Abstract

   The family of GRAS transcription factors plays an essential role in the
   regulation of plant development, the transmission of hormonal signals,
   and the adaptation to environmental stresses seen in numerous species.
   However, a comprehensive analysis of the GRAS family of Hemerocallis
   citrina (daylily) is lacking, despite its potential to help understand
   the stress content and developmental processes of the monospecies. This
   study identified 78 GRAS genes (HcGRAS) in H. citrina, which were
   classified into 15 subfamilies based on chromosomal location, gene
   structure, conserved motifs, and expression patterns. An analysis of
   promoter regions indicated a significant presence of elements related
   to hormones and stress, showcasing these genes’ role in adapting to
   environmental pressures. The GO and KEGG analyses indicated that HcGRAS
   genes were engaged in pathways associated with developmental processes
   and responses to environmental stress. Notably, HcGRAS38 was identified
   as a key interacting protein for SHR, SCR, and DELLA subfamily members,
   which suggested its central role in coordinating stress response and
   developmental signaling. Comparative genomic mapping with seven
   representative monocot and dicot species underscored evolutionary
   conservation and divergence in functions of the GRAS family. The
   expression profiling of 22 HcGRAS genes across different daylily
   tissues provided insights into their tissue-specific roles. This
   research may promote the further exploration of the functional
   characteristics of HcGRAS genes.

1. Introduction

   The GRAS transcription factors (TFs) represent a significant
   plant-specific TF family that consists of three members, which are
   gibberellin acid-insensitive (GAI), repressor of GA1 (RGA) and
   scarecrow (SCR) [[32]1]. GRAS is involved in almost the whole process
   of plant growth and development, which contributes much to the
   preservation of shoot meristems, the initiation of lateral meristems,
   male gametogenesis, root development, gibberellin (GA) and light signal
   transduction and stress resistance [[33]2,[34]3,[35]4]. All the GRAS
   genes are monoexonic, meaning they encode proteins with a length of
   360–850 amino acids [[36]1]. The GRAS protein family is characterized
   by a relatively conserved C-terminal domain paired with a more diverse
   N-terminal domain. The C-terminal consists of five extremely conserved
   fragments: SAW, PFYRE, VHIID, LHRI, and LHRII [[37]4,[38]5]. Between
   LHRI and LHRII, VHIID serves as a conserved domain in nearly each GRAS
   protein. The LHRI-VHIID-LHRII pattern is crucial to the binding of
   proteins to nucleic acids or other proteins [[39]6,[40]7,[41]8].
   Additionally, SAW and PFYRE motifs are associated with the structural
   integrity of GRAS proteins [[42]8]. Sequences at the N-terminal vary
   and are involved in distinct signal transduction pathways, which can
   decide their functions’ specificity [[43]5].

   The GRAS family exists in a variety of species, such as thale cress
   (Arabidopsis thaliana) [[44]2,[45]9], rice (Oryza sativa) [[46]2],
   soybean (Glycine max) [[47]10], foxtail millet (Setaria italica L.)
   [[48]10], sugar beet (Beta vulgaris) [[49]11], and six Rosaceae species
   [[50]8]. The classification of the GRAS family includes several
   subfamilies, including DELLA, LS (lateral suppressor), LISCL (Lilium
   longiflorum SCR-like), NSP (nodulation signaling pathway), PAT1
   (phytochrome a signal transduction 1), HAM (hairy meristem), SCR
   (scarecrow), SCL3 (scarecrow-like 3), and SHR (short root)
   [[51]12,[52]13]. However, a different number of subfamilies were
   reported [[53]14,[54]15,[55]16,[56]17], whose respective phylogenetic
   results were defined [[57]18]. GRAS proteins exhibit different
   structures and play multiple roles in the growth and development of
   plants [[58]19]. For instance, AtSCR participates in the process of
   cell division in the root cortex and endocortex, while SHR activates
   SCR proteins to regulate the asymmetric division of roots
   [[59]14,[60]15,[61]16,[62]17]. NSP1 and NSP2 are required for
   nodulation induced by the rhizobium Nod factor in Medicago truncatula
   [[63]20]. It has been reported that AtLAS, OsMOC1 and SlyLs are
   associated with axillary formation [[64]21,[65]22,[66]23]. HAM family
   members contribute much to shoot meristem development [[67]24]. The
   PAT1 subfamily is a well-known branch of the GRAS family with multiple
   regulatory functions in transcriptional regulation and phytochrome
   signal transduction [[68]25]. DELLA proteins act as crucial negative
   modulators of GA signaling [[69]26]. BraRGL1 mutants (a
   loss-of-function mutation) lead to the rapid differentiation of flower
   bud and time of bolting [[70]27]. After employing RNAi technology to
   silence the strawberry GRAS gene FveRGA1, many runners were produced in
   naturally non-running YW and RG strains, indicating that FveRGA1 plays
   a crucial role in the regulation of runner formation in woodland
   strawberry [[71]28]. FveLAM acts with GA in sequence from bud
   initiation to runner outgrowth, which cannot be separated from the
   formation of stamen and runner [[72]29].

   Transcription factors, including GRAS, are essential for plant
   adaptation to biotic and abiotic stresses when responding to
   environmental signals to regulate growth and development
   [[73]11,[74]29,[75]30,[76]31,[77]32,[78]33]. Given the current
   challenges of climate change, including temperature fluctuations and
   soil variations, the need for stress-resistant crop varieties is more
   urgent than ever [[79]30]. Open-field agricultural crops are especially
   vulnerable to these shifting conditions, affecting yields and overall
   crop resilience. Thus, understanding the function of GRAS genes in
   stress responses is a significant step in breeding programs aimed at
   creating high-yielding, resilient crops.

   The flower buds of Hemerocallis citrina Baroni (daylily) are valuable
   and widely cultivated in horticulture due to their edibility, and they
   are extensively enjoyed as a healthy vegetable option across Asia
   [[80]34]. Daylily is native to China, with its natural distribution
   spanning regions south of the Qinling Mountains and Hebei, Shanxi, and
   Shandong provinces. It has also been documented in Japan and Southeast
   Asia. This species exhibits strong cold resistance, drought tolerance,
   and adaptation to semi-shaded environments. Its soil requirements are
   highly adaptable, thriving in diverse conditions such as sandy soils,
   clay substrates, alluvial plains, and mountainous terrains [[81]34].
   The Datong region in Shanxi, one of China’s major production areas,
   provides unique growth conditions, including selenium-rich soil and
   extreme diurnal temperature variation, which contribute to the quality
   and resilience of daylily. The Datong yellow flower planting area
   belongs to the semi-arid to arid region, with an average annual
   rainfall of 300–400 mL [[82]35]. A study of GRAS TFs in daylily can
   provide insights into the genetic foundation of this adaptability and
   may inform breeding efforts aimed at enhancing stress resistance and
   yield. Moreover, these findings could have broader applications for
   other crops grown in similar environments, facilitating an
   understanding of how GRAS TFs contribute to crop resilience.

