Genome-Wide Identification of CaGA20ox Gene Family Members Related to Floral Organ Development in Pepper (Capsicum annuum) at Different Temperatures

Abstract

Environmental temperature significantly affects plant growth and development, particularly flower development. In pepper (Capsicum annuum), the molecular mechanisms underlying temperature-mediated floral organ development remain unclear. Gibberellins (GAs) are key plant hormones regulating growth and development, including flower development, and the CaGA20ox gene family may play a crucial role in this process due to its involvement in GA biosynthesis. In this study, we comprehensively analyzed the CaGA20ox gene family across six pepper genomes (‘Zhangshugang’, ‘Zunla’, ‘Chiltepin’, ‘CM334’, ‘Ca59’, and ‘T2T’) to explore their roles in flower development and temperature stress response, identifying five to six genes per genome. These genes exhibited distinct expression patterns across different tissues and developmental stages, with some members showing higher expression in specific floral organs, particularly pistils. Our results revealed that temperature significantly impacts pepper flower development and GA content, with lower temperatures enhancing antioxidant capacity and increasing GA levels. Specifically, the expression levels of four CazGA20ox genes (CazGA20ox1, CazGA20ox2, CazGA20ox4, and CazGA20ox6) were significantly influenced by temperature changes. Our systematic analysis of the role of the CaGA20ox gene family in temperature-mediated pepper flower development provides a foundation for further studies on the molecular mechanisms as well as the development of improved pepper varieties.

1. Introduction

Flower development is a critically important developmental event in higher plants, marking the transition from vegetative to reproductive growth. Moreover, temperature-induced disturbances to the flower development process can severely impact yield [1,2]. The regulatory network underlying flower development is not limited to floral morphogenesis, but also impacts fruit ripening and seed development [3]. The molecular genetic mechanism underlying flower development has been studied in detail in model species such as Arabidopsis thaliana [4,5,6]. However, less is known about flower development in many important crop plants such as peppers. Tang et al. [7] analyzed the gene expression profiles of peppers and identified 17 transcription factors (TFs) involved in flower development, including CaAP1, CaAP2, CaAP3, CaAG, and CaSTK. In peppers, exogenous gibberellic acid (GA) alters the expression patterns of GA metabolism and response genes (e.g., CaGA2ox1, CaGA20ox1, and CaGID1b), thereby increasing the thickness, cell size, and cell number of the ovary wall; the size and number of the inner ovary layers; and the overall fruit size [8].

GAs are a particularly important class of phytohormones that play diverse roles in both plant development and environmental stress responses [9,10,11,12]. Additionally, GAs play a significant role in flower development and fertility. In A. thaliana, the gibberellin (GA)-induced degradation of DELLA proteins, such as RGA, RGL1, and RGL2, is a critical regulatory mechanism that promotes the formation of petals and stamens. This process involves GA-mediated interaction with the GID1 receptor, leading to the ubiquitination and subsequent proteasomal degradation of DELLA proteins. By relieving the growth-repressive effects of DELLA proteins, GA facilitates the elongation of stamen filaments and the progression of microsporogenesis, ultimately contributing to the development of mature pollen grains and functional floral organs [13]. Both the A. thaliana GA biosynthesis mutant ga1-3 and the rice GA receptor mutant gid1 are sterile [14,15]. In many cases, GAs act in concert with other phytohormones to regulate plant growth and development. For example, GA induces jasmonic acid (JA) biosynthesis, thereby enhancing the expression of several MYB TFs (e.g., MYB21, MYB24, and MYB57) and enabling proper stamen formation and male fertility development [16]. Notably, the effects of GA on plant growth and development are often linked to stress responses. In rice, chilling represses the expression of GA20ox3 and GA3ox1, thereby increasing the expression of the SLR1 DELLA protein, decreasing endogenous GA production, hindering pollen production, and ultimately resulting in sterility [17]. Exogenous GA₃ has been found to both improve chilling tolerance and regulate anther and pollen development [18]. In Brassica campestris, exposure to high temperatures induces male sterility by disrupting the GA signaling pathway [19].

The biosynthesis of GAs is catalyzed by a series of enzymes. Among these, GA20ox and GA3ox are key enzymes for the final step in the conversion of GA₁₂ and GA₅₃ into bioactive GA₁ and GA₄, respectively [20]. In contrast, GA2oxs inactivate GAs and their precursors, thus modulating GA activity [21]. The knockout of GA20ox or GA3ox and the overexpression of GA2ox results in dwarfing [22,23,24,25]. Notably, the well-known Green Revolution Gene sd1 was generated from the loss of function in OsGA20ox2, and has been critically important in enhancing rice production [26]. In A. thaliana, GA20ox1 and GA20ox2 loss-of-function mutants exhibit partial functional redundancy, and the expression profiles of both genes partially overlap throughout the developmental cycle [27]. Similarly, Plackett et al. [28] found that AtGA20ox1, AtGA20ox2, and AtGA20ox3 were largely redundant during most developmental stages, including during floral transition. In addition, their absence results in extreme dwarfism and an almost complete loss of fertility. These results indicate that the GA20ox gene family plays an important role in the regulation of plant growth and development. In addition, their direct involvement in key processes contributing to crop yield and quality make genes in this family a critical research priority.

Peppers, annual or short-lived perennial plants in the genus Capsicum (Solanaceae), are important cash crops. The growth and development of the pepper industry has resulted in changes to both the cultivation mode and planting habits of pepper plants. In addition, the main production area has changed according to the growing market demand, resulting in the optimization of the variety structure and planting mode to highlight regional characteristics. Unfortunately, climate change is beginning to seriously affect pepper production, leading to slower vegetative growth, impaired photosynthesis, abnormal flowering and reproductive development, and significantly reduced yield and/or quality. Sub-optimal temperatures can disrupt the plant hormone balance and affect reproductive processes [17,18,19]. Given the importance of GAs in plant development and the potential impact of temperature on GA biosynthesis and flower development, it is imperative to comprehensively study the intertwined effects of temperature and phytohormone dynamics on floral organ development in pepper.

