Characterization of the Heat Shock Transcription Factor Family in Lycoris radiata and Its Potential Roles in Response to Abiotic Stresses

Heat shock transcription factors (HSFs) are an essential plant-specific transcription factor family that regulates the developmental and growth stages of plants, their signal transduction, and their response to different abiotic and biotic stresses. The HSF gene family has been characterized and systematically observed in various species; however, research on its association with Lycoris radiata is limited. This study identified 22 HSF genes (LrHSFs) in the transcriptome-sequencing data of L. radiata and categorized them into three classes including HSFA, HSFB, and HSFC, comprising 10, 8, and 4 genes, respectively. This research comprises basic bioinformatics analyses, such as protein sequence length, molecular weight, and the identification of its conserved motifs. According to the subcellular localization assessment, most LrHSFs were present in the nucleus. Furthermore, the LrHSF gene expression in various tissues, flower developmental stages, two hormones stress, and under four different abiotic stresses were characterized. The data indicated that LrHSF genes, especially LrHSF5, were essentially involved in L. radiata development and its response to different abiotic and hormone stresses. The gene–gene interaction network analysis revealed the presence of synergistic effects between various LrHSF genes’ responses against abiotic stresses. In conclusion, these results provided crucial data for further functional analyses of LrHSF genes, which could help successful molecular breeding in L. radiata.

The Lycoris radiata (L'Her.)Herb is a perennial bulb plant that is a member of the Amaryllidaceae family and is found in Northeast Asia, including China, South Korea, and Japan [52].It is markedly employed for preparing traditional medicine.Currently, >110 potent, structurally distinct Amaryllidaceae alkaloids have been identified or isolated for phytochemical and pharmacological research.Furthermore, it is primarily utilized for its perennial herbaceous flowers as they have good ornamental properties.It is barrenand drought-resistant, water-saving, and cold-tolerant.Additionally, L. radiata has significant tolerance against abiotic stress, including cold, drought, and soil impoverishment stresses.However, a comprehensive analysis of drought resistance in Lycoris has not been performed.Currently, sucrose degradation as well as Amaryllidaceae alkaloid and anthocyanin biosynthesis has been studied in Lycoris plants via transcriptome sequencing [52][53][54][55][56][57].There are few studies on stress-resistant genes, and they are limited to the detection of gene expression, and the function of gene has not been verified in Lycoris Our previous transcriptome research identified 22 LrHSF genes in L. radiata [52] and analyzed their motif pattern as well as the phylogenetic association of Arabidopsis with L. radiata.The assessment of subcellular localization indicated that LrHSF proteins were primarily present in the nucleus.Furthermore, the expression LrHSF gene under stress and hormone treatment was assessed by qRT-PCR, which indicated different expressions in different tissues, validating the LrHSF genes' biological role in L. radiata.In addition, the probable PPI of LrHSFs was also predicted.This research comprehensively assesses the LrHSF genes in L. radiata to provide novel data for screening crucial LrHSF genes during stress treatment and different Plants 2024, 13, 271 3 of 20 development stages in L. radiata and to furnish a theoretical foundation for assessing the functions of HSF family genes in other species.

Identification and Characterization of LrHSF Proteins in L. radiata
The prediction of HSF superfamily genes was vastly duplicated in L. radiata.BLASTP and HMMER 3.0 were employed to predict potential L. radiata's LrHSF protein sequences in the transcriptome database with an E-value threshold of <1 × 10 −5 .Using NCBI, all the identified sequences were verified, and with the help of SMART, conserved complete HSF domains were determined.Altogether 22 LrHSF proteins (LrHSF1 to LrHSF22) were identified (Table S1a).The lengths of LrHSF genes' CDS varied between 648 and 1407 bp.The amino acid length of LrHSF proteins ranged from 215 to 468 aa; their molecular weight was 24.92 to 51.35 kDa, whereas the isoelectric points were 4.77 to 8.95.The Plant-mPLoc, ProtComp 9.0, and WOLF PSORT were utilized to predict LrHSF proteins' subcellular localization, which revealed that most of these proteins had nuclear localization, while LrHSF9 was located in the cytoplasm.The NCBI was utilized to predict the homologous alignment, which revealed that 8 LrHSF proteins had the highest sequence homology with HSF proteins in Asparagus officinalis.Moreover, LrHSF5 and LrHSF19 indicated the highest sequence similarity with the Narcissus tazetta subsp.Chinensis homologous protein.These results indicated that significant physical and chemical differences exist between LrHSF proteins.

Multiple Sequence Alignment and Conserved Motifs of LrHSF Proteins
To further elucidate LrHSF proteins' conservation and diversification, their conservative motifs were predicted via MEME Version 5.5.5, which indicated that LrHSF proteins comprised 15 different conserved motifs (Figures 2 and 3).Motifs 1, 2, 4, and 5 were observed among the 22 LrHSF family members, whereas motifs 2 and 5 were identified in LrHSF22 only displayed, and LrHSF21 only displayed motif 5 (Figure 2).Furthermore, motifs 7 and 8 were exclusively identified in the HSFB, whereas 4, 10, 14, and 15 were only detected in HSFA.All HSFA members indicated motifs 1, 2, 3, 4, and 5 (Figure 3).Moreover, the HSFB members consistently indicated motifs 1, 2, 4, 5, and 8 and exclusively contained motifs 2 and 4. Each subgroup of LrHSF members mostly exhibited similar motifs; however, marked differences were observed between different subgroups.In the same subgroup, some proteins indicated similar motifs but unknown functions, suggesting a conserved protein structure in a specific subfamily of the LrHSF family.Motifs' conserved pattern and phylogenetic assessment of the same group of proteins can be essentially utilized for basing protein classification.The protein secondary structure predicted that the DBD structural domain comprises four reverse parallel folds and three helix bundles.Additionally, the 3D structure of the L. radiata HSF protein was predicted, and the starting position of the one DBD structural domain was labeled (Figure 4).

