Genome-wide identification, expression analysis, and potential roles under low-temperature stress of bHLH gene family in Prunus sibirica

The basic helix-loop-helix (bHLH) family is one of the most well-known transcription factor families in plants, and it regulates growth, development, and abiotic stress responses. However, systematic analyses of the bHLH gene family in Prunus sibirica have not been reported to date. In this study, 104 PsbHLHs were identified and classified into 23 subfamilies that were unevenly distributed on eight chromosomes. Nineteen pairs of segmental replication genes and ten pairs of tandem replication genes were identified, and all duplicated gene pairs were under purifying selection. PsbHLHs of the same subfamily usually share similar motif compositions and exon-intron structures. PsbHLHs contain multiple stress-responsive elements. PsbHLHs exhibit functional diversity by interacting and coordinating with other members. Twenty PsbHLHs showed varying degrees of expression. Eleven genes up-regulated and nine genes down-regulated in −4°C. The majority of PsbHLHs were highly expressed in the roots and pistils. Transient transfection experiments demonstrated that transgenic plants with overexpressed PsbHLH42 have better cold tolerance. In conclusion, the results of this study have significant implications for future research on the involvement of bHLH genes in the development and stress responses of Prunus sibirica.


Introduction
The bHLH transcription factor (TF) is one of the largest gene families shared by all three eukaryotic kingdoms (Li et al., 2020) and is widely distributed in plants, animals, and fungi (An et al., 2022).bHLH transcription factors include a highly conserved bHLH domain in their sequence that includes the basic, loop, and two helix regions (Zhuang et al., 2019).The basic region is composed of 10-20 amino acids and is located at the N-terminus of the bHLH domain, which recognizes and binds to DNA (Atchley and Fitch, 1997).The His-Glu-Arg sequence that binds to the cis-element Ebox (CANNTG) in the plant bHLH domain's basic region controls the capacity of bHLH transcription factors to bind DNA (Massari and Murre, 2000;Toledo-Ortiz et al., 2003).Two amphipathic ahelices are connected by a loop to form the HLH structure.Approximately 40-50 amino acids make up the HLH domain, which is found at the C-terminus of the bHLH domain.(Wu et al., 2021).Additionally, the HLH loop has a variable length and can form homo-or hetero-dimers, depending on its interaction with the hydrophobic amino acid region of another helix (Matsushima et al., 2004).bHLH TF was first discovered in plants in 1989 (Ludwig et al., 1989).Currently, bHLH superfamily genes have been found and studied in a variety of plant species, including Arabidopsis thaliana (Bailey et al., 2003), Setaria italica (Fan et al., 2021), Prunus avium (Sabir et al., 2022), and Malus domestica (Li et al., 2022).Based on conserved domains, phylogenetic relationships, and sequence homology, plant bHLH TFs are usually classified into 15-26 subfamilies (Buck and Atchley, 2003;Pires and Dolan, 2010), or up to 35 subfamilies if they contain atypical bHLH proteins (Carretero-Paulet et al., 2010).
The bHLH family has a large number of members and performs diverse and complex regulatory functions.It participates in a wide range of biological processes, including light signal regulation (Wang et al., 2020) and hormone signal transduction (Huang et al., 2022).bHLH TFs play important roles in different stages of plant growth and development, especially floral organ growth, anther development, and floral transition.Peach MYB10.1 is overexpression in tobacco plants causes changes in anther filament and pistil length, and developmental defects in male and female gametophytes by repressing NtMYB305 during flower development (Rahim et al., 2019).The SlbHLH22 gene can promote the early flowering of tomatoes (Waseem et al., 2019), whereas SmbHLH13 delays flowering by downregulating key flowering genes (Chen et al., 2023).FcbHLHs are specifically expressed in female flower tissues (Song et al., 2021).This indicates that bHLH is largely involved in floral organ development in plants.Some bHLH TFs may be activated in response to various stresses and bind to the promoters of downstream genes involved in various signaling cascades in order to control plant stress tolerance (Mao et al., 2017), such as responding to high salt, drought, and oxidative stress (Sun et al., 2018).An increasing number of bHLH TFs families have been identified with respect to their functions under low-temperature stress in plants.For example, PubHLH1 in Pyrus ussuriensis activates the transcription and expression of stress response genes to significantly enhance the resistance of transgenic plants to low temperatures and freezing stress (Jin et al., 2016).PtrbHLH improves Poncirus trifoliata's resistance to low temperatures (Geng et al., 2019).The most well-known low-temperature response route in plants is ICE-CBF-COR (Dong et al., 2022).MdCIbHLH1 enhances the cold tolerance of Malus pumila through the C-repeat-binding factor (CBF) pathway (Gao et al., 2019).The MYC-type bHLH transcription factor ICE is recognized as a core positive regulator of cold stress response.The expression level of CsICE1 in Camellia sinensis significantly increased after lowtemperature induction (Wang et al., 2012), ICE1 and its homologous gene ICE2 significantly enhanced the lowtemperature resistance of A. thaliana (Xu et al., 2014).
P. sibirica, a deciduous shrub of Rosaceae, is native to Mongolia, eastern Siberia, and northern and northeastern China (Ma et al., 2020a).P. sibirica has excellent characteristics and significant ecological value that prevent wind damage, promote sand fixation, and soil and water conservation (Yin et al., 2019).In addition, P. sibirica has uses in food, medicine, and timber, and is an important ecological and economic tree species unique to Asia.Frost damage during early spring in northern China can easily disrupt the flowering, pollination, and fruiting of P. sibirica.This causes serious loss of flowers and fruits, adversely affecting the ornamental value, fruit yield, and quality of P. sibirica (Ma et al., 2020a).To prevent the reduction in yield and quality of P. sibirica due to cold damage during the flowering period, detecting coldresistance genes is considered an important strategy for improving its resistance in P. sibirica breeding.
Over the past few decades, bHLH genes have undergone extensive functional analysis and genome-wide identification.However, there hasn't been any reported functional study on the role of bHLH TFs in P. sibirica's ability to withstand low temperatures.In the present study, we conducted a comprehensive genome-wide analysis of bHLH TFs in P. sibirica.The expression patterns under low-temperature stress and the tissue-specific expression of PsbHLHs were estimated using Quantitative Real-time PCR (qRT-PCR).Additionally, the gene function of PsbHLH42 in cold tolerance was verified via transient transformation.The results of this study will help predict the function of PsbHLH members and provide a theoretical basis and technical support for the realization of P. sibirica's stable yield, high-quality cultivation, and provide gene resources for future improvement of the germplasm through genetic engineering.