To explore the roles of moderate, non-stressful temperatures (28 °C and 18 °C) and the CaGA20ox gene family in pepper floral organ development, we first identified the CaGA20ox genes across six pepper genomes, including ‘Zhangshugang’, ‘Zunla’, ‘Chiltepin’, ‘CM334’, ‘Ca59’, and ‘T2T’. We then analyzed the physicochemical properties, sequence structures, phylogenies, and tissue-specific expression levels of the identified CaGA20ox genes using bioinformatics techniques. Furthermore, considering that temperature is a crucial environmental factor that can influence plant developmental processes, including flowering, we aimed to elucidate the impact of temperature on flower development and the expression patterns of the CaGA20ox gene family. Thus, we conducted transcriptome sequencing of pepper flower samples grown at 28 °C and 18 °C. Our results revealed that CaGA20oxs were found to possess extensive biological functions in regulating flower development. These findings provide a foundation for the continued functional characterization of CaGA20ox genes, as well as a theoretical basis for pepper breeding and variety identification.

2. Materials and Methods

2.1. Plant Materials and Growth Conditions

This research was conducted using pepper cultivar ‘Zhangshugang’ (S8), chosen for its wide cultivation, economic significance in our study region, typical pepper-growing conditions, and to ensure practical applicability and maintain experimental consistency. Pepper plants were grown in an artificial growth chamber at Hunan Agricultural University (Changsha, China). The “high temperature” growth condition consisted of an 8000 lx light intensity, 16 h/8 h light/dark cycle, 28 °C/28 °C day/night temperature, and 65% humidity. The “low temperature” growth condition consisted of an 8000 lx light intensity, 16 h/8 h light/dark cycle, 18 °C/18 °C day/night temperature, and 65% humidity. Tobacco (Nicotiana benthamiana) was employed as a model plant for in vitro validation experiments. Pest control and water management were carried out following the standard protocols.

2.2. Identification and Characterization of CaGA20ox Genes

The ‘Zunla’ (v2.0, GCA_000710875.1) [29], ‘Chiltepin’ (v2.0, GCA 000950795.1) [29], and ‘CM334’ (GCA_000512255.2) [30] whole-genome sequences were downloaded from the National Center for Biotechnology Information (NCBI, https://www.ncbi.nlm.nih.gov/ (accessed on 13 January 2025)). The ‘Zhangshugang’ genome was downloaded from the Pepper Genomics Database (http://ted.bti.cornell.edu/cgi-bin/pepper/index (accessed on 13 January 2025)) [31]. The ‘Ca59’ genome (CNP0001129) was downloaded from the CNGB Sequence Archive (CNSA) of the China National GeneBank Database (CNGBdb) (https://db.cngb.org/search/project/CNP0001129/ (accessed on 13 January 2025)) [32]. The gap-free ‘T2T’ genome and its associated annotation files were downloaded from the Capsicum Genome Database (http://www.pepperbase.site/node/3/ (accessed on 13 January 2025)) [33].

The Pfam models (2OG-FeII_Oxy [PF03171] and DIOX_N [PF14226]) were downloaded from the Pfam database (http://pfam-legacy.xfam.org/ (accessed on 13 January 2025)) [34]. The HMMER package was used for whole-genomic and proteomic alignments. Verification was performed using the HMMER web server (https://www.ebi.ac.uk/Tools/hmmer/ (accessed on 13 January 2025)) [35] with a cutoff value set to 0.01, as well as the NCBI Conserved Domain Database (CDD, https://www.ncbi.nlm.nih.gov/cdd/ (accessed on 13 January 2025)), with the E-value parameter set to 0.01 and Simple Modular Architecture Research Tool (SMART, http://smart.embl.de (accessed on 13 January 2025)). AtGA20ox genes were obtained from the A. thaliana reference genome (https://www.arabidopsis.org/ (accessed on 13 January 2025)).

2.3. Phylogenetic, Gene Structure, Conserved Motif, and Cis-Acting Element Analyses

The phylogenetic tree was constructed based on the amino acid sequences of pepper and A. thaliana GA20ox proteins. The gene accession numbers are listed in Supplementary Table S3. MEGA X was used to predict the best evolution model (JTT + G + I) to build the phylogenetic tree using 1000 bootstrap reiterations [36]. The phylogenetic tree was visualized using the Interactive Tree of Life online tool (https://itol.embl.de/ (accessed on 15 January 2025)) [37].

The CaGA20ox gene structures were analyzed using TBtools-II V2.210 [38]. Conserved motifs were analyzed using MEME (https://meme-suite.org/meme/ (accessed on 15 January 2025)) [39] and visualized using TBtools-II V2.210 [38]. The 2000 bp upstream sequences of the GA20ox genes were extracted using TBtools [38], analyzed using Plantcare [40], and visualized using TBtools-II V2.210 [38]. The physicochemical properties of each gene, including the isoelectric point (pI), molecular weight (MW), and amino acid (aa) number, were analyzed using ExPASy (https://www.expasy.org/ (accessed on 15 January 2025)) [41]. Protein subcellular localization predictions were conducted using WoLF PSORT (https://wolfpsort.hgc.jp/ (accessed on 15 January 2025)).

2.4. Chromosome Mapping and Synteny Analysis

The CaGA20ox gene structure files were extracted using TBtools-II V2.210 to determine their chromosomal starting and ending positions [38]. The protein database was constructed using the ‘Zhangshugang’, ‘Zunla’, ‘Chiltepin’, ‘CM334’, ‘Ca59’, and ‘T2T’ genomic protein files. Synteny analysis was performed using MCScanX (V1.0) [42]. The chromosomal map and synteny results were visualized using TBtools-II V2.210 [38].

2.5. Characterization of CauGA20oxs Gene Expression in Pepper

Using ‘Zunla-1’ as the reference genome, the gene expression data were obtained from the PepperHub (http://lifenglab.hzau.edu.cn/PepperHub/index.php (accessed on 17 January 2025)) [43], and the FPKM were normalized (log2(FPKM+1)). Experimental treatment and data analysis were conducted as described in Liu et al. [43]. Briefly, 40-day-old pepper seedlings were subjected to various stress treatments, including NaCl (200 mM), D-mannitol (400 mM), H₂O₂ (30 mM), heat (42 °C), and cold (10 °C). For hormone treatments, seedlings were treated with 2 μM IAA, 2 μM GA3, 30 μM ABA, 2 mM SA, and 10 μM JA. Leaf and root tissues were collected at 0, 0.5, 1, 3, 6, 12, and 24 h post-treatment. All samples were collected in quadruplicate and frozen in liquid nitrogen for RNA extraction. For spatiotemporal expression analysis, plants were grown in a natural greenhouse with a temperature of 25–29 °C during the day and 16–20 °C at night. Fruits were collected at various developmental stages, and floral buds were sampled at different sizes. Leaf samples were collected at 2, 5, 10, 15, 20, 25, 30, 40, 50, and 60 days after emergence. Gene expression was visualized using TBtools-II V2.210 [38].