   This research comprehensively characterized GRAS TFs in daylily at the
   genome-wide level for the first time, including an analysis of gene
   duplication, conserved structure, phylogeny, and physicochemical
   properties across different flower development stages, salicylic acid
   (SA) and jasmonic acid (JA) treatment, and thrips disease and rust
   treatment. Such findings are likely to not only offer support for
   future functional explorations of GRAS genes in daylily but also guide
   breeding efforts to enhance the adaptability and nutritional value of
   this crop and potentially others.

2. Materials and Methods

2.1. Plant Materials and Data Resources

   The daylily variety in this study was H. citrina Baroni cv.
   Datonghuanghua, which was collected in July 2024 from the Tangjiapu
   Organic Daylily Standardized Planting Base, Yunzhou District, Datong,
   Shanxi Province, China (40°2′1″ N, 113°36′4″ E, altitude ≈ 1157 m). The
   cultivation base for daylily is characterized by a temperate
   continental semi-arid monsoon climate, with an average annual
   temperature of 6.4 °C and annual precipitation of 391.5 mm. Its
   volcanic ash-derived soil is notably enriched with zinc and selenium.
   The research samples included leaves, roots, stems, and flowers in the
   developmental stage: F1 (1–2 cm), F3 (3–4 cm), F5 (5–6 cm), F7 (7–8
   cm), F9 (9–10 cm) and stage F (full opening). Additional information
   can be found in [83]Supplementary Figure S1. For each development
   stage, three samples were collected. For each type of sample, we took
   random samples from different plants in the daylily planting areas.
   Before being stored at −80 °C, all samples were flash frozen in liquid
   nitrogen.

   The genome sequences of A. thaliana and H. citrina Baroni were obtained
   from TAIR ([84]http://www.arabidopsis.org, accessed on 23 April 2024)
   and the NCBI
   ([85]https://www.ncbi.nlm.nih.gov/Traces/wgs/JACEHZ01?val=JACEHZ01,
   accessed on 23 April 2024) [[86]34]. In addition, those from O. sativa
   (GCF_034140825.1), Brachypodium distachyon (GCF_000005505.3), Sorghum
   bicolor (GCA_039639735.1), S. italica (GCF_000263155.2), Solanum
   lycopersicum (GCA_025759795.1) and Zea mays (GCA_001644905.2) were
   sourced from Phytozome 13.0 ([87]https://phytozome-next.jgi.doe.gov/,
   accessed on 23 April 2024). The RNA-seq data from different tissues of
   daylily (SRX7583022, SRX7583021, SRX7583020, SRX7583019, SRX7583018,
   SRX7583017, SRX7583016, SRX7583015, and SRX7583014) were systematically
   retrieved from the NCBI SRA database.

2.2. Identification and Characterization of GRAS Genes

   This research first retrieved the GRAS genes in species using BLASTP
   (v2.16.0) at the threshold E-value of 1 × 10^−5. Then, 56 Oryza sativa
   GRAS proteins ([88]Table S1) were used as query sequences to choose the
   minimum alignment coverage of 50%. Through the BLASTP analysis, every
   selected candidate protein was employed to perform a search within the
   Swissprot database ([89]https://www.uniprot.org/, accessed on 28 May
   2024), thus exploring its corresponding ortholog at the specified
   threshold along with the minimum alignment coverage parameters. These
   findings were verified by repeating the aforementioned steps. The
   sequence number of the GRAS genes was obtained through summarizing the
   aforementioned results. The protein sequences were sourced from the
   genomic protein dataset of daylily through the utilization of TBtools
   (v2.310) [[90]36]. The GRAS domain (PF03514) retrieved from Pfam
   ([91]http://pfam.xfam.org, accessed on 28 May 2024) was employed to
   identify the potential GRAS genes of H. citrina Baroni [[92]37]. To
   further verify the domain of GRAS, the Conserved Domain Database (CDD)
   in the NCBI
   ([93]https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi, accessed
   on 28 May 2024) was used [[94]38]. The HcGRAS gene structure was
   manually corrected by employing IGV-GSAman software (version 0.8.4,
   [95]https://gitee.com/CJchen/IGV-sRNA, accessed on 29 May 2024), with
   the aid of RNA sequencing data. The updated GFF annotation file was
   used for further analysis.

   Moreover, this research renamed the identified GRAS TFs in accordance
   with their chromosomal locations via the nomenclature by utilizing the
   GRAS + sequence number. Through employing the ProtParam
   ([96]https://web.expasy.org/protparam/, accessed on 28 May 2024) online
   software, physicochemical properties were explored for their
   theoretical pI, amino acid number, molecular weight (MW), and
   instability coefficient (aliphatic index). Through utilizing
   DeepLoc-2.0
   ([97]https://services.healthtech.dtu.dk/service.php?DeepLoc-2.0,
   accessed on 28 May 2024), the subcellular location analysis of GRAS
   genes was completed [[98]39].

2.3. Phylogenetic Relationship Analysis

   The amino acid sequences of the GRAS protein found in H. citrina
   Baroni, A. thaliana and O. sativa ([99]Tables S1 and S2) were processed
   using MEGA11.0 software for sequence alignment. Following this,
   phylogenetic trees were created utilizing the neighbor-joining (NJ)
   technique within MEGA11, based on 1000 bootstrap resampling iterations
   in MEGA11.0 software [[100]36,[101]40]. These trees were visualized
   using the Evolview v3
   ([102]http://www.evolgenius.info/evolview/#/treeview, accessed on 14
   June 2024) online software [[103]41].

2.4. Analysis of Gene Structure, Structural Domains, and Conserved Motifs

   The genome annotation file of daylily, formatted in GFF3, was taken as
   the basis for choosing TBtools software to explore all genes’
   structures [[104]36]. By using the NCBI CD-search online tool
   ([105]https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi,
   accessed on 28 May 2024) with default parameters, predictions were then
   conducted on their structural domains [[106]38]. The conserved motifs
   of GRAS proteins were explored through utilizing the MEME
   ([107]http://meme-suite.org/, accessed on 29 May 2024) online software
   [[108]42], which indicated differences between the HcGRAS family
   members. The parameters for the conserved motifs were optimized as
   follows: the number of motifs was set to a maximum of 20, and the
   optimal width of residues was between 6 and 200. These findings
   mentioned before were ultimately visualized through TBtools [[109]36].

2.5. Analysis of cis-Acting Elements

   After obtained gene IDs were identified, TBtools software (v2.310)
   facilitated the extraction of 2000 bp upstream nucleotide sequences for
   each gene from the GFF3 file. Subsequently, the promoter regions were
   analyzed using the online tool PlantCARE
   ([110]https://bioinformatics.psb.ugent.be/webtools/plantcare/html/,
   accessed on 5 June 2024) in order to identify the elements that act as
   cis-regulatory sequences in the promoter region [[111]43].

2.6. Chromosomal Distribution and Gene Duplication

   The locations of HcGRAS genes within the genome were assessed visually
   using TBtools software, with the data sourced from the daylily genome
   [[112]36]. To explore the gene duplication events of the GRAS genes,
   default parameters were used to adopt the Multiple Collinear Scanning
   Toolkits (MCScanX) [[113]44]. The homology of GRAS genes between H.
   citrina Baroni and seven other plants (Z. mays, B. distachyon, A.
   thaliana, S. italica, S. bicolor, O. sativa and S. lycopersicum) was
   explored through the dual synteny plotter project within TBtools
   software. Non-synonymous (Ka) and synonymous substitution sites (Ks) of
   all of the duplicated GRAS genes were calculated through the use of the
   Simple Ka/Ks Calculator module of TBtools.

