Abstract

   Drought stress is a significant environmental factor that can impede
   plant growth and ornamental quality. Hemerocallis middendorffii, a
   drought-tolerant garden plant, has attracted attention for its
   ornamental value and application prospects. To investigate the
   molecular mechanism of drought stress resistance of H. middendorffii,
   this study employed 20% polyethylene glycol (PEG) 6000 to simulate
   drought stress. Leaves and roots of H. middendorfii were subjected to
   24 h treatment and followed by transcriptome sequencing. Analysis
   revealed 8796 and 3401 differentially expressed genes (DEGs) in leaves
   and roots. The major biological processes and key molecular pathways
   activated under drought stress in H. middendorffii were revealed by
   Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG)
   enrichment analyses. The focus of this analysis was on the gene
   expression changes within plant hormone signal transduction pathway.
   Additionally, drought-associated transcription factor families such as
   AP2/ERF, WRKY, MYB, bHLH, NAC, and bZIP were identified among DEGs.
   Furthermore, potential regulatory relationships of the above
   transcription factors (TFs) with functional genes in the abscisic acid
   (ABA) and jasmonic acid (JA) signalling pathways were analysed using
   correlation network prediction. This research establishes the
   groundwork for subsequent exploration of drought-responsive gene
   expression and regulatory patterns in H. middendorfii and provides an
   importance for the systematic study of its drought-resistant molecular
   mechanism.

   Keywords: Hemerocallis middendorffii, drought stress, transcriptome,
   transcription factors, plant hormone signal transduction

1. Introduction

   Hemerocallis middendorffii is a perennial herbaceous ornamental plant
   that belongs to the genus Hemerocallis in the family Asphodelaceae, and
   it serves as an important hybrid parent for the breeding of modern
   daylily cultivars. The plant material is widely distributed in the
   three northeastern provinces and has abundant wild plant resources. It
   features excellent qualities such as strong drought resistance, vivid
   flower colours, an early flowering period, and tolerance to rough
   management. H. middendorffii is extensively utilized in flower beds,
   flower borders, and woodland edges. It is regarded as an ideal choice
   for urban landscaping and beautiful countryside construction [[38]1].
   At this stage, research on Hemerocallis plant mainly focuses on genetic
   diversity [[39]2], floral organ development [[40]3], aromatic compounds
   [[41]4], and physiological mechanisms of stress tolerance
   [[42]1,[43]5,[44]6]. However, there exists a paucity of research at the
   transcriptome level concerning the molecular mechanisms involved in
   drought stress in the H. middendorffii.

   Drought is now widely acknowledged to be among the primary abiotic
   stresses exerting an influence on plant growth and ornamental traits.
   This results from several elements, encompassing the gradual increase
   in global temperature, the increasing scarcity of freshwater resources,
   and the multiple challenges posed by human activities [[45]7]. The
   growth and development of plants is restricted by drought, which has
   serious consequences for the stability of the ecosystem and the
   sustainability of agricultural production. To cope with and withstand
   changes in extreme environments, plants have evolved sophisticated
   mechanisms for sensing unfavourable conditions and are capable of
   responding promptly for regulating their growth and development
   [[46]8,[47]9]. Plants have evolved a range of mechanisms to cope with
   the adverse effects of drought stress, including changes in their own
   morphological structure, physiological and biochemical aspects,
   drought-resistant gene expression, hormone synthesis, and osmotic
   regulatory substances. The plant exhibits several adaptive features,
   including a reduction in growth rate, partial stomatal closure to
   minimise the loss of water, and the accumulation of free radicals
   [[48]10,[49]11,[50]12,[51]13].

