Genome-wide identification and expression analysis of GA20ox and GA3ox genes during pod development in peanut

Identification of AhGA20ox and AhGA3ox gene family members in peanut

All sequences were downloaded from four databases: TAIR (Arabidopsis Information Resource, http://www.arabidopsis.org/), Rice (Rice Information Resource, http://www.rice.plantbiology.msu.edu/), Soybean Genome Annotation Project Database (http://www.phytozome.net/soybean/) and NCBI Genome Database (https://www.ncbi.nlm.nih.gov/). The Arabidopsis GA20ox and GA3ox protein sequences were first downloaded from TAIR website. Subsequently, the amino acid sequences of GA20oxs and GA3oxs of Arabidopsis were searched against peanut genome using NCBI database. The resulting sequences were further validated in SMART (http://smart.embl.de) and Pfam (http://pfam.xfam.org) to obtain all members of AhGA20ox and AhGA3ox with the 2OG-Fe (II) oxygenase domain (PF03171) in peanut genome. Previous study showed that the allotetraploid A. hypogaea (AABB, 2n = 4x = 40) is from hybridization between diploids A. duranensis (AA genome) and A. ipaensis (BB). One to ten (A01–A10) chromosomes originate from A subgenome, and eleven to twenty (B01–B10) chromosomes originate from B subgenome. In this study, gene members were ordered according to the order of chromosomes numbering from A and B subgenome. For example, AhGA20ox1 and AhGA20ox2 are from chr. 2 (A02), while AhGA20ox3 and AhGA20ox4 are from corresponding chr.12 (B02) (Table 1). The isoelectric point (pI), molecular weight (MW), and amino acid (aa) number of GA20ox and GA3ox proteins were predicted using the ProtPram tool in ExPASy (https://www.expasy.org/).

Phylogenetic analysis of GA20ox and GA3ox

Multiple sequence alignment of AhGA20ox and AhGA3ox amino acid sequences with the 2OG-Fe (II) oxygenase domain from peanut and homologous sequences from Arabidopsis thaliana, rice, and soybean was performed using the DNAMAN software (Vers7; Lynnon Corporation, Montreal, QC, Canada). Then, phylogenetic analysis was performed using AhGA20ox and AhGA3ox protein sequences together with other plant species by using neighbor-joining method in MEGA (Vers7; Pennsylvania State University, PA, USA) with bootstrap value of 1000 (Kumar, Stecher & Tamura, 2016). Finally, iTOL (https://itol.embl.de/) online software was used for the display, annotation and management of phylogenetic trees.

Chromosomal distribution and gene structure analysis of AhGA20ox and AhGA3ox

The loci of GA20ox and GA3ox genes were downloaded from the genome annotation file from Peanutbase in order to obtain chromosome location information. Then, the chromosome map was generated using Mapchart software (version 2.2) (http://mg2c.iask.in/mg2c_v2.1/). The online tool GSDS (version 2.0) (http://gsds.cbi.pku.edu.cn) was employed to analyze the genomic sequences of AhGA20ox and AhGA3ox for gene structure analysis.

Analysis of the conserved protein motifs and cis-acting elements

The online webserver of MEME software was used to analyze the conserved protein motifs of peanut AhGA20ox and AhGA3ox family members. The occurrence of top 8 conserved protein motifs in AhGA20ox and AhGA3ox sequences were further screened and analyzed.

The 2000 bp sequence upstream of the start codon of the AhGA20ox and AhGA3ox genes was downloaded from the peanut genome database (https://www.ncbi.nlm.nih.gov/), and the cis-acting elements were predicted and analyzed by PlantCARE online (http://bioinformatics.psb.ugent.be/).

AhGA20ox and AhGA3ox protein interaction networks prediction

The online webtool of STRING network (https://string-db.org/) was utilized to predict the functional protein interaction network of AhGA20ox and AhGA3ox. All possible interacting proteins constituting a hierarchical network with AhGA20ox and AhGA3ox were further classified and shown graphically in STRING-generated network.