Expression Patterns Analysis of LrHSF Genes in Different Tissues
To evaluate LrHSF proteins' biological role during various organs'/tissues' developmental stages of L. radiata, the spatial specificity expression of 22 LrHSF genes in 8 L. radiata organs was assessed via qRT-PCR.As Figure 5A indicates, within each of the 8 tissues, some LrHSF genes exhibited differential expression, whereas in diverse tissues, similar expression patterns were observed, attributing the functional variation of these genes during plant development.For example, four LrHSFs (i.e., LrHSF4, LrHSF9, LrHSF11, and LrHSF16) had relatively increased expression in leaves.LrHSF7 was specifically indicated in the petal; LrHSF2 had elevated expression in the gynoecium, whereas LrHSF4 and LrHSF6 had relatively enhanced levels in the bulb.Additionally, LrHSF18 was substantially expressed in stamen tissues.In particular, predominantly expression of LrHSF19 and LrHSF20 was observed in roots, of LrHSF8 and LrHSF12 in seeds, and of LrHSF5 and LrHSF10 in the flower stalk.The bulb and leaves indicated the least expression of LrHSFs, and some genes were specifically not expressed.In particular, it was observed that LrHSF9 and LrHSF11 had similar expression patterns, suggesting their functional similarity.These data suggested that LrHSFs might be essentially involved in the growth and development of L. radiata.Furthermore, the tissue-specific expression patterns of most LrHSFs were diverse, implying their diverse functions in different organs.

Multiple Sequence Alignment and Conserved Motifs of LrHSF Proteins
To further elucidate LrHSF proteins' conservation and diversification, their conservative motifs were predicted via MEME Version 5.5.5, which indicated that LrHSF proteins comprised 15 different conserved motifs (Figures 2 and 3).Motifs 1, 2, 4, and 5 were observed among the 22 LrHSF family members, whereas motifs 2 and 5 were identified in LrHSF22 only displayed, and LrHSF21 only displayed motif 5 (Figure 2).Furthermore, motifs 7 and 8 were exclusively identified in the HSFB, whereas 4, 10, 14, and 15 were only detected in HSFA.All HSFA members indicated motifs 1, 2, 3, 4, and 5 (Figure 3).Moreover, the HSFB members consistently indicated motifs 1, 2, 4, 5, and 8 and exclusively contained motifs 2 and 4. Each subgroup of LrHSF members mostly exhibited similar motifs; however, marked differences were observed between different subgroups.In the same subgroup, some proteins indicated similar motifs but unknown

Expression Patterns Analysis of LrHSF Genes in Different Tissues
To evaluate LrHSF proteins' biological role during various organs'/tissues' developmental stages of L. radiata, the spatial specificity expression of 22 LrHSF genes in 8 L. radiata organs was assessed via qRT-PCR.As Figure 5A indicates, within each of the 8 tissues, some LrHSF genes exhibited differential expression, whereas in diverse tissues, similar expression patterns were observed, attributing the functional variation of these genes during plant development.For example, four LrHSFs (i.e., LrHSF4, LrHSF9, LrHSF11, and LrHSF16) had relatively increased expression in leaves.LrHSF7 was specifically indicated in the petal; LrHSF2 had elevated expression in the gynoecium, whereas LrHSF4 and LrHSF6 had relatively enhanced levels in the bulb.Additionally, LrHSF18 was substantially expressed in stamen tissues.In particular, predominantly expression of LrHSF19 and LrHSF20 was observed in roots, of LrHSF8 and LrHSF12 in seeds, and of LrHSF5 and LrHSF10 in the flower stalk.The bulb and leaves indicated the least expression of LrHSFs, and some genes were specifically not expressed.In particular, it was observed that LrHSF9 and LrHSF11 had similar expression patterns, suggesting their functional similarity.These data suggested that LrHSFs might be essentially involved in the growth and development of L. radiata.Furthermore, the tissue-specific expression patterns of most LrHSFs were diverse, implying their diverse functions in different organs.Based on the tissue-specificity of LrHSFs, its expression in the flowering de mental stages was assessed with the help of previous RNA-seq literature an qRT-PCR technique (Figures 5B and 6, Table S1b).During the L. radiata flowering opment stage FB, FL1, FL2, and R indicated floral bud stage, partially opening fl stage, fully opened flower stage, and senescent flower stage, respectively (Figur During the FB stage, more than half of LrHSF genes indicated increased expre which notably reduced at the FL1 stage.LrHSF9 indicated a gradual increase in Based on the tissue-specificity of LrHSFs, its expression in the flowering developmental stages was assessed with the help of previous RNA-seq literature and the qRT-PCR technique (Figures 5B and 6, Table S1b).During the L. radiata flowering development stage FB, FL1, FL2, and R indicated floral bud stage, partially opening flower stage, fully opened flower stage, and senescent flower stage, respectively (Figure 5B).During the FB stage, more than half of LrHSF genes indicated increased expression, which notably reduced at the FL1 stage.LrHSF9 indicated a gradual increase in its expression during flower development, whereas the LrHSF7 gene indicated increased expression during the FL1 stage, suggesting that it may have diverse activities.Furthermore, the expression of LrHSF2, LrHSF4, LrHSF6, LrHSF8, LrHSF12, LrHSF17, LrHSF18, and LrHSF20 was also relatively increased during the early flower development stages.As shown in Figure 6, the expression levels of 22 LrHSF genes were normalized to the expression of reference gene LrTIP41 by qRT-PCR analysis, and comparisons between the RNA-Seq data and qRT-PCR results were also conducted at different flower developmental stages of L. radiata, showing their good agreements (Figure 6).

