Study of the Function and Expression of the AhRUVBL2 Promoter in Arabidopsis

Abstract

Background:

Cultivated peanut (Arachis hypogaea L.) is a major oil and economic crop. Pod size is one of the important agronomic traits of peanut variety, with a direct impact on peanut yield.

Methods:

In a previous study, AhRUVBL2 was identified by map-based cloning technology as a candidate gene that regulates peanut pod size.

Results:

Overexpression of AhRUVBL2 in transgenic Arabidopsis significantly increased plant height, branch number, leaf size, silique size, seed size, and thousand-seed weight. Further examination revealed an increase in the area of silique exocarp cells, and the number and area of endocarp lignified cells. A total of 337 differentially expressed genes, including PRX (Periaxin) , SAUR (Small Auxin-up RNA), and PYL (Pyrabactin Resistance 1-like), were identified by transcriptome analysis of transgenic Arabidopsis silique. proAhRUVBL2-GUS was found to be expressed explicitly in seeds, and the expression activity of proAhRUVBL2-D893 was significantly greater than that of proAhRUVBL2-79266. Exogenous ABA (abscisic acid) and IAA (indole acetic acid) treatment of proAhRUVBL2-GUS-transformed tobacco leaves revealed that the AhRUVBL2 promoter was hormone-responsive.

Conclusions:

This study sheds light on the function of AhRUVBL2 in regulating plant growth and development. Moreover, characterization of the AhRUVBL2 promoter provides a valuable genetic resource for enhancing peanut yield.

Graphical abstract

Keywords

peanut / AhRUVBL2 / promoter / expression characteristics / pod size

1. Introduction

The cultivated peanut or groundnut (Arachis hypogaea L.) is one of the most important oil and economic crops, and is widely grown in semiarid tropical and subtropical regions. Global annual peanut production has risen rapidly, from 45.2 million tons in 2015 to 54.3 million tons in 2022 (http://www.fao.org/faostat, 2025). However, this still does not satisfy the demands of a growing global population, and increasing the peanut yield per unit area remains a major challenge for peanut breeders. Pod and seed size are direct determinants of peanut yield, with this harvest organ regulated by a variety of plant hormones [1]. Seed size is a typical quantitative trait regulated by many genes and significantly influenced by the environment [2, 3, 4]. Therefore, exploration of the function and mechanism of genes that regulate pod size is important for improving the breeding of peanuts.

In many crops, pod size is an economically important trait that affects the crop yield and quality. Researchers have made great progress in the study of genes related to crop improvement, as well as the various molecular mechanisms involved [5]. For example, by using 156 recombinant inbred lines constructed from ND_S and ND_L parents and 88 peanut germplasm resources, Zhao et al. [6] identified the gene PSW1 that regulates peanut pod size. The different stages of pod growth and development are known to be associated with regulation of a variety of phytohormones, including auxin, abscisic acid, ethylene, cytokinin, and gibberellins [7, 8, 9]. Auxin is a key phytohormone that regulates plant pod development, and many of the genes involved in growth hormone signaling are involved in regulating pod size [10, 11, 12]. Previous studies have focused on the mechanism that regulates pod development [6, 13]. With the progress of technology, it has been found that tissue-specific promoters exert a targeted expression and regulation effect on exogenous genes, thereby regulating gene expression in specific organs and tissues.

RUVBL (Recombinant human RuvB-like) proteins, named RuvA and RuvB, were first isolated from Escherichia coli and later shown to interact with TATA-binding proteins (TIP49a and TIP49b) in humans, rats, and yeast [14, 15, 16]. Both RUVBL2 and its homologous protein RUVBL1 are members of the AAA+ superfamily, which are evolutionarily highly conserved AAA+ ATPases involved in various cellular processes [17, 18]. RUVBL2 and RUVBL1 share very similar structures and conserved structural domains, and both form a complex that plays a central role in regulating the cell cycle and mitotic progression [19, 20, 21]. Silencing of the RUVBL1 gene in Arabidopsis results in delayed plant development, dwarf plants, and abnormal leaf development [22]. RUVBL2 proteins are highly conserved ATPases involved in the regulation of cell division and expansion, but less is known about these proteins in plants. A major QTL (Quantitative trait locus) related to pod size, qAHPS07, was fine mapped to a 36.46 kb interval on chromosome A07 using F₂, recombinant inbred line (RIL) and secondary F₂ populations derived from a cross between variety 79266 (as the female, small pod) and D893 (as the male, big pod). The results of forward and reverse genetics strongly indicated that AhRUVBL2 (Arahy.TSR8I7) was the most likely candidate gene for qAHPS07. AhRUVBL2 may regulate the proliferation of pod shell cells, which ultimately affects the pod size [23].

