Wild-type (WT) Sophora davidii and radiation-induced plant height mutants grown in Guiyang, Guizhou, China, were used in this study, similar to previous studies (Zhao et al., 2022b). These plants were grown under natural light conditions with regular irrigation. Arabidopsis thaliana ecotype Columbia (Col-0) was used as the WT and grown alongside the second generation (T2) of pure transgenic Arabidopsis in a growth chamber set at 22 ± 2°C with 70% humidity and 1200 Lux light intensity, following a 16-hour daytime and 8-hour darkness cycle. For sampling, S. davidii and Arabidopsis samples were immediately immersed in liquid nitrogen and stored at -80°C.

Three-month-old S. davidii plant height mutants with similar growth status were selected for various treatments. Hormone treatments included 10 μM Gibberellin A3 (GA3, CAS: 77-06-5), 5 μM brassinolide (BR, CAS: 72962-43-7), 10 μM indole-3-acetic acid (IAA, CAS: 87-51-4), 10 μM uniconazole (CAS: 83657-22-1), and 10 μM Ethephon (substitute for ethylene, ETH, CAS: 16672-87-0) (Xie et al., 2023). All hormone solutions were first dissolved in a small amount of ethanol and then diluted with distilled water to the appropriate concentration. The hormones were sprayed on the plants until the solution started to run off the leaves. Stems from the various treatments were sampled at the indicated time points (0, 3, 6, 9, 12, 24, 36, 48 hours) for qRT-PCR analysis.

Total RNA was extracted from plant tissues as described previously (Zhao et al., 2022a). Reverse transcription was performed using the HiFiScript cDNA synthesis kit (Cwbiotech), and gene-specific primers were synthesized using the PerlPrimer program. qRT-PCR was performed on an Applied Biosystems CFX ConnectTM real-time PCR detection system using UltraSYBR^® Mixture (Low ROX). The qRT-PCR thermal cycling parameters were: 95°C for 10 minutes followed by 40 cycles of 95°C for 15 seconds and 60°C for 1 minute, in 20 μL volumes. Expression data were analyzed using the 2-ΔΔCt method. Each reaction was performed with three independent biological replicates and three technical replicates per sample. All primers used for qRT-PCR are listed in Supplementary Table S1 .

Total RNA was extracted from S. davidii as described in Zhao et al. (2022b), and the coding region of SdGA20ox1 was amplified according to the manufacturer’s instructions based on the transcriptomic data previously obtained in our laboratory. The PCR products were subsequently cloned into the pGEM-T vector (Promega Corporation, Madison, WI, USA) and sequenced. Specific primer information for gene cloning is listed in Supplementary Table S1 .. The 40 μL RT-PCR solution contained 5.0 μL of 2× Taq PCR Master Mix (Tian-gen, China), 1 μL forward and 1 μL reverse primers, 3.0 μL cDNA, and 15.0 μL deionized water. RT-PCR was performed with 30 cycles of 95°C for 30 seconds, 58°C for 30 seconds, and 72°C for 30 seconds, followed by 72°C for 10 minutes. The PCR product was then recovered and ligated into the pEASY-Blunt cloning vector (TransGen, Beijing, China), and sequenced by Sangon Biotech (Shanghai, China).

Amino acid sequences were obtained from the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov), with the gene name SdGA20ox1 and GenBank accession number ON229923. MEGA 11 software (https://www.megasoftware.net/megamac.php) and the online tool Evolview (https://www.evolgenius.info) were used to create phylogenetic trees (Subramanian et al., 2019). A neighbor-joining phylogenetic tree was generated using MEGA 11 with the Poisson model, gamma-distributed rates, and 1,000 bootstrap replicates.

The BamHI (5’) and PstI (3’) restriction sites were added to the ends of the SdGA20ox1 CDS fragment, respectively, and the PCR products were cloned into the pHB vector and sequenced. The overexpression vector plasmid was transformed into Agrobacterium GV3101, and positive transgenic plants were identified with 50 μg/mL of kanamycin. The Agrobacterium strain carrying the pHB::SdGA20ox1 vector was transformed into Arabidopsis using the flower dip method (Clough and Bent, 1998). PCR primer sequences for transgenic plants are shown in Supplementary Table S1 .. The transgenic Arabidopsis was screened for resistance using 50 μmol/L glufosinate until stable genetically pure T2 generations (OE2, OE5, and OE7) were obtained for subsequent experiments.

