity, and high temperature [17]. Five CBF/DREB1 transcription factors were identified in rice, where OsDREB1A and OsDREB1B expression is induced by cold stress, while OsDREB2A expression is induced by drought and high salinity stress, suggesting that OsDREB may contribute to tolerance to drought, salinity, and cold [18]. In maize, ZmDREB1A expression is related not only to cold and high salinity stress but also to transient expression following mechanical injury [19].

Pennisetum hybrid (Pennisetum glaucum x Pennisetum purpureum), a perennial forage grass of the Poaceae family, it has a well-developed root system, strong tillering ability, high biomass yield, and rich nutritional content, making it a premium forage grass. Due to its drought tolerance, salt resistance, low soil requirements, and rapid growth, it has been widely cultivated in tropical regions. However, Pennisetum genera grasses are sensitive to low-temperature stress and lacks the ability to adapt to cold environments, severely limiting its cultivation in temperate and high-altitude regions. Therefore, studying the cold resistance mechanisms in Pennisetum genera grasses has become a pressing issue. Previous studies showed that glutathione-encoding genes (GRX) have been shown to play a conserved role in promoting root cell elongation and enhancing pearl millet's adaptability to drought condition [20]. Expression analysis of PgNAC genes in pearl millet across different tissues and under salt and drought stress indicates that PgNACs are involved in stress response [21]. Members of the RWP-RK gene family in Napier grass also play positive roles in heat stress response [22]. Recently, researchers have developed a comprehensive and user-friendly millets multi-omics database (Milletdb, http://milletdb.novogene.com), comprising voluminous data with browsing and analytical tools. The Milletdb contains genomes of seven genera and 1800 sets of diverse omics data including graph-based pan-genomics, transcript tomics, epigenomics, variomics and phenomics data [23]. Referring to this database, researchers had explored to research the AgNPs-mediated amelioration of salt stress resistance/tolerance in pearl millet through transcriptome analyses and physiological profiling [24]. Additionally, previous studies have shown that CBF family members play crucial roles in regulating plant growth, development, and response to environmental stresses. However, research on the PgCBF family members is lacking. This study conducts a systematic analysis of the PgCBF family members, and finds PgCBF21 has the capacity to enhance cold tolerance, providing new insights for cold tolerance research in Pennisetum spp grasses.

Identification and analysis of PgCBF family members

The amino acid sequences of AtCBF family members were downloaded from the NCBI database (https://www.ncbi.nlm.nih.gov/), including AtCBF1 (NP_567721.1), AtCBF2 (NP_567719.1), AtCBF3 (NP_567720.1), AtCBF4 (NP_200012.1), AtDDF1 (NP_172721.1), and AtDDF2 (NP_176491.1). The amino acid sequences of PgCBF family members were obtained from the Pennisetum multi-omics database (Milletdb, http://milletdb.novogene.com/), specifically from the PI 583800 accession. To identify PgCBF family members within the Pennisetum glaucum genome (PI 583800), BLASTP 2.12.0⁺ was utilized, setting the E-value threshold at 1e-20. The AP2 domain HMM file (PF00847) was downloaded from the Pfam database (http://pfam.xfam.org/) and used for alignment against the whole genome protein sequences with HMMER3v3.2.1 (http://hmmer.org) [25]. The results from both BLASTP and HMMER alignments were compared, and amino acid sequences common to both methods were selected as CBF transcription factors in the Pennisetum glaucum genome. ExPASy tool (https://web.expasy.org/protparam/) was used to analyze the physicochemical properties of the PgCBF family members, including the number of amino acids, molecular weight, and isoelectric point. The subcellular localization of PgCBF proteins was predicted using the WoLF PSORT online tool (https://wolfpsort.hgc.jp/) [26].

Phylogenetic analysis

The amino acid sequences of CBF from Arabidopsis and rice were aligned using Muscle in MEGA 7, and the amino acid sequences of CBF21 from fifteen different plant species. were also aligned using Muscle in MEGA 7 [27]. The neighbor-joining method was employed to construct the phylogenetic tree, with bootstrap values set to 1000. The evolutionary tree was visualized using Chiplot (https://www.chiplot.online/). For conserved domains of PgCBF21 protein, DNAMAN was used for multiple sequence alignment of five CBF21 protein sequences.

Protein conserved motif analysis and chromosome localization analysis

The conserved motifs of the PgCBF family members were analyzed using MEME Version (https://meme-suite.org/meme/tools/meme), with parameters set as nmotif = 10, minw = 6, and maxw = 50. The results were further visualized using the MAST motif Pattern tool in TBtools software. Amino acid conserved motifs of PgCBF family members were identified using the NCBI Conserved Domain Database (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) [28]. The chromosome distribution map of PgCBF family genes was constructed using TBtools software (Version 1.120) [29].

