Genomic Organization and Expression Profiling of GOLDEN2-like Transcription Factor Genes in Eggplant and Their Role in Heat Stresses

Abstract

GOLDEN2-like (GLK) transcription factor genes are involved in chloroplast biogenesis during all stages of plant growth and development, as well as in the response to biotic and abiotic stresses. However, little is known about this transcription factor family in eggplant. In this study, we identified 54 GLK genes in the eggplant genome (S. melongena L.) and classified them into seven groups (G1–G7). Structural analysis illustrated that the SmGLK proteins of specific groups are relatively conserved. Cis-acting elements indicated that these genes are likely to be involved in multiple responses stimulated by light, phytohormones, and abiotic stress. Collinear analysis indicated that expansion of the SmGLK gene family primarily occurred through segmental duplication. Tissue-specific expression analysis revealed that SmGLKs were preferentially expressed in leaves, fruits, and seeds. Further screening of SmGLK genes revealed their differential expression under various treatments. Notably, SmGLK18 was significantly responsive to multiple phytohormones and stress treatments, whereas SmGLK3 and SmGLK12 were highly induced by ABA, IAA, SA, and drought treatments. Our study provides new information on the eggplant GLK family systematically and comprehensively. For the first time, we propose that SmGLK18 may play a key role in improving heat resistance. This study provides valuable candidate gene resources for further functional research and will benefit eggplant molecular breeding.

1. Introduction

GOLDEN2-LIKE (also known as G2-Like and GLK) is a transcription factor that is widely present in plants and belongs to the class of GARP (named Golden2, ARR-B, and Psr1 proteins) superfamilies of Myb transcription factors [1,2]. Historically, the oldest identified function, golden2 mutant, was reported to manifest a golden color phenotype in maize leaves [3]. Subsequently, G2 was recognized as a transcriptional regulator that governs the photosynthetic identity of maize leaves [4]. Recently, GLK genes have been characterized in diverse plant species including Arabidopsis [5], maize [6], moso bamboo [7], soybeans [8], tomato [9], wheat [10], etc. In general, GLK proteins contain two conserved structures: a Myb-DNA-binding domain (containing an HLH motif) and a GLK/C-terminal box (GCT box) [11], and the partial GLK subfamily has a conserved MYB-CC-LHEQLE motif [12].

GLK transcription factors function as key integrators of light and hormone signaling and have been reported to play a role in the regulation of chloroplast development [13]. GOLKENLIKE1/2 plays a positive regulatory role in the activation of nuclear genes (HEMA1, CHLH, GUN4, CAO, PORA, PORB, and PORC) associated with chloroplasts and photosynthesis, facilitating chloroplast biogenesis and division [13]. In a previous study, g2 mutants exhibited smaller chloroplasts and a reduced number of thylakoid lamellae compared to the wild type [14]. In general, GLK genes exist in pairs. AtGLK1 and AtGLK2 function in the formation and development of chloroplasts redundantly in Arabidopsis [15,16]. Functional redundancy of GLK genes has also been reported in rice [17]. In maize, ZmGLK1 and ZmGLK2, two homologous genes, share essentially identical functions and play a central role in the chloroplast formation of mesophyll cells in C4 plant tissues [18]. Beyond their function in leaves, GLK genes also mediate fruit color changes during ripening. Overexpression of SLGLK1 and SLGLK2 induces a darker green hue in immature tomato fruits than in normal fruits, ultimately leading to an enhanced nutritional value upon maturation [19,20,21]. In pepper, CaGLK2 regulates natural variations in chlorophyll content and fruit color [22]. The role of GLK in growth and development is beyond the scope of this study. It enhances the anthocyanin content of Arabidopsis seedlings [23], increases rice yield [24], delays flowering time [25], and promotes leaf senescence [26].

GLK has also been shown to have different functions under biotic and abiotic stress. In fungal diseases, overexpression of AtGLK1 enhances resistance to the non-host fungal pathogen Fusarium graminearum [27,28] and the necrotrophic pathogen Botrytis cinerea. In addition, it resulted in an increased sensitivity to Hyaloperonospora arabidopsidis Noco2 [29]. GLK transcription factors positively regulate plants infected with Cucumber Mosaic Virus [30]. Regarding abiotic stress, Yuan et al. reported that the elevation of SlGLK1 transcriptional levels increased chlorophyll accumulation and promoted robust photosynthesis in tomato plants. This enhancement may confer heightened tolerance to abiotic stresses such as drought and heat [31]. In Arabidopsis thaliana, GOLDEN 2-LIKE related to chloroplast development affects ozone tolerance by regulating stomatal movement [32]. Although certain GLKs have been extensively examined in various species, those associated with abiotic stress have received little attention, with only a limited number of published scientific studies available. Characterization of GLK function under abiotic stress remains particularly limited for economically important horticultural plants, especially eggplants.