2.7. Gene Ontology Function of GRAS Genes and Kyoto Encyclopedia of Genes and
Genomes Pathway Enrichment Analysis

   Function annotations for the protein sequences of 78 HcGRAS TF family
   genes were performed based on the eggNOG-MAPPER database
   ([114]http://eggnog-mapper.embl.de/, accessed on 5 June 2024)
   [[115]45]. TBtools software was leveraged to conduct the Gene Ontology
   (GO) function of the HcGRAS gene and Kyoto Encyclopedia of Genes and
   Genomes (KEGG) pathway enrichment analysis on the HcGRAS genes.
   Furthermore, the online tool ChiPlot ([116]https://www.chiplot.online/,
   accessed on 18 June 2024) was also used for mapping and visualization.

2.8. Protein–Protein Interaction (PPI) Network Analysis and Subcellular
Localization

   The interaction data of O. sativa were first downloaded from the STRING
   protein–protein interaction database (PPD) ([117]http://string-db.org/,
   accessed on 20 June 2024) [[118]46]. Subsequently, the representative
   transcriptional protein sequences were extracted from the genome of
   daylily using TBtools. Moreover, to obtain the protein interaction
   relationship in dayliy, the abovementioned obtained files were dragged
   into the TBtools PPI Predict module [[119]36]. After screening, a
   network interaction map was established for the daylily’s GRAS
   proteins. Next, Cytoscape software (v3.9.1) was leveraged to visualize
   and analyze the interaction [[120]47].

   The coding sequence of HcGRAS38, excluding the termination codon, was
   cloned into the vector pGreenII 62-SK-GFP, resulting in the creation of
   pGreenII 62-SK-HcGRAS38-GFP using Not I and Spe I enzymes (TaKaRa,
   Dalian, China). The co-infiltration of the appropriate vectors into the
   leaves of Nicotiana benthamiana (40 d old) was facilitated by the
   strain GV3101. The GFP fluorescence signal was examined using confocal
   fluorescence microscopy (Leica TCS SP8, Wetzlar, Germany) 48 h
   following the transient transformation conducted in darkness.

2.9. Analysis of Tissue Expression Characteristics and RT–PCR

   The gene expression data of daylily were collected from the NCBI
   ([121]https://www.ncbi.nlm.nih.gov, accessed on 20 May 2024).
   Transcriptome changes in daylily at various flower development stages
   (SRP243199) were measured through performing the RNA-seq experiment
   [[122]48]. In this experiment, the RNA samples were divided into six
   stages according to their sizes, which were stage A (2.1–2.5 cm), stage
   B (4.1–4.5 cm), stage C (5.1–5.5 cm), stage D (7.1–8.0 cm), stage E
   (full opening) and stage F (flower senescence) [[123]48]. Transcriptome
   changes in daylily by spraying exogenous JA were measured through
   performing the RNA-seq experiment [[124]49]. When the seedlings grew to
   the seven-leaf stage in spring, we selected disease-free seedlings with
   roughly the same growth conditions and sprayed 1 mg/L exogenous JA
   evenly on each plant with about 200 mL until the solution drips from
   the leaves. Then, we covered the entire plant with a transparent
   plastic bag for 8 h to prevent the treatment solution from evaporating.
   The fifth to seventh leaves (new leaves) of each daylily seedling were
   selected for each treatment at 1, 3, 5, 7, and 9 days after spraying
   (named JA-1d, JA-3d, JA-5d, JA-7d, JA-1d, and JA-9d), and the control
   check (CK) was sprayed with equal amounts of 0 mmol/L JA prepared by
   adding equal amounts of anhydrous ethanol and distilled water, with the
   samples designated ck-1d, ck-3d, ck-5d, ck-7d, and ck-9d (PRJNA1094559
   and PRJNA1097583). Transcriptome changes in the 5-leaf stage daylily by
   spraying 2 mgL^−1 SA were measured through performing the RNA-seq
   experiment (PRJNA1056505) [[125]50]. This transcriptomic information
   facilitated the assessment of the differential expression of GRAS genes
   after 0, 3, 6, 8, and 12 d to investigate the response mechanism of H.
   citrina (we selected healthy plants of daylily that had been replanted
   for two weeks) to rust infection (PRJNA891118) [[126]51]. The
   transcriptome data of daylily exposed to Thrips palmi (T. palmi)
   feeding stress were also measured. T. palmi was starved for 12 h prior
   to the test to ensure adequate feeding induction on plants. On the day
   of the experiment, T. palmi was transported to the net room and
   released to the seven-leaf stage daylily such that there were ~90
   individuals per plant. Daylily in this treatment group was individually
   covered with a 60-mesh insect-proof net to prevent T. palmi from
   escaping. Plant leaves were collected at 1, 3, 5, 7, and 9 days after
   the introduction of T. palmi (named thirps-1d, thirps-3d, thirps-5d,
   thirps-7d, and thirps-9d), and the leaves harvested concurrently from
   the CK group were utilized as controls (designated ck-1d, ck-3d, ck-5d,
   ck-7d, and ck-9d 5) (PRJNA1094559) [[127]52]. The GRAS genes were
   filtered to determine their potential functions in flower development.
   The expression was normalized via the “Normalized” scaling approach in
   TBtools, and this research also employed TBtools to generate heatmaps
   in accordance with the transformed data of log2 (FPKM + 1) values
   [[128]36].

   The RNA was isolated from various parts of the daylily using a kit
   specifically designed for RNA extraction (Tiangen Biotech, Beijing,
   China). First-strand cDNA was synthesized using a PrimeScriptTM RT
   reagent kit (TaKaRa, Dalian, China). PCR was conducted utilizing SYBR^®
   Premix Ex Taq^TM II (TaKaRa, Dalian, China) with gene-specific primer
   sets on the ABI QuantStudio (TM) 6 Flex System (Applied Biosystems,
   Foster City, CA, USA). The gene expression levels were quantified
   through an analysis of three separate biological samples, along with an
   assessment of three technical replicates, to determine the relative
   expression levels using the comparative Ct (2^−ΔΔCt) [[129]53] method,
   with Actin (used by [[130]54]) serving as a control ([131]Table S3). An
   analysis of statistical significance and data was conducted using the
   GraphPad Prism6 (GraphPad Software, Inc., San Diego, CA, USA). The data
   are presented in the form of “mean ± SD”. The least significant
   difference test (LSD) was utilized to determine the comparison between
   various samples, and various letters exhibit significant differences at
   the level of p < 0.05.