2.6. RNA Extraction and cDNA Synthesis

Flower buds were collected at six different stages according to their size (Figure 5B,D). Sepals (S), petals (P), stamens (STA), and ovaries (O) of S8 flowers were sampled at the blooming stage for tissue-specific expression analysis. The total RNA was isolated using a FastPure Plant Total RNA Isolation Kit (Vazyme Biotech Co., Ltd, Nanjing, China), following the manufacturer’s guidelines. Three biological replicates were maintained per time point. The RNA integrity was evaluated using agarose gel electrophoresis. RNA samples with bright and clear 28S and 18S bands, and for which the 28S band was twice as bright as the 18S band, were qualified. RNA was reverse transcribed into cDNA with a HiScript II 1st Strand cDNA Synthesis Kit (+gDNA wiper) (Vazyme Biotech Co., Ltd., Nanjing, China).

2.7. RNA-Seq Analysis

The total RNA was extracted from F4-stage ‘Zhangshugang’ flowers using TRlzol Reagent (Life Technologies, Carlsbad, CA, USA). The RNA integrity and concentration were checked using an Agilent 2100 Bioanalyzer (Agilent Technologies, Inc., Santa Clara, CA, USA). The cDNA library was constructed using a NEBNext Ultra RNA Library Prep Kit for Illumina (E7530) and NEBNext Multiplex Oligos for Illumina (E7500; New England Biolabs, Inc., Ipswich, MA, USA) following the manufacturer’s instructions. Finally, the constructed cDNA libraries were paired-end sequenced on an Illumina HiSeq™ X-Ten platform (BGI, Shenzhen, China), with three biological replicates for each sample. Low-quality reads were removed using a Perl script and clean reads were mapped to the ‘Zhangshugang’ genome using Tophat2 (V2.1.1) with the parameters --dta-p 6 and --max-intronlen 5000000 [44]. The gene expression levels were estimated from FPKM (fragments per kilobase of exon per million fragments mapped) values using Cufflinks (V2.1.1) [45]. DESeq2 V1.30.1 [46] was used to evaluate differential gene expression according to the |log2fold change (FC)| ≥ 2 and FDR < 0.01 criteria. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed using the clusterProfiler (v3.12) package in R [47].

2.8. Real-Time Fluorescence Quantitative PCR (qPCR) Analysis of Gene Expression

Gene expression was quantified via qPCR at different temperatures (28 °C and 18 °C) and developmental stages (F1-F6). Primers were designed using the Real Time PCR Primer Design Tool (https://www.genscript.com/tools/real-time-pcr-taqman-primer-design-tool (accessed on 23 January 2025)). All primers are listed in Supplementary Table S5. qPCR was performed on a LightCycler 96 Real-Time PCR System (Roche, Basel, Switzerland) using ChamQ Universal SYBR qPCR Master Mix (Vazyme Biotech Co., Ltd., Nanjing, China) in a 20 μL reaction volume. CaUBI-1 (Caz04g03620) was selected as the reference gene [48]. Three biological replicates and three technical replicates were utilized for each experiment. The 2^−ΔΔCt method [49] was used to calculate the relative expression level of each gene.

2.9. Pearson Correlation Coefficient Analysis of GA Metabolism Pathway and Flower Development Genes

The Pearson correlation coefficient (r) method was used to calculate the correlation between GA metabolism pathway genes and flower development-related genes in the “HT-S8_F4 vs. LT-S8_F4” transcriptome sequencing data. The larger the absolute value of r, the stronger the correlation. The data were visualized using ChiPlot (https://www.chiplot.online/ (accessed on 29 January 2025)).

2.10. Pollen Viability and Germination Assays

Pollen grains were collected from S8 flowers at the anthesis stage from pepper plants grown at 28 °C and 18 °C and immersed in potassium iodide (KI) staining solution (Beijing Coolaber Technology Co., Ltd., Beijing, China) for five minutes to evaluate their viability using a light microscope (IX71; Olympus, Tokyo, Japan). The in vitro germination medium contained 5% PEG-6000, 10% sucrose, 0.01% boric acid, and 0.04% calcium chloride. All reagents were purchased from Shanghai Macklin Biochemical Technology Co., Ltd., (Shanghai, China). After placing the pollen in this medium for six hours, germination was assessed by observing the growth of pollen tubes under a light microscope (IX71; Olympus, Tokyo, Japan). The experiment was conducted in triplicate.

2.11. Evaluation of Antioxidant Enzyme Activities and Oxidative Stress in S8 Flowers at Different Temperatures

To evaluate the antioxidant defenses and oxidative stress levels of S8 flowers at different temperatures at the blooming stage, a series of biochemical assays were conducted. Superoxide dismutase (SOD) activity, peroxidase (POD) activity, and malondialdehyde (MDA) content were quantified using commercial assay kits (Nanjing Jiancheng Biotechnology Co., Ltd., Nanjing, China).

2.12. Determination of GA Content in Pepper Flowers

F3, F4, and F5 ‘Zhangshugang’ flowers were sampled for analysis via an ultra-high performance liquid chromatography system coupled with electrospray ionization and tandem mass spectrometry (UPLC-ESI-MS/MS) (ExionLC AD; QTRAP 6500+). A Waters ACQUITY UPLC HSS T3 C18 column (100 mm × 2.1 mm i.d., 1.8 µm) was used with water containing 0.04% acetic acid (A) and acetonitrile containing 0.04% acetic acid (B), and run at a flow rate of 0.35 mL/min at 40 °C, and an injection volume of 2 μL [50,51,52]. The gradient program was as follows: start at 5% B (0–1 min), increase to 95% B (1–8 min), hold at 95% B (8–9 min), and decrease to 5% B (9.1–12 min). To acquire linear ion trap (LIT) and triple quadrupole (QQQ) scans, the QTRAP 6500+ LC-MS/MS system was equipped with an ESI Turbo Ion Spray interface operating in both positive (ESI+) and negative (ESI−) ion modes and controlled via Analyst 1.6.3 software (Sciex). The ESI source operation parameters were as follows: ion source, ESI+/−; source temperature, 550 °C; ion spray voltage (IS), 5500 V/−4500 V; and curtain gas (CUR), 35 psi. The GA content was analyzed using scheduled multiple reaction monitoring (MRM). A specific set of MRM transitions were monitored for each period according to the metabolites eluted within each period [53,54]. Three biological replicates and three technical replicates were utilized for each experiment.