   The recurrent occurrence of drought has profoundly impacted the global
   ecological balance, necessitating the investigation of
   drought-resistant adaptive mechanisms and the cultivation of novel
   drought-tolerant plant varieties. This has become a demanding and
   urgent scientific research endeavour. The leaves of the above-ground
   part of the plant are the first to sense the loss of water, while the
   roots of the underground part are the first organs affected by drought
   [[52]14]. Consequently, research concerning plant drought resistance
   has primarily concentrated on the analysis of plant organs,
   specifically leaves and roots [[53]15]. Abscisic acid (ABA) is a
   pivotal phytohormone for plant growth, development, and the regulation
   of abiotic stresses [[54]16]. The stimulation of ABA production and
   accumulation in plant organs is induced by drought stress, with the
   resultant activation of downstream signalling pathways [[55]17].
   Furthermore, a number of other phytohormones have also been recognized
   as having a fundamental influence on drought stress response. These
   include brassinosteroids (BR), cytokinin (CTK), and auxin (IAA), which
   have been found to crosstalk with ABA in order to regulate that
   response [[56]18,[57]19]. Transcription factors (TFs) serve a
   significant function in signal transduction pathways, interacting with
   the promoter regions of target genes to modulate their expression. The
   families of TFs most implicated in the realm of plant drought stress
   are chiefly bZIP, AP2/ERF, MYB, and WRKY, amongst others
   [[58]20,[59]21]. The aforementioned TFs have been demonstrated to
   regulate the expression of genes associated with the response to
   drought stress, thereby improving drought tolerance in plants. TFs
   respond to different adversity responses by regulating related genes in
   different hormone signalling pathways. The present study set out the
   finding that OsbZIP62 improves drought tolerance in rice by
   participating in the ABA signalling pathway and interacting with
   protein kinases (SAPKs) to regulate the expression of related genes
   [[60]22]. GbWRKY1 depends on the interaction of JAZ1 and ABI1 to
   negatively regulate ABA signalling and inhibit salt and drought
   tolerance in plants [[61]23].

   Transcriptome sequencing technology has become a significant instrument
   for elucidating the molecular mechanisms of abiotic stresses. It has
   been extensively utilised in research endeavours pertaining to drought
   stress signalling pathways and the prediction of gene function in
   plants [[62]24]. At the present time, the molecular mechanisms
   underlying drought stress responses have been analysed using
   transcriptome sequencing in many plant species, including Lanzhou lily
   [[63]25], rose [[64]26], and iris [[65]27]. Certain advancements have
   occurred in the investigation of molecular mechanisms underlying plant
   response to drought stress, but the change rule of genes and response
   mechanisms of H. middendorffii under drought stress are still unknown.
   The objective of this study was to ascertain the alterations in the
   expression profiles of differentially expressed genes (DEGs) in leaves
   and roots of H. middendorffii by simulating drought stress with
   polyethylene glycol (PEG) 6000 and to reveal the key metabolic pathways
   in response to drought stress. A particular focus was on exploring the
   potential regulatory relationship between drought-resistant TFs and the
   functional genes within ABA and jasmonic acid (JA) signalling pathways.
   The findings of this research will offer a conceptual foundation for
   the comprehensive understanding of the molecular mechanism of drought
   tolerance in leaves and roots of H. middendorffii. Simultaneously, the
   study will establish a framework for the excavation of drought-tolerant
   key genes and the further elucidation of molecular regulatory
   mechanisms.

2. Results

2.1. Transcriptome Sequencing Data and Assembly Statistics

   In the present study, the gene expression patterns of H. middendorffii
   under drought stress conditions were analysed using transcriptome
   sequencing technology. The 12 cDNA libraries were constructed from six
   samples of leaf tissue (PL: leaf tissue under drought stress, CL:
   control leaf tissue) and six samples of root tissue (PR: root tissue
   under drought stress, CR: control root tissue). A total of 277,689,578
   raw reads were obtained. The subsequent removal of low-quality data
   from the original sequence of data resulted in the attainment of
   271,604,426 clean reads. The range of the GC content of the samples was
   from 44.05% to 47.03%, with an error rate of only 0.03%; Q20 was higher
   than 97.43% and Q30 was higher than 92.73% ([66]Table S1). Trinity
   assembled a total of 194,376 unigenes, with an average length of 829 bp
   and an N50 of 1057 bp ([67]Table S2). In conclusion, the data derived
   from the sequencing of transcriptomes were of a high quality and could
   be utilised for further subsequent analyses.

2.2. Analysis of DEGs

   In order to investigate alterations in genetic expression profiles in
   leaves and roots of the H. middendorffii under drought stress, DEGs
   were screened and analysed. The results demonstrated that 8796 and 3401
   DEGs were obtained in leaves and roots, of which 680 genes were
   differentially expressed in both leaf and root tissues. Furthermore,
   there were 8116 and 2721 DEGs with specificity in leaves and roots
   ([68]Figure 1A). Among these genes which demonstrated differential
   expression, 4184 genes were found to be upregulated, whilst 4612 genes
   were downregulated in the leaves. For the roots, 1764 genes were
   upregulated, and 1637 genes were downregulated ([69]Figure 1B). The
   findings suggest that the leaves and roots of H. middendorffii may
   adopt different response mechanisms and strategies in resisting drought
   stress.