Identification of the bHLH gene family members in P. sibirica
In total, 104 sequences with conserved bHLH domains were identified as members of the P. sibirica bHLH family.The physical and chemical properties of the PsbHLHs are shown in Table 1.The lengths of the PsbHLHs range from 91-1159 amino acids (aa), and most of these proteins (74%) had lengths of 200-500 aa.The total number of atoms ranged from 1444 (PsbHLH16) to 17840

Multiple sequence alignment and phylogenetic analysis of the PsbHLHs family
Multiple sequence alignments were performed to analyze the sequence features and the presence of conserved amino acid residues in PsbHLHs.The bHLH structural domain of P. sibirica consisted of 66 amino acids.Visualizing the results of multiple sequence alignment allowed the identification of four conserved regions, one basic region (ERRR), two helical regions (IRLLLVP or ASLEAIYKLQQL), and a loop region (KD) (Figure 1).The size of the amino acid letters corresponding to each point represents the conservation of amino acids at each point in the bHLH transcription factor gene domain.The conservation of amino acid residues at 19 sites is greater than 50%.The conservation of Arg-11, Arg-12, Pro-27, Leu-22 and Leu-59 residues was greater than or equal to 90% implying the site was highly conserved.In addition, Helix1 is more conserved than Helix2.
Using the protein sequences of the 104 PsbHLHs and 135 A. thaliana bHLHs, a neighbor-joining phylogenetic tree was created to examine the evolutionary relationships and classification of PsbHLHs members.All of the bHLH proteins were divided into 26 subfamilies based on how they were categorized in A. thaliana's bHLH proteins.The nomenclature of the PsbHLH subfamilies was consistent with that of the A. thaliana bHLH group.Among them, there were multiple members of AtbHLHs, in the Orphans, XIV, and XIII groups, but there were none of the PsbHLHs.As shown in Figure 2, the Ib subfamily had the largest number of family members, containing 12 PsbHLHs; and the smallest were subfamilies VI and XV, each containing only one PsbHLH.According to the statistics of the number of AtbHLH and PsbHLH members in each subfamily, the number of bHLHs in Va, XV, VIIIa, VIIIb, VIIIc,VI, IVb, IVd, III(d+e), IIIb, and III(a+c) were the same.

