Arabidopsis (Col-0), tobacco, and peanut varieties D893 (large pod) utilized in this study were provided by the College of Agronomy, Shandong Agricultural University. The peanut varieties 79266 (small pod) was provided by the Shandong Peanut Research Institute. AhRUVBL2 transgenic Arabidopsis was obtained through a previous investigation conducted by our research group [23]. All plant materials were cultivated inside an artificial climate chamber. Tobacco plants were grown in a mixture of nutrient soil and vermiculite at a 3:1 ratio and maintained at 22 ℃ with a 16-h light/8-h dark photoperiod, 70% relative humidity, and an illumination intensity of 2000 Lux. Each treatment was carried out with a minimum of three biological replicates.

Genomic DNA from T₅ transgenic Arabidopsis seeding stage leaves was extracted using the CTAB (Hexadecyltrimethylammonium bromide) method [24]. PCR (Polymerase Chain Reaction) was performed using specific primers, and the expression of AhRUVBL2 was evaluated by qRT-PCR (Quantitative reverse transcription polymerase chain reaction). cDNA sequence information for the AhRUVBL2 gene was reported previously [23]. The specific primers for qRT-PCR were designed using Beacon Designer 8.0 software (Palo Alto, CA, USA). Arabidopsis UBQ5 (NP_191784, AT3G62250) was used as an internal control gene [25]. Three biological replicates and three technical replicates were performed for each sample. Relative gene expression was calculated using the 2^{-Δ⁢Δ⁢Ct} method [26].

The phenotypic characteristics of wild-type and AhRUVBL2 transgenic Arabidopsis lines grown under equivalent conditions were recorded, including plant height, rosette leaves, number of branches, silique and seeds. The results for bolting, flowering, and yellow fruiting stages of wild-type (WT) and transgenic Arabidopsis were assessed under the same growth conditions. WT and transgenic Arabidopsis silique were taken for cytological observations at 4 d, 6 d, 9 d, and 12 d after flowering [1]. Plant phenotypes and cytological features were photographed with a camera attached to microscope (Nikon D850, Tokyo, Japan), and ImageJ software (ImageJ 1.54, National Institutes of Health, Bethesda, MD, USA) was used for measurements.

Silique from WT and overexpressing AhRUVBL2 transgene Arabidopsis were used for transcriptome sequencing at 6 d after anthesis under normal growth conditions, with three biological replicates for each sample (Novogene Co., Ltd, Beijing, China). WT Arabidopsis silique was labelled WT-1, WT-2, and WT-3, while transgene Arabidopsis silique was labelled OE-1, OE-2, and OE-3 (OE, Overexpression). Total RNA was extracted using the FastPure Universal Plant Total RNA Isolation Kit (Vazyme, Nanjing, China), and RNA integrity was tested using the Agilent 2100 bioanalyzer. Samples that met the requirements for cDNA library construction were used in subsequent sequencing. Clean reads were compared for similarity to the Arabidopsis reference genome (TAIR10.1) using HISAT2 software (2.2.1, Johns Hopkins University, Baltimore, MD, USA). The expression levels of individual genes were normalised using Fragments Per Kilobase Million (FPKM). Differential gene analysis between WT and transgenic Arabidopsis was carried out using DESeq2 software (1.44.0, University of North Carolina, Chapel Hill, NC, USA), and differentially expressed genes (DEGs) were screened with the following parameters: adjusted p $\le$ 0.05 and $|$log2 (fold change)$|$ $\ge$1. Functional annotation and enrichment analysis of DEGs was carried out using the Gene Ontology (GO, http://geneontology.org/docs/download-ontology/) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases (https://www.kegg.jp/kegg/pathway.html), with the threshold for significant enrichment being p 0.05.