In this study, phenotypic and cytological analyses of AhRUVBL2 transgenic Arabidopsis were carried out to study the regulatory mechanism of AhRUVBL2 in plants. The AhRUVBL2 promoter sequences of 79266 and D893 were cloned to allow construction of proAhRUVBL2-GUS expression vectors. These were transformed into Arabidopsis plants and tobacco leaves to analyse specific expression of the promoter in tissues. The regulatory mechanism of RUVBL2 protein expression in plants was clarified, thus providing a theoretical reference for transgenic breeding of the peanut.

2. Materials and Methods

2.1 Plant Materials

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.

2.2 Identification of Transgenic Arabidopsis

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].

2.3 Phenotyping and Cytological Observations of Arabidopsis

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.

2.4 Transcriptome Sequencing of AhRUVBL2 in Arabidopsis Silique

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)| \ge1. 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.

2.5 Cloning and Expression Vector Construction of the AhRUVBL2 Promoter

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].

2.6 Genetic Transformation of the Expression Vectors pBI121-proAhRUVBL2::GUS

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].

2.7 Effect of Exogenous Hormones on Expression of the AhRUVBL2 Promoter in Tobacco

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].

2.8 Statistical Analysis

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).

3. Results

3.1 Identification of AhRUVBL2 Transgenic Arabidopsis

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.

3.2 Phenotypic Analyses of T₅-Generation AhRUVBL2-Overexpressing Arabidopsis Lines

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).

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).

3.3 Cytological Analysis of Silique Development in Transgenic and WT Arabidopsis

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).

3.4 Transcriptome Analysis of AhRUVBL2-Overexpressing Arabidopsis

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.

3.5 GO and KEGG Enrichment Analysis of DEGs

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).

3.6 Identification of DEGs

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).

3.7 Prediction of Proteins Interacting With RUVBL2 in Arabidopsis

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).

3.8 Expression Characterization of the AhRUVBL2 Promoter

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).

3.9 Regulation of the AhRUVBL2 Promoter by Exogenous Hormones

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).

4. Discussion

Pod size is the most important yield trait of the peanut, and one of the main traits studied in current research [31, 32]. The pod size is closely related to the number and area of cells [33, 34]. A large number of QTLs have been identified, and many important genes have been cloned [1, 35]. In Arabidopsis, silencing of the RUVBL1 gene leads to retardation of plant growth, shorter plants, and deformity of leaf development [22]. Lu et al. [36] conducted genome-wide association studies (GWAS) on 28 agronomic traits across 390 peanut accessions. They identified the candidate gene AhANT associated with pod and seed weight, and within an 886.7 kb interval on chromosome B06. Functional validation in transgenic Arabidopsis revealed that AhANT negatively regulates cell number in organs. Given the limitations of peanut transgenic systems and the advantages of Arabidopsis as a model plant due to its short life cycle, researchers have increasingly utilized Arabidopsis for functional gene studies. Building on the prior work of Yang et al. [23] who identified the AhRUVBL2 gene, the present study systematically analyzed phenotypic traits in AhRUVBL2-overexpressing Arabidopsis lines to investigate its biological roles. AhRUVBL2 overexpression not only promoted the growth of Arabidopsis plants, but also significantly increased the area of exocarp cells in siliques, and the number and area of lignification cells in endocarp. In addition, RUVBL2 and RUVBL1 have very similar structures and conserved domains, with this complex playing a central role in regulating the cell cycle and mitosis [20]. Therefore, we speculated the AhRUVBL2 gene regulates plant growth and development by controlling cell expansion and thus affecting pod size.

Plant promoters are critical regulatory elements that facilitate the binding of RNA polymerase and transcription factors, thereby playing a pivotal role in initiating and regulating gene transcription. Tissue-specific plant promoters are of particular interest due to their ability to drive the expression of exogenous genes in specific tissues or organs of transgenic plants. This targeted expression not only increases the efficiency of genetic transformation in desired plant parts, but it also has significant potential for improving crop quality through precise spatial and temporal regulation of transgene expression [37]. Seed-specific promoter is one of the tissue-specific promoters. Transcription factors may bind to conserved motifs in the promoter, thereby activating the specific expression of genes in seeds [38]. Seed-specific promoters generally have special regulatory elements, such as RY-motif, GCN4 (General Control Nonderepressible 4), AACA, CCAA and TATAA [39, 40]. Analysis of the AhRUVBL2 promoter sequence revealed the presence of seed-specific expression elements, namely AACC and CCAA motifs. Additionally, this promoter region contains conserved cis-acting elements such as the CAAT-box and G-box, which are commonly found in seed-specific promoters. Furthermore, the AhRUVBL2 promoter exhibits characteristics of hormone-responsive promoters, which classifies it as an inducible promoter. Notably, ABA-inducible promoters typically harbor ABRE (abscisic acid-responsive element) cis-regulatory elements that are critical for mediating responses to abscisic acid signaling. In wheat and rice seeds, ABRE elements mainly regulate gene expression during the late stage of development [41]. Auxin-inducible promoters have a variety of auxin response elements, including AuxRE, TGA-box, As-1-box and Ocs-element. When the auxin concentration is high, this leads to degradation of the transcription inhibitor Aux/IAA. The auxin response factor (ARF) can then bind to the AuxRE cis-acting element to promote the expression of auxin response genes [42].