Chromosomal localization and gene duplication of PsbHLHs
According to the genome annotation information, the 104 PsbHLHs were randomly distributed on eight chromosomes and numbered PsbHLH1-PsbHLH104 based on their chromosomal locations, as shown in Figure 3.The number of PsbHLHs on the chromosomes ranged from seven to 22. Chromosome 1 contained the highest number of PsbHLHs (22).The lowest number of PsbHLHs was present on chromosome 3, which had only seven PsbHLHs.Chromosomes 2, 7, and 8 contained 13 PsbHLHs each, whereas chromosomes 4, 5, and 6 contained 9, 15, and 12 PsbHLHs, respectively (Figure 3).In addition, except for chromosome 3, all the chromosomes exhibited member aggregation.
Gene duplication is considered the primary driving force in the evolution of genomes.Analysis of the segmental and tandem replication of the gene family plays a significant role in explaining the process of gene family expansion.In this study, we investigated the duplication events in the PsbHLH gene family.Gene duplication events were observed on all the chromosomes.Genes with segmental duplications were most frequently found on chromosome 1 (11 genes), followed by chromosome 5 (8 genes).In total, 48 gene duplication events representing approximately 46% (48 of 104) of the total PsbHLHs were found.A total of 34 genes underwent segmental duplication, which accounted for 65% of all syntenic relationships and played an essential role in the evolution of the PsbHLHs.These segmentally duplicated PsbHLHs formed 19 gene clusters that were linked to each other by colored lines, as presented in Figure 4.Among them, PsbHLH10, PsbHLH41, and PsbHLH65 are duplicate gene pairs with each other.PsbHLH18 forms two duplicate genes with PsbHLH86 and PsbHLH93.Moreover, 16 (35%) PsbHLHs, including PsbHLH16/17, PsbHLH32/33, PsbHLH33/34, PsbHLH55/56, PsbHLH56/57, PsbHLH57/58, PsbHLH58/59, PsbHLH73/74, PsbHLH80/81, and PsbHLH97/98, were demonstrated to be tandem duplicated genes.Gene family members formed by tandem replication are closely arranged on the same chromosome and form gene clusters with  2).The divergence time of these gene pairs ranged from 12.22 to 78.54 Mya.The earliest gene duplication event occurred at 78.54 Mya.
To learn more about the evolutionary process of the PsbHLH gene family, a syntenic map of the bHLH genes of four Rosaceae species    (Prunus salicina, P. mume, P. persica, and P. avium) and the model plant A. thalian was constructed.Red lines represent homologous gene pairs in P. sibirica and other plant genomes.Between P. sibirica and P. avium, 93 orthologous gene pairs were identified, followed by P.
persica, P. mume, P. salicina, and A. thalian, with 89, 88, 86, and 66 homologous gene pairs, respectively.P. sibirica had more homologous genes with the four plants in the Rosaceae family, which is indicative of the taxonomic relationships between the species (Figure 5).Synteny analysis of the bHLH genes between P. sibirica and five representative plant species.Gray lines are indicative of the collinear blocks within the genomes of P. sibirica and other plants, while red lines are indicative of the syntenic bHLH gene pairs.The plants named with the different prefixes "At," "Psa," "Pm," "Pp," and "Pa" represent A. thaliana, P. salicina, P. mume, P. persica, and P. avium, respectively.

Gene structure and conserved motif analysis
The prediction of gene structure plays an important role in understanding the evolution of gene family members.The exonintron structures within PsbHLHs were investigated to determine their genetic features and structural characteristics.Different subfamilies had different intron/exon patterns, but the PsbHLHs sequences within the same subfamily had similar numbers of exons and introns, especially for PsbHLHs on homologous branches (Figure 6).The number of introns ranged from 0 to 17.No introns were present in PsbHLH19, PsbHLH40, PsbHLH69, PsbHLH75, or PsbHLH89.Exon analysis revealed that the number of exons ranged from 1 to 16.In addition, 34 PsbHLHs had no untranslated region (UTR).Gene structure analysis revealed differences in the structural composition of PsbHLHs.
Twenty motifs were identified in the PsbHLHs using Motif Elicitation (MEME).As shown in Figure 6, different PsbHLHs featured various amounts of conserved motifs, ranging from 1 to 12. Motif 1 and motif 2 constitute the conserved domain of bHLH, motif 1 contains one basic and one helical region, whereas motif2 contains one loop and another helical region (Table S1).Except for PsbHLH2, PsbHLH3, PsbHLH16, PsbHLH27, and PsbHLH103, almost all of the PsbHLHs contained motif1 and motif 2, and both of these conserved domains were close to one another.Interestingly, PsbHLH18 has two conserved domains (motif 1 and motif 2).PsbHLHs grouped in the same subfamily frequently shared consistent motif patterns.In addition, motifs 19, 12, 13, and 17 occurred only in subfamilies XII, VIIIb, III (d+e), and Ib, respectively.

Cis-acting element analysis
Cis-regulatory elements play an important role in regulating plant growth, development, and stress responses.To predict the regulatory pattern of PsbHLHs, the −2000 bp sequence upstream of ATG were analyzed in the promoter region of 104 PsbHLHs using PlantCARE (Figure 7).The discovery of numerous hormone-and that was responsive to the abscisic acid (ABRE), and 67 members had cis-acting regulatory element that was responsive to the MeJA (TGACG-motif and CGTCA-motif).In addition, PsbHLH9 had the most cis-elements ( 22), followed by PsbHLH86 (20).

Functional prediction of PsbHLHs based on GO annotation
To better understand the function of PsbHLHs as TFs, the EggNog website was used for gene ontology (GO) annotation analysis.Among the 104 PsbHLHs, 72 were annotated using GO (Figure 8).The results showed that 72 PsbHLHs were annotated to eight cellular components, 70.83% of the genes were located in cells and cell parts, 69.44% in organelles and less than 20% in other cellular components.Molecular functions include transcriptional regulator activity, binding, molecular transducer activity, and catalytic activity.PsbHLHs were annotated in The −2000 bp sequence upstream of ATG was used as the promoter regions to identify the cis-elements.Ten cis-elements were represented in different colored blocks.17 biological processes.All PsbHLHs were involved in the regulation of biological processes, and 71 genes (98.6%) were involved in cellular and metabolic processes.Thirty genes were involved in response to stimuli, 22 genes in the developmental processes, and 15 genes in reproductive processes.In addition, PsbHLH16 had the most annotations ( 16), whereas PsbHLH91 was annotated for 11 biological processes.