Genomic DNA from peanut 79266 and D893 leaves was extracted using the CTAB method [24]. DNA concentration was estimated with a NanoDrop 2000 spectrophotometer, and DNA purity assessed by electrophoresis with 1% agarose gels. The sequence 2527 bp upstream of the start codon (ATG) of AhRUVBL2 (https://www.peanutbase.org/) was used as the promoter sequence. PCR primers for cloning of the AhRUVBL2 promoter were designed using Primer 5 (5.0, PREMIER Biosoft International, Palo Alto, CA, USA) and SnapGene (6.3, GSL Biotech LLC, Chicago, IL, USA) (see Supplementary Table 1). Gel recovery of the PCR products was performed using the E.Z.N.A.® Gel & PCR Clean Up Kit (Omega, Norcross, GA, USA). Target fragments of AhRUVBL2 promoter were cloned into the plasmid vector pUC18. The expression vectors pBI121-proAhRUVBL2::GUS were transformed into Agrobacterium tumefaciens GV3101 and used to infect Arabidopsis with the floral dip method [27].

Arabidopsis seeds were disinfected with 1 mL of 1% sodium hypochlorite for 5~10 min, uniformly spotted into 1/2 MS medium, and sealed with sealing film. The T₁ generation of Arabidopsis seeds harvested after infestation with Agrobacterium tumefaciens GV3101 (pBI121-proAhRUVBL2::GUS) were screened with a kanamycin plate. Green cotyledon plants were selected as the pure transgenic lines, and then continued to obtain the T₂ and T₃ generations of Arabidopsis [28]. Observations of the WT and transgenic Arabidopsis stems, leaves, flowers, fruit skin, and seeds were made following GUS ($\beta$-glucuronidase) staining [29].

Healthy, non-flowering tobacco plants in good growth condition and with up to four leaves were selected for infestation using a syringe with the needle removed. The tobacco leaves were injected with equal amounts of a pBI121-proAhRUVBL2 -79266::GUS and pBI121-proAhRUVBL2 -D893::GUS recombinant plasmid-transformed Agrobacterium tumefaciens GV3101 resuspension. pBI121-35S::GUS was used as a positive control, and resuspension as a negative control. Tobacco leaves that had been transiently transformed for 48 h were sprayed with ddH₂O as the control and two phytohormones, ABA (abscisic acid, 100 µM) and IAA (indole acetic acid, 100 µM), and then incubated for 24 h. Three replicates were used for each treatment. Tobacco Actin2 (NC_003074.8, AT3G18780.2) was used as an internal reference gene. The qRT-PCR-specific primer sequences for the GUS gene reference were previously reported by Anwar et al. [30].

All experiments were conducted with three replicates. Excel 2016 (16.0.4266.1001, Microsoft Corporation, Redmond, WA, USA) and SPSS 26.0 software (26.0, IBM, Armonk, NY, USA) were used for statistical analysis of data, with one-way analysis of variance (Student’s t-test). All values were calculated as the mean $\pm$ standard deviation (SD). Asterisks were used to indicate a significant difference as follows: * p 0.05, **p 0.01. All images and photos were annotated using ImageJ software (ImageJ 1. 54, National Institutes of Health, USA).

PCR was performed on DNA from T₅ generation Arabidopsis strains using AhRUVBL2 gene-specific primers. The target band of 1398 bp was amplified from the AhRUVBL2 transgenic strain, but not the non-transgenic plants (Supplementary Fig. 1). Expression analysis performed using qRT-PCR showed the relative expression of AhRUVBL2 in transgenic plants was significantly higher than that of WT Arabidopsis (Supplementary Fig. 1). The expression of AhRUVBL2 in WT Arabidopsis was similar to that of the internal reference gene. The expression of AhRUVBL2 in OE2 and OE3 transgenic Arabidopsis was 493-fold and 659-fold higher than that of the internal reference gene, respectively. These two transgenic lines, identified in the T₅ generation, were used in the subsequent experiments.