Expression Analysis for LrHSFs Genes under Different Abiotic Stresses
To investigate whether different abiotic stresses restricted the expression of LrHSF genes, the LrHSF genes' expression patterns under cold, drought, heat, and salt stresses were analyzed with qRT-PCR (Figures 7 and 8).The results showed that the functional genes in L. radiata during various stresses were slightly different than the control.Furthermore, some LrHSF genes exhibited marked up-and down-regulated expression under different stresses.Most LrHSF genes also indicated marked differences in leaves during the treatment period.For instance, cold stress induced most LrHSF genes, whereas during heat stress, most genes were expressed in leaves.Notably, LrHSF2, LrHSF4, LrHSF6, LrHSF8, LrHSF10, LrHSF15, LrHSF16, and LrHSF21 indicated alternate expression patterns compared to heat and cold stress in leaves (Figure 7).The expression of LrHSF5, LrHSF9, LrHSF11, LrHSF20, and LrHSF21 was markedly up-regulated under cold stress and mostly concentrated at 24 h treatment time (Figure 7A).During heat

Expression Analysis for LrHSFs Genes under Different Abiotic Stresses
To investigate whether different abiotic stresses restricted the expression of LrHSF genes, the LrHSF genes' expression patterns under cold, drought, heat, and salt stresses were analyzed with qRT-PCR (Figures 7 and 8).The results showed that the functional genes in L. radiata during various stresses were slightly different than the control.Furthermore, some LrHSF genes exhibited marked up-and down-regulated expression under different stresses.Most LrHSF genes also indicated marked differences in leaves during the treatment period.For instance, cold stress induced most LrHSF genes, whereas during heat stress, Plants 2024, 13, 271 8 of 20 most genes were expressed in leaves.Notably, LrHSF2, LrHSF4, LrHSF6, LrHSF8, LrHSF10, LrHSF15, LrHSF16, and LrHSF21 indicated alternate expression patterns compared to heat and cold stress in leaves (Figure 7).The expression of LrHSF5, LrHSF9, LrHSF11, LrHSF20, and LrHSF21 was markedly up-regulated under cold stress and mostly concentrated at 24 h treatment time (Figure 7A).During heat stress, LrHSF5, LrHSF8, LrHSF9, LrHSF10, LrHSF11, LrHSF12, LrHSF15, LrHSF16, LrHSF18, and LrHSF19 were notably up-regulated at 24 h in leaves (Figure 7B).Moreover, most genes also indicated a markedly increased expression under salt and drought stresses (Figure 8).In L. radiate, LrHSF5 and LrHSF10 responded to drought, whereas LrHSF1 and LrHSF21 responded to salt stress at 12 h in leaves.Additionally, some LrHSF genes were notably up-regulated and down-regulated during different stressors, and most of these genes were substantially different in leaves with the treatment period.
Furthermore, the expression of LrHSFs in L. radiata leaves under ABA and MeJA treatment was assessed to further elucidate the genes' functions, indicating differences in expression patterns after hormone treatments (Figure 9).For example, after ABA treatment, most genes were down-regulated, while LrHSF5, LrHSF8, LrHSF13, LrHSF16, and LrHSF18 were up-regulated at 24 h in L. radiata leaves.Moreover, during MeJA treatment, LrHSF1 and LrHSF5 indicated the highest expression at 12 h, while LrHSF1 and LrHSF5 were downregulated at 12 h and 24 h in L. radiata leaves.Additionally, the expression of LrHSF10 and LrHSF16 was increased during MeJA treatment.

LrHSF Proteins' PPI Networks
The STRING database predicted various LrHSF proteins linked with each other on the basis of the orthologs of Arabidopsis, consistent with the literature suggesting that the binding activity of HSF proteins relies on the formation of homo-or hetero-dimers among HSF proteins (Figure 10, Tables S2 and S3).Several key interactions were then predicted.Using STRING 11.5 e-software, an HSF PPI network was established to identify PPIs.Altogether, 8 high-confidence interacting HSF family proteins were identified in the Arabidopsis.For instance, LrHSF22 is substantially homologous to HSFA2 of Arabidopsis, suggesting that it may potentially interact with plant defense proteins APX2 (ascorbate peroxidase 2), ClpB1 (Caseinolytic protease B1), DREB2A (dehydration-responsive elementbinding 2A), HSP70 (heat shock protein 70), HSP90 (heat shock protein 90), and MPK3 (mitogen-activated protein kinase 3) more strongly.Similarly, the LrHSF03, LrHSF15, and LrHSF16 proteins were highly homologous to HSFB2A, the LrHSF12 and LrHSF17 proteins were highly homologous to HSFA1A and HSFA1B, LrHSF13 was to HSFA1A, and LrHSF21 was to HSFA3 of Arabidopsis.It is presumed that these proteins have a stronger interaction with the internal HSF family members.In this investigation, the HSF members indicated an expanded regulation network, suggesting that these genes might be crucial for sensing and responding to abiotic stresses.These results partially confirmed the hypothesized interaction networks and suggested their comparable roles in L. radiata.