Expression analysis of PsbHLHs
Twenty candidate genes were identified by integration and overall consideration of the results from the phylogenetic analysis, GO annotation, and cis-element analysis of PsbHLHs to further study the expression patterns and regulation processes in P. sibirica.These 20 genes expression levels were quantified indifferent tissues (roots, stems, leaves, pistils, and stamens) using the expression levels in petals as reference.Nine genes in pistils and 11 genes in the roots were highly expressed.Four genes were highly expressed in the stems and stamens.While, only two genes (PsbHLH54 and PsbHLH88) were highly expressed in leaves.PsbHLH39 was highly expressed in all five tissues.Five PsbHLHs (PsbHLHs19/29/38/44/66) were expressed in one tissue (Figure 9).PsbHLH39 was highly expressed (>60 times) in all tissues except the stamens.The expression levels of seven PsbHLHs were upregulated by more than 60 times in the roots, PsbHLH5/29/60 exceeded by 80 times; PsbHLH11 was upregulated by 80 times in the pistil; PsbHLH35 was upregulated by more than 60 times in the pistil and stem; and PsbHLH73 was upregulated by 90 times in the stamen.

Protein interactions of PsbHLHs
Based on these syntenic relationships, orthologous AtbHLHs from 104 PsbHLHs were selected in A. thaliana to predict interactions.Most of the PsbHLHs were predicted to exhibit interactions (Figure 11).Sixty bHLHs interacted with more than one bHLH protein, with PYE (PsbHLH63) having the largest number of interacting partners (24).Detailed information on the interactions is summarized in Table 3.In addition, the gene function of the homolog PsbHLHs was detected based on its verified gene function in A. thaliana.Since PsbHLH42 is homologous to the cold-inducible gene ICE1, it is very likely to take part in P. sibirica's reaction to low-temperature stress.

Characterization of the role of PsbHLH42 in Populus ussuriensis under low-temperature stress
Based on bioinformatics studies, expression pattern analysis, and homology with ICE1, the involvement of PsbHLH42 in the cold Gene ontology (GO) annotation analysis of PsbHLHs.GO annotation of PsbHLHs was performed by three categories including cellular component, molecular function and biological process.
response was hypothesized.To investigate the function of PsbHLH42 in low-temperature stress, the pRI101-PsbHLH42-GFP vector was constructed and transiently transformed tissue culture seedlings of P. ussuriensis by the Agrobacterium-mediated method.An empty vector was transiently transformed into P. ussuriensis as the control.The qRT-PCR results demonstrated that the expression level of PsbHLH42 in the transgenic plants was significantly higher than that in the control plants after transient transformation, indicating that PsbHLH42 was successfully transferred into P. ussuriensis and further analysis could be carried out.The seedlings of the control and transgenic plants were subjected to 4°C stress for 6 h to evaluate the cold resistance of PsbHLH42.Before low-temperature treatment, the physiological signs did not alter significantly between the transgenic and control strains (Figure 12).After low-temperature treatment, compared with the control plants, the transgenic plants that overexpressed PsbHLH42 had higher soluble sugar and soluble protein contents, higher Superoxide dismutase (SOD) and peroxidase (POD) activities, and lower electrical conductivity and Malondialdehyde (MDA) content.