Cis -acting element analysis

The upstream 2000 bp promoter sequences of PgCBF genes were analyzed for cis-acting elements using Plant CARE (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/) [30]. The cis-acting element analysis diagram of PgCBF genes was generated using the GSDS 2.0 website.

Plant materials and cold stress treatment

Plant material of Pennisetum hybrid 'Bond 1' (Pennisetum glaucum x Pennisetum purpureum, 3n = 21) was obtained from China agriculture university, Beijing, China. Seeds were germinated in growth chambers (30°C, 16-hour light/8-hour dark cycle) for 7 days, then transferred to a greenhouse (25°C, 16-hour light/8-hour dark cycle) for 21 days. Uniform and vigorous plants were subjected to cold stress treatment. Seedlings were exposed to 4°C for 8, 16, 24, and 32 hours, respectively. After cold stress, leaf, stem, and root of Pennisetum hybrid 'Bond 1' were immediately collected, frozen in liquid nitrogen, and stored at -80°C.

Total RNA extraction and RT-qPCR analysis

Total RNA from various tissues of Pennisetum hybrid 'Bond 1' after cold stress was extracted using the TRIzol reagent method. Reverse transcription was performed using the HiScript III 1st Strand cDNA Synthesis Kit (gDNA wiper, R312, Vazyme). The relative expression levels of genes were quantified using the 2^{^−ΔΔCT} method. The actin 1 gene (PpACT1, NCBI accession number: MT784734) was used as an internal reference (Table S1).

The function analysis of PgCBF21 under cold stress

The two vectors, pYES2-NTB and pYES2-NTB-PgCBF21, were individually transformed into the yeast strain BY4741. After shaking the cultures for 3 days, the yeast cell suspensions were diluted (10⁰, 10^− 1,10^− 2 and 10^− 3) and plated onto SG-Ura agar plates, then they were treated at 4℃, 0℃, -10℃, -20℃ for 24 h, 48 h, 72 h and 96 h, respectively, and cultured in an incubator at 30℃. After 3–4 days, the growth of yeast colonies was subsequently observed and documented.

Statistical analysis

Statistical analysis and graphical representation were performed using GraphPad Prism 8.3.0 and SPSS 20.0. Significant differences were determined using one-way ANOVA followed by Duncan's multiple range test, with significance set at P < 0.05.

Identification and phylogenetic analysis of PgCBF family members

Based on Pennisetum multi-omics database, a total of 23 CBF family members were identified and named PgCBF1 to PgCBF23. Using ExPASy, the physicochemical properties of these 23 PgCBF proteins were predicted (Table S2). The amino acid numbers ranged from 187 to 324, and the molecular weights ranged from 19,688.36 to 34,271.70 Da. The theoretical isoelectric points (pI) of PgCBF proteins ranged from 4.57 to 9.82, with only PgCBF13 and PgCBF23 having pI values greater than 7, indicating alkaline proteins, while the others were acidic. All 23 PgCBF proteins were predicted as unstable (instability index > 40) and showed a range of lipid solubility coefficients from 50.90 to 77.30. The average hydrophilicity of these proteins was negative, suggesting they are hydrophilic. Additionally, subcellular localization predictions indicated that most PgCBF genes are localized in the nucleus. For phylogenetic analysis of PgCBF family members, amino acid sequences of CBF family members from Arabidopsis were downloaded from the NCBI database. Using MEGA7, a phylogenetic tree was constructed to analyze the evolutionary relationship between PgCBF and AtCBF family members (Fig. 1). The results revealed that the CBF family members could be classified into 4 groups: Group A included 8 PgCBFs, Group B included 10 PgCBFs, Group C included 3 PgCBFs, and Group D included 2 PgCBFs, respectively.

Conserved motif of PgCBF family members

To further investigate the amino acid structural features of PgCBF family members, MEME online was used to analyze the conserved motifs (Fig. 2). A total of 10 conserved motifs were identified among the 23 PgCBF family members. The analysis revealed significant variation in motif numbers among different family members, ranging from 6 to 9 motifs. All PgCBF members contained motifs 1, 2, 3, 4, and 6 in the same order, indicating high conservation of these 5 motifs. Some motifs were specific to certain PgCBF members; for instance, motif 9 was present only in PgCBF14, PgCBF19, PgCBF20, and PgCBF21, while motif 10 was found in PgCBF2, PgCBF3, and PgCBF13. Further amino acid conserved domain analysis revealed that all PgCBF family members contained the AP2 domain (Fig. 2). Specifically, 18 PgCBF members had the AP2 domain, and 5 PgCBF members (PgCBF4, PgCBF14, PgCBF16, PgCBF22, and PgCBF23) had the AP2 superfamily domain.