LrHSF Proteins' PPI Networks
The STRING database predicted various LrHSF proteins linked with each other on the basis of the orthologs of Arabidopsis, consistent with the literature suggesting that the binding activity of HSF proteins relies on the formation of homo-or hetero-dimers among HSF proteins (Figure 10, Tables S2 and S3).Several key interactions were then predicted.Using STRING 11.5 e-software, an HSF PPI network was established to identify PPIs.Altogether, 8 high-confidence interacting HSF family proteins were identified in the Arabidopsis.For instance, LrHSF22 is substantially homologous to HSFA2 of Arabidopsis, suggesting that it may potentially interact with plant defense proteins APX2 (ascorbate peroxidase 2), ClpB1 (Caseinolytic protease B1), DREB2A (dehydration-responsive element-binding 2A), HSP70 (heat shock protein 70), HSP90 (heat shock protein 90), and MPK3 (mitogen-activated protein kinase 3) more strongly.Similarly, the LrHSF03, LrHSF15, and LrHSF16 proteins were highly homologous to HSFB2A, the LrHSF12 and LrHSF17 proteins were highly homologous to HSFA1A and HSFA1B, LrHSF13 was to HSFA1A, and LrHSF21 was to HSFA3 of Arabidopsis.It is presumed that these proteins have a stronger interaction with the internal HSF family members.In this investigation, the HSF members indicated an expanded regulation network, suggesting that these genes might be crucial for sensing and responding to abiotic stresses.These results partially confirmed the hypothesized interaction networks and suggested their comparable roles in L. radiata.

Subcellular Localization of LrHSF Proteins
The online software WolfPsort, ProtComp 9.0, and Plant-mPLoc predicted LrHSF proteins were present in the nucleus, whereas LrHSF9 was located in the plasmic (Table S1a).To validate the subcellular localization of certain LrHSF pro

Characterization of the LrHSF Gene Family in L. radiata
Recent rapid advancements in metagenomics have identified and characterized HSF genes in multiple plants, such as A. thaliana [15], rice [8], potato [9], Brachypodium distachyon [58], Triticum aestivum [59], and Zea mays [60].However, the LrHSF family remains to be elucidated.This research identified 22 LrHSF genes in L. radiata with HSF proteins comprising 215 to 468 amino acids (Table S1a).According to the conserved structural domains, Arabidopsis was categorized into three subgroups (Figure 1).Additionally, in L. radiate, all the HSF fragment duplication genes were from the same subfamily, suggesting that they may have similar functions.The literature suggests that the HSFs of class A were the primary modulatory factors during heat stress, while classes B

Characterization of the LrHSF Gene Family in L. radiata
Recent rapid advancements in metagenomics have identified and characterized HSF genes in multiple plants, such as A. thaliana [15], rice [8], potato [9], Brachypodium distachyon [58], Triticum aestivum [59], and Zea mays [60].However, the LrHSF family remains to be elucidated.This research identified 22 LrHSF genes in L. radiata with HSF proteins comprising 215 to 468 amino acids (Table S1a).According to the conserved structural domains, Arabidopsis was categorized into three subgroups (Figure 1).Additionally, in L. radiate, all the HSF fragment duplication genes were from the same subfamily, suggesting that they may have similar functions.The literature suggests that the HSFs of class A were the primary modulatory factors during heat stress, while classes B and C members indicated no transcriptional activity [39,61].Numerous researches validated that in the HSF gene family, HSFA2 and HSFA7 are the most heat-sensitive transcription factors [62]; they respond to heat stress by modulating plant hormones [63], protein synthesis [64], and ROS signaling pathways [65].The phylogenetic tree predicted the biological role of unknown genes using known gene activities.A substantial HSF gene collinearity was observed between L. radiata and Arabidopsis, such as LrHSF1/AtHSFA2, LrHSF8/AtHSFA2, and LrHSF12/AtHSFA2 (Figure 1; Supplementary Table S3).It has been observed that AtHSFA2 modulates the ABA pathway to negatively control A. thaliana heat resistance [66], inferring that LrHSF1, LrHSF8, and LrHSF12 might be linked with heat stress.Additionally, LrHSF8 and LrHSF12 corresponded to rice HSF genes (OsHsfB2b), which negatively modulated salt and drought tolerance [67].The direct homologous gene of LrHSF7, LrHSF13, and LrHSF17 is OsHSFA1, which promotes plants' reproductive development and improves stress resistance [68].Therefore, these LrHSF homologs may have similar functions under stress.Protein homology was found through the normal mode option for 3D modeling (Figure 4).The N-terminal of HSF [69] mainly consists of an α-helix structure.A study about chaperonins demonstrated that their α-helical structure facilitates the folding of a protein faster [69].The results of the recent study are compatible with rice [8], sunflower [69], and jujuba [70].