3. Results

3.1. Identification of GRAS Genes in Daylily

   The GRAS members were renamed HcGRAS1 to HcGRAS78 ([132]Table S2)
   according to the chromosomal location. A systematic analysis was
   conducted on basic characteristics, such as subcellular localization,
   the isoelectric point, the protein molecular weight, and the gene
   coding sequence. Among the 78 HcGRAS proteins, HcGRAS71 was the
   smallest with 199 amino acids and HcGRAS30 was the largest with 771
   amino acids. In these proteins, the range of the molecular weight was
   from 23.21 kDa (HcGRAS71) to 84.23 kDa (HcGRAS30). The range of the
   isoelectric point was from 4.74 (HcGRAS75) to 9.51 (HcGRAS44), in which
   the median was 5.54. The instability index was 32.33 to 64.18, the
   aliphatic index was 70.08 to 95.37, and the hydropathicity grand
   average was −0.549 to 0.08. The GRAS domain was included in each
   putative protein that was encoded by HcGRAS genes. In accordance with
   predicted subcellular localization, 68 HcGRASs were located in the
   nucleus, and (HcGRAS20 and HcGRAS71) and 8 (HcGRAS3, HcGRAS13,
   HcGRAS51, HcGRAS57, HcGRAS58, HcGRAS61, HcGRAS70 and HcGRAS77) were
   located in the cytoplasm/nucleus ([133]Table S2).

3.2. Phylogenetic Tree of HcGRASs

   For the purpose of revealing the evolutionary relationship of GRAS
   genes between A. thaliana, O. sativa and H. citrina, this research set
   up a neighbor-joining phylogenetic tree in accordance with the 33
   AtGRASs from A. thaliana, 56 OsGRASs ([134]Table S1) from O. sativa,
   and 78 HcGRASs ([135]Table S2) from H. citrina Baroni ([136]Figure 1).
   The results showed that 78 HcGRASs were clustered into 15 subfamilies:
   DELLA, SCL3, RAD1, RAM1, HAM, SCL4/7, NSP2, LS, DLT, SCR, SCLA, PAT,
   SHR, NSP1, and LISCL. HcGRAS1, HcGRAS24, HcGRAS28, HcGRAS38, HcGRAS40
   and HcGRAS55 are DELLA subfamily members. HcGRAS2, HcGRAS23, HcGRAS31
   and HcGRAS62 are DLT subfamily members. HcGRAS12, HcGRAS22, HcGRAS27,
   HcGRAS74, HcGRAS48 and HcGRAS59 are HAM subfamily members. The LS
   subfamily has four members, including HcGRAS4, HcGRAS45, HcGRAS46and
   HcGRAS57. HcGRAS3, HcGRAS6, HcGRAS7, HcGRAS11, HcGRAS20, HcGRAS44 and
   HcGRAS71 are LISCL subfamily members. The NSP1 subfamily has eight
   members, including HcGRAS41, HcGRAS54, HcGRAS17, HcGRAS35, HcGRAS50,
   HcGRAS52, HcGRAS61, and HcGRAS70. HcGRAS56, HcGRAS75, HcGRAS9,
   HcGRAS10, HcGRAS14, HcGRAS15, HcGRAS18, HcGRAS39 and HcGRAS77 are
   members of the NSP2 subfamily. HcGRAS34, HcGRAS67 and HcGRAS72 are SCLA
   subfamily members. HcGRAS13, HcGRAS29, and HcGRAS37 are RAD1 subfamily
   members. HcGRAS16, HcGRAS21 HcGRAS25, HcGRAS69, HcGRAS32, HcGRAS36,
   HcGRAS47, HcGRAS58, HcGRAS65 and HcGRAS78 are PAT subfamily members.
   HcGRAS26 and HcGRAS30 are RAM1 subfamily members. HcGRAS19, HcGRAS43
   and HcGRAS68 are SCL3 subfamily members. HcGRAS51 is an SCL4/7
   subfamily member. HcGRAS21, HcGRAS25 and HcGRAS69 are SCL8 subfamily
   members. HcGRAS5, HcGRAS8, HcGRAS42, HcGRAS53, HcGRAS63 and HcGRAS64
   are SCR subfamily members. HcGRAS33, HcGRAS49, HcGRAS60, HcGRAS66,
   HcGRAS73 and HcGRAS76 are SHR subfamily members.

Figure 1.

   [137]Figure 1
   [138]Open in a new tab

   The phylogenetic tree of daylily, Arabidopsis, and rice GRAS proteins
   constructed by MEGA11.0. The tree shows 16 phylogenetic subfamilies
   marked with different colored fonts on a white background. The GRAS
   proteins derived from A. thaliana and O. sativa are prefixed ‘At’ and
   ‘Os’, respectively.

3.3. Gene Structure, Domain, and Conserved Motif Composition Analysis of
HcGRAS Genes

   Based on phylogenetic analysis, the identified 78 HcGRAS proteins were
   divided into 14 subfamilies ([139]Figure 2A). Through leveraging the
   MEME online tool, an analysis of the HcGRAS protein sequences revealed
   the presence of 20 different conserved motifs ([140]Figure 2B). Changes
   in different proteins were indicated by the number and distribution of
   these motifs and 20 different conserved motifs were found ([141]Figure
   2B and [142]Figure S2). It is found that motifs 1, 2, 3, 9, and 4 found
   distribution within the HcGRAS family. A similar motif composition was
   often found in HcGRAS members of the same subfamily. The DELLA
   subfamily included motifs 9, 7, 17, 1, 15, 8, 11, 5, 3, 6, 10, 14 and
   4; the SHR subfamily included motifs 9, 7, 2, 1, 8, 11, 5, 3, 13, 6, 14
   and 4; and the PAT1 subfamily included motifs 9, 7, 2, 1, 15, 11, 5, 3,
   10, 14, 16 and 4. Certain motifs were only distributed in the pattern’s
   specific locations. An example is that motif 9 (73, ~93.59%) and motif
   7 (69, ~88.46%) were distributed at the pattern’s starting all the time
   and motif 4 (72, ~92.31%) at the pattern’s end. Conserved domain
   analysis showed that HcGRAS proteins consisted of GRAS, the GRAS
   superfamily, DELLA and the DELLA superfamily ([143]Figure 2C),
   suggesting significant levels of preservation throughout their
   evolutionary history. Additionally, a structural map containing the CDS
   and UTR was constructed based on the genome sequence ([144]Figure 2D),
   contributing to an understanding of the structural composition of
   HcGRAS genes. The coding sequences of 78 HcGRASs are shown in
   [145]Table S2. Gene structure analysis revealed that a similar gene
   structure was observed in members of the same evolutionary clade.

Figure 2.

   [146]Figure 2
   [147]Open in a new tab

   The analysis of phylogenetic relationships, gene structure, and
   distribution of motifs among HcGRAS genes. The phylogenetic tree was
   generated using the NJ method with 1000 replicates on each node (A).
   The conserved motif distribution of GRAS proteins (B); motifs 1–20 are
   represented by boxes of different colors. The conserved functional
   domains of HcGRASs (C). Gene structures of HcGRAS genes (D).

3.4. Chromosomal Localization, Collinearity and Evolutionary Analysis of
HcGRAS Gene Members

   The results indicated that 67 HcGRAS members were not evenly
   distributed on 11 Chrs and 11 HcGRAS members were evenly distributed at
   unanchored points. Chr 8 had the most distribution with nine members,
   followed by Chrs 2 and 7 with eight members, Chr 3 with seven members,
   Chrs 4, 9 and 11 with six members, Chr 1 with five members, and Chrs 5,
   6 and 10 with four members ([148]Figure 3).

Figure 3.