2.13. Subcellular Localization in N. benthamiana

The full-length coding sequence (CDS) of CazGA20ox1 (Caz01g13200), CazGA20ox2 (Caz03g21260), CazGA20ox4 (Caz03g36530), and CazGA20ox6 (Caz11g05440), without the stop codon, was amplified via PCR and subsequently cloned into the KpnI and HindIII sites of the 35s::pCAMBIA1300-GFP vector to produce the pCAMBIA1300::CazGA20ox1-eGFP, pCAMBIA1300::CazGA20ox2-eGFP, pCAMBIA1300::CazGA20ox4-eGFP, and pCAMBIA1300::CazGA20ox6-eGFP fusion construct. These constructs and the empty pCAMBIA1300-eGFP control vector were injected into four-week-old N. benthamiana leaves via Agrobacterium tumefaciens strain ‘GV3101’ (Weidi Biotechnology Co., Ltd., Shanghai, China). NF-YA4-mCherry was used as a nuclear marker. Following a three-day incubation period, the fluorescence signal was observed under a confocal laser-scanning microscope (Zeiss LSM510 microscope, Oberkochen, Germany) at the excitation/emission wavelengths 488/510 nm (GFP) and 561/610 nm (mCherry). All primers are listed in Supplementary Table S5.

2.14. Statistical Analysis

All independent experiments were performed using at least three biological replicates and three technical replicates. Statistically significant differences were calculated using two-tailed Student’s t-tests and two-way ANOVAs at the * p < 0.05, ** p < 0.01, and *** p < 0.001 levels. All data are shown as means ± standard deviation (SD).

3. Results

3.1. Identification and Characterization of CaGA20ox Genes

The CaGA20ox genes encode enzymes critical for GA biosynthesis and thereby regulate plant growth and development. In this study, we utilized two conserved domains (2OG-FeII_Oxy [PF03171] and DIOX_N [PF14226]) to identify candidate CaGA20ox genes across six pepper genomes. After removing redundant sequences and confirming the presence of GA20ox domains, a total of six, five, five, five, five, and five CaGA20ox genes were retained in the ‘Zhangshugang’, ‘Zunla’, ‘Chiltepin’, ‘CM334’, ‘Ca59’, and ‘T2T’ genome databases, respectively (

Table 1. The basic information of identified CaGA20ox genes in six pepper genomes.

Genome	Name	Gene ID	Amino Acid Length (aa)	pI	Molecular Weight (kDa)	Instability Index	GRAVY
Zhangshugang	CazGA20ox1	Caz01g13200	379	6.34	43.41	37.31	−0.354
	CazGA20ox2	Caz03g21260	375	6.01	42.39	40.92	−0.369
	CazGA20ox3	Caz03g36510	375	5.63	43.11	38.11	−0.388
	CazGA20ox4	Caz03g36530	294	6.75	33.60	35.83	−0.254
	CazGA20ox5	Caz03g36550	262	4.97	29.98	42.28	−0.315
	CazGA20ox6	Caz11g05440	364	5.90	41.46	31.11	−0.373
Zunla	CauGA20ox1	Capana01g001332	365	6.34	41.95	36.65	−0.381
	CauGA20ox2	Capana03g002435	375	6.01	42.39	40.92	−0.369
	CauGA20ox3	Capana03g003972	321	6.20	36.54	34.42	−0.269
	CauGA20ox4	Capana03g003973	179	6.19	20.62	38.96	−0.425
	CauGA20ox5	Capana11g000564	267	6.24	30.62	28.69	−0.36
Chiltepin	CacGA20ox1	Capang01g001151	365	6.34	41.95	36.65	−0.381
	CacGA20ox2	Capang02g001466	376	8.23	43.47	43.61	−0.387
	CacGA20ox3	Capang03g002357	375	6.11	42.43	40.26	−0.379
	CacGA20ox4	Capang11g000521	384	5.86	43.70	30.28	−0.361
	CacGA20ox5	Capang00g003932	375	5.64	43.16	38.95	−0.393
CM334	CamGA20ox1	PHT93425	377	6.34	43.20	37.45	−0.352
	CamGA20ox2	PHT87225	375	6.01	42.39	40.92	−0.369
	CamGA20ox3	PHT78363	367	8.05	42.41	39.05	−0.320
	CamGA20ox4	PHT68898	384	5.86	43.70	30.28	−0.361
	CamGA20ox5	PHT63838	375	5.75	43.19	40.54	−0.406
CA59	CaaGA20ox1	Capann_59V1aChr01g016000	365	6.34	41.95	36.65	−0.381
	CaaGA20ox2	Capann_59V1aChr03g024430	375	6.01	42.39	40.92	−0.369
	CaaGA20ox3	Capann_59V1aChr03g043420	375	5.63	43.11	38.11	−0.388
	CaaGA20ox4	Capann_59V1aChr03g043430	321	6.20	36.54	34.42	−0.269
	CaaGA20ox5	Capann_59V1aChr11g006860	348	6.06	39.56	29.53	−0.312
T2T	CatGA20ox1	CaT2T01g01562	379	6.34	43.41	37.31	−0.354
	CatGA20ox2	CaT2T03g02508	334	6.41	37.63	39.66	−0.342
	CatGA20ox3	CaT2T03g04651	321	5.65	36.82	36.90	−0.321
	CatGA20ox4	CaT2T03g04653	321	6.20	36.55	35.26	−0.252
	CatGA20ox5	CaT2T11g00667	372	5.85	42.34	29.62	−0.366

; Supplementary Table S1). The CaGA20ox genes were named according to their chromosomal positions. The CaGA20ox protein lengths ranged from 179 aa (CauGA20ox4) to 384 aa (CacGA20ox5). In addition, their molecular weights varied from 20.62 to 43.70 kDa, and their isoelectric points (pIs) ranged from 4.97 to 8.23. The grand averages of the hydropathicity (GRAVY) values of the CaGA20ox proteins were all negative, indicating that they are hydrophilic. Subcellular localization was predicted using WoLF PSORT, and the results suggested that CaGA20ox proteins may be diversely localized to the nucleus, chloroplast, cytoplasm, peroxisome, mitochondria, vacuole, cytoskeleton, extracellular space, plasma membrane, and endoplasmic reticulum. Additional information for each of the CaGA20ox genes is listed in Supplementary Table S1.