Plant materials and treatment conditions

The peanut cultivar Jihua8 with high oleic acid developed by our lab was used in this study. Jihua8 plants were grown in the field condition from May to September 2022 at Jiyang Experimental Station of Shandong Academy of Agricultural Sciences, Shandong, China (36°58′34.53″N, 116°59′1.29″E). In the growing period, the temperature ranged from 20 °C to 37 °C. Stems, leaves, flowers, inflorescences and pods at different stages of development were collected with six biological replicates from plants. Pegs and pod samples were collected at 0, 3, 9, 12, 20, and 30 days after soil penetration (DAP), representing the S1, S2, S3, S4, S5, and S6 stages, respectively (Fig. 1). All the materials were frozen immediately in liquid nitrogen and stored at −80 °C for RNA extraction and gene expression analysis.

RNA extraction and expression analysis of AhGA20ox and AhGA3ox

Total RNA was extracted by using FastPure Plant Total RNA isolation kit (AG RNAex Pro Reagent, Changsha, China). The extracted RNA was used as template for reverse transcription reaction using Hiscrip II Q RT SuperMix for qRT-PCR (Vazyme, Nanjing, China). The primers of qRT-PCR were designed by Primer Premier software (Premier Biosoft International, Palo Alto, CA, USA) (Table S1). An ABI 7500 real-time PCR instrument (Thermo Fisher Scientific, Waltham, USA) was used for subsequent quantitative reaction. The reaction mixture consisted of 10 µL ChamQ SYBR qPCR Master Mix (Vazyme, Nanjing, China), 0.5 µL of each primer (10 µM), 2 µL cDNA template, and 7 µL RNase-free H₂O. Reaction conditions included pre-denaturation (95 °C, 30 s), cyclic reaction (95 °C, 10 s; 60 °C, 30 s; 40 cycles). The relative expression level of these genes was analyzed by the 2^−ΔΔCT method (Livak & Schmittgen, 2001), using Ahactin gene as the internal control.

Identification and characterization of AhGA20ox and AhGA3ox genes

BLASTP (e-value ≦ 0.001) search was performed using the amino acid sequences of five Arabidopsis GA20ox and four GA3ox. After removing redundant sequences and confirming the presence of gibberellin-dioxygenases domains by SMART and Pfam, 20 gibberellin-dioxygenases genes including 15 GA20ox (AhGA20ox1-15) and five GA3ox (AhGA3ox1-5) were finally retained and used for further analysis. Further analysis showed that the length of amino acid encoded by AhGA20ox genes varied from 363 aa to 428 aa, and the molecular weight was 41.3 kDa-49.2 kDa. The isoelectric point (pI) values were between 5.14 and 7.09. The amino acid length of GA3oxs varied from 352 aa to 375 aa, the molecular weight was 40 kDa–41.7 kDa, and the pI values varied from 6.49 to 8.11 (Table 1).

Phylogenetic analysis of AhGA20ox and AhGA3ox

In order to analyze the phylogenetic relationship of GA20ox and GA3ox gene family, the phylogenetic tree was constructed using the protein sequences of GA20ox and GA3ox from peanut, Arabidopsis thaliana, rice, and soybean (Table S2). The results showed that the GA20oxs and GA3oxs were divided into three groups (group A-C). The largest group was group C containing 32 members of GA20ox and all peanut GA20oxs were in this group. Groups A was the smallest one only containing four members of rice GA20ox (Fig. 2). Group B contained 17 members, all of which were GA3ox. Moreover, in each group, genes from the same species showed higher similarity than those from different species. Further analysis revealed that GA20ox and GA3ox family members in peanut were closely related to those in soybean, but distantly related to those in rice (Fig. 2).

Gene structure and conserved motifs of AhGA20ox and AhGA3ox genes

The exon-intron structure analysis of 15 AhGA20ox genes showed that AhGA20ox genes contained three exons and two introns except that AhGA20ox4 had four exons and three introns. The closer genes in the phylogenetic tree showed more similarity in structure, for example, AhGA20ox13/14 and AhGA20ox9/11. However, AhGA20ox4 and AhGA20ox1 were closely related in the phylogenetic tree, but very different in gene structures. It may due that AhGA20ox4 gene acquired a long intron in the first exon. Five AhGA3ox genes contained two exons and two introns, and the length of the introns varied greatly. These differences in exon-intron structure may suggest the functional differentiation of these genes (Fig. 3).