   [149]Figure 3
   [150]Open in a new tab

   Chromosome distribution of GRAS genes on daylily (Chrs 1–11). Each
   HcGRAS gene named is exhibited on daylily Chrs, in which the Chr number
   is marked at each strip’s edge. The scale ranging from 0 to 500 Mb
   denotes the chromosome length, while the internal lines within the
   chromosomes reflect the density of the genes.

   The intra-species collinear analysis was carried out for the purpose of
   deciding if gene duplication resulted in the HcGRAS gene family’s
   evolution ([151]Figure 4 and [152]Figure 5). More pairs of duplicated
   segments were found within HcGRASs, which suggested that segmental
   duplication played a significant role in genetic diversity. Then,
   intra-species syntenic maps of H. citrina Baroni that have seven
   species were constructed to identify orthologous genes using TBtools
   ([153]Figure 5 and [154]Table S4). A total of 44 HcGRAS genes exhibited
   collinear relationships with A. thaliana (7), B. distachyon (33), Oryza
   sativa (32), S. bicolor (33), S. italica (32), S. lycopersicum (31),
   and Z. mays (27). The orthologous gene pairs identified between daylily
   and the remaining seven species totaled, including B. distachyon, S.
   bicolor, S. italica, Arabidopsis, tomato, rice, and maize, were 15, 50,
   6, 53, 52, and 55, respectively.

Figure 4.

   [155]Figure 4
   [156]Open in a new tab

   The chromosomal positions and the relationships among different
   chromosomes concerning GRAS family genes in H. citrina Baroni. Red
   lines indicate the events of duplication that have been identified.

Figure 5.

   [157]Figure 5
   [158]Open in a new tab

   Collinearity analysis between the GRASs of H. citrina, A. thaliana, B.
   distachyon, O. sativa, S. bicolor, S. italica, S. lycopersicum and Z.
   mays.

3.5. Identification and Distribution of cis-Regulatory Elements in the HcGRAS
Promoter

   Specifically, this research extracted a total of 2000 base pairs of the
   transcription start codon of HcGRAS genes in order to analyze their
   potential biological functions through the use of the PlantCARE
   database. Three major responsive elements in total were identified,
   such as plant growth and development, response to abiotic stress, and
   hormone response ([159]Figure 6 and [160]Table S5). Among these, 1 to
   22 elements responsive to light were detected in 78 genes, which
   indicated that a significant portion of the HcGRAS genes is influenced
   by light signaling pathways. Each of the 78 genes included cis-acting
   elements influencing hormonal response and developmental processes.
   Elements related to hormones contained GA response, SA, JA, auxin, and
   abscisic acid (ABA). In contrast, elements related to development
   contained MYB binding site response, circadian control, anoxic specific
   inducibility, meristem expression, endosperm, cell cycle regulation,
   anaerobic induction, seed-specific regulation, and zein metabolism
   regulation. Furthermore, numerous HcGRAS genes exhibited cis-acting
   elements associated with abiotic stress factors, including responses to
   low temperatures, combined environmental stress, and wounds. As the
   promoter regions of 78 HcGRASs contained various types of cis-elements,
   they may be involved in diverse regulatory pathways, environmental
   stresses, and biological processes.

Figure 6.

   [161]Figure 6
   [162]Open in a new tab

   A prediction of transcription factor binding sites in the promoters of
   HcGRAS (A); boxes of different colors represent different transcription
   factor families. The number of functional elements in HcGRASs (B).

3.6. Functional Annotation of HcGRAS TFs

   In order to further explore biological functions of the HcGRAS TF
   family, this research annotated 78 HcGRAS genes’ proteins ([163]Figure
   7 and [164]Table S6). It was found that 69 HcGRAS genes were annotated
   and assigned to three broad categories, which were biological process
   (BP), cellular component (CC), and molecular function (MF). For the
   high enrichment of HcGRAS genes for organic cyclic compound binding and
   nucleic acid binding, the HcGRAS TF family is found to contribute much
   to affecting transcription regulation. Among cellular components,
   HcGRAS genes were enriched in the nucleus and membrane. In biological
   processes, mediated signaling pathways, responses to hormonal and
   environmental stimuli, growth and development processes, and biomass
   synthesis were enriched with more HcGRAS genes. Among them, growth and
   development processes, including dormancy processes, tissue development
   and plant organ development, environmental stimulus responses,
   including responses to symbiotic fungus and phosphate starvation
   responses, and hormonal stimulus responses, including SA, GA, ABA,
   brassinosteroid and JA signaling pathways, all indicate that the HcGRAS
   TF family is likely to adapt plants to different environments.
   Furthermore, KEGG enrichment analysis demonstrated that 32 out of the
   78 identified GRAS genes were significantly associated with the
   specific signaling pathway related to plant hormones ([165]Table S7).

Figure 7.

   [166]Figure 7
   [167]Open in a new tab

   GO enrichment analysis of HcGRAS genes.

3.7. Protein–Protein Interaction Analysis of the HcGRAS TF Family

   An analysis of the interaction network among 78 HcGRAS proteins
   indicated that 25 of these genes established interactions, while
   interaction relationships were not formed in 53 genes ([168]Figure 8).
   [169]Figure 8A implies that those proteins are crucial and the DELLA
   subfamily members HcGRAS1, HcGRAS24, HcGRAS28, HcGRAS38, HcGRAS40 and
   HcGRAS55, which is a family of proteins that regulate an adverse
   environment by interacting with GA. The other proteins, all of which
   belong to the SCR, OS4, LS, and SHR subfamilies, interact with them,
   particularly the HcGRAS38 protein, serving as the core connecting DELLA
   proteins, which indicates that its role is necessary for the DELLA
   subfamily. HcGRAS21, HcGRAS25 and HcGRAS69, which belong to the SCL8
   subfamily, are the key factors of protein interaction in [170]Figure
   8B, and they can interact with many proteins in daylily. In [171]Figure
   8C, HcGRAS5 and HcGRAS8, which belong to the SCR subfamily, play an
   important role in the PPI network. Meanwhile, HcGRAS33, HcGRAS49,
   HcGRAS60, HcGRAS66, HcGRAS73, and HcGRAS76, which belong to the SHR
   subfamily, suggest that there is a protein interaction relationship
   between SHR and SCR subfamilies.

Figure 8.

   [172]Figure 8
   [173]Open in a new tab

   Diagram illustrating reciprocal interactions among proteins. Each
   circle represents a protein. PPI between DELLA subfamily members and
   other proteins (A). PPI between SCL8 subfamily members and other
   proteins (B). PPI between SCR subfamily members and SHR subfamily
   members (C).

3.8. Subcellular Localization of HcGRAS18 Protein

   In order to confirm the expected subcellular localization of HcGRAS38,
   daylily HcGRAS38-GFP was co-expressed with the nucleus marker (mCherry)
   vector, subsequently transferring the recombinant plasmid into
   Agrobacterium tumefaciens, and we injected it into N. benthamiana.
   After a period of 48 h, observations were made with a confocal laser
   scanning microscope, revealing that HcGRAS38 was localized to the
   nucleus ([174]Figure 9).

Figure 9.