Figure 1.

   [70]Figure 1
   [71]Open in a new tab

   Quantity and distribution of DEGs in leaves and roots of H.
   middendorffii under drought stress. (A) Venn diagram analysis of DEGs;
   (B) Venn diagram analysis of upregulated and downregulated DEGs.

2.3. Gene Ontology (GO) Enrichment Analysis of DEGs

   To enhance comprehension of the prospective biological functions of
   DEGs in H. middendorffii in the context of drought stress, GO
   functional annotation and enrichment analyses were conducted on DEGs in
   leaf and root tissues. The objective of this analysis was to reveal
   significant variations in biological processes, cellular components,
   and molecular functions. A statistical analysis was conducted on the
   top 30 GO terms, which were ranked for both leaf and root tissues. The
   screening criterion for significant enrichment was p-value ≤ 0.05. GO
   enrichment analyses demonstrated that 4494 DEGs in leaves and 2450 DEGs
   in roots were annotated to significantly enriched GO terms. In leaves,
   drought-responsive genes were significantly enriched mainly for protein
   phosphorylation, phosphorylation, polysaccharide metabolic process, and
   carbohydrate metabolic process in biological processes, followed by
   protein kinase activity, hydrolase activity, hydrolysing O-glycosyl
   compounds, hydrolase activity, acting on glycosyl bonds in molecular
   functions and mainly transcription regulator complex, and so on, in
   cellular components ([72]Figure 2A). In roots, drought-responsive genes
   were significantly enriched mainly in ribosome biogenesis and
   ribonucleoprotein complex biogenesis of biological processes,
   structural constituent of the ribosome, oxidoreductase activity, and
   structural molecule activity of molecular functions and the ribosome,
   and so on, in cellular components ([73]Figure 2B). In summary, the
   drought stress response mechanism of H. middendorffii is primarily
   focused on signal transduction, energy metabolism, and other biological
   functions.

Figure 2.

   [74]Figure 2
   [75]Open in a new tab

   GO enrichment analysis of DEGs under drought stress; (A) leaves; (B)
   roots.

2.4. Kyoto Encyclopedia of Genes and Genomes (KEGG) Enrichment Analysis of
DEGs

   This research annotated and analysed KEGG metabolic pathways of DEGs in
   response to drought stress in H. middendorffii. KEGG pathway enrichment
   analysis demonstrated that 1529 DEGs were annotated to 117 KEGG
   pathways in leaves, which exhibited significant enrichment in pathways
   such as plant hormone signal transduction, photosynthesis–antenna
   proteins, plant–pathogen interaction, cyanoamino acid metabolism, and
   starch and sucrose metabolism ([76]Figure 3A). In roots, a sum of 865
   DEGs were annotated to 100 KEGG pathways, with the main significantly
   enriched pathways being ribosome, cutin, suberine and wax biosynthesis,
   tyrosine metabolism, cysteine, and methionine metabolism and protein
   processing in endoplasmic reticulum ([77]Figure 3B). The findings
   suggest that leaves and roots may employ divergent mechanisms to
   withstand drought stress, with the significantly enriched metabolic
   pathways displaying a strong correlation with the response to drought
   stress in H. middendorffii.

Figure 3.

   [78]Figure 3
   [79]Open in a new tab

   KEGG enrichment analysis of DEGs under drought stress; (A) leaves; (B)
   roots.