3.2. Phylogenetic, Chromosomal Location, and Collinearity Analyses of CaGA20ox Genes

Following the identification of CaGA20ox genes, we performed phylogenetic, chromosomal location, and collinearity analyses to explore the evolutionary relationships and genomic distribution of these genes, which was crucial for understanding the functional divergence and conservation within the CaGA20ox gene family in pepper, as well as for identifying potential candidate genes for further functional validation. The CaGA20ox genes were unevenly distributed among the pepper chromosomes. Overall, the CaGA20ox genes were primarily distributed on Chr01, Chr03, and Chr11 in ‘Zhangshugang’, ‘Zunla’, ‘Ca59’, and ‘T2T’, with the most CaGA20ox genes found on Chr03. Chr03 of ‘Zhangshugang’ contained four CaGA20ox genes, while the same chromosome in three other genomes contained only three CaGA20ox genes (Figure 1A,B,E,F). In the ‘Chiltepin’ genome, Chr00, Chr01, Chr02, Chr03, and Chr11 each contained one CaGA20ox gene (Figure 1C). Similarly, one CaGA20ox gene was found on each of these five chromosomes in the ‘CM334’ genome, one of which was on the unassembled chromosome Scaffold681 (Figure 1D). Three, two, two, and two CaGA20ox genes were found to be similar in sequence and located close to each other on Chr03 in ‘Zhangshugang’, ‘Zunla’, ‘Ca59’, and ‘T2T’, respectively. Given this information, we analyzed tandem duplications within the CaGA20ox gene family across these four genomes. However, the results of the collinearity analysis revealed that these genes are not tandem repeats. Some of these genes belonged to the same phylogenetic branch, while others belonged to different phylogenetic branches, suggesting that the CaGA20ox gene family has experienced both functional differentiation and conservation (Figure 2).

A genome-wide synteny analysis was performed on the CaGA20ox genes in the ‘Zhangshugang’, ‘Zunla’, ‘Chiltepin’, ‘CM334’, ‘T2T’, and ‘Ca59’ genomes. ‘Zhangshugang’ and ‘Zunla’ shared co-linkages in three gene pairs (Figure 1G,H), while ‘Zunla’ and ‘Chiltepin’ shared four gene pairs (Figure 1H,I). Similarly, ‘Chiltepin’ and ‘CM334’ (Figure 1I,J), as well as ‘CM334’ and ‘T2T’ (Figure 1J,K), each shared three and four gene pairs, respectively. Lastly, ‘T2T’ and ‘Ca59’ shared four gene pairs with co-linkages (Figure 1K,L). CazGA20ox1 (Caz01g13200) and CazGA20ox6 (Caz11g05440) were found to co-link across the six genomes, suggesting that these two genes are highly conserved and may have similar functions.

3.3. Conserved Motif, Cis-Element, and Gene Structure Analyses of CazGA20ox Genes in ‘Zhangshugang’ Pepper

To further characterize the CaGA20ox genes and predict their potential functions, we conducted analyses of conserved motifs, cis-acting elements, and gene structures. As subsequent experiments were performed using sequences from the Zhangshugang genome, the results of structural characterization analysis of CazGA20oxs were primarily presented here. The analyses of the remaining five genomes were provided in Supplementary Figures S1–S5. Both CazGA20ox3 and CazGA20ox2 contained the greatest number of motifs (ten), while CazGA20ox5 contained the least (seven) (Figure 3B). All of the CazGA20ox proteins contained the typical conserved 2OG-FeII_Oxy and DIOX_N domains (Figure 3C,F). The majority of the genes contained three exons, with the exception of CazGA20ox5 (two exons) and CazGA20ox2, which contained an additional untranslated region (UTR) (Figure 3E).

Analysis of the 2000 bp upstream sequences revealed that the CazGA20ox genes contained an array of phytohormone-responsive cis-acting elements, including abscisic acid (ABA)-responsive elements (ABRE), auxin-responsive elements (TGA-element and AuxRR-core), salicylic acid (SA)-responsive elements (TCA-element), GA-responsive elements (GARE-motif, TATC-box, and P-box), and methyl jasmonate (MeJA)-responsive elements (CGTCA-motif and TGACG-motif). Several light-responsive cis-acting elements were also discovered, including G-box, GT1-motif, I-box, and TCT-motif. Finally, the CazGA20ox genes also contained MBS, a MYB binding site involved in drought-responsiveness; MRE, a MYB binding site involved in light-responsiveness; and LTR, a cis-acting element involved in low temperature-responsiveness (Figure 3D; Supplementary Table S2). These results indicate that the CazGA20ox genes may be involved in both phytohormone signaling and stress response.

3.4. CauGa20ox Genes Exhibit Distinct Tissue- and Developmental Stage-Specific Expression Profiles

The unique expression pattern of a gene is often indicative of its biological function. In the absence of a published expression profile for ‘Zhangshugang’ pepper, we used publicly available RNA-seq fragments per kilobase million (FPKM) data from the PepperHub database as a reference to investigate the tissue- and growth stage-specific expression profiles of CauGA20ox genes [43]. Specifically, the expression levels of five CauGA20ox genes were evaluated across various tissues and developmental stages, including developing roots, developing stems, mature leaves, closed flower buds, open flowers, and fruits at different developmental stages (Figure 4A). Overall, each of the CauGA20ox genes exhibited distinct tissue- and growth-stage-specific expression profiles. Notably, CauGA20ox5 showed the highest expression in developing stems, while CauGA20ox1, CauGA20ox3, and CauGA20ox4 were predominantly expressed in closed flower buds and open flowers. However, CauGA20ox2 displayed consistently low expression levels across most tissues, but showed high expression levels in stems.

Under the individual effects of five hormones (GA₃, ABA, JA, SA, and IAA), CauGA20ox3 and CauGA20ox4 were only responsive to GA₃ treatment and showed no expression in the other four hormones. The specific response of CauGA20ox3 and CauGA20ox4 to GA₃ suggests that they may play a crucial role in gibberellin-mediated physiological processes and functions independently of the other hormones (Figure 4B–F). Additionally, CauGA20ox3 showed increased expression during the GR3 development stage, while CauGA20ox4 was highly expressed in the GL3 development stage (Figure 4B). Here, “GR3” and “GL3” denote the third developmental stage of roots and leaves following GA₃ treatment, as visually illustrated in Supplementary Figure S6. In response to ABA (30 μM) treatment, the expression of CauGA20ox1 and CauGA20ox2 was up-regulated in leaves, whereas it was down-regulated in roots. CauGA20ox3 and CauGA20ox4 were not expressed in both leaves and roots, while CauGA20ox5 expression was up-regulated in roots, particularly during the middle growth stages (Figure 4C). In response to indole acetic acid (IAA, 2 μM) treatment, CauGA20ox2 expression was up-regulated in leaves and was down-regulated in roots. CauGA20ox1 expression was down-regulated at the early stage of root development and up-regulated at the middle and late stages, while CauGA20ox5, on the contrary, was up-regulated mainly at the middle and late stages of leaf development (Figure 4F).