   [175]Figure 9
   [176]Open in a new tab

   A fusion protein of HcGRAS38 with GFP was analyzed for its subcellular
   distribution in the leaves of N. benthamiana.

3.9. Expression Analysis of HcGRAS Genes

   Transcriptome data from different developmental stages of daylily were
   analyzed to understand HcGRAS genes’ expression patterns ([177]Figure
   10A). Certain HcGRAS genes exhibited a consistent pattern of high
   expression at various flower development stages, including HcGRAS21,
   HcGRAS2, HcGRAS77, HcGRAS18, HcGRAS6, and HcGRAS1. Each OS19 subfamily
   member exhibited high expression at different stages, but there were
   great differences in the number of transcripts from members of other
   subfamilies. An example is that the SCL32 subfamily’s HcGRAS61 almost
   cannot be detected in transcripts, with the overexpression of other
   members. The SCL8 subfamily’s HcGRAS25 was highly expressed at the
   flower stages A to D.

Figure 10.

   [178]Figure 10
   [179]Open in a new tab

   Analysis of expression patterns in HcGRASs. (A) Heatmap illustrating
   the expression profiles of 78 HcGRAS genes at various stages of floral
   development. (B) Expression patterns of HcGRASs under salicylic acid
   treatment. (C) Expression patterns of HcGRASs under jasmonic acid
   treatment. Different colored boxes exhibit row-scaled FPKM values, with
   expression gradually increasing from blue to yellow to red at a time.

   To investigate the expression patterns of HcGRAS genes under hormone
   stress, a heatmap of the expression profiles was performed for their
   predicted cis-acting elements associated with SA and JA. The
   transcriptomic analysis of daylily exposed to SA was conducted to
   elucidate the expression patterns of HcGRAS genes ([180]Figure 10B).
   After SA treatment, the level of expression of some genes increased
   significantly including HcGRAS35, HcGRAS17, HcGRAS47, and HcGRAS28.
   Some other genes showed decreased expression levels after the SA
   treatment of HcGRAS53, HcGRAS48, and HcGRAS9. The gene expression
   heatmap results after JA treatment was shown in [181]Figure 10C.
   Notably, HcGRAS16, HcGRAS36, HcGRAS11, HcGRAS6, and HcGRAS7 showed high
   expression under JA treatment. Additionally, HcGRAS55, HcGRAS53,
   HcGRAS40, HcGRAS1, and HcGRAS28 were down-regulated. These observations
   underscore the significant functions of HcGRAS genes in regulating
   plant development and their responses to stressors mediated through SA
   and JA.

   An investigation was conducted to assess the involvement of HcGRAS
   genes when subjected to biotic stress. The expression of HcGRAS genes
   was assessed following stress from Puccinia hemerocallidis infection
   and feeding by T. palmi. The expression levels of HcGRAS11 and HcGRAS65
   gradually increased after rust infection, and the expression level of
   HcGRAS9 gradually decreased ([182]Figure 11A). For example, genes such
   as HcGRAS21, HcGRAS25, HcGRAS40, HcGRAS41, and HcGRAS48 have the
   highest expression levels after 12d of treatment, while genes such as
   HcGRAS17, HcGRAS24, HcGRAS34, HcGRAS35, and HcGRAS69 show the highest
   levels of expression at 0 d and then decrease. Some genes, such as
   HcGRAS16, HcGRAS19, HcGRAS55, HcGRAS58, and HcGRAS59, showed higher
   expression levels at 3d and 6d after rust infection. After thrips
   stress treatment, the expression levels of HcGRAS5, HcGRAS9, HcGRAS14,
   HcGRAS24, HcGRAS49 and HcGRAS53 were higher in the control group
   ([183]Figure 11B). The expression levels of HcGRAS3, HcGRAS10,
   HcGRAS11, HcGRAS25, HcGRAS32, HcGRAS39, HcGRAS51 and HcGRAS76 were
   relatively high in the treatment group ([184]Figure 11B).

Figure 11.

   [185]Figure 11
   [186]Open in a new tab

   Analysis of expression patterns in HcGRASs under biotic stress. (A)
   Expression patterns of HcGRASs after rust infection. (B) Expression
   patterns of HcGRASs exposed to Thrips palmi feeding stress. Different
   colored boxes exhibit row-scaled FPKM values, with expression gradually
   increasing from blue to yellow to red at a time.

   The expression levels of 22 members in four organs (flower, root, stem,
   and leaf) and five flower stages were detected using real-time PCR
   ([187]Figure 12) to explore the physiological functions of GRAS genes
   within daylily. There was the highest expression in the leaf for seven
   genes (HcGRAS41, HcGRAS46, HcGRAS47, HcGRAS48, HcGRAS51, HcGRAS55 and
   HcGRAS74) and in the root for five genes (HcGRAS1, HcGRAS2, HcGRAS29,
   HcGRAS33 and HcGRAS77). Significant differences were identified in the
   expression levels of HcGRAS genes at various flower development stages.
   Compared to the full-open flower stage, there was higher gene
   expression before the early flower stage. Nine genes (HcGRAS8,
   HcGRAS21, HcGRAS29, HcGRAS33, HcGRAS40, HcGRAS43, HcGRAS48, HcGRAS50
   and HcGRAS55) exhibited the highest expression at the F1 stage
   (including F1, F3, F5, F7, F9 and flower), while three genes (HcGRAS6,
   HcGRAS38, and HcGRAS67) exhibited the highest expression at the
   full-open flower stage (flower).

Figure 12.

   [188]Figure 12
   [189]Open in a new tab

   Expression analysis of HcGRASs in different tissues. Different
   lowercase letters indicate significant differences by LSD (p < 0.05).

4. Discussion

4.1. HcGRAS Gene Structure and Evolutionary Analysis

   The identification of gene families has been conducted in an increasing
   number of species with the progress of genome sequencing technology.
   The molecular mechanisms of plant GRAS TFs for addressing environmental
   stress have been determined after the identification and functional
   analysis of GRAS TFs in multiple crops and cash crops. However, no
   relevant studies have been conducted in daylily. In this study, the
   bioinformatics method was used to identify 78 GRAS TFs from the daylily
   genome. The number of GRAS genes in daylily was higher compared to that
   within A. thaliana (34) and rice (57) [[190]2] and was close to that in
   apple (78) [[191]8] and M. acuminata var. DH-Pahang (73) [[192]11].
   There were differences between 78 HcGRAS genes in the theoretical
   isoelectric point, molecular weight and nucleotide sequence length,
   suggesting the complexity of the daylily genome and the functional
   difference in HcGRAS genes in various microenvironments.