Eggplants (Solanum melongena L.) are a vegetable crop cultivated globally. It is a thermophilic plant, exhibiting optimal growth between temperatures of 25 °C and 30 °C. The escalating greenhouse effect is contributing to an annual increase in global temperature. The expanding cultivation of vegetable facilities exacerbates the impact of extremely high temperatures during summer on eggplant production, resulting in considerable damage and a notable reduction in economic benefits [33,34,35]. The GLK gene plays a crucial role in regulating plant growth, development, and stress responses; however, there is a lack of relevant reports on its function in eggplant. This study aimed to identify the members of the eggplant GLK gene family within the latest high-quality genomes. Bioinformatics technology was employed to analyze the evolutionary relationships, gene structure, conserved motifs, and promoter elements of these genes. We also investigated their responses to abiotic stress. The observed expression patterns are intended to lay the foundation for an in-depth exploration of eggplant GLK function and provide valuable genetic resources for genetic improvement.

2. Materials and Methods

2.1. Plant Materials and Stress Treatments

The test materials used in this study were planted in the Germplasm Resource Nursery of the Academy of Agricultural Sciences in the Guangxi Zhuang Autonomous Region. For tissue-specific expression, roots, young stems, old stems, young leaves, mature leaves, old leaves, flower buds, flowers, young fruits, expansion-stage fruits, breaker-stage fruits, ripe-stage fruits, and seeds were collected from ‘177’ healthy and disease-free cultivars.

For hormone treatment, solution of 100 µM abscisic acid (ABA), 200 µM ethephon (ET), 100 µM salicylic acid (SA), 100 µM indoleacetic acid (IAA), and 100 µM 2,4-Epibrassinolide (EBR) were sprayed onto ‘177’ cultivars leaves, while free-water was used as a control, Leaf samples were collected randomly at 1, 3, 6, 12, and 24 h after the hormone treatments.

Five-week-old ‘177’ cultivar plants were subjected to different abiotic stress treatments: salt stress (irrigation with 200 mM NaCl), cold stress (placement in a 4 °C growth chamber), drought stress (cessation of irrigation), and oxidation stress (irrigation with 10 μM methyl viologen, MV). For heat treatment, ‘177’ and ‘ZS149’ cultivars plants were placed in a 45 °C growth chamber. Leaf samples from plants subjected to abiotic stress treatments were collected after 1, 3, 6, 12, and 24 h of stress imposition, among them, for heat treatment, after stopping the heat treatment, transplant them to normal growth conditions (24 °C) to resume growth, and collect the leaves at 1, 3, 6, 12, and 24 h after recovery of growth.

Three biological replicates of samples were established for different tissues and each treatment used a completely randomized design. Samples were collected, immediately frozen in liquid nitrogen, and then stored in −80 °C refrigerator for subsequent experiments.

2.2. Measurement of Relative Electrolyte Leakage and Relative Water Content

Relative electrolyte leakage is an important indicator of the integrity of plant cell membranes and is typically used to evaluate plant stress tolerance [36]. Briefly, the second fully expanded leaf from the upper part of both the heat-tolerant plant ‘177’ and the heat-sensitive plant ‘ZS149’ was excised to produce leaf discs with a diameter of 8 mm. Three replicates were prepared for each line with 20 leaves per replicate. The leaf discs were placed in 50-mL centrifuge tubes containing 25 mL of distilled deionized water and shaken at 60 rpm for 12 h at 25 °C in the dark. Electrolyte leakage (R1) in the solutions was measured at 25 °C using a portable conductivity meter (DDB-303A, Shanghai, China). The solution was then boiled for 30 min and cooled to room temperature. The electrolyte leakage (R2) of the boiled solutions was then determined. The relative electrolyte leakage rate (%) was calculated as follows:

relative electrolyte leakage rate (%) = (R1/R2) × 100

Relative water content (RWC) was determined using the Yamasaki-described methods [37]. Briefly, 10 test cultivar leaves were immediately weighed to determine their fresh weight (FW) after harvesting, and immersed in distilled water for 4 h to measure their turgid weight (TW). Finally, the leaves were dried in an oven at 80 °C for 24 h to determine their dry weight (DW). The RWC was calculated as follows:

RWC (%) = [(FW − DW)/(TW − DW)] × 100

2.3. Phenotypic Identification of Heat Tolerance in Eggplant at Seedling Stage

‘177’ and ‘ZS149’ cultivars plants were placed in a 45 °C growth chamber for 7 days and after that were counted using the heat-injury index. The heat injury grading standard for eggplant plants [38]: Level 0: normal plants, leaves are not damaged by heat; Level 1: only a few leaves turn yellow; Level 2: less than half of the leaves wilt and die; Level 3: more than half of the leaves wilt and die; Level 4: all leaves wilt and die, but the main stem and growth point are not dead, and new leaves can still grow after returning to normal temperature; Level 5: plant death.

The heat-injury index was calculated as follows:

heat-injury index = 100 × (x1a1 + x2a2 + … + xnan)/nT

where x1, x2, and xn represent the number of plants at each heat damage level; a1, a2, and an represent each heat damage level; and T represents the total number of plants investigated.