The conserved motifs of AhGA20ox and AhGA3ox proteins were predicted. In the predicted eight motifs, the E value of each motif was significant, and the length of motif was 36-50 aa. Motif 1, 2, 3 and 5 were distributed in all AhGA20ox and AhGA3ox sequences (Fig. 4), suggesting that these four motifs may be the core conserved domain of these gene families. This indicated that the AhGA20ox and AhGA3ox families were highly conserved and presumably had some degree of functional redundancy. In addition, motif 8 existed only in the AhGA3ox gene family (Fig. 4), while motif 4 and 6 existed only in the AhGA20ox gene family (Fig. 4).

Cis-element analysis of AhGA20ox and AhGA3ox genes

The analysis of cis-elements was performed using the 2000 bp promoter region of AhGA20ox and AhGA3ox genes. The results showed the presence of widely known eight key cis-elements in the promoter of these genes (Fig. 5, Tables S3 and S4). The most abundantly presented cis-elements mainly included light-responsive units such as Box 4, G-box, TCT-motif, GA-motif, GATA-motif, AT1-motif and GT1-motif. Hormone-responsive units such as ABA responsive-motif (ABRE), auxin-responsive element (TGA-element), gibberellin-responsive element (TATC-box, GARE-motif and P-box) and salicylic acid responsiveness (TCA-element) were also detected. Low-temperature responsive units (LTR), drought-inducibility (MBS) and defense and stress responsiveness (TC-rich repeats) elements were also identified. The occurrence of these cis-elements suggested that AhGA20ox and AhGA3ox genes are strongly linked to plant growth, development, and tolerance to varied stresses, as well as other crucial signaling pathways in peanut.

Chromosome location and selective pressure analysis of AhGA20ox and AhGA3ox genes

Based on the peanut genome information, AhGA20ox and AhGA3ox genes were distributed on 14 chromosomes. AhGA20ox1 and AhGA20ox2 were localized at chr.2. AhGA20ox9 and AhGA20ox10 were localized at chr.5, whereas AhGA20ox12 and AhGA3ox2 were mapped to chr.8. AhGA20ox13 and AhGA3ox4 were positioned at chr.9, while AhGA20ox3 and AhGA20ox4 were found at chr.12. AhGA20ox14 and AhGA3ox5 were located at chr.19. AhGA20ox5, AhGA20ox7, AhGA3ox1, AhGA20ox6, AhGA20ox8, AhGA20ox11, AhGA20ox15 and AhGA3ox3 were localized at chr.3, chr.4, chr.7, chr.13, chr.14, chr.15, chr.17 and chr.18, respectively. Notably, most of AhGA20ox and AhGA3ox genes located at the distal ends of chromosomes (Fig. 6, Table S5).

TBtools was used to calculate the Nonsynonymous (Ka)/synonymous (Ks) ratios for each gene pair, to explore the evolutionary constraints of the peanut GA20ox and GA3ox genes (Table S6). The Ka/Ks ratios of gene pairs from peanut (Ah-Ah), soybean (Gm-Gm), and peanut and soybean (Ah-Gm) were all less than 1, indicating that the orthologs in the related species experienced purification selection, meanwhile, the paralogs in the species were highly conserved.