Expression Patterns and Function Prediction Analysis of LrHSFs
The HSF genes are markedly linked with plants' development and growth.Here, the expression of selected 22 LrHSF genes was assessed in various tissues and developmental stages.It was indicated that the LrHSF genes were markedly expressed in L. radiata.It has been reported that most HSF transcription factors regulate Arabidopsis development.For example, AtHSFB2A from subgroup B2 modulates its gametophyte development [68].LrHSF18 and AtHSFB2A were clustered in subgroup B2, and both were highly expressed in stamen (Figure 5), suggesting their similar functionality.Moreover, the overexpressing transgenic lines of AtHSFB4 indicated a shorter root length phenotype than the wild type, suggesting its involvement in the negative root development modulation [71].Furthermore, in subgroup B4, AtHSFB2A was also clustered with LrHSF2, LrHSF4, and LrHSF6, all of which showed reduced expression in roots (Figures 5 and 6), suggesting that LrHSF2, LrHSF4, and LrHSF6 might have similar biological functions in L. radiata roots.
In plants, HSF proteins essentially regulate responses against various abiotic stresses, for instance, the response against drought, cold, salt, and high temperature [19].This research found that most LrHSF genes were markedly up-regulated in leaves during cold and heat treatments, while some were up-regulated during drought and salt treatments (Figures 7 and 8).This elaborates on the increased adaptability of L. radiata in alpine or arid regions.In Arabidopsis, most heat stress-response gene expressions are modulated by AtHsfA1 [61,72,73].It has been observed that AtHsfA1b and AtHsfA1d increase heat and drought tolerance, respectively [74,75], whereas AtHsfA2 and AtHsfA3 modulate heat tolerance, and AtHsfA2 also elevates anoxia tolerance [76][77][78][79][80]. Furthermore, in L. radiate, 10 LrHSF genes (LrHSF5, LrHSF8, LrHSF9, LrHSF10, LrHSF11, LrHSF12, LrHSF15, LrHSF16, LrHSF18, and LrHSF19) were observed to be markedly up-regulated under heat tolerance (Figure 7B).Some studies indicated that in plants, HSF genes also react against cold stress.For example, 6 PvHsf genes in common bean and 5 VviHsf genes in wild Chinese grapevine respond to cold stress [81,82].This investigation identified 5 LrHSF genes (LrHSF5, LrHSF9, LrHSF11, LrHSF20, and LrHSF21) that responded to cold stress in L. radiata (Figure 7A).In Tamarix hispida, salt tolerance was positively modulated by ThHSFA1, which directly stimulated ThWRKY4 [83].In rice, OsHsfB2b has been observed to negatively control salt and drought tolerances [66].An investigation on HSF genes in carrots identified that 33 of these genes were down-regulated during drought stress, whereas during salt stress, three genes were up-regulated, suggesting that these genes might be associated with salt and drought stresses [17].Here, 18 and 22 LrHSF genes were identified to respond to salt and drought stresses, respectively, whereas 18 genes responded to both these stresses (Figure 8).Additionally, LrHSF5 expression was observed under all stresses, validating it as a potential candidate gene for improving crop breeding.Altogether, these data suggest that in plants HSF genes crucially modulate responses to various abiotic stresses.

PPI Network Prediction and Validation of LrHSFs
Numerous researches have revealed that HSF proteins play an important function for replaying to respond abiotic stress [31,84].For example, HSFA1A (homolog of LrHSF13) and HSFA1B (homolog of LrHSF17) directly induce the expression of heat-stress-responsive transcription factors DREB2A, HSFA2 (homolog of LrHSF22), HSFA7s, and MBF1, further activating HSFA3 (homolog of LrHSF21) expression to maintain the heat stress response (Figure 10) [15,85].HSFA1 also negatively regulated the nuclear localizations and activities by HSP70 and HSP90, whereas heat stress induces the accumulation of unfolded proteins that interact competitively with HSP70 and HSP90, thereby causing HSFA1s to be released from the HSP70 and HSP90 complex and become active [86].ClpB1 interacted with Hsp70, Hsps, and proteasomes during heat stress [87].The survival tare of AtClpB1 mutant hot1-3 is lower compared to wild type plants under heat tolerance [88].MBF1, HSP70, HSP90, and DREB2A proteins were associated with abiotic stress responses, and ClpB1 protein is a critical component in governing tolerance against heat tolerance [88][89][90].Furthermore, LrHSF13 (HSFA1A homologs) and LrHSF17 (HSFA1B homologs) were observed to be markedly up-regulated under heat tolerance at 24h in L. radiata.LrHSF13 and LrHSF17 might have a similar function in heat stress response.HSFA2 is a regulator of multiple environmental stress responses required for stress acclimation, highly induced by HSFA1s following heat exposure, and strongly reduced the expression of HSP18, HSP21, HSP22, HSA32 (heat-stress-associated 32 KD protein), and APX2 [80,91,92].APX2 proteins regulated plant oxidative damage and induced the expression of the ROS-scavenging-related gene for response against abiotic stress in Arabidopsis [93].LrHSF22 may have the similar functions with HSFA2 and play conserved roles in the response to heat stress in plants.Plant HSF TFs are downstream components of the signal transduction pathway and maintain regulatory roles for stress-related gene expression [94].It was hypothesized that nearly all LrHSFs localize to the nucleus so that they may perform transcriptional functions (Figure 11).Additional research is necessary to demonstrate the comprehensive interaction network of the LrHSF TFs during L. radiata growth and development.