Wild-type and transgenic plants were grown under identical conditions, and seeds were collected simultaneously. Seed germination tests were conducted as described by Yan et al. (2017). Thirty seeds were sown on half-strength MS solid medium and incubated in the dark at 4°C for 4 days before being transferred to a growth chamber at 22°C. Seeds that completed germination were counted every 24 hours. The experiment was repeated three times. For seedling hypocotyl and root length determination, seeds were sown on half-strength MS solid medium. Plates were refrigerated at 4°C in the dark for 4 days, then moved to 22°C with a 16-hour light/8-hour dark cycle. On day 7, the hypocotyl lengths of approximately 10 seedlings were measured. On day 14, the root lengths of about 10 seedlings were measured. Arabidopsis seedlings grown on half-strength medium for 7 days were transplanted to pots for 45 days, and morphological characteristics (leaf length, plant height) were measured during the growing season.

One gram of leaf tissue from 45-day-old Arabidopsis and 3-month-old S. davidii plants was collected and ground to a powder in liquid nitrogen. The powdered leaves were placed in 10 ml of extraction solution (95% ethanol: 80% acetone = 1:1 by volume), mixed, and stored overnight at 4°C in the dark. Total chlorophyll (Chl) was measured according to the method described by Yang et al. (2020), and each measurement was repeated three times.

Samples of wild-type and transgenic Arabidopsis were collected and weighed for further analysis. Young stems (from stem tip to node) were collected for GA content determination. The assay was performed as described previously by Chen et al. (2012), with three independent biological replicates and three technical replicates measured for each sample.

To study the subcellular localization of SdGA20ox1, specific primers with the enzymatic cut site BsaI/Eco31I were designed. The pBWA(V)HS-SdGA20ox1-Glosgfp vector was constructed, transformed into Escherichia coli (DH5α), and then transformed into tobacco leaves. The fluorescence signal was observed using confocal laser scanning microscopy (CLSM, Leica TCS SP2) with excitation and emission light at 511 nm and 525 nm, respectively. For the bimolecular fluorescence complementation (BiFC) assay, full-length SdGA20ox1, 11430582, and 11416005 genes were amplified using primers ( Supplementary Table S1 ), cloned into the PpSm35s vector (Engler et al., 2008), and transiently transformed into the lower epidermis of tobacco leaves. After 48 hours of transfection, interactions were observed using CLSM with excitation at 511 nm and emission at 525 nm.

To prevent self-activation of SdGA20ox1, a self-activation assay was performed. The decoy plasmid transformed recipient strain AH109 was cotransformed with pGBKT7-SdGA20ox1 and pGBKT7 using Saccharomyces cerevisiae. The transformed AH109 strain was plated on SD/-Trp plates deficient in tryptophan. Six randomly selected spots were verified by PCR with vector primers of pGBKT7. Three cloning sites were randomly selected and applied to culture medium deficient in tryptophan, histidine, and adenine, supplemented with X-α-Gal blue-displaying substrate (5-Bromo-4-chloro-3-indolyl-alpha-D-galactopyranoside), namely SD/-Trp, SD/-Trp/-His, SD/-Trp/-His/-Ade, and SD/-Trp/-His/-Ade+X-α-gal plates. Plates were incubated at 30°C for 3-5 days. pGBKT7 was used as the negative control, and the diameter and color of the colonies were observed and recorded.

To identify candidate interacting proteins of SdGA20ox1, a cDNA library of S. davidii mutants was constructed, and transformed cells were plated on SD/-Trp plates and cultured. Cells were then transferred to YPDA and cultured for 4–5 hours. Twenty microliters of culture were diluted and plated on SD/-TLH plates, incubated at 30°C for 3-7 days. Single colonies were picked and transferred to SD/-TLH plates for 3-5 days. Positive yeast clones were obtained by screening on SD/-TLH plates. Positive clones were amplified from yeast cells and sequenced. The positive clones were diluted and plated on SD/-TL, SD/-TLH, SD/-TLHA, and SD/-TLHA/+X-α-gal, respectively, and incubated at 30°C for 3 days. pGBKT7-p53 and pGADT7-largeT were co-transformed as positive controls, and pGBKT7-laminC and pGADT7-largeT were cotransformed as negative controls.

All experiments were performed at least three times. Error bars in each graph indicate the mean values ± SE of three replicates. Statistical significance between control and treatment groups was determined using Tukey’s test and Student’s t-test in SPSS Statistics software version 19 (IBM, USA). Different lowercase letters indicate significant differences (P < 0.05), with ** indicating P < 0.01 and * indicating P < 0.05.

Based on the S. davidii transcriptome data (SRA315988) previously obtained by our group, we isolated the full-length cDNA of SdGA20ox1 (GenBank accession number ON229923) from S. davidii plant height mutants using reverse transcriptase-polymerase chain reaction (RT-PCR). The full-length cDNA contained a coding sequence (CDS) of 1143 bp ( Supplementary Figure S1 ). The primary structure of the SdGA20ox1 protein was deduced to contain 380 amino acids, with a predicted molecular mass of 43.16 kDa and an isoelectric point (pI) of 5.96. According to the National Center for Biotechnology Information (NCBI), the SdGA20ox1 protein contains a conserved 2OG-FeII_Oxy structural domain. Using the NCBI Conserved Domain database, we identified that the protein includes a 2OG-FeII_Oxy structural domain spanning amino acids 225–324, classifying SdGA20ox1 as part of the 2OG-FeII_Oxy protein superfamily ( Figure 1A ).