2.5. Identification and Analyzation of Differentially Expressed TFs

   TFs represent pivotal regulatory elements that modulate the expression
   of genes responsive to drought stress, playing a critical role in plant
   responses to this environmental stressor [[80]28]. Overall, 131 TFs
   belonging to 28 distinct families of TFs were identified through
   analysis of the PlantTFDB database and transcriptome sequence
   alignment. Among the identified transcription factor families, AP2/ERF
   was the most abundant with 17 members, followed by WRKY (16), MYB (15),
   bHLH (12), NAC (11), bZIP (7), GRAS (6), C2H2 (6), and Dof (5)
   ([81]Figure 4A). We analysed the expression patterns in the form of
   heatmaps for the most numerous AP2/ERF, WRKY, MYB, bHLH, NAC, and bZIP
   families. The AP2/ERF family showed three DEGs upregulated and 12 DEGs
   downregulated for expression in leaves, and all three DEGs were
   upregulated for expression in roots ([82]Figure 4B). The WRKY family
   exhibited a single DEG that was upregulated, with the remaining 11
   being downregulated for expression in leaves. Moreover, four DEGs
   showed upregulated expression, while one exhibited downregulated
   expression in roots ([83]Figure 4C). In the MYB family, there were six
   DEGs upregulated and six DEGs downregulated in leaves, while in roots,
   three DEGs were upregulated, and two DEGs were downregulated
   ([84]Figure 4D). The bHLH and NAC families exhibited two and four DEGs
   being upregulated, and seven and five DEGs being downregulated, for
   expression in leaves, respectively. Moreover, three DEGs were
   upregulated for expression in both roots ([85]Figure 4E,F). The bZIP
   family was observed to comprise a DEG that was upregulated and two DEGs
   that were downregulated for expression in leaves. In addition, three
   DEGs were upregulated and one DEG was downregulated for expression in
   roots ([86]Figure 4G). In summary, it can be seen that the majority of
   the DEGs of the AP2/ERF, WRKY, MYB, bHLH, NAC, and bZIP families
   exhibited a decreasing trend of expression in leaves and an increasing
   trend of expression in roots.

Figure 4.

   [87]Figure 4
   [88]Open in a new tab

   Distribution and expression patterns of TFs in response to drought
   stress; (A) classification of identified transcription factor families;
   (B) heatmap of AP2/ERF expression; (C) heatmap of WRKY expression; (D)
   heatmap of MYB expression; (E) heatmap of bHLH expression; (F) heatmap
   of NAC expression; (G) heatmap of bZIP expression.

2.6. Analysis of the Expression Pattern of Relevant DEGs on the Plant Hormone
Signal Transduction Pathway

   An analysis of the expression patterns of DEGs associated with the
   plant hormone signal transduction pathway of the H. middendorffii
   revealed the identification of 74 and 22 DEGs in the leaves and roots.
   These genes were found to be enriched in eight distinct hormonal
   signalling pathways, including IAA, gibberellin (GA), ABA, and so on.
   The greatest count of DEGs were involved in the IAA, ABA, and JA
   signalling pathways. Overall, 25 DEGs were enriched in the IAA
   signalling pathway, with most of the DEGs clustered in two genes, IAA
   and SAUR, and of the 12 IAA genes, only one IAA gene
   (Cluster-24263.69525) was significantly upregulated in both leaf and
   root tissues, while the remaining genes were upregulated in seven and
   downregulated in four cases in leaves. The SAUR gene exhibited one
   upregulated expression in roots, while leaves exhibited two upregulated
   and five downregulated expressions ([89]Figure 5A). All 19 DEGs
   involved in the JA signalling pathway presented differential expression
   patterns in leaves, including an upregulated COI-1 gene, 17
   downregulated JAZ genes, and one downregulated MYC2 gene ([90]Figure
   5B). The present study identified a total of 17 DEGs involved in the
   ABA signalling pathway, including a PYL gene that was upregulated in
   leaves and two and four PP2C genes that were upregulated in both leaves
   and roots. Among the five SnRK2 genes, only one SnRK2
   (Cluster-24263.80843) was found to be downregulated in terms of
   expression in both roots and leaves. ABF genes exhibited divergent
   patterns of expression, with two upregulated and two downregulated
   expressions in both leaves and roots. Specifically, Cluster-24263.67512
   and Cluster-24263.46033 were identified as being upregulated and
   downregulated in both tissues, respectively ([91]Figure 5C).

Figure 5.

   [92]Figure 5
   [93]Open in a new tab

   Expression pattern of DEGs associated with the plant hormone signal
   transduction pathway under drought stress; (A) the IAA signalling
   pathways; (B) the JA signalling pathways; (C) the ABA signalling
   pathways; (D) the CTK signalling pathways; (E) the SA signalling
   pathways; (F) the ET signalling pathways; (G) the GA signalling
   pathways; (H) the BR signalling pathways.