Chromosomal localization analysis

Visualization analysis of the chromosomal positions of 23 PgCBF genes was conducted using TBtools software, revealing uneven distribution across 7 chromosomes (Fig. S1). Specifically, PgCBF1 is located on chromosome 1; PgCBF2, PgCBF3, and PgCBF4 are on chromosome 2. Chromosome 3 harbors 7 PgCBF genes, the highest among all chromosomes containing PgCBF genes. Chromosome 4 contains 3 PgCBF genes, including PgCBF13, PgCBF14, and PgCBF15. Chromosome 5 hosts 2 PgCBF genes, PgCBF16 and PgCBF17. Chromosome 6 only includes PgCBF18 gene. Chromosome 7 accommodates 5 PgCBF genes: PgCBF19, PgCBF20, PgCBF21, PgCBF22, and PgCBF23, forming a gene cluster at the upper region of the chromosome.

Cis -acting element analysis

Cis-acting elements play a crucial role in determining gene function and expression patterns. Using the Plant CARE website, we analyzed all cis-acting elements within the 2000 bp upstream promoter regions of PgCBF genes. The promoter regions of the PgCBFs contain multiple cis-acting elements primarily associated with light response, plant response to stress conditions, plant hormones, and plant growth and development. As shown in Fig. 3, all PgCBF genes possess light-responsive elements, stress-responsive elements (including anaerobic induction, drought-responsive elements, and low-temperature responsive elements), plant hormone-related elements (abscisic acid, auxin, gibberellin, and salicylic acid-responsive elements), and elements related to plant growth and development (including seed-specific regulation and endosperm expression elements). These results indicate that PgCBF genes play roles throughout the growth and development processes. Additionally, the abundance of stress-related cis-elements in the promoter regions of the PgCBF genes suggests their involvement in complex mechanisms during plant responses to stress conditions.

Tissue-specific expression patterns of PgCBF family genes

To explore the tissue-specific expression pattern of PgCBF genes, Pennisetum hybrid 'Bond 1' seedlings at different stages were used as materials, and RT-qPCR was employed to detect the expression levels of 6 PgCBF genes in leaves, stems, and roots at four-week-old seedlings (Fig. 4). The results indicate significant differences in the expression patterns of PgCBF genes across different tissues. PgCBF10, PgCBF11, PgCBF19, and PgCBF20 show markedly higher expression level in leaves compared to stems and roots. PgCBF12 and PgCBF21 exhibit highest expression in roots. Interestingly, PgCBF21 shows the lowest expression in leaves, other genes generally exhibit the lowest expression in stems. Analysis of the tissue-specific expression patterns of PgCBF genes reveals differential expression patterns during different stages of plant growth, suggesting their involvement across various developmental stages of plant growth and development.

Expression patterns of PgCBF genes under cold stress

To investigate whether PgCBF genes are induced under cold stress, Pennisetum hybrid 'Bond 1' seedlings were subjected to 4°C for 8 h, 16 h, 24 h, and 32 h to simulate low temperature stress condition. We measured the expression levels of 6 PgCBF genes in roots, stems, and leaves of seedlings under cold stress, respectively (Fig. 5–7). Specifically, after 32 h of cold treatment, the expression level of PgCBF10 showed significant upregulation in stems, with expression increasing more than 7-fold, while its expression in leaves showed a downward trend. The expression level of PgCBF11 exhibited significant upregulation in stems and roots after cold stress treatment, with expression in stems increasing by over 40-fold after 32 h of cold stress treatment and in roots increasing by around 20-fold after 8 h of cold stress treatment. PgCBF12 showed highest expression in leaves and stems after 8 h of cold stress treatment, with expression increasing by more than 3-fold and 5-fold, respectively, compared to untreated condition. After 16 h of cold stress treatment, the expression level of PgCBF12 in roots reached its peak, increasing by more than 15-fold. PgCBF19 exhibited highest expression in leaves and stems after 8 h of cold stress treatment, with expression increasing by approximately 3-fold and 2.2-fold, respectively, compared to untreated conditions. PgCBF20 showed highest expression in leaves and stems after 32 h of cold stress treatment, with expression increasing by approximately 3-fold and 2.8-fold, respectively, compared to untreated condition. Interestingly, the expression level of PgCBF21 in leaf exhibited significant changes after 8 h of cold stress treatment, showing upregulation by more than 37-fold, while its expression in roots showed a downward trend. Under cold stress condition, significant expression differences of the 6 PgCBF genes were observed in roots, stems, and leaves, suggesting their potential roles in various aspects of cold stress response.