Interactive protein network of AhGA20ox and AhGA3ox proteins

The protein–protein interaction (PPI) network of AhGA20ox and AhGA3ox was investigated using the STRING database. The major interacting partners of AhGA20ox AhGA3ox were predicted as Fe2OG dioxygenase domain containing protein, which is a key component of iron-ascorbate dependent oxidoreductase family. Fe2OG enzyme is known to catalyze a wide range of oxidative reactions crucial to plant metabolisms. The interactions between GA20ox, GA3ox and GA2ox in peanut were very close. The lines in the figure indicated the confidence of the interaction between the two proteins, and the line thickness indicates the strength of data support. GO classification results indicated that these genes were mainly involved in short-day photoperiodism, flowering, GA metabolism, and GA-mediated signal transduction pathway. A0A444XWP4 had 18 interacting proteins, GA3ox1/4/5, A0A444Y5G9, A0A445BYM0 and A0A444YUS4 all had 17 interacting proteins (Table S7). A0A444XWP4, A0A444Y5G9 and A0A445BYM0 were speculated as GA2ox, a key enzyme catalyzing GA degradation. Amino acid sequences blasting result indicated that A0A444YUS4 was GA3ox. No interacting protein was found in AhGAox5/6/13/15, while AhGAox12 only had a weak interaction with A0A444XWP4. (Fig. 7). The prediction of PPI network of these GA biosynthesis enzymes in peanut provided important information for understanding the regulatory mechanism underlying gibberellin biosynthesis.

Expression analysis of AhGA20ox and AhGA3ox genes during pod development

The expressions of GA20ox and GA3ox genes during peanut pod development were investigated using qRT-PCR. Peanut pod development was divided into six stages in this study. In S1 stage, the green or purple pegs were aerial-grown. In S2 stage, pegs had been embedded in the soil for 3 days and the color of the pegs is white. Pod enlargement in S2 pegs was not detected. In S3 stage, pegs had been buried in the soil for 9 days and pod enlargement was initiated. In S4 stage, pegs had been buried in the soil for 12 days. In S5 stage, pegs had been buried in the soil for 20 days. In S6 stage, pegs had been buried in the soil for 30 days (Fig. 1).

Both AhGA20ox and AhGA3ox gene families were expressed ubiquitously at different stages of pod development, but showed different expression patterns. The expressions of AhGA20ox2/AhGA20ox3, AhGA20ox6/AhGA20ox5, AhGA20ox7/AhGA20ox8 GA20ox8 and GA20ox10 gradually increased with the development of pod, and reached the highest level at S6 (Fig. 8, Table S8). The expression levels of AhGA20ox15/AhGA20ox12 and AhGA3ox1 gradually decreased with the development of peanut pod.Theses results indicated that different members of AhGA20ox and AhGA3ox families had different functions in pod development. AhGA20ox1/AhGA20ox4, AhGA20ox12/AhGA20ox15, AhGA3ox1 and AhGA3ox4/AhGA3ox5 showed high expression levels in S1 (Fig. 8), suggesting their possible roles in regulating peg elongation. Other AhGA3ox genes may be involved in regulating the expansion and growth of pod, even though they showed different expression patterns during pod development. For example, AhGA3ox1 and AhGA3ox2/AhGA3ox3 showed high expression levels in S2, and AhGA20ox13/AhGA20ox14 showed the highest expression level in S3. The expression levels of AhGA20ox1/AhGA20ox4 and AhGA20ox9/11 were the highest in S4 (Fig. 8, Table S8).

Expression analysis of AhGA20ox and AhGA3ox genes in different tissues

The expressions of AhGA20ox and AhGA3ox genes in peanut leaves, main stems, flowers and inflorescences were analyzed. The results showed that AhGA20ox and AhGA3ox were expressed in different tissues with varied levels. The expression levels of AhGA20ox9/AhGA20ox11 and AhGA3ox4/AhGA3ox5 were the highest in the main stem, while the expression levels of AhGA20ox12/AhGA20ox15 were the lowest in the main stem (Fig. 9, Table S9). Except AhGA20ox9/AhGA20ox11, AhGA3ox1 and AhGA3ox4/AhGA20ox5, other genes showed high expression levels in flowers (Fig. 9), implying that these genes may be involved in the regulation of flower development. AhGA20ox2/AhGA20ox3, AhGA20ox5/AhGA20ox6, AhGA20ox7/AhGA20ox8, AhGA20ox13/AhGA20ox14 and AhGA3ox2/AhGA3ox3 were highly expressed in inflorescence (Fig. 9). AhGA20ox1/ AhGA20ox4, AhGA20ox12/AhGA20ox15 and AhGA3ox1 showed high expression levels in leaves (Fig. 9, Table S9).