2.4. Identification of GLK Genes in Eggplant Genome

High-quality eggplant genome sequence files were procured from our previous study [39]. The GLK protein sequences of Arabidopsis (AT2G20570.2 and AT5G44190.1) and rice (LOC4340977 and LOC4326363) were obtained from the TAIR database (https://www.arabidopsis.org/, accessed on 10 November 2023) and the National Center for Biotechnology Information (https://www.ncbi.nlm.nih.gov/, accessed on 10 November 2023), respectively. These sequences were then used as queries to identify GLK genes in eggplant using TBtools local_BLASTP (E-value: 1 × 10⁻⁵) [40]. The Hidden Markov Model (HMM) of PF00249 was downloaded from InterPro (https://www.ebi.ac.uk/interpro/, accessed on 10 November 2023), and HMMER3 (http://hmmer.janelia.org/, accessed on 10 November 2023) was employed to identify genes (e-value ≤ 1 × 10⁻¹⁰ as the output). The results obtained from HMMER3 and BLASTP were combined and short sequences (less than 150 amino acids in length) were removed. To confirm the identity of the genes and assess whether they contained completely conserved domains, the SMART database (http://smart.embl-heidelberg.de, accessed on 15 November 2023) and InterPro were consulted. The ProtParam web tool (http://web.expasy.org/protparam/, accessed on 16 November 2023) was used to determine the molecular weight (MW) and isoelectric point (pI) of SmGLK genes. Protein subcellular localization was predicted using the WoLF PSORT online server (https://wolfpsort.hgc.jp/, accessed on 16 November 2023).

2.5. Phylogenetic Tree Analysis

The full amino acid sequence of Arabidopsis [5] was acquired from the TAIR database. Multiple sequence alignment was performed using the ClustalW program with default settings, and phylogenetic trees were constructed using Maximum Likelihood methods and the Jones–Taylor–Thornton (JTT) matrix-based model with 1000 bootstrap replicates using MEGA 10 software [41].

2.6. Gene Structure Analysis, Motif Analysis, and Cis Regulatory Element (CREs) Analysis of GLK Genes in Eggplant

Gene structure analysis was performed using the online Gene Structure Display Server 2.0 (http://gsds.gao-lab.org/index.php, accessed on 13 November 2023). The analysis of conserved motifs in the GLKs was performed using the Expectation Maximization for Motif Elicitation (MEME) online software (version 5.5.4) (http://meme-suite.org/meme/tools/meme, accessed on 15 November 2023), and the parameters were set to a maximum of 15 motifs, while the others were set to default. InterPro was used to determine the motif annotations. The 2000 bp promoter region upstream of the genes in question was extracted using SPDE software version 9.3 [42]. Subsequently, the Plant CARE database [43] was then utilized to predict the cis-regulatory elements.

2.7. Chromosomal Location and Gene Duplication Analysis of SmGLK Genes

In the present study, all SmGLKs were mapped to their respective chromosomes, based on the physical locations of the putative genes. Tandem duplications, defined as regions containing two or more genes within 200 kb, were identified [44]. Synteny analysis of the SmGLKs gene was conducted using MCscanX in TBtools. KaKs_Calculator 2.0 [45] was employed to calculate the nonsynonymous substitution rate (Ka), synonymous substitution rate (Ks), and selection pressure (Ka/Ks) using the NY model. Ka/Ks < 1 indicated purifying selection, Ka/Ks = 1 indicated neutral selection, and Ka/Ks > 1 indicated positive selection.

2.8. RNA Isolation and Real-Time Quantitative PCR (RT-qPCR) Analysis

RNA was extracted using the OmniPlant RNA Kit (CWBIO, Beijing, China). The OD value of the RNA was determined using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), and the quality of the RNA samples was assessed by agarose gel electrophoresis. Prime ScriptRTase (Trans Gen Biotech, Beijing, China) was utilized to remove gDNA, and the UEIris II RT-PCR System for First-Strand cDNA Synthesis Kit (US Everbright^® Inc., Suzhou, China) was used to synthesize cDNA. Specific primers for qRT-PCR were designed using Primer3web (https://primer3.ut.ee/, accessed on 28 November 2023, version 4.1.0). The primer sequences are shown in Table S1. Actin (NC_012010) was used as an internal reference gene. Each qRT-PCR reaction had a total volume of 20 μL, containing 1 μL of each primer, 1 μL of cDNA template, 10 μL of 2× Fast Super EvaGreen^® qPCR Mastermix (US Everbright Inc., Suzhou, China), and 7 μL of ddH₂O. For each sample, three biological and three technical replicates were established. A LightCycler^®96 (Roche, Shanghai, China) was utilized for RT-qPCR, with the following amplification parameters: 95 °C for 120 s, followed by 45 cycles of 95 °C for 5 s and 59 °C for 30 s. The relative gene expression levels were calculated using the 2^−ΔΔCT method [46]. Heat maps were generated using TBtools.