Additionally, the expression levels of CauGA20ox3 were up-regulated in the FL1 development stage under low-temperature stress (Figure 4G), while the expression levels of CauGA20ox4 were up-regulated in the ML6 development stage under drought stress (Figure 4H). In response to heat and high-salt stress, CauGA20ox1, CauGA20ox2, and CauGA20ox5 all showed responsiveness (Figure 4I,J). These results highlight the importance of the CauGA20ox genes to the abiotic stress response in pepper plants.

3.5. Pepper Flower Development at Different Temperatures

To investigate the impact of temperature on pepper physiology, biochemistry, and floral organ development, Zhangshugang (S8) plants were cultivated at 28 °C (HT-S8) and 18 °C (LT-S8). Plants grown at 28 °C grew more densely and had more leaves and shoots than plants grown at 18 °C (Figure 5A–D). In addition, plants grown at 28 °C developed flowers faster and entered the blooming stage earlier than plants grown at 18 °C.

To understand how temperature-induced oxidative stress affects flower development, we measured the activities of the antioxidant enzymes POD and SOD, and the content MDA. POD activity was significantly higher in LT-S8 than in HT-S8 across all developmental stages (F1 to F6) (Figure 5E). SOD activity was significantly higher in LT-S8 than in HT-S8 from F1 to F2. Conversely, SOD activity was significantly higher in HT-S8 than in LT-S8 from F3 to F4, and insignificantly higher from F5 to F6 (Figure 5F). LT-S8 had a lower MDA content than HT-S8 from F1 to F3, but a higher MDA content from F4 to F6 (Figure 5G). Notably, LT-S8 exhibited stronger antioxidant capacity under low-temperature conditions, particulalry during early floral development (F1 to F3), enabling LT-S8 plants to more effectively scavenge reactive oxygen species (ROS) and reduce oxidative damage.

The significant differences in enzyme activity observed from F3 to F5 prompted us to measure the total GA content of S8 flowers during these key stages. The total GA content of both LT-S8 and HT-S8 flowers significantly increased from F3 to F5, with LT-S8 exhibiting a significantly higher GA content than HT-S8 at the F4 stage (Figure 5H). This suggests that low temperatures may influence the growth and development of pepper flowers by regulating GA dynamics.

In summary, low temperatures significantly affect pepper flower development by modulating antioxidant enzyme activity and GA content. Low-temperature-induced increases in antioxidant enzyme activity effectively reduce oxidative damage to cellular membranes. Finally, changes in the GA content appear to regulate growth in response to low-temperature conditions.

3.6. RNA-Seq Analysis of Pepper Flowers at Different Temperatures

To further investigate the impact of temperature on the morphological and structural development of pepper floral organs and to gain insights into the molecular mechanisms underlying temperature-mediated flower development in pepper, we conducted the following experiments. We focused on the ovary, a key structure directly related to fruit formation and seed development. Paraffin sectioning of ovary tissues was performed at the critical developmental stages between F3 and F5. As shown in Figure 6A, there were no significant differences in ovary morphology between HT-S8 and LT-S8 from F3 to F5. However, the cells of the ovary wall (OW) in LT-S8 were more tightly arranged during F4 and F5, with more orderly cellular structures and a thicker placenta (P). These observations suggest that under low-temperature conditions, plants may enhance their cold resistance by adjusting the compactness of their cellular arrangements.

Pollen viability and germination rates are key factors affecting fertilization success and fruit yield. We measured the viability and germination rates of pollen under different temperature treatments (Figure 6B) and observed no significant differences in pollen viability between HT-S8 and LT-S8. However, the pollen grains of HT-S8 were more uniform in size, whereas LT-S8 exhibited irregularly shaped pollen grains (Supplementary Figure S7). It is important to note that KI staining primarily assesses the starch content within pollen grains and may not fully distinguish between mature but non-viable and immature pollen grains. To complement this assay, we also assessed pollen germination in vitro. HT-S8 had a significantly higher pollen germination rate and longer pollen tubes than LT-S8 (Supplementary Figure S7), indicating that high temperatures result in stronger pollen development and germination capabilities. Overall, these results indicate that low temperatures can affect pollen quality and ovary development.

Given the significant differences in the GA content observed at stage F4, we chose this stage for transcriptome analysis to explore the underlying molecular mechanisms. GO and KEGG analyses of the differentially expressed genes (DEGs) (Supplementary Tables S6 and S8) showed that the DEGs were mainly enriched in the GO terms ‘photosynthesis’, ‘carbohydrate metabolic process’, and ‘pollen tube guidance’ (Figure 6C, Supplementary Table S8), as well as the KEGG terms ‘plant hormone signal transduction’, ‘carotenoid biosynthesis’, and ‘starch and sucrose metabolism’ (Figure 6C, Supplementary Table S9). Additionally, we conducted an in-depth analysis of the transcriptome data, focusing on correlations between known genes related to pepper flower development and CazGA20ox family members. By calculating the Pearson correlation coefficient (r), it was found that CazGA20ox1 was positively correlated with eight out of seventeen genes and negatively correlated with six out of seventeen genes related to pepper flower development (Figure 6D, Supplementary Figure S8). These results suggest that CazGA20ox1 may play an important role in regulating pepper flower development and may cooperate with other key genes to affect the morphological and structural development of floral organs.

3.7. Four Candidate CazGA20ox Genes May Be Involved in Temperature-Driven Flower Development

According to the Pearson correlation analysis, CazGA20ox1 was significantly positively correlated with most genes related to pepper flower development. This discovery prompted us to further investigate the expression patterns of CazGA20ox gene family members during different flower development stages at 28 °C and 18 °C. We conducted a detailed analysis of the expression of six CazGA20ox family members across different developmental stages and tissues. Under different temperature conditions, CazGA20ox1 expression initially increased and then decreased. CazGA20ox1 expression was highest at F3 in HT-S8, but highest at F4 in LT-S8 (Figure 7A). Regardless of temperature, CazGA20ox1 expression was consistently highest in pistils, followed by anthers, and lowest in flower stalks (Figure 7B). Further analysis revealed that CazGA20ox2, CazGA20ox4, and CazGA20ox6 expression initially increased and then decreased, with CazGA20ox6 showing a more gradual decline. At both high and low temperatures, the expression of each of these genes was highest at F4 (Figure 7C,E,G). Notably, CazGA20ox2 and CazGA20ox4 expression was highest in pistils, while CazGA20ox6 expression was highest in petals (Figure 7D,F,H). Notably, CazGA20ox4 exhibited significantly higher expression in LT-S8 compared to HT-S8, suggesting that this gene may be more sensitive to lower temperatures (Figure 7F).