   In addition, the response of H. middendorffii to drought stress
   involved the CTK, salicylic acid (SA), ethylene (ET), GA, and BR
   signalling pathways, with nine, eight, six, and two DEGs. Within the
   CTK signalling pathway, an ARR-B gene (Cluster-24263.111216) was found
   to be upregulated expression in leaves and downregulated expression in
   roots, and five ARR-A genes had downregulated expression in leaves
   ([94]Figure 5D). In the SA signalling pathway, the highest number of
   DEGs enriched TGA genes was found, with one showing upregulated and
   three showing downregulated expression in leaves, and two showing
   upregulated and one showing downregulated expression in roots
   ([95]Figure 5E). In the ET signalling pathway, three EBF1_2 genes
   showed upregulated expression in roots, and an ERF1 gene demonstrated
   downregulated expression in leaves but upregulated expression in roots
   ([96]Figure 5F). Within the GA signalling pathway, it was observed that
   a GID2 gene was upregulated in leaves, and a GID2 gene was upregulated
   in roots, while a DELLA gene was specifically upregulated in leaves
   ([97]Figure 5G). In the BR signalling pathway, two BSK genes were
   identified, one of which was downregulated for expression in leaves,
   and the other upregulated for expression in roots ([98]Figure 5H).

2.7. Correlational Network Analysis of Functional Genes Associated with ABA
and JA Signal Transduction Pathways in Correlation with TFs

   As demonstrated by related studies, the signalling pathways of ABA and
   JA have been shown to induce stomatal closure and improve drought
   tolerance in plants [[99]29,[100]30]. The present study employed
   AP2/ERF, WRKY, MYB, bHLH, NAC, and bZIP transcription factors for the
   purpose of correlation network analysis with functional genes on the
   ABA and JA pathways. The prediction results showed that a total of 62
   TFs had possible regulatory relationships with 32 functional genes in
   leaves ([101]Figure 6A). Major functional genes regulated by AP2/ERF,
   WRKY, MYB, bHLH, NAC, and bZIP included SnRK2 (Cluster-24263.80843),
   JAZ (Cluster-24263.79304), JAZ (Cluster-24263.114243), JAZ
   (Cluster-24263.87273), JAZ (Cluster-24263.52226), ABF
   (Cluster-24263.40019), and others. A total of 54 TFs in the root had
   probable regulatory relationships with 35 DEGs ([102]Figure 6B). It has
   been determined that the major functional genes regulated by AP2/ERF,
   WRKY, MYB, bHLH, NAC, and bZIP in roots include PP2C
   (Cluster-24263.80587), SnRK2 (Cluster-24263.131043), PP2C
   (Cluster-24263.25249), JAZ (Cluster-24263.106559), and others.

Figure 6.

   [103]Figure 6

   [104]Figure 6
   [105]Open in a new tab

   Network analysis of correlations between AP2/ERF, WRKY, MYB, bHLH, NAC,
   bZIP, and functional genes connected to the ABA and JA signal
   transduction pathways. (A) leaves; (B) roots.

2.8. Validation of RNA-Seq Data by qRT-PCR of DEGs

   To ensure the authenticity of the leaves and roots transcriptome data
   of H. middendorffii under 20% PEG-6000 simulated drought stress,
   qRT-PCR validation analyses of 16 randomly selected DEGs associated
   with drought stress were performed. The findings indicated that the
   selected DEGs showed similar gene expression trends in the qRT-PCR
   results and transcriptome sequencing results ([106]Figure 7). The
   aforementioned results suggest that the RNA-Seq data are of a higher
   quality and thus suitable for subsequent analyses.

Figure 7.

   [107]Figure 7
   [108]Open in a new tab

   Expression patterns of 16 DEGs screened by qRT-PCR validation.

3. Discussion

   Drought is widely regarded as a major stress factor, impacting plant
   growth, development, and appearance quality [[109]31]. With the
   accelerated development of the global economy and the continuous
   expansion of urban areas, the water consumption and cost of landscape
   management are increasing year by year. Consequently, the selection of
   plants with superior ornamental characteristics, along with those
   exhibiting water-saving and drought-resistant qualities, is poised to
   become the prevailing trend in future garden construction. H.
   middendorffii is a drought-tolerant ornamental plant, and it is of
   great significance to reveal its drought-resistant molecular mechanism
   and add new plant resources for the construction of water-saving
   gardens.