In the current study, bioinformatics analysis of the promoter sequence located 2527 bp upstream of ATG in the AhRUVBL2 gene revealed that it contained hormone responsive cis-elements, including two ABA response elements and one auxin response element. We first transferred GUS expression vectors containing the AhRUVBL2 promoter sequences D893 and 79266 into Agrobacterium by transformation assay. WT Arabidopsis was then transformed by the floral dip method, followed by histochemical staining and real-time fluorescence quantification of GUS. The AhRUVBL2 promoters D893 and 79266 were mainly expressed in the seeds of Arabidopsis siliques. By spraying exogenous hormones, ABA treatment was shown to significantly down-regulate the expression of proAhRUVBL2-D893, and significantly up-regulate the expression of proAhRUVBL2-79266. IAA treatment had no significant effect on the expression of proAhRUVBL2-D893, but significantly down-regulated the expression of proAhRUVBL2-79266. There is currently a dearth of information on the regulation of AhRUVBL2 by plant hormones, and the role of auxin and ABA in the regulation of this gene in plants requires further study.

Plant hormones play important roles in regulating plant growth and development, as well as their adaptation to various abiotic and biotic stresses. The SAUR gene family is involved in plant-specific auxin responses and plays a vital role in seed development in many plants. In Arabidopsis, overexpression of AtSAUR63, a homologous gene of SAURs, leads to the elongation of hypocotyls, petals, stamen and filaments [43]. PRX is involved in regulating the hydrogen peroxide level during the cell cycle, which in turn is linked to signal transduction during cell proliferation, differentiation and apoptosis [44]. The plant peroxidase genes PRX71 (AT5G64120), PRX66 (AT5G51890) and PRX25 (AT2G41480) are key genes in the phenylpropanoid biosynthesis pathway that regulate Arabidopsis siliques [45, 46]. In the present study, AhRUVBL2-overexpression significantly promoted the growth of Arabidopsis and the development of silique cells. Transcriptome sequencing of the siliques of AhRUVBL2-overexpressing and WT Arabidopsis showed that AhRUVBL2 can affect the growth and development of plants, and fruit ripening, by promoting the expression of genes associated with phenylpropanoid biosynthesis and plant hormone signal transduction. Previous transcriptome sequencing results [23] show that the expression pattern of ARP4 (Arahy.1T6SIZ) in peanut is highly consistent with that of AhRUVBL2. Furthermore, ARP4 was up-regulated in transgenic Arabidopsis, suggesting the protein encoded by this gene may interact with the AhRUVBL2 protein. In Arabidopsis, the loss of function of AtARP4 leads to smaller leaves, demonstrating that ARP4 is involved in regulating plant growth [47]. Therefore, we speculate that AhRUVBL2 protein mainly interacts with ARP4 in the regulation of plant growth and development.

5. Conclusions

In this study, overexpression of AhRUVBL2 in transgenic Arabidopsis was found to significantly increase plant height, branch number, leaf size, pod size, seed size, thousand-seed weight, and pericarp thickness, while significantly shortening the bolting and flowering stages. In addition, overexpression of AhRUVBL2 increased the area of silique exocarp cells, and the number and area of lignified cells in endocarp. A total of 337 DEGs were identified in transgenic Arabidopsis. These included the key genes PRX, SAUR and PYL that regulate development of Arabidopsis siliques and are closely related to AhRUVBL2 expression, as well as the ARP4 protein that interacts with AhRUVBL2. The AhRUVBL2 promoter is a seed-specific and hormone-induced promoter, with the promoter activity of AhRUVBL2 D893 being significantly greater than that of AhRUVBL2 79266. These results should prove useful for the breeding of high-yield peanut varieties and for the study of molecular mechanisms in peanut growth.

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Funding

National Key Research and Development Programs of China(2023YFD1202800)

Peanut Seed Industry Project in Shandong Province of China(2022LZGC007)

Key R&D Program of Shandong Province, China(2024LZGC031)

Taishan Industrial Experts Program(tscx202408152)

Earmarked fund for the Agriculture Research System in Shandong Province(SDAIT-04-03)

Natural Science Foundation of China(31571711)