Phenotypic analyses were performed on plants from the T₅-generation AhRUVBL2-overexpressing Arabidopsis lines OE2 and OE3 grown normally for 4 weeks, with WT plants used as the control. AhRUVBL2-overexpressing plants were larger in size and had leaves of significantly greater length and width compared to the WT (Fig. 1A). The leaves from the OE2 and OE3 lines were 37.78% and 46.81% longer than the WT, respectively, and 25.69% and 42.21% wider, respectively (Fig. 2A,B). Overexpression of the AhRUVBL2 gene accelerated the growth of Arabidopsis (Fig. 1B), with OE2 and OE3 plants being 46.18% and 49.95% taller, respectively, than WT plants during the growth period (Fig. 2C). Moreover, the number of branches per plant was increased by 0.74 and 0.47 in OE2 and OE3, respectively, compared to the WT (Fig. 2D).

Fig. 1.

Phenotype of wild-type and AhRUVBL2-overexpressing Arabidopsis lines. (A) Seedling stage phenotype of WT and AhRUVBL2-overexpressing Arabidopsis. 1 to 3, three replicates. Scale bar = 1 cm. (B) WT and AhRUVBL2-overexpressing Arabidopsis during the growth period. (C) The siliques of WT, OE2 and OE3 from 4 d to 12 d after flowering. Scale bar = 1 mm. (D) Phenotype of Arabidopsis seeds from WT, OE2 and OE3. Scale bar = 500 µm. WT, wild-type; OE2 and OE3, AhRUVBL2-overexpressing Arabidopsis lines.

Fig. 2.

Phenotypic analyses of wild type and AhRUVBL2-overexpressing Arabidopsis lines. (A,B) Seedling stage leaf phenotype (length and width) analysis. (C,D) Plant height and branches per plant. (E,F) Silique length and width analysis, from 4 d to 12 d after flowering. (G–I) Phenotype analysis of seed length, width, and thousand-seed weight. (J–L) Time for bolting stage, flowering stage, and yellow fruit stage. *, p 0.05; **, p 0.01. WT, wild-type; OE2 and OE3, AhRUVBL2-overexpressing Arabidopsis lines.

Silique from WT, OE2 and OE3 were sampled at 4 d, 6 d, 9 d and 12 d after flowering, and their length and width measured. The silique length and width from OE2 and OE3 were significantly greater than those of WT at 4 d and 12 d after flowering (Fig. 1C). At 4 d after flowering, the silique length in OE2 and OE3 was 13.49% and 4.00% greater, and the silique width was 11.10% and 11.21% greater, respectively, compared to WT. At 12 d after flowering, the silique length was 3.71% and 6.18% greater, and the width 7.20% and 7.14% greater, respectively, compared to WT (Fig. 2E,F). Seeds from WT, OE2, and OE3 were harvested at maturity, dried at 37 ℃, and photographed under a microscope (Fig. 1D). The length of WT, OE2, and OE3 seeds was 228.5 $\pm$ 7.5 µm, 270.9 $\pm$ 8.9 µm, and 276.1 $\pm$ 9.6 µm, respectively, and the width was 146.6 $\pm$ 5.8 µm, 167.8 $\pm$ 4.4 µm, and 161.6 $\pm$ 4.3 µm, respectively (Fig. 2G,H). Therefore, the length and width of seeds from OE2 and OE3 were significantly greater than those from WT. The length of seeds from OE2 and OE3 was 18.54% and 20.84% greater, respectively, and their width was 14.44% and 10.19% greater, respectively, compared to those from WT. The thousand-seed weight for seeds from OE2 and OE3 was 45.97% and 66.02% heavier, respectively, than that of WT (Fig. 2I). In addition, OE2 and OE3 exhibited faster growth, entered the bolting and flowering stages earlier than WT, and exhibited early bolting and early flowering. The bolting stage for OE2 and OE3 was 2.5 d and 1 d shorter, respectively, than that of WT (Fig. 2J), while the flowering stage was 3 d and 1.7 d shorter, respectively (Fig. 2K). The time required to reach the yellow fruit stage in OE2 and OE3 was 0.3 d and 0.6 d longer, respectively, than that of WT (Fig. 2L).