Plant Materials and Plant Treatments
The Lycoris radiata (L'Her.)Herb was cultivated at the Institute of Botany, Jiangsu Province and the Chinese Academy of Sciences, Nanjing, China.The similar diameter size (2.8-3.2 cm) seedlings were transferred into plastic pots containing soil and vermiculite (v/v, 1:1) and incubated in a plant growth chamber with 120 µmol m −2 s −1 irradiation at 22 • C for an 8 h dark and 25 • C for a 16 h light cycle.The expression pattern of 22 LrHSF genes under different stresses was determined.Briefly, L. radiata seedlings were subjected to different abiotic stress treatments, including drought (20% PEG 6000), high-temperature (42 • C), salt (200 mM NaCl), and low-temperature (4 • C) stresses.For high-temperature and low-temperature stress, L. radiata seedlings were transferred into a plant light incubator set as 42 • C and 4 • C, respectively.The control seedlings incubated in the distilled water were maintained in the plant growth chamber under normal growth conditions.For hormonal treatments, a solution comprising abscisic acid (ABA, 0.1 mM) and methyl jasmonate (MeJA, 0.1 mM) was sprayed on the seedlings while the control was sprayed with distilled water.Post 12 and 24 h of treatment, all the treated leaves were sampled for RNA isolation.All the experiments were repeated thrice.Furthermore, using the flower stalks, petals, seeds, leaves, stamens, roots, gynoeciums, and bulbs, the tissue-specific transcription data of 22 LrHSF genes of these plants were analyzed.Before storage at −80 • C, all the samples were snapping frozen in liquid nitrogen.

Identification and Sequence Analysis of LrHsf genes in L. radiata
To screen the potential LrHSF, the L. radiata transcriptome dataset comprising 87,584 unigenes associated with the four flower development stages was used [52].AtHSF proteins were imported from TAIR (Arabidopsis Information Resource database, https://www.arabidopsis.org/)(accessed on 7 June 2023) to elucidate database-acquired L. radiata transcripts' sequence homology via the basic local alignment (BLASTn).Furthermore, the PFAM protein family database (http://pfam.sanger.ac.uk) (accessed on 9 June 2023) was employed to generate a Hidden Markov Model (HMM) comprising HSF domains (PF0047).The HMM model cutoff value of 0.01 was set in HMMER 3.0 to compare LrHSF protein sequences of L. radiata (http://plants.ensembl.org/hmmer/index.html)(accessed on 10 June 2023).Subsequently, the predicted LrHSF transcription factors' HSF domain was verified via the NCBI Batch CD-Search Tool (https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi)(accessed on 10 June 2023) under default settings.It was suggested that this characteristic will have an increased confidence relation with the conserved domain.For further assessment, the sequences that were identified as specific hits were selected (Table S1).Moreover, the databases SMART and PFAM (http://smart.embl-heidelberg.de/)(accessed on 11 June 2023) were employed to verify the HSF domain in each selected protein sequence.Lastly, with the help of the ExPASy (https://web.expasy.org/protparam)(accessed on 11 June 2023), the molecular sizes (MW), protein instability index, isoelectric points (PI), and the complete amino acid sequences were determined.

Total RNA Extraction, cDNA Reverse Transcription, and qRT-PCR Analysis
For whole RNA isolation, the RNA prep Pure Plant Kit (BC508, Huayueyang, Beijing, China) was employed per the kit's guide.cDNA was generated via the PrimeScript™ II 1st Strand cDNA Synthesis Kit (TaKaRa Bio, Dalian, China), which was then used for relative gene expression levels assessment via qRT-PCR utilizing SYBR ® Premix Ex Taq™ II (Takara Bio, Dalian, China) on a Bio-Rad iQ5 Real-Time PCR System (Bio-Rad, Hercules, CA, USA).A prepared reaction was 15 µL and comprised ddH 2 O (5.9 µL) of 20 µM reverse and forward primers (0.6 µL, respectively), cDNA (1 µL), and 2×TransStart ® Top Green qPCR SuperMix (7.5 µL).The RT-qPCR protocol included the following: the PCR reaction conditions were at 95 • C for 5 min; denaturation 5 s at 95 • C; 60 • C for 30 s; 40 cycles.To normalize relative levels of target gene expression via the 2 −∆∆Ct method [95], the LrTIP41 gene [52] was chosen to serve as the reference gene according to a previous study on L. aurea [96].Supplementary Table S4 enlists the primer sequences used.

