Four different cultivars of Osmanthus fragrans (‘Tianxiang Taige’, ‘Rixiang Gui’, ‘Sijigui’, and ‘Tiannu Sanhua’) plants were produced in a natural environment and provided through the resource garden at Zhejiang A&F University (Wang et al., 2022). The roots, stems, leaves, flower buds, leaf buds, petioles, stem segments, and other tissues of ‘Sijigui’ were gathered and kept at -80°C for subsequent experiments. The Zhejiang Provincial Key Laboratory of Garden Plant Germplasm Innovation and Utilization preserves tobacco of the wild type. Plants of the transgenic varieties and the wild type were cultivated at 23°C in a greenhouse with a 12 hours of light followed by 12 hours of darkness.

There is a DELLA protein in Osmanthus fragrans, which is called OfRGA. The ORF sequence of OfRGA was obtained from transcriptome data. Gene-specific primers (listed in Supplementary Table S1 ) were used to amplify OfRGA. First-strand cDNA synthesized from O. fragrans ‘Sijigui’ was used as the PCR template. The amplification process consisted of an initial denaturation at 95°C for 3 minutes, followed by 32 cycles of denaturation at 95°C for 15 seconds, annealing at 60°C for 15 seconds, and extension at 72°C for 2 minutes. A final extension step at 72°C for 5 minutes completed the process.

The PCR product that had been gel-purified was cloned into the pMD18-T vector (TaKaRa, Dalian, China) and validated by forwarding it to the company for sequencing (Zhejiang Youkang Biotechnology Co. Ltd., Hangzhou, China).

Gene sequence homology was further analyzed using BLAST in the NCBI GenBank database (https://blast.ncbi.nlm.nih.gov). The phylogenetic tree was made with the MEGA 7.0 software. With the DNAMAN program, multiple sequence alignments and analyses were carried out (https://www.lynnon.com/dnaman.html) (Kumar et al., 2018).

The roots, flower buds, stems, leaf buds, leaves, petioles, and shoot apex of O. fragrans ‘Sijigui’ were collected as previously described. Total RNA was individually isolated from each tissue through the RNAprep Pure Plant Kit (Vazyme, Nanjing, China). Next, HiScript^® Reverse Transcriptase was utilized to generate the cDNA of the first strand (Vazyme, Nanjing, China) in accordance with the instructions provided by the manufacturer. Supplementary Table S1 lists the specific primers that were created. RT-qPCR was carried out with a 10 uL PCR mixture that included 5 μL of SYBR Premix Ex Taq, 2 μL of cDNA, 0.4 μL each of PCR forward primer and reverse primer, and 2.2 uL of double-distilled (ddH2O). Then the internal reference gene and the target gene were added independently to a 96-well plate with three copies of each sample. The relative gene expression was computed using the 2-Ct method (Livak and Schmittgen, 2001).

The ORF of OfRGA lacking the stop codon was ligated into the pORER4-35S-GFP vector. And using the freeze-thaw method, the control, nuclear marker, and OfRGA-GFP expression plasmids were transferred into the Agrobacterium rhizogenes strain GV3101, where Agrobacterium expressing the plasmid and the control plasmid were mixed with Agrobacterium with the nuclear marker (35S::D53-RFP), respectively, and co-injected into the back of the tobacco leaves. The GFP fluorescence of tobacco leaves was detected after infiltration for 2 days to identify the protein’s position in the cells using a confocal microscope (Olympus Corporation, Tokyo, Japan) (Liu et al., 2023).

Similarly, the ORF of OfRGA had been inserted into the vector pORER4 behind the 35S promoter, and the resulting construct 35S:OfRGA was transferred into the Agrobacterium tumefaciens strain GV3101 and then transformed into tobacco by the leaf disk method (Sparkes et al., 2006). Leaves were cultured in MS medium for 3 days. They were then moved to MS Medium, which included the following: 250 mg/L of carbenicillin disodium (Carb) + 2.25 mg/L of 6-benzyladenine (6-BA) + 0.3 mg/L of 1-naphthylacetic acid (NAA) + 100 mg/L of kana (a pH of 5.8). After being separated from the callus, the regenerated shoots were placed on the rooting media (MS + 100 mg/L Kana + 250 mg/L Carb) (a pH of 5.8). After rooting, the seedlings were moved to soil pots for cultivation. In order to observe phenotypic traits and compile data, transgenic plants that have been identified and WT control plants were grown in identical environments.

To observe the upper epidermal cells of the petals, the petals were collected and preserved for 24 hours at 4°C using a formaldehyde-acetic acid-alcohol (FAA) fixative (5% acetic acid, 5% formaldehyde, and 70% alcohol), followed by an alcohol rinse. Petal cells were then photographed using a confocal microscope (Olympus Corporation, Tokyo, Japan). The upper portion of the tobacco corolla tube was excised and transversely sectioned into several thin slices using a clean double-sided blade. These slices were then observed under the previously described confocal microscope. The area of the AbsE cells was measured using the ImageJ software (Rueden et al., 2017).