   This research set up a phylogenetic tree by selecting A. thaliana
   AtGRAS, rice OsGRAS and daylily HcGRAS to explore the evolutionary
   relationships of GRAS TFs in different species. The results of A.
   thaliana and rice allowed for the classification of the HcGRAS
   subfamily, indicating that GRAS TFs were classified into 15
   subfamilies: DELLA, SCL3, RAD1, RAM1, HAM, SCL4/7, NSP2, LS, DLT, SCR,
   SCLA, PAT, SHR, NSP1, and LISCL ([193]Figure 1). Therefore, it is
   speculated that these subfamilies may be present in most
   monocotyledonous plants and have fundamental physiological functions
   that are conserved throughout evolution. In each Arabidopsis subfamily,
   the identification of at least one HcGRAS protein suggests that these
   GRAS subfamilies may play certain basic biological functions and are
   not lost during long-term evolution [[194]2]. This study supports the
   hypothesis that separating the GRAS family is likely to precede the
   separation of daylily and A. thaliana [[195]2,[196]31]. Thus, further
   investigation is required for unique physiological functions of HcGRAS
   proteins. Among them, LS has the highest number of members (13,
   16.67%), while SCL4/7 (HcGRAS51) has the lowest number of members.
   Similarly to the GRAS subfamilies found in other species, like A.
   thaliana [[197]2], rice [[198]18], soybean [[199]10], millet [[200]31],
   sugar beet [[201]11], and S. bicolor [[202]32], different subfamilies
   are likely to possess various differentiation capabilities throughout
   the course of evolution. The variations observed in these genes may
   hold considerable importance for the distinct roles they play across
   different species. However, limited research indicates that the
   mechanisms of adapting to environmental factors are linked to
   variations in differentiation among the subfamilies.

   IDRs (intrinsically disordered regions) are constituted by the highly
   variable N-terminus of GRAS proteins, which include molecular
   recognition features and gene fragments that are easily exchanged.
   Through ordered and disordered transformations, the specific binding
   objects can be recognized in these functional regions
   [[203]19,[204]55]. Most HcGRAS genes have five major conserved domains:
   SAW, PFYRE, VHIID, LHR I, and LHR II ([205]Figure 2B). The
   characteristic sequences of Asp, Ile, His and Val are included in the
   core VHIID domain. The HcGRAS proteins possess a region near their N
   terminus that is notably disordered, yet there are observed
   similarities across various subfamilies. A case in point is the
   involvement of the DELLA domain by the proteins of the DELLA subfamily
   at the N terminus ([206]Figure 2C), which contributes to the functional
   diversity observed in GRAS proteins. In these regions, some residues
   are found to be conserved in various subfamilies, which may be
   necessary for the functions of GRAS proteins in various subfamilies.
   For instance, within woodland strawberry, alterations in the SAW domain
   have led to phenotypic variations [[207]56].

   The survey on the exons of HcGRAS genes shows that 1–4 exons are
   contained in each gene ([208]Figure 2D). About 75.6% of HcGRAS genes
   have no intron, which is seen to be higher compared to those in millet
   (64.9%) [[209]57] and sorghum (66.7%) [[210]58] but lower than those in
   buckwheat (87%) [[211]59]. Some subfamily members have a compact gene
   structure because SHR, LS, and SCL15 have only one intron or do not
   contain any introns. Also, genes with no intron are found in other
   families, such as the SAUR family [[212]60], and DEAD box RNA helicase
   [[213]61]. Genes that do not have an intron usually possess lower
   expression levels within plants [[214]62]. Nonetheless, GRAS genes may
   directly originate from prokaryotes by means of horizontal gene
   transfer and duplication events [[215]63,[216]64]. Hence, the gene
   structure may be compact in the majority of GRAS members [[217]65].
   Genes with no intron continue to encode proteins in the transcription
   and translation process, which enables them to quickly respond to
   various environments [[218]58,[219]66]. Additionally, the expression of
   genes might not be influenced by the quantity of introns present within
   those genes [[220]67].

   The increase and development of the GRAS gene family can be attributed
   to the process of gene duplication, which serves as a driving
   evolutionary mechanism [[221]68]. This research shows the uneven
   distribution of HcGRAS genes on 11 chromosomes, the majority of which
   were on chromosome 8. This phenomenon is potentially attributed to the
   localization characteristics of gene replication events, the structural
   and functional heterogeneity of chromosomes, and the disparities in
   evolutionary selection pressures [[222]69,[223]70]. Interestingly,
   there was no tandem duplication of GRAS genes on the chromosome of
   daylily. The majority of GRAS genes originated from segmental
   duplications, suggesting that this form of duplication serves as a
   primary mechanism for the expansion of gene families, which aligns with
   findings for Benincasa hispida [[224]71]. Furthermore, the covariance
   of daylily with five monocotyledonous plants and two dicotyledonous
   plants was compared in this study. The daylily with a monocotyledon
   showed superior covariance compared to that with a dicotyledon.
   Compared to the interspecific collinearity, there is weaker
   intraspecific collinearity of GRAS genes in Arabidopsis and daylily,
   which indicates that gene replication is not the source of GRAS genes
   in various species ([225]Figure 5) [[226]72].

   The Ka/Ks ratio is considered a crucial standard for measuring gene
   selection pressure [[227]73]. Prior studies have shown that powerful
   purifying selection stress is experienced by HcGRAS genes involved in
   sea buckthorn duplication [[228]74]. As shown in [229]Table S8, all the
   HcGRAS genes are subject to purifying selection. In each gene, the
   Ka/Ks value is below 1, which indciates the purification state of each
   gene in the peach, possibly facilitating the elimination of harmful
   mutations and the maintenance of gene functions [[230]75].

4.2. Predicting the Potential Functions of HcGRAS Genes

   Cis-acting elements, such as enhancers and silencers, are crucial for
   the regulation of gene transcription in adjacent regions by engaging
   with trans-acting factors, which in turn affects the patterns and
   levels of gene expression [[231]76]. The primary cis-acting components
   found within the initiation region of the HcGRAS TF family include
   elements responsive to hormones, those influencing light responses, and
   ones that react to environmental stress ([232]Figure 6). There is the
   existence of many cis-acting hormones, including auxin, GA, SA and ABA.
   For gene promoters, except for a few genes, 80% involve GA response
   elements. The SCL32 and DLT subfamilies have witnessed a relatively
   good distribution of ABA response elements. Prior studies indicate that
   the structure of the N-terminal DELLA protein, belonging to the GRAS
   family of transcription factors, is pivotal in controlling various
   aspects of plant growth and development, as it functions within GA
   signaling pathways [[233]77]. Although ABA can often promote stem
   elongation, seed germination and flower development, decreased cellular
   activity is observed in plants deficient in ABA and increased factor
   activity concerning ABA synthesis is also observed under adversity
   stress [[234]78]. High salt stress activates ABA signaling, for
   example, leading to an accumulation of DELLA proteins, which inhibit
   plant growth and increase resistance [[235]79]. Auxin has the functions
   of regulating stem growth rates, inhibiting lateral buds and promoting
   rooting, and some studies have found that ethylene and auxin interact
   with DELLA to regulate plant growth [[236]80,[237]81]. This implies
   that GRAS TFs in daylily can contribute much to regulating plant growth
   and development in the case of negative stress conditions as well.

   The GO enrichment analysis revealed a significant number of genes that
   are responsive to various hormonal signals, such as pathways associated
   with SA, ABA, and JA, various environmental stimuli, such as reactions
   to cold temperatures, increased salinity levels, adaptations to other
   stress factors and processes related to growth and development,
   including the formation of plant organs, tissue differentiation, and
   dormancy. This is consistent with the findings observed in sugar beet
   [[238]11]. By contrast, KEGG enrichment analysis just included a
   phytohormone signaling pathway, which suggests that HcGRAS
   transcription factors influence the regulation of plant growth and
   development by modulating hormone expression levels to adapt to
   challenging conditions. All of these findings further aligned with the
   analysis of the PPI network.