To further characterize the SdGA20ox1 gene within the GA20oxs family, we performed a phylogenetic analysis of the amino acid sequences of GA20oxs from various plant species. The phylogenetic tree revealed that the amino acid sequences of GA20oxs from S. davidii, Lupinus luteus, and Lupinus angustifolius clustered together, indicating a higher homology of SdGA20ox1 with LaGA20ox1 and LlGA20ox1 proteins ( Figure 1B ). The SdGA20ox1 protein showed the highest identity with LaGA20ox1 from Lupinus angustifolius and LlGA20ox1 from Lupinus luteus ( Figure 1C ). Additionally, The SdGA20ox1 protein contains three types of helical structures, with alpha helices accounting for 33.68%, irregular coils accounting for 52.11%, and extended chains accounting for 14.21%, which indicated that its main components are α-helices and irregular coils ( Figure 1C ). Based on these findings, we named this gene SdGA20ox1.

The quantitative real-time fluorescence PCR (qRT-PCR) analysis results showed significant differences in SdGA20ox1 expression across various parts of S. davidii, influenced by exogenous phytohormones ( Figure 2 ). The gene was significantly more expressed in leaves and stems than in roots (P < 0.05), with the highest expression in stems ( Figure 2A ). The expression of SdGA20ox1 decreased significantly after exogenous spraying of the gibberellin synthesis inhibitor uniconazole; however, it began to increase again after 12 hours, reaching a level not significantly different from 0 hours ( Figure 2B ). Following exogenous ETH treatment, the expression of SdGA20ox1 showed a trend of decreasing and then increasing, and it was significantly higher than at 0 hours after 24 hours of treatment ( Figure 2C ). After exogenous BR treatment, SdGA20ox1 expression peaked at 12 hours, then decreased, but remained not significantly different from pre-treatment levels ( Figure 2D ). After exogenous spraying of GA3, SdGA20ox1 expression continued to decline, eventually decreasing significantly at 48 hours ( Figure 2E ). Conversely, following exogenous spraying of IAA, SdGA20ox1 expression first increased and then decreased, peaking at 24 hours, which was significantly higher than at other treatment times ( Figure 2F ).

The open reading frame (ORF) of SdGA20ox1 was fused to the N-terminal of the GFP protein in the CaMV35S promoter-driven pBWA(V)HS-SdGA20ox1-Glosgfp vector to construct the 35S::SdGA20ox1 vector, which was transiently transformed into tobacco leaves. Microscopic observation revealed that SdGA20ox1-GFP was distributed in the nucleus and cytoplasm, with weaker expression in the cell membrane and more concentration in the nucleus compared to the distribution of the GFP control ( Figure 3 ).

PCR amplification of target fragments in Arabidopsis resistant plants showed consistency with the positive control band, but the target fragment was not detected in wild-type Arabidopsis ( Supplementary Figure S2 ), indicating the successful acquisition of transgenic Arabidopsis. The T2 generation homozygous OE2, OE5, and OE7 lines were further screened for subsequent experiments. During seed germination, the germination rate of seeds from transgenic lines overexpressing (OE) SdGA20ox1 was significantly higher than that of the wild type (WT), with the germination time also earlier ( Figures 4A, B ). On the second day, the transgenic Arabidopsis seeds germinated in large quantities, with the germination rate of OE7 close to 80%, while the WT lines had a germination rate of less than 20%, significantly lower than that of the transgenic lines (P < 0.05).

Compared to the WT, transgenic Arabidopsis seeds sown on half-strength MS agar medium exhibited significantly faster vegetative growth ( Figures 4C, D ). After 7 days of growth on half-strength MS medium, the hypocotyl length of transgenic Arabidopsis seedlings was significantly greater than that of the WT ( Figures 4C, E ), and after 14 days, the root length of transgenic Arabidopsis was significantly greater than that of the WT ( Figure 4F ), indicating that overexpression of SdGA20ox1 resulted in increased hypocotyl and root lengths in Arabidopsis seedlings.