3. Results

3.1. Identification of GLK Family Members in Eggplant

Analysis of the latest high-quality eggplant genome identified 54 GOLDEN2-LIKE transcription factor genes using a bioinformatics approach. These genes were systematically renamed SmGLK1–SmGLK54 based on their respective chromosomal locations. Furthermore, the fundamental characteristics of SmGLKs were ascertained. The protein prediction lengths of SmGLKs exhibited a range from 204 aa (SmGLK5) to 793 aa (SmGLK2), with corresponding molecular weights spanning from 22,504.46 Da (SmGLK5) to 88,500.67 Da (SmGLK2). The isoelectric points varied from 5.00 (SmGLK16) to 9.42 (SmGLK5). Detailed information on SmGLK genes, including gene ID, chromosome location, coding sequence length, and number of exons, is presented in

Table 1. Details of eggplant GLK genes.

Name	Gene ID	Chr ID	Start	End	pI	MW (Da)	CDS Length (bp)	Protein (aa)	Exons	Subcellular Localization
SmGLK1	EGP04115	chr1	68,643,071	68,646,335	6.61	60,615.06	1647	548	6	nucl
SmGLK2	EGP05116	chr1	95,471,528	95,480,561	5.56	88,500.67	2382	793	15	nucl
SmGLK3	EGP06342	chr1	108,107,510	108,110,509	7.17	44,925.73	1218	405	5	nucl
SmGLK4	EGP29953	chr2	60,788,782	60,792,579	8.88	40,016.61	1023	340	6	nucl
SmGLK5	EGP30651	chr2	69,219,561	69,221,293	9.42	22,504.46	615	204	6	nucl
SmGLK6	EGP30658	chr2	69,308,009	69,309,947	6.45	26,915.78	714	237	6	nucl
SmGLK7	EGP31411	chr2	76,116,607	76,119,195	7.16	40,238.87	1077	358	5	pero
SmGLK8	EGP12000	chr3	42,770,529	42,776,676	6.01	65,332.49	1761	586	5	nucl
SmGLK9	EGP26827	chr4	4,028,213	4,030,290	9.23	23,642.87	630	209	3	nucl
SmGLK10	EGP28551	chr4	79,927,110	79,931,297	7.02	35,226.93	942	313	6	cyto
SmGLK11	EGP24129	chr5	1,630,169	1,637,808	7.79	44,398.56	1197	398	6	nucl
SmGLK12	EGP24145	chr5	1,804,572	1,808,269	7.79	33,260.82	870	289	4	nucl
SmGLK13	EGP24440	chr5	5,613,154	5,620,386	5.69	72,812.13	1977	658	6	nucl
SmGLK14	EGP24597	chr5	7,563,196	7,569,245	5.60	45,814.18	1248	415	7	nucl
SmGLK15	EGP26270	chr5	83,294,033	83,298,987	6.37	32,857.76	897	298	6	nucl
SmGLK16	EGP26358	chr5	84,224,489	84,227,940	5.00	44,165.10	1179	392	7	nucl
SmGLK17	EGP15888	chr6	2,113,619	2,124,522	5.13	50,749.64	1395	464	7	nucl
SmGLK18	EGP15695	chr6	5,839,487	5,841,423	5.91	34,094.85	921	306	1	nucl
SmGLK19	EGP15014	chr6	54,881,706	54,883,465	7.15	40,025.95	1047	348	5	nucl
SmGLK20	EGP14289	chr6	81,275,437	81,280,149	6.21	61,361.55	1656	551	11	nucl
SmGLK21	EGP14253	chr6	81,787,799	81,793,799	9.15	43,622.27	1167	388	6	nucl
SmGLK22	EGP14239	chr6	82,118,616	82,120,799	8.48	28,372.73	765	254	6	nucl
SmGLK23	EGP14133	chr6	83,555,681	83,563,561	6.31	35,907.49	999	332	6	nucl
SmGLK24	EGP13249	chr6	91,911,599	91,914,246	5.47	35,110.80	939	312	1	nucl
SmGLK25	EGP02563	chr7	2,910,686	2,916,581	6.24	75,391.20	2064	687	6	nucl
SmGLK26	EGP01139	chr7	93,632,358	93,637,356	6.83	25,491.09	669	222	5	mito
SmGLK27	EGP00675	chr7	104,675,122	104,680,177	6.08	48,777.46	1341	446	6	nucl
SmGLK28	EGP20881	chr8	3,370,310	3,376,379	6.93	40,606.18	1086	361	6	nucl
SmGLK29	EGP19297	chr8	82,506,447	82,509,595	6.98	31,661.35	852	283	6	nucl
SmGLK30	EGP19254	chr8	83,141,169	83,147,055	9.24	32,473.04	864	287	4	nucl
SmGLK31	EGP19168	chr8	84,417,636	84,423,522	6.16	62,160.30	1674	557	11	nucl
SmGLK32	EGP18241	chr9	4,886,189	4,898,278	5.64	54,731.15	1449	482	5	nucl
SmGLK33	EGP18238	chr9	4,957,579	4,961,351	6.05	49,000.02	1287	428	4	nucl
SmGLK34	EGP18065	chr9	7,912,729	7,919,511	8.36	59,083.64	1584	527	5	E.R
SmGLK35	EGP17937	chr9	12,229,872	12,232,841	7.17	26,882.06	702	233	6	nucl
SmGLK36	EGP17858	chr9	16,429,096	16,431,149	8.36	35,911.84	969	322	5	nucl
SmGLK37	EGP16748	chr9	84,207,659	84,216,589	5.04	48,823.09	1338	445	7	nucl
SmGLK38	EGP16179	chr9	91,964,731	91,969,446	5.86	31,901.78	864	287	6	nucl
SmGLK39	EGP21185	chr10	331,005	332,779	6.48	32,238.59	882	293	1	nucl
SmGLK40	EGP21245	chr10	819,985	822,961	6.11	45,635.78	1212	403	7	nucl
SmGLK41	EGP21361	chr10	1,736,637	1,742,936	5.80	44,354.21	1173	390	7	nucl
SmGLK42	EGP21794	chr10	6,478,648	6,481,704	9.00	55,889.28	1482	493	7	nucl
SmGLK43	EGP22032	chr10	13,141,129	13,143,574	6.56	38,290.57	1026	341	5	nucl
SmGLK44	EGP22321	chr10	18,625,031	18,626,906	8.99	33,405.03	888	295	5	nucl
SmGLK45	EGP23367	chr10	77,444,097	77,449,402	6.93	42,472.29	1149	382	8	nucl
SmGLK46	EGP23752	chr10	85,369,460	85,375,848	5.91	77,539.63	2121	706	6	nucl
SmGLK47	EGP23800	chr10	85,902,577	85,905,785	6.84	42,768.62	1170	389	5	nucl
SmGLK48	EGP06828	chr11	1,453,235	1,460,575	7.90	51,124.93	1374	457	6	nucl
SmGLK49	EGP07802	chr11	29,542,927	29,557,840	5.82	51,410.97	1407	468	7	nucl
SmGLK50	EGP08969	chr11	97,459,423	97,468,863	6.30	70,201.99	1944	647	5	nucl
SmGLK51	EGP09012	chr11	98,353,223	98,358,334	6.57	34,346.06	897	298	6	nucl
SmGLK52	EGP09258	chr11	101,893,426	101,898,259	6.20	34,847.76	960	319	6	nucl
SmGLK53	EGP33729	chr12	1,379,693	1,382,695	6.85	49,594.35	1341	446	4	nucl
SmGLK54	EGP33116	chr12	21,405,470	21,415,883	7.70	71,609.34	1935	644	11	cyto_nucl