Using cDNA created from RNA extracted from tobacco flower bracts, differentially expressed genes (DEGs) between WT and transgenic lines of OfGRA were found using an Illumina sequencing platform (Beijing Bemac Biotechnology Co., Ltd., Beijing, China). Next, the Osmanthus fragrans reference geneome was mapped to the clean reads, and DEGs were examined for GO and KEGG enrichment. On the NCBI sequence read archive (SRA), the clean reads were submitted. (http://www.ncbi.nlm.nih.gov/sra/), accession number: PRJNA1122233. (https://www.ncbi.nlm.nih.gov/sra/PRJNA1122233).

Statistical significance was assessed using *p < 0.05, **p < 0.01, and ***p < 0.001 after data were processed with SPSS statistics and Excel software. There were three biological duplicates for each experiment.

The ORF of the OfRGA gene is 1776 bp in length and encodes 592 amino acids. Multiple sequence comparisons using DNAMAN software showed that all DELLA protein sequences have DELLA and TVHYNP motifs at the n-terminus and GRAS structural domains at the C-terminus, suggesting high consistency among DELLA proteins ( Figure 1A ). To further explore the evolutionary connection between OfRGA and other DELLA protein members, we compared 16 protein sequences from eight representative plant species and constructed a phylogenetic tree. According to the phylogenetic tree, OfRGA and the evolutionary history of OeDELLA1 in Olea europaea are tightly linked ( Figure 1B ).

In order to further elucidate the role of OfRGA, qRT-PCR is used to detect the expression of OfRGA in the stems, roots, flower buds, petioles, leaves, leaf buds, and shoot apex of O. fragrans ‘Sijigui’ ( Figure 2A ). The results indicate that OfRGA has the maximum degree of expression in the shoot apex. According to subcellular localization experiments, the control GFP signal was dispersed throughout the cell, whereas OfRGA-GFP showed strong fluorescence signals in the nucleus. The finding demonstrated that the nucleus was where the OfRGA fusion protein was located ( Figure 2B ).

Under the same growing environment, we observed a significant difference in petal size among four different cultivars of ‘Sijigui’. In order to understand this difference, four varieties, ‘Tianxiang Taige’, ‘Rixiang Gui’, ‘Putong Sijigui’, and ‘Tiannu Sanhua’, were selected for observation and measurement ( Figures 3A, C ). The results showed that the ‘Tianxing Taige’ variety had the largest petals, followed by ‘Rixiang Gui’, while ‘Putong Sijigui’ and ‘Tiannu Sanhu’ had approximately the same petal size. In order to investigate whether petal size is associated with the OfRGA gene, we quantitatively analyzed the petals of four different varieties of ‘Sijigui’ ( Figure 3B ). The analysis showed varying levels of OfRGA gene expression among the petals of the four different varieties, with a negative correlation between OfRGA expression and petal size. These findings suggest that OfRGA may play a role in limiting cell proliferation, thereby contributing to reduced petal size.

Agrobacterium-mediated transformation was used to create tobacco transgenic overexpression lines in order to further explore the gene function of OfRGA. From each construct, over ten distinct transgenic tobacco lines (35S::OfRGA) were produced, the vast majority of which had significantly altered phenotypes. For phenotyping purposes, we chose two transgenic lines that exhibit elevated levels of expression. The transgenic lines of OfRGA were all found to exhibit phenotypes of increased plant height, increased internode spacing, shorter leaf length, wider leaf width, and varying degrees of curling of the leaf margins when comparing the transgenic lines to the wild type ( Figures 4A–G ). These findings indicate that the overexpression of OfRGA promotes plant development and growth.

After blooming, the phenotypes were further observed in the transgenic tobacco lines, and the outcomes demonstrated that the petals of the OfRGA transgenic line were significantly smaller and lighter in color than those of the wild type, further verification of the hypothesis that OfRGA inhibits cell expansion and reduces petal size ( Figures 5A, B ). In addition, to determine whether different floral organs’ growth and development were impacted, we also measured the width and length of the wild-type and transgenic corolla tubes, as well as the lengths of the stamens, pistils, and flower stalks ( Figures 5C–F ). We found that compared to the wild type, the petal diameter of OE-3 and OE-4 was reduced by 0.92 cm and 0.74 cm, the corolla tube diameter was reduced by 0.86 mm and 0.76 mm, the length of the corolla tube was reduced by 0.68 cm and 0.64 cm, the length of the pedicel was reduced by 2.7 mm and 3.17 mm, the length of the pistil was reduced by 0.91 cm and 0.82 cm, and the length of the stamen was reduced by 0.88 cm and 0.80 cm. The results indicate that the corolla tube of the transgenic strain became shorter and narrower, with reduced lengths of stamens, pistils, and pedicels compared to the wild type ( Figures 5G–L ). These findings confirm that the OE-OfRGA transgenic line exhibited significant reductions in the size and dimensions of these floral structures. In summary, the overexpression of OfRGA led to the shortening of floral organs.