Gene Cloning and Expression Vector Construction
On the basis of the RNA-seq database acquired, the unigenes' putative ORFs, LrHSFs were cloned.For ORF sequence amplification, primers were prepared using Tks GflexTM DNA Polymerase (Takara, Dalian, China) from L. radiata petal cDNA (Table S4).Reaction conditions were: 5 min of 95 • C, 40 cycles for 30 s at 95 • C, 30 s at 58 • C, 2 min at 72 • C, with extension at 72 • C for 10 min.The obtained PCR products were then cloned into pTOPO001 simple vectors (Genesand, Beijing, China).Thereafter, for amplification, the T-vectors were transfected into TOP10 competent cells (Genesand, Beijing, China).Using the One Step Cloning Kit (Genesand, Beijing, China), the LrHSFs overexpression vectors were established, and their ORFs were linked into a pBIN-MCS-GFP4 plant transformation vector.Lastly, Agrobacterium tumefaciens EHA105 competent cells were transfected with the 35S: LrHSFs recombinant vectors.

PPI Network Prediction of LrHSF Proteins
The potential PPI networks were predicted by the STRING.410database (https://cn.string-db.org)(accessed on 11 August 2023) and on the basis of A. thaliana homologous proteins.Then, selecting A. thaliana as the comparative organism, sequences of 22 LrHSF proteins was submitted to the server (Table S2).The interaction network of LrHSF genes was established after blasting with the highest bitscore.

Statistical Analysis
All analyses were repeated thrice, and the data acquired were presented as mean ± SD.For data analysis, a Student t-test was carried out.* p < 0.05 and ** p < 0.01 were deemed significant.

Conclusions
In summary, identification and systematic analysis of HSF genes in L. radiata showed that the 22 LrHSF genes were classified into three subfamilies.A comprehensive assessment of conserved motifs of these 22 genes demonstrated similar motifs in the same, suggesting that they might have functional similarities.Furthermore, a preliminary structural analysis of LrHSF genes further detailed their expression pattern and indicated that LrHSF genes are crucial for L. radiata growth and development and respond to the hormonal and abiotic stresses during its development.

Supplementary Materials:
The following supporting information can be downloaded at https: //www.mdpi.com/article/10.3390/plants13020271/s1,Table S1.Characteristics of LrHSF genes identified from L. radiata transcriptome; Table S2.The full-length amino acids sequence of LrHSF proteins; Table S3.Summary information for LrHSF proteins in the STRING database; Table S4.Primers used in this study.
Author Contributions: N.W. and Z.W. were major contributors to writing the manuscript.X.S. and F.Z. contributed to plant sample collection, DNA/RNA preparation, library construction, and sequencing.G.S. worked on gene assembly and annotation.N.W. and Z.W. conducted transcriptome analysis and identified functional genes involved in petal color formation.N.W. and Z.W. analyzed the gene family and constructed the evolutionary tree.Z.W. conceived of the study, participated in its design and data interpretation, and revised the manuscript critically.All authors have read and agreed to the published version of the manuscript.

Figure 1 .
Figure 1.The phylogenetic assessment of HSF proteins in L. radiata.Using the neighbor-joining method, the phylogenetic tree was generated based on the HSF domain alignment.To verify the reliability, the numbers were computed via 1000 bootstrap replicates.The branches indicate replicate trees with >26%.The black font on a colored background depicts the subfamilies.

Figure 1 .
Figure 1.The phylogenetic assessment of HSF proteins in L. radiata.Using the neighbor-joining method, the phylogenetic tree was generated based on the HSF domain alignment.To verify the reliability, the numbers were computed via 1000 bootstrap replicates.The branches indicate replicate trees with >26%.The black font on a colored background depicts the subfamilies.

Figure 2 .
Figure 2. The phylogenetic relationships and conserved motifs analysis of LrHSF proteins.(A) Neighbor-joining LrHSFs phylogenetic tree (with bootstrap values calculated from 1000 replicates); (B) Distribution of the conserved motifs in LrHSF proteins.Various motifs are denoted by distinct colored boxes, where the length of each box corresponds to the length of the respective motif.

Figure 3 .
Figure 3. Motifs' diversity and conservation and diversity in LrHSF proteins.Using MEME, 15 motifs in the HSF family were schematically represented.

Figure 2 . 21 Figure 2 .
Figure 2. The phylogenetic relationships and conserved motifs analysis of LrHSF proteins.(A) Neighbor-joining LrHSFs phylogenetic tree (with bootstrap values calculated from 1000 replicates); (B) Distribution of the conserved motifs in LrHSF proteins.Various motifs are denoted by distinct colored boxes, where the length of each box corresponds to the length of the respective motif.

Figure 3 .
Figure 3. Motifs' diversity and conservation and diversity in LrHSF proteins.Using MEME, 15 motifs in the HSF family were schematically represented.

Figure 3 .
Figure 3. Motifs' diversity and conservation and diversity in LrHSF proteins.Using MEME, 15 motifs in the HSF family were schematically represented.

Figure 4 .
Figure 4. Protein structure of L. radiata HSFs.The confidence level of the prediction model is present at the bottom.

Figure 4 .Figure 5 .
Figure 4. Protein structure of L. radiata HSFs.The confidence level of the prediction model is present at the bottom.Plants 2024, 13, x FOR PEER REVIEW

Figure 5 .
Figure 5.The expression of LrHSF genes in various L. radiata tissues.(A) Heatmap of expression profile with hierarchal clustering of LrHSFs in various L. radiata tissues and (B) at different flower developmental stages.The color intensity of each field depicts each gene's relative expression.Red = higher values, and blue = lower values.FB: floral bud stage, FL1: partially opening flower stage, FL2: fully opened flower stage, R: senescent flower stage.