Paraffin-embedded tissue sections of silique from WT, OE2 and OE3 at 4 d, 6 d, 9 d and 12 d after flowering were prepared in order to observe the number and size of cells. OE2 and OE3 showed greater silique pericarp thickness at 4 d to 12 d after flowering compared to WT (Fig. 3A). At 6 d after flowering, the pericarp was 16.70% and 41.92% thicker in OE2 and OE3, respectively, than in WT. The thickest pericarp was found at 12 d after flowering in OE2, and at 9 d after flowering in OE3, both of which were significantly greater than WT (Fig. 4A). The number and area of cells in a cross-section of silique were also evaluated from 4 d to 12 d after flowering (Fig. 3B). The number of exocarp cells did not differ significantly between the transgenic plants and WT. However, the exocarp cell areas in OE2 and OE3 from 4 d to 9 d after flowering were significantly larger than those of WT. The fastest growth in exocarp cell area was observed at 6 d after flowering in OE2 and OE3, with increases of 27.83 and 40.00%, respectively (Fig. 4B,C). The exocarp cell areas of OE2 and OE3 were still larger than those of WT at 12 d after flowering. The number of endocarp lignified cells at 6 d after flowering was 8.58 and 11.82% greater in OE2 and OE3, respectively, than in WT, while the area of endocarp lignified cells was 20.78 and 25.14% greater, respectively. The number of endocarp lignified cells at 4 d, 6 d, and 12 d after flowering was significantly higher in OE2 and OE3 than in WT. The fastest increase in the number of endocarp lignified cells was 10.76 and 13.16% in OE2 and OE3, respectively, at 4 d after flowering. The number of endocarp lignified cells increased by 8.58% in OE2 and 11.82% in OE3 at 6 d after flowering. Only a small change in the number of endocarp lignified cells was observed in OE2 and OE3 at 9 d to 12 d after flowering. Both OE2 and OE3 had significantly larger endocarp lignified cell areas than WT at 6 d after flowering. The fastest growth of exocarp cell area was observed at 4 d after flowering in OE2 (31.15% increase) and at 9 d after flowering in OE3 (36.42%) (Fig. 4D,E).

Fig. 3.

Cytological analysis of Arabidopsis silique. (A) Cross section of pod shell from 4 d to 12 d after flowering. Scale bar = 100 µm. (B) Cross section of silique from 4 d to 12 d after flowering. Scale bar = 100 µm. WT, wild-type; OE2 and OE3, AhRUVBL2-overexpressing Arabidopsis lines.

Fig. 4.

Statistical analysis of Arabidopsis silique phenotypic. (A) Silique thickness from 4 d to 12 d after flowering. (B,C) The number and area of exocarp cells. (D,E) The number and area of endocarp lignification cells. *, p 0.05; **, p 0.01. WT, wild-type; OE2 and OE3, AhRUVBL2-overexpressing Arabidopsis lines.

WT and AhRUVBL2-overexpressing Arabidopsis silique at 6 d after anthesis and under normal growth conditions were selected for transcriptome sequencing. The clean bases for each sample were above 6.22 G, the Q30 content was 94.27–97.08%, and the comparison rate with the Arabidopsis reference genome was 96.05–97.76%. Hence, the data displayed a high degree of accuracy and satisfied the requirements for subsequent bioinformatics analysis (Supplementary Table 2). A total of 38,345 expressed genes were detected in the six sequenced samples, including 38,299 known genes and 46 new genes. There were 337 DEGs between the silique of WT and OE, including 127 up-regulated and 210 down-regulated genes. There are a total of 18,646 DEGs between wt and OE silique in the Venn diagrams (Supplementary Fig. 2). Of the 18,464 DEGs, 453 were expressed only in WT silique and 422 only in OE silique. In WT, 44 DEGs were down-regulated, while 35 DEGs were up-regulated in OE, all of which were associated with cellular metabolic processes.