After transplanting plants grown in half-strength medium for 14 days into nutrient soil, we observed the time of first bud appearance in both WT and transgenic Arabidopsis, along with their plant heights at this time. The experiment showed that transgenic Arabidopsis flowered earlier, with the first bud appearing about 5 days earlier than in the WT ( Figure 5A ). Additionally, when the first bud appeared, the plant height of the transgenic Arabidopsis was significantly higher than that of the WT (P < 0.05) ( Figure 5B ). We also measured the leaf size and leaf chlorophyll content of 3-month-old S. davidii plant height mutants and Arabidopsis after 45 days of growth. The results showed that the leaf length and plant height of the S. davidii plant height mutant were significantly higher than those of the WT S. davidii ( Figures 5F, G , 6A, B ), and the leaf length of transgenic Arabidopsis was also significantly higher than that of the WT ( Figures 5C, D ). However, the chlorophyll content of the leaves in both the S. davidii plant height mutant and transgenic Arabidopsis was not significantly different from the WT (P > 0.05), although it was lower ( Figures 5E, H ). After 45 days of growth in nutrient soil, the plant height of transgenic Arabidopsis was significantly higher (P < 0.05) than that of WT Arabidopsis across the three overexpression lines (OE2, OE5, and OE7) ( Figures 6C, D ). The plant heights of the OE2, OE5, and OE7 lines increased by 37.0%, 34.8%, and 27.7%, respectively, compared to WT Arabidopsis.

We measured the biologically active GA1, GA3, and GA4 contents in young transgenic Arabidopsis (OE2, OE5, and OE7) stems grown for 40 days ( Figure 7 ). The results showed that GA1 and GA3 contents in transgenic plants were significantly higher than in WT (P < 0.05), approximately 1.8-fold, 2.6-fold, and 1.6-fold for GA1 and 1.2-fold, 1.5-fold, and 1.2-fold for GA3, respectively. While GA4 content in transgenic plants was higher than in WT, only OE5 showed a significant difference (P < 0.05), being about 1.4-fold higher than WT.

We constructed a cDNA library with a capacity of 5.84 × 10⁷ CFU/ml and a total number of clones of 1.168 × 10⁸ CFU ( Supplementary Figure S3 ). To analyze the band distribution structure of the constructed library, 24 randomly selected clones were subjected to colony PCR using T7-F/AD-R primers ( Supplementary Table S1 .). PCR electrophoresis results showed that the average size of the library fragments was 1000 bp ( Supplementary Figure S4 ).

We used Y2H to screen for interacting proteins. Autoactivation analysis showed that pGBKT7-SdGA20ox1 had no autoactivation ( Figure 8A ). The AH109 yeast strain containing the pGBKT7-SdGA20ox1 decoy plasmid was used as a receptor to prepare the sensory state, into which the library plasmid pGADT7-S. davidii cDNA was subsequently transferred and plated on SD-TLH plates. Screening on SD-TLH plates yielded 192 positive yeast clones ( Supplementary Figures S5 , S6 ). We then amplified and DNA sequenced the 192 positive clones from yeast cells. BLAST analysis of the sequences in the GenBank database resulted in 28 reciprocal protein gene sequences ( Supplementary Figure S7 ; Supplementary Table S2 ).

To further validate the possible interaction of candidate proteins with SdGA20ox1, we selected two genes from the 28 candidate genes with predicted functions related to plant growth and development: rbcS (11416005) and EIN4 (11430582). We constructed pGADT7-11416005 and pGADT7-11430582 vectors for point-to-point yeast transformation with pGBKT7-SdGA20ox1. The results showed that the positive control could grow on SD/-Trp/-Leu, SD/-Trp/-Leu/-His, SD/-Trp/-Leu/-His/-Ade, and SD/-Trp/-Leu/-His/-Ade/+X-α-gal plates, and displayed a blue color on SD/-Trp/-Leu/-His/-Ade/+X-α-gal plates. The negative control could grow only on SD/-Trp/-Leu plates. In the experimental group, the growth of pGBKT7-SdGA20ox1 + pGADT7-11416005 and pGBKT7-SdGA20ox1 + pGADT7-11430582 was consistent with the positive control, indicating that both 11416005 and 11430582 physically interact with SdGA20ox1 in yeast ( Figure 8B ).

11416005 is involved in plant photosynthesis, while 11430582 may function in the early stages of the ethylene signaling pathway. We used the BiFC assay to confirm the interaction between SdGA20ox1 and 11416005 and 11430582. A distinct YFP yellow fluorescent signal was observed in tobacco leaf pulp protoplast cells after cotransformation with N-terminal YFPN fusion 11430582 and C-terminal YFPC fusion SdGA20ox1, as well as after cotransformation with N-terminal YFPN fusion SdGA20ox1 and C-terminal YFPC fusion 11416005. However, no YFP signal was detected in the negative controls (YFPN and YFPC-SdGA20ox1, YFPN and 11430582-YFPC, YFPN and YFPC-SdGA20ox1, and YFPN and 11416005-YFPC) ( Figure 8C ). These results suggest that the SdGA20ox1 protein can physically interact with 11416005 and 11430582 in plants, and the three may affect endogenous gibberellin production through these interactions.