To look into why the corolla tube is shorter and the petals are smaller, we conducted measurements on the cell area of both wild-type (WT) and transgenic petals, as well as corolla tubes ( Figures 6A, E–J ). We found that petal cell area decreased by 33.04% and 20.60%, as well as cell length decreased by 19.45% and 10.13% in OE-3 and OE-4, compared with the wild type ( Figures 6B–D ). It suggests that OfRGA may inhibit the growth of petals and corolla tubes by inhibiting cell expansion. Suggesting that OfRGA may inhibit petal and corolla tube growth by suppressing cell expansion.

To gain additional insight on the phenomenon through which OfRGA may inhibit the growth of petals and corolla tubes, as well as the causes of the lightening of flower color and the related genes through which OfRGA acts, differentially expressed genes (DEGs) were found using RNA-seq analysis. Transcriptomes from three biological replicates of WT and OfRGA overexpression transgenic plants were obtained for further bioinformatics analysis. Nine samples yielded 54 G of clean data, which was then used for additional bioinformatic analysis. 96.73% of the readings were successfully mapped to the Nicotiana genome. The DEG screen was conducted based on the criteria of a log2 fold change ≥ 1 and a Q value ≤ 0.05. 2,676 genes showed up-regulation and 2,074 genes showed down-regulation in WT compared to the OE-3 group, according to the analysis. Similarly, 4,331 genes showed up-regulation and 3,521 genes showed down-regulation in WT compared to the OE-4 group ( Figures 7A, D ). Taken together, genes that were up-regulated outnumbered those that were down-regulated. The GO analysis of WT versus OE-3 and OE-4 revealed that the up-regulated genes showed a high enrichment in molecular functions such as ‘DNA-binding transcription factor activity’ and ‘cytoskeletal motor activity’. In terms of cellular components, these genes were significantly enriched in the ‘nucleus’ and ‘cell wall’. The most significant enrichment was observed in the biological processes of ‘cell cycle’ and ‘multicellular organism development’ ( Figure 7B ). The molecular functions of the down-regulated genes were primarily abundant in ‘transporter activity’, the cellular components were primarily abundant in ‘plasma membrane’ and ‘extracellular region’, and the biological processes were mainly enriched in ‘transport’ and ‘cellular homeostasis’ ( Figure 7E ). According to KEGG enrichment analysis, the majority of the up-regulated genes were enriched in ‘Chromosome and associated proteins’, ‘Cytoskeleton proteins’, ‘Transcription factors’, and ‘Protein families: signaling and cellular processes’ ( Figure 7C ). while the down-regulated genes were gratetly enriched in these pathways, such as ‘Starch and Sucrose Metabolism’, ‘Carbohydrate Metabolism’, ‘Transporters’, and ‘Protein Families: Signaling and Cellular Processes’ ( Figure 7F ). These results indicate that genes and DNA-binding transcription factors involved in regulating the cell wall and cell cycle could be crucial in the organ development and cell expansion of transgenic tobacco flowers.

A total of 13 genes associated with the development of floral organs were identified from differentially expressed genes (DEGs), including the MADS-box gene family, AP2/ERF gene family, xyloglucan endotransglucosylase/hydrolase (XTH), Cellulose Synthase A1 (CESA1), Expansin B1 (EXPB1), NAC56, and Plasma Membrane Intrinsic Protein2-7 (PIP2-7). Among these, the MADS-box gene family and AP2/ERF gene family-related genes were significantly upregulated, while XTH, CesA1, EXPB1, NAC56, PIP2-7, and other genes participating in cell wall synthesis and cell expansion were significantly down-regulated ( Figure 8A ). These findings indicate that genes related to cell expansion are essential to the regulation of floral organ development. Furthermore, several DEGs associated with anthocyanin synthesis and regulation were identified. Genes involved in anthocyanin synthesis, such as Chalcone Isomerase (CHI), 4-Coumarate: Coenzyme A Ligase 3 (4CL3), MYB transcription factors MYB12, and MYB57 were significantly down-regulated, whereas genes related to anthocyanin regulation, like MYB transcription factors MYB26, MYB5, MYB32, MYB3, and bHLH transcription factor bHLH9, were markedly up-regulated ( Figure 8B ). These results suggest that the color fading of transgenic plants may be due to the declined expression of anthocyanin biosynthesis-related genes and the upregulation of transcription factors such as MYB and bHLH. The exact results still need to be further verified through more experiments.