Discussion
Basic helix-loop-helix (bHLH) TFs play a crucial roles in a variety of processes of plant growth, metabolism, and abiotic stress response (Zuo et al., 2023).However, the characterization of bHLH genes in P. sibirica has not been reported.In the present study, we identified 104 PsbHLHs.The number of bHLH TFs varied greatly among the different plants.The number of members in the P. sibirica bHLH gene family is smaller than that of many other plants, such as Oryza sativa (183) (Wei and Chen, 2018), Triticum aestivum (225) (Guo and Wang, 2017), Gossypium hirsutum (437) (Liu et al., 2019), and Zea mays (208) (Zhang T. et al., 2018).However, it is similar to members of the Rosaceae family, such as P. salicina (95) (Zhang C. et al., 2018), P. mume (100) (Wu et al., 2022), and Rosa chinensis (100) (Zhou et al., 2020).This indicates that the evolution of the bHLH gene family is highly conserved from herbaceous plants to woody plants.The fact that 98.08% of PsbHLHs (102) were located in the nucleus, indicates that PsbHLHs mainly function as TFs in the nucleus.Meanwhile, PsbHLH47 is located in the chloroplasts, and PsbHLH71 is located in the mitochondria.It has been speculated that these genes are mainly involved in the transcriptional regulation of photosynthesis or functional mitochondrial transcription, such as respiration, in P. sibirica.
Structural variation plays a crucial role in the process of gene evolution.Introns evolve with the evolution of the Genome evolution mainly represented in the gain, loss, insertion, or deletion of introns and exons (Zhao et al., 2022).The functions of genes in the identical subfamily can also be comparable or there may additionally be functional differentiation, according to the results of gene structure analysis, which revealed similarities and differences among members of the same subfamily (Zhang et al., 2022).The five genes identified without introns were all distributed in the VIIIb subfamilies, which proved that the subfamily classification was accurate.Except for   Protein interaction network for PsbHLHs according to bHLH orthologs in A. thaliana.The online tool STRING was used to predict the network.Liu et al. 10.3389/fpls.2023.1267107Frontiers in Plant Science frontiersin.org PsbHLH2/3/16/27/103, all other PsbHLHs contained complete conserved bHLH domain (Motif 1 and Motif 2), suggesting that these five proteins have bHLH domains but are incomplete and belong to special bHLH family members (Carretero-Paulet et al., 2010).In addition, PsbHLH18 has two conserved domains (double motif 1 and motif 2), suggesting that it has undergone certain mutations or replication of the conserved domain during evolution, which may have improved its binding (interaction) ability with downstream genes.In addition to the bHLH domain, other conserved motifs also perform other key functions that are essential for each subfamily.Proteins in the same subfamily have similar conserved motif patterns, indicating that they may have similar functions (Eulgem et al., 2000).Some motifs are unique, with motifs 12, 19, 13, and 17 present in only one subfamily.The uniqueness and conservation of these motifs are also evidence of the evolutionary classification of the PsbHLH gene family.Interestingly, PsbHLH28 and PsbHLH98 belong to the III (d+e) and III (a+c) subfamilies but are closer to the IIIf subfamily in motif patterns.These results indicate both functional conservation and differentiation in PsbHLHs.
Gene duplication plays a crucial role in the acquisition of new genes and supplies the building blocks for the development of genetic diversity (Ma et al., 2021).Duplication analysis indicated that the PsbHLH gene family expanded through segmental (65%) and tandem (35%) duplication events, with segmental duplication being the main mechanism of amplification.The duplicated genes take new functions, whereas the original function of the other gene copy is maintained in the homologous genes (Baldini et al., 2022).Except for PsbHLH18 (subfamily Ib) and PsbHLH86 (subfamily VII (a+b)), all duplication events occurred within the same subfamily, suggesting that after segmental replication, new genes (PsbHLH18 or PsbHLH86) may undergo functional differentiation.Ka/Ks values can be used as indicators of the selection pressure on a gene during evolution (Chen et al., 2022).Due to the degeneracy of the code, sites within codons were defined as synonymous substitutions (Ks) when they did not result in an amino acid change, and as a non-synonymous substitutions (Ka) when they did (Harouaka et al., 2016).Generally, Ka/Ks >1 indicates positive selection, Ka/Ks =1 indicates neutral selection, and Ka/Ks <1 indicates purified selection (Lin et al., 2021).Based on the Ka/Ks ratio, all PsbHLH pairs were subjected to purifying selection.
Due to segmental or tandem duplication and gene loss events, various species have variable numbers of subgroups and genes per subfamily (Sheng et al., 2022).Phylogenetic tree analysis showed that the 104 PsbHLHs could be divided into 23 subfamilies, consistent with the results for Carthamus tinctorius (23) (Hong et al., 2019).The lack of PsbHLHs in the three subgroups (Orphans, XIV, and XIII), when compared to A. thaliana, may be due to gene loss during the evolution of P. sibirica or natural selection leading to elimination; this reflects that the bHLH gene is highly conserved during the evolution from herbs to woody plants.There was only one member each in subclasses XV (PsbHLH104) and VI (PsbHLH101), suggesting that the members of these subclasses evolved relatively slowly, were relatively functionally conserved, and may play an important role in P. sibirica.
Cis-regulatory elements (CREs) in promoter regions exert a significant influence on gene functions (Hou et al., 2021).The promoter prediction results indicated that PsbHLHs contained a large number of hormone and stress response elements.PsbHLH16 contains eight types of elements, PsbHLH37 and PsbHLH86 contains 10 MeJA elements, and PsbHLH9 contains 10 ABA elements, indicating that these genes are most likely to be induced by different hormones to affect development or stress in P. sibirica.GO annotation revealed that the regulatory functions of PsbHLHs were diverse and complex.According to annotations, 69% of PsbHLHs were participating in the regulation of biological processes.Anthocyanins are naturally occurring plant pigments with antioxidant properties (Taghavi et al., 2022).Previous research has demonstrated that TT8 regulates anthocyanin biosynthesis (Ahn et al., 2022), and it has been speculated that the homologous gene PsbHLH103 participates in anthocyanin synthesis in P. sibirica.In addition, PsbHLH44 and PsbHLH97 interact and are both annotated to be involved in reproductive and developmental processes; their homologs, DYT1 (PsbHLH44) and AMS (PsbHLH44), are both involved in stamen development in Arabidopsis, suggesting that PsbHLH44 and PsbHLH97 are most likely to interact and regulate stamen development in P. sibirica.