21 Figure 6 .
Figure 6.The expression of LrHSF genes at different flower developmental stages by qRT-PCR.FB: floral bud stage, FL1: partially opening flower stage, FL2: fully opened flower stage, R: senescent flower stage.

Figure 6 .
Figure 6.The expression of LrHSF genes at different flower developmental stages by qRT-PCR.FB: floral bud stage, FL1: partially opening flower stage, FL2: fully opened flower stage, R: senescent flower stage.

Plants 2024 ,
13,  x FOR PEER REVIEW 9 of 21 ment, most genes were down-regulated, while LrHSF5, LrHSF8, LrHSF13, LrHSF16, and LrHSF18 were up-regulated at 24 h in L. radiata leaves.Moreover, during MeJA treatment, LrHSF1 and LrHSF5 indicated the highest expression at 12 h, while LrHSF1 and LrHSF5 were down-regulated at 12 h and 24 h in L. radiata leaves.Additionally, the expression of LrHSF10 and LrHSF16 was increased during MeJA treatment.

Figure 7 .
Figure 7.The expression of the LrHSF genes under cold and heat stresses in L. radiata.(A) Heatmap with hierarchical cluster assessment of differentially expressed LrHSF genes that were cold-responsive.(B) Heatmap of LrHSF gene expression in L. radiata during heat treatment.Leaf of seedling grown in distilled water under normal growth conditions for 12 h (CK-12 h) or 24 h (CK-24 h) was sampled as control.Red = higher values, and blue = lower values.

Figure 7 .
Figure 7.The expression of the LrHSF genes under cold and heat stresses in L. radiata.(A) Heatmap with hierarchical cluster assessment of differentially expressed LrHSF genes that were cold-responsive.(B) Heatmap of LrHSF gene expression in L. radiata during heat treatment.Leaf of seedling grown in distilled water under normal growth conditions for 12 h (CK-12 h) or 24 h (CK-24 h) was sampled as control.Red = higher values, and blue = lower values.

Figure 7 .
Figure 7.The expression of the LrHSF genes under cold and heat stresses in L. radiata.(A) Heatmap with hierarchical cluster assessment of differentially expressed LrHSF genes that were cold-responsive.(B) Heatmap of LrHSF gene expression in L. radiata during heat treatment.Leaf of seedling grown in distilled water under normal growth conditions for 12 h (CK-12 h) or 24 h (CK-24 h) was sampled as control.Red = higher values, and blue = lower values.

Figure 8 .
Figure 8.The expression pattern of LrHSF genes was tested during drought and salt stresses in L. radiata.(A) Heatmap of LrHSF gene expression during drought treatment.(B) Heatmap with hierarchical cluster assessment of differentially expressed LrHSF genes that were salt-responsive.Leaf of seedling grown in distilled water under normal growth conditions (22 • C for 8 h dark and 25 • C for 16 h light cycle) for 12 h (CK-12 h) or 24 h (CK-24 h) was set as the control.Red = higher values, and blue = lower values.

Figure 8 .
Figure 8.The expression pattern of LrHSF genes was tested during drought and salt stresses in L. radiata.(A) Heatmap of LrHSF gene expression during drought treatment.(B) Heatmap with hierarchical cluster assessment of differentially expressed LrHSF genes that were salt-responsive.Leaf of seedling grown in distilled water under normal growth conditions (22 °C for 8 h dark and 25 °C for 16 h light cycle) for 12 h (CK-12 h) or 24 h (CK-24 h) was set as the control.Red = higher values, and blue = lower values.

Figure 9 .
Figure 9.The expression of 22 LrHSF genes in seedling-stage leaves under ABA (A) and MeJA (B) treatments was assessed via qRT-PCR.Red = higher values, and blue = lower values.

Figure 9 .Figure 10 .
Figure 9.The expression of 22 LrHSF genes in seedling-stage leaves under ABA (A) and MeJA (B) treatments was assessed via qRT-PCR.Red = higher values, and blue = lower values.024, 13, x FOR PEER REVIEW 11

Figure 10 .
Figure 10.The STRING database predicted protein-protein interaction networks between LrHSFs.Different colors indicate distinct interactions.Arabidopsis HSF names are marked, whereas in the parentheses their L. radiata homologs are indicated.

Plants 2024 , 21 Figure 11 .
Figure 11.The L. radiata petal cells indicated transient expression of LrHSFs that had subcellular localization using GFP as a control.(A,B) The images were acquired via bright channel, green channel (GFP fluorescence), and their combination under a confocal microscope.Merged green fluorescence, bright-field, and green-bright fluorescence images.Scale bar = 10 µm.

Figure 11 .
Figure 11.The L. radiata petal cells indicated transient expression of LrHSFs that had subcellular localization using GFP as a control.(A,B) The images were acquired via bright channel, green channel (GFP fluorescence), and their combination under a confocal microscope.Merged green fluorescence, bright-field, and green-bright fluorescence images.Scale bar = 10 µm.

Funding:
This research was financially supported by the Jiangsu Provincial Crop Germplasm Resource Bank for Conservation (Grand No. 2023-SJ-013), the Jiangsu Provincial Forestry Science and Technology Promotion Project (Grand No. LYKJ[2022]08), and the Open Fund of Jiangsu Key Laboratory for the Research and Utilization of Plant Resources (JSPKLB202205).Data Availability Statement: All data are displayed in the manuscript.