To evaluate the subcellular localization of these genes, we conducted experiments in tobacco leaves. The results indicated that CazGA20ox1, CazGA20ox2, CazGA20ox4, and CazGA20ox6 were all localized to the nucleus and cytoplasm (Figure 7I).

4. Discussion

This study aimed to explore how temperature affects pepper flower development through its influence on the GA biosynthesis pathway, with a particular focus on the CaGA20ox gene family. We systematically identified and characterized CaGA20ox genes across six pepper genomes, analyzing their physical and chemical properties, sequence structures, phylogenies, and tissue-specific expression patterns. Our comprehensive analysis of the CaGA20ox gene family provides a foundation for further studies on the roles of these genes in regulating flower development at different temperatures. In addition, we identified four candidate CazGA20ox genes (CazGA20ox1, CazGA20ox2, CazGA20ox4, and CazGA20ox6). Their distinct expression patterns suggest that these genes may play crucial roles in regulating flower development in response to temperature changes, potentially through modulating GA biosynthesis.

4.1. Characteristics of the CaGA20ox Gene Family

As key enzymes related to GA synthesis and metabolism, GA-dioxygenases (GAoxs) are involved in many critical development processes. For example, GA20ox and GA3ox are involved in the final step of GA biosynthesis, and GA2ox inactivates GAs and their precursors [20,21]. However, GAoxs are relatively poorly characterized in peppers compared to other crops. Therefore, we sought to study the characteristics and functions of the GA20ox gene family in pepper. We systematically identified six, five, five, five, five, and five CaGA20ox genes across six pepper genomes (‘Zhangshugang’, ‘Zunla’, ‘Chiltepin’, ‘CM334’, ‘Ca59’, and ‘T2T’). The CaGA20ox genes were unevenly distributed among the pepper chromosomes, with the majority distributed on Chr03. An additional two to three genes shared similar positions and similar sequences. According to the intraspecies collinearity analysis (Supplementary Table S4), these genes did not exhibit tandem duplication events, suggesting that they have evolved independently and may exhibit divergent functions in response to different environmental conditions.

The CaGA20ox gene family appears to have experienced both functional differentiation and conservation. Although these genes may share some functional redundancy, they are still relatively functionally dissimilar. For example, the absence of AtGA20ox1, AtGA20ox2, and AtGA20ox3 in A. thaliana can cause male and female sterility [28]. GA20ox genes have been identified in many plant species, including five GA20ox genes in A. thaliana [55], eleven GA20ox genes in maize [56], fifteen GA20ox genes in peanut [57], and seven GA20ox genes in grape [58], suggesting that the number of GA20ox genes is unrelated to genome size. Phylogenetic analysis of CaGA20ox proteins from six pepper genomes compared with AtGA2ox, AtGA3ox, and AtGA20ox proteins from A. thaliana revealed that the CaGA20oxs were all clustered with the AtGA20oxs, suggesting strong conservation across species. The hydrophilic CaGA20ox proteins were predicted to be localized to the nucleus and cytoplasm, and this was confirmed by the subcellular localization verification (Figure 7I), suggesting that these proteins function across multiple cellular compartments where GA biosynthesis and regulatory processes occur. Such ubiquity suggests that CaGA20ox proteins effectively coordinate GA signaling and biosynthesis in response to temperature changes, thereby influencing flower development. Because each of these genes appears to have special functions, this requires further confirmation.

Cis-acting elements regulate gene transcription initiation and signal transduction in response to a variety of biotic and abiotic factors. The CazGA20ox genes appear to be responsive to phytohormones, light, and temperature. These results are consistent with previous studies on the GA20ox gene family in other species, such as A. thaliana [55], maize [56], and peanut [57]. CazGA20ox3 and CazGA20ox5 contain the same GA-responsive element (GARE-motif), while CazGA20ox4 contains a different GA-responsive element (TATC-box). However, CazGA20ox1, CazGA20ox2, and CazGA20ox6 do not contain GA-responsive elements, similar to VvGA20ox1 and VvGA20ox7 in grape [58], suggesting that these genes may regulate the endogenous GA content through other means, such as through other hormonal signaling pathways. This finding implies that temperature-regulated GA biosynthesis in pepper flowers may involve complex interactions between multiple signaling pathways. Finally, the CazGA20ox genes contain low temperature-responsive LTR elements. These results are in good agreement with our phenotypic observations (Figure 5) and subsequent qPCR experimental results (Figure 7).

Overall, the CauGA20ox genes exhibited tissue- and developmental stage-specific expression in pepper. For example, the high expression of CauGA20ox5 in developing stems indicates a potential role in stem elongation, which is a well-known function of GA [10]. Similarly, the high expression of CauGA20ox1, CauGA20ox3, and CauGA20ox4 in closed flower buds and open flowers suggests that these genes are involved in floral organ development, which is consistent with the role of GAs in promoting flowering and floral organ formation [11,13].

We further analyzed CauGA20ox gene expression in response to phytohormone treatment and found that these genes are responsive to GA₃, ABA, and IAA. The up-regulation of CauGA20ox3 and CauGA20ox4 under GA₃ treatment indicated that these genes are directly involved in GA signaling, most likely in the feedback regulation of GA biosynthesis [23]. The particular responsiveness of the CauGA20ox3 and CauGA20ox4 genes to GA₃, compared with ABA, IAA, JA, or SA, suggests that they play a special role in GA-mediated processes. These findings highlight the cross-talk between GA and other phytohormone signaling pathways in regulating plant growth and environmental stress responses [13]. The tissue-specific expression and stress responsiveness of CauGA20ox genes are further supported by the presence of various cis-acting elements in their promoters, including hormone-, stress-, and light-responsive elements. These expression patterns suggest that the CauGA20ox genes are involved not only in normal developmental processes, but also in environmental stress responses, particularly the low temperature response. The up-regulation of these genes in response to low temperature may form part of an adaptation mechanism to ensure normal growth and development under stress.