GO and KEGG enrichment analyses were performed on the DEGs between WT, OE1 and OE2. GO enrichment analysis showed that DEGs were mainly enriched in cellular processes, metabolic processes and bioregulation of biological processes, binding and catalytic activities of molecular functions, and cellular components of cellular organisms and protein complexes. Among the top 30 metabolic pathways enriched with DEGs in GO analysis were stress response to hypoxia, transmembrane transporter protein activity, and glycosyltransferase activity (Supplementary Fig. 3). KEGG enrichment analysis showed that DEGs were mainly enriched in plant-pathogen interaction and phenylpropanoid biosynthesis, followed by MAPK (Mitogen-activated Protein Kinase) signaling pathway, amino sugar and nucleotide sugar metabolism, and ABC (ATP-binding Cassette) transporter protein pathways. Further KEGG analysis of DEGs expressed specifically in OE angiosperms showed enrichment in plant hormone signal transduction and plant-pathogen interaction pathways (Supplementary Fig. 4).

Six DEGs related to the phenylpropane biosynthesis pathway were identified in silique: PRX71 (AT5G64120), PRX66 (AT5G51890) and PRX25 (AT2G41480), PER4 (AT1G14540) and ERF (AT1G24735) and UGT84A2 (AT3G21560) (Table 1). PRX71, PRX66 and PRX25 are plant peroxidase genes with very specific functions in plant growth and development, and in stress conditions. These functions include cell elongation, cell wall metabolism, lignification, growth hormone and anthocyanin metabolism, oxidation, and biotic stress. They were significantly up-regulated in OE silique, with more than 2-fold higher expression than WT silique. PRX (Periaxin) is also involved in cell proliferation, differentiation and apoptosis during cellular life processes. In contrast, the glycosyltransferase UGT84A2 affects plant growth and development by delaying the onset of flowering. Nine phytohormone signaling pathway-related DEGs were identified: PYL7 (AT4G01026), two IAA (AT4G14560 and AT4G14550), four SAUR (AT1G56150, AT5G18060, AT2G21200, AT1G75590) and two IAA (AT1G52830 and AT4G32280) (Table 2). A total of 203 differentially expressed transcription factors were identified amongst the DEGs of AhRUVBL2-overexpressing Arabidopsis, with 81 of them up-regulated in OE. The transcription factor families with the most up-regulated genes were NAC (9 genes), MYB-related (9), and FAR1 (6) (Fig. 5).

Fig. 5.

Up-regulated transcription factor families in AhRUVBL2-overexpressing Arabidopsis silique. The x-axis indicates the transcription factor family, while the y-axis indicates the number of up-regulated genes in each family.

The STRING database predicted 10 protein interactions with RUVBL2 in Arabidopsis. These included RUVBL2 protein (AT5G67630) interactions with ARP5/ARP4 (actin), SWC2 (DNA-binding proteins), PIE1 (proteins of the deconjugating enzyme structural domains), EEN (subunits of the chromatin remodeling complex), TRA1A (phosphatidylinositol kinase family proteins), SWC4 (myb-like transcription factor family proteins), TRA1B (phosphotransferase), RIN1 (RuvB-like proteins), and SEF (HIT-type zinc finger family proteins) (Fig. 6). Five interacting protein-related genes were predicted to be upregulated in OE silique, including ARP4 (AT1G18450), PIE1 (AT3G12810), TRA1A (AT2G17930), SWC4 (AT2G47210) and TRA1B (AT4G36080), combined with transcriptome sequencing data for AhRUVBL2-overexpressing Arabidopsis (Supplementary Table 3).

Fig. 6.

Protein interactions with RUVBL2 in Arabidopsis.

Positive plants were selected by kanamycin screening and transplanted into substrates for culture. Arabidopsis positive plants transformed with 35S, 79266, and D893 promoter-conjugated GUS expression vectors were named pBI121-35S::GUS, pBI121-proAhRUVBL2-79266::GUS, and pBI121-proAhRUVBL2-D893::GUS, respectively. Target fragments were amplified from both pBI121-proAhRUVBL2-D893::GUS and pBI121-proAhRUVBL2-79266::GUS positive plants (Supplementary Fig. 5). The T₁ generation of transgenic Arabidopsis at 6 d after flowering was selected for qPCR. GUS gene expression was detected in all specimens, with the activity of the 35S promoter found to be significantly higher than that of the AhRUVBL2 79266 and D893 promoters (Supplementary Fig. 5). Two pBI121-35S::GUS lines, three pBI121-proAhRUVBL2-79266::GUS pure lines, and four pBI121-proAhRUVBL2-D893::GUS pure lines were selected for further study.