Function validation of PgCBF21 in yeast

Based on the expression patterns of six PgCBF genes under cold Stress, PgCBF21 showed higher expression level, so PgCBF21 was used as a candidate gene to analyze its biological function. To further scrutinize the evolutionary relationships, amino acid sequences of CBF21 from fifteen different plant species were utilized to construct a phylogenetic tree. The resulting cladogram delineated two major groups, with the amino acid sequence of PgCBF21 displaying a high degree of identity to SiCBF21 from Setaria italica (Fig. S2A). Next, we analyze the conserved domains of PgCBF21 protein and found that PgCBF21 protein harbors AP2 conserved domain (Fig. S2B).

In addition, we transferred the pYES2-NTB-PgCBF21 vector into yeast and investigated the function of PgCBF21 under cold stress. We observed that the bacterial solution of the negative control (pYES2-NTB) was able to grow under conditions of SG-Ura treated with at 4℃, 0℃, -10℃, -20℃ for 24 h, 48 h, respectively. However, it failed to thrive on the SG-Ura treated with at -20℃ for 72 h and 96 h, respectively. In contrast, the growth pattern of the bacterial solution from the experimental group (pYES2-NTB-PgCBF21) differed from that of the negative control group, as it exhibited growth on the SG-Ura treated with at -20℃ for 72 h and 96 h, respectively (Fig. 8).

In recent years, researchers have been actively analyzing the molecular mechanisms of plant cold acclimation, with the ICE-CBF-COR signaling pathway emerging as crucial in this adaptive process. Upon exposure to cold stress, CBF transcription factors are activated and regulate approximately 12% of cold-responsive transcription factors to mitigate this extreme environment [31]. So far, CBF genes have been identified in various plant species including Arabidopsis thaliana [5], perennial ryegrass [32], wheat [33], barley [34], and maize [19]. However, information regarding the PgCBF family members in Pennisetum genera grasses is limited. This study utilized bioinformatics methods to identify and analyze the PgCBF family members in Pennisetum glaucum, including phylogenetic trees, chromosomal positions, motifs, conserved domains, cis-acting elements, and gene expression levels.

Previous studies have reported 6 AtCBF genes in Arabidopsis, where AtCBF1, AtCBF2, and AtCBF3 are induced under low temperature conditions to enhance plant cold tolerance [35]. AtCBF4 is induced by drought and ABA, and AtDDF1 and AtDDF2 are induced under salt stress [16]. In this study, 23 PgCBF genes were identified, more closely resembling cereals such as barley with 20 CBF genes [36], and wheat with 15 CBF genes [37]. These differences may be attributed to variations in genome size among species. The CBF family members belong to AP2/ERF-type transcription factor group, characterized by a highly conserved AP2 domain. In this study, alignment of amino acid sequences from 23 PgCBF family members revealed two types of AP2 domains present in all members: 18 PgCBFs contained the AP2 domain, while 5 PgCBFs contained the AP2 superfamily domain, both exhibiting high conservation in structure. Analysis of conserved amino acid motifs across the 23 PgCBFs identified 10 conserved motifs. Motifs 1, 2, 3, 4, and 6 were present in all PgCBF members, with identical arrangement, indicating their high conservation. Furthermore, clustering analysis revealed that 5 PgCBF genes (PgCBF19, PgCBF20, PgCBF21, PgCBF22, PgCBF23) on chromosome 7 exhibit clustered distribution, similar to findings in alfalfa where 12 MsCBFs clustered on chromosome 6 [38]. This clustering suggests potential synergistic or redundant regulation of CBF genes in response to cold stress. Cis-acting elements play a crucial role in plant growth and response to abiotic stresses, determining gene expression patterns [39]. In Pennisetum glaucum, various elements related to plant growth and stress responses have been identified through cis-acting element analysis, including elements responsive to low temperature, drought, light, and hormones such as abscisic acid, gibberellins, methyl jasmonate, and salicylic acid. These findings are consistent with reports in species such as birch (Betula platyphylla Sukaczev) [40] and walnut (Juglans regia L.) [41]. Studies have demonstrated that CBF genes rapidly respond to cold stress signals [42]. For example, LpCBFs are significantly expressed under cold conditions in perennial ryegrass [43], AtCBF1 plays a crucial role in cold acclimation and freezing tolerance in Arabidopsis [44], and OsCBF3 is involved in low temperature-induced expression in rice [45]. Moreover, the expression levels of CBF genes vary with different plant tissues. In Arabidopsis, AtCBF1 and AtCBF3 are expressed in leaves and sepals after cold stress, while AtCBF2 shows expression not only in leaves and sepals but also in stems [46]. In Populus tremula, PtCBF (PtCBF 1, PtCBF2, PtCBF3, PtCBF4) genes are significantly induced in leaves, with PtCBF1 and PtCBF3 induced only in stems under low temperature condition [47]. Similarly, PgCBF11, PgCBF19, PgCBF20, and PgCBF21 show the strongest expression response in leaves under cold stress, indicating differential expression levels of various PgCBF genes across different organs in Pennisetum hybrid 'Bond 1'. These responses to cold stress observed in PgCBFs are supported by cis-acting element analysis of PgCBF promoters, suggesting an important role for PgCBF genes in responding to low temperature stress.