To a certain extent, gene expression patterns reflect their function.bHLH family members are expressed to varying degrees in different organs of P. sibirica.Many PsbHLHs are highly expressed in the roots, which is in line with the conclusions in P. mume (Wu et al., 2022).PsbHLH38 is expressed in roots, and the homologous gene RHD6 guides root hair development in Arabidopsis through processes involving auxin and ethylene, suggesting that PsbHLH38 is also involved in root development.According to previous reports, MYC participates in the Jasmonic acid (JA) signaling pathway and directly or indirectly controls stamen development through the ABA-JA pathway (Li et al., 2023).PsbHLH40/73 contains five ABAs and 4/6 MeJA, respectively, suggesting that they may be involved in the development of P. sibirica stamens.AtHEC1 is involved in female development and fertility (Gremski et al., 2007), and its homologous gene, PsbHLH19, is highly expressed, and is speculated to regulate pistil development.Therefore, PsbHLHs play an important role in the growth and development of different organs in P. sibirica.
Low-temperature stress (late frost damage) severely affects the yield and quality of P. sibirica.To further investigate the function of PsbHLHs in low temperature regulation, the expression profiles of PsbHLHs under low temperature stress were analyzed.Twenty PsbHLHs exhibited different expression patterns under lowtemperature treatment.For example, MYC2 (homolog of PsbHLH29) is a core transcription factor of the JA signaling pathway that reduces damage to plants during abiotic stress by participating in the regulation of JA-inducible genes (Oña Chuquimarca et al., 2020;Xu et al., 2022).Studies have shown that MeJA can improve plants' freezing tolerance by reducing membrane lipids and maintaining their stability (Ma et al., 2020b).In this study, PsbHLH29 was highly expressed after 15 min and 2 h of cold stress.This may be the result of plasma membrane damage in P. sibirica during the early stages of cold induction.Initially, JA was induced to regulate membrane permeability after reducing plant damage, after that decrease expression; again, JA was expressed as the stress time increased and the degree of cell membrane damage increased.This suggests that PsbHLH29 may regulate cold resistance in plants by regulating JA.AtAIB positively regulates the ABA response in Arabidopsis (Li et al., 2007).PsbHLH53(MYC3)/95(AIB) contained ABA response cis-elements, suggesting that PsbHLH53/95 may also participate in P. sibirica cold response by regulating ABA.PPI analysis revealed that PsbHLH29 (MYC2) interacts with PsbHLH53/95, suggesting that ABA and JA operate together to regulate the freezing tolerance of flowering organs of P. sibirica.The number and kind of cisacting elements on the promoter may also have a bearing on how well a gene functions, with PsbHLH44 containing three gibberellinsresponsive elements that were upregulated 80-fold in response to low-temperature stress.Meanwhile, 89 contained eight MeJAresponsive elements that were also up-regulated 30-fold early (within the first 1 h), suggesting that these two genes are induced by gibberellins and MeJA to regulate the freezing resistance of P. sibirica flowering organs.PsbHLH35, 38, 46, and 60 expression reached its highest level after 15 min of cold treatment.This phenomena could have several causes, one of which is that during the initial phases of cold treatment, active regulatory factors are quickly engaged and operate on the bHLH protein to adapt to the stressful environment, which causes rapid upregulation of bHLH.To avoid an excessive amplification of stress signals, plants must maintain a balance between normal development and adaptation to cold settings when the cold treatment time is extended.At this moment, negative regulators become active and encourage the breakdown of the bHLH protein, delaying the expression of the bHLH gene (Tan et al., 2021).
Studies have demonstrated that ICE1 and ICE2 improve the freezing tolerance of transgenic plants (Liu et al., 2018;Ma et al., 2023).Here, PsbHLH42 (ICE1) was significantly upregulated after −4°C treatment, suggesting that it may play an important role in the cold stress response of P. sibirica.PsbHLH60 interacts with PsbHLH42 (ICE1) and contains a low-temperature response element, whose homologous gene SCRM2 encodes ICE2, suggesting that PsbHLH42 and PsbHLH60 may co-regulate the P. sibirica cold response.In addition, nine genes were downregulated after lowtemperature stress, which may have been caused by the brief stress period (2 h) that was insufficient to induce gene expression.In conclusion, the above results suggest that the PsbHLH gene family may play a significant part in the cold stress response pathway.
The homolog of ICE1 gene, PsbHLH42, was used to verify its function in resisting low temperatures.Transient genetic transformation assays can rapidly identify gene function (Han et al., 2023).Because the lack of mature genetic transformation and regeneration systems in P. sibirica, we used the ideal model tree plant P. ussuriensis as the genetic transformation material (Jansson and Douglas, 2007;Movahedi et al., 2014).In this experiment, the PsbHLH42 transgenic strain showed reduced membrane damage, increased osmoregulatory substance content, and increased SOD and POD activities after low-temperature treatment compared to the control.Overexpression of the ICE1 homologous gene PsbHLH42 improved the low-temperature tolerance of P. ussuriensis.In addition, PsbHLH42 interacts with PsbHLH5, and MUTE (PsbHLH5) participates in plant stomatal development (Lampard and Bergmann, 2007); therefore, it is speculated that they may be involved in the low-temperature response by regulating stomata.To further verify the function of this gene, stable genetic transformation experiments will be conducted in the future.