.

3.2. Evolutionary and Structural Analysis

To understand the evolutionary relationship of SmGLKs, a phylogenetic tree based on the protein sequences of Arabidopsis and eggplant was constructed using Maximum Likelihood methods (Figure 1). The results revealed that the 54 SmGLK proteins in Arabidopsis and eggplant could be classified into seven subgroups (14 in G1, 4 in G2, 17 in G3, 3 in G4, 5 in G5, 4 in G6, and 7 in G7).

To further understand the evolutionary process of SmGLK genes, a singular phylogenetic tree, exon-intron distribution patterns, and motif distribution patterns were analyzed (Figure 2). As indicated by the results, the phylogenetic trees were divided into seven subgroups. Each subgroup contained a varying number of exons, whereas some subgroups had identical exon counts. For instance, G1, G2, G4, and G7 contained 4–6, 4–5, 4–5, and 5–7, respectively, suggesting a higher level of conservation in the evolutionary process for these subgroups. The G6 subgroup, except for SmGLK2, contained only one exon, whereas the G3 subgroup, except for SmGLK54, only contained 6–8 introns, indicating a certain degree of conservation in the evolutionary process.

In the analysis of motif distribution patterns, all SmGLK proteins contain motif1 and motif2, defined as the Myb_DNA-binding domain of a typical GLK protein. Subgroups G1 and G2 contained only contain motif1 and motif2. Additionally, the G7 subgroup contained motifs 4 and 5 (both defined as the Response_reg domain), the G3 subgroup mostly contained motif3 (defined as the Myb_CC_LHEQLE domain), and the G4 subgroup contained all the motifs defined as functional. Interestingly, the G5 subgroup exhibited distinct exon-intron and motif distribution patterns. For instance, it contains 3–11 exons and two types of functional motifs (Myb_DNA-binding domain and Response_reg domain). These results suggest that G5 subgroup members underwent more pronounced changes during the evolutionary process than the other subgroups.