Different CBF family members exhibit varying efficiencies in response to cold stress. Three grapevine varieties were under cold treatment, the expression level of CBF1 began to increase at 0.5 h, reaching peak levels after 2 hours in 'Shahroodi' and 'Riparia', while reaching peak levels after 4 hours in 'Khalili-Danedar'; CBF2 reached peak expression at 8 hours in all three grapevine varieties [48]. In Arabidopsis, AtCBF1 and AtCBF3 reach peak expression in roots and cotyledons approximately 1 hour after cold treatment [13]. In Pennisetum hybrid 'Bond 1', PgCBF11 shows significant upregulation in stems and roots after cold treatment, with expression in stems increasing by over 40-fold after 32 hours of treatment and in roots by around 20-fold after 8 hours of treatment. PgCBF21 exhibits notable changes in leaf expression, with expression increasing by more than 37-fold after 8 hours of cold treatment.

In this study, 23 CBF transcription factor family members were identified in Pennisetum glaucum, classified into 4 clades. Cis-acting element analysis of PgCBF family members identified various stress-responsive elements related to light response, plant hormones, and cold stress. Six PgCBF genes exhibit sensitivity to cold stress treatment, and we found PgCBF21 can improve the cold tolerance in yeast.

CBF: C-repeat binding factor

AP2/ERF: APETALA2/ethylene-responsive element-binding factor

CRT/DRE: C-repeat/drought-responsive elements

COR: cold-regulated

ICE1: Inducer of CBF expression1

PIF3: Phytochrome-interacting factors

GRX: glutathione-encoding genes

pI: theoretical isoelectric points

Authors’ contributions

Conceived and designed the experiments: CG. Performed the experiments: WL, TL, YD, JYH and BW. Analyzed the data: WL, HW, and CG. Wrote the paper: WL and CG. All authors reviewed and approved the final manuscript.

Funding

This study was financially supported by the projects of National Key Research and Development Program of China (2023YFD2001400), China Agriculture Research System of MOF and MARA (CARS-34), Shandong Academy of Agricultural Sciences Agricultural Science and Technology Innovation Engineering (CXGC2024G16), National Natural Science Foundation of China (32201455), Science and technology development special fund project of central government guiding local government (YDZX2022146).

Availability of data and materials

All the data used in this study are included in the article with its supplementary material.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Acknowledgements

Not applicable.

Competing interests

The authors declare that they have no conflicts of interest to report regarding the present study.

Author details

1 Institute of Leisure Agriculture, Shandong Academy of Agricultural Science, Jinan 250100, China

2 Shandong Engineering Research Center of Ecological and Horticultural Plant Breeding, Jinan 250100, China

3 College of Grassland Science and Technology, China Agricultural University, No.2 Yuan Mingyuan West Road, Beijing 100193, China

4 Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, China.

Correspondence: [email protected]

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Genome-wide analysis of C-repeat binding factor (CBF) family members in Pennisetum glaucum and the function of PgCBF21 under cold stress

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Version 1

Abstract

Background

Results

Conclusion

Figures

Introduction

Materials and Methods

Identification and analysis of PgCBF family members

Phylogenetic analysis

Protein conserved motif analysis and chromosome localization analysis

Plant materials and cold stress treatment

Total RNA extraction and RT-qPCR analysis

The function analysis of PgCBF21 under cold stress

Statistical analysis

Results

Identification and phylogenetic analysis of PgCBF family members

Conserved motif of PgCBF family members

Chromosomal localization analysis

Discussion

Conclusion

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1