   Transcriptome sequencing provides comprehensive and rapid access to all
   transcript information and has become an effective tool for analysing
   plant response mechanisms to abiotic stresses [[110]32]. In instances
   where genomic information is lacking, the employment of transcriptome
   sequencing technology enables the acquisition of a substantial volume
   of transcript sequence data. And based on this, functional gene mining
   of plants was undertaken in order to disclose the underlying mechanisms
   of desirable plant traits [[111]33]. The present study analysed the
   DEGs of H. middendorffii under drought stress. Significant changes in
   DEGs were observed in both leaves and roots. This phenomenon may be
   attributable to a series of stress responses exhibited by plants, which
   are initiated in order to mitigate water loss in response to drought
   stress. The number of DEGs was found to be greater in leaves than in
   roots, suggesting that there may be differences in the mechanism of
   response to drought stress in these two organs. Concurrently, analysis
   revealed 680 DEGs shared between the two tissues, thereby further
   suggesting that these DEGs may serve a pivotal role in the response of
   leaves and roots to drought stress.

   GO and KEGG pathway enrichment analyses of the DEGs in response to
   drought stress in H. middendorffii were performed to identify key
   pathways associated with this response. In this current study, the
   pathways that were identified as highly enriched with DEGs in leaves
   primarily included plant hormone signal transduction,
   photosynthesis-antenna proteins, cyanoamino acid metabolism, and starch
   and sucrose metabolism. This finding suggests that signal transduction,
   energy supply, and depletion may be the primary strategies for coping
   with drought stress in H. middendorffii. The plant hormone signal
   transduction pathway was identified as the most prominently enriched
   pathway, and it is hypothesised that this pathway is the most important
   one for drought tolerance in the H. middendorffii. In root tissues, the
   pathway with the highest enrichment of DEGs is the ribosome, which
   contains the highest number of DEGs. The process of translating mRNA
   into protein by ribosomes represents a pivotal step in gene expression
   [[112]34]. As demonstrated in previous studies, there is a differential
   expression of ribosomal protein genes in plants in response to drought
   stress, with enhanced resistance being observed in transgenic plants
   [[113]35]. In this particular study, it was found that the number of
   DEGs enriched in this pathway in roots was significantly higher than in
   leaves. This phenomenon may be attributable to the capacity of the H.
   middendorffii roots to accumulate greater quantities of nutrients,
   thereby supplying energy to the ribosomal pathway.

   The plant hormone signal transduction pathway exerts a primary
   regulatory influence over plant physiology by means of a complex
   network of hormone signals, including ABA, IAA, JA, and CTK, thereby
   enabling plants to cope with drought stress [[114]36]. ABA-mediated
   signalling pathways have been demonstrated to be of significance in
   plants drought stress response, exerting an important influence on
   stomatal closure and the expression of resistance genes in plants
   [[115]37]. Within this pathway, the upstream PYR/PYL (ABA receptor)
   interaction with PP2C results in the inhibition of the negative
   regulator PP2C, thus reducing its inhibitory effect on SnRK2 [[116]38].
   It has previously been demonstrated that the over-expression of the
   BdSnRK2.9 gene leads to an enhancement in drought tolerance in
   transgenic plants [[117]39]. In the current study, five PP2C genes were
   identified in both leaves and roots. All of these were found to be
   significantly upregulated, and most of the SnRK2 genes were similarly
   upregulated. This is probably due to the fact that SnRK2 genes are
   regulated by TFs in response to drought stress. IAA signal transduction
   primarily regulates processes such as plant root growth, leaf
   development, and lightward growth [[118]40]. In this paper, five SAUR
   and four IAA genes were downregulated for expression in leaves, likely
   due to the inhibition of growth hormone synthesis, which resulted in
   slowing down of biomass accumulation, thus improving the drought
   tolerance of the plants. It has been established that the JA signalling
   pathway is intimately associated with the mitigation of drought stress.
   The JAZ protein family is a negative regulator in this pathway
   [[119]41]. Earlier research has established that the overexpression of
   OsJAZ1 results in a reduction in drought tolerance in rice through the
   regulation of the ABA and JA signalling pathways [[120]42]. In
   parallel, the MYC2 has been shown to significantly reduce the response
   to dehydration stress [[121]43]. We found that 17 JAZ and one MYC2 were
   downregulated expression under drought stress in H. middendorffii
   leaves. It is hypothesised that the JAZ gene may have a significant
   role in the response of H. middendorffii to drought stress.