3.3. Chromosome Distribution and Duplication Events of SmGLK Genes

Based on the eggplant genome annotations, the chromosomal locations of all SmGLK genes were identified and mapped onto individual eggplant chromosomes (Figure 3). The 54 SmGLK genes exhibited an uneven distribution across 12 chromosomes, with chromosome 10 containing the highest number of SmGLK genes (9). In the collinearity analysis, 16 gene pairs were identified as segmental duplications, and three gene pairs were identified as tandem duplications (Figure 4 and Table S2). Chromosome 11 did not show any SmGLK duplications. Interestingly, 26 of the 54 SmGLK genes were associated with segmental duplications, indicating a significant role for segmental duplication in the expansion of the SmGLK family. Further Ka and Ks analyses showed that 18 collinear gene pairs underwent purifying selection (Ka/Ks ratios ranged from 0.13 to 0.83), and one collinear gene pair (SmGLK5 and SmGLK6) underwent positive selection (Ka/Ks ratios were 2.97).

To delve deeper into the evolutionary relationships of the SmGLK family, a synteny analysis between eggplant and Arabidopsis was conducted and mapped (Figure 5). Forty orthologous gene pairs have been identified between eggplant and Arabidopsis. Interestingly, 10 SmGLK genes (SmGLK1, SmGLK16, SmGLK17, SmGLK18, SmGLK24, SmGLK27, SmGLK30, SmGLK44, SmGLK47, and SmGLK51) were paired with two or more Arabidopsis genes (Table S3). Based on these results, it can be inferred that these 10 SmGLK genes may have undergone a higher rate of mutation, indicating an adaptation to environmental changes.

3.4. Promoter Analysis of SmGLK Genes

The composition and types of cis-acting elements in gene promoters can potentially aid in determining gene function. The 2000 bp upstream of SmGLK genes’ start site (ATG) was analyzed. As illustrated in Figure 6, the 19 cis-acting elements in SmGLK gene promoters can be categorized into four groups: hormone-responsive, light-responsive, stress-responsive, and participation in plant development. All gene promoters were predicted to contain distinct cis-acting elements, except for SmGLK26.

Eight hormonal regulatory elements, including the CGTCA-motif, TGACG-motif, ABRE, TGA-element, AuxRR-core, TCA-element, P-box, and TATC-box, were identified in the promoter regions of SmGLKs. A significant number of ABRE elements associated with abscisic acid responsiveness were widely distributed within the SmGLKs genes. The SmGLK29 gene, in particular, contained the maximum number (nine) of ABRE elements, suggesting that SmGLK29 may participate in the abscisic acid signaling pathway. Additionally, stress regulation-related elements, including defense and stress (TC-rich repeats), anaerobic (ARE), drought (MBS), and cold (LTR), were also identified. The SmGLK42 gene exhibited the highest number (seven) of stress regulation-related elements, indicating that the SmGLK42 gene potential role in stress response. Light regulation (ACE and G-box elements) and plant development (GCN4_motif, CAT-box, RY-element, and circadian and O2-site elements) (Table S4) were also detected.

3.5. Expression Pattern of SmGLK Genes across Different Developmental Stages in Different Eggplant Tissues

The expression patterns of selected 26 SmGLK genes during different developmental stages in various eggplant tissues (roots, stems, leaves, buds, flowers, fruits, and seeds) were analyzed by RT-qPCR. As shown (Figure 7), compared to the expression levels in other tissues, SmGLK genes exhibited a wide range of high expression in leaves, particularly in mature and older leaves, while displaying lower expression levels in mature fruits. In contrast to their expression levels in the roots, SmGLK28 and SmGLK31/SmGLK38 demonstrated entirely divergent expression trends across all tissues. Some SmGLK genes exhibited tissue-specific expression profiles. For example, SmGLK12 is highly expressed in mature and older leaves, SmGLK29 is highly expressed in breaker-stage fruits and seeds, and SmGLK52 is highly expressed in older stems.

3.6. Expression Patterns of SmGLK Genes in Response to Hormone Treatments

The transcriptional response of genes to hormonal stimuli often signifies the underlying biological effects. This study verified the response of SmGLKs to hormone treatments (including abscisic acid (ABA), epibrassinolide (EBR), ethylene (ET), indoleacetic acid (IAA), and salicylic acid (SA)) by RT-qPCR. The results are presented in Figure 8. Overall, most SmGLK genes responded to different hormone treatments and showed an upregulated expression. Notably, SmGLK29 demonstrated rapid response to hormone treatment, with a significant increase in expression after 1 h of treatment, followed by subsequent downregulation over time. Conversely, SmGLK18 displayed hysteresis in response to hormone treatment, exhibiting pronounced upregulation of expression at 12 h post-treatment, which returned to relatively low expression levels at 24 h. Furthermore, certain genes maintained elevated expression levels following hormone treatment. For instance, SmGLK12/SmGLK47 expression was influenced by ABA treatment, SmGLK17 expression by EBR treatment, SmGLK23 expression by ET treatment, SmGLK25 expression by IAA treatment, and SmGLK39 expression by SA treatment.