Phylogenetic tree and multiple sequence alignment
MEGA software (version 11.0) and Jalview software (version 2.11.2.0) were used to visualize the sequences of the conserved domains in PsbHLHs.The sequence logo for the bHLH domain was created by submitting multiple alignment sequences to WebLogo (http://weblogo.berkeley.edu/logo.cgi)(Crooks et al., 2004).
The full-length bHLHs protein sequences from P. sibirica and A. thaliana were aligned using ClustalX (version 2.1).Thereafter, a phylogenetic tree was constructed based on the neighbor-joining (NJ) method using MEGA with the following parameter settings: Poisson's model, pairwise deletion option, and 1000 bootstrap replicates provided for statistical reliability.A phylogenetic tree was constructed using iTOL (https://itol.embl.de/)(Letunic and Bork, 2021).The A. thaliana bHLH protein sequences were downloaded from TAIR (https://www.arabidopsis.org/)(Berardini et al., 2015).Subfamily grouping of PsbHLHs was performed according to the AtbHLH classification scheme.

Chromosomal distribution, gene replication, and synteny
Chromosomal locations were identified and plotted using TBtools and the P. sibirica genomic database.The nonsynonymous (Ka) and synonymous (Ks) substitution ratios were calculated using the Simple Ka/Ks Calculator in TBtools software to acquire the natural purification selection between target gene pairs.The divergence time was calculated using the formula T = Ks/2r, with Ks being the synonymous substitutions per site, and the r of dicotyledonous plants being 1.5×10 −8 synonymous substitutions per site per year (Gaut and Doebley, 1997).Finally, sequences with complete bHLH domains were preserved and numbered PsbHLH1 through PsbHLH104 to correspond with their locations extending from top to bottom on the chromosome.
The GFF and genome data of A. thaliana, P. persica, P. mume, P. persica, and P. avium were derived from the GDR.Syntenic analysis maps of P. sibirica and other representative plants were constructed using TBtools.

Analysis of gene structure and conserved motif
The structure of PsbHLHs was analyzed using TBtools.The conserved motifs of PsbHLHs were identified using the online tool MEME (http://meme-suite.org)(Bailey et al., 2015).The sequence of each PsbHLHs was retrieved by TBtools according to the genomic full-length DNA sequences of PsbHLHs.

Prediction of the protein-protein interaction network
All PsbHLH sequences were submitted to STRING (version 11.0, http://string-db.org)(Szklarczyk et al., 2019), and A. thaliana was chosen as the reference organism.After the BLAST analysis, the orthologous genes of A. thaliana with the highest scores were used to construct a network.

Plant materials and stress treatments
P. sibirica clone No. 453 (Appraisal of Improved Cultivar of Forest Trees, S-SV-PS-002-2021, Liaoning Provincial Department of Forest and Grassland) from the National Forest Germplasm Resource Preservation Repository for Prunus species at Shenyang Agricultural University (Kazuo, Liaoning, China) was used as the experimental material.The clone No. 453 sprays were placed in an artificial climate box during their peak flowering period and subjected to stress.Samples were collected at 0 h (Control), 15 min, 30 min, 1 h, and 2 h for −4°C low temperature stress.Six different tissue samples (roots, stems, leaves, stamens, pistils, and petals) in a natural environment were collected.All samples were set up with three biological replicates, immediately frozen in liquid nitrogen, and stored at −80°C until required for further analysis.

RNA extraction and qRT-PCR analysis
Total RNA was extracted from the samples using RNA Extraction Kit (Takara, Beijing, China) following the manufacturer's instructions.The extracted RNA was then reverse transcribed to cDNA using the PrimeScript ™ RT Master Mix Perfect Real Time (Takara, Beijing, China), and stored at −80°C.Primer software (version 5.0) was used to design specific primers (Table S2), which were synthesized by SYNBIO Technologies (Suzhou, China).The 18S rRNA gene of P. sibirica was used as the internal reference (Kuchipudi et al., 2012).The qRT-PCR was performed using PerfectStart® Green qPCR SuperMix (TransGen Biotech, Beijing, China) in StepOnePlus ™ Real-Time PCR System (Applied Biosystems, Foster City, CA, United States) following the manufacturer's instructions.The qRT-PCR conditions were as follows: 94°C for 30 s, followed by 45 cycles of 94°C for 5 s and 60°C for 15 s, and 72°C for 10 s.The 2 −DDCT method was employed to calculate the relative expression levels of PsbHLHs (Livak and Schmittgen, 2001).

Construction of PsbHLH42 overexpression vector and transient transformation in Populus ussuriensis
The full-length coding sequence of PsbHLH42 was cloned into a pRI101-GFP vector.The construct was transformed into Agrobacterium tumefaciens GV3101, which was used to transform the tissue culture seedlings of P. ussuriensis (Liu et al., 2021).