   As evidenced by numerous studies, the TF families AP2/ERF, bZIP, MYB,
   NAC, WRKY, and bHLH have been identified as being pivotal regulators in
   the context of drought stress responses [[122]44,[123]45,[124]46]. In
   our investigation, 131 DEGs encoding TFs were identified in leaves and
   roots. These DEGs were predominantly distributed among the AP2/ERF,
   WRKY, MYB, bHLH, NAC, and bZIP families. The results indicate that the
   expression of numerous TF families is enhanced by drought stress, and
   the expression of the common TF families plays a significant function
   in the response of H. middendorffii to drought stress. A related study
   reported that the PtoERF15 directly regulated the expression of PtoMYC2
   to modulate the JA pathway and enhance drought tolerance in Populus
   [[125]47]. GmWRKY54 directly bonded to the promoters of PYL8, SRK2A,
   CIPK11, and CPK3 and activated their expression, which improved drought
   tolerance in soybean [[126]48]. The MdMYB44-like has been shown to
   repress MdPP2CA transcription and enhance ABA mediated drought and salt
   tolerance [[127]49]. OsbHLH148 enhanced drought tolerance in rice by
   interacting with OsJAZ protein [[128]50]. RtNAC055 was subjected to
   direct regulation by RtMYC2, with the result that it promoted plant
   drought tolerance by regulating stomatal closure through JA/hydrogen
   peroxide signalling [[129]51]. OsbZIP23 positively regulated OsPP2C49
   and regulated ABA signalling [[130]52]. Through the correlation network
   prediction, it was found that AP2/ERF, WRKY, MYB, bHLH, NAC, and bZIP
   may influence the plant’s drought tolerance by regulating the
   expression of downstream genes such as PP2C, JAZ and SnRK2. However,
   further investigation is required to elucidate the intricate regulatory
   relationships among these factors.

4. Materials and Methods

4.1. Material and Drought Stress Treatment

   The experimental material employed in this study was the H.
   middendorffii, which was supplied by the Ornamental Plant Resource
   Nursery of Jilin Agricultural University. In the greenhouse, single
   plants displaying robust and uniform growth were selected for planting
   in 10 cm × 13 cm pots. The cultivation substrate was composed of a
   blend of peat, garden soil, and perlite, with a proportion of 3:1:1.
   Subsequent to the cessation of the establishment period, the plants
   were arbitrarily allocated to two groups. Equal quantities (250 mL) of
   piped water and 20% PEG-6000 were administered to the roots of the
   plants, as determined by the preliminary experiment, with piped water
   serving as the control treatment. A total of 60 plants were utilised in
   the experimental setup, with 10 pots per treatment configured with
   three biological replicates. After the completion of a 24 h period of
   treatment, the leaves and roots were collected separately. They were
   then rapidly snap-frozen using liquid nitrogen, after which they were
   stored in a −80 °C refrigerator for subsequent use.

4.2. Total RNA Extraction, cDNA Library Construction, Sequencing and De Novo
Assembly from the Hemerocallis middendorffii

   The extraction of total RNA from leaves and roots of H. middendorffii
   was conducted in accordance with the TransZol Up Plus RNA Kit (TransGen
   Biotech, Wuhan, China) as outlined by the manufacturer. Subsequently,
   an Agilent 2100 bioanalyzer (Agilent, Santa Clara, CA, USA) was
   employed to assess the integrity of the extracted RNA. After passing
   the test, double-stranded cDNA was synthesised by reverse
   transcription, and suitable fragment sizes were screened for PCR
   amplification to obtain the final library. Following the completion of
   the testing phase, the libraries were then subjected to the Illumina
   platform for the purpose of conducting high-throughput sequencing.

   To improve the accuracy of sequencing results, clean reads were
   obtained for the purpose of subsequent analysis by the following
   processes: the removal and filtering of connectors, N-containing reads,
   and low-quality reads in the raw data. In view of the paucity of
   genomic data available for H. middendorffii, the resulting clean reads
   were spliced using Trinity software (2.4.0) in order to generate
   transcript sequence data [[131]53], which could thus be utilised as
   references in subsequent analyses. Finally, we obtained the Unigene