4.3. Gene Expression Profiles and RT-PCR Unveiled the Potential Roles of GRAS
Genes in Flower Development, Hormone Response and Biotic Stress

   It is reported that GRAS transcription factors are essential to flower
   development [[239]31,[240]32]. For Arabidopsis, a pentuple (ga1–3
   gai-t6 rga-t2 rgl1–1 rgl2–1) DELLA mutant flowered earlier, which
   suggested that these transcription factors represent flowering
   repressors [[241]82]. DELLA proteins function through repressing FT and
   SPL3 included in regulating flowering onset, since early flowering
   mutant displays an increase in FLOWERING LOCUS T (FT) and SQUAMOSA
   PROMOTER BINDING PROTEIN-LIKE 3 (SPL3) expression [[242]82]. In
   tomatoes, a significant change occurred in the expression of genes
   included within pollen development and hormonal signaling for the
   flower transcriptome at anthesis [[243]33]. A GA-constitutive response
   phenotype with increasing flowers was exhibited in the procera (pro)
   tomato mutant (SlDELLA) [[244]83]. In grapes, there was an
   up-regulation of VviRGA3 and VviRGA5 in the process of fruit set and
   they were likely to regulate the transition from inflorescence to
   flower [[245]84]. In this study, HcGRAS1 was highly expressed in each
   stage, and HcGRAS40 was expressed during the first four flower
   development stages, with low expression levels in the last two stages.
   HcGRAS24 exhibited low expression levels during the first two stages
   and was downregulated in the subsequent four stages. Meanwhile,
   HcGRAS55 was not detected or had low expression levels. This aligned
   with prior studies, indicating that the DELLA subfamily of GRAS genes
   plays a regulatory role in flower development.

   Both DELLA and other subfamilies are included during the flower
   development process. In apples, there was significant modulation of the
   expression of MdGRAS6 (LS), MdGRAS122 (DELLA), MdGRAS44 (PAT1),
   MdGRAS53, MdGRAS107, MdGRAS26 (SHR), and MdGRAS64 (LISCL) in the bud,
   which implies that they may contribute much to flowering. In the flower
   induction under hormonal treatment, through an observation of
   expression patterns of PAT1, LS, SCL, LISCL, SHR, and DELLA members,
   they were identified to be included in 6-benzyladenine, gibberellic
   acid and/or sugar-mediated flowering in apple trees as well [[246]85].
   It was also found the LAM gene had expression in all the floral
   meristematic tissues and young floral organs in strawberries [[247]29].
   These findings also showed HcGRAS33 (SHR) and HcGRAS74 (HAM) expressed
   at all stages of flower development, but the expression level showed a
   decreasing trend ([248]Figure 10 and [249]Figure 12). All these results
   indicate that daylily GRAS genes are likely to be included in flower
   development. Based on SA and JA treatment, as well as thrips disease
   and rust treatment, we have identified some specific expressed genes
   ([250]Figure 10 and [251]Figure 11), such as HcGRAS25 (PAT) and HcGRAS1
   (DELLA) which provide some basis for future applications. The altered
   expression of HcGRAS25 (PAT) aligns with trends observed in other
   species [[252]25], indicating that the activation of PAT subfamily
   genes facilitates physiological adjustments in daylily, enabling
   adaptation to stress conditions and the maintenance of normal growth
   and metabolic activities.

5. Conclusions

   This study focuses on identifying and characterizing the GRAS gene
   family within the daylily genome, using bioinformatics approaches to
   predict its structural and functional roles. The quality of the data
   could be enhanced as the majority were sourced from publicly available
   databases and the existing daylily data repository lacks completeness.
   A total of 78 HcGRAS transcription factors were identified, exhibiting
   an uneven distribution across 11 chromosomes. Transcriptional
   expression profiling, coupled with RT-PCR validation, confirmed the
   involvement of numerous GRAS genes in flower organ development and
   their response to SA and JA and resistance against rust and thrips
   pests. These findings provide a foundation for future research delving
   into the biological role of HcGRAS and offer a theoretical framework
   for its application in molecular breeding. Ultimately, this study
   contributes to the development of more efficient and precise breeding
   strategies for daylily, which may have broader implications for global
   agricultural advancements.

Supplementary Materials

   The following supporting information can be downloaded at
   [253]https://www.mdpi.com/article/10.3390/biology14070770/s1, Figure
   S1: Morphology of different tissue samples of daylily; Figure S2:
   Conserved sequence logo of GRAS proteins in daylily; Table S1: List of
   AtGRAS and OsGRASs used in this study; Table S2: List of 78
   Hemerocallis citrina Baroni GRAS genes identified in this study; Table
   S3: Primer information for quantitative real-time PCR; Table S4:
   One-to-one orthologous relationships between Hemerocallis citrina
   Baroni and seven species; Table S5: Cis-regulatory elements in the
   promoter region of HcGRAS genes; Table S6: The GO enrichment analysis
   of the GRAS genes; Table S7: The KEGG pathway enrichment analysis of
   the GRAS genes; Table S8: Ka/Ks ratio distribution of gene pairs.
   [254]biology-14-00770-s001.zip^ (19.9MB, zip)

Author Contributions

   Conceptualization, W.L. and Y.Z.; methodology, W.L. and H.S.; formal
   analysis, W.L., Z.C. and Y.Z.; writing—original draft preparation,
   W.L.; writing—review and editing, Y.Z.; supervision, J.Z. and Y.Z.;
   funding acquisition, Y.Z. All authors have read and agreed to the
   published version of the manuscript.

Institutional Review Board Statement

   Not applicable.

Informed Consent Statement

   Not applicable.

Data Availability Statement

   All of the datasets supporting the results of this article are included
   within the article and its additional files. RNA-seq data from various
   tissue parts and treatments of daylily (accessions: SRX7583022,
   SRX7583021, SRX7583020, SRX7583019, SRX7583018, SRX7583017, SRX7583016,
   SRX7583015, SRX7583014, SRX24114404, SRX24114427, SRX24114432,
   SRX24114433, SRX24114408, SRX24114411, SRX24114415, SRX24114420,
   SRX24114423, SRX24114426, SRX24187491, SRX24187493, SRX24187496,
   SRX24187486, SRX24187490, SRX17912432, SRX17912437, SRX17912456,
   SRX17912459, SRX17912460, SRX23017183, SRX23017182, SRX23017181,
   SRX23017180, SRX23017179, SRX23017178) were systematically retrieved
   from the NCBI’s Sequence Read Archive (SRA) database.

Conflicts of Interest

   The authors declare that this research was conducted in the absence of
   any commercial or financial relationships that could be construed as
   potential conflicts of interest.

Funding Statement

   This research was funded by the Applied Basic Research Programs of
   Shanxi Province (202303021221171, 202203021222295); the Science and
   Technology Innovation Project of Higher Education Institutions in
   Shanxi Province (2021L398); the Applied Basic Research Programs of
   Datong City (2020059, 2023063); and the Doctoral Research Project of
   Shanxi Datong University (2018-B-13).

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References