Determination of physiological parameters
The physiological characteristics of pRI101-GFP empty vector and pRI101-PsbHLH42-GFP seedlings (leaves) were determined after 6 h of 4°C stress treatment.SOD and POD activities were quantified using nitrogen blue tetrazolium (NBT), and guaiacol, respectively.MDA levels were determined using the thiobarbituric acid-reactive substances method.The soluble sugar content (SS) was determined using anthrone colorimetry.Soluble protein content (SP) was determined using the Coomassie Brilliant Blue G-250 staining method.Cell membrane permeability (CMP) was determined using a conductivity meter.The CMP was first determined using a conductivity meter after mixing fresh leaf tissue (0.3 g) with distilled water (15 mL) had been mixed for 4 h.The test tubes were then placed in a boiling water bath for 30 min.After standing for 4 h, measurements were performed again using the same conductivity meter (Wang et al., 2021).

Conclusion
In our experience, this is the first genome-wide survey of the bHLH gene family in P. sibirica.A total of 104 PsbHLHs were screened in the P. sibirica genome and grouped into 23 subfamilies.PsbHLHs were randomly distributed across eight chromosomes.Conserved motifs and exon-intron structures are likely to be the same or similar within the identical subfamily.The duplication of segments is a crucial factor in the expansion of the PsbHLH gene family, and the evolution of PsbHLHs is influenced by selective pressure for purification.PsbHLHs are primarily involved in biological regulation processes and contain various lowtemperature stress-and hormone-related elements.Twenty PsbHLHs exhibited diverse expression patterns in response to different lowtemperature stresses and different tissues.Overexpression of PsbHLH42 improved the low-temperature tolerance of P. ussuriensis.In summary, these results improve our knowledge of the functional characterization of the PsbHLH gene family and provide genetic resources for cultivating high-quality P. sibirica germplasm resources with cold resistance, high yield, and quality.
FIGURE 1 Characterization of PsbHLHs domains.(A) Visualization of conserved amino acids of bHLH domains.(B) Multiple sequence alignments of the bHLH domains using the Clustal color scheme.

FIGURE 2
FIGURE 2Phylogenetic analysis of the bHLH proteins of P. sibirica and A. tatiana.Different colors represent different groups of P. sibirica bHLH subfamilies.The black stars at the ends of branches represent P. sibirica, and the empty stars represent A. thaliana.

FIGURE 4
FIGURE 4 Analysis of PsbHLHs chromosomal distribution and duplication event.The 8 chromosomes of P. sibirica are represented by chr1-chr8.Duplication segments identified were connected by gray ribbons.Different colors are used to connect segmental duplicate PsbHLH gene pairs according to different clades.Positions are in Mb.
FIGURE 6 Phylogenetic relationships, conserved motifs, and gene structural analyses of the PsbHLH gene family.(A) Neighbor-joining phylogenetic tree of the PsbHLHs.(B) Gene structure of the PsbHLHs.The green box indicates the exons, the yellow box the untranslational regions (UTRs), and the gray line the introns.(C) PsbHLH proteins conserved motif distribution.Different motifs are represented by different colored boxes.

FIGURE 9
FIGURE 9Expression patterns of the 20 PsbHLHs in different tissues (pistil, stamen, root, stem, and leaf) were examined by qRT-PCR.The expression level of the 18S rRNA gene was used as the internal control.Error bars were obtained from three biological replicates and represent standard error.Significant differences achieved by Duncan's test (p < 0.05) using SPSS are represented by different letters.

FIGURE 10
FIGURE 10Expression patterns of 20 PsbHLHs in response to low-temperature stress treatment were examined by qRT-PCR.The expression level of the 18S rRNA gene was used as the internal control.Error bars were obtained from three biological replicates and represent standard error.Significant differences achieved by Duncan's test (p < 0.05) using SPSS are represented by different letters.

FIGURE 11
FIGURE 11 FIGURE 12 PsbHLH42 can improve plant resistance to low temperature.Physiological indicators were determined in empty vector control and PsbHLH42 overexpressed plants after 0 h and 6 h at 4°C low-temperature treatment.pRI101-PsbHLH42-GFP and pRI101-GFP indicate transgenic plants and empty vector transgenic plants, respectively.(A) Control, empty vector control plants; OE1, OE2 and OE3, three PsbHLH42 transgenic plants.Relative expression levels of PsbHLH42 in the transgenic and control P. ussuriensis plants.18S rRNA was used as an internal control.(B) Relative conductivity.(C) Soluble sugar content.(D) POD activity.(E) MDA content.(F) Soluble protein content.(G) SOD activity.Error bars were obtained from three biological replicates.The statistical analyses were performed using Student's t tests (**p < 0.01).

TABLE 1
Information on physicochemical properties and prediction of subcellular localization.

TABLE 2 The
Ka/Ks ratios and estimated divergence times for duplicate pairs of PsbHLHs.

TABLE 3
Protein-protein interaction information of PsbHLHs.