4.2. Temperature Significantly Affects Peppers’ Floral Organ Development and GA Content

Here, we observed that exposure to low temperatures (18 °C) delayed floral bud initiation, reduced pollen germination rates, and resulted in structural alterations in ovules. These findings are consistent with previous reports in crops such as rice, where lower temperatures negatively affect floral organ formation and lead to sterility [1,2,59]. To understand how temperature-induced oxidative stress influences flower development, we analyzed the antioxidant enzyme activities and MDA content in pepper flowers at different temperatures. We observed that POD activity was significantly higher in LT-S8 than in HT-S8 across all developmental stages (F1 to F6). Furthermore, SOD activity was significantly higher in LT-S8 than in HT-S8 during early floral development (F1 to F2), but the trend reversed in later stages (F3 to F6), during which SOD activity was slightly higher in HT-S8. These results suggest that low temperatures may enhance the antioxidant capacity of pepper flowers, especially during the early developmental stages (F1 to F3), effectively reducing oxidative damage to cell membranes. Similar findings have been reported in tomato and alfalfa, where exposure to low temperatures increases antioxidant enzyme activities and improves cold tolerance [60,61,62]. Finally, the MDA content was lower in LT-S8 during early floral development (F1 to F3), suggesting reduced oxidative damage. However, the MDA content was significantly higher in LT-S8 during later stages (F4 to F6), implying increased stress-induced damage as floral organs matured. These results indicate that the antioxidant system in LT-S8 was highly efficient during the early stages of flower development, thereby protecting cellular structures. However, as development progressed, prolonged exposure to lower temperatures may have led to cumulative oxidative stress, causing cellular damage.

The impact of temperature on GA content and flower development has been well documented in various plant species. For instance, in chrysanthemum, CmMAF2 has been shown to bind to the promoter of the GA biosynthesis gene CmGA20ox1 in response to low temperatures, thereby directly regulating the synthesis of bioactive GA₁ and GA₄. These elevated bioactive GA levels upon returning to warm temperatures subsequently activated LEAFY (CmLFY) expression, ultimately initiating floral transition [1]. Similarly, in pak choi, low temperatures (4 °C) significantly increase GA content, which is essential for vernalization and flower bud development [63]. In our study, the low-temperature treatment (18 °C) significantly increased the GA content in pepper flowers, particularly during the F4 stage, which coincides with the critical period of floral organ development. These results suggest that GA plays a pivotal role in mediating the effects of temperature on flower development in pepper.

Furthermore, the differential expression of CazGA20ox genes under different temperature conditions highlights their importance in temperature-responsive GA biosynthesis. Expression profiling of CazGA20ox genes in different floral organs (sepals, petals, stamens, and pistils) revealed that several members, such as CazGA20ox1, CazGA20ox2, CazGA20ox4, and CazGA20ox6 (Figure 7A–H), exhibit tissue-specific expression patterns, particularly in stamens and pistils. Previous studies have shown that GA20ox genes are essential for stamen development in A. thaliana and rice, where mutations in these genes lead to male sterility [28]. Our findings further support the crucial role of GA20ox genes in floral organ development, indicating that their tissue-specific expression patterns are closely associated with the regulation of GA biosynthesis in specific floral tissues, thereby influencing the development and function of these organs.

4.3. Four Candidate CazGA20ox Genes May Be Involved in Floral Organ Development

Based on the results of the RNA-seq, Pearson correlation, and qPCR analyses, we identified four candidate CazGA20ox genes (CazGA20ox1, CazGA20ox2, CazGA20ox4, and CazGA20ox6) that may play significant roles in the development of floral organs in pepper. CazGA20ox1 exhibited strong positive correlations with the majority of genes associated with flower development, suggesting that it might be a central regulator in the genetic network controlling floral organ development, especially in pistils. This aligns with studies on other plants where GA20ox genes are closely linked to floral organogenesis. For instance, in A. thaliana, GA20ox genes influence flower development by regulating GA levels, which in turn affect the formation of petals, stamens, and ovaries [27,28,64]. CazGA20ox2 and CazGA20ox6 also exhibited interesting expression patterns that hint at their specialized functions. Specifically, each had a relatively broad expression profile in floral organs, which might indicate a role in establishing foundational floral structures. On the other hand, CazGA20ox4 exhibited a more pronounced response to temperature changes, with higher expression at 18 °C. Such temperature sensitivity suggests that CazGA20ox4 may play a crucial role in helping pepper plants adapt their floral development to different environmental conditions. Specifically, CazGA20ox4 may be involved in regulating floral GA biosynthesis at different temperatures, potentially affecting reproductive success. In rice, low temperatures reduce GA levels, in turn disrupting pollen development. This occurs because the expression levels of GA biosynthesis genes decrease when plants are exposed to low temperatures, leading to a reduction in bioactive GAs and subsequent male sterility [17].

In conclusion, CazGA20ox1, CazGA20ox2, CazGA20ox4, and CazGA20ox6 may be involved in floral organ development in pepper. In future studies, we plan to verify the functions of these four CazGA20ox genes to clarify their roles in floral development and GA-mediated responses in pepper.

5. Conclusions

In this study, we identified six, five, five, five, five, and five CaGA20ox genes in the ‘Zhangshugang’, ‘Zunla’, ‘Chiltepin’, ‘CM334’, ‘Ca59’, and ‘T2T’ genomes, respectively. The identified CaGA20ox genes were comprehensively analyzed to clarify their potential roles in plant growth and development, particularly in floral organ development and temperature stress responses. The unique expression patterns exhibited by the CaGA20ox genes in response to different phytohormone and abiotic stress treatments underscore the complexity of their regulatory network. Notably, the identified CaGA20ox genes exhibited distinct expression patterns and functional characteristics, indicating both conservation and divergence within the CaGA20ox family. Temperature significantly impacts pepper flower organ development by regulating GA content and antioxidant enzyme activities. The observed enhanced antioxidant capacity and increased GA content at low temperatures suggests that a complex regulatory network underlies these processes. Future research should focus on the functional characterization of individual CazGA20ox genes, particularly CazGA20ox1, CazGA20ox2, CazGA20ox4, and CazGA20ox6, to clarify their specific roles in floral development and GA-mediated responses in pepper. This research offers valuable insights for improving pepper cultivation practices and breeding programs, particularly in the context of environmental adaptation and yield enhancement.