3.7. Expression Patterns of SmGLK Genes in Response to Abiotic Stress Treatment

To investigate the potential role of the SmGLK genes, RT-qPCR was employed to assess its expression in response to cold, drought, MV, and salt stress (Figure 9). Cold stress revealed that the expression of most SmGLK genes was repressed by cold stress, whereas salt stress elicited an overall upregulation in SmGLK gene expression. Specifically, SmGLK3, SmGLK18, and SmGLK47 were significantly upregulated after 12 h of cold stress. Conversely, under drought stress, SmGLK12 and SmGLK18 were upregulated post-treatment, with pronounced increases at 12 and 24 h after treatment, whereas SmGLK11, SmGLK28, SmGLK38, and SmGLK48 were downregulated at all time points. Following MV treatment, SmGLK29 displayed a rapid response with expression gradually decreasing over time, whereas SmGLK8 exhibited rapid downregulation, followed by a gradual return to baseline expression levels.

To ensure differences in heat tolerance among the tested plants, we analyzed the variations in phenotypic and physiological indices. The results are presented in Figure S1. Compared with the heat-sensitive cultivars ‘ZS149’, the heat-tolerant cultivars ‘177’ did not exhibit wilting symptoms after 7 days of heat treatment (Figure S1a–f), and stress-resistance related indexes (Figure S1e,g) in cultivars ‘177’ were significantly better than those in ‘ZS149’. These results confirm the reliability of the subsequent tests.

To gain a comprehensive understanding of the SmGLK gene response to high-temperature treatment, two distinct genotypes were chosen: the heat-tolerant variety ‘177’ and the heat-sensitive variety ‘ZS149’. The genotypes were subjected to heat treatment at 45 °C. After 24 h of treatment, the plants were returned to their normal growth temperature to resume growth. Some SmGLK genes with similar expression trends were found in different genotypes, as shown in Figure 10. During heat treatment, the expressions of SmGLK3, SmGLK8, SmGLK18, and SmGLK52 were upregulated. Notably, SmGLK18 exhibited a particularly pronounced increase in expression amplitude compared to the others. Furthermore, its expression levels remained consistently elevated during the subsequent recovery period, indicating a robust and sustained response, suggesting that SmGLK18 plays a significant role in enhancing heat resistance. The expression patterns of certain genes in the heat-tolerant varieties exhibited an inverse trend compared to those observed in the heat-sensitive varieties, as exemplified by SmGLK29 and SmGLK48. At normal growth temperatures, certain genes exhibit slight upregulation in heat-tolerant varieties or return to baseline expression levels, whereas they demonstrate consistent downregulation in heat-sensitive varieties, as exemplified by SmGLK11, SmGLK28, and SmGLK43. The aforementioned differentially expressed genes could potentially contribute to the varietal disparities observed in heat tolerance.

4. Discussion

With the advent of next-generation sequencing (NGS) technology, genome assembly is becoming increasingly popular in plants, and assembling high-quality genome data will be highly beneficial for researchers studying specific species. As reported in previous studies, GOLDEN2-LIKE transcription factors play important roles in plant growth and development, particularly in chloroplasts and photosynthesis [1,13,17,47]. Subsequently, GLK has been characterized in several important plant species [5,8,20,21,23,29]. Eggplant holds a significant position among Solanaceae crops, ranking third in both total production and economic value within the genus [39], and often suffers from adverse environmental stress. However, systematic genome-wide identification and analysis of GLK in eggplants are limited. In this study, based on the latest high-quality genome assembly of eggplant [39], we identified 54 full-length GLK coding sequences in eggplant (S. melongena L.) genome (Table 1), renamed based on the chromosomal location, and further analyzed their evolutionary relationships, gene structures, conserved motifs, chromosome locations, cis-acting elements, duplication events, tissue-specific expression, hormone responses, and expression patterns under abiotic stress.

Although eggplant has a higher chromosome count (12) than Arabidopsis (5), the number of GLK genes appeared to be comparable between the two species. This observation indicated that the number of GLK genes did not show an apparent relationship with genome size. Based on phylogenetic relationships and conserved motif analysis, the identified 54 SmGLK can be classified into seven groups (G1–G7), which is consistent with previous reports on Arabidopsis [5] and maize [6]. Among these groups, G3 contained the most SmGLK genes (17), whereas G4 contained the fewest (3). Groups G1, G2, G4, and G7 exhibited conversed motif distribution patterns and exon-intron structures, suggesting that these groups were conserved during the evolutionary process. In contrast, other groups (G3, G5, and G6) displayed variations in exon–intron numbers and conversed motif distribution patterns, indicating the emergence of diversity during evolution. Interestingly, members of the G6 group, such as SmGLK18, SmGLK24 and SmGLK39, lack introns, a feature also observed in tobacco (Nicotiana tabacum L.) [48], wheat (Triticum aestivum L.) [10], and tomato (Solanum lycopersicum L.) [9], suggesting that these genes originated from more recent ancestors and possess similar functions or might be derived from retrotransposition events. Furthermore, intronless genes are believed to evolve rapidly through processes such as duplication or reverse transcription [49].