Seedling and above-ground tissue samples from T₃ generation pBI121-35S::GUS, pBI121-proAhRUVBL2- 79266::GUS, and pBI121- proAhRUVBL2-D893::GUS transgenic Arabidopsis plants underwent GUS staining (Fig. 7A). WT Arabidopsis showed no blue stain in all tissues. Arabidopsis transformed with pBI121-35S::GUS stained distinctly blue in the stems, leaves, flowers, pericarp, and seeds. Arabidopsis transformed with pBI121-proAhRUVBL2-79266::GUS or pBI121-proAhRUVBL2-D893::GUS displayed stems, leaves, flowers and pericarp that were devoid of blue staining, stamens that stained light blue, and significant blue staining in the seeds. PCR of WT and transgenic Arabidopsis cDNAs showed bright specific bands for both target and internal reference genes, with no primer dimers observed (Supplementary Fig. 6). The silique, stems, leaves, and flowers of T₃ generation trans proAhRUVBL2::GUS Arabidopsis plants were analyzed by qPCR. The AhRUVBL2 promoter showed the highest expression in silique, with lower expression visible in the stems, leaves, and flowers (Fig. 7B).

Fig. 7.

GUS expression vectors constructed using different promoters in Arabidopsis. (A) GUS expression vectors constructed by different promoters in Arabidopsis. 1, Wild-type Arabidopsis; 2, pBI121-35S::GUS transgenic Arabidopsis; 3, pBI121-proAhRUVBL2-79266::GUS transgenic Arabidopsis; 4, pBI121-proAhRUVBL2-D893::GUS transgenic Arabidopsis; a, seedling plant; b, stem; c, leaf; d, flower; e, seed; f, silique. Scale bar = 500 µm. (B) Relative expression of the AhRUVBL2 D893 and 79266 promoters in transgenic Arabidopsis tissues at days 3, 6, 9 and 12 of silique development after flowering; S, stem; L, leaves; F, flowers. **, p 0.01.

The proAhRUVBL2::GUS vectors 893 and 79266 were injected into tobacco leaves by agrobacterium-mediated transformation. Injection of pBI121-35S::GUS empty vector was used as the positive control, and tobacco injected with buffer was used as the negative control. With the exception of the negative control, GUS activity was detectable as blue colour in all transgenic tobacco leaves. pBI121-35S::GUS displayed the strongest transcriptional activity, followed by pBI121-proAhRUVBL2-D893::GUS, and pBI121-proAhRUVBL2-79266::GUS showing weaker promoter activity (Fig. 8). Transiently transformed tobacco leaves were treated with IAA and ABA, and the expression of GUS genes detected by qPCR. The expression of proAhRUVBL2-D893 was significantly down-regulated by ABA treatment, while that of proAhRUVBL2-79266 was significantly up-regulated. IAA treatment had no significant effect on the expression of proAhRUVBL2-D893, but significantly down-regulated the expression of proAhRUVBL2-79266 (Fig. 9).

Fig. 8.

Detection of GUS ($\beta$-glucuronidase) activity in tobacco leaves after transient promoter transformation. (a) Negative control. (b) Positive control. (c) pBI121-proAhRUVBL2-79266::GUS. (d) pBI121-proAhRUVBL2-D893::GUS. Scale bar = 1000 µm.

Fig. 9.

GUS expression of proAhRUVBL2 after hormone treatment (IAA, indole acetic acid and ABA, abscisic acid). (A) proAhRUVBL2-D893. (B) proAhRUVBL2-79266. *, p 0.05; **, p 0.01.