Gene duplication events are ubiquitous biological phenomena that play a pivotal role in the expansion of gene families and serve as critical mechanisms for neofunctionalization and functional divergence during evolution [50,51]. In this study, collinearity analysis revealed 16 pairs of segmental duplications and 3 pairs of tandem duplications, and similar results were reported by other studies in tomatoes [9]. Notably, 32 of the 54 SmGLK genes with an uneven distribution across eggplant chromosomes were found to be involved in duplication events. (Table S3). In general, paralogous genes may exhibit sequence and structural similarities, but their functions may have undergone distinct differentiation. SmGLK3/7, SmGLK5/6, SmGLK15/38, SmGLK18/24, SmGLK17/37, SmGLK20/31, SmGLK21/30, SmGLK40/41, and SmGLK46/25 exhibited similarities in exon–intron numbers and conserved motif distribution patterns. In contrast, SmGLK1/16, SmGLK9/27, SmGLK11/12, SmGLK13/16, SmGLK13/24, SmGLK28/30, SmGLK32/33, and SmGLK54/23 exhibited diversity in exon–intron numbers and conversed motif distribution patterns. It appears that the loss and insertion of new introns are frequent events that play significant roles in gene evolution [48]. However, the determination of functional similarities or differences requires further testing. Furthermore, Ka/Ks analysis revealed that all SmGLK duplication gene pairs, except SmGLK5/6, were under purifying selection. This suggests that gene duplication events were subject to robust purifying constraints during evolution, underscoring the impact of such constraints on duplication events. Synteny analysis revealed 40 orthologous gene pairs in Arabidopsis and eggplant (Table S3). In particular, members of the G4 group showed no orthologous genes in Arabidopsis, suggesting divergence between Arabidopsis and eggplant.

Previous studies demonstrated that the GLK gene is associated with chloroplast development and plays a regulatory role in chloroplast biogenesis [11,15]. In this study, tissue-specific expression levels were detected. Compared with other tissues, the 26 selected SmGLK genes exhibited predominantly high expression levels in the leaves, spanning the three leaf developmental stages. This is consistent with previous findings in moso bamboo (Phyllostachys edulis) [7]. Additionally, our experiment identified significant expression of SmGLK genes in flowers and seeds. Phytohormones, including auxins and brassinosteroids, have been implicated in fruit chloroplast development and upregulation of SLGLK expression [17]. Our analysis revealed that certain SmGLK genes (SmGLK2 and SmGLK25) were also upregulated by IAA and EBR treatments. Furthermore, SmGLK31 and SmGLK38 displayed high expression levels across all the tested materials, suggesting their significant roles in eggplant growth and development.

Gene expression patterns in plants subjected to various stressors offer valuable insights for gene function analysis. In this study, the expression profiles of eggplant GLK genes under drought, low temperature, oxidation, salt, and heat stress were analyzed using RT-qPCR. The results showed that the expression pattern of the SmGLK gene under cold stress differed markedly from its response to other stressors, as it exhibited comprehensive inhibition. This finding was corroborated by Liu et al.’s experiments on maize [6], suggesting a relatively special response mechanism for GLK genes to low temperatures. Previous studies have shown that the expression of GLK genes (ZmGLK3 and SlGLK7) is upregulated under drought and salt stress conditions in maize [6] and tomato [9]. Our experiments demonstrated that SmGLK18 exhibits significant expression levels under drought and salt stress. Additionally, under ABA treatment, which is closely associated with drought stress, SmGLK18 displayed substantial upregulation of expression at 12 h post-treatment. This suggests a potential role for SmGLK18 in enhancing plant drought tolerance via the ABA signaling pathway. Our study presents, for the first time, the expression pattern of the GLK gene family under oxidative stress, in which most SmGLK genes are induced/repressed. SmGLK29 exhibited a rapid response to treatment, suggesting its potential role in oxidative stress resistance. For the heat treatment, a comprehensive expression analysis was conducted. Broadly speaking, the SmGLK gene exhibits a tendency for upregulation in the heat-tolerant variety ‘177’, with continuous upregulation observed during the recovery period. Conversely, some down-regulated expression trends are evident in the sensitive variety ‘ZS149’, among which SmGLK18 was notably induced by heat treatment in both varieties. Taken together, SmGLK18 plays a pivotal role in abiotic stress tolerance, including heat tolerance; however, the specific mechanism of action requires further experimental validation.

5. Conclusions

In this study, 54 GOLDEN2-like transcription factors in the eggplant genome were characterized and classified into seven groups based on their distinct structures and motif compositions. These genes were unevenly distributed across the 12 eggplant chromosomes, with 32 SmGLKs participating in gene duplication. Tissue-specific expression analysis revealed that most SmGLK genes were highly expressed in the leaves compared to other tissues, and SmGLK38 may play a significant role in eggplant growth and development. The expression patterns of SmGLK genes under different treatments provided insights into their functions. Consequently, SmGLK18 was selected for further investigation into its role in abiotic stress responses.